Proliferation of cardiomyocytes derived from human embryonic stem cells is mediated via the IGF/PI 3-kinase/Akt signaling pathway - PubMed (original) (raw)

Comparative Study

Proliferation of cardiomyocytes derived from human embryonic stem cells is mediated via the IGF/PI 3-kinase/Akt signaling pathway

Todd C McDevitt et al. J Mol Cell Cardiol. 2005 Dec.

Abstract

Cardiomyocytes from common experimental animals rapidly exit the cell cycle upon isolation, impeding studies of basic cell biology and applications such as myocardial repair. Here we examined proliferation of cardiomyocytes derived from human and mouse embryonic stem (ES) cells. While mouse ES cell-derived cardiomyocytes showed little proliferation, human cardiomyocytes were highly proliferative under serum-free conditions (15-25% BrdU+/sarcomeric actin+). The cells exhibited only a small serum dose-response, and proliferation gradually slowed with increasing differentiation of the cells. Neither cell density nor different matrix attachment factors affected cardiomyocyte proliferation. Blockade of phosphatidylinositol 3-kinase (PI 3-kinase) and Akt significantly reduced cardiomyocyte proliferation, whereas MEK inhibition had no effect. Antibody blocking of the insulin-like growth factor-1 (IGF-1) receptor significantly inhibited cardiomyocyte proliferation, while addition of IGF-1 or IGF-2 stimulated cardiomyocyte proliferation in a dose-dependent manner. Thus, cardiomyocytes derived from human ES cells proliferate extensively in vitro, and their proliferation appears to be mediated primarily via the PI 3-kinase/Akt signaling pathway, using the IGF-1 receptor as one upstream activator. This system should permit identification of regulatory pathways for human cardiomyocyte proliferation and may facilitate expansion of cardiomyocytes from human ES cells for therapeutic purposes.

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Conflict of interest statement

Conflict-of-interest disclosure

Dr Murry’s laboratory receives sponsorship from Geron Corporation for cardiac repair studies involving human ES cells. The current study however was not sponsored by Geron, but instead was paid for by grants from the National Institutes of Health.

Figures

Fig. 1

Fig. 1

A and B. Comparison of mouse and human ES-cell derived cardiomyocyte proliferation. Mouse ES cells were differentiated for 12 days (A) and human ES cells were differentiated for 14 days (B). The cultures were pulsed with BrdU and sections from cell pellets were immunostained for myosin heavy chain (mouse) or sarcomeric actin (human) to detect cardiomyocytes; cell nuclei were counterstained with methyl green. BrdU+ cardiomyocytes (brown cytoplasm, blue nuclei) were rarely seen in differentiating mouse ES cultures, whereas numerous BrdU+ cardiomyocytes were detected in differentiating human ES cultures, as noted by the arrows. Scale bar = 50 µm. C and D. Mitotic figures and BrdU incorporation in human ES cell-derived cardiomyocytes. H7 human ES cells were differentiated for 19 days before Percoll separation and fraction IV cells were cultured for an additional 4 days with 20% FBS. Cells were stained for sarcomeric actin (red) and BrdU (brown), and nuclei were counterstained with hematoxylin (blue). Multiple BrdU+ cardiomyocytes were evident; mitotic cardiomyocytes in metaphase (C) and telophase (D) are shown by the arrows.

Fig. 2

Fig. 2

Serum-dependence of human ES cell-derived cardiomyocyte proliferation. H7 human ES cells were differentiated for varying times (19, 33, or 47 days) before Percoll separation, and fraction IV cells were cultured for an additional 4 days at various FBS concentrations (0%, 1% or 20%). The percentage of BrdU+ cardiomyocytes was moderately sensitive to the concentration of FBS and decreased as the cells were differentiated for longer periods of time.

Fig. 3

Fig. 3

Signaling pathway inhibition of human ES cell-derived cardiomyocyte proliferation. H7 human ES cells were differentiated for 19 days before Percoll separation, and fraction IV cells were cultured for an additional 4 days in serum-free media with various signaling inhibitors. The inhibitors were added for the final 48 hours of culture prior to fixation. Only the PI 3-kinase inhibitor (LY294002) significantly inhibited human ES cell-derived cardiomyocyte proliferation (* = P < 0.05).

Fig. 4

Fig. 4

PI 3-kinase inhibition of human ES cell-derived cardiomyocyte proliferation. H7 human ES cells were differentiated for 18 days before Percoll separation, and fraction IV cells were cultured for an additional 4 days in serum-free media with various signaling inhibitors. The inhibitors were added for the final 48 hours of culture prior to fixation. Cells were stained for sarcomeric actin (red) and BrdU (brown), and nuclei were counterstained with hematoxylin (blue). A–D) Significantly fewer BrdU+ cardiomyocyte nuclei were apparent in the treated cultures (panels B–D) as opposed to the control (DMSO vehicle alone; panel A). Scale bar = 50 µm. E) Quantification of the data indicated that the PI 3-kinase inhibitors, LY294002 and wortmannin, and the Akt inhibitor significantly inhibited human ES cell-derived cardiomyocyte proliferation compared to the control (* = P < 0.05).

Fig. 5

Fig. 5

Antibody blocking of growth factor receptors. H7 human ES cells were differentiated for 20 days before Percoll separation, and fraction IV cells were cultured for an additional 4 days in serum-free media with various signaling inhibitors. The inhibitors were added for the final 48 hours of culture prior to fixation. Only the PI 3-kinase inhibitor (LY294002) and the IGF-1 receptor blocking antibody significantly inhibited human ES cell-derived cardiomyocyte proliferation (* = P < 0.05), whereas antibodies to the EPO ligand or receptor had no effect.

Fig. 6

Fig. 6

Stimulation of human ES cell-derived cardiomyocyte proliferation. H7 human ES cells were differentiated for 17 days before Percoll separation, and fraction IV cells were cultured for an additional 4 days in serum-free media. Various concentrations of recombinant human IGF-1 or IGF-2 were added for the final 48 hours of culture prior to fixation. IGFs increased cardiomyocyte proliferation in a dose-dependent manner to levels greater than treatment with 20% FBS (* = P < 0.05).

Fig. 7

Fig. 7

IGF stimulation of human ES cell-derived cardiomyocyte proliferation is mediated via PI 3-kinase. H7 human ES cells were differentiated for 21 days before Percoll separation, and fraction IV cells were cultured for an additional 4 days in serum-free media with various treatments for the final 48 hours of culture prior to fixation. The PI 3-kinase inhibitor LY294002 significantly inhibited and IGF-1 significantly stimulated hESC-derived cardiomyocyte proliferation (** = P < 0.01 and * = P < 0.05, respectively), similar to previous results. Dual treatment with IGF-1 and LY294002 significantly inhibited proliferation (P < 0.05) compared to treatment with IGF-1 alone, suggesting that IGF-1 stimulated proliferation is mediated via the PI 3-kinase pathway.

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