Abstract
BACKGROUND: In tissue regeneration, as well as in post-traumatic recovery or in treating pathological alterations, mesenchymal stromal cells (MSCs) and their products for cell-free treatments are increasingly attractive and applicable. For this reason, there is an urgent need to thoroughly investigate MSCs of different origins, especially those readily available and with no ethical concerns obtained from healthy donors. METHODS: Human MSCs were derived from discarded adipose tissue of four donors (ADSCs; 8 cell populations isolated by enzymatic digestion and mechanical fragmentation) and dental pulp of two donors (DPSCs; 4 cell populations from radicular and coronal compartments by mechanical fragmentation). Cells were characterized by differentiation, proliferation, and morphological features. Conditioned media (CM) were collected, and the secretome profile analyzed. RESULTS: The trilineage differentiation assay and CD immunophenotyping showed that all primary cell lines possessed typical MSC characteristics, apart from the inability of DPSCs to perform adipogenesis. Significant CD differences were found mainly due to tissue source and regional compartments regarding coronal vs. radicular dental pulp. Notably, DPSCs were consistently smaller, Nestin-positive, and had a higher proliferation rate than ADSCs. Secretome analysis regarding anti-inflammatory and pro-inflammatory cytokines, chemokines, and growth factors accumulating in the CM throughout the culture showed significant variations among MSC lines from the two tissues and within ADSCs obtained with different extraction methods. All MSC populations release a comparable number of extracellular vesicles (EVs), although ADSCs appeared to produce a significantly higher number of smaller exosomes than DPSCs. Depending on the tissue of origin, MSCs released specific sets of microRNAs, either free or enclosed in EVs, impacting many cellular processes. The microRNAs more expressed from DPSCs are mainly involved in oxidative stress and apoptosis pathways, while those of ADSCs play a regulatory role in cell cycle and proliferation. CONCLUSIONS: The results support the notion that, despite their common characteristics, MSCs can differ in many aspects related to their ontogeny, extraction method, and, to a lesser extent, regionalization and donor heterogeneity. These findings pose challenges for the clinical translation of MSCs, their CMs, and derivatives and underline the importance of standardizing protocols to obtain MSC products from their secretome. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s13062-025-00697-w.