Efficient and scalable purification of cardiomyocytes from human embryonic and induced pluripotent stem cells by VCAM1 surface expression

Abstract

Rationale: Human embryonic and induced pluripotent stem cells (hESCs/hiPSCs) are promising cell sources for cardiac regenerative medicine. To realize hESC/hiPSC-based cardiac cell therapy, efficient induction, purification, and transplantation methods for cardiomyocytes are required. Though marker gene transduction or fluorescent-based purification methods have been reported, fast, efficient and scalable purification methods with no genetic modification are essential for clinical purpose but have not yet been established. In this study, we attempted to identify cell surface markers for cardiomyocytes derived from hESC/hiPSCs.

Method and result: We adopted a previously reported differentiation protocol for hESCs based on high density monolayer culture to hiPSCs with some modification. Cardiac troponin-T (TNNT2)-positive cardiomyocytes appeared robustly with 30-70% efficiency. Using this differentiation method, we screened 242 antibodies for human cell surface molecules to isolate cardiomyocytes derived from hiPSCs and identified anti-VCAM1 (Vascular cell adhesion molecule 1) antibody specifically marked cardiomyocytes. TNNT2-positive cells were detected at day 7-8 after induction and 80% of them became VCAM1-positive by day 11. Approximately 95-98% of VCAM1-positive cells at day 11 were positive for TNNT2. VCAM1 was exclusive with CD144 (endothelium), CD140b (pericytes) and TRA-1-60 (undifferentiated hESCs/hiPSCs). 95% of MACS-purified cells were positive for TNNT2. MACS purification yielded 5-10×10(5) VCAM1-positive cells from a single well of a six-well culture plate. Purified VCAM1-positive cells displayed molecular and functional features of cardiomyocytes. VCAM1 also specifically marked cardiomyocytes derived from other hESC or hiPSC lines.

Conclusion: We succeeded in efficiently inducing cardiomyocytes from hESCs/hiPSCs and identifying VCAM1 as a potent cell surface marker for robust, efficient and scalable purification of cardiomyocytes from hESC/hiPSCs. These findings would offer a valuable technological basis for hESC/hiPSC-based cell therapy.

Figure 1. Efficient induction of cardiomyocytes from hiPSCs.

(A) Schematic representation of cardiomyocyte induction protocol. (B) Expression profiles of cardiac troponin-T (TNNT2) during differentiation. Open line indicates isotype control and blue line indicates TNNT2 staining. (C–F) qPCR of differentiation stage-specific genes. (C) Pluripotency marker genes (NANOG, POU5F1); normalized to d0 expression (D) Mesodermal marker genes (T, MESP1); normalized to d2 expression (E) Cardiac progenitor genes (KDR, ISL1); normalized to d5 expression (F) Cardiac marker genes (NKX2-5, MYH6, MYH7, MYL7, MYL2); normalized to d11 expression. Mean±SD, n = 3.

Figure 2. Cell surface marker screening.

(A) Classification of cell surface markers and schematic dot plot diagrams for each class. (B) Exclusive expression pattern of TNNT2 and PDGFRβ (Class 3) (C) Concordant expression pattern of TNNT2 and VCAM1 (Class 4). (D) qPCR of TNNT2 and VCAM1. VCAM1 was expressed concordantly with the cardiac gene. (E–I) Representative flow-cytometry analysis. (E) Expression time course of TNNT2 and VCAM1. (F) VCAM1 expression in TNNT2-positive cells. (G–I) Dot plots of VCAM1 and TRA-1-60 (undifferentiated hESC/hiPSC marker) at day 0 and 11 (G), PDGFRβ (mesoderm/pericyte marker) (H), and VE-cadherin (endothelial marker) (I) at day 11. These markers were exclusively expressed.

Figure 3. Purification of cardiomyocytes with VCAM1.

(A) Representative flow-cytometry histogram of pre- and post-MACS purification with VCAM1 at d11. (B) Purity of VCAM1- and TNNT2-positive cells after MACS purification with VCAM1. 98.2±1.3% and 95.6±2.5% of sorted cells were positive for VCAM1 and TNNT2, respectively (n = 6). (C) qPCR for cardiac marker genes. Open bars: MACS-purified VCAM1-positive cells. Closed bars: control (FACS-purified VCAM1-negative cells). (D) Fluorescent staining of purified VCAM1 stained with TNNT2 or α-actinin. Clear sarcomere structures were observed. (E) Electrophysiological study of single VCAM1-positive cell. Action potentials with ventricular-like pattern (left) and pacemaker-like pattern (right).

Figure 4. Robust expression of VCAM1 on cardiomyocytes.

Representative plots of flow-cytometry for TNNT2 and VCAM1 in various hESC/hiPSC lines at day 11. Left panels: dot plots for TNNT2 and VCAM1. Middle panels: histograms for VCAM1 expression in TNNT2-positive cells. Right panels: histograms for TNNT2 expression in VCAM1-positive cells.