STEM CELL-DERIVED RETIN RETINAL PIGMENT EPITHELIAL CELLS

• US 20110027333A1
• Filed: 04/27/2008
• Published: 02/03/2011
• Est. Priority Date: 04/18/2007
• Status: Active Grant

• Current Assignee: Hadasit Medical Research Services & Development Ltd (Hadassah, The Women's Zionist Organization of America, Inc.)
• Original Assignee: Hadasit Medical Research Services & Development Ltd (Hadassah, The Women's Zionist Organization of America, Inc.)
• Inventors: Reubinoff, Benjamin, Idelson, Masha, Alper-Pinus, Ruslana, Obolensky, Alex, Banin, Eyal
• Granted Patent: US 8,956,866 B2
• Time in Patent Office: Days
• US Class Current: 424/423
• CPC Class Codes: A61K 35/30 Nerves; Brain; Eyes; Corneal cells; Cerebrospinal fluid; Neuronal stem cells; Neuronal precursor cells; Glial cells; Oligodendrocytes; Schwann cells; Astroglia; Astrocytes; Choroid plexus; Spinal cord tissue
• CPC Class Codes: A61K 35/44 Vessels; Vascular smooth muscle cells; Endothelial cells; Endothelial progenitor cells
• CPC Class Codes: A61K 35/48 Reproductive organs
• CPC Class Codes: A61K 9/0051 Ocular inserts, ocular implants
• CPC Class Codes: A61P 27/02 Ophthalmic agents
• CPC Class Codes: A61P 9/10 for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
• CPC Class Codes: C12N 2500/38 Vitamins
• CPC Class Codes: C12N 2500/90 Serum-free medium, which may still contain naturally-sourced components
• CPC Class Codes: C12N 2500/99 Serum-free medium
• CPC Class Codes: C12N 2501/115 Basic fibroblast growth factor (bFGF, FGF-2)
• CPC Class Codes: C12N 2501/15 Transforming growth factor beta (TGF-β)
• CPC Class Codes: C12N 2501/155 Bone morphogenic proteins [BMP]; Osteogenins; Osteogenic factor; Bone inducing factor
• CPC Class Codes: C12N 2501/16 Activin; Inhibin; Mullerian inhibiting substance
• CPC Class Codes: C12N 2501/999 Small molecules not provided for elsewhere
• CPC Class Codes: C12N 2502/13 connective tissue cells; generic mesenchyme cells, e.g. so-called "embryonic fibroblasts"
• CPC Class Codes: C12N 2506/02 from embryonic cells
• CPC Class Codes: C12N 2533/52 Fibronectin; Laminin
• CPC Class Codes: C12N 5/0621 Eye cells, e.g. cornea, iris pigmented cells photoreceptors C12N5/062
• CPC Class Codes: C12N 5/0696 Artificially induced pluripotent stem cells, e.g. iPS

Abstract

The present invention concerns RPE cells obtainable by directed differentiation from stem cell, particularly, human stem cells. It has been specifically found that culturing stem cells in the presence of one or more member of the TGFβ superfamily, such as Activin A) induced directed differentiation into mature and functional RPE cells. This was evidenced by the expression of markers specific to mature RPE cells, including MiTF-A, RPE65 or Bestrophin). In accordance with one particular embodiment, the cells are a priori cultured with nicotinamide (NA) which was found to augment the cells'"'"' response to the inductive effect of the one or more member of the TGFβ superfamily. The invention also provides methods of performing the directed differentiation, as well as methods for use of the resulting RPE cells.

Claims

1. A method for promoting directed differentiation of human stem cells (hSCs) into retinal pigment epithelium (RPE) fate, the method comprising:

a. providing a cell culture comprising hSCs;

b. culturing cells in a culture system comprising a basic medium supplemented with one or more members of transforming growth factor β

(TGF β

) superfamily whereby said hSCs are induced to differentiate into RPE fate.

4. The method of claim 1, wherein said culturing comprises a first culture stage with a culture system comprising nicotinamide (NA) and a second culture stage with a culture system comprising said one or more members of said TGFβ

superfamily.

5. The method of claim 1, comprising culturing the hSCs in a culture system comprising the basic medium supplemented with NA for at least two days prior to culturing said cell culture in a basic medium supplemented with said one or more members of said TGFβ

superfamily.

6. The method of claim 1, wherein said culture system further comprises NA.

8. The method of claim 1, wherein said member of the TGFβ

superfamily is TGFβ

1, TGFβ

3 or activin A.

9. The method of claim 1, wherein said cells are cultured in a suspension.

16. The RPE cells of claim 15, obtained by the method of claim 1.

39. The method of claim 1, wherein said hSCs are human embryonic stem cells.

2-3. -3. (canceled)

7. (canceled)

10-14. -14. (canceled)

15. RPE cells obtained by directed differentiation of hSC in the presence of one or more members of the TGFβ

superfamily.

17. The RPE cells of claim 15 being terminally differentiated RPE cells.

18. The RPE cells of claim 15, expressing one or more of a marker selected from Bestrophin, and RPE65

19. The RPE cells of claim 18, wherein expression of said one or more marker is elevated as compared to expression of the marker in RPE cells obtained by spontaneous differentiation from SCs.

20. The RPE cells of claim 15, differentiated from human embryonic stem cells.

31. A method of treating or preventing in a subject a retinal disease or disorder comprising dysfunction, injury, and/or loss of retinal pigment epithelium, the method comprises intraocular transplantation to said subject REP cells of claim 15 or obtained by the method of.

32. The method of claim 31, comprising transplantation of said RPE cells to the subretinal space of the eye.

33. The method of claim 31, wherein said RPE cells are transplanted when in a suspension, or as a monolayer of cells immobilized on a matrix or a substrate.

34. The method of claim 31, wherein said retinal disease or disorder is selected from at least one of retinitis pigmentosa, lebers congenital amaurosis, hereditary or acquired macular degeneration, age related macular degeneration (AMD), Best disease, retinal detachment, gyrate atrophy, choroideremia, pattern dystrophy, RPE dystrophies, Stargardt disease, RPE and retinal damage due to damage caused by any one of photic, laser, inflammatory, infectious, radiation, neovascular or traumatic injury.

35. A therapeutic composition comprising the hSCs-derived RPE cells of claim 15 in a dosage form suitable for intraocular transplantation into a subject.

36. A therapeutic implant comprising the hSCs-derived RPE cells of claim 15, said RPE cells being in a suspension, or immobilized as a monolayer of cells on a matrix or a substrate.

37. The therapeutic composition of claim 35, for the treatment or prevention of a retinal disorder or disease.

21. (canceled)

22. A method of transplanting RPE cells into a subject'"'"'s eye, the method comprises a. providing a cell culture comprising hSCs;

b. culturing cells in the culture in a culture system comprising a basic medium supplemented with one or more members of the TGFβ

superfamily whereby the hSCs are induced to differentiate into RPE cells;

c. harvesting from said cell culture of (b) RPE cells; and

d. transplanting said differentiated RPE cells into said subject'"'"'s eye.

29. The method of claim 22, comprising transplantation of said RPE cells at the subretinal space of the eye.

30. The method of claim 22, wherein said RPE cells are transplanted in a suspension, or as a monolayer of cells immobilized on a matrix or a substrate.

23-28. -28. (canceled)

38. (canceled)