Hescs h9

hESCs (H9, WiCell, Madison) were maintained in a chemically defined medium (CDM-BSA) containing Activin-A (10 ng/ml, R&D Systems) and FGF2 (12 ng/ml, R&D Systems) as previously described (13 (link)). Chemically defined medium consisted of IMDM (250 ml, Life Technologies), Ham’s F12 (250 ml, Life Technologies), Pen/Strep (5 ml, Life Technologies), Insulin (350 μl, Roche), Transferin (250 μl, Roche), chemically defined 100x lipid concentrate (5 ml, Life Technologies) and monothioglycerol (20 μl, Sigma). Differentiation to intermediate lineages and smooth muscle cells was performed as previously described in CDM-PVA, containing polyvinyl alcohol (PVA, 1 mg/ml, Sigma)(14 (link)). In brief, early mesoderm differentiation was started with a combination of CDM-PVA, FGF2 (20 ng/ml), LY294002 (10 μM, Sigma) and BMP4 (10 ng/ml, R&D) for 1.5 days. Consequently either lateral mesoderm differentiation was started in CDM-PVA, FGF2 (20 ng/ml) and BMP4 (50 ng/ml) for 3.5 days or paraxial mesoderm differentiation in CDM-PVA, FGF2 (20 ng/ml) and LY294002 (10 μM) for 3.5 days. To induce neuroectoderm differentiation, cells were cultured in CDM-PVA, FGF (12 ng/ml) and SB431542 (10 μM, Tocris) for 7 days. For smooth muscle cell differentiation, LM-, PM- and NE-cells were resuspended as single cells in CDM-PVA, PDGF-BB (10 ng/ml, Peprotech) and TGF-β1 (2 ng/ml, Peprotech) for 6 days.