Stemlite pluripotency kit

The pluripotency tests and the germ-layer-specific detection was performed as previously described (Linta et al., 2012 (link); Stockmann et al., 2013 (link)) or done according to the manufacturer’s protocol using the StemLite Pluripotency Kit (Cell Signaling, Danvers, MA, USA). For in vitro differentiation, hiPSC colonies were mechanically lifted by dispase (Stemcell Technologies) digested and transferred in T75 low-attachment flasks (Corning, Corning, NY, USA). Embryoid body (EB) formation was performed in suspension in knockout DMEM supplemented with 2 mM GlutaMAX, 20% Knockout Serum Replacement (Invitrogen, Carlsbad, CA, USA), 1% Antibiotic-Antimycotic (Invitrogen), 100 μM nonessential amino acids (Invitrogen), and 100 μM ß-mercaptoethanol (Invitrogen) for 10 days. Afterwards, EBs were plated on 35 mm dishes (Ibidi, Munich, BY, Germany) and kept in culture for up to 14 days. Differentiated EBs were stained 1:1000 chicken anti-Tubulin beta-III (Millipore, Billerica, MA, USA); 1:150 mouse anti-Actinin (Sigma Aldrich) and 1:100 goat anti-AFP (Santa Cruz, CA, USA). Alexa fluor secondary antibodies were used from Invitrogen.

Immunofluorescent antibody staining of adherent iPSC colonies was conducted using the StemLite Pluripotency kit from Cell Signaling Technology, providing antibodies for the nuclear antigens OCT4, NANOG, SOX2 and surface antigens TRA1-60, TRA1-81 and SSEA4. Stainings were done according to manufacturer’s instructions. Antibodies and dilutions are listed in Supplementary Table S7.