Research Article

Definitive Endoderm Formation from Plucked Human Hair-Derived Induced Pluripotent Stem Cells and SK Channel Regulation

Figure 1

Generation of human induced pluripotent stem cells. (a) Scheme of reprogramming keratinocytes from human plucked hair into induced pluripotent stem cells (hiPSCs). Bright field microscopy images from outgrowing keratinocytes, in detail, the outer root sheath of a plucked human hair. Keratinocytes were infected with a lentiviral construct containing the reprogramming factors OCT4, SOX2, KlF4, and cMyc on two subsequent days. On the following day keratinocytes were transferred onto a monolayer of irradiated REFs (rat embryonic fibroblasts) and after some days small hiPSC colonies could be detected. For later passaging hiPSCs were cultured, under feeder-free conditions, on Matrigel-coated dishes in FTDA medium. Scale bars are 20  m. (b) hiPSCs express the nuclear factors OCT4, SOX2, and NANOG as well as the pluripotent surface markers SSEA4, TRA-1-60, and TRA-1-81 (all red). Scale bars as indicated. (c) hiPSCs used in the present study are capable of differentiating into cells of all 3-germ layers represented by -tubulin (beta-tubulin 3 in green, neurons—ectoderm), actinin (alpha-actinin in green, myocytes—mesoderm), and AFP (alpha-fetoprotein in green, liver cells—endoderm). Nuclei are stained with DAPI in blue. (d) Transcript levels of pluripotent markers such as OCT4, SOX2, and NANOG were highly expressed whereas markers for definitive endoderm (SOX17 and FOXA2) and markers for pancreatic progenitor cells (PTFA1 and PDX1) were not expressed at all. (e) Polymerase chain reaction to detect the STEMCCA cassette in iPS cell subclones before and after treatment with recombinant Cre protein (actin band ~480 bp, STEMCCA band ~400 bp).
360573.fig.001a
(a)
360573.fig.001b
(b)
360573.fig.001c
(c)
360573.fig.001d
(d)
360573.fig.001e
(e)