Hepatic Differentiation of Murine Disease-Specific Induced Pluripotent Stem Cells Allows Disease Modelling In Vitro

Figure 5

PiZ iPS cells. (a) iPSCs generated from ear fibroblasts isolated from PiZ mice using lentiviral vectors expressing human OCT4, KLF4, and SOX2. (b) Staining for alkaline phosphatase expression of PiZ-iPSCs. (c) Karyotype analysis of DAPI stained metaphase spreads. (d) qRT PCR analysis for endogenous expression of pluripotency markers Oct4, Nanog and Sox2 in PiZ-iPS and OG2 ES. (e)–(g) PiZ iPS were able to form tissues from all three germ layers when transplanted subcutaneously into NOD/SCID mice. (h) qRT PCR analysis for hepatic marker genes Afp, Alb, Ck18, Abcc2, Ttr, and Hnf4α. Undifferentiated cells served as negative control, in which no hepatic gene expression was determined until cycle 45 (↓). (i) Functional analysis of iPSC-derived hepatic cells: albumin secretion detected by ELISA, urea production, and CYP450 activity assay. (j)-(k) Day 5 + 9 + 14 hepatic differentiated PiZ-iPSCs transduced on day 5 + 9 + 3 with a lentiviral Alb-eGFP reporter construct (l) FACS-analysis on day 5 + 9 + 14 of hepatic differentiation for Alb-GFP positive cells. (m) qRT PCR analysis for expression of human SERPINA1 and Albumin on d 5 + 9 + 14 of cytokine-based differentiation, compared to undifferentiated PiZ-iPSCs (grey bars).