Extracellular vesicles (EVs) play key roles in cell biology and may provide new clinical diagnostics and therapies. However, it has proven difficult to develop protocols for their purification and characterization. One of the major barriers in the field has been a lack of convenient assays for their bioactivity. Developing assays has not been a trivial matter, because of the heterogeneity of EVs, the multiple activities they demonstrate, and the uncertainty about their modes of action. Therefore, it is likely that multiple assays for their activities are needed. One important assay will be for the anti-inflammatory activity observed in mice after administration of the small EVs commonly referred to as exosomes. We developed an assay for the anti-inflammatory activity of exosomes with a line of mouse macrophages. The assay makes it possible to rank different preparations of exosomes by their anti-inflammatory activity, and their ranking predicts their efficacy in suppressing LPS-stimulated inflammation in mice. The assay is convenient for comparing multiple samples and, therefore, should be useful in developing protocols for the purification and characterization of anti-inflammatory exosomes.

We were interested in evaluating the ability of the mesenchymal stromal cell (MSC) population, human umbilical cord perivascular cells (HUCPVCs), to undergo cardiomyocyte reprogramming in an established coculture system with rat embryonic cardiomyocytes. Results were compared with human bone marrow-derived (BM) MSCs. The transcription factors GATA4 and Mef 2c were expressed in HUCPVCs but not BM-MSCs at baseline and, at 7 days, increased 7.6- and 3.5-fold, respectively, compared with BM-MSCs. Although cardiac-specific gene expression increased in both cell types in coculture, upregulation was more significant in HUCPVCs, consistent with Mef 2c-GATA4 synergism. Using a lentivector with eGFP transcribed from the α-myosin heavy chain (α-MHC) promoter, we found that cardiac gene expression was greater in HUCPVCs than BM-MSCs after 14 days coculture (52 ± 17% vs. 29 ± 6%, respectively). A higher frequency of HUCPVCs expressed α-MHC protein compared with BM-MSCs (11.6 ± 0.9% vs. 5.3 ± 0.3%); however, both cell types retained MSC-associated determinants. We also assessed the ability of the MSC types to mediate cardiac regeneration in a NOD/SCID γ mouse model of acute myocardial infarction (AMI). Fourteen days after AMI, cardiac function was significantly better in cell-treated mice compared with control animals and HUCPVCs exhibited greater improvement. Although human cells persisted in the infarct area, the frequency of α-MHC expression was low. Our results indicate that HUCPVCs exhibit a greater degree of cardiomyocyte reprogramming but that differentiation for both cell types is partial. We conclude that HUCPVCs may be preferable to BM-MSCs in the cell therapy of AMI.

Human umbilical cord perivascular cells (HUCPVCs) are a readily available source of mesenchymal stromal cells (MSCs) for cell therapy. We were interested in understanding how differences from human bone marrow (BM)-derived MSCs might yield insights into MSC biology. We found that HUCPVCs exhibited increased telomerase activity and longer telomeres compared with BM-MSCs. We also observed enhanced expression of the pluripotency factors OCT4, SOX2, and NANOG in HUCPVCs. The methylation of OCT4 and NANOG promoters was similar in both cell types, indicating that differences in the expression of pluripotency factors between the MSCs were not associated with epigenetic changes. MSC methylation at these loci is greater than reported for embryonic stem cells but less than in dermal fibroblasts, suggesting that multipotentiality of MSCs is epigenetically restricted. These results are consistent with the notion that the MSC population (whether BM- or HUCPV-derived) exhibits higher proliferative capacity and contains more progenitor cells than do dermal fibroblasts.