The naive human embryonic stem cell line LIS2 was derived from the inner cell mass of a pre-implantation human embryo by culturing the inner cell mass on fibroblast feeder cells 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. The LIS2 naive human embryonic stem cell line has a normal 46,XX karyotype. 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.
The inner cell masses of in vitro-fertilized embryos, 6-7 days following fertilization, were mechanically isolated and plated on an irradiated DR4 mouse embryonic fibroblast feeder layer, and cultured in naive human stem cell medium (NHSM). 6-10 days following plating, outgrowths of proliferating embryonic stem cells were evident.
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.
EMBRYONIC DEVELOPMENT & STEM CELL COMPENDIUM
