WO2008002329A2 - Isolement et extension de cellules animales dans des cultures cellulaires - Google Patents

Isolement et extension de cellules animales dans des cultures cellulaires Download PDF

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WO2008002329A2
WO2008002329A2 PCT/US2006/049500 US2006049500W WO2008002329A2 WO 2008002329 A2 WO2008002329 A2 WO 2008002329A2 US 2006049500 W US2006049500 W US 2006049500W WO 2008002329 A2 WO2008002329 A2 WO 2008002329A2
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Prior art keywords
cells
epithelial
cell
limbal
stem
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PCT/US2006/049500
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English (en)
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WO2008002329A3 (fr
Inventor
Scheffer Tseng
Hua He
Wei Li
Ying-Ting Chen
Yasutaka Hayashida
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Tissuetech, Inc.
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Priority claimed from US11/476,376 external-priority patent/US20070010008A1/en
Application filed by Tissuetech, Inc. filed Critical Tissuetech, Inc.
Publication of WO2008002329A2 publication Critical patent/WO2008002329A2/fr
Publication of WO2008002329A3 publication Critical patent/WO2008002329A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0618—Cells of the nervous system
    • C12N5/0621—Eye cells, e.g. cornea, iris pigmented cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0603—Embryonic cells ; Embryoid bodies
    • C12N5/0605—Cells from extra-embryonic tissues, e.g. placenta, amnion, yolk sac, Wharton's jelly
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/90—Serum-free medium, which may still contain naturally-sourced components

Definitions

  • the embodiments described herein relate generally to the fields of developmental biology, cell culture, and tissue culture.
  • tissue can then be utilized for therapeutic uses.
  • cellular or cell-based therapy is the replacement of unhealthy, damaged, or diseased cells or tissues with new cells or tissues.
  • Blood transfusions and bone marrow transplantation are prime examples of the successful application of cell-based therapeutics, but recent advances in cellular and molecular biology have expanded the potential applications of this approach to a wide variety of clinical disorders. The realization of these applications, however, depends on obtaining or culturing the cell type of interest in sufficient numbers for transplantation into the damaged or diseased tissue or organ. Theoretically, cells of interest can be explanted from an animal or human subject and introduced into a primary cell culture system for expansion.
  • tissue engineering stem cells and stem-cell-like cells including methods for purifying/isolating stem cells and stem-cell-like cells, and methods for expanding stem cells and stem- cell-like cells. Also described herein are methods for using isolating/purified and/or expanded stem cells and stem- cell-like cells in therapeutic, cell culture, and tissue culture applications.
  • methods for preferentially purifying stem cells and/or stem-cell-like cells from a population of cells including, by way of example only, a sheet of limbal epithelial cells.
  • Such isolation/purification methods can be combined with any of the other tissue engineering methods described herein.
  • the method comprises differential dispase digestion.
  • the differential dispase digestion uses an enzymatic solution such as Dispase II (also known as Dispase 2) solution.
  • Dispase II also known as Dispase 2
  • purified cells can be expanded using the aforementioned method (or indeed any method for expanding described herein).
  • the Dispase II solution comprises SHEM, a polyhydroxy alcohol, a sugar, or any combination thereof.
  • a culture solution containing polyhydroxy alcohol can include sorbitol, mannitol, or galactitol.
  • the temperature for enzymatic (e.g., dispase) digestion is (a) about ambient temperature, (b) above about ambient temperature, (c) about 20 0 C; (d) about 22 0 C; (e) about 25 0 C; (f) about 27 ⁇ C; (g) about 30 0 C; (h) about 32 0 C; (i) about 35 0 C; (j) about 37 0 C; or (k) about 39 0 C.
  • the duration of enzymatic (e.g., dispase) digestion is shorter for higher temperatures and longer for lower temperatures.
  • the differential dispase digestion cells are expanded while preventing differentiation using any of the methods described herein.
  • the stem cells or stem-cell-like cells are purified by methods comprising rapid adhesion.
  • methods for purifying stem cells or stem-cell-like cells from a population of cells comprising the steps of contacting a population of cells comprising stem cells and stem- cell-like cells with a surface comprising collagen; incubating the population of cells; and removing non-adherent cells.
  • the incubating step is less than I hour and the collagen is collagen I.
  • the "rapid-adherent cells" are expanded while preventing differentiation using any of the methods described herein.
  • the population of cells is selected from a sheet of cells, a tissue sample, or a modified tissue sample.
  • the surface is a plastic surface.
  • the collagen is collagen I.
  • the incubating step can be for a relatively quick period of time, including less than about 1 hour, less than about 30 minutes, less than about 20 minutes, less than about 15 minutes, less than about 10 minutes and even less than about 5 minutes.
  • the non-adherent cells can be removed using the methods described herein or by standard techniques for removing non-adherent cells from a surface while not killing the adherent cells.
  • the rapid adherent cells are enriched in stem cells or stem-cell-like cells and can be further expanded using the methods described herein or by other methods known in the art.
  • the non-adherent cells can be further used as described herein, and in particular, the non-adherent cells acquired from the aforementioned rapid adhesion methods can have additional slow adherent cells that exhibit relatively reduced proliferative potential (i.e., relative to rapid adherent cells).
  • Such slow-adherent cells can be purified from the non-adherent cells by providing a prolonged incubation time on a collagen coated surface, followed by removal of the non-adherent cells and expanded.
  • methods comprising differential dispase digestion and rapid adhesion and/or slow-adhesion.
  • the purified cells are expanded while preventing differentiation using any of the methods described herein.
  • One method for preventing differentiation while expanding animal cells comprises modulating TGF- ⁇ signaling in the animal cells.
  • Cells or tissues, isolated from an animal subject, are placed in an ex vivo culture system in which the conditions are such as to downregulate TGF- ⁇ signaling in the cells.
  • methods for expanding limbal epithelial progenitor cells (isolated or in tissues) while preventing cellular differentiation comprising modulating p38 MAP kinase activity (and in one embodiment, such modulation is by means of an inhibitor of p38 MAP kinase).
  • HAEC human amniotic epithelial cells
  • TGF- ⁇ signaling is modulated using various agents.
  • Types of cells that can be expanded using TGF- ⁇ modulation include, but are not limited to, differentiated cells, keratocytes, stem-cell-like cells, and stem cells (including limbal epithelial progenitor cells, umbilical cord epithelial cells, and amniotic membrane epithelial cells).
  • the culture system for expanding the animal cells comprises a culture vessel, a matrix and a medium.
  • the medium is essentially free of amniotic membrane (or "AM"; the terms AM and amniotic membrane are used interchangeably herein) and non-human mesenchymal feeder cells and the conditions are such as to downregulate TGF- ⁇ signaling in the cells.
  • AM amniotic membrane
  • non-human mesenchymal feeder cells and the conditions are such as to downregulate TGF- ⁇ signaling in the cells.
  • the media of the culture comprises low amounts of Ca 2+ and is essentially serum-free.
  • TGF- ⁇ can be downregulated by utilizing Ca 2+ concentrations less than about 0.1 mM Ca 2+ (e.g., using KSFM).
  • TGF- ⁇ can be downregulated when serum is in the media and Ca 2+ concentrations are greater than about 1.0 mM (as high as about 1.8 mM in media such as DMEM and SHEM) during or before the cultured cells are in contact with an agent that downregulates TGF- ⁇ signaling.
  • a means for preventing differentiation of an expanded cell line occurs by contacting the cells in the cell line with agents that suppress TGF- ⁇ or by removing an agent/condition that upregulates TGF- ⁇ signaling.
  • Agents that downregulate TGF- ⁇ signaling to promote cell expansion without differentiation include, but are not limited to: agents that specifically bind TGF- ⁇ ; agents that antagonize a receptor for TGF- ⁇ ; AM derivatives; purified components of AM; isolated AM; AM stromal matrices; processed AM; AM extracts (AME); components derived from AM such as hyaluronic acid (HA), HA-inter- ⁇ -trypsin-inhibitor heavy chain (HA-ITI), lumican, TSG- 6, Pentraxin (PTX3) and Thrombospondin; anti-TGF- ⁇ antibodies; and inhibitors of components in the TFG- ⁇ signaling pathway such as serine/threonine kinase inhibitors and agents that prevent Smad protein translocation
  • a vessel includes animal cells (isolated or in tissues) whose differentiation state is controllable by modulating TGF- ⁇ signaling. Further, the animal cells have been expanded by culturing the cells in a medium free of amniotic membrane (AM) under conditions which downregulate TGF- ⁇ signaling in the cells to allow the cells to proliferate without changes to their phenotype.
  • AM amniotic membrane
  • ex vivo methods are described for preferentially expanding limbal epithelial progenitor cells (isolated or in tissues) in a cell culture.
  • Limbal epithelial progenitor cells are expanded by combining such cells with transient amplifying cells (TACs) in an ex vivo culture system.
  • the culture system described further comprises a culture vessel, a matrix, and a medium.
  • the mixture of cells is then seeded in the culture system at a sufficiently low cell density (e.g., between about 10 and 1,000 cells/cm 2 ) for a time period exceeding the lifespan of the TACs (e.g., greater than about 3 weeks).
  • a sufficiently low cell density e.g., between about 10 and 1,000 cells/cm 2
  • Such a time frame prevents TACs from having a negative paracrine effect on limbal epithelial progenitor cells.
  • methods for facilitating the outgrowth of epithelial cell sheet formation from limbal explants (isolated cells or tissues), while inhibiting abnormal epidermal cell differentiation. These methods comprise culturing the cells on a matrix in a culture medium with an effective amount of a p ' 38 MAP kinase inhibitor.
  • the p38 MAP kinase inhibitor is a pyridinylimidazole compound or a 2,4,5- triarylimidazole.
  • the p38 MAP kinase inhibitor is chosen from at least one of: SB 203580, L-167307, SKF 86002, SB 220025, SB 235699, VX-745, RWJ 68354, SB 202190, SB 239063, and 2-(4-Chloro- phenyl)-4-(4-fluoro-phenyl)-5-pyridin-4-yl-l,2-dihyrdro-pyrazoI-3-one.
  • ex vivo method for expanding limbal epithelial stem cells (isolated or in tissues) obtained from the limbus from a donor eye.
  • the limbus is combined with an enzymatic solution in order to separate the limbal epithelial cells from the stroma of the limbus.
  • the limbal epithelial sheet is then put in contact with an amniotic membrane (including isolated AM or AM stromal matrices).
  • the limbal epithelial sheet-amniotic membrane composite is then cultured for a period of time and under conditions sufficient to enable the epithelial stem cells to expand.
  • an ex vivo method for expanding mesenchymal cells is disclosed.
  • the method disclosed includes contacting an amniotic membrane with at least one type of mesechymal cell in order to form a composite of the mesenchymal cell and the amniotic membrane.
  • the composite can then be cultured in a medium for a period of time and under conditions sufficient to enable the mesenchymal cells to expand.
  • the conditions allow for the expansion of the mesenchymal cells, yet the conditions enable the cells to maintain their current differentiated state without further unwanted differentiation.
  • keratocytes are the cells to be expanded using this method.
  • surgical grafts comprising expanded cells or tissues, prepared or cultured as described herein, including expanded limbal epithelial cells or tissues and mesenchymal cells or tissues.
  • methods for in vitro expansion of animal cells comprising seeding such animal cells on human amniotic epithelial cell feeder layers obtained from the amnion layer of human placenta.
  • the human amniotic epithelial cell feeder layers can be grown by culturing the cells in a serum-containing media.
  • such a media is supplemented hormonal epithelial media (SHEM).
  • SHEM hormonal epithelial media
  • this media can contain a calcium concentration of about 1.05 mM.
  • the human amniotic epithelial cell feeder layers can be made fromMMC-treated human amniotic epithelial cells.
  • the cells types that can be grown on these amniotic epithelial cell feeder layer include by way of example only stem cells, stem-cell-like cells, limbal epithelial progenitor cells, umbilical cord epithelial cells, and amniotic membrane epithelial cells.
  • Limbal epithelial progenitor cells can be obtained from the corneoscleral tissue.
  • the term "cell" refers to an isolated cell, to a cell in an isolated tissue, to a cell being cultured as described herein, or to a cell in a tissue being cultured as described herein.
  • stem cell means a cell that retains the ability to divide and differentiate into other cell types.
  • a stem cell can be totipotent, pluripotent, or multipotent; the term “stem cell” includes progenitor cells.
  • stem-cell-like cells includes cells that are equivalent to progenitor cells and differentiated cells that can undergo further differentiation.
  • One, non-limiting example of a stem-cell-like cell are keratocytes that can further differentiate into other cell types such as fibroblasts.
  • amniotic membrane derivatives refers to agents, materials, and compositions derived from amniotic membrane, including the jelly portion of amniotic tissue.
  • the phrase includes the following: extracts of amniotic membrane and purified components of amniotic membranes; isolated AM; AM stromal matrices; processed AM; AM extracts (AME); and components derived from AM such as hyaluronic acid (HA), HA-inter- ⁇ -trypsin-inhibitor heavy chain (HA-ITI), lumican, TSG-6, Pentraxin (PTX3) and Thromb ospondin.
  • HA hyaluronic acid
  • HA-ITI HA-inter- ⁇ -trypsin-inhibitor heavy chain
  • lumican TSG-6
  • PTX3 Pentraxin
  • differentiated cell is meant a cell that is more differentiated than the stem cell from which it originated.
  • An example of a differentiated cell is a keratocyte, which expresses cellular markers not expressed by the stem cells from which the keratocytes originated.
  • a cell culture system essentially free of a substance (e.g., intact AM or feeder cells) is meant that that substance is not present in a sufficient amount to exert a detectable effect on the cells in the culture system (e.g., to cause or prevent a phenotypic change in the cells).
  • a substance e.g., intact AM or feeder cells
  • an "antibody” is an intact immunoglobulin or an antigen-binding fragment or derivative thereof.
  • purified means separated from components that naturally accompany such molecules, cells, tissues or samples. Typically, a molecule is purified when it is at least 30% (e.g., 40%, 50%, 60%, 70%, 80%, 90%, and 100%), by weight, free from the proteins or other naturally-occurring organic molecules with which it is naturally associated. Purity can be measured by any appropriate method, e.g., column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis. An isolated cell or tissue is a cell or tissue that has been removed from an animal using methods described herein or known in the art.
  • FIG. 1 is a non-limiting example of a microphotograph at 40Ox magnification of an immunohistochemistry stained explant cultured on dAM in a SHEM medium with addition of p3S inhibitor
  • Fig. 2 is a non-limiting example of a microphotograph at 10Ox magnification (A, B, C, and D), and 40Ox magnification (E and F) of immunohistochemistry stained explants cultured in SHEM medium in the airlift manner without p38 inhibitor (Figs. 2A, 2C, and 2E) and with p38 inhibitor (Figs 2B, 2D, and 2F).
  • Figs. 3 is a non-limiting example of a microphotograph of cultured peripheral corneal epithelial cells.
  • Figs. 3A-3C are microphotogxaphs of stained cultured peripheral corneal epithelial cells showing normal differentiation in the submerge manner.
  • Fig. 3D is a microphotograph of stained cultured peripheral corneal epithelial cells using the airlift method with a p38 MAPK inhibitor.
  • Fig.4 is a non-limiting example of a microphotograph of cultured peripheral corneal epithelial cells.
  • Figs 4A-4C are microphotographs of stained cultured peripheral corneal epithelial cells showing normal differentiation in the submerge manner (Fig. 4A) and in the airlift manner with p38 (Fig.4C); and some abnormal differentiation when cultured in the airlift manner without ⁇ 38 (Fig. 4B).
  • Fig. 5 is a non-limiting example of a bar graph demonstrating the growth capacity of human amniotic epithelial cells in all experimental conditions. HAEC cells growth was most proliferative in SHEM medium containing FBS and Ca 2+ .
  • CK12 is a marker for corneal epithelium; absence of this marker for cells seeded on HAEC feeder layers demonstrates that there was less differentiation in these cells as compared to cells seeded on murine feeder layers.
  • Fig. 7 is a non-limiting example of a series of bar graphs demonstrating the percentages of ⁇ 63-,
  • p63-, Musashi-1, and ABCG2 are all stem cell markers.
  • the higher percentage of stem cell markers on cells seeded on HAECs demonstrates that cells seeded on HAEC feeder layers inhibits differentiation of stem cells to a greater extent than the same cells seeded on murine feeder layers.
  • tissue engineering stem cells and/or stem-cell-like cells comprising (a) methods for isolating/purifying stem cells and/or stem-cell-like cells, (b) methods for expanding stem cells and/or stem-cell-like cells, and (c) combinations of such methods.
  • methods for preferentially purifying a population of stem cells or stem-cell-like cells from a sample e.g., a tissue sample.
  • Such methods include differential dispase digestion, rapid adhesion methods, and combinations thereof.
  • systems and methods for expanding primary animal cells (isolated or in tissues) without undesired differentiation of their phenotype i.e., preventing undesired differentiation).
  • methods disclosed herein involve manipulating cell signaling to control the differentiation of cells (including stem cells and stem-cell-like cells) from one phenotype to another.
  • methods disclosed herein include at least one of the following steps: downregulation of TGF- ⁇ signaling to prevent the differentiation of a cell during expansion; methods for controlling differentiation by inhibiting the ⁇ 38 MAP kinase pathway; expansion of epithelial cells or mesenchymal cells using AM derivatives; controlling expansion of epithelial stem cells through the use of human amniotic epithelial cell (HAEC) feeder layers; adjusting Ca 2+ levels in the media; control of cell seeding density; or a combination of the foregoing.
  • HAEC human amniotic epithelial cell
  • One aspect described herein includes methods of expanding animal cells ex vivo whose differentiation state is controllable by modulating TGF- ⁇ signaling.
  • any cell type can be used in which TGF-/3 signaling modulation affects its differentiation state.
  • Such cells might be stem cells, stem-cell-like cells or differentiated cells.
  • stem cells include totipotent stem cells, pluripotent stem cells, and multipotent stem cells.
  • a number of adult, embryonic, arid cord blood stem cells are known, including hematopoietic stem cells, pancreatic stem cells, mesenchymal stem cells, bone marrow stromal stem cells, adipose derived adult stem cells, olfactory stem cells, gastrointestinal stem cells, mammary gland stem cells, umbilical cord epithelial cells, amniotic membrane epithelial cells, and limbal epithelial progenitor cells.
  • Differentiated cells might include epithelial cells, fibroblasts, myocytes, pancreatic ⁇ cells, blood cells, neurons, smooth muscle cells, fat cells, oligodendrocytes, alveolar cells, epidermal cells, and keratocytes.
  • a typical method of isolating keratocytes from an animal subject includes removing an anterior corneoscleral segment from the globe of the animal subject's eye by cutting near the limbus with Wescott's scissors or other appropriate cutting implement (see Kawakita et al., Invest Ophthalmol. Vis. Sci. 47:1918-1927, 2006 and Espana et al., Invest Ophthalmol. Vis. Sci. 46:4528-4535, 2005).
  • a central comea can be obtained with an 8.0 mm Hessburg-Barron trephine or other suitable trephine system and transferred to an appropriate medium (e.g., KSFM).
  • an appropriate medium e.g., KSFM.
  • the remaining corneal stroma is incubated at 37°C for a suitable amount of time (e.g., 16 h) in medium (e.g., DMEM) containing collagenase and any other appropriate components for digestion (e.g., HEPES, gentamicin, amphotericin) on a suitable culture substrate or vessel (e.g., multi-well plate, plastic dish).
  • cells are resuspended in a suitable medium (e.g., KSFM), centrifuged to remove residual matrices, resuspended again, and seeded on an appropriate culture substrate or vessel (e.g., multi-well plate, plastic dish) in a suitable medium such as KSFM or DMEM containing ITS or 10% FBS.
  • a suitable medium such as KSFM or DMEM containing ITS or 10% FBS.
  • a typical method of isolating limbal epithelial progenitor cells includes first isolating a corneoscleral ring from a cornea (as described in He et al., Invest. Ophthalmol. Vis. Sci 47:151-157, 2005; Kawakita et al., Am. J. Pathol. 167:381-393, 2005). Then, limbal corneal epithelial sheets are isolated from the corneoscleral ring by digestion with a suitable protease (e.g., 10 mg/ml Dispase II in KSFM at 37°C for 2 hours).
  • a suitable protease e.g. 10 mg/ml Dispase II in KSFM at 37°C for 2 hours.
  • the limbal corneal epithelial cells can be isolated from the corneoscleral ring by treatment with cell dissociation buffer prior to culturing in an appropriate medium (e.g., SHEM and KSFM+S with or without 3T3 cells).
  • an appropriate medium e.g., SHEM and KSFM+S with or without 3T3 cells.
  • the sheets are trypsinized and cultured on a suitable culture substrate or vessel (e.g., multi- well plate or plastic with or without 3T3 fibroblast feeder layers) in an appropriate medium (e.g., SHEM).
  • a suitable culture substrate or vessel e.g., multi- well plate or plastic with or without 3T3 fibroblast feeder layers
  • an appropriate medium e.g., SHEM.
  • the use of differential dispase digestion based on temperature and duration also provides sheets of cells that are enriched in a desired cell type. For example, dispase digestion at higher temperatures for shorter periods of time provides sheets of cells (following mechanical separation) in which superficial and suprabasal cells have been depleted while not depleting the most primitive stem cells and stem-cell-like cells.
  • Methods of expanding animal cells (isolated or in tissues) ex vivo whose differentiation state is controllable by modulating TGF- ⁇ signaling as described herein include placing the cells in an ex vivo culture system including a culture vessel, a matrix, and a medium, wherein the medium is essentially free of AM and non- human mesenchymal feeder cells.
  • an ex vivo culture system including a culture vessel, a matrix, and a medium, wherein the medium is essentially free of AM and non- human mesenchymal feeder cells.
  • any suitable vessel can be used. Examples of suitable vessels include traditional tissue culture substrates such as 6-, 24-, and 96-well plates, Petri dishes, flasks, bottles, plastic, and coverslips.
  • any culture media that enables the proliferation of stem cells or stem-cell-like cells while maintaining the "sternness" or stem cell qualities of the stem cells, and/or maintaining already differentiated cells in their current state type are particularly useful.
  • a culture medium that inhibits TGF-/3 signaling in the cells is preferred.
  • isolated animal cells or tissues are expanded in a culture system comprising a culture vessel, a matrix and a medium, wherein the medium is essentially free of amniotic membrane and non- human mesenchymal feeder cells, under conditions that downregulate TGF- ⁇ signaling in the cells to allow the cells to proliferate without undergoing a change in their differentiation state.
  • Examples of suitable media for use in ex vivo culture systems include a medium essentially free of AM and non-human mesenchymal feeder cells.
  • the cells can be cultured in a serum-free medium (e.g., KSFM) having less than about 10 ng/ml (e.g., less than 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.5, 0.25, 0.1, 0.05, and 0.01 ng/ml) of TGF- ⁇ and less than about 0.10 mM (e.g., less than 0.11, 0.10, 0.09, 0.08, 0.07, 0.06, 0.05, 0.04, 0.03, 0.02, 0.01, and 0.005 mM) Ca 2+ .
  • a typical medium is KSFM (cat. no.
  • limbal epithelial progenitor cells can be cultured in (a) KSFM, (b) seeded on AM derivatives and cultured in KSFM, (c) cultured in KSFM to which AM derivatives have been added, and (d) cultured in serum-containing medium to which AM derivatives have been added.
  • the cells [0055] In some methods of an ex vivo culture systems in which TGF-/3 signaling is downregulated, the cells
  • conditions which downregulate TGF- ⁇ signaling in the cells include seeding the cells at a cell density sufficiently low to prevent transient amplifying cells (TACs) from having a negative paracrine effect (e.g., secretion of TGF- ⁇ l or TGF ⁇ 2) on the limbal epithelial progenitor cells and for a time period that is greater than that of the TACs.
  • TACs transient amplifying cells
  • the cell density is typically between about 10 and 500 cells/cm 2 and the time period is greater than about 3 weeks.
  • any suitable agent for downregulati ⁇ g TGF-jS signaling in a cell can be used.
  • agents that downregulate TGF-/3 signaling include those that downregulate transcription of TGF- ⁇ gene in the cells.
  • the agent may specifically bind to TGF- ⁇ (e.g., an antibody), while in other cases, the agent may antagonize a receptor for TGF- ⁇ .
  • Small molecule TGF- ⁇ signaling inhibitors such as SB-431542 (Hjelmand et al., MoI Cancer Ther. 3(6):737-745, 2004) and those described below might be used to downregulate TGF- ⁇ signaling in cells.
  • a serine/threonine protein kinase inhibitor a molecule that prevents translocation of a Smad protein from the cytoplasm of the cell to its nucleus, AM derivatives, AME, processed non-intact AM, and a purified component of AM (e.g., HA, HA-ITI, lumican, TSG-6, pentraxin and thrombospondin) are further examples of agents that can be used to downregulate TGF- ⁇ signaling in the cells at the transcriptional level.
  • HA, HA-ITI lumican
  • TSG-6 pentraxin and thrombospondin
  • AM might take the form of a powder (e.g., lyophilized and ground or pulverized AM) or other suitable form of AM.
  • portions of AM might be used such as extracts of AM (see, e.g., U.S. provisional patent application 60/657,399) or purified components of AM such as extracellular matrix components such as HA, HA-ITI, and lumican (see, e.g., U.S. provisional patent application 60/657,399).
  • Methods of culturing cells on AM e.g., AM stromal matrix
  • culture medium containing serum that prevent the differentiation of the cells are described herein.
  • a number of additional agents that downregulate TGF-/3 signaling are known and can be used in ex vivo culture systems and in methods described herein.
  • Typical agents for modulating expression (and thus signaling) of intracellular proteins are mutants proteins, nucleic acids, and small organic or inorganic molecules.
  • proteins that can modulate TGF-/3 expression and/or activity in a cell include variants or native TGF-/3 proteins or receptors thereof that can compete with a native TGF-/3 protein or receptor thereof.
  • Such protein variants can be generated through various techniques known in the art. For example, protein variants can be made by mutagenesis, such as by introducing discrete point mutation(s), or by truncation.
  • TGF-/3 signaling is a TGF-/3 based or TGF-/S receptor-based non- peptide mimetic or chemically modified form of a TGF- ⁇ or a TGF-/3 receptor that disrupts binding of between a TGF-/3 protein and its receptor.
  • TGF-/3 based or TGF-/S receptor-based non- peptide mimetic or chemically modified form of a TGF- ⁇ or a TGF-/3 receptor that disrupts binding of between a TGF-/3 protein and its receptor See, e.g., Freidinger et al. in Peptides: Chemistry and Biology, G. R. Marshall ed., ESCOM Publisher: Leiden, Netherlands, 1988).
  • TGF-/3 proteins or receptors thereof may, for example, be chemically modified to create protein derivatives by forming covalent or aggregate conjugates with other chemical moieties, such as glycosyl groups, lipids, phosphate, acetyl groups and the like.
  • Covalent derivatives of a protein can be prepared by linking the chemical moieties to functional groups on amino acid side chains of the protein or at the N-terminus or at the C-terminus of the polypeptide.
  • the agent that directly reduces TGF-/3 signaling can also be a nucleic acid that modulates expression of a TGF- ⁇ protein or receptor thereof.
  • the nucleic acid can be an antisense nucleic acid that hybridizes to mRNA encoding the TGF-/? or receptor thereof.
  • Antisense nucleic acid molecules for use herein are those that specifically hybridize (e.g. bind) under cellular conditions to cellular mRNA and/or genomic DNA encoding the protein of interest in a manner that inhibits expression of the protein, e.g., by inhibiting transcription and/or translation.
  • Antisense constructs can be delivered using an expression vector plasmid or any other suitable means.
  • Ribozyme molecules designed to catalytkally cleave TGF-/? or TGF-/S receptor mRNA transcripts can also be used to prevent translation of and expression of these proteins (see, e.g., PCT Publication No. WO 90/11364, published Oct. 4, 1990; Sarver et al., Science 247:1222-1225, 1990 and U.S. Pat. No. 5,093,246).
  • endogenous TGF-/J or TGF-/3 receptor gene expression might be reduced by targeting deoxyribonucleotide sequences complementary to the regulatory region of the TGF-j3 or TGF-/3 receptor gene (i.e., the TGF-
  • deoxyribonucleotide sequences complementary to the regulatory region of the TGF-j3 or TGF-/3 receptor gene i.e., the TGF-
  • RNA interference RNA interference
  • An example of a protein that can modulate TGF-/3 signaling is an antibody that specifically binds a TGF-j3 or a TGF - ⁇ receptor. Such an antibody can be used to interfere with the interaction of the TGF-/3 and its receptor or to directly antagonize the receptor.
  • ex vivo method of expanding limbal epithelial progenitor cells (isolated or in tissues) in a cell culture are disclosed.
  • the cell culture is initiated with a mixture of limbal progenitor cells and transient amplifying cells (TACs).
  • TACs transient amplifying cells
  • This method comprises placing the mixture of limbal epithelial progenitor cells and TACs in an ex vivo culture system including a culture vessel, a matrix, and a medium, and culturing the cells in the ex vivo culture system at a sufficiently low cell density for a time period exceeding the lifespan of the TACs under conditions suitable for expanding the limbal epithelial progenitor cells.
  • the mixture of limbal progenitor cells and TACs is obtained from donor (e.g., human) limbal corneal epithelial sheets isolated by digestion with an appropriate protease (e.g., Dispase II) in an appropriate medium (e.g., KSFM at 37°C for 2 h) from the corneoscleral ring (see Espana et al., Invest. Ophthalmol. Vis. Sci. 44:4275-4281, 2003).
  • donor e.g., human
  • an appropriate protease e.g., Dispase II
  • an appropriate medium e.g., KSFM at 37°C for 2 h
  • This isolated mixture of limbal epithelial progenitor cells and TACs are seeded in an appropriate medium at a density of about 10 — 1,000 cells/cm 2 and cultured for a time period greater than about 3 weeks.
  • the mixture of cells is seeded in the culture system at a cell density sufficiently low to prevent the TACs from having a negative paracrine effect in the limbal epithelial progenitor cells.
  • the cells are incubated under appropriate conditions for cell expansion (e.g., at 37°C, in a 5% CO 2 humidified incubator, with medium changes as necessary). Expanded cells can be re-seeded into new culture vessels for further expansion. Once expanded to desired numbers, cells can be harvested for use.
  • most TACs can be eliminated by isolating cells from the remaining limbal stroma that is surgically dissected and then digested by 2 mg/ml collagenase A solution in serum-free KSFM medium at 37°C for 16 h (see Kawakita et al., Am J Pathol.
  • Another aspect includes methods of expanding animal cells (isolated or in tissues) ex vivo whose differentiation state is controllable by modulating a mitogen-activated protein kinase (MAPK), using a p38 MAP kinase inhibitor.
  • MAPK mitogen-activated protein kinase
  • Such a method includes culturing cells derived from limbal explants on a matrix in a culture medium comprising a ⁇ 38 MAP kinase inhibitor in an amount effective to facilitate outgrowth of the epithelial sheet while inhibiting abnormal epidermal differentiation of the cells relative to the cells grown without p38 MAP kinase inhibitor.
  • Non-limiting examples of p38 MAP kinase inhibitors are provided in Table 1 below:
  • VX-745 C 19 H J a 1 F 2 N 3 OS 209410-46-8
  • any suitable agent for inhibiting p38 is any suitable agent for inhibiting p38
  • MAP kinase in a cell can be used.
  • agents that inhibit the MAPK pathway include those that ⁇ can prevent MAPK fromphosphorylating transcription factors and cystolic targets in cells.
  • the agent may inhibit the catalytic activity of a kinase by competitive binding in the ATP pocket.
  • a small molecular weight inhibitor of p38 MAP kinase such as SB2O358O, L- 167307, SKF 86002, SB 220025, SB 235699, VX-745, RWJ 68354, SB 202190, SB 239063, and 2-(4-chloro-phenyl)-4-(4-fluoro-phenyl)-5-pyridin-4-yl-l,2- dihydro-pyrazol-3-one, promotes the synthesis, deposition, formation, and ex vivo expansion of basement membrane components during epithelial outgrowth by limbal epithelial progenitor cells on denuded amniotic membrane (dAM).
  • dAM denuded amniotic membrane
  • the epithelial sheet is cultured on a collagen coated insert following an airlift method in SHEM medium with a ⁇ 38 inhibitor, such as a pyridinylimidazole compound or a 2,4,5-triarylimidazole compound or SB203580, L- 167307, SKF 86002, SB 220025, SB 235699, VX-745, RWJ 68354, SB 202190, SB 239063, and 2-(4-chloro- phenyl)-4-(4-fiuoro-phenyl)-5-pyridin-4-yl-l,2-dihydro-pyrazol-3-one.
  • a ⁇ 38 inhibitor such as a pyridinylimidazole compound or a 2,4,5-triarylimidazole compound or SB203580, L- 167307, SKF 86002, SB 220025, SB 235699, VX-745, RWJ 68354, SB 202190, SB 239063, and 2-
  • the method includes isolating an intact limbus from a biopsy of a donor eye of a living individual, an eye of a living, related individual, or from a cadaveric eye.
  • the limbus obtained from the eye consists of the limbal epithelium and an underlying stroma.
  • the limbus is then put in contact with a solution comprising Dispase II for a period of time and under conditions sufficient to substantially loosen a limbal epithelial sheet from the stroma, thereby forming a loosely adherent limbal epithelial sheet.
  • the limbal epithelial sheet can then be mechanically separated from the underlying stroma, thereby isolating a substantially intact, viable, limbal epithelial sheet.
  • the solution contacting the limbus accordingly comprises a substance chosen from either SHEM, a polyhydroxy alcohol, a sugar, or combinations thereof.
  • polyhydroxy alcohol suitable for use in this embodiment examples include sorbitol, mannitol, and galactitol.
  • the limbus/solution is maintained at a temperature from about 0°C to about 37°C for at least half an hour.
  • the limbal epithelial sheet separated from the limbus is put in contact "with the basement membrane side of an amniotic membrane. The limbal epithelial sheet and amniotic membrane thereby form a composite. This composite is then cultured for a period of time and under conditions sufficient to enable the epithelial cells to expand.
  • a method of expanding mesenchymal cells ex vivo, while maintaining the phenotype of the mesenchymal cells includes contacting the stromal side of the amniotic membrane with at least one type of mesenchymal cell, thereby forming a composite of the mesenchymal cells and the amniotic membrane. The composite is then cultured in a serum containing medium for a period of time and under conditions sufficient to enable the epithelial stem cells to expand.
  • Another method for expanding limbal epithelial cells described herein is an in vitro expansion of animal cells comprising expanding such limbal epithelial cells on human amniotic epithelial cell feeder layers isolated from the amnion layer.
  • animal cells include, but are not limited to, stem cells or stem-cell-like cells, such as limbal epithelial progenitor cells, umbilical cord epithelial cells, and amniotic membrane epithelial cells.
  • stem cells or stem-cell-like cells such as limbal epithelial progenitor cells, umbilical cord epithelial cells, and amniotic membrane epithelial cells.
  • limbal epithelial progenitor cells can be obtained from corneoscleral tissue.
  • the human amniotic epithelial cells used as feeder layers for animal cells are mitomycin C (MMC)-treated human amniotic epithelial cells.
  • MMC mitomycin C
  • HAEC human amniotic epithelial cells
  • SHEM culture medium containing 5% FBS together with EGF and insulin
  • feeder layers that are more effective in promoting clonal growth of human amniotic epithelial cells.
  • Limbal epithelial progenitor cells expanded on these HAEC feeder layers evidence more stem cell markers for longer periods of time than similar progenitor cells expanded on murine feeder layers.
  • Animal cells expanded ex vivo according to systems and methods described herein can be transplanted into an animal subject suffering from any of a number of disease states in which stem cells or stem-cell-like cells are dysfunctional or lacking.
  • an animal subject suffering from HIV or cancer in which stem cells or stem- cell-like cells are dysfunctional can receive a transplantation of stem cells or stem-cell-like cells expanded ex vivo to restore the function of the dysfunctional stem cells or stem-cell-like cells.
  • cells expanded according to the methods described above can be used to replace cells lost due to HIV infection or cancer or due to the side effects of treatment for those conditions (e.g., cell death caused by anti-viral or anti-neoplastic drugs or ionizing radiation).
  • a further aspect of this disclosure is surgical grafts, comprising cells or tissues cultured as described herein.
  • the surgical graft comprises an isolated, substantially intact, viable, limbal epithelial sheet or mesenchymal cells such as keratocytes.
  • Surgical grafts can also be formed from expanded fetal mesenchymal cells, fibroblasts, endothelial cells, melanocytes, cartilage cells, bone cells, hematopoietic stem cells, bone marrow mesenchymal stem cells, adult mesenchymal stem cells or combinations thereof.
  • the surgical graft may further comprise an amniotic membrane.
  • the examples set forth below describe methods for controlling the proliferation and differentiation of primary cells explanted from an animal or human subject such as corneal keratocytes and limbal epithelial progenitor cells from a variety of mammalian species including human beings.
  • Example 1- Preservation and Expansion of Primate Keratocvte Phenotvpe by Downregulating TGF-3 Signaling in a Low Calcium Serum-free Medium
  • the corneal epithelium was removed by dispase digestion for 16 h at 4 0 C and the remaining corneal stroma was incubated at 37°C for 16 h in 2.5 ml of DMEM containing 1 mg/ml collagenase A, 20 mM HEPES, 50 ⁇ g/ml gentamicin and 1.25 ⁇ g/ml amphotericin in a plastic dish.
  • corneal stromal cells were resuspended in 1 ml of KSFM, centrifuged to remove residual matrices, resuspended in KSFM, and seeded on plastic dishes in KSFM or DMEM containing insulin, transferrin, and selenium supplement (DMEM/ITSG) (cat# 41400-045, GIBCO, Carlsbad, CA) or 10% FBS (DMEM/10%FBS).
  • DMEM/ITSG insulin, transferrin, and selenium supplement
  • FBS DMEM/10%FBS
  • KSFM To verify cell proliferation in KSFM, primary cells in DMEM ⁇ TS, DMEM/10%FBS and KSFM were subcultured at a density of 3,000 cells per 96-well plastic dish, and submitted at day 3 and day 7 to MTT assay (Promega Corporation, Madison, WI) according to the manufacturer's instructions. Using the culture medium alone as the negative control, this assay was validated by establishing a linear correlation between 2,500 and 10,000 passage 2 murine corneal fibroblasts. Cells at day 7 were also immunostained using an anti-Ki67 antibody ( 1 : 100).
  • the number of Ki 67 positive nuclei was randomly measured in 10 fields under high magnification (400 x) for each culture, and the ratio of positive cells/total cells at each field was calculated. Experiments were performed in triplicate. Statistical analysis was performed by using Student's Mest: P ⁇ 0.05 was considered statistically significant.
  • cells were transfected for 24 h with self engineered aden-track-Kerapr3.2-intron-ECFP/BpA adenovirus at an MOI of 200 (see Kawakita et al., J Biol. Chem. 280:27085-27092, 2005).
  • the medium of Pl cells was replaced with fresh KSFM, DMEM/ITS, or DMEM/10%FBS 5 h before being transfected with replication-defective adenoviruses containing TGF-/31 or TGF-/3RII promoters, each linked with luciferase (100 MOI) and containing CMV- ⁇ - galactosidae (30 MOI) for 48 hours.
  • the promoter activity was measured by the Luciferase Assay System ® (Promega, Madison, WI) and normalized with the / 3-galactosidase activity.
  • TGF- ⁇ l and — ⁇ RII promoter activity of Pl cells was measured in cells cultured in KSFM, in which the [Ca 2+ ] was increased to 1.8 mM (identical to DMEM) with or without 10% FBS. / ⁇ -galactosidase activity was measured and relative transfection was normalized.
  • the monkey corneal stroma was subjected to collagenase digestion.
  • the resultant cell suspension yielded approximately 1.5 x 10 5 cells per cornea.
  • cells attached well in DMEM/ITS, DMEM/10%FBS, or KSFM but exhibited a distinctly different morphology.
  • Cells cultured in DMEM/ITS for 7 days did not grow and showed a mixture of flattened and dendritic cells, while cells cultured in DMEM/10%FBS for 7 days reached confluence and showed a flattened fibroblastic morphology.
  • KSFM for 7 days had a higher cell density than DMEM/ITS and maintained a dendritic morphology.
  • DMEM/10%FBS from day 3 to day 7 (p ⁇ 0.01).
  • the cell number in KSFM estimated by MTT was between that of DMEM/ITS and DMEM/10% FBS. (p ⁇ 0.0S, between day 3 and day 7).
  • cellular proliferation in KSFM was also between that in DMEM/10%FBS and that in DMEM/ITS (p ⁇ 0.05, both between KSFM and DMEM/10%FBS or DMEM/ITS).
  • Cells maintained in DMEM/ITS could not be subcultured to P 1. They immediately adopted a flattened morphology when subcultured in DMEM/10%FBS at P 1.
  • KSFM is culture medium supplemented by growth factors including EGF and bFGF, and differs from
  • DMEM-base medium in many aspects; the major features of KSFM are a low [Ca 2+ ] and the lack of FBS. Whether high [Ca 2+ ] or addition of 10% FBS or a combination of both might modulate the keratocyte phenotype determined by expression of keratocan was thus examined. To do so, the promoter activities following transient transfection of Aden-track-Kerapr3.2-intron-ECFP/BpA adenovirus containing CMV promoter-driven EGFP and keratocan promoter-driven ECFP in Pl cells was measured. In a given cell, expression of EGFP reflects the background transfection while expression of ECFP reflects the keratocan promoter activity. The protein expression of keratocan and CD34 was also monitored by immunostaining.
  • Such a percentage decreased to 62.0 ⁇ 9.6%, 33.3 ⁇ 5.4% and 29.8 ⁇ 4.5% when cells were cultured in KSFM with 1.8 mM [Ca 2+ ], in KSFM with 10% FBS, and KSFM with 1.8 mM [Ca 2+ ] and 10% FBS, respectively (p ⁇ 0.01 for KSFM vs. KSFM + FBS or KSFM + [Ca 2+ ] + FBS). There was no significant difference in those percentages between KSFM + [Ca 2+ ] + FBS and DMEM/10%FBS nor between KSFM and KSFM + [Ca 2+ ].
  • TGF-/? signaling was also similarly modulated by increasing [Ca 2+ ] or addition of 10% FBS, or a combination of both in KSFM was examined by measuring the promoter activity of TGF-/31 and — ⁇ RII after transient adenoviral transfection.
  • the control i.e., cells cultured in DMEM/FBS10% and adjusted by background transfection with CMV-j3 Gal
  • the promoter activity of TGF-/31 and - ⁇ RII was both significantly decreased in cells cultured in KSFM (p ⁇ 0.05). There was no significant difference in the promoter activity between KSFM and DMEM/ITS.
  • the majority of cells cultured in DMEM/ITS or KSFM showed cytoplasmic localization of Smad2 and Smad4, while the majority of cells cultured in DMEM/10% FBS or KSFM with increased [Ca 2+ ] and addition of 10% FBS showed nuclear localization of Smad2 and Smad4.
  • Keratocytes display a dendritic morphology and express keratocan. When cultured using conventional methods, however, keratocytes lose their dendritic morphology and cease expression of keratocan. As described below, keratocytes were expanded on AM and examined to determine if they maintained their characteristic phenotype, including the expression of keratocan.
  • the corneal stroma was separated from the sclera at the corneoscleral limbus by pressing down the limbus with a 27 G needle while the eye was held with a forcep. Isolated corneal stromas were incubated overnight at 37 0 C in DMEM containing 1.25 mg/ml collagenase A (Roche, Indianapolis, IN), 50 ⁇ g/ml gentamicin and 20 mM HEPES in a non-coated plastic dish until the tissue became "smeared" onto the dish bottom. Digested corneal stromas in collagenase A were centrifuged at 800 x g for 5 min.
  • Keratocytes were resuspended in DMEM containing 20 mM HEPES, ITS (5 ⁇ g/r ⁇ l insulin, 5 ⁇ g/ml transferrin and 5 ng/ml sodium selenite), 50 ⁇ g/ml gentamicin and 1.25 ⁇ g/ml amphotericin B with or without 10% FBS.
  • This keratocyte-containing cell suspension was then seeded on plastic dishes or on the stromal side of the AM (Bio-Tissue, Miami, FL) fastened to a culture insert as previously described (see Meller et al., Br J Ophthalmol 86, 463-471, 2002).
  • cells were resuspended in DMEM/10% FBS and seeded on a plastic dish or AM stroma. They were cultured in DMEM containing 10% FBS, 20 mM HEPES, 50 ⁇ g/ml gentamicin and 1.25 ⁇ g/r ⁇ l amphotericin B.
  • TGF- ⁇ l Challenge and Neutralizing Antibody - To assess whether TGF-/31 affected the cell phenotype, triggered Smad 2 and Smad 4 nuclear translocation, and differentiated keratocytes into myofibroblasts, 10 ng/ml human recombinant TGF-/31 (Sigma, St Louis, MO) was added to serum-free DMEM ⁇ TS cells for 3 h or 5 days when cells expanded on AM were passed to 24 well plastic dishes and AM inserts, respectively.
  • DMEM/ITS or DMEM/10% FBS primary keratocytes were seeded and cultured on AM or plastic for 3 days in DMEM/ITS or DMEM/10% FBS for 24 h, of which the latter was treated with or without 10 ⁇ g of a monoclonal antibody neutralizing TGF- ⁇ l, -/32, and -03 (R&D Systems, Minneapolis, MN) per ml of DMEM medium for 48 h before adenoviral transfection.
  • Keratocytes in the stroma expressed keratocan as evidenced by positive staining with an affinity-purified antibody against mouse keratocan peptide.
  • corneal epithelial cells or endothelial cells were not stained.
  • DMEM/10%FBS dendritic cells expanded on AM in DMEM/10%FBS maintained keratocan expression, and did not express oi-SMA.
  • CD34 was not expressed by cells cultured on plastic, but expressed by cells cultured on AM.
  • fibronectin was expressed extracellularly and intracellularly by cells cultured on plastic, but not expressed by cells cultured on AM.
  • keratocan transcript (of the size of 1065 bp) was expressed by cells cultured on plastic at passage 0, but lost at passage 1 and thereafter. In contrast, the keratocan transcript was continuously expressed in an abundant amount from passage 0 to passage 3 and up to passage 8 when cultured on AM.
  • insoluble matrix proteins were extracted by 4 M guanidine HCl and subjected to Western blot analysis using an antibody against the core protein of keratocan.
  • the sample from the normal murine corneal stroma which was used as the positive control, showed a dense smearing in the high molecular weight region.
  • TGF-/3 was indeed responsible for Smad signaling in DMEM/10%FBS.
  • a neutralizing antibody to three TGF-/3 isoforms was added to the plastic cultures. Nuclear translocation of Smad 4 was prevented.
  • ⁇ - SMA expression was quantified in parallel. Thirty-nine percent of cells cultured on plastic differentiated into a-
  • keratocan promoter-driven ECFP expression was observed in 30 - 40% of cells cultured on plastic in DMEM/ITS, 15 - 20% of cells cultured on AM in DMEM/10%FBS, but less than 2% of cells cultured on plastic in DMEM/10%FBS.
  • a flat mount preparation of freshly isolated intact human limbal epithelial sheet showed that p63- positive (p63 being an epithelium-specific transcription factor) basal cells are grouped in clusters, indicating that progenitor cells are intermixed with TACs in the limbal basal epithelium. Because TACs are known to have a negative paracrine influence on limbal epithelial progenitor cell renewal, it was hypothesized that elimination of TACs paracrine influence by seeding at a low density and prolonging the culturing time beyond TACs life span (about 3 weeks) would improve clonal initiation and expansion of limbal epithelial progenitor cells.
  • Single cells dissociated from isolated mouse corneal/limbal sheets by trypsin/EDTA were seeded at a density of 40 cells/cm 2 in a defined keratinocyte serum-free medium (KSFM) (Gibco-BRL, Carlsbad, CA) containing 0.07 mM (low) Ca 2+ but supplemented with insulin, bFGF, EGF and cholera toxin.
  • KSFM keratinocyte serum-free medium
  • Two types of clonal growth could be generated from a single cell derived from these holoclones by limiting dilutions in a 96 well culture plate.
  • Several such single-cell generated clones were expanded, and each proved to be non-transformed. These single cell-generated clones could be cryopreserved and then recovered more than once.
  • An alternative method to eliminate most of the TACs in the example of expanding human limbal epithelial progenitor cells is to surgically dissect the remaining limbal stroma from the sclera after dispase digestion as stated above, and then to digest it by 2 mg/ml collagenase A solution in KSFM medium at 37 °C for 16 h (Kawakita et al., Am J Pathol. 167:381-393, 2005). Cells thus isolated were then cultured on plastic dishes in KSFM at a seeding density of 10,000 cells/cm 2 . For human amniotic epithelial progenitor cells, the same results were obtained using the above technique with the exception that Ca 2+ concentrations could be elevated as high as 1.0 mM.
  • KSFM a low-calcium, serum-free medium
  • adenoviral vectors containing the promoters of TGF- ⁇ l, TGF- ⁇ 2, TGF- ⁇ 3, or TGF- ⁇ RII and the reporter gene luciferase were constructed and used to monitor the transcriptional activity of these four TGF- ⁇ genes in transiently transfected human limbal epithelial progenitor cells.
  • TGF- ⁇ l and TGF- ⁇ RII were found to be markedly downregulated inhuman limbal epithelial cells when the cells were maintained in KSFM medium when compared to SHEM which contains 5% FBS and high calcium.
  • SHEM which contains 5% FBS and high calcium.
  • Addition of 5% FBS to and elevation Of[Ca 2+ ] to 0.9 mM in KSFM markedly upregulated TGF- ⁇ l promoter activity to the same level as SHEM in human limbal epitfielial progenitor cells and monkey.
  • Conditioned media was collected from these three cultures after 3 days of culturing in KSFM and subjected to a Bio-Plex machine (Bio-Rad, Hercules, CA) using Beadlyte® TGF-/31, /32, ⁇ 3 detection system (Upstate, Waltham, MA).
  • TGF- ⁇ 1 and 5.1 ⁇ 0.4 ng/ml TGF- ⁇ 2 were detected only in the conditioned media of high density cultures seeded at 5,000 cells/cm 2 but not in those of low density cultures seeded at 50 or 500 cells/cm 2 . No TGF- ⁇ 3 was detected. Because the detection limit of this system is down to 0.1 ng/ml, the above data indicated that the levels of TGF- ⁇ l or /32 in the low density cultures should be less than 0.1 ng/ml.
  • umbilical cord epithelial cells were expanded ex vivo while maintaining their stem cell phenotype.
  • Example 4- Amniotic Epithelial Cells Help Maintain HA-containing Stromal Matrix in Intact AM to Support Li ⁇ nbal Epithelial Progenitor Cell Renewal by Downregulating TGE-g Signaling
  • HABP biotinylated HA binding protein
  • the HA-ITI complex was not only present in the insoluble extracts (obtained by 1 M NaCl and 4M guanidine HCl, respectively) but also in soluble AM extract (obtained by homogenization in PBS).
  • the covalent linkage of HA with ITI stabilizes high MW status of HA, preventing HA degradation to small MW in part because ITI is a natural inhibitor of HAse.
  • AM extracts were digested with or without 50 ⁇ g/ml HAse. The presence of ITI in these extracts and their interactions with HA were examined by Western blot. ITI was present in all extracts before HAse digestion, but there were extra bands appearing after digestion.
  • Such a method includes several steps.
  • the first step includes isolating HAECs from human placenta. An intact layer of the amnion was mechanically peeled, off of the placenta and individual HAECs are isolated from the amnion layer.
  • the next step is to seed individual progenitor cells on the HAECs and to incubate the cell cultures in medium.
  • the cells seeded on the HAECs can be any cell type which can differentiate.
  • Such cells might be stem cells or differentiated cells.
  • stem cells include totipotent stem cells, pluripotent stem cells, and multipotent stem cells.
  • Differentiated cells might include epithelial cells, fibroblasts, myocytes, pancreatic ⁇ cells, blood cells, neurons, smooth muscle cells, fat cells, oligodendrocytes, alveolar cells, epidermal cells, and keratocytes.
  • any suitable method may be used.
  • a typical method of isolating HAECs includes peeling the amnion off from the chorion after the placenta has been washed to remove blood. Once the amnion is removed, 5x5 cm 2 pieces are cut and rinsed in HBSS. The amnion is then digested with an appropriate protease (e.g., Dispase II at 37°C for 15 rnin), and incubated in the appropriate medium (e.g., KSFM) to generate a 5 loose single layer of the amniotic epithelium, which can then be separated from the underlying stroma by gentle stripping off with jewelry forceps.
  • an appropriate protease e.g., Dispase II at 37°C for 15 rnin
  • Isolated limbal epithelial sheets are rendered into single cells by digestion (e.g., 0.05% trypisn/0.53, Mm EDTA at 37°C for 15 min), and then seeded in triplicate at a density of 50 cells/cm 2 on HAEC feeder layers. Cells are then incubated at 37°C under 5% CO 2 and 95% humidity, and the medium was changed every 2-3 days.
  • protease e.g. 10 mg/ml Dispase II in SHEM at 4°C for 16 h.
  • Isolated limbal epithelial sheets are rendered into single cells by digestion (e.g., 0.05% trypisn/0.53, Mm EDTA at 37°C for 15 min), and then seeded in triplicate at a density of 50 cells/cm 2 on HAEC feeder layers. Cells are then incubated at 37°C under 5% CO 2 and 95% humidity, and the medium was changed every 2-3 days.
  • HAECs cultured in SHEM was subsequently subcultured in each of the above-mentioned 9 media and subjected to MTT assay, which is based on the cleavage of yellow tetrazolium salt MTT to purple formazan crystals by metabolically-active cells (see Kamiya et al., Exp Eye Res. 80:671-679, 2005). After cells were incubated for 4 h, formation of formazan dye in the microtiterplate was spectrophotometrically quantified with an ELISA plate reader
  • clonal assay was performed using a method reported by Rheinwald and Green (see Rheinwald & Green, 6:331 -337, 1975) and modified by us for limbal epithelial cells (see Tseng et al., Curr Eye Res. 15:973-984, 1996).
  • Rheinwald and Green see Rheinwald & Green, 6:331 -337, 1975
  • fibroblasts grown in DMEM containing 10% newborne calf serum at 80% subconfluence, and the 8 th
  • Isolated limbal epithelial sheets were rendered into single cells by digestion with 0.05% trypsin/0.53 mM EDTA at 37 0 C for 15 min, and seeded in triplicate at a density of 50 cells/cm 2 (500 cells/well) on either HAEC or 3T3 fibroblast feeder layers in six-well culture plates containing SHEM. Cultures were incubated at 37 C C under 5% CO 2 and 95% humidity, and the medium was changed every 2-3 days.
  • CFE colony-forming efficiency
  • each sample was fixated in cold methanol for 10 min at -20 °C, permeabilized in 0.2% Triton X-IOO for 15 min and blocked with 2% bovine serum albumin for 30 min at room temperature. Cells were then incubated with the primary antibody for 16 h at 4 0 C. After 3 times of wash with PBS, specific binding was detected by a FITC-conjugated anti-mouse or anti- rabbit secondary antibody incubated for 30 min at room temperature. Finally, the sample was counterstained with Hoescht 33342 and mounted in anti-fading solution (Vector Labs, Burlingame, CA, USA).
  • HAECs isolated immediately from amniotic epithelial sheets as well as from cultures of Passages 0, 1, and 6 in SHEM were quantitatively compared for their cytoskeleton protein expression using Western blots.
  • Cultured cells were collected and solubilized in the lysis buffer containing 50 mM Tris-HCl (PH 8.0), 1% NP-40, 0.5% DOC, 0.1% SDS, 150 mM NaCl, 5 mM EDTA, 1 mM PMSF and a protease inhibitor cocktail.
  • Total cell proteins were extracted from fresh amniotic sheets by gentle scraping with the same lysis buffer. Proteins in these lysates were denatured by boiling for 5 min with the equal volume of 2X Tris-glycine SDS sample buffer, separated by 4% to 15% gradient SDS-PAGE, and transferred to nitrocellulose membranes.
  • Limbal epithelial progenitor cells form clones in HAEC feeder layers.
  • clones grown on HAEC feeder layers emerged slower, i.e., after at least 5 days, and were fewer and smaller.
  • limbal cells grown on murine 3T3 feeder layers emerged as early as day 3, and were more and larger.
  • the border of limbal clones on 3T3 feeder layer was smooth, presumably because 3T3 fibroblasts were pushed by expanding limbal epithelial cells.
  • the border of limbal clones on HAEC was not smooth because the entire clone was growing on the top of their underlying feeder cells.
  • limbal clones supported by HAEC feeder layers were uniformly compact and the border was irregular.
  • clones supported by 3T3 feeder layers had less cellularity in the center of the clone and were round with a smooth border.
  • HAEC-supported limbal clones consisted of uniformly small and oval epithelial cells.
  • 3T3-supported clones were composed of cells with variable sizes with those in the center being large and squamous.
  • expression of differentiated cell markers was different in these two types of clones.
  • a corneal epithelium-specific marker, CK12 was not expressed by the amniotic epithelium, but expressed by human limbal suprabasal cells. Except for few single cells in the center of the clone, expression of
  • HAEC-supported clones were less differentiated than that of 3T3-sup ⁇ orted clones, it was also determined whether HAEC-supported clones actually expressed such stem cell markers as p63, Musashi-1, and ABCG2 proteins. Expression of p63 is found to be in epidermal and limbal epithelial progenitor cells. On HAEC feeder layers, positive nuclear p63 immunostaining was found in nearly all cells in the entire clone except for very few cells in the center of clone. On 3T3 feeder layers, positive nuclear p63 staining was restricted to peripheral small cells, and large squamous cells in the center of the clone were negative.
  • Musashi-1 a neural stem cell marker, was found in the nucleus of the subventrical zone of the mouse brain. In the human limbus, strong nuclear staining of Musashi-1 was found in basal epithelial cells with a faint staining noted throughout the whole epithelium. Nuclear expression of Musashi-1 was found in most cells in HAEC-'supported clones.
  • ⁇ -SMA was not expressed in the center, but was expressed in the periphery of the single cell-derived clone after being expanded more than 3 weeks.
  • Example 7 A TGF-/3 Promoter Assay That Demonstrates the Suppressing Effect of TGF-ff Signaling by AM Derivatives In Both Human Limbal Epithelial Progenitor Cells and Human Corneal Fibroblasts
  • Human corneal fibroblasts were transiently transfected with the aforementioned adenoviral promoter constructs. In these cells, TGF- ⁇ 1 promoter activity was significantly suppressed by an AME prepared according to the method described in U.S.
  • TGF- ⁇ 1 promoter activity was significantly suppressed by 25 ⁇ g/ml AME in both KSFM and SHEM media.
  • the suppressive activity of AME which contained -0.8 ⁇ g/ml HA was more potent than 125 mg/ml high MW pure HA alone.
  • the suppressive effect of both AME and HA alone was lost after pretreatment with HAse. No suppressive effect was noted in the control when HAse alone was added together with BSA. A similar result was obtained for TGF- ⁇ RII promoter activity.
  • Example 8- Signaling Transduction Pathways Required for Ex Vivo Expansion of Human Lirribal Explants on Intact AM The results described below show that ex vivo expansion of human limbal epithelial progenitor cells on intact AM is mediated by the survival signaling pathway mediated by PBK-Akt-FKHRLl and the mitogenic MAPK pathway mediated by p44/42, but not by p38 and INK.
  • the limbal ring was separated by a 7.5 mm trephine from donor human corneas. Each limbal ring was rinsed 3 times with SHEM media. The limbal ring was then exposed for 10 min to 1.2 units/ml Dispase II in Mg 2+ - and Ca 2+ -free HBSS at 37°C under 95% humidity and 5% CO 2 . Following three rinses with SHEM medium, each limbal ring was subdivided into two halves and each half further subdivided into 6 pieces of 1 x 1.5 x 2.5 mm explants.
  • explants from the corresponding position of the same donor cornea were selected for the control and the experimental group, respectively.
  • An explant was placed on the center of intact AM or plastic with the epithelial side facing up and cultured in SHEM medium.
  • the experimental group was added with the inhibitor of desired concentration, while the control group was added with the same concentration of DMSO as the vehicle which was used to dissolve each inhibitor.
  • the culture was maintained at 37°C under 95% humidity and 5% CO 2 , the medium was changed every other day, and their outgrowth was monitored daily for 17 days using an inverted phase microscope (Nikon, Japan).
  • the outgrowth area was digitized every other day by Adobe Photoshop 5.5 and analyzed by NIH ImageJ 1.3Ov (NIH, Bethesda, MD).
  • the culture was terminated before reaching confluence to avoid possible underestimation caused by cell contact inhibition.
  • the outgrowth rate of the control showed a consistent pattern as a group.
  • the outgrowth rate was gradually increased from day 5 to day 9, but rapidly increased from day 9 to day 13, and gradually slowed down from day 13 to day 17.
  • LY294002 is a specific inhibitor of PI3K, and one of the downstream target of PI3K is to phosphorylate and activate Akt kinase.
  • Addition of 10 ⁇ M of SRl 3668, a potent phosphor-Akt inhibitor resulted in 50 % reduction of the outgrowth rate from day 5 to day 11, and 60 % reduction from then on. Addition of 50 ⁇ M of SR13668 completely inhibited the epithelial outgrowth.
  • SB203580 and JNK inhibitor 1 are specific inhibitors for MAPK p38 kinase and JNK kinase, respectively.
  • compositions and methods of use for inhibitors of p38 MAP kinase for improved ex vivo culture of cells enhance cell outgrowth and differentiation relative to cells grown without inhibitors of p38 MAP kinase.
  • the AM was thawed, washed three times with sterile PBS, and cut into pieces approximately 2.5x2.5 cm in size.
  • dAM denuded AM
  • membranes were deprived of their devitalized amniotic epithelial cells by incubation with the appropriate media (0.02% EDTA at 37°C for 1 h) to loosen the cellular adhesion, followed by gentle scraping with a cell scraper.
  • the tissue was then rinsed three times with DMEM containing 50 ⁇ g/ml gentamicin and 1.25 ⁇ g/ml amphotericin B. After careful removal of excessive sclera, conjunctiva, iris, and corneal endothelium, the corneoscleral rim is trimmed to obtain limbal tissue cubes 1.5x2.5 mm size. Afterwards, limbal tissue cubes are placed on inserts with intact AM (iAM) or dAM in SHEM medium made of an equal volume of HEPES-buffered DMED containing bicarbonate and Ham's/F12.
  • iAM intact AM
  • SHEM medium made of an equal volume of HEPES-buffered DMED containing bicarbonate and Ham's/F12.
  • the medium is then supplemented with 5% FBS, 0.5% dimethyl sulfoxide, 2ng/ml mouse EFG, 5 ⁇ g/ml insulin, 5 ⁇ g/ml transferrin, 5 ng/ml selenium, 0.5 ⁇ g/ml hydrocortisone, InM cholera toxin, 50 ⁇ g/ml gentamicin, and 1.25 ⁇ g/ml amphotericin B.
  • 2.5 ml medium was added in each insert and the medium was changed every 2 days.
  • Human limbal tissue cubes can be placed in the alternative on the center of type I collagen coated inserts and cultured in SHEM medium under submerge and airlift manners, of which the later was also added with ⁇ 38 inhibitor.
  • the submerge manner 4 ml culture medium was added to the culture dish to cover the entire limbal explant.
  • the airlift manner 2 ml culture medium was added so as to let the insert bottom sit on the air-liquid interface. That is, the explant was exposed to air, but the remaining explant stroma was submerged in the medium meniscus.
  • p 38 inhibitor SB 203580 was added to culture medium at a concentration of 10 ⁇ M.
  • results [00151] Human corneal limbal explants were cultured on intact amniotic membrane (iAM) or denuded amniotic membrane (dAM) for 2 and 4 weeks. Results showed that outgrowth epithelial cells on iAM started to express type IV collagen intracellurly after 2 weeks of culturing, and deposited the collagen to the epithelial-stromal interface after 4 weeks of culturing (Fig.l). In contrast, outgrowth epithelial cells on dAM showed negative staining after 2 weeks of culturing, and showed intracellular staining after 4 weeks of culturing.
  • the outgrowth rate was not inhibited by the p38 inhibitor, SB203580, based on the outgrowth surface area.
  • SB203580 the outgrowth epithelial cells exhibited a migratory appearance, which showed a heterogeneous cell shape, a loose outgrowth sheet with space between cells, and an interrupted leading edge. Furthermore, there were stratified cell aggregates in the peripheral area of the outgrowth.
  • the outgrowth epithelial cells were uniformly smaller and had a homogeneous cell shape; the outgrowth sheet was very compact and had a very smooth leading edge.
  • epithelial cells migrating to the cut edge of the cornea and on the bare sclera were all K12 positive, while limbal basal epithelial cells still kept Kl 2 negative.
  • peripheral corneal epithelial cells still kept positive Kl 2 expression, however, limbal epithelial cells showed island-like groups of cells expressing K12 staining mainly located in the suprabasal epithelium.
  • the p38 MAP kinase inhibitor is a pyridinylimidazole compound. In another embodiment, the p38 MAP kinase inhibitor is a 2,4,5-triarylimidazole.
  • the p38 MAP kinase inhibitor is chosen from at least one of: SB203580, L 167307, SKF 86002, SB 220025m SB 235699, VX 745, RWJ 68354, SB 202190, SB 239063, and 2-(4-chloro-phenyl)-4-(4-fluoro-phenyl)-5-pyridin-4-yl-l,2-dihydro-pyrazol-3- one.
  • an epithelial sheet outgrowth in a culture medium comprising p38 inhibitor can be used to facilitate engineering of corneal epithelial tissues, both in vitro and in vivo.
  • An intact limbal epithelial sheet can be consistently and reproducibly isolated and contains stem cell characteristics in the basal epithelium by degrading laminin 5 and part of collagen IV, and disassembling collagen VH.
  • Plastic cell culture dishes 60 mm were from Falcon (Franklin Lakes, NJ, USA). Amphotericin B, Dulbecco's modified Eagle's medium (DMEM), F-12 nutrient mixture, fetal bovine serum (FBS), gentamicin, Hank's balanced salt solutions (HBSS), HEPES-buffer, neomycin, penicillin, streptomycin, phosphate buffered saline (PBS), TRIZOL® and 0.05% trypsin/O.53mM EDTA were purchased from Gibco-BRL (Grand Island, NY, USA). A LIVE/DEAD® viability/cytotoxity kit was from Molecular Probes (Eugene, OR, USA).
  • Dispase 2 powder was obtained from Roche (Indianapolis, IN, USA). Tissue-Tek OCT compound and cryomolds were from Sakura Finetek (Torrance, CA, USA). Other reagents and chemicals including bovine serum albumin (BSA), cholera-toxin (subunit A), collagenase A, dimethyl sulfoxide, hydrocortisone, insulin-transferrin-sodium selenite (ITS) media supplement, mouse-derived epidermal growth factor (EGF), pre-stained broad band SDS-PAGE standard and sorbitol were purchased from Sigma (St. Louis, MO, USA).
  • BSA bovine serum albumin
  • cholera-toxin subunit A
  • collagenase A dimethyl sulfoxide
  • hydrocortisone insulin-transferrin-sodium selenite
  • ITS insulin-transferrin-sodium selenite
  • EGF mouse-derived epidermal growth factor
  • An immunoperoxidase staining kit (Vecstatin ⁇ ) and DAPI containing mounting media (Vectashield®) were obtained from Vector Laboratories (Burlingame, CA, USA).
  • keratin 3 (AE5) (ICN, Aurora, OH, USA), integrin D4 (Chemicon, Temeluca, CA, USA), laminin 5 (Accurate Chemicals, Westbury, NY ,USA) 3 mouse and collagen VII antibody, rhodamine conjugated rabbit anti-goat antibody and fluorescein-conjugated goat anti-mouse antibody (Sigma, St.
  • SHEM medium which was made of an equal volume of HEPES-bufFered DMEM and Ham's F12 containing bicarbonate, 0.5% dimethyl sulfoxide, 2 ng/ml mouse derived EGF, 5 ⁇ g/ml insulin, 5 ⁇ g/ml transferrin, 5 ng/ml sodium selenite, 0.5 ⁇ g/ml hydrocortisone, 30 ng/ml cholera toxin A subunit, 5% FBS, 50 ⁇ g/ml gentamicin, and 1.25 ⁇ g/ml amphotericin B.
  • the primary antibody was detected using a fluorescein conjugated secondary antibody except for collagen W in which a rhodamine conjugated antibody was used.
  • Sections were mounted in anti-fading solution containing DAPI VECTASHIELD® (Vector Laboratories, Burlingame, CA, USA), and analyzed with a NikonTe-2000u Eclipse epi-fluorescent microscope (Nikon, Tokyo, Japan).
  • DAPI VECTASHIELD® Vector Laboratories, Burlingame, CA, USA
  • keratin 3 which is regarded as a corneal differentiation marker and p63 nuclear protein, that is a presumed corneal SC marker
  • proteins of confluent cultures were extracted by TRIZOL®, and precipitated by centrifuging at 12000 x g in 100% isopropyl alcohol. After washing and centrifuge for three times, the protein pellet was precipitated with a solution of 95% ethanol containing 0.3 M guanidine hydrochloride. A final wash was performed with 100% ethanol and the protein pellet was air dried for 10 min.
  • Pre-stained broad band SDS- PAGE standard and protein samples were dissolved into Ix SDS loading buffer: 50 mM Tris Cl, pH 6.8, 100 mM dithiothreitol, 2% SDS, 1% bromophenol blue and 10% glycerol.
  • Ten ⁇ g of total proteins were electrophoresed in a 7.5% gradient polyacrylamide gel.
  • TTBS which contained 0.1% (v/v) TWEEN 20TM in 100 mM Tris, 0.9% NaCl, pH 7.5, followed by 1 h blocking with 5% low fat dry milk in TTBS.
  • TWEEN 20TM also known generically as Polysorbate 20, is a surfactant and spreading agent.
  • Membranes were incubated for 1 h at room temperature with primary antibody against p63 (1:250 dilution) and keratin 3 (1:1000 dilution). After washing with TTBS, each membrane was transferred to a 1 :200 diluted solution of biotinylated goat anti-mouse antibody in TTBS containing 1% horse serum. After incubating for 30 min, the membrane was incubated with 1:50 diluted VECTASTAIN ELITE® ABC reagent conjugated with peroxidase for 30 min and developed in diaminobenzidine (DAB) (DAKO, Carpintera, CA, USA) between one and three min.
  • DAB diaminobenzidine
  • This stratified epithelium consisted of superficial large squamous cells, intermediate wing cells, and small basal epithelium, which was associated with pigmentation. The superficial surface was smooth, while the basal surface was undulating. Immunostaining of the isolated limbal epithelial sheet showed strong intracytoplasmic staining to the AE-5 antibody, which recognizes keratin 3, in the full thickness stratified epithelium corresponding to the peripheral corneal epithelium, and suprabasal cell layers of the limbal epithelium. This AE-5 staining pattern showing the basal negativity of keratin 3 has been reported as a proof of limbal epithelial SC.
  • Hematoxylin staining showed that the limbal epithelium was loosely adherent to the underlying stroma as evidenced by the spaces created in between.
  • the staining to collagen IV was positive in the blood vessels and the superficial stroma of the limbus with discontinuous staining in the basement membrane area of the basal surface of the loose limbal epithelial sheet.
  • the staining to collagen VII was linearly positive in the superficial stroma of the corneal portion, but was weak in the superficial stroma of the limbus after digestion. Under a higher magnification, the strong lineal pattern of staining was located in the basement membrane zone of the peripheral cornea. Nevertheless, the staining was diffuse in the superficial stroma of the limbus. Staining to laminin 5 was negative in the basement membrane zone of the entire region, suggesting total digestion of this protein during the 18-hour incubation.
  • Human keratocytes were isolated from central corneal buttons by digestion in 1 mg/ml of collagenase A in DMEM and seeded on plastic or the stromal matrix of human amniotic membrane (AM) in DMEM with different concentrations of FBS. Upon confluency, cells on AM were continuously subcultured for 6 passages on AM or plastic. In parallel, cells cultured on plastic at passages 3 and 11 were seeded back to AM. Cell morphology and intercellular contacts were assessed by phase contrast microscopy and LIVE AND DEATH assay, respectively. Expression of keratocan was determined by RT-PCR and Western blotting. Results
  • Trephined stroma yielded 91 ,600 ⁇ 26,300 cells (ranging from 67,000 to 128,000 cells per corneal button). Twenty-four hours after seeding, cells appeared dendritic on AM but fibroblastic on plastic even in 10% FBS. Such a difference in morphology correlated with expression of keratocan assessed by RT-PCR and Western blot, which was high and continued at least to passage 6 on AM even in 10% FBS, but was rapidly lost each time when cells on AM were passaged on plastic. Fibroblasts continuously cultured on plastic to passage 3 and 11 did not revert their morphology or synthesize keratocan when re-seeded on plastic in 1% FBS or on AM. Human keratocytes maintain their characteristic morphology and keratocan expression when subcultured on AM stromal matrix even in the presence of high serum concentrations. This method can be used to engineer a new corneal stroma.
  • the remaining corneal stroma was cut into 0.5 mm x 0.5 mm pieces. These stromal pieces ( ⁇ 12 per cornea) were then incubated at 37 0 C for 45 min in DMEM containing 1 mg/ml collagenase A in a plastic dish. After incubation, collagenase A was removed by pipetting and the digested stromal pieces were incubated in a second aliquot of collagenase A for another 45 min or until the tissue became "smeared" onto the bottom of the dish.
  • the digested tissue was then centrifuged at 800 x g for 5 min and resuspended in 1.5 ml of DMEM containing 20 mM HEPES, 50 ⁇ g/ml gentamicin and 1.25 ⁇ g/ml amphotericin per cornea. This keratocytes-containing cell suspension was then seeded on plastic dishes or the stromal side of the AM.
  • Epithelially denuded AM with the stromal side facing up was tightened to a small plastic insert -32 mm diameter- using a rubber band in a manner similar to what has been reported. See Grueterich M, Espana E, Tseng SC, "Connexin 43 expression and proliferation of human limbal epithelium on intact and denuded amniotic membrane," Invest Ophthalmol Vis Sd 2002;43:63-71.
  • the keratocyte cell suspension prepared from one corneal button was seeded on each 32 mm insert or a 35 mm plastic dish. They were cultured in a medium containing DMEM supplemented with 10% FBS, and the medium was changed every 2 - 3 days.
  • cells were re-suspended in DMEM containing 10% FBS, subdivided into 2 equal parts, with one being seeded onto AM stroma and the other on a plastic dish. They were cultured in DMEM containing 10% FBS.
  • the AM culture was subcultured to either AM or plastic culture in the same manner as described above for a total of 6 passages.
  • cells grown on plastic in DMEM containing 10% FBS were continuously subcultured at 1:3 split on plastic.
  • Cells on plastic at passage 3 and 11 were seeded on plastic in DMEM containing 1%, 5%, or 10% FBS or on AM stromal matrix in DMEM containing 10% FBS to see if there was any reversibility in morphology and keratocan expression.
  • RNA was extracted by TRIZOL® reagent from two 8 mm central corneal buttons, which had been minced with a blade and sonicated at 6000 rpm using a TISSUE TEARORTM sonicator (Biospec Products INC, Racine, WI) as a positive control.
  • Total RNA was similarly extracted from cells cultured on plastic or AM.
  • Total RNA equivalent to 1 x 10 5 cultured cells or one corneal button was subjected to RT-PCR based on a protocol recommended by Promega.
  • the final concentration of RT reaction was 10 mM Tris-HCl (pH 9.0 at 25 °C), 5 mM MgCl 2 , 50 mM KCl, 0.1 % Triton X- 100, 1 mM each dNTP, 1 unit/ml recombinant RNase in ribonucleases inhibitor, 15 units AMV reverse transcriptase, 0.5 mg OHgo(dT)15 primer and total RNA in a total volume of 20 ml.
  • the reaction was kept at 42 0 C for 60 min.
  • PCR reaction 10 mM Tris-HCl (pH 8.3 at 25 0 C), 50 mM KCl, 1.5 mM Mg(O Ac)2, 1.25 units of Taq DNA polymerase in a total volume of 50 ml using primers shown in Table 1.
  • the PCR mixture was first denatured at 94 0 C for 5 min then amplified for 30 cycles (94 0 C, 1 min; 60 0 C, lmin; 72 0 C, 1 min) using PTC-100 Programmable thermal Controller (MJ Research Inc, USA).
  • bp 1059 base pair
  • cells grown on plastic barely expressed keratocan transcript in 1% FBS, but rapidly lost keratocan expression in 5% or 10% FBS.
  • keratocan transcript (1 ,059 bp) was expressed in all AM cultures but largely lost when subcultured from AM to plastic cultures (Prior Art), especially after passage 3.
  • expression of lumican transcript (1,015 bp) and collagen III-al transcript (568bp) was detected in both AM and (Prior art) plastic cultures.
  • normal corneal stroma (K) expressed keratocan and lumican but not collagen III-a 1.
  • transcripts of lumican (1015 base pair (bp)) and collagen III-al (568 bp) were uniformly expressed by cells grown on AM and plastic for up to passage 5 (Fig. 12).
  • the normal cornea stroma (K) expressed keratocan, lumican but not collagen III-al.
  • collagen Hl-al is not expressed by normal corneal stroma, but expressed in wounded cornea has been reported.28
  • compositions and methods of use for generating human amniotic epithelial cell feeder layers are compositions and methods of use for generating human amniotic epithelial cell feeder layers.
  • the methods provide for ways of generating a feeder layer that allows for animal cells to be grown on it without causing differentiation of the present state of those cells.
  • DMEM Dulbecco's modified Eagle's medium
  • F12 F-12 nutrient mixture
  • HBSS Hank's balanced salt solutions
  • KSFM defined keratinocyte serum-free medium with supplement
  • PBS phosphate buffered saline
  • trypsin/lmM EDTA were purchased from Gibco-BR
  • DMEM/F-12 mixture Calcium-free DMEM/F-12 mixture, cholera toxin, dimethyl sulfoxide, hydrocortisone, insulin-transferrin-sodium selenite media supplement and mouse-derived epidermal growth factor (EGF) were obtained from Sigma- Aldrich (St. Louis, MO).
  • CK4 mouse-anti-human antibodies to CK4 (Sigma, St. Louis, MO), CK5/6 (Santa Cruz Biotechnology, Santa Cruz, CA), CK8 (ICN Biomedicals, Irvine, CA), CK14 (Chemicon, Temecula, CA), CKl 7 (Sigma, St.
  • CKl 8 (DakoCytomation, Carpinteria, CA), CK19 (DakoCytomation, Carpinteria, CA), pan-cytokeratin (AE1/AE3, DakoCytomation, Carpinteria, CA), Ki67 (DakoCytomation, Carpinteria, CA), p63 (DakoCytomation, Carpinteria, CA), and ABCG2 (Chemicon, Temecula, CA); rabbit-anti-human polyclonal antibodies to vimentin (Abeam, Cambridge, MA), connexin 43 (Zymed, San Francisco, CA) and Musashi-1 (Abeam, Cambridge, MA); goat-anti- human polyclonal antibody to CK12 (Santa Cruz Biotechnology, Santa Cruz, CA). For cell proliferation assay, a MTT kit was purchased from Roche (Indianapolis. IN).
  • Isolated epithelial sheets were further digested with 0.25% Trypsin/lmM EDTA at 37 0 C for 15 min; dissociated cells were collected after centrifugation at 2,000 rpm for 5 min. Cell viability was determined by exclusion of trypan blue dye and counted with a hemocytometer.
  • HAECs immediately harvested from the fresh placenta could be cultured in all 9 different media that varied in [Ca 2+ ], FBS concentration, and supplements of different growth factors.
  • serum-free, calcium-free or low calcium media HAECs became small and round with a high nucleus-to-cytoplasm (N/C) ratio, and were scattered around without formation of cell-cell junction.
  • N/C nucleus-to-cytoplasm
  • the proliferation rate indicated by the number of Ki67-positive cells, was extremely low.
  • some cells gradually lost epithelial markers, i.e., pan-CK over time and turned into Vim-expressing cells.
  • HAECs In serum-free, high-calcium media HAECs remained small and round, and had a high N/C ratio, but gathered to form a cluster or a small sheet. Cell proliferation was still largely halted. Intriguingly, HAECs also maintained their in vivo phenotype, showing as a mixture of pan-CK(+)/Vim(-) cells and pan-CK(+)/Vim(+) cells.
  • HAECs changed to a cobble-stone-like shape with a low N/C ratio, and formed a sheet with obviously discernible intercellular junctions.
  • the proliferation activity was notably increased, as indicated by numerous Ki-67 positive cells.
  • Immunostaining showed that cells expressed both pan-CK and vimentin at the same time with the majority of HAECs exhibiting colocalization of pan-CK and Vim while a minority showing a pan-CK-only phenotype.
  • a fibrillar staining pattern was noted in plethora of cells, while a few cells had a unique perinuclear "vimentin ring", suggestive of de novo synthesis of vimentin filaments in those cells.
  • Example 16 Preferential Isolation of Limbal Epithelial Stem Cells and Stem-like Cells by Rapid Adhesion to Collagen.
  • the stem cell population of the corneal epithelium is located in the limbus, the anatomic junction between the cornea and the conjunctiva.
  • the other type of proliferating epithelial cells are transit-amplifying cells, which are destined to undergo terminal differentiation aftdrjt few rounds of division. Both types of proliferating cells are located in the basal limbal epithelium in contact with the underlying basement membrane, while cells undergoing terminal differentiation migrate to suprabasal cell layers.
  • Plastic cell culture dishes (6 well-plates) were purchased from Falcon (Franklin Lakes, NJ). Rat tail collagen, type I was from BD Biosciences (Bedford, MA). Keratinocyte serum-free medium (KSFM), Dulbecco's modified Eagle's medium (DMEM), Ham F-12, fetal bovine serum (FBS), HEPES-buffer, Dulbecco's phosphate- buffered saline (D-PBS), amphotericin B, gentamicin and 0.25% trypsin/EDTA solution were from Invitrogen- GIBCO BRL (Grand Island, NY).
  • KSFM Keratinocyte serum-free medium
  • DMEM Dulbecco's modified Eagle's medium
  • FBS fetal bovine serum
  • HEPES-buffer Dulbecco's phosphate- buffered saline
  • amphotericin B gentamicin and 0.25% trypsin/EDTA solution
  • Dispase II powder was obtained from Roche (Basel, Switzerland), and mitomycin C (MMC), ITS (bovine insulin, human transferrin, sodium selenite), hydrocortisone, human EGF, cholera toxin, dimethyl sulfoxide (DMSO), heat-denatured bovine serum albumin (BSA), and Hoechst 33342 were from Sigma (St Louis, MO).
  • MMC mitomycin C
  • ITS bovine insulin, human transferrin, sodium selenite
  • hydrocortisone human EGF
  • cholera toxin cholera toxin
  • dimethyl sulfoxide (DMSO) dimethyl sulfoxide
  • BSA heat-denatured bovine serum albumin
  • Hoechst 33342 Hoechst 33342
  • KSFM KSFM containing 10 mg/ml Dispase II at 4 °C for 16 hours or at 37 0 C for 2 hours, and the limbal epithelial sheet removed using a spatula and forceps under a dissecting microscope.
  • slow adherent cells Some of these unattached cells, termed slow adherent cells, attach to the collagen-coated dish in 24 hours, at which time the conditioned medium was collected after non-adherent cells were cleared by centrifugation (4°C x lOOOrpm for 5min). For comparison, control cultures, in which slow adherent cells were not removed, were performed in parallel. The media were changed every 2 to 3 days. After the optimal duration for maximizing the recovery of most undifferentiated progenitor cells was determined, rapid adherent cells, separated from slow adherent cells, were cultured in KSFM on collagen I coated dishes. Upon reaching confluency, these cells were terminated for further characterization.
  • Subculture of the epithelial cells The primary cultured cells were subcultured into Pl passage in the following two ways: (a) using the rapid adhesion method (i.e, at every passage, the collagen coated dishes were used and the unattached cells were removed; R+/R+); (b) on collagen coated dishes without removal of the floating cells (R-/R-).
  • the phenotype and characteristics of the rapid adherent cells (R+/R.+) and non-selected cells (R-/R-) were evaluated based on adult stem cell criteria at every passages.
  • the cells were seeded on a 3T3 feeder layer to assess their growth potential. Approximately 4x10 3 cells per chamber from each group were seeded into wells of 8- chamber culture slides, incubated at 37°C overnight, and then fixed for immunofluorescent staining.
  • Immunofluorescent staining Limbal epithelial cultures and selected cell populations on 8-chamber slides and 24-well plate were fixed with cold methanol (for cytoplasmic and nuclear protein staining) for 15 min. After blocking with 2% normal goat serum in PBS for 30 min, primary monoclonal antibodies against cytokeratin 12 (1:100), nuclear p63 (1:40), a-SMA (1:100), Ki-67 (1:100), vimentin (1:20), and Pan-cytokeratin (1:100) diluted with 1% normal goat serum in PBS were applied and incubated overnight at 4°C.
  • Appropriate secondary antibodies ( 1 : 100) were applied and incubated in a dark chamber for 1 hr at RT, followed by counterstaining with Hoechst 33342 DNA binding dye. After washing with PBS, slides were mounted in antifading solution (Vectashield; VectorbLaboratories) and covered with the cover slip. Slides were examined and photographed with an epifluorescent microscope (TE-2000U Eclipse; Nikon, Japan). The percentages of positively stained cells were calculated by point counting through a Nikon TE-2000U inverted microscope using a 2Ox objective lens and a 1 Ox subjective lens. A total of 561-1383 nuclei were counted in 6-9 representative fields.
  • Colony forming efficiency - MMC treated 3T3 fibroblast feeder layers were used to evaluate proliferative potential of the cell populations selected by adhesion to collagen I.
  • NIH 3T3 cells were cultured in DMEM 10% FBS medium and treated with MMC (4 ⁇ g/ml) at 37°C for 2 hours when they reached to confluence and then trypsinized and plated at a density of 2xlO 4 cells/cm 2 in 6-well plates.
  • Each cell population was seeded at the density of 500 cells/well onto the sub-confluent 3T3 feeder layer into 6-well culture plates at least in triplicate.
  • the colony forming efficiency (CFE) was calculated at day 12 as a percentage of the number of colonies divided by the number of seeding cells, and the CFE was evaluated by using the staining with crystal violet.
  • Rapid Adherent Cells Indeed Had A higher Proliferative Potential Than Slow Adherent Cells in KSFM -Based on the above results, a 5 min incubation time was used to select rapid adherent cells, while the non-adherent cells were transferred to a new collagen I coated plastic dish. After incubation for 24 hours, the non-adherent cells were removed, and the remaining adherent cells were termed "slow adherent cells".
  • rapid adherent cells proliferate to form large epithelial clones in 1 week (Fig. 9A). On the contrary, slow adherent cells formed smaller and less organized epithelial clones under the same condition (Fig. 9B).
  • immunofluorescent staining with antibodies against vimentin and pan-cytokeratins was performed. As shown in Fig.
  • Rapid adherent cells have significant proliferative potential -
  • rapid and slow adherent cells were co-cultured with MMC-treated 3T3 fibroblast feeder layer in triplicate at a density of 500 cells / 6-well in SHEM medium.
  • the colony forming efficiency (CFE) on day 12 from both rapid and slow adherent cells was 12.4 ⁇ 1.7 % and 6.7 ⁇ 0.4 %, respectively.
  • Culturing limbal epithelial cells in low-calcium serum-free KSFM medium also had excellent CFE, suggesting that this system promotes good clonal growth of limbal progenitor cells (Fig. 1OA ⁇ D).
  • CFE was maintained with an increase in the passage number.
  • the CFE of limbal epithelial cells co-cultured with 3T3 fibroblasts was significantly lower, indicating that the 3T3 system promotes less clonal growth of subcultured limbal progenitor cells.
  • Example 17 Limbal Epithelial Progenitors That Lie Deeper Than Dispase AloneCan Isolate Require Contact with Limbal Niche Cells for Clonal Growth.
  • Limbal epithelial stem cells located in the limbal basal layer, are presumably under regulation by the limbal niche.
  • Single epithelial cells obtained from dispase-isolated human limbal epithelial sheets were fractionated by rapid adhesion on collagen I-coated dishes for 12 min before being cultured in KSFM medium. Cells isolated from the remaining limbal stroma by collagenase were briefly treated with or without 0.25% trypsin/EDTA.
  • PCK pan-cytokeratins
  • Vim vimentin
  • p63 p63
  • ABCG2 pan-cytokeratins
  • Example 18 Human Amniotic Epithelial Cells as Novel Feeder Layers for Ex Vivo Expansion of Multipotent Limbal Epithelial Profienitor Cells.
  • Intact cryopreserved amniotic membrane with devitalized human amniotic epithelial cells (HAECs) can, but epithelially-denuded cryopreserved amniotic membrane cannot, help expand human limbal epithelial progenitor cells (HLECs) without murine 3T3 fibroblast feeder layers.
  • HLECs human limbal epithelial progenitor cells
  • HLECs human limbal epithelial progenitor cells
  • HLECs human limbal epithelial progenitor cells
  • HLECs human limbal epithelial progenitor cells
  • HAECs were isolated from a fresh amnion by enzymatic digestion and serially cultured in different media varying in [Ca 2+ ], FBS concentration, and supplements of different growth factors. Feeder cells culturing conditions were investigated by immunostaining to pan-cytokeratins(CK)/vimentin(Vim) and MTT assay. HLEC clonal cultures and subcultures in different media or feeder layers on mitomycin C-treated HLAEC feeder layers were compared to those on traditional 3T3 fibroblast feeder layers regarding colony-forming efficiencies, differentiation and stem cell-associated markers. [00223] HAECs uniformly expressed pan-CK and heterogeneously expressed Vim.
  • Pan-CK(+)/Vim(+) and Pan-CK(+)/Vim(-) cells were maintained in serum-free media with high calcium, but some HAECs turned into pan-CK(-)/Vim(-) only in serum-free media with low or no calcium. In contrast, all HAECs became pan-CK(+)/Vim(+) in serum-containing media with an increase in proliferation, and could be subcultured for at least 8 passages in SHEM medium supplemented with EGF and insulin.
  • Mitomycin C-treated HAEC feeder layers were significantly more effective in promoting clonal growth of HLEC progenitor cells than 3T3 feeder layers as judged by a smaller cell size, less Kl 2 keratin expression, lack of connexin 43 expression, and higher percentages of stem cell-associated markers such as p63, Musashi-1 and ABCG2.
  • Clonally expanded HLECs from HAEC feeder layers could further differentiate into neurons and nestin-positive neuronal progenitors when subcultured in serum-free, feeder cell-free KSFM medium or HAEC feeder layers, while those on 3T3 feeder layers might changed into fibroblasts.

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Abstract

La présente invention concerne des procédés d'isolement, de purification et d'extension de cellules souches animales et de cellules de type souche. Les procédés d'isolement incluent des conditions permettant de digérer préférentiellement des cellules n'étant pas des cellules souches ou de type souche dans une population et de provoquer préférentiellement l'adhérence de cellules souches et de cellules de type souche dans une population. Les procédés d'extension consistent à cultiver de telles cellules dans des conditions permettant de moduler le signal TGF-β, d'inhiber le signal cellulaire médié par la p38 MAP kinase au moyen d'inhibiteurs de petite masse moléculaire, de provoquer l'extension des cellules sur des cellules épithéliales amniotiques humaines utilisées comme couches nourricières, de réguler la densité de l'ensemencement des cellules, de réguler les niveaux de Ca2+ dans les milieux de culture, de provoquer l'adhérence rapide sur un substrat, ou à cultiver les cellules dans lesdites conditions combinées. Plus particulièrement, l'invention concerne des procédés et des systèmes permettant l'extension de cellules animales dans des cultures cellulaires ex vivo tout en prévenant une différenciation cellulaire, et en enrichissant de manière sélective des cellules souches. Les modes de réalisation portent également sur un système de culture permettant l'extension ex vivo de cellules épithéliales ou de cellules mésenchymateuses limbiques, aussi bien que des greffes chirurgicales pratiquées au départ de ces cellules.
PCT/US2006/049500 2006-05-17 2006-12-28 Isolement et extension de cellules animales dans des cultures cellulaires WO2008002329A2 (fr)

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US80149106P 2006-05-17 2006-05-17
US60/801,491 2006-05-17
US11/476,376 US20070010008A1 (en) 2005-06-29 2006-06-28 Ex vivo expansion of primary animal cells
US11/476,376 2006-06-28

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2513297A1 (fr) * 2009-12-18 2012-10-24 Sino-American Icell (Shanghai) Biotechnology Co., Ltd. Matériels et méthodes pour la production de cellules souches pluripotentes
WO2014158089A1 (fr) * 2013-03-28 2014-10-02 Ge Healthcare Bio-Sciences Ab Méthode de culture cellulaire
CN104781389A (zh) * 2012-08-23 2015-07-15 日产化学工业株式会社 蛋白质生产促进剂
US9664671B2 (en) 2012-07-24 2017-05-30 Nissan Chemical Industries, Ltd. Culture medium composition and method of culturing cell or tissue using thereof
US9944895B2 (en) 2013-03-28 2018-04-17 Ge Healthcare Bio-Sciences Ab Method for cell culture
US10017805B2 (en) 2012-08-23 2018-07-10 Nissan Chemical Industries, Ltd. Enhancing ingredients for protein production from various cells
EP3553169A1 (fr) * 2011-12-28 2019-10-16 Kyoto Prefectural Public University Corporation Normalisation de culture de cellules endothéliales cornéennes

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007002900A1 (fr) * 2005-06-29 2007-01-04 Tissue Tech, Inc. Expansion ex vivo de cellules animales primaires

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007002900A1 (fr) * 2005-06-29 2007-01-04 Tissue Tech, Inc. Expansion ex vivo de cellules animales primaires

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
BONFIELD W. ET AL.: 'Designing porous scaffolds for tissue engineering' PHILOSOPHICAL TRANSACTIONS. SERIES A, MATHEMATICAL, PHYSICAL, AND ENGINEERING SCIENCES vol. 364, no. 1838, January 2006, pages 227 - 232 *
KAWAKITA T. ET AL.: 'Keratocan Expression of Murine Keratocytes Is Maintained on Amniotic Membrane by Down-regulating Transforming Growth Factor-beta Signaling' THE JOURNAL OF BIOLOGICAL CHEMISTRY vol. 280, no. 29, 2005, pages 27058 - 27092 *
KAWAKITA T. ET AL.: 'Preservation and Expansion of the Primate Keratocyte Phenotype by Downregulating TGF-beta Signaling in a Low-Calcium, Serum-Free Medium' INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE vol. 47, no. 5, May 2006, pages 1918 - 1927 *
YAM H.F. ET AL.: 'Growth Factor Changes in Ex Vivo Expansion of Human Limbal Epithelial Cells on Human Amniotic Membrane' CORNEA vol. 21, no. 1, 2002, pages 101 - 105 *
YEH L.K. ET AL.: 'Soluble Lumican Glycoprotein Purified from Human Amniotic Membrane Promotes Corneal Epithelial Wound Healing' INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE vol. 46, no. 2, 2005, pages 479 - 486 *

Cited By (18)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2513297A4 (fr) * 2009-12-18 2013-07-31 Sino American Icell Shanghai Biotechnology Co Ltd Matériels et méthodes pour la production de cellules souches pluripotentes
US9234179B2 (en) 2009-12-18 2016-01-12 Shanghai Icell Biotechnology Co., Ltd. Materials and methods for generating pluripotent stem cells
EP2513297A1 (fr) * 2009-12-18 2012-10-24 Sino-American Icell (Shanghai) Biotechnology Co., Ltd. Matériels et méthodes pour la production de cellules souches pluripotentes
EP3553169A1 (fr) * 2011-12-28 2019-10-16 Kyoto Prefectural Public University Corporation Normalisation de culture de cellules endothéliales cornéennes
US9664671B2 (en) 2012-07-24 2017-05-30 Nissan Chemical Industries, Ltd. Culture medium composition and method of culturing cell or tissue using thereof
US11371013B2 (en) 2012-07-24 2022-06-28 Nissan Chemical Industries, Ltd. Culture medium composition and method of culturing cell or tissue using thereof
US10590380B2 (en) 2012-07-24 2020-03-17 Nissan Chemical Industries, Ltd. Culture medium composition and method of culturing cell or tissue using thereof
US10017805B2 (en) 2012-08-23 2018-07-10 Nissan Chemical Industries, Ltd. Enhancing ingredients for protein production from various cells
EP2889368A4 (fr) * 2012-08-23 2016-06-15 Nissan Chemical Ind Ltd Accélérateur de production de protéine
CN104781389A (zh) * 2012-08-23 2015-07-15 日产化学工业株式会社 蛋白质生产促进剂
JP2016517686A (ja) * 2013-03-28 2016-06-20 ジーイー・ヘルスケア・バイオサイエンス・アクチボラグ 細胞培養方法
US9714415B2 (en) 2013-03-28 2017-07-25 Ge Healthcare Bio-Sciences Ab Method for cell culture
US9944895B2 (en) 2013-03-28 2018-04-17 Ge Healthcare Bio-Sciences Ab Method for cell culture
US10011816B2 (en) 2013-03-28 2018-07-03 Ge Healthcare Bio-Sciences Ab Method for cell culture
US10125349B2 (en) 2013-03-28 2018-11-13 Ge Healthcare Bio-Sciences Ab Method for cell culture
CN105102610A (zh) * 2013-03-28 2015-11-25 通用电气健康护理生物科学股份公司 用于细胞培养的方法
CN105102610B (zh) * 2013-03-28 2020-10-13 通用电气健康护理生物科学股份公司 用于细胞培养的方法
WO2014158089A1 (fr) * 2013-03-28 2014-10-02 Ge Healthcare Bio-Sciences Ab Méthode de culture cellulaire

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