Fig. 2

Establishment of proliferative trophoblast cells from hiPSC-induced cysts. a Analysis of pluripotency and trophoblast gene expression by qRT-PCR in hiPSCs and cysts. Cysts were collected on day 46, and hiPSCs were grown on laminin-coated dishes for 3 days. Expression levels were calculated relative to those of GAPDH and normalized to those of control hiPSCs. Values are the means ± SEMs (n = 4). Significance of differences was determined by Student’s t tests. *P < 0.05 versus the hiPSC group. b Immunofluorescence images of a representative cyst stained for E-cadherin. Scale bar = 100 μm (two left images) and 20 μm (two right enlarged images). Similar results were obtained for three independent samples from separate dishes. c Stereomicroscope images of separated cysts from 4-mm micromesh culture on day 46. Left: scale bar = 1 mm. Right: nuclei were stained with DAPI (blue). Scale bar = 20 μm. d Phase-contrast images of time-lapse monitoring of a single cyst on days 1–5. The cyst was isolated from the micromesh culture and transferred to 24-well plates on day 0. Cells derived from cysts in the area within the red-dotted box were recovered for reseeding of trophoblast cells. Scale bar = 100 μm. e Phase-contrast images showing the growth of trophoblast cells derived from cysts after 14 passages. Scale bar = 100 μm. f Proliferation rate of trophoblast cells derived from cysts. Left, population doubling levels (PDLs) versus days of culture; middle, PDL/day versus passage number; and right, doubling time (h) versus passage number