Background <p>Several studies have suggested that adult human dermal fibroblasts (HDFa) may be a potential alternative source to mesenchymal stem cells for cell therapies. This study aims to characterize HDFa, adipose-derived stem cells (ADMSCs) and dental pulp stem cells (DPSCs) to investigate their proliferation, differentiation potential, mitochondrial respiration, and metabolomic profile. We identified molecules and characteristics that would differentiate MSCs from different sources or confirm their uniformity.</p> Methods <p>Differentiation was induced using osteogenic and adipogenic differentiation media. Proteins specific to each differentiation process were monitored by immunofluorescence staining. High-resolution respirometry and targeted metabolomic analysis using the AbsoluteIDQ^®p180 kit (Biocrates Life Science) were applied to identify the essential properties of the studied cells.</p> Results <p>HDFa cells, ADMSCs, and DPSCs demonstrated morphological characteristics of mesenchymal stem cells (MSCs). In general, DPSCs and HDFa showed significantly higher proliferation than ADMSCs. Osteogenic and adipogenic capacities were similar for all cell origins after 21 days, but ADMSCs exhibited earlier calcium deposit formation. FoxO1 and adiponectin as osteogenic and adipogenic-related proteins confirmed differentiation processes. High-resolution respirometry and metabolomic analysis showed potential distinguished characterization, mainly for DPSC cells. Our results also demonstrated that lipid profiling could be a promising tool for MSC characterization.</p> Conclusions <p>Our analyzed data suggest that HDFa have properties similar to DPSCs and ADMSCs. However, each cell type has been shown to have unique specific characteristics. The similarities and differences in the characteristics of HDFa, ADMSCs, and DPSCs should be studied in detail and in a larger cohort when planning stem cell-based therapy.</p> Graphical Abstract <p></p>