274 Chapter 10 Supplmenetary Figure 2 – Example of hiPSC Datasheet Data Sheet: hiPSC line generated by the RMCU (PLN foundation) Contact: Renee Maas (r.g.c.maas-4@umcutrecht.nl) ; PLN foundation (Annet@plnheart.org) Product Information hiPSC line Gender Genotype Phenotype Age at PBMC withdraw (2018) Sendai RMCU_PF6B_C13 M PLN R14del mutation Yes 61 Description The patient-derived human induced pluripotent stem cells (hiPSC) lines are generated from peripheral blood mononuclear cells (PBMCs). Reprogramming was performed using the Cytotune 2.0 Sendai Virus Kit (Thermofisher Scientific at the appropriate MOI (i.e., KOS MOI=5, hc-Myc MOI=5, hKlf4 MOI=3). The resulting hiPSC were selected using morphological criteria. One cell vial contains enough cells to start culturing in 12well format (± 0.5-1.0 x 106 iPSC). For thawing and culturing instructions please see supplementary protocol 1 and 2. hiPSC line Quality information Pluripotency testing: Expression of pluripotency markers are quantified using PCR and immunofluorescent stainings. The PF6B_C13 hiPSC line displays a healthy iPSC morphology (Figure 1A) and is positive for the pluripotency markers Alkaline phosphathase (AP), TRA-160, NANOG, OCT3/4 and SOX2 by immunofluorescent (IF) and immunohistochemistry (IHC) stainings (Table 1, Figure 1B, scale bar; 200 µM). The PF6B_C13 hiPSC line is positive for the pluripotency markers NANOG, MYC, SOX2, ZFP42, OCT4 by RT-qPCR (Table 1, Figure 1C). Table 1: Pluripotency expression confirmation Myocplasma testing: PF6B_C13 hiPSC line is regularly tested for sterility for mycoplasma every 1-2 months. Karyotyping: ddPCR analysis on the gDNA by the iCS-digital™ PSC test kit (Stem genomics) allows analysis of the most common genomic defects occurring in hiPSC lines. In the PF6B_C13 hiPSC line (Passage 21) no CMVs were detected (Page 2). hiPSC-CM differentiation: We obtained hiPSC derived cardiomyocytes (hiPSC-CMs) using the original RPMI/B27 monolayer directed differentiation protocol in this hiPSC line.1,2 6-8 µM CHIR09921 was used for cardiac differentiation. The most optimal CHIR concentration for the PF6B_C13 hiPSC line is displayed in table 2, -; no beating observed, +/-; some beating observed, +; over 50% beating observed, ++; over 70% beating observed, +++; over 90% beating observed. 1 Lian X. Robust cardiomyocyte differentiation from human pluripotent stem cells via temporal modulation of canonical wnt signaling. Proc. Natl. Acad. Sci. U S A. 2012;109:1848–1857. 2 Maas, R.G.C et al. Massive expansion and cryopreservation of functional human induced pluripotent stem cell-derived cardiomyocytes. Cell STAR protocols 2021 Feb 9;2(1):100334. doi: 10.1016/j.xpro.2021.100334. Shipping, storage, and handling The shipping will take place on dry ice. After shipment either thaw for immediate use or store in liquid nitrogen. Cells stored for more than 1 day at −80°C quickly lose viability. This product is stable for at least 6 months from the date of receiving when stored as directed. Biosafety Level 2 Users should treat all human and animal cells as potential pathogens. Wear protective clothing and eyewear. Practice appropriate disposal techniques for potentially pathogenic or biohazardous materials. Supplementary Protocols Supplementary protocol 1: hiPSC thawing protocol Supplementary protocol 2: hiPSC culturing protocol Supplementary protocol 3: hiPSC-CM differentiation protocol Terms and Conditions This product is property of the PLN foundation and for research/ pharma use only; not intended for human or animal diagnostic or therapeutic uses. SOX2 MYC NANOG EIF1AY OCT4 ZFP42 0 5 10 15 20 25 200 400 600 Pluripotency (RT-qPCR) Relative expression Normalized for RPL32 1= hiPSC-CFs, (F) Assay Genes Positive #P qPCR NANOG Yes 21 qPCR OCT4 Yes 21 qPCR ZFP42 Yes 21 qPCR MYC Yes 21 qPCR SOX2 Yes 21 qPCR EIF1AY Yes, Male 21 IF Tra-160 Yes 21 IF AP Yes 21 IHC NANOG Yes 21 IHC OCT3/4 Yes 21 CHIR ( µM) Differentiation #1 Differentiation #2 Differentiation #3 6 ++ + ++ 7 +++ +++ ++ 8 +++ +++ +++ Figure 1 – hiPSC characterization Hoechst NANOG AP Tra-160 OCT3/4 Hoechst A B C
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