BC1 and TNC1 hiPSCs cultured in spinner flasks for more than 10 passages were tested for their pluripotency by in vitro and in vivo differentiation methods. For in vitro assay, by simply replacing the entire E8 medium with differentiation medium of day-2 spinner flask suspension culture, BC1 and TNC1 aggregates transformed into EBs and started spontaneous differentiation under the stimulation of 10% serum (Supplementary Fig. S4). After 8 days in spinner flask followed by 4 days on gelatin-coated tissue culture plates in differentiation medium, cells of all three germ layers could be detected by immunofluorescent staining of specific markers (Fig. 5a). In contrast, undifferentiated hiPSCs on day 0 showed negative staining of all germ markers (data not shown). For in vivo assay, BC1 and TNC1 harvested from suspension culture in spinner flasks were injected into immune-deficient mice and were able to form teratomas containing cells of all germ layers, including glandular epithelium, chondrocytes, and neural rosettes (Fig. 5b). Directed hematopoietic differentiation potential was also tested as previously described (Fig. 5c). Differentiating BC1 and TNC1 cells contained 43.57% ± 4.35% (n = 3) and 43.22% ± 7.13% (n = 3) CD34+CD45+ HPCs on day 14 of differentiation, respectively. The CFU assay measuring hematopoietic progenitors showed that hiPSCs cultured in spinner flasks were able to generate colonies of different hematopoietic cell lineages. The total number of CFUs was comparable or significantly larger (P = 0.0307, n = 3) than cells cultured in parallel in adhesion cultures (Fig. 5d).
BC1 and TNC1 hiPSCs
cultured in spinner flasks
more than 10 passages
passages
serum
10%
%
cells of all three germ layers
spinner flask
After 8 days
days
cells of all three germ layers
gelatin-coated tissue culture plates in differentiation medium
4 days
days
BC1
CD34+CD45+ HPCs
43.57% ± 4.35%
%
BC1
n
3
TNC1
CD34+CD45+ HPCs
43.22% ± 7.13%
%
TNC1
n
3
total number of CFUs
P
0.0307
total number of CFUs
n
3