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. 2016 Mar 17;6(4):592–606. doi: 10.1016/j.stemcr.2016.02.009

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© 2016 The Authors

This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

PMC Copyright notice

Establishment of Sarcoma-Derived iPSCs and Differentiation of Sarcoma iPSCs into Osteogenic Cells

(A) iPSCs-like cells were established from sarcoma cells by introducing reprogramming transcription factors. Scale bars, 200 μm.

(B) qRT-PCR revealed that the expression levels of pluripotency-related genes in sarcoma-derived iPSC-like cells were equivalent to those of ESCs. Data are presented as means ± SD (three technical replicates). The expression level of ESCs was set to 1.

(C) Bisulfite sequencing analyses revealed that the Nanog promoter and the Oct3/4 distal enhancer region are demethylated in sarcoma-derived iPSC-like cells. White and black circles indicate non-methylated and methylated cytosine at CpG sites, respectively.

(D) Sarcoma iPSCs gave rise to teratomas consisting of ectodermal, mesodermal, and endodermal tissue in the subcutaneous tissue of immunocompromised mice. Scale bars, 50 μm.

(E) Schematic illustrations of in vitro osteogenic differentiation.

(F) qRT-PCR analysis of osteogenic differentiation-related genes. Wild-type ESCs (V6.5), EWS-FLI1-inducible ESCs (Rosa-M2rtTA/Rosa::tetO-EWS-FLI1), and two independent fibroblast-derived iPSCs with Rosa-M2rtTA/Rosa::tetO-EWS-FLI1 alleles were used as controls in the osteogenic differentiation experiments. Sarcoma-derived iPSCs and control ESCs/iPSCs on day 0 and day 17 during osteogenic differentiation were examined for the expression of osteogenic differentiation-related genes. The mean ± SD is shown (three technical replicates per n; n = 3 biological replicates). The mean expression level of ESCs on day 17 was set to 1.

(G) Alizarin red staining revealed extracellular calcium deposits stained in blight reddish orange (day 28 after the induction of osteogenic differentiation). Scale bars, 20 μm.

(H) Histological analysis of an osteogenic region with osteoid production in teratomas. Ki67 immunohistochemistry revealed that osteoid-producing cells derived from sarcoma iPSCs have higher proliferative activities than those derived from control ESCs/iPSCs. Scale bars, 50 μm.

(I) Ki67 positive ratio of osteogenic regions in teratomas derived from sarcoma iPSCs or control ESCs/iPSCs. The mean ± SD of six independent osteogenic regions in two independent sarcoma iPSCs teratomas, five independent osteogenic regions in the control iPSC teratomas, and nine independent osteogenic regions in two independent ESC teratomas are shown. The ANOVA test was used for the statistical analysis. Sarcoma iPSCs vs control iPSCs, p < 0.05; sarcoma iPSCs vs control ESCs, p < 0.01; control ESCs vs control iPSCs, p > 0.05.