Bone morphogenetic protein 4 (bmp4)

For WNT ligands treatment during chondrogenesis, pellets were treated with either 10 ng ml⁻¹ TGF-β3 (control group) or a combination of 10 ng ml⁻¹ TGF-β3 and 100 ng ml⁻¹ individual WNT ligand (WNT2B, WNT3A, WNT4, WNT5B, or WNT7B, all from R&D system) in chondrogenic medium from d0 to d42 as appropriate. For WNT ligands treatment during the Cp stage, cells were supplemented with either 20 ng ml⁻¹ BMP4 (R&D Systems, #314-BP-010) alone (control group), a combination of 20 ng ml⁻¹ BMP4 and 1 μM C59, or a combination of 20 ng ml⁻¹ BMP4 and 100 ng ml⁻¹ WNT3A (R&D Systems, #5036-WN-010) in mesodermal differentiation medium from d7 to d12.

Differentiation to specific embryonic lineages was performed in CDM-PVA, which has the same composition as CDM-BSA, with polyvinyl alcohol (PVA, 1 mg/ml, Sigma) instead of BSA. For early mesoderm differentiation, cells were grown in CDM-PVA with FGF2 (20 ng/ml), LY294002 (10 μM, Sigma) and BMP4 (10 ng/ml, R&D) for 36 h then treated with CDM-PVA with FGF2 (20 ng/ml) and BMP4 (50 ng/ml) for 3.5 days to generate LM as previously described (Cheung et al., 2012 (link)). To generate epicardium, LM cells were differentiated in CDM-PVA with BMP4 (50 ng/ml), recombinant human WNT3A (25 ng/ml, R&D systems) and RA (4 μM, Sigma) at a seeding density of 2.5×10³/cm² for 10 days. To investigate the role of WNT pathway, LM cells were differentiated with IWP2 (2 μM, Tocris) along with BMP4 and RA. Subsequently, epicardial cells were re-suspended in CDM-PVA with PDGF-BB (10 ng/ml, Peprotech) and TGFβ1 (2 ng/ml, Peprotech), designated as PT for 12 days. Epicardial cells were differentiated with PT in the presence of Y27632 (2 μg/ml, Calbiochem) to study the role of RhoA/RhoK in EPI-SMC differentiation. To generate EPI-CFs, epicardial cells were differentiated in CDM-PVA with VEGF (50 ng/ml, Peprotech) and FGF2 (50 ng/ml) for 12 days. HESCs were grown in CDM-PVA with FGF2 (12 ng/ml) and SB431542 (10 μM, Tocris) for 7 days to generate neuroectoderm (Vallier et al., 2009 (link)).

For granulosa cell differentiation, human iPSCs were manually split and plated onto ultra-low attachment multiwell plates (Corning) to form embryonic bodies (EBs). Cells were cultured in maintenance medium (80% DMEM/F12, 20% knockout serum replacement, 4 ng/mL bFGF, 0.1 mM -mercaptoethanol, and 1% NEAA (Gibco)) for 48 h. Cells were then treated with 6 ng/mL BMP4 (R&D systems) for 24 h. On day 3, cells were then cultured in 10 ng/mL BMP4, 6 ng/mL WNT3A, 6 ng/mL activin-A, and 5 ng/mL bFGF (R&D Systems) for 72 h. Then EB-derived mesoderm progenitors were transferred to gelatin-coated plates and incubated in the maintenance medium supplemented with 10 ng/mL BMP4, 5 ng/mL bFGF, and 25 ng/mL follistatin (R&D Systems) for 6 days.