Distinct iPS Cells Show Different Cardiac Differentiation Efficiency

Abstract

Patient-specific induced pluripotent stem (iPS) cells can be generated by introducing transcription factors that are highly expressed in embryonic stem (ES) cells into somatic cells. This opens up new possibilities for cell transplantation-based regenerative medicine by overcoming the ethical issues and immunological problems associated with ES cells. Despite the development of various methods for the generation of iPS cells that have resulted in increased efficiency, safety, and general versatility, it remains unknown which types of iPS cells are suitable for clinical use. Therefore, the aims of the present study were to assess (1) the differentiation potential, time course, and efficiency of different types of iPS cell lines to differentiate into cardiomyocytes in vitro and (2) the properties of the iPS cell-derived cardiomyocytes. We found that high-quality iPS cells exhibited better cardiomyocyte differentiation in terms of the time course and efficiency of differentiation than low-quality iPS cells, which hardly ever differentiated into cardiomyocytes. Because of the different properties of the various iPS cell lines such as cardiac differentiation efficiency and potential safety hazards, newly established iPS cell lines must be characterized prior to their use in cardiac regenerative medicine.

Figure 1

Endogenous pluripotent gene and transgene expression profiles of undifferentiated pluripotent cells. Expression levels of the four transcription factors. Semiquantitative RT-PCR analyses were performed for endogenous pluripotent stem cell transcription factors and transgenes.

Figure 2

Cardiomyocyte differentiation efficiency of pluripotent stem cells and temporal gene expression patterns during cardiomyocyte differentiation. (a) Percentage of beating colonies on days 6–15 in mouse embryonic stem (ES) cells, Nanog induced pluripotent stem (iPS) cells, and Fbx15-iPS cells. Data are the mean ± SEM (n = 5 in all groups). ((b)–(g)) Quantitative RT-PCR analyses showing temporal gene expression patterns of the mesodermal marker Brachyury T (b), the early cardiac mesodermal marker Mesp1 (c), the cardiac-specific transcription factors Nkx2.5 (d) and Gata4 (e), and the cardiac-specific proteins Nppa (f) and Myl2 (g) in EB3 ES cells (closed squares), 20D17 Nanog-iPS cells (open circles), and WT-1 Fbx15-iPS cells (open triangles). Data are the mean ± SEM (n = 5 in all groups).

Figure 3

Transgene expression profiles of purified cardiomyocytes derived from Nanog and Fbx15 induced pluripotent stem (iPS) cells. Total RNA was isolated from purified cardiomyocytes derived from mouse embryonic stem (ES) cells, three Nanog-iPS cell clones (20D17, 38C2, and 38D2), two Fbx15-iPS cell clones (WT-1 and 4-3), undifferentiated Fbx15-iPS cells, and the mouse heart. RT-PCR analyses were performed to determine the transgene expression of the four transcription factors (Oct3/4, Sox2, Klf4, and c-Myc), with GAPDH used as an internal control.

Figure 4

Cardiomyocyte (CM) specific gene expression profiles, as determined by RT-PCR and immunostaining, of CM derived from mouse embryonic stem (ES) cells, Nanog induced pluripotent stem (iPS) cells, and Fbx15-iPS cells. (a) CM-associated structural protein gene expression profiles. The expression of Actn1, Myh6, Myh7, Myl7, Myl2, and Nppb was analyzed by semiquantitative RT-PCR analysis. GAPDH was used as an internal control. (b) Immunofluorescent staining of typical CM-specific proteins on day 15 of differentiation in CM derived from ES, Nanog-iPS, and Fbx15-iPS cells. Cells were stained with Nkx2.5 (green), GATA4 (green), atrial natriuretic peptide (ANP; green), myosin heavy chain (MHC; red), and α-actinin (red). Scale bar = 50 μm.

Figure 5

Electrophysiological studies of cardiomyocytes (CM) derived from mouse embryonic stem (ES) cells, Nanog induced pluripotent stem (iPS) cells, and Fbx15-iPS cells. Representative action potentials of (a) ES cell-derived CM, (b) Nanog-iPS cell-derived CM, and (c) Fbx15-iPS cell-derived CM showing sinus node-like (top), fetal atrial-type (middle), and fetal ventricular-type (bottom) action potentials.

Figure 6

Pharmacological studies of cardiomyocytes (CM) derived from mouse embryonic stem (ES) cells, Nanog induced pluripotent stem (iPS) cells, and Fbx15-iPS cells. ((a)–(c)) Multielectrode array (MEA) recordings in CM derived from ES (a), Nanog-iPS (b), and Fbx15-iPS cells (c) showing chronotropic responses to increasing doses of isoproterenol. (d) Summary of the positive chronotropic changes induced by isoproterenol. Data are the mean ± SEM. *P < 0.05 and **P < 0.01 compared with 0 mM isoproterenol. ((e)–(g)) MEA recordings of negative chronotropic responses to increasing doses of verapamil in CM derived from ES (d), Nanog-iPS (e), and Fbx15-iPS cells (f). (h) Summary of the negative chronotropic changes induced by verapamil. Data are the mean ± SEM. *P < 0.05 and **P < 0.01 compared with 0 mM verapamil.