Fig. 2.

Work flowcharts for developing iPSC-derived cell culture platforms from thirteen-lined ground squirrels and other unconventional model organisms. It is worth noting that iPSCs from non-mammalian vertebrate species and invertebrate species have been successfully developed. (A) A work flowchart for effective differentiation of thirteen-lined ground squirrel iPSCs into neurons. Maintenance medium: DMEM/F12, 1× MEM non-essential amino acids, 40 ng ml⁻¹ FGF-2, 15% (v/v) KSR (knockout serum replacement), 1× antibiotic-antimycotic. NPC (neural precursor cell) medium: DMEM, 5% KSR, B27 without vitamin A, 2 mmol l⁻¹ l-glutamine, 1× antibiotic-antimycotic, 10 ng ml⁻¹ NOGGIN, 20 ng ml⁻¹ EGF and 10 ng ml⁻¹ FGF-2. ND1 medium: DMEM, 5% KSR, B27 without vitamin A, 2 mmol l⁻¹ l-glutamine, 1× antibiotic-antimycotic, 10 ng ml⁻¹ BDNF, 2.5 ng ml⁻¹ GDNF and 10 ng ml⁻¹ activin A; ND2 medium: ND1 without activin A; ND3 medium: ND2 without KSR. For further information on cell culture-related reagents, see Ou et al. (2018). (B) Based on our experience working with thirteen-lined ground squirrel iPSCs and others' experience with iPSCs of naked mole rats and conventional species, this flowchart can act as a guide to developing iPSCs or other culturable cell types from unconventional model organisms. Note that a–f describe key factors that could greatly impact the outcome of the whole procedure – these steps require optimization for each cell type and each species; c–e are interactive factors that may require lots of trial-and-error optimization.