Figure 1.

Conversion of primed hPSCs (hESCs, hiPSCs) toward a naive state in different culture media conditions. (A) Overview of primed to naive conversion protocol in the feeder and feeder-free culture systems. In feeder conditions, primed hPSCs were maintained in PCM1 (hES medium). Passages and cells used for qRT-PCR and immunofluorescence analysis were mechanically picked up. For flow cytometry assay, cell colonies were disaggregated using 0.05% trypsin following manual picking. Finally, feeder primed hPSCs were cultured in NHSM or t2iLGö media for the primed to naive transition, and cell detachment was performed by 0.05% trypsin and TrypLE solution, respectively. In feeder-free conditions, primed hPSCs were cultured in PCM2 (E8 medium). Colonial cell disaggregation was performed by manual picking in all procedures, except inflow cytometry workflow, in which Accutase solution was used. Lastly, feeder-free primed hPSCs were maintained in NHSM or FINE media for naive conversion, and cells were detached with 0.5 mM EDTA and TrypLE solution, respectively. (B) Morphological differences between primed and naive hPSCs colonies in different culture media conditions. Scale bar, 200 µm. (C) Flow cytometry analysis of cell viability and CD90 stemness marker expression comparing several culture media conditions. All comparisons are relative to primed media (a* compared to a and b* compared to b. * p < 0.1, ** p < 0.01 and *** p < 0.001). Data are represented as mean ± SD.