Method for generating mesoderm and/or endothelial colony forming cell-like cells having in vivo blood vessel forming capacity

Abstract

The present disclosure relates generally to methods and compositions useful in cell and tissue biology and therapeutics. In particular, an in vitro method for differentiating pluripotent stem cells into KDR + NCAM + APLNR + mesoderm cells and/or SSEA5 − KDR + NCAM + APLNR + mesoderm cells is provided. The disclosed mesoderm cells may be used to generate blood vessels in vivo and/or further differentiated in vitro into endothelial colony forming cell-like cells (ECFC-like cells). Purified human cell populations of KDR + NCAM + APLNR + mesoderm cells and ECFC-like cells are provided. Test agent screening and therapeutic methods for using the cell populations of the present disclosure are provided.

1 - 14 . (canceled) 
     
     
         15 . The method of  claim 22 , wherein the endothelial inducing step is carried out in the absence of one or more of: co-culture cells, embryoid body formation and exogenous TGF-β inhibition. 
     
     
         16 . The method of  claim 22 , wherein the isolated ECFC-like cells have a capacity to form blood vessels when implanted into a mammal in the absence of co-implanted cells. 
     
     
         17 . An isolated population of human KDR + NCAM + APLNR +  mesoderm cells, wherein the isolated KDR + NCAM + APLNR +  mesoderm cells have a capacity to form blood vessels when implanted into a mammal and wherein the isolated KDR + NCAM + APLNR +  mesoderm cells were derived in vitro from human pluripotent stem cells in a method carried out in the absence of co-culture cells. 
     
     
         18 . The isolated population of  claim 17 , wherein the KDR + NCAM + APLNR +  mesoderm cells are SSEA5 − . 
     
     
         19 . The isolated population of  claim 17 , wherein the KDR + NCAM + APLNR +  mesoderm cells exhibit increased expression of one or more lateral plate-extra-embryonic mesoderm markers relative to PSCs, wherein the lateral plate-extra-embryonic mesoderm markers are selected from BMP4, WNT5A, NKX2-5 and HAND1. 
     
     
         20 . The isolated population of  claim 19 , wherein the KDR + NCAM + APLNR +  mesoderm cells lack increased expression of one or more axial mesoderm markers, paraxial mesoderm markers and/or intermediate mesoderm markers, relative to PSCs, wherein the axial mesoderm markers are selected from CHIRD and SHH, the paraxial mesoderm markers are selected from PAX1, MEOX1, and TCF15, and the intermediate mesoderm markers are selected from GOSR1, PAX2 and PAX8. 
     
     
         21 . An isolated population of human KDR + NCAM + APLNR +  mesoderm cells obtained according to a method comprising:
 (a) providing pluripotent stem cells (PSCs): 
 (b) inducing the pluripotent stem cells to undergo mesodermal differentiation, wherein the mesodermal induction is carried out in the absence of co-culture cells and further comprises:
 i) culturing the pluripotent stem cells for 24 hours in a mesoderm differentiation medium comprising Activin A, bone morphogenetic protein-4 (BMP-4), vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF-2); 
 ii) replacing the medium of step i) with a mesoderm differentiation medium comprising BMP-4, VEGF and FGF-2 every 24-48 hours thereafter for 72 hours; and 
 
 (c) isolating from the cells induced to undergo differentiation to mesoderm cells, wherein the isolation of mesoderm cells comprises:
 iii) sorting the mesoderm cells to select for KDR+NCAM+APLNR+ cells. 
 
 
     
     
         22 . An isolated population of human NRP-1+CD31+ endothelial colony forming cell-like cells (ECFC-like cells) obtained according to a method comprising generating an isolated population of human KDR+NCAM+APLNR+ mesoderm cells, wherein the generation of human KDR+NCAM+APLNR+ mesoderm cells population comprises:
 (a) providing pluripotent stem cells (PSCs);   (b) inducing the pluripotent stem cells to undergo mesodermal differentiation, wherein the mesodermal induction comprises:
 i) culturing the pluripotent stem cells for 24 hours in a mesoderm differentiation medium comprising Activin A, BMP-4, VEGF and FGF-2; 
 ii) replacing the medium of step i) with a mesoderm differentiation medium comprising BMP-4, VEGF and FGF-2 every 24-48 hours thereafter for 72 hours; and 
   (c) isolating from the cells induced to undergo differentiation the mesoderm cells, wherein the isolation of mesoderm cells comprises:
 iii) sorting the mesoderm cells to select for KDR+NCAM+APLNR+ cells; 
   wherein the method further comprising inducing the said population of mesoderm cells to undergo endothelial differentiation, wherein the induction of endothelial differentiation comprises:   (d) culturing the isolated mesoderm cells in an endothelial differentiation medium comprising BMP-4, VEGF and FGF-2 for 6-8 days; and   (e) isolating from the cells induced to undergo endothelial differentiation endothelial colony forming cell-like (ECFC-like) cells, wherein the ECFC-like cells are CD31+ NRP-1+ and exhibit a cobblestone morphology.   
     
     
         23 . A pharmaceutical composition comprising the KDR + NCAM + APLNR +  mesoderm cells of  claim 17 . 
     
     
         24 . A pharmaceutical composition comprising the endothelial colony forming cell-like cells (ECFC-like cells) of  claim 22 . 
     
     
         25 . A method of examining a test agent for its ability to modify cellular activity, the method comprising:
 exposing at least one of the cells of the population of cells of  claim 17  to a test agent and;   observing the effect of the test agent on one or more of cell growth and cell viability.   
     
     
         26 . A method for transplantation in a subject in need thereof, the method comprising:
 providing to the subject the isolated population of cells of  claim 17 .   
     
     
         27 . A method of treating a subject in need of epithelial repair, the method comprising:
 providing to the subject a therapeutically effective amount of a population of cells according to  claim 17 .