Human mesenchymal stem cells (hMSCs) are widely used in regenerative medicine, but large-scale in vitro expansion alters their function, impacting proliferation and differentiation potential. Currently, a predictive marker to assess these changes is lacking. Here, we used dielectrophoresis (DEP) to characterize the electrical phenotype of hMSCs derived from bone marrow (BM), adipose tissue (AT), and umbilical cord (UC) as they aged in vitro from passage 4 (P4) to passage 9 (P9). The electrical phenotype was defined by the DEP spectra, membrane capacitance, and cytoplasm conductivity. Cell morphology and size, growth characteristics, adipogenic differentiation potential, and osteogenic differentiation potential were assessed alongside label-free biomarker membrane capacitance and cytoplasm conductivity. Differentiation was confirmed by histological staining and RT-qPCR. All hMSCs exhibited typical morphology, though cell size varied, with UC-hMSCs displaying the largest variability across all size metrics. Growth analysis revealed that UC-hMSCs proliferated the fastest. The electrical phenotype varied with cell source and in vitro age, with high passage hMSCs showing noticeable shifts in DEP spectra, membrane capacitance, and cytoplasm conductivity. Correlation analysis revealed that population doubling level (PDL) correlated with membrane capacitance and cytoplasm conductivity, indicating PDL as a more precise marker of in vitro aging than passage number. Additionally, we demonstrate that membrane capacitance correlates with the osteogenic marker COL1A1 and that cytoplasm conductivity correlates with the adipogenic markers ADIPOQ and FABP4, suggesting that DEP-derived electrical properties serve as label-free biomarkers of differentiation potential. While DEP has previously been applied to BM-hMSCs and AT-hMSCs, and more recently to UC-hMSCs, few studies have provided a direct comparison across all three sources or tracked changes across continuous expansion. These findings underscore the utility of DEP as a label-free approach for assessing hMSC aging and function, offering practical applications for optimizing stem cell expansion and stem cell banking in clinical settings.

Cellular heterogeneity, an inherent feature of biological systems, plays a critical role in processes such as development, immune response, and disease progression. Human mesenchymal stem cells (hMSCs) exemplify this heterogeneity due to their multi-lineage differentiation potential. However, their inherent variability complicates clinical use, and there is no universally accepted method for detecting and quantifying cell population heterogeneity. Dielectrophoresis (DEP) has emerged as a powerful electrokinetic technique for characterizing and manipulating cells based on their dielectric properties, offering label-free analysis capabilities. Quantitative information from the DEP spectrum, such as transient slope, measure cells’ transition between negative and positive DEP behaviors. In this study, we employed DEP to estimate transient slope of various cell populations, including relatively homogeneous HEK-293 cells, heterogeneous hMSCs, and cancer cells (PC3 and DU145). Our analysis encompassed hMSCs derived from bone marrow, adipose, and umbilical cord tissue, to capture tissue-specific heterogeneity. Transient slope was assessed using two methods, involving linear trendline fitting to different low-frequency regions of the DEP spectrum. We found that transient slope serves as a reliable indicator of cell population heterogeneity, with more heterogeneous populations exhibiting lower transient slopes and higher standard deviations. Validation using cell morphology, size, and stemness further supported the utility of transient slope as a heterogeneity metric. This label-free approach holds promise for advancing cell sorting, biomanufacturing, and personalized medicine.