Peripheral blood mononuclear cells PBMCs were obtained from

Peripheral blood mononuclear Phenyl sulfate (PBMCs) were obtained from a 43-year-old male with familial HCM. The patient fulfilled clinical criteria for HCM with a maximum left ventricular wall thickness of 28mm (normal 7–10mm) in the absence of loading conditions. The patient is heterozygous for the pathogenic variant p.Val698Ala in MYH7. This variant segregates with HCM in an additional 5 family members. Two additional members of this family carry this variant however remain clinically unaffected. Reprogramming of patient PBMCs from whole blood to iPSCs was performed according to an established protocol by STEMCELL Technologies (Vancouver, CAN). In brief, PBMCs were transfected by electroporation with non-integrative episomal vectors carrying reprogramming factors OCT4, SOX2, LIN28, KLF4 and L-MYC. iPSC colonies were picked 16days post transfection, cryostored at passage 5 and harvested for analysis at passage 12. The pluripotent nature of the iPSC line was demonstrated through immunofluorescence analysis of pluripotency marker expression and trilineage differentiation potential (Fig. 1A and B). The identity of the cell line was confirmed through identification of the pathogenic variant p.Val698Ala in MYH7 by Sanger sequencing DNA isolated from the iPSC culture (Fig. 1C). PCR amplification, using primers targeting episomal vector DNA, was utilised to verify the absence of the episomal reprogramming vector in the cell line (Fig. 1E). The genome was shown to be intact through molecular karyotyping (Fig. 1D and Supplementary Fig. 1).
Materials and methods
Acknowledgments
Resource table Resource details Peripheral blood CD34+ hematopoietic progenitor cells (HPs) were collected from a 31year old healthy woman. The peripheral blood CD34+ HPs were then reprogrammed into iPSC lines by co-electroporation of episomal plasmids expressing hOCT4, hSOX2, hL-MYC, hKLF4, hNANOG, hLIN28, a short hairpin RNA against TP53 and Epstein-Barr nuclear antigen-1 (EBNA-1) (Okita et al., 2011). The iPSC-like colonies were picked 21–28days post electroporation (Fig. 1A). The absence of the reprograming plasmids in the genome was verified by PCR after 10 passages in the MUi019-A cell line (Fig. 1B and Supplementary Fig. 1). The cells expressed the pluripotency markers DNMT3B, OCT4, NANOG, GABRB3, SOX2, TDGF1, and GDF3 the same range as MU011.A-hiPSC, a control iPSC line described previously (Tangprasittipap et al., 2015) (Fig. 1C). Expression of the pluripotency markers OCT4 NANOG, TRA1-60, and SSEA4 at the protein level was confirmed by immunofluorescence staining (Fig. 1D). In vitro differentiation followed by immunofluorescent staining analysis with the ectodermal marker beta-III-Tubulin (TUJ1), the mesodermal marker smooth muscle actin (SMA) and the endodermal marker alpha-fetoprotein (AFP) demonstrated the potential for differentiation into cells derived from all three germ layers (Fig. 1E). In addition, the iPSCs presented a normal female karyotype (46, XX) (Fig. 1F). DNA fingerprinting of MUi019-A cell line is identical to their parental cells (Supplementary Table 1) and mycoplasma infection was negative by PCR (Supplementary Fig. 2).
Materials and methods
Acknowledgements We would like to thank Dr. Keisuke Okita and Prof. Shinya Yamanaka for providing the plasmids. Furthermore, we would like to thank Prof. Duncan R. Smith for proof reading the article. This work was supported by the Thailand Research Fund (MRG-5980187), National Research Council of Thailand (NRCT; DPAR/2559-158), the Office of the Higher Education Commission and Mahidol University under the National Research University Initiative, a Research Chair Grant from the National Science and Technology Development Agency (NSTDA-P-11-00435) and Mahidol University, and European Union Seventh Framework Programme (PIAPP-GA-2012-324451-STEMMAD). Wasinee Wongkummool is supported by the Thailand Graduate Institute of Science and Technology (TGIST; TG-22-14-57-036D to WW) scholarship, NSTDA.