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

• US 7,794,704 B2
• Filed: 01/24/2005
• Issued: 09/14/2010
• 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
• Primary Examiner(s): Kolker; Daniel E
• Assistant Examiner(s): Emch; Gregory S
• Application Number:
  US11/041,382
• Publication Number:
  US 20060031951A1
• Time in Patent Office: 2,059 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 treating retinal degeneration in a subject in need thereof, comprising:

(a) producing an enriched population of human retinal epithelium (RPE) cells by;

(i) providing a multilayer population of human embryonic stem (hES) cells;

(ii) culturing said multilayer population of hES cells under conditions that do not maintain the undifferentiated state of said hES cells for a sufficient time for the appearance of putative human RPE cells, wherein said putative human RPE cells comprise brown pigment dispersed within their cytoplasm;

(iii) selecting one or more of said putative human RPE cells from the culture of step (ii) to obtain human RPE cells; and

(iv) culturing said human RPE cells selected in step (iii) to form a cell monolayer containing cells that are Pax6− and

bestrophin+ and exhibit a characteristic cobblestone, polygonal, epithelial-like appearance and comprise brown pigment dispersed within their cytoplasm; and

(b) administering to an eye of a subject in need thereof the human RPE cells produced in step (a), thereby treating retinal degeneration in said subject.

2. The method of claim 1, wherein the condition of retinal degeneration is selected from the group consisting of:

Stargardt'"'"'s Macular Dystrophy, retinitis pigmentosa, and macular degeneration.

3. The method of claim 2, wherein the subject with retinitis pigmentosa is an animal.

4. The method of claim 3, wherein the animal 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.

5. The method of claim 4, 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.

6. The method of claim 1, wherein said subject is human.

7. The method of claim 1, wherein said administering step comprises vitrectomy surgery into the subretinal space of the eye.

8. The method of claim 7, wherein the cells are transplanted in a suspension, matrix, or substrate.

9. The method of claim 1, wherein during step (iv) the cultured cells temporarily lose their epithelial appearance and pigmentation after plating, and then regain their epithelial appearance and pigmentation upon further culturing.

10. The method of claim 1, wherein said culturing in step (ii) comprises culturing the multilayer population of hES cells in media lacking exogenously added FGF.

11. The method of claim 1, wherein said culturing in step (ii) comprises culturing the multilayer population of hES cells in media lacking exogenously added FGF and lacking exogenously added LIF.

12. The method of claim 1, wherein said culturing in step (ii) comprises culturing the multilayer population of hES cells in media 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).

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

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

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

16. The method of claim 1, wherein the cell monolayer of step (iv) contains cells that are Pax6-, bestrophin+, CRALBP+, PEDF+, and express RPE65.

17. The method of claim 1, wherein the cell monolayer of step (iv) contains cells that are Pax6-, bestrophin+, CRALBP+, PEDF+, and express RPE65, and have the absence of at least one ES cell marker selected from the group consisting of Oct4, Sox2, and TDGF-1.

18. The method of claim 1, wherein said multilayer population of hES cells in step (i) has been cultured in media containing exogenously added FGF.

19. The method of claim 18, wherein said media containing exogenously added FGF in step (i) further comprises exogenously added LIF.

20. The method of claim 18, wherein said media containing exogenously added FGF in step (i) further comprises 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 0.004M 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).

21. A method for treating retinal degeneration in a subject in need thereof, comprising:

(a) producing an enriched population of human RPE cells by;

(i) providing a culture of hES cells;

(ii) culturing the hES cells to produce one or more embryoid bodies;

(iii) culturing said one or more embryoid bodies under conditions that do not maintain the undifferentiated state of said hES cells for a sufficient time for the appearance of putative human RPE cells within at least one of said one or more embryoid bodies, wherein said putative human RPE cells comprise brown pigment dispersed within their cytoplasm;

(iv) selecting and dissociating one or more of said embryoid bodies containing putative human RPE cells from the culture of step (iii) to obtain human RPE cells; and

(v) culturing said human RPE cells selected in step (iv) to form a cell monolayer containing cells that are Pax6− and

bestrophin+ and exhibit a characteristic cobblestone, polygonal, epithelial-like appearance and comprise brown pigment dispersed within their cytoplasm; and

(b) administering to an eye of a subject in need thereof the human RPE cells produced in step (a), thereby treating retinal degeneration in said subject.

22. The method of claim 21, wherein the condition of retinal degeneration is selected from the group consisting of:

Stargardt'"'"'s Macular Dystrophy, retinitis pigmentosa, and macular degeneration.

23. The method of claim 22, wherein the subject with retinitis pigmentosa is an animal.

24. The method of claim 23, wherein 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.

25. The method of claim 24, 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.

26. The method of claim 21, wherein said subject is human.

27. The method of claim 21, wherein said administering step comprises vitrectomy surgery into the subretinal space of the eye.

28. The method of claim 27, wherein the cells are transplanted in a suspension, matrix, or substrate.

29. The method of claim 21, wherein during step (v) the cultured cells temporarily lose their epithelial appearance and pigmentation after plating, and then regain their epithelial appearance and pigmentation upon further culturing.

30. The method of claim 21, wherein the culturing of one of more embryoid bodies in step (iii) comprises culturing the embryoid bodies in media lacking exogenously added FGF.

31. The method of claim 21, wherein the culturing of one of more embryoid bodies in step (iii) comprises culturing the embryoid bodies in media lacking exogenously added FGF and lacking exogenously added LIF.

32. The method of claim 21, wherein the culturing of one or more embryoid bodies in step (iii) comprises culturing the embryoid bodies in media 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).

33. The method of claim 21, wherein the duration of culturing in step (iii) is at least about 6 weeks.

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

35. The method of claim 21, wherein the duration of culturing in step (iii) is between about 3 months and about 5 months.

36. The method of claim 21, wherein the cell monolayer of step (v) contains cells that are Pax6-, bestrophin+, CRALBP+, PEDF+, and express RPE65.

37. The method of claim 21, wherein the cell monolayer of step (v) contains cells that are Pax6-, bestrophin+, CRALBP+, PEDF+, and express RPE65, and have the absence of at least one ES cell marker selected from the group consisting of Oct4, Sox2, and TDGF-1.

38. The method of claim 21, wherein said culture of hES cells in step (i) have been cultured in media containing exogenously added FGF.

39. The method of claim 38, wherein said media containing exogenously added FGF in step (i) further comprises exogenously added LIF.

40. The method of claim 38, wherein said media containing exogenously added FGF in step (i) further comprises 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).

41. A method for treating retinal degeneration in a subject in need thereof, comprising:

(a) producing an enriched population of human RPE cells by;

(i) providing a multilayer population of hES cells, wherein said multilayer population of hES cells has been cultured in media containing exogenously added FGF;

(ii) culturing said multilayer population of hES cells under conditions that do not maintain the undifferentiated state of said hES cells for a sufficient time for the appearance of putative human RPE cells, wherein said putative human RPE cells comprise brown pigment dispersed within their cytoplasm, wherein said conditions that do not maintain the undifferentiated state of said hES cells comprise media lacking exogenously added FGF and the duration of culturing is at least 6 weeks;

(iii) selecting one or more of said putative human RPE cells from the culture of step (ii) to obtain human RPE cells; and

(iv) culturing said human RPE cells selected in step (iii) to form a cell monolayer containing cells that are Pax6−

, bestrophin+, CRALBP+, PEDF+, and express RPE65, have the absence of at least one ES cell marker selected from the group consisting of Oct4, Sox2, and TDGF-1, and exhibit a characteristic cobblestone, polygonal, epithelial-like appearance and comprise brown pigment dispersed within their cytoplasm, wherein during said culturing the cultured cells temporarily lose their epithelial appearance and pigmentation after plating, and then regain their epithelial appearance and pigmentation upon further culturing; and

(b) administering to an eye of a subject in need thereof the human RPE cells produced in step (a), thereby treating retinal degeneration in said subject.

42. The method of claim 41, wherein the condition of retinal degeneration is selected from the group consisting of:

Stargardt'"'"'s Macular Dystrophy, retinitis pigmentosa, and macular degeneration.

43. The method of claim 42, wherein the subject with retinitis pigmentosa is an animal.

44. The method of claim 43, wherein 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.

45. The method of claim 44, 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.

46. The method of claim 41, wherein said subject is human.

47. The method of claim 41, wherein said administering step comprises vitrectomy surgery into the subretinal space of the eye.

48. The method of claim 47, wherein the cells are transplanted in a suspension, matrix, or substrate.

49. The method of claim 41, wherein said media lacking exogenously added FGF in step (ii) further lacks exogenously added LIF.

50. The method of claim 41, wherein said media lacking exogenously added FGF in step (ii) further lacks exogenously added LIF and lacks 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).

51. The method of claim 41, wherein the duration of culturing in step (ii) is between about 6 weeks and about 8 weeks.

52. The method of claim 41, wherein the duration of culturing in step (ii) is between about 3 months and about 5 months.

53. The method of claim 41, wherein said media containing exogenously added FGF in step (i) further comprises exogenously added LIF.

54. The method of claim 41, wherein said media containing exogenously added FGF in step (i) further comprises 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).

55. A method for treating retinal degeneration in a subject in need thereof, comprising:

(a) producing an enriched population of human RPE cells by;

(i) providing a culture of hES cells;

(ii) culturing the hES cells to produce one or more embryoid bodies, wherein said one or more embryoid bodies or the cells from which said one or more embryoid bodies are formed have been cultured in media containing exogenously added FGF;

(iii) culturing said one or more embryoid bodies for a sufficient time for the appearance of putative human RPE cells within at least one of said one or more embryoid bodies, wherein said putative human RPE cells comprise brown pigment dispersed within their cytoplasm, wherein the one or more embryoid bodies are cultured in a media lacking exogenously added FGF, and the duration of culturing is at least 6 weeks, whereby one or more embryoid bodies containing putative human RPE cells are formed;

(iv) selecting and dissociating one or more of said embryoid bodies containing putative human RPE cells from the culture of step (iii) to obtain human RPE cells; and

(v) culturing said human RPE cells selected in step (iv) to form a cell monolayer containing cells that are Pax6−

, bestrophin+, CRALBP+, PEDF+, and express RPE65, have the absence of at least one ES cell marker selected from the group consisting of Oct4, Sox2 and TDGF-1, and exhibit a characteristic cobblestone, polygonal, epithelial-like appearance and comprise brown pigment dispersed within their cytoplasm, wherein during said culturing the cultured cells temporarily lose their epithelial appearance and pigmentation after plating, and then regain their epithelial appearance and pigmentation upon further culturing; and

(b) administering to an eye of a subject in need thereof the human RPE cells produced in step (a), thereby treating retinal degeneration in said subject.

56. The method of claim 55, wherein the condition of retinal degeneration is selected from the group consisting of:

Stargardt'"'"'s Macular Dystrophy, retinitis pigmentosa, and macular degeneration.

57. The method of claim 56, wherein the subject with retinitis pigmentosa is an animal.

58. The method of claim 57, wherein 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.

59. The method of claim 58, 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.

60. The method of claim 55, wherein said subject is human.

61. The method of claim 55, wherein said administering step comprises vitrectomy surgery into the subretinal space of the eye.

62. The method of claim 61, wherein the cells are transplanted in a suspension, matrix, or substrate.

63. The method of claim 55, wherein said media lacking exogenously added FGF in step (iii) further lacks exogenously added LIF.

64. The method of claim 55, wherein said media lacking exogenously added FGF in step (iii) further lacks exogenously added LIF and lacks 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).

65. The method of claim 55, wherein the duration of culturing in step (iii) is between about 6 weeks and about 8 weeks.

66. The method of claim 55, wherein the duration of culturing in step (iii) is between about 3 months and about 5 months.

67. The method of claim 55, wherein said media containing exogenously added FGF in step (ii) further comprises exogenously added LIF.

68. The method of claim 55, wherein said media containing exogenously added FGF in step (ii) further comprises 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).