Purification of small molecule‐induced cardiomyocytes from human induced pluripotent stem cells using a reporter system

Abstract

In order to realize the practical use of human pluripotent stem cell (hPSC)-derived cardiomyocytes for the purpose of clinical use or cardiovascular research, the generation of large numbers of highly purified cardiomyocytes should be achieved. Here, we show an efficient method for cardiac differentiation of human induced pluripotent stem cells (hiPSCs) in chemically defined conditions and purification of hiPSC-derived cardiomyocytes using a reporter system. Regulation of the Wnt/β-catenin signaling pathway is implicated in the induction of the cardiac differentiation of hPSCs. We increased cardiac differentiation efficiency of hiPSCs in chemically defined conditions through combined treatment with XAV939, a tankyrase inhibitor and IWP2, a porcupine inhibitor and optimized concentrations. Although cardiac differentiation efficiency was high (>80%), it was difficult to suppress differentiation into non-cardiac cells, Therefore, we applied a lentiviral reporter system, wherein green fluorescence protein (GFP) and Zeocin-resistant gene are driven by promoter activation of a gene (TNNT2) encoding cardiac troponin T (cTnT), a cardiac-specific protein, to exclude non-cardiomyocytes from differentiated cell populations. We transduced this reporter construct into differentiated cells using a lentiviral vector and then obtained highly purified hiPSC-derived cardiomyocytes by treatment with the lowest effective dose of Zeocin. We significantly increased transgenic efficiency through manipulation of the cells in which the differentiated cells were simultaneously infected with virus and re-plated after single-cell dissociation. Purified cells specifically expressed GFP, cTnT, displayed typical properties of cardiomyocytes. This study provides an efficient strategy for obtaining large quantities of highly purified hPSC-derived cardiomyocytes for application in regenerative medicine and biomedical research.

Comparative Proteomic Analysis Reveals the Upregulation of Ketogenesis in Cardiomyocytes Differentiated from Induced Pluripotent Stem Cells

“…In addition, we analyzed the marker gene expression profiles during cardiac differentiation using quantitative PCR analysis in each stage (Figure C). The expression of OCT4 and NANOG was highest in hiPSCs, whereas the highest levels of ISL1 and MESP1 were observed in the CPC stage . In addition, the mRNA expression levels of cTnT and MLC2A were maximum in CMs.…”

“…To characterize the molecular mechanisms and identify novel regulators of cardiac development involved in the differentiation of hiPSCs to CMs, we quantified the proteome alteration of hiPSCs and differentiated cells, namely, CPCs and beating CMs (Figure A). The differentiation of hiPSCs to CMs were induced as previously reported by sequential treatment with small molecules such as CHIR99021 for GSK‐3 inhibition, XAV939 for tankyrase inhibition, and IWP2 for WNT/β‐catenin signaling inhibition. To confirm the efficiency of differentiation hiPSCs to CMs, we stained the cells with DAPI and cTNT for the nucleus and muscle, respectively (Figure B).…”

“…The cardiomyogenic differentiation efficiency was also confirmed by comparative proteomic data (Table S1, Supporting Information). Protein ratios of two markers were monitored, OCT4 for hiPSCs and MYL7 for CMs . OCT4 levels decreased by 0.33 and 0.14‐fold in CPCs and CMs, respectively.…”

“…Additionally, many studies reported that hPSC‐derived cardiomyocytes commonly show phenotypes of fetal cardiomyocytes in morphological or functional aspects . As we described, we used small molecule‐induced differentiation protocol which sequentially regulates Wnt signaling pathway and the differentiated cardiomyocytes showed normal physiological function and cardiomyocyte‐specific protein expression . Therefore, we considered that there would be a few proteomic phenotypical differences between cardiomyocytes differentiated by different protocols and we conducted the experiments.…”

“…hiPSCs (ATCC‐BYS0112) were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). Differentiation of hiPSCs to CMs was induced as previously reported . hiPSCs were cultured on 10 µg mL −1 mitomycin‐C‐treated mouse embryonic fibroblasts and were maintained in undifferentiated ESC culture medium (20% knockout serum replacement, 10 ng mL −1 basic fibroblast growth factor (FGF), 0.1 m m β‐mercaptoethanol, 1% penicillin/streptomycin, 0.1 m m non‐essential amino acids in Dulbecco's modified Eagle medium (DMEM)/F‐12) (Life Technologies Carlsbad, CA, USA) for 4–5 days.…”