Methods and compositions for generating epicardium cells

Abstract

Methods and products for obtaining cardiovascular lineage cells from hPSCs. The method comprises one or more of the following steps: (a) contacting BMP component primed hPSCs with a cardiovascular mesoderm programming cocktail and culturing the contacted hPSCs for a period of time to generate a KDR+ and PDGFRalpha+ cardiovascular mesoderm cell population; (b) contacting the cardiovascular mesoderm cell population with a cardiovascular progenitor specification cocktail and culturing the contacted cardiovascular mesoderm cell population for a period of time to generate a NKX2-5+ or WT1+ cardiovascular progenitor cell population; and (c) contacting the cardiovascular progenitor cell population with a maturation cocktail and culturing the contacted cardiovascular progenitor population for a period of time to produce a cardiovascular population optionally cardiomyocyte lineage cells expressing cardiac troponin T (cTnT) and/or SIRPA and/or epicardial lineage cells expressing WT1.

1 . A method of obtaining a cardiomyocyte lineage or an epicardial lineage cell population from human pluripotent stem cells (hPSCs) comprising the steps: (a) contacting BMP component primed hPSCs with a cardiovascular mesoderm programming cocktail suitable for inducing the hPSCs to differentiate to a cardiovascular mesoderm cell population under conditions suitable for the programming cocktail to penetrate the hPSCs and culturing the contacted hPSCs for a period of time to generate a KDR+ and PDGFRalpha+ cardiovascular mesoderm cell population; (b) contacting the cardiovascular mesoderm cell population with a cardiovascular progenitor specification cocktail suitable to specify a NKX2-5+ or WT1+ cardiovascular progenitor cell population under conditions suitable for the specification cocktail to penetrate the cardiovascular mesoderm cell population and culturing the contacted cardiovascular mesoderm cell population for a period of time to generate a NKX2-5+ or WT1+ cardiovascular progenitor cell population; and (d) contacting the cardiovascular progenitor cell population with a maturation cocktail under conditions suitable for the maturation cocktail to penetrate the cardiovascular progenitor cell population and culturing the contacted cardiovascular progenitor population for a period of time to produce a cardiovascular population, optionally cardiomyocyte lineage cells expressing cardiac troponin T (cTnT) and/or SIRPA and/or epicardial lineage cells expressing WT1. 
     
     
         2 . The method of  claim 1  wherein the BMP component primed hPSCs are prepared by contacting the hPSCs with BMP component for about 1 to about 2 days, optionally wherein the BMP component is BMP4 and/or BMP2. 
     
     
         3 . The method of  claim 1 , wherein the cardiovascular mesoderm programming cocktail comprises a BMP component and an activin component and optionally a FGF component and the cardiovascular mesoderm programming cocktail is contacted with the hPSCs for about 3 to about 5 days. 
     
     
         4 . The method of  claim 3 , wherein the FGF component is bFGF. 
     
     
         5 . The method of  claim 3  wherein the BMP component is BMP4. 
     
     
         6 . The method of  claim 3 , wherein the activin component is Activin A. 
     
     
         7 . The method of  claim 1 , wherein the hPSCs are comprised in embryoid bodies. 
     
     
         8 . A method for obtaining a NKX2-5+ or WT1+ cardiovascular progenitor cell population from hPSCs comprising the steps: (a) obtaining a KDR+ and PDGFRalpha+ cardiovascular mesoderm cell population from hPSCs optionally as defined in step a of  claim 1 ; (b) contacting the KDR+ and PDGFRalpha+ cardiovascular mesoderm cell population with a cardiovascular progenitor specification cocktail under conditions suitable for the specification cocktail to penetrate the cardiovascular mesoderm cell population and culturing the contacted cardiovascular mesoderm cell population for a period of time sufficient to generate a NKX2-5+ or WT1+ cardiovascular progenitor cell population. 
     
     
         9 . The method of  claim 8 , wherein the KDR+ and PDGFRalpha+ cardiovascular mesoderm cell population is dissociated prior to contacting with the cardiovascular progenitor specification cocktail. 
     
     
         10 . The method of  claim 8 , wherein the cardiovascular mesoderm cell population is contacted with the cardiovascular progenitor specification cocktail for at least 12 hours to about 48 hours. 
     
     
         11 - 19 . (canceled) 
     
     
         20 . A method of producing a WT1+ epicardial lineage cell population, comprising the steps: (a) obtaining a KDR+ and PDGFRalpha+ cardiovascular mesoderm cell population from hPSCs optionally as defined in step a of  claim 1 ; (b) contacting the cardiovascular mesoderm cell population with a cardiovascular progenitor specification cocktail comprising an epicardial lineage promoting component under conditions suitable for the specification cocktail to penetrate the cardiovascular mesoderm cell population and culturing the contacted cardiovascular mesoderm cell population for a period of time sufficient to generate a WT1+ cardiovascular progenitor cell population. 
     
     
         21 . The method of  claim 20 , wherein the cardiovascular progenitor specification cocktail comprises an epicardial cell promoting component, optionally wherein the epicardial cell promoting component comprises BMP4 in a suitable concentration for promoting epicardial cell development. 
     
     
         22 . The method of  claim 20 , wherein the epicardial cell promoting component comprises BMP4 at a concentration of at least 1.25 ng/mL, at least 2.5 ng/mL, at least 5 ng/mL or at least 10 ng/mL. 
     
     
         23 . The method of  claim 21 , wherein the epicardial cell promoting component further comprises a Wnt component, optionally CHIR99021. 
     
     
         24 . The method of  claim 20 , wherein the epicardial cell promoting component comprises BMP4 and a Wnt component optionally CHIR 99021. 
     
     
         25 - 35 . (canceled) 
     
     
         36 . The method of  claim 1 , wherein the maturation cocktail contacted WT1+ epicardial lineage cell population and/or the WT1+ZO1+ epicardial lineage cell population is cultured for a period of time to obtain a retinol dehydrogenase expressing epicardial lineage cell population, optionally wherein the retinol dehydrogenase expressing epicardial lineage cell population is ALDH1A2 expressing or Aldefluor positive staining, optionally wherein the cell population is at least 50% Aldefluor™ positive. 
     
     
         37 . The method of  claim 36 , wherein the vascular smooth muscle lineage cell population, a fibroblast lineage cell population and/or a retinol dehydrogenase expressing epicardial lineage cell population are isolated. 
     
     
         38 . The method of  claim 37 , wherein cardiovascular progenitor specification cocktail further comprises an activin/nodal inhibitor, optionally SB431542. 
     
     
         39 . The method of  claim 36 , wherein the hPSCs are an induced pluripotent stem cell (iPSC) line and/or a human embryonic stem cell (hESC) line. 
     
     
         40 . The method of  claim 39 , wherein the iPSC is a fibroblast derived iPSC line. 
     
     
         41 - 43 . (canceled)