Lab Anim Res.  2011 Sep;27(3):205-212. 10.5625/lar.2011.27.3.205.

Chemical compound 31002 stimulates cardiomyogenic differentiation of embryonic stem cells

• Kim EK
• Son MY
• Kang Y
• Lee CH
• Kim HR
• Won Y
• Yoon W
• Kim HC
• Nam KH

• KMID: 1850047
• DOI: http://doi.org/10.5625/lar.2011.27.3.205

Abstract

Embryonic stem cells (ESCs) are an emerging source for cell-based therapies aimed at repairing damaged organ tissues; however, the efficiency of directed differentiation is low and refinement of differentiation protocols is hampered by incomplete understanding of the mechanisms involved in this process. To find new compounds which can improve the efficiency of directed differentiation of ESCs to cardiomyocytes, we screened several thousand chemical compounds and identified a promising group. All of the compounds found have a common structure of 1H-pyrrole,2,2'-(phenylmethylene)bis. Here we report the potential mechanism of action for 31002 which showed the strongest activity among the compounds selected. In the presence of 31002, 15 times more cardiomyocytes differentiated from ESCs, i.e., 3.5% to 52% of total differentiated cells. Moreover, the cardiomyocytes showed functional characteristics including rhythmic beating and marker gene expression. 31002 inhibited the down-regulation of genes related to the three germ layers in the late stage of ESCs differentiation, implying that 31002 supports a continuous fate commitment of undifferentiated ESCs to the cardiac lineage by prolonging the three germ layer stages. Therefore, compounds in this group, including 31002, might be useful as directed cardiomyogenic differentiation-inducers to produce cells for use in cell therapy aimed at restoring damaged heart tissue.

Keyword

Embryonic stem cells; 31002; cardiomyocytes; differentiation

MeSH Terms

Down-Regulation
Embryonic Stem Cells
Gene Expression
Germ Layers
Heart
Myocytes, Cardiac
Tissue Therapy

Figure

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  Figure 1 Chemical structure of 31002 and the structural derivatives showing activity propensity for cardiomyocyte-directed differentiation of embryonic stem cells (ESCs).

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  Figure 2 Activity of test compounds for cardiomyogenic differentiation induction of ESCs. KH2 ESCs were differentiated with one of the compounds at a final concentration of 1or 10 mM as described in Materials and Methods. On day 15 of differentiation, the cells were trypsinized, fixed with 1% formaldehyde and subjected to fluorescence activated cell sorter (FACS) analysis to detect enhanced green fluorescent protein (EGFP) expressing cells. Three independent experiments were repeated and the representative result is shown. All the experiments were done in triplicate.

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  Figure 3 Co-expression of cardiomyocyte marker proteins in EGFP-expressing cells. (A) Representative flow cytometric plots of differentiated KH2 cells in the presence of 10 mM 31002. KH2 ESCs were differentiated with 10 mM 31002 as described in Materials and Methods. On day 15 of differentiation, the cells were trypsinized, fixed with 1% formaldehyde and subjected to FACS analysis to detect EGFP expressing cells. (B) Differentiated cells on day 15 were fixed and immunostained for troponin T (b and d; red) or α-actinin (f; red) as described in Materials and Methods. EGFP (green) in the cells was observed under a fluorescence microscope (a, c and e). Three independent experiments were repeated and the representative results are shown. Troponin T (×100), α-actinin (×200).

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  Figure 4 Time-dependent increase of EGFP-positive cells. KH2 ESCs were differentiated with 10 mM 31002 as described in Materials and Methods. On the differentiation days indicated, the cells were trypsinized, fixed with 1% formaldehyde, and subjected to FACS analysis to detect EGFP expressing cells. Three independent experiments were repeated and the representative results are shown. All experiments were performed in triplicate.

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  Figure 5 The effects of periodic 31002 treatments on cardiomyogenic differentiation of ESCs. KH2 cells were differentiated for 15 days according to the differentiation protocol described in Materials and Methods. The cells were treated with 10 µM 31002 during only the indicated period. On day 15, flow cytometric analysis was performed for all cell groups. Three independent experiments were repeated and representative results are shown. All experiments were performed in triplicate. *Significant difference at P<0.05 compared to Group 1.

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  Figure 6 Marker gene expressions of differentiating ESCs. KH2 ESCs were differentiated in the presence of 10 µM 31002 as described in Materials and Methods. Total RNA was obtained from the differentiating cells collected at the indicated times. Marker genes for three germ layers (A) and cardiac lineage cells (B) were analyzed by semi-quantitative RT-PCR. The plots in the right panel show the relative expression levels of target genes which were normalized to β-actin expression. Open- and filled-symbols indicate dimethyl sulfoxide (DMSO) and 31002 treatments, respectively.

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