BackgroundAdipose-derived stem cells (ASCs) are being increasingly recognized for their potential to promote tissue regeneration and wound healing. These effects appear to be partly mediated by paracrine signaling pathways, and are enhanced during hypoxia. Mass spectrometry (MS) is a valuable tool for proteomic profiling of cultured ASCs, which may help to reveal the identity of the factors secreted by the cells under different conditions. However, serum starvation which is essentially required to obtain samples compatible with secretome analysis by MS can have a significant influence on ASCs. Here, we present a novel and optimized culturing approach based on the use of a clinically relevant serum-free formulation, which was used to assess the effects of hypoxia on the ASC proteomic profile.MethodsHuman ASCs from three human donors were expanded in StemPro® MSC SFM XenoFree medium. Cells were cultured for 24 h in serum- and albumin-free supplements in either normoxic (20 %) or hypoxic (1 %) atmospheres, after which the cells and conditioned medium were collected, subfractionated, and analyzed using MS. Prior to analysis, the secreted proteins were further subdivided into a secretome (>30 kDa) and a peptidome (3–30 kDa) fraction.ResultsMS analysis revealed the presence of 342, 98, and 3228 proteins in the normoxic ASC secretome, peptidome, and proteome, respectively. A relatively small fraction of the proteome (9.6 %) was significantly affected by hypoxia, and the most regulated proteins were those involved in extracellular matrix (ECM) synthesis and cell metabolism. No proteins were found to be significantly modulated by hypoxic treatment across all cultures for the secretome and peptidome samples.ConclusionsThis study highlights ECM remodeling as a significant mechanism contributing to the ASC regenerative effect after hypoxic preconditioning, and further underscores considerable inter-individual differences in ASC response to hypoxia. The novel culture paradigm provides a basis for future proteomic studies under conditions that do not induce a stress response, so that the best responders can be accurately identified for prospective therapeutic use.Data are available via ProteomeXchange with identifier PXD003550.Electronic supplementary materialThe online version of this article (doi:10.1186/s13287-016-0310-7) contains supplementary material, which is available to authorized users.

Since the discovery of adipose-derived stem cells (ASCs), there have been high expectations of their putative clinical use. Recent advances support these expectations, and it is expected that the transition from pre-clinical and clinical studies to implementation as a standard treatment modality is imminent. However ASCs must be isolated and expanded according to good manufacturing practice guidelines and a basic assurance of quality, safety, and medical effectiveness is needed for authorisation by regulatory agencies, such as European Medicines Agency and US Food and Drug Administration. In this review, a collection of studies investigating the influence of different steps of the isolation and expansion protocol on the yield and functionality of ASCs has been presented in an attempt to come up with best recommendations that ensure potential beneficial clinical outcome of using ASCs in any therapeutic setting. If the findings confirm the initial observations of beneficial effects of ASCs, the path is paved for implementing these ASC-based therapies as standard treatment options.

Adipose-derived stem cells (ASCs) are being tested in clinical trials related to cell-based regenerative therapies. Although most of the current expansion protocols for ASCs use fetal calf serum (FCS), xenogeneic-free medium supplements are greatly desired. This study aims to compare the effect of FCS, human platelet lysate (hPL), and a fully defined medium on the initiation and maintenance of ASC cultures. ASCs obtained from five donors were cultured in five different media: StemPro, Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% hPL, or α-minimum essential medium (A-MEM) supplemented with 5% hPL, 10% hPL, or 10% FCS. The effect of media on proliferation, colony-forming units (CFUs), attachment, and morphology was assessed along with cell size, granularity, and immunophenotype. StemPro greatly compromised the initiation of ASC cultures, which could not survive more than a few passages. Cells cultured in A-MEM proliferated at a faster rate than in DMEM, and hPL significantly enhanced cell size, granularity, and proliferation compared with FCS. All media except StemPro supported CFUs equally well. Analysis of surface markers revealed higher levels of CD73 and CD105 in FCS-cultured ASCs, whereas increased levels of CD146 were found in hPL-cultured cells. Multiparametric flow cytometric analysis performed after seven passages revealed the existence of four distinct ASC subpopulations, all positive for CD73, CD90, and CD105, which mainly differed by their expression of CD146 and CD271. Analysis of the different subpopulations might represent an important biological measure when assessing different medium formulations for a particular clinical application.

BackgroundHuman adipose stem cells (hASCs) can promote angiogenesis through secretion of proangiogenic factors such as vascular endothelial growth factor (VEGF). In other cell types, it has been shown that induction of VEGF is mediated by both protease activated receptor 2 (PAR2) and hypoxia inducible factor 1(HIF-1). The present study hypothesized that PAR2 stimulation through activation of kinase signaling cascades lead to induction of HIF-1 and secretion of VEGF.Methodology/Principal FindingsImmunohistochemistry revealed the expression of PAR2 receptors on the surface of hASCs. Blocking the PAR2 receptors with a specific antibody prior to trypsin treatment showed these receptors are involved in trypsin-evoked increase in VEGF secretion from hASCs. Blocking with specific kinase inhibitors suggested that that activation of MEK/ERK and PI3-kinase/Akt pathways are involved in trypsin-eveoked induction of VEGF. The effect of the trypsin treatment on the transcription of VEGF peaked at 6 hours after the treatment and was comparable to the activation observed after keeping hASCs for 24 hours at 1% oxygen. In contrast to hypoxia, trypsin alone failed to induce HIF-1 measured with ELISA, while the combination of trypsin and hypoxia had an additive effect on both VEGF transcription and secretion, results which were confirmed by Western blot.ConclusionIn hASCs trypsin and hypoxia induce VEGF expression through separate pathways.

BackgroundComplex immunophenotypic repertoires defining discrete adipose-derived stem cell (ASC) subpopulations may hold a key toward identifying predictors of clinical utility. To this end, we sorted out of the freshly established ASCs four subpopulations (SPs) according to a specific pattern of co-expression of six surface markers, the CD34, CD73, CD90, CD105, CD146, and CD271, using polychromatic flow cytometry.MethodUsing flow cytometry-associated cell sorting and analysis, gating parameters were set to select for a CD73+CD90+CD105+ phenotype plus one of the four following combinations, CD34−CD146−CD271− (SP1), CD34−CD146+CD271− (SP2), CD34+CD146+CD271− (SP3), and CD34−CD146+CD271+ (SP4). The SPs were expanded 700- to 1000-fold, and their surface repertoire, trilineage differentiation, and clonogenic potential, and the capacity to support wound healing were assayed.ResultsUpon culturing, the co-expression of major epitopes, the CD73, CD90, and CD105 was maintained, while regarding the minor markers, all SPs reverted to resemble the pre-sorted population with CD34−CD146−CD271− and CD34−CD146+CD271− representing the most prevalent combinations, followed by less frequent CD34+CD146−CD271− and CD34+CD146+CD271− variants. There was no difference in the efficiency of adipo-, osteo-, or chondrogenesis by cytochemistry and real-time RT-PCR or the CFU capacity between the individual SPs, however, the SP2CD73+90+105+34-146+271- outperformed others in terms of wound healing.ConclusionsOur study shows that ASCs upon culturing inherently maintain a stable distribution of immunophenotype variants, which may potentially disguise specific functional properties of particular downstream lines. Furthermore, the outlined approach suggests a paradigm whereby discrete subpopulations could be identified to provide for a therapeutically most relevant cell product.Electronic supplementary materialThe online version of this article (doi:10.1186/s13287-016-0435-8) contains supplementary material, which is available to authorized users.