Isolation, cultivation, and characterization of human mesenchymal stem cells.

Mesenchymal stem cells (MSC) exhibit a high self-renewal capacity, multilineage differentiation potential and immunomodulatory properties. This set of exceptional features makes them an attractive tool for research and clinical application. However, MSC are far from being a uniform cell type, which makes standardization difficult. The exact properties of human MSC (hMSC) can vary greatly depending on multiple parameters including tissue source, isolation method and medium composition. In this review we address the most important influence factors. We highlight variations in the differentiation potential of MSC from different tissue sources. Furthermore, we compare enzymatic isolation strategies with explants cultures focusing on adipose tissue and umbilical cords as two relevant examples. Additionally, we address effects of medium composition and serum supplementation on MSC expansion and differentiation. The lack of standardized methods for hMSC isolation and cultivation mandates careful evaluation of different protocols regarding efficiency and cell quality. MSC characterization based on a set of minimal criteria defined by the International Society for Cellular Therapy is a widely accepted practice, and additional testing for MSC functionality can provide valuable supplementary information. The MSC secretome has been identified as an important signaling mechanism to affect other cells. In this context, extracellular vesicles (EVs) are attracting increasing interest. The thorough characterization of MSC-derived EVs and their interaction with target cells is a crucial step toward a more complete understanding of MSC-derived EV functionality. Here, we focus on flow cytometric approaches to characterize free as well as cell bound EVs and address potential differences in the bioactivity of EVs derived from stem cells from different sources. © 2017 International Society for Advancement of Cytometry.

Ex vivo expansion of haematopoietic stem/progenitor cells from human umbilical cord blood on acellular scaffolds prepared from MS-5 stromal cell line.

Lineage-specific expansion of haematopoietic stem/progenitor cells (HSPCs) from human umbilical cord blood (UCB) is desirable because of their several applications in translational medicine, e.g. treatment of cancer, bone marrow failure and immunodeficiencies. The current methods for HSPC expansion use either cellular feeder layers and/or soluble growth factors and selected matrix components coated on different surfaces. The use of cell-free extracellular matrices from bone marrow cells for this purpose has not previously been reported. We have prepared insoluble, cell-free matrices from a murine bone marrow stromal cell line (MS-5) grown under four different conditions, i.e. in presence or absence of osteogenic medium, each incubated under 5% and 20% O2 tensions. These acellular matrices were used as biological scaffolds for the lineage-specific expansion of magnetically sorted CD34⁺ cells and the results were evaluated by flow cytometry and colony-forming assays. We could get up to 80-fold expansion of some HSPCs on one of the matrices and our results indicated that oxygen tension played a significant role in determining the expansion capacity of the matrices. A comparative proteomic analysis of the matrices indicated differential expression of proteins, such as aldehyde dehydrogenase and gelsolin, which have previously been identified as playing a role in HSPC maintenance and expansion. Our approach may be of value in identifying factors relevant to tissue engineering-based ex vivo HSPC expansion, and it may also provide insights into the constitution of the niche in which these cells reside in the bone marrow.