Comparative phenotypic analysis of primary aortic VSMCs versus TGF - β/PDGF - BB differentiated MSCs in rat

In this investigation, two distinct strategies for generating vascular smooth muscle cells (VSMCs) from adult Wistar rats were systematically compared, viz: direct enzymatic isolation from the thoracic aorta and differentiation from bone marrow-derived mesenchymal stem cells (MSCs). Thoracic aorta-derived VSMCs, harvested by stepwise enzymatic (Collagenase Type 1 (3 mg/mL) and Elastase (1 mg/mL)) digestion for 60-90 min (until the tissue was fully dissolved), consistently exhibited classic spindle-shaped morphology and robust expression of contractile protein markers, namely, Smoothelin, and α-SMA. In parallel, MSCs isolated from rat bone marrow were expanded and then driven towards a smooth muscle phenotype through targeted exposure to TGF-β (10 ng/mL) and PDGF-BB (20 ng/mL) for 12-14 days. These differentiated cells acquired distinctive VSMC-like features (spindle shape, hill-and-valley growth) over time and showed negligible differences in VSMC-specific and contractile protein markers along with cellular behaviour compared to their aortic counterparts, albeit increased number of passages. Migration assays showed minor differences in migratory capacities between the two VSMC groups at P4, whereas, aortic VSMCs showed significantly faster colony forming capacities than the MSC-derived ones. Current findings highlight the critical influence of isolation and derivation method on the functional properties of VSMCs, emphasizing the need for tailored protocols in cardiovascular disease modelling and tissue engineering applications in atherosclerosis, vascular remodelling, and restenosis, etc. Moreover, this study will guide researchers with a framework for selecting reproducibly functional VSMCs for research applications and informed strategies for the scalable expansion and long-term banking (cryo-preservation) of either VSMC population. All animal experiments were conducted in compliance with the ARRIVE guidelines, the U.K. Animals (Scientific Procedures) Act (1986), EU Directive 2010/63/EU, and the NIH Guide for the Care and Use of Laboratory Animals.