For the accurate determination of gene expression changes during growth and differentiation studies on adipose-derived stem cells (ASCs), quantitative real-time RT-PCR has become a method of choice. The technology is very sensitive, however, without a proper selection of reference genes, to which the genes of interest are normalized, erroneous results may be obtained.

Studies of mesenchymal stromal cells (MSC) from BM and adipose tissue have demonstrated similar differentiation potentials along the adipo-, osteo- and chondrogenic lineages. While most clinical trials have been performed using BM-derived MSC, the focus is shifting toward the use of stem cells derived from fat tissue. The aim of the current investigation was to define optimal culture conditions that would facilitate clinical use of adipose-derived stem cells (ASC). Different types and concentrations of serum replacers and basal media were tested with respect to the optimal expansion and subsequent differentiation of primary human ASC. The effect of initial seeding density on the growth of ASC was also determined. While several of the serum replacements proved to be clearly inferior to fetal calf serum (FCS) in promoting ASC growth, the knockout serum replacement (KOSR) had expansion properties similar to those of FCS. However, with respect to the capacity to support adipo-, osteo- and chondrogenic differentiation, KOSR proved to be less consistent than FCS. Among the media formulations, modified Eagle medium alpha supported a significantly faster cell expansion than the other basal media while still maintaining the full differentiation potential of ASC. Regarding the plating density most favorable for rapid expansion, we found that initial plating densities ranging from 100 to 200 cell/cm² resulted in significantly shorter doubling times than plating densities both below and above that range. Identification of the optimal basal medium and serum replacer, together with the most favorable plating density, will facilitate cell-based and tissue-engineering applications employing ASC in pre-clinical and clinical settings.

Ex vivo engineering of skeletal muscle represents an exciting new area of biotechnology. Although the ability of skeletal muscle cells to sense and respond to mechanical forces is well known, strategies based on the use of mechanical stimuli to optimize myogenesis in vitro remain limited. In this work, we describe a simple but powerful method based on uniaxial cyclic tensile strain (CTS) to induce assembly and differentiation of skeletal myocytes in vitro. Confluent mouse myoblastic precursors cultured on flexible-bottomed culture plates were subjected to either uniaxial or equibiaxial CTS. The uniaxial CTS protocol resulted in a highly aligned array of cross-striated fibers, with the major axis of most cells aligned perpendicularly to the axis of strain. In addition, a short period of myogenin activation and significant increase in the myotube/myoblast ratio and percentage of myosin-positive myotubes was found, indicating an enhanced cell differentiation. In contrast, cells under equibiaxial strain regimen had no clear orientation and displayed signs of membrane damage and impaired differentiation. These results, thus, demonstrate that the selection of a proper paradigm is a key element when discussing the relevance of mechanical stimulation for myogenesis in vitro. This study provides a rational framework to optimize engineering of functional skeletal muscle.

The synthesis and deposition of extracellular matrix (ECM) plays an important role in the healing of acute and chronic wounds. Consequently, the use of ECM as treatment for chronic wounds has been of special interest—both in terms of inducing ECM production by resident cells and applying ex vivo produced ECM. For these purposes, using adipose tissue-derived stem cells (ASCs) could be of use. ASCs are recognized to promote wound healing of otherwise chronic wounds, possibly through the reduction of inflammation, induction of angiogenesis, and promotion of fibroblast and keratinocyte growth. However, little is known regarding the importance of ASC-produced ECM for wound healing. In this review, we describe the importance of ECM for wound healing, and how ECM production by ASCs may be exploited in developing new therapies for the treatment of chronic wounds.

Background: Adipose-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. Methods: Human 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. Results: MS 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. Conclusions: This 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.