Exploring the Potential Effects of Cryopreservation on the Biological Characteristics and Cardiomyogenic Differentiation of Rat Adipose-Derived Mesenchymal Stem Cells

Abstract

Cryopreservation is essential for the broad clinical application of mesenchymal stem cells (MSCs), yet its impact on their cellular characteristics and cardiomyogenic differentiation potential remains a critical concern in translational medicine. This study aimed to evaluate the effects of cryopreservation on the biological properties and cardiomyogenic capacity of rat adipose-derived MSCs (AD-MSCs). We examined their cellular morphology, surface marker expression (CD29, CD90, CD45), trilineage differentiation potential (adipogenic, osteogenic, chondrogenic), and gene expression profiles for the pluripotency marker <i>REX1</i> and immunomodulatory markers <i>TGFβ1</i> and <i>IL-6</i>. After inducing cardiomyocyte differentiation, we assessed cardiac-specific gene expressions (<i>Troponin I</i>, <i>MEF2c</i>, <i>GSK-3β</i>) using quantitative RT-qPCR, along with live/dead cell staining and immunofluorescence for cardiac-specific proteins (Troponin T, α-actinin, Myosin Heavy Chain). Cryopreserved AD-MSCs preserved their morphology, surface markers, and differentiation potential, but exhibited a reduced expression of <i>REX1</i>, <i>TGFβ1</i>, and <i>IL-6</i>. Additionally, cryopreservation diminished cardiomyogenic differentiation, as indicated by the lower levels of <i>Troponin I</i>, <i>MEF2c</i>, and <i>GSK-3β</i> seen compared to non-cryopreserved cells. Despite this, high cell viability (>90%) and maintained cardiac protein expression were observed post-cryopreservation. These findings highlight the necessity of optimizing cryopreservation protocols to ensure the full therapeutic potential of AD-MSCs, particularly in applications related to cardiac regenerative medicine.