Teratoma formation frequency and teratoma size of late-passage Ser-iPS cells are similar to MEF-iPS cells.

<p>Frequency and size of teratomas of late-passage Ser-iPS cells in B6 mice. Late-passage MEF-iPS cells and ES cells are shown as controls. Average values of Ser-iPS cells (clones 1, 2 and 3) and MEF-iPS cells (clones 1 and 2) are as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106110#pone-0106110-g001" target="_blank">Figure 1D</a>. Late-passage: p35–38. **P<0.01. There is no statistical difference in teratoma size. Bars represent mean ± standard deviation.</p

Immunogenicity of syngeneic Ser-iPS cells.

<p>(A) CD3 T cell infiltration in Ser-iPS cell teratomas in B6 mice by immunohistochemistry. Teratomas of MEF-iPS cells and ES cells are shown as controls. Ser-iPS cells (OSKM, clone 1), MEF-iPS cells (OSK, clone 2) and ES cells (JM8) of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106110#pone-0106110-g001" target="_blank">Figure 1C</a>. Images are representative for all Ser-iPS cells and MEF-iPS cells analyzed. CD3 positive T cells, brown. Scale bar, 200 µm. (B) Tissue damage and necrosis are detected in Ser-iPS cell teratomas by HE staining. MEF-iPS cell and ES cell teratomas, controls as in (A). Scale bar, 200 µm. (C) Expression of T cell (CD3), B cell (B220) and DC (CD11c) genes in Ser-iPS cell teratomas by qRT-PCR analysis. MEF-iPS cell and ES cell teratomas, controls as in (A). Average values of Ser-iPS cells (clones 1, 2 and 3) and MEF-iPS cells (clones 1 and 2) are as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106110#pone-0106110-g001" target="_blank">Figure 1D</a>. Spleen is shown as a further control. The number of B6 teratomas analyzed: Ser-iPS cells, n = 19; MEF-iPS cells, n = 8; ES cells, n = 10. Relative gene expression was normalized to β-actin. Average mRNA level in ES cell teratomas was arbitrarily set to 1. All Ser-iPS cells and MEF-iPS cells are passage 9–15 (early-passage). *P<0.05. Bars represent mean ± standard deviation.</p

Ser-iPS cells form more teratomas than MEF-iPS cells.

<p>(A) Schematic representation of Ser-iPS cell generation from B6 Sertoli cells by Oct4, Sox2 and Klf4 transfection with and without c-Myc (OSKM or OSK) and teratoma assay in B6 mice. (B) AP staining and analysis of pluripotency markers (Oct4 and SSEA1, red). DAPI staining, blue. Scale bars, 200 µm and 35 µm, respectively. AP staining, Ser-iPS cells (OSKM, clone 1), MEF-iPS cells (OSKM, clone 1) and ES cells (JM8). Oct4 and SSEA1 staining, Ser-iPS cells (OSK, clone 3), MEF-iPS cells (OSK, clone 2) and ES cells (JM8). iPS cell data are representative of all Ser-iPS cells and MEF-iPS cells analyzed. (C) Gene expression in undifferentiated and differentiated Ser-iPS cells (EB assays) determined by qRT-PCR is shown in heatmap format. Expression in undifferentiated ES cells (JM8) was arbitrarily set to 1. Ser-iPS 1 refers to 4F iPS cells (OSKM, clone 1) and Ser-iPS 2 and 3 to 3F iPS cells (OSK, clones 2 and 3); MEF-iPS 1 and 2 refer to 4F and 3F iPS cells (OSKM, clone 1 and OSK, clone 2, respectively). (D) Number and size of teratomas of Ser-iPS cells in B6 mice. B6 MEF-iPS cells and B6 ES cells are shown as controls. Average values of Ser-iPS cells (clones 1, 2 and 3) and MEF-iPS cells (clones 1 and 2) are shown. All Ser-iPS cells and MEF-iPS cells are passage 9–15 (early-passage). *P<0.05, **P<0.01. Bars represent mean ± standard deviation.</p

EBs from Ser-iPS cells exhibit reduced CD4 T cell stimulation potential <i>in</i><i>vitro</i>.

<p>(A) Schematic representation of T cell proliferation assay <i>in</i><i>vitro</i>. iPS cells or ES cells were induced to differentiate in EB assays (14 days). EBs were co-cultured with splenic CD4 T cells to assess T cell proliferation. (B) Proliferation of CFSE labeled CD4 T cells co-cultured with Ser-iPS cells (day 12–17) in T cell medium. MEF-iPS cells and ES cells were used as controls. Ser-iPS cells (OSKM, clone 1), MEF-iPS cells (OSKM, clone 1) and ES cells (JM8) of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106110#pone-0106110-g001" target="_blank">Figure 1C</a>. PMA and ionomycin activated T cells, positive control. T cell proliferation refers to the percentage of dividing T cells after 5 days of co-culture. (C) T cell proliferation data after 5 days of co-culture of (B) (n = 3). Average values of Ser-iPS cells (clones 1, 2 and 3) and MEF-iPS cells (clones 1 and 2) are as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106110#pone-0106110-g001" target="_blank">Figure 1D</a>. All Ser-iPS cells and MEF-iPS cells are passage 9–15 (early-passage). *P<0.05; ***P<0.001. Bars represent mean ± standard deviation.</p