Figure 4. Workflow diagram of hESC derivation.

(a) Female mice of selected genotypes are selected, superovulated and used for the harvest of oocytes. The oocytes are then activated by the use of strontium chloride (SrCl₂) and passed through rounds of selection for identification of blastocysts that can be used for zona pellucida removal and later transferred individually in NDiff-N2B27/2i/LIF culture medium in 96 well plates (steps 1 to 52). (b) The individual clones are allowed to expand to form defined colonies that are disrupted by trypsinisation and grown and expanded into individual cell lines (steps 53 to 62). The established cell lines are then quality checked including: haploid status (for cell cycle analysis see Box 1), karyotyped (see Box 2), mycoplasma tested and banked. (c and d) The haploid cell lines can be adapted to DMEM/2i/LIF culture or only DMEM/LIF culture while enriching for the haploid content by sorting (see Box 3).