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Fig. 3 | BMC Biology

Fig. 3

From: Hmga2 is necessary for Otx2-dependent exit of embryonic stem cells from the pluripotent ground state

Fig. 3

Induced pluripotent stem cells (iPSCs) derived from Hmga2 knockout (KO) embryonic fibroblasts (MEFs) are unable to properly differentiate. a iPSC clones derived from wildtype (wt; #1 and #2) and Hmga2 KO (#1, #2, #3) MEFs and grown under 2i + leukemia inhibitory factor (LIF) conditions are induced to differentiate into neuroectoderm through serum-free embryoid body (SFEB) formation for 4 days. Sectioned SFEBs were stained for neuroectodermal (Sox1) and stemness (Oct4) markers. Scale bars: 100 μm. b Western blot analysis to evaluate the expression of the indicated proteins in 4 day-differentiated SFEBs derived from different wt and Hmga2 KO iPSC clones and qPCR analysis of the indicated mRNAs. qPCR data are reported as means of relative mRNA expression ± SEM of two biological replicates for each of the indicated wt or Hmga2 KO iPSC clones. c The expression of the endodermal marker Gata 4 was evaluated by qPCR in wt and KO Hmga2 iPSCs after 4 days of differentiation as SFEBs. qPCR data represent mean ± SD (n = 2) for each of the indicated wt or Hmga2 KO iPSC clones. d Different clones of wt and Hmga2 KO iPSCs were induced to differentiate into cardiomyocytes through the hanging drops method. Histogram reports the percentage of embryoid bodies showing rhythmically contracting areas (beating hearts) on day 13 of differentiation. e Immunofluorescence analysis showing the extent of the areas positive for the cardiac ventricular marker myosin Mlc2v or the stemness marker Oct4 in wt and Hmga2 KO iPSCs differentiated for 13 days through the hanging drop method. f The expression of the stemness markers was measured by qPCR analysis in wt and Hmga2 KO iPSCs differentiated for 13 days through the hanging drop method. qPCR data represent mean ± SD (n = 2) for each of the indicated wt or Hmga2 KO iPSC clones. g The expression of Oct4 and Nanog was analyzed by western blot in wt and Hmga2 KO clones induced to differentiate into EpiLCs. The relative expression of the indicated genes was measured by qPCR analysis in EpiLCs obtained from wt and Hmga2 KO iPSC clones. qPCR data represent means of independent experiments (n = 2; in the case of Oct6 n = 3) ± SD for each of the indicated wt or Hmga2 KO iPSC clones. h The relative expression of Pax6, Sox1, and Gata4 was measured by qPCR analysis in SFEBs obtained from wt and Hmga2 KO iPSC clones, stably transfected with an Hmga2 or GFP (Mock) under the control of β-actin gene promoter. qPCR data represent the mean of three independent experiments (n = 3) ± SEM for each of the indicated wt or Hmga2 KO iPSC clones. *P <0.05. The expression levels of endogenous Hmga2 (>) and of the transfected form (#) are reported in the western blot. i The relative expression of Fgf5 and Oct6 was measured by qPCR analysis in EpiLCs obtained from wt and Hmga2 KO iPSC clones, stably transfected with Hmga2 or GFP (Mock) under the control of β-actin gene promoter. qPCR data represent mean ± SEM of three independent experiments (n = 3) for each of the indicated wt or Hmga2 KO iPSC clones. *P <0.05. j Wt and Hmga2 KO iPSCs, transfected or not with Hmga2 cDNA, were induced to differentiate into cardiomyocytes through the hanging drops method. The histogram reports the percentage of embryoid bodies showing rhythmically contracting areas (beating hearts) on day 13 of differentiation

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