Fig. 4

Influence of long-term, matrix-free suspension culture on pluripotency and genetic stability. A Exemplary flow cytometry analysis plots for surface markers associated with an undifferentiated state TRA-1-60, SSEA-3 and SSEA-4 as well as transcriptions factors OCT-3/4 and NANOG and proliferation marker KI67. Cells harvested at process endpoints of D3 (5 passages; 15 days) and D4 (5 passages; 20 days) were compared to monolayer-derived cells used for inoculation of the processes. B Representative immunocytological stainings of single cell-dissociated, seeded hPSC aggregates derived after 5 passages in D4 (20 days) stained for markers TRA-1-60, OCT-3/4, SSEA-4 and SOX2 (scale bar = 100 µm). C Undirected differentiation of D4 process-derived aggregates after 5 passages revealed expression of markers representing the three germ layers ectoderm (based on TUBB3), endoderm (based on SOX17 and FOXA2) and mesoderm (based on Vimentin). Scale bar = 100 µm. D Exemplary flow cytometry analysis plots of cardiomyocyte-specific markers NKX2.5, MHC, SA and cTNT after directed differentiation of D4-derived cells at the end of passage 5. E Karyotype of cells cultivated for 5 passages in D4 approach (20 days)