Comparison of the depolarization response of human mesenchymal stem cells from different donors

HK-induced suppression of osteogenic differentiation was seen in three out of five donors as measured by IBSP expression, and five out of five donors as measured by calcium content (Figs 2A–E and 3A–E). When osteogenic cells were depolarized after pre-differentiation, four out of five donors exhibited lowered IBSP expression and four out of five donors exhibited lower calcium content compared to non-depolarized cells in four out of five donors. Interestingly, the donor that responded atypically was different for IBSP and calcium in this latter study, indicating that heterogeneity can be seen in the response of individual differentiation markers, rather than the entire osteogenic profile.

During adipogenic differentiation with HK, LPL suppression was observed in all five donors, while PPARG and FABP4 suppression were observed in four out of five donors. The atypical response for PPARG and FABP4 were both found in Donor E, where HK upregulated transcript expression instead of suppressing it. Evaluation of lipid accumulation by Oil Red O staining also revealed that Donor E cells did not exhibit an appreciable difference in staining between cells differentiated with or without HK. Thus, in contrast to osteogenic differentiation, the outlier donor for adipogenic differentiation was the same donor for all markers. After adipogenic pre-differentiation, HK-induced suppression was observed in three out of five donors for PPARG expression, and five out of five donors for both LPL and FABP4 expression. It is worth noting that PPARG is considered an early adipogenic marker, while LPL and FABP4 are markers for later stages of differentiation. This could explain why PPARG is less responsive to late depolarization in some donors: in these cells, perhaps the window of time for significant PPARG modulation has passed, and the cell instead modulates expression of the markers that are more relevant to the adipogenic stage at which HK is introduced (e.g., LPL and FABP4).

The present study focuses exclusively on inter-donor stem cell heterogeneity. However, additional studies in the literature have reported not only inter-donor stem cell variability, but also variability within a stem cell population from a single donor source. In vitro differentiation studies of bone marrow-derived hMSCs have shown that this population consists of a mixture of undifferentiated stem/progenitor cells and lineage-restricted precursors which have different capacities for differentiation toward osteogenic, adipogenic, and chondrogenic lineages17,19,49,50. Other adult stem cell populations exhibiting heterogeneity include intestinal stem cells and hematopoietic stem cells51. Similarly, cell populations differentiated from embryonic stem cells display heterogeneous phenotypes despite stimulation with specific growth factors52,53,54. Electrophysiological characterization of the phenotypically-distinct subpopulations within these stem cell populations may provide clues about how ion channels and currents contribute to the differences in their differentiation capacities and lineage biases, as has been shown in neuroblastoma cells55. Another source of heterogeneity may be the specific organ or anatomical location from which the cells are derived. Studies that compared mesenchymal stem cells derived from the bone marrow, adipose tissue, placenta, and umbilical cord56,57,58 reported differences in their proliferative and differentiation capacities. Similarly, fetal and adult human fibroblasts derived from skin at different anatomical locations displayed different characteristic gene expression patterns59.

While the data in the present study do not provide specific information about intra-population variability in hMSCs or about variability arising from different tissue sources, they do suggest that the assays that performed most consistently across donors may be the most useful for future studies addressing other sources of variability in hMSCs.

Future studies may also examine whether three-dimensional (3D) cultures of hMSCs would exhibit similar levels of heterogeneity during depolarization as the 2D cultures in this study. We previously developed a 3D culture system to study the effect of membrane potential stimulation on wound healing60. Interestingly, high K+ treatment had different effects depending on spatial location within the wound model (wound center vs. surrounding tissue). One potential explanation for the different observed responses is that the spatial variables introduced by the 3D culture system may intensify any intra-population differences. From these observations, if 3D models were used to compare different donors’ cells, we would expect that in addition to seeing variability among different donors, as in the current study, we would also observe increased intra-population variability within a single 3D culture system caused by spatial effects.

Overall, this study provided an analysis of hMSC heterogeneity across multiple donor sources, specifically with respect to their inherent differentiation potential and their differentiation response to electrophysiological perturbations. Our results indicate that, consistent with the literature, there was a baseline degree of variability among the donor cells’ ability to express tissue-specific markers. The specifics of this heterogeneity cannot always be summarized by identifying one outlier donor or one outlier assay, suggesting the need to systematically characterize each cell line that will be used for further analysis. Furthermore, there was additional variability in donor cell response to Vmem depolarization, and that this variability was not always predictable based on the inherent heterogeneity of hMSC differentiation capacity. However, for each donor, there was at least one osteogenic marker and one adipogenic marker that responded to depolarization in the expected manner. Based on this screen, we suggest that IBSP expression, calcium deposition, LPL expression, and FABP4 expression are the preferred markers for assessing differentiation response to Vmem depolarization, but a full characterization of a cell source with all markers is preferred. Donor-specific characterization of primary stem cells will be increasingly important as stem cell transplantation and personalized medicine strategies are being developed for clinical therapies.

How to cite this article: Sundelacruz, S. et al. Comparison of the depolarization response of human mesenchymal stem cells from different donors. Sci. Rep. 5, 18279; doi: 10.1038/srep18279 (2015).

We thank the NIH for support through the Tissue Engineering Resource Center (P41 EB002520), R01 AR005593, and R01 AR061988; the G. Harold and Leila Y. Mathers Charitable Foundation; and the W. M. Keck Foundation. Acknowledgement of grants: NIH (Tissue Engineering Resource Center (P41 EB002520), R01 AR005593, and R01 AR061988), G. Harold and Leila Y. Mathers Charitable Foundation, W. M. Keck Foundation.