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Fig. 4 | Stem Cell Research & Therapy

Fig. 4

From: Restoration of keratinocytic phenotypes in autonomous trisomy-rescued cells

Fig. 4

Purification of iPSC-derived keratinocytes, based on differential reactions to dispase. a Phase-contrast photomicrographs of keratinocytes derived from D21-KC#1 at passage 5. Two types of colonies could be observed: stratified keratinocyte colonies (left) and non-keratinocyte-like cell colonies (right). Scale bars, 200 μm. b Immunocytochemical analysis of D21-KC#1 colony with epithelial markers, i.e., KRT14 and KRT10. Scale bars, 200 μm. c Selection of keratinocytes with dispase digestion. Non-keratinocytes (non-KC) detached earlier than keratinocytes with dispase treatment. Non-keratinocytes and feeder cells were removed at each passage. Keratinocytes were then passaged. d Phase-contrast photomicrographs of D21-KC#1 during the dispase selection. There were insular colonies, which were formed by stratified epithelial cells, and another area contained non-keratinocyte-like cells. Dispase-based selection at each passage caused non-keratinocyte-like cells to decrease. Scale bars, 500 μm. e Real-time qPCR analysis of KRT14 at each passage. Values are shown as means ± SD from two or three independent experiments. f Real-time qPCR analysis of TP63 at each passage. Values are shown as means ± SD from two or three independent experiments. g Real-time qPCR analysis of Involucrin at each passage. Values are shown as means ± SD from two or three independent experiments. h Immunocytochemical analysis of epithelial markers, i.e., KRT14, KRT10, involucrin, and loricrin after colony isolations (T21-KC) at passage 7 and dispase-based selection (D21-KC#1) at passage 13. Scale bars, 100 μm. i Epithelial sheets of keratinocyte-like cells derived from D21-iPSC#1 at passage 8. Cell sheets were harvested by dispase treatment. Scale bar, 1 cm

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