METHODS OF GENERATING CORNEAL CELLS AND CELL POPULATIONS COMPRISING SAME

• US 20130251692A1
• Filed: 12/01/2011
• Published: 09/26/2013
• Est. Priority Date: 12/02/2010
• Status: Active Grant

• Current Assignee: Institut National De La Sante Et De La Recherche Medicale (Inserm) (Allianz Global Investors GmbH), TECHNION RESEARCH AND DEVELOPMENT FOUNDATION, LTD. (Technion-Israel Institute Of Technology)
• Original Assignee: Institut National De La Sante Et De La Recherche Medicale (Inserm) (Allianz Global Investors GmbH), TECHNION RESEARCH AND DEVELOPMENT FOUNDATION, LTD. (Technion-Israel Institute Of Technology)
• Inventors: Itskovitz-Eldor, Joseph, Aberdam, Daniel, Shalom-Feuerstein, Ruby
• Granted Patent: US 9,574,171 B2
• Time in Patent Office: Days
• US Class Current: 424/93.7
• 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: A61P 27/02 Ophthalmic agents
• CPC Class Codes: C12N 2501/11 Epidermal growth factor [EGF]
• CPC Class Codes: C12N 2501/155 Bone morphogenic proteins [BMP]; Osteogenins; Osteogenic factor; Bone inducing factor
• CPC Class Codes: C12N 2501/33 Insulin
• CPC Class Codes: C12N 2502/085 eye cells
• CPC Class Codes: C12N 2502/1323 Adult fibroblasts
• CPC Class Codes: C12N 2506/02 from embryonic cells
• CPC Class Codes: C12N 2506/45 from artificially induced pluripotent stem cells
• CPC Class Codes: C12N 2533/52 Fibronectin; Laminin
• CPC Class Codes: C12N 2533/54 Collagen; Gelatin
• CPC Class Codes: C12N 2533/90 Substrates of biological origin, e.g. extracellular matrix, decellularised tissue
• CPC Class Codes: C12N 5/0062 General methods for three-dimensional culture
• CPC Class Codes: C12N 5/0621 Eye cells, e.g. cornea, iris pigmented cells photoreceptors C12N5/062
• CPC Class Codes: G01N 33/5023 on expression patterns
• CPC Class Codes: G01N 33/5026 on cell morphology
• CPC Class Codes: G01N 33/5058 Neurological cells
• CPC Class Codes: G01N 33/5073 Stem cells
• CPC Class Codes: G01N 33/53 Immunoassay; Biospecific binding assay; Materials therefor

Abstract

A method of generating a population of corneal epithelial cells is disclosed. The method comprises culturing human pluripotent stem cells in corneal fibroblast-conditioned medium on a solid surface comprising an extracellular matrix component thereby generating the population of corneal epithelial cells. Isolated cell populations and corneal tissues are also disclosed, as well as uses thereof.

Claims

1. A method of generating a population of corneal epithelial cells comprising culturing human pluripotent stem cells in corneal fibroblast-conditioned medium on a solid surface comprising an extracellular matrix component thereby generating the population of corneal epithelial cells.

2. The method of claim 1, wherein said corneal fibroblast-conditioned medium comprises bone morphogenetic protein-4 (BMP-4).

3. The method of claim 1, wherein said human pluripotent stem cells comprise human embryonic stem cells (hESCs) or human induced pluripotent stem cells (hIPSCs).

4. The method of claim 1, wherein said corneal fibroblast-conditioned medium comprises at least one agent selected from the group consisting of insulin, hydrocortisone and epidermal growth factor (EGF).

5. The method of claim 1, wherein said corneal fibroblast-conditioned medium comprises insulin, hydrocortisone and epidermal growth factor (EGF).

6. The method of claim 1, wherein said extracellular matrix component is selected from the group consisting of collagen IV, laminin, fibronectin and Matrigel®

.

7. The method of claim 1, further comprising analyzing the population of corneal epithelial cells for at least one marker selected from the group consisting of keratin 3 (K3), keratin 12 (K12), paired box gene 6 (pax6), keratin 18 (K18) and Connexin 43.

8. The method of claim 1, wherein said corneal fibroblast-conditioned medium is devoid of limbal fibroblasts.

9. An isolated population of human corneal epithelial cells generated according to the method of claim 1.

10. The isolated population of corneal epithelial cells of claim 9, not comprising skin cells.

11. The isolated population of corneal epithelial cells of claim 9, wherein at least 70% of the cells of the population co-express K3 and Pax6.

12. The isolated population of corneal epithelial cells of claim 9, wherein less than 10% of the cells of the population express nanog and Oct4.

14. A method of treating an eye disorder in a subject in need thereof, the method comprising transplanting to the subject a therapeutically effective amount of corneal epithelial cells generated according to the method of claim 1, thereby treating the eye disorder in the subject.

15. A pharmaceutical composition comprising the isolated population of corneal epithelial cells of claim 9 and a pharmaceutically acceptable carrier.

16. A method of generating corneal tissue, the method comprising:

(a) dissociating the isolated population of human corneal epithelial cells of claim 9 to generate a population of dissociated human corneal epithelial cells; and

(b) culturing said dissociated human corneal epithelial cells on a scaffold under conditions that generate corneal tissue.

17. The method of claim 16, wherein said scaffold comprises Matrigel®

and collagen I.

18. The method of claim 16, wherein said scaffold comprises a human amniotic membrane.

19. An isolated human corneal tissue generated according to the method of claim 16.

21. A method of treating an eye disorder in a subject in need thereof, the method comprising transplanting to the subject a therapeutically effective amount of corneal tissue generated according to the method of claim 16, thereby treating the eye disorder in the subject.

22. A pharmaceutical composition comprising the isolated corneal tissue of claim 19 and a pharmaceutically acceptable carrier.

13. (canceled)

20. (canceled)

23. A method of screening for an agent which enhances differentiation towards a corneal epithelial lineage, the method comprising:

(a) culturing human pluripotent stem cells in corneal fibroblast-conditioned medium on a solid surface comprising an extracellular matrix component in a presence of said agent; and

(b) analyzing a differentiation status of said human pluripotent stem cells, wherein an increase in differentiation compared to a differentiation in an absence of said agent is indicative of an agent which enhances differentiation towards a corneal epithelial lineage.

24. The method of claim 23, wherein said pluripotent stem cells comprise iPS cells.

25. The method of claim 23, wherein said iPS cells are derived from healthy patients.

26. The method of claim 23, wherein said iPS cells are derived from diseased patients.

27. The method of claim 23, wherein said medium further comprises BMP-4.

28-31. -31. (canceled)