Metastable Reprogramming State of Single Transcription Factor-Derived Induced Hepatocyte-Like Cells

Figure 1

Generation of integration-free iHeps using Hnf1a. (a) Schematic diagram depicting the procedure of e-iHep generation using an episomal vector encoding Hnf1a. (b) Gene expression patterns of hepatocyte- and fibroblast-specific markers in e-iHeps and r-iHeps were analyzed by RT-PCR. (c) Fluorescence microscopy images of e-iHeps immunostained with antibodies raised against albumin, Aat, CK18, and E-cadherin. Scale bars: 100 μm. (d) Flow cytometry analysis describing the percentage of albumin (-axis) and E-cadherin (-axis) double-positive cells on day 30 after transfection. (e) Functional analysis of e-iHeps including Periodic acid-Schiff (PAS) staining, intake of acetylated low-density lipoprotein (Ac-LDL), Oil-red-O staining, and indocyanine green (ICG) uptake. Scale bars: 100 μm. (f and g) Both albumin secretion (f) and urea production (g) were determined in e-iHeps. MEFs and primary hepatocytes were used as negative and positive controls, respectively. Error bars indicate the standard deviation of triplicate values. and . (h) Expression levels of CYP450 genes in e-iHeps were analyzed by qPCR upon treatment of CYP inducers (3-methylcholanthrene, rifampicin, and dexamethasone). Expression levels were normalized to those of MEFs. Error bars indicate the standard deviation of triplicate values. , , and .

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