Effects of LiCl and Shh on paraxial mesodermal differentiation of mouse iPS cells.

<p>(<b>A</b>) A scheme for paraxial mesodermal differentiation of mouse iPS cells using various combinations of LiCl and Shh from D3 to D6. The cells were analyzed on D6 in (B–F). The cultures also contained Activin A (5 ng/ml), BMP4 (10 ng/ml), and IGF-1 (10 ng/ml). (<b>B</b>) Total number of mouse iPS cells after differentiation with the protocol shown in (<b>A</b>) (n = 3). Both LiCl and Shh enhanced proliferation of mouse iPS cells. (<b>C</b>) The expression of PDGFR-α in mouse iPS cells after differentiation. The percentage indicates the proportion of PDGFR-α⁺ cells (n = 3). LiCl prominently induced generation of PDGFR-α⁺ cells. (<b>D</b>) Gene expression profiles of PDGFR-α⁺ cultured as shown in (<b>A</b>) (n = 3). LiCl treatment enhanced expression of <i>Myf-5</i>. (<b>E</b>) Differentiation of various types of mouse iPS cell clones into PDGFR-α⁺ cells in serum-free induction culture. 3F/TTF: iPS cells induced by 3 factors (Oct3/4, Sox2, and Klf4) using retroviral transduction from tail-tip fibroblasts. 4F/MEF and 4F-Plasmid/MEF: iPS cells induced by 4 factors (Oct3/4, Sox2, Klf4, and c-Myc) from MEFs using retroviral transduction (4F/MEF), or plasmid transduction (4F-Plasmid/MEF), respectively. The percentage indicates the proportion of PDGFR-α⁺ cells (n = 3). (<b>F</b>) Expression profiles of 2 mesodermal markers, PDGFR-α and Flk-1, after mouse iPS cell differentiation. Up to 90% of PDGFR-α⁺ cells cultured under these conditions were PDGFR-α⁺/Flk-1⁻ paraxial progenitors. PDGFR-α⁺/Flk-1⁺ and PDGFR-α^−/Flk-1⁺ populations were barely induced under these conditions. *p<0.05, **p<0.01 between selected two samples.