Fig. 5

Characterisation of the hiPSCs following expansion in the PBS MINI 0.1 bioreactor. a-b Immunocytochemistry staining for pluripotency markers OCT4, SOX2, TRA-1-60 and SSEA-4 of cell aggregates harvested from the PBS MINI 0.1 bioreactor at day 7 post-expansion (a; scale bars = 100 μm), and of cells which were dissociated with Accutase and replated on 2D tissue culture plates (b; scale bars = 100 μm). c Immunocytochemistry staining of cells harvested from the bioreactor and left to form EBs for 5 weeks. The cells were stained for germ layer markers TUJ1 (ectoderm), α-SMA (mesoderm) and SOX17 (endoderm; scale bars = 50 μm). d-e Immunocytochemistry staining of cells harvested from the bioreactor and differentiated to d cardiomyocytes and e neural progenitors (scale bars = 50 μm). Cardiomyocytes were stained for cTNT, while neural progenitors were stained for PAX6 and NESTIN. f Flow cytometry analysis of cells harvested from the bioreactor after 7 days of expansion in the PBS MINI 0.1 bioreactor. The cells were labelled for pluripotency (OCT4 and TRA-1-60) and early differentiation (SSEA-1) markers. g qRT-PCR analysis of cells prior to (day 0) and following (day 7) expansion in the PBS MINI 0.1 bioreactor. The cells were tested for pluripotency (OCT4 and NANOG) and germ layer (SOX1, T/BRACHYURY and SOX17, representing ectoderm, mesoderm and endoderm, respectively) marker expression. RNA levels are relative to expression of GAPDH and were computed as 2^–ΔCT