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  • br Materials and methods br Resource

    2018-11-12


    Materials and methods
    Resource table Resource details This line was derived from a 62-year-old female with familial hypertrophic cardiomyopathy (HCM). HCM is a genetic heart disease with a prevalence of up to 1 in 200 people and is characterised by unexplained hypertrophy of the left ventricle, resulting in a maximum left ventricular wall thickness≥15mm (Semsarian et al., 2015). A genetic basis of HCM is identified in approximately 40% of cases; the majority of disease-causing variants are found in myosin-binding protein C (MYBPC3) and beta-myosin heavy chain (MYH7). Recent advances in next generation sequencing and induced pluripotent stem cell (iPSC) technology provide a unique opportunity to further explore the genetic causes of HCM and other inherited heart diseases (Bagnall et al., 2016; Ross et al., 2016). The patient has a maximum left ventricular wall thickness of 16mm (normal 7–10mm), was diagnosed at 56years of age and is 1 of 4 affected individuals in her family. Comprehensive genetic testing including a 46 cardiac gene panel, whole exome and genome sequencing was performed on DNA from multiple affected members of this family. A number of variants of interest were found, however no disease-causing variants in known HCM genes were identified. Rare variants of interest that segregate with disease in this family include a p.Glu847Lys variant in calcium channel, voltage-dependent, L-type, Alpha 1D Subunit (CACNA1D) and a p.Arg2Gln variant in ubiquinol-cytochrome c reductase binding protein (UQCRB). The iPSC line was generated from peripheral blood mononuclear StemRegenin 1 Supplier (PBMCs) isolated from whole blood. Non-integrative episomal vectors carrying reprogramming factors OCT4, SOX2, LIN28, KLF4 and L-MYC were used to reprogram patient PBMCs according to the established STEMCELL Technologies (Vancouver, CAN) protocol. Analysis of the iPSC line included DNA sequencing to confirm the identity of the line, PCR amplification to confirm the absence of episomal reprogramming vector expression, immunohistochemistry confirmation of pluripotency marker expression, trilineage differentiation potential and molecular karyotyping to ensure genome integrity (Fig. 1).
    Materials and methods
    Acknowledgments
    Resource table Resource details Peripheral blood CD34+ haematopoietic progenitor (HP) cells were collected from a 32year old male patient who had coinherited a homozygous β°-thalassemia deletion mutation at codon 41/42 (-TCTT) with a heterozygous α-thalassemia 4.2 deletion. The peripheral blood CD34+ HPs were then reprogrammed into a human induced pluripotent stem cell line 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 MUi009-A cell line (Fig. 1B and Supplementary fig. 1). Coinheritance of the homozygous codon 41/42 (-TCTT) deletion and heterozygous α-thalassemia 4.2kb deletion in the MUi009-A were confirmed by direct sequencing and multiplex Gap-polymerase chain reaction respectively (Fig. 1C and D). The cells expressed the pluripotency markers NANOG, OCT4, TDGF1, DNMT3B, GABRB3, GDF3, and SOX2 in the same range as MU011.A-hiPSC, a control iPSC line described previously (Fig. 1E) (Tangprasittipap et al., 2015). Expression of the pluripotency markers, OCT4, NANOG, TRA1-60, and SSEA4 at the protein level was confirmed by immunofluorescence staining (Fig. 1F). 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. 1G). In addition, the iPSCs presented a normal male karyotype (46, XY) (Fig. 1H). DNA fingerprinting of the MUi009-A cell line is identical to their parental cells (Supplementary table 1) and mycoplasma infection was negative by PCR (Supplementary fig. 2).