Co-culture with neonatal cardiomyocytes enhances the proliferation of iPSC-derived cardiomyocytes via FAK/JNK signaling

Abstract

Background: We previously reported that the pluripotent stem cells can differentiate into cardiomyocytes (CMs) by co-culture with neonatal CMs (NCMs) in vitro. However, the involving mechanism is not clear.

Methods: Mouse induced pluripotent stem cells (iPSCs) were cultured in hanging drops to form embryoid bodies (EBs) and to induce myocardial differentiation. Co-culture of EBs and NCMs was established in a transwell insert system, while EBs grown alone in the wells were used as controls.

Results: Co-culture with NCMs markedly increased the generation of functional CMs from iPSCs. The focal adhesion kinase (FAK) phosphorylation, and c-Jun N-terminal kinase (JNK) phosphorylation in co-culture were higher than that in EBs grown alone. Treating FAK small interfering RNA (FAK siRNA) or specific inhibitor for JNK (SP600125) to iPSCs significantly reduced the phosphorylation of JNK and the expressions of Mef2c and Bcl-2. The expressions of cTnT and MLC-2V were also decreased. Our results revealed that co-culture with NCMs significantly enhance the differentiation ability of iPSCs by increasing Mef2c and Bcl-2 expressions concomitantly with a marked augment on cell proliferation through JNK signaling pathways.

Conclusions: These findings indicated that co-culture of EBs with NCMs induces genes expressed in a mature pattern and stimulates the proliferation of iPSC-derived CMs (iPS-CMs) by activating FAK/JNK signaling.

Keywords: Cardiomyocytes; Co-culture; FAK/JNK; Induced pluripotent stem cells; Proliferation.

Fig. 1

Myocardial differentiation of induced pluripotent stem cells (iPSCs) in the indirect co-culture model. a the culture of undifferentiated iPSC colonies with Oct4-GFP⁺ expression (green) on feeder cells. b The indirect co-culture system: a facile cell expansion and stem cell differentiation system with continuous medium conditioning while preventing mixing by hanging culture inserts. Two cell populations that are co-cultured in different compartments (insert and well) but can communicate via paracrine signaling through the pores of the membrane. c, d, e show the development of embryoid bodies (EBs) from 5 to 10 days in grown alone, co-culture with mouse embryo fibroblasts (MEFs), and co-culture with neonatal cardiomyocytes (NCMs) (c, 5-day-old EB cultured in suspension. d, 7-day-old EB. e, 10-day-old EB). Hanging inserts removed when photographed). There are few remaining GFP+ colonies (green) after 10 days. Scale bar =300 μm

Fig. 2

Effect of co-culture on the differentiation efficiency of iPSCs. Time course quantification of spontaneous beating activity of differentiated CMs was expressed as the percentage of beating EBs. *: P < 0.05 versus control or MEFs co-culture

Fig. 3

The effects of co-culture on the myocardial differentiation. Reverse Transcription-PCR analysis of cardiac marker genes (Mef2c, Cardiac Troponin T, and MLC-2 V) during the myocardial differentiation of induced pluripotent stem cells (iPSCs) (days 6–20). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a normalizing internal control. a, the results of Semi-quantitative RT-PCR. b, c, d show the quantitative analysis of cardiac marker gene expression: relative expression over the control at day 6. (b, Mef2c. c, Cardiac Troponin T. d, MLC-2 V). **: p < 0.01 versus control

Fig. 4

Expression of cardiac specific proteins in induced pluripotent stem cell-derived cardiomyocytes (iPS-CMs) on day 12. a, double immunostaining of cells by antibodies against cTnT (red) and CX43 (green). b, double immunofluorescent staining for cTnT (red) and MLC-2 V (green), c, double immunofluorescent staining for cTnT (red) and α-actinin (green). Nuclei in the same field were stained with DAPI (blue). Merged figures were made by FV10-ASW Systems. d, growth curves of cells in co-culture and control analyzed by a MTT assay. The growth of the cells in co-culture was significantly better than that in control after day 6. Scale bar =20 μm,*: P < 0.05 versus control, **: P < 0.01 versus control

Fig. 5

Focal adhesion kinase (FAK) and c-Jun N-terminal kinase (JNK) are involved in coculture-mediated myocardial differentiation. a, the phosphorylation of FAK and JNK were performed. b, cells were transfected with FAK siRNA or treated with specific JNK inhibitor (SP600125), the double immunostainings for cTnT (red) and CX43 (green) was examined, nuclei in the same field were stained with DAPI (blue). Note the presence of gap-junctions (immunostaining for Cx43, green) at the interphase (arrow heads) between the cells. c, West blot showed that the cells transfected with FAK siRNA expressed lower levels of FAK protein than controls. In addition, the p-JNK protein level was also significant decreased after transfected with FAK siRNA, compared to the control at the same time. Scale bar =100 μm, *: P < 0.05 versus control, #: P < 0.01 versus control

Fig. 6

The effects of focal adhesion kinase (FAK) siRNA or SP600125 on myocardial differentiation. a, the expression of Mef2c, cTnT, MLC-2 V and BCL-2 after pretreation of FAK siRNA or SP600125 was examined by RT-PCR. b, c, d, e, the quantitative analysis on Mef2c, cTnT, MLC-2V, and Bcl-2 expressions after pretreation of FAK siRNA or SP600125 by a real-time PCR. *: P < 0.01 versus control, **: P < 0.01 versus co-culture

Fig. 7

The proliferative capacity of the induced pluripotent stem cell-derived cardiomyocytes (iPS-CMs). a, double immunostainings for Ki67 (a marker for cycling cells, green) and cTnT (a marker for CM, red), nuclei in the same field were stained with DAPI (blue). b, the percentage of proliferating iPS-CMs in control (7.9 ± 1.1 %), co-culture (10.4 ± 1.2 %), FAK siRNA (4.1 ± 0.5 %), and SP600125 (1.8 ± 0.2 %). Scale bar =100 μm, *: P < 0.01 versus control, **: P < 0.01 versus co-culture