TW201836623A - Treatment of retinal degeneration using progenitor cells - Google Patents

Abstract

Methods and compositions for treating and reducing retinal degeneration using progenitor cells and conditioned media from progenitor cells, such as postpartum-derived cells are disclosed. Trophic factors and other agents secreted by the progenitor cells that protect retinal cells and inhibit apoptosis of retinal cells such as photoreceptor cells are also disclosed.

Description

   Using precursor cells to treat retinal degeneration    [Cross Reference of Related Applications]

This application is a partial continuation of US Patent Application No. 14 / 960,006 filed on December 4, 2015, and it claims US Provisional Application No. 62 / 092,658 filed on December 16, 2014 and 2015 The rights and interests of US Provisional Application No. 62 / 236,732 filed on October 2, 2003, the entire contents of the above cases are incorporated by reference.

The present invention relates to the field of cell-based or regenerative therapies for ophthalmic diseases and disorders. In detail, the present invention provides methods and compositions for regenerating or repairing eye cells and tissues using precursor cells (such as umbilical cord tissue-derived cells and placental tissue-derived cells) and conditioned medium prepared from these cells.

The eye is a complex and sensitive body organ that can suffer from many diseases and other harmful conditions that affect its ability to function normally. Many of these pathologies are associated with damage or degeneration of specific eye cells and tissues composed of these cells. For example, diseases and degenerative conditions of the optic nerve and retina are the leading causes of blindness worldwide. Damage or degeneration of the cornea, crystals and related eye tissues is another significant cause of vision loss worldwide.

The retina contains seven layers of alternating cells and the process of converting light signals into neural signals. The retinal photoreceptor forms a functional unit with the adjacent retinal pigment epithelium (RPE), which in many disorders becomes unbalanced due to genetic mutations or environmental conditions (including age). This leads to the loss of photoreceptors through apoptosis or secondary degeneration, leading to progressive deterioration of vision and in some cases blindness (for a review, see, for example, Lund, RD et al., Progress in Retinal and Eye Research , 2001; 20: 415-449). Two types of eye disorders with this pattern are age-related macular degeneration (AMD) and retinitis pigmentosa (RP).

AMD is the most common cause of vision loss in the 50-year-old or older population in the United States and its prevalence increases with age. The primary disease of AMD appears to be due to abnormal RPE function and changes in Bruch's membranes, which is characterized by, inter alia, lipid deposition, protein cross-linking, and reduced nutrient permeability (see Lund et al., See above in 2001). Various factors can cause macular degeneration, including genetic makeup, age, nutrition, smoking, and exposure to sunlight or other oxidative stress. Non-exudative, or "dry" forms of AMD account for 90% of AMD cases; the other 10% are exudative, neovascularized forms ("wet" AMD). In patients with dry AMD, the gradual disappearance of retinal pigment epithelium (RPE) results in surrounding atrophic areas. Because the loss of photoreceptors follows the disappearance of RPE, there is little or no visual function left in the affected retinal area.

AMD's current therapies involve procedures such as, for example, laser therapy and medical intervention. The laser beam transfers heat energy to destroy the leaking blood vessels under the macula to delay the rate of vision loss. The disadvantage of laser therapy is that the high thermal energy delivered by the beam also destroys nearby healthy tissue. The fourth edition of Neuroscience (Purves, D, et al. 2008) states that "there is currently no treatment for dry AMD."

RPE transplantation has not been successful in humans. For example, Zarbin, M, 2003 pointed out that "under normal aging, human Bruch's membrane (especially in the submacular region) undergoes many changes (eg, increased thickness, deposition of ECM and lipids, protein exchange Non-enzymatic formation of the final product of oligomerization and advanced glycation. These changes and additional changes due to AMD may reduce the bioavailability of ECM ligands (eg, laminin, fibronectin, and collagen IV), and Causes extremely poor RPE cell survival in AMD eyes. Therefore, although human RPE cells exhibit the integrin required to attach to these ECM molecules, the survival of RPE cells on human Bruch's membrane under the aging macula is still impaired. "(Zarbin, MA, Trans Am Ophthalmol Soc, 2003; 101: 493-514).

Retinitis pigmentosa is primarily considered to be an inherited disease in which more than 100 mutations are associated with loss of photoreceptors (see Lund et al., 2001, see above). Although most mutations target photoreceptors, some mutations directly affect RPE cells. In summary, these mutations affect processes such as molecular transport and light transmission between photoreceptors and RPE cells.

Other less common but also detrimental health retinopathy can also involve progressive cell degeneration that leads to vision loss and blindness. Such retinopathy includes, for example, diabetic retinopathy and choroidal neovascular membrane (CNVM).

The emergence of stem cell-based therapies for tissue repair and regeneration provides potential treatments for many of the aforementioned cell degeneration and other eye disorders. Stem cells are capable of self-renewal and differentiation to produce a variety of mature cell lineages. The transplantation of such cells can be used as a clinical tool to reconstruct target tissues, thereby restoring physiological and structural functions. Stem cell technology has a wide range of applications, including tissue engineering, gene therapy delivery, and cell therapy, that is, the delivery of these agents to target locations through the exogenous supply of live cells or cell components that can produce or contain biotherapeutics . (For a review, see, for example, Tresco, PA et al., Advanced Drug Delivery Reviews , 2000, 42: 2-37).

Recently, postpartum-derived cells have been shown to improve retinal degeneration (US 2010/0272803). Royal College of Surgeons (RCS) rats have defects in the tyrosine receptor kinase (Mertk) that affects phagocytosis of the outer segment, causing photoreceptor cell death. (Feng W. et al., J Biol Chem., 2002, 10: 277 (19): 17016-17022). It was found that transplantation of retinal pigment epithelium (RPE) cells into the subretinal space of RCS rats limits the progress of photoreceptor loss and preserves visual function. (US 2010/0272803). It has also been shown that postpartum-derived cells can be used in the RCS model to promote photoreceptor rescue and thus retain photoreceptors. (US 2010/0272803). Human umbilical cord tissue-derived cells (hUTC) were injected subretinally into the eyes of RCS rats to improve visual acuity and improve retinal degeneration (US 2010/0272803; Lund RD et al., Stem Cells. 2007; 25 (3): 602- 611). In addition, treatment with conditioned medium (CM) derived from hUTC can restore the phagocytosis of ROS by in vitro dystrophic RPE cells. (US 2010/0272803).

The removal of apoptotic cells by phagocytes is an indispensable part of normal life. Defects in this process will have a significant impact on self-tolerance and autoimmunity (Ravichandran et al. Cold Spring Harb Perspect Biol. , 2013 , 5 (1): a008748.doi: 10.1101 / cshperspect.a008748.Review). Identification and removal of apoptotic cells are mainly caused by occupational phagocytes (receptors bind pathogens for phagocytosis) such as macrophages, monocytes, and other white blood cells, and non-professional phagocytic cells (phagocytosis is not the main function) such as Mediated by epithelial cells, RPE cells, and endothelial cells. Many "eat me" signals have been identified so far, including changes in glycosylation of surface proteins or changes in surface charge (Ravichandran et al., Cold Spring Harb Perspect Biol., 2013). Phosphatidylserine (PS) is externalized as a sign of apoptosis and is the most studied "eat me" signal (Wu et al., Trends. Cell Biol., 2006 , 16 (4): 189-197). The "eat me" signal is directly recognized by the engulfment receptors of phagocytes (such as by PS receptors), or indirectly via bridge molecules and co-receptors (Erwig et al., Cell Death.Differ. , 2008; 15; : 243-250). Bridge molecule milk fat globule EGF factor 8 (MFG-E8), growth arrest specific 6 (growth arrest-specific 6, Gas6), protein S, thrombospondin (thrombospondin; TSP), apolipoprotein H (formerly known as β2 -Glycoprotein, β2-GPI) all bind to PS on the surface of apoptotic cells. Next, MFG-E8 can be identified through its RGD motif by αvβ3 and αvβ5 integrins (Hanayama et al., Science , 2004, 304: 1147-1150; Borisenko et al., Cell Death Differ. , 2004: 11: 943-945 ), Gas6 can be identified by the receptor tyrosine kinases of the Axl, Tyro3 and Mer families (Scott et al., Nature , 2001; 411: 207-211), and apolipoprotein H can be identified by the β2-GPI receptor ( Balasubramanian et al., J Bio Chem , 1997; 272: 31113-31117). Other bridge molecules are involved in the identification of altered sugars and / or lipids on the surface of apoptotic cells, such as the interface-active proteins A and D of members of the collectin family (Vandivier et al., J Immunol, 2002; 169: 3978 -398).

The molecular gelin family was subsequently identified through the interaction of its collagen tail with calreticulin (CRT), which in turn was passed through low density lipoprotein (LDL) receptor-related protein (LRP) -1 / CD91) conducts uptake signals (Gardai et al., Cell, 2003; 115: 13-23). As another example, the identified first bridge molecule is thrombospondin (TSP) -1 (Savill et al., J Clin Invest, 1992; 90: 1513-1522), which is used as an extracellular matrix glycoprotein and is considered It binds to the TSP-1 binding site on apoptotic cells and then to the receptor complex on the phagocytes, which contains αvβ3 and αvβ5 integrin and scavenger receptor CD36. Annexin I belongs to the annexin family of Ca2 + -dependent phospholipid binding proteins and is preferentially localized on the cytosolic surface of cell membranes. Annexin I is shown to co-localize with PS.

The phagocytosis of ROS by RPE is necessary for retinal function (Finnemann et al., PNAS , 1997; 94: 12932-937). Receptors involved in RPE phagocytosis include scavenger receptor CD36, integrin receptor αvβ5, receptor tyrosine kinase called Mertk, and mannose receptor (MR) (CD206) (Kevany et al., Physiology , 2009; 25: 8-15). Finnemann found that the isolated ROS possessed externalized PS, and the blocking or removal of the externalized PS reduced the binding and engulfing of ROS in culture by RPE (Finnemann et al., PNAS, 2012; 109 (21) : 8145-8148). However, there is still little understanding of RPE engulfment.

The present invention provides compositions and methods based on cell-based or regenerative therapies suitable for ophthalmic diseases and disorders. In particular, the invention is characterized by methods and compositions for treating ophthalmic diseases or conditions, including the use of precursor cells such as postpartum-derived cells and conditioned medium produced by these cells to regenerate or repair ocular tissues. The postpartum-derived cells may be umbilical cord tissue-derived cells (UTC) or placental tissue-derived cells (PDC).

One aspect of the present invention is a method for treating ophthalmic diseases, which comprises administering precursor cells or a conditioned medium prepared from a population of precursor cells to an individual, wherein the cells secrete bridge molecules. In one embodiment of the invention, the bridge molecule is secreted by the cell population in the conditioned medium. In a further embodiment, the bridge molecule system is selected from MFG-E8, Gas6, thrombospondin (TSP) -1 and TSP-2. In the embodiments of the present invention, the cells are precursor cells. In a specific embodiment of the present invention, the cells are postpartum-derived cells. In an embodiment of the invention, the postpartum-derived cell lines are isolated from human umbilical cord tissue or placental tissue that is substantially free of blood.

In an embodiment, a population of precursor cells (eg, postpartum-derived cells) secretes bridge molecules. In one embodiment, the conditioned medium prepared from a population of precursor cells (eg, postpartum-derived cells) contains bridge molecules secreted by the population of cells. Such bridge molecules secreted by these cells and secreted in the conditioned medium are selected from MFG-E8, Gas6, TSP-1 and TSP-2. Postpartum-derived cells are umbilical cord tissue-derived cells (UTC) or placental tissue-derived cells (PDC).

In one embodiment, the bridge molecule inhibits apoptosis of photoreceptor cells. In another embodiment, the bridge molecule secreted by the precursor cells and secreted in the conditioned medium reduces photoreceptor cell loss. In one embodiment, the loss of photoreceptor cells is reduced by phagocytosis of the photoreceptor fragments by bridge molecules.

In another embodiment, the cell population described above or the conditioned medium prepared from the cell population described above modifies the rod outer segment (ROS) to facilitate phagocytosis. In a further embodiment, the bridge molecule enhances the binding and internalization of retinal pigment epithelium (RPE) cells to ROS.

In another embodiment, the cell population described above or the conditioned medium prepared from the cell population described above contains a receptor tyrosine kinase (RTK) trophic factor secreted by the cell population. In a specific embodiment, the nutritional factors are BDNF, NT3, HGF, PDGF-CC, PDGF-DD, and GDNF. In an embodiment, RTK trophic factor mediates phagocytosis in retinal pigment epithelium (RPE) cells.

In certain embodiments, RTK trophic factor-mediated RPE cells phagocytose to engulf shed photoreceptor fragments (photoreceptor fragments shed by cells). In further embodiments, RTK trophic factors activate receptors on RCE cells to stimulate phagocytosis.

Another aspect of the present invention is characterized by a method for reducing the loss of photoreceptor cells in retinal degeneration, the method comprising administering to the eye a precursor cell population or an amount Conditioned medium for group preparation. In one embodiment of the present invention, the precursor cells are postpartum-derived cells. In a specific embodiment, the postpartum-derived cell line is isolated from human umbilical cord tissue or placental tissue that is substantially free of blood. As in other embodiments, the postpartum-derived cell line secretes bridge molecules. In embodiments, the conditioned medium contains bridge molecules secreted by cell populations, such as postpartum-derived cell populations. Such bridge molecules secreted by postpartum-derived cells are selected from MFG-E8, Gas6, TSP-1 and TSP-2.

In another embodiment, the conditioned medium line is produced from isolated postpartum-derived cells or a population of postpartum-derived cells derived from human umbilical cord tissue or placental tissue that is substantially free of blood. In an embodiment, the postpartum-derived cells are capable of expanding in culture and have the potential to differentiate into neuronal phenotype cells; wherein the cells require L-valine for growth and can grow in at least about 5% oxygen . This cell further includes one or more of the following characteristics: (a) the potential to perform at least about 40 doublings in culture; (b) attachment and expansion on coated or uncoated tissue culture vessels, wherein The coated tissue culture container contains a coating of gelatin, laminin, collagen, polyguanylic acid, glass connexin, or fibronectin; (c) production of tissue factor, vimentin, and α- At least one of alpha-smooth muscle actin; (d) Production of CD10, CD13, CD44, CD73, CD90, PDGFr-α, PD-L2 and HLA-A, HLA-B, HLA-C At least one of; (e) does not produce at least one of CD31, CD34, CD45, CD80, CD86, CD117, CD141, CD178, B7-H2, HLA-G, and HLA-DR, HLA-DP, HLA-DQ One, as detected by flow cytometry; (f) for the expression of genes encoding at least one of the following genes (relative to human cells of fibroblasts, mesenchymal stem cells, or iliac bone marrow cells). Increase: Interleukin 8; Reticulon 1; Chemokine (C--X--C motif) ligand 1 (melanoma growth stimulation) Sex, α); chemokine (C--X--C motif) ligand 6 (granulocyte chemoattractant protein 2); chemokine (C--X--C motif) ligand 3 ; Tumor necrosis factor, alpha-evoked protein 3; C-type lectin superfamily member 2; Wilms tumor 1; aldehyde dehydrogenase 1 family member A2; renin; oxidized low-density lipoprotein Receptor 1; Homo sapiens strain IMAGE: 4179671; protein kinase C ζ; hypothetical protein DKFZp564F013; ovarian cancer down-regulatory factor 1; and the strain Homo sapiens gene DKFZp547k1113; The gene expression of a gene (human cells relative to fibroblasts, mesenchymal stem cells, or iliac bone marrow cells) is reduced: short stature homeobox 2; heat shock 27kDa protein 2; chemokine ( C--X--C motif) ligand 12 (stromal cell-derived factor 1); elastin (supervalvular aortic stenosis, Williams-Beuren syndrome); Homo sapiens mRNA; cDNA DKFZp586M2022 (from strain DKFZp586M2022); mesenchymal homeobox 2 (growth arrest-specific homeo box )); Sine oculis homology box homolog 1 (Drosophila); αB aquatic protein; morphogenesis disorder associated activator 2 (disheveled associated activator of morphogenesis 2); DKFZP586B2420 protein; similar to neuralin 1; binding Tetranectin (plasminogen binding protein); src homolog 3 (SH3) and polycysteine rich domain (cysteine rich domain); cholesterol 25-hydroxylase; dwarf related transcription factor 3 ( runt-related transcription factor 3); interleukin 11 receptor, α; procollagen C-endopeptidase enhancer; frizzled homolog 7 (Drosophila); Hypothetical gene BC008967; type III collagen α1; tenascin C (tenascin C, hexabrachion); iroquois homeobox protein 5 (iroquois homeobox protein 5); hephaestin (hephaestin); integrin β8 ; Synaptic vesicle glycoprotein 2; Neuroblastoma, tumorigenic inhibitory factor 1; Insulin-like growth factor binding protein 2, 36 kDa; Homo sapiens cDNA FLJ12280 fis, strain MAMMA10017 44; Interleukin-like receptor factor 1; potassium intermediate / small conductivity calcium activated channel, subfamily N, member 4; integrin β7; transcription coactivator (TAZ) with PDZ-binding motif; sine oculis Source box homolog 2 (Drosophila); KIAA1034 protein; vesicle-associated membrane protein 5 (myobrevin); EGF-containing fibulin-like extracellular matrix protein 1 (EGF-containing fibulin-like extracellular matrix protein) 1; Early growth response protein 3; distant-less homeo box 5; hypothetical protein FLJ20373; aldehyde-keto reductase family 1, member C3 (3-α-hydroxysteroid dehydrogenation) Enzyme (3-alpha hydroxysteroid dehydrogenase), type II); biglycan; transcription coactivator (TAZ) with PDZ-binding motif; fibronectin 1; proenkephalin; Integrin, β1-like (with EGF-like repeat region); Homo sapiens mRNA full-length insert cDNA strain EUROIMAGE 1968422; EphA3; KIAA0367 protein; natriuretic peptide receptor C (natriuretic peptide receptor C) / guanylate cyclase C (guanylate cyclase C) (atrial natriuretic peptide receptor C (a trionatriuretic peptide receptor C)); hypothetical protein FLJ14054; Homo sapiens mRNA; cDNA DKFZp564B222 (from the strain DKFZp564B222); BCL2 / adenovirus E1B 19kDa interacting protein 3 (adenovirus E1B 19kDa interacting protein 3-like); AE binding protein 1; cytochrome c oxidase subunit VIIa polypeptide 1 (muscle); similar to neuralin 1; B cell translocation gene 1; hypothetical protein FLJ23191; and DKFZp586L151; and (h) does not express hTERT or telomerase . In one embodiment, umbilical cord tissue-derived cells further have the following characteristics: (i) secretion of MCP-1, IL-6, IL-8, GCP-2, HGF, KGF, FGF, HB-EGF, BDNF, TPO, MIP1b , I309, MDC, RANTES, and TIMP1; (j) does not secrete at least one of TGF-β2, MIP1a, ANG2, PDGFbb, and VEGF, as detected by ELISA. In another embodiment, the placental tissue-derived cells further have the following characteristics: (i) secretion of MCP-1, IL-6, IL-8, GCP-2, HGF, KGF, HB-EGF, BDNF, TPO, MIP1a, At least one of RANTES and TIMP1; (j) does not secrete at least one of TGF-β2, MIP1b, ANG2, PDGFbb, FGF, and VEGF, as detected by ELISA.

In a particular embodiment, the postpartum-derived cells have all the identifying characteristics of the following cell types: cell type UMB 022803 (P7) (ATCC access number PTA-6067); cell type UMB 022803 (P17) (ATCC access number PTA-6068 ), Cell type PLA 071003 (P8) (ATCC access number PTA-6074); cell type PLA 071003 (P11) (ATCC access number PTA-6075); or cell type PLA 071003 (P16) (ATCC access number PTA -6079). In one embodiment, the postpartum-derived cells derived from umbilical tissue have all of the cell type UMB 022803 (P7) (ATCC access number PTA-6067) or cell type UMB 022803 (P17) (ATCC access number PTA-6068) Identify features. In another embodiment, postpartum-derived cells derived from placental tissue have all the identifying characteristics of the following cell types: cell type PLA 071003 (P8) (ATCC accession number PTA-6074); cell type PLA 071003 (P11) (ATCC Accession number PTA-6075); or cell type PLA 071003 (P16) (ATCC accession number PTA-6079).

In certain embodiments, the postpartum-derived cells are isolated in the presence of one or more enzyme activities, including metalloproteinase activity, mucus decomposition activity, and neutral protease activity. Preferably, the postpartum-derived cells have a normal karyotype, which is maintained during the subculture of the cells. In a preferred embodiment, the postpartum-derived cells express each of CD10, CD13, CD44, CD73, and CD90. In the examples, the postpartum-derived cells express each of CD10, CD13, CD44, CD73, CD90, PDGFr-α, and HLA-A, B, and C. In a preferred embodiment, the postpartum-derived cells do not express CD31, CD34, CD45, CD117. In an embodiment, the postpartum-derived cells do not express CD31, CD34, CD45, CD117, CD141, or HLA-DR, DP, DQ, as detected by flow cytometry. In an embodiment, these cells do not express hTERT or telomerase.

In the embodiment as above, the cell population is a substantially homogeneous postpartum-derived cell population. In certain embodiments, the population is a homogeneous postpartum-derived cell population. In an embodiment of the invention, the postpartum-derived cell line is derived from human umbilical cord tissue or placental tissue that is substantially free of blood.

In certain embodiments, the postpartum-derived cell population as described above or a conditioned medium prepared from the cell population is administered with at least one other cell type, such as stellate cells, oligodendrocyte , Neurons, neural precursors, neural stem cells, retinal epithelial stem cells, corneal epithelial stem cells, or other pluripotent or pluripotent stem cells. In these embodiments, other cell types may be administered simultaneously, before, or after the postpartum-derived cell population or the conditioned medium prepared from the postpartum-derived cell population.

Similarly, in these and other embodiments, the postpartum-derived cell population or the conditioned medium prepared from the postpartum-derived cell population as described above is combined with at least one other agent such as a drug for eye treatment, or another beneficial Adjuvants such as anti-inflammatory agents, anti-apoptotic agents, antioxidants or growth factors are administered together. In these embodiments, other agents may be administered simultaneously, before, or after the postpartum-derived cell population or the conditioned medium prepared from the postpartum-derived cell population.

In the various embodiments described herein, the postpartum-derived cell population (umbilical or placenta) or conditioned medium produced by the postpartum-derived cells is administered to the eye, such as the surface of the eye, or to the interior of the eye or to a location near the eye, such as Give later. The postpartum-derived cell population or the conditioned medium prepared from the postpartum-derived cell population can be administered through a cannula or from a device implanted in or near a patient in the eye, or can be implanted with a cell population or conditioned medium Substrate or scaffold to administer.

Another aspect of the present invention is characterized by a composition for reducing the loss of photoreceptor cells in a retinal degeneration pathology, the composition comprising a precursor cell population or a precursor cell population effective to reduce the amount of photoreceptor cell loss Prepared conditioned medium. Preferably, the precursor cells are post-natally derived cells as described above. More preferably, the postpartum-derived cell line is isolated from a postpartum umbilical cord or placenta that is substantially free of blood as described above. A degenerative condition can be acute, chronic, or progressive.

In certain embodiments, the composition as described above includes at least one other cell type, such as stellate cells, oligodendritic cells, neurons, neural precursors, neural stem cells, retinal epithelial stem cells, corneal epithelial stem cells, or other Pluripotent or pluripotent stem cells. In these or other embodiments, the composition contains at least one other medicament, such as a medicament for treating an ocular degenerative disorder or other beneficial auxiliary agents, such as anti-inflammatory agents, anti-apoptotic agents, antioxidants or growth factors.

In the embodiments as described above, the composition is a pharmaceutical composition further comprising a pharmaceutically acceptable carrier. In certain embodiments, the pharmaceutical composition is formulated for administration to the surface of the eye. Alternatively, it can be formulated for administration into or near the eye (eg, behind the eye). The pharmaceutical composition can also be formulated as a matrix or scaffold containing precursor cells as described above or a conditioned medium prepared from the precursor cells.

According to another aspect of the present invention, there is provided a kit for treating patients with ocular degeneration. The kit contains a pharmaceutically acceptable carrier, precursor cells or conditioned medium produced by the precursor cells (such as cells isolated from postpartum tissues, preferably the postpartum derived cells described above), and methods for treating patients Use the instruction manual of the set. The kit may also contain one or more additional components, such as reagents and instructions for producing conditioned medium, or at least one other cell type population, or one or more agents that can be used to treat ocular degeneration.

Other aspects of the invention include a method of reducing the loss of photoreceptor cells in retinal degeneration, the method comprising administering a composition of conditioned medium comprising or prepared from a precursor cell population in an amount effective to reduce the loss of photoreceptor cells Thing. Preferably, the precursor cells are postpartum-derived cells or the conditioned medium is prepared from the postpartum-derived cell population as described herein. In an embodiment of the invention, the postpartum-derived cell line is isolated from umbilical cord tissue or placental tissue that is substantially free of blood. In certain embodiments, the postpartum-derived cells or conditioned medium prepared from the postpartum-derived cell population contains bridge molecules secreted by the cell population. Such bridge molecules secreted by the postpartum-derived cells or secreted in the conditioned medium are selected from MFG-E8, Gas6, TSP-1, and TSP-2.

In some embodiments, the present invention provides a method for reducing the loss of photoreceptor cells in retinal degeneration, the method comprising administering to the eye a postpartum-derived cell in an amount effective to reduce or prevent loss of photoreceptor cells or derived from postpartum Conditioned medium prepared from cell populations. The postpartum-derived cell line is derived from umbilical cord tissue or placental tissue that is substantially free of blood. In some embodiments, the postpartum-derived cell population is a substantially homogeneous population. In a specific embodiment, the cell population is homogeneous.

In a further aspect of the invention described herein, the postpartum-derived cell population (umbilical or placenta) or conditioned medium produced by the postpartum-derived cells protects retinal cells or improves retinal damage caused by oxidative stress or oxidative damage. In one embodiment, the invention is a method of reducing retinal degeneration, the method comprising administering to the eye a postpartum-derived cell population or a conditioned medium produced from the postpartum-derived cell population in an amount effective to reduce or prevent oxidative stress or damage. In an embodiment of the invention, the postpartum-derived cell line is isolated from umbilical cord tissue or placental tissue that is substantially free of blood. In an embodiment, retinal cells and tissues are exposed to oxidative stress or oxidative damage. In the embodiments herein, the retinal cells and tissues are photoreceptor cells or retinal epithelium, including retinal pigment epithelium (RPE) cells. In the embodiments herein, the oxidative stress or oxidative damage is high oxygen tension, sun exposure, including long-term sun exposure.

One embodiment is a method of reducing the loss of photoreceptor cells in retinal degeneration, the method comprising administering to the eye a postpartum-derived cell population or a conditioned medium produced from the postpartum-derived cell population in an amount effective to reduce or prevent oxidative stress or damage. In an embodiment, the postpartum-derived cell line is isolated from umbilical cord tissue or placental tissue that is substantially free of blood. In an embodiment, retinal cells and tissues are exposed to oxidative stress or oxidative damage. In the embodiments herein, the retinal cells and tissues are photoreceptor cells or retinal epithelium, including retinal pigment epithelium (RPE) cells. In the embodiments herein, the oxidative stress or oxidative damage is selected from high oxygen tension, solar exposure (including long-term solar exposure), free radical stress, photo-oxidation, and photo-induced damage.

In one embodiment, the invention is a method for reducing the loss of photoreceptor cells in retinal degeneration, the method comprising administering a postpartum-derived cell population or cells derived from postpartum in an amount effective to reduce or prevent the loss of photoreceptor cells A conditioned medium produced by a population, wherein the cell population is isolated from a postpartum tissue that is substantially free of blood, and wherein the population of cells can be expanded in culture, has the potential to differentiate into cells with at least one neurophenotype, Maintain normal karyotype after generation and have the following characteristics: a) Potential for 40 population doublings in culture; b) Production of CD10, CD13, CD44, CD73, and CD90; and c) No production of CD31, CD34, CD45 , CD117, and CD141, and the postpartum-derived cell population secretes bridge molecules, and the conditioned medium prepared from the postpartum-derived cell population contains bridge molecules secreted by the cell population. In some embodiments, the bridge molecule secreted by the postpartum-derived cells is selected from MFG-E8, Gas6, TSP-1, and TSP-2. In some embodiments, the cell population is a substantially homogeneous population. In a specific embodiment, the cell population is homogeneous. Postpartum-derived cells are umbilical cord tissue-derived cells or placental tissue-derived cells. In one embodiment, the umbilical cord tissue-derived cell population secretes MCP-1, IL-6, IL-8, GCP-2, HGF, KGF, FGF, HB-EGF, BDNF, TPO, MIP1b, I309, MDC, RANTES, And TIMP1. In an embodiment, the umbilical cord tissue-derived cell population does not secrete TGF-β2, MIP1a, ANG2, PDGFbb, and VEGF, as detected by ELISA. In another embodiment, the placental tissue-derived cell population secretes MCP-1, IL-6, IL-8, GCP-2, HGF, KGF, HB-EGF, BDNF, TPO, MIP1a, RANTES, and TIMP1. In an embodiment, the placental tissue-derived cell population does not secrete TGF-β2, MIP1b, ANG2, PDGFbb, FGF, and VEGF, as detected by ELISA. In the embodiment, the umbilical-derived cells or placenta-derived cells are positive for HLA-A, HLA-B, HLA-C, and negative for HLA-DR, HLA-DP, HLA-DQ. In further embodiments, umbilical derived cells do not express hTERT or telomerase.

In one embodiment, the present invention is a method for reducing photoreceptor cell loss in retinal degeneration, the method comprising administering or prepared from a postpartum-derived cell population effective to reduce the amount of photoreceptor cell loss Conditioned medium, wherein the cell population is isolated from human umbilical cord tissue that is substantially free of blood, and wherein the cell population can be expanded in culture, has the potential to differentiate into cells with at least one neurophenotype, and is passaged The normal karyotype is maintained afterwards and has the following characteristics: a) the potential for 40 population doublings in culture; b) production of CD10, CD13, CD44, CD73, and CD90; c) no production of CD31, CD34, CD45, CD117 , And CD141, and d) Compared with human cells of fibroblasts, mesenchymal stem cells, or iliac bone marrow cells, the expression of genes encoding interleukin 8 and endoplasmic reticulum protein 1 is increased, and the post-natal derived cell population Is a secreted bridge molecule, or a conditioned medium prepared from a postpartum-derived cell population contains a bridge molecule secreted by the cell population. In an embodiment, the bridge molecule secreted by the postpartum-derived cell population or the bridge molecule in the conditioned medium secreted by the postpartum-derived cell population is selected from MFG-E8, Gas6, TSP-1, and TSP-2. In an embodiment, the cell population secretes MCP-1, IL-6, IL-8, GCP-2, HGF, KGF, FGF, HB-EGF, BDNF, TPO, MIP1b, I309, MDC, RANTES, and TIMP1. In some embodiments, the cell population does not secrete TGF-β2, MIP1a, ANG2, PDGFbb, and VEGF, as detected by ELISA. In the examples, the cell population is positive for HLA-A, HLA-B, HLA-C, and negative for HLA-DR, HLA-DP, HLA-DQ. In some embodiments, the cell population is a substantially homogeneous population. In a specific embodiment, the cell population is homogeneous. In addition, the cell population did not express hTERT or telomerase.

In certain embodiments, the present invention provides a method for reducing photoreceptor cell loss in retinal degeneration, the method comprising administering an umbilical-derived cell population in an amount effective to reduce or prevent photoreceptor cell loss or derived from the umbilical cord A conditioned medium prepared by a cell population, wherein the cell population is isolated from human umbilical cord tissue that is substantially free of blood, and wherein the cell population can be expanded in culture and has the potential to differentiate into cells with at least one neurophenotype, And has the following characteristics: a. Potential for 40 times population doubling in culture; b. Production of CD10, CD13, CD44, CD73, and CD90; and c. Relative to fibroblasts, mesenchymal stem cells, or iliac bones Human cells of bone marrow cells increase the expression of genes encoding interleukin 8 and endoplasmic reticulum protein 1, and the postpartum-derived cell population secretes bridge molecules, in which the conditioned medium prepared from the postpartum-derived cell population contains the Bridge molecule. In an embodiment, the bridge molecule secreted in the postpartum-derived cell population secreting the bridge molecule, or the bridge molecule secreted in the conditioned medium is selected from MFG-E8, Gas6, TSP-1, and TSP-2. In the examples, these cells are not produced or are negative for CD31, CD34, CD45, CD117, and CD141. In one embodiment, the umbilical cord tissue-derived cell population secretes MCP-1, IL-6, IL-8, GCP-2, HGF, KGF, FGF, HB-EGF, BDNF, TPO, MIP1b, I309, MDC, RANTES, And TIMP1, and does not secrete TGF-β2, MIP1a, ANG2, PDGFbb, and VEGF, as detected by ELISA. In the examples, the umbilical-derived cell population is positive for HLA-A, HLA-B, HLA-C, and negative for HLA-DR, HLA-DP, HLA-DQ. In addition, the cell population did not express hTERT or telomerase. In some embodiments, the cell population is a substantially homogeneous population. In a specific embodiment, the cell population is homogeneous.

Another aspect of the invention is a method of making a conditioned medium, the method comprising culturing a cell population, wherein the conditioned medium contains bridge molecules secreted by the cell population. In one embodiment of the invention, the bridge molecule is secreted by the cell population in the conditioned medium. In a further embodiment, the bridge molecule system is selected from MFG-E8, Gas6, thrombospondin (TSP) -1 and TSP-2. In the embodiments of the present invention, the cells are precursor cells. In a specific embodiment of the present invention, the cells are postpartum-derived cells. In an embodiment of the invention, the postpartum-derived cell lines are isolated from human umbilical cord tissue or placental tissue that is substantially free of blood.

In the embodiment of the present invention as described above, the postpartum-derived cell population has the following characteristics: it is attached and expanded on the coated or uncoated tissue culture container, wherein the coated tissue culture container contains gelatin, lamellar adhesive Coating of protein, collagen, polyguanylic acid, vitronectin, or fibronectin; production of vimentin and α-smooth muscle actin; and positive for HLA-A, HLA-B, HLA-C, And negative for HLA-DR, HLA-DP, HLA-DQ.

In the embodiments of the present invention as described above, the retinal degeneration, retinopathy, or retinal / macular disorder is age-related macular degeneration. In an alternative embodiment, the retinal degeneration, retinopathy, or retinal / macular disorder is dry age-related macular degeneration.

In the embodiments of the present invention as described above, the loss of photoreceptor cells is reduced or prevented by inhibiting apoptosis of photoreceptor cells. In an embodiment, photoreceptor cell loss is reduced or prevented by stimulating phagocytosis of shed photoreceptor fragments.

Figures 1A-1B show the effect of pre-cultivation of hUTC CM1 preparation with serum on phagocytosis of deferent RPE. Figure 1A. Pigment-dystrophic RPE is pre-cultured with CM1 preparation (with serum). As for the control group, the brown hooded normal and pigment-dystrophic RPE were pre-cultured in a control medium (DMEM: F12 medium + 10% FBS + Pen (50U / ml) / Strep (50 μg / ml)). The cultivation time is 16 hours. (tan N: brown cape-shaped normal RPE; pig D: pigment-dystrophic RPE; con: control group; M: medium; CM: conditioned medium; w: with). The value is the average value of the number of ROS phagocytized by a sample in the counting field ± SD (n = 11 or 12 per sample; n: number of fields counted); compared with the pig D control group, p <0.05. Figure 1B. The dystrophic RPE line is pre-cultured with CM1 (with serum). As for the control group, normal and deferent RPE were pre-cultured in control medium with serum. The cultivation time is 24 hours. (N: normal RPE; D: dystrophy RPE; D (1): triple repeat 1; D (2): triple repeat 2; D (3): triple repeat 3; con: control group; M: medium; CM: Conditioned medium; w: with). The value is the average value of the number of ROS phagocytosed by a sample in the counting field ± SD (n = 11, 12, or 13 per sample; n: number of fields counted). Compared with the D control group, p <0.05.

Figure 2 shows the effect of pre-cultured serum-free hUTC CM1 preparation on phagocytosis of deferent RPE. The pigment-dystrophic RPE is pre-cultured with CM1 (without serum). As for the control group, the brown hooded normal and pigment-dystrophic RPE were pre-cultured in a control medium without serum. The cultivation time is 24 hours. (tan N: brown hood-like normal RPE; pig D: pigment-dystrophic RPE; con: control group. M: medium; CM: conditioned medium; wo: no. The value is the number of ROS phagocytosed by a sample in the counting field The mean ± SD (n = 9, 10, 11 or 12 per sample; n: number of fields counted); compared with the pig D control group, p <0.05.

Figures 3A to 3D show the effect of CM pre-cultured in hUTC on phagocytosis. (Figure 3A) The effect of CM2 pre-cultured in hUTC on phagocytosis. The pigment-dystrophic RPE line is pre-cultured with CM2. As for the control group, the brown hooded normal and pigment-dystrophic RPE were pre-cultured in the control medium. The cultivation time is 24 hours. (tan N: brown hooded normal RPE; pig D: pigment-dystrophic RPE; con: control group. M: medium; CM: conditioned medium). The value is the average of the number of ROS phagocytosed by a sample in the counting field ± SD (n = 8 or 10 per sample; n: number of fields counted). (Figure 3B to Figure 3D) The effect of CM3 pre-cultured on phagocytosis. Infertile RPE is pre-cultured with CM3. As for the control group, normal and deferent RPE were pre-cultured in the control medium. The cultivation time is 24 hours. Figure 3B. (N: normal RPE; D: defertile RPE; con: control group; M: medium; CM: conditioned medium). Values are the average of the number of ROS phagocytosed by the replicate samples in the counting field ± SD (n = 5 to 12 per sample; n: number of fields counted); compared with the D + con M control group, p <0.05 . Figure 3C. Normal RPE control line from CM3 test 1. (N: normal RPE; D: dystrophic RPE; con: control group; M: medium; CM: conditioned medium). The value is the average value of the number of ROS phagocytosed by a sample in the counting field ± SD (n = 11 or 14 per sample; n: number of fields counted); compared with the D + con M control group, p <0.05. Figure 3D. (N: normal RPE; N1: normal RPE from culture; N2: normal RPE from N2 culture; tan N: brown hooded normal RPE; D: defertile RPE; con: control group; M: Medium; CM: conditioned medium). The value is the average value of the number of ROS phagocytized by a sample in the counting field ± SD (n = 12 or 13 per sample; n: number of fields counted); compared with the D + con M control group, p <0.05.

4A to 4C show the effect of hUTC on phagocytosis in RCS RPE cells in vitro. RCS RPE cells were co-cultured with hUTC seeded in transwell (Figure 4A) or with hUTC CM (Figure 4B) for 24 hours and then subjected to phagocytosis assay. N: normal RPE; D: defertile RCS RPE; CM, conditioned medium. Increased phagocytosis was observed in RCS RPE co-cultured with hUTC or cultured with hUTC CM. (Figure 4C) The photoreceptor OS line was cultured with hUTC CM for 24 hours and then fed to RCS RPE cells for phagocytosis assay in the absence of hUTC CM. The OS treated with hUTC CM restored RCS RPE phagocytosis. The data represent the mean ± SEM (n = 3). **** p <0.0001.

5A to 5B show the results of the RTK ligand BDNF or HB-EGF test. The dystrophic RPE cell line was cultured in MEM + 5% FBS (MEM5) for 24h with BDNF (200ng / ml) ( Figure 5A ) or HB-EGF (200ng / ml) ( Figure 5B ). As for the positive control group, the dystrophic RPE cells were cultured in CM3 for 24h. As for the other control groups, normal and deferent RPE were cultured in MEM5 for 24 hours and subjected to phagocytosis test. (N: normal RPE; D: dystrophic RPE; con: control group; M: medium; CM: conditioned medium). The value is the average of the number of ROS phagocytosed in the field of counts of duplicate or triple repeat samples ± SD (n = 10, 11 or 12 per sample; n: number of counted fields); compared with the D control group, p <0.05.

6A to 6E show the test results of RTK ligand PDGF-DD, ephrin A4, and HGF. The dystrophic RPE cell line was cultured in MEM5 with PDGF-DD ( Figure 6A, Figure 6B ), Pterin A4 ( Figure 6C ) or HGF (200ng / ml) ( Figure 6D, Figure 6E ) for 24h and then added ROS to the PDGF containing -DD5, Pterin A4 or HGF in MEM5 for phagocytosis assay (the medium was not changed when ROS was added). As for the positive control group, the dystrophic RPE cells were cultured in CM3 for 24h. As for the other control groups, normal and deferent RPE were cultured in MEM5 for 24 hours and subjected to phagocytosis test. (N: normal RPE; D: dystrophic RPE; con: control group; M: medium; CM: conditioned medium). The value is the average of the number of ROS engulfed in the counting field of the duplicate or triple samples ± SD (n = 10, 11 or 12 per sample; n: the number of counted fields); compared with the D control group, p <0.05.

7A to 7B show the results of RTK ligand Pterin B2 assay. The dystrophic RPE cell line was cultured with Pterin B2 (200ng / ml). As for the positive control group, the dystrophic RPE cells were cultured in CM3. As for the other control groups, normal and deferent RPE were cultured in MEM5 for 24h and subjected to phagocytosis test. (N: normal RPE; D: dystrophic RPE; con: control group; M: medium; CM: conditioned medium). The value is the average of the number of ROS engulfed in the counting field of the duplicate or triple samples ± SD (n = 10, 11 or 12 per sample; n: the number of counted fields); compared with the D control group, p <0.05.

8A to 8C show dystrophic RPE cells treated with endothelin-1, TGF-β1, or IL-6. (Figure 8A) Verification of phagocytosis of endothelin-1 or TGF-β1 (at 200 ng / mL) compared to the normal control group. (Figure 8B, Figure 8C) The dystrophic RPE cell line was cultured with 200ng / mL recombinant human endothelin-1, TGF-β1, or IL-6 and tested for phagocytosis. The hUTC CM3 line was used as a positive control group. As for other controls, normal and deferent RPE were cultured in MEM5 for 24h and subjected to phagocytosis assay. (N: normal RPE; D: infertile RPE). The value is the average value of the number of ROS phagocytosed by each sample in the counting field ± SD (n = 10 per sample; n: number of fields counted).

Fig. 9 shows that dystrophic RPE cells were fed with ROS pre-cultured with various concentrations of vitronectin, and the phagocytosis of the normal control group was examined. ROS lines were pre-cultured with control medium (DMEM + 10% FBS) or CM3. At the same time, ROS was pre-cultured in MEM20 with various concentrations of human recombinant glass connexin (4, 2, 1, 0.5ug / ml). As for the control group, normal RPE alone or deprived RPE alone was cultured in MEM20, and then replaced with MEM5 in the presence of untreated ROS (resuspended in MEM20 and fed to RPE cells) for phagocytosis assay. (N: normal RPE; D: dystrophic RPE; Con M: control medium; V: glass connexin). The value is the average value of the number of ROS phagocytosed by each sample in the counting field ± SD (n = 10 per sample; n: number of fields counted).

Figure 10 shows the gene expression level of RTK ligands identified in hUTC. Total mRNA was extracted from hUTC and RNA-Seq was performed to identify and quantify RTK ligand gene expression in hUTC. The identified RTK matching system is classified based on the corresponding RTK subfamily and graded according to its FPKM value. Detect the gene expression of multiple RTK ligands in 15 RTK subfamilies in hUTC.

Figures 11A to 11G show the selected RTK ligand levels measured in hUTC CM. (FIG. 11A to FIG. 11F) RTK ligand levels are compared with those in NHDF and ARPE-19 conditioned media. Compared with NHDF and ARPE-19 conditioned medium, the BDNF line was secreted at high levels in hUTC conditioned medium ( Figure 11A ). Compared with NHDF CM, the level of NT3 in hUTC CM is high, however, the amount of NT3 in ARPE-19 conditioned medium and control medium is undetectable ( Figure 11B ). Compared with NHDF and ARPE-19 conditioned medium, HGF lines are secreted at high levels in hUTC CM ( Figure 11C ). Compared with NHDF and ARPE-19 conditioned medium, the levels of PDGF-CC and PDGF-DD in hUTC conditioned medium are low ( Figure 11D and Figure 11E, respectively ). The GDNF line is secreted in hUTC and NHDF conditioned medium, but only in trace amounts in ARPE-19 conditioned medium ( Figure 11F ). All values are the mean ± SD of triplicate samples, except NT3 is the mean ± SD of duplicate samples. (FIG. 11G) RTK ligand level measured by ELISA. (The data shown is the mean ± SEM; n = 3).

Figures 12A to 12E show the level of bridge molecules measured in hUTC CM. (Figures 12A to 12E). The level of bridge molecules is compared with NHDF and ARPE-19 conditioned medium. Figure 12A shows MFG-E8 levels in hUTC, ARPE-19 and NHDF conditioned medium. The value is the mean ± SD of duplicate or triple samples. Figure 12B shows Gas6 levels in hUTC, ARPE-19 and NHDF conditioned medium. Values are the mean ± SD of duplicate samples. Figure 12C shows the levels of TSP-1 in conditioned medium of hUTC, ARPE-19 and NHDF. Values are the mean ± SD of duplicate samples. Figure 12D shows TSP-2 levels in hUTC, ARPE-19 and NHDF conditioned medium. Values are the mean ± SD of duplicate samples. (Fig. 12E) The level of bridge molecules measured by ELISA. The data shown is the mean ± SEM (n = 2 for TSP-1 and TSP-2; n = 3 for all MFG-E8).

Figures 13A to 13D show the effect of precultivation of dystrophic RPE cells with hUTC conditioned medium (CM) on phagocytosis. For the control group, normal RPE alone or deprived RPE alone were pre-cultured in their conventional growth medium MEM20 (MEM + 20% FBS). At the same time, dystrophic RPE was also pre-cultured with CM3. In addition, ROS pre-cultured with hUTC CM3 was tested. The value is the mean ± SD of the number of phagocytized ROS of each sample in the counting field. Figure 13A , n = 10 to 20 per sample; Figure 13B is the original data. Figure 13C , n = 12 per sample and each sample is duplicated. (n: number of counted fields of view); Figure 13D is the original data.

Figures 14A to 14K show the effect of bridge molecules and RTK ligands on RCS RPE cells phagocytosis of the rod outer segment (ROS). ROS lines were pre-cultured with control medium (DMEM + 10% FBS) or hUTC conditioned medium. At the same time, ROS lines were pre-cultured in control medium with various concentrations of human recombinant MFG-E8 ( Figure 14A ), Gas6 ( Figure 14B ), TSP-1 ( Figure 14C ), or TSP-2 ( Figure 14D ). As for the control group, normal RPE alone or deprived RPE alone were cultured for phagocytosis assay. (N: normal RPE; N + ROS: normal RPE cells are fed with untreated ROS during the phagocytosis test; D: dystrophic RPE cells; D + ROS: dystrophic RPE cells are fed with untreated ROS during the phagocytosis test; Con M: control medium; D + ROS + Con: defertile RPE cells were fed with ROS pre-cultured in control medium; CM4: batch 4 hUTC conditioned medium). D + ROS + CM4: dystrophic RPE cells fed ROS pre-cultured with CM4; D + ROS + MFG-E8: dystrophic RPE cells fed ROS pre-cultured with MFG-E8. The value is the average value of the number of ROS phagocytosed by each sample in the counting field ± SD (n = 10 per sample; n: number of fields counted). * P <0.001, compared with D + ROS and D + ROS + ConM, and D + ROS pretreated with MFG-E8, Gas6, TSP-1 or TSP-2; ** P <0.0001, D + ROS + CM4 and Comparison of D + ROS and D + ROS + ConM. (FIG. 14E to FIG. 14H) The photoreceptor OS line was cultured with recombinant human MFG-E8 (FIG. 14E) , Gas6 (FIG. 14F) , TSP-1 (FIG. 14G) , or TSP-2 (FIG. 14H) for 24 hours and then Feed to RCS RPE cells for phagocytosis assay in the absence of hUTC CM. The OS pre-cultured with hUTC CM is used as a positive control for the test. The phagocytosis of OS by RCS RPE cells was rescued by MFG-E8, Gas6, TSP-1 or TSP-2 in a dose-dependent manner. ( FIG. 14I to FIG. 14K) RCS RPE cell lines were cultured with recombinant human BDNF ( FIG. 14I ), HGF ( FIG. 14J ), or GDNF ( FIG. 14K ) for 24 hours, and then subjected to phagocytosis assay. RCS RPE cultured with hUTC CM is used as a positive control for the test. BDNF, HGF or GDNF dose-dependently increased phagocytosis in RCS RPE cells. The data represent the mean ± SEM (n = 3). **** p <0.0001, *** p <0.001, ** p <0.01, * p <0.05, ns is not significant.

Figures 15A to 15C. RTK ligands and bridge molecules used for hUTC-induced phagocytosis rescue in RCS RPE. (Figure 15A) ELISA of cell culture supernatant collected from untransfected hUTC and siRNA transfected hUTC. (Figure 15B) The performance of RTK ligands BDNF, HGF and GDNF was silenced by hUTC siRNA transfection. Collect Knockdown (Knockdown, KD) hUTC CM. The RCS RPE line was cultured with KD hUTC CM for 24 hours and then subjected to phagocytosis assay. When BDNF, HGF or GDNF is eliminated, the effect of hUTC on RCS RPE phagocytosis is abolished. (Figure 15C) The performance of the bridge molecules MFG-E8, TSP-1 and TSP-2 in hUTC was silenced by siRNA transfection. Collect KD hUTC CM. RCS RPE was fed with OS pre-cultured with KD hUTC CM for 24 hours and subjected to phagocytosis test. The elimination of MFG-E8, TSP-1, or TSP-2 reduces OS phagocytosis rescue of hUTC media in RCS RPE. CM lines prepared from untransfected and scrambled siRNA transfected hUTC were used as a control group. The data represent the mean ± SEM, n = 3 for (B) and (C), and n = 6 for hUTC CM ELISA (A) transfected with untransfected, simulated, and messy siRNA. **** p <0.0001, *** p <0.001, ** p <0.01, * p <0.05, ns is not significant.

Figures 16A-16D. The bridge molecule secreted by hUTC binds to the photoreceptor OS. Immunofluorescence (IF) staining of OS, OS was individually recombinant human MFG-E8 (124ng / mL), Gas6 (8.75ng / mL), TSP-1 (1.2ng / mL) or TSP-2 (238ng / mL) ) ( Figure 16A ), or hUTC CM ( Figure 16B ), or control medium ( Figure 16C ) for 24 hours, and then double IF staining of rhodopsin and bridge molecules, rhodopsin is conjugated by Alexa Fluor 568 The rhodopsin antibody was stained and the bridge molecule was stained with Alexa Fluor 488 conjugated anti-MFG-E8, anti-Gas6, anti-TSP-1, anti-TSP-2, mouse IgG2A or mouse IgG2B isotype control antibody. The rhodopsin-stained OS particles also showed positive staining for each of the recombinant bridge molecule proteins or secretory bridge molecules present in hUTC CM. ( Figure 16D ) The specificity of anti-rhodopsin antibodies was confirmed by double IF staining of OS with Alexa Fluor 568 conjugated anti-rhodopsin antibody and Alexa Fluor 488 conjugated mouse IgG2b, x isotype control antibody. Upper panel ( Figures 16A-16D ), bridge molecules and isotype antibody staining; lower panel (Figures 16A-16D), rhodopsin antibody staining.

17A to 17F show that hUTC and hUTC conditioned medium prevent oxidative stress or damage. 17A to 17B illustrate that hUTC conditioned medium prevents APE-containing RPE cells from surviving after 430 nm irradiation. (Figure 17A). Cell death is detected using a two-color fluorescent test. The hUTC conditioned medium and unconditioned control medium (250 μL / well) were cultured with A2E-rich ARPE-19 cells for 7 days. The percentage of non-viable cells was determined by two-color fluorescence assay; 5 repeats. Figure 17B shows pooled data from 5% and 10% FBS processing. Values are average +/- SEM. p <0.05; single factor ANOVA and Newman Keuls multiple comparison test. Figures 17C to 17D illustrate that hUTC conditioned medium prevents ARPE-19 cells from reducing cell viability associated with A2E photooxidation. (Figure 17C). Cell viability was tested by MTT. hUTC conditioned medium and unconditioned control medium (250 μL / well) were cultured with ARPE-19 cells rich in A2E (7 days, 37 ° C, 5% CO ₂ , 5% FBS). Bar height indicates MTT absorbance and reflects cell viability. Figure 17D shows pooled data from 5% and 10% FBS processing. Values are mean +/- SEM; 4 replicates / 2 experiments. * p >0.05; ** p <0.05; single factor ANOVA and Newman Keuls multiple comparison test. 17E to 17F illustrate that hUTC conditioned medium prevents ARPE-19 cells from reducing cell viability associated with acute H ₂ O ₂ . Cell viability was verified by MTT (Figure 17E) and crystal violet (Figure 17F) . The y-axis represents the corrected OD reading at 550nm. The data system is expressed as mean ± standard deviation. p <0.05, using two-factor ANOVA.

Figure 18 shows immunofluorescence micrographs of human RPE and rat RPE phagocytosis.

Figure 19 shows immunofluorescence micrographs of the time course of human RPE and rat RPE phagocytosis.

Figure 20 shows immunofluorescence micrographs of human ROS engulfed by rat RPE.

Figure 21 shows cytokeratin immunostaining of human RPE (sample 15-02-032).

Figure 22 shows the cytokeratin immunostaining of San Diego 1 "dry" AMD RPE.

Figure 23 shows cytokeratin immunostaining of human wet AMD RPE (sample 15-10-021).

Figure 24 shows cytokeratin immunostaining of ND08333 "AMD" RPE.

Figures 25A and 25B show the comparison of RPE phagocytic levels from individuals of different ages.

Figure 26 shows the comparison of phagocytosis of ROS on 15-10-021 by normal RPE and AMD (SD1 L & R ("dry"), 15-10-021 ("wet")) RPE.

Figure 27 shows the human ROS dose response test results for human RPE (sample 15-08-074).

Figure 28 shows the human ROS dose response test results for human RPE (Samples 15-11-098).

Figure 29 shows the phagocytosis of ROS on human eye 15-09-027 (including human eye 15-04-001 RPE) by AMD RPE and normal RPE with or without CM.

Figure 30 shows the effect of RTK ligand GDNF on AMD RPE phagocytosis.

Figure 31 shows the effect of RTK ligand HGF on "wet" AMD RPE (15-10-021) phagocytosis.

Figure 32 shows the effect of RTK ligand BDNF on phagocytosis of "wet" AMD RPE (15-10-021).

Figure 33 shows the effect of RTK ligand GDNF on "wet" AMD RPE (15-10-021) phagocytosis.

Figure 34 shows the effect of RTK ligand BDNF on AMD RPE phagocytosis.

Figure 35 shows the effect of RTK ligand HGF on AMD RPE (ND08333) phagocytosis.

Figure 36 shows the effect of RTK ligand GDNF on AMD RPE (ND08333) phagocytosis.

Figure 37 shows the effect of RTK ligand GDNF on AMD RPE (ND08626) phagocytosis.

Figure 38 shows the effect of RTK ligand BDNF on AMD RPE (ND08626) phagocytosis.

Figure 39 shows the effect of RTK ligand HGF on AMD RPE (ND08626) phagocytosis.

Figure 40 shows the effect of the bridge molecule MFG-E8 on AMD RPE (SD1 L) phagocytosis.

Figure 41 shows the effect of the bridge molecule Tsp 1 on AMD RPE (SD1 L) phagocytosis.

Figure 42 shows the effect of the bridge molecule Tsp 2 on AMD RPE (SD1 L) phagocytosis.

Figure 43 shows the effect of the bridge molecule Tsp 1 on phagocytosis of "wet" AMD RPE (15-10-021).

Figure 44 shows the effect of the bridge molecule MFG-E8 on phagocytosis of "wet" AMD RPE (15-10-021).

Figure 45 shows the effect of the bridge molecule Tsp 2 on phagocytosis of "wet" AMD RPE (15-10-021).

Figure 46 shows the effect of the bridge molecule MFG-E8 on AMD RPE (ND08333) phagocytosis.

Figure 47 shows the effect of the bridge molecule Tsp 1 on AMD RPE (ND08333) phagocytosis.

Figure 48 shows the effect of bridge molecule Tsp 2 on phagocytosis of AMD RPE (ND08333).

Figure 49 shows the effect of the bridge molecule MFG-E8 on AMD RPE (ND08626) phagocytosis.

Figure 50 shows the effect of bridge molecule Tsp 1 on AMD RPE (ND08626) phagocytosis.

Figure 51 shows the effect of the bridge molecule Tsp 2 on phagocytosis of AMD RPE (ND08626).

Other features and advantages of the present invention will be apparent from the following embodiments and examples.

Various patents and other publications are mentioned in this specification. The entire contents of each of these publications are incorporated herein by reference. In the following detailed description of the illustrative embodiments, reference is made to the accompanying drawings that form part of this specification. These embodiments are described in sufficient detail to allow those skilled in the art to implement the present invention, and will understand that other embodiments can be utilized, and logical structures and machinery can be made without departing from the spirit or scope of the present invention , Electrical and chemical changes. In order to avoid details that are not necessary for those with ordinary knowledge in the technical field to implement the embodiments described herein, this specification may omit certain information known to those with ordinary knowledge in the technical field. Therefore, the following embodiments should not be construed in a limiting sense, and the scope of the illustrative examples is defined by the scope of the accompanying patent applications.

definition

The various terms used in this specification and the scope of patent application are defined as set forth below and are intended to clarify the present invention.

Stem cells are undifferentiated cells, which are defined by the ability of a single cell to simultaneously renew and differentiate to produce progeny cells, including self-renewing progenitors and non-renewing progenitors ( non-renewing progenitor) and terminally differentiated cells. Stem cells are also characterized by their ability to differentiate into functional cells of various cell lineages of multiple germ layers (endoderm, mesoderm and ectoderm) in vitro, and the ability to form multiple germ layer tissues after transplantation The injection of blastocysts substantially contributes to the formation of most, if not all, tissues.

Stem cells are classified according to their developmental potential as follows: (1) totipotent; (2) pluripotent; (3) multipotent; (4) oligopotent; and (5 ) Unipotent. Totipotent cells can form all embryonic and extraembryonic cell types. Pluripotent cells can form all embryonic cell types. Pluripotent cells include those cells that can form a subset of cell lineages, but all are within a specific tissue, organ, or physiological system (eg, hematopoietic stem cells (HSC) can produce progeny, including HSC (self-renewal), blood cell restriction The precursor of dysfunction, and all cell types and components that are normal components of blood (eg, platelets). Pluripotent cells can form a more restricted subset of cell lineages than pluripotent stem cells; and unipotent cells can form a single cell lineage (eg spermatogenic stem cells).

Stem cells are also classified according to the source from which they were obtained. Adult stem cells are usually pluripotent undifferentiated cells, which are found in tissues containing multiple differentiated cell types. Adult stem cells can renew themselves. Under normal circumstances, it can also differentiate to produce a specialized cell type of its original tissue, and may also produce other tissue types. Induced pluripotent stem cells (iPS cells) are adult cells transformed into pluripotent stem cells. (Takahashi et al., Cell , 2006; 126 (4): 663-676; Takahashi et al., Cell , 2007; 131: 1-12). Embryonic stem cells are pluripotent cells derived from the inner cell mass of embryos at the embryonic cell stage. Fetal stem cells are stem cells derived from fetal tissues or membranes. Postpartum stem cells are pluripotent or pluripotent cells, which are essentially derived from extra-embryonic tissues available after production, that is, the placenta and umbilical cord. These cells have been found to possess the characteristics of pluripotent stem cells, including the potential for rapid proliferation and differentiation into multiple cell lineages. Postpartum stem cells can be blood-derived (such as those derived from cord blood) or non-blood-derived (such as non-blood tissue derived from the umbilical cord and placenta).

Embryonic tissue is generally defined as tissue derived from an embryo (in humans, a period of about six weeks from fertilization to development). Carcass tissue refers to tissue derived from the carcass, which refers to the period from about six weeks of development to delivery in humans. Extra-embryonic tissue is tissue that is related to the embryo or carcass but not derived from it. Extra-embryonic tissues include the extra-embryonic membranes (chorionic membrane, amniotic membrane, yolk sac, and allantoic), umbilical cord, and placenta (which themselves form from the chorion and maternal decidua).

Differentiation is the process by which unspecified ("uncommitted") or less specialized cells acquire characteristics of specialized cells (such as, for example, nerve cells or muscle cells). Differentiated cells are cells that occupy higher specialized ("committed") positions within the cell lineage. The term "committed" when applied to the differentiation process means that the cell has reached a point in the differentiation path. Under normal circumstances, the cell that reaches this point will continue to differentiate into a specific cell type or subtype of cell type, and it is normal Under the environment, it cannot differentiate into different cell types or revert to lower differentiated cell types. De-differentiation refers to the process by which cells revert to lower specialized (or directed) positions within the cell lineage. As used herein, the lineage of a cell defines the inheritance of the cell, ie which cells the cell line comes from and which cells the cell can form. The lineage of cells places the cells in a hereditary scheme for development and differentiation.

Broadly speaking, a progenitor cell (progenitor cell) is a cell that can produce a more differentiated descendant than itself, but retains the ability to replenish the precursor pool. In this definition, stem cells themselves are also precursor cells, like more immediate precursors of terminally differentiated cells. When referring to the cells of the present invention, as described in more detail below, this broader definition of precursor cells can be used. In a narrow sense, a precursor cell is usually defined as a cell that acts as an intermediary in the differentiation pathway, that is, it is formed by stem cells and acts as an intermediary in the production of mature cell types or subpopulations of cell types. This type of prodromal cell is usually unable to renew itself. Therefore, if this type of cell is mentioned in this article, it will be referred to as a non-renewing progenitor cell or an intermediate progenitor or precursor cell.

As used herein, the phrase "differentiates into an ocular lineage or phenotype" refers to cells that become partially or fully directed to a specific ocular phenotype, including but not limited to retinal and corneal stem cells, Pigment epithelial cells of the retina and iris, photoreceptors, retinal ganglia, and other optic nerve lineages (eg, retinal glial cells, microglia cells, stellate cells, Mueller cells), water crystal forming cells, and Sclera, cornea, limbus (limbus) and conjunctival epithelial cells. The phrase "differentiates into a neural lineage or phenotype" refers to a specific neural phenotype in which cells become partially or completely directed to the CNS or PNS, that is, neurons or glial cells, the latter Categories include, but are not limited to, stellate cells, oligodendritic cells, Schwann cells, and micelles.

The cells exemplified herein and preferably used in the present invention are generally referred to as postpartum-derived cells (or PPDC). It may sometimes be more specifically referred to as umbilical derived cells or placental derived cells (UDC or PDC). In addition, these cells may be described as stem cells or progenitor cells, and the latter term is used in a broad manner. The term derived is used to indicate that the cell line is derived from its biological source, and is grown in vitro or otherwise regulated (eg, cultured in growth medium to expand populations and / or cell lines). The in vitro regulation of umbilical stem cells and placental stem cells and the unique characteristics of the umbilical derived cells and placental derived cells of the present invention are described in detail later. Cells isolated from the postpartum placenta and umbilicus by other means are also considered suitable for use in the present invention. These other cells are referred to herein as postpartum cells (not postpartum derived cells).

Various terms are used to describe the cells in culture. Cell culture generally refers to cells taken from living organisms and grown under controlled conditions ("in culture" or "cultured"). Primary cell culture is a culture of cells, tissues, or organs taken directly from the organism and before the first subculture. When cells are placed in growth medium and under conditions favorable for cell growth and / or division, they expand in culture resulting in a larger cell population. When cells are expanded in culture, the rate of cell proliferation is sometimes measured by the amount of time required to double the number of cells. This is called the doubling time.

A cell line is a cell population formed by one or more subcultivations of primary cell cultures. Each round of subculture is called a passage. When cells are subcultured, they are said to have been passaged. Certain cell populations or cell lines are sometimes referred to or characterized by their number of passages. For example, a cultured cell population subcultured ten times may be referred to as a P10 culture. The primary culture (the first culture after the cells were separated from the tissue) was named P0. After the first subculture, these cell lines were described as secondary cultures (P1 or subculture 1). After the second subculture, the cells became a three-culture (P2 or subculture 2), and so on. Those of ordinary skill in the art will understand that there will be multiple population doublings during the subculture; therefore, the number of population doublings of the culture is greater than the number of subcultures. The expansion of cells during the passage interval (ie, the number of population doublings) depends on many factors, including but not limited to seeding density, substrate, culture medium, growth conditions, and passage interval time.

The term growth medium generally refers to a medium sufficient to cultivate PPDC. In detail, one currently preferred medium for culturing the cells of the present invention includes Dulbecco's Modified Essential Media (also abbreviated as DMEM herein). Particularly preferred is DMEM-low glucose (also referred to herein as DMEM-LG) (Invitrogen, Carlsbad, Calif.). DMEM-low glucose is preferably supplemented with 15% (v / v) fetal bovine serum (such as defined fetal bovine serum (Hyclone, Logan Utah)), antibiotics / antimycotics (preferably 50 to 100 Units / ml penicillin, 50 to 100 μg / ml streptomycin, and 0 to 0.25 μg / ml amphotericin B; Invitrogen, Carlsbad, Calif.), And 0.001% (v / v) 2-mercaptoethanol (Sigma , St. Louis Mo.). As used in the following examples, the growth medium refers to DMEM-low glucose with 15% fetal bovine serum and antibiotics / antimycotics (when penicillin / streptomycin is included, it is preferably 50U / ml and 50 micrograms / ml, respectively) ; When penicillin / streptomycin / amphotericin is used, it is preferably 100 U / ml, 100 μg / ml, and 0.25 μg / ml, respectively). In some cases, different growth media are used, or different supplements are provided, which are usually indicated in the text as supplements for growth media.

A conditioned medium is a medium in which specific cells or cell population lines are cultured and then removed. When cells are cultured in a medium, they can secrete cytokines that can provide nutritional support to other cells. Such nutritional factors include, but are not limited to hormones, interleukins, extracellular matrix (ECM), proteins, vesicles, antibodies, and particles. The medium containing cytokines is conditioned medium.

Generally, trophic factors are defined as substances that promote cell survival, growth, differentiation, proliferation, and / or maturation, or substances that stimulate cell activity. The interaction between cells via trophic factors can occur between different types of cells. Cell interactions with the help of trophic factors are found in essentially all cell types, and are a particularly significant means of communication for nerve cell types. Nutritional factors can also act in an autocrine manner, that is, cells can produce nutritional factors that affect their own survival, growth, differentiation, proliferation, and / or maturation.

When referring to cultured vertebrate cells, the term senescence (replicative senescence or cellular senescence is also the same) refers to properties attributable to limited cell culture; that is, they are incapable of Growth exceeds the limited number of population doublings (sometimes called Hayflick's limit). Although cell senescence was first described using fibroblast-like cells, most normal human cell types that can successfully grow in culture undergo cell senescence. Different cell types have different life spans in vitro, but the maximum life span is usually less than 100 population doublings (this is the number of doublings in which all cells in the culture become senescent and therefore make the culture unable to divide). Aging does not depend on time sequence, but is measured by the number of cell divisions or population doublings experienced by the culture.

The terms ocular, ophthalmic, and optic are used interchangeably herein to define "of, or about, or related to the eye." The term ocular degenerative condition (or disorder) is an inclusive term that covers acute and chronic conditions and disorders involving the eyes (including the neural connection between the eye and the brain) of cell damage, degeneration or loss Or disease. Eye degeneration symptoms may be related to age, or may be caused by injury or trauma, or may be related to a specific disease or condition. Acute ocular degenerative conditions include but are not limited to conditions related to cell death or damage to the eye, including conditions caused by: cerebrovascular insufficiency, focal or diffuse brain trauma, diffuse brain injury, infection Sexual or inflammatory eye conditions, split or detached retina, intraocular lesions (contusion penetration, compression, tearing) or other physical injuries (eg, physical or chemical burns). Chronic ocular degeneration conditions (including progressive conditions) include but are not limited to retinopathy and other retinal / macular diseases, such as retinitis pigmentosa (RP), age-related macular degeneration (AMD), choroidal neovascular membrane (CNVM); Retinopathy, such as diabetic retinopathy, occlusive retinopathy, sickle cell retinopathy and hypertensive retinopathy, central retinal vein occlusion, carotid artery stenosis, optic neuropathy, such as glaucoma and related syndromes; water lens and external eye disorders such as cornea Limbry stem cell deficiency (LSCD), also known as limbal epithelial cell deficiency (LECD), such as occurs in chemical or thermal injuries, Steven-Johnson syndrome, contact lens-induced corneal lesions, eye scarring Pemphigoid, congenital absence of iris or ectodermal dysplasia, and multiple endocrine deficiency related keratitis.

The term treatment of an ocular degenerative condition or treatment of an ocular degenerative condition refers to the effect of relieving an ocular degenerative condition as defined herein, or delaying, suspending or reversing the eye The progress of degenerative symptoms, or delay or prevent the occurrence of ocular degenerative symptoms.

The term effective amount refers to the concentration or amount of an agent or pharmaceutical composition such as growth factors, differentiation agents, trophic factors, cell populations, or other agents that is effective to produce the desired result, including as described herein Cell growth and / or differentiation in vitro or in vivo, or treatment of ocular degeneration. With regard to growth factors, the effective amount may range from about 1 ng / ml to about 1 μg / ml. With regard to PPDC administered to a patient, the effective amount may range from as few as hundreds or less to as many as millions or more. In a particular embodiment, an effective amount can range from 10 ³ to 11 ¹¹ cells, the more specific words of at least about ¹⁰⁴ cells. It should be understood that the number of cells to be administered will depend on the details of the condition to be treated, including but not limited to the size or total volume / surface area to be treated, the proximity of the administration site to the area to be treated, and medical biology The home is familiar with other factors.

The terms effective period (or time) and effective condition refer to the period of time or other controllable conditions necessary or better for the agent or pharmaceutical composition to achieve its expected result (For example, the temperature and humidity of the in vitro method).

The terms patient or subject refer to animals treated with the pharmaceutical composition described herein or according to the methods described herein, including mammals, preferably humans.

The term pharmaceutically acceptable carrier (or medium) can be used interchangeably with the term biologically compatible carrier or medium, which refers to reagents, cells, compounds, Materials, compositions, and / or dosage forms are not only compatible with the cells and other agents to be administered therapeutically, but also suitable for contact with human and animal tissues without excessive toxicity within the scope of reasonable medical judgment , Irritation, allergic reactions, or other complications in a reasonable benefit / risk ratio.

Several terms related to cell replacement therapy are used herein. The terms autologous transfer, autologous transplantation, autograft and similar terms refer to the treatment in which the cell donor is also the recipient of cell replacement therapy. The terms allogeneic transfer (allogeneic transfer), allogeneic transplantation (allogeneic transplantation), allograft (allograft) and similar terms refer to the treatment in which the cell donor and the recipient of the cell replacement therapy are the same species, but not the same individual . Donor cells and recipients' cell compatibility matching cell transfer is sometimes referred to as syngeneic transfer (syngeneic transfer). The terms xenogeneic transfer, xenogeneic transplantation, xenograft, and similar terms refer to the treatment in which the cell donor and the recipient of cell replacement therapy are different species. Transplantation as used herein refers to the introduction of autologous, or allogeneic donor cell replacement therapy to the recipient.

As used herein, the term "about" when referring to a measurable value (such as quantity, duration, and the like) is intended to cover between ± 20% and ± 0.1% of the specified value, preferably Variations of ± 20% or ± 10%, more preferably ± 5%, even better ± 1%, and still more preferably ± 0.1%, if such variations are suitable for performing the disclosed method.

Explanation

The ocular degeneration pathology covers acute, chronic, and progressive disorders and diseases with different causes. The common feature is the abnormal or loss of function of specific or vulnerable eye cell populations. This commonality enables the development of similar treatments for repairing or regenerating vulnerable, damaged or lost eye tissue, one of which is cell-based therapy. The development of cell therapies for ocular degenerative diseases is limited to relatively few types of stem cells or precursor cells, including eye-derived stem cells themselves (eg, retinal and corneal stem cells), embryonic stem cells, and a few types of adult stem cells or precursor cells (eg , Nerve, mucosal epithelium and bone marrow stem cells). Cells isolated from the postpartum umbilical cord and placenta have been identified as a significant new source of precursor cells suitable for this purpose. (US 2005-0037491 and US 2010-0272803) In addition, conditioned medium produced from cells isolated from postpartum placenta and umbilical cord tissue provides another new source for the treatment of ocular degeneration. Therefore, in the various embodiments described herein, the present invention is characterized by a method and composition (including a pharmaceutical composition) for repairing and regenerating eye tissue, which uses precursor cells isolated from postpartum tissue And conditioned medium for cell populations. The present invention is applicable to ocular degenerative conditions, but it is expected to be particularly applicable to some ocular disorders that are difficult to treat or cure or have no available treatment or therapy. These eye conditions include, but are not limited to, age-related macular degeneration, retinitis pigmentosa, diabetic, and other retinopathy.

It is expected that conditioned medium derived from precursor cells, such as cells isolated from the postpartum umbilical cord or placenta according to any method known in the art, is suitable for use in the present invention. However, in one embodiment, the present invention uses a conditioned medium derived from umbilical cord tissue-derived cells (hUTC) or placental tissue-derived cells (PDC) as defined above, these cell lines being derived preferably from the following The method of making it substantially free of umbilical cord tissue or placenta without blood. hUTC or PDC can expand in culture and have the potential to differentiate into cells of other phenotypes. Certain embodiments are characterized by a conditioned medium prepared from such precursor cells, a composition containing the conditioned medium, and a method of using a composition such as a pharmaceutical composition to treat patients with acute or chronic ocular degenerative diseases. The postpartum-derived cells of the present invention are characterized by their growth properties in culture, their cell surface markers, their gene expression, their ability to produce certain biochemical trophic factors, and their immune properties. The conditioned medium derived from postpartum-derived cells is characterized by trophic factors and bridge molecules secreted by these cells.

Preparation of precursor cells

Cells, cell populations, and formulations containing cell lysates, conditioned media, and the like used in the compositions and methods of the present invention are described herein, and are described in detail in U.S. Patent Nos. 7,524,489, and 7,510,873, and In US Published Application No. 2005/0058634, all of which are incorporated herein by reference. According to these methods, the mammalian umbilical cord and placenta are recovered at the end of a full-term or short-term pregnancy or shortly after the end, for example, after the birth-birth is discharged. Postpartum tissues can be transported from the production site to the laboratory in sterile containers such as flasks, beakers, petri dishes or bags. The container may have a solution or culture medium, including but not limited to a saline solution such as Dulbecco's Modified Eagle's Medium (DMEM) or phosphate buffered saline (PBS), or any organ used for transportation for transplantation Such as the University of Wisconsin solution (University of Wisconsin solution) or perfluorochemical solution (perfluorochemical solution). One or more antibiotics and / or antimycotics (such as but not limited to penicillin, streptomycin, amphotericin B), gentamicin and nystatin ( nystatin)) to this medium or buffer. Postpartum tissues can be rinsed with anticoagulant solutions such as heparin-containing solutions. Preferably, the tissue is kept at about 4 to 10 ° C before PPDC extraction. Even better is that the tissue is not frozen before PPDC extraction.

The separation of PPDC is preferably carried out in a sterile environment. The umbilical cord can be separated from the placenta by means known in the art. Alternatively, the umbilical cord and placenta can be used without being separated. It is preferable to remove blood and debris from postpartum tissues before separating PPDC. For example, postpartum tissues can be washed with buffers, such as but not limited to phosphate buffers. The washing buffer may also contain one or more anti-fungal agents and / or antibiotics, such as but not limited to penicillin, streptomycin, amphotericin B, itanomycin and nystatin.

Postpartum tissues that contain intact placenta or umbilical cord, or fragments or sections thereof are disintegrated by mechanical force (shredding or shearing force). In the presently preferred embodiment, the separation procedure also uses enzyme digestion. Many enzymes are known in the art to isolate individual cells from complex tissue matrices to facilitate growth in culture. Such enzymes are commercially available and range from weak digestibility (such as deoxyribonuclease and neutral protease dispase) to strong digestibility (such as papain and trypsin). A non-exhaustive list of enzymes compatible with this article includes mucolytic enzyme activity, metalloprotease, neutral protease, serine protease (e.g. trypsin, chymotrypsin or elastase) and deoxyribose Nuclease. Currently preferred are enzyme actives selected from metalloproteinases, neutral proteases and mucolytic activity. For example, collagenase is known to isolate various cells from tissues. Deoxyribonuclease can digest a single strand of DNA and can minimize cell clumping during separation. Preferred methods involve enzymatic treatment with, for example, collagenase and dispase, or collagenase, dispase, and hyaluronidase, and such methods are provided, wherein in certain preferred embodiments, collagenase and neutral protease dispase The mixture is used in the dissociation step. More preferably, it is a method of digesting in the presence of at least one of collagenase from Clostridium histolyticum and protease actives, dispase and thermolysin. Even better is a method of digestion using collagenase and dispase enzyme actives at the same time. It is also preferable to include a method of digesting with hyaluronidase actives in addition to collagenase and dispase actives. Those skilled in the art will understand that many such enzyme treatments are well known in the art and can be used to isolate cells from various tissue sources. For example, the LIBERASE ^™ Blendzyme 3 (Roche) series of enzyme combinations is suitable for use in the method of the present invention. Other sources of enzymes are known, and those skilled in the art can also obtain these enzymes directly from the natural sources of such enzymes. Those skilled in the art also have sufficient knowledge to evaluate the utility of new or other enzymes or enzyme combinations in the isolation of cells of the invention. The preferred enzyme treatment takes 0.5, 1, 1.5 or 2 hours or more. In other preferred embodiments, the tissue is cultured at 37 ° C during the enzyme treatment in the dissociation step.

In some embodiments of the invention, the postpartum tissue is divided into sections containing various tissue morphologies such as, for example, neonates, placenta / maternal and maternal morphology of the placenta. The separated sections are then dissociated by mechanical and / or enzymatic dissociation according to the methods described herein. The cells of the newborn or maternal lineage can be identified by any means known in the art, such as by karyotype analysis or in situ hybridization of the Y chromosome.

The isolated cells or the postpartum tissue from which PPDC is grown can be used to initiate or inoculate cell cultures. Transfer the separated cells to a sterile tissue culture container, and the container is not coated or extracellular matrix or ligands such as laminin, collagen (native, denatured or cross-linked), gelatin, fibronectin and other cells Outer matrix protein coating. PPDC is cultured in any medium that can maintain the growth of these cells, such as but not limited to DMEM (high or low glucose), advanced DMEM, DMEM / MCDB 201, Eagle's basal medium, Ham F10 Medium (Ham's F-10 medium, F10), Ham F-12 medium, Iscove's modified Dulbecco's medium, Mesenchymal Stem Cell Growth Medium (MSCGM), DMEM / F12, RPMI 1640 and cellgro FREE ^TM . The culture medium can be supplemented with one or more components, including, for example, fetal bovine serum (FBS) (preferably about 2-15% (v / v)); horse serum (ES); human serum (HS); β-mercaptoethanol ( beta-mercaptoethanol, BME or 2-ME), preferably about 0.001% (v / v)); one or more growth factors, such as platelet-derived growth factor (PDGF), epidermal growth factor (EGF), fibroblast growth Factor (FGF), vascular endothelial growth factor (VEGF), insulin-like growth factor-1 (IGF-1), leukocyte inhibitory factor (LIF) and erythropoietin; amino acids, including L-valine; and one or A variety of antibiotics and / or anti-fungal agents to control microbial contamination, such as penicillin G, streptomycin sulfate, amphotericin B, itanomycin and nystatin, whether used alone or in combination. The culture medium preferably contains growth medium (DMEM-low glucose, serum, BME, and antibiotics).

The cell line is seeded in the culture vessel at a density that allows the cells to grow. In a preferred embodiment, the cells are cultured in air at about 0 to about 5 volume percent CO ₂ . In some preferred embodiments, the cells are cultured at about 2 to about 25 percent O ₂ in air, preferably at about 5 to about 20 percent O ₂ in air. The preferred cell line is cultured at about 25 to about 40 ° C, and the more preferred line is cultured at 37 ° C. The cells are preferably cultured in an incubator. The culture medium in the culture vessel may be stationary or stirred, for example, using a bioreactor. Preferably, the PPDC is grown under low oxidative stress (for example, adding glutathione, vitamin C, catalase, vitamin E, N-acetylcysteine). As used herein, "low oxidative stress" refers to the condition of no or minimum free radical damage to the cultured cells.

Methods for selecting the most appropriate medium, medium preparation, and cell culture technology are well known in the art, and are described in various sources, including Doyle et al. (Eds.), 1995, Cell & TISSUE CULTURE: LABORATORY PROCEDURES , John Wiley & Sons, Chichester; and Ho and Wang (eds.), 1991, ANIMAL CELL BIOREACTORS, Butterworth-Heinemann, Boston, all of which are incorporated herein by reference.

After culturing the separated cells or tissue fragments for a sufficient period of time, PPDC will grow out, which is caused by tissue migration from postpartum or cell division, or both. In some embodiments of the invention, the PPDC is subcultured or removed to a separate culture vessel containing fresh medium of the same or different type as the medium initially used, where the cell population can be expanded by mitosis. The cells of the present invention can be used at any point between passage 0 and senescence. Preferably, the cells are passaged between about 3 and about 25 times, more preferably about 4 to about 12 times, and more preferably 10 or 11 times. Cloning and / or subcloning can be performed to confirm the clonal population of isolated cells.

In some aspects of the invention, the different cell types present in the postpartum tissue are fractionated into subpopulations from which PPDCs can be separated. This can be achieved using standard cell separation techniques, including but not limited to enzymatic treatment to dissociate postpartum tissue into its component cells, followed by colonization and selection of specific cell types, such as but not limited to selection based on morphology and / or biochemical markers ; Selective growth of desired cells (positive selection), selective destruction of non-desired cells (negative selection); separation according to the agglutinability of differential cells in mixed populations, for example using soybean lectin; freezing- Thawing procedures; differential adherence of cells in mixed populations; filtration; traditional and zonal centrifugation; centrifugal elutriation (counter-streaming centrifugation); unit gravity separation; countercurrent Countercurrent distribution; electrophoresis; and fluorescence activated cell sorting (FACS). For a review of plant selection and cell isolation techniques, see Freshney, 1994, CULTURE OF ANIMAL CELLS: A MANUAL OF BASIC TECHNIQUES, 3rd edition, Wiley-Liss, Inc., New York, which is incorporated herein by reference.

If necessary, replace the culture medium, for example, carefully aspirate the medium from the dish using, for example, a pipette, and then make up with fresh medium. Continue culturing until sufficient cell number or density has accumulated in the dish. The original explanted tissue sections can be removed and then trypsinized using standard techniques or using a cell spatula. After trypsin digestion, the cells are collected, transferred to fresh medium and then cultured as described above. In some embodiments, the medium is changed at least once about 24 hours after trypsin digestion to remove any floating cells. The remaining cells in the culture are considered PPDC.

PPDC can be stored frozen. Therefore, in the preferred embodiment described in more detail below, PPDCs used for self-transfer (whether for mothers or infants) can be derived from appropriate postpartum tissues after the infant is born, and then cryopreserved so that subsequent transplantation is required These cells are available.

Characteristics of precursor cells

The precursor cells of the present invention, such as PPDC, can be characterized by: for example, growth characteristics (eg, population doubling ability, doubling time, number of passages to aging), karyotype analysis (eg, normal karyotype; maternal or newborn lineage), flow Cytometry (eg FACS analysis), immunohistochemistry and / or immunocytochemistry (eg detection of epitopes), gene expression profiles (eg gene chip array analysis; polymerase chain reaction (eg reverse transcriptase PCR, Real-time PCR and traditional PCR)), protein array analysis, protein secretion method (e.g. by plasma coagulation assay or PDC conditioned medium analysis (e.g. by Enzyme Linked ImmunoSorbent Assay (ELISA))), mixed lymphocytes Spherical response (e.g. as a measure of PBMC stimulation) and / or other methods known in the art.

An example of PPDC derived from umbilical tissue was deposited with the American Type Culture Preservation Center (ATCC, 10801 University Boulevard, Manassas, VA, 20110) on June 10, 2004, and the assigned ATCC access number is as follows: (1 ) The cell line code UMB 022803 (P7) is assigned the access number PTA-6067; and (2) the cell line code UMB 022803 (P17) is assigned the access number PTA-6068. Examples of PPDCs derived from placental tissues are deposited with the American Type Culture Collection (ATCC, Manassas, Va.), And the ATCC access numbers assigned are as follows: (1) Cell line code PLA 071003 (P8) was in 2004 Deposited on June 15, 2014 and assigned access number PTA-6074; (2) Cell line code PLA 071003 (P11) was deposited on June 15, 2004 and assigned access number PTA-6075 ; And (3) The cell line code PLA 071003 (P16) was deposited on June 16, 2004 and the assigned access number was PTA-6079.

In various embodiments, PPDC possesses one or more of the following growth characteristics: (1) L-valine is required for growth in culture; (2) They can grow to contain about 5% to at least about 20% In the atmosphere of oxygen; (3) they have the potential of at least about 40 doublings before reaching senescence in culture; and (4) they attach and expand on coated or uncoated tissue culture vessels, where The coated tissue culture container contains a coating of gelatin, laminin, collagen, polyguanylic acid, glass connexin, or fibronectin.

In certain embodiments, PPDC possesses a normal karyotype, which is maintained during cell passage. Karyotyping is particularly useful for identifying and distinguishing neonates and maternal cells derived from the placenta. The karyotype analysis method can be used and known by those with ordinary knowledge in the technical field.

In other embodiments, PPDC can be characterized by producing certain proteins, including: (1) producing at least one of vimentin and alpha-smooth muscle actin; and ( 2) Production of at least one of CD10, CD13, CD44, CD73, CD90, PDGFr-α, PD-L2 and HLA-A, HLA-B, HLA-C cell surface markers, such as by flow cytometry Detect. In other embodiments, PPDC can be characterized by not producing at least one of the following: CD31, CD34, CD45, CD80, CD86, CD117, CD141, CD178, B7-H2, HLA-G, and HLA-DR, HLA- DP, HLA-DQ cell surface markers, as detected by flow cytometry. Particularly preferred are cells that produce vimentin and α-smooth muscle actin.

In other embodiments, PPDC can be characterized by an increase in gene expression (relative to human cells of fibroblasts, mesenchymal stem cells, or iliac bone marrow cells) encoding genes encoding at least one of: Retinol 8; Reticulon 1; Chemokine (C--X--C motif) ligand 1 (melanoma growth stimulating active substance, alpha); Chemokine (C--X-- C motif) ligand 6 (granulocyte chemoattractant protein 2); chemokine (C--X--C motif) ligand 3; tumor necrosis factor, alpha-induced protein 3; C-type lectin super Family member 2; Wilms tumor 1; aldehyde dehydrogenase 1 family member A2; renin; oxidized low-density lipoprotein receptor 1; Homo sapiens strain IMAGE: 4179671; Protein kinase C ζ; hypothetical protein DKFZp564F013; ovarian cancer down-regulator 1; and Homo sapiens gene of strain DKFZp547k1113. In one embodiment, PPDC derived from umbilical cord tissue can be increased by gene expression (relative to human cells of fibroblasts, mesenchymal stem cells, or iliac bone marrow cells) encoding genes encoding at least one of the following To characterize: interleukin 8; reticulon 1 (reticulon) 1; or chemokine (C--X--C motif) ligand 3. In another embodiment, PPDC derived from placental tissue may be expressed by a gene targeting at least one gene encoding renin or oxidized low density lipoprotein receptor 1 (relative to fibroblasts, mesenchymal stem cells, or iliac Human cells of bone marrow cells are characterized by increase.

In other embodiments, PPDC can be characterized by a reduction in gene expression (relative to human cells of fibroblasts, mesenchymal stem cells, or iliac bone marrow cells) that encodes at least one of the following genes: dwarf Source box 2; heat shock 27kDa protein 2; chemokine (C--X--C phantom) ligand 12 (stromal cell-derived factor 1); elastin (superior valve aortic stenosis, Williams-Polen (Williams) -Beuren) syndrome; Homo sapiens mRNA; cDNA DKFZp586M2022 (from the strain DKFZp586M2022); mesenchymal homeobox 2 (growth arrest-specific homeo box); sine oculis homeobox Homolog 1 (Drosophila); αB aquacris; morphogenetic disorder associated activator 2 (disheveled associated activator of morphogenesis 2); DKFZP586B2420 protein; similar to neuralin 1; bound tetranectin (tetranectin, fibrinolytic) Plasminogen binding protein); src homolog 3 (SH3) and cysteine rich domain; cholesterol 25-hydroxylase; runt-related transcription factor 3; Albumin 11 receptor, alpha; procollagen C-endopeptidase enhancer; frizzled homolog 7 (Drosophila); hypothetical gene BC008967; type III collagen α1; Tenascin C (hexabrachion); Iroquois homeobox protein 5 (iroquois homeobox protein 5); Hephaestin (hephaestin); integrin β8; synaptic vesicle glycoprotein (synaptic vesicle glycoprotein) 2; neuroblastoma, tumorigenic inhibitory factor 1; insulin-like growth factor binding protein 2, 36 kDa; Homo sapiens cDNA FLJ12280 fis, strain MAMMA1001744; cytokinin receptor factor 1; potassium intermediate / Small conductivity calcium-activated channel, subfamily N, member 4; integrin β7; transcription coactivator (TAZ) with PDZ-binding motif; sine oculis homeobox homolog 2 (Drosophila); KIAAI034 protein; capsule Follicle-associated membrane protein 5 (myobrevin); EGF-containing fibulin-like extracellular matrix protein 1; early growth response protein 3; no dorsal end Source box (distal-less homeo box) 5; hypothetical protein FLJ20373; aldehyde-keto reductase family 1, member C3 (3-alpha hydroxysteroid dehydrogenase, type II); disaccharide ( biglycan); transcription coactivator (TAZ) with PDZ-binding motif; fibronectin 1; proenkephalin; integrin, β1 like (with EGF-like repeat); Homo sapiens mRNA full length Insert cDNA strain EUROIMAGE 1968422; EphA3; KIAA0367 protein; natriuretic peptide receptor C (guantriate cyclase C) (guanylate cyclase C) (atrionatriuretic peptide C (atrionatriuretic peptide receptor C)); hypothetical protein FLJ14054; Homo sapiens mRNA; cDNA DKFZp564B222 (from strain DKFZp564B222); BCL2 / adenovirus E1B 19kDa interacting protein 3 (adenovirus E1B 19kDa interacting protein 3-like); AE Binding protein 1; and cytochrome c oxidase subunit VIIa polypeptide 1 (muscle).

In other embodiments, PPDC can be characterized by secreting a bridge molecule selected from MFG-E8, Gas6, TSP-1, and TSP-2. In addition, PPDC derived from umbilical cord tissue can be secreted by MCP-1, IL-6, IL-8, GCP-2, HGF, KGF, FGF, HB-EGF, BDNF, TPO, MIP1b, I309, RANTES, MDC, And at least one of TIMP1. In some embodiments, PPDC derived from umbilical cord tissue can be characterized by not secreting at least one of TGF-β2, ANG2, PDGFbb, MIP1a, and VEGF, as detected by ELISA. In alternative embodiments, PPDC derived from placental tissue can be secreted by MCP-1, IL-6, IL-8, GCP-2, HGF, KGF, HB-EGF, BDNF, TPO, MIP1a, RANTES, and TIMP1 At least one of them is not characterized by secretion of at least one of TGF-β2, MIP1b, ANG2, PDGFbb, FGF, and VEGF, as detected by ELISA. In a further embodiment, PPDC does not express hTERT or telomerase.

In a preferred embodiment, the cell contains two or more of the characteristics listed above for growth, protein / surface marker production, gene expression, or substance secretion. More preferably, the cells contain three, four, or five or more of these characteristics. Even better is a PPDC containing six, seven, or eight or more of these characteristics. Even better are those cells that contain all the above characteristics.

In a particularly preferred embodiment, the cell line is isolated from human umbilical cord tissue that is substantially free of blood. These cells can be expanded in culture, do not produce CD117 or CD45, and do not express hTERT or telomerase. In one embodiment, the cells do not produce CD117 and CD45 and optionally also do not express hTERT and telomerase. In another embodiment, the cells do not express hTERT and telomerase. In another embodiment, the cell line is isolated from human umbilical cord tissue that is substantially free of blood, can be expanded in culture, does not produce CD117 or CD45, and does not express hTERT or telomerase, and has one of the following characteristics Or more: express CD10, CD13, CD44, CD73, and CD90; do not express CD31 or CD34; relative to human fibroblasts, mesenchymal stem cells, or iliac bone marrow cells, exhibit increased levels of interleukin 8 or endoplasmic Reticulin 1; and has the potential to differentiate.

The currently preferred cells used in several aspects of the present invention are post-natal cells with the above characteristics, and especially those in which these cells have a normal karyotype and maintain the normal karyotype at subsequent passages, and further The cells showed the following markers: CD10, CD13, CD44, CD73, CD90, PDGFr-α and HLA-A, HLA-B, HLA-C, and the production of these cells was immunologically detectable and corresponding to List of labeled proteins. Even better are those cells that do not produce proteins corresponding to any of the following markers except for the aforementioned: CD31, CD34, CD45, CD117, CD141 or HLA-DR, HLA-DP, HLA-DQ as determined by flow cytometry Detected. In a further preferred embodiment, these cells do not express hTERT or telomerase.

Certain cell lines with various phenotypes have the potential for lineage differentiation and are therefore unstable and therefore can differentiate on their own. The cells currently preferred for use in the present invention are cells that do not differentiate themselves, for example, along the neural lineage. When the preferred cells are grown in growth medium, the cell markers produced on their surface, and the expression patterns of their various genes (as determined using Affymetrix GENECHIP) are substantially stable. Through multiple population doublings, these cells remain substantially constant, for example, in terms of their surface marking characteristics.

However, a characteristic of PPDC is that it can be intentionally induced to differentiate into various lineage phenotypes by subjecting it to differentiation-inducing cell culture conditions. In the treatment of certain ocular degenerative conditions, one or more methods known in the art can be used to induce the differentiation of PPDC into a neurophenotype. For example, as exemplified herein, PPDC can be inoculated into Neurobasal-A containing B27 (B27 supplement, Invitrogen), L-glutamic acid, and penicillin / streptomycin in a culture bottle coated with laminin Medium (Invitrogen, Carlsbad, Calif.), The combination of which is referred to herein as neural precursor expansion (NPE) medium. The NPE medium can be further supplemented with bFGF and / or EGF. Alternatively, in vitro differentiation of PPDC can be induced by: (1) co-culturing PPDC with neural precursor cells; or (2) growing PPDC in conditioned medium of neural precursor cells.

The differentiation of PPDC into a neurophenotype can be demonstrated by the morphology of bipolar cells with elongated processes. The induced cell population can be stained positive for the presence of nestin. Differentiated PPDC can be evaluated by detecting nestin, TuJ1 (BIII tubulin), GFAP, tyrosine hydroxylase, GABA, 04 and / or MBP. In some embodiments, PPDC exhibits the ability to form the three-dimensional features of neurosphere stem cells that form neurospheres.

Cell population

Another aspect of the invention is characterized by a population of precursor cells such as postpartum derived cells. Postpartum-derived cells can be isolated from placenta or umbilical tissue. In a preferred embodiment, the cell populations include PPDC as described above, and these cell population lines are described in the next section.

In some embodiments, the cell population is heterogeneous. The heterogeneous cell population of the present invention may comprise at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% cells. The heterogeneous cell population of the present invention may further include precursor cells (postpartum-derived cells), or other precursor cells, such as epithelial or neural precursor cells, or it may further include fully differentiated cells.

In some embodiments, the ethnic group is substantially homogenous, that is, it substantially contains only PPDC (preferably at least about 96%, 97%, 98%, 99% or more cells). In some embodiments, the cell population line is homogeneous. In embodiments, the homogeneous cell population of the present invention may comprise umbilical derived cells or placental derived cells. The homogeneous population of umbilical derived cells is preferably free of maternal lineage cells. The homogeneous population of placental-derived cells can be neonatal or maternal lineage. The homogeneity of the cell population can be achieved by any method known in the art, for example, by cell sorting (eg, flow cytometry) or by plant expansion according to conventional methods. Therefore, it is preferred that the homogeneous PPDC population can contain a colony cell line of postpartum-derived cells. Such ethnic groups are particularly useful when isolating cell strains with highly desired functionality.

Also provided herein are cell populations cultured in the presence of one or more factors, or under conditions that stimulate the differentiation of stem cells along a desired path (eg, nerve, epithelium). Such factors are known in the art and those skilled in the art will understand that the determination of appropriate differentiation conditions can be accomplished through routine experiments. The optimization of such conditions can be accomplished by statistical experiment design and analysis, such as response surface methodology allowing simultaneous optimization of multiple variables, such as in biological cultures. Currently preferred factors include, but are not limited to, factors such as growth or trophic factors, demethylating agents, co-cultivation with nerve or epithelial lineage cells, or culture in nerve or epithelial lineage cell conditioned medium, and are known in the art Other conditions used to stimulate stem cells to differentiate along these pathways (for factors that can be used for neural differentiation, see, for example, Lang, KJD et al., 2004, J. Neurosci. Res. 76: 184-192; Johe, KK et al., 1996 , Genes Devel. 10: 3129-3140; Gottleib, D., 2002, Ann. Rev. Neurosci. 25: 381-407).

Conditioned medium

In one aspect, the invention provides a conditioned medium from cultured precursor cells such as postpartum-derived cells, or other precursor cells, which is used in vitro and in vivo as described below. The use of such conditioned medium allows the beneficial nutrient factors secreted by the cells to be used allogeneically in patients without introducing whole cells that may trigger rejection or other adverse immune reactions. The conditioned medium is prepared by culturing cells (such as cell populations) in the medium, and then removing the cells from the medium. In some embodiments, the postpartum cells are UTC or PDC, more preferably hUTC.

The conditioned medium prepared from the cell population described above can be used as is, further concentrated, for example by ultrafiltration or lyophilization, or even dried, used after partial purification, as known in the technical field Used in combination with a pharmaceutically acceptable carrier or diluent, or in combination with other compounds such as biological products such as pharmaceutically usable protein compositions. The conditioned medium can be used alone in vitro or in vivo, or for example with autologous or syngeneic living cells. If the conditioned medium is introduced into the body, it can be introduced into a local treatment site or distally to provide, for example, the desired cell growth or nutritional factors to the patient.

It has previously been shown that human umbilical cord tissue-derived cells improve visual function and improve retinal degeneration (see US 2010/0272803). It has also been shown that postpartum-derived cells can be used in the RCS model to promote photoreceptor rescue and thus retain photoreceptors. (See US 2010/0272803). Subretinal injection of hUTC into the eyes of RCS rats improved visual acuity and relieved retinal degeneration.

As provided herein, various formulations of hUTC conditioned medium were prepared and evaluated for phagocytosis rescue activity. It was found that inoculation density and culture conditions affect the activity level of conditioned medium. In terms of hUTC in serum (CM1), inoculate hUTC at 5,000 viable cells / cm ² in hUTC growth medium (DMEM low glucose + 15% FBS + 4mM L-glutamic acid) in T75 cell culture flasks And cultivate for 24 hours. The medium was replaced with 21 mL of DMEM / F12 complete medium (DMEM: F12 medium + 10% FBS + Pen (50U / ml) / Strep (50 μg / ml)), the cells were cultured for 54 hours, and the culture supernatant was collected and Freeze (freeze) at -70 ° C.

As for the serum-free medium (CM1 serum-free), the medium was supplemented with 21 mL of DMEM / F12 serum-free medium (DMEM: F12 medium + Pen (50 U / ml) / Strep (50 μg / ml)) on the second day. CM1 with or without serum restored phagocytic activity (Figure 1A to Figure 1B and Figure 2). Another conditioned medium (CM2) was prepared in the same procedure as CM1 with serum, except that the cell lines were cultured in T225 bottles with 63 mL of medium per bottle, and the culture time was 48 hours after changing the medium. However, this medium is not active. (Figure 3A). CM3 was prepared under the same conditions as CM2, but 10,000 viable cells / cm ^{2 were used} , and CM3 stimulated phagocytosis in the deferent RPE. (Figures 3B to 3D).

In the tested conditions, CM2 was found to lack activity (Figure 3A). Compared with CM1, which has a cultivation time of 54 hours, the cultivation time of CM2 is shortened to 48 hours. Compared to CM2, CM3 was prepared by doubling the cell seeding density and maintaining the same incubation time after changing the medium, and CM3 was found to be active. Therefore, in order to obtain active CM, the initial cell seeding density and the cell culture time after medium replacement are two important conditions.

Retinal pigment epithelium (RPE) cells from the Royal College of Surgeons (RCS) rats have defective phagocytosis of the rod outer segment (ROS) due to mutations in Mertk gene. Mertk is a member of the receptor tyrosine kinase (RTK) family and is thought to have its role in RPE phagocytosis. Basic fibroblast growth factor (bFGF) is a ligand for FGF RTK and has been shown to induce phagocytic ability in cultured RPE cells from RCS rats (McLaren et al., FEBS Letters , 1997; 412: 21-29) . In one embodiment of the present invention, the phagocytosis of hUTC to rescue defertile RPE is through the secretion of RTK ligands, activation of RTK signaling, and enhancement of signaling by other phagocytic related receptors.

In one embodiment of the present invention, the RTK ligands BDNF, HB-EGF, PDGF-DD, ephrin A4, HGF, and Pterin B2 have a rescue effect on phagocytosis of RCS-dystrophic RPE cells. In a specific embodiment, BDNF, PDGF-DD, and Pterin B2 have a positive rescue effect. (See FIGS. 5A, 6A to 6B and 7A to 7B).

The non-RTK ligand activates a receptor different from RTK and does not have a similar effect on phagocytosis as the RTK ligand (Figures 8A to 8C and Figure 9). hUTC has been shown to secrete vitronectin, endothelin-1, TGF-β1, and IL-6. Receptors for vitronectin include αvβ3 and αvβ5 integrin. Finnemann et al. Reported that RPE cells require αvβ5 integrin for phagocytosis of ROS (Finnemann et al., 1997, see above). Although hUTC CM increased the phagocytosis of dystrophic RPE cells, endothelin-1, TGF-β1 or IL-6 (concentration 200 ng / mL), and vitronectin (various concentrations) had no effect on RCS RPE phagocytosis (Figures 8A to 8C , Figure 9).

Gene expression profiles performed using RNA analysis of conditioned medium-treated and untreated dystrophic RPE showed that hUTC expressed multiple RTK ligand genes within 15 RTK subfamilies (Figure 10 and Table 1-1). hUTC also expresses bridge molecule genes, including MFG-E8, Gas6, protein S, TSP-1, and TSP-2 (Table 1-3). RCS RPE expresses genes in 18 RTK subfamilies (Table 1-2). Among the 18 RTK subfamilies, 15 subfamilies correspond to the RTK ligand genes expressed in hUTC. RCS RPE also expresses receptor genes for bridge molecule binding, including integrin αvβ3, αvβ5, Axl, Tyro3, MerTK, and CD36 (Table 1-4).

The transcript profile of both RCS RPE cells and hUTC using RNA-Seq followed by informatics data analysis showed that RCS RPE cells expressed multiple RTK genes, while hUTC expressed multiple RTK ligand genes (Tables 1-1 to 1 -4 and Table 2-1). In particular, the RTK ligands of the seven RTK subfamilies have a relatively high level of gene expression. These ligands include BDNF (brain-derived neurotrophic factor) and NT3 (neurotrophin 3)-ligands of the Trk family, HGF (hepatocyte growth factor)-ligands of the Met family, PDGF-DD (platelet-derived growth factor Type D) and PDGF-CC (platelet-derived growth factor type C) -ligands of PDGF family, ligands of Pterin B2-Eph family, HB-EGF (heparin-binding epidermal growth factor) -ligands of ErbB family, GDNF (Glial cell-derived neurotrophic factor)-ligand of the Ret family, and ligand of the agrin-Musk family.

In one embodiment of the present invention, BDNF, NT3, HGF, and GDNF are secreted in conditioned medium of hUTC, and their levels are compared with those from normal human skin fibroblasts (NHDF) and ARPE-19 cells. High, as measured in the ELISA test. (Figures 11A to 11C and 11F). In another embodiment, hUTC secretes low levels of PDGF-CC and PDGF-DD compared to NHDF or ARPE-19 (Figures 11D, 11E). In a further embodiment, Pterin B2 and HB-EGF were not detected in the conditioned medium of hUTC, NHDF, and ARPE-19, as measured in the ELISA test. These cells do not secrete these two proteins, or the secretion level is below the detection limit of the ELISA test. The levels of agrin in hUTC, NHDF, and ARPE-19 conditioned medium are similar to the control medium; the agrin detected in all conditioned medium samples may come from this medium.

The RNA-Seq-based transcript profile analysis of RCS RPE cells based on the level of bridge molecules and other factors secreted in the hUTC conditioned medium further proved the effect on phagocytosis, and therefore on apoptosis. As shown, RCS RPE cells express the genes of many receptors that have been identified so far to recognize the "eat me" signal on apoptotic cells. These receptors include scavenger receptors (SR-A, LOX-1, CD68, CD36, CD14), integrin (αvβ3 and αvβ5), Axl and Tyro3 receptor tyrosine kinase, LRP-1 / CD91, And PS receptor stabilizer 1 (Table 3-1; adapted from Erwig LP and Henson PM, Cell Death and Differentiation 2008; 15: 243-250). In addition, hUTC expresses many bridge molecule genes, including TSP-1, TSP-2, interface active protein D (SP-D), MFG-E8, Gas6, apolipoprotein H (apolipoprotein H), and annexin 1 (annexin 1). In one embodiment of the present invention, hUTC secretes MFG-E8, Gas6, TSP-1, TSP-2 in hUTC conditioned medium. (Figures 12A to 12E and Table 3-2). In another embodiment, hUTC does not secrete apolipoprotein H, SP-D, or annexin I (Table 3-2). In certain embodiments, hUTC secretes MFG-E8 and TSP-2 at levels significantly higher than NHDF and ARPE-19. (Figures 12A and 12D).

In one aspect of the invention, when RCS RPE cells are fed with ROS pre-cultured with hUTC CM, hUTC conditioned medium stimulates phagocytosis of ROS. The phagocytosis of dystrophic RPE cells is completely rescued. As shown in Figures 13A to 13D, untreated defertile RPE cells have reduced phagocytosis compared to normal RPE cells. In one embodiment of the present invention, the dystrophic RPE cells are pre-cultured with hUTC CM to rescue phagocytosis. This will happen even if there is no hUTC CM during the verification period. In an embodiment of the present invention, phagocytosis-related receptors and their signaling pathways are up-regulated during pre-culture. When hUTC CM was present during the phagocytosis assay, steadily enhanced phagocytosis was observed regardless of whether the dystrophic RPE cells were pretreated with hUTC CM. In one embodiment, please restore or rescue phagocytosis with trophoblastic RPE cells pre-treated with hUTC CM. This will happen even if there is no hUTC CM during the phagocytosis test. In specific embodiments of the invention, hUTC CM can prime or modify ROS, which enhances ROS binding and internalization via, for example, bridge molecules / opsonins that advantageously promote phagocytosis.

In an embodiment of the present invention, the bridge molecules MFG-E8, Gas6, TSP-1 and TSP-2 mediate RCS RPE cells to phagocytose ROS. The dystrophic RPE cells were fed with ROS pre-cultured with various concentrations of MFG-E8, Gas6, TSP-1 or TSP-2 and tested for phagocytosis, showing rescue phagocytosis of ROS (Figure 14A to 14H). In a specific embodiment, the hUTC conditioned medium is phagocytosed and rescued by RCS RPE that secretes bridge molecules such as MFG-E8, Gas6, TSP-1 and TSP-2.

In one embodiment, RTK ligands such as BDNF, HGF, and GDNF stimulate hUTC-mediated phagocytosis rescue in RCS RPE. RTK ligands BDNF, HGF and GDNF rescue phagocytosis in RCS RPE (Figures 14I to J). Recombinant RTK ligands and bridge molecule proteins can mimic the role of hUTC CM and restore RCS RPE phagocytosis, and involve hUTC-mediated phagocytosis rescue in RCS RPE.

Silencing of siRNA vector gene proved that BDNF, HGF, GDNF, MFG-E8, Gas6, TSP-1 and TSP-2 in hUTC were eliminated (silent). Simulated or scrambled siRNA transfection has no effect on hUTC secretion of these factors. SiRNAs targeting MFG-E8, TSP-1, TSP-2, and HGF produced almost 100% knockout efficiency; BDNF and GDNF were observed to be 80% and 65% knockouts, respectively (Figure 15A to Figure 15B). Removal of each of the bridge molecules MFG-E8, TSP-1, TSP-2 reduces the phagocytosis of OS by RCS RPE (Figure 15C). In a specific embodiment, RTK ligands BDNF, HGF and GDNF are required for phagocytosis rescue of hUTC media in RCS RPE. In another embodiment, bridge molecules such as MFG-E8, Gas6, TSP-1, and TSP-2 are required for phagocytosis rescue of hUTC media in RCS RPE.

Cell modification, components and products

Precursor cells such as postpartum cells can also be genetically modified to produce therapeutically useful gene products, or to produce anti-cancer drugs for the treatment of tumors. Gene modification can be achieved using any of a variety of vectors, including but not limited to integrated viral vectors, for example, retroviral vectors or adeno-associated viral vectors; non-integrated replication vectors, for example, papillomavirus vectors , SV40 vector, adenovirus vector; or replication-defective virus vector. Other methods of introducing DNA into cells include the use of liposomes, electroporation, particle guns, or by direct DNA injection.

The host cell is preferably transformed or transfected with DNA that is subjected to one or more suitable expression control elements such as promoter or enhancer sequences, transcription terminators, polyadenylation sites and other elements, and optionally Mark control or operable connection with it. Any promoter can be used to drive the performance of the inserted gene. For example, viral promoters include, but are not limited to, CMV promoter / enhancer, SV40, papillomavirus, Epstein-Barr virus, or elastin gene promoter. In some embodiments, the control elements used to control the expression of the gene of interest may allow the regulated performance of the gene so that the product is synthesized in vivo only when needed. If transient performance is required, constitutive promoter lines are preferably used in non-integrating and / or replication-defective vectors. Alternatively, inducible promoters can be used to drive the expression of inserted genes when needed. Inducible promoters include but are not limited to promoters related to metallothionein and heat shock proteins.

After the introduction of foreign DNA, the engineered cells can be allowed to grow in concentrated medium and then switched to selective medium. Selectable markers in foreign DNA confer selective resistance and allow cells to stably integrate foreign DNA, such as plastids, into their chromosomes and grow to form cell communities (foci), which in turn can be colonized and expanded Into a cell line. This method can be advantageously used to engineer cell lines that express gene products.

Cells can be genetically engineered to "knock out" or "knock down" the expression of factors that promote inflammation or rejection at the implantation site. The following discusses the negative regulation techniques used to reduce the target gene expression level or target gene product activity level. As used herein, "negative modulation" refers to reducing the level and / or activity of a target gene product relative to the level and / or activity of the target gene product in the absence of a modulation process. The expression of natural genes in neurons or glial cells can be reduced or knocked out using many techniques, including, for example, suppression of expression by inactivating genes using homologous recombination techniques. In general, exons (or exons located on the 5 'side of the region) that encode important regions of the protein are interrupted by a positively selectable marker (such as neo) to prevent normal mRNA production from the target gene and cause gene Deactivated. Genes can also be inactivated by creating a deletion of a part of the gene, or by deleting the entire gene. By using a construct that has two regions homologous to the target gene and the two regions are far apart in the genome, the sequence between the two regions can be deleted (Mombaerts et al., 1991, Proc. Nat. Acad. Sci. USA88: 3084-3087). Antisense, DNase, ribonuclease, small interfering RNA (siRNA) and other such molecules that inhibit the expression of target genes can also be used to reduce the level of target gene activity. For example, antisense RNA molecules that inhibit the expression of major histocompatibility gene complexes (HLA) have been shown to be the most diverse in terms of immune response. In addition, triple helix molecules can be used to reduce the level of target gene activity. These techniques are described in detail by L.G. Davis et al. (Eds.), 1994, BASIC METHODS IN MOLECULAR BIOLOGY, 2nd edition, Appleton & Lange, Norwalk, CT.

In other aspects, the present invention provides cell lysates and cell soluble parts prepared from postpartum cells. Postpartum cells are preferably PPDC, or a heterogeneous or homogeneous cell population comprising PPDC cells, and genetically modified or stimulated Take PPDC or its ethnic groups that differentiate along a neurological path. Such lysates and their parts have many uses. The use of a soluble portion of the cell lysate in the body (ie, substantially free of membranes), for example, allows the beneficial intracellular milieu to be used allogeneically in patients without the need to introduce the most likely cause of rejection or other adverse effects A detectable amount of cell surface protein in the immune response. Methods for lysing cells are well known in the art and include various means of mechanical rupture, enzymatic rupture, or chemical rupture, or a combination thereof. Such cell lysates can be prepared directly from the cells in the growth medium, and therefore contain secreted growth factors and the like, or can be prepared from cells washed without culturing medium, such as PBS or other solutions. The washed cells can be resuspended at a concentration greater than the original population density (if preferred).

In one embodiment, the whole cell lysate is prepared, for example, by rupturing the cells without later separating the cell parts. In another embodiment, the cell membrane portion is separated from the soluble portion of the cell by conventional methods known in the art, such as centrifugation, filtration, or the like.

The cell lysate or cell-soluble fraction prepared from a population of precursor cells (such as postpartum-derived cells) can be used as is, further concentrated, for example by ultrafiltration or lyophilization, or even dried, used after partial purification, As known in the technical field, it is used in combination with a pharmaceutically acceptable carrier or diluent, or in combination with other compounds such as biological products such as pharmaceutically usable protein compositions. The cell lysate or part thereof can be used alone in vitro or in vivo, or for example with autologous or syngeneic living cells. If the lysate is introduced into the body, it can be introduced into the local treatment site or distally to provide, for example, the required cell growth factor to the patient.

In a further embodiment, the postpartum cells, preferably PPDC, can be cultured in vitro to produce high-yield biological products. For example, whether such cells naturally produce specific biological products of interest (eg, trophic factors), or are genetically engineered to produce biological products, they can be colonized and expanded using the culture techniques described herein. Alternatively, the cells can be expanded in medium that induces differentiation into the desired lineage. In either case, biological products produced by the cells and secreted into the culture medium can be easily separated from the conditioned medium using standard separation techniques, for example, such as differential protein precipitation, ion exchange chromatography, gel filtration chromatography, electrophoresis, and HPLC, etc. Can't wait. A "bioreactor" can be used to feed, for example, an in vitro three-dimensional culture using a flow method. Basically, as fresh medium passes through the three-dimensional culture, biological products are washed out of the culture and can then be separated from the effluent as described above.

Alternatively, the biological product of interest may remain within the cell, so its collection may require the cell to be lysed as described above. The biological product can then be purified using any one or more of the techniques listed above.

In another embodiment, the extracellular matrix (ECM) produced by culturing postpartum cells (preferably PPDC) on a liquid, solid or semi-solid substrate is prepared, collected and used as implanted living cells to the desired tissue Replacement in the repaired or replaced entity. The cells are cultured in vitro on a three-dimensional architecture as described elsewhere herein, and the culture conditions will allow the required amount of ECM to be secreted onto the architecture. The cells and architecture are removed, and the ECM is processed for further use, for example as an injectable preparation. To achieve this, the cells on the architecture are killed and any cell debris is removed from the architecture. This process can be carried out in many different ways. For example, living tissue can be placed in liquid nitrogen without cryopreservation for rapid freezing, or the tissue can be immersed in sterile distilled water to cause the cells to burst due to osmotic pressure.

Once the cells are killed, the cell membrane may rupture and cell debris is removed by cleaning with a mild detergent such as EDTA, CHAPS, or zwitterionic detergent. Alternatively, the tissue may be digested with enzymes and / or extracted with reagents that disrupt the cell membrane and allow the removal of cell contents. Examples of such enzymes include but are not limited to hyaluronidase, dispase, protease, and nuclease. Examples of cleaning agents include nonionic cleaning agents such as, for example, alkylaryl polyether alcohols (TRITON X-100), octoxyphenoxypolyethoxyethanol (Rohm and Haas Philadelphia, Pa.), BRIJ-35, poly Ethoxyethanol lauryl ether (Atlas Chemical Co., San Diego, Calif.), Polysorbate 20 (TWEEN 20), polyethoxyethanol desorbitan monolaurate (Rohm and Haas), polyethylene Lauryl ether (Rohm and Haas); and ionic cleaners such as, for example, sodium lauryl sulfate, sulfated higher aliphatic alcohols, sulfonated alkanes containing 7 to 22 carbon atoms in branched or unbranched chains, and Sulfonated alkyl aromatics.

The collection of ECM can be achieved in many ways, depending on, for example, the formation of new tissues on a biodegradable or non-biodegradable three-dimensional structure. For example, if the architecture is non-biodegradable, the ECM can be removed by subjecting the architecture to ultrasonic treatment, high-pressure water jets, mechanical scraping, or mild treatment with detergents or enzymes, or any combination of the above.

If the framework is biodegradable, the ECM can be collected, for example, by degrading or dissolving the framework in solution. Alternatively, if the biodegradable framework is composed of materials that can be injected together with the ECM, the framework and the ECM can all be processed together for later injection. Alternatively, the ECM can be removed from the biodegradable architecture by any method described above for collecting ECM from the non-biodegradable architecture. All collection processes are better designed so that the ECM is not denatured.

After collecting the ECM, it can be further processed. For example, the ECM can be homogenized into fine particles using techniques well known in the technical field, such as ultrasound processing, so that it can pass through a surgical needle. If necessary, the components of ECM can be cross-linked by gamma irradiation. Preferably, the ECM can be irradiated between 0.25 and 2 million Reid to sterilize and cross-link the ECM. Chemical interactions using toxic agents such as glutaraldehyde may be but usually poor.

The amount and / or ratio of proteins such as various types of collagen present in the ECM can be adjusted by mixing the ECM produced by the cells of the invention with one or more ECMs of other cell types. In addition, biologically active substances such as proteins, growth factors, and / or pharmaceuticals can be incorporated into the ECM. Exemplary biologically active substances include tissue growth factors, such as TGF-β, and the like, which promote healing of the injection site and tissue repair. Such additional agents can be used in any of the embodiments described above, for example with whole cell lysates, soluble cell fractions, or further purified components and products produced by cells.

Pharmaceutical composition

In another aspect, the present invention provides a pharmaceutical composition that uses precursor cells such as postpartum cells (preferably PPDC), its cell population, produced by such cells in various methods for treating ocular degenerative conditions Conditioned medium, and cell components and products produced by such cells. Certain embodiments encompass pharmaceutical compositions that include living cells (eg, PPDC alone or PPDC mixed with other cell types). Other embodiments encompass pharmaceutical compositions comprising PPDC conditioned medium. Other embodiments may use cellular components of PPDC (eg, cell lysate, soluble cell fraction, ECM, or components of any of the foregoing) or products (eg, naturally produced by cells or produced by genetic modification Nutritional factors and other biological factors, conditioned medium for culturing these cells). In either case, the pharmaceutical composition may further contain other active agents, such as anti-inflammatory agents, anti-apoptotic agents, antioxidants, growth factors, neurotrophic factors, or nerve regeneration, neuroprotection, or ophthalmic drugs known in the art .

Examples of other components that can be added to the pharmaceutical composition include, but are not limited to: (1) other neuroprotective or neurobeneficial drugs; (2) selected extracellular matrix components, such as one or more known in the art Types of collagen, and / or growth factors, platelet-rich plasma, and medicines (or, PPDC can be genetically engineered to express and produce growth factors); (3) Anti-apoptotic agents (eg, erythropoietin (EPO ), EPO mimetibody, thrombopoietin, insulin-like growth factor (IGF) -I, IGF-II, hepatocyte growth factor, apoptosis protease inhibitor); (4) anti-inflammatory compounds (for example, p38 MAP kinase inhibitors, TGF-β inhibitors, statins, IL-6 and IL-1 inhibitors, PEMIROLAST, TRANILAST, REMICADE, SIROLIMUS, and non-steroidal anti-inflammatory drugs (NSAIDS) (such as TEPOXALIN , TOLMETIN, and SUPROFEN)); (5) immunosuppressive or immunomodulatory agents, such as calcineurin inhibitors, mTOR inhibitors, antiproliferative agents, corticosteroids, and various antibodies; (6) antioxidants, such as Probucol ), Vitamins C and E, coenzyme Q-10, glutathione, L-cysteine and N-acetylcysteine; and (6) local anesthetics are not enough.

The pharmaceutical composition of the present invention comprises precursor cells such as postpartum cells (preferably PPDC), conditioned medium produced by these cells, or a component or product thereof, which is formulated with a pharmaceutically acceptable carrier or medium. Suitable pharmaceutically acceptable carriers include water, saline solutions (such as Ringer's solution), alcohols, oils, gelatin, and carbohydrates, such as lactose, amylose, or starch, fatty acid esters, hydroxymethyl fiber Element, and polyvinylpyrrolidine. Such preparations can be sterilized and, if necessary, mixed with auxiliary agents such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for affecting osmotic pressure, buffers, and coloring agents. Usually, but not exclusively, pharmaceutical compositions containing cellular components or products rather than living cells are formulated as liquids. Pharmaceutical compositions containing live PPDC cells are generally formulated as liquids, semi-solids (eg, gels) or solids (eg, matrices, scaffolds, and the like, suitable for ophthalmic tissue engineering).

The pharmaceutical composition may contain auxiliary components familiar to medical chemists or biologists. For example, it may contain antioxidants in a range that varies depending on the type of antioxidant used. A reasonable range of commonly used antioxidants is about 0.01 wt / vol% to about 0.15 wt / vol% EDTA, about 0.01 wt / vol% to about 2.0 wt / vol% sodium sulfite, and about 0.01 wt / vol% to about 2.0 wt / vol% sodium metabisulfite. Those of ordinary skill in the art can use the above-mentioned concentrations at a concentration of about 0.1% by weight / volume. Other representative compounds include mercaptopropionylglycine, N-acetylcysteine, β-mercaptoethylamine, glutathione, and similar substances, but other antioxidants suitable for ocular administration may also be used , Such as ascorbic acid and its salts or sulfites or sodium metabisulfite.

Buffers can be used to maintain the pH of the eye drop formulations in the range of about 4.0 to about 8.0; in order to minimize eye irritation. For direct intravitreal or intraocular injection, the formulation should be at pH 7.2 to 7.5, preferably at pH 7.3 to 7.4. The ophthalmic composition may also include tonicity agents suitable for administration to the eye. The suitable one is sodium chloride to make the formula approximately isotonic with 0.9% saline solution.

In some embodiments, the pharmaceutical composition is formulated with a viscosity enhancer. Exemplary agents are hydroxyethyl cellulose, hydroxypropyl cellulose, methyl cellulose, and polyvinylpyrrolidone. If necessary, a co-solvent can be added to the pharmaceutical composition. Suitable co-solvents may include glycerin, polyethylene glycol (PEG), polysorbate, propylene glycol, and polyvinyl alcohol. May also include preservatives, for example, alkyl dimethyl benzyl ammonium chloride, phenyl thorium chloride, chlorobutanol, phenylmercuric acetate, phenylmercuric nitrate, thimerosal, methyl paraben, or paraben Propyl ester.

The formulation for injection is preferably designed for single-use administration and does not contain preservatives. The injectable solution should have an isotonicity equal to 0.9% sodium chloride solution (290 to 300 mOsmol). This can be obtained by adding sodium chloride or other co-solvents as listed above, or excipients such as buffers and antioxidants as listed above.

The tissue in the anterior chamber of the eye is bathed in the anterior chamber fluid, while the retina is continuously exposed to the vitreous. These fluids / gels exist in a highly reduced redox state because they contain antioxidant compounds and enzymes. Therefore, it may be advantageous to include a reducing agent in the ophthalmic composition. Suitable reducing agents include N-acetylcysteine, ascorbic acid or salt forms, and sodium sulfite or sodium metabisulfite, wherein ascorbic acid and / or N-acetylcysteine or glutathione are particularly suitable for use in Injectable solution.

The pharmaceutical composition comprising cells or conditioned medium, or cell components or cell products can be delivered to the patient's eye by one or more of several delivery methods known in the art. In one embodiment that may be suitable for use in some situations, the composition is delivered locally to the eye in the form of eye drops or eye wash. In another embodiment, the composition can be delivered to various locations in the eye via periodic intraocular injection or by infusion in a lavage solution such as BSS or BSS PLUS (Alcon USA, Fort Worth, Tex.). Alternatively, the composition may be applied in other ophthalmic dosage forms known to those of ordinary skill in the art, such as preformed or in situ formed gels or liposomes, such as disclosed in US Patent No. 5,718,922 to Herrero-Vanrell. In another embodiment, the composition may be delivered to or through the lens of the eye in need of treatment via contact lenses (eg Lidofilcon B, Bausch & Lomb CW79 or DELTACON (Deltafilcon A)) or other objects temporarily resting on the surface of the eye . In other embodiments, supports such as collagen corneal shields (eg BIO-COR soluble corneal shields, Summit Technology, Watertown, Mass.) May be used. The composition can also be administered by infusion into the eyeball, the infusion is through a cannula of an osmotic pump (ALZET, Alza Corp., Palo Alto, Calif.) Or by implantation of a time-release capsule (OCCUSENT) or biological Degradation disk (OCULEX, OCUSERT) any one. These routes of administration have the advantage of providing a continuous supply of pharmaceutical composition to the eye. This may facilitate local delivery of the cornea.

The pharmaceutical composition of living cells contained in a semi-solid or solid carrier is generally formulated for surgical implantation at the site of eye injury or pain. It should be understood that liquid compositions can also be administered by surgical procedures, such as conditioned medium. In a specific embodiment, the semi-solid or solid pharmaceutical composition may include a semi-permeable gel, lattice, cell scaffold, and the like, which may be non-biodegradable or biodegradable. For example, in certain embodiments, it may be desirable or appropriate to isolate exogenous cells from their surrounding environment, but still allow these cells to be secreted and deliver biomolecules to surrounding cells. In these embodiments, the cells can be formulated into autonomous implants containing live PPDC or PPDC-containing cell populations, which are made of non-degradable, selective The permeable barrier surrounds the physical separation of transplanted cells and host tissue. Such implants are sometimes referred to as "immunoprotective" because of their ability to prevent immune cells and macromolecules from killing transplanted cells in the absence of medically induced immunosuppression (a review of such devices and methods) , See, for example, PATresco et al., 2000, Adv. Drug Delivery Rev. 42: 3-27).

In other embodiments, different types of degradable gels and networks can be used in the pharmaceutical composition of the present invention. For example, biodegradable materials that are particularly suitable for sustained release formulations include biocompatible polymers such as poly (lactic acid), poly (milk-co-glycolic acid), methyl cellulose, hyaluronic acid, collagen, and the like Thing. The structure, selection, and use of degradable polymers in drug delivery vehicles have been reviewed in several publications, including A. Domb et al., 1992, Polymers for Advanced Technologies 3: 279-291. US Patent No. 5,869,079 to Wong et al. Discloses a combination of biodegradable sustained release hydrophilic and hydrophobic entities in ocular implants. In addition, US Patent No. 6,375,972 by Guo et al., US Patent No. 5,902,598 by Chen et al., US Patent No. 6,331,313 by Wong et al., US Patent No. 5,707,643 by Ogura et al., US Patent No. 5,466,233 by Weiner et al. No. 6, and Avery et al. US Patent No. 6,251,090 each describe an intraocular implant device and system that can be used to deliver pharmaceutical compositions.

In other embodiments, such as for repairing neurological lesions such as damaged or severed optic nerves, cells delivered on or in a biodegradable (preferably bioresorbable or bioresorbable) scaffold or matrix may be desirable Or appropriate. These general three-dimensional biomaterials contain living cells attached to the scaffold, dispersed within the scaffold, or incorporated into the extracellular matrix embedded in the scaffold. Once implanted into the target area of the body, these implants become integrated with the host tissue, where the transplanted cells gradually become established (see, for example, PATresco et al., 2000, see the foregoing; see also DWHutmacher, 2001, J. Biomater. Sci. Polymer Edn. 12: 107-174).

Examples of scaffold or matrix (sometimes collectively referred to as "framework") materials that can be used in the present invention include non-woven mats, porous foams, or self-assembling peptides. Non-woven mats can be formed, for example, using fibers containing synthetic absorbable copolymers of glycolic acid and lactic acid (PGA / PLA), which are sold under the trade name VICRYL (Ethicon, Inc., Somerville, N.J). It is also possible to use a foam composed of, for example, poly (ε-caprolactone) / poly (glycolic acid) (PCL / PGA) copolymer, which is formed by a process such as freeze drying or freeze drying, as described in Discussed in 6,355,699. Hydrogels such as self-assembling peptides (eg, RAD16) can also be used. Degradable webs formed in situ are also suitable for use in the present invention (see, for example, Anseth, KS et al., 2002, J. Controlled Release 78: 199-209; Wang, D. et al., 2003, Biomaterials 24: 3969- 3980; He et al. US Patent Publication 2002/0022676). These materials are formulated into fluids suitable for injection, and can then be induced to form a degradable hydrogel network in situ or in vivo by various means (eg, changing temperature, pH, exposure to light).

In another embodiment, the framework is a felt, which may be multifilament yarn made from bioabsorbable materials (eg, PGA, PLA, PCL copolymer or blend, or hyaluronic acid) composition. The yarn is made into felt using standard textile processing techniques consisting of crimping, cutting, carding and needle punching. In another embodiment, the cell line is seeded onto a foam scaffold that can be a composite structure.

In many of the embodiments mentioned above, the framework can be molded into useful shapes. In addition, it should be understood that PPDC can be cultured on a pre-formed, non-degradable surgical or implantable device, which corresponds, for example, to the method used to prepare, for example, fibroblast-containing GDC intravascular coils (Marx, WF et al. , 2001, Am. J. Neuroradiol. 22: 323-333).

Prior to seeding cells, the matrix, scaffold or device can be treated to enhance cell attachment. For example, before inoculation, the nylon matrix may be treated with 0.1 mol acetic acid and cultured in polyamic acid, PBS, and / or collagen to coat nylon. Polystyrene can be similarly treated using sulfuric acid. The outer surface of the framework can also be modified to improve cell attachment or growth and tissue differentiation, such as by coating the framework with plasma or adding one or more proteins (eg, collagen, elastic fibers, mesh fibers), glycoproteins, Glycosaminoglycans (eg, heparin sulfate, chondroitin-4-sulfate, chondroitin-6-sulfate, dermatan sulfate, keratin sulfate), cell matrix, and / or other materials such as but not limited to gelatin, algae Salts, agar, agarose, vegetable gums and others.

The framework containing living cells is prepared according to methods known in the art. For example, cells can grow freely in a culture vessel until they are overgrown or overgrown, lifted from the culture, and seeded onto the framework. If necessary, growth factors can be added to the medium before, during, or after seeding the cells to induce differentiation and tissue formation. Alternatively, the architecture itself can be modified to enhance the growth of cells on it, or to reduce the risk of repelling the implant. Therefore, one or more biologically active compounds, including but not limited to anti-inflammatory agents, immunosuppressive agents, or growth factors can be added to the framework for local release.

Instructions

Precursor cells such as postnatal cells (preferably hUTC or PDC), or their cell populations, or conditioned medium or other components or products produced by such cells can be used in a variety of ways to support and facilitate the repair and regeneration of eye cells and tissues . Such uses include in vitro, ex vivo, and in vivo methods. The methods described below are related to PPDC, but other postpartum cells can also be applied to these methods.

In vitro and ex vivo methods

In one embodiment, precursor cells such as postpartum cells (preferably hUTC or PDC), and the conditioned medium produced therefrom can be used in vitro to screen the effectiveness of various compounds and pharmaceutical agents, growth factors, regulatory factors and the like Cytotoxicity. For example, such screening can be performed on a substantially homogeneous PPDC population to assess the efficacy or toxicity of candidate compounds to be formulated with PPDC or co-administered for the treatment of eye disorders. Alternatively, for the purpose of evaluating the effectiveness of new pharmaceutical drug candidates, such screening may be performed on PPDCs stimulated to differentiate into the cell types found in the eye, or their precursors. In this example, PPDC was maintained in vitro and exposed to the compound to be tested. The activity of potentially cytotoxic compounds can be measured by their ability to damage or kill cells in culture. This can be easily assessed by in vivo staining techniques.

As discussed above, PPDC can be cultured in vitro to produce biological products that are produced naturally by cells, or when cells are induced to differentiate into other lineages, or by genetic modification of cells. For example, TIMP1, TPO, KGF, HGF, FGF, HBEGF, BDNF, MIP1b, MCP1, RANTES, I309, TARC, MDC, and IL-8 were found to be secreted from umbilical-derived cells grown in growth medium. It was found that TIMP1, TPO, KGF, HGF, HBEGF, BDNF, MIP1a, MCP-1, RANTES, TARC, Eotaxin, and IL-8 are all secreted from placenta-derived PPDC cultured in growth medium (see examples ).

In this regard, embodiments of the present invention are characterized by the use of PPDC to produce conditioned medium. The conditioned medium produced from PPDC can be derived from undifferentiated PPDC or from PPDC cultured under conditions that stimulate differentiation. Such conditioned media are considered for use in, for example, in vitro or ex vivo culture of epithelial or neural precursor cells, or in vivo to support transplanted cells of a homogeneous population comprising PPDC or a heterogeneous population comprising PPDC and other precursors.

Cell lysates, soluble cell parts or components of PPDC, or their ECMs or components can be used for a variety of purposes. As mentioned above, some of these components can be used in pharmaceutical compositions. In other embodiments, the cell lysate or ECM is used to coat or otherwise treat substances or devices intended for surgery, or for implantation, or for ex vivo purposes to facilitate in such treatment procedures Healing or survival of the contacted cells or tissues.

As described in Examples 22 and 24, when PPDC is grown in co-culture with adult neural precursor cells, PPDC has the ability to support the survival, growth and differentiation of these cells. Similarly, previous studies have pointed out that PPDC can play a role in supporting retinal cells through nutritional mechanisms. (US 2010-0272803). Therefore, PPDC is advantageously used in in vitro co-cultures to provide nutritional support to other cells, especially nerve cells and nerve and eye precursors (eg, neural stem cells and retinal or corneal epithelial stem cells). As for the co-cultivation, it may be desirable to co-culture the PPDC with the desired other cells under conditions in which the two cell types are in contact. This can be achieved, for example, by seeding the cells in a medium in the form of a heterogeneous cell population or onto a suitable culture substrate. Alternatively, the PPDC can first be grown to fullness and then serve as a substrate for the second desired cell type in culture. In this latter embodiment, after the co-cultivation period, the cells can be further physically separated, such as by a membrane or similar device, so that the other cell types can be removed and used alone. The use of PPDC co-culture to promote the expansion and differentiation of neural or ocular cell types may have applicability in research and clinical / therapeutic fields. For example, PPDC co-culture can be used to facilitate the growth and differentiation of such cells in culture, for example for basic research purposes or for drug screening tests. PPDC co-culture can also be used for ex vivo expansion of nerve or eye precursors for later administration for therapeutic purposes. For example, nerve or ocular precursor cells can be collected from an individual, co-cultured with PPDC, expanded in vitro, and then returned to the individual (autologous transfer) or another entity (syngeneic or allogeneic transfer). In these embodiments, it should be understood that after ex vivo expansion, a mixed cell population comprising PPDC and precursors can be administered to patients in need of treatment. Alternatively, where autologous transfer is appropriate or desirable, the co-cultured cell population can be physically separated in the culture, allowing the autologous precursor to be removed for administration to the patient.

In vivo method

As illustrated in the examples, conditioned medium can be effectively used to treat ocular degenerative conditions. Once transplanted into the target location in the eye, conditioned medium from precursor cells (such as PPDC) provides nutritional support to the eye cells in situ.

Conditioned medium from precursor cells (such as PPDC) can be administered together with: other beneficial drugs, biomolecules (such as growth factors, trophic factors), conditioned medium (from precursor cells or differentiated cell cultures), or other active agents (Such as anti-inflammatory agents, anti-apoptotic agents, antioxidants, growth factors, neurotrophic factors or nerve regeneration or neuroprotective drugs as known in the art). When the conditioned medium is administered with other agents, it can be administered together in the form of a single pharmaceutical composition, or separately or simultaneously with other agents (before or after the administration of other agents).

Examples of other components that can be administered with precursor cells (such as PPDC) and conditioned medium products include, but are not limited to: (1) other neuroprotective or neurobeneficial drugs; (2) selected extracellular matrix components, such as One or more types of collagen, and / or growth factors, platelet-rich plasma, and medicines known in the technical field (or, cells can be genetically engineered to express and produce growth factors); (3) Anti-apoptosis Agents (eg, erythropoietin (EPO), EPO mimetibody, thrombopoietin, insulin-like growth factor (IGF) -I, IGF-II, hepatocyte growth factor, apoptosis protease inhibitor) ; (4) Anti-inflammatory compounds (eg, p38 MAP kinase inhibitors, TGF-β inhibitors, statins, IL-6 and IL-I inhibitors, PEMIROLAST, TRANILAST, REMICADE, SIROLIMUS, and non-sterols Anti-inflammatory drugs (NSAIDS) (such as TEPOXALIN, TOLMETIN, and SUPROFEN); (5) Immunosuppressive or immunomodulatory agents, such as calcineurin inhibitors, mTOR inhibitors, antiproliferative agents, corticosteroids and various Antibody; (6) Antioxidants, such as probucol, vitamins C and E, coenzyme Q-10, glutathione, L-cysteine and N-acetylcysteine; and (6) local anesthetics, etc. Can't wait.

The liquid or fluid pharmaceutical composition can be administered to a more general location in the eye (eg, locally or intraocularly).

Other embodiments encompass methods for treating ocular degenerative conditions by administering pharmaceutical compositions that include conditioned medium from precursor cells (such as PPDC), or are naturally produced by these cells or genetically modified by the cells Nutrients and other biological factors for quality production. In addition, these methods may further comprise the administration of other active agents, such as growth factors, neurotrophic factors, or nerve regeneration or neuroprotective drugs as known in the art.

The dosage forms and protocols for the administration of conditioned medium from precursor cells (such as PPDC), or any other pharmaceutical composition described herein, are developed according to good medical practice, taking into account the conditions of individual patients, for example, ocular The nature and degree of degenerative conditions, age, gender, weight and general medical conditions, and other factors known to medical practitioners. Therefore, the effective amount of the pharmaceutical composition to be administered to the patient is determined by these considerations as known in the art.

Before starting cell therapy, it may be desirable or appropriate to suppress the patient with medical immunosuppression. This can be achieved by using systemic or local immunosuppressants, or can be achieved by cells delivered in an encapsulated device, as described above. These and other means for reducing or eliminating the immune response to transplanted cells are known in the art. Alternatively, the conditioned medium may be prepared from PPDC that has been genetically modified to reduce its immunogenicity, as mentioned above.

The survival of transplanted cells in living patients can be determined by using various scanning techniques, for example, computerized tomography (CAT or CT) scanning, magnetic resonance imaging (MRI) or positron emission tomography (PET) scanning. The survival of the transplant can also be determined by removing the tissue and visually or by examining the tissue through a microscope for post-mortem analysis. Alternatively, the cells can be treated with stains specific for nerve or eye cells or their products (eg, neurotransmitters). Transplanted cells can also be obtained by incorporating tracer dyes such as rose red or luciferin-labeled microspheres, fast blue, iron particles, bisbenzamide, or gene-introduced reporter gene products, such as Lactosidase or β-glucuronidase to recognize.

The functional integration of transplanted cells or conditioned medium into ocular tissues or individuals can be assessed by examining the recovery of damaged or diseased eye function. For example, the effectiveness of treating macular degeneration or other retinopathy can be judged by the improvement of visual acuity and the evaluation of abnormalities and grading of stereoscopic fundus color photos. (Age-related eye disease research investigation team, NEI, NIH, AREDS Report No. 8, 2001, Arch. Ophthalmol. 119: 1417-1436).

Set and Bank (Bank)

In another aspect, the present invention provides a kit that utilizes precursor cells (such as PPDC), and cell populations, by these cells (preferably PPDC) in various methods for eye regeneration and repair as described above The prepared conditioned medium and its components and products. When used to treat ocular degenerative conditions, or other planned treatments, the kits may include one or more cell populations or conditioned medium (including at least postpartum cells or conditioned medium derived from postpartum cells), and pharmaceutically acceptable Carrier (liquid, semi-solid or solid). The kit may optionally also include means for administering the cells and conditioned medium by injection, for example. The kit may further include instructions for using cells and conditioned medium. Prepared for field hospital use, such as military use kits may include a full-procedure supply, which includes tissue scaffolds, surgical sutures, and the like, in which cells or conditioned medium and repair of acute injuries In conjunction with. The kit used for the assay and in vitro methods as described herein may contain, for example, one or more of the following: (1) PPDC or its components, or conditioned medium or other products of PPDC; (2) for practice in vitro Reagents for the method; (3) other cells or cell populations selected as appropriate; and (4) instructions for in vitro methods.

In another aspect, the invention also provides tissues, cells, cell populations, conditioned medium, and cell components for banking the invention. As discussed above, cells and conditioned medium are easy to freeze. Therefore, the present invention provides a method for cryopreserving cells in storage, wherein the cells are stored frozen and associated with the complete characterization of cells based on the immune, biochemical, and genetic properties of the cells. Frozen cells can be thawed and expanded or directly used for autologous, allogenic, or allogeneic treatment, depending on the requirements of the procedure and the needs of the patient. Preferably, the information about each frozen sample is stored in a computer, which can be searched based on the requirements of the surgeon, the procedure, and the patient, where the appropriate match is obtained based on the characterization of the cell or population. Preferably, the cells of the present invention are grown and expanded to the desired amount of cells, and the therapeutic cell composition is prepared separately or in the form of co-culture in the presence or absence of a matrix or support. Although for some applications, it may be preferable to use freshly prepared cells, the remaining material can be frozen and stored by freezing the cells and entering the information in a computer to associate the computer entry with the sample. Even in cases where there is no need to match the source or donor to the recipient of such cells, the storage system makes it easy to match, for example, the desired biochemical or genetic properties of the stored cells with the treatment needs. After finding a stock sample that matches the desired properties, a sample can be taken and prepared for treatment. Cell lysates, ECM or cell components prepared as described herein can also be frozen or otherwise retained (eg, by lyophilization) and stored in accordance with the present invention.

The following examples are provided to describe the invention in more detail. These examples are intended to illustrate rather than limit the invention.

The following abbreviations may appear elsewhere in the examples and specifications and in the scope of patent applications: ANG2 (or Ang2) is Angiopoietin 2; APC is an antigen presenting cell; BDNF is brain-derived neurotrophic factor; bFGF Basic fibroblast growth factor (basic fibroblast growth factor); bid (BID) is "twice a day" (twice a day); CK18 is cytokeratin 18; CNS is the central nervous system; CXC Body 3 is chemokine receptor ligand 3; DMEM is the minimum essential medium for Dulbecco's; DMEM: lg (or DMEM: Lg, DMEM: LG) is DMEM with low glucose; EDTA Ethylenediaminetetraacetic acid; EGF (or E) is epidermal growth factor; FACS is fluorescent activated cell sorting; FBS is fetal bovine serum; FGF (or F) is fiber Fibroblast growth factor; GCP-2 is granulocyte chemotactic protein-2; GDNF is a glial cell-derived neurotrophic factor; GF AP is glial fibrillary acidic protein; HB-EGF is heparin-binding epidermal growth factor; HCAEC is human coronary artery endothelial cell; HGF is Hepatocyte growth factor; hMSC is Human mesenchymal stem cell; HNF-1α is hepatocyte-specific transcription factor; HVVEC is human umbilical vein endothelial cell (Human umbilical vein endothelial cell); I309 is a ligand for chemokine and CCR8 receptor; IGF-1 is insulin-like growth factor 1; IL-6 is interleukin 6; IL- 8 is interleukin 8; K19 is keratin 19; K8 is keratin 8; KGF is keratinocyte growth factor; LIF is leukemia inhibitory factor; MBP is myelin base Myelin basic protein; MCP-1 is a monocyte chemotactic protein 1; MDC is a macrophage-derived chemokine (ma crophage-derived chemokine); MIP1α is macrophage inflammatory protein 1α; MIP1β is macrophage inflammatory protein 1β; MMP is matrix metalloprotease (MMP); MSC is mesenchymal stem cell ); NHDF is Normal Human Dermal Fibroblast; NPE is Neural Progenitor Expansion media; NT3 is neurotrophin 3; 04 is oligodendrocyte ) Or glial differentiation marker 04; PBMC is peripheral blood mononuclear cell; PBS is phosphate buffered saline; PDGF-CC is platelet derived growth factor (platelet derived growth factor) growth factor) C; PDGF-DD is platelet-derived growth factor D; PDGFbb is platelet-derived growth factor bb; PO is "per os" (via mouth); PNS is peripheral nervous system; Rantes (Or RANTES) is to regulate activation, normal T cell performance and secretion (regulated on activat ion, normal T cell expressed and secreted); rhGDF-5 is a recombinant human growth and differentiation factor 5; SC is subcutaneously; SDF-1α is a stromal-derived factor 1α; SHH for sonic hedgehog; SOP for standard operating procedures; TARC for thymus and activation-regulated chemokine; TCP for tissue culture plastic; TCPS for tissue culture Polystyrene; TGF β2 is transforming growth factor β2; TGF β-3 is transforming growth factor β-3; TIMP1 is tissue inhibitor of matrix metalloproteinase 1; TPO is platelets Thrombopoietin; TUJ1 is BIII tubulin; VEGF is vascular endothelial growth factor; vWF is von Willebrand factor; and αFP is α-fetoprotein .

The following examples further illustrate but do not limit the invention.

Example 1 Effect of umbilical-derived cell conditioned medium on rescue phagocytic activity of infertile RPE cells in vitro

It has been well established that retinal pigment epithelium (RPE) cells from the Royal College of Surgeons (RCS) rats exhibit impaired phagocytic extracellular section (ROS) phagocytosis due to invalid mutations in the Mertk gene. (Feng W. et al., J Biol Chem. 2002, 10: 277 (19): 17016-17022). It has been shown that human umbilical tissue-derived cells (hUTC) are injected subretinally into the eyes of RCS rats to improve visual acuity and appear to improve retinal degeneration. (US 2010/0272803). In this example, treatment with conditioned medium (CM) derived from hUTC restores phagocytosis of ROS in in vitro dystrophic RPE cells.

The hUTC conditioned medium was tested to: 1) evaluate the effect of using the new hUTC CM preparation on phagocytosis of deferent RPE; 2) separate RNA of acceptable quality from CM-treated and untreated deferent RPE for use by RNA-Seq Gene expression analysis; 3) Test whether the selected RTK ligand can increase the RPE phagocytosis level of RCS rats that cannot use the Mertk signaling pathway; and 4) Test whether other non-RTK ligands that activate different receptors other than RTK can show similar Features.

Materials and Method

Human umbilical tissue-derived cells (hUTC) are obtained from the methods described in Examples 14 to 26 below and described in detail in US Patent Nos. 7,524,489 and 7,510,873, and US Published Application No. 2005/0058634 The way of quotation is incorporated herein. In short, the donor agreed that the human umbilical cord donated after live birth was obtained from the National Disease Research Interchange (Philadelphia, PA). The tissue was minced and digested with enzymes. After almost complete digestion with Dulbecco's modified Eagle's medium (DMEM) -low glucose (Lg) (Invitrogen, Carlsbad, CA) medium containing a mixture of 50 U / mL collagenase (Sigma, St. Louis), the cells were allowed to The suspension was filtered through a 70 [tm filter, and the supernatant was centrifuged at 350 g. The separated cells were washed several times in DMEM-Lg, and they were seeded at a density of 5,000 cells / cm ² in 15% (v / v) FBS (Hyclone, Logan, Utah) and 4 mM L-glutamine Acid (Gibco, Grand Island, NY) in DMEM-Lg medium. When the cells reached approximately 70% overgrowth, trypLE (Gibco, Grand Island, NY) was used for subculture. Cells were collected and pooled after several passages.

Cultured RPE cells beginning: RPE cell lines were obtained from 6-11 days of age normal dye (RCS rdy + / p +) ( homologous allogeneic control) or sexual dystrophy (RCS rdy- / p +) rats. The front of the eye before the limbus is removed. Carefully remove the retina and place the eye cup at 4% (w / v) dispase ( 0.8 U / mg, Roche Diagnostics, Mannheim, Germany) for 20 to 30 minutes. The RPE sheet was removed, suspended in growth medium (DMEM + 10% FBS [new paper 20%] + Pen (200U / ml) / Strep (200 μg / ml)), developed by trypsin treatment, and inoculated in 8 wells Chamber slide holes or inoculate on round glass coverslips placed in the wells of a 24-well plate. The cells were cultured in 5% v / v CO ₂ at 37 ° C.

RPE cell sulforhodamine (sulforhodamine) staining: maintain RPE culture in sulforhodamine (40 μg / ml final concentration) growth medium containing 24h to 72h. Cells were stained for 36h to 48h before adding ROS. Between 6h and 18h before the addition of ROS, the sulforhodamine-containing medium was removed, and the culture was maintained in fresh growth medium that was replaced several times.

Isolation of rat photoreceptor OS : Eyes were obtained from Long Evans rats 2 to 4 weeks old or 6 to 8 weeks old several hours after the initiation of light. The retina was separated, homogenized with a Polytron (8mm homogenizer) or Dounce glass homogenizer, layered on top of a 27% to 50% linear sucrose gradient, and centrifuged at 38,000rpm in a SW41 rotor (240,000xg) at 4 ° C hour. The top two ROS bands were collected, diluted with HBSS, and centrifuged at 7000 rpm for 10 minutes in an HB-4 rotor (8000xg) to make ROS clumps.

FITC staining of ROS: the ROS pellet of about 1ml per pellet was resuspended in serum-free medium (MEM basal medium only) on. FITC stock solution (2 mg / ml in 0.1 M sodium bicarbonate, pH 9.0 to 9.5) was added to a final concentration of 10 μg / ml, and incubated at room temperature for 1 h. FITC-stained ROS were pelleted by centrifugation at 8000 rpm for 10 minutes in a microcentrifuge, resuspended in growth medium (MEM20), counted, and diluted to a final concentration of 107 / ml.

hUTC conditioned medium (CM) : Three sets of hUTC conditioned medium (CM) and control medium were prepared and used in phagocytosis testing.

1. CM1 preparation with serum

On day 1, hUTC was seeded at 5,000 viable cells / cm ² in hUTC growth medium (DMEM low glucose + 15% FBS + 4 mM L-glutamic acid) in T75 cell culture flasks. The cells were cultured in a 5% CO ₂ incubator at 37 ° C for 24 hours. On the second day, the medium was aspirated, washed twice with DPBS, and supplemented with 21 mL of DMEM / F12 complete medium (DMEM: F12 medium + 10% FBS + Pen (50U / ml) / Strep (50 μg / ml)). The cells were incubated for an additional 54 hours. A separate control medium (DMEM: F12 medium + 10% FBS + Pen (50U / ml) / Strep (50 μg / ml)) was also cultured for 54h. On the 4th day, the cell culture supernatant and control medium were collected and centrifuged at 250 g for 5 min at RT, aliquoted in 3 mL / tube into cryopreservation tubes, and immediately frozen in a -70 ° C refrigerator.

2. Serum-free CM1 preparation

On day 1, hUTC was seeded at 5,000 viable cells / cm ² in hUTC growth medium (DMEM low glucose + 15% FBS + 4 mM L-glutamic acid) in T75 cell culture flasks. The cells were cultured in a 5% CO ₂ incubator at 37 ° C for 24 hours. On Day 2, the medium was aspirated, washed twice with DPBS, and supplemented with 21 mL of DMEM / F12 serum-free medium (DMEM: F12 medium + Pen (50U / ml) / Strep (50 μg / ml)). The cells were cultured for 54h. A serum-free control medium (DMEM: F12 medium + Pen (50U / ml) / Strep (50 μg / ml)) was also cultured for 54 h. On the 4th day, the cell culture supernatant and control medium were collected and centrifuged at 250 g for 5 min at RT, aliquoted in 3 mL / tube into cryopreservation tubes, and immediately frozen in a -70 ° C refrigerator.

3. CM2 preparation

The preparation was the same as the CM1 preparation with serum and hUTC was inoculated at 5,000 viable cells / cm ² , except that the cell culture flask was a T225 flask with 63 mL of culture medium per flask, and the culture time after the medium replacement was 48 hours.

4. CM3 preparation

The same formulation as CM2, except that the hUTC seeding density was increased to 10,000 viable cells / cm ² and the culture time after the medium was changed was 48 hours.

Phagocytosis test: Prior to the test, 5 × 10 ⁴ RPE cells stained with sulforhodamine were seeded in multi-well plates, maintained in MEM + 20% (v / v) FBS for 6 days, and then maintained in MEM + 5 % (v / v) 24h in FBS (2 or more repeats per sample). Fresh medium was added 3h before the addition of ROS, and the assay was performed by covering the culture with FITC-ROS (10 ⁷ / ml in MEM + 20% (v / v) FBS) and incubating at 37 ° C for 3 to 19h (usually 8h). At the end of the culture, the cells were vigorously washed to remove unbound ROS and fixed with 2% (w / v) paraformaldehyde (Sigma, St. Louis, MO).

RPE phagocytosis of ROS is optimized in terms of the following procedures: preparation and cultivation of primary RPE, preparation of ROS, and the phagocytosis test itself.

RTK ligands: The RTK ligands used were: recombinant human butterfly B2 (catalogue number pro-937, batch number 1112PEFNB2, ProSpec-Tany TechnoGene Ltd., Israel), recombinant human BDNF (catalogue number 248-BD-025 / CF , Batch number NG4012051, R & D Systems, Inc., Minneapolis, MN), recombinant human HB-EGF (catalogue number 259-HE-050 / CF, batch number JI3012021, R & D Systems, Inc., Minneapolis, MN), recombinant human HGF (catalog PHG0254, batch number 73197181A, Life Technologies, Carlsbad, CA), recombinant human butterfly A4 (catalog number E199, batch number 1112R245, Leinco Technologies, Inc., St. Louis, MO), and recombinant human PDGF-DD (catalog number 1159 -SB-025 / CF, lot number OTH0412071, R & D Systems, Inc., Minneapolis, MN). The reconstitution of individual RTK ligand stock solutions was based on the supplier's data sheet: recombinant human BDNF and HB-EGF were reconstituted in sterile PBS at 100 μg / mL and 250 μg / mL, respectively. Recombinant human HGF was reconstituted in sterile distilled water at 500 μg / mL. Recombinant human butterfly A4 was reconstituted in sterile PBS at 100 μg / mL. Recombinant human PDGF-DD was reconstituted in sterile 4mM HCl at 100μg / mL. The reconstituted stock solution was aliquoted and frozen in the refrigerator at -70 ° C.

The medium was changed to MEM + 5% (v / v) FBS (MEM5), and the ligand was added to the dystrophic cells at 200ng / ml, cultured for 24h, then ROS was added in the presence of the ligand, and the cells were subjected to phagocytosis assay . Normal RPE repeats were pre-cultured in MEM5 and phagocytosis assay was used as a control.

Non- RTK ligands: The non-RTK ligands used are: recombinant human vitreous connexin (catalog number 2308-VN-050, batch number NBH0713021, R & D Systems, Inc., Minneapolis, MN), recombinant human TGF-β1 (catalog number 240-B-010 / CF, batch number AV5412113, R & D Systems, Inc., Minneapolis, MN), recombinant human IL-6 (catalog number 206-IL-010 / CF, batch number OJZ0712041, R & D Systems, Inc., Minneapolis, MN ), And human endothelin-1 (catalog number hor-307, batch number 1211PEDN112, ProSpec-Tany TechnoGene Ltd., Israel). The reconstitution of individual non-RTK ligand stock solutions was reconstituted in sterile PBS at 100 μg / mL of recombinant human vitronectin and IL-6 according to the supplier's data sheet. Recombinant human TGF-β1 was reconstituted in sterile 4mM HCl at 100μg / mL. Recombinant human endothelin-1 was reconstituted in sterile 18 MΩ-cm H ₂ O at 100 μg / mL. The reconstituted stock solution was aliquoted and frozen in the refrigerator at -70 ° C.

The phagocytosis rescue activity test of conditioned medium: the repeat of dystrophic (D) RPE was cultured for about 24 hours in 1 ml of conditioned medium each. As for the control group, the homologous allogeneic control (N) RPE was repeatedly cultured in the control medium (DMEM: F12 medium + 10% FBS + Pen (50U / ml) / Strep (50 μg / ml)) at the same time. After the cultivation, the conditioned medium and the control medium were removed, replaced with fresh MEM5, and RPE was subjected to phagocytosis assay.

Assay for detecting the effect of non- RTK ligands on phagocytosis of RCS RPE cells: hUTC CM3 is used as a positive control for the assay. In the test of endothelin-1, TGF-β1, and IL-6, the dystrophic (D) RPE was cultured in 1 ml of conditioned medium for 24 hours. Then, the conditioned medium was removed, replaced with fresh MEM + 5% (v / v) FBS (MEM5), and a phagocytosis test was performed (feed ROS 8h in MEM5). As for other controls, normal and deferent RPE were cultured in MEM5 for 24h and subjected to phagocytosis assay. The dystrophic RPE cell line was cultured in MEM5 at 200 ng / mL with recombinant human endothelin-1, TGF-β1 or IL-6 for 24 h, and then added ROS to cells containing recombinant human endothelin-1, TGF-β1 or IL-6 Perform phagocytosis assay in MEM5 (do not change medium when adding ROS).

For the test of vitronectin, ROS was pre-cultured in CO ₂ cell incubator with control medium (DMEM + 10% FBS) or conditioned medium at 37 ° C for 24h. At the same time, ROS were cultured in MEM + 20% (v / v) FBS (MEM20) with various concentrations of human recombinant glass connexin (4, 2, 1, 0.5μg / ml) at 37 ℃ in CO ₂ cells Pre-incubated in the instrument for 24h. After culturing, the ROS was centrifuged without washing, resuspended in MEM20, and fed to the dystrophic RPE cells in the presence of MEMS for phagocytosis assay. As for the control group, normal RPE alone or deprived RPE alone was cultured in MEM20, and then replaced with MEM5 in the presence of untreated ROS (resuspended in MEM20 and fed to RPE cells) for phagocytosis assay.

Imaging and quantification: By phase contrast and fluorescence microscopy, live RPE cells are detected using an inverted microscope equipped with epi-fluorescence optics, fluorescence microscope, and digital camera. The recognition of FITC-ROS bound to the cell surface, FITC-ROS ingested, and phagolysosome is as described in McLaren et al . (Investor Ophthalmol Vis Sci., 1993; 34 (2): 317-326.) definition. Quantification of bound and ingested ROS is performed on fixed cells on the coverslip. Count at 250x magnification with an appropriate filter and grid (field size, 40 × 40 μm). Cells of various types in representative fields (10 to 15 fields per culture) were counted, and the data was expressed as the average of pooled values obtained from each time point of 2 to 3 separate experiments. The statistical significance of the paired data was tested by Student's t-test, and the statistical significance was determined at p <0.05.

Acceptance Criteria for Testing: The absolute level of phagocytosis varies in the experiment depending on various factors, including the quality of the isolated RPE and the prepared ROS. In particular, RPEs of the same lineage (ie, the same collection and preparation time) are used to compare the effects of different treatments on cells. If the relationship between the normal phagocytosis level and the dystrophy phagocytosis level is approximately 1: 0.3, the test is judged to be reasonable.

Relative phagocytosis: Relative phagocytosis is the level of phagocytic RPE phagocytosis compared to the reference (normal) allogeneic control (normal) phagocytosis level. The level of phagocytosis can be expressed as mean ROS / field of view, or mean ROS / cell.

RNA isolation. Inoculate hUTC at 5,000 cells / cm ² in DMEM-Lg medium containing 15% (v / v) FBS (Hyclone, Logan, Utah) and 4 mM L-glutamic acid (Gibco, Grand Island, NY) After growing for 24 hours, the medium was then replaced with DMEM: F12 medium containing 10% (v / v) FBS and regenerated for 48 hours. Cells were then collected for total RNA extraction and DNA removal, using Qiagen RNAeasy extraction and on-column DNAse kits (Qiagen, Valencia, CA). A NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific, 'Waltham, MA) and an Agilent 2100 bioanalyzer (Agilent Technologies, Santa Clara, CA) were used to determine the integrity and amount of RNA in the sample. Library preparation and sequencing were performed by Expression Analysis Inc., Durham, NC. The RNA library was prepared using Illumina's TruSeq RNA-Seq sample preparation kit according to the manufacturer's instructions, and sequenced with Illumina's HiSeq 2000. The software ArrayStudio version 6.1 was used to map the sequenced readings to the reference human genome (GRCh37 patch 8). Gene performance was calculated using fragments per kilobase transcript (FPKM) per million enantiomeric readings.

After separation, the RCS RPE cells were first seeded in 96-well plates in triplicate for about one week, and the time point when the medium was replaced with hUTC conditioned medium or control medium was regarded as 0h. According to the manufacturer's protocol, Trizol (Life Technologies, Carlsbad, CA) was used to extract RNA at 2, 4, 8, and 24 h, respectively. The RNA extracted from the triplicate at each time point was pooled into one sample. The ratio of the sample concentration to the 260/280 absorbance is determined spectrophotometrically.

The duplication of defertile RPE (about 2.4 × 10 ^{4 each} ) was treated with conditioned medium or not, as shown in the following scheme:

result Conditioned medium test

As described in the method, three conditioned medium preparations (CM1, CM2, CM3) were tested for phagocytic rescue activity on deferent RPE and compared with normal RPE.

CM1: The CM1 series was tested twice (Figure 1A and Figure 1B), once using brown normal RPE and once using pigment RPE. Observe the phagocytosis rescue activity. It should be noted that in FIG. 1A, the basal level of phagocytosis of RPE is almost 50% of the normal RPE level of tan hooded, which exceeds the acceptance range (<30% of normal phagocytosis level). The effect of serum-free CM1 medium was also tested (Figure 2). The difference between the dystrophic cells with and without serum-free CM1 is statistically significant.

CM2: CM2 was tested and found to lack activity (Figure 3).

CM3: After the medium was changed on the first day, the cultivation time of CM2 (inactive) was 48h, and the cultivation time of CM1 (active) was 54h. In order to obtain an active conditioned medium, the initial cell seeding density and the cell culture time after changing the medium are two aspects that should be considered. Compared to CM2, CM3 is prepared by doubling the cell seeding density and maintaining the same incubation time after changing the medium. CN3 was tested multiple times to confirm the presence of activity (Figure 4A, Figure 4B, Figure 4C), showing up to 100% phagocytosis rescue activity.

Devour in RCS RPE

The RPE cell line isolated from RCS rat eyes was cultured in vitro for phagocytosis assay. Untreated RPE cells from normal homeologous control rat eyes were used as controls to show normal phagocytic levels. When RCS RPE was co-cultured with hUTC (Figure 4A) or treated with hUTC conditioned medium (CM) (Figure 4B), the defective phagocytosis in RCS RPE returned to normal RPE levels. When RCS RPE was fed with OS pre-cultured with hUTC CM and phagocytosed in the absence of hUTC CM, the OS phagocytosis of these cells was rescued (Figure 4C). As demonstrated, hUTC secretes specific factors to promote RPE phagocytosis.

RTK ligand test

BDNF and HB-EGF test: Repeat the treatment of dystrophy RPE with BDNF and HB-EGF twice, and test their phagocytosis and normal control group as described in the method (Figure 5A, 5B). Each sample is observed ten to twelve times. The dystrophic cells tend to show a higher phagocytic rate than normal cells, but this does not hinder the interpretation of the results. BDNF showed higher phagocytosis rescue activity than CM3.

PDGF-DD, Pterin A4, and HGF assay: The number of cells counted per sampling is expressed per field (Figure 6A, 6C, 6D) and per cell (Figure 6B, 6E). The results were not affected because the number of cells counted in each box was constant. Compared to untreated controls, PDGF-DD (Figure 6A), Pterin A4 (Figure 6C), and HGF (Figure 6D) all upregulate phagocytosis in dystrophic RPE cells. PDGF-DD showed the highest rescue effect (higher than CM3).

Pterin B2 test: Pterin B2 showed extremely high phagocytosis rescue activity higher than CM3. Two results per field (Figure 7A) and per cell (Figure 7B) were determined.

Non- RTK ligand testing

The effect of endothelin-1, TGF-β1, or IL-6 on RCS RPE phagocytosis: the dystrophic RPE cells are treated with endothelin-1, TGF-β1, or IL-6, and tested as described in the materials and methods And the phagocytosis of the normal control group (Figure 8A to Figure 8C). Each sample was observed ten times. Figures 8A and 8B show two separate tests. The basic phagocytic level of normal and deferent RPE cells in Figure 8B is lower than that in Figure 8A, which is attributed to the variation of cells isolated from rats and the different preparation of ROS; due to the relationship between normal phagocytic levels and deferent phagocytic levels It is roughly 1: 0.3, so the test is considered valid. For comparison with the results in FIG. 8A, the data in FIG. 8C is normalized so that the phagocytic levels of normal and deferent RPE cells are the same as in FIG. 8A. hUTC CM3 increased the phagocytosis of dystrophic RPE cells, and the concentration (200ng / mL) of endothelin-1, TGF-β1, or IL-6 tested in the assay had no effect on RCS RPE phagocytosis.

The effect of vitronectin on RCS RPE phagocytosis: The dystrophic RPE cells were fed with ROS pre-cultured with various concentrations of glass connexin, and their phagocytosis was examined as described in the materials and methods and normal control groups (Figure 9). Each sample was observed ten times.

As for the control medium and hUTC conditioned medium used in this study, the control medium contains 10% FBS, and the amount of glass connexin in the control medium is about 500 ng / ml. Since hUTC continuously secretes vitronectin, hUTC conditioned medium may contain more glass connexin relative to the control medium. In contrast to ROS pre-treated with hUTC CM3, ROS pre-treated with control medium appeared to have no effect on the phagocytosis of deprived RPE. All test concentrations of glass connexin have a similar effect to the control medium. These results are consistent with those reported in previous publications (Edwards et al., J Cell Physiol. 1986, 127: 293-296; Miceli et al., Invest Ophthalmol Vis Sci., 1997; 38 (8): 1588-1597 ). Vitronectin is the active component of serum and causes serum from isolated human donor RPE cells to stimulate ROS uptake (Miceli et al., 1997). However, in rat RPE cells, the effect of serum on the phagocytosis of normal RPE is different from that of deferent RCS RPE. Edwards et al. Showed that cultured RCS rat RPE cells and normal homeologous control RPE cells phagocytosed in serum-free medium can be compared with a small amount of ROS. The presence of 20% serum in the medium significantly increased (6 times) the phagocytosis in normal RPE cells, but had no effect on RCS RPE cells (Edwards et al., J Cell Physiol. 1986, 127: 293-296).

The current results indicate that vitronectin does not involve enhanced phagocytosis of hUTC CM-mediated RCS RPE cells.

RNA isolation of dystrophic RPE treated with conditioned medium

As described in the method, several rounds of this experiment were performed to obtain the necessary RNA samples. RNA samples are used for RNA sequencing by Expression Analysis, Inc. In Experiment 1, the RIN scores of samples 8 and 9 did not meet the RNA sequencing criteria. Remove the two samples from the sequencing list. In Experiment 2, the RIN score of Sample 1 did not meet the RNA sequencing criteria and was removed from the sequencing list. (The sequencing results are as follows).

    Experiment 1    

    Experiment 2    

hUTC expression RTK ligand gene and bridge molecule gene line were analyzed by RNA-Seq-based hUTC transcript analysis. The gene expression of multiple RTK ligands in 15 RTK subfamilies was detected (Table 1-1). The performance levels of RTK ligand genes of each RTK subfamily are classified and plotted based on the number of fragments per kilobase transcript (FPKM) per million enantiomeric readings (Figure 10; Table 1-1). The gene expression of bridge molecules including MFG-E8, Gas6, protein S, TSP-1 and TSP-2 was also detected (Table 1-3).

The gene expression of the RTK subfamily and bridge molecule receptor in RCS RPE shows that the RTK superfamily can be grouped into 20 subfamilies based on the kinase domain sequence (Robinson DR et al., Oncogene. 2000; 19 (49): 5548-5557 ). The gene expression of 18 out of 20 RTK subfamilies in RCS RPE was detected (Table 1-2). Among the 18 RTK subfamilies, 15 subfamilies correspond to the RTK ligand genes expressed in hUTC. The gene expression of receptors reported to be used for bridge molecule binding (Kevany BM et al., Physiology, 2010; 25 (1): 8-15) was also detected in RCS RPE (Table 1-4), including integrins avP3, avP5, Axl, Tyro3, MerTK, and CD36.

Example 2 Receptor tyrosine kinase (RTK) ligand measurement in hUTC conditioned medium

The transcript profiles of both RCS RPE cells and hUTC analyzed using RNA-Seq and informatics data showed that RCS RPE cells expressed multiple RTK genes, while hUTC expressed multiple RTK ligand genes (Table 2-1). The RTK formulations of seven RTK subfamilies with relatively high levels of gene expression in hUTC were measured in conditioned medium of hUTC to compare with the conditioned medium of normal human skin fibroblasts (NHDF) and ARPE-19 cells. These ligands include BDNF and NT3 (Trk family ligands), HGF (Met family ligands), PDGF-DD and PDGF-CC (PDGF family ligands), Pterin B2 (Eph family ligands) , HB-EGF (ErbB family ligand), GDNF (Ret family ligand), and Aggregate (Musk family ligand).

Materials and Method

hUTC (batch number NB12898P7 was prepared from the PDL20 research library, page 7), ARPE-19 cells (passage 3) and NHDF (passage 10) were used in this study.

Human BDNF ELISA kit (Cat. No. DBD00, Lot No. 311655, Standard detection range: 62.5-4000pg / mL; Sensitivity: 20pg / mL), Human HGF ELISA kit (Cat. No. DHG00, Lot No. 307319, Standard detection range: 125 -8000pg / mL; sensitivity: <40pg / mL), human PDGF-CC ELISA kit (catalog number DCC00, batch number 309376, standard detection range: 62.5-4000pg / mL; sensitivity: 4.08pg / mL), human PDGF- The DD ELISA kit (catalog number DDD00, batch number 310518, standard detection range: 31.3-2000 pg / mL; sensitivity: 1.67 pg / mL) is from R & D Systems, Inc., Minneapolis, MN. HB-EGF ELISA kit (catalog number ab100531, batch number GR135979-1, standard detection range: 16.4-4000pg / mL; sensitivity: <20pg / mL) and NT3 ELISA kit (catalog number ab100615, batch number GR141281-1, standard Detection range: 4.12-3000pg / mL; sensitivity: <4pg / mL) from abcam, Cambridge, MA. The human GDNF ELISA kit (catalogue number RAB0205, batch number 0919130270, standard detection range: 2.74-2000pg / mL; sensitivity: 2.74pg / mL) is from Sigma, St. Louis, MI. Human Pterin B2 ELISA kit (catalogue number MBS916324, batch number R21199424, standard detection range: 15.6-1000pg / mL; sensitivity: 15.6pg / mL) and agrin ELISA kit (catalog number MBS454684, batch number EDL201310110, standard detection Range: 31.2-2000 pg / mL; sensitivity: <13.6 pg / mL) from MyBioSource, Inc., San Diego, CA.

Preparation of conditioned medium for hUTC , ARPE-19 , and NHDF : On day 1, hUTC, ARPE-19, and NHDF were inoculated into 15mL hUTC growth medium (DMEM) in T75 cell culture flasks at 10,000 viable cells / cm ² respectively Low glucose + 15% v / v FBS + 4mM L-glutamic acid). The cells were cultured in a 5% CO ₂ incubator at 37 ° C for 24h. On Day 2, the medium was aspirated and supplemented with 21 mL of DMEM / F12 complete medium (DMEM / F12 medium + 10% v / v FBS + 50 U / ml Pen / 50 μg / ml Strep). The cells were cultured for 48h. The control medium alone (DMEM / F12 complete medium) was also cultured for 48h. On day 4, the cell culture supernatant and the control medium, and at 250 g at 4 deg.] C was centrifuged 5min, at 0.5mL / tube aliquots in cryotubes, and immediately frozen at -70 ℃ refrigerator . Frozen samples were thawed for ELISA.

result

The selected RTK ligand levels measured in hUTC conditioned medium are summarized in Table 2-2.

Table 2-2. Concentrations of BDNF, NT3, HGF, PDGF-CC, PDGF-DD, GDNF, Pterin B2, HB-EGF, and Aggregate in hUTC conditioned medium.

The selected RTK ligand levels measured in hUTC conditioned medium were also compared with NHDF and ARPE-19 conditioned medium (normalized with pg / mL / 1 × 10 ⁶ cells). 72.7 pg / mL BDNF was secreted per million hUTC cells every 48 h, compared to 20.4 pg / mL and 16.2 pg / mL BDNF secreted per million NHDF and ARPE-19 cells every 48 h (Figure 11A). The amount of BDNF in the control medium is not detectable. (Figure 11G).

hUTC and NHDF secrete a small amount of NT3 (Figure 11B). The amount of NT3 in ARPE-19 conditioned medium and control medium is undetectable.

Every 48h, 23.7 pg / mL HGF was secreted per million hUTC cells, compared with 3.9 pg / mL and 0.4 pg / mL NHDF, respectively, compared to NHDF and ARPE-19 (Figure 11C). The amount of HGF in the control medium is not detectable. (Figure 11G).

The amounts of PDGF-CC and PDGF-DD in hUTC CM compared to NHDF and ARPE-19 CM are shown in FIGS. 11D and 11E. 0.3 pg / mL PDGF-CC and 3.1 pg / mL PDGF-DD were detected in the control medium, respectively.

Every 48h, every million hUTC cells secrete 9.5pg / mL GDNF, compared with 8.5pg / mL GDNF secreted by NHDF. Only trace amounts of 1.3 pg / mL GDNF were released per million ARPE-19 cells every 48h (Figure 11F). The amount of GDNF in the control medium is not detectable. (Figure 11G).

The levels of pterin B2 and HB-EGF in the conditioned medium of hUTC, NHDF, and ARPE-19 were below the detection limit of ELISA (15.6 pg / mL and 20 pg / mL, respectively). The levels of aggregated protein in conditioned medium of hUTC, NHDF and ARPE-19 were similar to the control medium.

In the RCS RPE phagocytosis test, tests performed with BDNF, HGF, PDGF-DD, pterin-B2, and HB-EGF at a dose of 200 ng / mL all showed a positive effect on rescue phagocytosis of RCS RPE cells. (Example 1). However, the actual concentration of these ligands secreted by hUTC in the conditioned medium is lower than the level tested.

Example 3 Bridge molecule in hUTC conditioned medium

The transcript profiles of both RCS RPE cells and hUTC show that RCS RPE cells express receptor genes that recognize the "eat me" signal on apoptotic cells. (Erwig L-P, Cell Death and Differentiation 2008; 15: 243-250). These receptors include scavenger receptors (SR-A, LOX-1, CD68, CD36, CD14), integrin (αvβ3 and αvβ5), Axl and Tyro3 receptor tyrosine kinase, LRP-1 / CD91, And PS receptor stabilizer 1. In addition, hUTC expresses many bridge molecule genes, including TSP-1, TSP-2, interface active protein D (SP-D), MFG-E8, Gas6, apolipoprotein H (apolipoprotein H), and annexin 1 (annexin 1). The secretion of bridge molecules in hUTC conditioned medium was detected and the levels were compared with those from ARPE-19 and normal human skin fibroblasts (NHDF).

Materials and Method

hUTC (PDL20, main cell bank number 25126057), ARPE-19 cells (passage 3) (ATCC, Manassas, VA) and NHDF (passage 11) (Lonza, South Plainfield, NJ) were used in this study.

Human Gas6 ELISA kit (Cat. No. SK00098-01, batch number 20111218) and human SP-D ELISA Kit (Cat. No. SK00457-01, batch number 20111135) were from Aviscora Bioscience, Santa Clara, CA. The standard detection range of the human Gas6 ELISA kit is 62.5-8000pg / mL, and the sensitivity is 31pg / ml. The standard detection range of the human SP-D ELISA kit is 78-5000pg / mL, and the sensitivity is 30pg / ml. Human MFG-E8 ELISA kit (Cat. No. DFGE80, Lot No. 307254, Standard detection range: 62.5-4000pg / mL; Sensitivity: 4.04pg / mL), Human TSP-1 ELISA kit (Cat. No. DTSP10, Lot No. 307182, Standard Detection range: 7.81-500ng / mL; sensitivity: 0.355ng / mL), human TSP-2 ELISA kit (catalogue number DTSP10, batch number 307266, standard detection range: 0.31-20ng / mL; sensitivity: 0.025ng / mL ) Is from R & D Systems, Inc., Minneapolis, MN. The apolipoprotein H human ELISA kit (catalog number ab108814, batch number GR126938, standard detection range: 0.625-40ng / mL, sensitivity: 0.6ng / mL) was from Abcam, Cambridge, MA. Human Annexin I (ANX-I) ELISA kit (catalog number MBS704042, batch number N10140947, standard detection range: 0.312-20ng / mL; sensitivity: 0.078ng / mL) is from MyBioSource, Inc., San Diego, CA .

Preparation of conditioned medium for hUTC, ARPE-19 , and NHDF : On day 1, hUTC, ARPE-19, and NHDF were inoculated into 15mL hUTC growth medium (DMEM) in T75 cell culture flasks at 10,000 viable cells / cm ² respectively Low glucose + 15% FBS + 4mM L-glutamic acid). Incubate at 37 ° C in a 5% CO ₂ incubator for 24h. On the second day, the medium was aspirated and supplemented with 18 mL of DMEM / F12 complete medium (DMEM: F12 medium + 10% FBS + Pen (50 U / ml) / Strep (50 μg / ml)). The cells were cultured for 48h. A separate control medium (DMEM: F12 medium + 10% FBS + Pen (50U / ml) / Strep (50 μg / ml)) was also cultured for 48h. On Day 4, the cell culture supernatant and control medium were collected and centrifuged at 250g for 5 min at 4 ° C, aliquoted in 0.5mL / tube in cryopreservation tubes, and immediately frozen in a -70 ° C refrigerator. Frozen samples were thawed for ELISA.

result

77.2ng MFG-E8 is secreted per million hUTC cells every 48h, compared to 7.6ng and 17.5ng MFG-E8 secreted per million ARPE-19 and NHDF cells every 48h (Figure 12A). The concentration of MFG-E8 in hUTC conditioned medium is 15.5 ng / mL. The amount of MFG-E8 in the control medium is undetectable. (Figure 12E).

Every 48h, 352.8 pg of Gas6 was secreted per million hUTC cells, compared with 183.9 pg of ARPE-19 and 1101 pg of NHDF (Figure 12B). The concentration of Gas6 in hUTC conditioned medium is 70.6 pg / mL. The amount of Gas6 in the control medium is undetectable. (Figure 11G).

Every 48h, 759.2ng TSP-1 was secreted per million hUTC cells, compared with 4748.68ng TSP-1 secreted per million ARPE-19 cells per 48h and 2487.55ng TSP-1 secreted by NHDF cells (Figure 12C). The concentration of TSP-1 in hUTC conditioned medium is 151.8ng / mL. 4.0ng / mL TSP-1 was detected in the control medium. (Figure 12E).

Every 48h, 44.2ng TSP-2 was secreted per million hUTC cells, compared to 30ng TSP-2 secreted by NHDF. Every 48h, a negligible amount of 0.08ng TSP-2 was released per million ARPE-19 cells (Figure 12D). The concentration of TSP-2 in hUTC conditioned medium is 8.8 ng / mL. Trace amounts of TSP-2 (0.02ng / mL) were detected in the control medium. (Figure 12E).

The level of apolipoprotein H in the conditioned medium of hUTC, ARPE-19 and NHDF is similar to the level in the control medium (6.8ng / mL). The levels of SP-D and Annexin I in conditioned medium of hUTC, ARPE-19 and NHDF and control medium were below the detection limit of ELISA (<30pg / mL and <78pg / mL, respectively). These cells do not secrete these two proteins, or the secretion level is below the detection limit.

A summary of the bridge molecules and their concentrations detected in hUTC conditioned medium are listed in Table 3-2.

Among the measured bridge molecules, MFG-E8, Gas6, TSP-1 and TSP-2 are candidate bridge molecules involved in hCSC-mediated phagocytosis rescue in RCS RPE cells.

The combination of ROS regulated by the bridge molecule can activate the integrin and RTK communication path, which can compensate for the absence of Mertk communication and cause phagocytosis rescue.

Example 4 The effect of hUTC conditioned medium and bridge molecules on RCS RPE cells phagocytosis of rod outer segment (ROS)

The direct effect of hUTC conditioned medium on phagocytosis of ROS was detected by feeding RCS RPE cells with ROS pre-cultured with hUTC conditioned medium. (US 2010/0272803). The phagocytosis of dystrophic RPE cells is completely rescued. Here, the role of bridge molecules present or secreted in hUTC conditioned medium is studied. Currently, the only "eat me" signal recognized on ROS is phospholipid amide serine (PS) (Finnemann et al., PNAS , 2012; 109 (21): 8145-8148).

Materials and Method

The procedures for RPE isolation, primary culture of RPE cells, sulforadamine staining of RPE cells, isolation of rat ROS, FITC staining of ROS, phagocytosis assay, imaging and quantification, acceptance criteria for assay, and relative phagocytosis level are described In Example 1.

hUTC conditioned medium ( CM ): hUTC CM3 is used in this study. On day 1, hUTC was seeded at 10,000 viable cells / cm ² in hUTC growth medium (DMEM low glucose + 15% FBS + 4 mM L-glutamic acid) in T75 cell culture flasks. Incubate at 37 ° C in a 5% CO ₂ incubator for 24 hours. On the second day, the medium was aspirated and supplemented with 21 mL of DMEM / F12 complete medium (DMEM: F12 medium + 10% FBS + Pen (50 U / ml) / Strep (50 μg / ml)). The cells were incubated for another 48 hours. A separate control medium (DMEM: F12 medium + 10% FBS + Pen (50 U / ml) / Strep (50 μg / ml)) was also cultured for 48 h. On the 4th day, the cell culture supernatant and control medium were collected and centrifuged at 250g for 5 min at RT, aliquoted in 3mL / tube into cryovials, and immediately frozen in a -70 ° C refrigerator.

Recombinant human bridge molecules: recombinant human MFG-E8 (catalog number 2767-MF-050, batch number MPP2012061), recombinant human Gas6 (catalog number 885-GS-050, batch number GNT5013011), recombinant human TSP-1 (catalog number 3074-TH -050, batch number MVF3613041), recombinant human TSP-2 (catalogue number 1635-T2-050, batch number HUZ1713021) were all obtained from R & D Systems, Inc., Minneapolis, MN. The reconstitution of individual protein stock solutions was based on the supplier's data sheet: recombinant human MFG-E8, TSP-1 and TSP-2 were reconstituted in sterile PBS at 100 μg / mL, respectively. Recombinant human Gas6 was reconstituted in sterile water at 100 μg / mL. The reconstituted stock solution was aliquoted and frozen in the refrigerator at -70 ° C.

Effect of bridge molecule on phagocytosis of RCS RPE cells: ROS line was pre-cultured in CO ₂ cell incubator with control medium (DMEM + 10% FBS) or CM3 at 37 ℃ for 24h. Meanwhile, ROS was pre-cultured in various concentrations of human recombinant MFG-E8, Gas6, TSP-1, or TSP-2 in a CO ₂ cell culture incubator at 37 ° C for 24h. After culturing, ROS were centrifuged to settle without washing, resuspended in MEM5, and fed to the dystrophic RPE cells in the presence of MEM5 for phagocytosis assay. As for the control group, normal RPE alone or deprived RPE alone was cultured in MEM20, and then replaced with MEM5 in the presence of untreated ROS (resuspended in MEM20 and fed to RPE cells) for phagocytosis assay.

Effect of RTK ligand on phagocytosis of RCS PRE cells: RCS RPE was cultured with recombinant human BDNF, HGF, and GDNF individually for 24 hours, and then OS was added for phagocytosis assay. The RCS RPE cultured with hUTC CM was used as a positive control.

siRNA Removal: On-TARGETplus human siRNA-SMARTpool and ON-TARGETplus Non-targeting pool for human BDNF, HGF, GDNF, MFG-E8, Gas6, TSP-1, and TSP-2 were purchased from GE Dharmacon (Lafayette, CO). Using DharmaFECT transfection reagent (GE Dharmacon), each siRNA pool of 25 nM was incorporated into hUTC.

Immunofluorescent stained antibodies: unconjugated monoclonal antibodies of bridge molecules (human MFG-E8, Gas6, TSP-1, and TSP-2), and mouse IgG2A and IgG2B isotype control antibody systems were obtained from R & D Systems, Inc ., Minneapolis, MN. These antibodies were conjugated to Alexa Fluor 488 fluorophore by Life Technologies (Eugene, OR). The unconjugated single anti-rhodopsin antibody (EMD Millipore Corp., Temecula CA) was conjugated to Alexa Fluor 568 by Life Technologies (Eugene, OR). The unconjugated mouse IgG2b, x isotype control antibody system was obtained from Biolegend Corporation (San Diego, CA) and conjugated with Alexa Fluor 488 fluorophore by Life Technologies (Eugene, OR). It is used as an isotype control antibody for Alexa Fluor 568 conjugated anti-rhodopsin antibody. Alexa Fluor 488 conjugated mouse IgG2A line is used as an isotype control antibody for Alexa Fluor 488 conjugated anti-human MFG-E8, Gas6, or TSP-2 antibody. Alexa Fluor 488 conjugated mouse IgG2B line is used as isotype control antibody of Alexa Fluor 488 conjugated human TSP-1 antibody.

Immunofluorescence: 10 × 10 ⁶ OS cells at 37 ° C in 1 mL hUTC CM, 1 mL control medium, or 1 mL containing 124 ng / mL MFG-E8, 8.75 ng / mL Gas6, 1.2 μg / mL TSP-1, or Incubate for 24 hours in 238ng / mL TSP-2 control medium. The OS was agglomerated, washed and embedded in Tissue-Tek OCT compound (Sakura Finetek USA, Inc., Torrance, CA). A cryostat (Leica CM1950, Leica Microsystems, Inc., Buffalo Grove, Illinois) was used to obtain 10 iAm series of slices. These slices were transferred to slides for immunofluorescence staining. Use the blocking buffer (10% (v / v) goat serum, 1% (v / v) BSA, and 0.1% (v / v) Triton × 100 in PBS) for 1 hour, and then at 4 ° C with Alexa Fluor 568 conjugated anti-rhodopsin antibody and Alexa Fluor 488 conjugated anti-MFG-E8, anti-Gas6, anti-TSP-1, anti-TSP-2, Or mouse IgG ₂ A or IgG2 _B isotype control antibody double stained for 2 hours. After washing three times with PBS, the sections were fixed in Vectashield mounted tablets (Vector Laboratories, Inc., Burlingame, CA) and evaluated with Zeiss Photomicroscope III (Carl Zeiss, Oberkochen, Germany) equipped with epi-fluorescence. Images were captured using a Kodak 290 digital camera and analyzed using Kodak Microscopy Documentation System 290 Photoshop image analysis software (Eastman Kodak, Rochester, NY). The image is obtained with an appropriate filter at 250x magnification.

result

As shown in FIGS. 13A and 13B (Experiment 1) and FIGS. 13C and 13D (Experiment 2), untreated defermented RPE cells reduced phagocytosis compared to normal RPE cells. In the absence of hUTC conditioned medium during the test period, precultivation of dystrophic RPE cells with hUTC conditioned medium completely rescues phagocytosis. When hUTC conditioned medium is present during the phagocytosis assay, whether or not the defermented RPE cells are pretreated with hUTC conditioned medium, a more stable and enhanced phagocytosis is observed. The dystrophic RPE cells fed with ROS pretreated with hUTC conditioned medium showed recovery of phagocytosis in the absence of hUTC conditioned medium during the phagocytosis assay.

The dystrophic RPE cells were fed with various concentrations of MFG-E8 (15.5ng / mL; 31ng / mL; 62ng / mL; 124ng / mL), Gas6 (70pg / mL; 350pg / mL; 1750pg / mL; 8750pg / mL ), TSP-1 (152ng / mL; 304ng / mL; 608ng / mL; 1216ng / mL), or TSP-2 (8.8ng / mL; 26.4ng / mL; 79.2ng / mL; 237.6ng / mL) pre-culture ROS, and tested for phagocytosis (Figure 14A to Figure 14D). Each sample was observed ten times. Phagocytosis of ROS is rescued by feeding RCS RPE cells with ROS pre-cultured with MFG-E8, Gas6, TSP-1 or TSP-2.

Each of MFG-E8, Gas6, TSP-1, and TSP-2 dose-dependently increased the level of phagocytosis in RCS RPE cells (Figures 14E to 14H). Similarly, BDNF, HGF and GDNF dose-dependently increased phagocytic misalignment in RCS RPE cells, with HGF still having the strongest effect even at the lowest dose. When applied at higher concentrations, BDNF, HGF and GDNF were able to rescue phagocytosis in RCS RPE (Figures 14I to J). These results show that the recombinant RTK ligand and bridge protein can mimic the role of hUTC CM and restore RCS RPE phagocytosis, and involve hUTC-mediated phagocytosis rescue in RCS RPE.

BDNF, HGF, GDNF, MFG-E8, Gas6, TSP-1 and TSP-2 were eliminated in hUTC by siRNA-mediated gene silencing. A messy siRNA pool that does not target any genes is used as a gene knockout control. The removal efficiency of each factor was detected by measuring the level of each factor in the cell culture supernatant, which was collected from hUTC transfected with siRNA (Figure 15A). Simulated or messy siRNA transfection has no effect on the secretion of hUTC from these factors. SiRNAs targeting MFG-E8, TSP-1, TSP-2, and HGF produced almost 100% rejection efficiency; BDNF and GDNF were observed to be 80% and 65% removed, respectively (Figure 15A). siRNAs targeting Gas6 in hUTC will not work (data not shown). CM is produced from hUTC transfected with siRNA and applied to RCS RPE to recognize the role of RTK ligand and bridge molecule knockout. Incubate RCS RPE with CM produced from hUTC transfected with siRNA targeting BDNF, HGF or GDNF (Figure 15B), or feed with siRNA produced from MFG-E8, TSP-1 or TSP-2 CM pretreated OS of stained hUTC (Figure 15C). The CM prepared from untransfected and messy siRNA transfected hUTC was used as a control CM. The individual knockout of each RTK ligand abolished the effect of hUTC CM on phagocytosis rescue compared to the knockout control CM (Figure 15B). Removal of each of the bridge molecules reduces the phagocytosis of OS by RCS RPE (Figure 15C). These RTK ligands and bridge molecules are required for hUTC-mediated phagocytosis rescue in RCS RPE.

Double staining of each individual bridge molecule and rhodopsin ensures that OS is evaluated. Rhodopsin is a visual pigment located in the photoreceptor OS and is a sign of OS staining (Szabo K et al., Cell Tissue Res. 2014; 356 (1): 49-63). The rhodopsin staining (Alexa Fluor 568 conjugated, red) particles pre-cultured with individual recombinant human bridge molecules were stained positive by each of the four bridge molecule antibodies (Alexa Fluor 488 conjugated, green), but Alexa Fluor 488 Conjugated mouse IgG2A or IgG2B isotype control antibody staining was not positive (Figure 16A). Similar results were obtained for OS cultured with hUTC CM (Figure 16B), however, OS bridge cultured with the control medium did not observe any bridge molecule staining (Figure 16C). The specificity of anti-rhodopsin antibodies was confirmed by double staining of OS particles with Alexa Fluor 568 conjugated anti-rhodopsin antibody and Alexa Fluor 488 conjugated mouse IgG2b, x isotype control antibody. Only OS stained with anti-rhodopsin antibody was positive (Figure 16D). The bridge molecules MFG-E8, Gas6, TSP-1 and TSP-2 in hUTC CM co-localize with rhodopsin on the OS, proving that the bridge molecules bind to the OS.

Example 5 hUTC prevents oxidative damage to RPE

Oxidative stress can affect the health of retinal pigment epithelium. To study the effect of hUTC and hUTC conditioned medium on improving the health of RPE cells exposed to oxidative damage.

Materials and Method

Hydrogen peroxide (H ₂ O ₂ ), crystal violet and thiazolyl blue tetrazolium bromide (MTT) were obtained from Sigma-Aldrich (St Louis, MO).

Ham F10 medium, penicillin-streptomycin solution (5000 units / mL penicillin / 5000 μg / mL streptomycin), trypsin-EDTA solution (0.05%), low glucose DMEM, L-glutamic acid 200 mM were obtained from Life Technologies.

Hyclone FBS and formaldehyde were purchased from Thermo Scientific. Isopropyl alcohol, glacial acetic acid and hydrochloric acid were purchased from Fisher Scientific (Pittsburgh, PA). Ethanol was obtained from Decon Labs Inc. (King of Prussia, PA). PBS was obtained from Lonza (South Plainfield, NJ).

ARPE growth medium: DMEM (with 4.5g / L glucose and sodium pyruvate without L-glutamic acid and phenol red) (Mediatech, Inc. A Corning Subsidiary, Manassas, VA) supplemented with 5% or 10% heat loss Live fetal bovine serum (FBS, Life Technologies, Grand Island, NY), 1X minimum essential medium-non-essential amino acids (MEM-NEAA, Life Technologies), and 0.01 mg / mL itamycin reagent solution (Life Technologies) .

5% ARPE growth medium containing 10 μM A2E : DMEM (Mediatech, Inc. A Corning Subsidiary) supplemented with 5% heat-inactivated FBS (Life Technologies), 1X MEM-NEAA (Life Technologies), 0.01 mg / mL citracin Reagent solution (Life Technologies), and 10 μM A2E (prepared by Dr. Janet Sparrow's laboratory).

hUTC complete medium: DMEM low glucose (Life Technologies) supplemented with 15% Hyclone® FBS (Thermo Scientific, Logan, Utah) and 4mML-glutamic acid (Life Technologies).

hUTC FBS medium: DMEM (Mediatech, Inc.) supplemented with 5% or 10% heat-inactivated FBS (Life Technologies), 1X MEM-NEAA (Life Technologies), 0.01 mg / mL itanomycin reagent solution (Life Technologies) , And 4mM L-glutamic acid (Mediatech, Inc.).

hUTC conditioned medium: hUTC (Research Library NB12898P6, PDL20) was inoculated into hUTC complete medium (15 mL) in 2 T75 culture flasks at 37 ° C and 5% CO ₂ at 5000 cells / cm ² . Twenty-four hours after seeding, the medium was removed from each flask, and the cells were washed 3 times with 15 mL of 1X Dulbecco's phosphate buffered saline (DPBS). After the third wash, 15 mL of 5% or 10% FBS hUTC medium was added to each bottle. Fifteen mL of 5% or 10% FBS hUTC medium was also added to 2 empty T75 bottles and served as a control group. Return all bottles to 37 ° C, 5% CO _{2 for} 48 hours. After 48 hours, the medium was removed from each flask and centrifuged at 250 × g for 5 minutes at 4 ° C. The medium was placed on ice and then aliquoted and stored at -80 ° C.

ARPE-19 cell culture for A2E research: On day 1, ARPE-19 cells were seeded at a density of 40,000 cells / well on 8-well Nunc ^™ Lab-Tek ^™ II chamber slides (Nalge Nunc International Corporation, Rochester, NY) in a final volume of 300 μL of 10% ARPE growth medium. 24 hours after seeding (day 2), the medium on the cells was removed and replaced with 300 μL of 5% ARPE growth medium. One week later (Day 9), the medium was removed again and replaced with fresh 5% ARPE growth medium. On day 14, the medium was removed and replaced with fresh 5% ARPE growth medium containing 10 μM A2E. On days 17 and 21, the medium was removed again and replaced with fresh A2E-containing medium. On day 24, the A2E-containing medium was removed and replaced with fresh 5% ARPE growth medium, and the cells were quiescent for five days.

On day 29, the medium was removed from each well and replaced with 250 μL of 5 or 10% hUTC conditioned medium or control medium (5% and 10% FBS hUTC medium not exposed to cells). On days 32 and 35, the conditioned medium and control medium were removed from the cells and replaced with fresh conditioned medium or control medium.

On day 36, all media was removed from each well and the cells were washed once with IX DPBS. Later, 200 μL of fresh DPBS was added to each well and the cells were exposed to 430 nm light delivered by a tungsten halogen source for 20 minutes. After light exposure, DPBS was removed and a cell viability test was performed.

MTT assay for A2E research: by metabolic (MTT, 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide) colorimetric microtitration assay ( Roche Diagnostics Corporation, Indianapolis, IN) measures cytotoxicity. For MTT assay, 20 μL of MTT labeling reagent (Roche Diagnostics Corporation) was added to 0.2 mL of 5% medium in each well. After culturing for 4 hours, another 200 μL of solubilizing solution was added to each well for overnight culture. After centrifugation at 13,000 rpm for 2 minutes, the supernatant was measured spectrophotometrically at 570 nm (SpectaMax MJ, Molecular Devices, Sunnyvale, CA). The reduced absorbance of reduced MTT at 570 nm indicates a decrease in cell viability. Analyze the data with Prism software.

Dead Red test for A2E research: After labeling by fluorescence exclusion assay allowing labeling of apoptotic nuclei, quantify non-surviving cells, allowing labeling of apoptotic nuclei due to the late stage of cell death Caused by loss of cell membrane integrity. After light exposure, the cells were put back into 5% ARPE growth medium. Eight hours after blue light exposure, the nuclei of dead cells were stained with membrane-impermeable dye Dead Red (Life Technologies; 1/500 dilution, 15 min incubation), and all nuclei were stained with 4 ', 6'-diamine-2 -Phenylindole (DAPI) (Life Technologies) staining. Briefly, cells were washed twice with preheated (37 ° C) Hanks' balanced salt solution (1X) HBSS (Life Technologies). 250 μL Dead Red working solution (8 μL Dead Red stock solution + 4 mL HBSS) was added to each well at room temperature for 15 minutes. After 15 minutes, the cells were washed twice with HBSS. 300 μL of 4% formaldehyde-made 1X DPBS was added to each well at room temperature for 30 minutes. The cells were washed 3 times with 1X DPBS and incubated with a working solution of DAPI (prepared in 1X DPBS at 1: 300) at room temperature for 5 minutes. The cells were washed 3 times with 1X DPBS. Slides were fixed and covered with slides, and duplicates were verified by counting DAPI stained and Dead-Red stained nuclei in at least 5 microscope fields in the illuminated area in each well. Values are expressed as Dead-Red stained nuclei / DAPI stained nuclei × 100.

Cell culture for H ₂ O ₂ research: ARPE-19 cells (American Bacteria Conservation Center; Manassas, VA) were grown in a single layer at 37 ° C and 5% CO ₂ in a T75 flask containing 10% FBS and 50 units / mL penicillin / 50 μg / mL streptomycin in Ham's F10 Medium. In experiments co-cultivated with hUTC, ARPE19 cells were grown in T-75 flasks to 80-90% overgrown and later seeded in 24-well cell culture plates. ARPE-19 cells were grown in 10% FBS growth medium until day 3 after inoculation, at which time the medium was changed to basal medium (Hamm F10 medium supplemented with 2% FBS and 50 units / mL penicillin / 50 μg / mL chain Amycin).

HUTC was seeded onto cell culture inserts (well size 1 μm) in hUTC complete growth (low glucose DMEM supplemented with 15% FBS and 4 mM L-glutamic acid) at 5000 cells / cm ² for 24 hours. The insert was transferred to ARPE-19 cells grown on cell culture plates for 72 hours and grown in hUTC complete medium. The insert was removed and ARPE-19 cells prepared in serum-free Ham F10 medium were treated with H ₂ O ₂ (0 to 1500 μM) for 9 hours.

Crystal Violet Cell Viability Assay for H ₂ O ₂ research: Relative cell viability was determined by crystal violet uptake. After the treatment, the cells were fixed in 4% paraformaldehyde in PBS and stained in a solution of 0.1% crystal violet and 10% ethanol. After washing with water, the remaining stain is dissolved in 10% acetic acid and the absorbance at 550 nm is measured with a microplate reader.

MTT assay for H ₂ O ₂ research: Cells were incubated with MTT at 0.25 mg / mL in serum-free medium at 37 ° C for 3 hours. The medium was then removed and acidic isopropanol (1 μL concentrated HCl per 1 mL isopropanol) was added to solubilize the blue (formazan) (MTT metabolite). Blue armor The density is measured using a microplate reader at 550nm, and the background wavelength is 630nm.

result

The hUTC conditioned medium protects A2E-rich RPE cells from blue light-induced damage. ARPE-19 viability was evaluated after irradiation by labeling cells with the membrane-impermeable dye Dead Red and labeling all nuclei with DAPI staining. The nuclei counted in the digital image provide the percentage of viable and non-viable cells (Figure 17A-17B). In the absence of 430 nm illumination, 10 μM A2E had no effect on ARPE-19 viability. Cells cultured with control medium and subjected to 430 nm illumination showed high levels of non-viable cells (about 50%). Conversely, treatment with hUTC conditioned medium resulted in a reduction in the number of non-viable cells (about 20%).

The cell viability was also quantified by MTT assay (Figures 17C to 17D), which cut off the yellow tetrazolium salt MTT into purple formazan based on healthy cells The power of crystals. A The production is proportional to the number of surviving cells in the culture. ARPE-19 cells treated with conditioned medium and exposed to light showed reduced viability compared to ARPE-19 cells added with 10 μM A2E and not exposed to light. Cells treated with 5 or 10% hUTC conditioned medium showed higher viability than cells exposed to control medium.

After total H ₂ O ₂ treatment, ARPE19 cell viability was determined by crystal violet and MTT assay (Figure 17E to Figure 17F). ARPE-19 cells co-cultured with hUTC showed improved viability after treatment with 1500 μM H ₂ O ₂ compared to untreated control cells.

Example 6 Isolation of RPE and ROD cells I. Isolation of RPE cells from human donor eyes

Human eyes obtained in a wet room were rinsed in PBS with 4% Pen-Strep (Life Technologies, penicillin-streptomycin, 10,000 U / mL, catalog number 15140122), and the anterior segment was removed at the limbus ( If it has not been removed) to open it. After necropsy and photography, the retina was removed and the posterior pole was cut into small pieces (about 1 cm) and cultured in 4 ml of 2% dispase solution (w / v in DMEM, 25 mM HEPES, 200 U / mL) at 37C. Pen-Strep) (Roche Diagnostic Corp, catalog number 04942078001) for 5 minutes. At least two volumes of DMEM (Life Technologies, catalog number 12430054), 25 mM HEPES (Sigma-Aldrich Corp., catalog number H4034-100G), 200U / mL Pen-Strep were added to stop the culture. Place the tissue in a 100 mm Petri dish with sufficient fresh DMEM, 25 mM HEPES, 2% Pen-Strep, and allow it to stand at 37 ° C for 6 to 12 hours. The tissue was transferred to RPMI medium (Life Technologies, catalog number 22400089) in a Petri dish, and RPE cells were teased off by gently scraping or spraying with a pipette tip. The cells were rinsed twice with HBSS (GIBCO, catalog number 310-4170), resuspended in 0.5 ml 0.1% trypsin (pH 8.0, Life Technologies, catalog number 25300054), and incubated at 37C for 2 minutes. The cell mixture was triturated with Pasteur pipette until the RPE cells were dispersed and 5x excess MEM / 20% FBS (minimum essential medium, Lift Technologies, catalog number 1095098; fetal bovine serum) was added , Life Technologies, catalog number 16000036) to stop enzyme treatment. The cells were centrifuged at 1000 xg, resuspended in an appropriate volume of MEM / 20 (MEM containing 20% FBS), counted, and cultured in an appropriate container (24-well dish, Thomas Scientific, catalog number 6901A11; 25ml bottle, VWR, Catalog number 29185-298). Roughly, 50,000 cells / well were seeded in a 24-well dish. As for the cells cultured in the 25ml bottle, one-fifth to one-third of the collected amount is inoculated into the 25ml bottle for the purpose of passage. Using this protocol, human RPE was isolated from the donors listed in Table 6-1 below.

Table 6-1. Donors used

Table 6-2 shows additional information about the donor eye used.

II. Culture of primary human RPE cells

After isolation, the following procedure was used to subculture the previous RPE cells. Next, in the studies in Examples 7 to 13, these cell cultures were used.

Human RPE cells were cultured in 25ml flasks or 24-well dishes to achieve fullness. When collecting human RPE cells, in addition to seeding cells in a 24-well format, a 25 ml bottle was also seeded to obtain a large number of cells for storage, seeding an additional 24 wells, and subculture. When the flask is full, the cells are passaged. Continue this process to carry out about 6 generations. The number of days between generations and the number of doublings depend on how many cells are seeded in the flask, but the doubling time seems to be about 2 days. Bottles often grow up in about 10 days.

As for the subculture, the culture was rinsed twice with PBS, trypsinized with 0.1% trypsin (pH 8.0, Life Technologies, catalog number 25300054), and collected. Passage the cells to another flask or dish (approximately 1/5 to 1/3 full volume), or resuspend at least 3 × 10 ⁵ cells in 0.5 to 1 ml cryopreservation medium (Life Technologies, catalog number 12648010) Medium, gradually cooled in Mr. Frosty ^TM Freezing Container, and stored in liquid nitrogen. For regeneration, the cryotubes of the cells were warmed in a 37 ° C water bath, and the cells were transferred to the wells of a 24-well plate or 60 mm petri dishes, and cultured until they were overgrown, and then subcultured.

III. Isolation and storage of rod outer segment (ROS) from human eyes

Human eye systems were obtained from different eye banks (Florida Lions Eye Bank, Bascom Palmer Eye Institute, Miami, FL; San Diego Eye Bank, San Diego, CA; National Disease Research Interchange (NDRI), Philadelphia, PA). Specifically, the human eyes of the donors in Table 6-1 are used. The retina was separated, homogenized with a Polytron (5mm homogenizer), layered on top of a 27% to 50% linear sucrose gradient, and centrifuged at 38,000 rpm in a SW41 rotor at 4 ° C for 1 hour. Collect ROS bands that represent homogenized ROS particles of different sizes (possibly up to three bands, namely upper band, middle band, and lower band), dilute them with HBSS, and centrifuge at 7000 rpm in HB-4 rotor for 10 minutes to make ROS Become clumps. Resuspend the ROS pellet in serum-free medium. FITC stock solution (2 mg / ml in 0.1 M sodium bicarbonate, pH 9.0 to 9.5) (Sigma-Aldrich Corp., catalog number F7250-50MG) was added to a final concentration of 10 μg / ml and incubated at room temperature for 4 hours. FITC-stained ROS were pelleted by centrifugation in a microcentrifuge and resuspended in MEM / 20. Estimate its concentration with a hemocytometer and record its appearance under the microscope (should show a generally spherical or rod-like structure). Store the "active" part at 4 ° C for daily use, and freeze the frozen part as it is at -20 ° C for storage.

IV. Isolation and storage of rod extracellular section (ROS) from rat and pig eyes

Eyes were also obtained from 6-8 weeks old Long Evans rats and adult pigs (slaughterhouses). Using the process described above for human ROS, the outer segments of rod cells were isolated and classified from the eyes of rats and pigs.

As discussed, Examples 1 to 5 demonstrate that hUTC will rescue phagocytosis, thereby slowing retinal degeneration in the RCS rat model of retinal degeneration. Examples 7 to 13 show the results of similar tests using human RPE cells and ROS obtained by the method in this example. In some examples, rat and pig ROS were used for comparison.

The following abbreviations are used in Examples 6 to 13: TSP thrombospondin

In addition, as used in Examples 6 to 13, ROS and RPE cells will be identified by their donor identification. For example, 14-06-040 RPE refers to RPE cells isolated from donor 14-06-040. Furthermore, as used in Examples 6 to 13, 1 'means the first generation, 2' means the second generation, and the like.

Example 7 Phagocytosis test using isolated human RPE and ROS

A phagocytosis test using isolated human RPE and ROS was performed.

I. Immunofluorescence of RPE cells

RPE cells fixed in wells (24-well plate) with 4% paraformaldehyde were washed thoroughly with PBS. Cells were treated with 1% BSA (Sigma-Aldrich catalog number A-7030) + 10% newborn goat serum (Life Technologies, catalog number 16210-064) + 0.1% TritonX-100 (Sigma-Aldrich, catalog number T-8787) in Incubate in PBS for 0.5 to 1 hour, then incubate with primary antibody (Pan-keratin C11 Mouse mAb, Cell Signaling, catalog number 4545S, 1: 400 dilution with PBS containing 1% BSA) at RT for 2 hours or at 4C Incubate overnight, wash, and finally incubate with appropriate Alexa Green or Red conjugated secondary antibodies (diluted 1:50 in PBS, 1 hour at RT) (Life Technologies, catalog number A21202). The control group was treated with secondary antibodies only after blocking. After the final washing, the preparation was fixed in Vectashield mounting solution (VWR, catalog number 101098-042) and checked under Photomicroscope III equipped with a digital camera.

II. Phagocytosis test

Prior to the assay, 5 × 10 ⁴ human RPE cells (isolated in Example 6) were seeded on round glass coverslips in each well of a 24-well plate, maintained in MEM / 20 for at least 6 days, and then maintained in MEM + 5% FBS. The assay was started by covering the culture with FITC-ROS (0.5 to 5 × 10 ⁶ ) and incubating at 37 ° C. for 8 to 12 hours. At the end of the culture, the cells were vigorously washed with PBS to remove uningested ROS, and 2% paraformaldehyde (Paraformaldehyde, 4% in PBS, diluted with PBS to 2%, used within 1 week after dilution ) (Paraformaldehyde, 4% in PBS, VWR, catalog number AAJ61899-AK) fixed. Using fluorescence microscopy, Zeiss Fluorescence Photomicroscope III was used to visualize the ingested ROS at 312x magnification. Check at least 10 representative visual fields (field size, 0.021 mm ² ), and count the ROS absorbed into the fluorescent light.

III. Phagocytosis of human RPE and rat RPE

To demonstrate the ability of cultured human RPE to engulf rat ROS, two human (14-06-040) (1 ', day 13) cultures and two N rat RPE (6/4/14, 6 / 13/14) Culture to perform phagocytosis test on rat ROS (7.5 × 10 ⁶ , 6/18/14) (8 h). Immunofluorescence micrographs (see Figure 18) show evidence of human cell phagocytosis, however autofluorescence hinders good quantitative evaluation. (Autofluorescence may come from lipofuscin in RPE.)

IV. Phagocytosis time course of human RPE and rat RPE

Previously, it was shown that the phagocytosis of ROS by rat RPE cells in vitro followed a different pattern, in which the added ROS was bound to these cells and remained relatively stationary until about 8 hours, at which time violent uptake occurred. In 15 to 16 hours, the first cycle of phagocytosis is about to end, and a second cycle of similar binding, waiting, and ingestion begins. To understand whether human RPE exhibits the same pattern of phagocytosis, in the case of using rat ROS, examine the phagocytosis time course of human RPE and rat RPE. Use the same cells as above. A mixed preparation of rat ROS (6/18/14, 6/30/14) was used. Rat ROS was added to the cultured cells, and phagocytosis was stopped and analyzed at 1, 2, 3, 6, 8, 9, 10, 11, 12, 13, 15, and 16 hours. The expected phagocytosis time course appeared in rat RPE, ie, it continued to bind for 1 to 6 hours but exhibited low phagocytic activity. Phagocytosis started at 8 hours, continued until 15 hours, and slowed down at 16 hours with evidence of the next cycle (see also Figure 19). A substantially identical pattern appears in human RPE, which confirms the similarity in phagocytic characteristics between rat RPE and human RPE.

V. Repeat the phagocytosis time course of human RPE and rat RPE

Using another preparation of rat ROS, the phagocytosis time course experiment was repeated. Use human RPE (14-06-040) (1 ', day 28), rat N RPE (7/3/14, 7/11/14), and rat ROS (2 × 10 ⁶ , 7/10 / 14). ROS was added to the cells, and phagocytosis was stopped at 6, 8, 10, 11, 13, 15, and 16h for analysis. The same pattern as observed in previous experiments has been observed, which confirms the similarity between rat RPE and human RPE phagocytosis. Perform partial phagocytosis (see Table 7-1).

VI. Rat RPE phagocytosis of human ROS

Two rat RPE (8-14-14, 9-9-14) cultures were used to test phagocytosis for 8 hours using human ROS preparation (5 × 10 ⁶ ). The test results are shown in Figure 20. Evidence of phagocytosis has been observed by rat RPE. The upper band ROS represents homogenized ROS particles smaller than the middle and lower band materials, and appears to show better phagocytosis.

VII. Human ND04760 RPE phagocytosis of human ROS and rat ROS

The culture of human ND04760 RPE (day 11) was used for 8h phagocytosis of upper and lower human ROS (made from ND04760 retina, 9-18-14) and rat ROS (9-25-14) . The effect of engulfing human upper and lower ROS is better than that of ROS in rats. The quantification of human ROS phagocytosis is shown below (see Table 7-2).

Table 7-2. Quantification of human ROS phagocytosis

VIII. Human 15-02-032 RPE phagocytosis test of frozen ND04760 human ROS

The ND04760 human ROS (9/18/14) (10 × 10 ⁶ each) frozen on the top and bottom bands as they were or frozen with cryopreservation medium were tested. The test is for human RPE (15- 02-032) 8h phagocytosis efficiency. All frozen ROS samples were effectively phagocytosed. These samples have been stored for about 6 months. The quantification of human ROS phagocytosis is shown below (see Table 7-3).

IX. Human ( 15-02-032 ) and rat RPE phagocytosis of human ROS and rat ROS

Use 15-02-032 human RPE and N rat RPE (1/23/15) cultures for 8h of human ROS (15-02-032, 2/13/15, 5 × 10 ^{6 each} ) and Phagocytosis test of rat ROS (1/27/15, each 10 × 10 ⁶ ). Both cells showed low phagocytic levels for rat ROS, but both showed good phagocytosis of human ROS, so it confirmed that human ROS is more efficient than previously observed rat ROS. Quantification was performed on human 15-02-032 results (see Table 7-4).

X. 15-02-032 Cytokeratin immunostaining of human RPE

15-02-032 human RPE (day 24) in culture was immunostained with Pan-keratin (C11) monoclonal antibody (Cell Signaling) (1: 400) to confirm that it was RPE cells (see Figure 21 ). Secondary Antibody System Alexa 488 conjugated anti-mouse IgG antibody. The control group was not treated with primary antibody. Good staining of RPE cells was obtained, while the control group was not stained, which confirmed that the cell type was RPE.

XI. Human 15-03-027 and rat RPE phagocytosis of human ROS and rat ROS

Human 15-03-027 RPE cultures (1 'day 12) and rat N RPE cultures (2/15/15) in a 24-well dish were subjected to rat ROS (1/27/15, 8 × 10 ⁶ phagocytosis test for 10 × 10 ^{6 each} ) and human ROS (15-03-027, 3/12/15, 5 × 10 ^{6 each} ). Note: 1 'means the first generation, 2' means the second generation, and so on. Good phagocytosis was observed above. Both human RPE and rat RPE have higher levels of phagocytosis of human ROS than rat ROS. The test results are shown in Table 7-5 below.

XII. Human 15-03-027 RPE culture subculture

The primary 15-03-027 RPE culture of multiple wells was collected on day 14 and subcultured into the wells and observed. The 2 &apos; cells grew well and had grown up on the 15th day, and were ready to be used in experiments. After the cell has reached fullness, proceed to its sub-generation and proceed to the 5 'sub-generation stage.

XIII. 3 ' human RPE 15-03-027 phagocytosis of ROS in rats, humans, and pigs

In order to test the ability of phagocytosis of subcultured human RPE and the efficiency of different ROS in phagocytosis, the 20-day 3 'culture of human RPE 15-03-027 was used to treat rats (4/24/15 5 × 10 ⁶ ), humans (15-03-027, 3/12/15, each 5 × 10 ⁶ ; and 15-04-001, 4/3/15, each 5 × 10 ⁶ ), and pigs (3/26/15, each 20 × 10 ⁶ ) ROS were phagocytosed for 8 h. The test results are shown in Table 7-6.

All samples showed good phagocytosis; except for human ROS (15-04-001), the results did not perform as expected. The phagocytosis level of the 3 'human RPE after successive generations is within the same range as the phagocytosis level of the first generation RPE in the previous similar tests. The relative phagocytosis efficiency of different ROS. The result is the same as before. Again, human ROS showed higher phagocytosis levels than rat ROS and porcine ROS. A consistent pattern has been observed with regard to the phagocytosis levels of different ROS preparations and the primary and secondary human RPE.

XIV. 2 ' human RPE 15-03-027 phagocytosis of ROS in rats, humans, and pigs

In order to test the ability of phagocytosis of subcultured human RPE and the efficiency of different ROS in phagocytosis, the 21-day 2 'culture of human RPE 15-03-027 was used to treat rats (4/24/15, each 7 × 10 ⁶ ), humans (15-03-027, 3/12/15, each 5 × 10 ⁶ ; and 15-04-001, 4/3/15, each 5 × 10 ⁶ ), and pigs (3/26/15, each 20 × 10 ⁶ ) ROS were phagocytosed for 8 h. The test results are shown in Table 7-7.

Good phagocytosis was observed in all samples, and the results were very similar to the results of 3'15-03-027 cells in Example 5. Therefore, the phagocytic level observed for the 2 'human 15-03-027 RPE culture was again within the same range as the phagocytic level observed for the 3' and primary human RPE, and the ROS of rats, humans, and pigs were phagocytic The relative efficiencies in Xia are again as before; that is, human ROS are superior to mouse and pig ROS. Porcine ROS showed the lowest efficiency in human RPE phagocytosis. Therefore, consistency was again observed in the phagocytosis efficiency of the first generation of human RPE and different ROS preparations. The level of phagocytosis between the first generation and subsequent generations of human RPE does not seem to change much. Human ROS seems to be the best substrate for human RPE.

XV. 3 ' human RPE 15-03-027 phagocytosis of ROS in rats, humans, and pigs

In order to test the ability of phagocytosis of subcultured human RPE and the efficiency of different ROS in phagocytosis, the 40-day 3 'culture of human RPE 15-03-027 was used to treat rats (4/24/15, respectively 7 × 10 ⁶ ), humans (15-03-027, 3/12/15, each 5 × 10 ⁶ ; and 15-04-001, 4/3/15, each 5 × 10 ⁶ ), and pigs (3/26/15, each 20 × 10 ⁶ ) ROS were phagocytosed for 8 h. The test results are shown in Table 7-8.

The results are similar to those obtained at 20 days 3'15-03-027 human RPE, except that the level of phagocytosis against rat ROS is higher. However, it maintains the basic relative relationship of 3 different types of ROS, of which human ROS is still the best compared to rat ROS and porcine ROS in terms of human 15-03-027 RPE phagocytosis. Pig ROS is again the worst. The phagocytic level of these 3'RPEs is also in the same range as the previous 3 'cells.

XVI. 3'15-03-027 Phagocytosis of frozen human ROS preparation by human RPE

Freezing of human RPE ( 2 ' , 3' , 15-03-027 ). Collect multiple wells of passaged 15-03-027 human RPE cells (2 'on day 23, 3' on day 42, approximately 10 ^{6 each} ), and resuspend in 1 ml of cryopreservation medium in. Divide the sample into two, gradually cool down, and store in liquid nitrogen. The viability and functionality of these frozen storage cells will be tested later.

Regeneration of frozen human RPE (7/22/15) . Part of 2 'and 3' human 15-03-027 RPE cells frozen and stored from 5/21/15 were thawed and allowed to re-grow. Cell regeneration takes approximately 3 weeks.

To test the ability of frozen human ROS preparations for phagocytosis, frozen samples of various ROS preparations were thawed and used for phagocytosis assays. The frozen ROS preparations tested include: 1.NDRI # 1 04760 ROS (9/18/14), re-frozen on 3/10/15; 2.15-02-032 human ROS (2/13/15), 3/6 / 15 frozen; 3.15-03-027 human ROS (3/12/15), 3/24/15 frozen; 4.15-04-001 human ROS (4/3/15), 4/27/15 frozen. After thawing, 5 × 10 ⁶ ROS were used each for phagocytosis of 15-03-027 3 ′ (day 103) human RPE cultures for 8 hours. The test results are shown in Table 7-9.

All frozen and thawed human ROS preparations showed good phagocytosis levels, which were within the expected range. Therefore, frozen and thawed human ROS preparations seem completely acceptable for phagocytosis experiments.

XVII. Human RPE 15-04-001 phagocytosis of ROS in rats, humans, and pigs

To confirm the ability of 15-04-001 human RPE to engulf, and to test the efficiency of human RPE to engulf ROS in rats, humans, and pigs, 17-04-001 human RPE 17-day cultures were used for 5 × 10 ⁶ rat ROS (4/24/15), 15-03-027 human ROS (3/12/15), 15-04-001 human ROS (4/3/15), and 20 × 10 ⁶ swine ROS (3/26/15) 8h phagocytosis test. This experiment was also carried out on the day 32 culture of the primary 15-04-001 human RPE and the day 55 culture of the primary 15-04-001 human RPE. The test results are shown in Table 7-10 (17-day culture), Table 7-11 (32-day culture), and Table 7-12 (55-day culture).

Results of the 17- day culture. The expected phagocytosis pattern was observed. The phagocytic levels for the three different types of ROS are in the same range as previously observed. Again, human ROS showed the best phagocytosis, while rat ROS was about 50% of human, and porcine ROS showed the lowest activity. Therefore, consistency is maintained.

The results of the 32- day culture. It has no effect on 15-04-001 phagocytosis of human ROS. The remaining samples showed good phagocytosis, and the expected phagocytosis pattern was as previously shown in terms of the phagocytic level range and relative efficiency of ROS in rats, humans, and pigs. In terms of the phagocytosis patterns that appeared, the differences between the cultures of day 17 and day 32 of the primary human RPE did not seem to differ much.

Results of the 55- day culture. Results similar to the primary RPE cultures on days 17 and 32 of 15-04-001 were obtained, which confirmed a consistent pattern in terms of phagocytosis level, relative efficiency of different ROS preparations, and equivalence of cells in different culture periods.

XVIII. Human RPE 15-04-001 phagocytosis of ROS in rats, humans, and pigs (collected on day 30 ( 2 ' ) culture)

In order to check the phagocytosis efficiency of different generations of human RPE, the above experiment was repeated using the 30th day culture of the next generation 15-04-001 human RPE (see Table 7-13 below). The phagocytosis pattern obtained with the secondary culture was basically the same as that seen in the primary RPE culture of 15-04-001, which confirmed a consistent pattern in terms of phagocytosis level and the relative efficiency of different ROS preparations. 15-04-001 cells exhibited equal phagocytosis of different passages of human RPE culture.

XIX. 2'15-04-001 phagocytosis of frozen human ROS preparation by human RPE

To test the ability of frozen human ROS preparations for phagocytosis, frozen samples of various human ROS preparations were thawed and used for phagocytosis assays. The frozen ROS preparations tested include: 1.NDRI # 1 04760 ROS (9/18/14), re-frozen on 3/10/15; 2.15-02-032 human ROS (2/13/15), 3/6 / 15 frozen; 3.15-03-027 human ROS (3/12/15), 3/24/15 frozen; 4.15-04-001 human ROS (4/3/15), 4/27/15 frozen. After thawing, 5 × 10 ⁶ ROS were used each for phagocytosis of 15-04-001 2 ′ (day 85) human RPE cultures for 8 hours. The same experiment was also performed using 3'15-03-027 human RPE. The test results are shown in Table 7-14.

All frozen and thawed human ROS preparations showed good phagocytosis. 15-02-032 and 15-03-027 The phagocytosis level of ROS is higher than usual, which may be because these ROS preparations show smaller particles than usual, leading to more visible uptake of ROS particles in the cells. As shown by 3'15-03-027 cells, frozen and thawed ROS preparations seem to be completely acceptable for phagocytosis experiments.

XX. Human 15-04-001 RPE phagocytosis test of 15-11-098 ROS upper, middle and lower bands

In order to check the phagocytosis efficiency of different preparations of human ROS (different bands in sucrose gradient centrifugation), 15-11-098 ROS upper band, middle band, and lower band are for 3 'day 44 and 4' day 42 15- 04-001 RPE phagocytosis has been tested. The test results are shown in Table 7-15.

The level of phagocytosis obtained with mid-band ROS is roughly within the normal range. However, the level of phagocytosis obtained with top band ROS is higher than others. Again, the reason may be that the upper band is rich in smaller ROS particles; it causes the concentration to be underestimated because they are more difficult to visualize in bright fields and there are more visible particles in the cells under fluorescence. The level obtained in the lower band is somewhat variable, and the results of other RPE cultures demonstrate this, as shown below. The variability of the total phagocytosis level may exist, depending on the nature of the ROS particles, but consistency can be obtained by using the same ROS preparation across samples.

XXI. Effect of different cleaning agents on the phagocytosis of 15-07-072 RPE and ROS (8/4/15)

To test the effect of different types of cleaning on human phagocytosis of human RPE that may be contaminated, 15-07-072 primary RPE cultures (suspicion of possible bacterial contamination) were washed with Betadine for 3 minutes or with PBS containing 2x Pen-Strep 5 minutes, and allowed to phagocytose 15-07-072 ROS (7/28/15) for 8x each of 5 × 10 ⁶ upper or lower bands. The test results are shown in Table 7-16 (upper band) or Table 7-17 (lower band).

The second repeat of this experiment was also conducted. The results of this test are shown in Table 7-18 (upper belt and lower belt (second experiment)).

Washing with Betadine seemed too severe for the cells, while washing with PBS containing 2x P-S did not harm the cells, and the cells showed the expected level of phagocytosis. 2x P-S cleaning also seems to eliminate bacterial contamination in the culture.

Table 7-17. Effect of different cleaning agents on phagocytosis (lower band)

XXII. Human 15-07-072 phagocytosis test of 2 ' and 3' RPE cultures

To compare the phagocytic levels of human 2 'and 3' RPE (the latter concentration is 50k or less), the 15-07-072 2 'and 3' (50k and less than 50k per culture) RPE cultures were paired to 2.5 × 10 ⁶ 8-hour phagocytosis tests with ROS on 15-07-072. The test results are shown in Table 7-19.

The expected level of phagocytosis was observed in all 3 samples, which proved the equality of the 2 'and 3' cultures, and also proved the equality of the cultures with a concentration of 50k and less than 50k.

XXIII. Human 15-07-072 RPE phagocytosis test of 15-11-098 ROS upper, middle and lower bands

In order to check the phagocytosis efficiency of different preparations of human ROS (different bands in sucrose gradient centrifugation), the 15-11-098 ROS upper, middle, and lower bands were targeted at 5 'day 62 and 6' day 24 07-072 RPE phagocytosis has been tested. The test results are shown in Table 7-20.

As previously shown, the level of phagocytosis obtained with mid-band ROS is roughly within the normal range. The level of phagocytosis obtained with top band ROS is again higher than the others. (Again, the reason is most likely because the upper zone is rich in smaller ROS particles.) In the case of using 15-07-072 cells, the phagocytosis level obtained with the lower zone is higher than expected, which reflects The variability of this band mentioned earlier.

XXIV. Normal human primary RPE and SD1 "dry" AMD primary RPE phagocytosis test

At this time, the primary culture of SD1 AMD RPE is rapidly deteriorating, so the final phagocytosis test is performed, which compares two normal human primary RPE cultures (15-09-027, 1 ', day 41; 15-08- 074, 1 ', day 62) with San Diego 1 "dry" AMD primary RPE cultures (L, 1', day 48; R, 1 ', day 48) for 5 × 10 ⁶ humans each The phagocytosis level of ROS (15-10-021, top) is 8 hours. The test results are shown in Table 7-21.

The same reduction in the phagocytic level of the SD1 AMD primary RPE compared to normal human primary RPE was observed, which confirmed that the AMD RPE phagocytic level was reduced. Both R and L AMD RPE showed a decrease compared to the two normal cells (15-09-027 and 15-08-074) at this time, and as previously appeared, the level of reduction was 60% Digits.

XXV. San Diego 1 "Dry" AMD RPE Cytokeratin Immunostaining

The SD1 AMD L 5 'RPE (day 69) in the culture was immunostained with Pan-keratin (C11) monoclonal antibody (Cell Signaling) (1: 400) to confirm that these AMD cells were RPE cells. Secondary Antibody System Alexa 488 conjugated anti-mouse IgG antibody. The control group was not treated with primary antibody. Good staining of RPE cells was obtained, while the control group was not stained, which confirmed that the cell type was RPE (see FIG. 22).

XXVI. Cytokeratin immunostaining of wet AMD 15-10-021 RPE

The 4'15-10-021 wet AMD RPE (day 56) in the culture was immunostained with Pan-keratin (C11) monoclonal antibody (Cell Signaling) (1: 400) to confirm these AMD cells RPE cells. Secondary Antibody System Alexa 488 conjugated anti-mouse IgG antibody. The control group was not treated with primary antibody. Good staining of RPE cells was obtained, while the control group was not stained, which confirmed that the cell type was RPE (see FIG. 23).

XXVII. ND08333 " AMD " RPE cytokeratin immunostaining

The 2'ND08333 "AMD" RPE (day 15) in the culture was immunostained with Pan-keratin (C11) monoclonal antibody (Cell Signaling) (1: 400) to confirm that these AMD cells were RPE cells. Secondary Antibody System Alexa 488 conjugated anti-mouse IgG antibody. The control group was not treated with primary antibody. Good staining of RPE cells was obtained, while the control group was not stained, which confirmed that the cell type was RPE (see FIG. 24).

XXVIII. Human 15-08-074 RPE phagocytosis test of 15-11-098 ROS upper, middle and lower bands

In order to check the phagocytosis efficiency of different preparations of human ROS (different bands in sucrose gradient centrifugation), 15-11-098 ROS upper band, middle band, and lower band are for 15 days of 4 'day 51 and 5' day 31 -08-074 The phagocytosis of RPE culture was tested. As previously shown, the level of phagocytosis obtained with mid-band ROS is roughly within the normal range. The level of phagocytosis obtained with top band ROS is again higher than the others. In the case of using 15-08-074 cells, the phagocytic level obtained with this lower band is higher than expected, which reflects the variability of this band as mentioned previously. Therefore, variability in the total phagocytosis level may exist, depending on the nature of the ROS particles, but consistency can be obtained by using the same ROS preparation across samples. The test results are shown in Table 7-22 below.

Example 8 RNA level of human RPE

The RNA levels of several human RPEs isolated in Example 6 were obtained. Specifically, these RNA levels are determined using the following procedure.

I. RNA extraction from human RPE cells

RPE cultures in 24-well dishes will be washed 3x with PBS. 1 ml Trizol (Life Technologies, catalog number 15596026) was added to the cells, and RNA was isolated according to the manufacturer's protocol. According to the manufacturer's protocol, DNAFree (Applied Biosystems, catalog number AM1906) was used to remove contaminated genomic DNA from the preparation. Using BioRad spectrophotometer or Nanodrop 2000, determine the concentration of RNA preparation.

II. Isolation of RNA from 15-03-027 RPE

RNA was isolated from a 2-well 15-03-027 RPE culture (day 13).

1. 0.15μg / μl, 260/280 = 3.04,18μl

2. 0.08μg / μl, 260/280 = 1.94,18μl

III. RNA isolation from 15-04-001 primary human RPE culture

RNA was isolated from the 3-well day 24-04-001 primary human RPE culture (in a 24-well dish).

Results:

0.05μg / μl, 260/280 = 2.50,18μl

0.18μg / μl, 260/280 = 4.21,18μl

0.13μg / μl, 260/280 = 1.58,18μl

IV. Isolation of RNA from the original 15-04-001 RPE

RNA was isolated from the primary culture of 15-04-001 human RPE (1 ', day 217).

Results:

0.04μg / μl, 260/280 = 2.48,18μl

V. RNA isolation from the first generation 15-07-072 RPE

RNA was isolated from two 15-07-072 RPE culture (at day 16) samples.

Results:

1. 0.2μg / μl, 260/280 = 1.45,18μl

2. 0.2μg / μl, 260/280 = 1.89,18μl

VI. RNA isolation from primary cultures of wet AMD 15-10-021 RPE

RNA was isolated from the primary culture on day 75 of the wet AMD 15-10-021 RPE.

Results: 1 ’, Day 75, 0.06μg / μl, 260/280 = 1.80,18μl

VII. RNA isolation from ND08626 AMD RPE

RNA was isolated from two of the following samples: (a) 1 'day 26 ND08626 AMD RPE culture; and (b) 1' day 26 ND08626 AMD RPE culture.

Results:

1 ’Day 16, 0.04μg / μl, 260/280 = 1.92,18μl

1’Day 16, 0.03μg / μl, 260/280 = 2.00,18μl

1 ’Day 26, 0.05μg / μl, 260/280 = 2.14, 18μl

Day 26 on 1 ’, 0.08μg / μl, 260/280 = 1.89,18μl

VIII. Isolation of RNA from 15-08-074 1 'RPE

RNA was isolated from 2-well 15-08-074 1 'day 17 RPE culture.

Results:

1. 0.17μg / μl, 260/280 = 1.78,18μl

2. 0.15μg / μl, 260/280 = 2.64,18μl

Example 9 ROS phagocytosis level of human RPE separated from donors of different ages, and normal human RPE and AMD RPE I. Phagocytosis of human RPE of different ages (including 15-04-001 RPE ).

In order to compare the phagocytosis level of RPE from individuals of different ages, the human eye was 5 '(day 200) of 15-07-072 (from a 39-year-old donor), and 5 of 15-04-001 (from a 59-year-old donor) '(Day 63), 15-08-074 (from a 61-year-old donor) 5' (Day 48), 15-11-098 (71-year-old) 4 '(Day 20), and 15-09 -027 (from a 79-year-old donor) 5 '(day 82) of normal RPE was performed on the upper, middle, and lower ROS preparations from the human eye 15-11-098 (12/2/15, each It is 5 × 10 ⁵ ) for 8 hours. Roughly, 50,000 cells / well were seeded in a 24-well dish. For additional comparison, the results of phagocytosis experiments from RPE isolated earlier from human eye 15-02-032 (from a 31-year-old donor) were also included. (Note: 5 'means the 5th generation) The test results are shown in Figure 25A and Figure 25B. The data shows that the phagocytic function of human RPE decreases with age (especially after age 60).

II. Comparison of normal RPE and AMD ( SD1 L & R ("dry"), 15-10-021 ("wet")) RPE phagocytosis of ROS on 15-10-021

To compare the phagocytic levels of AMD and normal RPE, San Diego 1 AMD left 4 'day 20 and right 3' day 11, 15-10-021 "wet" AMD 3 'day 18, 15-09-027 N 3 'Day 31 and 4' Day 19, 15-08-074 N 3 'Day 41 and 4' Day 34, 15-07-072 N 5 'Day 45, and 15-04-001 N 8 hours phagocytosis test was carried out on 15-10-021 with 2 × 106 RPEs at 1 ′ day 209, 3 ′ day 27, and 4 ′ day 25. As can be seen from FIG. 26, the phagocytic level of all AMD RPEs is significantly lower than that of normal RPE. (The phagocytosis of 15-04-001 1 ’has no effect. This may be due to the state of the cells in the culture, because these cells have reached senescence and stop phagocytosis.)

III. Engulfing of ND08333 AMD and normal RPE

In order to compare the phagocytic levels of AMD ND08333 and normal RPE, the RPE of ND08333 AMD 3 'day 42, 15-09-027 normal 5' day 96, and 15-08-074 normal 5 'day 62 5 × 10 ⁵ 8-11 hours phagocytosis test with ROS on 15-11-098. The phagocytosis level of AMD ND08333 RPE is much lower than the normal (15-09-027 and 15-08-074) RPE phagocytosis level, which again confirms the observed pattern.

In order to compare the phagocytic levels of AMD ND08333 and normal RPE again, the RPE of ND08333 AMD 3 'day 43, 15-04-001 normal 5' day 78, and 15-07-072 normal 6 'day 115 were compared. 5 × 105 8-hour phagocytosis test with ROS on 15-11-098. The reduced phagocytosis level of AMD ND08333 compared to normal (15-04-001 and 15-07-072) RPE has been confirmed again.

IV. Phagocytosis test of AMD ND08626 and normal RPE

In order to compare the phagocytosis level of AMD ND08626 and normal RPE, the RPE of ND08626 AMD 1 'day 17, 15-09-027 N 5' day 88, and 15-08-074 N 5 'day 55 were compared 5 × 10 ⁵ 8-11 hours phagocytosis test with ROS on 15-11-098. The phagocytosis level of AMD ND08626 RPE is significantly lower than that of normal RPE (15-09-027 and 15-08-074), which confirms the pattern observed consistently.

In order to compare the phagocytosis levels of AMD ND08626 and normal RPE again, the RPE of ND08626 AMD 1 'day 17 and 15-11-098 N 3' day 40 and 4 'day 24 were 5 × 10 ⁵ each 8-hour phagocytosis test with ROS on 15-11-098. The phagocytosis level of AMD ND08626 RPE is much lower than the normal phagocytosis level of 15-11-098 RPE, which again confirms the pattern observed consistently.

In order to compare the phagocytosis levels of AMD ND08626 and normal RPE again, the RPE of ND08626 AMD 1 'day 24, 15-04-001 N 5' day 77, and 15-07-072 N 6 'day 123 were compared It is an 8-hour phagocytosis test with 5 × 10 ⁵ 15-11-098 with ROS. The phagocytosis level of AMD ND08626 RPE is much lower than the normal phagocytosis levels of 15-04-001 and 15-07-072 RPE, which again confirms the pattern observed consistently.

In order to compare the phagocytosis level of AMD ND08626 and normal RPE again, the RPE of ND08626 AMD 1 'day 25, and 15-08-074 N 4' day 111 and 6 'day 82 were 5 × 10 ⁵ each 8-hour phagocytosis test with ROS on 15-11-098. The phagocytosis level of AMD ND08626 RPE is again much lower than the normal 15-08-074 RPE phagocytosis level, which again confirms the pattern observed consistently.

V. Human 15-08-074 1'RPE engulfed 15-08-074 ROS

Two-well human 15-08-074 1 'day 24 RPE cultures were subjected to an 8-hour phagocytosis test for 5 × 10 ⁶ each of 15-08-074 upper and lower band ROS preparations. Phagocytosis is observed, but the level is low; it may be due to very small ROS particles, which are difficult to visualize and count, resulting in missed counts.

VI. 15-08-074 ROS phagocytosis test in different human RPE cultures (including 15-08-074 RPE )

In order to test the phagocytosis efficiency of 15-08-074 upper and lower band ROS preparations (8/25/15), 15-03-027 5 'freezing / regeneration, 15-07-072 3' day 43, 15- 04-001 2 'Day 134 and 4' Freeze / Regeneration, and 15-08-074 1 'Day 15 RPE cultures were phagocytosed for ROS preparations. Engulfing was observed, but the level was low. (Very small ROS particles are difficult to visualize and count.)

VII. Comparison of phagocytosis of human RPE cultures of different passages

In order to compare the phagocytosis levels of different generations of human RPE cultures, 15-08-074 2 ′ day 46, 3 ′ day 20, and 4 ′ day 13 RPE were performed for 2 × 10 ⁶ 15-10-021 Engulfed with ROS on humans. Equal phagocytic levels were observed in RPE cultures of different passages, which confirmed the equality of human RPE cultures of different passages.

VIII. Normal RPE and AMD (SD1 L, 15-10-021 "wet")) RPE phagocytosis comparison with the ROS on 15-09-027

To compare AMD RPE (San Diego 1 L wet (no longer R) 4 'day 21; 15-10-021 wet 3' day 19) with normal RPE (15-09-027 3 'day 32 , 4 '20th day; 15-08-074 3' 42nd day, 4 '35th day; 15-07-072 5' 46th day; 15-04-001 1 '210th day, 3' 28th Day, 4 'Day 26) phagocytosis level, RPE cultures were used for 8h phagocytosis test, fixation and analysis of 2 × 10 ⁶ ROS on 15-09-027. As previously shown, the phagocytosis level of AMD RPE is lower than that of all normal RPE.

IX. Comparison of phagocytosis between normal ( 15-11-098 ) and AMD ( SD1 L , 15-10-021 ) RPE (with and without CM treatment)

In order to compare normal (15-11-098 1 'day 48, 2' day 12) and AMD (San Diego 1 L 5 'day 60; 15-10-021 wet 5' day 40) RPE (after And the phagocytosis level without CM11 treatment for 24 hours), so that the RPE cultures were each subjected to 8x phagocytosis assay, fixation, and analysis on 1 × 10 ⁶ ROS of 15-11-098 medium.

The phagocytic levels of the two AMD RPEs (SD1 L, 15-10-021 wetness) are much lower than the phagocytic levels of normal RPE (15-11-098). CM11 treatment basically normalized the reduced phagocytosis level of AMD RPE. The results of both confirm the previously observed situation.

X. 15-08-074 RPE human ROS dose response test

In order to determine the optimal amount of ROS to be used for the phagocytosis test of 15-11-098 ROS preparations (upper band, middle band, lower band), 15-08-074 5 'day 43 RPE cultures were tested for 1 15-15-098 ROS preparation phagocytosis test for × 10 ⁵ , 5 × 10 ⁵ , and 1 × 10 ⁶ upper, middle, and lower bands. The absolute phagocytosis level reflects the above results, that is, the upper and lower bands are higher, and the middle band is lower. The expected dose response to different amounts of individual ROS was observed, which indicated that 5 × 10 ⁵ could be a suitable dosage for this ROS preparation. The results of the dose response test are shown in Figure 27.

XI. 15-11-098 RPE human ROS dose response test

In order to determine the optimal amount of ROS for the phagocytosis test of the 15-11-098 ROS preparation (upper band, middle band, lower band), the 15-11-098 4 'day 14 RPE cultures were tested for 1 15-15-098 ROS preparation phagocytosis test for × 10 ⁵ , 5 × 10 ⁵ , and 1 × 10 ⁶ upper, middle, and lower bands. The absolute level of phagocytosis reflects the above results of 15-09-027, that is, the upper and lower bands are higher, and the middle band is lower. The expected dose response for different amounts of individual ROS was observed, indicating that 5 × 10 ⁵ may be a suitable dosage for this ROS preparation (U, L). The results of the dose response test are shown in Figure 28.

XII. Human 15-09-027 RPE phagocytosis test of 15-11-098 ROS upper, middle and lower bands

In order to check the phagocytosis efficiency of different preparations of human ROS (different bands in sucrose gradient centrifugation), the upper, middle, and lower bands of 15-11-098 ROS were directed at 3'day 48, 4'day 36, and 5 'Day 7 15-09-027 RPE cultures were tested for phagocytosis. The test results are shown in Table 9-1. As previously shown, the level of phagocytosis obtained with mid-band ROS is roughly within the normal range. The level of phagocytosis obtained with top band ROS is again higher than the others. (Again, the reason is most likely because the upper band is rich in smaller ROS particles.) In the case of using 15-09-027 cells, the phagocytosis level obtained with the lower band was higher than expected, which reflects The variability of this band mentioned earlier. The variability of the total phagocytosis level may exist, depending on the nature of the ROS particles, but consistency can be obtained by using the same ROS preparation across samples.

Even when compared to normal age-matched normal RPE, various phagocytosis tests from RPE from AMD and non-AMD patients in this example showed reduced phagocytosis of AMD RPE.

Example 10 Effect of hUTC conditioned medium (CM) added to AMD RPE on phagocytosis

The effect of hUTC conditioned medium added to AMD RPE on phagocytosis has been evaluated.

I. hUTC conditioned medium ( CM ) preparation

HUTC CM 5 (5th preparation: CM5 10k and CM5 11k), CM 10 (10th preparation), CM11 (11th preparation), CM13 (13th preparation), CM14 and CM15 (CM14 and CM15 in one preparation (Preparation) for experiment. Use conditioned medium and control medium. To prepare CM5, hUTC was inoculated into hUTC growth medium (DMEM low) in T225 cell culture flasks at 10,000 viable cells / cm ² (for CM5 10k) or 11,000 viable cells / cm ² (for CM5 11k) on day 1. Glucose + 15% FBS + 4mM L-glutamic acid). To prepare CM10, CM11, CM13, CM14, and CM15, hUTC was inoculated into hUTC growth medium in T75 cell culture flasks at 10,000 viable cells / cm ² on day 1. After the cells were seeded, they were cultured in a 5% CO ₂ incubator at 37 ° C for 24 hours. On the second day, aspirate the medium and supplement with 63mL / T225 bottles of DMEM / F12 complete medium (DMEM: F12 medium + 10% FBS + Pen (50 U / ml) / Strep (50 μg / ml)) , In the case of CM10, CM11 supplemented with 20mL / T75 bottles of DMEM / F12 complete medium, and in the case of CM13 supplemented with 21mL / T75 bottles of DMEM / F12 complete medium. The cells were incubated for another 48 hours. A separate control medium (DMEM: F12 medium + 10% FBS + Pen (50U / ml) / Strep (50 μg / ml)) was also cultured for 48h. On the fourth day, the cell culture supernatant and control medium were collected and centrifuged at 250g for 5 min at 4 ° C, then aliquoted in 1.8mL cryovials at 1mL / tube, and immediately frozen at -70 ° C refrigerator.

II. Normal 1 ' and San Diego AMD 1' RPE (with and without CM treatment) phagocytosis test

To compare the phagocytic capacity of normal human primary RPE and AMD ("dry") primary RPE (with and without CM treatment), San Diego # 1 "dry AMD" primary RPE culture (L, day 19; R, day 19 days), normal human 15-09-027 primary RPE culture (day 21), and normal human 15-08-074 primary RPE culture (day 42) (overnight with and without CM10 treatment) 8 hours phagocytosis test for 5 × 10 ⁶ human 15-09-027 ROS. The test results are shown in Table 10-1 below.

Both L and R AMD RPE (particularly R) showed lower phagocytic levels than normal (15-09-027), and CM10 treatment increased the phagocytic levels of all others (including normal RPE). 15-08-074 The phagocytosis of normal subjects has no effect. Overall, the intake level of 15-08-074 is abnormally low, which indicates that this RPE culture cannot be phagocytosed.

III. Phagocytosis test and RNA isolation of normal 1 ' and SD1 1' RPE (with / without CM treatment)

Perform experiments to characterize normal human primary RPE (15-09-027, 1 ', day 29) and San Diego # 1 "dry" AMD primary RPE (L, 1', day 27; R, 1 ', day 27 Days) (with and without CM10 treatment for 24 hours), and RNA was also isolated from each sample. For phagocytosis, 5 × 10 ⁶ human ROS (15-09-027, top tape) were used.

The RPE culture flakes during the experiment, thus destroying the phagocytosis assay. However, RNA was still isolated from some of the samples as follows: normal 15-09-027 RPE, 0.18 μg / μl, 260/280 =? , 18μl

Normal 15-09-027 RPE, treated with CM10 for 24h, 0.15μg / μl, 260/280 = 6.59, 18μl

SD1 "dry" AMD RPE, L, 0.1 μg / μl, 260/280 =? , 18μl

SD1 "dry" AMD RPE, L, treated with CM10 for 24h, 0.1μg / μl, 260/280 =? , 18μl

Some question marks after the 260/280 measurement indicate that the BioRad spectrophotometer failed when measuring these samples.

IV. Phagocytosis test and RNA isolation of normal 1 ' and SD1 1' RPE (with / without CM treatment)

Basically, the same experiment as above was performed, except that all primary cultures were 1 day old, and the CM treatment was 6 hours. Since there were not enough primary cultures of SD1 R RPE cells, RNA was not isolated from these cells.

These samples suffered the same situation as in the previous experiment, that is, the flaking of the culture, thus ruining the phagocytosis test. Nonetheless, RNA isolation of these samples is still performed. The results are as follows: normal 15-09-027 RPE, 0.08μg / μl, 260/280 = 1.80, 18μl

Normal 15-09-027 RPE, treated with CM10 for 6h, 0.3μg / μl, 260/280 = 1.61,18μl

SD1 "Dry" AMD RPE, L, 0.18μg / μl, 260/280 = 2.38, 18μl

SD1 "dry" AMD RPE, L, treated with CM10 for 6h, 0.1μg / μl, 260/280 = 1.47,18μl

    V. Phagocytosis test of normal 15-09-027 and 15-08-074 and wet AMD 15-10-021 RPE (with / without CM treatment for 24h), and RNA isolation from AMD cells    

To compare the phagocytic level of wet AMD with normal human RPE (with and without CM treatment for 24 hours), and to isolate RNA from wet AMD cells, the first 14 days of "wet" AMD 15-10-021 RPE, The first generation 37 days normal 15-09-027 RPE, and the second generation 35 days and third generation 9 days normal 15-08-074 RPE (with and without CM10 addition) were performed on 15-09-027 of 5 × 10 ⁶ each 8 hours phagocytosis test with ROS. RNA was also isolated from "wet" AMD 15-10-021 RPE (also added with and without CM10). The results of the phagocytosis test are shown in Table 10-2.

Many samples experienced flaking during the experiment, so that only the "wet" AMD 15-10-021 (with and without CM) and normal 15-08-074 3 '(without CM addition) survived and could be analyzed. However, these results show that AMD RPE once again showed a lower phagocytosis level than normal (as shown by San Diego's "dry" AMD RPE), and that it was "reverted" to normal levels by adding CM10.

RNA isolated from 15-10-021 wet AMD RPE (with and without CM10 treatment for 24 hours) is as follows: those without CM10 treatment, 0.08 μg / μl, 260/280 = 1.69, 18 μl

CM10 treated for 24 hours, 0.02μg / μl, 260/280 = 0.36,18μl

VI. " Humid " AMD 15-10-021 RPE (with / without CM6 hours) compared with normal 15-08-074 1 ' , 2' , 3 ' RPE phagocytosis, and RNA isolation from AMD cells

In order to compare the phagocytosis level of "wet" AMD RPE and normal RPE, the "wet" AMD 15-10-021 RPE (with and without CM10 treatment for 6 hours) on day 19 of 1 'and normal 15-08-074 RPE cultures of 1'day 63, 2'day 40, and 3'day 14 were subjected to an 8h phagocytosis test for 15-10-021 ROS of 2 × 10 ⁶ each. RNA was also isolated from AMD RPE with and without CM10 treatment. The results of the phagocytosis test are shown in Table 10-3.

As previously observed, approximately equal phagocytic levels were observed in 1 ', 2', and 3 '15-08-074 RPE, which confirmed the equivalence of different subcultures of RPE. The absolute count is very high, which may be attributed to the 15-10-021 ROS preparation which is mainly composed of very small ROS particles. (Small ROS particles seem to cause excessive phagocytosis and high variability, which is because it is difficult to visualize small ROS particles.) It may be attributed to this. In this experiment, the "wet" AMD 15-10-021 RPE phagocytosis level was only Those slightly lower than normal are in contrast to the significantly lower levels observed many times. Adding CM10 to AMD RPE increased the phagocytosis level as previously appeared, while maintaining the observed pattern.

RNA isolated from wet AMD 15-10-021 RPE with and without CM10 treatment for 6 hours: 0.2μg / μl, 260/280 = 1.43,18μl without CM10 treatment

After treated by CM10 for 6h, 0.08μg / μl, 260/280 = 1.19,18μl

VII. AMD RPE and normal RPE (with or without CM ) phagocytosis of ROS on human eye 15-09-027 (including human eye 15-04-001 RPE )

In order to compare the phagocytic level of AMD and normal (N) RPE, and to check the effect of treatment with hUTC CM, the following RPE (with and without CM11 treatment) were subjected to several ROS preparations (in this case 15-09- 027 with ROS preparation) 8 hours phagocytosis test: San Diego 1 AMD left 4 '(day 21); 15-10-021 "wet" AMD 3' (day 19); 15-09-027 N 3 '(Day 19) and 4' (Day 20); 15-08-074 N 3 '(Day 42), 4' (Day 35), and 5 '(Day 15); 15-07- 072 N 5 '(day 46); and 15-04-001 N 2' (day 210), 3 '(day 28), and 4' (day 26).

Comparison of the phagocytosis levels of 2 AMD RPEs and 8 normal RPEs (15-04-001, 4 'seems to have no effect) without CM treatment showed that the phagocytosis levels of RNA isolated from the eyes of two AMD donors were significant It is lower than the average level of phagocytosis of RNA isolated from the eyes of eight normal donors. This is consistent with the observation that AMD RPE reduces phagocytosis compared to normal RPE. When comparing the AMD RPE phagocytosis level with the age-matched normal RPE, a significant reduction was observed in the 15-10-021 "wet" AMD sample, but not in the SD1 AMD left sample (see bottom calculation ). The test results are shown in Figure 29.

The comparison of the phagocytic levels of all samples (with and without CM treatment) showed that the phagocytic levels after hUTC CM treatment increased significantly. In AMD samples, phagocytosis decreased more than normalized after CM treatment. Even in normal samples, hUTC CM treatment promoted its phagocytic level (except for one sample (15-07-072, 5 ')). This is also consistent with the pattern of CM treatment increasing human RPE phagocytosis.

VIII. AMD RPE and normal RPE (with or without hUTC CM treatment) phagocytosis of ROS (including 15-04-001 RPE ) on 15-10-021

Repeat the previous experiment using RPE cells from another donor. In order to compare the phagocytic levels of AMD and N RPE, and to check the effect of treatment with hUTC CM, the following RPE (with and without CM11 treatment) were subjected to several ROS preparations (in this case 15-10-021 on the belt ROS preparation) 8 hours phagocytosis test: San Diego 1 AMD left 4 '(day 21); 15-10-021 "wet" AMD 3' (day 19); 15-09-027 N 3 '(day 19 days) and 4 '(day 20); 15-08-074 N 3' (day 42), 4 '(day 35), and 5' (day 15); 15-07-072 N 5 '(Day 46); and 15-04-001 N 2' (day 210), 3 '(day 28), and 4' (day 26).

The same results as the same experiment performed with the ROS preparation were obtained. When examining the results without hUTC CM treatment, the phagocytosis levels of the two AMD RPEs (ie, SD1 "Dry AMD" L and 15-10-021 "Wet" AMD) were significantly lower than the average of all 9 normal RPEs. This is again consistent with the observed pattern. When comparing the AMD RPE phagocytosis level with the age-matched normal samples, both AMD samples showed significantly lower phagocytosis levels compared to the normal samples. CM11 treatment increased the phagocytic level of all RPEs (including AMD RPE) (data not shown). After CM11 treatment, the reduced phagocytosis level in AMD RPE was basically normalized. This is again consistent with the observed pattern.

IX. AMD RPE and normal RPE (with or without hUTC CM ) phagocytosis of SD1 with ROS (including 15-04-001 RPE )

Repeat the previous experiment with RPE cells from the third donor. In order to compare the phagocytosis level of AMD and normal (N) RPE, and to check the effect of treatment with CM, the following RPE (with and without CM11 treatment) were subjected to several ROS preparations (in this case, SD1 with ROS preparations) ) Of 8 hours phagocytosis test: San Diego 1 AMD left 4 '(day 21); 15-10-021 "wet" AMD 3' (day 19); 15-09-027 N 3 '(day 19) ) And 4 '(day 20); 15-08-074 N 4' (day 35), and 5 '(day 15); 15-07-072 N 5' (day 46); and 15- 04-001 N 3 '(Day 28).

The same results as previous experiments performed with other ROS preparations were obtained. When examining the results without CM treatment, the phagocytic levels of the two AMD RPEs (ie, SD1 "Dry AMD" L and 15-10-021 "Wet" AMD) were significantly lower than the average phagocytic levels of all six normal RPE ( Data not shown). This is again consistent with the observed pattern. When comparing the AMD RPE phagocytosis level with the age-matched normal samples, both AMD samples significantly reduced the phagocytosis level compared to the normal samples.

CM11 treatment increased the phagocytic level of all RPEs (including AMD RPE) (data not shown). After CM11 treatment, the reduced phagocytosis level in AMD RPE was basically normalized. This is again consistent with the pattern.

X. ND08333 AMD and N 15-11-098 RPE (with / without CM ) phagocytosis ( 1/21/16 )

In order to compare the phagocytosis level of RPE between AMD (ND08333) and normal (15-11-098) (with and without CM treatment), ND08333 AMD 1 'day 34 and 2' day 8 and normal 15-11- 098 2 'Day 14 RPE (with and without CM11 treatment) was tested for 8 hours phagocytosis with ROS in 15-11-098 of 2 × 10 ⁶ each. Again, as seen previously, the phagocytosis level of AMD ND08333 RPE is lower than the normal 15-11-098 RPE. CM11 has a stimulating effect on all RPE, but its effect on AMD RPE seems to be less than that previously observed.

XI. Repeated phagocytosis test of ND08333 AMD and N 15-11-098 RPE (with or without hUTC CM treatment)

In order to compare the phagocytosis level of AMD (ND08333) and normal (15-11-098) RPE again (with and without hUTC CM treatment), ND08333 AMD 2 'day 19 and 3' day 6 and normal 15- 11-098 RPE (with and without hUTC CM11 treatment) on 2 'day 25 and 3' day 11 were tested for 8 hours phagocytosis with ROS on 15-11-098 of 1 × 10 ⁶ each. The phagocytosis level of AMD ND08333 RPE is significantly lower than the normal 15-11-098 RPE (data not shown), which confirms the pattern that has emerged. The hUTC CM11 treatment had a significant stimulating effect on all RPE phagocytosis, which normalized the reduced phagocytic level in AMD RPE (data not shown).

XII. Phagocytosis test of AMD ND08626 RPE and normal RPE (with or without hUTC CM treatment)

In order to compare the phagocytosis level of AMD ND08626 and normal RPE (with and without hUTC CM treatment), the following RPE (with and without CM8 10k treatment) were performed on 15-11-098 of 5 × 10 ⁵ each 8-hour phagocytosis test with ROS: ND08626 AMD 2 'day 21 and 3' day 3; 15-11-098 N 3 'day 60 and 4' day 47; 15-09-027 N 5 'day 109 Days; 15-08-074 N 4 'day 123 and 5' day 75; and 15-04-001 N 5 'day 90. The phagocytosis level of AMD ND08626 2 ', 3' RPE is much lower than normal RPE (15-11-098 3 ', 4'; 15-09-027 5 '; 15-08-074 4', 5 '; 15-04 -001 5 '). CM8 treatment increases the phagocytosis of all RPEs, including AMD RPE that is substantially normalized. The observed pattern is maintained.

XIII. Comparison of phagocytosis of AMD (ND08626) and normal ( 15-11-098 , 15-09-027 , 15-08-074 , and 15-04-001 ) RPE (with / without CM )

In order to compare the phagocytic levels of AMD (ND08626) RPE and normal RPE (15-11-098, 15-09-027, 15-08-074, 15-04-001) (with and without CM treatment), the following 8h phagocytosis of ROS on 15-11-098 with 5 × 10 ⁵ each: ND08626 2 'Day 24 and 3' Day 6 RPE; and 15-11-098 N 3 'Day 63, 4' Day 50; 15-09-027 N 5 'day 112; 15-08-074 N 4' day 126, 5 'day 78; and 15-04-001 N 5' day 93 RPE culture (With and without CM5 10k treatment for 24h). As previously shown, the phagocytosis level of AMD ND08626 RPE is lower than the normal 15-11-098, 15-09-027, 15-08-074 and 15-04-001 RPE. As also seen before, treatment of AMD ND08626 RPE with CM5 10k will normalize the reduced phagocytosis level. Treatment of normal RPE with CM also stimulated all its phagocytic levels to a variable degree.

The test in this example confirmed that CM has a stimulating effect on both the phagocytosis of both AMD and normal RPE, which substantially normalizes the level of AMD RPE.

Example 11 Effect of adding hUTC conditioned medium (CM) on RPE RNA level

The effect of adding hUTC conditioned medium on RPE RNA levels has been evaluated.

I. RNA isolation from 4'15-04-001 human RPE (with and without CM incubation for 6 and 24h )

In order to examine the effect of CM on the gene expression of normal human RPE through RNA, 15-04-001 human RPE (4 ', day 20) cultures were treated with CM11 for 6 and 24 hours, and from these cultures and control samples Isolate RNA. Control samples were incubated in MEM5 for 6 hours.

Results:

1. Control group, 0.4μg / μl, 260/280 = 1.64,18μl

2. If treated with CM11 for 6h, 0.1μg / μl, 260/280 = 3.28, 18μl

3. For 24h treated by CM11, 0.21μg / μl, 260/280 = 2.28, 18μl

II. RNA isolation from 15-07-072 human RPE cultures (with or without CM treatment)

RNA was isolated from the 6 'day 21 culture of 15-07-072 RPE (with and without CM11 treatment for 6 or 24 hours).

Results:

Without CM11 treatment, 0.81μg / μl, 260/280 = 1.21,18μl

For CM11 treatment for 6h, 0.52μg / μl, 260/280 = 1.23,18μl

For CM11 treatment for 24h, 1.07μg / μl, 260/280 = 1.21,18μl

III. RNA isolation from human RPE (with or without CM treatment)

RNA was isolated from different generations of human RPE cultures, including SD1 "dry" AMD L 5 'day 20 RPE cultures, with or without CM11 treatment for 6 and 24 hours. Other human RPE cultures include normal 15-08-074 4 'day 20 culture, normal 15-09-027 5' day 7 culture, and "wet" AMD 15-10-021 4 'day 10 Cultures.

Results:

SD1 AMD L 5 ’Day 20, 0.1μg / μl, 260/280 = 1.64,18μl

SD1 AMD L 5 ’Day 20, treated with CM11 for 6 hours, 0.07μg / μl, 260/280 = 1.84,18μl

SD1 AMD L 5 ’Day 20, treated with CM11 for 24 hours, 0.09μg / μl, 260/280 = 1.56,18μl

IV. RNA isolation from SD1 "dry" AMD R 3'RPE culture

Since it is not possible to isolate RNA from SD1 right (R) RPE (due to poor growth and limited supply), attempts have been made to breed some surviving 3 'cultures. The surviving 3 'cultures were combined to isolate any RNA (with and without CM11 treatment for 24 hours).

Results:

1. SD1 AMD R 3 ’Day 56 RPE, 0.4μg / μl, 260/280 = 1.29,18μl

2. SD1 AMD R 3 ’Day 56 RPE, treated with CM11 for 24 hours, 0.25μg / μl, 260/280 = 1.18

V. RNA isolation from wet AMD 15-10-021 4'RPE (with and without CM treatment)

RNA was isolated from the 4 'day 10 culture of wet AMD 15-10-021 RPE (with and without CM11 treatment for 6 and 24 hours).

Results:

Without CM11 treatment, 0.03μg / μl, 260/280 = 0.58,18μl

After CM11 treatment for 6h, 0.09μg / μl, 260/280 = 1.23,18μl

After treated by CM11 for 24h, 0.11μg / μl, 260/280 = 1.36,18μl

VI. RNA isolation from ND08333 " AMD " RPE ( 1/21/16)

RNA was isolated from the 2 'day 8 culture of ND08333 "AMD" RPE (with and without CM11 treatment for 6 and 24 hours).

Results:

1. Without CM11 treatment, 0.04μg / μl, 260/280 = 1.77,18μl

2. For CM11 treatment for 6h, 0.03μg / μl, 260/280 = 1.77, 18μl

3. For CM11 treatment for 24h, 0.04μg / μl, 260/280 = 1.94,18μl

VII. RNA isolation from ND08333 RPE (with or without hUTC CM treatment)

RNA was isolated from ND08333 3 'day 106 and 4' day 97 RPE cultures (with and without CM13 treatment for 6 and 24 hours).

Results:

1. 3 ’without CM treatment, 0.02μg / μl, 260/280 = 1.845,17μl

2. 3’6h CM13, 0.12μg / μl, 260/280 = 2.022,17μl

3. 3’23h CM13, 0.13μg / μl, 260/280 = 2.393,17μl

4. 4 ’without CM treatment, 0.21μg / μl, 260/280 = 2.027, 17μl

5. 4’6h CM13, 0.28μg / μl, 260/280 = 2.056,17μl

6. 4’24h CM13, 0.31μg / μl 260/280 = 2.064,17μl

VIII. RNA isolation from ND08626 " AMD " RPE (with or without hUTC CM treatment)

RNA was isolated from ND08626 AMD RPE 1 'day 39, 2' day 23, and 3 'day 5 cultures (with and without CM5 10k treatment for 6 and 24 hours).

Results:

1. Day 39 of 1 ’, without CM5 treatment, 0.02μg / μl, 260/280 = 1.96,18μl

2. Day 39 of 1 ’, treated with CM5 for 6h, 0.04μg / μl, 260/280 = 3.03, 18μl

3. Day 39 on 1 ’, treated with CM5 for 24h, 0.03μg / μl, 260/280 = 1.69, 18μl

4. On day 23 of 2 ’, without CM5 treatment, 0.1μg / μl, 260/280 = 1.90,18μl

5. On day 23 of 2 ’, treated with CM5 for 6h, 0.08μg / μl, 260/280 = 1.92,18μl

6. On day 23 of 2 ’, treated with CM5 for 24h, 0.09μg / μl, 260/280 = 1.84,18μl

7. On the 5th day of 3 ’, without CM5 treatment, 0.08μg / μl, 260/280 = 1.72,18μl

8. On the 5th day of 3 ’, treated with CM5 for 6h, 0.16μg / μl, 260/280 = 1.53,18μl

9. On the 5th day of 3 ’, treated with CM5 for 24h, 0.03μg / μl, 260/280 = 1.87,18μl

IX. RNA isolation from 15-08-074 human RPE cultures (with or without hUTC CM treatment)

RNA was isolated from the 4 'day 17 culture of 15-08-074 RPE (with and without CM11 treatment for 6 or 24 hours).

Results:

1. Without CM11 treatment, 0.15μg / μl, 260/280 = 9.17,18μl

2. For CM11 treatment for 6h, 0.18μg / μl, 260/280 = 1.32, 18μl

3. If treated with CM11 for 24h, 0.18μg / μl, 260/280 = 2.82, 18μl

X. RNA isolation from human 15-08-074 RPE (with and without CM treatment)

RNA was isolated from 15-08-074 4 'day 20 RPE cultures (with and without CM11 treatment for 6 and 24 hours).

Results:

1. Without CM11 treatment, 0.05μg / μl, 260/280 = 0.86,18μl

2. For CM11 treatment for 6 hours, 0.06μg / μl, 260/280 = 1.60, 18μl

3. After 24 hours of CM11 treatment, 0.05μg / μl, 260/280 = 1.30, 18μl

XI. RNA isolation from human 15-04-001 RPE and human 15-07-072 RPE (with and without CM treatment)

RNA was prepared from many normal and AMD RPE samples (with and without CM15 treatment for 6 and 24 hours). The concentration was determined by Nanodrop technique.

Results:

XII. Ppp separation from human San Diego 1 AMD L 5 'RPE , 15-10-021 AMD 4' RPE , and 15-08-074 AMD 4 'RPE (with or without CM treatment)

RNA was prepared from many normal and AMD RPE samples (with and without CM15 treatment for 6 and 24 hours). The concentration was determined by Nanodrop technique.

Results:

XIII. ND08626 of from 1 ', 2' and 3 'RPE (without CM treatment and after 6 and 24 hours) the RNA isolation

RNA was isolated from RPE cultures (with and without CM13 treatment for 6 and 24 hours) on day 1 'day 60, 2' day 44 and 3 'day 26 of NDRI AMD ND08626 eyes.

Results:

XIV. RNA isolation from human RPE (with or without CM treatment) (12/10/15 )

RNA was isolated from human normal 15-09-027 5 'day 7 cultures (with and without CM11 treatment for 6 and 24 hours).

Results:

XV. RNA isolation from 15-11-098 RPE (with and without CM treatment)

RNA was isolated from 15-11-098 1 'day 50 and 2' day 14 RPE cultures (with and without CM11 treatment for 24 hours).

Results:

XVI. RNA isolation from 15-11-098 RPE (with and without CM treatment)

RNA was isolated from 15-11-098 2 'day 26 RPE (untreated) and 3' day 12 RPE (with and without CM11 treatment for 6 and 24 hours).

Results:

Example 12 Effect of RTK ligand on RPE phagocytosis

The effect of various RTK ligands on RPE phagocytosis was tested. For testing, the recombinant RTK ligand was exposed to various RPE isolates obtained in Example 6.

I. Recombinant human RTK ligand for testing

Recombinant human BDNF (catalog number 248-BD-025 / CF, batch number NG6515031) and human GDNF (catalog number 212-GD-010 / CF, batch number VQ2215081) are from R & D Systems, Inc., Minneapolis, MN. It was reconstituted in sterile PBS at 100 μg / mL, aliquoted and frozen in a refrigerator at -70 ° C. Recombinant human HGF (Cat. No. GF116, Lot No. 2651986) is from EMD Millipore Corp., Temecula, CA. It was reconstituted in water at 0.5 mg / mL, aliquoted and frozen at -20 ° C.

II. Method to check the effect of RTK ligand on RPE phagocytosis

Combine AMD RPE cells with various concentrations of recombinant human BDNF (405pg / mL, 2ng / mL, 10ng / mL, 50ng / mL, 200ng / mL), or recombinant human HGF (792pg / mL, 4.75ng / mL, 28.5ng / mL, 200 ng / mL), or recombinant human GDF (52.8 pg / mL, 422.4 pg / mL, 3.4 ng / mL, 27 ng / mL, 200 ng / mL) were cultured for 24 hours, and then phagocytosis assay was performed without changing the medium. The AMD RPE cell line cultured with hUTC CM was used as a positive control for the assay.

III. Effect of RTK ligand BDNF on SD1 "dry" AMD L 3'RPE phagocytosis

In order to examine the effect of RTK ligand BDNF on SD1 "dry" AMD RPE phagocytosis, the 3 'day 6 culture of SD1 AMD L RPE was cultured with 2 normal human RPEs with or without CM10 or different amounts of BDNF Phagocytosis test. The test results are shown in Table 12-1.

There has been a dose response of phagocytosis to increased levels of BDNF, except for the 2ng / ml result. The low phagocytosis level of SD1 AMD RPE compared to normal (15-09-027 and 15-08-074) RPE was confirmed again. The addition of CM10 increases the phagocytosis level of SD1 AMD RPE.

IV. Effect of RTK ligand HGF on SD1 "dry" AMD L 4'RPE phagocytosis

To examine the effect of RTK ligand HGF on the phagocytosis of SD1 "dry" AMD RPE, the 4 'day 10 culture of SD1 AMD L RPE was cultured with 2 normal human RPEs with or without CM10 or different amounts of HGF Phagocytosis test. The test results are shown in Table 12-2.

HGF does not show a dose effect on SD1 AMD 4'RPE phagocytosis, but it generally increases phagocytosis. The phagocytosis level of SD1 AMD RPE was again lower than normal (15-09-027 and 15-08-074) RPE, while the addition of CM10 increased the phagocytosis level.

Table 12-2. RTK ligand HGF phagocytosis of SD1 "dry" AMD L 4'RPE

V. Effect of RTK ligand GDNF on phagocytosis of AMD RPE (SD1 L)

In order to examine the effect of RTK ligand GDNF on AMD RPE (San Diego # 1 L) phagocytosis, in addition to and without CM10, perform SD1 L 3 'Day 11 RPE phagocytosis for increased levels of GDNF (52.8pg / ml, 422.4 pg / ml, 3.4ng / ml, 27ng / ml, 200ng / ml) dose response. As for the normal control group, 15-09-027 N 3 'day 11 and 15-08-074 N 3' day 21 RPE were also carried on 15-10-021 with 2 × 10 ⁶ each 8h phagocytosis analysis. Obtain the results of the typical patterns that appear in most of these dose response experiments. That is: 1) AMD RPE (SD1 L) engulfed a clear dose response to RTK ligand (GDNF); 2) AMD RPE (SD1 L) compared to normal RPE (15-09-027, 15-08-074) Reduced phagocytosis level; and 3) Reduced phagocytosis level of AMD RPE (SD1 L) is normalized by CM (CM10) treatment.

VI. Effect of RTK ligand HGF on phagocytosis of "wet" AMD RPE (15-10-021)

In order to examine the effect of RTK ligand HGF on AMD RPE (15-10-021, "wet") phagocytosis, in addition to and without CM10, perform 15-10-021 3 'day 13 RPE phagocytosis for increased levels The dose response of HGF (792pg / ml, 4.75ng / ml, 28.5ng / ml, 200ng / ml). As for the normal control group, 15-09-027 N 3 'day 26 and 15-08-074 N 3' day 36 RPE were also performed on 15-10-021 of 2 × 10 ⁶ each with ROS 8h phagocytosis analysis. Get slightly atypical results. That is: 1) AMD RPE (15-10-021) phagocytosis level clearly increased RTK ligand HGF, but no dose effect; 2) AMD RPE (15-10-021) compared to normal RPE (15-09 -027, 15-08-074) Reduced phagocytosis level; and 3) Reduced phagocytosis level of AMD RPE (15-10-021) is normalized by CM (CM10) treatment (see FIG. 31).

VII. Effect of RTK ligand BDNF on "wet" AMD RPE ( 15-10-021 ) phagocytosis

In order to check the effect of RTK ligand BDNF on AMD RPE (15-10-021, "wet") phagocytosis, in addition to being with or without CM13, perform 15-10-021 4 'day 206 RPE phagocytosis for increasing levels Dose response of BDNF (405pg / ml, 2ng / ml, 10ng / ml, 50ng / ml, 200ng / ml). As for the normal control group, RPE on 15-09-027 N5 'day 203 and 15-08-074 N 4' day 216 were also tested on 15-11-098 with 5 × 10 ⁵ pieces each 8h phagocytosis analysis. Get slightly typical results. That is: 1) AMD RPE (15-10-021) phagocytosis level has some dose effect on RTK ligand BDNF; 2) AMD RPE (15-10-021) compared to normal RPE (15-09-027; 15 -08-074 did not work) Reduced phagocytosis level; and 3) Reduced phagocytosis level of AMD RPE (15-10-021) was normalized by CM (CM13) treatment (see Figure 32).

VIII. Effect of RTK ligand GDNF on phagocytosis of "wet" AMD RPE (15-10-021)

In order to examine the effect of RTK ligand GDNF on AMD RPE (15-10-021, "wet") phagocytosis, in addition to and without CM11, perform 15-10-021 4 'day 7 RPE phagocytosis for increased levels Dose response of GDNF (52.8pg / ml, 422.4pg / ml, 3.4ng / ml, 27ng / ml, 200ng / ml). As for the normal control group, 15-09-027 N 4 'day 33 and 15-08-074 N 4' day 17 RPE were also carried on the 15-10-021 with 2 × 10 ⁶ each. 8h phagocytosis analysis. Obtain the results of the typical patterns that appear in most of these dose response experiments. That is: 1) AMD RPE (15-10-021) engulfed a clear dose response to RTK ligand (GDNF); 2) AMD RPE (15-10-021) compared to normal RPE (15-09-027, 15-08-074) Reduced phagocytosis level; and 3) Reduced phagocytosis level of AMD RPE (15-10-021) is normalized by CM (CM11) treatment (see Figure 33).

IX. Effect of RTK ligand BDNF on phagocytosis of AMD RPE (ND08333)

In order to examine the effect of RTK ligand BDNF on AMD RPE (ND08333) phagocytosis, in addition to and without CM11, perform ND08333 2 'day 20 RPE phagocytosis on increased levels of BDNF (405pg / ml, 2ng / ml, 10ng / ml , 50ng / ml, 200ng / ml) dose response. As for the normal control group, 15-11-098 N 3 'day 12 and 15-08-074 N 4' day 74 RPE were also performed on 15-11-098 with 1 × 10 ⁶ ROS on each 8h phagocytosis analysis. Get slightly typical results. That is: 1) AMD RPE (ND08333) phagocytosis level has some dose effect on RTK ligand BDNF; 2) AMD RPE (ND08333) phagocytosis is reduced compared to normal RPE (15-11-098 and 15-08-074) Level; and 3) The reduced phagocytosis level of AMD RPE (ND08333) is normalized by CM (CM11) treatment (see Figure 34).

X. Effect of RTK ligand HGF on AMD RPE (ND08333) phagocytosis

In order to examine the effect of RTK ligand HGF on AMD RPE (ND08333) phagocytosis, in addition to and without CM11, perform ND08333 2 'day 22 RPE phagocytosis on increased levels of HGF (792pg / ml, 4.75ng / ml, 28.5ng / ml, 200ng / ml) dose response. As for the normal control group, 15-11-098 N 3 'day 14 and 15-08-074 N 4' day 76 RPE were also performed on 15-11-098 of 1 × 10 ⁶ each with ROS 8h phagocytosis analysis. Obtain results for atypical patterns. That is: 1) AMD RPE (ND08333) phagocytic level responds to some dose of RTK ligand HGF; 2) AMD RPE (ND08333) reduced phagocytosis compared to normal RPE (15-11-098, 15-08-074) Level; but 3) the reduced phagocytosis level without AMD RPE (ND08333) is normalized by CM (CM11) treatment, which may be attributed to lack of CM11 activity (see Figure 35).

XI. Effect of RTK ligand GDNF on phagocytosis of AMD RPE (ND08333)

In order to examine the effect of RTK ligand GDNF on AMD RPE (ND08333) phagocytosis, in addition to and without CM11, perform ND08333 2 'Day 15 RPE phagocytosis for increased levels of GDNF (52.8pg / ml, 422.4pg / ml, 3.4 ng / ml, 27ng / ml, 200ng / ml) dose response. As for the normal control group, 15-11-098 N 2 'on the 21st day and 15-08-074 N 4' on the 69th day of RPE were performed on 15-11-098 with 1 × 10 ⁶ ROS on each 8h phagocytosis analysis. Obtain the results of the typical patterns that appear in most of these dose response experiments. That is: 1) AMD RPE (ND08333) phagocytosis of RTK ligand (GDNF) a clear dose response; 2) AMD RPE (ND08333) compared to normal RPE (15-11-098, 15-08-074) reduced Phagocytosis level; and 3) The reduced phagocytosis level of AMD RPE (ND08333) is normalized by CM (CM11) treatment (see Figure 36).

XII. Effect of RTK ligand GDNF on phagocytosis of AMD RPE (ND08626)

In order to examine the effect of RTK ligand GDNF on AMD RPE (ND08626) phagocytosis, in addition to and without CM5 10k, perform ND08626 2 'day 28 RPE phagocytosis for increased levels of GDNF (52.8pg / ml, 422.4pg / ml, 3.4ng / ml, 27ng / ml, 200ng / ml) dose response. As for the normal control group, 15-11-098 N 5 'day 116 and 15-11-098 N 3' day 67 RPE were also performed on 15-11-098 with 0.5 × 10 ⁶ each carrying ROS 8h phagocytosis analysis. Obtain the results of the typical patterns that appear in most of these dose response experiments. That is: 1) The clear dose response of AMD RPE (ND08626) phagocytosis to RTK ligand (GDNF); 2) The decrease in AMD RPE (ND08626) compared to normal RPE (15-09-027, 15-11-098) Phagocytosis level; and 3) The reduced phagocytosis level of AMD RPE (ND08626) is normalized by CM (CM5 10k) treatment (see Figure 37).

XIII. Effect of RTK ligand BDNF on phagocytosis of AMD RPE (ND08626)

In order to examine the effect of RTK ligand BDNF on AMD RPE (ND08626) phagocytosis, in addition to and without CM5 10k, perform ND08626 2 'day 25 RPE phagocytosis on increased levels of BDNF (405pg / ml, 2ng / ml, 10ng / ml, 50ng / ml, 200ng / ml) dose response. As for the normal control group, 15-09-027 N 5 'day 113 and 15-11-098 N 3' day 64 RPE were also performed on each 15-11-098 with 0.5 × 10 ⁶ ROS 8h phagocytosis analysis. Get slightly typical results. That is: 1) AMD RPE (Nd08626) phagocytosis level is equal to the effect of RTK ligand BDNF; 2) AMD RPE (ND08626) reduced phagocytosis compared to normal RPE (15-09-027, 15-11-098) Level; and 3) Normalization of the reduced phagocytosis level of AMD RPE (ND08626) by CM (CM5 10k) treatment (see Figure 38).

XIV. Effect of RTK ligand HGF on phagocytosis of AMD RPE (ND08626) (3/29/16)

In order to examine the effect of RTK ligand HGF on phagocytosis of AMD RPE (ND08626), in addition to and without CM5 10k, perform ND08626 3 'day 11 RPE phagocytosis on increased levels of HGF (792pg / ml, 4.75ng / ml, 28.5 ng / ml, 200ng / ml) dose response. As for the normal control group, 15-09-027 N 5 'day 117 and 15-11-098 N 3' day 68 RPE were also performed on 15-11-098 with 0.5 × 10 ⁶ ROS on each 8h phagocytosis analysis. Get slightly atypical results. That is: 1) AMD RPE (ND08626) phagocytic level has a slight dose effect on RTK ligand HGF; 2) AMD RPE (ND08626) is lower than normal RPE (15-09-027, 15-11-098) Phagocytosis level; and 3) The reduced phagocytosis level of AMD RPE (ND08626) is normalized by CM (CM5 10k) treatment (see Figure 39).

Example 13 The effect of bridge molecules on RPE phagocytosis

The effect of various bridge molecules on RPE phagocytosis was also tested. For testing, the recombinant bridge molecule was exposed to various RPE isolates obtained in Example 6.

I. Recombinant bridge molecule

Recombinant human MFG-E8 (catalog number 2767-MF-050, batch number MPP2415081), recombinant human TSP-1 (catalog number 3074-TH-050, batch number MVF4114111), recombinant human TSP-2 (catalog number 1635-T2-050, Lot number HUZ2014121) were all obtained from R & D Systems, Inc., Minneapolis, MN. The reconstitution of individual protein stock solutions was based on the supplier's data sheet: recombinant human MFG-E8, TSP-1 and TSP-2 were reconstituted in sterile PBS at 100 μg / mL, respectively. Recombinant human Gas6 was reconstituted in sterile water at 100 μg / mL. The reconstituted stock solution was aliquoted and frozen in the refrigerator at -70 ° C.

II. Methods to examine the effect of bridge molecules on RPE phagocytosis

ROS were pre-incubated with hUTC CM control medium (DMEM with 10% FBS and 1% P / S: F12 medium) or hUTC CM in a CO ₂ cell culture incubator at 37 ° C for 24 h. At the same time, the ROS line was pre-cultured in a CO ₂ cell culture incubator at 37 ° C for 24h in a control medium containing various concentrations of human recombinant MFG-E8 (15.5ng / mL, 31ng / mL, 62ng / mL, 124ng / mL), TSP-1 (152ng / mL, 304ng / mL, 608ng / mL, 1216ng / mL), or TSP-2 (8.8ng / mL, 26.4ng / mL, 79.2ng / mL, 237.6ng / mL) ). After culturing, the ROS was centrifuged to settle, resuspended in MEM5 (MEM medium with 5% FBS), and fed to dystrophic RPE cells in the presence of MEM5 for phagocytosis assay. As for the control group, normal RPE alone or deprived RPE alone were cultured in MEM20 (MEM medium with 20% FBS), and then in the presence of untreated ROS (resuspended in MEM20 and fed to RPE cells) Replace with MEM5 for phagocytosis test.

III. Effect of bridge molecule MFG-E on phagocytosis of AMD RPE (SD1 L)

In order to examine the effect of the bridge molecule (MFG-E8) on the phagocytosis of AMD RPE (SD1 L), 15-09-027 of 5 × 10 ⁶ each were provided with ROS to increase the level of the bridge molecule MFG-E8 (15.5ng / ml, 31ng / ml, 62ng / ml, 124ng / ml) treatment (also treated with and without CM10) for 24 hours, and the treated and untreated ROS are used for AMD SD1 L 4 'RPE on day 10 and normal The control group 15-09-027 N 4 'day 8 and 15-08-074 N 4' day 24 RPE 8h phagocytosis test. Obtain the results of the typical patterns that appear in most of these dose response experiments. That is: 1) AMD RPE (SD1 L) engulfed a clear dose response to ROS treated with bridge molecules (MFG-E8); 2) AMD RPE (SD1 L) compared to normal RPE (15-09-027, 15 -08-074) Reduced phagocytosis level; and 3) Reduced phagocytosis level of AMD RPE (SD1 L) is normalized by treatment of ROS with CM (CM10) (see FIG. 40).

IV. The effect of bridge molecule ( Tsp 1 ) on AMD RPE (SD1 L) phagocytosis

In order to check the effect of the bridge molecule (Tsp 1) on the phagocytosis of AMD RPE (SD1 L), 2 × 10 ⁶ pieces of 15-09-027 were added with ROS to increase the level of bridge molecule Tsp 1 (152ng / ml, 304ng / ml, 608ng / ml, 1216ng / ml) treatment (also with and without CM10 treatment) for 24 hours, and the treated and untreated ROS were used for AMD SD1 L 4 'RPE on day 16 and normal control group 15 -09-027 N 4 'day 14 and 15-08-074 N 4' day 29 RPE 8h phagocytosis test. Get slightly atypical results. That is: 1) AMD RPE (SD1 L) phagocytosis level has a clear increase in ROS treated with bridge molecules (Tsp 1), but there is no dose effect; 2) AMD RPE (SD1 L) compared to normal RPE (15-09 -027, 15-08-074) Reduced phagocytosis level; and 3) Reduced phagocytosis level of AMD RPE (SD1 L) is normalized by treatment of ROS with CM (CM10) (see FIG. 41).

V. Effect of bridge molecule ( Tsp 2 ) on AMD RPE (SD1 L) phagocytosis

In order to check the effect of the bridge molecule (Tsp 2) on the phagocytosis of AMD RPE (SD1 L), 15-09-027 with 2 × 10 ⁶ each were added with ROS to increase the level of the bridge molecule Tsp 2 (8.8ng / ml, 26.4ng / ml, 79.2ng / ml, 237.6ng / ml) treatment (also with and without CM10 treatment) for 24 hours, and the treated and untreated ROS were used for AMD SD1 L 4 '14th day RPE and The normal control group 15-09-027 N 4 'day 13 and 15-08-074 N 4' day 28 RPE 8h phagocytosis test. Obtain the results of the typical patterns that appear in most of these dose response experiments. That is: 1) AMD RPE (SD1 L) engulfed a clear dose response to ROS treated with bridge molecules (Tsp 2); 2) AMD RPE (SD1 L) compared to normal RPE (15-09-027, 15- 08-074) Reduced phagocytosis level; and 3) Reduced phagocytosis level of AMD RPE (SD1 L) is normalized by treatment of ROS with CM (CM10) (see Figure 42).

VI. Effect of bridge molecule ( Tsp 1 ) on phagocytosis of "wet" AMD RPE (15-10-021)

In order to check the effect of the bridge molecule (Tsp 1) on the phagocytosis of AMD RPE (15-10-021, "wetness"), 15-09-027 with 5 × 10 ⁶ each were added with ROS to increase the level of bridge molecules Tsp 1 (152ng / ml, 304ng / ml, 608ng / ml, 1216ng / ml) treatment (also treated with and without CM10) for 24 hours, and treated and untreated ROS are used for AMD 15-10-021 RPE of 3'day 15 and normal control group 15-09-027 N 3 'day 28 and 15-08-074 N 3' day 38 RPE 8h phagocytosis test. Obtain results in a fairly typical model that has appeared in most of these dose response experiments. That is: 1) AMD RPE (15-10-021) engulfed in a dose response to ROS treated with bridge molecule (Tsp 1); 2) AMD RPE (15-10-021) compared to normal RPE (15- 09-027, 15-08-074) Reduced phagocytosis level; and 3) Reduced phagocytosis level of AMD RPE (15-10-021) is normalized by treatment of ROS with CM (CM10) (see FIG. 43).

VII. The effect of bridge molecule ( MFG-E8 ) on the phagocytosis of "wet" AMD RPE (I5-10-021) .

In order to check the effect of the bridge molecule (MFG-E8) on the phagocytosis of AMD RPE (15-10-021 "wetness"), 2 × 10 ⁶ pieces of 15-09-027 were added with ROS to increase the level of bridge molecules MFG-E8 (15.5ng / ml, 31ng / ml, 62ng / ml, 124ng / ml) treatment (also with and without CM10 treatment) for 24 hours, and the treated and untreated ROS are used for AMD 15-10 -021 3 'Day 12 RPE and normal control group 15-09-027 N 3' Day 25 and 15-08-074 N 3 'Day 35 RPE 8h phagocytosis test. Obtain results close to typical patterns that appear in most of these dose response experiments. That is: 1) AMD RPE (15-10-021) phagocytosis dose-response (although low level) to ROS treated with bridge molecule (MFG-E8); 2) AMD RPE (15-10-021) compared Reduced phagocytosis level in normal RPE (15-09-027, 15-08-074); and 3) Reduced phagocytosis level in AMD RPE (15-10-021) normalization by treating ROS with CM (CM10) (See Figure 44).

VIII. The effect of bridge molecule ( Tsp 2 ) on phagocytosis of "wet" AMD RPE (15-10-021)

In order to check the effect of the bridge molecule (Tsp 2) on the phagocytosis of AMD RPE (15-10-021 "wetness"), 2 × 10 ⁶ pieces of 15-09-027 were added with ROS to increase the level of bridge molecule Tsp 2 (8.8ng / ml, 26.4ng / ml, 79.2ng / ml, 237.6ng / ml) treatment (also with and without CM10 treatment) for 24 hours, and the treated and untreated ROS are used for AMD 15- 10-021 3 'day 13 RPE and normal control group 15-09-027 N 3' day 26 and 15-08-074 N 3 'day 36 RPE 8h phagocytosis test. Obtain the results of the typical patterns that appear in most of these dose response experiments. That is: 1) AMD RPE (15-10-021) engulfed a clear dose response to ROS treated with bridge molecules (Tsp 2); 2) AMD RPE (15-10-021) compared to normal RPE (15-021) 09-027, 15-08-074) Reduced phagocytosis level; and 3) Reduced phagocytosis level of AMD RPE (15-10-021) is normalized by treatment of ROS with CM (CM10) (see FIG. 45).

IX. Effect of bridge molecule (MFG-E8) on the phagocytosis of AMD RPE (ND08333)

In order to check the effect of the bridge molecule (MFG-E8) on the phagocytosis of AMD RPE (ND08333), 15-11-098 of 1 × 10 ⁶ each were provided with ROS to increase the level of bridge molecule MFG-E8 (15.5ng / ml , 31ng / ml, 62ng / ml, 124ng / ml) treatment (also treated with and without CM11) for 24 hours, and the treated and untreated ROS were used for AMD ND08333 2 'day 20 RPE and normal control group 15-11-098 N 3 'day 12 and 15-08-074 N 4' day 74 RPE 8h phagocytosis test. Obtain results close to typical patterns that appear in most of these dose response experiments. That is: 1) AMD RPE (ND08333) phagocytosis response to ROS treated with bridge molecule (MFG-E8); 2) AMD RPE (ND08333) compared to normal RPE (15-11-098, 15-08 -074) Reduced phagocytosis level; and 3) Reduced phagocytosis level of AMD RPE (ND08333) is normalized by treatment of ROS with CM (CM11) (see Figure 46).

X. Effect of bridge molecule ( Tsp 1 ) on AMD RPE (ND08333) phagocytosis

In order to check the effect of the bridge molecule (Tsp 1) on the phagocytosis of AMD RPE (ND08333), 15-11-098 with 0.5 × 10 ⁶ each were added with ROS to increase the level of the bridge molecule Tsp 1 (152ng / ml, 304ng / ml, 608ng / ml, 1216ng / ml) treatment (also treated with and without CM13) for 24 hours, and the treated and untreated ROS were used for AMD ND08333 3 'day 56 RPE and normal control group 15-11 -098 N 3 'Day 61 and 15-08-074 N 4' Day 124 RPE 8h phagocytosis test. Get slightly atypical results. That is: 1) AMD RPE (ND08333) phagocytosis level slightly increased ROS treated with bridge molecule (Tsp 1), but no real dose effect; 2) AMD RPE (ND08333) compared to normal RPE (15-11- 098, 15-08-074) Reduced phagocytosis level; and 3) Reduced phagocytosis level of AMD RPE (ND08333) is normalized by treatment of ROS with CM (CM13) (see FIG. 47).

XI. Effect of bridge molecule ( Tsp 2 ) on AMD RPE (ND08333) phagocytosis

In order to check the effect of the bridge molecule (Tsp 2) on the phagocytosis of AMD RPE (ND08333), 15-11-098 with 1 × 10 ⁶ each were added with ROS to increase the level of the bridge molecule Tsp 2 (8.8ng / ml, 26.4 ng / ml, 79.2ng / ml, 237.6ng / ml) treatment (also treated with and without CM11) for 24 hours, and the treated and untreated ROS were used for AMD ND08333 2 'day 20 RPE and normal controls Group 15-11-098 N 3 'day 12 and 15-08-074 N 4' day 74 RPE 8h phagocytosis test. Obtain the results of the typical patterns that appear in most of these dose response experiments. That is: 1) AMD RPE (ND08333) engulfed a clear dose response to ROS treated with bridge molecules (Tsp 2); 2) AMD RPE (ND08333) compared to normal RPE (15-11-098, 15-08- 074) Reduced phagocytosis level; and 3) Reduced phagocytosis level of AMD RPE (ND08333) is normalized by treatment of ROS with CM (CM11) (see Figure 48).

XII. Effect of bridge molecule (MFG-E8) on the phagocytosis of AMD RPE (ND08626)

In order to examine the effect of the bridge molecule (MFG-E8) on the phagocytosis of AMD RPE (ND08626), 15-11-098 with 0.5 × 10 ⁶ each were added with ROS to increase the level of bridge molecule MFG-E8 (15.5ng / ml , 31ng / ml, 62ng / ml, 124ng / ml) treatment (also with and without CM5 10k treatment) for 24 hours, and the treated and untreated ROS were used for AMD ND08626 3 'day 5 RPE and normal controls Group 15-09-027 N 5 'day 111 and 15-11-098 N 3' day 62 RPE 8h phagocytosis test. Obtain the results of the typical patterns that appear in most of these dose response experiments. That is: 1) AMD RPE (ND08626) engulfed a clear dose response to ROS treated with bridge molecule (MFG-E8); 2) AMD RPE (ND08626) compared to normal RPE (15-09-027, 15-11 -098) Reduced phagocytosis level; and 3) Reduced phagocytosis level of AMD RPE (ND08626) is normalized by treatment of ROS with CM (CM5 10k) (see Figure 49).

XIII. Effect of bridge molecule ( Tsp 1 ) on AMD RPE (ND08626) phagocytosis

In order to check the effect of the bridge molecule (Tsp 1) on the phagocytosis of AMD RPE (ND08626), 15-11-098 with 0.5 × 10 ⁶ each were added with ROS to increase the level of the bridge molecule Tsp 1 (152ng / ml, 304ng / ml, 608ng / ml, 1216ng / ml) treatment (also with and without CM5 10k treatment) for 24 hours, and the treated and untreated ROS were used for AMD ND08626 3 '14th day RPE and normal control group 15- 9-027 N 5 'day 120 and 15-11-098 N 3' day 71 RPE 8h phagocytosis test. Obtain results in a fairly typical model that has appeared in most of these dose response experiments. That is: 1) AMD RPE (ND08626) phagocytosis response to ROS treated with bridging molecule (Tsp 1); 2) AMD RPE (ND08626) compared to normal RPE (15-09-027, 15-11- 098) Reduced phagocytosis level; and 3) Reduced phagocytosis level of AMD RPE (ND08626) is normalized by treatment of ROS with CM (CM5 10k) (see FIG. 50).

XIV. Effect of bridge molecule ( Tsp 2 ) on AMD RPE (ND08626) phagocytosis

In order to check the effect of the bridge molecule (Tsp 2) on the phagocytosis of AMD RPE (ND08626), 15-11-098 with 0.5 × 10 ⁶ each were added with ROS to increase the level of the bridge molecule Tsp 2 (8.8ng / ml, 26.4 ng / ml, 79.2ng / ml, 237.6ng / ml) treatment (also with and without CM5 10k treatment) for 24 hours, and the treated and untreated ROS are used for AMD ND08626 3 'day 5 RPE and normal The control group 15-09-027 N 5 'day 111 and 15-11-098 N 3' day 62 RPE 8h phagocytosis test. Get slightly atypical results. That is: 1) AMD RPE (ND08626) engulfed a clear dose response to ROS treated with bridge molecule (Tsp 2); 2) AMD RPE (ND08626) compared to normal RPE (15-09-027, 15-11- 098) Reduced phagocytosis level; and 3) The reduced phagocytosis level of AMD RPE (ND08626) is normalized by the partial treatment of ROS with CM (CM5 10k), which may be attributed to the decline in CM activity (see Figure 51).

Examples 6 to 13 demonstrate the following points:

A. Confirm that AMD RPE has reduced phagocytic function compared to normal RPE (both dry and wet AMD).

B. Confirm the stimulating effect of hUTC CM on the phagocytosis of human RPE, which includes AMD and even normal RPE (the former has more effects than the latter), so that the reduced phagocytosis in AMD RPE is normalized in most cases .

C. Confirm that the RTK ligand and the bridge molecule stimulate the AMD RPE phagocytosis in a dose-dependent manner as seen in rat RCS RPE.

Despite the differences in the diseases and species involved (retinal degeneration based on the MERTK gene defect in the RCS rat model, and AMD, the main human eye disease in the elderly), there are defects in RPE phagocytosis and hUTC in Examples 6 to 13 CM rescues similarities in the effectiveness of phagocytosis defects. The effectiveness of hUTC CM to correct abnormal phagocytosis proves the effectiveness of hUTC as a treatment for retinal degeneration and AMD.

Example 14 Tissue-derived cells

This example describes the preparation of postpartum-derived cells from placenta and umbilical cord tissue. Obtain the postpartum umbilical cord and placenta after full-term or insufficient-term delivery. The cell line was collected from the umbilical cord and placental tissue of five different donors. Test the ability of different cell separation methods to produce cells with the following potentials: 1) the potential to differentiate into cells with different phenotypes, which is a common feature of stem cells; or 2) the potential to provide nutritional factors that can be used in other cells and tissues .

Methods and materials

Umbilical cell isolation: The umbilical cord was obtained from the National Disease Research Exchange Center (NDRI, Philadelphia, Pa.). The tissue system is obtained after normal delivery. The cell separation procedure is performed aseptically in a laminar flow hood. To remove blood and debris, place the umbilical cord in phosphate buffer (PBS; Invitrogen, Carlsbad, Calif.) With antimycotics and antibiotics (100 units / ml penicillin, 100 μg / ml streptomycin, 0.25 Amphotericin B) in the presence of micrograms / ml was washed down. The tissue was then mechanically dissociated on a 150 cm ² tissue culture dish and in the presence of 50 ml of medium (DMEM-low glucose or DMEM-high glucose; Invitrogen) until the tissue was minced into fine mud. Transfer the minced tissue to a 50 ml conical tube (approximately 5 grams of tissue per tube).

The tissue is then digested in DMEM-low glucose medium or DMEM-high glucose medium, each medium containing the antifungal agent and antibiotic as described above. In some experiments, an enzyme mixture of collagenase and dispase ("C: D") (collagenase (Sigma, St Louis, Mo.), 500 units / ml; and dispase (Invitrogen), 50 units were used / Ml in DMEM-low glucose medium). In other experiments, a mixture of collagenase, dispase and hyaluronidase ("C: D: H") (collagenase, 500 units / ml; dispase, 50 units / ml; and hyaluronidase (Sigma) , 5 units / ml in DMEM-low glucose). A conical tube containing tissue, culture medium, and digestive enzymes was incubated at 37 ° C. for 2 hours at 225 rpm in a rotary shaker (Environ, Brooklyn, N.Y).

After digestion, the tissue was centrifuged at 150 ^x g for 5 minutes, and the supernatant was aspirated. Resuspend the pellet in 20 ml of growth medium (DMEM-low glucose (Invitrogen), 15% (v / v) fetal bovine serum (FBS; special bovine serum); batch number AND18475; Hyclone, Logan, Utah ), 0.001% (v / v) 2-mercaptoethanol (Sigma), 1 ml per 100 ml of antibiotic / antifungal agent as described above. The cell suspension was filtered through a 70 micron nylon cell filter (BD Biosciences). An additional 5 ml of rinse medium containing growth medium was passed through the filter. The cell suspension was then passed through a 40 micron nylon cell strainer (BD Biosciences) and then extra 5 ml of growth medium rinse was used to drive out.

The filtrate was resuspended in growth medium (total volume 50 ml) and then centrifuged at 150 ^x g for 5 minutes. Aspirate the supernatant and resuspend the cells in 50 ml of fresh growth medium. Repeat this process two more times.

After the final centrifugation, the supernatant was aspirated and the cell pellet was resuspended in 5 ml of fresh growth medium. Trypan blue staining was used to determine the number of viable cells. The cells are then cultured under standard conditions.

Cells isolated from the umbilical cord were seeded at 5,000 cells / cm ² on gelatin-coated T-75cm ² culture flasks (Corning Inc., Corning, NY) and on growth medium with antibiotics / antifungals as described above in. After 2 days (in various experiments, the cells were cultured for 2 to 4 days), the used medium was aspirated from the flask. Wash cells three times with PBS to remove debris and blood-derived cells. The cells were then supplemented with growth medium and allowed to grow to fullness (from passage 0 to passage 1 for about 10 days). At subsequent passages (since passages 1 to 2, etc.), the cells reach the second overgrowth after 4 to 5 days (75 to 85 percent overgrowth). For these subsequent passages, cells were seeded at 5000 cells / cm ² . The cells were grown in a humidified incubator at 37% carbon dioxide and atmospheric oxygen.

Isolation of placental cells: Placental tissue lines were obtained from NDRI (Philadelphia, Pa.). These tissues are from pregnant women and are obtained during normal surgical delivery. The placental cell line is isolated as described for umbilical cell isolation.

The following examples apply to the separation of maternally-derived and neonate-derived separate cell populations from placental tissue.

The cell separation procedure is performed aseptically in a laminar flow hood. Placental tissue was washed in phosphate buffer (PBS; Invitrogen, Carlsbad, Calif.) In the presence of antifungal agents and antibiotics (as described above) to remove blood and debris. Subsequently, the placental tissue was dissected into three areas: top line (neonatal side or appearance), midline (mixed cells separating neonate and mother) and bottom line (maternal side or appearance).

The separated areas were washed several times in PBS with antibiotics / antimycotics to further remove blood and debris. Next, the zones were mechanically dissociated on a 150 cm ² tissue culture plate in the presence of 50 ml of DMEM-low glucose until the zones were chopped into fine mud. Transfer the mud to a 50 ml conical tube. Each tube contains approximately 5 grams of tissue. The tissues were digested in DMEM-low glucose or DMEM-high glucose medium containing antifungal agents and antibiotics (100 U / ml penicillin, 100 μg / ml streptomycin, 0.25 μg / ml amphotericin B) and digestive enzymes. In some experiments, an enzyme mixture of collagenase and dispase ("C: D") was used, which contained 500 units / ml collagenase (Sigma, St Louis, Mo.) and 50 in DMEM-low glucose medium Units / ml of dispase (Invitrogen). In other experiments, a mixture of collagenase, dispase and hyaluronidase (C: D: H) (collagenase, 500 units / ml; dispase, 50 units / ml; and hyaluronidase (Sigma), 5 Units / ml in DMEM-low glucose). A conical tube containing tissue, culture medium, and digestive enzymes was incubated at 225 rpm for 2 hours at 37 ° C in a rotary shaker (Environ, Brooklyn, NY).

After digestion, the tissue was centrifuged at 150 ^x g for 5 minutes, and the resulting supernatant was aspirated. The pellet was resuspended in 20 ml of growth medium with penicillin / streptomycin / amphoterin B. The cell suspension was filtered through a 70 micron nylon cell strainer (BD Biosciences) and chased out with an additional 5 ml of growth medium rinse. The total cell suspension was passed through a 40 micron nylon cell strainer (BD Biosciences), followed by an additional 5 ml of growth medium as a rinse.

The filtrate was resuspended in growth medium (total volume 50 ml) and then centrifuged at 150 ^x g for 5 minutes. Aspirate the supernatant and resuspend the cell pellet in 50 ml of fresh growth medium. Repeat this process two more times. After the final centrifugation, the supernatant was aspirated and the cell pellet was resuspended in 5 ml of fresh growth medium. The trypan blue exclusion test was used to determine the cell count. The cells are then cultured under standard conditions.

LIBERASE cell isolation: in DMEM with LIBERASE (Boehringer Mannheim Corp., Indianapolis, Ind.) (2.5 mg per ml, Blendzyme 3; Roche Applied Sciences, Indianapolis, Ind.) And hyaluronidase (5 units / ml, Sigma) -Separation of cells from umbilical tissue in low glucose medium. Tissue digestion and cell separation are as described above for other protease digestions, but use LIBERASE / hyaluronidase mixture instead of C: D or C: D: H enzyme mixture. Digestion of tissues with LIBERASE allows the separation of rapidly expanding cell populations from postpartum tissues.

Cell separation using other enzyme combinations: Comparison of procedures using different enzyme combinations to isolate cells from the umbilical cord. Enzymes used for digestion comparison include: i) collagenase; ii) dispase; iii) hyaluronidase; iv) collagenase: dispase mixture (C: D); v) collagenase: hyaluronidase mixture (C: H) ; Vi) dispase: hyaluronidase mixture (D: H); and vii) collagenase: dispase: hyaluronidase mixture (C: D: H). Observe the differences in cell separation using these different enzyme digestion conditions (Table 14-1).

Separation of cells from residual blood in the umbilical cord: Other attempts were made to separate the cell pool from the umbilical cord by different means. In one example, the umbilical cord is sliced and then washed with growth medium to expel blood clots and gel-like material. The mixture of blood, gel-like material and growth medium was collected and centrifuged at 150 ^x g. The pellet was resuspended and inoculated into growth medium on gelatin-coated culture flasks. Through these experiments, a population of cells that would rapidly expand was isolated.

Isolate cells from cord blood: Isolate cells from a cord blood sample obtained from NDR1. The separation protocol used here is Ho International's International Patent Application WO 2003/025149 (Ho, TW, et al., "Cell Populations Which Co-Express CD49C and CD90", application number PCT / US02 / 29971). Cord blood (NDRI, Philadelphia PA) samples (50 ml and 10.5 ml, respectively) and lysis buffer (155 mM ammonium chloride sterilized by filter, 10 mM potassium bicarbonate, 0.1 mM EDTA, buffered to pH 7.2 (all components are from Sigma, St. Louis, Mo.)) mixed. The cells were lysed at a ratio of 1:20 cord blood to lysis buffer. The resulting cell suspension was vortexed for 5 seconds and then incubated at ambient temperature for 2 minutes. The lysate was centrifuged (centrifuged at 200 ^x g for 10 minutes). The cell pellet was resuspended in complete minimum essential medium (Gibco, Carlsbad, Calif.) Containing 10% fetal bovine serum (Hyclone, Logan Utah), 4 millimolar branamide (Mediatech, Herndon, Va .), 100 units of penicillin per 100 ml and 100 micrograms of streptomycin per 100 ml (Gibco, Carlsbad, Calif.). Centrifuge the resuspended cells (centrifugation at 200 ^x g for 10 minutes), aspirate the supernatant and wash the cell pellet in complete medium. Cells were directly seeded into T75 flasks (Corning, NY), T75 laminin coated flasks or T175 fibronectin coated flasks (both from Becton Dickinson, Bedford, Mass.).

Separation of cells using different enzyme combinations and growth conditions: To determine whether the cell population can be isolated under different conditions and whether it can be expanded immediately under various conditions after separation, use the C: D: H enzyme combination according to the procedure provided above The cells were digested in growth medium with or without 0.001% (v / v) 2-mercaptoethanol (Sigma, St. Louis, Mo.). The placenta-derived cells thus isolated were seeded under various conditions. All cells are grown in the presence of penicillin / streptomycin. (Table 14-2).

Cells were separated using different enzyme combinations and growth conditions: under all conditions, the cells attached and expanded well between passages 0 and 1 (Table 14-2). The cells in conditions 5 to 8 and 13 to 16 all showed good proliferation up to 4 passages after inoculation, and these cells were frozen and stored at this point.

result

Cell separation using a combination of different enzymes: the combination of C: D: H provides the best post-separation cell yield and produces cells that are expanded in more generations in culture than other conditions (Table 14-1). Collagen protease or hyaluronidase alone cannot obtain an expandable cell population. No attempt was made to determine whether this result was specific to the collagen tested.

Cells were isolated using different enzyme combinations and growth conditions: under all conditions tested for enzyme digestion and growth, cells adhered and expanded well between passages 0 and 1 (Table 14-2). The cells in experimental conditions 5 to 8 and 13 to 16 all proliferated well up to 4 passages after seeding, and these cells were frozen at this point. All cells were cryopreserved for further study.

Isolate cells from residual blood in the umbilical cord: nucleated cells attach and grow rapidly. These cells were analyzed by flow cytometry, and these cells were similar to those obtained by enzyme digestion.

Separation of cells from cord blood: The preparation contains red blood cells and platelets. There was no attachment and division of nucleated cells during the first 3 weeks. The culture medium was changed 3 weeks after inoculation but cell attachment and growth were still not observed.

Summary: Enzyme combinations of collagenase (matrix metalloproteinase), dispase (neutral protease), and hyaluronidase (mucolytic enzyme that breaks down hyaluronic acid) can be used to effectively derive cell populations from the umbilical cord and placental tissue. LIBERASE called Blendzyme can also be used. In particular, Blendzyme 3 can also be used in conjunction with hyaluronidase to isolate cells. Blendzyme 3 is collagenase (4 Wunsch units / g) and thermolysin (1714 casein units / g). When cultured in growth medium on gelatin-coated plastics, these cells rapidly expanded many passages.

Cells were also isolated from the residual blood in the umbilical cord, but the residual blood was not cord blood. The reason why the blood clot washed out of the tissue exists and adheres and grows under the conditions used may be because the cells are released during the dissection process.

Example 15 Karyotype analysis of postpartum derived cells

The cell line used for cell therapy is preferably homogeneous and does not contain any contaminating cell types. Cells used in cell therapy should have a normal chromosome number (46) and structure. In order to identify homogeneous placenta and umbilical cord-derived cell lines that do not contain cells of non-postpartum tissue origin, the karyotype of the cell samples was analyzed.

Methods and materials

PPDCs from postnatal tissues of male neonates were cultured in growth medium containing penicillin / streptomycin. Postnatal tissues from male neonates (X, Y) were selected to be able to distinguish neonate-derived cells from maternal-derived cells (X, X). Cells were seeded at 5,000 cells per square centimeter in medium in T25 flasks (Corning Inc., Corning, N.Y.) and expanded to 80% overgrown. Fill the T25 flask containing cells to the neck with growth medium. The courier will send the sample to the clinical cytogenetics laboratory (estimated laboratory to laboratory transportation time is one hour). The cells are analyzed in metaphase, at which time the staining system is best visualized. Of the twenty cells in the metaphase that were counted, five were analyzed for the number of normal homogeneous karyotypes (two). If two karyotypes are observed, the cell sample is classified as homogeneous. If more than two karyotypes are observed, the cell sample is classified as heterogeneous. When the number of heterogeneous karyotypes (four) is identified, additional metaphase cells are counted and analyzed.

result

All cell samples submitted for chromosome analysis are interpreted as showing normal appearance. Three of the 16 analyzed cell lines exhibited a heterogeneous phenotype (XX and XY), indicating the presence of cells derived from both neonatal and maternal sources (Table 15-1). Cells derived from tissue placenta-N were isolated from the neonatal appearance of the placenta. At passage zero, this cell line exhibited homogeneous XY. However, in generation nine, the cell line was heterogeneous (XX / XY), indicating the presence of maternal-derived cells not previously detected.

Summary: Chromosome analysis identified placenta and umbilical-derived cells whose karyotype appeared normal (interpreted by the clinical cytogenetics laboratory). Karyotype analysis also identified cell lines that did not contain maternal cells, as determined by the homogeneous karyotype.

Example 16 Evaluation of human postpartum-derived cell surface markers by flow cytometry

Characterization of cell surface proteins or "markers" by flow cytometry can be used to determine the identity of cell lines. The consistency of performance can be determined by multiple donors and in cells exposed to different treatment and culture conditions. The postpartum-derived cell (PPDC) lines isolated from the placenta and navel are characterized (by flow cytometry) to provide an overview for identifying these cell lines.

Methods and materials

Culture medium and culture vessel: The cells were cultured in a growth medium (Gibco Carlsbad, Calif.) With penicillin / streptomycin. The cells were cultured in plasma-treated T75, T150, and T225 tissue culture flasks (Corning Inc., Corning, N.Y.) until they were overgrown. The growth surface of the culture flask was coated with gelatin by incubating 2% (w / v) gelatin (Sigma, St. Louis, Mo.) at room temperature for 20 minutes.

Antibody staining and flow cytometry analysis: The attached cells in the bottle were washed in PBS and desorbed with trypsin / EDTA. The cells were collected, centrifuged, and resuspended in 3% (v / v) FBS in PBS at a cell concentration of 1 × 10 ⁷ per ml. According to the manufacturer's specifications, the antibody labeled on the cell surface of interest (see below) is added to one hundred microliters of the cell suspension, and then the mixture is incubated at 4 ° C in the dark for 30 minutes. After incubation, the cells were washed with PBS and centrifuged to remove unbound antibody. The cells were resuspended in 500 microliters of PBS and analyzed by flow cytometry. Flow cytometry analysis was performed with a FACScalibur ^™ instrument (Becton Dickinson, San Jose, Calif.). Table 16-1 lists the cell surface labeled antibodies used.

Comparison of placenta and umbilical cord: Placenta-derived cells are compared with umbilical cord-derived cells at the 8th passage.

Comparison of sub-generation to sub-generation: placenta-derived cells and umbilical-derived cells were analyzed at sub-generations 8, 15, and 20.

Donor-to-donor comparison: To compare the differences between donors, placental-derived cells from different donors are compared with each other, and umbilical-derived cells from different donors are compared with each other.

Surface coating comparison: compare placental-derived cells cultured on gelatin-coated culture flasks with placenta-derived cells cultured on uncoated culture flasks. Umbilical-derived cells cultured on gelatin-coated culture flasks were compared with umbilical-derived cells cultured on uncoated culture flasks.

Digestive enzyme comparison: compare the four treatments used to isolate and prepare cells. It was isolated from the cell line of placenta treated with 1) collagenase; 2) collagenase / dispase; 3) collagenase / hyaluronidase; and 4) collagenase / hyaluronidase / dispase.

Placental layer comparison: Compare the cells derived from the maternal form of placental tissue with the cells derived from the villi region of the placental tissue and the cells derived from the neonatal fetus of the placenta.

result

Comparison of placenta and umbilicus: Placenta-derived cells and umbilical-derived cells analyzed by flow cytometry showed positive expression of CD10, CD13, CD44, CD73, CD90, PDGFr-α and HLA-A, HLA-B, HLA-C This is indicated by the increase in fluorescence value relative to the IgG control group. The detectable performance of CD31, CD34, CD45, CD117, CD141, HLA-DR, HLA-DP, and HLA-DQ of these cells is negative, which is indicated by the comparison of the fluorescence value with the IgG control group. Consider the variation of the fluorescence value of the positive curve. The mean (ie CD13) and range (ie CD90) of the positive curve showed some variation, but the curve appeared normal, confirming a homogeneous population. The two curves individually show values greater than the IgG control group.

Passage-to-passage comparison-placental-derived cells: placental-derived cells analyzed by flow cytometry at passage 8, 15, and 20 were positive for CD10, CD13, CD44, CD73, CD90, PDGFr-α, and HLA -A, HLA-B, HLA-C, as reflected by the increase in fluorescence value relative to the IgG control group. The CD31, CD34, CD45, CD117, CD141 and HLA-DR, HLA-DP, and HLA-DQ of these cells were negative, and had the same fluorescence value as the IgG control group.

Passage to Passage Comparison-Umbilical Derived Cells: Umbilical derived cells analyzed by flow cytometry at passage 8, 15, and 20 all express CD10, CD13, CD44, CD73, CD90, PDGFr-α and HLA- A, HLA-B, HLA-C, this is indicated by the increase in fluorescence relative to the IgG control group. These cells are negative for CD31, CD34, CD45, CD117, CD141 and HLA-DR, HLA-DP, HLA-DQ, which is indicated by the fluorescence value consistent with the IgG control group.

Donor-to-donor comparison-placenta-derived cells: analysis of the expression of CD10, CD13, CD44, CD73, CD90, PDGFr-α and HLA-A, HLA- by placental-derived cells isolated from different donors by flow cytometry B. HLA-C, which has an increased fluorescence value relative to the IgG control group. These cells were CD31, CD34, CD45, CD117, CD141 and negative for HLA-DR, HLA-DP, HLA-DQ, which is indicated by the fluorescence value consistent with the IgG control group.

Donor-to-donor comparison-umbilical derived cells: analysis of umbilical derived cells isolated from different donors by flow cytometry showed CD10, CD13, CD44, CD73, CD90, PDGFr-α and HLA-A, HLA- B. The positive performance of HLA-C is reflected in the increased fluorescence value relative to the IgG control group. The expression of CD31, CD34, CD45, CD117, CD141 and HLA-DR, HLA-DP, and HLA-DQ of these cells were negative, and they had the same fluorescence value as the IgG control group.

The effect of gelatin surface coating on placenta-derived cells: analysis of placenta-derived cells expanded on gelatin-coated or uncoated culture flasks by flow cytometry showed CD10, CD13, CD44, CD73, CD90, PDGFr -α and HLA-A, HLA-B, HLA-C, this is reflected in the increased fluorescence value relative to the IgG control group. These cells were CD31, CD34, CD45, CD117, CD141 and negative for HLA-DR, HLA-DP, HLA-DQ, which is indicated by the fluorescence value consistent with the IgG control group.

Effect of gelatin surface coating on umbilical-derived cells: analysis of umbilical-derived cells expanded on gelatin and uncoated culture flasks by flow cytometry showed positive expression CD10, CD13, CD44, CD73, CD90, PDGFr-α and HLA-A, HLA-B, and HLA-C have increased fluorescence values relative to the IgG control group. The expression of CD31, CD34, CD45, CD117, CD141 and HLA-DR, HLA-DP, and HLA-DQ of these cells were negative, and they had the same fluorescence value as the IgG control group.

Effect of enzymatic digestion procedures used to prepare cells on cell surface marker profiles: Placenta-derived cells isolated using various digestive enzymes analyzed by flow cytometry showed CD10, CD13, CD44, CD73, CD90, PDGFr-α and HLA-A, HLA-B, HLA-C, as indicated by the increase in fluorescence value relative to the IgG control group. These cells were negative for CD31, CD34, CD45, CD117, CD141 and HLA-DR, HLA-DP, HLA-DQ, as indicated by the fluorescence values consistent with the IgG control group.

Placental layer comparison: Cells isolated from the maternal, villi, and neonatal layers of the placenta analyzed by flow cytometry showed CD10, CD13, CD44, CD73, CD90, PDGFr-α, and HLA-A, HLA-B , The positive performance of HLA-C, as indicated by the increase in fluorescence value relative to the IgG control group. These cells were negative for CD31, CD34, CD45, CD117, CD141 and HLA-DR, HLA-DP, HLA-DQ, as indicated by the fluorescence values consistent with the IgG control group.

Summary: Flow cytometry analysis of placenta-derived cells and umbilical-derived cells has established the identity of these cell lines. Placenta-derived cells and umbilical-derived cells are positive for CD10, CD13, CD44, CD73, CD90, PDGFr-α, HLA-A, HLA-B, HLA-C, and are positive for CD31, CD34, CD45, CD117, CD141 and HLA -DR, HLA-DP, HLA-DQ negative. This identity is consistent among the variables including donor, sub-generation, culture vessel surface coating, digestive enzymes, and placental layer variation. Some variation in the average and range of individual fluorescence value histogram curves was observed, but all positive curves under all test conditions were normal and exhibited fluorescence values greater than the IgG control group, thus confirming that these cells contained A homogeneous group with positive expressions of these markers.

Example 17 Immunohistochemical characteristics of postpartum tissue phenotype

The phenotypes of cells found in human postpartum tissues (ie, umbilical cord and placenta) were analyzed by immunohistochemistry.

Methods and materials

Tissue preparation: Human umbilical cord and placental tissues were collected and immersed and fixed in 4% (w / v) paraformaldehyde at 4 ° C overnight. Immunohistochemistry was performed using antibodies against the following epitopes: vimentin (1: 500; Sigma, St. Louis, Mo.), intermycin (desmin, 1: 150, generated against rabbits; Sigma; or 1: 300, generated against mice; Chemic on, Temecula, Calif.), Α-smooth muscle actin (SMA; 1: 400; Sigma), cytokeratin 18 (CK18; 1: 400; Sigma), Feng Weili Factor (vWF; 1: 200; Sigma) and CD34 (human CD34 Class III; 1: 100; DAKOCytomation, Carpinteria, Calif). In addition, the following markers were tested: anti-human GROα-PE (1: 100; Becton Dickinson, Franklin Lakes, NJ), anti-human GCP-2 (1: 100; Santa Cruz Biotech, Santa Cruz, Calif), anti-human oxidized LDL Receptor 1 (ox-LDL R1; 1: 100; Santa Cruz Biotech) and anti-human NOGO-A (1: 100; Santa Cruz Biotech). The fixed sample was trimmed with a scalpel and then placed in an OCT embedding compound (Tissue-Tek OCT; Sakura, Torrance, Calif) on a dry ice bath containing ethanol. Next, the frozen embedded block was sliced (10 μm thick) using a standard cryostat (Leica Microsystems) and then fixed on a glass slide for staining.

Immunohistochemistry: Immunohistochemistry is performed similar to previous studies (for example, Messina et al., 2003, Exper. Neurol. 184: 816-829). Wash tissue sections with phosphate buffered saline (PBS) and then expose them to PBS, 4% (v / v) goat serum (Chemic on, Temecula, Calif) and 0.3% (v / v) Triton (Triton X-100 ; Sigma) protein blocking solution for 1 hour to obtain intracellular antigen. In the case where the epitope of interest will be located on the cell surface (CD34, ox-LDL R1), Triton is omitted in all steps of the procedure to avoid epitope loss. Furthermore, in the case where the primary antibody system was generated against goats (GCP-2, ox-LDL R1, NOGO-A), 3% (v / v) donkey serum was used in place of goat serum in the entire procedure. Next, primary antibodies (diluted in blocking solution) were applied to the slices at room temperature for 4 hours. The primary antibody solution was removed, the culture was washed with PBS, and then a secondary antibody solution (1 hour at room temperature) was applied. The secondary antibody solution contained a blocking agent and goat anti-mouse IgG--Texas Red (1: 250; Molecular Probes, Eugene, Oreg.) And / or goat anti-rabbit IgG-Alexa 488 (1: 250; Molecular Probes) or donkey anti-goat IgG-FITC (1: 150; Santa Cruz Biotech). The culture was washed, and then 10 micromolar DAPI (Molecular Probes) was applied for 10 minutes to visualize the nucleus.

After immunostaining, the fluorescence is visualized using an appropriate fluorescent filter on an Olympus inverted epifluorescence microscope (Olympus, Melville, N.Y.). The fluorescent signal higher than the staining of the control group represents positive staining. Use a digital color camera and ImagePro software (Media Cybernetics, Carlsbad, Calif.) To capture the table image. For triple-stained samples, only one emission filter was used to shoot each image at a time. Then use Adobe Photoshop software (Adobe, San Jose, Calif.) To prepare layered composite images (Layered montage).

result

Characterization of the umbilical cord: Vimentin, intermuscular fibroin, SMA, CK18, vWF, and CD34 markers are expressed in the subpopulations of cells found in the umbilical cord. Specifically, the performance of vWF and CD34 is limited to the blood vessels contained in the umbilical cord. The CD34 + cell line is located on the innermost layer (lumen side). Vimentin expression is found throughout the umbilical cord matrix and blood vessels. SMA is limited to the matrix and outer wall of arteries and veins, but the blood vessels themselves are not. CK18 and intermuscular fibroin were only observed in blood vessels, and intermuscular fibroin was limited to the middle and outer layers.

Characterization of the placenta: vimentin, intermuscular protein, SMA, CK18, vWF, and CD34 are all observed in the placenta and are region-specific.

GROα , GCP-2 , ox-LDL RI , and NOGO-A tissue performance: none of these markers were observed in the umbilical cord or placental tissue.

Summary: Cells in the human umbilical cord and placenta exhibit vimentin, intermuscular fibroin, α-smooth muscle actin, cytokeratin 18, von Willie's factor, and CD 34.

Example 18 Analysis of postpartum tissue-derived cells using oligonucleotide arrays

The Affymetrix GENECHIP array is used to compare gene expression profiles of umbilical-derived and placental-derived cells and fibroblasts, human mesenchymal stem cells, and another cell line derived from human bone marrow. This analysis provides the characteristics of postpartum-derived cells and unique molecular markers that identify these cells.

Methods and materials

Isolation and cultivation of cells: The human umbilical cord and placenta were obtained from the National Center for Disease Research and Exchange (NDRI, Philadelphia, Pa.), Which was derived from normal full-term delivery and obtained patient consent. The tissue was received and the cells were isolated as described in Example 14. Cells were cultured in growth medium (using DMEM-LG) on gelatin-coated tissue culture plastic culture flasks. The culture line was cultured at 37 ° C with 5% CO ₂ .

Human skin fibroblast cell lines were purchased from Cambrex Incorporated (Walkersville, Md .; batch number 9F0844) and ATCC CRL-1501 (CCD39SK). The two cell lines were cultured in DMEM / F12 medium (Invitrogen, Carlsbad, Calif.) Containing 10% (v / v) fetal bovine serum (Hyclone) and penicillin / streptomycin (Invitrogen). Allow cells to grow on standard tissue processing plastic.

Human mesenchymal stem cells (hMSC) were purchased from Cambrex Incorporated (Walkersville, Md .; batch numbers 2F1655, 2F1656 and 2F1657) and cultured in MSCGM medium (Cambrex) according to the manufacturer's specifications. The cells were grown on standard tissue culture plastics at 37 ° C with 5% CO ₂ .

The human iliac bone marrow system was received from NDRI and obtained patient consent. Bone marrow is processed according to the method outlined by Ho et al. (W003 / 025149). Bone marrow was mixed with lysis buffer (155 mM NH4Cl, 10 mM KHCO ₃ and 0.1 mM EDTA, pH 7.2), and the mixing ratio was 1 part bone marrow to 20 parts lysis buffer. The cell suspension was vortexed, incubated at ambient temperature for 2 minutes and then centrifuged at 500 ^x g for 10 minutes. The supernatant was discarded, and the cell pellet was resuspended in the minimum essential medium α (Invitrogen) supplemented with 10% (v / v) fetal bovine serum and 4 mM glutamic acid. The cells were centrifuged again and the cell pellet was resuspended in fresh medium. The trypan blue exclusion method (Sigma, St. Louis, Mo.) was used to count viable monocytes. Mononuclear cells were seeded in tissue culture plastic bottles at 5 × 10 ⁴ cells / cm ² . The cells were cultured at 37 ° C with 5% CO ₂ , standard atmospheric O _2, or 5% O ₂ . The cells were cultured for 5 days without medium replacement. After 5 days of cultivation, the medium and non-attached cells were removed. The adherent cells are maintained in culture.

Isolation of mRNA and GENECHIP analysis: Actively growing cell culture was removed from the culture flask in cold PBS using a cell spatula. Centrifuge the cells at 300 ^x g for 5 minutes. The supernatant was removed, then the cells were resuspended in fresh PBS and centrifuged again. The supernatant was removed, and immediately the cell pellet was frozen and stored at -80 ° C. Cell mRNA is extracted and transcribed into cDNA, which is then transcribed into cRNA and labeled with biotin. The biotin-labeled cRNA was hybridized to the HG-U133A GENECHIP oligonucleotide array (Affymetrix, Santa Clara, Calif.). Hybridization and data collection are performed according to the manufacturer's specifications. The analysis was performed using "Significance Analysis of Microarrays" (SAM) version 1.21 computer software (Stanford University; Tusher, VG et al., 2001, Proc. Natl. Acad. Sci. USA 98: 5116-5121).

result

Fourteen different cell populations were analyzed. Table 18-1 lists the cells and information on the subcultures, culture media and culture medium series.

The data was evaluated by Principle Component Analysis, which analyzed 290 genes with differential performance in cells. This analysis allows relative comparison of similarities between ethnic groups.

Table 18-2 shows the calculation of Euclidean distance for cell pair comparison. The Euclidean distance is based on the comparison of cells based on 290 genes, which have different performances among different cell types. The Euclidean distance is inversely proportional to the similarity between the performance of 290 genes (that is, the greater the distance, the less similarity exists).

Tables 18-3, 18-4, and 18-5 show increased performance in placenta-derived cells (Table 18-3), increased performance in umbilical-derived cells (Table 18-4), and decreased performance in umbilical and placenta-derived cells (Table 18-5) Genes. The column titled "Probe Set ID" refers to the manufacturer's identification codes for several oligonucleotide probe sets located at specific locations on the chip. These probe sets and the listed genes ("genes "Name" column) hybridization. These genes contain sequences that can be found in the NCBI (GenBank) database with the specified access number ("NCBI Access Number" column).

Tables 18-6, 18-7, and 18-8 show genes that increase expression in human fibroblasts (Table 18-6), ICBM cells (Table 18-7), and MSC (Table 18-8).

Summary: This test was performed to provide molecular characterization of postnatal cells derived from the umbilical cord and placenta. This analysis included cells derived from three different umbilical cords and three different placenta. The test also included two different skin fibroblast cell lines, three mesenchymal stem cell lines, and three iliac bone marrow cell lines. The mRNA expressed by these cells was analyzed using an oligonucleotide array containing probes of 22,000 genes. The results showed that 290 genes performed differently in these five different cell types. These genes include ten genes that are specifically increased in placenta-derived cells, and seven genes that are specifically increased in umbilical cord-derived cells. It was found that forty-four genes have specifically lower performance levels in the placenta and umbilical cord compared to other cell types. The performance of selected genes has been confirmed by PCR (see example below). These results prove that postpartum-derived cells have a unique gene expression profile, for example, compared to bone marrow-derived cells and fibroblasts.

Example 19 Cell markers for postpartum derived cells

In the foregoing examples, the similarity and difference of cells derived from human placenta and human umbilical cord were evaluated by comparing their gene performance profiles with those of cells derived from other sources (using oligonucleotide arrays). Identify six "signature" genes: oxidized LDL receptor 1, interleukin-8, rennet, endoplasmic reticulum protein, chemokine receptor ligand 3 (CXC ligand 3), and granularity Cell chemotactic protein 2 (GCP-2). These "signature" genes are expressed at a relatively high level in postpartum derived cells.

Perform the procedure described in this example to verify the microarray data and find the consistency / inconsistency between gene and protein performance, and establish a series of reliable tests for detecting the uniqueness of placenta-derived cells and umbilical-derived cells Identifier.

Methods and materials

Cells: Placenta-derived cells (three isolates, including one isolate identified by karyotyping as mainly neonates), umbilical-derived cells (four isolates), and normal human skin fibroblasts (NHDF; neonates And adults), grown in growth medium with penicillin / streptomycin in T75 bottles coated with gelatin. Mesenchymal stem cells (MSCS) lines were grown on the Mesothelial stem cell growth medium Bullet set (MSCGM; Cambrex, Walkerville, Md.).

As for the IL-8 protocol, cells were thawed from liquid nitrogen and seeded in gelatin-coated bottles at 5,000 cells / cm ² , grown in growth medium for 48 hours and then in 10 ml of serum starvation medium [DMEM-low Glucose (Gibco, Carlsbad, Calif.), Penicillin / streptomycin (Gibco, Carlsbad, Calif.) And 0.1% (w / v) bovine serum albumin (BSA; Sigma, St. Louis, Mo.)) Grow for 8 hours. After this treatment, RNA was extracted and the supernatant was centrifuged at 150 ^x g for 5 minutes to remove cell debris. The supernatant was then frozen at -80 ° C for ELISA analysis.

ELISA- tested cell culture: Post-natal cells derived from placenta and umbilicus, and human fibroblasts derived from human neonatal foreskin are cultured in growth medium in gelatin-coated T75 culture flasks. The cells were frozen in liquid nitrogen at 11th passage. Thaw the cells and transfer to a 15 ml centrifuge tube. After centrifugation at 150 ^x g for 5 minutes, the supernatant was discarded. The cells were resuspended in 4 ml of medium and counted. The cells were grown at 375,000 cells / flask in a 75 cm ² flask containing 15 ml of growth medium for 24 hours. The medium was replaced with serum starvation medium for 8 hours. At the end of the culture, centrifuge at 14,000 ^x g for 5 minutes to collect serum starvation medium (and stored at -20 ° C).

In order to estimate the number of cells in each flask, 2 mL of trypsin / EDTA (Gibco, Carlsbad, Calif) was added to each flask. After the cells were detached from the culture flask, trypsin activity was neutralized with 8 ml of growth medium. Transfer cells to a 15 ml centrifuge tube and centrifuge at 150 ^x g for 5 minutes. The supernatant was removed and 1 ml of growth medium was added to each tube to resuspend the cells. Estimate the cell number using a hemocytometer.

ELISA assay: The amount of IL-8 secreted from cells to serum starvation medium was analyzed using ELISA assay (R & D Systems, Minneapolis, Minn.). All verifications are tested according to the instructions provided by the manufacturer.

Total RNA isolation: RNA is extracted from overgrown postpartum-derived cells and fibroblasts, or in terms of IL-8 performance from cells treated as described above. The cells were lysed with 350 μl of buffer RLT containing β-mercaptoethanol (Sigma, St. Louis, Mo.) according to the manufacturer's instructions (RNeasy ^® Mini Kit; Qiagen, Valencia, Calif). RNA was extracted according to the manufacturer's instructions (RNeasy ^® Mini Kit; Qiagen, Valencia, Calif) and subjected to DNase treatment (2.7 U / sample) (Sigma St. Louis, Mo.). RNA was extracted with 50 microliters of DEPC-treated water and stored at -80 ° C.

Reverse transcription: RNA is also extracted from the human placenta and navel. The tissue (30 mg) was suspended in 700 microliters of 2-mercaptoethanol buffer RLT. The samples were mechanically homogenized and RNA extraction was performed according to the manufacturer's specifications. RNA was extracted with 50 microliters of DEPC-treated water and stored at -80 ° C. Using random hexamers reverse transcription reagents to TaqMan ^® (Applied Biosystems, Foster City, Calif .) By reverse transcription of RNA, anti-transcribed over at 25 ℃ 10 minutes duration at 37 ℃ 60 minutes and at 95 deg.] C over 10 minutes. Store samples at -20 ° C.

Identification of genes uniquely regulated in postpartum cells by cDNA microarrays (signature genes--including oxidized LDL receptors, interleukin-8, rennet, and endoplasmic reticulum proteins) was further performed using real-time and traditional PCR the study.

Real-time PCR : Use Assays-on-Demand ^® gene expression products to perform PCR on cDNA samples: use the 7000 sequence detection system (Applied Biosystems, Foster City, Calif.) With ABI Prism 7000 SDS software, according to the manufacturer ’s instructions (Applied Biosystems, Foster City, Calif.), Will oxidize LDL receptor (Hs00234028); rennet (Hs00166915); endoplasmic reticulum protein (Hs003825 15); CXC ligand 3 (Hs00171061); GCP-2 (Hs00605742); IL -8 (Hs00174103); (. Applied Biosystems, Foster City, Calif) , and mixed with GAPDH cDNA master mix and TaqMan ^® Universal PCR. The thermal cycle conditions were to start 50 ° C for 2min and 95 ° C for 10min, followed by 40 cycles of 95 ° C for 15sec and 60 ° C for 1min. Analyze the PCR data according to the manufacturer's specifications (Applied Biosystems user notice # 2 on the ABI Prism 7700 sequence detection system).

Traditional PCR : ABI PRISM 7700 (Perkin Elmer Applied Biosystems, Boston, Mass., USA) was used to perform traditional PCR to confirm the results of real-time PCR. PCR was performed using 2 microliters of cDNA solution, 1 ^x AmpliTaq Gold Universal Mix PCR reaction buffer (Applied Biosystems, Foster City, Calif.) And initial denaturation at 94 ° C for 5 minutes. The amplification line is optimized for each primer set. For IL-8, CXC ligand 3, and endoplasmic reticulum protein (15 seconds at 94 ° C, 15 seconds at 55 ° C, and 30 seconds at 72 ° C for 30 cycles); for rennet (15 seconds at 94 ° C, 53 ° C for 15 seconds and 72 ° C for 30 seconds for 38 cycles); for oxidized LDL receptor and GAPDH (94 ° C for 15 seconds, 55 ° C for 15 seconds and 72 ° C for 30 seconds for 33 cycles). The primers used for amplification are listed in Table 11-1. The primer concentration in the final PCR reaction was 1 micromolar, except for GAPDH which was 0.5 micromolar. The GAPDH primer is the same as real-time PCR, except that the manufacturer's TaqMan ^® probe is not added to the final PCR reaction. The samples were run on a 2% (w / v) agarose gel and stained with ethidium bromide (Sigma, St. Louis, Mo.). Images were captured using a 667 Universal Twinpack film (VWR International, South Plainfield, NJ) and a focal length Polaroid camera (VWR International, South Plainfield, NJ).

Immunofluorescence: PPDC was fixed with cold 4% (w / v) paraformaldehyde (Sigma-Aldrich, St. Louis, Mo.) at room temperature for 10 minutes. One isolate using the umbilical-derived cells and placenta-derived cells at passage 0 (P0) (used directly after isolation), and the passage 11 (P 11) (two placenta-derived cells) Isolates, two umbilical-derived cell isolates) and fibroblasts (P 11). Immunocytochemistry was performed using antibodies against the following epitopes: vimentin (1: 500, Sigma, St. Louis, Mo.), intermyosin (1: 150; Sigma--generated against rabbits; or 1: 300 ; Chemicon, Temecula, Calif-produced against mice), α-smooth muscle actin (SMA; 1: 400; Sigma), cytokeratin 18 (CK18; 1: 400; Sigma), von Willie ’s factor (vWF ; 1: 200; Sigma) and CD34 (human CD34 Class III; 1: 100; DAKOCytomation, Carpinteria, Calif). In addition, the following markers were tested on 11 post-natal cells: anti-human GRO α-PE (1: 100; Becton Dickinson, Franklin Lakes, NJ), anti-human GCP-2 (1: 100; Santa Cruz Biotech, Santa Cruz , Calif), anti-human oxidized LDL receptor 1 (ox-LDL R1; 1: 100; Santa Cruz Biotech) and anti-human NOGA-A (1: 100; Santa Cruz, Biotech).

The culture was washed with phosphate buffered saline (PBS) and then exposed to PBS, 4% (v / v) goat serum (Chemic on, Temecula, Calif) and 0.3% (v / v) Triton (Triton X-100 ; Sigma, St. Louis, Mo.) protein blocking solution for 30 minutes to obtain intracellular antigens. When the epitope of interest is located on the cell surface (CD34, ox-LDL R1), Triton X-100 in all steps of the procedure is omitted to avoid epitope loss. Furthermore, in the case where the primary anti-system is generated against goats (GCP-2, ox-LDL R1, NOGO-A), 3% (v / v) donkey serum will be used instead of goat serum throughout. Next, primary antibody (diluted in blocking solution) was applied to the culture at room temperature for 1 hour. The primary antibody solution was removed, the culture was washed with PBS, and then a secondary antibody solution (1 hour at room temperature) was applied. The secondary antibody solution contained a blocking agent and goat anti-mouse IgG--Texas Red (1: 250; Molecular Probes, Eugene, Oreg.) And / or goat anti-rabbit IgG-Alexa 488 (1: 250; Molecular Probes) or donkey anti-goat IgG-FITC (1: 150, Santa Cruz Biotech). The culture was subsequently washed, and then 10 micromolar DAPI (Molecular Probes) was applied for 10 minutes to visualize the nucleus.

After immunostaining, use an appropriate fluorescent filter on an Olympus® inverted epifluorescence microscope (Olympus, Melville, N.Y.) to visualize the fluorescence. In all cases, positive staining represented a fluorescent signal higher than that of the control stain, in which all procedures outlined above were followed, except for the application of primary antibody solution. Use a digital color camera and ImagePro® software (Media Cybernetics, Carlsbad, Calif) to capture the table image. For triple-stained samples, only one emission filter was used to shoot each image at a time. Then use Adobe Photoshop® software (Adobe, San Jose, Calif) to prepare layered composite images (Layered montage).

Cells were prepared for FACS analysis: adherent cells in culture flasks were washed in phosphate buffered saline (PBS) (Gibco, Carlsbad, Calif) and desorbed with trypsin / EDTA (Gibco, Carlsbad, Calif). The cells were collected, centrifuged, and resuspended in 3% (v / v) FBS in PBS at a cell concentration of 1 × 10 7 per ml. One hundred microliter aliquots were delivered into conical tubes. The stained cell lines with intracellular antigens were penetrated with Perm / Wash buffer (BD Pharmingen, San Diego, Calif). The antibody was added to aliquots according to the manufacturer's specifications and the cells were incubated at 4 ° C in the dark for 30 minutes. After incubation, the cells were washed with PBS and centrifuged to remove excess antibody. Resuspend cells that require secondary antibody in 100 μl of 3% FBS. Secondary antibodies were added according to the manufacturer's specifications and the cells were incubated in the dark at 4 ° C for 30 minutes. After incubation, the cells were washed with PBS and centrifuged to remove excess secondary antibody. The washed cells were resuspended in 0.5 ml PBS and analyzed by flow cytometry. The following antibodies were used: oxidized LDL receptor 1 (sc-5813; Santa Cruz, Biotech), GROa (555042; BD Pharmingen, Bedford, Mass.), Mouse IgG1κ (P-4685 and M-5284; Sigma), donkey anti Goat IgG (sc-3743; Santa Cruz, Biotech.). Flow cytometry analysis was performed with FACScalibur ^™ (Becton Dickinson San Jose, Calif.).

result

The results of real-time PCR for selected "signature" genes performed on cDNA from cells derived from human placenta, adult and neonatal fibroblasts and mesenchymal stem cells (MSC) indicate that the oxidized LDL receptor and curd Both enzymes perform at a higher level in placenta-derived cells than other cells. The data obtained from real-time PCR was analyzed by the AACT method and expressed on a log scale. The expression levels of endoplasmic reticulum protein and oxidized LDL receptor in umbilical cord-derived cells are higher than those in other cells. No significant differences were found in the performance levels of CXC ligand 3 and GCP-2 between the postpartum-derived cells and the control group. The results of real-time PCR are confirmed by conventional PCR. The sequencing of PCR products further validates these observations. Using the conventional PCR CXC ligand 3 primers listed in Table 19-1 above, it was found that there was no significant difference in the performance level of CXC ligand 3 between postpartum-derived cells and the control group.

The production of postpartum cytokine IL-8 is increased in both the postpartum-derived cells cultured in growth medium and serum-starved. All real-time PCR data is verified using conventional PCR and by sequencing PCR products.

When detecting the presence of IL-8 in the supernatant of cells grown in serum-free medium, the highest amount was detected in the culture medium of some isolates derived from umbilical cells and placental cells (Table 19-2). IL-8 was not detected in the culture medium derived from human skin fibroblasts.

The production of oxidized LDL receptor, GCP-2 and GROα in placental-derived cells was also detected by FACS analysis. The tested cells were positive for GCP-2. This method did not detect oxidized LDL receptor and GRO.

The production of selected proteins of placenta-derived cells is also tested by immunocytochemical analysis. Immediately after isolation (subsequence 0), cells derived from human placenta were fixed with 4% paraformaldehyde and exposed to antibodies against six proteins: von Willie's factor, CD34, cytokeratin 18, intermycin, α-smooth muscle actin and vimentin. The stained cells were positive for α-smooth muscle actin and vimentin. This mode remains until generation 11. At passage 0, only some cells (<5%) were positive for cytokeratin 18 staining.

Cells derived from human umbilical cord were probed for the production of selected proteins by immunocytochemical analysis at 0 generations. Immediately after isolation (passage 0), the cells were fixed with 4% paraformaldehyde and exposed to antibodies against six proteins: von Willich's factor, CD34, cytokeratin 18, intermuscular protein, α-smooth muscle actin Protein, and vimentin. Umbilical-derived cells were positive for α-smooth muscle actin and vimentin, and the staining pattern remained consistent until passage 11.

Summary : For the following four genes, the consistency between the gene expression levels measured by microarray and PCR (both real-time and traditional) has been established: oxidized LDL receptor 1, rennet, endoplasmic reticulum protein , And IL-8. The expression of these genes is differentially regulated at the mRNA level in PPDC, and IL-8 is also differentially regulated at the protein level. By FACS analysis, the presence of oxidized LDL receptors was not detected at the protein level in cells derived from the placenta. By FACS analysis, the differential performance of GCP-2 and CXC ligand 3 in placenta-derived cells at the mRNA level was not confirmed, but GCP-2 was detected at the protein level. Although this result is not reflected in the data originally obtained from the microarray experiment, it may be due to the different sensitivity of the method.

Immediately after isolation (Sub 0), cells derived from human placenta were stained positive for α-smooth muscle actin and vimentin. This pattern was also observed in cells of passage 11. Vimentin and α-smooth muscle actin expression can be retained in the subcultured cells (in the growth medium and under the conditions utilized in these procedures). The cell lines derived from human umbilical cord at passage 0 were tested for alpha-smooth muscle actin and vimentin, and both were positive. This staining pattern remains until generation 11.

Example 20 In vitro immune evaluation of postpartum-derived cells

The immune characteristics of postpartum-derived cells (PPDC) are evaluated in vitro to predict the immune response (if any) that these cells may elicit after transplantation in vivo. The presence of HLA-DR, HLA-DP, HLA-DQ, CD80, CD86, and B7-H2 in PPDC was verified by flow cytometry. These proteins are expressed by antigen presenting cells (APe) and are required for direct stimulation of naïve CD4 + T cells (Abbas and Lichtman, CELLULAR AND MOLECULAR IMMUNOLOGY, 5th edition (2003) Saunders, Philadelphia, p. 171). Cell lines were also analyzed by flow cytometry for HLA-G (Abbas and Lichtman, 2003, see above), CD 178 (Coumans et al., (1999) Journal of Immunological Methods 224, 185-196), and PD-L2 ( Abbas and Lichtman, 2003, see above; Brown et al. (2003) The Journal of Immunology, 170: 1257-1266). It is believed that these proteins mediated by the cells present in the placental tissues mediate the immune exemption status of the intrauterine placental tissues. To predict the extent to which the placenta and umbilical-derived cell lines elicit an immune response in vivo, the cell lines were tested in a one-way mixed lymphocyte reaction (MLR).

Methods and materials

Cell culture: Cells were grown to fullness in growth medium containing penicillin / streptomycin in T75 flasks (Corning Inc., Corning, N.Y.) coated with 2% gelatin (Sigma, St. Louis, Mo.).

Antibody staining: cells were washed in phosphate buffered saline (PBS) (Gibco, Carlsbad, Calif.) And desorbed with trypsin / EDTA (Gibco, Carlsbad, Mo.). The cells were collected, centrifuged, and resuspended in 3% (v / v) FBS in PBS at a cell concentration of 1 × 10 ⁷ per ml. The antibody (Table 20-1) was added to one hundred microliters of cell suspension according to the manufacturer's specifications, and incubated at 4 ° C in the dark for 30 minutes. After incubation, the cells were washed with PBS and centrifuged to remove unbound antibody. The cells were resuspended in five hundred microliters of PBS and analyzed by flow cytometry using a FACSCalibur ^™ instrument (Becton Dickinson, San Jose, Calif.).

Mixed lymphocyte reaction: Freeze vials of 10 umbilical-derived cells labeled as cell line A and 11 placental-derived cells labeled as cell line B to CTBR (Senneville, Quebec) on dry ice to use CTBR SOP No.CAC-031 Perform mixed lymphocyte reaction. Peripheral blood mononuclear cells (PBMC) were collected from multiple male and female voluntary donors. The stimulator (donor) allogeneic PBMC, autologous PBMC, and postpartum cell lines were treated with mitomycin C. Autologous cells and stimulator cells treated with mitomycin C were added to the responder (recipient) PBMC and cultured for 4 days. After incubation, [ ³ H] -thymidine was added to each sample and incubated for 18 hours. After collecting the cells, the radiolabeled DNA was extracted, and [ ³ H] -thymidine incorporation was measured using a scintillation counter.

The allogeneic donor stimulation index (SIAD) is calculated as the average hyperplasia of the recipient plus the average hyperplasia of the allogeneic donor treated with mitomycin C divided by the baseline hyperplasia of the recipient. The PPDC stimulation index is calculated as the average proliferation of the recipient plus the average proliferation of the postpartum cell line treated with mitomycin C divided by the baseline proliferation of the recipient.

result

Mixed Lymphocyte Response-Placenta-derived cells: Seven human voluntary blood donors were screened to identify a single allogeneic donor who would exhibit a solid hyperplastic response when mixed lymphocyte response was performed with six other blood donors This donor was selected as the allogeneic positive control group donor. The remaining six blood donors were selected as recipients. Allogeneic positive control donors and placenta-derived cell lines were treated with mitomycin C and cultured in a mixed lymphocyte reaction with six individual allogeneic recipients. The reaction system was repeated three times using two cell culture plates, each with three recipients (Table 20-2). The average stimulation index ranged from 1.3 (disc 2) to 3 (disc 1), and the positive control group of allogeneic donors ranged from 46.25 (disc 2) to 279 (disc 1) (Table 20-3).

Mixed Lymphocyte Response-Umbilical Derived Cells: Six human voluntary blood donors were screened to identify a single allogeneic donor who exhibited a solid hyperplastic response when mixed lymphocyte responses were performed with five other blood donors. This donor was selected as the allogeneic positive control group donor. The remaining five blood donors were selected as recipients. Allogeneic positive control donors and placental cell lines were treated with mitomycin C and cultured in a mixed lymphocyte reaction with five individual allogeneic recipients. The reaction system was repeated three times using two cell culture plates, each with three recipients (Table 12-4). The average stimulation index ranged from 6.5 (disc 1) to 9 (disc 2), and the positive control group of allogeneic donors ranged from 42.75 (disc 1) to 70 (disc 2) (Table 12-5).

Table 20-5. Umbilical cord-derived cells and allogeneic donors are connected to five individual allogeneic

Antigen-presenting cell markers-placenta-derived cells: Histograms of placenta-derived cells analyzed by flow cytometry showed negative performance of HLA-DR, HLA-DP, HLA-DQ, CD80, CD86, and B7-H2 As indicated by the fluorescence values consistent with the IgG control group, it indicates that the placental cell line lacks the cell surface molecules required to directly stimulate CD4 + T cells.

Immunomodulatory markers-placenta-derived cells: Histograms of placenta-derived cells analyzed by flow cytometry showed a positive manifestation of PD-L2, as illustrated by the increase in fluorescence relative to the IgG control group, and CD178 And the negative performance of HLA-G, as illustrated by the fluorescence value consistent with the IgG control group.

Antigen-presenting cell markers-umbilical derived cells: Histogram of umbilical derived cells analyzed by flow cytometry showed negative performance of HLA-DR, HLA-DP, HLA-DQ, CD80, CD86, and B7-H2 As indicated by the fluorescence values consistent with the IgG control group, it indicates that the umbilical cell line lacks the cell surface molecules required to directly stimulate CD4 + T cells.

Immune Regulatory Cell Marker-Umbilical-derived cells: The histogram of umbilical-derived cells analyzed by flow cytometry shows a positive performance of PD-L2, as illustrated by the increase in fluorescence value relative to the IgG control group, and The negative performance of CD178 and HLA-G was explained by the fluorescence values consistent with the IgG control group.

Summary: In the mixed lymphocyte reaction with placenta-derived cell lines, the average stimulation index ranged from 1.3 to 3, and the allogeneic positive control group had an average stimulation index range from 46.25 to 279. In the mixed lymphocyte reaction with the umbilical-derived cell line, the average stimulation index ranged from 6.5 to 9, and the allogeneic positive control group had an average stimulation index ranging from 42.75 to 70. The placenta and umbilical-derived cell lines are negative for the stimulation proteins HLA-DR, HLA-DP, HLA-DQ, CD80, CD86, and B7-H2, as measured by flow cytometry. The placenta and umbilical derived cell lines were negative for the immunomodulatory proteins HLA-G and CD178 and positive for PD-L2, as measured by flow cytometry. Allogeneic donor PBMC contains antigen-presenting cells expressing HLA-DR, HLA-DQ, CD8, CD86, and B 7-H2, thereby allowing the stimulation of pure CD4 + T cells. There is no antigen present on the placenta and umbilical-derived cells that directly stimulates pure CD4 + T cells to present cell surface molecules and the presence of the immunomodulatory protein PD-L2. The index is low.

Example 21 Nutritional factors secreted by postpartum derived cells

The secretion of selected nutrient factors from placenta and umbilical derived cells was measured. Selected factors detected include: (1) Factors known to have angiogenic activity, such as hepatocyte growth factor (HGF) (Rosen et al. (1997) Ciba Found. Symp. 212: 215-26), monocytes Chemotactic protein 1 (MCP-1) (Salcedo et al. (2000) Blood 96; 34-40), interleukin-8 (IL-8) (Li et al. (2003) J. Immunol. 170: 3369-76 ), Keratinocyte growth factor (KGF), basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF) (Hughes et al. (2004) Ann. Thorac. Surg. 77: 812-8), matrix metal Protease 1 (TIMP1), angiopoietin 2 (ANG2), platelet-derived growth factor (PDGF-bb), thrombopoietin (TPO), heparin-binding epidermal growth factor (HB-EGF), matrix-derived factor 1α (SDF-1α ); (2) Factors known to have neurotrophic / neuroprotective activity, such as brain-derived neurotrophic factor (BDNF) (Cheng et al. (2003) Dev. Biol. 258; 319-33), interleukin-6 ( IL-6), Granular Cell Chemotactic Protein-2 (GCP-2), Transforming Growth Factor β2 (TGFβ2); and (3) Factors known to have chemokine activity, such as macrophage inflamed protein 1α ( MIP1a), macrophage inflammation protein 1 β (MIP1b), monocyte chemokine-1 (MCP-1), Rantes (regulatory activation, normal T cell expression and secretion), I309, thymus and activation-regulated chemokine (TARe), erythropoietin ( Eotaxin), macrophage-derived chemokine (MDC), IL-8.

Methods and materials

Cell culture: PPDC from placenta and umbilical cord and human fibroblasts derived from human neonatal foreskin are cultured in growth medium with penicillin / streptomycin in gelatin-coated T75 flasks. The cells were frozen at 11 o'clock and stored in liquid nitrogen. After thawing the cells, the growth medium was added to the cells, then transferred to a 15 ml centrifuge tube and the cells were centrifuged at 150 ^x g for 5 minutes. Discard the supernatant. The cell pellet was resuspended in 4 ml of growth medium, and the cells were counted. The cells were seeded at 375,000 cells / 75 cm ² in a culture flask containing 15 ml of growth medium and cultured for 24 hours. The medium was replaced with serum-free medium (DMEM-low glucose (Gibco), 0.1% (w / v) bovine serum albumin (Sigma), penicillin / streptomycin (Gibco)) for 8 hours. At the end of the culture, centrifuge at 14,000 ^x g for 5 minutes to collect serum-free conditioned medium, and then store at -20 ° C.

To assess the number of cells in each flask, wash the cells with PBS and then use 2 ml of trypsin / EDTA for desorption. Trypsin activity was inhibited by adding 8 ml of growth medium. Centrifuge the cells at 150 ^x g for 5 minutes. The supernatant was removed and the cells were resuspended in 1 ml of growth medium. Estimate the cell number using a hemocytometer.

ELISA test: cells are grown in 5% carbon dioxide and atmospheric oxygen at 37 ° C. Placental-derived cells (batch 101503) were also grown in 5% oxygen or β-mercaptoethanol (BME). The amount of MCP-1, IL-6, VEGF, SDF-1α, GCP-2, IL-8 and TGF-β2 produced by each cell sample was measured by ELISA (R & D Systems, Minneapolis, Mn.). All verifications are performed according to the manufacturer's instructions.

SearchLight ^TM multiplex ELISA test: chemokines (MIP1a, MIP1b, MCP-1, Rantes, 1309, TARC, Eotaxin, MDC, IL8), BDNF, and angiogenic factors (HGF, KGF, bFGF , VEGF, TIMP1, ANG2, PDGF-bb, TPO, HB-EGF) were measured using searchLight ^™ Proteome Arrays (Pierce Biotechnology Inc.). Proteome Arrays is a multiplex sandwich ELISA for quantitative measurement of two to 16 proteins per well. Arrays were produced by spotting four to 16 different capture antibodies into each well of a 96-well plate in a 2 × 2, 3 × 3, or 4 × 4 pattern. After the sandwich ELISA procedure, the entire disk was imaged to capture the chemiluminescent signal generated at each spot in each well on the disk. The amount of signal generated at each spot is proportional to the amount of target protein in the original standard or sample.

result

ELISA test: MCP-1 and IL-6 lines are secreted by placental-derived cells, umbilical-derived cells, and skin fibroblasts (Table 21-1). SDF-1α is secreted by placental-derived cells and fibroblasts cultured in 5% O ₂ . GCP-2 and IL-8 are secreted by navel-derived cells and placental-derived cells cultured in the presence of BME or 5% O ₂ . GCP-2 is also secreted by human fibroblasts. TGF-β2 was not detected by ELISA test.

SearchLight ^TM multiplex ELISA test: TIMP1, TPO, KGF, HGF, FGF, HBEGF, BDNF, MIP1b, MCP1, RANTES, I309, TARC, MDC, and IL-8 are all secreted from umbilical derived cells (Table 21-2 and Table 21-3). TIMP1, TPO, KGF, HGF, HBEGF, BDNF, MIP1a, MCP-1, RANTES, TARC, Eotaxin, and IL-8 are all secreted from placental-derived cells (Tables 21-2 and 21-3). No Ang2, VEGF or PDGF-bb were detected.

Example 22 Short-term neural differentiation of postpartum-derived cells

The ability of placenta and umbilical derived cells (collectively called postpartum derived cells or PPDC) to differentiate into neural lineage cells was tested.

Methods and materials

Isolation and expansion of postpartum cells: PPDCs from placenta and umbilical tissue were isolated and expanded as described in Example 14.

Modified Woodbury-Black procedure (A) : This test is adapted from the test that was originally performed to test the nerve-inducing potential of bone marrow stromal cells (1). Umbilical-derived cells (022803) P4 and placenta-derived cells (042203) P3 were thawed and culture-expanded at 5,000 cells / cm ² in growth medium until reaching sub-fullness (75%). The cells were then trypsinized and seeded on Titretek II slides (VWR International, Bristol, Conn.) At 6,000 cells per well. In the control group, mesenchymal stem cells (P3; 1F2155; Cambrex, Walkersville, Md.), Osteoblasts (P5; CC2538; Cambrex), fat-derived cells (Artecel, US Patent No. 6,555,374 B1) (P6; donor 2) Inoculated under the same conditions as neonatal human skin fibroblasts (P6; CC2509; Cambrex).

Initially all cells were expanded for 4 days in DMEM / F12 medium (Invitrogen, Carlsbad, Calif.) Containing 15% (v / v) fetal bovine serum (FBS; Hyclone, Logan, Utah), basic fiber mother Cell growth factor (bFGF; 20 ng / ml; Peprotech, Rocky Hill, NJ), epidermal growth factor (EGF; 20 ng / ml; Peprotech) and penicillin / streptomycin (Invitrogen). After four days, the cells were rinsed in phosphate buffer (PBS; Invitrogen) and then cultured in DMEM / F12 medium + 20% (v / v) FBS + penicillin / streptomycin for 24 hours. After 24 hours, cells were rinsed with PBS. The cells were then cultured for 1 to 6 hours in an induction medium containing DMEM / FI2 (serum-free) containing 200 mM butylated hydroxymethoxybenzene, 10 μM potassium chloride, 5 mg / ml insulin, and 10 μM forskolin ( forskolin), 4 μM valproic acid, and 2 μM hydrocorticosterone (all chemicals are from Sigma, St. Louis, Mo.). Cells were then fixed in 100% ice-cold methanol and immunocytochemistry (see method below) was performed to evaluate human nestin performance.

Modified Woodbury-Black protocol (B) : PPDC (umbilical (022803) P11; placental (042203) P11) and adult human skin fibroblasts (1F1853, P11) are thawed and cultured at 5,000 cells / cm ² Amplify in growth medium until reaching sub-fullness (75%). The cells were then trypsinized and seeded at a similar density as in (A), but inoculated to (1) 24-well tissue culture treated discs (TCP, Falcon brand, VWR International), (2) TCP wells + 2% (w / v) gelatin (adsorbed for 1 hour at room temperature), or (3) TCP well +20 μg / ml for adsorbed mouse laminin (adsorbed at 37 ° C for a minimum of 2 hours; Invitrogen).

As in (A), the cells were initially expanded and the medium was switched in the aforementioned time frame. A group of cultures was fixed as described above at 5 days and 6 hours, but this time using ice-cold 4% (w / v) paraformaldehyde (Sigma) at room temperature for 10 minutes. In the second set of cultures, the medium was removed and converted to neural precursor expansion medium (NPE), which consisted of Neurobasal-A medium (Invitrogen), which contained B27 (B27 supplement; Invitrogen ), L-glutamic acid (4mM), and penicillin / streptomycin (Invitrogen). The NPE medium was further supplemented with retinoic acid (RA; 1 μM; Sigma). After 4 days, the medium was removed, and the culture was fixed with ice-cold 4% (w / v) paraformaldehyde (Sigma) for 10 minutes at room temperature, and stained for nestin, GFAP, and TuJ1 protein expression (See Table 22-1).

Two-stage differentiation protocol: PPDC (umbilical (042203) P11, placental (022803) P11), adult human skin fibroblasts (P11; 1F1853; Cambrex) are thawed and cultured at 5,000 cells / cm ² in growth medium Medium until reaching the second overgrown (75%). The cells were then trypsinized and seeded at 2,000 cells / cm ² , but tied to a 24-well laminin-coated disk (BD Biosciences, Franklin Lakes, NJ) inoculated with NPE medium supplemented with this NPE medium There are bFGF (20 ng / ml; Peprotech, Rocky Hill, NJ) and EGF (20 ng / ml; Peprotech) [the entire medium composition is further referred to as NPE + F + E]. At the same time, the adult rat neural precursor (P4; 062603) isolated from the hippocampus was also inoculated into a 24-well laminin-coated disk (in NPE + F + E medium). All cultures were maintained under such conditions for a period of 6 days (the cells were fed once during this period), at which time the medium was switched to the differentiation conditions listed in Table 14-2 for a period of another 7 days. Cultures were fixed with ice-cold 4% (w / v) paraformaldehyde (Sigma) for 10 minutes at room temperature, and stained for rat nestin, GF AP, and TuJ1 protein performance.

Multiple growth factor protocol: Umbilical derived cells (P11; (042203)) were thawed and cultured at 5,000 cells / cm ² in growth medium until reaching the second fullness (75%). The cells were then trypsinized and seeded at 2,000 cells / cm ² into a 24-well laminin-coated dish (BD) in the presence of NPE + F (20 ng / ml) + E (20 ng / ml) Biosciences). In addition, some cells contain NPE + F + E + 2% FBS or 10% FBS. After four days of "pre-differentiation" conditions, all media was removed and the samples were converted to NPE media supplemented with acoustic hedgehog (SHH; 200 ng / ml; Sigma, St. Louis, Mo. ), FGF8 (100 ng / ml; Peprotech), BDNF (40 ng / ml; Sigma), GDNF (20 ng / ml; Sigma), and retinoic acid (1 μM; Sigma). Seven days after the medium change, the culture was fixed with ice-cold 4% (w / v) paraformaldehyde (Sigma) for 10 minutes at room temperature, and targeted to human nestin, GFAP, TuJ1, intermyosin, and alpha -Staining of smooth muscle actin performance.

Neural precursor co-cultivation protocol: Inoculate adult rat hippocampus hippocampus precursor (062603) with neurospheres or single cells (10,000 cells / well) onto NPE + F (20 Nanograms / ml) + E (20 nanograms / ml).

Separately, umbilical-derived cells (042203) P11 and placenta-derived cells (022803) P11 were thawed and cultured and expanded at 5,000 cells / cm ² in NPE + F (20 ng / ml) + E (20 ng / ml) ) In the 48-hour period. The cells were then trypsinized and seeded at 2,500 cells / well onto existing neural precursor cultures. At this time, the existing medium is exchanged for fresh medium. Four days later, the culture was fixed with ice-cold 4% (w / v) paraformaldehyde (Sigma) for 10 minutes at room temperature and stained for human nuclear protein (hNuc; Chemicon) (Table 14-1 above) ) To identify PPDC.

Immunocytochemistry: Immunocytochemistry was performed using the antibodies listed in Table 14-1. The culture was washed with phosphate buffered saline (PBS) and then exposed to PBS, 4% (v / v) goat serum (Chemicon, Temecula, Calif) and 0.3% (v / v) Triton (Triton X-100; Sigma) protein blocking solution for 30 minutes to obtain intracellular antigens. Next, primary antibody (diluted in blocking solution) was applied to the culture at room temperature for 1 hour. Next, the primary antibody solution was removed, the culture was washed with PBS, and then a secondary antibody solution (1 hour at room temperature) was applied. The secondary antibody solution contained a blocking solution and goat anti-mouse IgG--Texas Red ( 1: 250; Molecular Probes, Eugene, OR) and goat anti-rabbit IgG-Alexa 488 (1: 250; Molecular Probes). The culture was subsequently washed, and then 10 micromolar DAPI (Molecular Probes) was applied for 10 minutes to visualize the nucleus.

After immunostaining, the fluorescence is visualized using an appropriate fluorescent filter on an Olympus inverted epifluorescence microscope (Olympus, Melville, N.Y.). In all cases, positive staining represented a fluorescent signal higher than that of the control stain, in which all procedures outlined above were followed, except for the application of primary antibody solution. Use a digital color camera and ImagePro software (Media Cybernetics, Carlsbad, Calif) to capture the table image. For triple-stained samples, only one emission filter was used to shoot each image at a time. Then use Adobe Photoshop software (Adobe, San Jose, Calif) to prepare layered composite images (Layered montage).

result

Modified Woodbury-Black protocol (A) : After culturing in this nerve-inducing composition, all cell types are transformed into cells with bipolar morphology and elongated processes. Other larger non-bipolar patterns have also been observed. In addition, the induced cell population was positive for nestin staining, and nestin was a marker for pluripotent neural stem cells and precursor cells.

Modified Woodbury-Black protocol (B) : When repeated on a tissue culture plastic (TCP) plate, no nestin performance was observed, unless laminin was pre-adsorbed to the culture surface. To further assess whether nestin-expressing cells can subsequently continue to produce mature neurons, PPDC and fibroblasts were exposed to NPE + RA (1 μM), which is known to induce neural stem cells and precursor cells to differentiate into such cells (2 , 3, 4) medium composition. The cells were stained for TuJ1 (marker of immature and mature neurons), GFAP (marker of stellate cells), and nestin. TuJ1 was not detected under such conditions, and cells with neuronal morphology were not observed. In addition, PPDC no longer expressed nestin and GF AP, as determined by immunocytochemistry.

Two-stage differentiation: inoculate the umbilical and placental PPDC isolates (and human fibroblasts and rodent neural precursors as negative and positive control cell types, respectively) on discs coated with laminin (neuropromotion) and exposed to Thirteen different growth conditions (and two control conditions) known to promote neural precursor differentiation into neurons and stellate cells are known. In addition, two conditions were added to examine the effect of GDF5 and BMP7 on PPDC differentiation. In general, a two-step differentiation method is adopted, in which cells are first placed in a neural precursor expansion condition for a period of 6 days, and then placed in a fully differentiated condition for 7 days. Morphologically, both umbilical and placental-derived cells exhibit basic changes in cell morphology throughout the course of this procedure. However, cells in the shape of neurons or stellate cells were not observed, except in the conditions of control group, nerve precursor inoculation. Immunocytochemistry (negative to human nestin, TuJ1, and GFAP) confirmed morphological observations.

Multiple growth factors: After one week of exposure to various neural differentiation agents, cells were stained for markers indicating neural precursors (human nestin), neurons (TuJ1), and stellate cells (GFAP). The cells grown in the serum-free medium in the first stage have a different form from those in the serum-containing medium (2% or 10%), indicating potential neural differentiation. Specifically, after exposing umbilical-derived cells to EGF and bFGF, followed by two-step procedures of SHH, FGF8, GDNF, BDNF, and retinoic acid, the cells showed elongation similar to the cultured stellate cells Protruding. When 2% FBS or 10% FBS was included in the first stage of differentiation, the cell number increased and the cell morphology was no different from the high-density control culture. Immunocytochemical analysis of human nestin, TuJ1, or GFAP did not demonstrate potential neural differentiation.

Co-culture of neural precursors and PPDC : PPDC was inoculated to the culture of rat neural precursors in neural expansion conditions (NPE + F + E) two days before inoculation. Although visual confirmation of the inoculated PPDC proved that these cell lines were inoculated with a single cell, human specific nuclear staining (hNuc) 4 days after inoculation (total 6 days) showed that it tended to ball up and Avoid contact with neural precursors. In addition, when PPDC is attached, these cells expand and appear to be innervated by rat-derived differentiated neurons, indicating that PPDC may have differentiated into myocytes. This observation is based on the morphology under a phase contrast microscope. Another observation is that the general large cell body (larger than the neural precursor) has a shape resembling a neural precursor, with fine protrusions that span in multiple directions. hNuc staining (found in half of the nucleus) shows that in some cases, these human cells can be fused with rat precursors and assume their phenotype. Control wells containing only neural precursors have fewer total precursors and significantly differentiated cells than co-culture wells containing umbilical or placental PPDC, which further indicates that both umbilical and placental-derived cells affect the differentiation of neural precursors and Behavior, its role is through the release of chemokines and cytokines, or through contact with mediators.

Summary: Several procedures were performed to determine the short-term potential of PPDC to differentiate into neural lineage cells. These include morphological phase contrast imaging and immunocytochemistry for nestin, TuJ1, and GFAP (proteins associated with pluripotent neural stem cells and precursor cells, immature and mature neurons, and stellate cells, respectively) combination.

Example 23 Long-term neural differentiation of postpartum-derived cells

To evaluate the ability of umbilical and placental-derived cells (collectively called postpartum-derived cells or PPDC) to differentiate into neural lineage cells over a long period of time.

Methods and materials

Isolation and amplification of PPDC : Isolate and amplify PPDC as described in the previous example.

Thawing and inoculation of PPDC cells: Frozen aliquots of PPDC previously grown in growth medium (umbilical (022803) P11; (042203) P11; (071003) P12; placenta (101503) P7) were thawed and inoculated at 5,000 cells / cm2 The T-75 culture flask coated with laminin (BD, Franklin Lakes, NJ) contains B27 (B27 supplement, Invitrogen), L-glutamic acid (4 mM), and penicillin / streptomycin (10 Ml) of Neurobasal-A medium (Invitrogen, Carlsbad, Calif.), The combination of which is referred to herein as a neural precursor expansion (NPE) medium. The NPE medium is further supplemented with bFGF (20 ng / ml, Peprotech, Rocky Hill, NJ) and EGF (20 ng / ml, Peprotech, Rocky Hill, NJ), which is referred to herein as NPE + bFGF + EGF.

Control cell inoculation: In addition, adult human skin fibroblasts (P11, Cambrex, Walkersville, Md.) And mesenchymal stem cells (P5, Cambrex) were thawed and inoculated with laminin-coated T at the same cell seeding density -75 NPE + bFGF + EGF on culture flask. As a further control group, fibroblasts, navel, and placental PPDC were grown in growth medium for the period specified by all cultures.

Cell expansion: Replace the culture medium of all cultures with fresh medium once a week, and observe the expansion of the cells. In general, each culture was only subcultured once in a month due to restricted growth in NPE + bFGF + EGF.

Immunocytochemistry: After a one-month period, all culture flasks were fixed with cold 4% (w / v) paraformaldehyde (Sigma) at room temperature for 10 minutes. The Department of Immunocytochemistry was performed using antibodies against: TuJ1 (BIII Tubulin; 1: 500; Sigma, St. Louis, Mo.) and GFAP (Glial Fibrillary Acidic Protein; 1: 2000; DakoCytomation, Carpinteria, Calif. ). Briefly, the culture was washed with phosphate buffered saline (PBS) and then exposed to PBS, 4% (v / v) goat serum (Chemic on, Temecula, Calif.) And 0.3% (v / v) Triton (Triton X-100; Sigma) protein blocking solution for 30 minutes to obtain intracellular antigens. The primary antibody (diluted in blocking solution) was then applied to the culture at room temperature for 1 hour. Next, the primary antibody solution was removed, the culture was washed with PBS, and then a secondary antibody solution (1 hour at room temperature) was applied. The secondary antibody solution contained a blocking agent and goat anti-mouse IgG--Texas Red ( 1: 250; Molecular Probes, Eugene, OR) and goat anti-rabbit IgG-Alexa 488 (1: 250; Molecular Probes). The culture was subsequently washed, and then 10 micromolar DAPI (Molecular Probes) was applied for 10 minutes to visualize the nucleus.

After immunostaining, the fluorescence is visualized using an appropriate fluorescent filter on an Olympus inverted epifluorescence microscope (Olympus, Melville, N.Y.). In all cases, positive staining represented a fluorescent signal higher than that of the control stain, in which all procedures outlined above were followed, except for the application of primary antibody solution. Use a digital color camera and ImagePro software (Media Cybernetics, Carlsbad, Calif.) To capture the table image. For triple-stained samples, only one emission filter was used to shoot each image at a time. Then use Adobe Photoshop software (Adobe, San Jose, Calif.) To prepare layered composite images (Layered montage).

result

NPE + bFGF + EGF medium delays the proliferation of PPDC and changes its morphology. Immediately after inoculation, a subset of PPDC was attached to the culture flask coated with laminin. This may be due to cell death due to changes in the freezing / thawing process or due to new growth conditions. The attached cells adopt a morphology different from that observed in the growth medium.

Umbilical-derived cell colonies express neuronal proteins: cultures were fixed one month after thawing / inoculation and stained for neuronal proteins TuJ1 and GFAP (intermediate filaments found in stellate cells). Although all control cultures grown in growth medium and human fibroblasts and MSCs grown in NPE + bFGF + EGF medium were found to be TuJ1- / GFAP-, TuJ1 was detected in umbilical and placental PPDC. Performance is observed in cells with and without neuron-like morphology. No performance of GFAP was observed in all cultures. The percentage of cells exhibiting TuJ1 and having neuron-like morphology is less than or equal to 1% of the total population (n = 3 tested umbilical-derived cell isolates). Although not quantified, the percentage of TuJ1 + cells without neuronal morphology in umbilical derived cell culture is higher than in placental derived cell culture. These results seem to be specific because the age-matched control group in growth medium does not exhibit TuJ1.

Summary: Methods for generating differentiated neurons (based on TuJ1 performance and neuronal morphology) from umbilical derived cells have been developed. Although TuJ1 performance was not detected in vitro cultures older than one month, it is clear that at least a small group of umbilical-derived cells can undergo default differentiation or through exposure to supplemented with L-glutamine, The minimal medium of basic FGF and EGF is induced for a month to form neurons.

Example 24 PPDC nutritional factor for neural precursor support

The effects of umbilical and placental-derived cells (collectively called postpartum-derived cells or PPDCs) on the survival and differentiation of adult neural stem cells and precursor cells through non-contact-dependent (nutrition) mechanisms have been tested.

Methods and materials

Isolation of adult neural stem cells and precursor cells: Fisher 344 adult rats were sacrificed by CO ₂ asphyxia followed by cervical dislocation. All brains were completely removed using bone forceps, and hippocampal anatomy was dissected based on a coronal incision after the movement and somatosensory areas of the brain (Paxinos, G. & Watson, C. 1997. The Rat Brain in Stereotaxic Coordinates). Tissues were washed in Neurobasal-A medium (Invitrogen, Carlsbad, Calif.) Containing B27 (B27 supplement; Invitrogen), L-glutamic acid (4 mM; Invitrogen), and penicillin / streptomyces Invitrogen, and the like, are referred to herein as neural precursor expansion (NPE) medium. The NPE medium is further supplemented with bFGF (20 ng / ml, Peprotech, Rocky Hill, NJ) and EGF (20 ng / ml, Peprotech, Rocky Hill, NJ), which is referred to herein as NPE + bFGF + EGF.

After washing, the overlying meninges are removed, and the tissue is minced with a scalpel. The minced tissue was collected and 75% of the total volume of trypsin / EDTA (Invitrogen) was added. DNase (100 μl / 8 ml total volume, Sigma, St. Louis, Mo.) was also added. Next, the tissue / medium was sequentially passed through the 18-gauge needle, the 20-gauge needle, and finally the 25-gauge needle (all needles were from Becton Dickinson, Franklin Lakes, N.J.). The mixture was centrifuged at 250g for 3 minutes. The supernatant was removed, fresh NPE + bFGF + EGF was added and the pellet was resuspended. The resulting cell suspension was passed through a 40 micron cell strainer (Becton Dickinson), seeded on a T-75 culture flask (Becton Dickinson) coated with laminin or a low-clustered 24-well dish (Becton Dickinson), and grown on NPE bFGF + EGF medium until the sufficient number of cells required for the summary study is obtained.

PPDC inoculation: postnatally derived cells (umbilical (022803) P12, (042103) P12, (071003) P12; placenta (042203) P12) previously grown in growth medium with 5,000 cells / transwell insert (Applies to the size of a 24-well plate) Inoculate and grow in the insert in the growth medium for a period of one week to achieve fullness.

Adult nerve precursor inoculation: a nerve precursor that grows as a neurosphere or single cell is inoculated at a density of approximately 2,000 cells / well into NPE + bFGF + EGF on a 24-well plate coated with laminin for a period of one day to Promote cell attachment. One day later, add a transwell insert containing postpartum cells according to the following protocol:

a. Cross-well (umbilical-derived cells in growth medium, 200 μl) + neural precursor (NPE + bFGF + EGF, 1 ml)

b. Trans-well (placenta-derived cells in growth medium, 200 μl) + neural precursor (NPE + bFGF + EGF, 1 ml)

c. Transwell (adult human skin fibroblasts [1 F 1853; Cambrex, Walkersville, Md.] P12 in growth medium, 200 μl) + neural precursors (NPE + bFGF + EGF, 1 ml)

d. Control group: neural precursor alone (NPE + bFGF + EGF, 1 ml)

e. Control group: nerve precursor alone (NPE only, 1 ml)

Immunocytochemistry: After 7 days in the co-culture, all conditions were fixed with cold 4% (w / v) paraformaldehyde (Sigma) for 10 minutes at room temperature. Immunocytochemistry was performed using antibodies against the epitopes listed in Table 14-1. Briefly, the culture was washed with phosphate buffered saline (PBS) and then exposed to PBS, 4% (v / v) goat serum (Chemic on, Temecula, Calif.) And 0.3% (v / v) Triton (Triton X-100; Sigma) protein blocking solution for 30 minutes to obtain intracellular antigens. The primary antibody (diluted in blocking solution) was then applied to the culture at room temperature for 1 hour. Next, the primary antibody solution was removed, the culture was washed with PBS, and then a secondary antibody solution (1 hour at room temperature) was applied. The secondary antibody solution contained a blocking solution and goat anti-mouse IgG--Texas Red ( 1: 250; Molecular Probes, Eugene, OR) and goat anti-rabbit IgG-Alexa 488 (1: 250; Molecular Probes). The culture was subsequently washed, and then 10 micromolar DAPI (Molecular Probes) was applied for 10 minutes to visualize the nucleus.

After immunostaining, the fluorescence is visualized using an appropriate fluorescent filter on an Olympus inverted epifluorescence microscope (Olympus, Melville, N.Y.). In all cases, positive staining represented a fluorescent signal higher than that of the control stain, in which all procedures outlined above were followed, except for the application of primary antibody solution. Use a digital color camera and ImagePro software (Media Cybernetics, Carlsbad, Calif.) To capture the table image. For triple-stained samples, only one emission filter was used to shoot each image at a time. Then use Adobe Photoshop software (Adobe, San Jose, Calif.) To prepare layered composite images (Layered montage).

Quantitative analysis of neural precursor differentiation: Quantitative analysis of hippocampal gyrus neural precursor differentiation has been tested. Each condition counts a minimum of 1000 cells, or if fewer, counts the total number of cells observed in that condition. The percentage of cells that are positive for a given stain is evaluated by dividing the number of positive cells by the total number of cells determined by DAPI (nuclear) staining.

Mass spectrometry and 2D gel electrophoresis: In order to identify unique factors secreted by co-cultivation, samples of the conditioned medium obtained before the culture was fixed were frozen at -80 ° C overnight. The sample was then applied to an ultrafiltration spin device (with a cut-off MW of 30 kD). Retentate is applied to immunoaffinity chromatography (anti-Hu albumin; IgY) (immunoaffinity does not remove albumin from the sample). The filtrate was analyzed by MALDI. Pass through was applied to Cibachron Blue affinity chromatography. The samples were analyzed by SDS-PAGE and 2D gel electrophoresis.

result

PPDC co-culture stimulates adult neural precursor differentiation: after culturing with umbilical or placental-derived cells, co-cultured neural precursor cells derived from adult rat hippocampus exhibit significant differentiation along all three major lineages in the central nervous system. This effect was clearly observed after five days of co-cultivation, in which many cells developed complex processes and lost the bright features that are characteristic of precursor cell division. Conversely, neural precursors that grow alone in the absence of bFGF and EGF appear unhealthy and have limited survival.

After the procedure is completed, the culture is targeted for markers indicating undifferentiated stem cells and precursor cells (nestin), immature and mature neurons (TuJ1), stellate cells (GFAP), and mature oligodendritic cells (MBP) dyeing. The differentiation lines along all three lineages were confirmed, although the control conditions did not show significant differentiation, as evidenced by the majority of cells retaining nestin-positive staining. Although both umbilical and placenta-derived cells induce cell differentiation, all three lineages in co-cultures with placenta-derived cells are less differentiated than in co-cultures with umbilical-derived cells.

The percentage of differentiated neural precursors after co-culture with umbilical derived cells was quantified (Table 24-2). Umbilical-derived cells significantly increased the number of mature oligodendrocytes (MBP) (24.0% compared to 0% for both control conditions). In addition, co-cultivation increased the number of GFAP + stellate cells and TuJ1 + neurons in the culture (47.2% and 8.7%, respectively). These results were confirmed by nestin staining, indicating the loss of the precursor state after co-cultivation (13.4% compared to 71.4% of control group condition 4).

Although differentiation also appears to be affected by adult human fibroblasts, such cells cannot promote the differentiation of mature oligodendrocytes or produce appreciable amounts of neurons. Although not quantified, fibroblasts do seem to enhance the survival of neural precursors.

Identification of unique compounds: The difference between the conditioned medium derived from the umbilical and placenta-derived co-cultures and the appropriate control group (NPE medium ± 1.7% serum, co-cultured with fibroblasts). Potentially unique compounds are identified and excised from their respective 2D gels.

Summary: Co-culture of adult neural precursor cells with umbilical or placental PPDC leads to the differentiation of these cells. The results presented in this example indicate that after co-cultivation with umbilical derived cells, the differentiation of adult neural precursor cells is particularly prominent. In particular, a significant percentage of mature oligodendritic cells are produced in co-cultures of umbilical derived cells.

Example 25 Transplantation of postpartum derived cells

Cells derived from the postpartum umbilical and placental can be used for regenerative treatment. The tissues produced by transplanting postpartum-derived cells (PPDC) into SCID mice with biodegradable materials were evaluated. The materials evaluated were Vicryl non-woven fabric, 35/65 PCL / PGA foam, and RAD 16 self-assembled peptide hydrogel.

Methods and materials

Cell culture: grow placenta-derived cells and umbilical-derived cells in growth medium (DMEM-low glucose (Gibco, Carlsbad Calif.), 15% (v / v) fetal bovine serum (Cat. # SH30070.03; Hyclone, Logan, Utah), 0.001% (v / v) β-mercaptoethanol (Sigma, St. Louis, Mo.), penicillin / streptomycin (Gibco)), the growth medium is in a gelatin-coated culture flask.

Sample preparation: Inoculate one million viable cells in 15 microliters of growth medium to 5 mm diameter, 2.25 mm thick Vicryl non-woven scaffold (64.33 mg / cc; batch number 3547-47-1) or 5 mm diameter 35/65 PCL / PGA foam (Lot No. 3415-53). The cells were allowed to attach for two hours, after which more growth medium was added to cover the scaffold. The cells were grown on the scaffold overnight. The scaffold without cells was also cultured in the medium.

RAD16 self-assembling peptides (3D Matrix, Cambridge, MA) based on the form of a solution in water of the obtained in sterile 1% (w / v), to 1: 1 mixed with 1 x 10 ⁶ cells in 10% (w / v ) Sucrose (Sigma, St Louis, Mo.), 10 mM HEPES in Dulbecco's modified medium (DMEM; Gibco), and used immediately thereafter. The final concentration of cells in the RAD 16 hydrogel was 1 × 10 ⁶ cells / 100 μl.

Test material (N = 4 / Rx)

a. Vicryl non-woven fabric + 1 × 10 ⁶ umbilical derived cells

b. 35/65 PCL / PGA foam + 1 × 10 ⁶ umbilical derived cells

c. RAD 16 self-assembling peptide + 1 × 10 ⁶ umbilical derived cells

d. Vicryl non-woven fabric + 1 × 10 ⁶ placenta-derived cells

e. 35/65 PCL / PGA foam + 1 × 10 ⁶ placenta-derived cells

f. RAD 16 self-assembling peptide + 1 × 10 ⁶ placental-derived cells

g. 35/65 PCL / PGA foam

h. Vicryl non-woven

Animal preparation: Operate and feed animals in accordance with the current regulations of the Animal Welfare Act. By complying with the Animal Welfare Regulations (9 CFR) and complying with the current standards published in the Guide for the Care and Use of Laboratory Animals, 7th edition, compliance with the above public laws is achieved.

Mice ( Mus Musculus) / Fox Chase SCID / Male ( Harlan Sprague Dawley, Inc., Indianapolis, Ind. ), 5 weeks old: All operations of SCID mice are performed in a fume hood. The mice were individually weighed and used 60 mg / kg KETASET (ketamine hydrochloride, Aveco Co., Inc., Fort Dodge, Iowa) and 10 mg / kg ROMPUN (xylazine, Mobay Corp., Shawnee, Kans.) And saline. The mixture is anesthetized by intraperitoneal injection. After the introduction of anesthesia, the animal's entire back hair is shaved from the back neck area to the back waist area using animal clippers. The area was then scrubbed with chlorhexidine diacetate, rinsed with alcohol, dried, and then smeared with a 1% aqueous solution of iodophor with available iodine. Ophthalmic ointment is applied to the eyes to prevent tissue drying during anesthesia.

Subcutaneous transplantation technique: Make four skin incisions on the back of the mouse, each approximately 1.0 cm long. The two cranial sites are located laterally in the dorsal lateral thoracic cavity, with the hand touching the region about 5-mm behind the lower edge of the scapula, one on the left side of the spine and the other on the right side of the spine. The other two sites are positioned laterally at the caudal sacro-Iumbar, with the hand touching the gluteal muscle area about 5-mm behind the iliac bone, one on each side of the midline. According to the test plan, the implants were randomly placed in these sites. Separate the skin from the underlying connective tissue to make a small pocket, and place (or inject RAD16 into) the implant about 1-cm behind the incision. Implant the appropriate test material into the subcutaneous space. The skin incision was closed with a metal clip.

Animal placement: During the entire study period, mice were individually housed in micro-isolated cages with a temperature range of 64 ° F to 79 ° F and a relative humidity of 30% to 70%, and maintained for approximately 12 hours of light / 12 hours of darkness cycle. Keep the temperature and relative humidity within the range as possible. Food consists of irradiated Pico Mouse Chow 5058 (Purina Co.) and unlimited drinking water.

The mice were euthanized with carbon dioxide during their designated period. The subcutaneous implantation site with its overlying skin was excised and frozen for histological study.

Histology: The excised skin together with the implant was fixed with 10% neutral buffered formalin (Richard-Allan Kalamazoo, Mich.). Cut the sample together with the overlying and adjacent tissues from the middle, using conventional methods to treat and embed the cut surface with paraffin. Using conventional methods, five micron tissue sections were obtained by a thin slicer and stained with hematoxylin and eosin (Poly Scientific Bay Shore, N.Y.).

result

After 30 days, there was minimal ingrowth in the foam (cell-free) implanted under the skin of SCID mice. Conversely, implanted foam containing umbilical-derived cells or placental-derived cells is filled with extensive tissue. Some tissue growth was observed in Vicryl non-woven scaffold. Non-woven scaffolds seeded with umbilical or placental-derived cells show increased matrix deposition and mature blood vessels.

Summary: Synthetic absorbable non-woven / foam discs (5.0mm diameter x 1.0mm thick) or self-assembling peptide hydrogels are inoculated with cells derived from human umbilicus or placenta and implanted bilaterally subcutaneously in SCID The back of the mouse. The results prove that postpartum-derived cells can significantly increase the formation of high-quality tissue in biodegradable scaffolds.

Example 26 Telomerase expression in umbilical tissue-derived cells

The function of telomerase is to synthesize telomere repeats, which are used to protect the integrity of chromosomes and extend the replication life of cells (Liu, K, et al., PNAS, 1999; 96: 5147-5152) . Telomerase is composed of two components, namely telomerase RNA template (hTER) and telomerase reverse transcriptase (hTERT). The regulation of telomerase is determined by hTERT rather than hTER transcription. The real-time polymerase chain reaction (PCR) of hTERT mRNA is therefore an approved method for measuring cell telomerase activity.

Cell isolation: Perform real-time PCR experiments to determine telomerase production of human umbilical cord tissue-derived cells. Human umbilical cord tissue-derived cells were prepared according to the examples described above. In general, umbilical cords obtained from the National Center for Disease Research and Exchange (Philadelphia, Pa.) Are washed after normal delivery to remove blood and debris and then mechanically dissociated. Next, the tissue was cultured in the culture medium at 37 ° C with digestive enzymes including collagenase, dispase, and hyaluronidase. Human umbilical cord tissue-derived cells were cultured according to the methods described in the examples above. Mesenchymal stem cells and normal skin fibroblasts (cc-2509 lot number 9F0844) were obtained from Cambrex, Walkersville, Md. The pluripotent human testicular embryonic carcinoma (teratoma) cell line nTera-2 cells (NTERA-2 cl. Dl) (see Plaia et al., Stem Cells, 2006; 24 (3): 531-546) were purchased from ATCC (Manassas, Va.) And cultivated according to the method described above.

Total RNA isolation: RNA was extracted from cells using RNeasy® kit (Qiagen, Valencia, Ca.). RNA was extracted with 50 microliters of DEPC-treated water and stored at -80 ° C. Reverse transcription of RNA using TaqMan® reverse transcription reagent (Applied Biosystems, Foster City, Ca.) using random hexamers, reverse transcription at 25 ° C for 10 minutes, 37 ° C for 60 minutes, and 95 ° C for 95 minutes 10 minutes. Store samples at -20 ° C.

Real-time PCR: Applied Biosystems Assays-On-Demand ^™ (also known as TaqMan® Gene Expression Assays) was used to perform PCR on cDNA samples according to the manufacturer's specifications (Applied Biosystems). This commercial kit is widely used to detect telomerase in human cells. In short, using the 7000 sequence detection system (equipped with ABI prism 7000 SDS software (Applied Biosystems)), hTert (human telomerase gene) (Hs00162669) and human GAPDH (internal control group) and cDNA and TaqMan® Universal PCR The main mixture is mixed. The thermal cycle conditions were initially 50 ° C for 2 minutes and then 95 ° C for 10 minutes, followed by 40 cycles of 95 ° C for 15 seconds and 60 ° C for 1 minute. Analyze the PCR data according to the manufacturer's specifications.

Human umbilical cord tissue-derived cells (ATCC accession number PTA-6067), fibroblasts and mesenchymal stem cell lines were tested against hTert and 18S RNA. As shown in Table 26-1, neither hTert nor telomerase was detected in human umbilical cord tissue-derived cells.

Both human umbilical cord tissue-derived cells (isolate 022803, ATCC accession number PTA-6067) and nTera-2 cells were tested, and the results showed that neither batch of umbilical cord tissue-derived cells showed telomerase expression, but teratomas The cell line showed high performance (Table 26-2).

Therefore, it can be concluded that the human umbilical tissue-derived cells of the present invention do not express telomerase.

Various patents and other publications are mentioned in this specification. The entire contents of each of these publications are incorporated herein by reference.

Although the various aspects of the present invention have been described above by examples and preferred embodiments, it should be understood that the scope of the present invention is not limited by the foregoing description, but is limited by the following patent application scope correctly interpreted based on the principles of patent law.

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Claims (27)

    A postpartum-derived cell population for the treatment of retinal degeneration, which comprises administering a postpartum-derived cell population to an individual's eye, wherein the cell population secretes bridge molecules, and wherein the bridge molecules are selected from MFG-E8, Gas6, TSP-1 and TSP-2.         A composition for the treatment of retinal degeneration, which comprises administering a composition comprising a postpartum-derived cell population to an individual's eye, wherein the cell population secretes bridge molecules, and wherein the bridge molecules are selected from MFG-E8, Gas6, TSP-1 and TSP-2.         The use according to claim 1, wherein the postpartum-derived cell population comprises human umbilical cord tissue-derived cells, and the human umbilical cord tissue-derived cell lines are isolated from human umbilical cord tissue that is substantially free of blood.         The use according to claim 3, wherein the cell population isolated from human umbilical cord tissue that is substantially free of blood can be expanded in culture, has the potential to differentiate into cells of at least one neurophenotype, after passage Maintain normal karyotype and have the following characteristics: a) Potential for 40 population doubling in culture; b) Production of CD10, CD13, CD44, CD73 and CD90; c) No production of CD31, CD34, CD45, CD117 and CD141 , And d) Increase the expression of genes encoding interleukin 8 and reticulon 1 relative to human cells of fibroblasts, mesenchymal stem cells, or iliac bone marrow cells.         The use according to claim 1, wherein the cell population secretes receptor tyrosine kinase (RTK) trophic factor.         The use according to claim 5, wherein the nutritional factors are BDNF, NT3, HGF, PDGF-CC, PDGF-DD and GDNF.         The use as claimed in claim 2, wherein the cell population secretes receptor tyrosine kinase (RTK) trophic factor.         The use according to claim 7, wherein the nutritional factors are BDNF, NT3, HGF, PDGF-CC, PDGF-DD and GDNF.         The use according to claim 4, wherein the cell population secretes receptor tyrosine kinase (RTK) trophic factors selected from the group consisting of BDNF, NT3, HGF, PDGF-CC, PDGF-DD and GDNF Formed into groups.         A use of a postpartum-derived cell population for reducing oxidative damage to retinal cells, which comprises administering a postpartum-derived cell population to an individual's eye or a conditioned medium produced by culturing the postpartum-derived cells, wherein the postpartum-derived cell lines are isolated Human umbilical cord tissue that is substantially free of blood.         A use of a postpartum-derived cell population to rescue retinal pigment epithelium (RPE) cell dysfunction in retinal degeneration, the method comprising administering a postpartum-derived cell population to an individual's eye, wherein the postpartum-derived cell line is isolated from substantially no Human umbilical cord tissue containing blood, wherein the cell population secretes bridge molecules, and wherein the bridge molecules are selected from MFG-E8, Gas6, TSP-1 and TSP-2.         A use of a postpartum-derived cell population for reducing photoreceptor cell loss in retinal degeneration, the method comprising administering to the individual's eye a postpartum-derived cell population effective to reduce the amount of photoreceptor cell loss, wherein the postnatal-derived cell line Isolated from human umbilical cord tissue substantially free of blood, wherein the postpartum-derived cell population secretes bridge molecules, and wherein the bridge molecules are selected from MFG-E8, Gas6, TSP-1, and TSP-2.         The use according to claim 11, wherein the postpartum-derived cell population secretes receptor tyrosine kinase trophic factors selected from BDNF, NT3, HGF, PDGF-CC, PDGF-DD and GDNF Group.         The use according to claim 12, wherein the postpartum-derived cell line secretes receptor tyrosine kinase trophic factors selected from the group consisting of BDNF, NT3, HGF, PDGF-CC, PDGF-DD and GDNF Group.         The use according to claim 10, wherein the postpartum-derived cell population is administered together with at least one other agent.         A composition for reducing the loss of photoreceptor cells in retinal degeneration, which comprises administering a composition containing a postpartum-derived cell population to an individual's eye, wherein the composition is administered in an amount effective to reduce the loss of photoreceptor cells Yu, wherein the postpartum-derived cell lines are isolated from human umbilical cord tissue that is substantially free of blood, wherein the postpartum-derived cell line secretes bridge molecules, and wherein the bridge molecules are selected from MFG-E8, Gas6, TSP-1 and TSP -2.         The use according to claim 16, wherein the postpartum-derived cell line secretes receptor tyrosine kinase trophic factors selected from the group consisting of BDNF, NT3, HGF, PDGF-CC, PDGF-DD and GDNF Group.         The use according to claim 17, wherein the nutritional factors are BDNF, NT3, HGF, PDGF-CC, PDGF-DD and GDNF.         The use according to claim 16, wherein the composition is a pharmaceutical composition.         The use according to claim 19, wherein the pharmaceutical composition contains a pharmaceutically acceptable carrier.         The use according to claim 1, wherein the retinal degeneration is age-related macular degeneration.         The use according to claim 21, wherein the age-related macular degeneration is dry age-related macular degeneration.         The use as claimed in claim 11, wherein the cell population isolated from human umbilical cord tissue that is substantially free of blood can be expanded in culture, has the potential to differentiate into cells of at least one neurophenotype, after passage Maintain normal karyotype and have the following characteristics: a) Potential for 40 population doubling in culture; b) Production of CD10, CD13, CD44, CD73 and CD90; c) No production of CD31, CD34, CD45, CD117 and CD141 , And d) Increase the expression of genes encoding interleukin 8 and endoplasmic reticulum protein 1 relative to human cells of fibroblasts, mesenchymal stem cells, or iliac bone marrow cells.         The use according to claim 4, wherein the cell population is positive for HLA-A, HLA-B, HLA-C, and negative for HLA-DR, HLA-DP, HLA-DQ.         The use according to claim 23, wherein the cell population is positive for HLA-A, HLA-B, HLA-C, and negative for HLA-DR, HLA-DP, HLA-DQ.         The use according to claim 1, wherein the administration to the eye is selected from administration to the inside of the eye or administration behind the eye.         A composition for reducing the loss of photoreceptor cells in retinal degeneration, which comprises administering a composition comprising a conditioned medium produced by culturing a postpartum-derived cell population to an individual's eye, wherein the composition is effective to reduce The amount of photoreceptor cell loss is administered, wherein the postpartum-derived cell lines are isolated from human umbilical cord tissue that is substantially free of blood, wherein the postpartum-derived cell line secretes bridge molecules, and wherein the bridge molecules are selected from MFG-E8 , Gas6, TSP-1 and TSP-2.