WO2018102174A1 - Traitement de la dégénérescence rétinienne à l'aide de cellules progénitrices - Google Patents

Traitement de la dégénérescence rétinienne à l'aide de cellules progénitrices Download PDF

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WO2018102174A1
WO2018102174A1 PCT/US2017/062526 US2017062526W WO2018102174A1 WO 2018102174 A1 WO2018102174 A1 WO 2018102174A1 US 2017062526 W US2017062526 W US 2017062526W WO 2018102174 A1 WO2018102174 A1 WO 2018102174A1
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cells
rpe
cell
phagocytosis
postpartum
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PCT/US2017/062526
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English (en)
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Ian R. Harris
Jing Cao
Nadine Dejneka
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Janssen Biotech, Inc.
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Priority claimed from US15/366,947 external-priority patent/US20170080033A1/en
Application filed by Janssen Biotech, Inc. filed Critical Janssen Biotech, Inc.
Publication of WO2018102174A1 publication Critical patent/WO2018102174A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/48—Reproductive organs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/48—Reproductive organs
    • A61K35/50—Placenta; Placental stem cells; Amniotic fluid; Amnion; Amniotic stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/48—Reproductive organs
    • A61K35/51—Umbilical cord; Umbilical cord blood; Umbilical stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0048—Eye, e.g. artificial tears
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents

Definitions

  • This invention relates to the field of cell-based or regenerative therapy for ophthalmic diseases and disorders.
  • the invention provides methods and compositions for the regeneration or repair of ocular cells and tissue using progenitor cells, such as umbilical cord tissue-derived cells and placenta tissue-derived cells, and conditioned media prepared from those cells.
  • progenitor cells such as umbilical cord tissue-derived cells and placenta tissue-derived cells
  • conditioned media prepared from those cells BACKGROUND
  • the eye can experience numerous diseases and other deleterious conditions that affect its ability to function normally. Many of these conditions are associated with damage or degeneration of specific ocular cells, and tissues made up of those cells.
  • diseases and degenerative conditions of the optic nerve and retina are the leading causes of blindness throughout the world. Damage or degeneration of the cornea, lens and associated ocular tissues represent another significant cause of vision loss worldwide.
  • the retina contains seven layers of alternating cells and processes that convert a light signal into a neural signal.
  • the retinal photoreceptors and adjacent retinal pigment epithelium (RPE) form a functional unit that, in many disorders, becomes unbalanced due to genetic mutations or environmental conditions (including age). This results in loss of photoreceptors through apoptosis or secondary degeneration, which leads to progressive deterioration of vision and, in some instances, to blindness (for a review, see, e.g., Lund, R. D. et al., Progress in Retinal and Eye Research, 2001; 20: 415-449).
  • Two classes of ocular disorders that fall into this pattern are age-related macular degeneration (AMD) and retinitis pigmentosa (RP).
  • AMD age-related macular degeneration
  • RP retinitis pigmentosa
  • Retinitis pigmentosa is mainly considered an inherited disease, with over 100 mutations being associated with photoreceptor loss (see Lund et al., 2001, supra). Though the majority of mutations target photoreceptors, some affect RPE cells directly. Together, these mutations affect such processes as molecular trafficking between photoreceptors and RPE cells and phototransduction.
  • retinopathies can also involve progressive cellular degeneration leading to vision loss and blindness. These include, for example, diabetic retinopathy and choroidal neovascular membrane (CNVM).
  • CNVM choroidal neovascular membrane
  • phagocytosis bind pathogen for phagocytosis
  • non-professional phagocytes phagocytosis is not the principal function
  • epithelial cells RPE cells
  • endothelial cells apoptotic cells.
  • Numerous“eat me” signals have been identified to date including changes in glycosylation of surface proteins or changes in surface charge (Ravichandran et al., Cold Spring Harb Perspect Biol., 2013).
  • PS phosphatidylserine
  • MFG-E8 milk-fat-globule-EGF-factor 8
  • Gas6 growth arrest-specific 6
  • protein S protein S
  • TSPs thrombospondins
  • apolipoprotein H previously known as ⁇ 2-glycoprotein I, ⁇ 2-GPI
  • MFG-E8 can then be recognized by ⁇ v ⁇ 3 and ⁇ v ⁇ 5 integrins through its RGD motif (Hanayama et al., Science, 2004, 304: 1147-1150; Borisenko et al., Cell Death Differ., 2004; 11:943-945), Gas6 by receptor tyrosine kinases of the Axl, Tyro3 and Mer family (Scott et al., Nature, 2001; 411:207-211) and apolipoprotein H to the ⁇ 2-GPI receptor (Balasubramanian et al., J Bio Chem, 1997; 272:31113-31117).
  • bridge molecules are linked to the recognition of altered sugars and/or lipids on the apoptotic cell surface, such as the members of the collectin family surfactant protein A and D (Vandivier et al., J Immunol, 2002; 169:3978-398).
  • the collectin family of molecules are then recognized through their interactions of their collagenous tails with calreticulin (CRT), which in turn signals for uptake by the phagocyte through the low-density lipoprotein (LDL)-receptor-related protein (LRP-1/CD91) (Gardai et al., Cell, 2003; 115:13-23).
  • CRT calreticulin
  • LDL low-density lipoprotein
  • LRP-1/CD91 low-density lipoprotein
  • Phagocytosis of ROS by RPE is essential for retinal function (Finnemann et al., PNAS, 1997; 94:12932-937).
  • Receptors reported to participate in RPE phagocytosis of ROS include a scavenger receptor CD36, integrin receptor ⁇ v ⁇ 5, a receptor tyrosine kinase known as Mertk, and the mannose receptor (MR) (CD206) (Kevany et al., Physiology, 2009; 25:8-15).
  • This invention provides compositions and methods applicable to cell-based or regenerative therapy for ophthalmic diseases and disorders.
  • the invention features methods and compositions for treating ophthalmic disease or condition, including the regeneration or repair of ocular tissue using progenitor cells, such as postpartum- derived cells, and conditioned media generated from those cells.
  • the postpartum-derived cells may be umbilical cord tissue-derived cells (UTCs) or placental tissue-derived cells (PDCs).
  • One aspect of the invention is a method of treating ophthalmic disease comprising administering to a subject progenitor cells, or a conditioned media prepared from a population of progenitor cells, wherein the cells secrete bridge molecules.
  • the bridge molecules are secreted by the cell population in the conditioned media.
  • the bridge molecules are selected from MFG-E8, Gas6, thrombospondin (TSP)-1 and TSP-2.
  • the cells are progenitor cells.
  • the cells are postpartum-derived cells.
  • the postpartum-derived cells are isolated from human umbilical cord tissue or placental tissue substantially free of blood.
  • a population of progenitor cells secretes bridge molecules.
  • conditioned media prepared from a population of progenitor cells, for example postpartum-derived cells contains bridge molecules secreted by the cell population.
  • bridge molecules secreted by the cells and secreted in the conditioned media are selected from MFG-E8, Gas6, TSP-1 and TSP-2.
  • the postpartum-derived cells are umbilical cord tissue-derived cells (UTCs) or placental tissue-derived cells (PDCs).
  • the bridge molecules inhibit the apoptosis of
  • bridge molecules secreted by progenitor cells, and secreted in conditioned media reduce the loss of photoreceptor cells.
  • the loss of photoreceptor cells is reduced by the bridge molecules stimulating phagocytosis of photoreceptor fragments.
  • the population of cells described above or conditioned media prepared from the population of cells described above modifies rod outer segment (ROS) to facilitate phagocytosis.
  • the bridge molecules enhance binding and internalization of ROS by retinal pigment epithelial (RPE) cells.
  • the population of cells described above or conditioned media prepared from the population of cells described above contains receptor tyrosine kinase (RTK) trophic factors secreted by the cell population.
  • RTK receptor tyrosine kinase
  • the trophic factors are BDNF, NT3, HGF, PDGF-CC, PDGF-DD, and GDNF.
  • the RTK trophic factors mediate phagocytosis in retinal pigment epithelial (RPE) cells.
  • the RTK trophic factors mediate phagocytosis by RPE cells to phagocytose shed photoreceptor fragments (photoreceptor fragments shed by the cells).
  • the RTK trophic factors activate receptors on RCE cells to stimulate phagocytosis.
  • Another aspect of the invention features a method for reducing the loss of photoreceptor cells in retinal degeneration, the method comprising administering to the eye a population of progenitor cells or a conditioned media prepared from the population of progenitor cells in an amount effective to reduce the loss of photoreceptor cells.
  • the progenitor cells are postpartum-derived cells.
  • the postpartum-derived cells are isolated from human umbilical cord tissue or placental tissue substantially free of blood.
  • the postpartum-derived cells secrete bridge molecules.
  • the conditioned media contains bridge molecules secreted by the cell population, such as a population of postpartum-derived cells. Such bridge molecules secreted by the postpartum-derived cells are selected from MFG-E8, Gas6, TSP-1 and TSP-2.
  • the conditioned media is generated from an isolated postpartum-derived cell or a population of postpartum-derived cells, derived from human umbilical cord tissue or placental tissue substantially free of blood.
  • the postpartum-derived cell is capable of expansion in culture and has the potential to differentiate into a cell of a neural phenotype; wherein the cell requires L-valine for growth and is capable of growth in at least about 5% oxygen.
  • This cell further comprises one or more of the following characteristics: (a) potential for at least about 40 doublings in culture; (b) attachment and expansion on a coated or uncoated tissue culture vessel, wherein the coated tissue culture vessel comprises a coating of gelatin, laminin, collagen, polyomithine, vitronectin, or fibronectin; (c) production of at least one of tissue factor, vimentin, and alpha-smooth muscle actin; (d) production of at least one of CD10, CD13, CD44, CD73, CD90, PDGFr-alpha, PD-L2 and HLA-A,B,C; (e) lack of production of at least one of CD31, CD34, CD45, CD80, CD86, CD117, CD141, CD178, B7-H2, HLA-G, and HLA-DR,DP,DQ, as detected by flow cytometry; (f) expression of a gene, which relative to a human cell that is a fibroblast, a mesenchymal stem cell
  • mesenchyme homeo box 2 growth arrest-specific homeo box
  • sine oculis homeobox homolog 1 Drosophila
  • crystallin, alpha B disheveled associated activator of morphogenesis 2
  • DKFZP586B2420 protein similar to neuralin 1; tetranectin (plasminogen binding protein); src homology three (SH3) and cysteine rich domain; cholesterol 25-hydroxylase; runt-related transcription factor 3; interleukin 11 receptor, alpha; procollagen C-endopeptidase enhancer; frizzled homolog 7 (Drosophila);
  • cytokine receptor-like factor 1 potassium intermediate/small conductance calcium- activated channel, subfamily N, member 4; integrin, beta 7; transcriptional co-activator with PDZ-binding motif (T AZ); sine oculis homeobox homolog 2 (Drosophila);
  • KIAA1034 protein vesicle-associated membrane protein 5 (myobrevin); EGF-containing fibulin-like extracellular matrix protein 1; early growth response 3; distal-less homeo box 5; hypothetical protein FLJ20373; aldo-keto reductase family 1, member C3 (3-alpha hydroxysteroid dehydrogenase, type II); biglycan; transcriptional co-activator with PDZ- binding motif (TAZ); fibronectin 1; proenkephalin; integrin, beta-like 1 (with EGF-like repeat domains); Homo sapiens mRNA full length insert cDNA clone EUROIMAGE 1968422; EphA3; KIAA0367 protein; natriuretic peptide receptor C/guanylate cyclase C (atrionatriuretic peptide receptor C); hypothetical protein FLJ14054; Homo sapiens mRNA; cDNA DKFZp564B222 (from clone DKFZp56
  • the umbilical cord tissue-derived cell further has the characteristics of (i) secretion of at least one of MCP-l, IL-6, IL-8, GCP-2, HGF, KGF, FGF, HB-EGF, BDNF, TPO, MIPlb, I309, MDC, RANTES, and TIMP1; (j) lack of secretion of at least one of TGF-beta2, MIP1a, ANG2, PDGFbb, and VEGF, as detected by ELISA.
  • the placenta tissue-derived cell further has the characteristics of (i) secretion of at least one of MCP-l, IL-6, IL-8, GCP-2, HGF, KGF, HB-EGF, BDNF, TPO, MIP1a, RANTES, and TIMP1; (j) lack of secretion of at least one of TGF-beta2, MIPlb, ANG2, PDGFbb, FGF, and VEGF, as detected by ELISA.
  • the postpartum-derived cell has all the identifying features of cell type UMB 022803 (P7) (ATCC Accession No. PTA-6067); cell type UMB 022803 (P17) (ATCC Accession No. PTA-6068), cell type PLA 071003 (P8) (ATCC Accession No. PTA-6074); cell type PLA 071003 (P11) (ATCC Accession No. PTA-6075); or cell type PLA 071003 (P16) (ATCC Accession No. PTA-6079.
  • the postpartum-derived cell derived from umbilicus tissue has all the identifying features of cell type UMB 022803 (P7) (ATCC Accession No. PTA-6067) or cell type UMB 022803 (P17) (ATCC Accession No. PTA-6068).
  • P7 ATCC Accession No. PTA-6067
  • cell type UMB 022803 P17
  • ATCC Accession No. PTA-6068 ATCC Accession No. PTA-6068
  • the postpartum-derived cell derived from placenta tissue has all the identifying features of cell type PLA 071003 (P8) (ATCC Accession No. PTA-6074); cell type PLA 071003 (P11) (ATCC Accession No. PTA-6075); or cell type PLA 071003 (P16) (ATCC Accession No. PTA-6079).
  • postpartum-derived cells are isolated in the presence of one or more enzyme activities comprising metalloprotease activity, mucolytic activity and neutral protease activity.
  • the postpartum-derived cells have a normal karyotype, which is maintained as the cells are passaged in culture.
  • the postpartum-derived cells express each of CD10, CD13, CD44, CD73, CD90.
  • the postpartum-derived cells express each of CD10, CD13, CD44, CD73, CD90, PDGFr-alpha, and HLA-A,B,C.
  • the postpartum-derived cells do not express CD31, CD34, CD45, CD117.
  • the postpartum-derived cells do not express CD31, CD34, CD45, CD117, CD141, or HLA-DR,DP,DQ, as detected by flow cytometry.
  • the cells lack expression of hTERT or telomerase.
  • the cell population is a substantially homogeneous population of postpartum-derived cells.
  • the population is a homogeneous population of postpartum-derived cells.
  • the postpartum-derived cells are derived from human umbilical cord tissue or placental tissue substantially free of blood.
  • the population of postpartum-derived cells or the conditioned medium prepared from the cell population as described above is administered with at least one other cell type, such as an astrocyte, oligodendrocyte, neuron, neural progenitor, neural stem cell, retinal epithelial stem cell, corneal epithelial stem cell, or other multipotent or pluripotent stem cell.
  • the other cell type can be administered simultaneously with, before, or after the population of postpartum- derived cells or the conditioned medium prepared from the population of postpartum- derived cells.
  • the population of postpartum- derived cells or the conditioned medium prepared from the population of postpartum- derived cells as described above is administered with at least one other agent, such as a drug for ocular therapy, or another beneficial adjunctive agent such as an anti- inflammatory agent, anti-apoptotic agents, antioxidants or growth factors.
  • the other agent can be administered simultaneously with, before, or after, the population of postpartum-derived cells or the conditioned medium prepared from the population of postpartum-derived cells.
  • the population of postpartum- derived cells (umbilical or placental) or the conditioned medium generated from postpartum-derived cells is administered to the eye, for example the surface of an eye, or to the interior of an eye or to a location in proximity to the eye, e.g., behind the eye.
  • the population of postpartum-derived cells or the conditioned medium prepared from the population of postpartum-derived cells can be administered through a cannula or from a device implanted in the patient's body within or in proximity to the eye, or may be administered by implantation of a matrix or scaffold with the population of cells or the conditioned media.
  • compositions for reducing the loss of photoreceptor cells in a retinal degenerative condition comprising a population of progenitor cells or a conditioned media prepared from a population of progenitor cells in an amount effective to reducing the loss of photoreceptor cells.
  • the progenitor cells are postpartum-derived cells as described above. More preferably, the postpartum- derived cells are isolated from a postpartum umbilical cord or placenta substantially free of blood as described above.
  • the degenerative condition may be an acute, chronic or progressive condition.
  • the composition above comprises at least one other cell type, such as an astrocyte, oligodendrocyte, neuron, neural progenitor, neural stem cell, retinal epithelial stem cell, corneal epithelial stem cell, or other multipotent or pluripotent stem cell.
  • the composition comprises at least one other agent, such as a drug for treating the ocular degenerative disorder or other beneficial adjunctive agents, e.g., anti-inflammatory agents, anti-apoptotic agents, antioxidants or growth factors.
  • the composition is a pharmaceutical composition further comprising a pharmaceutically-acceptable carrier.
  • the pharmaceutical compositions are formulated for administration to the surface of an eye.
  • compositions can be formulated for administration to the interior of an eye or in proximity to the eye (e.g., behind the eye).
  • the pharmaceutical compositions also can be formulated as a matrix or scaffold containing the progenitor cells or conditioned media prepared from the progenitor cells as described above.
  • kits for treating a patient having an ocular degenerative condition.
  • the kit comprises a pharmaceutically acceptable carrier, progenitor cells or a conditioned media generated from progenitor cells such as cells isolated from postpartum tissue, preferably the postpartum-derived cells described above, and instructions for using the kit in a method of treating the patient.
  • the kit may also contain one or more additional components, such as reagents and instructions for generating the conditioned medium, or a population of at least one other cell type, or one or more agents useful in the treatment of an ocular degenerative condition.
  • aspects of the invention include a method of reducing the loss of photoreceptor cells in retinal degeneration, the method comprising administering a composition comprising a population of progenitor cells or a conditioned media prepared from a population of progenitor cells, in an amount effective to reduce the loss of photoreceptor cells.
  • the progenitor cells are postpartum-derived cells or the conditioned media is prepared from a population of postpartum-derived cells as described herein.
  • the postpartum-derived cells are isolated from umbilical cord tissue or placental tissue substantially free of blood.
  • the postpartum-derived cells or the conditioned media prepared from a population of postpartum-derived cells contains bridge molecules secreted by the cell population.
  • bridge molecules secreted by the postpartum-derived cells or secreted in the conditioned media are selected from MFG-E8, Gas6, TSP-1 and TSP-2.
  • the present invention provides a method for reducing the loss of photoreceptor cells in retinal degeneration, the method comprising
  • the postpartum-derived cells are derived from umbilical cord tissue or placental tissue substantially free of blood.
  • the population of postpartum-derived cells is a substantially homogeneous population. In particular embodiments, the population of cells is homogeneous.
  • the population of postpartum-derived cells (umbilical or placental) or the conditioned medium generated from postpartum-derived cells protects retinal cells or improves retinal damage from oxidative stress or oxidative damage.
  • the present invention is a method of reducing retinal degeneration, the method comprising administering a population of postpartum-derived cells or conditioned media generated from a population of postpartum-derived cells to the eye in an amount effective to reduce or protect from oxidative stress or damage.
  • the postpartum-derived cells are isolated from umbilical cord tissue or placental tissue substantially free of blood.
  • retinal cells and tissue are exposed to oxidative stress or oxidative damage.
  • retinal cells and tissue are photoreceptor cells or retinal epithelium, including retinal pigment epithelial (RPE) cells.
  • oxidative stress or oxidative damage is high oxygen tension, sunlight exposure, including chronic sunlight exposure.
  • An embodiment is a method of reducing the loss of photoreceptor cells in retinal degeneration, the method comprising administering a population of postpartum- derived cells or conditioned media generated from a population of postpartum-derived cells to the eye in an amount effective to reduce or protect from oxidative stress or damage.
  • the postpartum-derived cells are isolated from umbilical cord tissue or placental tissue substantially free of blood.
  • retinal cells and tissue are exposed to oxidative stress or oxidative damage.
  • retinal cells and tissue are photoreceptor cells or retinal epithelium, including retinal pigment epithelial (RPE) cells.
  • oxidative stress or oxidative damage is selected from high oxygen tension, sunlight exposure, including chronic sunlight exposure, free radical stress, photoxidation, and light-induced damage.
  • the present invention is a method for reducing the loss of photoreceptor cells in retinal degeneration, the method comprising administering a population of postpartum-derived cells or conditioned media generated from a population of postpartum-derived cells in an amount effective to reduce or prevent the loss of photoreceptor cells, wherein the cell population is isolated from postpartum tissue substantially free of blood, and wherein the cell population is capable of expansion in culture, has the potential to differentiate into cells of at least a neural phenotype, maintains a normal karyotype upon passaging, and has the following characteristics: a) potential for 40 population doublings in culture;
  • the population of postpartum-derived cells secretes bridge molecules, wherein conditioned media prepared from a population of postpartum-derived cells contains bridge molecules secreted by the cell population.
  • the bridge molecules secreted by the postpartum-derived cells are selected from MFG-E8, Gas6, TSP-1 and TSP-2.
  • the population of cells is a substantially homogeneous population. In particular embodiments, the population of cells is homogeneous.
  • the postpartum-derived cells are umbilical cord tissue-derived cells or placental tissue-derived cells.
  • the umbilical cord tissue-derived cell population secretes MCP-l, IL-6, IL-8, GCP-2, HGF, KGF, FGF, HB-EGF, BDNF, TPO, MIPlb, I309, MDC, RANTES, and TIMP1.
  • the umbilical cord tissue- derived cell population lacks secretion of TGF-beta2, MIP1a, ANG2, PDGFbb, and VEGF, as detected by ELISA.
  • the placental tissue-derived cell population secretes MCP-l, IL-6, IL-8, GCP-2, HGF, KGF, HB-EGF, BDNF, TPO, MIP1a, RANTES, and TIMP1.
  • the placental tissue-derived cell population lacks secretion of TGF-beta2, MIPlb, ANG2, PDGFbb, FGF, and VEGF, as detected by ELISA.
  • the umbilicus-derived cells or placental-derived cells are positive for HLA-A,B,C, and negative for HLA-DR,DP,DQ.
  • the umbilicus-derived cells lack expression of hTERT or telomerase.
  • the present invention is a method for reducing the loss of photoreceptor cells in retinal degeneration, the method comprising administering a population of postpartum-derived cells, or a conditioned media prepared from a population of postpartum-derived cells, in an amount effective to reduce the loss of photoreceptor cells, wherein the cell population is isolated from human umbilical cord tissue substantially free of blood, and wherein the cell population is capable of expansion in culture, has the potential to differentiate into cells of at least a neural phenotype, maintains a normal karyotype upon passaging, and has the following characteristics: a) potential for 40 population doublings in culture; b) production of CD10, CD13, CD44, CD73, and CD90;
  • the population of postpartum-derived cells secretes bridge molecules, or wherein the conditioned media prepared from a population of postpartum-derived cells contains bridge molecules secreted by the cell population.
  • the bridge molecules secreted by the population of postpartum-derived cells, or bridge molecules in the conditioned media secreted by the population of postpartum-derived cells are selected from MFG-E8, Gas6, TSP-1 and TSP-2.
  • the cell population secretes MCP-l, IL-6, IL-8, GCP-2, HGF, KGF, FGF, HB-EGF, BDNF, TPO, MIPlb, I309, MDC, RANTES, and TIMP1.
  • the cell population lacks secretion of TGF-beta2, MIP1a, ANG2, PDGFbb, and VEGF, as detected by ELISA.
  • the cell population is positive for HLA-A,B,C, and negative for HLA- DR,DP,DQ.
  • the population of cells is a substantially
  • the population of cells is homogeneous.
  • the cell population lacks expression of hTERT or telomerase.
  • the present invention provides a method for reducing the loss of photoreceptor cells in retinal degeneration, the method comprising
  • a population of umbilicus-derived cells, or conditioned media prepared from a population of umbilicus-derived cells in an amount effective to reduce or prevent the loss of photoreceptor cells, wherein the cell population is isolated from human umbilical cord tissue substantially free of blood, and wherein the cell population is capable of expansion in culture, has the potential to differentiate into cells of at least a neural phenotype, and has the following characteristics:
  • the cells c. increased expression of genes, encoding interleukin 8 and reticulon 1 relative to a human cell that is a fibroblast, a mesenchymal stem cell, or an iliac crest bone marrow cell, and the population of postpartum-derived cells secretes bridge molecules, wherein the conditioned media prepared from a population of postpartum-derived cells contains bridge molecules secreted by the cell population.
  • the bridge molecules secreted in the population of postpartum-derived cells secretes bridge molecules, or bridge molecules secreted in conditioned media are selected from MFG-E8, Gas6, TSP-1 and TSP-2.
  • the cells lack production of, or are negative for CD31, CD34, CD45, CD117, and CD141.
  • the umbilical cord tissue-derived cell population secretes MCP-l, IL-6, IL-8, GCP-2, HGF, KGF, FGF, HB-EGF, BDNF, TPO, MIPlb, I309, MDC, RANTES, and TIMP1, and lacks secretion of TGF-beta2, MIP1a, ANG2, PDGFbb, and VEGF, as detected by ELISA.
  • the population of umbilicus-derived cells are positive for HLA-A,B,C, and negative for HLA- DR,DP,DQ. Further, the cell population lacks expression of hTERT or telomerase.
  • the population of cells is a substantially homogeneous population. In particular embodiments, the the population of cells is homogeneous.
  • Another aspect of the invention is a method of making a conditioned media comprising culturing a population of cells, wherein the conditioned media contains bridge molecules secreted by the cell population.
  • the bridge molecules are secreted by the cell population in the conditioned media.
  • the bridge molecules are selected from MFG-E8, Gas6, thrombospondin (TSP)-1 and TSP-2.
  • the cells are progenitor cells.
  • the cells are postpartum-derived cells.
  • the postpartum-derived cells are isolated from human umbilical cord tissue or placental tissue substantially free of blood.
  • the population of postpartum-derived cells have the following characteristics: attachment and expansion on a coated or uncoated tissue culture vessel, wherein the coated tissue culture vessel comprises a coating of gelatin, laminin, collagen, polyornithine, vitronectin, or fibronectin; production of vimentin and alpha-smooth muscle actin; and positive for HLA-A,B,C, and negative for HLA-DR,DP,DQ.
  • the retinal degeneration, retinopathy or retinal/macular disorder is age-related macular degeneration. In an alternate embodiment, the retinal degeneration, retinopathy or retinal/macular disorder is dry age-related macular degeneration.
  • the loss of photoreceptor cells is reduced or prevented by inhibiting the apoptosis of the photoreceptor cells. In embodiments, the loss of photoreceptor cells is reduced or prevented by stimulating the phagocytosis of shed photoreceptor fragments.
  • FIG. 1A shows the effect on dystrophic RPE phagocytosis with pre- incubation in a preparation of hUTC CM1 with serum.
  • FIG.1A Pigmented dystrophic RPE was preincubated with the CM1 preparation (with serum). For controls, tan hooded normal and pigmented dystrophic RPE were preincubated with control medium
  • Figure 2 shows the effect on dystrophic RPE phagocytosis with pre- incubation in a preparation of hUTC CM1 without serum.
  • Pigmented dystrophic RPE was preincubated with CM1 (without serum).
  • tan hooded normal and pigmented dystrophic RPE were preincubated in control media without serum.
  • FIG.3A Effect on phagocytosis with pre-incubation in hUTC CM2.
  • Pigmented dystrophic RPE was preincubated with CM2.
  • tan hooded normal and pigmented dystrophic RPE were preincubated in control media. Incubation time was 24 hours.
  • tan N tan hooded normal RPE
  • pig D pigmented dystrophic RPE
  • con control.
  • M medium
  • CM conditioned medium
  • N normal RPE
  • N1 normal RPE from culture
  • N2 normal RPE from N2 culture
  • tan N tan hooded normal RPE
  • D dystrophic RPE
  • con control
  • M medium
  • CM conditioned medium
  • FIG.4A shows the effect of hUTC on phagocytosis in RCS RPE cells in vitro.
  • RCS RPE cells co-cultured with hUTC plated in transwells (FIG.4A) or incubated with hUTC CM (FIG.4B) for 24 hours and then subjected to phagocytosis assay.
  • N normal RPE
  • D dystrophic RCS RPE
  • CM conditioned medium.
  • Increased phagocytosis was observed in RCS RPE co-cultured with hUTC or incubated with hUTC CM.
  • Dystrophic RPE cells were incubated with Ephrin B2 (200 ng/ml). For positive control, dystrophic RPE cells were incubated in CM3. For other controls, normal and dystrophic RPE were incubated in MEM5 for 24 h and subjected to phagocytosis assay.
  • N normal RPE
  • D dystrophic RPE
  • con control
  • M medium
  • CM conditioned medium
  • FIGS. 8A - 8C show dystropic RPE cells treated with endothelin-1, TGF- ⁇ 1, or IL-6.
  • FIG.8A Endothelin-1 or TGF- ⁇ 1 (at 200 ng/mL) assayed for phagocytosis compared to normal controls.
  • FIGGS.8B, 8C Dystrophic RPE cells incubated with recombinant human endothelin-1, TGF- ⁇ 1 or IL-6 at 200 ng/mL and assayed for phagocytosis.
  • hUTC CM3 was used as a positive control.
  • ROS was preincubated in MEM20 with various concentrations of human recombinant vitronectin (4, 2, 1, 0.5 ug/ml) respectively.
  • normal RPE alone or dystrophic RPE alone was cultured in MEM20, then changed to 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 vitronectin
  • Figure 10 shows gene expression level of RTK ligands identified in hUTC.
  • Total mRNAs were extracted from hUTC and RNA-Seq was performed to identify and quantify the RTK ligands gene expression in hUTC.
  • the identified RTK ligands were sorted based on the corresponding RTK subfamilies and ranked according to their FPKM value. Gene expression of multiple RTK ligands for 15 RTK subfamilies were detected in hUTC.
  • FIGS 11A– 11G show levels of selected RTK ligands measured in hUTC CM.
  • FIGS 11A-11F Levels of RTK ligands compared to those from NHDF and ARPE- 19 conditioned medium.
  • BDNF is secreted in high level in hUTC conditioned medium compared to NHDF and ARPE-19 conditioned medium (FIG.11A).
  • NT3 level is high in hUTC CM compared to NHDF CM, whereas the amount of NT3 in ARPE-19 conditioned medium and control medium was undetectable (FIG.11B).
  • HGF is secreted in high level in hUTC CM compared to NHDF and ARPE-19 conditioned medium (FIG. 11C).
  • PDGF-CC and PDGF-DD levels in hUTC conditioned medium are low compared to NHDF and ARPE-19 conditioned medium (FIG.11D and Fig.11E, respectively).
  • GDNF is secreted in hUTC and NHDF conditioned medium, with trace amount in ARPE- 19 conditioned medium (FIG.11F). All values are mean ⁇ SD of triplicate samples, except NT3 is mean ⁇ SD of duplicate samples.
  • FIG. 12A– 12 E show levels of bridge molecules measured in hUTC CM.
  • FIG.12A shows the MFG-E8 level in hUTC, ARPE-19 and NHDF conditioned medium. Values are mean ⁇ SD of duplicate or triplicate samples.
  • FIG.12B shows the Gas6 level in hUTC, ARPE-19 and NHDF conditioned medium. Values are mean ⁇ SD of duplicate samples.
  • FIG.12C shows TSP-1 level in hUTC, ARPE-19 and NHDF conditioned medium. Values are mean ⁇ SD of duplicate samples.
  • FIG.12D shows the TSP-2 level in hUTC, ARPE-19 and NHDF conditioned medium. Values are mean ⁇ SD of duplicate samples.
  • FIG. 13A– 13D demonstrate the effect on phagocytosis with preincubation of dystrophic RPE cells with hUTC conditioned medium (CM).
  • CM hUTC conditioned medium
  • normal RPE alone or dystrophic RPE alone was preincubated in its regular growth medium MEM20 (MEM+20% FBS).
  • dystrophic RPE was also preincubated with CM3.
  • ROS was tested to be preincubated with hUTC CM3.
  • Values are mean ⁇ SD of number of phagocytized ROS in the counted fields per sample.
  • FIG.13A, n 10-20 per sample;
  • FIG.13B is raw data.
  • FIG.13C, n 12 per sample and each sample is in duplicate. (n: number of fields counted);
  • FIG.13D is raw data.
  • FIGs 14A– 14K show the effect of bridge molecules and RTK ligands on rod outer segment (ROS) phagocytosis by RCS RPE cells.
  • ROS was preincubated with control medium (DMEM + 10% FBS) or hUTC conditioned media.
  • control medium DMEM + 10% FBS
  • ROS was preincubated in control medium with various concentrations of human recombinant MFG-E8 (FIG.14A), Gas6 (FIG.14B), TSP-1 (FIG.14C), or TSP-2 (FIG.14D).
  • DMEM + 10% FBS fetal bovine serum
  • N normal RPE; N+ROS: normal RPE cells fed with untreated ROS during phagocytosis assay; D: dystrophic RPE cells; D+ROS: dystrophic RPE cells fed with untreated ROS during phagocytosis assay; Con M: control medium; D+ROS+Con: dystrophic RPE cells fed with control medium pre-incubated ROS; CM4: the 4 th batch of hUTC conditioned medium). D+ROS+CM4: dystrophic RPE cells fed with CM4 pre-incubated ROS;
  • FIGS.14E-14H Photoreceptor OS were incubated with recombinant human MFG-E8 (FIG.14E), Gas6 (FIG.14F), TSP-1 (FIG.14G), or TSP- 2 (FIG.14H) for 24 hours and then fed to RCS RPE cells for phagocytosis assay in the absence of hUTC CM. OS pre-incubated with hUTC CM was used as a positive control for the assay. Phagocytosis of OS by RCS RPE cells was rescued in a dose-dependent manner by MFG-E8, Gas6, TSP-1 or TSP-2.
  • FIGS.14I-14K RCS RPE cells were incubated with recombinant human BDNF (FIG.14I), HGF (FIG.14J) or GDNF (FIG. 14K) for 24 hours, and then subject to phagocytosis assay.
  • RCS RPE incubated with hUTC CM was used as a positive control for the assay.
  • BDNF, HGF or GDNF dose- dependently increased the phagocytosis in RCS RPE cells.
  • FIG. 15A RTK ligands and bridge molecules for hUTC-induced phagocytosis rescue in RCS RPE.
  • FIG.15A ELISA of cell culture supernatants collected from untransfected hUTC and hUTC transfected with siRNA.
  • FIG.15B Expression of RTK ligands BDNF, HGF and GDNF were silenced by siRNA transfection of hUTC. Knockdown (KD) hUTC CM were harvested. RCS RPE were incubated with KD hUTC CM for 24 hours and then subject to phagocytosis assay.
  • FIG. 16A-16D hUTC-secreted bridge molecules bind to photoreceptor OS.
  • IF Immunofluorescence
  • the rhodopsin-stained OS particles also stained positively with each of the recombinant bridge molecule proteins or the secreted bridge molecules present in hUTC CM.
  • FIG.16D The specificity of anti-rhodopsin antibody 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 (FIGS.16A-16D), bridge molecule and isotype antibody staining; lower panel (FIGS.16A-16D), rhodopsin antibody staining.
  • FIGS.17A-17B illustrate hUTC conditioned media protected A2E-containing RPE cells from non-viability after 430 nm irradiation.
  • FIG.17A Cell death was assayed using a two-color fluorescence assay.
  • hUTC conditioned media and unconditioned control media 250 ⁇ L/well were incubated with A2E-laden ARPE-19 cells for 7 days. The percent of nonviable cells was determined by a two-color fluorescence assay; 5 replicates.
  • FIG.17B shows pooled data from the 5% and 10% FBS treatments. Values are mean +/- SEM.
  • FIGS.17C-17D illustrate hUTC conditioned media protected ARPE-19 cells against A2E photooxidation-associated reduced cell viability.
  • FIG.17C Cell viability was assayed by MTT.
  • hUTC conditioned media and unconditioned control media 250 ⁇ L/well were incubated with A2E-laden ARPE-19 cells (7 days, 37 °C, 5% CO 2 , 5% FBS). Bar height is indicative of MTT absorbance and reflects cell viability.
  • FIG.17D shows pooled data from the 5% and 10% FBS treatments. Values are mean +/- SEM; 4 replicates/2 experiments.
  • FIG.18 shows an immunofluorescence micrograph of phagocytosis by human and rat RPE.
  • FIG.20 shows an immunofluorescence micrograph of phagocytosis of human ROS with rat RPE.
  • FIG.23 shows cytokeratin immunostaining of human wet AMD RPE (sample 15-10-021).
  • FIG.24 shows cytokeratin immunostaining of ND08333“AMD” RPE.
  • FIG.27 shows the results for human ROS dose response test in human RPE (sample 15-08-074).
  • FIG.28 shows the results for human ROS dose response test in human RPE (sample 15-11-098).
  • FIG.29 shows phagocytosis in AMD and Normal RPEs with or without CM with human eye 15-09-027 upper ROS (includes human eye 15-04-001 RPE).
  • FIG.32 shows the effect of RTK ligand BDNF on phagocytosis by“wet” AMD RPE (15-10-021).
  • FIG.33 shows the effect of RTK ligand GDNF on phagocytosis by“wet” AMD RPE (15-10-021).
  • FIG.34 shows the effect of RTK ligand BDNF on phagocytosis by AMD RPE.
  • FIG.36 shows the effect of RTK ligand GDNF on phagocytosis by AMD RPE (ND08333).
  • FIG.37 shows the effect of RTK ligand GDNF on phagocytosis by AMD RPE (ND08626).
  • FIG.43 shows the effect of bridge molecule Tsp 1 on phagocytosis by“wet” AMD RPE (15-10-021).
  • FIG.44 shows the effect of bridge molecule MFG-E8 on phagocytosis by “wet” AMD RPE (15-10-021).
  • FIG.45 shows the effect of bridge molecule Tsp 2 on phagocytosis by“wet” AMD RPE (15-10-021).
  • FIG.46 shows the effect of bridge molecule MFG-E8 on phagocytosis by AMD RPE (ND08333).
  • FIG.47 shows the effect of bridge molecule Tsp 1 on phagocytosis by AMD RPE (ND08333).
  • FIG.48 shows the effect of bridge molecule Tsp 2 on phagocytosis by AMD RPE (ND08333).
  • FIG.49 shows the effect of bridge molecule MFG-E8 on phagocytosis by AMD RPE (ND08626).
  • FIG.50 shows the effect of bridge molecule Tsp 1 on phagocytosis by AMD RPE (ND08626).
  • FIG.51 shows the effect of bridge molecule Tsp 2 on phagocytosis by AMD RPE (ND08626).
  • Stem cells are undifferentiated cells defined by the ability of a single cell both to self-renew, and to differentiate to produce progeny cells, including self-renewing progenitors, non-renewing progenitors, and terminally differentiated cells. Stem cells are also characterized by their ability to differentiate in vitro into functional cells of various cell lineages from multiple germ layers (endoderm, mesoderm and ectoderm), as well as to give rise to tissues of multiple germ layers following transplantation, and to contribute substantially to most, if not all, tissues following injection into blastocysts.
  • ⁇ Stem cells are classified according to their developmental potential as: (1) totipotent; (2) pluripotent; (3) multipotent; (4) oligopotent; and (5) unipotent.
  • Totipotent cells are able to give rise to all embryonic and extraembryonic cell types.
  • Pluripotent cells are able to give rise to all embryonic cell types.
  • Multipotent cells include those able to give rise to a subset of cell lineages, but all within a particular tissue, organ, or physiological system (for example, hematopoietic stem cells (HSC) can produce progeny that include HSC (self-renewal), blood cell-restricted oligopotent progenitors, and all cell types and elements (e.g., platelets) that are normal components of the blood).
  • HSC hematopoietic stem cells
  • Cells that are oligopotent can give rise to a more restricted subset of cell lineages than multipotent stem cells; and cells that are unipotent are able to give rise to a single cell lineage (e.g., spermatogenic stem cells).
  • Stem cells are also categorized on the basis of the source from which they may be obtained.
  • An adult stem cell is generally a multipotent undifferentiated cell found in tissue comprising multiple differentiated cell types.
  • the adult stem cell can renew itself. Under normal circumstances, it can also differentiate to yield the specialized cell types of the tissue from which it originated, and possibly other tissue types.
  • Induced pluripotent stem cells iPS cells are adult cells that are converted into pluripotent stem cells.
  • An embryonic stem cell is a pluripotent cell from the inner cell mass of a blastocyst-stage embryo.
  • a fetal stem cell is one that originates from fetal tissues or membranes.
  • a postpartum stem cell is a multipotent or pluripotent cell that originates substantially from extraembryonic tissue available after birth, namely, the placenta and the umbilical cord. These cells have been found to possess features characteristic of pluripotent stem cells, including rapid proliferation and the potential for differentiation into many cell lineages.
  • Postpartum stem cells may be blood-derived (e.g., as are those obtained from umbilical cord blood) or non-blood-derived (e.g., as obtained from the non-blood tissues of the umbilical cord and placenta).
  • Embryonic tissue is typically defined as tissue originating from the embryo (which in humans refers to the period from fertilization to about six weeks of
  • Extraembryonic tissues include extraembryonic membranes (chorion, amnion, yolk sac and allantois), umbilical cord and placenta (which itself forms from the chorion and the maternal decidua basalis).
  • a specialized cell such as a nerve cell or a muscle cell, for example.
  • a differentiated cell is one that has taken on a more specialized (“committed”) position within the lineage of a cell.
  • the term committed when applied to the process of differentiation, refers to a cell that has proceeded in the differentiation pathway to a point where, under normal circumstances, it will continue to differentiate into a specific cell type or subset of cell types, and cannot, under normal circumstances, differentiate into a different cell type or revert to a less differentiated cell type.
  • De- differentiation refers to the process by which a cell reverts to a less specialized (or committed) position within the lineage of a cell.
  • the lineage of a cell defines the heredity of the cell, i.e. which cells it came from and what cells it can give rise to.
  • the lineage of a cell places the cell within a hereditary scheme of development and differentiation.
  • a progenitor cell is a cell that has the capacity to create progeny that are more differentiated than itself, and yet retains the capacity to replenish the pool of progenitors.
  • stem cells themselves are also progenitor cells, as are the more immediate precursors to terminally differentiated cells.
  • this broad definition of progenitor cell may be used.
  • a progenitor cell is often defined as a cell that is intermediate in the differentiation pathway, i.e., it arises from a stem cell and is intermediate in the production of a mature cell type or subset of cell types.
  • progenitor cell is generally not able to self-renew. Accordingly, if this type of cell is referred to herein, it will be referred to as a non-renewing progenitor cell or as an intermediate progenitor or precursor cell.
  • the phrase“differentiates into an ocular lineage or phenotype” refers to a cell that becomes partially or fully committed to a specific ocular phenotype, including without limitation, retinal and corneal stem cells, pigment epithelial cells of the retina and iris, photoreceptors, retinal ganglia and other optic neural lineages (e.g., retinal glia, microglia, astrocytes, Mueller cells), cells forming the crystalline lens, and epithelial cells of the sclera, cornea, limbus and conjunctiva.
  • retinal and corneal stem cells include retinal and corneal stem cells, pigment epithelial cells of the retina and iris, photoreceptors, retinal ganglia and other optic neural lineages (e.g., retinal glia, microglia, astrocytes, Mueller cells), cells forming the crystalline lens, and epithelial cells of the sclera, cornea, limbus and conjunctiva.
  • the cells After the second subculture, the cells become a tertiary culture (P2 or passage 2), and so on. It will be understood by those of skill in the art that there may be many population doublings during the period of passaging; therefore the number of population doublings of a culture is greater than the passage number.
  • the expansion of cells (i.e., the number of population doublings) during the period between passaging depends on many factors, including but not limited to the seeding density, substrate, medium, growth conditions, and time between passaging.
  • the term Growth Medium generally refers to a medium sufficient for the culturing of PPDCs.
  • one presently preferred medium for the culturing of the cells of the invention comprises Dulbecco's Modified Essential Media (also abbreviated DMEM herein).
  • DMEM-low glucose also DMEM-LG herein
  • the DMEM-low glucose is preferably supplemented with 15% (v/v) fetal bovine serum (e.g.
  • a conditioned medium is a medium in which a specific cell or population of cells has been cultured, and then removed. When cells are cultured in a medium, they may secrete cellular factors that can provide trophic support to other cells. Such trophic factors include, but are not limited to hormones, cytokines, extracellular matrix (ECM), proteins, vesicles, antibodies, and granules.
  • the medium containing the cellular factors is the conditioned medium.
  • Chronic ocular degenerative conditions include, but are not limited to, retinopathies and other retinal/macular disorders such as retinitis pigmentosa (RP), age-related macular degeneration (AMD), choroidal neovascular membrane (CNVM); retinopathies such as diabetic retinopathy, occlusive retinopathy, sickle cell retinopathy and hypertensive retinopathy, central retinal vein occlusion, stenosis of the carotid artery, optic
  • RP retinitis pigmentosa
  • AMD age-related macular degeneration
  • CNVM choroidal neovascular membrane
  • retinopathies such as diabetic retinopathy, occlusive retinopathy, sickle cell retinopathy and hypertensive retinopathy, central retinal vein occlusion, stenosis of the carotid artery, optic
  • treating (or treatment of) an ocular degenerative condition refers to ameliorating the effects of, or delaying, halting or reversing the progress of, or delaying or preventing the onset of, an ocular degenerative condition as defined herein.
  • an effective amount refers to a concentration or amount of a reagent or pharmaceutical composition, such as a growth factor, differentiation agent, trophic factor, cell population or other agent, that is effective for producing an intended result, including cell growth and/or differentiation in vitro or in vivo, or treatment of ocular degenerative conditions, as described herein.
  • a reagent or pharmaceutical composition such as a growth factor, differentiation agent, trophic factor, cell population or other agent, that is effective for producing an intended result, including cell growth and/or differentiation in vitro or in vivo, or treatment of ocular degenerative conditions, as described herein.
  • growth factors an effective amount may range from about 1 nanogram/milliliter to about 1 microgram/milliliter.
  • PPDCs as administered to a patient in vivo, an effective amount may range from as few as several hundred or fewer, to as many as several million or more. In specific embodiments, an effective amount may range from 10 3 to 11 11 , more specifically at least about 10 4 cells.
  • the number of cells to be administered will vary depending on the specifics of the disorder to be treated, including but not limited to size or total volume/surface area to be treated, as well as proximity of the site of administration to the location of the region to be treated, among other factors familiar to the medicinal biologist.
  • pharmaceutically acceptable carrier which may be used interchangeably with the term biologically compatible carrier or medium, refers to reagents, cells, compounds, materials, compositions, and/or dosage forms that are not only compatible with the cells and other agents to be administered therapeutically, but also are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other complication commensurate with a reasonable benefit/risk ratio.
  • autologous transfer, autologous transplantation, autograft and the like refer to treatments wherein the cell donor is also the recipient of the cell replacement therapy.
  • allogeneic transfer, allogeneic transplantation, allograft and the like refer to treatments wherein the cell donor is of the same species as the recipient of the cell replacement therapy, but is not the same individual.
  • a cell transfer in which the donor's cells and have been histocompatibly matched with a recipient is sometimes referred to as a syngeneic transfer.
  • Ocular degenerative conditions which encompass acute, chronic and progressive disorders and diseases having divergent causes, have as a common feature the dysfunction or loss of a specific or vulnerable group of ocular cells. This commonality enables development of similar therapeutic approaches for the repair or regeneration of vulnerable, damaged or lost ocular tissue, one of which is cell-based therapy.
  • stem or progenitor cells including ocular-derived stem cells themselves (e.g., retinal and corneal stem cells), embryonic stem cells and a few types of adult stem or progenitor cells (e.g., neural, mucosal epithelial and bone marrow stem cells).
  • ocular-derived stem cells themselves (e.g., retinal and corneal stem cells), embryonic stem cells and a few types of adult stem or progenitor cells (e.g., neural, mucosal epithelial and bone marrow stem cells).
  • ocular-derived stem cells themselves (e.g., retinal and corneal stem cells), embryonic stem cells and a few types of adult stem or progenitor cells (e.g., neural, mucosal epithelial and bone marrow stem cells).
  • conditioned media generated from cells isolated from the postpartum placenta and umbilical cord tissue provides another new source for treating ocular degenerative conditions.
  • Conditioned media derived from progenitor cells such as cells isolated from postpartum umbilical cord or placenta in accordance with any method known in the art is expected to be suitable for use in the present invention.
  • the invention uses conditioned media derived from umbilical cord tissue-derived cells (hUTCs) or placental-tissue derived cells (PDCs) as defined above, which are derived from umbilical cord tissue or placenta that has been rendered substantially free of blood, preferably in accordance with the method set forth below.
  • the hUTCs or PDCs are capable of expansion in culture and have the potential to differentiate into cells of other phenotypes.
  • Progenitor cells The cells, cell populations and preparations comprising cell lysates, conditioned media and the like, used in the compositions and methods of the present invention are described herein, and in detail in U.S. Patent Nos.7,524,489, and 7,510,873, and U.S. Pub. App. No.2005/0058634, both incorporated by reference herein. According to the methods, a mammalian umbilical cord and placenta are recovered upon or shortly after termination of either a full-term or pre-term pregnancy, for example, after expulsion of after-birth.
  • the postpartum tissue may be transported from the birth site to a laboratory in a sterile container such as a flask, beaker, culture dish, or bag.
  • the container may have a solution or medium, including but not limited to a salt solution, such as, for example, Dulbecco's Modified Eagle's Medium (DMEM) or phosphate buffered saline (PBS), or any solution used for transportation of organs used for transplantation, such as University of Wisconsin solution or perfluorochemical solution.
  • DMEM Dulbecco's Modified Eagle's Medium
  • PBS phosphate buffered saline
  • antibiotic and/or antimycotic agents such as but not limited to penicillin, streptomycin, amphotericin B, gentamicin, and nystatin, may be added to the medium or buffer.
  • the postpartum tissue may be rinsed with an anticoagulant solution such as heparin-containing solution. It is preferable to keep the tissue at about 4-10° C prior to extraction of PPDCs. It is even more preferable that the tissue not be frozen prior to extraction of PPDCs.
  • Isolation of PPDCs preferably occurs in an aseptic environment.
  • the umbilical cord may be separated from the placenta by means known in the art. Alternatively, the umbilical cord and placenta are used without separation.
  • Blood and debris are preferably removed from the postpartum tissue prior to isolation of PPDCs.
  • the postpartum tissue may be washed with buffer solution, such as but not limited to phosphate buffered saline.
  • the wash buffer also may comprise one or more antimycotic and/or antibiotic agents, such as but not limited to penicillin, streptomycin, amphotericin B, gentamicin, and nystatin.
  • Postpartum tissue comprising a whole placenta or umbilical cord, or a fragment or section thereof is disaggregated by mechanical force (mincing or shear forces).
  • the isolation procedure also utilizes an enzymatic digestion process.
  • Many enzymes are known in the art to be useful for the isolation of individual cells from complex tissue matrices to facilitate growth in culture. Ranging from weakly digestive (e.g. deoxyribonucleases and the neutral protease, dispase) to strongly digestive (e.g. papain and trypsin), such enzymes are available commercially.
  • a nonexhaustive list of enzymes compatible herewith includes mucolytic enzyme activities, metalloproteases, neutral proteases, serine proteases (such as trypsin, chymotrypsin, or elastase), and deoxyribonucleases.
  • enzyme activities selected from metalloproteases, neutral proteases and mucolytic activities.
  • collagenases are known to be useful for isolating various cells from tissues.
  • Deoxyribonucleases can digest singlestranded DNA and can minimize cell clumping during isolation.
  • 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, a mixture of collagenase and the neutral protease dispase are used in the dissociating step. More preferred are those methods that employ digestion in the presence of at least one collagenase from
  • Clostridium histolyticum and either of the protease activities, dispase and thermo lysin. Still more preferred are methods employing digestion with both collagenase and dispase enzyme activities. Also preferred are methods that include digestion with a hyaluronidase activity in addition to collagenase and dispase activities.
  • the skilled artisan will appreciate that many such enzyme treatments are known in the art for isolating cells from various tissue sources. For example, the LIBERASE TM Blendzyme 3 (Roche) series of enzyme combinations are suitable for use in the instant methods. Other sources of enzymes are known, and the skilled artisan may also obtain such enzymes directly from their natural sources.
  • Preferred enzyme treatments are 0.5, 1, 1.5, or 2 hours long or longer.
  • the tissue is incubated at 37° C during the enzyme treatment of the dissociation step.
  • postpartum tissue is separated into sections comprising various aspects of the tissue, such as neonatal, neonatal/maternal, and maternal aspects of the placenta, for instance.
  • the separated sections then are dissociated by mechanical and/or enzymatic dissociation according to the methods described herein.
  • Cells of neonatal or maternal lineage may be identified by any means known in the art, for example, by karyotype analysis or in situ hybridization for a Y chromosome.
  • Isolated cells or postpartum tissue from which PPDCs grow out may be used to initiate, or seed, cell cultures. Isolated cells are transferred to sterile tissue culture vessels either uncoated or coated with extracellular matrix or ligands such as laminin, collagen (native, denatured or crosslinked), gelatin, fibronectin, and other extracellular matrix proteins.
  • extracellular matrix or ligands such as laminin, collagen (native, denatured or crosslinked), gelatin, fibronectin, and other extracellular matrix proteins.
  • PPDCs are cultured in any culture medium capable of sustaining growth of the cells such as, but not limited to, DMEM (high or low glucose), advanced DMEM, DMEM/MCDB 201, Eagle's basal medium, Ham's F10 medium (F10), Ham's F-12 medium (F12), Iscove's modified Dulbecco's medium, Mesenchymal Stem Cell Growth Medium (MSCGM), DMEM/F12, RPMI 1640, and cellgro FREETM.
  • DMEM high or low glucose
  • advanced DMEM DMEM/MCDB 201
  • Eagle's basal medium Eagle's basal medium
  • Ham's F10 medium (F10) Ham's F-12 medium (F12)
  • Iscove's modified Dulbecco's medium Iscove's modified Dulbecco's medium
  • MSCGM Mesenchymal Stem Cell Growth Medium
  • DMEM/F12 RPMI 1640
  • cellgro FREETM cellgro FREETM.
  • the culture medium may be supplemented with one or more components including, for example, fetal bovine serum (FBS), preferably about 2-15% (v/v); equine serum (ES); human serum (HS); beta-mercaptoethanol (BME or 2-ME), preferably about 0.001% (v/v); one or more growth factors, for example, 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 more antibiotic and/or antimycotic agents to control microbial contamination, such as, for example, penicillin G, streptomycin sulfate, amphotericin B, gentamicin, and nystatin, either alone or in combination.
  • the culture medium preferably comprises Growth Medium (DMEM-low glucose, serum, BME
  • the cells are seeded in culture vessels at a density to allow cell growth.
  • the cells are cultured at about 0 to about 5 percent by volume CO2 in air.
  • the cells are cultured at about 2 to about 25 percent O2 in air, preferably about 5 to about 20 percent O2 in air.
  • the cells preferably are cultured at about 25 to about 40° C and more preferably are cultured at 37° C.
  • the cells are preferably cultured in an incubator.
  • the medium in the culture vessel can be static or agitated, for example, using a bioreactor.
  • PPDCs preferably are grown under low oxidative stress (e.g., with addition of glutathione, Vitamin C, Catalase, Vitamin E, N-Acetylcysteine).“Low oxidative stress”, as used herein, refers to conditions of no or minimal free radical damage to the cultured cells.
  • PPDCs After culturing the isolated cells or tissue fragments for a sufficient period of time, PPDCs will have grown out, either as a result of migration from the postpartum tissue or cell division, or both.
  • PPDCs are passaged, or removed to a separate culture vessel containing fresh medium of the same or a different type as that used initially, where the population of cells can be mitotically expanded.
  • the cells of the invention may be used at any point between passage 0 and senescence.
  • the cells preferably are passaged between about 3 and about 25 times, more preferably are passaged about 4 to about 12 times, and preferably are passaged 10 or 11 times. Cloning and/or subcloning may be performed to confirm that a clonal population of cells has been isolated.
  • the different cell types present in postpartum tissue are fractionated into subpopulations from which the PPDCs can be isolated.
  • This may be accomplished using standard techniques for cell separation including, but not limited to, enzymatic treatment to dissociate postpartum tissue into its component cells, followed by cloning and selection of specific cell types, for example but not limited to selection based on morphological and/or biochemical markers; selective growth of desired cells (positive selection), selective destruction of unwanted cells (negative selection); separation based upon differential cell agglutinability in the mixed population as, for example, with soybean agglutinin; freeze-thaw procedures; differential adherence properties of the cells in the mixed population; filtration; conventional and zonal centrifugation; centrifugal elutriation (counter-streaming centrifugation); unit gravity separation; countercurrent distribution; electrophoresis; and fluorescence activated cell sorting (FACS).
  • FACS fluorescence activated cell sorting
  • the culture medium is changed as necessary, for example, by carefully aspirating the medium from the dish, for example, with a pipette, and replenishing with fresh medium. Incubation is continued until a sufficient number or density of cells accumulates in the dish.
  • the original explanted tissue sections may be removed and the remaining cells trypsinized using standard techniques or using a cell scraper. After trypsinization, the cells are collected, removed to fresh medium and incubated as above. In some embodiments, the medium is changed at least once at approximately 24 hours post-trypsinization to remove any floating cells.
  • the cells remaining in culture are considered to be PPDCs.
  • PPDCs may be cryopreserved.
  • PPDCs for autologous transfer may be derived from appropriate postpartum tissues following the birth of a child, then cryopreserved so as to be available in the event they are later needed for transplantation.
  • the progenitor cells of the invention may be characterized, for example, by growth characteristics (e.g., population doubling capability, doubling time, passages to senescence), karyotype analysis (e.g., normal karyotype; maternal or neonatal lineage), flow cytometry (e.g., FACS analysis), immunohistochemistry and/or immunocytochemistry (e.g., for detection of epitopes), gene expression profiling (e.g., gene chip arrays; polymerase chain reaction (for example, reverse transcriptase PCR, real time PCR, and conventional PCR), protein arrays, protein secretion (e.g., by plasma clotting assay or analysis of PDC-conditioned medium, for example, by Enzyme Linked ImmunoSorbent Assay (ELISA)), mixed lymphocyte reaction (e.g., as measure of stimulation of PBMCs), and/or other methods known in the art.
  • growth characteristics e.g., population doubling capability, doubling time, passages to
  • Examples of PPDCs derived from umbilicus tissue were deposited with the American Type Culture Collection on (ATCC, 10801 University Boulevard, Manassas, VA, 20110) Jun.10, 2004, and assigned ATCC Accession Numbers as follows: (1) strain designation UMB 022803 (P7) was assigned Accession No. PTA-6067; and (2) strain designation UMB 022803 (P17) was assigned Accession No. PTA-6068.
  • Examples of PPDCs derived from placental tissue were deposited with the American Type Culture Collection (ATCC, Manassas, Va.) and assigned ATCC Accession Numbers as follows: (1) strain designation PLA 071003 (P8) was deposited Jun.15, 2004 and assigned Accession No.
  • strain designation PLA 071003 (P11) was deposited June 15, 2004 and assigned Accession No. PTA-6075; and (3) strain designation PLA 071003 (P16) was deposited Jun.16, 2004 and assigned Accession No. PTA-6079.
  • the PPDCs possess one or more of the following growth features: (1) they require L-valine for growth in culture; (2) they are capable of growth in atmospheres containing oxygen from about 5% to at least about 20%; (3) they have the potential for at least about 40 doublings in culture before reaching senescence; and (4) they attach and expand on a coated or uncoated tissue culture vessel, wherein the coated tissue culture vessel comprises a coating of gelatin, laminin, collagen,
  • polyomithine polyomithine, vitronectin or fibronectin.
  • the PPDCs possess a normal karyotype, which is maintained as the cells are passaged.
  • Karyotyping is particularly useful for identifying and distinguishing neonatal from maternal cells derived from placenta. Methods for karyotyping are available and known to those of skill in the art.
  • the PPDCs may be characterized by production of certain proteins, including: (1) production of at least one of vimentin and alpha-smooth muscle actin; and (2) production of at least one of CD10, CD13, CD44, CD73, CD90, PDGFr-alpha, PD-L2 and HLA-A,B,C cell surface markers, as detected by flow cytometry.
  • the PPDCs may be characterized by lack of production of at least one of CD31, CD34, CD45, CD80, CD86, CD117, CD141, CD178, B7-H2, HLA-G, and HLA-DR,DP,DQ cell surface markers, as detected by flow cytometry.
  • the PPDCs may be characterized by gene expression, which relative to a human cell that is a fibroblast, a mesenchymal stem cell, or an iliac crest bone marrow cell, is increased for a gene encoding at least one of interleukin 8; reticulon 1; chemokine (C--X--C motif) ligand 1 (melonoma growth stimulating activity, alpha); chemokine (C--X--C motif) ligand 6 (granulocyte chemotactic protein 2);
  • chemokine (C--X--C motif) ligand 3 tumor necrosis factor, alpha-induced 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 clone IMAGE:4179671; protein kinase C zeta; hypothetical protein DKFZp564F013;
  • the PPDCs derived from umbilical cord tissue may be characterized by gene expression, which relative to a human cell that is a fibroblast, a mesenchymal stem cell, or an iliac crest bone marrow cell, is increased for a gene encoding at least one of interleukin 8; reticulon 1; or chemokine (C--X--C motif) ligand 3.
  • the PPDCs derived from placental tissue may be characterized by gene expression, which relative to a human cell that is a fibroblast, a mesenchymal stem cell, or an iliac crest bone marrow cell, is increased for a gene encoding at least one of renin or oxidized low density lipoprotein receptor 1.
  • the PPDCs may be characterized by gene expression, which relative to a human cell that is a fibroblast, a mesenchymal stem cell, or an iliac crest bone marrow cell, is reduced for a gene encoding at least one of: short stature homeobox 2; heat shock 27 kDa protein 2; chemokine (C--X--C motif) ligand 12 (stromal cell-derived factor 1); elastin (supravalvular aortic stenosis, Williams-Beuren syndrome); Homo sapiens mRNA; cDNA DKFZp586M2022 (from clone
  • DKFZp586M2022 mesenchyme homeo box 2 (growth arrest-specific homeo box); sine oculis homeobox homolog 1 (Drosophila); crystallin, alpha B; disheveled associated activator of morphogenesis 2; DKFZP586B2420 protein; similar to neuralin 1; tetranectin (plasminogen binding protein); src homology three (SH3) and cysteine rich domain; cholesterol 25-hydroxylase; runt-related transcription factor 3; interleukin 11 receptor, alpha; procollagen C-endopeptidase enhancer; frizzled homolog 7 (Drosophila);
  • cytokine receptor-like factor 1 potassium intermediate/small conductance calcium- activated channel, subfamily N, member 4; integrin, beta 7; transcriptional co-activator with PDZ-binding motif (TAZ); sine oculis homeobox homolog 2 (Drosophila);
  • KIAAI034 protein vesicle-associated membrane protein 5 (myobrevin); EGF-containing fibulin-like extracellular matrix protein 1; early growth response 3; distal-less homeo box 5; hypothetical protein FLJ20373; aldo-keto reductase family 1, member C3 (3-alpha hydroxysteroid dehydrogenase, type II); biglycan; transcriptional co-activator with PDZ- binding motif (TAZ); fibronectin 1; proenkephalin; integrin, beta-like 1 (with EGF-like repeat domains); Homo sapiens mRNA full length insert cDNA clone EUROIMAGE 1968422; EphA3; KIAA0367 protein; natriuretic peptide receptor C/guanylate cyclase C (atrionatriuretic peptide receptor C); hypothetical protein FLJ14054; Homo sapiens mRNA; cDNA DKFZp564B222 (from clone DKFZp
  • the PPDCs may be characterized by secretion of bridge molecules selected from MFG-E8, Gas6, TSP-1 and TSP-2.
  • the PPDCs derived from umbilical cord tissue may be characterized by secretion of at least one of MCP-1, IL-6, IL-8, GCP-2, HGF, KGF, FGF, HB-EGF, BDNF, TPO, MIP1b, I309, RANTES, MDC, and TIMP1.
  • the PPDCs derived from umbilical cord tissue may be characterized by lack of secretion of at least one of TGF-beta2, ANG2, PDGFbb, MIP1a and VEGF, as detected by ELISA.
  • PPDCs derived from placenta tissue may be characteristized by secretion of at least one of MCP-l, IL-6, IL-8, GCP-2, HGF, KGF, HB-EGF, BDNF, TPO, MIP1a, RANTES, and TIMP1, and lack of secretion of at least one of TGF-beta2, MIP1b, ANG2, PDGFbb, FGF, and VEGF, as detected by ELISA.
  • the PPDCs lack expression of hTERT or telomerase.
  • the cell comprises two or more of the above-listed growth, protein/surface marker production, gene expression or substance-secretion characteristics. More preferred are those cells comprising, three, four, or five or more of the characteristics. Still more preferred are PPDCs comprising six, seven, or eight or more of the characteristics. Still more preferred presently are those cells comprising all of above characteristics.
  • the cells isolated from human umbilical cord tissue substantially free of blood, which are capable of expansion in culture lack the production of CD117 or CD45, and do not express hTERT or telomerase. In one embodiment, the cells lack production of CD117 and CD45 and, optionally, also do not express hTERT and telomerase. In another embodiment, the cells do not express hTERT and telomerase.
  • the cells are isolated from human umbilical cord tissue substantially free of blood, are capable of expansion in culture, lack the production of CD117 or CD45, and do not express hTERT or telomerase, and have one or more of the following characteristics: express CD10, CD13, CD44, CD73, and CD90; do not express CD31 or CD34; express, relative to a human fibroblast, mesenchymal stem cell, or iliac crest bone marrow cell, increased levels of interleukin 8 or reticulon 1; and have the potential to differentiate.
  • Certain cells having the potential to differentiate along lines leading to various phenotypes are unstable and thus can spontaneously differentiate.
  • Presently preferred for use with the invention are cells that do not spontaneously differentiate, for example along neural lines.
  • Preferred cells, when grown in Growth Medium, are substantially stable with respect to the cell markers produced on their surface, and with respect to the expression pattern of various genes, for example as determined using an Affymetrix GENECHIP. The cells remain substantially constant, for example in their surface marker
  • PPDCs may be deliberately induced to differentiate into various lineage phenotypes by subjecting them to differentiation- inducing cell culture conditions.
  • the PPDCs may be induced to differentiate into neural phenotypes using one or more methods known in the art. For instance, as exemplified herein, PPDCs may be plated on flasks coated with laminin in Neurobasal-A medium (Invitrogen, Carlsbad, Calif.) containing B27 (B27 supplement, Invitrogen), L-glutamine and
  • NPE Neural Progenitor Expansion
  • PPDCs may be induced to differentiate in vitro by: (1) co-culturing the PPDCs with neural progenitor cells; or (2) growing the PPDCs in neural progenitor cell-conditioned medium.
  • PPDCs Differentiation of the PPDCs into neural phenotypes may be demonstrated by a bipolar cell morphology with extended processes.
  • the induced cell populations may stain positive for the presence of nestin.
  • Differentiated PPDCs may be assessed by detection of nest in, TuJ1 (BIII tubulin), GFAP, tyrosine hydroxylase, GABA, 04 and/or MBP.
  • PPDCs have exhibited the ability to form three-dimensional bodies characteristic of neuronal stem cell formation of neurospheres.
  • Cell Populations [0148] Another aspect of the invention features populations of progenitor cells, such as postpartum-derived cells.
  • the postpartum-derived cells may be isolated from placental or umbilical tissue.
  • the cell populations comprise the PPDCs described above, and these cell populations are described in the section below.
  • the cell population is heterogeneous.
  • a heterogeneous cell population of the invention may comprise at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% of the cell.
  • the heterogeneous cell populations of the invention may further comprise the progenitor cells (postpartum-derived cells), or other progenitor cells, such as epithelial or neural progenitor cells, or it may further comprise fully differentiated cells.
  • the population is substantially homogeneous, i.e., comprises substantially only PPDCs (preferably at least about 96%, 97%, 98%, 99% or more of the cells).
  • the cell population is homogeneous.
  • the homogeneous cell population of the invention may comprise umbilicus- or placenta-derived cells. Homogeneous populations of umbilicus-derived cells are preferably free of cells of maternal lineage. Homogeneous populations of placenta-derived cells may be of neonatal or maternal lineage.
  • Homogeneity of a cell population may be achieved by any method known in the art, for example, by cell sorting (e.g., flow cytometry) or by clonal expansion in accordance with known methods.
  • preferred homogeneous PPDC populations may comprise a clonal cell line of postpartum- derived cells. Such populations are particularly useful when a cell clone with highly desirable functionality has been isolated.
  • populations of cells incubated in the presence of one or more factors, or under conditions, that stimulate stem cell differentiation along a desired pathway e.g., neural, epithelial.
  • a desired pathway e.g., neural, epithelial
  • factors are known in the art and the skilled artisan will appreciate that determination of suitable conditions for differentiation can be accomplished with routine experimentation. Optimization of such conditions can be accomplished by statistical experimental design and analysis, for example response surface methodology allows simultaneous optimization of multiple variables, for example in a biological culture.
  • Presently preferred factors include, but are not limited to factors, such as growth or trophic factors, demethylating agents, co-culture with neural or epithelial lineage cells or culture in neural or epithelial lineage cell-conditioned medium, as well other conditions known in the art to stimulate stem cell differentiation along these pathways (for factors useful in neural differentiation, see, e.g., Lang, K. J. D. et al., 2004, J. Neurosci. Res.76: 184-192; Johe, K. K. et al., 1996, Genes Devel.10: 3129-3140; Gottleib, D., 2002, Ann. Rev. Neurosci.25: 381-407).
  • factors such as growth or trophic factors, demethylating agents, co-culture with neural or epithelial lineage cells or culture in neural or epithelial lineage cell-conditioned medium, as well other conditions known in the art to stimulate stem cell differentiation along these pathways (for factors useful in neural differentiation, see, e.g., Lang, K. J.
  • the invention provides conditioned medium from cultured progenitor cells, such as postpartum-derived cells, or other progenitor cells, for use in vitro and in vivo as described below.
  • cultured progenitor cells such as postpartum-derived cells, or other progenitor cells
  • Use of such conditioned medium allows the beneficial trophic factors secreted by the cells to be used allogeneically in a patient without introducing intact cells that could trigger rejection, or other adverse
  • Conditioned medium is prepared by culturing cells (such as a population of cells) in a culture medium, then removing the cells from the medium.
  • the postpartum cells are UTCs or PDCs, more preferably hUTCs.
  • Conditioned medium prepared from populations of cells as described above may be used as is, further concentrated, by for example, ultrafiltration or lyophilization, or even dried, partially purified, combined with pharmaceutically-acceptable carriers or diluents as are known in the art, or combined with other compounds such as biologicals, for example pharmaceutically useful protein compositions.
  • Conditioned medium may be used in vitro or in vivo, alone or for example, with autologous or syngeneic live cells.
  • the conditioned medium, if introduced in vivo may be introduced locally at a site of treatment, or remotely to provide, for example needed cellular growth or trophic factors to a patient.
  • hUTC conditioned medium were prepared and evaluated for phagocytosis rescue activities. Seeding density and culture conditions were found to affect activity level for conditioned media. For hUTC in serum (CM1), hUTCs were seeded at 5,000 viable cells/cm 2 in T75 cell culture flask in hUTC growth medium (DMEM low glucose + 15% FBS + 4 mM L-glutamine), and cultured for 24 hours. Medium was replaced with 21 mL of DMEM/F12 complete medium
  • DMEM F12 medium + 10% FBS + Pen (50U/ml)/Strep (50 ⁇ g/ml)
  • cells were cultured for another 54 hours, and the culture supernatant was collected and frozen at -70 o C (cryopreserved).
  • CM1 serum-free medium was replenished at day 2 with 21 mL of DMEM/F12 serum-free medium (DMEM:F12 medium + Pen
  • CM1 with or without serum restored phagocytosis activity ( Figures 1A-1B and 2).
  • Another conditioned medium (CM2) was prepared under the same procedure as CM1 with serum, except the cells were cultured T225 flasks with 63 mL of medium per flask, and the incubation time after medium change was 48 hours. This media, however, had no activity.
  • Figure 3A A CM3 was prepared with the same conditions as CM2 but with 10,000 viable cells/cm 2 , and stimulated phagocytosis in dystrophic RPE. ( Figures 3B-3D).
  • CM2 was found to lack activity (Figure 3A).
  • CM2 had a shortened incubation time of 48 h, compared to the incubation time for CM1 of 54 hours.
  • CM3 was prepared by doubling the cell seeding density with the same incubation time after medium change, compared to CM2, and found to be active. Therefore, to obtain an active CM, initial cell seeding density and cell incubation time after medium change are two important conditions.
  • RPE Retinal pigment epithelium
  • RCS rod outer segment
  • bFGF Basic fibroblast growth factor
  • hUTC rescue of dystrophic RPE phagocytosis is through secretion of RTK ligands, activating RTK signaling and enhancing signaling of other phagocytosis-related receptors.
  • RTK ligands BDNF, HB-EGF, PDGF-DD, Ephrin A4, HGF, and Ephrin B2 have rescue effect on phagocytosis by the RCS dystrophic RPE cells.
  • BDNF, PDGF-DD, and Ephrin B2 have positive rescue effects. (See Figures 5A, 6A– 6B and 7A– 7B).
  • Non-RTK ligands activate different receptors from RTK, and do not have a similar effect on phagocytosis as RTK ligands ( Figures 8A-8C and Figure 9).
  • hUTC has been shown to secrete vitronectin, endothelin-1, TGF- ⁇ 1, and IL-6.
  • Receptors for vitronectin include ⁇ v ⁇ 3 and ⁇ v ⁇ 5 integrins.
  • Finnemann et al. reported that phagocytosis of ROS by RPE cells requires ⁇ v ⁇ 5 integrin (Finnemann et al., 1997, supra).
  • RNA analysis from conditioned media-treated and untreated dystrophic RPE for gene expression profiling show that hUTC express multiple genes of RTK ligands within 15 RTK subfamilies ( Figure 10, and Table 1-1). hUTC also express genes of bridge molecules, including MFG-E8, Gas6, protein S, TSP-1 and TSP-2 (Table 1-3). RCS RPE express genes in 18 RTK subfamilies (Table 1-2). Among the 18 are the 15 RTK subfamilies corresponding to the RTK ligand genes expressed in hUTC. RCS RPE also express receptor genes for bridge molecule binding, including integrin ⁇ v ⁇ 3, ⁇ v ⁇ 5, Axl, Tyro3, MerTK, and CD36 (Table 1-4).
  • ligands include BDNF (brain-derived neurotrophic factor) and NT3 (neurotrophin 3) -ligands of Trk family, HGF (hepatocyte growth factor) -a ligand of Met family, PDGF-DD (platelet-derived growth factor type D) and PDGF-CC (platelet- derived growth factor type C) -ligands of PDGF family, ephrin-B2 -a ligand of Eph family, HB-EGF (heparin-binding epidermal growth factor) -a ligand of ErbB family, GDNF (glial cell-derived neurotrophic factor) -a ligand of Ret family, as well as agrin -a ligand of Musk family.
  • BDNF brain-derived neurotrophic factor
  • NT3 neurotrophin 3
  • HGF hepatocyte growth factor
  • PDGF-DD platelet-derived growth factor type D
  • PDGF-CC platelet- derived growth factor type
  • BDNF, NT3, HGF, and GDNF are secreted in hUTC conditioned media, and at higher levels in comparison with those from normal human dermal fibroblast (NHDF) and ARPE-19 cells, as measured in ELISA assays.
  • NHDF normal human dermal fibroblast
  • GDNF GDNF secrete low levels of PDGF-CC and PDGF-DD compared to NHDF or ARPE-19
  • ephrin-B2 and HB-EGF are not detected in conditioned medium of hUTC, NHDF, and ARPE-19, as measured in ELISA assays.
  • agrin in hUTC, NHDF and ARPE-19 conditioned medium are similar to that in control medium; agrin detected in all the conditioned medium samples may be from the medium.
  • RNA-Seq-based transcriptome profile analysis of RCS RPE cells Based on the RNA-Seq-based transcriptome profile analysis of RCS RPE cells, the level of bridge molecules and other factors secreted in hUTC conditioned medium demonstrates further the effect on phagocytosis, and consequently, apoptosis. As shown, RCS RPE cells express genes of many receptors identified to date that recognize “eat me” signals on apoptotic cells.
  • scavenger receptors include scavenger receptors (SR-A, LOX-1, CD68, CD36, CD14), integrins ( ⁇ v ⁇ 3 and ⁇ v ⁇ 5), receptor tyrosine kinases of the Axl and Tyro3, LRP-1/CD91, and PS receptor Stabilin 1 (Table 3-1; adapted from Erwig L-P and Henson PM, Cell Death and Differentiation 2008; 15: 243-250).
  • hUTC expresses a number of bridge molecule genes including TSP-1, TSP-2, surfactant protein D (SP-D), MFG-E8, Gas6, apolipoprotein H, and annexin 1.
  • hUTC secrete MFG-E8, Gas6, TSP-1, TSP-2 in hUTC conditioned media.
  • hUTC do not secrete apolipoprotein H, SP-D or annexin I (Table 3-2).
  • hUTC secrete MFG-E8 and TSP-2 at significantly higher levels than NHDF and ARPE-19. ( Figures 12A and 12D).
  • hUTC conditioned media stimulates ROS phagocytosis when feeding RCS RPE cells with ROS preincubated with hUTC CM. Phagocytosis of the dystrophic RPE cells was completely rescued. As shown in Figures 13A-13D, untreated dystrophic RPE cells have reduced phagocytosis compared to normal RPE cells. In an embodiment of the invention, preincubation of dystrophic RPE cells with hUTC CM rescues phagocytosis. This occurs even without hUTC CM being present during the assay. In embodiments of the invention, phagocytic-related receptors and their signaling pathways are up-regulated during the preincubation period. Robust
  • hUTC CM may prime or modify ROS that enhances ROS binding and internalization, through for example, bridge molecules/opsonins that favorably facilitate phagocytosis.
  • bridge molecules MFG-E8, Gas6, TSP-1 and TSP-2 mediate ROS phagocytosis by RCS RPE cells.
  • Dystrophic RPE cells fed with ROS preincubated with various concentrations of MFG-E8, Gas6, TSP-1 or TSP-2 and assayed for phagocytosis showed rescue of ROS phagocytosis ( Figures 14A-14H).
  • hUTC conditioned media mediates RCS RPE phagocytosis rescue through secretion of bridge molecules, for example, MFG-E8, Gas6, TSP-1 and TSP-2.
  • RTK ligands such as BDNF, HGF and GDNF stimulate hUTC-mediated phagocytosis rescue in RCS RPE.
  • Recombinant RTK ligand and bridge molecule proteins can mimic the effect of hUTC CM and restore RCS RPE phagocytosis, and are involved in hUTC-mediated phagocytosis rescue in RCS RPE.
  • siRNA mediated gene silencing demonstrated BDNF, HGF, GDNF, MFG-E8, Gas6, TSP-1 and TSP-2 knocked down (silenced) in hUTC. Mock or scrambled siRNA transfection had no effect on hUTC secretion of these factors. siRNA targeting MFG-E8, TSP-1, TSP-2 and HGF yielded almost 100% knockdown efficiency; 80% and 65% knockdown were observed for BDNF and GDNF, respectively ( Figures 15A-15B).
  • each of the bridge molecules MFG-E8, TSP-1, TSP-2 decreased the phagocytosis of OS by RCS RPE (FIG.15C).
  • RTK ligands BDNF, HGF and GDNF are required for hUTC-mediated phagocytosis rescue in RCS RPE.
  • bridge molecules such as MFG-E8, Gas6, TSP-1 and TSP-2 are required for hUTC-mediated phagocytosis rescue in RCS RPE.
  • Cell Modifications, Components and Products [0169]
  • Progenitor cells, such as postpartum cells may also be genetically modified to produce therapeutically useful gene products, or to produce antineoplastic agents for treatment of tumors.
  • Genetic modification may be accomplished using any of a variety of vectors including, but not limited to, integrating viral vectors, e.g., retrovirus vector or adeno-associated viral vectors; non-integrating replicating vectors, e.g., papilloma virus vectors, SV40 vectors, adenoviral vectors; or replication-defective viral vectors.
  • integrating viral vectors e.g., retrovirus vector or adeno-associated viral vectors
  • non-integrating replicating vectors e.g., papilloma virus vectors, SV40 vectors, adenoviral vectors
  • replication-defective viral vectors e.g., papilloma virus vectors, SV40 vectors, adenoviral vectors
  • Other methods of introducing DNA into cells include the use of liposomes, electroporation, a particle gun, or by direct DNA injection.
  • Hosts cells are preferably transformed or transfected with DNA controlled by or in operative association with, one or more appropriate expression control elements such as promoter or enhancer sequences, transcription terminators, polyadenylation sites, among others, and a selectable marker.
  • Any promoter may be used to drive the expression of the inserted gene.
  • viral promoters include, but are not limited to, the CMV promoter/enhancer, SV40, papillomavirus, Epstein-Barr virus or elastin gene promoter.
  • the control elements used to control expression of the gene of interest can allow for the regulated expression of the gene so that the product is synthesized only when needed in vivo.
  • constitutive promoters are preferably used in a non-integrating and/or replication-defective vector.
  • inducible promoters could be used to drive the expression of the inserted gene when necessary.
  • Inducible promoters include, but are not limited to those associated with metallothionein and heat shock proteins.
  • engineered cells may be allowed to grow in enriched media and then switched to selective media.
  • the selectable marker in the foreign DNA confers resistance to the selection and allows cells to stably integrate the foreign DNA as, for example, on a plasmid, into their chromosomes and grow to form foci which, in turn, can be cloned and expanded into cell lines. This method can be advantageously used to engineer cell lines that express the gene product.
  • Cells may be genetically engineered to“knock out” or“knock down” expression of factors that promote inflammation or rejection at the implant site. Negative modulatory techniques for the reduction of target gene expression levels or target gene product activity levels are discussed below.“Negative modulation,” as used herein, refers to a reduction in the level and/or activity of target gene product relative to the level and/or activity of the target gene product in the absence of the modulatory treatment.
  • the expression of a gene native to a neuron or glial cell can be reduced or knocked out using a number of techniques including, for example, inhibition of expression by inactivating the gene using the homologous recombination technique.
  • an exon encoding an important region of the protein is interrupted by a positive selectable marker, e.g., neo, preventing the production of normal mRNA from the target gene and resulting in inactivation of the gene.
  • a gene may also be inactivated by creating a deletion in part of a gene, or by deleting the entire gene. By using a construct with two regions of homology to the target gene that are far apart in the genome, the sequences intervening the two regions can be deleted (Mombaerts et al., 1991, Proc. Nat. Acad. Sci. U.S.A.88:3084-3087).
  • Antisense, DNAzymes, ribozymes, small interfering RNA (siRNA) and other such molecules that inhibit expression of the target gene can also be used to reduce the level of target gene activity.
  • antisense RNA molecules that inhibit the expression of major histocompatibility gene complexes (HLA) have been shown to be most versatile with respect to immune responses.
  • triple helix molecules can be utilized in reducing the level of target gene activity.
  • the invention provides cell lysates and cell soluble fractions prepared from postpartum cells, preferably PPDCs, or heterogeneous or homogeneous cell populations comprising PPDCs cells, as well as PPDCs or populations thereof that have been genetically modified or that have been stimulated to differentiate along a neurogenic pathway.
  • PPDCs postpartum cells
  • PPDCs preferably PPDCs
  • PPDCs or populations thereof that have been genetically modified or that have been stimulated to differentiate along a neurogenic pathway.
  • Such lysates and fractions thereof have many utilities.
  • Use of the cell lysate soluble fraction i.e., substantially free of membranes
  • Methods of lysing cells are well known in the art and include various means of mechanical disruption, enzymatic disruption, or chemical disruption, or combinations thereof.
  • Such cell lysates may be prepared from cells directly in their growth medium and thus containing secreted growth factors and the like, or may be prepared from cells washed free of medium in, for example, PBS or other solution. Washed cells may be resuspended at concentrations greater than the original population density if preferred.
  • whole cell lysates are prepared, e.g., by disrupting cells without subsequent separation of cell fractions.
  • a cell membrane fraction is separated from a soluble fraction of the cells by routine methods known in the art, e.g., centrifugation, filtration, or similar methods.
  • Cell lysates or cell soluble fractions prepared from populations of progenitor cells, such as postpartum-derived cells, may be used as is, further concentrated, by for example, ultrafiltration or lyophilization, or even dried, partially purified, combined with pharmaceutically-acceptable carriers or diluents as are known in the art, or combined with other compounds such as biologicals, for example pharmaceutically useful protein compositions.
  • Cell lysates or fractions thereof may be used in vitro or in vivo, alone or for example, with autologous or syngeneic live cells.
  • the lysates, if introduced in vivo may be introduced locally at a site of treatment, or remotely to provide, for example needed cellular growth factors to a patient.
  • postpartum cells preferably PPDCs
  • PPDCs postpartum cells
  • such cells which either naturally produce a particular biological product of interest (e.g., a trophic factor), or have been genetically engineered to produce a biological product, can be clonally expanded using the culture techniques described herein.
  • cells may be expanded in a medium that induces differentiation to a desired lineage.
  • biological products produced by the cell and secreted into the medium can be readily isolated from the conditioned medium using standard separation techniques, e.g., such as differential protein precipitation, ion-exchange chromatography, gel filtration
  • A“bioreactor” may be used to take advantage of the flow method for feeding, for example, a three-dimensional culture in vitro. Essentially, as fresh media is passed through the three-dimensional culture, the biological product is washed out of the culture and may then be isolated from the outflow, as above.
  • a biological product of interest may remain within the cell and, thus, its collection may require that the cells be lysed, as described above.
  • the biological product may then be purified using anyone or more of the above-listed techniques.
  • an extracellular matrix (ECM) produced by culturing postpartum cells (preferably PPDCs) on liquid, solid or semi-solid substrates is prepared, collected and utilized as an alternative to implanting live cells into a subject in need of tissue repair or replacement.
  • the cells are cultured in vitro, on a three dimensional framework as described elsewhere herein, under conditions such that a desired amount of ECM is secreted onto the framework.
  • the cells and the framework are removed, and the ECM processed for further use, for example, as an injectable preparation.
  • cells on the framework are killed and any cellular debris removed from the framework.
  • This process may be carried out in a number of different ways.
  • the living tissue can be flash-frozen in liquid nitrogen without a cryopreservative, or the tissue can be immersed in sterile distilled water so that the cells burst in response to osmotic pressure.
  • the cellular membranes may be disrupted and cellular debris removed by treatment with a mild detergent rinse, such as EDTA, CHAPS or a zwitterionic detergent.
  • a mild detergent rinse such as EDTA, CHAPS or a zwitterionic detergent.
  • the tissue can be enzymatically digested and/or extracted with reagents that break down cellular membranes and allow removal of cell contents.
  • enzymes include, but are not limited to, hyaluronidase, dispase, proteases, and nucleases.
  • detergents include non-ionic detergents such as, for example, alkylaryl polyether alcohol (TRITON X-100), octylphenoxy polyethoxy-ethanol (Rohm and Haas Philadelphia, Pa.), BRIJ-35, a
  • polyethoxyethanollauryl ether (Atlas Chemical Co., San Diego, Calif.), polysorbate 20 (TWEEN 20), a polyethoxyethanol sorbitan mono laureate (Rohm and Haas), polyethylene lauryl ether (Rohm and Haas); and ionic detergents such as, for example, sodium dodecyl sulphate, sulfated higher aliphatic alcohols, sulfonated alkanes and sulfonated alkylarenes containing 7 to 22 carbon atoms in a branched or unbranched chain.
  • ionic detergents such as, for example, sodium dodecyl sulphate, sulfated higher aliphatic alcohols, sulfonated alkanes and sulfonated alkylarenes containing 7 to 22 carbon atoms in a branched or unbranched chain.
  • the collection of the ECM can be accomplished in a variety of ways, depending, for example, on whether the new tissue has been formed on a three- dimensional framework that is biodegradable or non-biodegradable.
  • the framework is non-biodegradable
  • the ECM can be removed by subjecting the framework to sonication, high-pressure water jets, mechanical scraping, or mild treatment with detergents or enzymes, or any combination of the above.
  • the ECM can be collected, for example, by allowing the framework to degrade or dissolve in solution.
  • the biodegradable framework is composed of a material that can itself be injected along with the ECM, the framework and the ECM can be processed in toto for subsequent injection.
  • the ECM can be removed from the biodegradable framework by any of the methods described above for collection of ECM from a non-biodegradable framework. All collection processes are preferably designed so as not to denature the ECM.
  • the amounts and/or ratios of proteins may be adjusted by mixing the ECM produced by the cells of the invention with ECM of one or more other cell types.
  • biologically active substances such as proteins, growth factors and/or drugs, can be incorporated into the ECM.
  • tissue growth factors such as TGF-beta, and the like, which promote healing and tissue repair at the site of the injection.
  • additional agents may be utilized in any of the embodiments described herein above, e.g., with whole cell lysates, soluble cell fractions, or further purified components and products produced by the cells.
  • compositions that use progenitor cells such as postpartum cells (preferably PPDCs), cell populations thereof, conditioned media produced by such cells, and cell components and products produced by such cells in various methods for treatment of ocular degenerative conditions.
  • progenitor cells such as postpartum cells (preferably PPDCs)
  • cell populations thereof conditioned media produced by such cells
  • cell components and products produced by such cells in various methods for treatment of ocular degenerative conditions.
  • Certain embodiments encompass pharmaceutical compositions comprising live cells (e.g., PPDCs alone or admixed with other cell types).
  • Other embodiments encompass pharmaceutical compositions comprising PPDC conditioned medium.
  • Additional embodiments may use cellular components of PPDC (e.g., cell lysates, soluble cell fractions, ECM, or components of any of the foregoing) or products (e.g., trophic and other biological factors produced naturally by the cells or through genetic modification, conditioned medium from culturing the cells).
  • the pharmaceutical composition may further comprise other active agents, such as anti-inflammatory agents, anti-apoptotic agents, antioxidants, growth factors, neurotrophic factors or neuroregenerative, neuroprotective or ophthalmic drugs as known in the art.
  • compositions examples 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 known in the art, and/or growth factors, platelet-rich plasma, and drugs
  • PPDCs may be genetically engineered to express and produce growth factors); (3) anti-apoptotic agents (e.g., erythropoietin (EPO), EPO mimetibody, thrombopoietin, insulin-like growth factor (IGF)-I, IGF-II, hepatocyte growth factor, caspase inhibitors); (4) anti-inflammatory compounds (e.g., p38 MAP kinase inhibitors, TGF-beta 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,
  • anti-apoptotic agents e.g., erythropoietin (EPO), EPO mi
  • antioxidants such as probucol, vitamins C and E, conenzyme Q-10, glutathione, L-cysteine and N- acetylcysteine; and (6) local anesthetics, to name a few.
  • compositions of the invention comprise progenitor cells, such as postpartum cells (preferably PPDCs), conditioned media generated from those cells, or components or products thereof, formulated with a pharmaceutically acceptable carrier or medium.
  • Suitable pharmaceutically acceptable carriers include water, salt solution (such as Ringer's solution), alcohols, oils, gelatins, and carbohydrates, such as lactose, amylose, or starch, fatty acid esters, hydroxymethylcellulose, and polyvinyl pyrolidine.
  • Such preparations can be sterilized, and if desired, mixed with auxiliary agents such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, and coloring.
  • compositions may comprise auxiliary components as would be familiar to medicinal chemists or biologists.
  • they may contain antioxidants in ranges that vary depending on the kind of antioxidant used.
  • Reasonable ranges for commonly used antioxidants are about 0.01 % to about 0.15% weight by volume of EDTA, about 0.01 % to about 2.0% weight volume of sodium sulfite, and about 0.01 % to about 2.0% weight by volume of sodium metabisulfite.
  • One skilled in the art may use a concentration of about 0.1 % weight by volume for each of the above.
  • representative compounds include mercaptopropionyl glycine, N-acetyl cysteine, beta- mercaptoethylamine, glutathione and similar species, although other antioxidant agents suitable for ocular administration, e.g. ascorbic acid and its salts or sulfite or sodium metabisulfite may also be employed.
  • a buffering agent may be used to maintain the pH of eye drop formulations in the range of about 4.0 to about 8.0; so as to minimize irritation of the eye.
  • formulations should be at pH 7.2 to 7.5, preferably at pH 7.3-7.4.
  • the ophthalmologic compositions may also include tonicity agents suitable for administration to the eye. Among those suitable is sodium chloride to make formulations approximately isotonic with 0.9% saline solution.
  • compositions are formulated with viscosity enhancing agents.
  • exemplary agents are hydroxyethylcellulose,
  • the pharmaceutical compositions may have cosolvents added if needed. Suitable cosolvents may include glycerin, polyethylene glycol (PEG), polysorbate, propylene glycol, and polyvinyl alcohol. Preservatives may also be included, e.g., benzalkonium chloride, benzethonium chloride, chlorobutanol, phenylmercuric acetate or nitrate, thimerosal, or methyl or propylparabens.
  • cosolvents may include glycerin, polyethylene glycol (PEG), polysorbate, propylene glycol, and polyvinyl alcohol.
  • Preservatives may also be included, e.g., benzalkonium chloride, benzethonium chloride, chlorobutanol, phenylmercuric acetate or nitrate, thimerosal, or methyl or propylparabens.
  • Formulations for injection are preferably designed for single-use
  • injectable solutions should have isotonicity equivalent to 0.9% sodium chloride solution (osmolality of 290-300 milliosmoles). This may be attained by addition of sodium chloride or other co-solvents as listed above, or excipients such as buffering agents and antioxidants, as listed above.
  • Suitable reducing agents include N-acetylcysteine, ascorbic acid or a salt form, and sodium sulfite or metabisulfite, with ascorbic acid and/or N-acetylcysteine or glutathione being particularly suitable for injectable solutions.
  • compositions comprising cells or conditioned medium, or cell components or cell products may be delivered to the eye of a patient in one or more of several delivery modes known in the art.
  • the compositions are topically delivered to the eye in eye drops or washes.
  • the compositions may be delivered to various locations within the eye via periodic intraocular injection or by infusion in an irrigating solution such as BSS or BSS PLUS (Alcon USA, Fort Worth, Tex.).
  • BSS or BSS PLUS Alcon USA, Fort Worth, Tex.
  • compositions may be applied in other ophthalmologic dosage forms known to those skilled in the art, such as pre-formed or in situ-formed gels or liposomes, for example as disclosed in U.S. Pat. No.5,718,922 to Herrero-Vanrell.
  • the composition may be delivered to or through the lens of an eye in need of treatment via a contact lens (e.g. Lidofilcon B, Bausch & Lomb CW79 or DELTACON (Deltafilcon A) or other object temporarily resident upon the surface of the eye.
  • a contact lens e.g. Lidofilcon B, Bausch & Lomb CW79 or DELTACON (Deltafilcon A) or other object temporarily resident upon the surface of the eye.
  • supports such as a collagen corneal shield (e.g. BIO-COR dissolvable corneal shields, Summit Technology, Watertown, Mass.) can be employed.
  • compositions can also be administered by infusion into the eyeball, either through a cannula from an osmotic pump (ALZET, Alza Corp., Palo Alto, Calif.) or by implantation of timed-release capsules (OCCUSENT) or biodegradable disks (OCULEX, OCUSERT).
  • AZAT osmotic pump
  • OCUSENT timed-release capsules
  • OCULEX biodegradable disks
  • compositions comprising live cells in a semi-solid or solid carrier are typically formulated for surgical implantation at the site of ocular damage or distress. It will be appreciated that liquid compositions also may be administered by surgical procedures, for example conditioned media.
  • semi- solid or solid pharmaceutical compositions may comprise semi-permeable gels, lattices, cellular scaffolds and the like, which may be non-biodegradable or biodegradable. For example, in certain embodiments, it may be desirable or appropriate to sequester the exogenous cells from their surroundings, yet enable the cells to secrete and deliver biological molecules to surrounding cells.
  • cells may be formulated as autonomous implants comprising living PPDCs or cell population comprising PPDCs surrounded by a non-degradable, selectively permeable barrier that physically separates the transplanted cells from host tissue.
  • Such implants are sometimes referred to as “immunoprotective,” as they have the capacity to prevent immune cells and
  • degradable materials particularly suitable for sustained release formulations include biocompatible polymers, such as poly (lactic acid), poly (lactic-co-glycolic acid), methylcellulose, hyaluronic acid, collagen, and the like.
  • biocompatible polymers such as poly (lactic acid), poly (lactic-co-glycolic acid), methylcellulose, hyaluronic acid, collagen, and the like.
  • U.S. Pat. No. 5,869,079 to Wong et al. discloses combinations of hydrophilic and hydrophobic entities in a biodegradable sustained release ocular implant.
  • U.S. Pat. No.6,375,972 to Guo et al. discloses combinations of hydrophilic and hydrophobic entities in a biodegradable sustained release ocular implant.
  • U.S. Pat. No.6,375,972 to Guo et al. discloses combinations of hydrophilic and hydrophobic entities in a biodegradable sustained release ocular implant.
  • U.S. Pat. No.5,902,598 to Chen et al. U.S. Pat. No.6,331,313 to Wong et al.
  • U.S. Pat. No.5,707,643 to Ogura et al. U.S. Pat. No.5,466,233 to Weiner et al.
  • a biodegradable, preferably bioresorbable or bioabsorbable, scaffold or matrix typically three-dimensional biomaterials contain the living cells attached to the scaffold, dispersed within the scaffold, or incorporated in an extracellular matrix entrapped in the scaffold. Once implanted into the target region of the body, these implants become integrated with the host tissue, wherein the transplanted cells gradually become established (see, e.g., P. A. Tresco et al., 2000, supra; see also D. W. Hutraum, 2001, J. Biomater. Sci. Polymer Edn.12: 107-174).
  • scaffold or matrix (sometimes referred to collectively as “framework”) material examples include nonwoven mats, porous foams, or self-assembling peptides.
  • Nonwoven mats may, for example, be formed using fibers comprised of a synthetic absorbable copolymer of glycolic and lactic acids (PGA/PLA), sold under the trade name VICRYL (Ethicon, Inc., Somerville, N.J).
  • Foams composed of, for example, poly (epsilon-caprolactone)/poly (glycolic acid) (PCL/PGA) copolymer, formed by processes such as freeze-drying, or lyophilized, as discussed in U.S. Pat.
  • No.6,355,699 also may be utilized.
  • Hydrogels such as self-assembling peptides (e.g., RAD16) may also be used.
  • In situ-forming degradable networks are also suitable for use in the invention (see, e.g., Anseth, K. S. et al., 2002, J. Controlled Release 78: 199-209; Wang, D. et al., 2003, Biomaterials 24: 3969-3980; U.S. Patent Publication 2002/0022676 to He et al.). These materials are formulated as fluids suitable for injection, and then may be induced by a variety of means (e.g., change in temperature, pH, exposure to light) to form degradable hydrogel networks in situ or in vivo.
  • means e.g., change in temperature, pH, exposure to light
  • the framework is a felt, which can be composed of a multifilament yarn made from a bioabsorbable material, e.g., PGA, PLA, PCL copolymers or blends, or hyaluronic acid.
  • the yarn is made into a felt using standard textile processing techniques consisting of crimping, cutting, carding and needling.
  • cells are seeded onto foam scaffolds that may be composite structures.
  • the framework may be molded into a useful shape.
  • PPDCs may be cultured on pre-formed, non-degradable surgical or implantable devices, e.g., in a manner corresponding to that used for preparing fibroblast-containing GDC endovascular coils, for instance (Marx, W. F. et al., 2001, Am. J. Neuroradiol.22: 323-333).
  • the matrix, scaffold or device may be treated prior to inoculation of cells in order to enhance cell attachment.
  • nylon matrices can be treated with 0.1 molar acetic acid and incubated in polylysine, PBS, and/or collagen to coat the nylon.
  • Polystyrene can be similarly treated using sulfuric acid.
  • the external surfaces of a framework may also be modified to improve the attachment or growth of cells and differentiation of tissue, such as by plasma coating the framework or addition of one or more proteins (e.g., collagens, elastic fibers, reticular fibers), glycoproteins, glycosaminoglycans (e.g., heparin sulfate, chondroitin-4-sulfate, chondroitin-6-sulfate, dermatan sulfate, keratin sulfate), a cellular matrix, and/or other materials such as, but not limited to, gelatin, alginates, agar, agarose, and plant gums, among others.
  • proteins e.g., collagens, elastic fibers, reticular fibers
  • glycoproteins e.g., glycoproteins, glycosaminoglycans (e.g., heparin sulfate, chondroitin-4-sulfate, chondroitin-6-sulfate, dermat
  • Frameworks containing living cells are prepared according to methods known in the art. For example, cells can be grown freely in a culture vessel to sub-confluency or confluency, lifted from the culture and inoculated onto the framework. Growth factors may be added to the culture medium prior to, during, or subsequent to inoculation of the cells to trigger differentiation and tissue formation, if desired. Alternatively, the frameworks themselves may be modified so that the growth of cells thereon is enhanced, or so that the risk of rejection of the implant is reduced. Thus, one or more biologically active compounds, including, but not limited to, anti-inflammatory agents,
  • immunosuppressants or growth factors may be added to the framework for local release.
  • Progenitor cells such as postpartum cells (preferably hUTCs or PDCs), or cell populations thereof, or conditioned medium or other components of or products produced by such cells, may be used in a variety of ways to support and facilitate repair and regeneration of ocular cells and tissues. Such utilities encompass in vitro, ex vivo and in vivo methods. The methods set forth below are directed to PPDCs, but other postpartum cells may also be suitable for use in those methods. In Vitro and Ex Vivo Methods
  • progenitor cells such as postpartum cells (preferably hUTCs or PDCs), and conditioned media generated therefrom may be used in vitro to screen a wide variety of compounds for effectiveness and cytotoxicity of pharmaceutical agents, growth factors, regulatory factors, and the like.
  • screening may be performed on substantially homogeneous populations of PPDCs to assess the efficacy or toxicity of candidate compounds to be formulated with, or co-administered with, the PPDCs, for treatment of a an ocular condition.
  • screening may be performed on PPDCs that have been stimulated to differentiate into a cell type found in the eye, or progenitor thereof, for the purpose of evaluating the efficacy of new pharmaceutical drug candidates.
  • the PPDCs are maintained in vitro and exposed to the compound to be tested.
  • the activity of a potentially cytotoxic compound can be measured by its ability to damage or kill cells in culture. This may readily be assessed by vital staining techniques.
  • PPDCs can be cultured in vitro to produce biological products that are either naturally produced by the cells, or produced by the cells when induced to differentiate into other lineages, or produced by the cells via genetic modification.
  • TIMP1, TPO, KGF, HGF, FGF, HBEGF, BDNF, MIP1b, MCP1, RANTES, I309, TARC, MDC, and IL-8 were found to be secreted from umbilicus-derived cells grown in Growth Medium.
  • TIMP1, TPO, KGF, HGF, HBEGF, BDNF, MIP1a, MCP-1, RANTES, TARC, Eotaxin, and IL-8 were found to be secreted from placenta-derived PPDCs cultured in Growth Medium (see Examples).
  • an embodiment of the invention features use of PPDCs for production of conditioned medium.
  • Production of conditioned media from PPDCs may either be from undifferentiated PPDCs or from PPDCs incubated under conditions that stimulate differentiation.
  • Such conditioned media are contemplated for use in in vitro or ex vivo culture of epithelial or neural precursor cells, for example, or in vivo to support transplanted cells comprising homogeneous populations of PPDCs or heterogeneous populations comprising PPDCs and other progenitors.
  • Cell lysates, soluble cell fractions or components from PPDCs, or ECM or components thereof, may be used for a variety of purposes. As mentioned above, some of these components may be used in pharmaceutical compositions. In other embodiments, a cell lysate or ECM is used to coat or otherwise treat substances or devices to be used surgically, or for implantation, or for ex vivo purposes, to promote healing or survival of cells or tissues contacted in the course of such treatments.
  • PPDCs As described in Examples 22 and 24, PPDCs have demonstrated the ability to support survival, growth and differentiation of adult neural progenitor cells when grown in co-culture with those cells. Likewise, previous studies indicate that PPDCs may function to support cells of the retina via trophic mechanisms. (US 2010-0272803).
  • PPDCs are used advantageously in co-cultures in vitro to provide trophic support to other cells, in particular neural cells and neural and ocular progenitors (e.g., neural stem cells and retinal or corneal epithelial stem cells).
  • neural cells and neural and ocular progenitors e.g., neural stem cells and retinal or corneal epithelial stem cells.
  • the PPDCs and the desired other cells may be co-cultured under conditions in which the two cell types are in contact. This can be achieved, for example, by seeding the cells as a heterogeneous population of cells in culture medium or onto a suitable culture substrate.
  • the PPDCs can first be grown to confluence, and then will serve as a substrate for the second desired cell type in culture.
  • the cells may further be physically separated, e.g., by a membrane or similar device, such that the other cell type may be removed and used separately, following the co-culture period.
  • a membrane or similar device e.g., a membrane or similar device
  • Use of PPDCs in co-culture to promote expansion and differentiation of neural or ocular cell types may find applicability in research and in clinical/therapeutic areas.
  • PPDC co-culture may be utilized to facilitate growth and differentiation of such cells in culture, for basic research purposes or for use in drug screening assays, for example.
  • PPDC co-culture may also be utilized for ex vivo expansion of neural or ocular progenitors for later administration for therapeutic purposes.
  • neural or ocular progenitor cells may be harvested from an individual, expanded ex vivo in co- culture with PPDCs, then returned to that individual (autologous transfer) or another individual (syngeneic or allogeneic transfer).
  • autologous transfer or another individual (syngeneic or allogeneic transfer).
  • the mixed population of cells comprising the PPDCs and progenitors could be administered to a patient in need of treatment.
  • the co-cultured cell populations may be physically separated in culture, enabling removal of the autologous progenitors for administration to the patient.
  • conditioned media may effectively be used for treating an ocular degenerative condition.
  • conditioned media from progenitor cells such as PPDCs provides trophic support for ocular cells in situ.
  • the conditioned media from progenitor cells may be administered with other beneficial drugs, biological molecules, such as growth factors, trophic factors, conditioned medium (from progenitor or differentiated cell cultures), or other active agents, such as anti-inflammatory agents, anti-apoptotic agents, antioxidants, growth factors, neurotrophic factors or neuroregenerative or neuroprotective drugs as known in the art.
  • beneficial drugs biological molecules, such as growth factors, trophic factors, conditioned medium (from progenitor or differentiated cell cultures), or other active agents, such as anti-inflammatory agents, anti-apoptotic agents, antioxidants, growth factors, neurotrophic factors or neuroregenerative or neuroprotective drugs as known in the art.
  • biological molecules such as growth factors, trophic factors, conditioned medium (from progenitor or differentiated cell cultures)
  • active agents such as anti-inflammatory agents, anti-apoptotic agents, antioxidants, growth factors, neurotrophic factors or neuroregenerative or neuroprotective drugs as known in the art.
  • active agents such as anti-inflammatory agents, anti-
  • Examples of other components that may be administered with progenitor cells, such as PPDCs, and conditioned media 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 known in the art, and/or growth factors, platelet- rich plasma, and drugs (alternatively, the cells may be genetically engineered to express and produce growth factors); (3) anti-apoptotic agents (e.g., erythropoietin (EPO), EPO mimetibody, thrombopoietin, insulin-like growth factor (IGF)-I, IGF-II, hepatocyte growth factor, caspase inhibitors); (4) anti-inflammatory compounds (e.g., p38 MAP kinase inhibitors, TGF-beta inhibitors, statins, IL-6 and IL-I inhibitors, PEMIROLAST, TRANILAST, REMICADE, SIROLIMUS, and non-steroidal
  • immunosuppressive or immunomodulatory agents such as calcineurin inhibitors, mTOR inhibitors, antiproliferatives, corticosteroids and various antibodies; (6) antioxidants such as probucol, vitamins C and E, conenzyme Q-10, glutathione, L-cysteine and N- acetylcysteine; and (6) local anesthetics, to name a few.
  • Liquid or fluid pharmaceutical compositions may be administered to a more general location in the eye (e.g., topically or intra-ocularly).
  • compositions comprising conditioned medium from progenitor cells, such as PPDCs, or trophic and other biological factors produced naturally by those cells or through genetic modification of the cells.
  • these methods may further comprise administering other active agents, such as growth factors, neurotrophic factors or neuroregenerative or neuroprotective drugs as known in the art.
  • Dosage forms and regimes for administering conditioned media from progenitor cells, such as PPDCs, or any of the other pharmaceutical compositions described herein are developed in accordance with good medical practice, taking into account the condition of the individual patient, e.g., nature and extent of the ocular degenerative condition, age, sex, body weight and general medical condition, and other factors known to medical practitioners. Thus, the effective amount of a pharmaceutical composition to be administered to a patient is determined by these considerations as known in the art.
  • conditioned media may be prepared from PPDCs genetically modified to reduce their immunogenicity, as mentioned above.
  • survival of transplanted cells in a living patient can be determined through the use of a variety of scanning techniques, e.g., computerized axial tomography (CAT or CT) scan, magnetic resonance imaging (MRI) or positron emission tomography (PET) scans. Determination of transplant survival can also be done post mortem by removing the tissue and examining it visually or through a microscope. Alternatively, cells can be treated with stains that are specific for neural or ocular cells or products thereof, e.g., neurotransmitters.
  • CAT or CT computerized axial tomography
  • MRI magnetic resonance imaging
  • PET positron emission tomography
  • Transplanted cells can also be identified by prior incorporation of tracer dyes such as rhodamine-or fluorescein-labeled microspheres, fast blue, ferric microparticles, bisbenzamide or genetically introduced reporter gene products, such as beta-galactosidase or beta-glucuronidase.
  • tracer dyes such as rhodamine-or fluorescein-labeled microspheres, fast blue, ferric microparticles, bisbenzamide or genetically introduced reporter gene products, such as beta-galactosidase or beta-glucuronidase.
  • Functional integration of transplanted cells or conditioned medium into ocular tissue of a subject can be assessed by examining restoration of the ocular function that was damaged or diseased.
  • effectiveness in the treatment of macular degeneration or other retinopathies may be determined by improvement of visual acuity and evaluation for abnormalities and grading of stereoscopic color fundus photographs. (Age-Related Eye Disease Study Research Group, NEI, NIH, AREDS Report No.8, 2001, Arch. Ophthalmol.119: 1417-1436). Kits and Banks
  • kits that utilize progenitor cells, such as PPDCs, and cell populations, conditioned medium prepared from the cells, preferably from PPDCs, and components and products thereof in various methods for ocular regeneration and repair as described above.
  • 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 a pharmaceutically acceptable carrier (liquid, semi-solid or solid).
  • the kits also optionally may include a means of administering the cells and conditioned medium, for example by injection.
  • the kits further may include instructions for use of the cells and conditioned medium.
  • Kits prepared for field hospital use such as for military use may include full-procedure supplies including tissue scaffolds, surgical sutures, and the like, where the cells or conditioned medium are to be used in conjunction with repair of acute injuries.
  • Kits for assays and in vitro methods as described herein may contain, for example, one or more of: (1) PPDCs or components thereof, or conditioned medium or other products of PPDCs; (2) reagents for practicing the in vitro method; (3) other cells or cell populations, as appropriate; and (4) instructions for conducting the in vitro method.
  • the invention also provides for banking of tissues, cells, cell populations, conditioned medium, and cellular components of the invention.
  • the cells and and conditioned medium are readily cryopreserved.
  • the invention therefore provides methods of cryopreserving the cells in a bank, wherein the cells are stored frozen and associated with a complete characterization of the cells based on immunological, biochemical and genetic properties of the cells.
  • the frozen cells can be thawed and expanded or used directly for autologous, syngeneic, or allogeneic therapy, depending on the requirements of the procedure and the needs of the patient.
  • the information on each cryopreserved sample is stored in a computer, which is searchable based on the requirements of the surgeon, procedure and patient with suitable matches being made based on the characterization of the cells or populations.
  • the cells of the invention are grown and expanded to the desired quantity of cells and therapeutic cell compositions are prepared either separately or as co-cultures, in the presence or absence of a matrix or support. While for some applications it may be preferable to use cells freshly prepared, the remainder can be cryopreserved and banked by freezing the cells and entering the information in the computer to associate the computer entry with the samples.
  • the bank system makes it easy to match, for example, desirable biochemical or genetic properties of the banked cells to the therapeutic needs.
  • the sample is retrieved and prepared for therapeutic use.
  • Cell lysates, ECM or cellular components prepared as described herein may also be cryopreserved or otherwise preserved (e.g., by lyophilization) and banked in accordance with the present invention.
  • ANG2 for angiopoietin 2
  • APC for antigen-presenting cells
  • BDNF brain-derived neurotrophic factor
  • bFGF for basic fibroblast growth factor
  • bid (BID) for“bis in die” (twice per day)
  • CK18 for cytokeratin 18
  • CNS for central nervous system
  • CXC ligand 3 for chemokine receptor ligand 3
  • DMEM Dulbecco's Minimal Essential Medium
  • DMEM:lg or DMEM:Lg, DMEM:LG
  • EGF for epidermal growth factor
  • FACS for fluorescent activated cell sorting
  • FBS for fetal bovine serum
  • FGF (or F) for fibroblast growth factor
  • GCP-2 for granulocyte chemotactic protein-2
  • HVVEC Human umbilical vein endothelial cells; I309 for a chemokine and the ligand for the CCR8 receptor; IGF-l for insulin-like growth factor 1; IL-6 for interleukin-6; IL-8 for interleukin 8;K19 for keratin 19; K8 for keratin 8; KGF for keratinocyte growth factor; LIF for leukemia inhibitory factor; MBP for myelin basic protein; MCP-l for monocyte chemotactic protein 1; MDC for macrophage-derived chemokine; MIPlalpha for macrophage inflammatory protein 1 alpha; MIP1beta for macrophage inflammatory protein 1 beta; MMP for matrix metalloprotease (MMP); MSC for mesenchymal stem cells; NHDF for Normal Human Dermal Fibroblasts; NPE for Neural Progenitor Expansion media; NT3 for neurotrophin 3; 04 for oligodendrocyte or glial differentiation marker 04
  • RPE retinal pigment epithelium
  • RCS Royal College of Surgeons
  • ROS rod outer segment
  • hUTC conditioned medium was examined: 1) to evaluate the effect on dystrophic RPE phagocytosis using new preparations of hUTC CM; 2) to isolate RNA of acceptable quality from CM-treated and untreated dystrophic RPE for use in gene expression profiling by RNA-Seq; 3) to examine whether selected RTK ligands can increase the level of phagocytosis in RPE from the RCS rat that cannot use the Mertk signaling pathway; and 4) to test whether other non-RTK ligands, which activate different receptors from RTK, could exhibit similar function.
  • Human umbilical tissue derived cell were obtained from the methods described in Examples 14 to 26 following, and in detail in U.S. Patent Nos.7,524,489, and 7,510,873, and U.S. Pub. App. No.2005/0058634, each incorporated by reference herein. Briefly, human umbilical cords were obtained with donor consent following live births from the National Disease Research Interchange (Philadelphia, PA). Tissues were minced and enzymatically digested.
  • DMEM Dulbecco's modified Eagle's medium
  • Lg low glucose
  • FBS Hyclone, Logan, Utah
  • 4 mM L-glutamine Gibco, Grand Island, NY
  • RPE cells were obtained from 6-11 day old pigmented normal (RCS rdy+/p+) (congenic control) or dystrophic (RCS rdy-/p+) rats. The anterior part of the eye was removed anterior to the limbus. The retina was gently removed and the eye cup was incubated in 4% (w/v) dispase ( ⁇ 0.8 U/mg, Roche
  • RPE culture were maintained for 24 h to 72 h in growth medium containing sulforhodamine (40 ⁇ g/ml final concentration). The cells were stained 36 h to 48 h before the addition of ROS. The sulforhodamine- containing medium was removed 6 h to 18 h before the addition of ROS, and the culture was maintained in several changes of fresh growth medium.
  • FITC Staining of ROS The ROS pellet was resuspended in serum-free culture medium (MEM basal medium only) at about 1 ml per pellet.
  • the FITC stock solution (2 mg/ml in 0.1M sodium bicarbonate, pH 9.0-9.5) was added to a final concentration of 10 ⁇ g/ml and incubated at room temperature for 1 h.
  • the FITC-stained ROS is pelleted by centrifugation in a microfuge at 8000 rpm, 10 minutes, resuspended in growth medium (MEM20), counted, and diluted to a final concentration of 107/ml.
  • CM hUTC Conditioned Medium
  • CM1 Three sets of hUTC conditioned medium (CM) and a control medium were prepared and used for testing in the phagocytosis assay. 1.
  • CM1 with serum preparation [0229] On day 1, hUTC was seeded at 5,000 viable cells/cm 2 in T75 cell culture flask in hUTC growth medium (DMEM low glucose + 15% FBS + 4 mM L-glutamine). Cells were cultured for 24 hours in 37 o C, 5% CO2 incubator.
  • hUTC was seeded at 5,000 viable cells/cm 2 in T75 cell culture flask in hUTC growth medium (DMEM low glucose + 15% FBS + 4 mM L-glutamine). Cells were cultured for 24 hours in 37 o C, 5% CO2 incubator. On day 2, medium was aspirated, washed twice with DPBS, and replenished with 21 mL of DMEM/F12 serum-free medium (DMEM:F12 medium + Pen (50U/ml)/Strep (50 ⁇ g/ml)). Cells were cultured for another 54 h.
  • DMEM low glucose + 15% FBS + 4 mM L-glutamine
  • Serum-free control medium (DMEM:F12 medium + Pen (50U/ml)/Strep (50 ⁇ g/ml)) alone was also cultured for 54 h. On day 4, cell culture supernatant and control medium were collected and centrifuged at 250 g, 5 min at RT, aliquoted in cryotube at 3 mL/tube, and frozen immediately at -70 o C freezer.
  • CM2 The same preparation as CM2, except the hUTC seeding density was increased to 10,000 viable cells/cm 2 , and the incubation time after medium change was 48 hours.
  • Phagocytosis Assay 5 x 10 4 sulforhodamine-stained RPE cells were plated in multi-well plate, maintained in MEM + 20% (v/v) FBS for 6 days, then in MEM + 5% (v/v) FBS 24 h before the assay (2 or more replicates per sample). Fresh medium was added 3 h before the addition of ROS, and the assay was started by overlaying the culture with FITC-ROS (10 7 /ml in MEM + 20% (v/v) FBS) and incubating at 37°C for 3 to 19 h (8 h typically). At the end of the incubation, the cells were vigorously washed to remove unbound ROS and fixed with 2% (w/v) paraformaldehyde (Sigma, St. Louis, MO).
  • RPE phagocytosis of ROS was optimized with respect to the protocols for preparation and culture of primary RPE, preparation of ROS, and the phagocytosis assay itself.
  • RTK ligands The RTK ligands used were: Recombinant Human Ephrin-B2 (Cat # pro-937, Lot # 1112PEFNB2, ProSpec-Tany TechnoGene Ltd., Israel),
  • Recombinant Human BDNF (Cat # 248-BD-025/CF, Lot # NG4012051, R&D Systems, Inc., Minneapolis, MN), Recominant Human HB-EGF (Cat # 259-HE-050/CF, Lot # JI3012021, R&D Systems, Inc., Minneapolis, MN), Recombinant Human HGF (Cat # PHG0254, Lot # 73197181A, Life Technologies, Carlsbad, CA), Recombinant Human Ephrin A4 (Cat # E199, Lot # 1112R245, Leinco Technologies, Inc., St.
  • Recombinant Human PDGF-DD (Cat # 1159-SB-025/CF, Lot # OTH0412071, R&D Systems, Inc., Minneapolis, MN). Reconstitution of individual RTK ligand stock solution followed the vendors’ data sheets: Recombinant Human BDNF and HB-EGF were reconstituted at 100 ⁇ g/mL and 250 ⁇ g/mL in sterile PBS, respectively. Recombinant Human HGF was reconstituted at 500 ⁇ g/mL in sterile, distilled water. Recombinant Human Ephrin A4 was reconstituted at 100 ⁇ g/mL in sterile PBS. Recombinant Human PDGF-DD was reconstituted at 100 ⁇ g/mL in sterile 4 mM HCl. The reconstituted stocks were aliquoted and frozen at -70 o C freezer.
  • the culture media was changed to MEM + 5% (v/v) FBS (MEM5), and ligands were added to the dystrophic cells at 200 ng/ml, incubated for 24 h followed by the addition of ROS in the presence of the ligands, and the cells were subjected to phagocytosis assay. Normal RPE replicates were preincubated in MEM5 and assayed for phagocytosis as controls.
  • Non-RTK ligands used were: Recombinant Human Vitronectin (Cat # 2308-VN-050, Lot # NBH0713021, R&D Systems, Inc., Minneapolis, MN), Recombinant Human TGF- ⁇ 1 (Cat # 240-B-010/CF, Lot # AV5412113, R&D Systems, Inc., Minneapolis, MN), Recombinant Human IL-6 (Cat # 206-IL-010/CF, Lot # OJZ0712041, R&D Systems, Inc., Minneapolis, MN), and Human Endothelin-1 (Cat # hor-307, Lot # 1211PEDN112, ProSpec-Tany TechnoGene Ltd., Israel).
  • Recombinant Human Vitronectin and IL-6 were reconstituted at 100 ⁇ g/mL in sterile PBS, respectively.
  • Recombinant Human TGF- ⁇ 1 was reconstituted at 100 ⁇ g/mL in sterile 4 mM HCl.
  • Recombinant Human Endothelin-1 was reconstituted at 100 ⁇ g/mL in sterile 18M ⁇ -cm H 2 O. The reconstituted stocks were aliquoted and frozen at -70 o C freezer.
  • hUTC CM3 was used as a positive control for the assays.
  • D dystrophic
  • MEM5 MEM + 5% (v/v) FBS
  • phagocytosis assay fed with ROS for 8 h in MEM5
  • normal and dystrophic RPE were incubated in MEM5 for 24 h and subject to phagocytosis assay.
  • Dystrophic RPE cells were incubated with recombinant human endothelin-1, TGF- ⁇ 1 or IL-6 at 200 ng/mL in MEM5 for 24 h and then subjected to phagocytosis assay with the addition of ROS in MEM5 containing recombinant human endothelin-1, TGF- ⁇ 1 or IL-6 (medium was not changed when adding ROS).
  • ROS was preincubated with control medium (DMEM + 10% FBS) or conditioned media for 24 h in CO2 cell culture incubator at 37 o C.
  • ROS was preincubated in MEM + 20% (v/v) FBS (MEM20) with various concentrations of human recombinant vitronectin (4, 2, 1, 0.5 ⁇ g/ml) respectively for 24 h in CO2 cell culture incubator at 37 o C.
  • MEM20 20% (v/v) FBS
  • human recombinant vitronectin 4, 2, 1, 0.5 ⁇ g/ml
  • the ROS was spun down without wash, resuspended in MEM20 and fed to the dystrophic RPE cells in the presence of MEM5 for phagocytosis assay.
  • normal RPE alone or dystrophic RPE alone was cultured in MEM20, then changed to MEM5 in the presence of untreated ROS (resuspended in MEM20 and fed to RPE cells) for phagocytosis as
  • Imaging and Quantitation The living RPE cells were examined by phase contrast and fluorescence microscopy using an inverted microscope equipped with epifluorescence optics, a fluorescence microscope, and a digital camera.
  • FITC-ROS bound to the cell surface, ingested FITC-ROS, and phagolysosomes were identified as defined in McLaren et al. (Invest Ophthalmol Vis Sci., 1993; 34(2):317-326.).
  • Quantitation of bound and ingested ROS was performed on fixed cells on cover slips. Counts were made at 250x magnification with the appropriate filters and a grid (field size, 40 x 40 ⁇ m). Representative fields (10 to 15 per culture) were counted for various types of cells, and the data were expressed as the mean of pooled values obtained per time point from 2-3 separate experiments. Statistical significance was assessed by Student’s t-test for paired data, and was considered at p ⁇ 0.05.
  • Assay acceptance criteria The absolute level of phagocytosis varies in experiments depending on a multitude of factors, including the quality of isolated RPE and prepared ROS. Effort was made to use RPE of the same lineage, i.e., time of harvest and preparation, for comparison of the effects of different treatments on the cells. The assay was judged to be legitimate if the relationship of the phagocytosis level in the normal compared to the dystrophic is approximately 1:0.3.
  • Relative phagocytosis is the level of phagocytosis shown by dystrophic RPE compared to the congenic control (normal) as the reference point. The level of phagocytosis could be expressed as a mean number of ROS/field, or as a mean number of ROS/cell.
  • RNA Isolation of RNA.
  • hUTC was seeded at 5,000 cells/cm 2 and grown in DMEM- Lg medium containing 15% (v/v) FBS (Hyclone, Logan, Utah) and 4 mM L-glutamine (Gibco, Grand Island, NY) for 24 hours followed by medium change to DMEM:F12 medium containing 10% (v/v) FBS and grown for another 48 hours. Cells were then collected for total RNA extraction and DNA removal using the Qiagen RNAeasy extraction and on-column DNAse kit (Qiagen, Valencia, CA).
  • RNA libraries were prepared using Illumina's TruSeq RNA- Seq Sample Preparationkit following manufacturer's instructions, and sequenced with Illumina's HiSeq 2000. Sequencing reads were mapped to the reference human genome (GRCh37 patch8) using the software ArrayStudio version 6.1. Fragments Per Kilobase of transcript per Million mapped reads (FPKM) was used to calculate gene expression.
  • CM1, CM2, CM3 Three conditioned media preparations (CM1, CM2, CM3) were tested for their phagocytosis rescue activities with dystrophic RPE and comparison with normal RPE as described in Methods.
  • CM1 was tested twice (FIGS.1A and 1B), once using tan normal RPE, and again using pigmented RPE. Phagocytosis rescue activity was observed. It should be noted that in FIG.1A, the basal level of phagocytosis in pigmented dystrophic RPE was almost 50% of the level of tan hooded normal RPE, which falls beyond the acceptance range ( ⁇ 30 % of normal phagocytosis level). Effect of CM1-serum free medium was also tested (FIG.2). The difference between dystrophic cells with and without CM1-serum free was statistically significant.
  • CM2 was tested and found to lack activity (FIG.3).
  • CM3 After the medium change on day 1, the incubation time for CM2 (not active) was 48 h whereas the incubation time for CM1 (active) was 54 h. To obtain an active conditioned media, initial cell seeding density and cell incubation time after medium change are two aspects to be considered. CM3 was prepared by doubling the cell seeding density with the same incubation time after medium change, compared to CM2. CM3 was assayed multiple times to confirm the presence of activity (FIGS.4A, 4B, 4C), and showed a phagocytosis rescue activity of up to 100%.
  • Isolated RPE cells from RCS rat eyes were cultured in vitro for phagocytosis assay. Untreated RPE cells from normal congenic control rat eye were used as control to show normal level of phagocytosis.
  • the defective phagocytosis in RCS RPE was restored to the level of normal RPE when the RCS RPE are co-cultured with hUTC (FIG. 4A) or treated with hUTC conditioned medium (CM) (FIG. 4B).
  • Phagocytosis of OS by RCS RPE was rescued when the cells were fed with OS pre-incubated with hUTC CM and subjected to phagocytosis in the absence of hUTC CM (FIG. 4C).
  • hUTC as demonstrated secrete specific factors to promote RPE phagocytosis.
  • BDNF and HB-EGF assays Duplicates of dystrophic RPE were treated with BDNF and HB-EGF and assayed for phagocytosis as described in Methods, along with normal controls (FIGS.5A, 5B). Ten to twelve observations were made per sample. The dystrophic cells tended to show a higher rate of phagocytosis than usual compared to normal cells, but it did not prevent the interpretation of the results. BDNF showed phagocytosis rescue activity, higher than that of CM3.
  • PDGF-DD The number of cells being counted for every sampling were expressed as both per field (FIGS.6A, 6C, 6D) and per cell (FIGS.6B, 6E). The results were not affected, since the number of cells counted for every frame was constant.
  • PDGF-DD (FIG.6A), Ephrin A4 (FIG.6C) and HGF (FIG. 6D) all upregulated phagocytosis in dystrophic RPE cells compared to untreated control.
  • PDGF-DD showed the most rescue effect, greater than that of CM3.
  • Ephrin B2 assay Ephrin B2 showed a very high phagocytosis rescue activity, higher than that of CM3. Both per field (FIG.7A) and per cell (FIG.7B) results were determined.
  • Dystropic RPE cells were treated with endothelin-1, TGF- ⁇ 1 or IL-6 and assayed for phagocytosis as described in Materials and Methods, along with normal controls (FIGS. 8A-8C). Ten observations were made per sample.
  • FIGS.8A and 8B show two separate assays. The basal phagocytosis level of normal and dystrophic RPE cells in FIG.8B is lower than that in FIG.8A due to the variation of cells isolated from rats and different preparation of ROS; the assay is considered valid as the relationship of the phagocytosis level in the normal compared to the dystrophic is approximately 1:0.3.
  • FIG.8C was normalized so that the phagocytosis level of the normal and dystrophic RPE cells were the same as those from FIG.8A.
  • hUTC CM3 increased phagocytosis in dystrophic RPE cells, while endothelin-1, TGF- ⁇ 1 or IL- 6, at the concentration (200 ng/mL) tested in the assays, had no effect on RCS RPE phagocytosis.
  • control medium and hUTC conditioned media used for the study contained 10% FBS, the amount of vitronectin in the control medium was ⁇ 500 ng/ml.
  • hUTC conditioned media may contain more vitronectin compared to control medium as hUTC constitutively secretes vitronectin.
  • ROS pretreated with hUTC CM3 ROS pretreated with control medium appears to have had no effect on dystrophic RPE phagocytosis. Vitronectin, at all the concentrations tested, had a similar effect to that of the control medium.
  • hUTC expression of the genes of RTK ligands and bridge molecules was shown by RNA-Seq-based transcriptome profiling of hUTC.
  • Gene expression of multiple RTK ligands for 15 RTK subfamilies were detected (Table 1-1).
  • the expression level of the RTK ligand genes for each RTK subfamily were sorted and graphed based on the values of Fragments Per Kilobase of transcript per Million mapped reads (FPKM) (FIG. 10; Table 1-1).
  • FPKM Fragments Per Kilobase of transcript per Million mapped reads
  • bridge molecules including MFG-E8, Gas6, protein S, TSP-1 and TSP-2, were also detected (Table 1-3).
  • RTK superfamily can be grouped into 20 subfamilies based on kinase domain sequences (Robinson DR, et al., Oncogene.
  • RCS RPE Gene expression of 18 out of 20 RTK subfamilies were detected in RCS RPE (Table 1-2). Among the 18 are the 15 RTK subfamilies corresponding to the RTK ligand genes expressed in hUTC. Gene expression of receptors reported for bridge molecule binding (Kevany BM, et al., Physiology, 2010;25(1):8-15) were also detected in RCS RPE (Table 1-4), including integrin avP3, avP5, Axl, Tyro3, MerTK, and CD36.
  • RTK Receptor tyrosine kinase
  • ligands include BDNF and NT3 - ligands of Trk family, HGF -a ligand of Met family, PDGF-DD and PDGF-CC -ligands of PDGF family, ephrin-B2 -a ligand of Eph family, HB-EGF -a ligand of ErbB family, GDNF -a ligand of Ret family, as well as agrin -a ligand of Musk family.
  • Table 2-1 Summary of RTK gene expression classification in RCS RPE cells and RTK ligands ene ex ression classification in hUTC from transcri tome rofile anal sis.
  • hUTC Lot NB12898P7 prepared from PDL20 Research Bank, page 7
  • ARPE-19 cells Passage 3
  • NHDF Passage 10
  • Human BDNF ELISA kit (catalog #DBD00, lot #311655, standard detection range: 62.5-4000 pg/mL; sensitivity: 20 pg/mL), human HGF ELISA kit (catalog #DHG00, lot #307319, standard detection range: 125-8000 pg/mL; sensitivity: ⁇ 40 pg/mL), human PDGF-CC ELISA kit (catalog #DCC00, lot # 309376, standard detection range: 62.5-4000 pg/mL; sensitivity: 4.08 pg/mL), human PDGF-DD ELISA kit (catalog #DDD00, lot # 310518, standard detection range: 31.3-2000 pg/mL; sensitivity: 1.67 pg/mL) were from R&D Systems, Inc., Minneapolis, MN.
  • HB-EGF ELISA kit (catalog #ab100531, lot #GR135979-1, standard detection range: 16.4-4000 pg/mL; sensitivity: ⁇ 20 pg/mL) and NT3 ELISA kit (catalog #ab100615, lot #GR141281-1, standard detection range: 4.12-3000 pg/mL; sensitivity: ⁇ 4 pg/mL) were from abcam, Cambridge, MA.
  • Human GDNF ELISA kit (catalog #RAB0205, lot #0919130270, standard detection range: 2.74-2000 pg/mL; sensitivity: 2.74 pg/mL) was from Sigma, St. Louis, MI.
  • Human ephrin-B2 ELISA kit (catalog #MBS916324, lot #R21199424, standard detection range: 15.6-1000 pg/mL; sensitivity: 15.6 pg/mL) and agrin ELISA kit (catalog #MBS454684, lot #EDL201310110, standard detection range: 31.2-2000 pg/mL; sensitivity: ⁇ 13.6 pg/mL) were from MyBioSource, Inc., San Diego, CA.
  • hUTC, ARPE-19 and NHDF conditioned media were seeded, respectively, at 10,000 viable cells/cm 2 in T75 cell culture flasks in 15 mL of hUTC growth medium (DMEM low glucose + 15% v/v FBS + 4 mM L-glutamine). Cells were cultured for 24 h in 37 o C 5% CO2 incubator. On day 2, media were aspirated and replenished with 21 mL of DMEM/F12 complete medium (DMEM/F12 medium + 10% v/v FBS + 50U/ml Pen/50 ⁇ g/ml Strep). Cells were cultured for another 48 h.
  • DMEM low glucose + 15% v/v FBS + 4 mM L-glutamine mM L-glutamine
  • Control medium (DMEM/F12 complete medium) alone was also cultured for 48 h. On day 4, cell culture supernatants and control medium were collected and centrifuged at 250 g, 5 min at 4 o C, aliquoted in cryotube at 0.5 mL/tube, and frozen immediately at -70 o C freezer. The frozen samples were thawed and used for ELISA. Results
  • Levels of selected RTK ligands measured in hUTC conditioned medium were also compared with those from NHDF and ARPE-19 conditioned medium, as normalized at pg/mL/1x10 6 cells.
  • hUTC secreted 72.7 pg/mL of BDNF per million cells per 48 h, compared to 20.4 pg/mL and 16.2 pg/mL of BDNF per million cells per 48 h from NHDF and ARPE-19, respectively (Figure 11A).
  • the amount of BDNF in control medium was undetectable. (FIG.11G).
  • hUTC secreted 23.7 pg/mL of HGF per million cells per 48 h, compared to 3.9 pg/mL and 0.4 pg/mL from NHDF and ARPE-19, respectively (Figure 11C). The amount of HGF in the control medium was undetectable. (FIG.11G).
  • hUTC secreted 9.5 pg/mL of GDNF per million cells per 48 h compared to 8.5 pg/mL of GDNF from NHDF.
  • ARPE-19 only released trace amount of 1.3 pg/mL of GDNF per million cells per 48 h ( Figure 11F). The amount of GDNF in control medium was undetectable. (FIG.11G).
  • concentrations of these ligands secreted from hUTC in conditioned medium appear lower than the level tested.
  • RCS RPE cells express genes of receptors that recognize“eat me” signals on apoptotic cells.
  • receptors include scavenger receptors (SR-A, LOX-1, CD68, CD36, CD14), integrins ( ⁇ v ⁇ 3 and ⁇ v ⁇ 5), receptor tyrosine kinases of the Axl and Tyro3, LRP-1/CD91, and PS receptor Stabilin 1.
  • hUTC expresses a number of bridge molecule genes including TSP-1, TSP-2, surfactant protein D (SP-D), MFG-E8, Gas6, apolipoprotein H, and annexin 1. Secretion of bridge molecules in hUTC conditioned medium was examined and the levels compared with those from ARPE-19 and normal human dermal fibroblast (NHDF). Materials and Methods
  • hUTC PDL20, master cell bank number 25126057
  • ARPE-19 cells ATC, Manassas, VA
  • NHDF Passage 11
  • Human Gas6 ELISA kit (catalog #SK00098-01, lot #20111218) and human SP-D ELISA kit (catalog #SK00457-01, lot #20111135) from Aviscera Bioscience, Santa Clara, CA.
  • the standard detection range of human Gas6 ELISA kit is 62.5-8000 pg/mL with the sensitivity of 31 pg/ml.
  • the standard detection range of human SP-D ELISA kit is 78-5000 pg/mL with the sensitivity of 30 pg/ml.
  • Human MFG-E8 ELISA kit (catalog #DFGE80, lot #307254, standard detection range: 62.5-4000 pg/mL; sensitivity: 4.04 pg/mL), human TSP-1 ELISA kit (catalog #DTSP10, lot #307182, standard detection range: 7.81-500 ng/mL; sensitivity: 0.355 ng/mL), and human TSP-2 ELISA kit (catalog #DTSP10, lot #307266, standard detection range: 0.31-20 ng/mL; sensitivity: 0.025 ng/mL) were from R&D Systems, Inc., Minneapolis, MN.
  • Apolipoprotein H human ELISA kit (catalog #ab108814, lot #GR126938, standard detection range: 0.625-40 ng/mL, sensitivity: 0.6 ng/mL) was from Abcam, Cambridge, MA.
  • Human Annexin I (ANX-I) ELISA kit (catalog #MBS704042, lot #N10140947, standard detection range: 0.312-20 ng/mL; sensitivity: 0.078 ng/mL) was from MyBioSource, Inc., San Diego, CA.
  • N ote that receptor tyrosine kinases Tyro3 and Axl are also PS-bridge molecule
  • hUTC, ARPE-19 and NHDF conditioned media were seeded, respectively, at 10,000 viable cells/cm 2 in T75 cell culture flasks in 15 mL of hUTC growth medium (DMEM low glucose + 15% FBS + 4 mM L-glutamine). Culture for 24 h in 37 o C 5% CO2 incubator. On day 2, media was aspirated and replenished with 18 mL of DMEM/F12 complete medium (DMEM:F12 medium + 10% FBS + Pen (50U/ml)/Strep (50 ⁇ g/ml)). Cells were cultured for another 48 h.
  • Control medium (DMEM:F12 medium + 10% FBS + Pen (50U/ml)/Strep (50 ⁇ g/ml)) alone was also cultured for 48 h. On day 4, cell culture supernatants and control medium were collected and centrifuged at 250 g, 5 min at 4 o C, aliquoted in cryotube at 0.5 mL/tube, and frozen immediately at -70 o C freezer. The frozen samples were thawed and used for ELISA. Results
  • Apolipoprotein H in the conditioned medium of hUTC, ARPE-19 and NHDF was similar to that in control medium (6.8 ng/mL).
  • Levels of SP-D and Annexin I in hUTC, ARPE-19 and NHDF conditioned media as well as control medium were under the detection limit of the ELISAs ( ⁇ 30 pg/mL and ⁇ 78 pg/mL, respectively). The cells either do not secrete the two proteins, or the levels are below the limit of detection.
  • MFG-E8 Gas6, TSP-1 and TSP-2 are bridging molecule candidates involved in hUTC-mediated phagocytosis rescue in RCS RPE cells.
  • ROS Phagocytosis by RCS RPE cells
  • hUTC Conditioned Medium CM3 was used for the study. On day 1, hUTC was seeded at 10,000 viable cells/cm 2 in T75 cell culture flask in hUTC growth medium (DMEM low glucose + 15% FBS + 4 mM L-glutamine). Culture for 24 hours in 37 o C 5% CO2 incubator. On day 2, medium was aspirated and replenished with 21 mL of DMEM/F12 complete medium (DMEM:F12 medium + 10% FBS + Pen (50U/ml)/Strep (50 ⁇ g/ml)). Cells were cultured for another 48 hours.
  • DMEM DMEM low glucose + 15% FBS + 4 mM L-glutamine
  • Control medium (DMEM:F12 medium + 10% FBS + Pen (50U/ml)/Strep (50 ⁇ g/ml)) alone was also cultured for 48 h. On day 4, cell culture supernatant and control medium were collected and centrifuged at 250 g, 5 min at RT, aliquoted in cryotube at 3 mL/tube, and frozen immediately at -70 o C freezer.
  • Recombinant human bridge molecules Recombinant Human MFG-E8 (Cat # 2767-MF-050, Lot # MPP2012061), recombinant Human Gas6 (Cat # 885-GS-050, Lot # GNT5013011), recombinant Human TSP-1 (Cat # 3074-TH-050, Lot # MVF3613041), recombinant Human TSP-2 (Cat # 1635-T2-050, Lot # HUZ1713021), were all obtained from R&D Systems, Inc., Minneapolis, MN.
  • Reconstitution of individual protein stock solution was to follow the vendor’s data sheets: Recombinant Human MFG-E8, TSP-1 and TSP-2 were reconstituted at 100 ⁇ g/mL in sterile PBS, respectively. Recombinant Human Gas6 was reconstituted at 100 ⁇ g/mL in sterile water. The reconstituted stocks were aliquoted and frozen at -70 o C freezer.
  • ROS was preincubated with control medium (DMEM + 10% FBS) or CM3 for 24 h in CO 2 cell culture incubator at 37 o C.
  • ROS was preincubated in control medium with various concentrations of human recombinant MFG-E8, Gas6, TSP-1 or TSP-2 for 24 h in CO2 cell culture incubator at 37 o C.
  • the ROS was spun down without wash, resuspended in MEM5 and fed to the dystrophic RPE cells in the presence of MEM5 for phagocytosis assay.
  • normal RPE alone or dystrophic RPE alone was cultured in MEM20, then changed to MEM5 in the presence of untreated ROS (resuspended in MEM20 and fed to RPE cells) for phagocytosis assay.
  • RCS RPE Effects of RTK ligands on RCS PRE cell phagocytosis: RCS RPE were incubated with recombinant human BDNF, HGF and GDNF individually for 24 hours, and then OS was added for phagocytosis assay. RCS RPE incubated with hUTC CM was used as a positive control.
  • siRNA knockdown The On-TARGETplus human siRNA-SMARTpools directed against human BDNF, HGF, GDNF, MFG-E8, Gas6, TSP-1 and TSP-2, as well as ON-TARGETplus Non-targeting pool (scrambled siRNA pool) were purchased from GE Dharmacon (Lafayette, CO).25 nM of each siRNA pool was incorporated into hUTC respectively, using DharmaFECT transfection reagent (GE Dharmacon).
  • Antibodies for immunofluorescence staining Unconjugated monoclonal antibodies for the bridge molecules (human MFG-E8, Gas6, TSP-1 and TSP-2), as well as mouse IgG2A and IgG2B isotype control antibodies were obtained from R&D Systems, Inc., Minneapolis, MN. These antibodies were conjugated with Alexa Fluor 488 fluorophore by Life Technologies (Eugene, OR). An unconjugated monoclonal anti- rhodopsin antibody (EMD Millipore Corp., Temecula CA) was conjugated with Alexa Fluor 568 by Life Technologies (Eugene, OR).
  • An unconjugated mouse IgG2b, x isotype control antibody was obtained from Biolegend Inc. (San Diego, CA) and conjugated with Alexa Fluor 488 fluorophore by Life Technologies (Eugene, OR). It was used as an isotype control antibody for Alexa Fluor 568 conjugated anti-rhodopsin antibody.
  • Alexa Fluor 488 conjugated mouse IgG2A was used as an isotype control antibody for Alexa Fluor 488 conjugated anti-human MFG-E8, Gas6, or TSP-2 antibodies.
  • Alexa Fluor 488 conjugated mouse IgG2B was used as an isotype control antibody for Alexa Fluor 488 conjugated human TSP-1 antibody.
  • the sections were transferred to glass slides for the immunofluorescence staining. Circled spots with the OS pieces were treated with blocking buffer (10% (v/v) goat serum, 1% (v/v) BSA, and 0.1% (v/v) Triton x 100 in PBS) for 1 hour at room temperature and then double stained 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 IgG2A or IgG2B isotype control antibody for 2 hours at 4°C.
  • blocking buffer 10% (v/v) goat serum, 1% (v/v) BSA, and 0.1% (v/v) Triton x 100 in PBS
  • Dystrophic RPE cells were fed with ROS preincubated with various concentrations of MFG-E8 (15.5 ng/mL; 31 ng/mL; 62 ng/mL; 124 ng/mL), Gas6 (70 pg/mL; 350 pg/mL; 1750 pg/mL; 8750 pg/mL), TSP-1 (152 ng/mL; 304 ng/mL; 608 ng/mL; 1216 ng/mL) or TSP-28.8 ng/mL; 26.4 ng/mL; 79.2 ng/mL; 237.6 ng/mL) and assayed for phagocytosis (FIGS.14 A-14D). Ten observations were made per sample. The ROS phagocytosis was rescued by feeding RCS RPE cells with ROS preincubated with MFG-E8, Gas6, TSP-1 or TSP-2.
  • BDNF, HGF, GDNF, MFG-E8, Gas6, TSP-1 and TSP-2 were knocked down in hUTC by siRNA mediated gene silencing. Scrambled siRNA pool that does not target any genes was used as knockdown control. The knockdown efficiency of each factor was examined by measuring the level of each factor in the cell culture supernatants collected from hUTC transfected with siRNA (FIG.15A). Mock or scrambled siRNA transfection had no effect on hUTC secretion of these factors. siRNA targeting MFG-E8, TSP-1, TSP- 2 and HGF yielded almost 100% knockdown efficiency; 80% and 65% knockdown were observed for BDNF and GDNF, respectively (FIG.15A).
  • CM was produced from siRNA-transfected hUTC and applied to RCS RPE to identify the effects of RTK ligands and bridge molecule knockdown.
  • RCS RPE were cultured with CM produced from hUTC transfected with siRNA targeting BDNF, HGF or GDNF (FIG.15B), or were fed with OS pre-treated with CM produced from hUTC transfected with siRNA targeting MFG-E8, TSP-1 or TSP-2 (FIG.15C).
  • CM prepared from untransfected and scrambled siRNA transfected hUTC were used as knockdown control CMs.
  • Oxidative stress can compromise the health of retinal pigment epithelium.
  • the effect of hUTC and hUTC conditioned media to improve the health of RPE cells exposed to oxidative damage was investigated.
  • Ethanol was obtained Decon Labs Inc. (King of Prussia, PA). PBS was obtained from Lonza (South Plainfield, NJ).
  • ARPE growth medium DMEM with 4.5 g/L glucose and sodium pyruvate without L-glutamine and phenol red) (Mediatech, Inc. A Corning Subsidiary, Manassas, VA) supplemented with 5% or 10% heat-inactivated fetal bovine serum (FBS, Life Technologies, Grand Island, NY), 1X Minimum Essential Medium - Non-Essential Amino Acids (MEM-NEAA, Life Technologies) and 0.01mg/mL Gentamicin Reagent Solution (Life Technologies).
  • 5% ARPE growth medium containing 10 ⁇ M A2E DMEM (Mediatech, Inc.) supplemented with 5% heat-inactivated FBS (Life Technologies), 1X MEM-NEAA (Life Technologies), 0.01mg/mL Gentamicin Reagent Solution (Life Technologies) and 10 ⁇ M A2E (prepared by the lab of Dr. Janet Sparrow).
  • hUTC complete medium DMEM low glucose (Life Technologies) supplemented with 15% Hyclone® FBS (Thermo Scientific, Logan, Utah) and 4mM L- glutamine (Life Technologies).
  • ARPE-19 Cell Culture for A2E Study On Day 1 ARPE-19 cells were seeded in 8-well NuncTM Lab-TekTM II chamber slides (Nalge Nunc International Corporation, Rochester, NY) at a density of 40,000 cells/well in a final volume of 300 ⁇ L 10% ARPE growth medium. 24-hours post-seeding (Day 2) the medium on the cells was removed and replaced with 300 ⁇ L 5% ARPE growth medium. One week later (Day 9), medium was again removed and replaced with fresh 5% ARPE growth medium. On Day 14, media was removed and replaced with fresh 5% ARPE growth medium containing 10 ⁇ M A2E. On Days 17 and 21, media was removed again and replaced with fresh A2E- containing medium.
  • the A2E containing medium was removed and replaced with fresh 5% ARPE growth medium, and the cells were allowed to quiesce for five days.
  • medium was removed from each well and replaced with 250 ⁇ L of 5 or 10% hUTC conditioned or control media (5% and 10% FBS hUTC media not exposed to cells).
  • the conditioned and control media were removed from the cells and replaced with fresh conditioned or control media.
  • all media were removed from each well and cells were washed once with 1X DPBS. 200 ⁇ L fresh DPBS was subsequently added to each well and cells were exposed to 430 nm light delivered from a tungsten halogen source for 20 minutes.
  • MTT Assay for A2E Study Cytotoxicity was measured by a metabolic (MTT, (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) colorimetric microtiter assay (Roche Diagnostics Corporation, Indianapolis, IN). To perform the MTT assay, 20 ⁇ L of MTT labeling reagent (Roche Diagnostics Corporation) was added to 0.2 mL of 5% culture medium in each well. After 4 hours of incubation, another 200 ⁇ L of MTT labeling reagent (Roche Diagnostics Corporation) was added to 0.2 mL of 5% culture medium in each well. After 4 hours of incubation, another 200 ⁇ L of MTT labeling reagent (Roche Diagnostics Corporation) was added to 0.2 mL of 5% culture medium in each well. After 4 hours of incubation, another 200 ⁇ L of MTT labeling reagent (Roche Diagnostics Corporation) was added to 0.2 mL of 5% culture medium in each well
  • solubilization solution was added to each well for an overnight incubation. After centrifugation at 13,000 rpm for 2 minutes, supernatants were measured
  • Nonviable cells were quantified after labeling by a fluorescence exclusion assay that allowed for the labeling of apoptotic nuclei because of a loss of plasma membrane integrity during the latter stages of cell death. Following light exposure cells were returned to 5% ARPE growth medium.
  • ARPE-19 cells (American Type Culture Collection; Manassas, VA) were grown in Ham's F10 Medium containing 10% FBS and 50 units/mL penicillin/50 ⁇ g/mL streptomycin as monolayers in T75 flasks at 37°C, 5% CO2.
  • the ARPE19 cells were grown to 80-90% confluency in T-75 flasks and subsequently seeded in 24-well cell culture plates.
  • ARPE- 19 cells were allowed to grow in 10% FBS growth medium until the 3rd day after seeding when the media was changed to basal medium (Ham’s F10 media supplemented with 2% FBS and 50 units/mL penicillin/50 ⁇ g/mL streptomycin).
  • hUTC were seeded onto cell culture inserts (pore size 1 ⁇ m) at 5000 cells/cm 2 in hUTC complete growth (low glucose DMEM supplemented with 15% FBS and 4mM L-glutamine) for 24 hours. Inserts were transferred to ARPE-19 cells growing on cell culture plates for 72 hours and grown in hUTC complete medium.
  • hUTC conditioned media protected A2E-laden RPE cells from blue light- induced damage.
  • ARPE-19 viability was assessed post-irradiation by labeling cells with the membrane-impermeant dye Dead Red and all nuclei with DAPI. Nuclei counted in digital images provided the percent of viable and non-viable cells (FIGS.17A-17B).
  • 10 ⁇ M A2E had no effect on ARPE-19 viability.
  • Cells that were incubated with control media and subjected to 430nm illumination showed high levels of nonviable cells ( ⁇ 50%).
  • treatment with hUTC conditioned media resulted in a reduction in the number of nonviable cells ( ⁇ 20%).
  • the human globe received in moist chamber, were rinsed in PBS with 4% Pen-Strep (Life Technologies, Penicillin-Streptomycin (10,000 U/mL, Cat#15140122), and the anterior segment was removed at the limbus, if not removed already, to open it up. After gross examination and photography, the retina was removed, and the posterior pole was cut into smaller pieces ( ⁇ 1 cm) and incubated in 4 ml of 2% Dispase solution (w/v, in DMEM, 25 mM HEPES, 200 U/mL Pen-Strep) (Roche Diagnostic Corp, Cat# 04942078001) for 5 minutes at 37C.
  • Pen-Strep Life Technologies, Penicillin-Streptomycin (10,000 U/mL, Cat#15140122)
  • the incubation was stopped with addition of at least double volume of DMEM (Life Technologies, Cat#12430054), 25 mM HEPES (Sigma- Aldrich Corp. Cat# H4034-100G), 200 U/mL Pen-Strep.
  • DMEM Life Technologies, Cat#12430054
  • 25 mM HEPES Sigma- Aldrich Corp. Cat# H4034-100G
  • Pen-Strep 200 U/mL Pen-Strep.
  • the tissues were put in a 100 mm petri dish with ample amount of fresh DMEM, 25 mM HEPES, 2% Pen-Strep, and allowed to sit at 37C for 6-12 hours.
  • the tissues were transferred to RPMI media (Life Technologies, Cat#22400089) in a petri dish, and the RPE cells were teased off the choroid by gentle scraping or a jet of liquid from a pipette tip.
  • the cells were rinsed with HBSS (GIBCO, Cat#310-4170) x2, and resuspended in 0.5 ml of 0.1 % Trypsin, pH 8.0 (Life Technologies, Cat#25300054) and incubated for 2 minutes at 37C.
  • the cell mixture was triturated with a Pasteur pipette until the RPE cells were dispersed, and the enzymatic treatment stopped with addition of 5x excess of MEM/20 % FBS (Minimum Essential Medium, Lift Technologies, Cat#1095098; Fetal Bovine Serum, Life Technologies, Cat#16000036).
  • the cells were spun down at 1000 xg, resuspended in appropriate volume of MEM/20 (MEM containing 20% FBS), counted, and cultured in appropriate vessel (24-well plate, Thomas Scientific, Cat#6901A11; 25 ml flask, VWR, Cat#29185- 298). In general, 50,000 cells/well were seeded in 24-well plate. For cells cultured in 25 mL flask, one-fifth to one-third of the harvested amount was seeded in a 25 mL flask for passaging purpose. Human RPE were isolated using this protocols from the donors listed in Table 6-1 below. Table 6-1. Donors used
  • Table 6-2 shows additional information for the donor eyes used. Table 6-2. Additional information on donors
  • the human RPE cells were cultured in 25 ml flask or 24-well plate to confluence. When harvesting human RPE, in addition to plating cells out in the 24-well format, a 25 ml flask was also seeded to obtain a large number of cells for stocking, plating out additional 24 wells, and passing. When the flask became confluent, the cells were passaged. This process was continued for ⁇ 6 passages or so. The days and doublings between passaging depended on how many cells were seeded into the flask, but the doubling time seemed to be ⁇ 2 days. A flask usually became confluent in ⁇ 10 days.
  • the cultures were rinsed 2x with PBS, trypsinized with 0.1 % Trypsin, pH 8.0 (Life Technologies, Cat#25300054), and harvested.
  • the cells were either passaged to another flask or plate (approximately 1/5 to 1/3 confluent amount) or at least 3 x 10 5 cells were resuspended in 0.5– 1 ml of cryomedia (Life Technologies, Cat#12648010), gradually chilled in a Mr. Frosty TM Freezing Container, and stored in liquid nitrogen.
  • the frozen tube of cells were warmed in a 37 o C water bath, and the cells were transferred to either a well in the 24-well plate or a 60 mm petri dish and cultured until confluent before passaging.
  • ROS Rod Outer Segments
  • Human eyes 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). In particular, the human eyes from the donors in Table 6-1 were used. Retinas were isolated, homogenized with Polytron (5 mm generator), layered on top of 27% - 50% linear sucrose gradient, and centrifuged at 38,000 rpm in SW41 rotor for 1 hour at 4°C.
  • Polytron 5 mm generator
  • the ROS bands (up to 3 bands are possible, upper, middle, lower) which represent homogenized ROS particles of different size were collected, diluted with HBSS, and centrifuged at 7000 rpm in HB-4 rotor for 10 minutes to pellet the ROS.
  • the ROS pellets were resuspended in serum-free culture medium.
  • the FITC stock solution (2 mg/ml in 0.1M sodium bicarbonate, pH 9.0- 9.5) (Sigma-Aldrich Corp. Cat#F7250-50MG) was added to a final concentration of 10 ⁇ g/ml and incubated at room temperature for 4 hours.
  • the FITC-stained ROS were pelleted by centrifugation in a microfuge, resuspended in MEM/20. Their concentrations were estimated with a hemocytometer, and their microscopic appearance recorded (should show typical spherical or rod structures).
  • The“active” portions were stored at 4oC for daily use, and the frozen portions were frozen as is
  • Eyes were also obtained from 6- to 8-week old Long Evans rats and adult pigs (slaughter house). Rod out segments were isolated and sorted from the rat and pig eyes using the process described above for human ROS.
  • Examples 1 to 5 demonstrate that hUTC rescue phagocytosis, thereby slowing down the retinal degeneration in the RCS rat model of retinal degeneration.
  • Examples 7 to 13 show the results of similar testing using human RPE cells and ROS obtained by the methods in this Example. In certain examples, rat and pig ROS were used for comparison.
  • ROS and RPE cells will be identified by their donor identification.
  • 14-06-040 RPE refers to RPE cells isolated from donor 14-06-040.
  • 1’ means primary, 2’ means secondary, etc.
  • the cells were vigorously washed with PBS to remove uningested ROS and fixed with 2% paraformaldehyde (Paraformaldehyde, 4 % in PBS, diluted to 2% with PBS, used within 1 week after dilution) (Paraformaldehyde, 4% in PBS VWR, Cat#AAJ61899-AK).
  • the ingested ROS were visualized by fluorescence microscopy at x312 magnification with a Zeiss fluorescence Photomicroscope III. At least 10 representative fields (field size, 0.021 mm 2 ) were examined and the fluorescent ingested ROS were counted.
  • the rat ROS was added to the cultured cells, and the phagocytosis was stopped at 1, 2, 3, 6, 8, 9, 10, 11, 12, 13, 15, and 16 hours and analyzed.
  • the expected time course of phagocytosis was seen in the rat RPE, i.e., binding but low phagocytosis activity for 1-6h, burst of phagocytosis starting at 8h, continuing through 15h, and a slowdown and evidence of the next cycle at 16h (see FIG.19).
  • Essentially the same pattern was seen in the human RPE, confirming the similarity of the phagocytic characteristic between the rat and human RPE.
  • the 15-02-032 human RPE and N rat RPE (1/23/15) cultures were used for phagocytosis assays with the human ROS (15-02-032, 2/13/15, 5 x 10 6 each) and rat ROS (1/27/15, 10 x 10 6 each) for 8h.
  • Low level of phagocytosis was seen with the rat ROS for both cells, but good phagocytosis was seen with the human ROS for both, thus confirming the higher efficiency of the human ROS compared to rat ROS previously observed.
  • Quantitation was performed for the human 15-02-032 results (see Table 7-4). Table 7-4. Quantitation of human RPE phagocytosis with human and rat ROS
  • the 15-02-032 human RPE in culture was immunostained with the Pan-keratin (C11) monoclonal antibody (Cell Signaling) (1:400) for confirmation that they are RPE cells (see FIG.21).
  • the secondary antibody was Alexa 488-conjugated anti- mouse IgG antibody.
  • Control was not treated with the primary antibody.
  • Good staining of the RPE cells was obtained with no staining in the control, confirming the cell type as RPE.
  • XI Human 15-03-027 and rat RPE phagocytosis with human and rat ROS
  • Results for 55 day culture Results similar to day 17 and day 32 primary RPE cultures of 15-04-001 were obtained, confirming the consistent patterns with respect to the level of phagocytosis, the relative efficiencies of the different ROS preparations, and the equivalence of different age of culture of the cells. Table 7-12. Phagocytosis of human RPE 15-04-001 with rat, human, and pig ROS (32 day culture)
  • the levels of phagocytosis obtained with the middle band ROS were generally in the usual ranges. Phagocytosis levels obtained with the upper band ROS, however, were higher than the others. Again, the reason may be the abundance of smaller ROS particles in the upper band; which causes an underestimate of the concentration since they are more difficult to visualize in bright field and more visible particles intracellularly by fluorescence.
  • the levels obtained with the lower band were somewhat variable, and this was borne out by results with other RPE cultures, as shown below. Variability in gross phagocytosis level can exist depending on the nature of the ROS particles, but consistency can be obtained by using the same ROS preparation across samples.
  • the SD1 AMD L 5’ RPE in culture (day 69) was immunostained with the Pan-keratin (C11) monoclonal antibody (Cell Signaling) (1:400) for confirmation that these AMD cells are RPE cells.
  • the secondary antibody was Alexa 488-conjugated anti- mouse IgG antibody. Control was not treated with the primary antibody. Good staining of the RPE cells was obtained with no staining in the control, confirming the cell type as RPE (see FIG.22).
  • RNA levels for several human RPE isolated in Example 6 were obtained. Specifically, these RNA levels were determined using the protocol below
  • ROS phagocytosis in human RPE isolated from donors of different age, and normal human and AMD RPE
  • ND08626 AMD 1’ day17, 15-09-027 N 5’ day88, and 15-08-074 N 5’ day55 RPEs were subjected to 8-hour phagocytosis assays with 5 x 10 5 each of 15-11-098 upper ROS.
  • the phagocytosis levels in AMD ND08626 RPEs were significantly lower than that of normal RPEs (15-09-027 and 15-08-074), confirming the pattern consistently observed.
  • ND08626 AMD 1’ day17, and 15-11-098 N 3’ day40 and 4’ day24 RPEs were subjected to 8-hour phagocytosis assays with 5 x 10 5 each of 15-11-098 upper ROS.
  • the phagocytosis level in the AMD ND08626 RPE was much lower than that of the normal 15-11-098 RPEs, again confirming the consistently observed pattern.
  • ND08626 AMD 1’ day24, 15-04-001 N 5’ day77, and 15-07-072 N 6’ day123 RPEs were subjected to 8-hour phagocytosis assays with 5 x 10 5 each of 15-11- 098 upper ROS.
  • the phagocytosis level of the AMD ND08626 RPE was much lower than that of the normal 15-04-001 and 15-07-072 RPEs, again confirming the consistently observed pattern.
  • ND08626 AMD 1’ day25, and 15-08-074 N 4’ day111 and 6’ day82 RPEs were subjected to 8-hour phagocytosis assays with 5 x 10 5 each of 15-11-098 upper ROS.
  • the phagocytosis level in the AMD ND08626 RPE was again much lower than that of the normal 15-08-074 RPEs, again confirming the consistently observed pattern.
  • the phagocytosis levels obtained with the lower band were higher than expected this time with the 15-09-027 cells, reflecting the variability of this band as mentioned before. Variability in gross phagocytosis level can exist depending on the nature of the ROS particles, but consistency can be obtained by using the same ROS preparation across samples.
  • CM hUTC conditioned medium
  • hUTC CM 5 (5 th preparation: CM510k and CM511k), CM 10 (10 th preparation), CM11 (11 th preparation), CM13 (13 th preparation), CM14 and CM15 (CM14 and CM15 are prepared in one preparation) were used for the experiments. Conditioned medium and control medium were used.
  • CM5 hUTC were seeded on day 1 at 10,000 viable cells/cm 2 (for CM510k) or 11,000 viable cells/cm 2 (for CM511k) in T225 cell culture flasks in hUTC growth medium (DMEM low glucose + 15% FBS + 4 mM L-glutamine).
  • hUTC were seeded on day 1 at 10,000 viable cells/cm 2 in T75 cell culture flasks in hUTC growth medium. After cell seeding, then cultured for 24 hours in 37 o C 5% CO2 incubator. On day 2, medium was aspirated and replenished with 63 mL/T225 flask of DMEM/F12 complete medium (DMEM:F12 medium + 10% FBS + Pen
  • CM11 increased the level of phagocytosis for all the RPEs, including the AMD RPEs (data not shown).
  • the reduced levels of phagocytosis in the AMD RPEs were essentially normalized after treatment with CM11. This was again consistent with the pattern.
  • CM8 increased phagocytosis in all RPEs, including the AMD RPEs which were essentially normalized. The patterns observed were maintained.
  • the phagocytosis levels of the AMD ND08626 RPE were lower than the normal 15-11-098, 15-09-027, 15-08-074, and 15-04- 001 RPE as seen before.
  • Treatment of the AMD ND08626 RPE with CM510k normalized the decreased level of phagocytosis as also seen before.
  • Treatment of the normal RPE with the CM also stimulated phagocytosis levels to variable degrees in all of them.
  • CM hUTC conditioned medium
  • CM11 for 6 and 24 hours
  • RNA was isolated from them, along with a control sample.
  • the control sample was incubated in MEM5 for 6 hours.
  • RNA from the SD1 Right (R) RPE could not be isolated because of poor growth and limited supply, it was attempted to nurture some surviving 3’ cultures. The surviving 3’ cultures were combined to isolate any RNA, with and without treatment with CM11 for 24 hours.
  • RNA from human 15-04-001 RPE, human 15-07-072 RPE treated or not treated with CM
  • Recombinant human BDNF (Cat # 248-BD-025/CF, Lot # NG6515031) and human GDNF (Cat # 212-GD-010/CF, Lot # VQ2215081) were from R&D Systems, Inc., Minneapolis, MN. They were reconstituted at 100 ⁇ g/mL in sterile PBS, aliquoted and frozen at -70 o C freezer.
  • Recombinant human HGF (Cat # GF116, Lot # 2651986) was from EMD Millipore Corp., Temecula, CA. It was reconstituted at 0.5 mg/mL in water, aliquoted and frozen at -20 o C.
  • HGF did not show a dose effect on phagocytosis by SD1 AMD 4’ RPE, although in general, it increased phagocytosis.
  • the phagocytosis level in the SD1 AMD RPE was again lower than in the normal (15-09-027 and 15-08-074) RPE, and addition of CM10 increased the phagocytosis level.
  • ND08626 phagocytosis to the RTK ligand BDNF; 2) a decreased phagocytosis level of the AMD RPE (ND08626) compared to that of the normal RPE (15-09-027, 15-11-098); and 3) normalization of the decreased phagocytosis level of the AMD RPE (ND08626) by treatment with CM (CM510k) (see FIG.38).
  • Recombinant Human MFG-E8 (Cat # 2767-MF-050, Lot # MPP2415081), recombinant Human TSP-1 (Cat # 3074-TH-050, Lot # MVF4114111), recombinant Human TSP-2 (Cat # 1635-T2-050, Lot # HUZ2014121), were all obtained from R&D Systems, Inc., Minneapolis, MN. Reconstitution of individual protein stock solution was to follow the vendor’s data sheets: Recombinant Human MFG-E8, TSP-1 and TSP-2 were reconstituted at 100 ⁇ g/mL in sterile PBS, respectively. Recombinant Human Gas6 was reconstituted at 100 ⁇ g/mL in sterile water. The reconstituted stocks were aliquoted and frozen at -70 o C freezer.
  • ROS was pre-incubated with hUTC CM control medium (DMEM:F12 medium with 10% FBS and 1% P/S) or hUTC CM for 24 h in CO 2 cell culture incubator at 37 o C.
  • ROS was pre-incubated in control medium containing various concentrations of human recombinant MFG-E8 (15.5 ng/mL, 31 ng/mL, 62 ng/mL, 124 ng/mL), TSP-1 (152 ng/mL, 304 ng/mL, 608 ng/mL, 1216 ng/mL) or TSP-2 (8.8 ng/mL, 26.4 ng/mL, 79.2 ng/mL, 237.6 ng/mL) for 24 h in CO 2 cell culture incubator at 37 o C.
  • the ROS was spun down, resuspended in MEM5 (MEM medium with 5% FBS) and fed to the dystrophic RPE cells in the presence of MEM5 for phagocytosis assay.
  • MEM5 MEM medium with 5% FBS
  • dystrophic RPE alone was cultured in MEM20 (MEM medium with 20% FBS), then changed to MEM5 in the presence of untreated ROS (resuspended in MEM20 and fed to RPE cells) for phagocytosis assay.
  • This example describes the preparation of postpartum-derived cells from placental and umbilical cord tissues.
  • Postpartum umbilical cords and placentae were obtained upon birth of either a full term or pre-term pregnancy.
  • Cells were harvested from five separate donors of umbilicus and placental tissue. Different methods of cell isolation were tested for their ability to yield cells with: 1) the potential to differentiate into cells with different phenotypes, a characteristic common to stem cells; or 2) the potential to provide trophic factors useful for other cells and tissues.
  • Umbilical cell isolation Umbilical cords were obtained from National Disease Research Interchange (NDR1, Philadelphia, Pa.). The tissues were obtained following normal deliveries. The cell isolation protocol was performed aseptically in a laminar flow hood. To remove blood and debris, the cord was washed in phosphate buffered saline (PBS; Invitrogen, Carlsbad, Calif.) in the presence of antimycotic and antibiotic (100 units/milliliter penicillin, 100 micrograms/milliliter streptomycin, 0.25
  • tissue culture plates were then mechanically dissociated in 150 cm 2 tissue culture plates in the presence of 50 milliliters of medium (DMEM-Low glucose or DMEM-High glucose; Invitrogen), until the tissue was minced into a fine pulp.
  • the chopped tissues were transferred to 50 milliliter conical tubes (approximately 5 grams of tissue per tube).
  • C:D collagenase (Sigma, St Louis, Mo.), 500 Units/milliliter; and dispase
  • the tissues were centrifuged at 150 x g for 5 minutes, and the supernatant was aspirated.
  • the pellet was resuspended in 20 milliliters of Growth Medium (DMEM-Low glucose (Invitrogen), 15 percent (v/v) fetal bovine serum (FBS; defined bovine serum; Lot#AND18475; Hyclone, Logan, Utah), 0.001% (v/v) 2- mercaptoethanol (Sigma), 1 milliliter per 100 milliliters of antibiotic/antimycotic as described above.
  • the cell suspension was filtered through a 70-micrometer nylon cell strainer (BD Biosciences). An additional 5 milliliters rinse comprising Growth Medium was passed through the strainer.
  • the cell suspension was then passed through a 40- micrometer nylon cell strainer (BD Biosciences) and chased with a rinse of an additional 5 milliliters of Growth Medium. [0507] The filtrate was resuspended in Growth Medium (total volume 50 milliliters) and centrifuged at 150 x g for 5 minutes. The supernatant was aspirated and the cells were resuspended in 50 milliliters of fresh Growth Medium. This process was repeated twice more.
  • BD Biosciences BD Biosciences
  • the cells isolated from umbilical cords were seeded at 5,000 cells/cm 2 onto gelatin-coated T-75 cm 2 flasks (Corning Inc., Corning, N.Y.) in Growth Medium with antibiotics/antimycotics as described above. After 2 days (in various experiments, cells were incubated from 2-4 days), spent medium was aspirated from the flasks. Cells were washed with PBS three times to remove debris and blood-derived cells. Cells were then replenished with Growth Medium and allowed to grow to confluence (about 10 days from passage 0) to passage 1. On subsequent passages (from passage 1 to 2 and so on), cells reached sub-confluence (75-85 percent confluence) in 4-5 days. For these subsequent passages, cells were seeded at 5000 cells/cm 2 . Cells were grown in a humidified incubator with 5 percent carbon dioxide and atmospheric oxygen, at 37° C.
  • Placental Cell Isolation Placental tissue was obtained from NDRI
  • the tissues were from a pregnancy and were obtained at the time of a normal surgical delivery. Placental cells were isolated as described for umbilical cell isolation.
  • the cell isolation protocol was performed aseptically in a laminar flow hood.
  • the placental tissue was washed in phosphate buffered saline (PBS; Invitrogen, Carlsbad, Calif.) in the presence of antimycotic and antibiotic (as described above) to remove blood and debris.
  • PBS phosphate buffered saline
  • the placental tissue was then dissected into three sections: top-line (neonatal side or aspect), mid-line (mixed cell isolation neonatal and maternal) and bottom line (maternal side or aspect).
  • the separated sections were individually washed several times in PBS with antibiotic/antimycotic to further remove blood and debris. Each section was then mechanically dissociated in 150 cm 2 tissue culture plates in the presence of 50 milliliters of DMEM-Low glucose, to a fine pulp. The pulp was transferred to 50 milliliter conical tubes. Each tube contained approximately 5 grams of tissue. The tissue was digested in either DMEM-Low glucose or DMEM-High glucose medium containing antimycotic and antibiotic (100 U/milliliter penicillin, 100 micrograms/milliliter streptomycin, 0.25 micrograms/milliliter amphotericin B) and digestion enzymes. In some experiments an enzyme mixture of collagenase and dispase (“C:D”) was used containing collagenase (Sigma, St Louis, Mo.) at 500 Units/milliliter and dispase (Invitrogen) at 50
  • the tissues were centrifuged at 150 x g for 5 minutes, the resultant supernatant was aspirated off.
  • the pellet was resuspended in 20 milliliters of Growth Medium with penicillin/streptomycin/amphotericin B.
  • the cell suspension was filtered through a 70 micrometer nylon cell strainer (BD Biosciences), chased by a rinse with an additional 5 milliliters of Growth Medium.
  • the total cell suspension was passed through a 40 micrometer nylon cell strainer (BD Biosciences) followed with an additional 5 milliliters of Growth Medium as a rinse.
  • the filtrate was resuspended in Growth Medium (total volume 50 milliliters) and centrifuged at 150 x g for 5 minutes. The supernatant was aspirated and the cell pellet was resuspended in 50 milliliters of fresh Growth Medium. This process was repeated twice more. After the final centrifugation, supernatant was aspirated and the cell pellet was resuspended in 5 milliliters of fresh Growth Medium. A cell count was determined using the Trypan Blue Exclusion test. Cells were then cultured at standard conditions.
  • LIBERASE Cell Isolation Cells were isolated from umbilicus tissues in DMEM-Low glucose medium with LIBERASE (Boehringer Mannheim Corp.,
  • Isolation of cells from residual blood in the cords Other attempts were made to isolate pools of cells from umbilical cord by different approaches. In one instance umbilical cord was sliced and washed with Growth Medium to dislodge the blood clots and gelatinous material. The mixture of blood, gelatinous material and Growth Medium was collected and centrifuged at 150 x g. The pellet was resuspended and seeded onto gelatin-coated flasks in Growth Medium. From these experiments a cell population was isolated that readily expanded.
  • Isolation of cells from cord blood Cells have also been isolated from cord blood samples attained from NDR1.
  • the isolation protocol used here was that of International Patent Application WO 2003/025149 by Ho et al. (Ho, T. W., et al.,“Cell Populations Which Co-Express CD49C and CD90,” Application No. PCT/US02/29971).
  • Samples (50 milliliter and 10.5 milliliters, respectively) of umbilical cord blood (NDRl, Philadelphia Pa.) were mixed with lysis buffer (filter-sterilized 155 mM ammonium chloride, 10 millimolar potassium bicarbonate, 0.1 millimolar EDT A buffered to pH 7.2 (all components from Sigma, St. Louis, Mo.)).
  • Cells were lysed at a ratio of 1:20 cord blood to lysis buffer. The resulting cell suspension was vortexed for 5 seconds, and incubated for 2 minutes at ambient temperature. The lysate was centrifuged (10 minutes at 200 x g). The cell pellet was resuspended in complete minimal essential medium (Gibco, Carlsbad, Calif.) containing 10 percent fetal bovine serum (Hyclone, Logan Utah), 4 millimolar glutamine (Mediatech, Herndon, Va.), 100 Units penicillin per 100 milliliters and 100 micrograms streptomycin per 100 milliliters (Gibco, Carlsbad, Calif.).
  • complete minimal essential medium Gibco, Carlsbad, Calif.
  • the resuspended cells were centrifuged (10 minutes at 200 x g), the supernatant was aspirated, and the cell pellet was washed in complete medium. Cells were seeded directly into either T75 flasks (Corning, N.Y.), T75 laminin-coated flasks, or T175 fibronectin-coated flasks (both Becton Dickinson, Bedford, Mass.).
  • Table 14-2 Isolation and culture expansion of postpartum cells under varying conditions
  • Isolation of cells from cord blood The preparations contained red blood cells and platelets. No nucleated cells attached and divided during the first 3 weeks. The medium was changed 3 weeks after seeding and no cells were observed to attach and grow.
  • Blendzyme 3 which is collagenase (4 Wunsch units/g) and thermolysin (1714 casein Units/g) was also used together with hyaluronidase to isolate cells. These cells expanded readily over many passages when cultured in Growth Medium on gelatin- coated plastic.
  • Cell lines used in cell therapy are preferably homogeneous and free from any contaminating cell type.
  • Cells used in cell therapy should have a normal chromosome number (46) and structure.
  • Cells were seeded at 5,000 cells per square centimeter in Growth Medium in a T25 flask (Corning Inc., Corning, N.Y.) and expanded to 80% confluence. A T25 flask containing cells was filled to the neck with Growth Medium. Samples were delivered to a clinical cytogenetics laboratory by courier (estimated lab to lab transport time is one hour). Cells were analyzed during metaphase when the chromosomes are best visualized. Of twenty cells in metaphase counted, five were analyzed for normal homogeneous karyotype number (two). A cell sample was characterized as homogeneous if two karyotypes were observed.
  • a cell sample was characterized as heterogeneous if more than two karyotypes were observed. Additional metaphase cells were counted and analyzed when a heterogeneous karyotype number (four) was identified. Results [0530] All cell samples sent for chromosome analysis were interpreted as exhibiting a normal appearance. Three of the 16 cell lines analyzed exhibited a heterogeneous phenotype (XX and XY) indicating the presence of cells derived from both neonatal and maternal origins (Table 15-1). Cells derived from tissue Placenta-N were isolated from the neonatal aspect of placenta. At passage zero, this cell line appeared homogeneous XY. However, at passage nine, the cell line was heterogeneous (XX/XY), indicating a previously undetected presence of cells of maternal origin.
  • Chromosome analysis identified placenta-and umbilicus-derived cells whose karyotypes appeared normal as interpreted by a clinical cytogenetic laboratory.
  • Karyotype analysis also identified cell lines free from maternal cells, as determined by homogeneous karyotype.
  • Characterization of cell surface proteins or“markers” by flow cytometry can be used to determine a cell line's identity. The consistency of expression can be determined from multiple donors, and in cells exposed to different processing and culturing conditions. Postpartum-derived cell (PPDC) lines isolated from the placenta and umbilicus were characterized (by flow cytometry), providing a profile for the identification of these cell lines.
  • PPDC Postpartum-derived cell
  • Placenta and umbilicus comparison Placenta-derived cells were compared to umbilicus-derive cells at passage 8.
  • Donor to donor comparison To compare differences among donors, placenta- derived cells from different donors were compared to each other, and umbilicus-derived cells from different donors were compared to each other.
  • Digestion enzyme comparison Four treatments used for isolation and preparation of cells were compared. Cells isolated from placenta by treatment with 1) collagenase; 2) collagenase/dispase; 3) collagenase/hyaluronidase; and 4)
  • Placental layer comparison Cells derived from the maternal aspect of placental tissue were compared to cells derived from the villous region of placental tissue and cells derived from the neonatal fetal aspect of placenta. Results
  • Placenta vs. umbilicus comparison Placenta-and umbilicus-derived cells analyzed by flow cytometry showed positive expression of CD10, CD13, CD44, CD73, CD90, PDGFr-alpha and HLA-A, B, C, indicated by the increased values of fluorescence relative to the IgG control. These cells were negative for detectable expression of CD31, CD34, CD45, CD117, CD141, and HLA-DR, DP, DQ, indicated by fluorescence values comparable to the IgG control. Variations in fluorescence values of positive curves were accounted. The mean (i.e. CD13) and range (i.e. CD90) of the positive curves showed some variation, but the curves appeared normal, confirming a homogenous population. Both curves individually exhibited values greater than the IgG control.
  • Passage to passage comparison--placenta-derived cells Placenta-derived cells at passages 8, 15, and 20 analyzed by flow cytometry all were positive for expression of CD10, CD13, CD44, CD73, CD90, PDGFr-alpha and HLA-A, B, C, as reflected in the increased value of fluorescence relative to the IgG control.
  • the cells were negative for expression of CD31, CD34, CD45, CD117, CD141, and HLA -DR, DP, DQ having fluorescence values consistent with the IgG control.
  • Donor to donor comparison--umbilicus derived cells Umbilicus-derived cells isolated from separate donors analyzed by flow cytometry each showed positive expression of CD10, CD13, CD44, CD73, CD90, PDGFr-alpha and HLA-A, B, C, reflected in the increased values of fluorescence relative to the IgG control. These cells were negative for expression of CD31, CD34, CD45, CD117, CD141, and HLA-DR, DP, DQ with fluorescence values consistent with the IgG control.
  • Placenta- derived cells expanded on either gelatin-coated or uncoated flasks analyzed by flow cytometry all expressed of CD10, CD13, CD44, CD73, CD90, PDGFr-alpha and HLA-A, B, C, reflected in the increased values of fluorescence relative to the IgG control. These cells were negative for expression of CD31, CD34, CD45, CD117, CD141, and HLA-DR, DP, DQ indicated by fluorescence values consistent with the IgG control.
  • Placental layer comparison Cells isolated from the maternal, villous, and neonatal layers of the placenta, respectively, analyzed by flow cytometry showed positive expression of CD10, CD13, CD44, CD73, CD90, PDGFr-alpha and HLA-A, B, C, as indicated by the increased value of fluorescence relative to the IgG control. These cells were negative for expression of CD31, CD34, CD45, CD117, CD141, and HLA-DR, DP, DQ as indicated by fluorescence values consistent with the IgG control.
  • Placenta-and umbilicus-derived cells are positive for CD10, CD13, CD44, CD73, CD90, PDGFr-alpha, HLA-A,B,C and negative for CD31, CD34, CD45, CD117, CD141 and HLA-DR, DP, DQ. This identity was consistent between variations in variables including the donor, passage, culture vessel surface coating, digestion enzymes, and placental layer.
  • Tissue Preparation Human umbilical cord and placenta tissue was harvested and immersion fixed in 4% (w/v) paraformaldehyde overnight at 4° C.
  • Willebrand Factor vWF; 1:200; Sigma
  • CD34 human CD34 Class III; 1:100; DAKOCytomation, Carpinteria, Calif.
  • antihuman GROalpha--PE 1: 100; Becton Dickinson, Franklin Lakes, N.J
  • antihuman GCP-2 1:100; Santa Cruz Biotech, Santa Cruz, Calif
  • anti-human oxidized LDL receptor 1 ox-LDL R1; 1:100; Santa Cruz Biotech
  • anti-human NOGO-A (1:100; Santa Cruz Biotech).
  • Triton was omitted in all steps of the procedure in order to prevent epitope loss. Furthermore, in instances where the primary antibody was raised against goat (GCP-2, ox-LDL R1, NOGO-A), 3% (v/v) donkey serum was used in place of goat serum throughout the procedure. Primary antibodies, diluted in blocking solution, were then applied to the sections for a period of 4 hours at room temperature.

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Abstract

L'invention concerne des méthodes et des compositions destinées à traiter et à réduire la dégénérescence rétinienne à l'aide de cellules progénitrices et de milieux conditionnés issus de cellules progénitrices, telles que les cellules dérivées du post-partum. L'invention concerne également des facteurs trophiques et d'autres agents sécrétés par les cellules progénitrices qui protègent les cellules rétiniennes et inhibent l'apoptose de cellules rétiniennes, telles que des cellules photoréceptrices.
PCT/US2017/062526 2016-12-01 2017-11-20 Traitement de la dégénérescence rétinienne à l'aide de cellules progénitrices WO2018102174A1 (fr)

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10744164B2 (en) 2003-06-27 2020-08-18 DePuy Synthes Products, Inc. Repair and regeneration of ocular tissue using postpartum-derived cells
US10758571B1 (en) 2019-04-09 2020-09-01 Combangio, Inc. Processes for making and using a mesenchymal stem cell derived secretome
US10881693B2 (en) 2019-04-09 2021-01-05 Combangio, Inc. Processes for making and using a mesenchymal stem cell derived secretome
US11129853B2 (en) 2019-04-09 2021-09-28 Combangio, Inc. Processes for making and using a mesenchymal stem cell derived secretome
US11654160B2 (en) 2019-04-09 2023-05-23 Combangio, Inc. Processes for making and using a mesenchymal stem cell derived secretome

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