The naive human embryonic stem cell (hESC) line H9 was generated by culturing H9 hESCs in naive human stem cell medium (NHSM). Cells express pluripotency markers and form teratomas in-vivo, which contain cells of all the three germ layers. When compared to non-naive or primed hESCs, naive hESCs display a substantial reduction in expression of epigenetic markers. In addition, they have a much shorter doubling time, compared to primed hESCs, and can form cross-species chimeras when injected into mouse morulas.
Cells were seeded on gelatin/vitronectin-coated plates and cultured in naive human stem cell medium (NHSM) for 4-8 days.
NHSM medium (for 500 ml medium): knockout DMEM, AlbuMAXI (1%), N2 supplement (1%), recombinant human insulin (12.5 μg/ml), recombinant human LIF (10 μg), recombinant bFGF (8 ng/ml), recombinant TGF-β1 (1 ng/ml), glutamine (1 mM), non-essential amino acids (1%), β-mercaptoethanol (0.1mM), Penicillin-Streptomycin, PD0325901 (1 μM), CHIR99021 (3 μM), SP600125 (10 μM), SB203580 (10 μM). Instead of SB203580, medium can be supplemented with SB202190 (5 μM), or BIRB796 (2 μM). Medium can be also supplemented with Y27632 (5 μM), and the protein kinase C inhibitor Go6983 (5 μM), to reduce apoptosis levels.