Methods of mammalian retinal stem cell production and applications
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.
1. An in vitro method for producing isolated mammalian primitive retinal stem cells (pRSCs) comprising:
(a) culturing isolated pluripotent stem cells (PSCs) from a mammal on a solid support in N2B27 cell culture medium that is free of feeder cells, feeder-conditioned medium or serum so as to produce and grow an adherent culture of the isolated PSCs; and
(b) contacting the adherent culture of the isolated PSCs so grown with a combination of an inhibitor of Wnt, an inhibitor of TGF-β, and an inhibitor of BMP signaling so as to differentiate the isolated PSCs of (b) into primitive retinal stem cells thereby producing isolated mammalian pRSCs.
2. The method of claim 1 , wherein in step (a) the culture so produced is grown to near confluency before step (b).
3. The method of claim 1 , wherein the mammal is a human, monkey, bear, rat, mouse, mink, rabbit, guinea pig, pig, dog, cat, goat, sheep, horse or cow.
4. The method of claim 1 , wherein the isolated mammalian primitive retinal stem cells (pRSCs) are positive for expression of PAX6, LHX2, RAX, OTX2, SIX3 and/or SIX6 typical early eye field transcription factors.
5. The method of claim 1 , wherein the isolated mammalian primitive retinal stem cells (pRSCs) which are positive for expression of stemness factors SOX2, and STAT3.
6. The method of claim 1 , further comprising the isolated mammalian primitive retinal stem cells which are positive for expression of Ki67.
7. The method of claim 1 , wherein the inhibitor of Wnt signaling is a small molecule inhibitor selected from the group consisting of Inhibitor of Wnt Production-1 (IWP-1), Inhibitor of Wnt Production-2 (IWP2), JW55, JW74, okadaic acid, tautomycin, SB239063, SB203580, ADP-HPD, 2-[4-(4-fluorophenyl) piperazin-1-yl]-6-methylpyrimidin-4(3H)-one, PJ34, cambinol, sulindac, 3289-8625, scaffold A for series of analogs designed to inhibit Dishevelled protein, scaffold B for series of analogs designed to inhibit Dishevelled protein, JO 1-017a, NSC668036, filipin, IC261, PF670462, Bosutinib, PHA665752, Imatinib, ICG-001, ethacrynic acid, ethacrynic acid derivative, PKF115-584, PNU-74654, PKF118-744, CGP049090, PKF118-310, ZTM000990, BC21, GDC-0941, Rp-8-Br-cAMP, LGK974, C59, Ant 1.4Br/Ant 1.4CI, niclosamide, apicularen, bafilomycin, XAV939, IWR1, pyrvinium, NSC668036, 2,4-diamino-quinazoline, quercetin, and PKF115-584, and combinations thereof.
8. The method of claim 1 , wherein the inhibitor of Wnt signaling is IWP2 or a Dkkl analog.
9. The method of claim 8 , wherein the Dkkl analog is N-(6-methyl-1,3-benzothiazol-2-yl)-2-[(4-oxo-3-phenyl-6,7-dihydrothieno[3,2-d]pyrimidin-2-yl)sulfanyl]acetamide having a chemical formula of C 22 H 18 N 4 O 2 S 3 and a chemical structure of:
10. The method of claim 1 ,
wherein the inhibitor of TGF-β signaling is a small molecule inhibitor selected from the group consisting of SB431542 (4-[4-(1,3-benzodioxol-5-yl)-5-(2-pyridinyl)-1H-imidazol-2-yl]benzamide), A 83-01 (3-(6-Methyl-2-pyridinyl)-N-phenyl-4-(4-quinolinyl)-1H-pyrazole-1-carbothioamide), SJN 2511 (2-(3-(6-Methylpyridine-2-yl)-1H-pyrazol-4-yl)-1,5-naphthyridine), D 4476 (4-[4-(2,3-Dihydro-1,4-benzodioxin-6-yl)-5-(2-pyridinyl)-1H-imidazol-2-yl]benzamide), LY 364947 (4-[3-(2-Pyridinyl)-1H-pyrazol-4-yl]-quinoline), SB 525334 (6-[2-(1,1-Dimethylethyl)-5-(6-methyl-2-pyridinyl)-1H-imidazol-4-yl]quinoxaline), SD 208 (2-(5-Chloro-2-fluorophenyl)-4-[(4-pyridyl)amino]pteridine); and
wherein the inhibitor of BMP signaling is LDN-193189 (4-(6-(4-(piperazin-1-yl)phenyl)pyrazolo[1,5-a]pyrimidin-3-yl)quinoline).
11. The method of claim 1 , wherein the inhibitor of TGF-β signaling is a small molecule inhibitor of the transforming growth factor-beta (TGF-β) superfamily type I activin receptor-like kinases ALK-4, -5, and -7.
12. The method of claim 11 , wherein the small molecule inhibitor of the transforming growth factor-beta (TGF-β) superfamily type I activin receptor-like kinases ALK-4, -5, and -7 is SB431542 (CAS No. 301836-41-9) or 4-[4-(1,3-benzodioxol-5-yl)-5-(2-pyridinyl)-1H-imidazol-2-yl] benzamide having a chemical formula of C 22 H 16 N 4 O 3 and a chemical structure of
13. The method of claim 1 , wherein the inhibitor of BMP signaling is a small molecule inhibitor of BMP type I receptors ALK-2 and ALK-3 or a noggin analog.
14. The method of claim 13 , wherein the small molecule inhibitor of BMP type I receptors ALK-2 and ALK-3 is 4-(6-(4-(piperazin-1-yl)phenyl)pyrazolo[1,5-a]pyrimidin-3-yl)quinoline having a chemical formula of C 25 H 22 N 6 and a chemical structure of
15. The method of claim 1 , wherein the combination of the inhibitor for Wnt, the inhibitor of TGF-β and the inhibitor of BMP signaling is a combination of: (a) one or more inhibitors of Wnt Production-2 or Dkkl analog; (b) one or more inhibitors of transforming growth factor-beta (TGF-β) superfamily type I activin receptor-like kinases ALK-4, -5, and -7; and (c) one or more inhibitors of BMP type I receptors ALK-2 and ALK-3 or a noggin analog, wherein the combination includes inhibitors for all three signaling pathways.
16. The method of claim 15 , wherein the inhibitors are small molecules.
17. The method of claim 15 , wherein the combination of inhibitors for Wnt, TGF-β and BMP signaling activities is a combination of IWP2 (CAS No. 686770-61-6), SB431542 (CAS No. 301836-41-9), and LDN-193189 (CAS No. 1062368-24-4) wherein the combination produces synergistic inhibition of Wnt and TGF-β and BMP signaling activities.
18. The method of claim 1 , wherein the solid support or coated with reduced growth factor basement membrane.
19. A method for production of isolated human primitive retinal stem cells (hpRSCs) from isolated human pluripotent stem cells (hPSCs) comprising:
(a) culturing isolated hPSCs on a solid support with N2B27 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 hPSCs 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 hPSCs of step b) on a solid support with a culture medium comprising a combination of small molecule inhibitors for Wnt, TGF-8 and BMP signaling so as to differentiate the isolated hPSCs to an isolated human primitive retinal stem cells, thereby, producing isolated hpRSCs.
20. The method of claim 19 , wherein the combination of small molecule inhibitors for Wnt, TGF-β and BMP signaling activities in step (c) is (i) a combination of SB 431542, LDN193189 and IWP2, (ii) a combination of SB 431542, a noggin analog and IWP2, (iii) a combination of SB 431542, LDN193189 and a Dkkl analog; or (iv) a combination of SB 431542, a noggin analog and a Dkkl analog; wherein the combination of any of (i)-(iv) produces synergistic inhibition of Wnt and TGF-β and BMP signaling activities.
21. The in vitro method of claim 1 , wherein the PSCs are selected from embryonic stem cells and induced pluripotent stem cells.