Isolation and transplantation of retinal stem cells
First Claim
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1. Retinal stem cells isolated from a neuroretina of a donor mammal.
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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.
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Citations
45 Claims
- 1. Retinal stem cells isolated from a neuroretina of a donor mammal.
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2. Retinal stem cells isolated from a neuroretina of a donor mammal, being capable of:
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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.
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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).
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13. A method of isolating and culturing retinal stem cells from a neuroretina of a donor mammal, comprising:
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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. - View Dependent Claims (15, 16, 17, 18, 23)
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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:
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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. - View Dependent Claims (19, 20, 21, 22, 24)
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- 27. A method of repopulating or rescuing a dystrophic eye, comprising introducing neuroretina-derived retinal stem cells into an eye of a mammalian recipient.
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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.
Specification