To confirm the pluripotency of the MIXL eGFP

To confirm the pluripotency of the MIXL1-eGFP hESCs, the expression of several pluripotency markers was analyzed by quantitative real-time PCR as well as immunocytochemsitry. Endogenous expression of NANOG, OCT4 and SOX2 was determined at the mRNA level by real-time PCR (Fig. 1C). Protein expression of OCT4, NANOG, and SOX2 was assayed by immunocytochemsitry (Fig. 1D). Three germ-layer differentiation ability was demonstrated by spontaneous in vitro differentiation of embryoid bodies with subsequent replating and immunocytochemical detection of smooth muscle cycloastragenol (SMA) for mesoderm, alpha-feto protein (AFP) for endoderm and beta-III tubulin (TUJI) for ectoderm (Fig. 1G). Correlation of eGFP expression with endogenous MIXL1 expression was confirmed by flow cytometry and real-time PCR during specific in vitro differentiation to the 3-germ layers (mesoderm, endoderm and neuroectoderm) (Fig. 1H & I). Expression was dynamic and restricted to specific times during differentiation that correlate with the emergence of mesodermal or endodermal precursors. Immunocytochemistry corroborated the flow cytometry and PCR results (Fig. 1J and data not shown).
Materials and methods
Acknowledgment This work was supported by the NY State Department of Health/Empire State Stem Cell Board (NYSTEM) grant (C024176) and the Black Family Stem Cell Institute at the Icahn School of Medicine at Mount Sinai. SLD and CS conceived the study. VA and SLD performed the experiments. VA, SLD and CS wrote the manuscript.
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Resource details Skin fibroblasts were reprogrammed using the CytoTune®-iPS 2.0 Sendai Reprogramming Kit (Thermo Fisher Scientific, USA), which contains reprogramming vectors with the four Yamanaka factors, Oct3/4, Sox2, Klf4 and c-Myc. These factors have been repeatedly shown to be sufficient for efficient reprogramming (Lieu et al., 2013; Takahashi et al., 2007). The identity of derived human iPS cell lines was confirmed by immunohistochemistry, using the following antibodies for pluripotency markers, TRA-1-60, Sox2, Oct3/4 and SSEA4 (Fig. 1A). To confirm trilineage differentiation potential, in vitro embryonic body (EB) formation assay was performed (Fig. 3A). Spontaneous differentiation induced the transcription of the following genes: AFP (endoderm), MSX1 (mesoderm) and Pax6 (ectoderm) (Fig. 3B). Additionally, the formation of the three germ layers was confirmed at the protein level by immunocytochemistry, which showed the expression of Nestin, TUJ1, SMA and AFP (Fig. 3C). Ploidy of the derived iPS cell lines was analyzed by low-pass whole-genome sequencing (Fig. 2) (Wells et al., 2014).
Materials and methods
Resource table
Resource details Urine epithelial cells underwent the reprogramming procedure using the CytoTune®-iPS 2.0 Sendai Reprogramming Kit (Thermo Fisher Scientific, USA) containing reprogramming vectors with the four Yamanaka factors, Oct4, Sox2, Klf4 and c-Myc. These factors have been repeatedly shown to be sufficient for very efficient reprogramming (Lieu et al. 2013; Takahashi et al. 2007). The identity of derived human iPS cell lines was confirmed by immunocytochemistry, using the following antibodies for pluripotency markers, TRA-1-60, Sox2, Oct3/4 and SSEA4 (Fig. 1a). Sendai virus transgenes were undetectable from passage 7 (Fig. 1c). To confirm trilineage differentiation potential, in vitro embryonic body (EB) formation assay was performed (Fig. 3a). Spontaneous differentiation induced the transcription of the following genes: AFP (endoderm), MSX1 (mesoderm) and PAX6 (ectoderm) (Fig. 3b). Additionally, the formation of the three germ layers was confirmed at the protein level by immunocytochemistry, which showed the expression of Nestin, Tuj1, SMA and AFP (Fig. 3c). Ploidy of the 3 derived iPS cell lines was analyzed by low-pass whole-genome sequencing (Fig. 2) (Wells et al. 2014).
Materials and methods