Methods for producing enriched populations of human retinal pigment epithelium cells

• US 7,795,025 B2
• Filed: 07/21/2006
• 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:
  US11/490,953
• Publication Number:
  US 20070031386A1
• Time in Patent Office: 1,516 Days
• Field of Search: None
• US Class Current: 435/377
• 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 for producing an enriched population of human retinal pigment epithelium (“

RPE”

) cells, the method comprising;

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

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

(c) selecting one or more of said putative human RPE cells from the culture of step (b); and

(d) culturing said one or more of said putative human RPE cells selected in step (c) to form a culture containing cells that are Pax6−

, bestrophin+, CRALBP+, PEDF+, and express RPE65, and exhibit a characteristic cobblestone, polygonal, epithelial-like appearance and comprise brown pigment dispersed within their cytoplasm.

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

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

4. The method of claim 1, wherein said culturing in step (b) comprises culturing the 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).

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. The method of claim 1, wherein the culture of step (d) 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.

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

10. The method of claim 9, wherein said media containing exogenously added FGF in step (a) further comprises exogenously added LIF.

11. The method of claim 9, wherein said media containing exogenously added FGF in step (a) 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).

12. A method for producing an enriched population of human RPE cells, the method comprising:

(a) providing a culture of hES cells;

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

(c) culturing said one or more embryoid bodies for a sufficient time to allow 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, whereby one or more embryoid bodies containing putative human RPE cells are formed;

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

(e) culturing said putative human RPE cells obtained in step (d) to form a culture containing cells that are Pax6−

, bestrophin+, CRALBP+, PEDF+, and express RPE65, and exhibit a characteristic cobblestone, polygonal, epithelial-like appearance and comprise brown pigment dispersed within their cytoplasm, thereby producing an enriched population of human RPE cells.

13. The method of claim 12, wherein the culturing of one of more embryoid bodies in step (c) comprises culturing the embryoid bodies in media lacking exogenously added FGF.

14. The method of claim 12, wherein the culturing of one of more embryoid bodies in step (c) comprises culturing the embryoid bodies in media lacking exogenously added FGF and lacking exogenously added LIF.

15. The method of claim 12, wherein the culturing of one or more embryoid bodies in step (c) comprises culturing the embryoid bodies in media lacking exogenously added FGF and 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).

16. The method of claim 12, wherein the duration of culturing in step (c) is at least 6 weeks.

17. The method of claim 12, wherein the duration of culturing in step (c) is between about 6 weeks and about 8 weeks.

18. The method of claim 12, wherein the duration of culturing in step (c) is between about 3 months and about 5 months.

19. The method of claim 12, wherein the culture of step (e) 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.

20. A method for producing an enriched population of human RPE cells, the method comprising:

(a) providing a multilayer population of hES cells;

(b) 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;

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

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

, bestrophin+, CRALBP+, PEDF+, and express RPE65, and exhibit a characteristic cobblestone, polygonal, epithelial-like appearance and comprise brown pigment dispersed within their cytoplasm.

21. The method of claim 20, wherein during step (d) the cultured cells temporarily lose their epithelial appearance and pigmentation after plating, and then regain their epithelial appearance and pigmentation upon further culturing.

22. The method of claim 20, wherein said culturing in step (b) comprises culturing the multilayer population of hES cells in media lacking exogenously added FGF.

23. The method of claim 20, wherein said culturing in step (b) comprises culturing the multilayer population of hES cells in media lacking exogenously added FGF and lacking exogenously added LIF.

24. The method of claim 20, wherein said culturing in step (b) 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).

25. The method of claim 20, wherein the duration of culturing in step (b) is between about 6 weeks and about 8 weeks.

26. The method of claim 20, wherein the duration of culturing in step (b) is between about 3 months and about 5 months.

27. The method of claim 20, wherein the culture of step (d) 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.

28. The method of claim 20, wherein said multilayer population of hES cells in step (a) has been cultured in media containing exogenously added FGF.

29. The method of claim 28, wherein said media containing exogenously added FGF in step (a) further comprises exogenously added LIF.

30. The method of claim 28, wherein said media containing exogenously added FGF in step (a) 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).

31. A method for producing an enriched population of human RPE cells, the method comprising:

(a) providing a culture of hES cells;

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

(c) 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, and the duration of culturing is at least 6 weeks, whereby one or more embryoid bodies containing putative human RPE cells are formed;

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

(e) culturing said human RPE cells obtained in step (d) to form a culture containing cells that are Pax6−

, bestrophin+, CRALBP+, PEDF+, and express RPE65, and exhibit a characteristic cobblestone, polygonal, epithelial-like appearance and comprise brown pigment dispersed within their cytoplasm, thereby producing an enriched population of human RPE cells.

32. The method of claim 31, wherein during step (e) the cultured cells temporarily lose their epithelial appearance and pigmentation after plating, and then regain their epithelial appearance and pigmentation upon further culturing.

33. The method of claim 31, wherein said culturing in step (c) comprises culturing the embryoid bodies in media lacking exogenously added FGF.

34. The method of claim 31, wherein said culturing in step (c) comprises culturing the embryoid bodies in media lacking exogenously added FGF and lacking exogenously added LIF.

35. The method of claim 31, wherein said culturing in step (c) 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).

36. The method of claim 31, wherein the duration of culturing in step (c) is between about 6 weeks and about 8 weeks.

37. The method of claim 31, wherein the duration of culturing in step (c) is between about 3 months and about 5 months.

38. The method of claim 31, wherein the culture of step (e) 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.

39. The method of claim 31, wherein said multilayer population of hES cells in step (a) have been cultured in media containing exogenously added FGF.

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

41. The method of claim 39, wherein said media containing exogenously added FGF in step (a) 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).