It has not been reported whether the significantly simplified E8 medium is sufficient to support the survival and proliferation of undifferentiated hPSCs in suspension culture. Thus our studies began by establishing a static suspension culture condition in E8 medium for serial passaging and expansion of hiPSCs. BC1 cells cultured on E8 feeder-free conditions (E8-Matrigel or E8-VNT-N) for at least 3 passages were seeded as a single cell suspension in E8 medium supplemented with Y27632 on the first day (Watanabe et al., 2007). The cells survived and formed convex dish-shaped aggregates within 24 h due to the gravity force (Fig. 1a). The size of the cell aggregates gradually increased corresponding to the cell number along the culture period. BC1 cells expanded in static suspension in E8 medium with an average rate of 3.7 ± 0.9 fold per passage, 3.7 × 106 fold increase in total with > 99% of the cells being TRA-1–60+ after 13 passages (Fig. 1b,c). We also examined the differentiation potential of expanded cells, using the spin-EB method (Ng et al., 2008, 2005; Yu et al., 2008). We found that under the hematopoiesis-inducing condition, leukocyte-like cells emerged around day 10 (Fig. 1d) with 46.8% ± 1.6% of the cells becoming CD34+CD45+ HPCs (Fig. 1e). These results indicate that undifferentiated hiPSCs can be expanded in suspension in E8 medium supplemented with one-day treatment of Y27632, and retain their differentiation potential.
BC1 cells
cultured on E8 feeder-free conditions
at least 3 passages
passages
cells
survived and formed convex dish-shaped aggregates
within 24 h
h
BC1 cells
rate
3.7 ± 0.9 fold per passage
fold per passage
BC1 cells
3.7 × 106 fold
fold
cells
TRA-1–60+
> 99%
%
passages
13
cells
CD34+CD45+ HPCs
46.8% ± 1.6%
%
undifferentiated hiPSCs
treatment
one-day
day