Activin a

Human iPS cells cultured on various substrates were differentiated into three germ layer-derived lineages as described previously with minor modifications [33 ]. Briefly, 201B6 cells maintained on R-Fc, NC-Fc, or Matrigel were treated with Accutase and were plated on the Matrigel-coated surface. Cells were treated with 1% DMSO for 1 day before differentiation, and differentiation was induced under the following conditions. Ectoderm: RPMI-1640 media supplemented with B27 (Life Technologies), 500 ng/ml Noggin (Peprotech, Rocky Hill, NJ), and 10 μM SB431542 (Cayman Chemical, Ann Arbor, MI) for 2 days. Mesoderm: RPMI-1640 media supplemented with B27 (without insulin) and 100 ng/ml Activin A (R&D Systems, Minneapolis, MN) for 1 day followed by 20 ng/ml bone morphogenetic factor 4 (BMP4; HumanZyme Inc., Chicago, IL) for 1 day. Endoderm: RPMI-1640 media containing B27 (without insulin), 20 ng/ml BMP4, 10 ng/ml basic fibroblast growth factor (bFGF; ReproCELL, Tokyo, Japan) and 100 ng/ml Activin A for 1 day followed by 100 ng/ml Activin A for 1 day.

hiPSC cells grow in mTeSR were dissociated into single cells using accutase and plated at density of 25–50 k cells/cm² on matrigel coated cell culture plates and subsequently differentiated into endoderm and mesoderm lineages (Loh et al., 2014 (link); Ang et al., 2018 (link)). For definitive endoderm induction, anterior primitive streak was first specified using 100 ng/ml Activin A (R and D systems, 338-AC-050), 3 μm CHIR (Tocris, 4423) and 20 ng/ml FGF2 (R and D Systems, 233-FB-01M) in CDM2 basal media. After 24 hr, the cells were washed with DMEM/F12 (1x, Thermo Fisher Scientific, 11320033), and definitive endoderm was induced using 100 ng/ml Activin A and 250 nM LDN (Reprocell, Yokohama, Japan; 04–0074) in CDM2 basal media for 24 hr. For lateral mesoderm induction, midprimitive streak was specified using 30 ng/ml Activin, 16 μM CHIR, 20 ng/ml FGF2 and 40 ng/ml BMP (R and D Systems, 314 BP-050) in CDM2 basal media for 24 hr. After 24 hr, cells were washed with DMEM/F12 (1x, Thermo Fisher Scientific, 11320033), and lateral mesoderm was induced using 1 μM A8301 (R and D Systems, 2939) 30 ng/ml BMP and 1 μM C59 (Tocris, Bristol, United Kingdom; 5148) for 24 hr in CDM2 basal media. On the third day, cells were lysed for RNA collection and purification.