WO2005084119A2 - Cellules souches de type embryonnaire multipotentes derivee de limbe, leurs procedes d'isolation et d'utilisation - Google Patents

Cellules souches de type embryonnaire multipotentes derivee de limbe, leurs procedes d'isolation et d'utilisation Download PDF

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WO2005084119A2
WO2005084119A2 PCT/IB2005/000516 IB2005000516W WO2005084119A2 WO 2005084119 A2 WO2005084119 A2 WO 2005084119A2 IB 2005000516 W IB2005000516 W IB 2005000516W WO 2005084119 A2 WO2005084119 A2 WO 2005084119A2
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cells
elscs
culture
pluripotent
isolated
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PCT/IB2005/000516
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WO2005084119A3 (fr
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Satish Mahadeorao Totey
Subhadra Devi Kashyap
Firdos Alam Khan
Rajarshi Pal
Aparna Khanna
Shabri Tipnis
Geeta Ravindran
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Reliance Life Sciences Pvt. Ltd.
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Priority to AU2005220066A priority Critical patent/AU2005220066B2/en
Priority to KR1020097002160A priority patent/KR100931887B1/ko
Priority to BRC10506536-4A priority patent/BRPI0506536C1/pt
Priority to EP05708629A priority patent/EP1758984A2/fr
Publication of WO2005084119A2 publication Critical patent/WO2005084119A2/fr
Priority to US11/502,338 priority patent/US8187875B2/en
Priority to IL177672A priority patent/IL177672A/en
Priority to IL177671A priority patent/IL177671A0/en
Publication of WO2005084119A3 publication Critical patent/WO2005084119A3/fr

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    • C12N5/0602—Vertebrate cells
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    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/03—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from non-embryonic pluripotent stem cells

Definitions

  • the present disclosure relates to purified preparations of mammalian pluripotent stem cells, preferably human pluripotent stem cells, derived from corneal limbus tissue.
  • the pluripotent limbal stem cell lines are self- renewing and have the ability to differentiate into tissues derived from all three embryonic germ layers (endoderm, mesoderm and ectoderm). Methods for isolating pluripotent limbal stem cell lines and methods of their use are also disclosed.
  • ES cells embryonic stage embryonic stem cells
  • ES cells embryonic stage embryonic stem cells
  • ectoderm mesoderm
  • endoderm The ectoderm germ layer forms the epidermis of the skin, sense organs, nervous system, and spinal cord.
  • the mesoderm germ layer forms smooth muscle, connective tissues, blood vessels, heart, blood cells and bone marrow, reproductive organ, excretory system, striated muscles, and skeletal muscles. Finally, the endoderm germ layer forms epithelial linings of respiratory and gastrointestinal tract, pharynx, esophagus, stomach, intestine, and other associated organs.
  • ES cells are referred to as pluripotent stem cells because they can differentiate into almost all cell types in an adult organism.
  • ES cells are often referred to as pluripotent stem cells because they are not fixed in their developmental potentialities and can differentiate into many different cell types in vitro.
  • Cultured ES cells that are highly pluripotent can form clumps of cells in suspension culture referred to as embryoid bodies.
  • ES cells were isolated from humans relatively recently (Thomson et al, (1998) Science 282:1145-1147; Gearhart, (1998) Science 282:1061-62). In embryoid bodies derived from human ES cells it is possible to discern differentiated cells bearing markers of a wide variety of cell types.
  • adult stem cells have not been found to be an adequate replacement for ES cells.
  • adult stem cells can be difficult to isolate because they are usually present only in minute quantities in tissues that are often not easily accessible, and their numbers appear to decrease with age.
  • adult stem cells appear to be a less desirable source of cultured tissue than ES cells because they have a shorter life span and less capacity for self-renewal.
  • adult stem cells are believed to be tissue specific and not pluripotent, generally capable of giving rise only to new cells of a few types closely related to their tissue of origin.
  • ES cells in suspension culture are capable of forming aggregates of cells known as embryoid bodies. These embiyoid bodies usually contain germ cells of all three lineages that differentiate into various lineage-committed tissues. Therefore, embryoid bodies can be useful in the preparation of different types of differentiated cells in culture. To date, no other isolated adult stem cell lines have been reported that are capable of forming structures similar to embryoid-like bodies in culture.
  • Pluripotent adult stem cells have also been recently described in many tissues such as skeletal muscle, brain, and intestinal epithelium (Howell et al, (2003) Ann. N.Y. Acad. Sci. 996:158-173.). Still, while these papers report isolated or identified adult stem cells that are pluripotent, these "pluripotent" adult stem cells, unlike ES cells, differentiate into only a few lineages. In addition, none of the isolated adult stem cells reported to date appear to be capable of fo ⁇ ning embryoid-like bodies in culture in a manner similar to ES cells.
  • adult stem cells are present in the corneoscleral limbus of the eye. These cells participate in the dynamic equilibrium of the corneal surface and replace superficial epithelial cells that are shed and sloughed off during eye-blinking. Severe damage to the limbal stem cells from chemical or thermal burns, contact lenses, severe mierobial infection, multiple surgical procedures, cryotherapy, or diseases such as Steven- Johnson syndrome or ocular cicatrical pemphigoid can lead to destruction of limbal stem cells and limbal stem cell deficiency which can lead to an abnormal corneal surface, photophobia, and reduced vision (Anderson et al, (2001) Br. J. Opthalmol.
  • the present disclosure describes the isolation of mammalian pluripotent embryonic-like stem cells (ELSCs) derived from non-embryonic tissue, preferably corneal limbal tissue.
  • ELSCs mammalian pluripotent embryonic-like stem cells
  • the present disclosure provides isolated m------mali--n pluripotent ELSCs which: (i) are capable of proliferating in an in vitro culture, (ii) m--intain the potential to differentiate into cells of endoderm, mesoderm, and ectoderm lineage in culture, and (iii) are capable of foraiing embryoid-like bodies when placed in suspension culture.
  • the isolated ELSCs are human ELSCs.
  • the ELSCs are derived from corneoscleral limbus tissue, preferably human tissue.
  • the isolated ELSCs remain substantially undifferentiated in an in vitro culture for at least about 20 passages, more preferably at least about 50 passages, and most preferably at least about 100 passages in culture.
  • the substantially undifferentiated ELSCs maintain normal karyotype and high telomerase activity.
  • the isolated ELSCs have the potential to terminally differentiate into cells or tissues of endoderm, mesoderm, or ectoderm lineage.
  • the present disclosure also provides isolated mammalian pluripotent ELSCs which: (i) are isolated from corneoscleral limbus, (ii) are capable of proliferating in an in vitro culture, and (iii) maintain the potential to differentiate into lineage-committed endodermal, ectodermal, or mesodermal cells.
  • the isolated ELSCs are human ELSCs, which are more preferably SSEA-4 positive.
  • the corneoscleral limbus is isolated from a human subject.
  • the isolated ELSCs are capable of fo ⁇ riing embryoid-like bodies when placed in suspension culture.
  • the isolated ELSCs remain substantially undifferentiated in an in vitro culture for at least about 20 passages, more preferably at least about 50 passages, and most preferably at least about 100 passages in culture.
  • the substantially undifferentiated ELSCs maintain normal karyotype and high telomerase activity.
  • the isolated ELSCs have the potential to terminally differentiate into cells or tissues of endoderm, mesoderm, or ectoderm lineage.
  • the present disclosure also provides methods of isolating a population of mammalian pluripotent embryonic-like stem cells (ELSCs), comprising the steps of: (a) isolating corneal limbal tissue from a donor; (b) culturing the corneal limbal tissue to expand corneal limbal cells in culture; and (c) isolating a population of pluripotent ELSCs from the cultured corneal limbal cells by sorting the corneal limbal cells to select for one or more undifferentiated cell-specific surface markers.
  • ELSCs mammalian pluripotent embryonic-like stem cells
  • the isolated population of pluripotent ELSCs are human ELSCs, which are more preferably SSEA-4 positive.
  • the donor of the corneal limbal tissue is human.
  • the corneal limbal tissue is cultured in culture media such as DMEM or F12, further supplemented with a nutrient serum and one or more soluble factors selected from the group consisting of dimethyl sulphoxide (DMSO), recombinant human epidermal growth factor (rhEGF), insulin, sodium selenite, transferrin, basic fibroblast growth factor (bFGF), and leukemia inhibitory factor (LIF).
  • DMSO dimethyl sulphoxide
  • rhEGF recombinant human epidermal growth factor
  • bFGF basic fibroblast growth factor
  • LIF leukemia inhibitory factor
  • the corneal limbal tissue is cultured until the corneal limbal cells in the culture become confluent.
  • the corneal limbal tissue is cultured on an extracellular matrix, for example MatrigelTM, laminin, collagen-IV, poly-L-lysine, gelatin, poly-L-o ⁇ nthin, fibronectin, and combinations thereof, or mammalian amniotic membrane.
  • the above methods preferably -nrther comprise the step of dissociating the cultured corneal limbal cells from the extracellular matrix prior to isolating the pluripotent ELSCs.
  • the corneal limbal cells are sorted using methods well known to those of skill in the art, for example magnetic-affinity cell sorting (MACS) or fluorescence-activated cell sorting (FACS) to isolate a population of pluripotent ELSCs.
  • the one or more undifferentiated cell-specific markers selected for to isolate pluripotent ELSCs include but are not limited to SSEA-4, SSEA-3, CD73, CD105, CD31, CD54, and CD117.
  • corneal limbal cells are sorted to select for SSEA-4 positive ELSCs.
  • the sorted ELSCs comprise at least about 80%, 90%, 95%, 98%, or 99% pluripotent ELSCs that are SSEA-4 positive.
  • the isolated population of pluripotent ELSCs comprise at least about 70%, 80%, 90%, 95%, 98%, or 99% pluripotent ELSCs.
  • the isolated population of pluripotent ELSCs are further cultured to produce an embryonic-like stem cell line.
  • the pluripotent ELSCs are cultured in culture media such as DMEM or F12, further supplemented with a nutrient serum and one or more soluble factors selected from the group consisting of DMSO, rhEGF, insulin, sodium selenite, transferrin, bFGF, and LIF.
  • pluripotent ELSCs isolated by the methods disclosed herein are capable of proliferating and maintaining the potential to differentiate in vitro or in vivo into cells or tissues of endoderm, mesoderm or endoderm lineage.
  • the isolated pluripotent ELSCs are also capable of forming embryoid-like bodies, for example when placed in suspension culture.
  • the isolated ELSCs remain substantially undifferentiated in an in vitro culture for at least about 20 passages, more preferably at least about 50 passages, and most preferably at least about 100 passages in culture.
  • the substantially undifferentiated ELSCs maintain normal karyotype and high telomerase activity.
  • the isolated ELSCs have the potential to terminally differentiate into cells or tissues of endoderm, mesoderm, or ectoderm lineage.
  • the isolated pluripotent ELSCs preferably human ELSCs, are further differentiated in culture into endodermal lineage-committed cells or tissues, mesodermal lineage-committed cells or tissues, or ectodermal lineage-committed cells or tissues.
  • the isolated pluripotent ELSCs are further differentiated into endodermal lineage-committed cells or tissues, mesodermal lineage-committed cells or tissues, or ectodermal lineage-committed cells or tissues in vivo.
  • these ELSCs are further differentiated by exposing the ELSCs to one or more agents known to induce differentiation of pluripotent embryonic stem (ES) cells, including but not limited to acidic fibroblast growth factor, bFGF, platelet-derived growth factor (PDGF), insulin, retinoic acid, transferrin, insulin-transferrin-selenious acid (ITS), dexamethasone, sodium butyrate, DMSO, nerve growth factor (NGF), Cytosine beta-d-Arabino Furanoside (Ara C), glial cell line-derived neurotrophic factor gene (GDNF), transforming growth factor ⁇ 3 (TGF- ⁇ 3), ascorbic acid, N-acetyl Cysteine, dibutaryl cyclic AMP, Neurturin, transforming growth factor ⁇ l (TGF- ⁇ l), insulin-like growth factor I or II (IGF-I or IGF-II), epidermal growth factor (EGF), bone morphogenic proteins 2 (B
  • FIG. 1 Limbal composite grafts (LCG): (a) H & E stained LCG (whole mount); (b) LCG probed using immunofluorescence for SSEA-4 antigen; (c) LCG analysis by RT-PCR for expression of the pluripotent stem cell markers Oct-4, Nanog, and Rex 1, as well as GAPDH as a positive control; and (d) SSEA-4 positive cells isolated from LCG by flow cytometry.
  • FIG. 1 Figure 2. -rnmunophenotyping of ELSCs: ELSCs cultured for 20 passages were labeled with FITC-coupled antibodies against SSEA-4, CD105, CD73, CD54, CD45, CD34, CD123, CD133, CD 123, and HLA-DR. The ELSCs demonstrated positive labeling with SSEA-4, CD105, CD73, and CD54 antibodies, indicating that the cells maintain pluripotency after 20 passages, and that ELSCs are not hematopoietic in origin.
  • FIG. 3 Gene expression profiling by RT-PCR of the undifferentiated stem cell markers Oct-4, Nanog, Rexl, and TDGF1 after 5, 10, 15, and 20 passages of the isolated ELSCs. GAPDH expression was also analyzed as a positive control. hEF cells were used as a negative control for expression of the undifferentiated stem cell markers, while NTERA (NT) cells were used as a positive control for expression of these markers.
  • FIG. 1 Karyotyping of ELSCs. ELSCs isolated after 13 passages maintained a normal karyotype (analysis by CYTO VISION software).
  • FIG. 10X Phase-contrast micrographs of ELSCs and ELBs (10X): (a) micrograph of passage 15 ELSCs; (b) micrograph of ELBs formed from ELSCs after 4 days of suspension culture; and (c) micrograph demonstrating initiation of differentiation from ELBs formed from ELSCs.
  • FIG. 6 Gene expression profiling by RT-PCR of the undifferentiated stem cell markers Oct-4, Nanog, Rexl, and TDGF1 in ELSCs (UD) and in ELBs collected on day 2 (2d), day 4 (4d), day 8 (8d), day 12 (12d), and day 14 (14d) of ELB formation. GAPDH expression was also analyzed as a positive control. hEF cells were used as a negative control for expression of the undifferentiated stem cell markers.
  • Figure 7 Gene expression profiling by RT-PCR of lineage markers NFH and Keratin (neuroectoderm lineage markers); c-Actin (mesoderm lineage marker); and AFP and Albumin (endoderm lineage markers) in ELSCs (UD) and in ELBs collected on day 2 (2d), day 4 (4d), day 8 (8d), day 12 (12d), and day 14 (14d) of ELB formation.
  • Positive controls (PC) for each marker were as follows: fetal-brain tissue extract for NFH and Keratin; fetal-heart tissue extract for c-Actin; and fetal-liver tissue extract for AFP and Albumin.
  • the negative controls (NC) were (-)RT products.
  • FIG. 8 Gene expression profiling by RT-PCR of the following markers in ELSCs (UD) and in ELBs collected on day 2 (2d), day 4 (4d), day 8 (8d), day 12 (12d), and day 14 (14d) of ELB formation: PECAM, which is an endothelial lineage marker; KGF and Collagen I, which are stromal cell markers; and p63, which is a corneal epithelial stem cell marker.
  • Positive controls (PC) for each marker were as follows: fetal- heart tissue extract for PECAM; hEF cells for KGF and Collagen I; and limbal tissue extract for p63.
  • the negative controls (NC) were (-)RT products.
  • FIG. 9 Immunofluorescence assays of neuronal cells differentiated from ELSCs through the formation of ELBs (10X). -mmunological characterization of neuronal cells differentiated from ELSCs showed positive immunofluorescence for the neuronal markers ⁇ -tubulin III, Neurofilament, O4, Glutamate, GABA, Tyrosine hydroxylase, Serotonin, Nestin.
  • FIG. 10 Cellular and functional characterization of various differentiated cell-types from ELSCs through the formation of ELBs: (a) initiation of differentiation from ELBs; (b) Von Kossa staining of osteoblasts; (c) Alcian Blue staining of chondrocytes; (d) Oil Red-O staining of adipocytes; (e) immunofluorescence of myocytes with anti-myogenin antibody; (f) phase contrast micrograph of beating cardiomyocytes (10X); (g) immunofluorescence of cardiomyocytes with anti-cTnT antibody; (h) phase contrast micrograph of mature hepatocytes derived from ELSCs; (i) immunofluorescence of hepatocytes with anti-albumin antibody; (j) PAS staining of mature hepatocytes showing insoluble glycogen deposits; (k) immunofluorescence of pancreatic beta-islet cells with anti-PDX-1 antibody.
  • FIG. 11 Gene expression profiling of differentiated cells of various lineages derived from ELSCs by RT-PCR. Positive expression of the following markers was found: c-Actin (cardiac cell marker); NCS (functional cardiomyocyte marker); Myogenin (myocyte marker); Alpha-Fetoprotein (AFP) (early mesendoderm marker); Albumin (hepatocyte marker); PECAM (endothelial cell marker); Insulin (pancreatic islet cell marker); Somatostatin (pancreatic islet cell marker); ⁇ -tubulin (neuronal cell marker); and Tyrosine hydroxylase (TH) (dopaminergic neuronal marker). GAPDH expression was also analyzed as a positive control.
  • the present disclosure relates to the isolation of mammalian pluripotent embryonic-like stem cells (ELSCs) from non-embryonic cells or tissues.
  • ELSCs mammalian pluripotent embryonic-like stem cells
  • the present disclosure utilizes mammalian eye structures as a source of ELSCs, preferably corneal limbus tissue.
  • the ELSCs are derived from corneoscleral or corneal limbus tissue of a human donor.
  • ELSCs of the present disclosure are particularly advantageous due to several unique properties of these cells: (1) ELSCs are capable of differentiating into cells of a variety of different lineage- committed or differentiated cell and tissue types, including cells and tissues derived from all three germ layers, endoderm, ectoderm, and mesoderm; (2) ELSCs are self-renewing and capable of propagating in culture for at least about 20 to about 100 population doublings or more while maintaining pluripotency, high telomerase activity, and normal karyotype; and (3) ELSCs are capable of forming embryoid-like bodies (ELBs).
  • ELBs embryoid-like bodies
  • the pluripotent ELSCs of the present disclosure are undifferentiated or substantially undifferentiated cells that have the potential to differentiate into almost any cell type. Morphological characteristics of undifferentiated cells are well known to those of skill in the art. For example, human ES cells may be morphologically identified by high nucleus to cytoplasm ratios, prominent nucleoli, and compact colony formation, with often distinct cell borders and colonies that are often flatter than mouse ES cells. Human ES cells are also preferably immunoreactive with markers for human pluripotent ES cells, for example SSEA-3, SSEA-4, GCTM-2 antigen, and TRA 1-60, as described by Thomson et al.
  • non-embryonic cells or tissues refer to any cells or tissues that are derived from cells or tissues other than embryonic cells, embryonic tissue, fetal primordial germ cells, or fetal gonadal ridge tissue.
  • non-embryonic cells or tissues include cells or tissues from adult mammals, and can also include cells or tissues from juvenile mammals.
  • ELSCs can differentiate into cells that are committed to a particular germ lineage but still able to give rise to various progeny cells of different cell types within that lineage, as well as cells that are terminally-differentiated.
  • ELSCs of the present disclosure can be propagated in an in vitro culture, where they are capable of differentiating cells that are derivitives of each of the three germ layers (ectoderm, mesoderm, and endoderm).
  • the term "differentiation” refers to a process whereby undifferentiated pluripotent stem cells or precursors cells acquire a more specialized fate.
  • endodermal cells include but are not limited to epithelial cells (e.g., corneal epithelial cells), hepatocytes, beta islet cells, pancreatic cells (e.g., islet, acinar and ductal cells), parenchymal cells of the trachea, bronchi, lungs, gastrointestinal tract, bladder, pharynx, thyroid, thymus, parathyroid glands, tympanic cavity, pharyngotympanic tube, tonsils, and the like; mesodermal cells include but are not limited to myocytes (e.g., smooth muscle, skeletal muscle, cardiac myocytes, cardiomyocytes), adipocytes (e.g., white fat and brown fat adipocytes), chondrocytes, hematopoietic cells (e.g., erythrocytes), lymphocytes, monocytes, macrophages, plasma cells, B cells, natural killer cells and mast cells, endothelial cells
  • ELSCs are also capable of forming embryoid-like bodies (ELBs) in culture, for example in suspension culture.
  • ELBs embryoid-like bodies
  • ELBs may also have undifferentiated cells in the aggregation of cells.
  • ELBs typically contain cells derived from all three germ layers, ectoderm, mesoderm and endoderm. Functionally, ELBs may be similar or identical to embryoid bodies generated in culture from ES cells, for example human ES cells.
  • Embryoid bodies and ELBs are distinguished from each other in the present disclosure primarily by source, i.e., embryoid bodies are derived from ES cells, while ELBs are derived from ELSCs.
  • the present disclosure further describes the isolation of pluripotent ELSCs and uses of those cell lines.
  • the use of ELSCs as a source of pluripotent stem cells has numerous advantages. First, the use of ELSCs does not raise many of the ethical concerns that are associated with research using cells derived from embryonic or fetal cells and tissue. Second, the use of ELSCs may make autologous pluripotent stem cells available for medical therapies as the source of differentiated cells and tissues without the intermediate step of cloning.
  • transplanted cells or tissues be genetically identical to the recipient of the transplant in order to avoid problems with tissue rejection.
  • ES cells that are genetically identical to a patient in need of treatment.
  • the use of ELSCs can surmount this problem if the donor of the ELSCs is also the recipient of transplanted cells or tissue derived from the ELSCs.
  • ELSCs are derived from corneal limbus tissue, which is a safe, simple, and efficient source of pluripotent ELSCs. Therefore, the present disclosure obviates the problems associated with conventional sources of pluripotent stem cells.
  • corneal limbal tissue as a source of ELSCs is the relative ease in obtaining corneal limbal tissue from a donor. The process requires only minor surgery, unlike the more invasive procedures that may be used to obtain other types of adult stem cells.
  • the corneal limbal tissue is found in the cornea, which is a transparent, avascular tissue that is located at the outer surface of the anterior eye. It provides protection from environmental insult, and allows for the efficient transmission of light into the eye.
  • the cornea is comprised of two main compartments: (1) the anterior non-cornified stratified squa ous epithelial layer and (2) the underlying substantia basement.
  • the human cornea harbors three known cell types: corneal epithelial cells; stromal keratocytes (corneal fibroblast); and an underlying layer of stromal associated corneal endothelial cells.
  • Corneal epithelium is a cellular multiplayer that is five to seven cells thick and covers the anterior surface of the cornea. Ordinarily, a natural turnover of corneal epithelial cells takes place in which superficial epithelial cells are shed from the epithelial surface and replaced by those from below. Basal epithelial cells, migrating inward from the periphery, replenish the population of deeper corneal epithelial cells.
  • Corneal limbus also known as corneoscleral limbus
  • Corneal limbus is an annular transitional zone approximately 1 mm wide between the cornea and the bulbar conjunctiva and sclera. It appears on the outer surface of the eyeball as a slight furrow marking the line between the clear cornea and the sclera. It is highly vascular and is involved in the metabolism of the cornea. Limbal and conjuctival epithelial cells, together with a stable pre-ocular tear film maintain the integrity of the cornea. While it is known that the source of the replenished corneal epithelial cells are adult stem cells, the exact location and properties of these cells were unknown.
  • the adult stem cells previously isolated from the eye are P-63 positive, and are responsible for maintaining corneal integrity (Pellegrini et al, (2001) Proc. Acad. Natl. Sci. USA, 98:3156-61).
  • the plasticity of these corneal stem cells was recently reported by Seigel et al. (Mol. Vis. 9:159-63, 2003).
  • the existence of a second population of stem cells that are pluripotent and have similar properties to ES cells was unknown.
  • the present disclosure describes the localization of ELSCs of the eye to the corneoscleral limbus.
  • a typical procedure for isolating corneal limbal tissue is to surgically remove a small biopsy consisting of 2-3 mm of limbal tissue from the superior or temporal quadrant of the corneal surface of the donor's eye. Procedures for obtaining such biopsies from the corneal limbus are known to those of skill in the art.
  • limbal tissue is biopsied from a donor, it is placed in culture, preferably on an extracellular matrix or bio-coated surface, for example extracellular matrix or bio-coated petri dishes.
  • extracellular matrices useful for culturing limbal tissue include but are not limited to MatrigelTM and its equivalents, mammalian -tn-uiiotic membrane, laminin, collagen-IV, poly-L-lysine, gelatin, poly-L-o- ⁇ thin, fibronectin, or platelet derived growth factor (PDGF), either alone or in combination with other extracellular matrix materials.
  • MatrigelTM and human amniotic membrane are particularly preferred for culturing biopsied limbal tissue.
  • a preferred method of culturing the limbal tissue is to subject the explants to dry incubation for several minutes on a bio-coated tissue culture plate. The explants are then affixed to the tissue culture dish with a small amount of culture medium so that they stick to the bio-coated tissue culture surface. After several hours to a day, media is gently added and cells are incubated for approximately 4-5 days at 37°C in a CO 2 incubator, changing the media every alternate day.
  • the preferred media used for culturing the cells of the limbal tissue is Dulbecco's Modified Eagles Medium (DMEM) or DMEM:F-12 (1 :1), preferably supplemented with a nutrient serum, for example a serum or serum-based solution that supplies nutrients effective for maintaining the growth and viability of the cells (e.g., knock-out serum or heat-inactivated human serum), as well as growth factors.
  • a nutrient serum for example a serum or serum-based solution that supplies nutrients effective for maintaining the growth and viability of the cells (e.g., knock-out serum or heat-inactivated human serum), as well as growth factors.
  • growth factor refers to proteins that bind to receptors on the cell surface with the primary result of activating cellular proliferation and differentiation.
  • the growth factors used for culturing limbal tissue are preferably selected from epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), leukemia inhibitory factor (LIF), insulin, sodium selenite, human transferrin, or human leukemia inhibitory factor (hLIF), as well as combinations thereof.
  • EGF epidermal growth factor
  • bFGF basic fibroblast growth factor
  • LIF leukemia inhibitory factor
  • insulin sodium selenite
  • human transferrin human transferrin
  • hLIF human leukemia inhibitory factor
  • ELSCs can isolated from the culture, h preferred embodiments, the limbal cells are first dissociated from the extracellular matrix, preferably through enzymatic digestion, for example using trypsin-EDTA or dispase solutions.
  • the pluripotent ELSCs can be isolated from the other limbal cells in the culture using a variety of the methods known to those of skill in the art such as immunolabeling and fluorescence sorting, for example solid phase adsorption, FACS, MACS, and the like.
  • the ELSCs are isolated through sorting, for example immunofluorescence sorting of certain cell-surface markers. Two methods of sorting well known to those of skill in the art are are magnetic-affinity cell sorting (MACS) and fluorescence-activated cell sorting (FACS).
  • MCS magnetic-affinity cell sorting
  • FACS fluorescence-activated cell sorting
  • Sorting techniques such as immunofluorescence-staining techniques involve the use of appropriate stem cell markers to separate ELSCs from other cells in the culture.
  • Appropriate stem cell markers that may be used to isolate ELSCs from cultured limbal cells include but are not limited to SSEA-4, SSEA-3, CD73, CD105, CD31, CD54, and CD117.
  • pluripotent ELSCs are isolated by MACS through the use of a cell surface marker such as SSEA-4.
  • enriched populations of cell-surface marker positive ELSCs are obtained from the mixed population of limbal cells.
  • the cells can be sorted to remove undesirable cells by selecting for cell-surface markers not found on the pluripotent ELSCs.
  • the ELSCs were found to be negative for the following cell-surface markers: CD34, CD45, CD14, CD133, CD106, CDllc, CD123, and HLA-DR.
  • the enriched ELSCs cultures obtained by sorting have at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% pluripotent ELSCs.
  • the isolated cells will be at least about 50%, 70%, 80%, 90%, 95%o, 98%, or 99% SSEA-4 positive ELSCs cells.
  • mixed cell cultures containing pluripotent ELSCs are screened for the presence of ELSCs that express certain gene markers.
  • populations of pluripotent ELSCs can be identified by the expression of gene markers such as OCT-4, Nanog, TDGF, UTX-1, FGF-4, Sox 2, Rex 1, as well as other gene marker of undifferentiated cells.
  • the ELSCs isolated from the limbal cell culture are cultured or passaged in an appropriate medium to allow the ELSCs to remain in a substantially undifferentiated state. Although colonies of undifferentiated ELSCs within the population may be adjacent to neighboring cells that are differentiated, the culture of ELSCs will nevertheless remain substantially undifferentiated when the population is cultured or passaged under appropriate conditions, and individual undifferentiated ELSCs constitute a substantial proportion of the cell population.
  • ELSCs cultures that are substantially undifferentiated contain at least about 20% undifferentiated ELSCs, and may contain at least about 40%, 60%, 80%, or 90% ELSCs.
  • ELSCs must be kept at an appropriate cell density and repeatedly dissociated and subcultured while frequently exchanging the culture medium to prevent them from differentiating.
  • ELSCs When ELSCs are passaged they may be dispersed into small clusters or into single-cell suspensions. Typically, a single cell suspension of cells is achieved and then seeded onto another tissue culture grade plastic dish.
  • the isolated ELSCs are cultured in an appropriate cell culture medium such as DMEM or DMEM:F-12 medium, preferably supplemented with a nutrient serum, for example a serum or serum-based solution that supplies nutrients effective for maintaining the growth and viability of the cells (e.g., knock-out serum or heat-inactivated human serum), as well as growth factors.
  • a nutrient serum for example a serum or serum-based solution that supplies nutrients effective for maintaining the growth and viability of the cells (e.g., knock-out serum or heat-inactivated human serum), as well as growth factors.
  • the medium is supplemented with growth factors such as EGF, basic FGF, LIF, insulin, transferrin, sodium selenite, and fibronectin.
  • the ELSCs are cultured on a feeder layer. Methods for culturing pluripotent stem cells on feeder layers are well known to those of skill in the art (U.S. Patent No.
  • the ELSCs are cultured on an extracellular matrix.
  • An extracellular matrix provides conditions for supporting cell growth, for example similar to the conditions provided by feeder cells.
  • ELSCs may also be grown in the presence of conditioned medium that can support growth of ELSCs cells, for example in a feeder-free culture.
  • Conditioned medium is prepared by culturing a first population of cells in a medium for a sufficient period of time to produce "conditioned" medium which will support the culturing of ELSCs without substantial differentiation.
  • the isolated ELSCs can be serially passaged for at least 20, 40, 60, 80, 100 or more passages, without substantially differentiating.
  • ELSCs also can be frozen for further use at various time points without loss of differential potential, i.e., the cells will retain the ability to differentiate into derivative of endodermal, ectodermal, or mesodermal lineage under appropriate conditions.
  • the isolated ELSCs retain high telomerase activity and normal karyotypes for at least 20, 40, 60, 80, 100 or more passages.
  • isolated pluripotent ELSCs are identified and characterized by the ability to form ELBs in culture, for example in suspension culture.
  • the ELBs can be further cultured to differentiate into cells of ectodermal, mesodermal, and endodermal lineages.
  • Methods for culturing pluripotent stem cells to generate embryoid bodies are disclosed in U.S. Patent No. 6,602,711, incorporated herein by reference. These same methods can also be used to generate ELBs from the ELSCs disclosed herein.
  • ELSCs are dissociated using trypsin, and cultured on bacteriological plates that have a non-adhesive surface, thereby preventing attachment of the ELSCs to the surface of the plate.
  • the ELSCs are preferably cultured in an appropriate cell culture medium such as knockout DMEM or DMEM:F-12 medium, preferably supplemented with a nutrient serum, for example a serum or serum-based solution that supplies nutrients effective for maintaining the growth and viability of the cells (e.g., fetal calf serum or fetal bovine serum), as well as growth factors.
  • the medium is supplemented with growth factors such as insulin, transferrin, or sodium selenite.
  • the cells are cultured until they form ELBs.
  • the ELBs are cultured until they reach sufficient size or desired differentiation, for example after 3-10 days of culture, preferably 4-14 days.
  • the ELBs are allowed to grow to a sufficient size to facilitate differentiation into the selected cell type.
  • the ELBs may be plated onto a substrate, for example a substrate coated with extracellular matrix components, including but not limited to poly-L-lysine, poly-L-o ⁇ -ithine, 1-mnnin, collagen, fibronectin, Matrigel®, or combinations thereof.
  • the ELBs may be plated directly onto a substrate with or without dispersing the cells.
  • ELSCs disclosed herein can be utilized for various applications, such as therapeutic and diagnostic applications, as well as for in vitro and in vivo assessment and screening of various compounds such as small molecule drugs for their effects on these cells, as well as differentiated cells derived from ELSCs.
  • the differentiated cells maybe either lmeage-committed progenitor cells, or terminally-differentiated cells.
  • Examples of differentiated cell types that may be derived from pluripotent ELSCs include but are not limited to neuronal cells, corneal cells, osteoblasts, chondrocytes, adipocytes, beta-islets, cardiomyocytes, hepatocytes, and the like.
  • the ELSCs and cells and tissues differentiated therefrom of the present disclosure can be used to treat any subject in need of treatment, including but not limited to humans, primates, and domestic, farm, pet, or sports animals, such as dogs, horses, cats, sheep, pigs, cattle, rats, mice, and the like. These cells can also be used to prepare cDNA expression libraries to analyze the expression patterns of ELSCs as well as cells derived therefrom, and to prepare monoclonal or polyclonal antibodies that are specific to markers for the particular cells used, using techniques that are well known to those of skill in the art.
  • These cells can also be use therapeutically to the benefit of individuals suffering from debilitating diseases, conditions, injuries, and disorders, for example in tissue reconstitution or regeneration in subjects such as human patients.
  • therapeutically refers to both therapeutic treatment and prophylactic or preventative measures.
  • Therapeutic treatment includes but is not limited to reducing or eliminating the symptoms of a particular disease, condition, injury or disorder, or slowing or attenuating the progression of, or curing an existing disease or disorder. Subjects in need of such therapy will be treated by a therapeutically effective amount of such cells to tissues to restore or regenerate function.
  • a "therapeutically effective amount" of cells or tissues is an amount sufficient to arrest or ameliorate the physiological effects in a subject caused by the loss, damage, malfunction, or degeneration of particular cell-types or tissue-types.
  • the therapeutically effective amount of cells or tissues used will depend on the needs of the subject, the subject's age, physiological condition and health, the desired therapeutic effect, the size of the area of tissue that is to be targeted for therapy, the site of implantation, the extent of pathology, the chosen route of delivery, and the treatment strategy.
  • These cells or tissues may be ac-ministered to the patient in a manner that permits the cells or tissue to graft to t e intended tissue site and reconstitute or regenerate the functionally deficient area.
  • neurodegenerative disorders and neuronal diseases such as Parkinson's disease, Alzheimer's disease, Huntington's disease, Lewy body dementia, pancreatic diseases such as diabetes and juvenile onset diabetes mellitus, cardiovascular and heart diseases such as cardiac infarcts, Acquired Immunodeficiency Disease Syndrome (AIDS), hematopoietic diseases such as lymphoma and leukemia, cerebellar ataxia, progressive supranuclear palsy, amyotrophic lateral sclerosis (ALS), epilepsy, multiple sclerosis, burns, stroke, ischemia, trauma to the nervous system, neurotoxic injury,and spinal cord injuries.
  • neurodegenerative disorders and neuronal diseases such as Parkinson's disease, Alzheimer's disease, Huntington's disease, Lewy body dementia, pancreatic diseases such as diabetes and juvenile onset diabetes mellitus, cardiovascular and heart diseases such as cardiac infarcts, Acquired Immunodeficiency Disease Syndrome (AIDS), hematopoietic diseases such as lymphoma and le
  • ELSCs of the present disclosure may be induced to differentiate by any appropriate method known to those of skill in the art. Many such methods are well known to those of skill in the art for differentiating ES cells or adult stem cells into specific cell types, for example neuronal precursor cells, neuronal cells, or glial cells (U.S. Serial No. 09/970,382, WO 01/88104, WO 03/000868, WO 01/68815, WO 01/83715, and U.S. Serial Nos. 10/157,288 and 10/127,740), hematopoietic cells (U.S. Patent No.
  • ELSCs can also be differentiated directly into committed precursor cells or fully differentiated cells, for example without forming ELBs as an intermediate step.
  • Preferred methods of inducing differentiation of ELSCs include the use of differentiation agents, including but not limited to progesterone, putrescine, 1- ⁇ minin, insulin, sodium selenite, transferrin, neurturin, sonic hedgehog (SHH), noggin, follistatin, retinoic acid, epidermal growth factor (EGF), any type of fibroblast growth factor, cytosine ⁇ -d-Arabino ruranoside (Ara-C), growth and differentiation factor 5 (GDF-5), members of the neurofrophin family (nerve growth factor (NGF), neurotrophin 3 (NT-3), neurotrophin 4 (NT-4), brain derived neurotropic factor (BDNF)), fr- sforming growth factor ⁇ (TGF- ⁇ ), transforming growth factor beta-1 (TGF ⁇ l), transforming growth factor beta-3 (TGF ⁇ 3), platelet-derived growth factor (PDGF), insulin-like growth factor (IGF-1), bone morphogenic proteins (BMP-2,
  • fibroblast growth factor refers to any suitable fibroblast growth factor, derived from any organism that expresses such factors, and functional fragments thereof.
  • FGFs include but are not limited to, FGF-1 (acidic fibroblast growth factor), FGF-2 (basic fibroblast growth factor), FGF- 3 (int-2), FGF-4 (hst/K-FGF), FGF-5, FGF-6, FGF-7,.FGF-8, and FGF-9.
  • Differentiation nutrient mediums may also contain additives that help sustain cultures of neural cells, for example N2 and B27 additives (Gibco).
  • Pluripotent ELSCs can be induced to differentiate in various available culture media, including but not limited to DMEM, DMEM-F-12, MCDB, Neurobasal medium, neurturin, N2, B27, and the like, or combinations thereof.
  • the presence of differentiated cells in a cell culture can be determined by any one of many methods known to those of skill in the art.
  • determination of differentiated cells can be accomplished by methods such as flow cytometry, irnmunochemistry, immunofluorescence staining, or other staining techniques, for example Von Kossa staining of osteoblasts, Alcian Blue staining of chondrocytes, or Oil Red-O staining of adipocytes, to detect the presence of cell surface markers, proteins, or other types of genetic markers.
  • identifying differentiated cells may be accomplished by detecting expression of certain genes or gene products such as RNA or proteins using RT-PCR, HPLC, and the like.
  • pluripotent ELSCs can be induced to differentiate into neurons by culturing the cells in a neurobasal medium supplemented with B-27, N2, insulin-transferrin, and selenite in the presence of retinoic acid, basic FGF, and Ara C for approximately 4 to 14 days.
  • pluripotent ELSCs can be induced to differentiate into hepatocytes directly or by exposing ELBs to acidic FGF, basic FGF, HGF, oncostatin, dexamethasone, insulin, transferrin-selenious acid (ITS), DMSO, 5-azacytidine, sodium butyrate, or combinations thereof.
  • pluripotent ELSCs can be differentiated into cardiomyocytes directly or by exposing ELBs to TGF- ⁇ l, IGF-I, IGF- II, BMP-4, basic FGF, FGF-4, PDGF-BB, 5-aza-deoxycytidine, insulin, EGF, or combinations thereof.
  • pluripotent ELSCs can be differentiated into beta-islet cells either directly or by exposing ELBs to N 2 , B 27 , nicotinamide, basic FGF, TGF- ⁇ l, or combinations thereof.
  • pluripotent ELSCs can be differentiated into chondrocytes either directly or by exposing ELBs to TGF ⁇ 3, ascorbic acid 2 phosphate, or combinations thereof.
  • pluripotent ELSCs can be differentiated into osteoblasts directly or by exposing ELBs to dexamethasone, ⁇ -glycerophosphate, ascorbic acid 2 phosphate, hydrocortisone, or combinations thereof.
  • pluripotent ELSCs can be differentiated into adipocytes directly or by exposing ELBs to dexamethasone, isobutylme ylx-mthine (IBMX), indomethacin, insulin, or combinations thereof.
  • pluripotent ELSCs can be differentiated into myocytes directly or by exposing ELBs to 5-Azacytidine, PDGF-BB, or combination thereof.
  • the present disclosure also provides a method of cryopreservation of pluripotent ELSCs, for example, wherein the cells are cryopreserved in 10% dimethyl sulfoxide (DMSO) or another appropriate medium and stored in liquid nitrogen.
  • DMSO dimethyl sulfoxide
  • the present disclosure also contemplates the use of pluripotent ELSCs for cell-based therapies.
  • Pluripotent ELSCs disclosed herein may be induced to terminally differentiate into appropriate cell or tissue types, or to differentiate into appropriate lineage-committed progenitor cells, which can then be administered or transplanted into a mammalian subject for cell replacement therapy or tissue regeneration.
  • ELSCs may be directly administered to a subject. Therefore, the methods of the present disclosure may be useful in the treatment of many diseases, injuries, or other detrimental condition.
  • Pluripotent ELSCs of the present disclosure can be induced to differentiate either in vitro or in vivo.
  • ELSCs generated according to the present disclosure can also be used to study the cellular and molecular biology of development, functional genomics, as well as the generation of differentiated cells for use in therapeutic or prophylactic transplantation, treatment, drug screening, or in vitro drug discovery.
  • the ELSCs can be used for genomic analysis, to produce mRNA, cDNA, or genomic libraries, to produce specific polyclonal or monoclonal antibodies, including but not limited to humanized monoclonal antibodies (WO 01/51616, specifically incorporated herein by reference), or to screen for the effects of different test compounds or biologically active molecules on ELSCs and cells or tissues derived therefrom, such as pharmaceutical compounds in drug research.
  • test compounds or biologically active molecules screened may be derived for example from plants, plant-based extracts, or synthetic sources.
  • ELSCs can also be used to screen for factors (such as small molecule drugs, peptides, polynucleotides, and the like) or conditions (such as cell culture conditions or manipulations) that affect the characteristics of ELSCs in culture, and the differentiation of ELSCs into various specific cell and tissue types.
  • Differentiated cells derived from ELSCs for example neuronal cells, beta-islets, cardiomyocyte, hepatocyte, corneal cells, osteoblasts, chondrocytes, and adipocytes, can be used to generate human body organs by 3-D reconstruction, for example tissues in the human brain may be reconstructed by 3-D culturing of the neurons derive from human ELSCs.
  • other human body organs or parts such as liver, heart, kidney, skin, eye, ear, and the like may be derived and reconstructed from pluripotent ELSCs.
  • ELSCs of the present disclosure may also be used as carrier vehicles for various therapetically active molecules or genes to be delivered at various sites of the human body, for example by genetically manipulating and differentiating the ELSCs as required, and delivering the cells or tissue to a target site in a donor for gene therapy.
  • the present disclosure therefore provides methods of using pluripotent ELSCs with their unique capability to differentiate into cells of all three germ layer lineages for pharmaceutical interventions and for human-based cell assays for drug discovery, analysis, and testing. * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * *
  • the transport medium consisted of Dulbecco's Modified Eagles Medium (DMEM) and Ham's F-12 Medium (DMEM.F-12; 1:1) supplemented with 5% fetal bovine serum (FBS) or 5% human serum collected from cord blood, 0.5% dimethyl sulphoxide (DMSO), 2 ng/ml recombinant human epidermal growth factor (rhEGF), 5 ⁇ gml insulin, 5 ⁇ g/ml fr-msferrrn, 5 ⁇ g/ml sodium selenite, 0.5 ⁇ g/ml hydrocortisone, 0.1 nmol/1 cholera toxin A, 50 ⁇ g/ml gentamycin, and 1.25 ⁇ g/ml amphotericin B.
  • DMEM Dulbecco's Modified Eagles Medium
  • FBS fetal bovine serum
  • DMSO dimethyl sulphoxide
  • rhEGF recombinant human epidermal growth factor
  • HBV Hepatitis B virus
  • HCV Hepatitis C virus
  • CMN CMN
  • suitable extracellular matrix carriers such as MatrigelTM, fibrinogen, PDGF, laminin, EGF, collagen N, or human amniotic membrane were used to culture limbal tissue biopsies.
  • the extracellular matrix carrier was treated with attachment factors such as laminin, collagen N, PDGF, EGF, or fibrinogen, either singly or in combination, along with growth factors such as EGF, insulin-like growth factor- 1 (IGF-1), insulin, either singly or in combination.
  • MatrigelTM (BD Biosciences) is the preferred extracellular matrix carrier.
  • the plates were allowed to set for 1-2 hours at room temperature or overnight at 4°C. After setting, any remaining MatrigelTM solution was removed from the plate prior to use, and the plates were washed with knockout DMEM prior to use.
  • Amniotic membrane cultures were used to culture limbal tissue biopsies isolated from human subjects. The preparation of these amniotic membrane culture began with the collection of human placental membranes. Placental membranes were collected from elective Cesarean section operations and transported to laboratory facilities in a transport medium consisting of Dulbecco's phosphate buffered saline (DPBS) supplemented with 50 unit/ml penicillin, 50 ⁇ g/ml streptomycin, 50 ⁇ g/ml neomycin, and 2.5 ⁇ g/ml amphotericin B. Placental membrane was transported to the laboratory within 3 hours of surgery. Blood samples were also collected from each donor and sent for infectious disease diagnostic tests as described above.
  • DPBS Dulbecco's phosphate buffered saline
  • the washing medium consisted of Dulbecco's phosphate buffered saline (DPBS) supplemented with 50 unit/ml penicillin, 50 ⁇ g /ml streptomycin, 100 ⁇ g/ml neomycin, and 2.5 ⁇ g/ml amphotericin B.
  • DPBS Dulbecco's phosphate buffered saline
  • Placental tissue was removed from the amniotic membrane using sterile scissors, and the amniotic membrane was washed thoroughly at least 7 times to remove substantially all blood clots.
  • the chorion was peeled off of the amniotic membrane with blunt forceps, and the epithelial side of the amniotic membrane was washed 5 times with the washing medium.
  • the amniotic membrane was then placed on a sterile nitrocellulose membrane with the epithelial side of the membrane facing up.
  • the membrane was cut into 5 cm x 5 cm area pieces and each piece was placed in a cryo-vial filled with freezing medium consisting of 50% glycerol in DMEM.
  • Each batch of processed amniotic membrane was checked for sterility, as well as the absence of mycoplasma or endotoxin contamination before being used for limbal culture.
  • the pieces of amniotic membrane were each stored at -80°C.
  • Amniotic membrane cultures for culturing limbal tissue biopsies were prepared from these pieces of amniotic membrane by first thawing the pieces at room temperature for 20 minutes. Each amniotic membrane was then carefully removed from the nitrocellulose membrane using blunt forceps, preferably without tearing the surface, and placed on a sterile glass slide in a 100 mm petri plate. Next, a small volume of trypsin (1.0-1.5 ml of 0.05% Trypsin-EDTA) was added to cover the amniotic membrane, and the membrane was incubated at 37°C for 30 minutes. After incubation, the epithelial layer of the amniotic membrane was scraped off with a cell scraper under sterile aseptic conditions.
  • the -unniotic membrane was then washed 3 times with washing solution.
  • the processed and treated amniotic membrane which functions as an extracellular carrier matrix in culture, was placed on a culture plate with a 0.4 ⁇ M track-etched polyethylene terephthalate (PET) membrane insert (Falcon, USA, 3090).
  • PET polyethylene terephthalate
  • the amniotic membrane was fastened to the PET insert, for example by using number 10 Ethilon non-absorbent suture or by using a medical grade silicon O-ring.
  • the amniotic membrane should be spread on the membrane insert in such a way that the denuded epithelial side of the membrane faces the inner side of the insert and the stromal side of the membrane faces out of the insert.
  • the amniotic membrane was stretched uniformly before being secured to the insert, for example by inserting the silicon O-ring into the bottom of the amniotic membrane, or suturing the amniotic membrane to the basement membrane of the insert.
  • the entire setup was incubated in a 6-well dish filled with culture medium for at least 2 hours in DMEM/F12 (Gibco-BRL) media supplemented with 10% FBS (ES tested) (Hyclone), 5 ⁇ g/ml transferrin (Gibco), 0.1 ⁇ g/ml cholera toxin (Sigma), 50U/ml penicillin- streptomycin (Sigma), 5 ⁇ g/ml gentamicin (Sigma), 5 ng/ml Na-selenite (Sigma), 10 ng/ml EGF (Sigma), and 0.5% DMSO (Sigma).
  • the amniotic membrane was washed two times with culture medium and again incubated in culture medium for 30 minutes, after which the amniotic membrane was ready for culturing limbal tissue biopsies.
  • Limbal tissue from the limbal biopsies was initially washed several times with culture medium DMEM/F12 (Gibco-BRL) media supplemented with 10% FBS (ES tested) (Hyclone), 5 ⁇ g/ml transferrin (Gibco), 0.1 ⁇ g/ml cholera toxin (Sigma), 50U/ml penicillin-streptomycin (Sigma), 5 ⁇ g/ml gentamicin (Sigma), 5 ng/ml Na-selenite (Sigma), 10 ng/ml EGF (Sigma), and 0.5% DMSO (Sigma).
  • the biopsies were then trimmed of any sclera and conjunctiva tissues and cut into 6 to 7 pieces. These limbal tissue pieces were subsequently cultured on MatrigelTM coated plates or on amniotic membrane cultures, which were prepared as described above. Some of the limbal tissue pieces were enzymatically treated with 0.25% trypsin-EDTA (Gibco-BRL, USA) for 30 minutes, while others were treated with 0.25% trypsin-EDTA overnight at 4°C.
  • the epithelial layer of each biopsy piece was removed using blunt forceps, and the stromal were cultured cells on MatrigelTM-coated 35 mm plates in culture medium consisting of Dulbecco's Modified Eagles Medium (DMEM) and F-12 (DMEM:F-12; 1:1) supplemented with 10% knock-out serum or 10% heat-inactivated human serum collected from cord blood, 0.5% dimethyl sulphoxide (DMSO), 2 ng/ml recombinant human epidermal growth factor (rhEGF), 5 ⁇ g/ml insulin, 5 ⁇ g/ml transferrin, 5 ⁇ g/ml sodium selenite, 0.5 ⁇ g/ml hydrocortisone, 4 ng/ml bFGF, 10 ng/ml hLIF, 50 ⁇ g/ml gentamycin, and 1.25 ⁇ g/ml amphotericin B.
  • the entire culture process to generate limbal composite grafts was carried out at 37°C in air (
  • FIG. 1(a) shows Hematoxylin and Eosin (H & E) staining of a LCG (whole mount). Hematoxylin stains negatively charged nucleic acids such as nuclei and ribosomes blue, while Eosin stains proteins pink.
  • the cultured cells were subjected to magnetic affinity cell sorting (MACS) to isolate pluripotent ELSCs.
  • the cultured cells were first dispersed using 0.05% trypsin-EDTA.
  • the trypsin was neutralized by adding an equal amount of culture medium that contained a trypsin -nhibitor or fetal calf serum.
  • the cells were subsequently pipeted into a single cell suspension, and counted using a hemocytometer. Next, the cells were spun down and resuspended to a concentration of 10 7 cells per 200 ⁇ l of PBS.
  • the cells were incubated for 30 minutes at 4°C with 1 ⁇ l of primary antibody SSEA-4 (DSHB, USA; 1:40). After incubation with SSEA-4 primary antibody, the cells were washed twice with PBS to remove any unbound antibody. A 20 ⁇ l suspension of secondary antibody beads (Miltenyi Biotech, Germany; 1 :4) that bind to the SSEA-4 primary antibody was added to 200 ⁇ l of the cell suspension, mixed well, and incubated at 4°C for 20 minutes. The cells were washed three times with PBS to remove any unbound secondary antibody.
  • the cell suspension were then passed through a MACS magnetic column according to the manufacturer's instructions (Miltenyi Biotech, Germany) to isolate SSEA-4 positive cells.
  • the negative fraction was collected first, and the column was washed twice with PBS. Next, the column was removed from the magnet and the positive fraction with SSEA-4 positive cells was collected.
  • the SSEA-4 positive cells are also pluripotent ELSCs, were washed twice and seeded on an extracellular matrix carrier in culture medium.
  • the extracellular matrix carrier was MatrigelTM- coated plates and the culture medium was DMEM and F-12 (DMEM:F-12; 1:1), supplemented with 10% knock-out serum or 10% heat-inactivated human serum collected from cord blood, DMSO (0.5%), rhEGF (2 ng/ml), insulin (5 ⁇ g/ml), transferrin (5 ⁇ g/ml), sodium selenite (5 ⁇ g/ml), gentamycin (50 ⁇ g/ml) and amphotericin B (1.25 ⁇ g/ml), hLIF (lO ⁇ g/ml), and bFGF (4 ⁇ g/ml).
  • the ELSCs were cultured for an additional week at 37°C in a CO 2 incubator or until the cultures became confluent.
  • the ELSCs in culture were dissociated and re-plated on fresh bio-coated tissue culture dishes at a plating dilution of 1:3.
  • the ELSCs were then expanded and serially passaged for at least 100 population doublings. ELSCs that were serially passaged could also be frozen for further use without any loss of differential potential. Telomerase activity was still detected in the cultured cells after 50 passages.
  • Example 2
  • pluripotent ELSCs were derived from limbal tissue biopsies. Although not wishing to be limited to any particular theory, it appears that corneal limbus has essentially two stem-cell types that are segregated into two zones. The top layer of the limbus is composed mainly of corneal epithelial stem cells that are P-63 positive, while the basal layer is composed mainly of stromal cells. It appears that the pluripotent ELSCs disclosed herein, predominantly reside in the stromal layer, and may migrate towards the epithelial zone as needed.
  • ELSCs pluripotent ELSCs derived from limbal tissue, and the undifferentiated status of these cells. ELSCs were analyzed using flow cytometry, immunofluorescence, and molecular analysis for the presence or absence of various cellular markers for undifferentiated and differentiated cells. Karyotype and telomerase activity were also analyzed at various passages to determine whether these cells maintain an undifferentiated state after serial passages.
  • Pluripotent ELSCs isolated in Example 1 were trypsinized after expansion using 0.25% trypsin-EDTA for 2-3 minutes. After inactivation of the trypsin, the cells were passed through a 40 micron filter mesh to remove any remaining cellular clumps that could interfere with staining. The cells were then centrifuged and resuspended in wash buffer at a concentration of lxl 0 6 cells/ml.
  • the wash buffer consisted of phosphate buffer supplemented with 1% fetal bovine serum.
  • Results of immunophenotyping pluripotent ELSCs cultured for 20 passaged are shown in Figure 2.
  • the ELSCs were labeled with FITC-coupled antibodies against SSEA-4, CD105, CD73, CD54, CD45, CD34, CD123, CD133, CD 123 and HLA-DR.
  • the ELSCs were analyzed using FACS-calibur.
  • the results in Figure 2 are consistent with the results shown in Table 1.
  • ELSCs are positive for SSEA-4, CD54, CD73, and CD105 markers, and negative for CD34, CD45, CD106, CD 123, CD 133, and HLA-DR markers.
  • Pluripotent ELSCs were found to have similar characteristics to previously isolated primate ES cells derived from the inner cell mass of a blastocyst (U.S. Patent No. 6,200,806), namely the ELSCs were positive for the stage-specific embryonic antigen markers SSEA-3 and SSEA-4, and negative for the SSEA-1 marker.
  • SSEA-3 and SSEA-4 and the lack of expression of SSEA-1, in limbal-tissue-derived pluripotent ELSCs is similar to that of human ES cells, indicating that these pluripotent cells may have similar properties.
  • Pluripotent ELSCs were also analyzed for cluster differentiation markers, and were found to be negative for CDllc, CD14, CD34, CD 45, CD106, CD123, CD133, and HLA-DR markers.
  • This data demonstrates that ELSCs isolated from limbus tissue are not hematopoietic in origin since they are negative for the CD34 and CD45 markers.
  • the remaining CD markers analyzed, CDllc, CD14, CD106, CD123, CD133 and HLA-DR markers are only known to be expressed in differentiated cells. Therefore, the absence of these markers on the ELSCs demonstrates that these cells are undifferentiated and are not generating cells of differentiated lineages while passaged in culture under the indicated conditions.
  • the expression pattern shown in Figure 2 also suggests that ELSCs can be maintained in an undifferentiated state, (i.e., maintain "sternness") for at least 20 passages, and that the ELSCs are not hematopoetic in origin.
  • ELSCs are mesenchyrnal in origin. Therefore, it is hypothesized that ELSCs are derived from the lower stromal cell layer of limbus tissue that contains mesenchyrnal and fibroblastic cells rather than the upper epithelial cell layer. ELSCs, however, also expressed the CD54 marker, which is known to be an endothelial cell marker. To further understand this finding, the cells were analyzed by RT-PCR for expression of another endothelial marker, the PECAM gene (see Figure 11).
  • Pluripotent ELSCs were further assayed for their pluripotency and undifferentiated status through the use of the cellular markers OCT-4, TRA-1-60, TRA-1-80, and Alkaline phosphatase.
  • cultured ELSCs were collected at day 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, and 18 and subjected to gene expression analysis. Strong expression of each of these undifferentiated markers was observed in pluripotent ELSCs. The results of this gene profiling again demonstrate that the pluripotent ELSCs of the present disclosure are undifferentiated stem cells.
  • RNA of pluripotent ELSCs was isolated at every passage, for example passage 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20, using the TRIzol method (Gibco-BRL).
  • 1 ⁇ g of total RNA treated with RNase- OUT ribonuclease inhibitor was used for cDNA synthesis by reverse-transcription using reverse transcriptase (Invitrogen Inc, USA) and oligo dT (Invitrogen Inc, USA) to prime the reaction.
  • PCR polymerase chain reaction
  • the PCR amplification conditions used in the thermal cycler (ABI Biosystems 9700) to amplify the PCR products were as follows: (1) 94°C, 1 minute; (2) 94°C, 30 seconds; annealing Tm °C, 45 seconds; 72°C, 1 minute, for 30 cycles of amplification; (3) 72°C, 5 minutes; and (4) final hold at 4°C until the samples were analyzed.
  • Table 2 The PCR amplification conditions used in the thermal cycler (ABI Biosystems 9700) to amplify the PCR products were as follows: (1) 94°C, 1 minute; (2) 94°C, 30 seconds; annealing Tm °C, 45 seconds; 72°C, 1 minute, for 30 cycles of amplification; (3) 72°C, 5 minutes; and (4) final hold at 4°C until the samples were analyzed. Table 2
  • telomerase activity of the presently disclosed ELSCs was evaluated over time.
  • ELSCs extracts from passage 5 (P5), 10 (PI 0), 15 (PI 5), and 20 (p20) were prepared, and protein concentration in each extract was estimated.
  • the extracts were next evaluated using the telomeric repeat amplification protocol (TRAP), which is an assay designed for highly sensitive qualitative detection of telomerase activity. Telomerase activity was then detected by photometric enzyme immunoassay. Briefly, after estimation of protein concentration, samples were placed in a thermocycler and PCR was performed per the protocol.
  • TRAP telomeric repeat amplification protocol
  • telomerase PCR ELISA was done according to manufacturer's protocol (Roche Molecular Biochemicals), and all proper positive and negative controls provided in the kit were used. The telomerase PCR ELISA allows highly specific amplification of telomerase-mediated elongation products combined with non-radioactive detection following the ELISA protocol. Care was taken during the protocol to remove inhibitors of Taq polymerase, which could result in a false negative result. In the first step of the protocol, telomerase in the extract adds telomeric repeats (TTAGGG) to the 3' end of biotin-labeled synthetic primer.
  • TTAGGG telomeric repeats
  • these elongation products are amplified by PCR using primers that generate PCR products that contain the telomerase-specific six nucleotide increments.
  • An aliquot of the PCR products are denatured and hybridized to a digoxigenin-(DIG)-labeled, telomeric repeat-specific detection probe.
  • the resulting product is immobilized via the biotin labeled primer to a strepatavidin-coated microtiter plate.
  • telomerase activity is detected by formation of a colored product. High expression of telomerase activity was seen in the extracts of all passages tested, indicating the high proliferative capacity of pluripotent ELSCs.
  • ELSCs were karyotyped using a standard G-banding technique (Genetics Lab., Reliance Life Sciences Pvt. Ltd., Mumbai) and compared to published human karyotypes. The karyotyping was performed using the CYTONISION software. ELSCs from passage 13 (PI 3) were karyotyped and found to have 'normal karyotype', that is, the cells at P13 were found to be euploid, all human chromosomes were present, and the chromosomes were not noticeably altered (Figure 4). ELSCs were also found to have normal karyotypes at passage 20.
  • PI 3 a standard G-banding technique
  • ELSCs were dissociated by briefly exposing them to a 0.05% trypsin-EDTA solution, and subsequently cultured as a suspension culture in ES cell medium containing DMEM:F-12 or knockout DMEM, supplemented with 10-20% fetal calf serum, cord blood serum, or knockout serum replacement.
  • the media was also supplemented with ⁇ -mercaptoethanol, L-glutamine, insulin, human transferrin, sodium selenite, but did not contain bFGF or hLIF.
  • the cells were incubated in suspension culture for about 4 days and the media was changed every other day.
  • the medium was changed by transferring the suspension of aggregates to a centrifuge tube, allowing the aggregates to settle down, aspirating the old medium, replacing it with fresh medium, and returning the aggregates and fresh medium to the culture dish.
  • FIG. 5 shows this differentiation process using phase contrast micrographic pictures (10X).
  • Figure 5(a) shows ELSCs grown in culture after 15 passages.
  • Figure 5(b) shows ELBS, which formed after culturing ELSCs for 4 days in suspension culture.
  • Figure 5(c) shows initiation of differentiation from the ELBs.
  • Example 2 To further characterize the process of differentiation from ELSCs to ELBs, the molecular analysis protocol described above in Example 2 was repeated on undifferentiated ELSCs (UD), as well as ELBs after 2 days (EB2), 4 days (EB4), 8 days (EB8), 12 days (EB12), and 14 days (EB14) of differentiation in suspension culture.
  • the cells were analyzed by RT-PCR for expression of the following pluripotent stem cell marker genes: Oct-4, Nanog, Rexl, and TDGFl, the expression of which are down regulated upon differentiation.
  • the primers used to amply Oct-4, Nanog, Rexl, and TDGFl cDNAs are set forth above in Table 2.
  • expression of the "housekeeping" gene GAPDH was also analyzed as a positive control. Expression of each of these markers was also analyzed in hEF, which functioned as a negative control. The identity of the RT-PCR products was confirmed by sequencing.
  • NFH Neurofilament Heavy Chain
  • Keratin ectodermal lineage markers
  • cardiac-Actin c-Actin
  • Alpha-Fetoprotein AFP
  • Albumin endodermal lineage markers
  • PECAM endothelial lineage marker
  • Keratinocyte Growth Factor KGF
  • Collagen I stromal cell markers
  • p63 corneal epithelial stem cell marker
  • Figures 7 and 8 shows the results of the gene expression pattern of different gene markers in the ELSCs (UD) and in ELBs collected after 2, 4, 8, 12, and 14 days of culture.
  • the positive controls used for each RT-PCR were fetal-brain tissue extract as a neuroectodermal lineage control (for NFH and Keratin), fetal-heart tissue extract as a mesodermal and endothelial lineage control (for c-Actin and PECAM), fetal-liver tissue extract as a endodermal lineage control (for AFP and Albumin), hEF cells as a stromal cell control (for Collagen I and KGF), and limbal tissue extract as a corneal epidermal stem cell control (for p63), while the negative control was (-)RT product.
  • the same data as shown in Figures 7 and 8 are reproduced in a tabulated manner in Table 6.
  • Table 6 The same data as shown in Figures 7 and 8 are reproduced in a tabulated manner in Table 6.
  • Keratin is an early ectodermal lineage marker, while expression of KGF and Collagen-I at nearly all stages is expected because they are both markers for fibroblastic cells, and ELSCs are isolated from the stromal layer. It is unclear why expression of Collagen-I appears to be upregulated at day 8 and 12 of ELB formation, but is not present at day 14 of ELB formation.
  • P-63 expression was found in the population of undifferentiated ELSCs, indicating that these cells also contain a population of corneal limbal stem cells. P-63 expression decreased as the ELSCs differentiate during ELB formation.
  • the lineage markers NFH, c-Actin, AFP, and Albumin do not appear to be expressed in the ELSCs, but are expressed at various stages of differentiation in the ELBs.
  • NFH which is an early neuro-ectodermal marker, is not expressed in undifferentiated ELSCs, but is expressed at day 4 and 8 of ELB formation. NFH was not expressed in later stages of ELB formation, however, because expression of this marker is downregulated upon maturation of neuronal cells.
  • c-Actin which is a mesodermal lineage marker, is highly expressed by day 12 of ELB formation.
  • AFP which is an early mesendodermal marker
  • AFP is highly expressed at day 2 of ELB formation, but is gradually downregulated as the ELBs differentiate.
  • Albumin which is a mature hepatic cell marker, is gradually upregulated as ELBs differentiate.
  • PECAM is expected to be expressed at all stages of embryonic development, and is closely correlated with the pluripotency of reported cells (Furusawa et al, (2004) Biol. Reprod. 70:1452-57). The differential expression of appropriate lineage markers in ELBs at various stages of differentiation indicates the ability of ELSCs to differentiate into cells of all three lineages.
  • ELBs derived from ELSCs were cultured for approximately 4-10 days in an appropriate medium and plated directly onto a suitable substrate with an extracellular matrix component such as polyornithine, laminin, or fibronectin.
  • the ELBs were cultured in a suitable nutrient medium adapted to promote differentiation of the cells into neuroprogenitor cells. Then the cells were further cultured under conditions that encouraged differentiation and maturation into specific neuronal phenotypes, including GABAergic and dopaminergic neurons.
  • ELBs were cultured in serum-free neuronal induction medium composed of basal medium DMEM:F12 along with additives that help sustain cultures of neural cells, for example N2 (1-15%) and B27 (1-20%).
  • the medium was also supplemented with one or more growth factors selected from Retinoic acid (20-80 ng/ml), GDNF (1-10 ⁇ g/ml), Ara-C (10-50 ng/ml), and Neurotrophin-3 (5-20 ⁇ g/ml).
  • the cells were grown for 6 days, which resulted in the differentiation of neuroprogenitor cells.
  • the neuroprogenitor cells were grown in a neural differentiation medium containing Neurobasal medium supplemented with N2 (1-15%), B27 (1-20%), and growth factors including insulin (5-20 ⁇ g/ml), Transferrin (4-10 ⁇ g/ml), FGF-8 (50-200 ng/ml), and Ara-C (10-50 ng ml) for 12 days with the media being change every second day.
  • the cells were grown in neural differentiation medium that was supplemented with other neuronal growth factors such as neutrotrophin-3 and GDNF.
  • Neurotrophin-3 (5-20 ⁇ g/ml) and GDNF (1-10 ⁇ g/ml) were added to the differentiation medium on day 6 to day 12 of differentiation.
  • the ELBs were cultured in serum-free defined medium composed of DMEM:F12 supplemented with N2 (1-15%) and B27 (1-20%), along with one or more antioxidants, such as DMSO (1-10%), Butylated hydroxyanisole (50-400 ⁇ M), and forskolin (5-20 ⁇ M), for initiation of neuronal induction.
  • one or a combination of growth factors such as Retinoic acid (20-80 ng/ml), GDNF (1-10 ⁇ g/ml), Shh (50-200 ng/ml), FGF-8 (50-200 ng/ml), or bFGF (10-50 ng/ml) were added to the medium to facilitate neuronal differentiation.
  • the cells were grown in this medium for an additional 7-10 days. Changes in cell morphology of the cultured cells were observed within 48 hours. The percentage of responsive cells increased progressively with incubation under antioxidant and serum-free conditions.
  • the neuroprogenitor cells were next grown in neuronal maturation media containing Neurobasal medium supplemented withN2 (1-15%), B27 (1-20%), GDNF (1-10 ⁇ g/ml), Retinoic acid (20-80 ng/ml), db-cAMP (10-200 ⁇ M), and IL-lb (1-5 ⁇ g/ml). Under these culture conditions, about 30-40% of the cells extended neurite processes and stained positive for ⁇ -tubulin, which evidenced their ability to form neurons.
  • the growth factors present in the neuronal induction medium contribute to the overall increase in percentage of neuronal cells, and further induce these precursor cells to adopt the dopaminergic phenotype.
  • the differentiated neuronal cell types generated according to the above protocols were evaluated both by the overall morphology of the cells, as well as the phenotypes identified by immunofluorescence. Immunofluorescence analysis was carried out at day 12 and day 25 of differentiation of GABAergic and dopaminergic neurons, respectively. First, the isolated cells were grown on 2-well chamber slides precoated with extracellular matrices, rinsed with PBS, and fixed for 10 minutes with 4% paraformaledyde at room temperature. .
  • the cells were permeabil ⁇ zed with 0.2% Triton X-100 in PBS for 5 minutes, blocked with 1% bovine serum albumin (BSAVPBS for 2 hours, and incubated with a primary antibody (antibody dilutions were made in 1% BSA/Tris-buffered saline) overnight at 4°C.
  • BSAVPBS bovine serum albumin
  • the cells were stained with the following primary antibodies: early neuronal marker ⁇ -tubulin III (1:500); late neuronal marker Microtubule associated protein 2 (MAP-2) (1:200); gamma aminobutyric acid (GAB A) (1:200); Glutamate (1:500); Nestin (1 :50); Neurofila ent (1:500); Tyrosine hydroxylase (TH) (1 :800); Serotonin (1 :500) and Oligodendrocyte (1:500). All primary antibodies were obtained from Chemicon Inc., USA. Next, the. cells were incubated with the appropriate FITC-labeled secondary antibody. After each step, the cells were washed three times with PBS.
  • the chamber slides were observed under a fluorescence microscope to evaluate the immunopositive areas.
  • This immunofluorescence analysis demonstrated that many of the differentiated cells were immunoreactive to the neuron specific markers MAP-2, ⁇ -tubulin III, and Neurofilament, as well as the phenotype specific markers TH (marker for dopaminergic neurons), GABA (marker for GABAergic neurons), Glutamate (marker for glutamatergic neurons), and Serotonin (marker for serotonergic neurons).
  • TH marker for dopaminergic neurons
  • GABA marker for GABAergic neurons
  • Glutamate marker for glutamatergic neurons
  • Serotonin marker for serotonergic neurons
  • ELSCs and differentiated neuronal cell types generated above were also analyzed for expression of ⁇ -tubulin and Tyrosine hydroxylase (TH) by RT-PCR as previously described above using the following primers: Table 7
  • FIG. 11 shows the results of RT-PCR analysis for the expression of cardiomyocyte specific markers in ELSCs and differentiated cardiomyocytes derived therefrom. As shown, both c-Actin and NCX are expressed in the differentiated cardiomyoctyes, and not in the undifferentiated ELSCs.
  • the housekeeping gene GAPDH was used as a positive control as previously described.
  • ELSC-derived dopaminergic neurons to produce dop-unine was evaluated by directly measuring the extracellular dopamine levels using Reverse Phase HPLC (RP-HPLC).
  • concentration of dopamine detected in culture supernatant was determined by comparison with a standard solution of dop -imine injected into the column immediately before and after each analysis. Approximately 5xl0 6 cells were trypsinized and pelleted by centrifugation. The cells were then sonicated in cold IN perchloric acid with antioxidants (0.2 g/l sodium metabisulphite), and centrifuged at 15,000 rpm/min for 20 minutes at 4°C.
  • the culture supernatant was immediately stabilized with 7.5% orfhophosphoric acid and sodium metabisulphite, and stored at -70°C for subsequent determination of the extracellular dopamine concentration by RP-HPLC.
  • Dopamine levels in the culture supernatant (48 hours after the last medium change) at day 25 of differentiation was approximately 70 ⁇ g/ml.
  • Differentiated neuronal cells derived from ELSCs may be utilized for various applications, such as therapeutic application, as well as in vitro and in vivo assessment and screening of various compounds such as small molecule drugs for their effects on neuronal cells.
  • the neuronal cells may be used, for example, to treat or prevent various neurological or neurodegenerative disorders or diseases including but limited to Parkinson's disease, Alzheimer's disease, Huntington's disease, Lewy body dementia, multiple sclerosis, cerebellar ataxia, progressive supranuclear palsy, spinal cord injury, amyotrophic lateral sclerosis (ALS), epilepsy, stroke, ischemia, injury or trauma to the nervous system, neurotoxic injury, and the like, in which neuronal cells, neurons, or glial cells are injured or die in the central nervous system or spinal cord.
  • Parkinson's disease Alzheimer's disease, Huntington's disease, Lewy body dementia, multiple sclerosis, cerebellar ataxia, progressive supranuclear palsy, spinal cord injury, amyotrophic lateral sclerosis (ALS), epilepsy, stroke, ischemia, injury or trauma to the nervous system, neurotoxic injury, and the like, in which neuronal cells, neurons, or glial cells are injured or die in the central nervous system
  • neuronal cells derived from pluripotent ELSCs can also used to treat neurological disorders associated with cognition and psychology including but not limited to anxiety disorders, mood disorders, obsessive-compulsive disorders (OCD), personality disorders, attention deficit disorder (ADD), attention deficit hyperactivity disorder (ADHD), and schizophrenia.
  • OCD obsessive-compulsive disorders
  • ADD attention deficit disorder
  • ADHD attention deficit hyperactivity disorder
  • ELBs derived from ELSCs were cultured for approximately 4-10 days in an appropriate medium and plated directly onto a suitable substrate with an extracellular matrix component such as polyo ⁇ iithine, laminin, or fibronectin.
  • the ELBs were cultured in a suitable nutrient medium adapted to promote differentiation of the cells into osteoblasts.
  • the ELBs were cultured in DMEM supplemented with 10-15% fetal bovine serum in the presence dexamethasone (10-100 nM), glycerophosphate, (1-10 mM), ascorbic acid 2 phosphate (0.1-0.5 mM), bone morphogenic protein 2 (BMP2) (1-10 ng'ml), and hydrocortisone (0.05-0.1 ⁇ M).
  • dexamethasone 10-100 nM
  • glycerophosphate (1-10 mM
  • ascorbic acid 2 phosphate 0.1-0.5 mM
  • bone morphogenic protein 2 (BMP2) (1-10 ng'ml)
  • hydrocortisone 0.05-0.1 ⁇ M
  • ELBs derived from ELSCs were cultured for approximately 4-10 days in an appropriate medium and plated directly onto a suitable substrate with an extracellular matrix component such as polyornit-oine, laminin, or fibronectin.
  • the ELBs were cultured in a suitable nutrient medium adapted to promote differentiation of the cells into chondrocytes.
  • the ELBs were cultured in DMEM supplemented with 10- 15% knockout serum and in the presence of TGF beta-3 (10-100 ng/ml), ascorbic acid (0.01-0.05 mM), IX ITS, and sodium pyruvate (1-5 mM).
  • the cells were cultured for approximately 21 days.
  • the cells isolated from the above differentiation protocol were analyzed to confirm the presence of chondrocytes in the culture.
  • the differentiated cells were analyzed for the presence of glycogen deposits, which are indicative of chondrocytes, by staining with Alcian Blue (Pittenger et al, (1999) Science 284:143-147).
  • Figure 10(c) shows Alcian Blue staining of sulfated proteoglycan deposits in chondrocytes derived from ELSCs after 17 days of differentiation culture.
  • ELBs derived from ELSCs were cultured for approximately 4-10 days in an appropriate medium and plated directly onto a suitable substrate with an extracellular matrix component such as polyornithine, laminin, or fibronectin.
  • the ELBs were cultured in a suitable nutrient medium adapted to promote differentiation of the cells into adipocytes.
  • the ELBs were cultured in DMEM supplemented with 10-15% knockout serum and in the presence of dexamethasone (1 ⁇ M-100 mM), isobutylmethylxanthine (IBMX) (10-50 ng/ml), insulin (10-20 ng/ml), indomethac (2-20 mM), and insulin-like growth factor (IGF) (10-100 ng ml).
  • dexamethasone (1 ⁇ M-100 mM
  • IBMX isobutylmethylxanthine
  • insulin 10-20 ng/ml
  • indomethac 2-20 mM
  • IGF insulin-like growth factor
  • ELBs derived from ELSCs were cultured for approximately 4-10 days in an appropriate medium and plated directly onto a suitable substrate with an extracellular matrix component such as polyornithine, laminin, or fibronectin.
  • the ELBs were cultured in a suitable nutrient medium adapted to promote differentiation of the cells into hepatocytes.
  • the ELBs were cultured in DMEM supplemented with 10- 15% knockout serum, EGF (10-100 ⁇ g/ml), hepatocyte growth factor (HGF) (5-50 ⁇ g/ml), bFGF (5-20 ⁇ g ml), FGF-4 (5-50 ⁇ g ml), IL-6 (10-100 ⁇ g/ml), acidic FGF (50- 100 ⁇ g ml), human oncostatin (10-50 ⁇ g ml), insulin-transferrin-selenious acid (ITS) (IX), dexamethasone (10-100 ⁇ M), sodium butyrate (1-5 mM), DMSO (0.5-1%), and 5-azacytidine (1-10 ⁇ M).
  • the growth factors were added together or at different time points to the cultured cells as early growth factors, mid-stage growth factors, or late stage growth factors. The cells were cultured for 20 days.
  • ELSC-derived hepatocytes As a continuation of extensive functional characterization of ELSC-derived hepatocytes, the potential of these hepatocytes as a prospective drug screening tool is being confirmed by evaluating the hepatocytes for glucose-6- phosphatase activity, uptake of LDL, and albumin production, as well as analyzing cytochrome p450 and Urea assays of the differentiated cells.
  • hepatocytes were prepared for immunofluorescence analysis. 21 -day old hepatocytes (oval-shaped) were first fixed with paraformaldehyde (Sigma-Aldrich) for 20 minutes. Next, the hepatocytes were rinsed once with PBS at room temperature (RT), and either stored at 4°C or directly permeabilized with 0.2% Triton X-100 for 5 minutes at RT. After aspirating the fixative, the hepatocytes were washed three times (5 minutes each) with PBS, and blocked with PBS containing 1% BSA for 1 hour at RT.
  • paraformaldehyde Sigma-Aldrich
  • the hepatocytes were incubated with a primary antibody solution diluted in IX PBS-1%BSA overnight at RT.
  • the primary antibody used was to cytokeratin 18 (CK18), 1:200 (Chemicon, Inc, USA). CK18 is expressed on hepatocyte plasma membrane surface (Wells et al, (1997) J. Biol. Chem. 272:28574-581).
  • CK18 is expressed on hepatocyte plasma membrane surface (Wells et al, (1997) J. Biol. Chem. 272:28574-581).
  • the hepatocytes were washed with IX PBS three times (10 minutes each) on a rocker, and incubated with a secondary antibody dilution cont-tining a fluorescent label FITC in IX PBS-1% BSA at RT for 1 hour on a rocker.
  • FIG. 10(e) shows that hepatocytes differentiated from ELSCs stain positive for the anti-CK18 antibody.
  • Hepatocytes differentiated from ELSCs were also analyzed for expression of AFP and Albumin by RT-PCR as described above.
  • Figure 11 shows expression of AFP and Albumin that is indicative of both early and mature hepatocyte formation.
  • ELBs derived from ELSCs were cultured for approximately 4- 10 days in an appropriate medium and plated directly onto a suitable substrate with an extracellular matrix component such as polyornithine, laminin, or fibronectin.
  • the ELBs were cultured in a suitable nutrient medium adapted to promote differentiation of the cells into pancreatic beta-islet cells.
  • the ELBs were cultured in DMEM supplemented with 10-15% knockout serum and in the presence of N2 supplement (1%), B27 supplement (2%), forskolin (10 ⁇ M), and cyclopamine (10 ⁇ M). The cells were cultured for approximately 12 days.
  • the cells isolated from the above differentiation protocol were analyzed to confirm the presence of pancreatic beta-islet cells in the culture using immunofluorescence analysis, as described above for differentiated hepatocytes.
  • the differentiated cells were analyzed using immunofluorescence for staining with an anti- PDX-1 antibody, which is indicative of beta-islet cells, tosulin-promoting factor-1 (PDX-1) is a transcription factor expressed by beta-islet cells of the pancreas.
  • Figure 10(k) shows positive immunofluorescence in beta-islet cells derived from ELSCs after staining with anti-PDX-1 antibody.
  • the beta-islet cells were also analyzed for gene expression of insulin and somatostatin using RT-PCR as previously described using the following primers: Table 8
  • Figure 11 shows expression of insulin and somatostatin by differentiated cells, which are indicative of beta-islet cells.
  • ELBs derived from ELSCs were cultured for approximately 4-10 days in an appropriate medium and plated directly onto a suitable substrate with an extracellular matrix component such as polyornithine, laminin, or fibronectin.
  • the ELBs were cultured in a suitable nutrient medium adapted to promote differentiation of the cells into cardiomyocytes.
  • the ELBs were cultured in DMEM/F-12 (1:1) supplemented with 15% knockout serum and 100 mM L-glutamine in the presence of EGF (50 ⁇ M), TGF beta-3 (10 ⁇ g/ml), bFGF (50 ⁇ g/ml), PDGF-BB (50 ⁇ g/ml), and ITS (IX).
  • EGF 50 ⁇ M
  • TGF beta-3 10 ⁇ g/ml
  • bFGF 50 ⁇ g/ml
  • PDGF-BB 50 ⁇ g/ml
  • ITS ITS
  • a well known cardiomyocyte inducing factor for human ES cells, 5- Azadeoxycitidine (5-10 ⁇ M) was not found to be useful for differentiating ELSCs into cardiomyocytes.
  • the cardiotropic factors were added together or at different time points to the differentiation media as early growth factors, mid-stage growth factors, or late stage growth factors.
  • the cells were cultured for approximately 21 days, and were carefully monitored for contracting embryoid-like bodies (i.e., beating cardiac cells), for example through a phase contrast microscope.
  • the cells isolated from the above differentiation protocol were evaluated morphologically to confirm the presence of ELSC-derived cardiomyoctyes, as shown in Figure 10(f).
  • the ELSC-derived cardiomyocytes were analyzed for expression of cardiac troponin T (cTnT), which is a marker characteristic of cardiomyocytes, using the anti-cTnT antibody (Santacruz, USA).
  • Figure 10(g) shows that these cells are recognized by anti-cTnT antibody, which is indicative of cardiomyocytes.
  • Both ELSCs and cardiomyocytes differentiated from ELSCs were analyzed for expression of c-Actin and Na-Ca exchanger (NCX) by RT-PCR as previously described above using the following primers: Table 9
  • FIG. 11 shows the results of RT-PCR analysis for the expression of cardiomyocyte specific markers in ELSCs and differentiated cardiomyocytes derived therefrom. As shown, both c-Actin and NCX are expressed in the differentiated cardiomyoctyes, and not in the undifferentiated ELSCs. Additionally, the potential of these ELSC-derived cardiomyocytes in cell therapy for cardiac diseases is being assessed by extensive functional characterization of the cardiomyocytes by electrophysiology, as well as testing using in vivo animal models.
  • ELBs derived from ELSCs were cultured for approximately 4-10 days in an appropriate medium and plated directly onto a suitable substrate with an extracellular matrix component such as polyornithine, laminin, or fibronectin.
  • the ELBs were cultured in a suitable nutrient medium adapted to promote differentiation of the cells into myocytes.
  • the ELBs were cultured in DMEM supplemented with 10-15% knockout serum and in the presence of 5-Azacytidine (5-10 ⁇ M) and PDGF-BB (10-50 ng/ml). The cells were cultured for approximately 12 days.
  • Myogenin is a member of the gene family encoding muscle-specific basic-helix-loop-helix transcription factors that is activated in myoblasts at the onset of differentiation.
  • Antibodies to Myogenin (Santacruz, USA) were used to confirm the presence of myocytes in the culture.
  • Figure 10(e) shows that these cells are recognized by anti-Myogenin antibody.
  • Both ELSCs and cells differentiated from ELSCs were also analyzed for expression of Myogenin by RT-PCR as previously described above.
  • Figure 11 shows expression of Myogenin in differentiated cells, which is indicative of myocytes.
  • ELBs derived from ELSCs were cultured for approximately 4-10 days in an appropriate medium and plated directly onto a suitable substrate with an extracellular matrix component such as polyornithine, 1-iminin, or fibronectin.
  • the ELBs were cultured in a suitable nutrient medium adapted to promote differentiation of the cells into endothelial cells.
  • the ELBs were cultured in DMEM supplemented with 10-15% knockout serum and in the presence of NEGF (20 ng/ml), bFGF (50 ng/ml), and BMP -4 (1-10 ng/ml). The cells were cultured for approximately 21 days.

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  • Developmental Biology & Embryology (AREA)
  • Neurology (AREA)
  • Rheumatology (AREA)
  • Neurosurgery (AREA)
  • Gynecology & Obstetrics (AREA)
  • Reproductive Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Veterinary Medicine (AREA)
  • Medicinal Chemistry (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Orthopedic Medicine & Surgery (AREA)
  • Public Health (AREA)
  • General Chemical & Material Sciences (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Cardiology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Psychology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)
  • Materials For Medical Uses (AREA)

Abstract

La présente invention a trait à des cellules souches mammaliennes de type embryonnaire multipotentes isolées du tissu limbique, un tissu non embryonnaire. Les cellules souches de type embryonnaire multipotentes de la présente invention sont capables de prolifération dans une culture in vitro, de maintien du potentiel de différenciation en des cellules de lignage d'endoderme, de mésoderme, et d'ectoderme, et sont capables de formation de corps de type embryoïde lorsqu'elles sont placées dans une culture en suspension. Ainsi. les cellules possèdent une différenciation à multiples lignages et une capacité d'autorenouvellement. Les cellules souches de type embryonnaire multipotentes peuvent constituer un outil thérapeutique prometteur et peuvent fournir des alternatives thérapeutiques pour diverses maladies, conditions, et lésions.
PCT/IB2005/000516 2004-02-26 2005-02-23 Cellules souches de type embryonnaire multipotentes derivee de limbe, leurs procedes d'isolation et d'utilisation WO2005084119A2 (fr)

Priority Applications (7)

Application Number Priority Date Filing Date Title
AU2005220066A AU2005220066B2 (en) 2004-02-26 2005-02-23 Pluripotent embryonic-like stem cells derived from corneal limbus, methods of isolation and uses thereof
KR1020097002160A KR100931887B1 (ko) 2004-02-26 2005-02-23 각막 윤부에서 유도된 다능성 배아 유사 줄기 세포, 분리 방법 및 이들의 용도
BRC10506536-4A BRPI0506536C1 (pt) 2004-02-26 2005-02-23 neurÈnios dopaminérgicos derivados do limbus corneal, métodos de isolamento e usos dos mesmos
EP05708629A EP1758984A2 (fr) 2004-02-26 2005-02-23 Cellules souches de type embryonnaire multipotentes derivees de limbe, leur procedes d'isolation et d'utilisation
US11/502,338 US8187875B2 (en) 2004-02-26 2006-08-10 Dopaminergic neurons derived from corneal limbus, methods of isolation and uses thereof
IL177672A IL177672A (en) 2004-02-26 2006-08-23 Dopaminergic neurons derived from corneal limbus, methods of isolation and uses thereof
IL177671A IL177671A0 (en) 2004-02-26 2006-08-23 Pluripotent embryonic-like stem cells derived from corneal limbus, methods of isolation and uses thereof

Applications Claiming Priority (4)

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IN240MU2004 2004-02-26
IN240/MUM/2004 2004-02-26
US62147604P 2004-10-22 2004-10-22
US60/621,476 2004-10-22

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WO2005084119A3 WO2005084119A3 (fr) 2010-06-17

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EP (1) EP1758984A2 (fr)
KR (3) KR20080036636A (fr)
AU (1) AU2005220066B2 (fr)
BR (1) BRPI0506536C1 (fr)
IL (2) IL177671A0 (fr)
WO (1) WO2005084119A2 (fr)

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* Cited by examiner, † Cited by third party
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EP1733026A2 (fr) * 2004-01-27 2006-12-20 Reliance Life Sciences Pvt. Ltd. Systeme tissulaire comprenant des cellules souches non differenciees derivees du limbe corneen
WO2008026226A2 (fr) * 2006-09-01 2008-03-06 Stempeutics Research Private Limited Cellules souches pluripotentes adultes maîtresses à autorenouvellement
JP2013520164A (ja) * 2010-02-22 2013-06-06 ユニベルシテ ピエール エ マリー キュリー（パリ シズエム） 造血系列の細胞を増殖及び分化させる細胞培養培地
WO2018094209A1 (fr) * 2016-11-18 2018-05-24 Rita Perlingeiro Marqueurs de surface pour l'isolement de cellules souches/progénitrices myogènes

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KR101323650B1 (ko) * 2011-05-04 2013-11-05 연세대학교 산학협력단 미분화 전능성 줄기세포의 제거방법
KR102038503B1 (ko) * 2015-08-12 2019-11-26 (주) 넥셀 고기능성 간세포의 분화방법
KR101645901B1 (ko) * 2015-12-31 2016-08-04 가톨릭대학교 산학협력단 양막 슬라이드 지지체를 이용한 윤부줄기세포 배양방법
KR101789917B1 (ko) 2016-03-29 2017-10-26 가톨릭대학교 산학협력단 윤부줄기세포의 증식 및 줄기세포능을 증대시키는 방법
CN107345897A (zh) * 2016-05-04 2017-11-14 上海米洋杨生物科技有限公司 一种视网膜感光细胞特异性表面蛋白的筛选方法

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1733026A2 (fr) * 2004-01-27 2006-12-20 Reliance Life Sciences Pvt. Ltd. Systeme tissulaire comprenant des cellules souches non differenciees derivees du limbe corneen
EP1733026A4 (fr) * 2004-01-27 2009-06-03 Reliance Life Sciences Pvt Ltd Systeme tissulaire comprenant des cellules souches non differenciees derivees du limbe corneen
WO2008026226A2 (fr) * 2006-09-01 2008-03-06 Stempeutics Research Private Limited Cellules souches pluripotentes adultes maîtresses à autorenouvellement
WO2008026226A3 (fr) * 2006-09-01 2008-04-24 Stempeutics Res Private Ltd Cellules souches pluripotentes adultes maîtresses à autorenouvellement
JP2013520164A (ja) * 2010-02-22 2013-06-06 ユニベルシテ ピエール エ マリー キュリー（パリ シズエム） 造血系列の細胞を増殖及び分化させる細胞培養培地
JP2016171811A (ja) * 2010-02-22 2016-09-29 ユニベルシテ ピエール エ マリー キュリー（パリ シズエム） 造血系列の細胞を増殖及び分化させる細胞培養培地
WO2018094209A1 (fr) * 2016-11-18 2018-05-24 Rita Perlingeiro Marqueurs de surface pour l'isolement de cellules souches/progénitrices myogènes

Also Published As

Publication number Publication date
EP1758984A2 (fr) 2007-03-07
AU2005220066B2 (en) 2010-01-21
KR100840979B1 (ko) 2008-06-24
KR100931887B1 (ko) 2009-12-15
KR20080036636A (ko) 2008-04-28
BRPI0506536A (pt) 2007-05-15
WO2005084119A3 (fr) 2010-06-17
AU2005220066A1 (en) 2005-09-15
BRPI0506536C1 (pt) 2007-05-22
KR20060114372A (ko) 2006-11-06
KR20090021229A (ko) 2009-02-27
IL177671A0 (en) 2008-03-20
IL177672A (en) 2011-03-31
IL177672A0 (en) 2008-03-20

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