Background <p>Cardiovascular diseases are a major cause of death, accounting for around 19.91 million deaths worldwide in 2021. Current treatment options temporarily address the disease’s progression but fail to restore heart functions. Recent developments in stem cells research have drawn attention to human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) due to their differentiation potential into cardiomyogenic lineage along with others.</p> Purpose <p>This study aims to investigate the effect of transcription factors GATA binding protein 4 (GATA-4) and myocyte enhancer factor 2C (MEF2C) in the differentiation of hUC-MSCs into myogenic lineage and cardiac regeneration after myocardial infarction (MI).</p> Methods <p>An animal model of MI was established by ligating the left anterior descending coronary artery (LAD); normal and pre-differentiated hUC-MSCs were transplanted in the peri-infarct region. To assess the regeneration, functional and histological examinations were performed. Moreover, immunohistochemistry was used to assess the heart-specific proteins for survival, homing, differentiation, and integration of implanted cells.</p> Results <p>Hearts transplanted with pre-differentiated hUC-MSCs had significantly improved cardiac systolic and diastolic functions. The fibrotic area was decreased, and left ventricular wall thickness was maintained in the pre-differentiated hUC-MSCs transplanted group compared to MI. Immunohistochemical staining revealed that transplanted cells survived, homed, differentiated, and integrated into the peri-infract region of the heart.</p> Conclusion <p>Cardiomyogenic transcription factors GATA-4 and MEF2C combination enhanced hUC-MSCs differentiation potential into cardiomyogenic cells. These cells survived in the infarcted region of the heart, matured into cardiomyocytes, and contributed to improvement in heart function. These cardiac transcription factors can effectively be used to improve cellular treatment for myocardial infarction.</p> Lay Summary <p>Heart diseases are one of the leading causes of death around the world. Current treatment options cannot revert the normal functions of the heart. Umbilical cord-derived mesenchymal stem cells (hUC-MSCs) have gained attention in the field of regenerative medicine, particularly in heart repair. Cardiac transcription factors (TFs), GATA-4, and MEF2C effectively contribute to the development of the embryonic heart. In this study, we investigated GATA-4 and MEF2C combination for the differentiation of hUC-MSCs into cardiac lineage and heart regeneration after myocardial infarction. The findings of our study demonstrated that GATA-4 and MEF2C-guided pre-differentiated hUC-MSCs show significant survival, homing, differentiation, and improvement in heart functions in vivo. This study presents an innovative approach for cardiac tissue regeneration and emphasizes the encouraging potential of the GATA-4 and MEF2C combination-based differentiation of MSCs.</p> Graphical Abstract <p></p>