Methods and applications of mammalian retinal stem cell production

Abstract

The invention provides an in vitro method for producing an isolated mammalian primitive retina stem cell (pRSC), comprising: (a) culturing an isolated embryonic stem cell (ESC) from a mammal in a cell culture medium that is free of feeder cells, feeder layer conditioned medium, or serum to produce and grow a culture of the isolated ESC; and (b) contacting the culture of the isolated ESC so grown with one or more of a Wnt or TGF-beta / BMP signaling inhibitor to differentiate the isolated ESC of (a) into a primitive retina stem cell, thereby producing an isolated mammalian pRSC.

Description

[0001] This application is a divisional application of Chinese invention patent application No. 201480066229.3, filed on October 9, 2014, entitled "Method and Application for Generating Mammalian Retinal Stem Cells", which was filed after PCT international application No. PCT / US2014 / 059951 entered the Chinese national phase.

[0002] This invention was made with government support from NIH grants R01 EY021374 and R01 EY018660. The government owns certain rights in this invention.

[0003] Various publications have been cited throughout this application. The disclosures of these publications are incorporated herein by reference in their entirety to provide a more complete description of the prior art to which this invention pertains. Technical Field

[0004] This disclosure generally relates to methods for the in vitro generation of stem cells and their applications, and more specifically to methods for the in vitro generation of mammalian retinal stem cells and their applications. Background Technology

[0005] The leading causes of irreversible blindness include age-related macular degeneration (AMD), retinitis pigmentosa, glaucoma, and retinal vascular disease, which causes the loss of retinal progenitor cells (RPEs), photoreceptors, retinal ganglion cells (RGCs), and supporting cells in the retina. One potential treatment is to restore visual function by transplanting healthy retinal cells to replace the lost ones. In recent decades, limited success has been achieved using primary retinal progenitor cells isolated from human fetal or adult retinal tissue. 1 This is due to the low expansion capacity and differentiation potential of adult progenitor cells, or the difficulty in obtaining sufficient human fetal retinal progenitor cells, raising ethical concerns. Human pluripotent stem cells (hPSCs) (including human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs)) represent a promising renewable donor source for cell-based replacement therapies. However, hPSCs themselves are not suitable for direct transplantation in clinical applications due to their tendency to form teratomas and their low efficiency in reconstructing host tissues in vivo with the desired reprogrammed cell types.

[0006] Therefore, the main efforts are focused on generating differentiation derivatives of hPSCs, such as neural retinal progenitor cells. 2,3 retinal pigment epithelium (RPE) 4-6 photosensors 7-9Although hESC-derived RPE transplantation has progressed to clinical trials for the treatment of patients with Stargardt's macular dystrophy and AMD. 10 However, if most photoreceptors have been lost, the effectiveness of RPE grafts may be limited. Therefore, there is a strong desire to develop a regenerative retinal stem cell product that has the potential to reconstruct both the RPE and photoreceptors in the degenerated retina, without posing a risk of tumor formation. Such a product could replace damaged cells and restore visual circuitry in conditions where multiple cell types are affected. Summary of the Invention

[0007] The present invention provides an in vitro method for generating isolated mammalian primitive retinal stem cells (pRSCs), comprising: (a) culturing isolated embryonic stem cells (ESCs) from mammals in a cell culture medium free of feeder cells, feeder-conditioned medium, or serum to generate and grow a culture of said isolated ESCs; and (b) contacting said culture of said isolated ESCs thus grown with one or more of Wnt or TGF-β / BMP signaling inhibitors to differentiate said isolated ESCs of (a) into primitive retinal stem cells, thereby generating isolated mammalian pRSCs.

[0008] The present invention further provides an in vitro method for generating isolated mammalian primitive retinal stem cells (pRSCs), comprising: (a) culturing isolated pluripotent stem cells (PSCs) from mammals in a cell culture medium free of feeder cells, feeder layer conditioned medium, or serum to generate and grow a culture of said isolated PSCs; and (b) contacting said culture of said isolated PSCs grown in this manner with one or more of Wnt or TGF-β / BMP signaling inhibitors to differentiate said isolated PSCs into primitive retinal stem cells, thereby generating isolated mammalian pRSCs.

[0009] The present invention further provides an in vitro method for generating isolated mammalian primitive retinal stem cells (pRSCs), comprising: (a) culturing isolated induced pluripotent stem cells (iPSCs) from mammals in a cell culture medium free of feeder cells, feeder layer conditioned medium, or serum to generate and grow a culture of said isolated iPSCs; and (b) contacting said culture of said isolated iPSCs grown in this manner with one or more of Wnt or TGF-β / BMP signaling inhibitors to differentiate said isolated iPSCs into primitive retinal stem cells, thereby generating isolated mammalian pRSCs.

[0010] The present invention further provides an in vitro method for generating isolated mammalian retinal ganglion cells (RGCs), comprising: (a) culturing isolated primitive retinal stem cells (pRSCs) from mammals in a cell culture medium free of feeder cells, feeder layer conditioned medium, or serum to generate and grow a culture of said isolated pRSCs; and (b) contacting said culture of said isolated pRSCs grown in this manner with one or more of Wnt, Notch, or VEGFR signaling inhibitors to differentiate said isolated pRSCs into isolated mammalian RGCs, thereby generating isolated mammalian RGCs.

[0011] The present invention also provides a method for generating isolated mammalian photoreceptors from isolated mammalian pRSCs, comprising: (a) culturing and growing dissociated pRSCs from mammals in a neural induction medium containing one or more inhibitors of TGF-β / activin receptor-like kinases ALK-4, ALK-5 or ALK-7, glycogen synthase kinase-3 (GSK-3), Notch or Wnt signaling, or hedgehog signaling activator, for a sufficient time to induce the pRSCs to a photoreceptor cell lineage fate with no visible morphological changes or no expression of photoreceptor-specific markers; and (b) thereafter culturing and growing the pRSCs of step (a) in a neural induction medium containing retinoic acid or taurine or both to differentiate the mammalian pRSCs into photoreceptors, thereby generating isolated mammalian photoreceptors; wherein the medium does not contain feeder cells, feeder layer conditioned medium, or serum.

[0012] Furthermore, the present invention provides a method for generating non-neural isolated mammalian retinal pigment epithelium (RPE) or isolated mammalian retinal pigment epithelial cells (RPEs), comprising: (a) culturing pRSCs from mammals in a medium containing nicotinamide or activin A or both, in the absence of SMAD signaling inhibitors, for a sufficient time to induce the pRSCs to the RPE fate; and (b) culturing the pRSCs in a medium containing one or more of N1 medium supplement, taurine, hydrocortisone, or triiodothyronine to differentiate the mammalian pRSCs into mammalian RPEs or RPEs, thereby generating isolated mammalian RPEs or RPEs, wherein the medium is free of feeder cells or feeder layer conditioned medium.

[0013] The present invention further provides a method for generating isolated human primitive retinal stem cells (hpRSCs) from isolated human embryonic stem cells (hESCs), comprising: (a) culturing the isolated hESCs on a solid support having a medium in the absence of feeder cells, feeder layer conditioned medium, or serum for a sufficient time to grow to near confluence, preferably to about 80% cell confluence; (b) culturing the thus-grown isolated hESCs on a solid support in a medium containing alkaline FGF (bFGF) for a sufficient time to grow to near confluence; and (c) culturing the isolated hESCs of step (b) on a solid support having a medium containing a combination of small molecule inhibitors of Wnt and TGF-β / BMP signaling activity to differentiate the isolated hESCs into isolated human primitive retinal stem cells, thereby generating isolated hpRSCs.

[0014] Furthermore, the present invention provides a method for generating isolated human primitive retinal stem cells (hpRSCs) from isolated human pluripotent stem cells (hPSCs), comprising: (a) culturing the isolated hPSCs on a solid support having a medium in the absence of feeder cells, feeder layer conditioned medium, or serum for a sufficient time to grow to near confluence, preferably to about 80% cell confluence; (b) culturing the thus-grown isolated hPSCs on a solid support in a medium containing bFGF for a sufficient time to grow to near confluence; and (c) culturing the isolated hPSCs of step (b) on a solid support having a medium containing a combination of small molecule inhibitors of Wnt and TGF-β / BMP signaling activity to differentiate the isolated hPSCs into isolated human primitive retinal stem cells, thereby generating isolated hpRSCs.

[0015] The present invention also provides a method for generating isolated human primitive retinal stem cells (hpRSCs) from isolated human induced pluripotent stem cells (iPSCs), comprising: (a) culturing the isolated human iPSCs on a solid support having a medium in the absence of feeder cells, feeder layer conditioned medium, or serum for a sufficient time to grow to near confluence, preferably to about 80% cell confluence; (b) culturing the thus-grown isolated iPSCs on a solid support in a medium containing bFGF for a sufficient time to grow to near confluence; and (c) culturing the isolated iPSCs of step (b) on a solid support having a medium containing a combination of small molecule inhibitors of Wnt and TGF-β / BMP signaling activity to differentiate the isolated iPSCs into isolated human primitive retinal stem cells, thereby generating isolated hpRSCs.

[0016] The present invention further provides a kit for generating mammalian primitive retinal stem cells (pRSCs), wherein the kit includes instructions for culturing mammalian embryonic stem cells (ESCs), pluripotent stem cells (PSCs), or induced pluripotent stem cells (iPSCs) in a culture medium with a chemical composition defined free of feeder cells, feeder layer conditioned medium, or serum, and instructions for using small molecule inhibitors of Wnt signaling or TGF-β / BMP signaling, or inhibitors of both Wnt signaling and TGF-β / BMP signaling.

[0017] This invention provides implementation schemes for, for example, the following items:

[0018] 1. An in vitro method for generating isolated mammalian primitive retinal stem cells (pRSCs), comprising:

[0019] (a) Cultures of isolated embryonic stem cells (ESCs) from mammals in a cell culture medium free of feeder cells, feeder layer conditioned medium, or serum to produce and grow cultures of said isolated ESCs; and

[0020] (b) The culture of the isolated ESC grown in this way is contacted with one or more of Wnt or TGF-β / BMP signaling inhibitors to differentiate the isolated ESC of (b) into primitive retinal stem cells, thereby producing isolated mammalian pRSCs.

[0021] 2. An in vitro method for generating isolated mammalian primitive retinal stem cells (pRSCs), comprising:

[0022] (a) Culture of isolated pluripotent stem cells (PSCs) from mammals in a cell culture medium free of feeder cells, feeder layer conditioned medium, or serum to produce and grow cultures of said isolated PSCs; and

[0023] (b) The culture of the isolated PSCs grown in this way is contacted with one or more of Wnt or TGF-β / BMP signaling inhibitors to differentiate the isolated PSCs into primitive retinal stem cells, thereby producing isolated mammalian pRSCs.

[0024] 3. An in vitro method for generating isolated mammalian primitive retinal stem cells (pRSCs), comprising:

[0025] (a) Culture of isolated induced pluripotent stem cells (iPSCs) from mammals in a cell culture medium free of feeder cells, feeder layer conditioned medium, or serum to produce and grow cultures of said isolated iPSCs; and

[0026] (b) The culture of the isolated iPSCs grown in this way is contacted with one or more of Wnt or TGF-β / BMP signaling inhibitors to differentiate the isolated iPSCs into primitive retinal stem cells, thereby producing isolated mammalian pRSCs.

[0027] 4. The method according to Project 1, wherein in step (a), the culture of the isolated ESC thus produced is a monolayer culture.

[0028] 5. The method according to Project 2, wherein in step (a), the culture of the isolated PSC thus produced is a monolayer culture.

[0029] 6. The method according to Project 3, wherein in step (a), the culture of the isolated iPSC thus produced is a monolayer culture.

[0030] 7. The method according to item 1, 2 or 3, wherein in step (b), one or more of the Wnt or TGF-β / BMP signaling inhibitors is a combination of Wnt and TGF-β / BMP signaling inhibitors.

[0031] 8. The method according to item 1, 2 or 3, wherein, prior to step (b), the culture thus produced is grown to near confluence in step (a).

[0032] 9. The method according to item 1, 2 or 3, wherein the mammal is a human, monkey, bear, rat, mouse, mink, rabbit, guinea pig, pig, dog, cat, goat, sheep, horse or cow.

[0033] 10. The method according to item 1, 2 or 3, wherein the isolated mammalian primitive retinal stem cells (pRSCs) are positive for expression of typical early ocular transcription factors PAX6, LHX2, RAX, OTX2, SIX3 and / or SIX6.

[0034] 11. The method according to item 1, 2 or 3, further comprising the isolated mammalian primitive retinal stem cells (pRSCs) that are positive for the expression of stemness factors SOX2 and STAT3.

[0035] 12. The method according to item 1, 2 or 3, further comprising the isolated mammalian primitive retinal stem cells that are positive for Ki67 expression.

[0036] 13. The method according to item 1, 2 or 3, wherein the Wnt signaling inhibitor is a small molecule inhibitor of Wnt signaling selected from the group consisting of: Wnt production inhibitor-1 (IWP-1), Wnt production inhibitor-2 (IWP2), JW55, JW74, okadaic acid, temmelin, SB239063, SB203580, ADP-HPD, 2-[4-(4-fluorophenyl)piperazin-1-yl]-6-methylpyrimidin-4(3H)-one, PJ34, cambinol, sulindac, 3289-8625, scaffold A for a series of analogues designed to inhibit Dishevelled proteins, scaffold B for a series of analogues designed to inhibit Dishevelled proteins, J01-0 17a, NSC668036, Ferulic acid, IC261, PF670462, Bosutinib, PHA665752, Imatinib, ICG-001, Ethacrylic acid, Ethacrylic acid derivatives, PKF115-584, PNU-74654, PKF118-744, CGP049090, PKF118-310, ZTM000990, BC21, GDC-0941, Rp-8-Br-cAMP, LGK974, C59, Ant1.4Br / Ant 1.4CI, Niclosamide, Apicularen, Bafloxacin, XAV939, IWR1, Pyrantel acetonide, NSC668036, 2,4-diamino-quinazoline, Quercetin, and PKF115-584, as well as their equivalents and combinations thereof.

[0037] 14. The method according to item 1, 2 or 3, wherein the Wnt signaling inhibitor is a small molecule inhibitor of Wnt production -2 or a Dkk1 analog.

[0038] 15. The method according to item 14, wherein the Wnt-generating small molecule inhibitor-2 or Dkk1 analog has the chemical formula C 22 H 18 N4O2S3 and the following chemical structures produce Wnt inhibitors -2 (IWP2; CAS No. 686770-61-6) or N-(6-methyl-1,3-benzothiazo-2-yl)-2-[(4-oxo-3-phenyl-6,7-dihydrothieno[3,2-d]pyrimidin-2-yl)thioalkyl]acetamide:

[0039] .

[0040] 16. The method according to Project 1, 2, or 3, wherein the TGF-β / BMP signaling inhibitor is a small molecule inhibitor of TGF-β / BMP signaling selected from the group consisting of: SB431542 (4-[4-(1,3-benzodioxane-5-yl)-5-(2-pyridyl)-1H-imidazol-2-yl]benzamide), A 83-01 (3-(6-methyl-2-pyridyl)-N-phenyl-4-(4-quinolinyl)-1H-pyrazole-1-thiocarboxamide), SJN 2511 (2-(3-(6-methylpyridin-2-yl)-1H-pyrazole-4-yl)-1,5-naphthidine), D 4476 (4-[4-(2,3-dihydro-1,4-benzodioxin-6-yl)-5-(2-pyridinyl)-1H-imidazol-2-yl]benzamide), LY 364947 (4-[3-(2-pyridinyl)-1H-pyrazol-4-yl]quinoline), SB525334 (6-[2-(1,1-dimethylethyl)-5-(6-methyl-2-pyridinyl)-1H-imidazol-4-yl]quinoxaline), SD 208 (2-(5-chloro-2-fluorophenyl)-4-[(4-pyridinyl)amino]pteridine) and LDN-193189 (4-(6-(4-(piperazin-1-yl)phenyl)pyrazolo[1,5-a]pyrimidin-3-yl)quinoline), and their equivalents and combinations thereof.

[0041] 17. The method described in accordance with Item 1, 2 or 3, wherein the TGF-β / BMP signaling inhibitor is a small molecule inhibitor of type I activin receptor-like kinases ALK-4, ALK-5 and ALK-7 of the transforming growth factor-β (TGF-β) superfamily.

[0042] 18. The method according to Project 17, wherein the small molecule inhibitors of the transforming growth factor-β (TGF-β) superfamily type I activin receptor-like kinases ALK-4, ALK-5, and ALK-7 are small molecule inhibitors having the chemical formula C 22 H 16 N4O3 and the following chemical structures: SB431542 (CAS No. 301836-41-9) or 4-[4-(1,3-benzodioxacyclopenten-5-yl)-5-(2-pyridyl)-1H-imidazol-2-yl]benzamide

[0043] .

[0044] 19. The method according to item 1, 2 or 3, wherein the TGF-β / BMP signaling inhibitor is a small molecule inhibitor or head protein analog of BMP type I receptors ALK-2 and ALK-3.

[0045] 20. The method according to item 19, wherein the small molecule inhibitor or head protein analog of the BMP type I receptors ALK-2 and ALK-3 is a compound having the chemical formula C 25 H 22 N6 and the following chemical structures are used in LDN-193189 (CAS No. 1062368-24-4) or 4-(6-(4-(piperazin-1-yl)phenyl)pyrazolo[1,5-a]pyrimidin-3-yl)quinoline

[0046] .

[0047] 21. The method according to Item 7, wherein the combination of Wnt and TGF-β / BMP signaling inhibitors is a combination of: (a) one or more Wnt production inhibitors-2 or Dkk1 analogs; (b) one or more inhibitors of transforming growth factor-β (TGF-β) superfamily type I activin receptor-like kinases ALK-4, ALK-5 and ALK-7; and (c) one or more inhibitors or head protein analogs of BMP type I receptors ALK-2 and ALK-3, wherein the combination comprises inhibitors of at least two signaling pathways or preferably inhibitors of all three signaling pathways.

[0048] 22. The method according to item 21, wherein the inhibitor is a small molecule.

[0049] 23. The method according to item 21, wherein the combination of Wnt and TGF-β / BMP signaling activity inhibitors is a combination or equivalent of at least two, preferably all three, of IWP2 (CAS No. 686770-61-6), SB431542 (CAS No. 301836-41-9), and LDN-193189 (CAS No. 1062368-24-4), wherein the combination produces synergistic inhibition of Wnt and TGF-β / BMP signaling activity; or comprises a combination or equivalent of at least two, preferably all three, of IWP2 (CAS No. 686770-61-6), SB431542 (CAS No. 301836-41-9), and LDN-193189 (CAS No. 1062368-24-4), wherein the combination produces synergistic inhibition of Wnt and TGF-β / BMP signaling activity.

[0050] 24. The method according to item 1, 2 or 3, wherein the cultured cells of step (a) are cultured on a solid support.

[0051] 25. The method according to item 24, wherein the solid support is a Matrigel reduced by growth factors. The basement membrane is coated or reduced by growth factors.

[0052] 26. An in vitro method for producing isolated mammalian retinal ganglion cells (RGCs), comprising:

[0053] (a) Culture isolated primitive retinal stem cells (pRSCs) from mammals in a cell culture medium free of feeder cells, feeder layer conditioned medium, or serum to produce and grow cultures of said isolated pRSCs; and

[0054] (b) The isolated pRSCs cultured in this manner are contacted with one or more of Wnt, Notch, or VEGFR signaling inhibitors to differentiate the isolated pRSCs into isolated mammalian RGCs, thereby producing isolated mammalian RGCs.

[0055] 27. The method according to item 26, wherein in step (a), the pRSC thus cultured is grown on a solid support.

[0056] 28. The method according to item 27, wherein the solid support is made of Matrigel. ® Or base film coating.

[0057] 29. The method according to item 28, wherein the Matrigel ® Or the basement membrane is Matrigel with reduced growth factors. ® Or a basement membrane with reduced growth factors.

[0058] 30. The method according to item 26, wherein in step (b), one or more of the Wnt, Notch, or VEGFR signaling inhibitors is a combination of Wnt, Notch, and VEGFR signaling inhibitors, wherein the combination comprises inhibitors of at least two signaling pathways or preferably inhibitors of all three signaling pathways.

[0059] 31. The method according to item 30, wherein the Wnt, Notch, or VEGFR signaling inhibitor is a small molecule inhibitor.

[0060] 32. The method according to Item 30, wherein the combination of Wnt, Notch and VEGFR signaling inhibitors is IWP2, DAPT and PD173074, respectively.

[0061] 33. The method according to Project 26, wherein the upregulation of RGC precursor-specific transcription factor genes includes any one or more of BRN3A, BRN3B, ISL-1, and MATH5.

[0062] 34. The method described in Project 26, wherein most cells in culture are positive for the marker of RGC.

[0063] 35. The method described in Item 34, wherein most cells in culture are positive for the RGC marker and further positive for TUJ1 and BRN3.

[0064] 36. A method for generating isolated mammalian photoreceptors from isolated mammalian pRSCs, comprising:

[0065] (a) Dissociated pRSCs from mammals were cultured and grown for a sufficient time in a neural induction medium containing one or more inhibitors of TGF-β / activin receptor-like kinases ALK-4, ALK-5, or ALK-7, glycogen synthase kinase-3 (GSK-3), Notch or Wnt signaling, or hedgehog signaling activator, to induce pRSCs to a photoreceptor cell lineage fate with no visible morphological changes or no expression of photoreceptor-specific markers; and

[0066] (b) Subsequently, the pRSCs from step (a) are cultured and grown in a neural induction medium containing retinoic acid or taurine or both to differentiate the mammalian pRSCs into photoreceptors, thereby producing isolated mammalian photoreceptors.

[0067] The culture medium contained in the culture medium does not contain feeder cells, feeder layer conditioned medium, or serum.

[0068] 37. A method for generating isolated mammalian photoreceptors from isolated mammalian pRSCs, comprising:

[0069] (a) Dissociated pRSCs from mammals were cultured and grown for a sufficient time in a neural induction medium containing one or more inhibitors of TGF-β / activin receptor-like kinases ALK-4, ALK-5, or ALK-7, glycogen synthase kinase-3 (GSK-3), Notch or Wnt signaling, or hedgehog signaling activator, to induce pRSCs to a photoreceptor cell lineage fate with no visible morphological changes or no expression of photoreceptor-specific markers; and

[0070] (b) Contact the cultured and grown pRSC of step (a) with retinoic acid or taurine or both to differentiate the mammalian pRSC into photoreceptors, thereby producing isolated mammalian photoreceptors.

[0071] The culture medium contained in the culture medium does not contain feeder cells, feeder layer conditioned medium, or serum.

[0072] 38. The method according to item 37, wherein in step (a), the pRSC thus cultured is grown on a solid support.

[0073] 39. The method according to item 38, wherein the solid support is made of Matrigel ® Or base film coating.

[0074] 40. The method according to item 39, wherein the Matrigel ® Or the basement membrane is Matrigel with reduced growth factors. ® Or a basement membrane with reduced growth factors.

[0075] 41. The method according to item 36, wherein in step (a), one or more inhibitors of TGF-β / activin receptor-like kinase ALK-4, ALK-5 or ALK-7, glycogen synthase kinase-3 (GSK-3), Notch or Wnt signaling are a combination of inhibitors of TGF-β / activin receptor-like kinase ALK-4, ALK-5 and ALK-7, glycogen synthase kinase-3 (GSK-3), Notch and Wnt signaling, wherein the combination comprises inhibitors of at least two signaling pathways or preferably inhibitors of all four signaling pathways.

[0076] 42. The method according to item 36, wherein in step (a), the one or more inhibitors or hedgehog signaling activators of TGF-β / activin receptor-like kinase ALK-4, ALK-5 or ALK-7, glycogen synthase kinase-3 (GSK-3), Notch or Wnt signaling are a combination of inhibitors of TGF-β / activin receptor-like kinase ALK-4, ALK-5 and ALK-7, glycogen synthase kinase-3 (GSK-3), Notch and Wnt signaling and hedgehog signaling activators, wherein the combination comprises regulators of at least two signaling pathways or preferably regulators of all five signaling pathways.

[0077] 43. The method according to Item 36, wherein the inhibitor of the TGF-β / activin receptor-like kinase ALK-4, ALK-5 or ALK-7, glycogen synthase kinase-3 (GSK-3), Notch or Wnt signaling is a small molecule inhibitor.

[0078] 44. The method according to item 36, wherein the hedgehog signal transduction activator is a small molecule activator.

[0079] 45. The method according to Item 36, wherein the isolated mammalian photoreceptor is positive for rhodopsin, rhodopsin kinase, and / or transmucin.

[0080] 46. ​​The method according to item 42, wherein the inhibitors of TGF-β / activin receptor-like kinases ALK-4, ALK-5 and ALK-7, glycogen synthase kinase-3 (GSK-3), Notch and Wnt signaling, and the hedgehog signaling activator are SB431542 (CAS No. 301836-41-9) or equivalent, CHIR99021 (CAS No. 252917-06-9) or equivalent, DAPT (CAS No. 208255-80-5) or equivalent, IWP2 (CAS No. 686770-61-6) or equivalent, and puromorphamine (CAS No. 483367-10-8) or equivalent.

[0081] 47. A method for producing non-neural, isolated mammalian retinal pigment epithelium (RPE) or isolated mammalian retinal pigment epithelial cells (RPEs), comprising:

[0082] (a) Incubating mammalian pRSCs in a medium containing nicotinamide or activin A or both, without SMAD signaling inhibitors, for a sufficient time to induce the pRSCs to the RPE fate; and

[0083] (b) The pRSCs are cultured in a medium containing one or more of N1 medium supplement, taurine, hydrocortisone or triiodothyronine;

[0084] To cause the mammalian pRSC to differentiate into mammalian RPEs or RPEs, thereby producing isolated mammalian RPEs or RPEs.

[0085] The culture medium contained in the culture medium did not contain feeder cells or feeder layer conditioned medium.

[0086] 48. A method for producing non-neural, isolated mammalian retinal pigment epithelium (RPE) or isolated mammalian retinal pigment epithelial cells (RPEs), comprising:

[0087] (a) Incubating mammalian pRSCs in a medium containing nicotinamide or activin A or both, without SMAD signaling inhibitors, for a sufficient time to induce the pRSCs to the RPE fate; and

[0088] (b) Contact the pRSC cultured in step (a) with one or more of N1 medium supplement, taurine, hydrocortisone or triiodothyronine;

[0089] To cause the mammalian pRSC to differentiate into mammalian RPEs or RPEs, thereby producing isolated mammalian RPEs or RPEs.

[0090] The culture medium contained in the culture medium did not contain feeder cells or feeder layer conditioned medium.

[0091] 49. The method according to item 47, wherein in step (b), the culture medium is an RPE medium containing N1 medium supplement, taurine, hydrocortisone and triiodothyronine.

[0092] 50. The method according to item 47, wherein the RPE or RPEs express RPE65, form polygonal actin bundles, and become colored.

[0093] 51. The method according to item 26, 36 or 47, wherein the mammal is a human, monkey, bear, rat, mouse, mink, rabbit, guinea pig, pig, dog, cat, goat, sheep, horse or cow.

[0094] 52. A method for generating isolated human primitive retinal stem cells (hpRSC) from isolated human embryonic stem cells (hESC), comprising:

[0095] (a) The isolated hESCs are cultured on a solid support in a medium free of feeder cells, feeder layer conditioned medium or serum for a sufficient time to grow to near confluence, preferably to 80% cell confluence.

[0096] (b) The isolated hESCs thus grown are incubated on a solid support in a medium containing alkaline FGF (bFGF) for a sufficient time to grow to near confluence.

[0097] (c) The isolated hESCs of step b) are cultured on a solid support in a medium containing a combination of small molecule inhibitors of Wnt and TGF-β / BMP signaling to differentiate the isolated hESCs into isolated human primitive retinal stem cells, thereby producing isolated hpRSCs.

[0098] 53. A method for generating isolated human primitive retinal stem cells (hpRSC) from isolated human pluripotent stem cells (hPSC), comprising:

[0099] (a) The isolated hPSCs are cultured on a solid support in a medium free of feeder cells, feeder layer conditioned medium or serum for a sufficient time to grow to near confluence, preferably to 80% cell confluence.

[0100] (b) The separated hPSCs thus grown are cultured on a solid support in a medium containing bFGF for a sufficient time to grow to near confluence.

[0101] (c) The isolated hPSCs of step b) are cultured on a solid support in a medium containing a combination of small molecule inhibitors of Wnt and TGF-β / BMP signaling to differentiate the isolated hPSCs into isolated human primitive retinal stem cells, thereby producing isolated hpRSCs.

[0102] 54. A method for generating isolated human primitive retinal stem cells (hpRSCs) from isolated human induced pluripotent stem cells (iPSCs), comprising:

[0103] (a) The isolated human iPSCs are cultured on a solid support in a medium without feeder cells, feeder layer conditioned medium or serum for a sufficient time to grow to near confluence, preferably to 80% cell confluence.

[0104] (b) The separated iPSCs thus grown are cultured on a solid support in a medium containing bFGF for a sufficient time to grow to near confluence;

[0105] (c) The isolated iPSCs of step (b) are cultured on a solid support in a medium containing a combination of small molecule inhibitors of Wnt and TGF-β / BMP signaling to differentiate the isolated iPSCs into isolated human primitive retinal stem cells, thereby producing isolated hpRSCs.

[0106] 55. The method according to items 52, 53 or 54, wherein the combination of small molecule inhibitors of Wnt and TGF-β / BMP signaling activity in step (c) is a combination of SB 431542, LDN193189 and IWP2 (Wnt production inhibitor-2); a combination of SB 431542, a head protein analog and IWP2; a combination of SB 431542, LDN193189 and a Dkk1 analog; a combination of SB 431542, a head protein analog and a Dkk1 analog; or equivalent combinations, wherein the combination produces synergistic inhibition of Wnt and TGF-β / BMP signaling activity.

[0107] 56. The method according to item 55, wherein the combination of SB 431542, LDN193189 and IWP2 comprises 5 μM SB 431542, 50 nM LDN193189 and 1 μM IWP2.

[0108] 57. The method according to item 52, 53 or 54, wherein the isolated human primitive retinal stem cells are positive for typical early ocular transcription factors PAX6, LHX2, RAX, OTX2, SIX3 and / or SIX6.

[0109] 58. The method according to Item 57, wherein the isolated human primitive retinal stem cells are positive for the expression of stemness factors SOX2, nestin and STAT3.

[0110] 59. The method according to item 57, wherein the isolated human primitive retinal stem cells are positive for Ki67 expression.

[0111] 60. The method according to Project 57, wherein the isolated human primitive retinal stem cells downregulate the transcription of hESC pluripotency transcription factors POU5F1 (OCT4), NANOG, KLF4 and TBX3 genes, as well as TBF-β superfamily genes SMAD1, SMAD2, TGFβ3, BMP3, BMP6, TGFBR1 and BMPR1B.

[0112] 61. The method according to Item 57, wherein the isolated human primitive retinal stem cells upregulate the transcription of the BMP4 and BMP7 genes as well as the OTX2, RAX, LHX2, SIX3 and SIX6 genes.

[0113] 62. The method according to Item 57, wherein the isolated human primitive retinal stem cells are positive for transcription of the SRFP1 and FZD3 / 5 genes.

[0114] 63. The method according to Item 57, wherein the isolated human primitive retinal stem cells downregulate the transcription of FGFR1 / 2 / 3 and FGF3 / 8 genes.

[0115] 64. A method for treating retinal degeneration in a subject in need, comprising administering a sufficient amount of primitive retinal stem cells (pRSC) to the eye of the subject to treat the retinal degeneration of the subject, wherein the pRSC is generated by the method according to item 1, 2, 3, 52, 53 or 54.

[0116] 65. A method for treating retinal degeneration in a subject of need, comprising:

[0117] (a) Obtaining tissue or cell samples from a subject to obtain or produce isolated PSCs or isolated iPSCs;

[0118] (b) Cultivating the isolated PSC or isolated iPSC of step (a) to produce by means of the method according to items 2, 3, 53 or 54; and

[0119] (c) Administering a sufficient amount of pristine retinal stem cells (pRSC) to the subject's eye to treat the subject's retinal degeneration.

[0120] 66. A method for treating retinal degeneration in a subject in need, comprising administering a sufficient amount of mammalian retinal ganglion cells (RGCs) to the eye of the subject to treat the retinal degeneration, wherein the RGCs are generated by the method according to item 26.

[0121] 67. A method for treating retinal degeneration in a subject of need, comprising:

[0122] (a) Obtaining tissue or cell samples from a subject to obtain or produce isolated PSCs or isolated iPSCs;

[0123] (b) Cultivate the isolated PSCs or isolated iPSCs of step (a) to produce isolated pRSCs;

[0124] (c) Cultivate the isolated pRSCs to produce isolated RGCs according to the method described in item 26; and

[0125] (d) Apply a sufficient amount of the mammalian retinal ganglion cells (RGCs) thus generated in step (c) to the subject's eye to treat the subject's retinal degeneration.

[0126] 68. A method for treating retinal degeneration in a subject in need, comprising applying a sufficient amount of a mammalian photoreceptor to the eye of the subject to treat the retinal degeneration, wherein the mammalian photoreceptor is generated by the method according to item 36.

[0127] 69. A method for treating retinal degeneration in a subject of need, comprising:

[0128] (a) Obtaining tissue or cell samples from a subject to obtain or produce isolated PSCs or isolated iPSCs;

[0129] (b) Cultivate the isolated PSCs or isolated iPSCs of step (a) to produce isolated pRSCs;

[0130] (c) Cultivate the isolated pRSCs to generate isolated photoreceptors according to the method described in item 36; and

[0131] (d) Apply a sufficient amount of the photoreceptor from step (c) to the subject's eye to treat the subject's retinal degeneration.

[0132] 70. A method for treating retinal degeneration in a subject in need, comprising administering mammalian retinal pigment epithelial cells (RPEs) in an adequate amount to the eye of the subject to treat the retinal degeneration of the subject, wherein the RPEs are generated by the method according to item 47.

[0133] 71. A method for treating retinal degeneration in a subject of need, comprising:

[0134] (a) Obtaining tissue or cell samples from a subject to obtain or produce isolated PSCs or isolated iPSCs;

[0135] (b) Cultivate the isolated PSCs or isolated iPSCs of step (a) to produce isolated pRSCs;

[0136] (c) Culturing the isolated pRSCs to produce isolated RPEs according to the method described in item 47; and

[0137] (d) Applying a sufficient amount of the mammalian retinal pigment epithelial cells (RPEs) from step (c) to the eye of the subject to treat the retinal degeneration of the subject.

[0138] 72. The method according to item 64, 66, 68 or 70, wherein the cells to be applied are in, on or mixed with the matrix before being delivered to the patient's eye.

[0139] 73. The method according to item 72, wherein the matrix is ​​a hydrogel comprising hyaluronic acid and methylcellulose or their salts and derivatives, a hydrogel having bioactive peptides, a biocompatible polymer or mixture of biocompatible polymers supporting cell viability and function of transplanted cells, an artificial biomimetic matrix, a bioactive scaffold derived from tissue or organ matrix, a biosynthesized extracellular matrix based on collagen and N-isopropylacrylamide copolymer, or a scaffold modified with adhesion molecules, laminins, growth factors, morphogenetic factors, survival factors, extracellular matrix or fragments or derivatives.

[0140] 74. The method according to item 64, 66, 68 or 70, wherein the cells are administered into the subretinal space of the subject's eye.

[0141] 75. The method described in items 64, 66, 68 or 70, wherein the retinal degeneration is associated with age-related macular degeneration (AMD), Staggart's macular dystrophy, retinitis pigmentosa, glaucoma, retinal vascular disease, viral infection of the eye or other retinal / eye diseases of known or unknown cause.

[0142] 76. The method according to items 64, 66, 68 or 70, wherein the subject is a human being.

[0143] 77. A method for delivering human primitive retinal stem cells (hpRSCs) to a subject in need, comprising administering to the subretinal space of the subject a monolayer cell suspension or single-cell suspension of hpRSCs generated by the method according to items 1, 2, 3, 52, 53 or 54 in a matrix solution or matrix suspension.

[0144] 78. A method for delivering human primitive retinal ganglion cells (hpRGCs) to a subject in need, comprising administering to the subretinal space of the subject a monolayer cell suspension or single-cell suspension of hpRGCs produced by the method according to item 26 in a matrix solution or matrix suspension.

[0145] 79. A method for delivering a human photoreceptor to a subject in need, comprising applying to the subretinal space of the subject a monolayer cell suspension or single-cell suspension of the human photoreceptor generated by the method according to item 36 in a matrix solution or matrix suspension.

[0146] 80. A method for delivering human retinal pigment epithelial cells (hRPEs) to a subject in need, comprising administering to the subretinal space of the subject a monolayer cell suspension or single-cell suspension of hRPEs generated by the method according to item 47 in a matrix solution or matrix suspension.

[0147] 81. A method for preventing or inhibiting tumors associated with the use of pluripotent stem cells to treat a subject with retinal degeneration, comprising:

[0148] (a) Inducing isolated pluripotent stem cells into isolated human primitive retinal stem cells (hpRSCs) by the methods described in accordance with items 1, 2, 3, 52, 53 or 54; and

[0149] (b) Administer the isolated hpRSC produced in (a) to the subject.

[0150] 82. A method for preventing or inhibiting tumors associated with the use of pluripotent stem cells to treat a subject with retinal degeneration, comprising:

[0151] (a) Inducing isolated pluripotent stem cells into isolated hPRSCs by the methods described in accordance with items 1, 2, 3, 52, 53 or 54;

[0152] (b) Furthermore, the isolated hPRSCs are differentiated into isolated human primitive retinal ganglion cells (hpRGCs) produced by the method described according to item 26; and

[0153] (c) Administer the isolated hpRGC produced in (b) to the subject.

[0154] 83. A method for preventing or inhibiting tumors associated with the use of pluripotent stem cells to treat a subject with retinal degeneration, comprising:

[0155] (a) Inducing isolated pluripotent stem cells into hPRSCs by the methods described in accordance with items 1, 2, 3, 52, 53 or 54;

[0156] (b) Furthermore, the separated hPRSCs are differentiated into separated human photoreceptors produced by the method described according to item 36; and

[0157] (c) Administer the isolated human photoreceptor generated in (b) to the subject.

[0158] 84. A method for preventing or inhibiting tumors associated with the use of pluripotent stem cells to treat a subject with retinal degeneration, comprising:

[0159] (a) Inducing isolated pluripotent stem cells into hPRSCs by the methods described in accordance with items 1, 2, 3, 52, 53 or 54;

[0160] (b) Furthermore, the isolated hPRSCs are differentiated into isolated human retinal pigment epithelium (hRPE) or isolated human retinal pigment epithelial cells (hRPEs) produced by the method described according to item 47; and

[0161] (c) Administer to the subject the isolated hPRE or hPREs produced in (b) as described in (b).

[0162] 85. The method described in item 81, 82, 83 or 84, wherein the retinal degeneration is associated with age-related macular degeneration (AMD), Staggart's macular dystrophy, retinitis pigmentosa, glaucoma, retinal vascular disease, viral infection of the eye or other retinal / eye disease of known or unknown cause.

[0163] 86. A composition comprising mammalian primitive retinal stem cells (hpRSCs) generated by the method according to items 1, 2, 3, 52, 53 or 54 and a suitable vector.

[0164] 87. A composition comprising mammalian retinal ganglion cells (RGCs) produced by the method according to item 26 and a suitable carrier.

[0165] 88. A composition comprising a mammalian photoreceptor and a suitable carrier generated by the method according to item 36.

[0166] 89. A composition comprising mammalian retinal pigment epithelium (RPE) or mammalian retinal pigment epithelial cells (RPEs) produced by the method according to item 47 and a suitable carrier.

[0167] 90. A kit for generating mammalian primitive retinal stem cells (pRSCs), wherein the kit comprises instructions for culturing mammalian embryonic stem cells (ESCs), pluripotent stem cells (PSCs), or induced pluripotent stem cells (iPSCs) in a culture medium with a chemical composition defined free of feeder cells, feeder layer conditioned medium, or serum, and instructions for using a small molecule inhibitor of Wnt signaling or TGF-β / BMP signaling, or an inhibitor of both Wnt signaling and TGF-β / BMP signaling.

[0168] 91. The kit according to item 90, wherein the kit further comprises a small molecule inhibitor of Wnt signaling or TGF-β / BMP signaling or both Wnt signaling and TGF-β / BMP signaling.

[0169] 92. The kit according to item 91, wherein the small molecule inhibitor of Wnt signaling is or includes a chemical formula C 22 H 18 The Wnt production inhibitor of N4O2S3 is either -2 (IWP2; CAS No. 686770-61-6) or N-(6-methyl-1,3-benzothiazol-2-yl)-2-[(4-oxo-3-phenyl-6,7-dihydrothieno[3,2-d]pyrimidin-2-yl)thioalkyl]acetamide.

[0170] 93. The kit according to item 91, wherein the small molecule inhibitor of TGF-β / BMP signaling is a small molecule inhibitor of transforming growth factor-β (TGF-β) superfamily type I activin receptor-like kinases ALK-4, ALK-5 and ALK-7.

[0171] 94. The kit according to item 93, wherein the small molecule inhibitors of the transforming growth factor-β (TGF-β) superfamily type I activin receptor-like kinases ALK-4, ALK-5, and ALK-7 are or include those having the chemical formula C 22 H 16 N4O3 is SB431542 (CAS No. 301836-41-9) or 4-[4-(1,3-benzodioxane-5-yl)-5-(2-pyridyl)-1H-imidazol-2-yl]benzamide.

[0172] 95. The kit according to item 91, wherein the small molecule inhibitor of TGF-β / BMP signaling is a small molecule inhibitor or head protein analog of BMP type I receptors ALK-2 and ALK-3.

[0173] 96. The kit according to item 95, wherein the small molecule inhibitor or head protein analog of the BMP type I receptors ALK-2 and ALK-3 is or includes a substance having the chemical formula C 25 H 22 N6 LDN-193189 (CAS No. 1062368-24-4) or 4-(6-(4-(piperazin-1-yl)phenyl)pyrazolo[1,5-a]pyrimidin-3-yl)quinoline.

[0174] 97. The kit according to item 91, wherein the small molecule inhibitors of both Wnt signaling and TGF-β / BMP signaling are a combination or equivalent combination of IWP2 (CAS No. 686770-61-6), SB431542 (CAS No. 301836-41-9) and LDN-193189 (CAS No. 1062368-24-4), wherein the combination produces synergistic inhibition of Wnt and TGF-β / BMP signaling activities; or comprising a combination or equivalent combination of IWP2 (CAS No. 686770-61-6), SB431542 (CAS No. 301836-41-9) and LDN-193189 (CAS No. 1062368-24-4), wherein the combination produces synergistic inhibition of Wnt and TGF-β / BMP signaling activities.

[0175] 98. The kit according to item 90 or 91, wherein the kit further comprises embryonic stem cells (ESCs), pluripotent stem cells (PSCs), or induced pluripotent stem cells (iPSCs) from mammals.

[0176] 99. The kit according to item 90, 91 or 98, wherein the kit further comprises a culture medium and / or supplements with a defined chemical composition. Attached Figure Description

[0177] Figure 1 hpRSC induction by hESC. (a) Schematic diagram of in vitro directed induction of human retinal cell fate from human pluripotent stem cells. (b) Confocal image of immunofluorescence labeling of typical early eye field transcription factors PAX6 (green) and LHX2 (red) in hpRSC derived from hESC after 1 week of induction. Nuclei were counterstained with Hoechst 33342 (blue). (c) Staining of hpRSC against the neural progenitor / stem cell marker nestin (white) and the proliferation marker Ki67 (green). (d) Real-time qPCR analysis of induced expression of typical early eye field transcription factors. Scale bar = 30 μm.

[0178] Figure 2 Transcriptome analysis of hESC-derived hpRSCs. Heatmap of differentially regulated gene expression in hfRPC, hpRSC, and the hESC line HuES9. Unsupervised clustering of cell types shows the transitional state of hESC-derived hpRSCs between hfRPC and the parental hESC line HuES9. (a) Genome-wide transcriptome analysis. (b) Pluripotency regulation. (c) TGF-β superfamily signaling pathway. (d) Wnt signaling pathway. Red, high expression; blue, low expression, relative to the other two cell types. (e) Pairwise scatter plot analysis between hpRSC and hfRPC. Shows the signal intensity of transcripts in hfRPC relative to hpRSC. Diagonal lines indicate equal expression levels of genes between the two cell types. Genes marked with * are important for hpRSC stemness and specification.

[0179] Figure 3 Under chemically defined culture conditions, hpRSCs can be specialized into retinal ganglion cells (RGCs). (a) hpRSCs can differentiate into RGCs efficiently. After two weeks of culture under RGC-inducing conditions, differentiated cells exhibit long neuronal processes and express typical RGC markers such as BRN3 (red) and TUJ1 (green). The nuclei were counterstained with Hoechst 33342 (blue). (b) Real-time qPCR analysis of the expression of RGC-specific transcription factors in cells differentiated from hpRSCs.

[0180] Figure 4Characterization of hpRSC differentiation into neuronal or non-neuronal retinal cell fate under defined culture conditions. (ad) Photoreceptor induction as demonstrated by immunocytochemical detection of recoverin (a), blue opsin (OPN1SW) (b), and rhodopsin (c), and expression of the photoreceptor-specific receptor IRBP-GFP (d); well-differentiated photoreceptors show a typical morphology with long external processes and short internal processes (d, inset). (eg) RPE induction; (e) RPE formation as demonstrated by early expression of RPE65 (green) and phalloidin staining of polygonal RPE cells (red); (f) Increased RPE65 expression (red) observed after 3 weeks of culture; (g) Pigmented RPEs appearing after prolonged maturation in culture. (af) Nuclei counterstained with Hoechst 33342 (blue).

[0181] Figure 5 In vivo integration and differentiation of transplanted hpRSCs in newborn nude rats. Confocal images of immunohistochemically stained frozen sections of the retina after hpRSC transplantation. Significant integration of transplanted GFP-expressing human cells (green) was observed in the recipient neuroretina. Some GFP-expressing cells (a, c, e) co-localized with recovery protein (white) positive cells; immunostaining of nestin (red), a marker of retinal progenitor cells, was detected in many transplanted GFP-expressing cells (b, d, f). Cell nuclei were counterstained with Hoechst 33342 (blue). Scale bar: ad = 30 μm; ef = 15 μm.

[0182] Figure 6 Integration and differentiation of transplanted hpRSCs in the photoreceptor layer of RCS rats two months post-transplantation. (a) Transplanted cells undergo immunocytochemical labeling with HSA antibody (red). Some transplanted cells receive the fate of photoreceptors (arrows) expressing recovery protein (green). (b) hpRSC-derived cones (arrows) are positive for both red / green opsin (green) and HSA (red). Nuclei are counterstained with Hoechst 33342 (blue). Scale bar = 20 μm.

[0183] Figure 7 Representative heatmaps of gene expression involved in FGF(a), Notch(b), and hedgehog signaling pathways(c) in hfRPC, HuES9, and HuES9-derived hpRSC. Genes marked with * are known to be important in retinogenesis.

[0184] Figure 8In vivo transplantation of hpRSCs in nude P21 rats with or without (a) and (b) HAMC hydrogel. Retinal sections were processed 6 weeks post-transplantation. In the absence of hydrogel, fewer hpRSCs survived and were frequently surrounded by scar tissue in the subretinal space. In the presence of HAMC hydrogel, transplanted hpRSCs diffused across the subretinal space and into the neuroretinal layer. Transplanted hpRSCs were labeled with human nuclear antigen staining (green). Nuclei were counterstained with Hoechst 33342 (blue). Detailed Implementation

[0185] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. All patents, applications, publications, and other publications cited herein are incorporated herein by reference in their entirety.

[0186] It is important to note that, unless the context clearly specifies otherwise, the singular forms “a (a, an)” and “the” as used herein and in the appended claims include a plural of indicators. Thus, for example, “a formulation” may refer to a variety of compounds.

[0187] When used before numerical names (e.g., temperature, time, quantity, concentration, and other such names, including range), the term “about” as used herein indicates an approximate value that may vary (+) or (-) by 10%, 5%, or 1%.

[0188] The method of the present invention

[0189] Methods for generating isolated mammalian primitive retinal stem cells (pRSCs)

[0190] This invention provides an in vitro method for generating isolated mammalian pRSCs. The advantages of this method include the ability to generate isolated mammalian primitive retinal stem cells in sufficient quantity and quality to be suitable for cell transplantation or grafting into the eyes of a subject, without the need for cell grading or purification prior to transplantation.

[0191] As used herein, isolated pRSC means, for example, a pRSC that is substantially separated from contaminants (e.g., cells that are not pRSCs).

[0192] In one embodiment of the invention, the method includes culturing isolated embryonic stem cells (ESCs) from mammals in a cell culture medium free of feeder cells, feeder layer conditioned medium, or serum to generate and grow cultures of the isolated ESCs; and contacting the cultures of the isolated ESCs with one or more of Wnt or TGF-β / BMP signaling inhibitors to differentiate the isolated ESCs into primitive retinal stem cells, thereby generating isolated mammalian pRSCs. In one embodiment, one or more of the Wnt or TGF-β / BMP signaling inhibitors is a combination of Wnt and TGF-β / BMP signaling inhibitors. The cultures of the isolated PSCs may be adherent cultures. In a preferred embodiment, the cultures of the isolated ESCs are monolayer cultures. In one embodiment, the cultures of the isolated ESCs are grown to near confluence and then contacted with one or more of Wnt or TGF-β / BMP signaling inhibitors or a combination of Wnt and TGF-β / BMP signaling inhibitors. In a preferred embodiment, the Wnt or TGF-β / BMP signaling inhibitors are small molecule inhibitors.

[0193] As used herein, a separated ESC means, for example, an ESC that is substantially separate from contaminants (e.g., cells that are not ESCs).

[0194] As used in the context of this invention, signal transduction refers to one or more signal transduction pathways in which a group of cellular proteins are involved in controlling one or more cellular functions. A signal (e.g., a ligand) activates a signal transduction pathway via a receptor of the signal, which acts as a first member of the pathway; this interaction between the signal and its receptor triggers a series of events that lead to subsequent changes in the biological activity or biological state of downstream members, ultimately resulting in alterations in cellular function. Therefore, changes in cellular function are a result of a signal activating a signal transduction pathway associated with that signal. A signal can also activate more than one signal transduction pathway, such as in Wnt or TGF-β signaling, for example. Furthermore, signal transduction pathways can exhibit crosstalk through shared members within two or more pathways.

[0195] Often, signal transduction pathways are named in relation to the ligand proteins or signals that activate the pathway, such as Wnt, TGF-β, BMP, or hedgehog ligands in Wnt, TGF-β, BMP, or hedgehog signal transduction, respectively. For example, the Wnt protein ligand can induce Wnt signal transduction upon binding to its receptor Frizzled; TGF-β protein ligands or members of the TGF-β ligand superfamily (including activin and BMP) can induce TGF-β signal transduction upon binding to TGF-β type II receptors and participate in ligand-specific signal transduction of the TGF-β superfamily ligands in combination with type I receptors; BMP can induce BMP signal transduction upon binding to bone morphogenetic protein receptor type 2 (BMPR2); and hedgehog protein ligands can induce hedgehog signal transduction upon binding to its receptor Patched. Alternatively, signal transduction pathways can be named in relation to the recipients of the signal or ligand, such as the Notch receptor in Notch signaling, which, upon activation, transmits its activation state downstream to control one or more cellular functions, such as controlling gene expression.

[0196] Furthermore, in TGF-β signaling, the binding of TGF-β superfamily ligands (such as TGF-β, activin, or BMP) to their respective type II receptors (such as TGF-β receptor type II, activin receptor type II, or BMP receptor type II) can lead to receptor activation and subsequent activation of one of the seven type I receptors, which are divided into two distinct groups: the TGF-β / activin subgroup, including the TGF-β superfamily type I receptor activin receptor-like kinases ALK-4, ALK-5, and ALK-7; and the BMP subgroup, including ALK-1, ALK-2, ALK-3, and ALK-6 type I receptors. Both of these subgroups are involved in the regulation of gene transcription by SMAD family transcription factors.

[0197] In the context of this invention, an equivalent refers to a compound or substitute that can perform the same or similar function as the substituted compound or substance. Such equivalence can be determined using assays similar to those used to characterize the compound, based on its ability to inhibit or activate a specific signaling pathway (such as the substituted compound or the preferred or most preferred substituted compound). The most preferred compounds of this invention are: IWP2 for inhibiting Wnt signaling, SB431542 for inhibiting TGF-β superfamily type I activin receptor-like kinases ALK-4, ALK-5, and ALK-7, LDN-193189 for inhibiting BMP type I receptors ALK-2 and ALK-3, DAPT for inhibiting Notch signaling, PD173074 for inhibiting VEGFR signaling, CHIR99021 for inhibiting glycogen synthase kinase-3, purmorphamine for activating hedgehog signaling, or retinoic acid for activating retinoic acid signaling (e.g., via the RAR complex).

[0198] A culture medium with defined composition or chemical composition refers to a culture medium that is substantially free of indeterminate components such as serum and feeder conditioned media. A culture medium with defined chemical composition can be produced using chemical components (including deionized or distilled water). It may include purified proteins (such as growth factors and serum proteins, such as albumin, preferably recombinant proteins), amino acids (essential and / or non-essential amino acids), vitamins, salts, lipids, sugars, pyruvate, buffers, trace metals, reducing agents, and indicator dyes. Furthermore, the culture medium may be supplemented with hormones, receptor ligands, metabolites, sulfamic acid, inhibitors, morphogenetics, cell signaling molecules, and activators or inhibitors of cell signaling or kinase activity. Using a culture medium with defined composition or chemical composition to culture cells is advantageous for cell-based alternative therapies.

[0199] According to the practice of the invention, monolayer cultures can be grown until they are nearly confluent, close to confluent, or confluent. In one embodiment, near confluence or close to confluence can be about 70% confluence, preferably about 80% confluence. Further culturing of cell cultures with about 80% confluence and the growth of such cultures can be described as near confluence or close to confluence, where confluence can be about 90% or greater. At confluence, cells crowd the entire cell-covered culture surface and all cells are in contact with each other. Furthermore, in one embodiment of the invention, the cultured cells can be cultured or grown using a solid support. Optionally, the solid support can be coated with Matrigel. ® Or base film. In addition, Matrigel ®Alternatively, the base membrane could be a Matrigel with reduced growth factors. ® Or a base film with reduced growth factors. In an alternative embodiment, the solid support may be coated with a base film with reduced growth factors and coated with Matrigel with reduced growth factors. ® (BD Biosciences), Geltrex-free ® LDEVs are grown in tissue culture plates, trays, flasks or microbeads in a base membrane matrix or equivalent or mixture thereof, with growth factors verified by hESC.

[0200] In one embodiment of the invention, the method includes culturing isolated pluripotent stem cells (PSCs) from mammals in a cell culture medium free of feeder cells, feeder layer conditioned medium, or serum to generate and grow cultures of the isolated PSCs; and contacting the cultures of the isolated PSCs with one or more of Wnt or TGF-β / BMP signaling inhibitors to differentiate the isolated PSCs into pRSCs, thereby generating isolated mammalian pRSCs. In one embodiment, one or more of the Wnt or TGF-β / BMP signaling inhibitors is a combination of Wnt and TGF-β / BMP signaling inhibitors. The cultures of the isolated PSCs may be adherent cultures. In a preferred embodiment, the cultures of the isolated PSCs are monolayer cultures. In one embodiment, the cultures of the isolated PSCs are grown to near confluence and then contacted with one or more of Wnt or TGF-β / BMP signaling inhibitors or a combination of Wnt and TGF-β / BMP signaling inhibitors. In a preferred embodiment, the Wnt or TGF-β / BMP signaling inhibitors are small molecule inhibitors. According to the practice of the present invention, induced pluripotent stem cells (iPSCs) from mammals can be treated in a similar manner to replace isolated PSCs to produce isolated mammalian pRSCs.

[0201] As used herein, isolated PSC means, for example, a PSC that is substantially separated from contaminants (e.g., cells that are not PSCs).

[0202] As used herein, isolated iPSCs mean, for example, iPSCs that are substantially separated from contaminants (e.g., cells that are not iPSCs).

[0203] In one embodiment, the method of the present invention produces isolated mammalian primitive retinal stem cells with an efficiency greater than about 90% over approximately 7 days of treatment, for example, with one or more of Wnt or TGF-β / BMP signaling inhibitors or a combination of Wnt and TGF-β / BMP signaling inhibitors. In a preferred embodiment, the Wnt or TGF-β / BMP signaling inhibitor is a small molecule inhibitor.

[0204] Small molecules are low molecular weight organic compounds with a molecular weight of less than 900 Daltons, preferably about 500 Daltons or less. For example, in some embodiments, the small molecules used in this invention have molecular weights of 466.6 Daltons (IWP2), 384.39 Daltons (SB431542), 406.48 Daltons (LDN-193189), 465.34 Daltons (CHIR-99021), 432.46 Daltons (DAPT), 523.67 Daltons (PD173074), or 520.62 Daltons (purometamorphine). Small molecules bind to specific proteins or nucleic acids to alter the activity or function of said proteins or nucleic acids. Therefore, small molecules possess biological activity and can regulate biological processes.

[0205] As used in the context of this invention, small molecule inhibitors are inhibitors of cell signaling pathways. Small molecule inhibitors act by competing with activating signals, such as the binding of ligands or protein ligands to their receptors. They act by blocking protein-protein interactions required for the propagation of activating signals. They can act to inhibit the activity of any member in a signaling pathway, thereby disrupting the transmission of activating signals through said pathway. For example, small molecule inhibitors such as SB431542 further block the signaling of activin or TGF-β to SMAD2 and SMAD3 transcription factors by selectively and efficiently inhibiting the activity of the TGF-β superfamily type I ALK-4, ALK-5, and ALK-7 receptors downstream of type II receptors that bind to activin or TGF-β.

[0206] Unlike small molecule inhibitors, small molecule activators of signal transduction pathways regulate these pathways by activating them. These small molecule activators are also referred to as small molecule activators of signal transduction (e.g., Wnt signaling, TGF-β / BMP signaling, Notch signaling, VEGFR signaling, hedgehog signaling, or SMAD signaling). Similar to small molecule inhibitors of signal transduction or signal transduction pathways, small molecule activators of signal transduction or signal transduction pathways can activate the pathway at any step or location within the pathway via various members of the pathway. While it may often be desirable to activate the pathway at its inception by interacting with an agonist ligand of a specific receptor, this is not necessarily required. In the case of small molecule activators of hedgehog signaling, purinemorphine activates downstream components of the hedgehog signaling pathway by acting as an agonist of Smoothened (a key component in hedgehog signaling).

[0207] Regulators of signal transduction pathways can be activators or inhibitors of the pathway. They can be small molecules.

[0208] In practice, the preferred embodiments of the inhibitors, activators or modulators of signal transduction or kinases are small molecule inhibitors, small molecule activators or small molecule modulators of signal transduction or kinases.

[0209] Co-inhibition of Wnt and TGF-β / BMP signaling or signaling activity refers to the result of inhibiting Wnt signaling and inhibiting TGF-β and BMP signaling, wherein the result is not merely the sum of the result of inhibiting Wnt signaling alone and the result of inhibiting TGF-β and BMP signaling alone. Co-inhibition can arise from interactions between Wnt signaling components and TGF-β and BMP signaling components, such as the formation of transcription factor complexes between β-catenin and Lef1 / Tcf (downstream components of Wnt signaling) and Smad4 (coSMAD and an essential mediator of TGF-β and BMP signaling), leading to co-activation of target genes (Nishita et al., Nature 403, 781-785 (2000)).

[0210] Examples of small molecule inhibitors of Wnt signaling include, but are not limited to, Wnt production inhibitor-1 (IWP-1), Wnt production inhibitor-2 (IWP2), JW55, JW74, and okadaic acid. acid), temmelin, SB239063, SB203580, ADP-HPD, 2-[4-(4-fluorophenyl)piperazin-1-yl]-6-methylpyrimidin-4(3H)-one, PJ34, cambinol, sulindac, 3289-8625, scaffold A for a series of analogues designed to inhibit Dishevelled proteins, scaffold B for a series of analogues designed to inhibit Dishevelled proteins, J01-017a, NSC668036, filipin, IC261, PF670462, bosutinib, PHA665752, imatinib, ICG-001, ethacrynic acid Acetaminophen, ethacrynic acid derivatives, PKF115-584, PNU-74654, PKF118-744, CGP049090, PKF118-310, ZTM000990, BC21, GDC-0941, Rp-8-Br-cAMP, LGK974, C59, Ant 1.4Br / Ant 1.4CI, niclosamide, apicularen, bafilomycin, XAV939, IWR1, pyrvinium, NSC668036, 2,4-diamino-quinazoline, quercetin, and PKF115-584, as well as their equivalents and combinations thereof.

[0211] By way of example only, Wnt signaling inhibitors can be small molecule inhibitor-2 or Dkk1 analogs derived from Wnt. Suitable examples of small molecule inhibitor-2 or Dkk1 analogs derived from Wnt include, but are not limited to, those with C 22 H 18 The chemical formula of N4O2S3 and the following chemical structures of Wnt production inhibitor-2 (IWP2; CAS No. 686770-61-6) or N-(6-methyl-1,3-benzothiazol-2-yl)-2-[(4-oxo-3-phenyl-6,7-dihydrothieno[3,2-d]pyrimidin-2-yl)thioalkyl]acetamide:

[0212] .

[0213] Suitable examples of small molecule inhibitors of TGF-β / BMP signaling include, but are not limited to, SB431542 (4-[4-(1,3-benzodioxane-5-yl)-5-(2-pyridyl)-1H-imidazol-2-yl]benzamide), A83-01 (3-(6-methyl-2-pyridyl)-N-phenyl-4-(4-quinolinyl)-1H-pyrazole-1-thiocarboxamide), SJN 2511 (2-(3-(6-methylpyridin-2-yl)-1H-pyrazole-4-yl)-1,5-naphthidine), D 4476 (4-[4-(2,3-dihydro-1,4-benzodioxane-6-yl)-5-(2-pyridyl)-1H-imidazol-2-yl]benzamide), LY 364947 (4-[3-(2-pyridinyl)-1H-pyrazol-4-yl]-quinoline), SB 525334 (6-[2-(1,1-dimethylethyl)-5-(6-methyl-2-pyridinyl)-1H-imidazol-4-yl]quinoxaline), SD 208 (2-(5-chloro-2-fluorophenyl)-4-[(4-pyridinyl)amino]pteridine), and LDN-193189 (4-(6-(4-(piperazin-1-yl)phenyl)pyrazolo[1,5-a]pyrimidin-3-yl)quinoline), as well as their equivalents and combinations.

[0214] In one embodiment, the TGF-β / BMP signaling inhibitor may also be a small molecule inhibitor of transforming growth factor-β (TGF-β) superfamily type I activin receptor-like kinases ALK-4, ALK-5, or ALK-7. In a preferred embodiment, the TGF-β / BMP signaling inhibitor may also be a small molecule inhibitor of transforming growth factor-β (TGF-β) superfamily type I activin receptor-like kinases ALK-4, ALK-5, and ALK-7.

[0215] In addition, small molecule inhibitors of transforming growth factor-β (TGF-β) superfamily type I activin receptor-like kinases ALK-4, ALK-5, and ALK-7 can be or may include those with C 22 H 16 The chemical formula of N4O3 and The chemical structure of SB431542 (CAS No. 301836-41-9) or 4-[4-(1,3-benzodioxane-5-yl)-5-(2-pyridyl)-1H-imidazol-2-yl]benzamide.

[0216] Additionally, the TGF-β / BMP signaling inhibitor may be, or may include, a small molecule inhibitor of BMP type I receptor ALK-2 or ALK-3, or a noggin analog. In a preferred embodiment, the TGF-β / BMP signaling inhibitor may be, or may include, a small molecule inhibitor of BMP type I receptor ALK-2 and ALK-3, or a noggin analog.

[0217] Furthermore, small molecule inhibitors or head protein analogs of BMP type I receptors ALK-2 and ALK-3 can be or may include those with C 25 H 22 The chemical formula of N6 and The chemical structure of LDN-193189 (CAS No. 1062368-24-4) is 4-(6-(4-(piperazin-1-yl)phenyl)pyrazolo[1,5-a]pyrimidin-3-yl)quinoline.

[0218] Furthermore, the combination of Wnt and TGF-β / BMP signaling inhibitors may be or may include the following combinations: (a) one or more Wnt production inhibitors-2 or Dkk1 analogs; (b) one or more inhibitors of transforming growth factor-β (TGF-β) superfamily type I activin receptor-like kinases ALK-4, ALK-5, and ALK-7; and (c) one or more inhibitors or head protein analogs of BMP type I receptors ALK-2 and ALK-3, wherein the combination comprises inhibitors of at least two signaling pathways or preferably inhibitors of all three signaling pathways. The inhibitors may be small molecules.

[0219] Furthermore, the combination of Wnt and TGF-β / BMP signaling activity inhibitors can be a combination or equivalent of at least two, preferably all three, of IWP2 (CAS No. 686770-61-6), SB431542 (CAS No. 301836-41-9), and LDN-193189 (CAS No. 1062368-24-4), wherein the combination produces synergistic inhibition of Wnt and TGF-β / BMP signaling activity; or a combination or equivalent of at least two, preferably all three, of IWP2 (CAS No. 686770-61-6), SB431542 (CAS No. 301836-41-9), and LDN-193189 (CAS No. 1062368-24-4), wherein the combination produces synergistic inhibition of Wnt and TGF-β / BMP signaling activity.

[0220] In one embodiment of the invention, isolated mammalian primitive retinal stem cells may constitute more than about 90% of the total cell population and be positive for both PAX6 and LHX2 transcription factors expressed by ocular progenitor cells.

[0221] In another implementation, isolated mammalian primitive retinal stem cells may be positive for expression of PAX6, LHX2, RAX, OTX2, SIX3 and / or SIX6, typical early ocular transcription factors.

[0222] In yet another implementation, isolated mammalian primitive retinal stem cells may be positive for expression of typical markers of primitive neuroepithelial stem cells, such as stem factor SOX2, nestin, and STAT3.

[0223] In another implementation, isolated mammalian primitive retinal stem cells may be positive for Ki67 (a cell proliferation marker).

[0224] In another implementation, compared to ESCs, isolated mammalian primitive retinal stem cells downregulated the transcription of ESC pluripotency transcription factors POU5F1 (OCT4), NANOG, KLF4 and TBX3 genes, as well as TBF-β superfamily genes SMAD1, SMAD2, TGF-β3, BMP3, BMP6, TGFBR1 and BMPR1B.

[0225] In yet another implementation, isolated mammalian primitive retinal stem cells maintain the transcription of the LIN28 and SALL4 transcription factor genes at the same levels as in ESCs, but significantly higher than in fetal retinal progenitor cells.

[0226] In another implementation, isolated mammalian primitive retinal stem cells upregulated the transcription of BMP4 and BMP7 genes, as well as OTX2, RAX, LHX2, SIX3, and SIX6 genes, compared to fetal retinal progenitor cells.

[0227] In yet another implementation, isolated mammalian primitive retinal stem cells were strongly positive for transcription of the SRFP1 and FZD3 / 5 genes.

[0228] In yet another implementation, isolated mammalian primitive retinal stem cells downregulated the transcription of FGFR1 / 2 / 3 and FGF3 / 8 genes compared to fetal retinal progenitor cells.

[0229] In yet another implementation, a small molecule differentiation inducer is used to direct isolated mammalian pRSCs to a specific retinal cell fate in vitro.

[0230] In yet another implementation, a specific retinal cell fate includes neuroretinal cells and non-neuronal cells.

[0231] In yet another implementation, the neural retinal cells include retinal ganglion cells (RGCs) and photoreceptors.

[0232] In yet another implementation, non-neuronal cells include retinal pigment epithelial (RPE) cells or retinal pigment epithelial cells (RPEs).

[0233] Examples of suitable cell culture media

[0234] In one embodiment, the culture medium, with or without supplements, comprises: DMEM / F12 or an equivalent medium; glutamine or L-alanyl-L-glutamine (GlutaMAX). ® StemPro ® hESC supplement or equivalent; bovine serum albumin or equivalent; basic fibroblast growth factor (bFGF-basic) or equivalent; and 2-mercaptoethanol or equivalent.

[0235] In another embodiment, the culture medium, with or without supplements, comprises: DMEM / F12 or equivalent medium; approximately 2.3 mM glutamine or L-alanyl-L-glutamine (GlutaMAX). ® ); 1 xStemPro ® hESC supplement or equivalent; approximately 1.8% bovine serum albumin or equivalent; approximately 8 ng / mL basic fibroblast growth factor (bFGF-basic) or equivalent; and approximately 0.1 mM 2-mercaptoethanol or equivalent.

[0236] In another embodiment, the culture medium, with or without a supplement, may be a serum-free N2B27 priming medium comprising: DMEM / F12 or equivalent medium; N-2 supplement or equivalent; B-27 serum-free supplement or equivalent; glutamine or L-alanyl-L-glutamine (GlutaMAX). ®The medium may contain: bovine serum albumin or equivalent; MEM non-essential amino acids or equivalent; and 2-mercaptoethanol or equivalent. Alternatively, one embodiment of the medium is a serum-free N2B27 starter medium supplemented with basic fibroblast growth factor (bFGF-basic) or equivalent. Furthermore, in another embodiment, the medium may contain DMEM / F12 or equivalent; 1 x N-2 supplement or equivalent; 1 x B-27 serum-free supplement or equivalent; and approximately 2 mM glutamine or L-alanyl-L-glutamine (GlutaMAX). ® The medium contains approximately 0.2% bovine serum albumin or equivalent; approximately 0.1 mM MEM non-essential amino acids or equivalent; and approximately 0.1 mM 2-mercaptoethanol or equivalent. Alternatively, another embodiment of the medium is serum-free N2B27 starter medium supplemented with approximately 20 ng / mL basic fibroblast growth factor (bFGF-basic) or equivalent.

[0237] In one embodiment, the culture medium with or without supplements may further comprise IWP2 (CAS No. 686770-61-6), SB431542 (CAS No. 301836-41-9), or LDN-193189 (CAS No. 1062368-24-4) or equivalents. In a preferred embodiment, the culture medium with or without supplements may further comprise at least two of IWP2 (CAS No. 686770-61-6), SB431542 (CAS No. 301836-41-9), or LDN-193189 (CAS No. 1062368-24-4), preferably all three or equivalents.

[0238] In one embodiment, the culture medium, with or without supplements, may further comprise about 1 μM IWP2, about 5 μM SB431542, or about 50 nM LDN193189. In a preferred embodiment, the culture medium, with or without supplements, may further comprise at least two of about 1 μM IWP2, about 5 μM SB431542, and about 50 nM LDN193189, preferably all three.

[0239] In another embodiment, the composition-defined culture medium may be a serum-free N2B27 starter medium supplemented with one or more of Wnt or TGF-β / BMP signaling inhibitors, or more preferably a combination of small molecule inhibitors of Wnt and TGF-β / BMP signaling. Furthermore, the culture medium may or may not contain supplements and may not contain feeder cells, feeder layer conditioned medium, or serum. Further, the culture medium may additionally contain a combination of small molecule inhibitors of Wnt and TGF-β / BMP signaling. In a preferred embodiment, the culture medium may contain DMEM / F12 or equivalent medium; N-2 supplement or equivalent; B-27 serum-free supplement or equivalent; glutamine or L-alanyl-L-glutamine (GlutaMAX). ® Bovine serum albumin or equivalent; MEM non-essential amino acids or equivalent; 2-mercaptoethanol or equivalent; and one or more of IWP2 (CAS No. 686770-61-6) or equivalent, SB431542 (CAS No. 301836-41-9) or equivalent, and LDN-193189 (CAS No. 1062368-24-4) or equivalent, preferably a combination of at least two, and most preferably a combination of all three.

[0240] In a more preferred embodiment, the composition-defined culture medium is a starter medium supplemented with one or more of Wnt or TGF-β / BMP signaling inhibitors, or more preferably a combination of small molecule inhibitors of Wnt and TGF-β / BMP signaling activity, and further comprises DMEM / F12 or equivalent medium; 1x N-2 supplement or equivalent; 1x B-27 serum-free supplement or equivalent; and approximately 2 mM glutamine or L-alanyl-L-glutamine (GlutaMAX). ® The following are ingredients: approximately 0.2% bovine serum albumin or equivalent; approximately 0.1 mM MEM non-essential amino acids or equivalent; approximately 0.1 mM 2-mercaptoethanol or equivalent; and one or more of approximately 1 μM IWP2 (CAS No. 686770-61-6) or equivalent, approximately 5 μM SB431542 (CAS No. 301836-41-9) or equivalent, and approximately 50 nM LDN-193189 (CAS No. 1062368-24-4) or equivalent, preferably a combination of at least two, and most preferably a combination of all three.

[0241] In one embodiment, isolated mammalian pRSCs can be maintained and / or amplified in serum-free N2B27 initiation medium supplemented with a combination of small molecule inhibitors of Wnt and TGF-β / BMP signaling activity of the present invention.

[0242] In vitro methods for producing isolated mammalian retinal ganglion cells (RGCs)

[0243] The present invention further provides an in vitro method for generating isolated mammalian retinal ganglion cells (RGCs). The method includes culturing isolated primitive retinal stem cells (pRSCs) from mammals in a cell culture medium free of feeder cells, feeder layer conditioned medium, or serum to generate and grow a monolayer culture of the isolated pRSCs; and contacting the culture of the isolated pRSCs with one or more of Wnt, Notch, or VEGFR signaling inhibitors to differentiate the isolated pRSCs into isolated mammalian RGCs. In one embodiment, the cells may be cultured on a solid support. The solid support may be coated with, for example, Matrigel. Or a base film or equivalent. Matrigel Alternatively, the base membrane could be a Matrigel with reduced growth factors. Or a basement membrane with reduced growth factors. In one embodiment of the invention, the cells can be cultured and allowed to grow and proliferate for more than two weeks.

[0244] As used herein, isolated RGCs mean, for example, RGCs that are substantially separated from contaminants (e.g., cells that are not RGCs).

[0245] The Wnt, Notch, or VEGFR signaling inhibitors can be small molecule inhibitors. In one embodiment, one or more of the Wnt, Notch, or VEGFR signaling inhibitors are a combination of inhibitors of at least two signaling pathways. Optionally, the combination may include inhibitors of all three signaling pathways.

[0246] In one embodiment, the culture of the isolated pRSCs is a monolayer culture. Furthermore, the monolayer culture may be nearly confluent or confluent.

[0247] In one embodiment, the cell culture medium that does not contain feeder cells, feeder layer conditioned medium, or serum is a chemically defined culture medium. A chemically defined culture medium may be N2B27 starter medium or an equivalent that does not contain feeder cells, feeder layer conditioned medium, or serum.

[0248] In one implementation, the combination of Wnt, Notch, and VEGFR signaling inhibitors can be IWP2, DAPT, and PD173074, respectively. IWP2, DAPT, and PD173074 can be used at concentrations of approximately 1 μM IWP2, approximately 10 μM DAPT, and approximately 200 nM PD173074.

[0249] In one embodiment, isolated mammalian pRSCs can be cultured for more than 2 weeks in a chemically defined medium supplemented with small molecule inhibitors of Wnt, Notch, and VEGFR signaling to obtain isolated mammalian RGCs.

[0250] In one implementation, the upregulation of RGC precursor-specific transcription factor genes can occur approximately 6 days after culturing in a chemically defined medium supplemented with small molecule inhibitors of Wnt, Notch, and VEGFR signaling. Compared to ESC, the upregulation of RGC precursor-specific transcription factor genes may include BRN3A, BRN3B, ISL-1, and MATH5.

[0251] In one implementation, most cultured cells may be positive for RGC markers after 2 weeks in a culture medium supplemented with a chemically defined composition of small molecule inhibitors of Wnt, Notch, and VEGFR signaling. Furthermore, most cultured cells that are positive for RGC markers may also be positive for TUJ1 and BRN3.

[0252] Methods for generating isolated mammalian photoreceptors

[0253] The present invention also provides a method for generating isolated mammalian photoreceptors from isolated mammalian pRSCs under chemically defined in vitro conditions free of feeder cells, feeder layer conditioned medium, or serum, wherein the method may include culturing and growing dissociated pRSCs from mammals in a neural induction medium supplemented with one or more inhibitors of TGF-β / activin receptor-like kinases ALK-4, ALK-5, or ALK-7, glycogen synthase kinase-3 (GSK-3), Notch or Wnt signaling, or hedgehog signaling activator. For example, culturing and growing dissociated pRSCs in a neural induction medium for a sufficient time may induce pRSCs to a photoreceptor cell lineage fate without visible morphological changes or expression of photoreceptor-specific markers. In one embodiment, the cells may be cultured on a solid support. The solid support may be coated with, for example, Matrigel. Or a base film or equivalent. Matrigel Alternatively, the base membrane could be a Matrigel with reduced growth factors. Or a basement membrane with reduced growth factors. In one embodiment of the invention, the cells can be cultured and allowed to grow and proliferate for approximately 6 days.

[0254] As used herein, a separate photoreceptor means, for example, a photoreceptor that is substantially separate from contaminants (e.g., cells that are not photoreceptors).

[0255] For example, one or more inhibitors of TGF-β / activin receptor-like kinases ALK-4, ALK-5, or ALK-7, glycogen synthase kinase-3 (GSK-3), Notch, or Wnt signaling, or hedgehog signaling activators, can be a combination of inhibitors of TGF-β / activin receptor-like kinases ALK-4, ALK-5, and ALK-7, glycogen synthase kinase-3 (GSK-3), Notch, and Wnt signaling, and hedgehog signaling activators, which may contain regulators of at least two signaling pathways. Optionally, and more preferably, the combination may contain regulators of all five signaling pathways. In cases involving one or more inhibitors of TGF-β / activin receptor-like kinases ALK-4, ALK-5, or ALK-7, glycogen synthase kinase-3 (GSK-3), Notch, or Wnt signaling, the inhibitors may be combinations of inhibitors of TGF-β / activin receptor-like kinases ALK-4, ALK-5, and ALK-7, glycogen synthase kinase-3 (GSK-3), Notch, and Wnt signaling, wherein the combination comprises inhibitors of at least two signaling pathways or preferably inhibitors of all four signaling pathways. Inhibitors of TGF-β / activin receptor-like kinases ALK-4, ALK-5, and ALK-7, glycogen synthase kinase-3 (GSK-3), Notch, or Wnt signaling, or hedgehog signaling activators, may be small molecule inhibitors.

[0256] Following this first culture step, the method further includes culturing the cells in a neural induction medium supplemented with retinoic acid or an equivalent, or taurine or an equivalent, or both, to induce differentiation of mammalian pRSCs into photoreceptor cells, thereby generating isolated mammalian photoreceptors from isolated pRSCs from mammals under in vitro cell culture conditions defined by the absence of feeder cells, feeder layer conditioned medium, or serum. In yet another embodiment, the cells may be cultured for approximately 7 days or longer, or for a period allowing pRSCs to differentiate into photoreceptor cells.

[0257] In a preferred embodiment, a method for generating isolated mammalian photoreceptors from isolated mammalian pRSCs under in vitro conditions defined by the chemical composition of the culture medium, which does not contain feeder cells, feeder layer conditioned medium, or serum, may include: [the process involves] coating Matrigel... or base film or equivalent (of which Matrigel) pRSCs were cultured on a solid support (or a basement membrane with reduced growth factors) in a neural induction medium supplemented with TGF-β / activin receptor-like kinases ALK-4, ALK-5 and ALK-7, glycogen synthase kinase-3 (GSK-3), small molecule inhibitors of Notch and Wnt signaling, and small molecule activators of hedgehog signaling; and subsequently cultured in a neural induction medium supplemented with retinoic acid or equivalent and taurine or equivalent; to allow the mammalian pRSCs to differentiate into photoreceptors, thereby generating isolated mammalian photoreceptors from isolated pRSCs from mammals under in vitro cell culture conditions defined by the absence of feeder cells, feeder layer conditioned medium, or serum.

[0258] In yet another preferred embodiment, the cells can be cultured for approximately 6 days, followed by a further culture for approximately 7 days or longer to differentiate mammalian pRSCs into photoreceptor cells. Following culture, photoreceptors can be derived from the pRSCs. The resulting photoreceptors may express pan-photoreceptor markers such as recovery protein, cone cell-specific markers such as OPN1SW or blue opsin, rod cell-specific markers such as rhodopsin, and / or interreceptor vitamin A class-binding protein (IRBP). Furthermore, the photoreceptors may exhibit cellular processes, short internal processes, and / or long extended external processes.

[0259] In one implementation, the isolated mammalian photoreceptors are positive for rhodopsin, rhodopsin kinase, and / or transmucin.

[0260] In an alternative embodiment, isolated mammalian photoreceptors can be obtained by culturing dissociated pRSCs from mammals in a neural induction medium containing one or more inhibitors of TGF-β / activin receptor-like kinases ALK-4, ALK-5, or ALK-7, glycogen synthase kinase-3 (GSK-3), Notch or Wnt signaling, or hedgehog signaling activator, for a sufficient time to induce the pRSCs to a photoreceptor cell lineage fate without visible morphological changes or expression of photoreceptor-specific markers. The cultured cells are then contacted with retinoic acid or taurine, or both, to differentiate the mammalian pRSCs into photoreceptor cells. The medium is free of feeder cells, feeder layer conditioned medium, or serum. The cells are then grown on a solid support, which may be coated with Matrigel. Or base film, preferably Matrigel with reduced growth factors. Or a basement membrane with reduced growth factors.

[0261] Suitable examples of neural induction media include the following:

[0262] Neurobasal medium may contain Advanced DMEM / F12 medium or equivalent; Culture medium or equivalent; N2 supplement or equivalent; B-27 serum-free supplement or equivalent; glutamine or L-alanyl-L-glutamine (e.g., GlutaMAX) ); bovine serum albumin or equivalent; and human leukemia inhibitory factor (hLIF) or equivalent.

[0263] Preferred neural induction media may contain Advanced DMEM / F12:Neurobasal (approximately 1:1) medium or equivalent; N2 supplement (e.g., approximately 1x N2 supplement) or equivalent; B-27 serum-free supplement (e.g., approximately 1x B-27 serum-free supplement) or equivalent; glutamine (e.g., approximately 1% glutamine) or L-alanyl-L-glutamine (GlutaMAX). Bovine serum albumin (e.g., about 5 ug / mL bovine serum albumin) or equivalents; and human leukemia inhibitory factor (hLIF) (e.g., about 10 ng / mL hLIF) or equivalents.

[0264] In culture media, examples of small molecule inhibitors of TGF-β / activin receptor-like kinases ALK-4, ALK-5 and ALK-7, glycogen synthase kinase-3 (GSK-3), Notch and Wnt signaling, and hedgehog signaling activators include, but are not limited to, SB431542 (CAS No. 301836-41-9) or equivalent, CHIR99021 (CAS No. 252917-06-9) or equivalent, DAPT (CAS No. 208255-80-5) or equivalent, IWP2 (CAS No. 686770-61-6) or equivalent, and puromorphamine (CAS No. 483367-10-8) or equivalent.

[0265] As a supplement to the neural induction medium, any one or more of the following may be added: SB431542 (CAS No. 301836-41-9) or equivalent, CHIR99021 (CAS No. 252917-06-9) or equivalent, DAPT (CAS No. 208255-80-5) or equivalent, IWP2 (CAS No. 686770-61-6) or equivalent, and puromorphine (CAS No. 483367-10-8) or equivalent. For example only, the supplement may be added at the following concentrations: about 2 μM SB431542 (CAS No. 301836-41-9) or equivalent; about 3 μM CHIR99021 (CAS No. 252917-06-9) or equivalent; about 10 μM DAPT (CAS No. 208255-80-5) or equivalent; about 1 μM IWP2 (CAS No. 686770-61-6) or equivalent; and about 100 nM purinemorphine (CAS No. 483367-10-8) or equivalent.

[0266] In yet another embodiment, retinoic acid or its equivalent and taurine or its equivalent may be used to supplement the neural induction medium at a concentration of about 500 nM retinoic acid or its equivalent and / or about 100 M taurine or its equivalent.

[0267] Methods for producing non-neural isolated mammalian retinal pigment epithelium (RPE) or isolated mammalian retinal pigment epithelial cells (RPEs)

[0268] The present invention further provides a method for generating non-neural isolated mammalian retinal pigment epithelium (RPE) in vitro from isolated mammalian pRSCs in the absence of feeder cells or feeder layer conditioned medium. Optionally, the method can be used to obtain isolated mammalian retinal pigment epithelial cells (RPEs) in vitro from isolated mammalian pRSCs in the absence of feeder cells and feeder layer conditioned medium. In one embodiment of the invention, the method includes culturing monolayer pRSCs from mammals in an RPE induction medium supplemented with nicotinamide or equivalent, or activin A or equivalent, or both; and subsequently culturing the pRSCs in a medium supplemented with N1 medium supplement or equivalent, taurine or equivalent, hydrocortisone or equivalent, or triiodothyronine or equivalent; to allow the mammalian pRSCs to differentiate in vitro from isolated mammalian pRSCs into mammalian RPEs or RPEs in the absence of feeder cells or feeder layer conditioned medium. For example, mammalian pRSCs can be cultured for a sufficient time in a medium containing nicotinamide or activin A or both, in the absence of SMAD signaling inhibition, to induce the pRSCs to the RPE fate.

[0269] As used herein, isolated RPEs mean, for example, RPEs that are substantially separated from contaminants (e.g., cells that are not RPEs).

[0270] In one embodiment, pRSCs can be cultured in RPE medium supplemented with N1 medium supplement, taurine, hydrocortisone and triiodothyronine.

[0271] In one embodiment of the method, pRSC cells (e.g., derived from mammals) can be cultured on a solid support. The solid support may be coated with Matrigel. Or a base film or equivalent.

[0272] Matrigel Alternatively, the base membrane could be a Matrigel with reduced growth factors. Or a basement membrane with reduced growth factors. The culture medium used for culturing can be an RPE-inducing medium supplemented with nicotinamide or its equivalent, or activin A or its equivalent, or both, without SMAD signaling inhibitors, for approximately 7 days to induce pRSCs to the RPE fate.

[0273] In another embodiment, cells can be cultured in RPE medium supplemented with N1 medium supplement or equivalent, taurine or equivalent, hydrocortisone or equivalent, or triiodothyronine or equivalent for approximately 12 days or longer. pRSCs then differentiate into RPE fate. In yet another embodiment, cells can be cultured in RPE induction medium supplemented with nicotinamide or equivalent and activin A or equivalent for approximately 7 days, followed by culture in RPE medium supplemented with N1 medium supplement or equivalent, taurine or equivalent, hydrocortisone or equivalent, and triiodothyronine or equivalent for approximately 12 days or longer. After culturing in RPE medium supplemented with N1 medium supplement or equivalent, taurine or equivalent, hydrocortisone or equivalent, and triiodothyronine or equivalent for approximately 12 days or longer, RPEs or RPEs can be obtained from pRSCs. RPEs or RPEs can express RPE65, form polygonal actin bundles, and become colored.

[0274] In a preferred embodiment, a method for generating non-neural, isolated mammalian retinal pigment epithelium (RPE) or isolated mammalian retinal pigment epithelial cells (RPEs) in vitro from isolated mammalian pRSCs without feeder cells and feeder layer conditioned medium may include: culturing monolayer pRSCs in an RPE induction medium supplemented with nicotinamide or equivalent and activin A or equivalent; and subsequently culturing them in an RPE maturation medium supplemented with N1 medium supplement or equivalent, taurine or equivalent, hydrocortisone or equivalent, and triiodothyronine or equivalent; to differentiate the mammalian pRSCs into mammalian RPEs or RPEs, thereby generating isolated mammalian RPEs or RPEs in vitro from isolated mammalian pRSCs without feeder cells and feeder layer conditioned medium.

[0275] In a separate embodiment, a method for generating non-neural isolated mammalian RPEs or RPEs in vitro from isolated mammalian pRSCs in the absence of feeder cells and feeder layer conditioned medium may include: culturing mammalian pRSCs in a medium containing nicotinamide or activin A or both, in the absence of SMAD signaling inhibitors, and contacting the pRSCs with one or more of N1 medium supplement, taurine, hydrocortisone, or triiodothyronine; and differentiating the mammalian pRSCs into mammalian RPEs or RPEs, thereby generating isolated mammalian RPEs or RPEs in vitro from isolated mammalian pRSCs in the absence of feeder cells and feeder layer conditioned medium.

[0276] Suitable examples of RPE induction media and RPE media include the following:

[0277] RPE induction medium may contain Glasgow Minimum Essential Medium (GMEM) or equivalent; KnockOut™ serum substitute or equivalent; MEM non-essential amino acids or equivalent; sodium pyruvate or equivalent; and β-mercaptoethanol or equivalent.

[0278] Preferred examples of RPE induction media include Glasgow Minimum Essential Medium (GMEM) or equivalent; KnockOut™ serum substitute (e.g., about 10% KnockOut™ serum substitute) or equivalent; MEM non-essential amino acids (e.g., about 0.1 mM MEM non-essential amino acids) or equivalent; sodium pyruvate (e.g., about 1 mM sodium pyruvate) or equivalent; and β-mercaptoethanol (e.g., about 0.1 mM β-mercaptoethanol) or equivalent.

[0279] Nicotinamide or its equivalents and / or activator A or its equivalents may be present in RPE induction medium at concentrations of, for example, about 10 mM nicotinamide or its equivalents and / or about 100 ng / mL activator A or its equivalents.

[0280] RPE medium may contain minimum essential medium (αMEM), α-modified medium or equivalent; fetal bovine serum or equivalent; L-glutamine or L-alanyl-L-glutamine (GlutaMAX). ); MEM non-essential amino acids or equivalents; and sodium pyruvate or equivalents.

[0281] Preferred examples of RPE media may include minimum essential medium (αMEM), α-modified medium, or equivalents; approximately 5% fetal bovine serum or equivalents; and approximately 2 mM L-glutamine or L-alanyl-L-glutamine (GlutaMAX). ); approximately 0.1 mM MEM non-essential amino acids or equivalents; and approximately 1 mM sodium pyruvate or equivalents.

[0282] In one embodiment, the RPE medium may contain an N1 medium supplement or equivalent, taurine or equivalent, hydrocortisone or equivalent, and triiodothyronine or equivalent. Furthermore, in a preferred embodiment, the RPE medium may contain 1 x N1 medium supplement or equivalent; about 0.25 mg / mL taurine or equivalent; about 20 ng / mL hydrocortisone or equivalent; and about 0.013 ng / mL triiodothyronine or equivalent.

[0283] Methods for generating isolated human primitive retinal stem cells (hpRSC)

[0284] The present invention further provides a method for generating isolated human primitive retinal stem cells (hpRSC) from isolated human embryonic stem cells (hESC), isolated human pluripotent stem cells (hPSC), or isolated human induced pluripotent stem cells (iPSC) under in vitro cell culture conditions with a defined composition that does not contain feeder cells, feeder layer conditioned medium, or serum. In one embodiment, the method includes (a) culturing isolated hESCs, isolated hPSCs, or isolated human iPSCs on a solid support coated with growth factor-reducing Matrigel (BD Bioscience) or its equivalent in StemPro hESC SFM medium (Invitrogen) or its equivalent in the absence of feeder cells, feeder layer conditioned medium, or serum until near confluence, preferably until about 80% cell confluence; (b) culturing in a starter medium supplemented with alkaline FGF (bFGF) for a sufficient time to grow to near confluence; and (c) culturing in a starter medium supplemented with a combination of small molecule inhibitors of Wnt and TGF-β / BMP signaling activity to differentiate the isolated hESCs, isolated hPSCs, or isolated human iPSCs into isolated human primitive retinal stem cells.

[0285] In a preferred embodiment, a method for generating isolated human primitive retinal stem cells (hpRSCs) may include: encapsulating growth factor-reducing Matrigel... ® Isolated hESCs, isolated hPSCs, or isolated human iPSCs were cultured on solid supports of (BD Bioscience) or equivalents in StemPro hESC SFM medium (Invitrogen) or equivalents in the absence of feeder cells, feeder layer conditioned medium, or serum. The StemPro hESC SFM medium, with approximately 80% cell confluence, was then converted to serum-free N2B27 starter medium supplemented with approximately 20 ng / mL bFGF. The serum-free N2B27 starter medium may contain: DMEM / F12 or equivalent, 1x N-2 supplement or equivalent, 1x B-27 serum-free supplement or equivalent, approximately 0.2% BSA or equivalent, and approximately 2 mM L-GlutaMAX. ®Or equivalent, about 0.1 mM MEM non-essential amino acids or equivalents and about 0.1 mM β-mercaptoethanol or equivalents); cultured in N2B27 starter medium supplemented with bFGF for about 1-2 days; the medium supplemented with bFGF in the near-confluent monolayer culture of hESCs in N2B27 starter medium was converted to N2B27 starter medium supplemented with a combination of small molecule inhibitors of Wnt and TGF-β / BMP signaling activity, so as to differentiate the isolated hESCs, isolated hPSCs or isolated human iPSCs into isolated human primitive retinal stem cells, thereby generating isolated hpRSCs from isolated hESCs, isolated hPSCs or isolated human iPSCs under in vitro cell culture conditions defined by the absence of feeder cells, feeder layer conditioned medium or serum.

[0286] In one embodiment, the combination of small molecule inhibitors of Wnt and TGF-β / BMP signaling activity in the culture medium may be a combination of SB 431542, LDN193189, and IWP2 (Wnt production inhibitor-2); a combination of SB 431542, a head protein analog, and IWP2; a combination of SB 431542, LDN193189, and a Dkk1 analog; a combination of SB 431542, a head protein analog, and a Dkk1 analog; or equivalent combinations, wherein the combination produces synergistic inhibition of Wnt and TGF-β / BMP signaling activity. In a preferred embodiment, the combination of SB 431542, LDN193189, and IWP2 may comprise about 5 μM SB 431542, about 50 nM LDN193189, and about 1 μM IWP2.

[0287] In one embodiment, isolated human primitive retinal stem cells, comprising more than approximately 90% of the total cell population, may be positive for typical early ocular region transcription factors PAX6, LHX2, RAX, OTX2, SIX3, and SIX6, expressed by ocular progenitor cells. In another embodiment, isolated human primitive retinal stem cells may be positive for expression of typical markers of stem cell characteristics, such as SOX2, nestin, and STAT3. In yet another embodiment, isolated human primitive retinal stem cells may be positive for expression of Ki67 (a cell proliferation marker).

[0288] In one implementation, compared to hESCs, isolated human primitive retinal stem cells can downregulate the transcription of hESC pluripotency transcription factors POU5F1 (OCT4), NANOG, KLF4 and TBX3 genes, as well as TBF-β superfamily genes SMAD1, SMAD2, TGF-β3, BMP3, BMP6, TGFBR1 and BMPR1B.

[0289] In one embodiment of the invention, isolated human progenitor retinal stem cells can maintain the transcription of the LIN28 and SALL4 transcription factor genes at the same level as in hESCs, but significantly higher than in human fetal retinal progenitor cells.

[0290] In another implementation, compared to human fetal retinal progenitor cells, isolated human primitive retinal stem cells can upregulate the transcription of BMP4 and BMP7 genes as well as OTX2, RAX, LHX2, SIX3, and SIX6 genes.

[0291] In one embodiment of the invention, isolated human primitive retinal stem cells can be strongly positive for transcription of the SRFP1 and FZD3 / 5 genes.

[0292] In one implementation, isolated human primitive retinal stem cells can downregulate the transcription of FGFR1 / 2 / 3 and FGF3 / 8 genes compared to human fetal retinal progenitor cells.

[0293] In one embodiment, isolated human pRSCs can be maintained and / or amplified in serum-free N2B27 starter culture medium supplemented with a combination of small molecule inhibitors of Wnt and TGF-β / BMP signaling activity of the present invention.

[0294] In another implementation, small molecule differentiation inducers can be used to direct isolated human pRSCs to a specific retinal cell fate in vitro.

[0295] In one implementation, a specific retinal cell fate can include neural retinal cells and non-neuronal cells. Neural retinal cells can include retinal ganglion cells (RGCs) and photoreceptors. Non-neuronal cells can include retinal pigment epithelial (RPE) cells.

[0296] Methods for generating mammalian primitive retinal stem cells (hpRSCs), more differentiated retinal progenitor cells, retinal neurons, retinal ganglion cells, photoreceptor precursors, or retinal pigment epithelium (RPE).

[0297] The present invention further provides a method for generating mammalian primitive retinal stem cells (hpRSC), more differentiated retinal progenitor cells, retinal neurons, retinal ganglion cells, photoreceptor precursors, or retinal pigment epithelium (RPE) from a subject, which requires the generation of induced pluripotent stem cells (iPSC) from the subject and the generation of pRSC from the iPSC by the method of the present invention, and / or further induction of differentiation by the method of the present invention.

[0298] Methods for treating retinal degeneration in subjects

[0299] The present invention further provides a method for treating retinal degeneration in a subject in need, comprising administering primitive retinal stem cells, retinal ganglion cells, photoreceptors, retinal pigment epithelial cells, and / or combinations thereof to the patient's eye. The primitive retinal stem cells, retinal ganglion cells, photoreceptors, and retinal pigment epithelial cells can be administered in sufficient quantities to treat the retinal degeneration in the subject and can be generated by the method of the present invention.

[0300] As used herein, the terms “subject” and “patient” are used interchangeably and refer to any living organism to which any derivative composition of the present invention may be applied. This term includes, but is not limited to, humans; non-human animals, such as non-human primates like chimpanzees and other apes and monkeys; farm animals such as cattle, sheep, pigs, goats, and horses; domesticated subjects such as dogs and cats; and laboratory animals, including rodents such as mice, rats, and guinea pigs. Therefore, it is intended to encompass adult and newborn subjects, both male and female, as well as fetuses.

[0301] As used herein, the term "effective amount" refers to, for example, the amount of cells and / or compositions of the present invention that are needed to delay, reduce, or improve at least one symptom of an eye-related disease or condition (e.g., retinal degeneration). For example, an effective amount of any cell of the present invention is an amount that effectively reduces or inhibits retinal degeneration of the cells of the present invention. Therefore, an effective amount is also an amount sufficient to prevent the development of symptoms of an eye-related disease or to alleviate or reduce the rate of symptom progression.

[0302] As used herein, the terms “application” and “introduction” are used interchangeably and refer to the placement of said cells or compositions into a subject by means of a method or route that at least partially directs said cells or compositions of the invention as disclosed herein to a desired site. The cells or compositions of the invention may be applied by any suitable route that produces an effective treatment for said subject.

[0303] In one implementation, the cells to be delivered may be combined with or contained in a matrix (e.g., a hydrogel) before being delivered to the patient's eye.

[0304] In one embodiment, the hydrogel may comprise hyaluronic acid and methylcellulose, or their salts and derivatives. Additionally, the hydrogel may comprise a hydrogel containing bioactive peptides. In one embodiment, the matrix may be a biocompatible polymer or mixture of biocompatible polymers supporting the cell viability and function of the transplanted cells, an artificial biomimetic matrix, a bioactive scaffold derived from a tissue or organ matrix, a biosynthesized extracellular matrix based on collagen and N-isopropylacrylamide copolymers, or a scaffold modified with adhesion molecules, laminins, growth factors, morphogenetic factors, survival factors, extracellular matrix fragments or derivatives. The hydrogel may comprise about 0.5% sodium hyaluronate (1400-1800 kDa) and about 0.5% methylcellulose (100 kDa) or equivalents thereof in a balanced salt solution.

[0305] In one implementation, the cells can be administered into the subretinal space of the subject's eye.

[0306] In one implementation, retinal degeneration may be associated with age-related macular degeneration (AMD), Staggart's macular dystrophy, retinitis pigmentosa, glaucoma, retinal vascular disease, viral infections of the eye, and other retinal / eye diseases of known or unknown etiology.

[0307] According to the practice of the present invention, the subject can be a mammal, such as a human. The subject can also be a mammal, such as a monkey, bear, rat, mouse, mink, rabbit, guinea pig, pig, dog, cat, goat, sheep, horse or cow.

[0308] In one embodiment, a method for treating retinal degeneration in a subject of need includes (a) obtaining a tissue or cell sample containing PSCs and somatic cells from the subject; (b) isolating the PSCs and somatic cells from the sample to obtain isolated PSCs and isolated somatic cells; (c) culturing the isolated PSCs to allow the isolated PSCs to grow; or reprogramming the isolated somatic cells to obtain iPSCs and isolating the iPSCs to obtain isolated iPSCs; (d) culturing the isolated PSCs or isolated iPSCs by the method of the present invention to generate pRSCs; and (e) administering a sufficient amount of primitive retinal stem cells (pRSCs) to the eye of the subject to treat the retinal degeneration in the subject.

[0309] In another embodiment, a method for treating retinal degeneration in a subject of need includes (a) obtaining a tissue or cell sample containing PSCs and somatic cells from the subject; (b) isolating the PSCs and somatic cells from the sample to obtain isolated PSCs and isolated somatic cells; (c) culturing the isolated PSCs to allow the isolated PSCs to grow; or reprogramming the isolated somatic cells to obtain iPSCs and isolating the iPSCs to obtain isolated iPSCs; (d) culturing the isolated PSCs or isolated iPSCs to generate pRSCs; (e) culturing the isolated pRSCs by the method of the present invention to generate isolated RGCs; and (f) administering the thus generated mammalian retinal ganglion cells (RGCs) to the eye of the subject in a sufficient amount to treat the retinal degeneration in the subject.

[0310] In yet another embodiment, a method for treating retinal degeneration in a subject of need comprises (a) obtaining a tissue or cell sample containing PSCs and somatic cells from the subject; (b) isolating the PSCs and somatic cells from the sample to obtain isolated PSCs and isolated somatic cells; (c) culturing the isolated PSCs to allow the isolated PSCs to grow; or reprogramming the isolated somatic cells to obtain iPSCs and isolating the iPSCs to obtain isolated iPSCs; (d) culturing the isolated PSCs or isolated iPSCs to generate pRSCs; (e) culturing the isolated pRSCs by the method of the present invention to generate isolated photoreceptors; and (f) administering the photoreceptors of step (e) to the eye of the subject in a sufficient amount to treat the retinal degeneration in the subject.

[0311] In another embodiment, a method for treating retinal degeneration in a subject of need includes (a) obtaining a tissue or cell sample containing PSCs and somatic cells from the subject; (b) isolating the PSCs and somatic cells from the sample to obtain isolated PSCs and isolated somatic cells; (c) culturing the isolated PSCs to allow the isolated PSCs to grow; or reprogramming the isolated somatic cells to obtain iPSCs and isolating the iPSCs to obtain isolated iPSCs; (d) culturing the isolated PSCs or isolated iPSCs to generate pRSCs; (e) culturing the isolated pRSCs by the method of the present invention to generate isolated RPEs; and (f) administering the thus generated mammalian retinal pigment epithelial cells (RPEs) to the eye of the subject in a sufficient amount to treat the retinal degeneration in the subject.

[0312] Methods for delivering human primitive retinal stem cells (hpRSC), human primitive retinal ganglion cells (hpRGC), human photoreceptors, or human retinal pigment epithelial cells (hRPEs) to subjects

[0313] The present invention further provides a method for delivering human primitive retinal stem cells (hpRSC), human primitive retinal ganglion cells (hpRGC), human photoreceptors, or human retinal pigment epithelial cells (hRPEs) to a patient or subject in need, wherein hpRSC, hpRGC, human photoreceptors, or hRPEs are administered into the subretinal space of the subject in a single-cell suspension in, for example, a BSS / HAMC (about 0.5 / 0.5% w / w) hydrogel solution.

[0314] In one embodiment, the present invention provides a method for treating retinal degeneration in a patient or subject in need, which requires delivering primitive retinal stem cells (pRSCs) to the eye of the patient or subject in need, wherein the pRSCs can be generated by the method of the present invention.

[0315] In one embodiment, the present invention provides a method for treating retinal degeneration in a patient or subject in need, which involves delivering mammalian retinal ganglion cells (RGCs) to the eye of the patient or subject in need, wherein the RGCs can be generated by the method of the present invention.

[0316] In one embodiment, the present invention provides a method for treating retinal degeneration in a patient or subject in need, which requires delivering a mammalian photoreceptor to the eye of the patient or subject in need, wherein the photoreceptor can be generated by the method of the present invention.

[0317] In one embodiment, the present invention provides a method for treating retinal degeneration in a patient or subject in need, which involves delivering mammalian retinal pigment epithelium (RPE) or mammalian retinal pigment epithelial cells (RPEs) to the eye of the patient or subject in need, wherein the RPEs can be generated by the method of the present invention.

[0318] Mammals can be humans, monkeys, bears, rats, mice, minks, rabbits, guinea pigs, pigs, dogs, cats, goats, sheep, horses, or cattle.

[0319] The subject or patient can be a human. In one implementation, the subject or patient can be a mammal, such as a human, monkey, bear, rat, mouse, mink, rabbit, guinea pig, pig, dog, cat, goat, sheep, horse, or cow.

[0320] This invention also provides a method for delivering human primitive retinal stem cells (hpRSC), human retinal ganglion cells (hpRGC), human photoreceptors, human retinal pigment epithelium (hRPE), or human retinal pigment epithelial cells (hRPEs) to a patient. In one embodiment, hpRSC, hpRGC, human photoreceptors, hRPE, or hRPEs can be administered as a monolayer cell suspension or a single-cell suspension in a matrix solution or matrix suspension to the subretinal space of a subject. The cells can be generated by the method of this invention. In one embodiment, a single-cell suspension of hpRSC in a BSS / HAMC (approximately 0.5 / 0.5% w / w) hydrogel solution can be administered to the subretinal space of a patient. The cells can be delivered to a subject in need, and the delivery of the cells can be used to treat retinal degeneration.

[0321] In one embodiment, the matrix is ​​a hydrogel containing hyaluronic acid and methylcellulose or their salts and derivatives, a hydrogel containing bioactive peptides, a biocompatible polymer or mixture of biocompatible polymers that support the cell viability and function of the transplanted cells, an artificial biomimetic matrix, a bioactive scaffold derived from a tissue or organ matrix, a biosynthesized extracellular matrix based on collagen and N-isopropylacrylamide copolymer, or a scaffold modified with adhesion molecules, laminins, growth factors, morphogenetic factors, survival factors, extracellular matrix or fragments or derivatives.

[0322] In one embodiment, the present invention provides a method for identifying molecular and cellular events that explain the pathogenesis of retinal degeneration in a subject, which requires generating human primitive retinal stem cells (hpRSCs), more differentiated retinal progenitor cells, retinal neurons, retinal ganglion cells, photoreceptor precursors, or retinal pigment epithelium (RPE) from the subject using the method of the present invention to obtain a "disease in a dish" model in order to be able to identify molecular and cellular events that explain the pathogenesis of retinal degeneration in the subject.

[0323] In one implementation scheme, the subjects are mammals, humans, monkeys, bears, rats, mice, minks, rabbits, guinea pigs, pigs, dogs, cats, goats, sheep, horses, or cattle.

[0324] In one embodiment, the present invention provides a method for preventing or inhibiting tumors associated with the use of pluripotent stem cells to treat a subject suffering from retinal degeneration. The method includes inducing isolated pluripotent stem cells into isolated pRSCs and administering the resulting isolated pRSCs to the subject, wherein the isolated pRSCs can be generated by the method of the present invention prior to the administration of stem cells to the subject's eye.

[0325] In another embodiment, the method includes inducing isolated pluripotent stem cells into isolated hpRSCs, further differentiating the isolated hpRSCs into isolated hpRGCs, isolated human photoreceptors, or isolated hRPEs, and administering the resulting isolated hpRSCs, isolated human photoreceptors, or isolated hRPEs to the subject.

[0326] The composition of the present invention

[0327] The present invention provides a composition comprising mammalian primitive retinal stem cells (hpRSC) generated by the method of the present invention and a suitable carrier.

[0328] As used herein, the phrase "suitable carrier" means a pharmaceutically acceptable material, composition, or medium, such as a liquid or solid filler, diluent, excipient, solvent, or encapsulating material, that participates in carrying or transporting the pharmaceutical agent of the present invention from one organ or part of the body to another organ or part of the body. Each carrier must be "acceptable" in the sense of compatibility with other components of the formulation, or bioinert.

[0329] Furthermore, the present invention provides a composition comprising mammalian retinal ganglion cells (RGCs) produced by the method of the present invention and a suitable carrier.

[0330] Furthermore, the present invention provides a composition comprising a mammalian photoreceptor produced by the method of the present invention and a suitable carrier.

[0331] The present invention also provides compositions comprising mammalian retinal pigment epithelium (RPE) or mammalian retinal pigment epithelial cells (RPEs) produced by the method of the present invention and a suitable carrier.

[0332] In one embodiment, the combination of small molecule inhibitors of Wnt and TGF-β / BMP signaling activity may be a combination or equivalent combination of IWP2 (CAS No. 686770-61-6), SB431542 (CAS No. 301836-41-9), and LDN-193189 (CAS No. 1062368-24-4), wherein the combination can produce synergistic inhibition of Wnt and TGF-β / BMP signaling activity. Alternatively, the combination may comprise IWP2 (CAS No. 686770-61-6), SB431542 (CAS No. 301836-41-9), and / or LDN-193189 (CAS No. 1062368-24-4), or equivalent combinations, wherein the combination can produce synergistic inhibition of Wnt and TGF-β / BMP signaling activity.

[0333] In one implementation, the mammal is a human, monkey, bear, rat, mouse, mink, rabbit, guinea pig, pig, dog, cat, goat, sheep, horse, or cow.

[0334] According to another aspect of the invention, a kit is provided. The kit according to the invention comprises packaging containing the compounds or compositions of the invention.

[0335] The phrase "packaging" means any container containing the compounds or compositions presented herein. In a preferred embodiment, the packaging may be a box or wrapping. Packaging materials used for packaging pharmaceutical products are well known to those skilled in the art.

[0336] The kit may also contain items not included in the packaging but attached to the outside of the packaging, such as pipettes.

[0337] The kit may optionally contain instructions for administering the compositions of the invention to a subject suffering from a condition requiring treatment. The kit may also include instructions for use of the compounds described herein approved by a regulatory agency such as the U.S. Food and Drug Administration. The kit may optionally contain a label or product insert regarding the compounds. The packaging and / or any product insert itself may be regulatory approved. The kit may contain the compounds in a solid or liquid phase (such as the buffer provided) within the package. The kit may also contain a buffer for preparing solutions for carrying out the methods, and a pipette for transferring liquid from one container to another.

[0338] The kit may also optionally contain one or more other compounds for use in combination with the therapies as described herein. In some embodiments, the packaging is a container for intravenous administration. In other embodiments, the compound is provided in an inhaler. In other embodiments, the compound is provided in a polymer matrix or in liposome form.

[0339] In one embodiment, the present invention provides a kit for generating mammalian primitive retinal stem cells (pRSCs) from a subject, which requires in vitro treatment of adherent monolayer pluripotent stem cells (PSCs) or induced pluripotent stem cells (iPSCs) from the subject with a chemically defined medium containing no feeder cells, feeder layer conditioned medium or serum, but supplemented with one or more of Wnt or TGF-β / BMP signaling inhibitors or more preferably a combination of small molecule inhibitors of Wnt and TGF-β / BMP signaling.

[0340] The present invention further provides a kit for generating mammalian primitive retinal stem cells (pRSCs), wherein the kit includes instructions for culturing mammalian embryonic stem cells (ESCs), pluripotent stem cells (PSCs), or induced pluripotent stem cells (iPSCs) in a culture medium with a chemical composition defined free of feeder cells, feeder layer conditioned medium, or serum, and instructions for using small molecule inhibitors of Wnt signaling or TGF-β / BMP signaling, or inhibitors of both Wnt signaling and TGF-β / BMP signaling.

[0341] In one embodiment, the kit further comprises a small molecule inhibitor of Wnt signaling or TGF-β / BMP signaling, or a small molecule inhibitor of both Wnt signaling and TGF-β / BMP signaling.

[0342] In one implementation, a small molecule inhibitor of Wnt signaling may include a C 22 H 18 The chemical formula of N4O2S3 is Wnt production inhibitor-2 (IWP2; CAS No. 686770-61-6) or N-(6-methyl-1,3-benzothiazol-2-yl)-2-[(4-oxo-3-phenyl-6,7-dihydrothieno[3,2-d]pyrimidin-2-yl)thioalkyl]acetamide.

[0343] In one embodiment, the small molecule inhibitor of TGF-β / BMP signaling can be a small molecule inhibitor of transforming growth factor-β (TGF-β) superfamily type I activin receptor-like kinases ALK-4, ALK-5, and ALK-7. The small molecule inhibitor of transforming growth factor-β (TGF-β) superfamily type I activin receptor-like kinases ALK-4, ALK-5, and ALK-7 can be a small molecule inhibitor with C 22 H 16 The chemical formula of N4O3 is SB431542 (CAS No. 301836-41-9) or 4-[4-(1,3-benzodioxane-5-yl)-5-(2-pyridyl)-1H-imidazol-2-yl]benzamide.

[0344] In one embodiment, the small molecule inhibitor of TGF-β / BMP signaling can be a small molecule inhibitor or head protein analog of BMP type I receptors ALK-2 and ALK-3. The small molecule inhibitor or head protein analog of BMP type I receptors ALK-2 and ALK-3 can be a protein containing C... 25 H 22N6 has the chemical formula LDN-193189 (CAS number 1062368-24-4) or 4-(6-(4-(piperazin-1-yl)phenyl)pyrazolo[1,5-a]pyrimidin-3-yl)quinoline.

[0345] In one embodiment, the small molecule inhibitors of both Wnt signaling and TGF-β / BMP signaling can be a combination or equivalent combination of IWP2 (CAS No. 686770-61-6), SB431542 (CAS No. 301836-41-9), and LDN-193189 (CAS No. 1062368-24-4), wherein the combination can produce synergistic inhibition of Wnt and TGF-β / BMP signaling activities. Alternatively, the combination may comprise IWP2 (CAS No. 686770-61-6), SB431542 (CAS No. 301836-41-9), and LDN-193189 (CAS No. 1062368-24-4), or equivalent combinations, wherein the combination can produce synergistic inhibition of Wnt and TGF-β / BMP signaling activities.

[0346] In one embodiment, the kit may further comprise embryonic stem cells (ESCs), pluripotent stem cells (PSCs), or induced pluripotent stem cells (iPSCs) from mammals. The kit may further comprise chemically defined culture media and / or supplements.

[0347] In one embodiment, a kit may be produced that includes instructions for use and is based on any method or composition used in the invention as disclosed in this application or claims. Such kits may include kits for generating RPGs, photoreceptors, RPEs, or RPEs from the pRGCs of the present invention and may contain culture media, supplements, small molecule inhibitors, and / or small molecule activators.

[0348] The following examples are intended to illustrate the invention and are not intended to limit the scope of the invention in any way.

[0349] Example

[0350] Example 1

[0351] method

[0352] Cell culture and differentiation. Human embryonic stem cells H9 (WA9, WiCell) and HuES9 (…) were cultured in StemPro hESC SFM medium (Invitrogen) on Matrigel (BD Biosciences) plates coated with reduced growth factors under feeder-free and serum-free conditions. http: / / grants.nih.gov / stem_cells / registry / current.htm?id=40(Paragraphs 25-40). Following the previously described procedure. 45 Human primary fetal retinal progenitor cells (hESCs) were isolated from 17-week-old human fetuses with informed consent and IRB approval and cultured. After undifferentiated hESCs reached approximately 80% confluence in culture, the medium was converted to serum-free N2B27 starter medium (DMEM / F12, N2, B27, 0.2% BSA, 2 mM L-GlutaMAX, 0.1 mM MEM non-essential amino acids, and 0.1 mM β-mercaptoethanol) supplemented with 20 ng / ml bFGF for 1–2 days. Near-confluenced monolayers of hESCs were further cultured in N2B27 starter medium supplemented with small molecule inhibitors (5 μM SB431542, 50 nM LDN193189, and 1 μM IWP2). The medium was changed daily for 6 days. hESC-derived hpRSCs could be maintained and expanded in this serum-free, inhibitor-supplemented starter medium. To induce RGC differentiation from hpRSCs, cells were cultured for more than two weeks in starter medium supplemented with a novel combination of small molecule inhibitors including 1 μM IWP2, 10 μM DAPT, and 200 nMPD173074. For photoreceptor precursor differentiation, dissociated hpRSCs were spread on Matrigel-coated plates and cultured as previously described. 18 The culture was then cultured for 6 days in a neural induction medium supplemented with 1 μM WP2, 10 μM DAPT, and 100 nM purinemorphine. Subsequently, the culture was transferred to a neural induction medium supplemented with 500 nM retinoic acid and 100 μM taurine, and cultured for another week. For RPE differentiation, hpRSC monolayer cultures were supplemented with RPE induction medium (GMEM, 10% KnockOut serum substitute, 0.1 mM MEM non-essential amino acids, 1 mM sodium pyruvate, and 0.1 mM β-mercaptoethanol) supplemented with 10 mM nicotinamide and 100 ng / ml activin A, and cultured for one week. Subsequently, the RPE precursor was matured in RPE medium, which consisted of MEM-a modified medium, 5% fetal bovine serum, 2 mM L-glutamine, 0.1 mM MEM non-essential amino acids, and 1 mM sodium pyruvate, supplemented with N1 and THT (taurine, hydrocortisone, and triiodothyronine). 46 .

[0353] PCR analysis. Total RNA was extracted from cells using the RNeasy kit (Qiagen), and cDNA was reverse transcribed using the iScript cDNA Synthesis Kit (Bio-Rad), both according to the manufacturer's instructions. Transcripts were amplified for 40 cycles on a 7500 real-time PCR system (Applied Biosystems) using gene-specific primers (Table 1) and Power SYBR Green PCR Master Mix, and their levels were quantified. Measurements were performed in triplicate and normalized to β-actin levels.

[0354] Table 1. Primers used for real-time qPCR

[0355] Transcriptome and pathway data analysis. Data generated in this study (hfRPC and hpRSC) and from the GEO database were analyzed. 47 (HuES9, GSM1001748) 48 The obtained gene expression data were normalized and processed using the Cluster 3.0 / Tree View software package. 49 Average linkage hierarchical clustering was performed. 4359 genes were used, based on the lowest log2=1.5 distance between the highest and lowest expressed samples. The total distance (similarity) between samples was calculated as the uncentered Pearson correlation coefficient. For each gene in the heatmap, red and blue represent high and low expression relative to other cells, respectively. For the analysis of individual pathways, previous studies were used... 50-54 Gene subsets from the TGF-β superfamily, Wnt pathway, Notch pathway, hESC pluripotency, hedgehog pathway, and FGF pathway were selected. Each gene subset was clustered separately using the same parameters.

[0356] Immunocytochemistry. Cells were fixed with 4% paraformaldehyde for 20 min, permeabilized with 0.3% Triton X-100-PBS for 5 min twice, and blocked in PBS containing 5% normal donkey serum and 0.3% Triton X-100%. Cells were then incubated overnight at 4°C in primary antibody solution. After washing three times in PBS, cells were incubated with Alexa fluorescently conjugated secondary antibody for 90 min. After rinsing and washing in PBS, the nuclei were counterstained with 100 ng / ml Hoechst 33342 for 10 min. The primary antibodies and their working dilutions are as follows: sheep anti-Chx10 (1:300, Exalpha), rabbit anti-Pax6 (1:600, Covance), goat anti-Lhx2 (1:200, Santa Cruz), rabbit anti-recovery protein (1:2000, Millipore), mouse anti-rhodopsin (1:250, Millipore), rabbit anti-red / green opsin (1:300, Millipore), goat anti-OPN1SW (1:300, Santa Cruz), and rabbit anti-GFP (1:1000, Invitrogen) or chicken anti-GFP (1:400, Invitrogen). The mouse anti-HSA antibody is a mixture of monoclonal antibodies against the human-specific marker TRA-1-85 (1:100, R&D Systems), human nuclear antigen (1:300, Millipore), and human mitochondria (1:100, Millipore). The secondary antibodies used were fluorescently labeled antibodies (1:1000, Invitrogen) of the corresponding Alexa-488, Alexa-555, Alexa-633, or Alexa-647. The labeled cells were imaged using a laser scanning confocal microscope (Olympus). The specific immunoreactivity of each antibody was confirmed by immunostaining with appropriate retinal tissue as a positive control under the same conditions.

[0357] Cell transplantation. Subretinal transplantation in rat eyes is adapted from a previously described method. 55 In short, hpRSCs were dissociated into single cells using StemPro Accutase (Invitrogen) and concentrated to 5 x 10⁻⁶ cells in a single balanced salt solution (BSS, Alcon) or BSS / HAMC (0.5 / 0.5% w / w) hydrogel solution. 4 density per μL 56To prepare the HAMC hydrogel solution, sodium hyaluronate (HA, 1400-1800 kDa, pharmaceutical grade 150, Novamatrix) was sterilized by dissolving it in water (HyClone, cell culture grade, Thermo Scientific) at 0.1% w / v and filtering it through a 0.22 μm tube-top vacuum filter (Corning). The solution was then freeze-dried under aseptic conditions by covering the tube with a 0.22 μm PVDF filter (Millex GV, Millipore). Methylcellulose (MC, 100 kDa, Methocel A4M Premium, Dow Chemical) was dissolved in water at 0.3% w / v by stirring in an ice bath, and then, as with HA, aseptically filtered and freeze-dried. The sterile HA and MC powders were resuspended in a BSS in a biosafety cabinet (e.g., for 0.5% / 0.5% HAMC, 30 mg HA and 30 mg MC were dissolved in 6 mL of BSS). The suspension was then vortexed, allowed to stand overnight at 4°C to dissolve, and vortexed again before use. Using a glass micropipette, 1–2 μl of hpRSC (5–10 x 10⁻⁶) of the suspension was transferred to the appropriate volume. 4 (Number of cells) were slowly injected into the subretinal space of nude rats or RCS rats. At different time points post-transplantation, animals were euthanized, and the eyes were removed, embedded in tissue cryopreservation medium, frozen sectioned, and co-immunostained, as described above. Animal procedures were performed with approval and under the supervision of the Institutional Animal Care and Use Committee at the University of California, San Diego.

[0358] result

[0359] hpRSC was induced by hESC using small molecules. Eye region specialization was induced under the influence of Wnt and BMP signaling gradients during the early stages of vertebrate embryonic development. 16,17 Previous research has shown that Wnt signaling gradients are important for establishing forebrain and midbrain characteristics during early development. Downregulation of Wnt signaling leads to the formation of the forebrain, in which the ocular region is located. Therefore, treatment of hESC-derived germ cells with Dkk1 (a potent Wnt inhibitor) and cephalin (a BMP antagonist) promotes the development of the ocular region. 3,19Additionally, under dual SMAD inhibition by exposure to SB431542 (a selective and potent inhibitor of transforming growth factor-β (TGF-β) superfamily type I activin receptor-like kinases ALK-4, ALK-5, and ALK-7) and LDN193189 (a selective inhibitor of BMPI-type receptors ALK-2 and ALK-3, a head protein analog), hESCs may transform into forebrain / ocular precursors that strongly express PAX6 and LHX2. 20,21 Therefore, we developed a chemically defined culture protocol to use the small molecule inhibitor IWP2 (Wnt production inhibitor-2, a Dkk1 analog). 22 LDN193189 and SB431542 (hereinafter referred to as IWP, LDN, and SB, respectively) induced hESC differentiation into hpRSCs under the synergistic inhibition of Wnt and TGF-β / BMP signaling activity. Pluripotent hESCs were plated on Matrigel-coated plates and cultured to near confluence without a feeder layer or serum. The cultures were then transferred to hpRSC initiation medium supplemented with the IWP-LDN-SB small molecule mixture for one week. Under this treatment, most hESCs transformed into tightly packed cells. Massive hpRSC induction was confirmed by immunocytochemical labeling of key early ocular transcription factors. 3,23 Over 90% of the cells were positive for both PAX6 and LHX2 (two key transcription factors expressed by progenitor cells in the ocular region). Figure 1 b). Most hESC-derived hpRSCs also express nestin (a typical marker of primitive neural epithelial stem cells) Figure 1 c). Furthermore, hESC-derived hpRSCs retain the proliferative activity demonstrated by strong Ki67 expression. Figure 1 c). Real-time PCR was used to further confirm hpRSC induction to assess the expression of typical early ocular transcription factor genes (including PAX6, RX, LHX2, SIX3, and SIX6). We found a more than 20- to 1200-fold increase in the expression of these retinal progenitor cell markers in hpRSCs after 6 days of induction. Figure 1 d). The expression levels of these genes are maintained in culture for more than two passages.

[0360] Transcriptomic profile of hpRSCs. To obtain a global view of hpRSC characteristics and investigate their molecular signature relative to their parent cells, undifferentiated hESCs, and more committed retinal progenitor cells, we compared the hpRSC transcriptome with those of hESCs and human 17-week fetal retinal progenitor cells (hfRPCs) using a microarray. The data suggest that hpRSCs represent a transitional state within the differentiation process from pluripotent hESCs to neuroretinal fate-restricted hfRPCs. Figure 2 a) Significant downregulation of key hESC pluripotency transcription factors such as POU5F1 (OCT4), NANOG, KLF4, and TBX3 was observed in hpRSC. Interestingly, LIN28 and SALL4 (two transcription factors that define stemness in embryonic stem cells and several tissue lineages) were also downregulated. 24-26 It is expressed at a similar level in hpRSC as in hESC, but at a significantly reduced level in hfRPC. Figure 2 b, 2e). Furthermore, elevated SOX2 expression was detected only in hpRSC ( Figure 2 b) indicates the primitive neuroectodermal state of hpRSC. 27 Based on known molecular tags associated with eye region formation. 23 hpRSC showed reduced expression of several genes in the TGF-β superfamily, such as SMAD1, SMAD2, TGF-β3, BMP3, BMP6, TGFBR1, and BMPR1B. Figure 2 c). It is worth noting that BMP4 and BMP7 (which are sufficient and essential for RPE in vivo development) 28 It exhibits higher expression levels in hpRSC than in hfRPC. This is consistent with previous studies. 17,29 Consistently, the expression of WNT4 and WNT11 (which are typically suppressed in the ocular region but elevated outside the posterior border) was decreased in hpRSC. Figure 2 d). Similarly, SRFP1 (an endogenous Wnt inhibitor) and FZD3 / 5 (receptors for Wnt signaling proteins) are known to be important for eye zone formation. 30 Strong expression in hpRSC ( Figure 2 d). Furthermore, pairwise comparisons of the hpRSC and hfRPC transcriptomes confirmed the transitional state of hpRSC during ocular specialization. Apart from PAX6, which remains active throughout retinogenesis, a typical group of early ocular transcription factor genes (including OTX2, RAX, LHX2, SIX3, and SIX6) were all significantly more expressed in hpRSC than in hfRPC. Figure 2e). Downregulation of members of the FGF signaling pathway was also observed, including FGFR1 / 2 / 3 and FGF3 / 8 (e). Figure 7 a) It is known that these two members promote retinal neurogenesis. 31 This indicates that hpRSCs are not yet oriented to become RPE or neuroretinal. Data confirm that hpRSCs derived from hESCs can recapitulate ocular specialization and that hpRSCs are ready to enter retinal sublineage differentiation.

[0361] Directed Differentiation of hESC-Derived hpRSCs into Retinal Ganglion Cells. To investigate the potential of hESC-derived hpRSCs to generate different retinal sublineage cell types in culture, we employed a small molecule-based approach to mimic the induction command (cue) of early eye development. We directed hpRSCs to differentiate into specific retinal cell fates in vitro. Retinal ganglion cells (RGCs) are the predominant type of retinal neuron and play a crucial role in transmitting visual signals from the retina to several regions of the brain. We first tested whether hESC-derived hpRSCs could be directed to differentiate into RGCs under chemically defined conditions. Previous results confirmed that inhibition of Notch and VEGFR signaling is important for RGC specialization. 32,33 Our transcriptional profiling analysis showed that the expression of several members of the Notch and VEGFR signaling pathways, such as NOTCH1, NOTCH2, NOTCH3, DLL1, DLL3, HES5, and HES1, was significantly upregulated in hpRSC. Figure 7 b). Therefore, we formulated a mixture of small molecule inhibitors, including IWP2, DAPT, and PD173074, which inhibit the activity of Wnt, Notch, and VEGFR signaling, respectively. This treatment rapidly oriented hpRSCs to RGC fate. After two weeks of induction in the presence of these three small molecule inhibitors, most cells were positive for both TUJ1 and BRN3 (RGC markers). Figure 3 a). Quantitative PCR analysis showed that after the first 6 days of induction, RGC precursor-specific transcription factor genes such as BRN3A, BRN3B, ISL-1, and MATH5 were significantly upregulated ( Figure 3 b).

[0362] hpRSCs derived from hESCs are directed to differentiate into photoreceptors. Photoreceptors are the main cell type in the retina and are responsible for initiating visual signal transduction. hpRSCs express several hedgehog (HH) signaling genes, such as GLI2, GLI3, and smoothed (SMO), at higher levels than hESCs and hfRPCs. Figure 7c) indicates that hpRSCs tend to differentiate into photoreceptors. Therefore, based on previous research... 7 An improved in vitro photoreceptor differentiation method was developed, utilizing small molecules to guide the differentiation process. A two-step method was used to restrict the fate of photoreceptors from hpRSCs. 7 During the initial phase, hpRSCs were treated with small molecule inhibitors SB, CHIR99021, DAPT, and IWP2 to inhibit ALK4 / 5 / 7, GSK-3, Notch, and Wnt signaling activities, respectively, and hpRSCs were treated with puromorphamine (a small molecule activator of the Shh signaling pathway). 34 As before 35 As previously reported, robust cell growth and proliferation were observed during the initial phase, but the expression of photoreceptor-specific markers was not observed. During the second phase, as previously reported... 7 The cultures were transferred to a medium supplemented with retinoic acid and taurine, which induced morphological changes after one week, including the extension of cellular processes in some cells. To identify the fate of these differentiated cells, we examined the expression of photoreceptor-specific markers by immunocytochemistry. By day 14 post-initial induction, the holophotoreceptor marker recovery protein (HRP) was detected. Figure 4 a) Cone cell-specific markers OPN1SW or blue opsin ( Figure 4 b) and rhodopsin, a rod cell-specific marker ( Figure 4 c). To determine whether photoreceptors differentiated in vitro from hpRSC also express human photoreceptor inter-vitamin A binding protein (IRBP) (a marker for both rod and cone photoreceptors). 36 We infected hpRSCs with IRBP-GFP lentivirus and induced the transduced cells to differentiate into photoreceptor fates. This method has been shown to specifically label photoreceptors in transgenic mice as well as in human, mouse, and chicken retinal explants. 37,38 After 12 days of differentiation, GFP-positive cells began to appear. By day 16, clusters of GFP-expressing photoreceptors were clearly visible. Figure 4 d). Upon further maturation, photoreceptors derived from hpRSC exhibit typical morphological features similar to those of the outer segment, such as short internal processes and long, extended external processes. Figure 4 d, illustration).

[0363] RPE (a monolayer of cells between the neuroretinal and choroidal vascular layers) is the first directed retinal cell type to appear in the outer layer of the early optic cup. 28To test whether hESC-derived hpRSCs could differentiate not only into neural retinal cells but also into non-neuronal RPE cells, we extracted small molecule inhibitors from adherent monolayer cultures of hpRSCs and transferred them to RPE-initiating medium, as previously described. 39 The removal of SMAD signaling inhibition and the addition of activin A are important for guiding hpRSCs to the RPE fate, as activin A and BMP activity are required for RPE specialization. 28,39,40 Twelve days after RPE differentiation, low expression levels of RPE65 (an RPE-specific isomerase required for the conversion of all-trans retinol to 11-cis retinaldehyde and for visual pigment regeneration) and the formation of polygonal actin bundles were observed in epithelioid cells. Figure 4 e). After further cultivation and maturation, increased expression of RPE65 was observed. Figure 4 f) and pigmentation of RPE ( Figure 4 g).

[0364] hpRSC transplantation. We next determined the ability of hESC-derived hpRSCs to survive and differentiate into photoreceptors after transplantation into the subretinal space of a murine eye. To avoid immune rejection of human cells, we used nude rats without a thymus for transplantation. Six weeks after transplantation of approximately 50,000 hpRSCs, transplanted human cells were observed in the subretinal tissue of the recipient's eye, but the number of human-specific antigen (HSA) positive cells was generally limited and surrounded by scar tissue. Figure 8 a). To overcome the tendency for cell aggregation and increase cell survival after subretinal transplantation, we chose to deliver cells in a hydrogel composed of hyaluronic acid and methylcellulose (HAMC) instead of saline. Recently, it has been demonstrated that transplanting HAMC hydrogels into retinal progenitor cells derived from murine eyes improves the survival, distribution, and differentiation of transplanted cells. 41,42 We injected hpRSCs mixed with HAMC (0.5 / 0.5% w / w) hydrogel into the subretinal space of nude rats. Six weeks post-transplantation, an increased number of transplanted human cells survived and were widely distributed throughout the subretinal region. Figure 8 b). Therefore, for subsequent transplantation experiments, unless otherwise specified, we delivered hpRSCs in HAMC hydrogels. Since the neuroretina is still developing in newborn animals, it provides a permissive environment for the integration and differentiation of transplanted hpRSCs. To test this hypothesis, we injected GFP-tagged hpRSCs into the subretinal space of newborn nude rats and observed significant integration of GFP-expressing hpRSCs into the neuroretina, including the outer nuclear layer (ONL) and inner nuclear layer (INL), 6 days post-transplantation. Figure 5 Some GFP-positive cells integrated into ONL colocalize with the typical photoreceptor marker recovery protein. Figure 5 a, 5e, 5f). Notably, many transplanted GFP-positive cells continued to express nestin (a, 5e, 5f). Figure 5 (b, 5d, 5f) (a marker of unoriented hpRSCs), indicating that these cells have not yet been oriented to a specific retinal cell fate. Next, we tested the ability of hpRSCs to differentiate into desired cell types, such as photoreceptors, in an animal model of retinal degeneration (Royal College of Surgeons (RCS) rats). RCS rats hold a mutation in the Mer receptor tyrosine kinase, which causes photoreceptor degeneration via defective RPE phagocytosis. 43,44 We injected hpRSC into the subretinal space of RCS rats on day 21 (P21) and examined differentiation and integration two months later. Frozen sections of the eyes to be transplanted were compared with HSA and photoreceptor markers recovery protein (HP). Figure 6 a) or red / green opsin ( Figure 6 b) Antibody co-immunostaining. Multilayered human cells were detected in the region adjacent to the ONL. Many recovery protein-positive cells and red / green opsin-positive cells were also positive for HSA. Figure 6 The presence of double-positive labeled cells indicates that some transplanted hpRSCs are oriented toward photoreceptor fate in the degenerating retina.

[0365] discuss

[0366] Here, we used a small molecule-based approach to recreate the in vivo retinal development process from undifferentiated hESCs and generated hpRSCs under chemically defined culture conditions. The hESC-derived hpRSCs exhibited typical characteristics of ocular neuroepithelial cells. Induction was achieved rapidly and effectively through synergistic inhibition of Nodal, BMP, and Wnt signaling in undifferentiated hESCs. The hESC-derived hpRSCs proliferated and actively expressed typical early ocular transcription factors (i.e., PAX6, LHX2, RAX, OTX2, and SIX3) and stemness factors (i.e., SOX2, LIN28, SALL4, and STAT3). When given specific differentiation instructions, these primitive retinal stem cells can be directed toward neuronal fates such as RGCs or photoreceptors or non-neuronal RPEs. Furthermore, we demonstrated that the transplanted hpRSCs could integrate, survive, and differentiate into the desired cell types in vivo. Encouragingly, two months after transplantation into RCS rats (a model of retinal degeneration where photoreceptors begin to be lost at P14 and disappear completely by the time the rats are 3 months old), some of the transplanted hpRSCs clearly differentiated into photoreceptors in the remaining ONLs. Furthermore, in the transplanted animals we have examined so far, we have not observed any tumor formation from the transplanted hpRSCs. Our results demonstrate the feasibility of inducing and expanding hpRSCs in vitro in a scale-up manner suitable for clinical trials. Additionally, this small-molecule-based approach could be used to generate hpRSCs derived from patient iPSCs, as well as more differentiated retinal progenitor cells or retinal neurons, which will have significant implications for modeling “disease in a petri dish” to investigate the molecular and cellular events that explain the pathogenesis of retinal degeneration.

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Claims

1. An in vitro method for generating isolated mammalian primitive retinal stem cells (pRSCs), comprising: (a) Culture of isolated embryonic stem cells (ESCs) from mammals in a cell culture medium that does not contain feeder cells, feeder layer conditioned medium or serum to produce and grow cultures of said isolated ESCs. and (b) The culture of the isolated ESC grown in this way is contacted with one or more of Wnt or TGF-β / BMP signaling inhibitors to differentiate the isolated ESC of (b) into primitive retinal stem cells, thereby producing isolated mammalian pRSCs.

2. An in vitro method for generating isolated mammalian primitive retinal stem cells (pRSCs), comprising: (a) Culture of isolated pluripotent stem cells (PSCs) from mammals in a cell culture medium free of feeder cells, feeder layer conditioned medium, or serum to produce and grow cultures of said isolated PSCs; and (b) The culture of the isolated PSCs grown in this way is contacted with one or more of Wnt or TGF-β / BMP signaling inhibitors to differentiate the isolated PSCs into primitive retinal stem cells, thereby producing isolated mammalian pRSCs.

3. An in vitro method for generating isolated mammalian primitive retinal stem cells (pRSCs), comprising: (a) Culture of isolated induced pluripotent stem cells (iPSCs) from mammals in a cell culture medium free of feeder cells, feeder layer conditioned medium, or serum to produce and grow cultures of said isolated iPSCs; and (b) The culture of the isolated iPSCs grown in this way is contacted with one or more of Wnt or TGF-β / BMP signaling inhibitors to differentiate the isolated iPSCs into primitive retinal stem cells, thereby producing isolated mammalian pRSCs.

4. The method according to claim 1, wherein in step (a), the culture of the isolated ESC thus produced is a monolayer culture.

5. The method according to claim 2, wherein in step (a), the culture of the isolated PSC thus produced is a monolayer culture.

6. The method of claim 3, wherein in step (a), the culture of the isolated iPSC thus produced is a monolayer culture.

7. The method according to claim 1, 2 or 3, wherein in step (b), one or more of the Wnt or TGF-β / BMP signaling inhibitors is a combination of Wnt and TGF-β / BMP signaling inhibitors.

8. The method according to claim 1, 2 or 3, wherein, prior to step (b), the culture thus generated is grown to near confluence in step (a).

9. The method according to claim 1, 2 or 3, wherein the mammal is a human, monkey, bear, rat, mouse, mink, rabbit, guinea pig, pig, dog, cat, goat, sheep, horse or cow.

10. The method according to claim 1, 2 or 3, wherein the isolated mammalian primitive retinal stem cells (pRSCs) are positive for expression of typical early ocular transcription factors PAX6, LHX2, RAX, OTX2, SIX3 and / or SIX6.

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