Isolation and transplantation of retinal stem cells

• US 20030207450A1
• Filed: 08/06/2002
• Published: 11/06/2003
• Est. Priority Date: 02/11/2000
• Status: Active Grant

• Current Assignee: Children's Hospital of Orange County, The Schepens Eye Research Institute Inc (Mass General Brigham, Inc.)
• Original Assignee: Children's Hospital of Orange County
• Inventors: Klassen, Henry J., Young, Michael J., Shatos, Marie A., Mizumoto, Keiko
• Granted Patent: US 7,514,259 B2
• Time in Patent Office: Days
• US Class Current: 435/368
• CPC Class Codes: A01K 67/0271 Chimeric vertebrates, e.g. comprising exogenous cells
• 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: A61P 27/02 Ophthalmic agents
• CPC Class Codes: A61P 29/00 Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• CPC Class Codes: A61P 31/00 Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
• CPC Class Codes: C12N 2501/11 Epidermal growth factor [EGF]
• CPC Class Codes: C12N 2501/115 Basic fibroblast growth factor (bFGF, FGF-2)
• CPC Class Codes: C12N 2501/135 Platelet-derived growth factor [PDGF]
• CPC Class Codes: C12N 2506/02 from embryonic cells
• CPC Class Codes: C12N 5/0621 Eye cells, e.g. cornea, iris pigmented cells photoreceptors C12N5/062
• CPC Class Codes: C12N 5/0623 Stem cells

Abstract

The present invention relates to the isolation, in vitro propagation, and transplantation and integration of non-pigmented retinal stem cells derived from the neuroretina of the eye, ex vivo and in vivo.

Claims

1. Retinal stem cells isolated from a neuroretina of a donor mammal.

3. The retinal stem cells of claim 1 or 2, wherein the retinal stem cells express nestin and are non-pigmented.

4. The retinal stem cells of claim 1 or 2, said cells requiring, when cultured in vitro, the presence at least one exogenous growth factor in a culture medium in order to proliferate.

6. The retinal stem cells of claim 1 or 2, which can differentiate into neurons, as evidenced by expression of a neurofilament protein, NF-200.

7. The retinal stem cells of claim 1 or 2, which can differentiate into astrocytes, as evidenced by expression of glial fibrillary acidic protein, GFAP.

8. The retinal stem cells of claim 1 or 2, which, when grafted onto a retina, onto a retinal explant, or into a mature eye, differentiate into photoreceptor cells expressing rhodopsin, recoverin, or both.

9. The retinal stem cells of claim 8, further being capable of integrating into and repopulating a diseased retina.

10. The retinal stem cells of claim 1 or 2, wherein the mammal was an embryo, a neonate, or an adult.

11. The retinal stem cells of claim 1 or 2, wherein the mammal was a primate, a rodent, or a domesticated animal.

12. The retinal stem cells of claim 1 or 2, wherein the mammal was a human, a mouse, a rat, a cat, a dog, a pig, a cow, a horse, a monkey, or a great ape.

25. The neuroretina-derived retinal stem cells of claim 1 or 2, wherein the mammal was a transgenic mouse expressing green fluorescent protein.

26. The neuroretina-derived retinal stem cells of claim 1 or 2, being clonally derived.

2. Retinal stem cells isolated from a neuroretina of a donor mammal, being capable of:

a) self-renewal in vitro;

b) differentiating into any one cell type of the group consisting of neurons, astrocytes, and oligodendrocytes;

c) integration into a host retina when grafted on the host retina; and

d) differentiation into photoreceptor cells when grafted onto a retina, onto a retinal explant, or into a mature eye.

5. The retinal stem cells of claim 5, wherein at least one exogenous growth factor is a member selected from the group consisting of epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), a combination of bFGF and EGF, and a combination of EGF and bFGF and platelet-derived growth factor (PDGF).

13. A method of isolating and culturing retinal stem cells from a neuroretina of a donor mammal, comprising:

a) isolating neuroretinal tissue from a donor eye, the isolated neuroretinal tissue being substantially free of vitreous humor, optic nerve head tissue, pars plana epithelial tissue, and retinal pigmented epithelial tissue;

b) dissociating the neuroretinal tissue into cells;

c) culturing the dissociated, neuroretina-derived cells in a culture vessel, in a first culture medium comprising serum, in mammalian cell culture conditions for about 24 hours;

d) removing the first culture medium from the culture vessel; and

e) thereafter maintaining the cultured neuroretina-derived cells in a second culture medium that is serum free and gentamycin-free and comprises at least one growth factor, under mammalian cell culture conditions.

15. The method of claim 13 or 14, further comprising, every 2-7 days, removing non-viable cells and a portion of the second culture medium from the culture vessel and replacing said portion with an equivalent amount of fresh second culture medium.

16. The method of claim 13 or 14, wherein the mammal was an embryo, a neonate, or an adult mammal.

17. The method of claim 13 or 14, wherein the mammal was an aged mammal.

18. The method of claim 13 or 14, wherein the culture vessel is a plastic tissue culture flask.

23. The method of claim 13 or 14, wherein the at least one growth factor is a member selected from the group consisting of EGF, bFGF, a combination of bFGF and EGF, a combination of EGF and bFGF and PDGF.

14. A method of isolating and culturing retinal stem cells from a neuroretina of a donor mammal, comprising the following steps in the order given:

a) isolating neuroretinal tissue from a donor eye, the isolated neuroretinal tissue being substantially free of vitreous humor, optic nerve head tissue, pars plana epithelial tissue, and retinal pigmented epithelial tissue;

b) passing the isolated neuroretinal tissue through a nylon screen having a pore size in a range of about 100 microns, to dissociate the isolated neuroretinal tissue into cells;

c) placing an aliquot of cells from step (b) in a culture vessel coated with a protein layer;

d) incubating the aliquot of cells in an amount of a first cell culture medium to provide a cell concentration within a range of about 1000-1,000,000 cells/ml, for about 24 hours at about 35-39°

C., and in an approximately 4-6% C0₂ atmosphere, wherein the first cell culture medium comprises a physiologically balanced salt solution containing a glucose content of about 0.5-2 mg/liter, and about 5-15% fetal calf serum;

e) after about 24 hours, removing the first culture medium from the culture vessel; and

f) adding to the culture vessel a second culture medium that is serum free and gentamycin-free, the second culture medium comprising a physiologically balanced salt solution containing a glucose content of about 0.5-2 mg/liter a neural progenitor cell-conditioned medium, at least one growth factor at a concentration of about 30-50 ng/ml per growth factor, an effective amount of L-glutamine 0.5-3 mM, and an effective amount of at least one antibiotic that is not gentamycin.

19. The method of claim 14, wherein the protein layer comprises polyornithine overlaid with fibronectin or laminin.

20. The method of claim 14, wherein the protein layer is of the same mammalian origin as the neuroretinal tissue from which the retinal stem cells are derived.

21. The method of claim 14, wherein the physiologically balanced salt solution is Dulbecco'"'"'s Minimal Essential Medium F-12 (DMEM/F-12).

22. The method of claim 14, wherein neuroprogenitor ecll-conditioned medium is N2 Supplement.

24. The method of claim 14, wherein the at least one antibiotic is penicillin, streptomycin, or both, in an effective amount.

27. A method of repopulating or rescuing a dystrophic eye, comprising introducing neuroretina-derived retinal stem cells into an eye of a mammalian recipient.

28. The method of claim 27, wherein the neuroretina-derived retinal stem cells are introduced into a retinal site, a subretinal space, an optic nerve, a vitreal cavity, a brain or a spinal cord.

29. The method of claim 27, further comprising administering to the mammalian recipient, a substance that stimulates differentiation of the neuroretina-derived stem cells into photoreceptors cells.

30. The method of claim 27, further comprising administering to the mammalian recipient, a substance that stimulates differentiation of the neuroretina-derived stem cells into neurons.

31. The method of claim 27, further comprising administering to the mammalian recipient, a substance that stimulates differentiation of the neuroretina-derived stem cells into astrocytes

32. The method of claim 27, further comprising administering to the mammalian recipient, a substance that stimulates differentiation of the neuroretina-derived stem cells into oligodendrocytes.

33. The method of claim 27, wherein the neuroretinal-derived retinal stem cells are clonally derived.

34. The method of claim 27, wherein the recipient is a member selected from the group consisting of an immature mammal or an adult mammal.

36. The method of claim 27, wherein said donor and said recipient are of different species.

37. The method of claim 27, wherein said donor and recipient pair is selected from the group consisting of the following pairs:

a rat donor and a mouse recipient;

a mouse donor and a rat recipient;

a pig donor and a human recipient.

38. The method of claim 27, wherein the donor and the recipient are of the same species.

39. The method of claim 27, wherein the donor and the recipient are allogeneic.

40. The method of claim 27, wherein the donor and the recipient are syngeneic.

41. The method of claim 27, wherein the dystrophic retinal tissue is a result of at least one member selected from the group consisting of photoreceptor degeneration;

retinal detachment;

retinal trauma;

a photic lesion;

a macular hole;

a macular edema;

night blindness;

color blindness;

ischemic retinopathy;

retinopathy due to premature birth;

infection;

inflammatory condition; and

an ocular neuropathy.

42. The method of claim 27, wherein said dystrophic retinal tissue is a result of an ocular neuropathy

35. The method of 26, wherein the recipient is a member selected from the group consisting of a human, a mouse, a rat, a cat, a dog, a pig, a cow, a horse, a monkey, or a great ape.

43. A method of studying integration and development of retinal stem cells in vivo, comprising transplanting a green fluorescent protein-expressing, neuroretina-derived retinal stem cell into a recipient mammal.

44. The method of claim 43, wherein the green fluorescent protein-expressing, neuroretina-derived retinal stem cells were derived from a transgenic donor mammal that expressed green fluorescent protein.

45. The method of claim 43, wherein the green fluorescent protein-expressing, neuroretina-derived retinal stem cells were derived by inserting a transgene for expressing green fluorescent protein into a clonally derived, neuroretina-derived retinal stem cell line.