Methods for producing enriched populations of human retinal pigment epithelium cells for treatment of retinal degeneration

• US 8,268,303 B2
• Filed: 08/17/2010
• Issued: 09/18/2012
• Est. Priority Date: 01/23/2004
• Status: Active Grant

• Current Assignee: President and Fellows of Harvard College (Harvard Corporation)
• Original Assignee: Advanced Cell Technology Incorporated (Astellas Pharma Incorporated)
• Inventors: Klimanskaya, Irina V., Lanza, Robert P.
• Primary Examiner(s): Kolker, Daniel E
• Assistant Examiner(s): Emch, Gregory S
• Application Number:
  US12/857,911
• Publication Number:
  US 20110117063A1
• Time in Patent Office: 763 Days
• Field of Search: None
• US Class Current: 424/93.7
• 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 35/36 Skin; Hair; Nails; Sebaceous glands; Cerumen; Epidermis; Epithelial cells; Keratinocytes; Langerhans cells; Ectodermal cells islets of Langerhans A61K35/39
• CPC Class Codes: A61K 35/44 Vessels; Vascular smooth muscle cells; Endothelial cells; Endothelial progenitor cells
• CPC Class Codes: A61K 35/545 Embryonic stem cells; Pluripotent stem cells; Induced pluripotent stem cells; Uncharacterised stem cells
• CPC Class Codes: A61P 25/16 Anti-Parkinson drugs
• CPC Class Codes: A61P 27/02 Ophthalmic agents
• CPC Class Codes: A61P 27/06 Antiglaucoma agents or miotics
• CPC Class Codes: C12N 2500/32 Amino acids
• CPC Class Codes: C12N 2500/44 Thiols, e.g. mercaptoethanol
• 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 2501/235 Leukemia inhibitory factor [LIF]
• CPC Class Codes: C12N 2501/33 Insulin together with transferrin C12N2500/25; Insulin-like growth factors C12N2501/105
• CPC Class Codes: C12N 2501/60 Transcription factors
• 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 for the differentiation of hES cells into RPE cells, said 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 thick multilayer of cells or EB for a sufficient time for the appearance of pigmented cells comprising brown pigment dispersed in their cytoplasm; and

c) isolating and culturing the pigmented cells from the resultant cell cultures, thereby obtaining a culture comprising human RPE cells, wherein said human RPE cells are Pax6−

, bestrophin+, CRALBP+, PEDF+, express RPE65.

2. The method of claim 1, where in the step of isolating the pigmented cells comprises contacting the cultured cells of step (b) with an enzyme selected from the group consisting of one or more of trypsin, collagenase, and dispase.

3. The method of claim 1, wherein said pigmented cells have a cobblestone, polygonal appearance characteristic of epithelial cells.

4. The method of claim 1, 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®

.

5. The method of claim 1, wherein the duration of culturing in step (b) is at least 6 weeks.

6. The method of claim 1, wherein the duration of culturing in step (b) is between about 6 weeks and about 8 weeks.

7. The method of claim 1, wherein the duration of culturing in step (b) is between about 3 months and about 5 months.

8. A method of administering human RPE cells to an individual in need thereof, 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 thick multilayer of cells or EB for a sufficient time for the appearance of pigmented cells comprising brown pigment dispersed in their cytoplasm;

c) isolating and culturing the pigmented cells from the resultant cell cultures, thereby obtaining a culture comprising human RPE cells, wherein said human RPE cells are Pax6−

, bestrophin+, CRALBP+, PEDF+, and express RPE65; and

d) introducing the human RPE cells into the individual in need thereof.

9. The method of claim 8, wherein the individual suffers from retinitis pigmentosa and/or macular degeneration.

10. The method of claim 8, which comprises transplantation of the RPE cells by vitrectomy surgery into the subretinal space of the eye of said individual.

11. The method of claim 10, wherein the cells are transplanted in a suspension, matrix, or substrate.

12. The method of claim 9, wherein the retinitis pigmentosa is associated with an animal model.

13. The method of claim 12, where in the animal model is selected from the group consisting of:

rd mouse, RPE-65 knockout mouse, tubby-like mouse, RCS rat, Abyssinian cat, cone degeneration “

cd”

dog, progressive rod-cone degeneration “

prcd”

dog, early retinal degeneration “

erd”

dog, rod-cone dysplasia 1, 2 &

3 “

rcd1, rcd2 and rcd3”

dogs, photoreceptor dysplasia “

pd”

dog, and Briard “

RPE-65”

dog.

14. The method of claim 13, wherein the outcome of the therapy in the animal model is evaluated using one or more of behavioral tests, fluorescent angiography, histology, and functional testing such as measuring the ability of the cells to perform phagocytosis (photoreceptor fragments), vitamin A metabolism, tight junctions conductivity, or evaluation using electron microscopy.

15. A method for the treatment of Parkinson'"'"'s disease in an individual in need thereof, 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 thick multilayer of cells or EB for a sufficient time for the appearance of pigmented cells comprising brown pigment dispersed in their cytoplasm;

c) isolating and culturing the pigmented cells from the resultant cell cultures, thereby obtaining a culture comprising human RPE cells, wherein said human RPE cells are Pax6−

, bestrophin+, CRALBP+, PEDF+, and express RPE65; and

d) introducing the human RPE cells into the individual in need thereof.

16. A method for the differentiation of hES cells into RPE cells, said method comprising:

a) allowing hES cell cultures to overgrow and form a thick multilayer of cells;

b) culturing the thick multilayer of cells for a sufficient time for the appearance of pigmented cells comprising brown pigment dispersed in their cytoplasm; and

c) isolating and culturing the pigmented cells from the resultant cell cultures, thereby obtaining a culture comprising human RPE cells, wherein said human RPE cells are Pax6−

, bestrophin+, CRALBP+, PEDF+, express RPE65.

17. The method of claim 16, where in the step of isolating the pigmented cells comprises contacting the cultured cells of step (b) with an enzyme selected from the group consisting of one or more of trypsin, collagenase, and dispase.

18. The method of claim 16, wherein said pigmented cells have a cobblestone, polygonal appearance characteristic of epithelial cells.

19. The method of claim 16, 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®

.

20. The method of claim 16, wherein the duration of culturing in step (b) is at least 6 weeks.

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

22. The method of claim 16, wherein the duration of culturing in step (b) is between about 3 months and about 5 months.

23. A method of administering human RPE cells to an individual in need thereof, comprising:

a) allowing hES cell cultures to overgrow and form a thick multilayer of cells;

b) culturing the thick multilayer of cells for a sufficient time for the appearance of pigmented cells comprising brown pigment dispersed in their cytoplasm;

c) isolating and culturing the pigmented cells from the resultant cell cultures, thereby obtaining a culture comprising human RPE cells, wherein said human RPE cells are Pax6−

, bestrophin+, CRALBP+, PEDF+, and express RPE65; and

d) introducing the human RPE cells into the individual in need thereof.

24. The method of claim 23, wherein the individual suffers from retinitis pigmentosa and/or macular degeneration.

25. The method of claim 23, which comprises transplantation of the RPE cells by vitrectomy surgery into the subretinal space of the eye of said individual.

26. The method of claim 25, wherein the cells are transplanted in a suspension, matrix, or substrate.

27. A method for the treatment of Parkinson'"'"'s disease in an individual in need thereof, comprising:

a) allowing hES cell cultures to overgrow and form a thick multilayer of cells;

b) culturing the thick multilayer of cells for a sufficient time for the appearance of pigmented cells comprising brown pigment dispersed in their cytoplasm;

c) isolating and culturing the pigmented cells from the resultant cell cultures, thereby obtaining a culture comprising human RPE cells, wherein said human RPE cells are Pax6−

, bestrophin+, CRALBP+, PEDF+, and express RPE65; and

d) introducing the human RPE cells into the individual in need thereof.

28. A method for the differentiation of hES cells into RPE cells, said method comprising:

a) forming an embryoid body (EB) from hES cells;

b) culturing the EB for a sufficient time for the appearance of pigmented cells comprising brown pigment dispersed in their cytoplasm; and

c) isolating and culturing the pigmented cells from the resultant cell cultures, thereby obtaining a culture comprising human RPE cells, wherein said human RPE cells are Pax6−

, bestrophin+, CRALBP+, PEDF+, express RPE65.

29. The method of claim 28, where in the step of isolating the pigmented cells comprises contacting the cultured cells of step (b) with an enzyme selected from the group consisting of one or more of trypsin, collagenase, and dispase.

30. The method of claim 28, wherein said pigmented cells have a cobblestone, polygonal appearance characteristic of epithelial cells.

31. The method of claim 28, 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®

.

32. The method of claim 28, wherein step (b) comprises culturing said EB in the absence of feeder cells.

33. The method of claim 28, wherein the duration of culturing in step (b) is at least 6 weeks.

34. The method of claim 28, wherein the duration of culturing in step (b) is between about 6 weeks and about 8 weeks.

35. The method of claim 28, wherein the duration of culturing in step (b) is between about 3 months and about 5 months.

36. The method of claim 28, wherein step (a) comprises culturing said hES cells under low adherent conditions.

37. The method of claim 28, wherein step (a) comprises culturing said hES cells on low adherent plates.

38. A method of administering human RPE cells to an individual in need thereof, comprising:

a) forming an embryoid body (EB) from hES cells;

b) culturing the EB for a sufficient time for the appearance of pigmented cells comprising brown pigment dispersed in their cytoplasm;

c) isolating and culturing the pigmented cells from the resultant cell cultures, thereby obtaining a culture comprising human RPE cells, wherein said human RPE cells are Pax6−

, bestrophin+, CRALBP+, PEDF+, and express RPE65; and

d) introducing the human RPE cells into the individual in need thereof.

39. The method of claim 38, wherein the individual suffers from retinitis pigmentosa and/or macular degeneration.

40. The method of claim 38, which comprises transplantation of the RPE cells by vitrectomy surgery into the subretinal space of the eye of said individual.

41. The method of claim 40, wherein the cells are transplanted in a suspension, matrix, or substrate.

42. A method for the treatment of Parkinson'"'"'s disease in an individual in need thereof, comprising:

a) forming an embryoid body (EB) from hES cells;

b) culturing the EB for a sufficient time for the appearance of pigmented cells comprising brown pigment dispersed in their cytoplasm;

c) isolating and culturing the pigmented cells from the resultant cell cultures, thereby obtaining a culture comprising human RPE cells, wherein said human RPE cells are Pax6−

, bestrophin+, CRALBP+, PEDF+, and express RPE65; and

d) introducing the human RPE cells into the individual in need thereof.