Purpose Stem cell transplantation aids in myocardial regeneration, but poor cell survival and differentiation impact their therapeutic potential. Hydrogen peroxide (H₂O₂) at low doses can activate differentiation and cell resistance in response to stress. The study explores the role of hydrogen peroxide (H2O2) in cardiac differentiation, both individually and in combination with 5-azacytidine (5-Aza), and the mechanisms through which H2O2 treatment enhances cardiac differentiation. Methods Human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) were characterized via immunocytochemistry. Cardiac differentiation, initiated with a non-toxic dose of H2O2 and 5-Aza for 24 h, was investigated via cardiac gene and protein expression analysis, while migration potential was assessed using wound healing assays. Results H2O2, 5-Aza, or combined treatment (H2O2 + 5-Aza) showed higher expression of redox sensitive genes, superoxide dismutase (SOD), Jun-proto-oncogene (c-Jun), Nuclear factor kappa B (NF-kB), NAPDH oxidase-4 (NOX-4), and Nuclear factor erythroid 2-related factor 2 (Nrf2). In addition, these groups showed significant expression of the cardiac genes GATA binding protein 4 (GATA-4), Connexin-43, Myocyte enhancer factor 2 C (MEF2C), and Cardiac myosin heavy chain (cMHC) as well as cardiac proteins Connexin-43, Cardiac troponin-I (cTnI) and GATA-4. However, the combined treatment (H2O2 + 5Aza) group indicated better differentiation of hUC-MSCs into cardiomyocytes. Moreover, wound healing assay demonstrated higher cell migration in the H2O2 and combined treatment (H2O2 + 5-Aza) groups. Conclusions This study concludes that H2O2 promotes cardiac differentiation by regulating redox and antioxidant genes, and when combined with 5-Aza improves the hUC-MSCs differentiation and migration. These cells can be utilized for better transplantation and therapeutic approaches to cardiac diseases. However, further research is needed to investigate the advantages of H2O2 treated MSCs in the in vivo settings. Lay Summary Umbilical cord-derived mesenchymal stem cells (hUC-MSCs) have become a key player in cardiac regenerative therapy. However, inflammatory myocardium affects their survival and therapeutic potential. H2O2 have been reported to promote cell resistance to stress and cardiac differentiation. In this study, hUC-MSCs treated with H2O2 alone and combined with 5-Aza show cardiac differentiation by regulating redox-sensitive and antioxidant genes. The current study demonstrates an efficient preconditioning method for stem cell therapy applications for heart diseases. Graphical Abstract

The Combined Effects of Hydrogen Peroxide and 5-Azacytidine on the Cardiac Differentiation of Human Umbilical Cord Derived Mesenchymal Stem Cells

期刊论文
发表日期：

2025年11月13日

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Anum Siraj,
Syeda Saima Razzaq,
Asmat Salim,
Nadia Naeem,
Kanwal Haneef

摘要

Purpose

Stem cell transplantation aids in myocardial regeneration, but poor cell survival and differentiation impact their therapeutic potential. Hydrogen peroxide (H₂O₂) at low doses can activate differentiation and cell resistance in response to stress. The study explores the role of hydrogen peroxide (H₂O₂) in cardiac differentiation, both individually and in combination with 5-azacytidine (5-Aza), and the mechanisms through which H₂O₂ treatment enhances cardiac differentiation.

Methods

Human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) were characterized via immunocytochemistry. Cardiac differentiation, initiated with a non-toxic dose of H₂O₂ and 5-Aza for 24 h, was investigated via cardiac gene and protein expression analysis, while migration potential was assessed using wound healing assays.

Results

H₂O₂, 5-Aza, or combined treatment (H₂O₂ + 5-Aza) showed higher expression of redox sensitive genes, superoxide dismutase (SOD), Jun-proto-oncogene (c-Jun), Nuclear factor kappa B (NF-kB), NAPDH oxidase-4 (NOX-4), and Nuclear factor erythroid 2-related factor 2 (Nrf2). In addition, these groups showed significant expression of the cardiac genes GATA binding protein 4 (GATA-4), Connexin-43, Myocyte enhancer factor 2 C (MEF2C), and Cardiac myosin heavy chain (cMHC) as well as cardiac proteins Connexin-43, Cardiac troponin-I (cTnI) and GATA-4. However, the combined treatment (H₂O₂ + 5Aza) group indicated better differentiation of hUC-MSCs into cardiomyocytes. Moreover, wound healing assay demonstrated higher cell migration in the H₂O₂ and combined treatment (H₂O₂ + 5-Aza) groups.

Conclusions

This study concludes that H₂O₂ promotes cardiac differentiation by regulating redox and antioxidant genes, and when combined with 5-Aza improves the hUC-MSCs differentiation and migration. These cells can be utilized for better transplantation and therapeutic approaches to cardiac diseases. However, further research is needed to investigate the advantages of H₂O₂ treated MSCs in the in vivo settings.

Lay Summary

Umbilical cord-derived mesenchymal stem cells (hUC-MSCs) have become a key player in cardiac regenerative therapy. However, inflammatory myocardium affects their survival and therapeutic potential. H₂O₂ have been reported to promote cell resistance to stress and cardiac differentiation. In this study, hUC-MSCs treated with H₂O₂ alone and combined with 5-Aza show cardiac differentiation by regulating redox-sensitive and antioxidant genes. The current study demonstrates an efficient preconditioning method for stem cell therapy applications for heart diseases.

Graphical Abstract