br Resource table Resource details Episomal plasmids harbori

Resource table. Resource details Episomal plasmids harboring the transgenes OCT4, SOX2, NANOG, LIN28, SV40LT, KLF4 and c-MYC were introduced into human mesenchymal stromal myeloperoxidase derived from the fetal femur of the age 55days post-conception (fetal hMSCs) by nucleofection. In addition, a combination of TGFβ receptor inhibitor — A-83-01, MEK inhibitor — PD325901, GSK3β inhibitor — CHIR99021 and ROCK inhibitor — HA-100 was used to enhance the efficiency of reprogramming (Yu et al., 2011). Two iPS-cell lines, MSC-iPS(fetal)#1 and MSC-iPS(fetal)#2 were characterized. The expression of pluripotency-associated markers was confirmed by immunofluorescence staining and microarray-based transcriptome profiling (Fig. 1A and B). The transcriptomes of MSC-iPS(fetal)#1 and MSC-iPS(fetal)#2 were more comparable to the transcriptome of the human embryonic stem cell line H1 (hESC H1) than to that of their parental cells with Pearson correlations of 0.953 and 0.952 respectively (Fig. 1C and D). In addition, the origin of the cell-types as well as the absence of episomal plasmids in the generated iPS cells was confirmed by PCR (Fig. 1D and E). Chromosome analyses of iPS(fetal)#1 and MSC-iPS(fetal)#2 revealed a normal male karyotype for both iPS-cell lines (Fig. 2A). Pluripotency in vitro was also confirmed by embryoid body-based assays (Fig. 2B). Both iPS-cell lines showed high transcriptome similarity with hESC H1 compared to the parental cells based on the in silico PluriTest (Fig. 2C) (Müller et al., 2011).
Materials and methods
Acknowledgments JA acknowledges support from the Medical Faculty, Heinrich-Heine-University, Düsseldorf, Germany and the BMBF grant number 01GN1005. RO is supported by grants from the BBSRC (LO21072/1) and MRC (MR/K026682/1). We thank Prof David Wilson, University of Southampton for access to fetal tissue and Dr Kelvin Cheung, University of Southampton for derivation of fetal femur explant cultures.
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Resource details
We generated KCL038 clinical grade hESC line following protocols, established previously (Ilic et al., 2012; Stephenson et al., 2012), and now adapted to cGMP conditions. The expression of the pluripotency markers was tested after freeze/thaw cycle (Fig. 1). Differentiation potential into three germ layers was verified in vitro (Fig. 2). Molecular karyotyping identified deletion of approximately 683kb from band q13.2 in the long arm of chromosome 5 (69,705,561 – 70,388,844). The deletion represents benign copy number variant.
Materials and methods
Author disclosure statement
Acknowledgments This work was supported by the UK Medical Research Council grants G0701172 and G0801061. We thank Dr Yacoub Khalaf, Director of the Assisted Conception Unit of Guy\'s and St Thomas\' NHS Foundation Trust and his staff for supporting the research program. We are especially indebted to Prof Peter Braude and patients who donated embryos.
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Resource details
We generated KCL037 clinical grade hESC line following protocols established previously (Ilic et al., 2012; Stephenson et al., 2012), and now adapted to cGMP conditions. The expression of the pluripotency markers was tested after freeze/thaw cycle (Fig. 1). Differentiation potential into three germ layers was verified in vitro (Fig. 2). Whole-genome single nucleotide polymorphism (SNP) array analysis revealed a 542kb gain on chromosome 18q23 in KCL037 containing two coding genes, SALL3 and ATP9B (Canham et al., 2015). A smaller duplication (nsv577794) covering the same two genes has been reported previously (Cooper et al., 2011).
Materials and methods
Author disclosure statement
Acknowledgments This work was supported by the UK Medical Research Council grants G0701172 and G0801061. We thank Dr. Yacoub Khalaf, Director of the Assisted Conception Unit of Guy\'s and St. Thomas\' NHS Foundation Trust and his staff for supporting the research program. We are especially indebted to Prof Peter Braude and patients who donated embryos.