Method of differentiating human pluripotent cells into cardiac fibroblasts
Methods for generating high-yield, high-purity cardiac fibroblasts are described. Differentiation methods comprising chemically defined culture conditions and methods for in vitro maintenance of human pluripotent stem cell-derived cardiac fibroblasts are also provided.
1. A method for generating a population of human GATA4+ FSP1+ collagen I+ fibronectin+ ISL1+ HAND2+ TCF21+ SNAI2+ cells that bind antibody TE-7, the method comprising:
a) culturing human pluripotent cells in chemically defined culture medium comprising a Gsk3 inhibitor for about 1 day such that a population of T+ SNAI2− cells forms;
b) culturing the T+ SNAI2− cells in the chemically defined culture medium without the Gsk3 inhibitor for about 1 day such that a population of T+ MESP1+ GATA4+ SNAI1+ SNAI2+ cells forms, and
c) culturing the T+ MESP1+ GATA4+ SNAI1+ SNAI2+ cells in a chemically defined culture medium free of exogenous Bone Morphogenetic Proteins (BMPs) and comprising a fibroblast growth factor for about 18 days such that a population of human GATA4+ FSP1+ collagen I+ fibronectin+ ISL1+ HAND2+ TCF21+ SNAI2+ cells that bind antibody TE-7 is generated.
2. The method of claim 1 , wherein the fibroblast growth factor is FGF2.
3. The method of claim 1 , wherein at least 90% of cells generated in step (c) bind antibody TE-7.
4. The method of claim 1 , wherein the method does not comprise a cell separation or selection step.
5. The method of claim 1 , wherein the human GATA4 + FSP1 + collagen I + fibronectin + ISL1 + HAND2 + TCF21 + SNAI2 + cells that bind antibody TE-7 are capable of undergoing at least 10 cell divisions.
6. The method of claim 1 , wherein the Gsk3 inhibitor is one or more of CHIR99021, CHIR98014, BIO-acetoxime, BIO, LiCI, SB216763, SB415286, AR A014418, 1-Azakenpaullone, and Bis-7-indolylmaleimide.
7. The method of claim 6 , wherein the Gsk3 inhibitor is CHIR99021 and is present in a concentration of about 0.2 μM to about 20 μM.
8. The method of claim 7 , wherein the Gsk3 inhibitor is CHIR99021 and is present in a concentration of about 12 μM.