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Combination of IGF‑1 gene manipulation and 5‑AZA treatment promotes differentiation of mesenchymal stem cells into cardiomyocyte‑like cells.

Li J, Zhu K, Wang Y, Zheng J, Guo C, Lai H, Wang C - Mol Med Rep (2014)

Bottom Line: Our results demonstrated that 5‑AZA treatment alone induced a limited cardiomyocyte‑like differentiation effect in vitro.Overexpression of the IGF‑1 gene in MSCs improved the induction effect of 5‑AZA, while knockdown of the IGF‑1 gene attenuated the differentiation.These results suggest that IGF‑1 is a significant stimulus affecting the cardiomyocyte‑like differentiation of porcine MSCs.

View Article: PubMed Central - PubMed

Affiliation: Department of Cardiac Surgery, Shanghai Institute of Cardiovascular Disease, Zhongshan Hospital, Fudan University, Shanghai 200032, P.R. China.

ABSTRACT
Mesenchymal stem cell (MSC) transplantation has been proposed as a promising therapeutic strategy for ischemic myocardium repair following myocardial infarction. Differentiation of MSCs into cardiomyocyte‑like cells prior to cell transplantation is advantageous in improving their potential clinical benefits for cardiac repair. In the present study, we isolated and cultured porcine MSCs and evaluated the synergistic effect of 5‑azacytidine (5‑AZA) treatment and insulin‑like growth factor‑1 (IGF‑1) gene manipulation on MSC differentiation into cardiomyocyte‑like cells. Our results demonstrated that 5‑AZA treatment alone induced a limited cardiomyocyte‑like differentiation effect in vitro. Overexpression of the IGF‑1 gene in MSCs improved the induction effect of 5‑AZA, while knockdown of the IGF‑1 gene attenuated the differentiation. These results suggest that IGF‑1 is a significant stimulus affecting the cardiomyocyte‑like differentiation of porcine MSCs. In addition, the combination of IGF‑1 gene manipulation and 5‑AZA treatment provides a new strategy to obtain more committed differentiated cardiomyocyte‑like cells from porcine MSCs prior to cell transplantation.

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Insulin-like growth factor-1 (IGF-1) expression and cardiomyocyte-specific marker expression. (A) The expression of IGF-1 in different mesenchymal stem cell (MSC) groups was determined by immunocytochemistry. Scale bars, 50 μm. (B) Cardiomyocyte-like differentiation of MSCs with 5-azacytidine (5-AZA) treatment was detected by quantitative polymerase chain reaction, which was demonstrated by upregulation of GATA-4, Nkx2.5, β-MHC and MEF2c mRNA expression on days 14 and 21. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a reference gene. (C) Cardiomyocyte-like differentiation of MSCs with 5-AZA treatment was detected by western blot analysis, which was demonstrated by upregulation of GATA-4, Nkx2.5, β-MHC and MEF2c protein expression on days 14 and 21. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a loading control. MSC, untreated MSCs; control, MSCs transfected with control lentivirus; oeIGF-1 group, MSCs transfected with lentivirus encoding IGF-1; siIGF-1 group, MSCs transfected with lentivirus encoding shRNA-IGF-1. *P<0.05 and **P<0.01, vs. the control group.
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f3-mmr-11-02-0815: Insulin-like growth factor-1 (IGF-1) expression and cardiomyocyte-specific marker expression. (A) The expression of IGF-1 in different mesenchymal stem cell (MSC) groups was determined by immunocytochemistry. Scale bars, 50 μm. (B) Cardiomyocyte-like differentiation of MSCs with 5-azacytidine (5-AZA) treatment was detected by quantitative polymerase chain reaction, which was demonstrated by upregulation of GATA-4, Nkx2.5, β-MHC and MEF2c mRNA expression on days 14 and 21. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a reference gene. (C) Cardiomyocyte-like differentiation of MSCs with 5-AZA treatment was detected by western blot analysis, which was demonstrated by upregulation of GATA-4, Nkx2.5, β-MHC and MEF2c protein expression on days 14 and 21. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a loading control. MSC, untreated MSCs; control, MSCs transfected with control lentivirus; oeIGF-1 group, MSCs transfected with lentivirus encoding IGF-1; siIGF-1 group, MSCs transfected with lentivirus encoding shRNA-IGF-1. *P<0.05 and **P<0.01, vs. the control group.

Mentions: IGF-1 expression in the oeIGF-1 group is shown in Fig. 3A. The combination of IGF-1 overexpression with 5-AZA treatment induced the cardiomyocyte-like differentiation of MSCs, which was demonstrated by the expression of specific cardiomyocyte markers (GATA-4, Nkx2.5, β-MHC and MEF2c). Compared with the MSC and control groups, the oeIGF-1 group expressed mRNA and protein of specific markers at a higher level on day 14 and day 21 following treatment with 5-AZA (P<0.05), but no significant difference was shown on day 7 (Fig. 3B and C).


Combination of IGF‑1 gene manipulation and 5‑AZA treatment promotes differentiation of mesenchymal stem cells into cardiomyocyte‑like cells.

Li J, Zhu K, Wang Y, Zheng J, Guo C, Lai H, Wang C - Mol Med Rep (2014)

Insulin-like growth factor-1 (IGF-1) expression and cardiomyocyte-specific marker expression. (A) The expression of IGF-1 in different mesenchymal stem cell (MSC) groups was determined by immunocytochemistry. Scale bars, 50 μm. (B) Cardiomyocyte-like differentiation of MSCs with 5-azacytidine (5-AZA) treatment was detected by quantitative polymerase chain reaction, which was demonstrated by upregulation of GATA-4, Nkx2.5, β-MHC and MEF2c mRNA expression on days 14 and 21. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a reference gene. (C) Cardiomyocyte-like differentiation of MSCs with 5-AZA treatment was detected by western blot analysis, which was demonstrated by upregulation of GATA-4, Nkx2.5, β-MHC and MEF2c protein expression on days 14 and 21. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a loading control. MSC, untreated MSCs; control, MSCs transfected with control lentivirus; oeIGF-1 group, MSCs transfected with lentivirus encoding IGF-1; siIGF-1 group, MSCs transfected with lentivirus encoding shRNA-IGF-1. *P<0.05 and **P<0.01, vs. the control group.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4262510&req=5

f3-mmr-11-02-0815: Insulin-like growth factor-1 (IGF-1) expression and cardiomyocyte-specific marker expression. (A) The expression of IGF-1 in different mesenchymal stem cell (MSC) groups was determined by immunocytochemistry. Scale bars, 50 μm. (B) Cardiomyocyte-like differentiation of MSCs with 5-azacytidine (5-AZA) treatment was detected by quantitative polymerase chain reaction, which was demonstrated by upregulation of GATA-4, Nkx2.5, β-MHC and MEF2c mRNA expression on days 14 and 21. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a reference gene. (C) Cardiomyocyte-like differentiation of MSCs with 5-AZA treatment was detected by western blot analysis, which was demonstrated by upregulation of GATA-4, Nkx2.5, β-MHC and MEF2c protein expression on days 14 and 21. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a loading control. MSC, untreated MSCs; control, MSCs transfected with control lentivirus; oeIGF-1 group, MSCs transfected with lentivirus encoding IGF-1; siIGF-1 group, MSCs transfected with lentivirus encoding shRNA-IGF-1. *P<0.05 and **P<0.01, vs. the control group.
Mentions: IGF-1 expression in the oeIGF-1 group is shown in Fig. 3A. The combination of IGF-1 overexpression with 5-AZA treatment induced the cardiomyocyte-like differentiation of MSCs, which was demonstrated by the expression of specific cardiomyocyte markers (GATA-4, Nkx2.5, β-MHC and MEF2c). Compared with the MSC and control groups, the oeIGF-1 group expressed mRNA and protein of specific markers at a higher level on day 14 and day 21 following treatment with 5-AZA (P<0.05), but no significant difference was shown on day 7 (Fig. 3B and C).

Bottom Line: Our results demonstrated that 5‑AZA treatment alone induced a limited cardiomyocyte‑like differentiation effect in vitro.Overexpression of the IGF‑1 gene in MSCs improved the induction effect of 5‑AZA, while knockdown of the IGF‑1 gene attenuated the differentiation.These results suggest that IGF‑1 is a significant stimulus affecting the cardiomyocyte‑like differentiation of porcine MSCs.

View Article: PubMed Central - PubMed

Affiliation: Department of Cardiac Surgery, Shanghai Institute of Cardiovascular Disease, Zhongshan Hospital, Fudan University, Shanghai 200032, P.R. China.

ABSTRACT
Mesenchymal stem cell (MSC) transplantation has been proposed as a promising therapeutic strategy for ischemic myocardium repair following myocardial infarction. Differentiation of MSCs into cardiomyocyte‑like cells prior to cell transplantation is advantageous in improving their potential clinical benefits for cardiac repair. In the present study, we isolated and cultured porcine MSCs and evaluated the synergistic effect of 5‑azacytidine (5‑AZA) treatment and insulin‑like growth factor‑1 (IGF‑1) gene manipulation on MSC differentiation into cardiomyocyte‑like cells. Our results demonstrated that 5‑AZA treatment alone induced a limited cardiomyocyte‑like differentiation effect in vitro. Overexpression of the IGF‑1 gene in MSCs improved the induction effect of 5‑AZA, while knockdown of the IGF‑1 gene attenuated the differentiation. These results suggest that IGF‑1 is a significant stimulus affecting the cardiomyocyte‑like differentiation of porcine MSCs. In addition, the combination of IGF‑1 gene manipulation and 5‑AZA treatment provides a new strategy to obtain more committed differentiated cardiomyocyte‑like cells from porcine MSCs prior to cell transplantation.

Show MeSH
Related in: MedlinePlus