Adipose tissue as a stem cell source is ubiquitously available and has several advantages compared to other sources. It is easily accessible in large quantities with minimal invasive harvesting procedure, and isolation of adipose-derived mesenchymal stromal/stem cells (ASCs) yields a high amount of stem cells, which is essential for stem-cell-based therapies and tissue engineering. Several studies have provided evidence that ASCs in situ reside in a perivascular niche, whereas the exact localization of ASCs in native adipose tissue is still under debate. ASCs are isolated by their capacity to adhere to plastic. Nevertheless, recent isolation and culture techniques lack standardization. Cultured cells are characterized by their expression of characteristic markers and their capacity to differentiate into cells from meso-, ecto-, and entodermal lineages. ASCs possess a high plasticity and differentiate into various cell types, including adipocytes, osteoblasts, chondrocytes, myocytes, hepatocytes, neural cells, and endothelial and epithelial cells. Nevertheless, recent studies suggest that ASCs are a heterogeneous mixture of cells containing subpopulations of stem and more committed progenitor cells. This paper summarizes and discusses the current knowledge of the tissue localization of ASCs in situ, their characterization and heterogeneity in vitro, and the lack of standardization in isolation and culture methods.

Initial in vitro investigations demonstrated type I interferons (IFNs: IFN-alpha, IFN-beta) to inhibit replication of SARS coronavirus (SARS-CoV), but found the nucleoside analogue ribavirin ineffective in Vero cells. In this report, ribavirin was shown to inhibit SARS-CoV replication in five different cell types of animal or human origin at therapeutically achievable concentrations. Since clinical anti-SARS-CoV activity of type I interferons or ribavirin is limited, we investigated the combination of IFN-beta and ribavirin. Determination of the virus yield indicated highly synergistic anti-SARS-CoV action of the combination suggesting the consideration of ribavirin plus IFN-beta for the treatment of SARS.

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Adipose tissue is a rich, ubiquitous and easily accessible source for multipotent stromal/stem cells and has, therefore, several advantages compared to other sources of mesenchymal stromal/stem cells. Several studies have tried to identify the origin of the stromal/stem cell population within adipose tissue in situ. This is a complicated attempt because no marker has currently been described which unambiguously identifies native adipose-derived stromal/stem cells (ASCs). Isolated and cultured ASCs are a non-uniform preparation consisting of several subsets of stem and precursor cells. Cultured ASCs are characterized by their expression of a panel of markers (and the absence of others), whereas their in vitro phenotype is dynamic. Some markers were expressed de novo during culture, the expression of some markers is lost. For a long time, CD34 expression was solely used to characterize haematopoietic stem and progenitor cells, but now it has become evident that it is also a potential marker to identify an ASC subpopulation in situ and after a short culture time. Nevertheless, long-term cultured ASCs do not express CD34, perhaps due to the artificial environment. This review gives an update of the recently published data on the origin and phenotype of ASCs both in vivo and in vitro. In addition, the composition of ASCs (or their subpopulations) seems to vary between different laboratories and preparations. This heterogeneity of ASC preparations may result from different reasons. One of the main problems in comparing results from different laboratories is the lack of a standardized isolation and culture protocol for ASCs. Since many aspects of ASCs, such as the differential potential or the current use in clinical trials, are fully described in other recent reviews, this review further updates the more basic research issues concerning ASCs' subpopulations, heterogeneity and culture standardization.

Mesenchymal stem/stromal cells (MSCs) are becoming increasingly important for the development of cell therapeutics in regenerative medicine. Featuring immunomodulatory potential as well as secreting a variety of trophic factors, MSCs showed remarkable therapeutic effects in numerous preclinical disease models. However, sustainable translation of MSC therapies to the clinic is hampered by heterogeneity of MSCs and non-standardized in vitro culture technologies. Moreover, potent MSC therapeutics require MSCs with maximum regenerative capacity. There is growing evidence that in vitro preconditioning strategies of MSCs can optimize their therapeutic potential. In the following we will discuss achievements and challenges of the development of MSC therapies in regenerative medicine highlighting specific in vitro preconditioning strategies prior to cell transplantation to increase their therapeutic efficacy.

After collagenase digestion and Percoll density gradient centrifugation of human renal tissue, tubular epithelial cells of the proximal and the distal segments were isolated with an immunomagnetic method using MACS microbeads. To enrich proximal tubular (PT) cells we used a monoclonal antibody (mAb) against aminopeptidase M (APM, CD 13), specific of the proximal tubule. Distal tubular (DT) cells were isolated through a mAb recognizing Tamm-Horsfall glycoprotein (THG), a specific antigen for the thick ascending limb and the early distal convoluted tubule. Cells of the proximal primary isolate were histochemically strongly positive for aminopeptidase M (98.6%), however, cells of the distal portion were negative (98.7%). Ultrastructural analysis of PTC primary isolates revealed highly preserved brush border microvilli, well-developed endocytosis apparati and numerous mitochondria, whereas DTC primary isolates showed smaller cells with basolateral invaginations and less apical microvilli. Characterization by immunofluorescence indicated the coexpression of cytokeratin and vimentin, whereas staining for desmin, smooth muscle actin, a fibroblast-specific marker and von Willebrand factor was negative. Cultured PT and DT cells displayed different adenylate cyclase responsiveness to hormonal stimulation. PTH (10(-6) M) increased cAMP production in distal cells up to 32.8-fold of the basal level and in proximal only up to 3.5-fold (10(-8) M, DT 14.4x and PT 2.25x). Calcitonin stimulated adenylate cyclase in DT in a dose dependent fashion (10(-6) M, 4.3x; 10(-8) M, 2.25x), whereas only a low calcitonin response was found in PT cells (10(-6) M, 1.6x; 10(-8) M, 1.4x). AVP (10(-6) M) activated the distal cAMP-production only up to 1.9x of the basal level, but the proximal cAMP-production was negligible (only 1.3x the basal level). The data of this study indicate the proximal and distal tubule origin of the cultured cells that were isolated according to their segment-specific antigens.

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The characterization of adipose-derived stromal/stem cells (ASCs) remains difficult due to the lack of a definitive and unique cellular marker. Therefore, a combination of markers is necessary to identify the cells. No comprehensive analysis of the immunophenotype of expanded plastic adherent ASCs has been published. Therefore, the aim of this study was to characterize the general phenotype of cultured ASCs and to further analyze cellular subsets. ASCs were isolated from lipoaspirates from patients undergoing cosmetic liposuction and cultured in standard cell culture. A comprehensive phenotype characterization was done with the BD Lyoplate™ Human Cell Surface Marker Screening Panel containing 242 antibodies and isotype controls. Cultured ASCs not only showed the characteristic expression profile of mesenchymal stem cells (MSCs), but also revealed donor-specific variability in the expression of 49 other markers. We further detected markers with a scattering in the fluorescence intensity, indicating subpopulations with different expression profiles. Therefore, a multi-color flow cytometric analysis was done after staining the cells with direct-labeled antibodies against CD73, CD90, CD105, and either CD34, CD140b, CD200, CD201, or CD36 to verify the selected subpopulations of ASCs. We detected no CD34-CD36 double-positive population, but CD34(+)-CD36(-) and CD34(-)CD36(+) subpopulations, both of which are positive for the 3 main MSC markers, CD73, CD90, and CD105. All other detected subpopulations also co-expressed the 3 main MSC markers, and therefore fulfill the minimal phenotypic criteria for the definition of cultured MSCs. Our study demonstrates the first comprehensive phenotypic characterization of ASCs and clearly highlights donor-specific variability in ASC preparations.