METHOD OF DIRECTED DIFFERENTIATION PRODUCING CORNEAL ENDOTHELIAL CELLS, COMPOSITIONS THEREOF, AND USES THEREOF
This disclosure generally relates to cell-based therapies for treatment of visual disorders, including disorders of the cornea. Methods are exemplified for directed differentiation of corneal cells from stem cells. Compositions of corneal endothelial cells and uses thereof are also provided. Exemplary compositions exhibit improved cell density and/or more “youthful” gene expression relative to cells obtained from donated tissue.
1 - 242 . (canceled)
243 . A method of producing corneal endothelial cells (CECs), comprising
(a) contacting neural crest stem cells (NCSCs) with at least one dickkopf-related protein 2(DKK2) agonist, at least one platelet-derived growth factor subunit B (PDGFB) agonist, or a combination of at least one DKK2 agonist and at least one PDGFB agonist, to induce differentiation of the NCSCs into CECs,
wherein the at least one DKK2 agonist is selected from the group consisting of: DKK2; Dkk 1; Dkk 3; Dkk 4; LRP5/6 antagonists; Kremen antagonists; Soggy; secreted frizzled related proteins (Frzb); Wnt inhibitor factor (WIF); a Wnt modulator; Casein Kinase 1-7 β catenin antagonists; LEF/TCF transcription factor members modulators; IWR; pyrvinium; ICG-001; PKF115-584; IWP; Ant1.4Br/Ant 1.4Cl; Niclosamide; apicularen; bafilomycin; XAV939; NSC668036; 2,4-diamino-quinazoline; Quercetin; Wnt proteins, nucleic acids encoding Wnt proteins, LiCl, Axin antagonists; APC antagonists; norrin; R-spondin2; (hetero) arylpyrimidines; IQ1; BIO (6-bromoindirubin-3′-oxime); 2-amino-4-[3,4-(methylenedioxy) benzyl-amino]-6-(3-methoxyphenyl) pyrimidine; WAY-316606; QS11; SB-216763; SB-216763; DCA; and any combination thereof; and/or wherein the at least one PDGFB agonist is selected from the group consisting of: PDGFB;
PDGFAA polypeptide; a nucleic acid encoding PDGFAA; PDGFAB polypeptide; a nucleic acid encoding PDGFAB; Phorbol 12-myristate 13-acetate (PMA); VEGF; and any combination thereof.
244 . The method of claim 243 , wherein the method comprises contacting NCSCs in vitro with at least one DKK2 agonist having a concentration of between 2 ng/ml and 20 ng/ml.
245 . The method of claim 243 , wherein the method comprises contacting NCSCs in vitro with at least one PDGFB agonist having a concentration of between 2 ng/ml and 20 ng/ml.
246 . The method of claim 243 , wherein step (a) comprises contacting NCSCs in vitro with a combination of at least one DKK2 agonist and at least one PDGFB agonist.
247 . The method of claim 243 , wherein the at least one DKK2 agonist is DKK2.
248 . The method of claim 243 , wherein the at least one PDGFB agonist is PDGFB.
249 . The method of claim 243 , wherein step (a) comprises contacting NCSCs in vitro with DKK2 and PDGFB.
250 . The method of claim 243 , further comprising culturing the CECs on a matrix selected from: laminin, fibronectin, vitronectin, proteoglycan, entactin, collagen, collagen I, collagen IV, collagen VIII, heparan sulfate, a soluble preparation from Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells (MATRIGEL®), a human basement membrane extract, and any combination thereof.
251 . The method of claim 250 , wherein the matrix is a soluble preparation obtained from EHS mouse sarcoma cells (MATRIGEL®).
252 . The method of claim 243 , wherein, subsequent to commencement of step (a), the differentiating NCSCs are passaged.
253 . The method of claim 252 , wherein the passaging is effected between 2 hours and 4 days subsequent to commencement of step (a).
254 . The method of claim 252 , wherein the passaging is effected about 3 days subsequent to commencement of step (a).
255 . The method of claim 252 , wherein the passaging is effected by a method comprising contacting the cells with a cell dissociation buffer, or by a method comprising mechanical dissociation of the cells or a subset thereof, or by a method comprising optical isolation of the cells or a subset thereof.
256 . The method of claim 255 , wherein the passaging is effected by a method comprising contacting the cells with a cell dissociation buffer and the cell dissociation buffer is non-enzymatic.
257 . The method of claim 255 , wherein the passaging is effected by a method comprising contacting the cells with a cell dissociation buffer and the cell dissociation buffer comprises ethylenediaminetetraacetic acid (EDTA).
258 . The method of claim 243 , wherein the duration of the contacting NCSCs with the at least one DKK2 agonist, at least one PDGFB agonist, or combination of at least one DKK2 agonist and at least one PDGFB agonist is between 2 and 25 days.
259 . The method of claim 243 , wherein step (a) further comprises culturing the NCSCs on a matrix selected from: laminin, fibronectin, vitronectin, proteoglycan, entactin, collagen, collagen I, collagen IV, collagen VIII, heparan sulfate, a soluble preparation from Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells (MATRIGEL®), a human basement membrane extract, and any combination thereof.
260 . The method of claim 259 , wherein the matrix is a soluble preparation obtained from EHS mouse sarcoma cells (MATRIGEL®).
261 . The method of claim 243 , further comprising harvesting the CEC as a sheet of cells, optionally wherein the CEC exhibit a culture density of at least 6000 cells/mm 2 .
262 . The method of claim 243 , wherein the NCSCs express Sox10 and p75 (NGFR).
263 . The method of claim 243 , wherein the NCSCs express Sox 10, AP2, HNK1, Pax3, PAX7 and p75 (NGFR).
264 . The method of claim 243 , wherein the CEC express one or more markers selected from the group consisting of: Na+/K+ ATPase, ZO-1, KLF13, AQP1, Collagen VIII, SLC16A3, CFTR, NBC1, CA2, AE2/SCL4A2, SCL16A1, CA12, CA4, FoxC1, and any combination thereof, optionally wherein the CECs do not express vWF and CD31.
265 . The method of claim 243 , wherein the NCSCs are produced from human pluripotent stem cells in vitro.
266 . The method of claim 265 , wherein the NCSCs are produced by contacting human pluripotent stem cells with at least one inhibitor of SMA/Mothers Against Decapentaplegic (SMAD) protein signaling to induce differentiation of the human pluripotent stem cells into NCSCs.
267 . The method of claim 266 , wherein the at least one inhibitor of SMAD protein signaling comprises Noggin, 4-[4-(1,3-benzodioxol-5-yl)-5-(2-pyridinyl)-1H-imidazol-2-yl]benzamide (SB431542), or a combination of Noggin and 4-[4-(1,3-benzodioxol-5-yl)-5-(2-pyridinyl)-1H-imidazol-2-yl]benzamide (SB431542).
268 . The method of claim 266 , wherein the NCSCs are produced by contacting the human pluripotent stem cells with Noggin and 4-[4-(1,3-benzodioxol-5-yl)-5-(2-pyridinyl)-1H-imidazol-2-yl]benzamide (SB431542) to induce differentiation of the human pluripotent stem cells into NCSCs.
269 . The method of claim 265 , wherein the human pluripotent stem cells are human ES cells.
270 . The method of claim 265 , wherein the human pluripotent stem cells are iPS cells.