Elf1 naive human embryonic stem cells were derived from blastocysts of frozen 6-8-cell embryos under 2iF conditions (see growth conditions section). Cells express pluripotency markers, form teratomas in-vivo with an extensive endoderm differentiation capacity and exhibit characteristic features of naive human embryonic stem cells such as two active X chromosomes, and an overall reduction of H3K27me3 at developmental gene promoters. In addition, DNase I hypersensitivity analysis revealed that, like other naive stem cells, Elf1 cells exhibit increased hypersensitivity at the distal OCT4 enhancer region. A microarray analysis revealed that naive Elf1 gene expression correlated with gene expression of previously reported naive stem cell lines, and that it resembled a gene expression pattern of mouse pre-implantation inner cell mass. Cells have a normal 22,XX karyotype.
Six-to-eight-cell embryos were thawed and cultured in blastocyst medium to expand blastocysts. The zona pellucida was removed 4 days after thawing, using Tyrode's Solution, and the remaining zona-free embryos were plated into a 96-well plate, pre-coated with feeder cells. The first passage was conducted mechanically 3 days following zona removal, and a second mechanical passage was performed 7 days later. All the subsequent passages were performed using trypsin/EDTA solution. Cells were derived and propagated in human embryonic stem cell medium comprising of high-glucose DMEM/F12 supplemented with GlutaMax, KnockOut serum replacer (SR, 20%), sodium pyruvate (1 mM), nonessential amino acids (0.1 mM), penicillin (50 U/mL ), streptomycin (50 mg/mL), and β-mercaptoethanol (0.1 mM). The medium was further supplemented with CHIR99021 (3 μM), PD0329501 (0.4 μm), and FGF2 (10-12 ng/ml). These culturing conditions are termed 2iF.