5-Azacytidine-Induced Cardiomyocyte Differentiation of Very Small Embryonic-Like Stem Cells

Abstract

The use of stem cells in generating cell-based pacemaker therapies for bradyarrhythmia is currently being considered. Due to the propensity of stem cells to form tumors, as well as ethical issues surrounding their use, the seed cells used in cardiac biological pacemakers have limitations. Very small embryonic-like stem cells (VSELs) are a unique and rare adult stem cell population, which have the same structural, genetic, biochemical, and functional characteristics as embryonic stem cells without the ethical controversy. In this study, we investigated the ability of rat bone marrow- (BM-) derived VSELs to differentiate in vitro into cardiomyocytes by 5-Azacytidine (5-AzaC) treatment. The morphology of VSELs treated with 10 μM 5-AzaC increased in volume and gradually changed to cardiomyocyte-like morphology without massive cell death. Additionally, mRNA expression of the cardiomyocyte markers cardiac troponin-T (cTnT) and α-sarcomeric actin (α-actin) was significantly upregulated after 5-AzaC treatment. Conversely, stem cell markers such as Nanog, Oct-4, and Sox2 were continuously downregulated posttreatment. On day 14 post-5-AzaC treatment, the positive expression rates of cTnT and α-actin were 18.41 ± 1.51% and 19.43 ± 0.51%, respectively. Taken together, our results showed that rat BM-VSELs have the ability to differentiate into cardiomyocytes in vitro. These findings suggest that VSELs would be useful as seed cells in exploring the mechanism of biological pacemaker activity.

Figure 1

Rat bone marrow- (BM-) derived very small stem cells (VSELs) were successfully isolated by flow cytometry. (a) The tibia and femur of rats were separated, erythrocytes were lysed, and total BM mononuclear cells were extracted. Fluorophore-conjugated antibodies specific to CD45, CD106, and hematopoietic markers (Lin: TCRαβ, CD3, CD11b, and CD45RA) were used. Flow cytometric sorting was carried out after incubation. (b) Blank control; (c) Lin control; (d) CD45 control; (e) CD106 control; (f) VSEL sorting.

Figure 2

Bone marrow- (BM-) derived very small stem cells (VSELs) retain embryonic stem cell (ESC) characteristics. (a) Rat BM-derived VSELs were obtained by flow cytometric sorting. (b) BM-VSELs were assessed for alkaline phosphatase (AKP) activity by staining for AKP. (c) Expression of Sox2, Oct-4, and Nanog in VSELs was assessed by immunostaining with Sox2-, Oct-4-, and Nanog-specific primary antibodies and a Cy3 secondary antibody.

Figure 3

5-AzaC induces cardiomyocyte-like morphology in BM-VSELs. (a) 5-AzaC-induced VSELs showed morphological changes at day 7, and the population of cardiomyocytes peaked at day 14. (b) Growth of VSELs treated with different concentrations of 5-AzaC (1 μM, 10 μM, and 30 μM). (c) MTT assay indicated VSEL growth induced by different concentrations of 5-AzaC over time. (d) Morphological changes in BM-VSELs after treatment with 10 μM 5-AzaC. The arrow indicates that VSELs developed embryoid body-like structures after 5-AzaC treatment.

Figure 4

5-AzaC treatment increased levels of cardiac troponin-T (cTnT) and α-sarcomeric actin (α-actin) in BM-VSELs. (a) Immunostaining for cTnT and α-actin in cardiomyocytes derived from 5-AzaC-induced differentiation of VSELs. The white arrow indicates positive cells. (b) Flow cytometric analysis demonstrated the percentages of cTnT- and α-actin-positive cells on different days.

Figure 5

Gene expression levels in BM-VSELs differentiated into cardiomyocytes after 5-AzaC treatment. Expression of (a) Sox2, Oct-4, and Nanog and (b) cTnT and α-actin was detected in 5-AzaC-treated cells via qRT-PCR. The gene expression levels of cardiomyocytes and pluripotent stem cell genes were normalized with GAPDH and compared to the relative control group and cardiomyocytes (n = 3, level of significance p < 0.05; ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001).