Abstract
BackgroundHuman mesenchymal stromal cells (hMSC) are multipotent cells with both regenerative and immunomodulatory activities making them an attractive tool for cellular therapy. In the last few years it has been shown that the beneficial effects of hMSC may be due to paracrine effects and, at least in part, mediated by extracellular vesicles (EV). EV have emerged as important mediators of cell-to-cell communication. Flow cytometry (FCM) is a routine technology used in most clinical laboratories and could be used as a methodology for hMSC-EV characterization. Although several reports have characterized EV by FCM, a specific panel and protocol for hMSC-derived EV is lacking. The main objective of our study was the characterization of hMSC-EV using a standard flow cytometer.MethodsHuman MSC from bone marrow of healthy donors, mesenchymal cell lines (HS-5 and hTERT) and a leukemic cell line (K562 cells) were used to obtain EV for FCM characterization. EV released from the different cell lines were isolated by ultracentrifugation and were characterized, using a multi-parametric analysis, in a conventional flow cytometer. EV characterization by transmission electron microscopy (TEM), western blot (WB) and Nano-particle tracking analysis (NTA) was also performed.ResultsEV membranes are constituted by the combination of specific cell surface molecules depending on their cell of origin, together with specific proteins like tetraspanins (e.g. CD63). We have characterized by FCM the EV released from BM-hMSC, that were defined as particles less than 0.9 μm, positive for the hMSC markers (CD90, CD44 and CD73) and negative for CD34 and CD45 (hematopoietic markers). In addition, hMSC-derived EV were also positive for CD63 and CD81, the two characteristic markers of EV. To validate our characterization strategy, EV from mesenchymal cell lines (hTERT/HS-5) were also studied, using the leukemia cell line (K562) as a negative control. EV released from mesenchymal cell lines displayed the same immunophenotypic profile as the EV from primary BM-hMSC, while the EV derived from K562 cells did not show hMSC markers. We further validated the panel using EV from hMSC transduced with GFP.Finally, EV derived from the different sources (hMSC, hTERT/HS-5 and K562) were also characterized by WB, TEM and NTA, demonstrating the expression by WB of the exosomal markers CD63 and CD81, as well as CD73 in those from MSC origin. EV morphology and size/concentration was confirmed by TEM and NTA, respectively.ConclusionWe described a strategy that allows the identification and characterization by flow cytometry of hMSC-derived EV that can be routinely used in most laboratories with a standard flow cytometry facility.Electronic supplementary materialThe online version of this article (doi:10.1186/s12964-015-0124-8) contains supplementary material, which is available to authorized users.
Highlights
Human mesenchymal stromal cells are multipotent cells with both regenerative and immunomodulatory activities making them an attractive tool for cellular therapy
Results Human mesenchymal stromal cells (hMSC) definition criteria hMSC were isolated and expanded from bone marrow (BM) samples and further characterized according to International Society for Cellular Therapy (ISCT) minimal definition criteria [28]. These cells did not express hematopoietic lineage markers such as CD14, CD19, CD34, CD45 and HLA-DR, and were positive for CD73, CD90, CD105, CD166 and CD44 thereby demonstrating a characteristic immunophenotype of hMSC In addition, their multilineage differentiation ability was demonstrated (Fig. 2)
HTERT and H-S5 were inmunophenotypically characterized using the same panel used for hMSC from BM
Summary
Human mesenchymal stromal cells (hMSC) are multipotent cells with both regenerative and immunomodulatory activities making them an attractive tool for cellular therapy. Human mesenchymal stromal cells (hMSC) are multipotent adult stem cells that are able of differentiate into most mesodermic cells [1] and display a potent immunomodulatory activity [2, 3]. These two properties make them an attractive potential therapeutic tool for cell therapy programs. Shedding vesicles are generated by budding from the plasma membrane They are heterogeneous since their size (80–1000 nm) depends on their cell of origin as well as the releasing stimuli [13]. EV membranes are enriched in cholesterol, sphingomyelin, and ceramide (lipid rafts) [16,17,18,19]
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