Methods of mammalian retinal stem cell production and applications
AssigneeThe Regents of the University of California
InventorsKang Zhang, Jiagang Zhao, Adah Almutairi
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Abstract
The invention provides an in vitro method for producing isolated mammalian primitive retinal stem cells (pRSCs) comprising: (a) culturing isolated embryonic stem cells (ESCs) from a mammal in a cell culture medium that is free of feeder cells, feeder-conditioned medium or serum so as to produce and grow a culture of the isolated ESCs; and (b) contacting the culture of the isolated ESCs so grown with one or more of an inhibitor for Wnt or TGF-β/BMP signaling so as to differentiate the isolated ESCs of (a) into primitive retinal stem cells thereby producing isolated mammalian pRSCs.
Claims51
An in vitro method for producing isolated mammalian primitive retinal stem cells (pRSCs) comprising:
- (a) culturing isolated embryonic stem cells (ESCs), pluripotent stem cells (PSCs), or induced pluripotent stem cells (iPSCs) from a mammal in a cell culture medium that is free of feeder cells, feeder-conditioned medium or serum so as to produce and grow a culture of the isolated ESCs, PSCs or iPSCs, and
- (b) contacting the culture of the isolated ESCs, PSCs or iPSCs so grown with one or more of an inhibitor for Wnt or TGF-β/BMP signaling so as to differentiate the isolated ESCs, PSCs, or iPSCs of (b) into primitive retinal stem cells thereby producing isolated mammalian pRSCs.
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A method for producing isolated mammalian photoreceptors from isolated mammalian pRSCs comprising:
- (a) culturing and growing dissociated pRSCs from a mammal in a neural induction medium comprising one or more of an inhibitor of a TGF-β/Activin receptor-like kinases ALK-4, -5 or -7, glycogen synthase kinase-3 (GSK-3), Notch or Wnt signaling or an activator of a hedgehog signaling for a sufficient time to induce pRSCs to a photoreceptor cell lineage fate without visible morphological changes or expression of photoreceptor-specific markers;
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- (b) culturing and growing pRSCs of step (a) in neural induction medium comprising retinoic acid or taurine or both so as to differentiate the mammalian pRSCs to photoreceptors, thereby producing isolated mammalian photoreceptors;
- wherein the culture medium is free of feeder cells, feeder-conditioned medium or serum.
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A method for producing non-neural isolated mammalian retinal pigment epithelium (RPE) or isolated mammalian retinal pigment epithelial cells (RPEs) comprising:
- (a) culturing pRSCs from a mammal in culture medium comprising nicotinamide or activin A or both in absence of SMAD signaling inhibitor for a sufficient time so as to direct pRSCs toward RPE fate;
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- (b) culturing the pRSCs in culture medium comprising one or more of a N1 medium supplement, taurine, hydrocortisone, or triiodo-thyronin;
- so as to differentiate the mammalian pRSCs to mammalian RPE or RPEs, thereby, producing isolated mammalian RPE or RPEs,
- wherein the medium is free of feeder cells or feeder-conditioned medium.
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A method for production of isolated human primitive retinal stem cells (hpRSCs) from isolated human embryonic stem cells (hESCs), isolated human pluripotent stem cells (hPSCs), or human induced pluripotent stem cells (iPSCs) comprising:
- (a) culturing isolated hESCs, hPSCs and iPSCs on a solid support with culture medium in the absence of feeder cells, feeder-conditioned medium or serum for a sufficient time so as to grow nearly confluent, preferably to 80% cellular confluence;
- (b) culturing the isolated hESCs, hPSCs and iPSCs so grown on a solid support in a culture medium comprising basic FGF (bFGF) for a sufficient time so as to grow nearly confluent;
- (c) culturing the isolated hESCs, hPSCs and iPSCs of step b) on a solid support with a culture medium comprising a combination of small molecule inhibitors for Wnt and TGF-β/BMP signaling so as to differentiate the isolated hESCs, hPSCs and iPSCs to an isolated human primitive retinal stem cells, thereby, producing isolated hpRSCs.
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Dependencies are inferred from the claim text.