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1. (WO2014121077) METHOD FOR GENERATING RETINAL PIGMENT EPITHELIUM (RPE) CELLS FROM INDUCED PLURIPOTENT STEM CELLS (IPSCs)
Note: Text based on automatic Optical Character Recognition processes. Please use the PDF version for legal matters

We claim:

1. A method for producing human retinal pigment epithelial cells, comprising:

producing embryoid bodies from human induced pluripotent stem cells;

culturing the embryoid bodies in a first medium comprising two Wnt pathway inhibitor, and a Nodal pathway inhibitor;

plating the embryoid bodies on a matrigel coated tissue culture substrate in a second medium that

(a) does not comprise beta fibroblast growth factor (bFGF) (b) comprises a basic fibroblast growth factor (bFGF) inhibitor, the two Wnt pathway inhibitors, and the Nodal pathway inhibitor; (c) comprises about 20 to about 90 ng of Noggin; and (d) comprises about 1 to about 3% knock out serum replacement to form differentiating retinal pigment epithelial cells;

culturing the differentiating retinal pigment epithelial cells in a third medium comprising ACTIVAN A and WNT3a;

then culturing the cells in retinal pigment epithelial (RPE) cell medium comprising about 5% fetal serum, a canonical WNT inhibitor, a non-canonical WNT inducer, and inhibitors of the Sonic and FGF pathway,

thereby producing human retinal pigment epithelial cells.

2. The method of claim 1, wherein one of the Wnt pathway inhibitors is N-(2-aminoethyl)-5-chloroisoquinoline-8-sulfonamide (CK1-7), 3,5,7,8-Tetrahydro-2-[4-(trifluoromethyl)phenyl]-4H-thiopyrano[4,3-d]pyrimidin-4-one (XAV939) , Secreted frizzled-related protein (SFRP) 1, SFRP-2, SFRP-3, SFRP-4 or SFRP-5.

3. The method of claim 1 or claim 2, wherein the first medium comprises about 3.5 to about 9 mM of CK1-7.

4. The method of claim 1, wherein the Nodal pathway inhibitor is 4-(5-Benzol[1,3]dioxol-5-yl-4-pyrldin-2-yl-1H-imidazol-2-yl)-benzamide hydrate, 4-[4-(1,3-Benzodioxol-5-yl)-5-(2-pyridinyl)-1H-imidazol-2-yl]-benzamide hydrate, 4-[4-(3,4-Methylenedioxyphenyl)-5-(2-pyridyl)-1H-imidazol-2-yl]-benzamide hydrate (SB-431542), left-right determination factor (Lefty) or 2-(5-Benzo[1,3]dioxol-5-yl-2-tert-butyl-3H-imidazol-4-yl)-6-methylpyridine hydrochloride hydrate (SB-505124).

5. The method of claim 1 or claim 4, wherein the first medium comprises about 3.5 to about 9 mM of SB43152.

6. The method of any one of claims 1-5, wherein the first medium is a retinal cell inducing medium comprising Dulbecco' s Modified Eagle's Medium and F12 at a ratio of about 1:1 in the absence of serum.

7. The method of any one of claims 1-5, wherein the inhibitor of bFGF comprises N-[(2R)-2,3-Dihydroxypropoxy]-3,4-difluoro-2-[(2-fluoro-4-iodophenyl)amino]-benzamide (PD0325901), N-[2-[[4-(Diethylamino)butyl]amino-6-(3,5-dimethoxyphenyl)pyrido[2,3-d]pyrimidin-7-yl]-N'-(1,1-dimethylethyl)urea (PD173074), 2-(2-Amino-3-methoxyphenyl)-4H-1-benzopyran-4-one (PD 98059), 1-tert-Butyl-3-[6-(2,6-dichlorophenyl)-2-[[4-(diethylamino)butyl]amino]pyrido[2,3-d]pyrimidin-7-yl]urea (PD161570), or 6-(2,6-Dichlorophenyl)-2-[[4-[2- (diethylamino)ethoxy]phenyl]amino]-8-methyl-pyrido[2,3-d]pyrimidin-7(8H)-one dihydrochloride hydrate (PD 166285).

8. The method of any one of claims 1-7, wherein the second medium comprises about 0.5 to about 2 mM of PD0325901.

9. The method of claim 1, wherein the first medium comprises about 50 ng/ml of Dickkopf-related protein 1 (DKK1).

10. The method of any one of claims 1-7, wherein the second medium comprises about 3.5 to about 9 mM of CK1-7 and about 3.5 to about 9 mM of SB431542.

11. The method of any one of claims 1-10, wherein the third medium comprises about 100 to about 200 ng/ml of ACTIVIN A.

12. The method of any one of claims 1-11, wherein the third medium comprises about 75 to 150 ng/ml of Wnt3a.

13. The method of any one of claims 1-12, wherein the canonical Wnt inhibitor is

Dickkopf-related protein 1 (DKK1).

14. The method of claim 13, wherein the third medium comprises about 75 to about 150 ng/ml of DKK1.

15. The method of any one of claims 1-14, wherein the non-canonical Wnt inducer is WNT5a.

16. The method of any one of claims 1-15, wherein the fourth medium comprises about 75 to about 150 ng/ml of WNT5a, 75 to about 150 ng/ml of DKK1, about 5 μΜ Cycolopamine and/or about 10 μΜ of SU5402.

17. The method of any one of claims 1-15, wherein step (a) comprises culturing the cells in the presence of about 1.5% knock out serum replacement.

18. The method of any one of claims 1-17, wherein the substrate is a transwell.

19. The method of any one of claims 1-18, comprising expressing OCT4, SOX2, LIN28 and Nanog in a human fetal retinal pigment epithelial cell to produce the human induced pluripotent stem cells.

20. The method of any one of claims 1-19, wherein the human induced pluripotent stem cells comprise a nucleic acid encoding a marker operably linked to a retinal pigment epithelial cell specific promoter.

21. The method of claim 20, wherein the retinal specific promoter is a tyrosinase promoter.

22. A method for determining if an agent of interest increases the differentiation of retinal pigment epithelial cells, comprising

culturing the embryoid bodies produced from human induced pluripotent stem cells comprising a nucleic acid encoding a first marker operably linked to a retinal pigment epithelial cell specific promoter, and comprising a second marker operably linked to a constitutive promoter in a first medium comprising two Wnt pathway inhibitor and a Nodal pathway inhibitor;

plating the embryoid bodies on a tissue culture substrate in a second medium that

(a) does not comprise beta fibroblast growth factor (bFGF) (b) comprises a basic fibroblast growth factor (bFGF) inhibitor, the two Wnt pathway inhibitors, and the Nodal pathway inhibitor; and (c) comprises about 20 to about 90 ng of Noggin to form differentiating retinal pigment epithelial cells;

culturing the differentiating retinal pigment epithelial cells in a third medium comprising ACTIVAN A and WNT3a;

culturing the cells in a fourth retinal pigment epithelial cell (RPE) medium comprising about 5% fetal serum, a canonical WNT inhibitor, a non-canonical WNT inducer, and inhibitors of the Sonic and FGF pathway; thereby producing human retinal pigment epithelial cells, and in the presence of the agent of interest; and

evaluating the expression of the first marker in the retinal pigment epithelial cells as compared to the expression of the second marker,

wherein an increase in expression of the first marker as compared to the second marker indicates that the agent increases the differentiation of retinal pigment epithelial cells.

23. A method for authenticating that a cell of interest is a retinal pigment epithelial cell, comprising

assaying expression of a set of markers comprising Μ ITF (NM_000248), PAX6

(NM_000280), LHX2 (NM_004789), TFEC (NM_012252), CDH1 (NM_004360), CDH3

(NM_001793), CLDN10 (NM_182848), CLDN16 (NM_006580), CLDN19 (NM_148960), BEST1 (NM_004183), TIMP3 (NM_000362), TRPM1 (NM_002420), TRPM3 (NM_020952), TTR (NM_000371), VEGFA (NM_003376), CSPG5 (NM_006574), DCT (NM_001922), TYRP1 (NM_000550), TYR (NM_000372), SILV (NM_006928), SIL1 (NM_022464), MLANA

(NM_005511), RAB27A (NM_183236), OCA2 (NM_000275), GPR143 (NM_000273), GPNMB (NM_002510), MYO6 (NM_004999), MYRIP (NM_015460), RPE65 (NM_000329), RBP1 (NM_002899), RBP4 (NM_006744), RDH5 (NM_002905), RDH11 (NM_016026), RLBP1 (NM_000326), MERTK (NM_006343), ALDH1A3 (NM_000693), FBLN1 (NM_001996), SLC16A1 (NM_003051), KCNV2 (NM_133497), KCNJ13 (NM_002242), CFTR (NM_000492), in the cell of interest;

assaying expression of miR204 and miR211 in the cell of interest;

assaying the polarizaton of the cell of interest;

assaying the resting potential of the cell using patch clamping; and

evaluating the fluid transport rate of the cell,

wherein expression of MITF, PAX6, LHX2, TFEC, CDH1, CDH3, CLDN10, CLDN16, CLDN19, BEST1, TIMP3, TRPM1, TRPM3, TTR, VEGFA, CSPG5, DCT, TYRP1, TYR, SILV, SIL1, MLANA, RAB27A, OCA2, GPR143, GPNMB, MYO6, MYRIP, RPE65, RBP1, RBP4, RDH5, RDH11, RLBP1, MERTK, ALDH1A3, FBLN1, SLC16A1, KCNV2, KCNJ13, CFTR, expression of miR204 and miR211, a resting potential of about -50 to about -60 mV, and a fluid transport rate of about 5 to about 10 μl cm-2h-1 indicates that the cell is a retinal pigment epithelial cell.

24. The method of claim 23, further comprising:

assaying expression of MITF, PAX6, LHX2, TFEC, CDH1, CDH3, CLDN10, CLDN16, CLDN19, BEST1, TIMP3, TRPM1, TRPM3, TTR, VEGFA, CSPG5, DCT, TYRP1, TYR, SILV, SIL1, MLANA, RAB27A, OCA2, GPR143, GPNMB, MYO6, MYRIP, RPE65, RBP1, RBP4, RDH5, RDH11, RLBP1, MERTK, ALDH1A3, FBLN1, SLC16A1, KCNV2, KCNJ13, CFTR in the cell of interest;

assaying expression of miR204 and miR211 in the cell of interest;

assaying the polarization of the cell of interest;

assaying the resting potential of the cell using patch clamping, and;

evaluating the fluid transport rate of the cell,

wherein expression of MITF, PAX6, LHX2, TFEC, CDH1, CDH3, CLDN10, CLDN16, CLDN19, BEST1, TIMP3, TRPM1, TRPM3, TTR, VEGFA, CSPG5, DCT, TYRP1, TYR, SILV, SIL1, MLANA, RAB27A, OCA2, GPR143, GPNMB, MYO6, MYRIP, RPE65, RBP1, RBP4, RDH5, RDH11, RLBP1, MERTK, ALDH1A3, FBLN1, SLC16A1, KCNV2, KCNJ13, CFTR, expression of miR204 and miR211, a resting potential of about -50 to about -60 mV and a fluid transport rate of about 5 to about 10 μl cm-2h-1 indicates that the cell is a retinal pigment epithelial cell.

25. The method of any one of claim 1-21, wherein the embryoid bodies are cultured in the first medium for about 48 hours.

26. The method of any one of claims 1-21 or 25, wherein the embryoid bodies are cultured in the second medium for about 18 to about 24 days.

27. The method of claim 26, wherein the embryoid bodies are cultured in the second medium for about three weeks.

28. The method of any one of claims 1-21 or 25-27, wherein the differentiating retinal pigment epithelial cells in the third medium for about 18 to about 24 days.

29. The method of claim 28, wherein the differentiating retinal pigment epithelial cells in the third medium for about three weeks.

30. The method of any one of claims 1-21 or 25-29, wherein the cells are cultured in the retinal pigment epithelial cell (RPE) medium for about 12 to about 16 days.

31. The method of claim 30, wherein the cells are cultured in the retinal pigment epithelial cell (RPE) medium for about two weeks.

32. The method of any one of claims 1-21 or 25-33, further comprising maintaining the retinal pigment epithelial cells in retinal pigment epithelial cell (RPE) medium comprising about 5% fetal serum.

33. The method of claim 32, wherein the cells comprising maintaining the retinal pigment epithelial cells in retinal pigment epithelial cell (RPE) medium comprising about 5% fetal serum for about six to about eight weeks.

34. The method of claim 23 or 24, wherein assaying expression comprises the use of a multiplex assay.

35. A retinal pigment epithelial cell produced by the methods of any one of claims 1-21 or

25-34.

36. The retinal pigment epithelial cell of claim 35, wherein the cell is a human cell.

37. A pharmaceutical composition comprising an effective amount of the retinal pigment epithelial cell of claim 35 or claim 36,

38. The composition of claim 37, for the manufacture of a medicament.

39. A method of treating a subject with a retinal degenerative disease, retinal dysfunction, retinal degradation, retinal damage, or loss of retinal pigment epithelium, comprising administering to the subject a therapeutically effect amount of the pharmaceutical composition of claim 37, thereby treating the subject.

40. The method of claim 39, wherein the retina degenerative disease is Stargardt's macular dystrophy, retinitis pigmentosa, age related macular degeneration, glaucoma, diabetic retinopathy, Lebers congenital amaurosis, acquired macular degeneration, heriditary macular degeneration, Best disease, retinal detachment, gyrate atrophy, choroideremia, pattern dystrophy.

41. The method of claim 39, wherein the retinal damage is caused by laser, inflammatory, infectious, radiation, neovascular or traumatic injury.

42. The method of any one of claims 39-41, wherein the retinal pigment epithelial cells are introduced in a subretinal space of the eye, a vitreal space, an inner or outer retina, a retinal periphery or within a choroid.

43. The method of any one of claims 39-42, wherein the retinal pigment epithelial cells are introduced into the target site in the form of a cell suspension.

44. The method of any one of claims 39-42, wherein the retinal pigment epithelial cells are adhered onto extracellular matrix.

45. The method of any one of claims 39-42, wherein the retinal pigment epithelial cells are provided on a substrate such as a biodegradable polymer.