Methods for producing enriched populations of human retinal pigment epithelium cells

• US 9,649,340 B2
• Filed: 08/17/2010
• Issued: 05/16/2017
• Est. Priority Date: 01/23/2004
• Status: Active Grant

• Current Assignee: President and Fellows of Harvard College (Harvard Corporation)
• Original Assignee: Astellas Institute for Regenerative Medicine (Astellas Pharma Incorporated)
• Inventors: Klimanskaya, Irina V., Lanza, Robert P.
• Primary Examiner(s): Emch, Gregory S
• Application Number:
  US12/857,866
• Publication Number:
  US 20110117062A1
• Time in Patent Office: 2,464 Days
• Field of Search: 514 208, 435366, 424 937
• CPC Class Codes: A61K 35/12 Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells vaccines or medicinal preparations containing antigens or antibodies A61K39/00
• 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 9/0048 Eye, e.g. artificial tears
• CPC Class Codes: A61P 25/16 Anti-Parkinson drugs
• CPC Class Codes: A61P 27/00 Drugs for disorders of the senses
• CPC Class Codes: A61P 27/02 Ophthalmic agents
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• 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: A61P 9/14 Vasoprotectives; Antihaemorrhoidals; Drugs for varicose therapy; Capillary stabilisers
• CPC Class Codes: C12N 2501/01 Modulators of cAMP or cGMP, e.g. non-hydrolysable analogs, phosphodiesterase inhibitors, cholera toxin
• 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 2501/33 Insulin together with transferrin C12N2500/25; Insulin-like growth factors C12N2501/105
• CPC Class Codes: C12N 2506/02 from embryonic cells
• CPC Class Codes: C12N 5/062 Sensory transducers, e.g. photoreceptors; Sensory neurons, e.g. for hearing, taste, smell, pH, touch, temperature, pain
• CPC Class Codes: C12N 5/0621 Eye cells, e.g. cornea, iris pigmented cells photoreceptors C12N5/062

Abstract

This invention relates to methods for improved cell-based therapies for retinal degeneration and for differentiating human embryonic stem cells and human embryo-derived into retinal pigment epithelium (RPE) cells and other retinal progenitor cells.

Claims

1. A method of treating retinal disease or condition in an human subject, comprising transplanting into the eye of said subject a composition comprising an allogeneic human pigmented cell population comprising bestrophin+/CRALBP+ cells obtained by in vitro differentiation of human pluripotent cells that express Oct-4, alkaline phosphatase, SSEA-3, SSEA-4, TRA-I-60, and TRA-I-81, wherein the human pigmented cell population has a normal karyotype.

2. The method of claim 1, wherein said human pigmented cell population comprises cells that are bestrophin+, CRALBP+, PEDF+, and express RPE65.

3. The method of claim 1, wherein said human pigmented cell population comprises cells that are Pax6+.

4. The method of claim 1, wherein said retinal disease or condition is a disease of retinal degeneration.

5. The method of claim 1, wherein the retinal disease or condition comprises pathologic myopia, retinitis pigmentosa, RPE detachment, retinal dysplasia, retinal atrophy, retinopathy, macular dystrophy, cone dystrophy, cone-rod dystrophy, Malattia Leventinese, Doyne honeycomb dystrophy, Sorsby'"'"'s dystrophy, Stargardt disease, pattern/butterfly dystrophies, Best vitelliform dystrophy, North Carolina dystrophy, central areolar choroidal dystrophy, angioid streaks, or a toxic maculopathy.

6. The method of claim 1, which comprises transplantation of the human pigmented cell population by vitrectomy surgery into the subretinal space of the eye of said human subject.

7. The method of claim 1, wherein said human pigmented cell population is transplanted into the eye of said human subject in a suspension format or on a substrate or matrix having said human pigmented cell population disposed thereon.

8. The method of claim 1, wherein the retinal disease or condition is macular degeneration.

9. The method of claim 1, wherein the retinal disease or condition is Stargardt disease.

10. The method of claim 1, wherein the retinal disease or condition is age-related macular degeneration.

11. The method of claim 1, wherein the retinal disease or condition is retinitis pigmentosa.

12. The method of claim 1, wherein said human pigmented cell population comprises pigmented cells that exhibit a cobblestone, polygonal appearance characteristic of epithelial cells in culture.

13. The method of claim 1, wherein said human pigmented cell population is prepared by a method comprising:

a) allowing hES cell cultures to overgrow on MEF and form a thick multilayer of cells, or forming an embryoid body (EB) from hES cells;

b) culturing the multilayer of cells or EB for a sufficient time for the appearance of pigmented cells; and

c) isolating and culturing the pigmented cells from the resultant cell cultures, thereby obtaining a human pigmented cell population comprising CRALBP+/bestrophin+cells.

14. The method of claim 13, wherein said culturing in step (b) comprises culturing the hES cells in a medium lacking exogenously added FGF, lacking exogenously added LIF and lacking exogenously added PLASMANATE®

(an aqueous solution containing 5 g plasma proteins per 100 mL, buffered with sodium carbonate and stabilized with 0.005 M sodium caprylate and 0.004 M acetyltryptophan, said plasma proteins comprising approximately 88% normal human albumin, 12% alpha and beta globulins, and not more than 1% gamma globulin, and containing sodium 145 mEq/L, potassium 0.25 mEq/L, and chloride 100 mEq/L).

15. The method of claim 13, wherein the duration of culturing in step (b) is at least 6 weeks.

16. The method of claim 13, wherein the duration of culturing in step (b) is between about 6 weeks and about 8 weeks.

17. The method of claim 13, wherein the duration of culturing in step (b) is between about 3 months and about 5 months.

18. The method of claim 1, wherein the method restores visual acuity in the subject.

19. The method of claim 18, wherein visual acuity is restored to a moderate level.