Simple, low-cost in vitro protocol for differentiating mesenchymal stem cells into cardiomyocyte-like cells

Congenital heart disease (CHD) remains the foremost cause of mortality in children under 20 years of age. Given their ease of harvest, robust proliferative capacity and multilineage differentiation potential, stem cells (SCs) have emerged as a promising therapeutic alternative. In particular, mesenchymal stem cells (MSCs) derived from Wharton's jelly in the umbilical cord, i.e., an abundant byproduct of childbirth, especially in lowand middle-income settings, can be induced toward a cardiomyocyte lineage, making them an attractive cell source for cardiac regeneration. Although MSCs can be directed toward a cardiomyocyte lineage using growth factors or chemical cues, most in vitro-differentiated cells remain developmentally immature, with only a small fraction achieving the structural, functional and metabolic maturity required for therapeutic use. In resource-limited laboratories, the primary challenge is to develop a simple, cost-effective protocol that reliably differentiates MSCs into structurally, functionally and metabolically mature cardiomyocytes. This review presents a streamlined, cost-effective in vitro differentiation protocol for umbilical-cord MSCs into cardiomyocytes, designed for laboratories with minimal resources, involving 3 sequential stages. First, induce cardiac mesoderm commitment by treating umbilical cord-derived MSCs (UC-MSCs) with 5-azacytidine (5-Aza) or bone morphogenetic protein (BMP). Next, specify cardiac progenitor cells by adding a Wnt-pathway inhibitor (e.g., IWP-2). Finally, drive cardiomyocyte maturation by supplementing the culture with insulin-like growth factors (IGFs). In laboratories lacking complex bioreactors, seeding the cells onto a simple biocompatible scaffold, such as a collagen or fibrin hydrogel, can further boost differentiation efficiency and promote tissue-like organization.