Mass spectrometry analysis of adipose-derived stem cells reveals a significant effect of hypoxia on pathways regulating extracellular matrix

Abstract

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.

Histone Variants as Stem Cell Biomarkers for Long‐Term Injection Medialization Laryngoplasty

“…An initial set of validating experiments (not shown) was performed to compare the ADSC protein yield with results from the literature. Typically, ∼3,000 proteins were identified for rabbit ADSCs in tissue culture, in agreement with previously reported data for human ADSCs . Furthermore, a similar number of proteins with > two unique peptides was identified in tissue culture and excised laryngeal ellipsoids at 12 weeks post‐IML ADSC injection (tissue culture: 1,883 ± 279; rabbit larynges: 1,327 ± 204).…”

“…Typically, 3,000 proteins were identified for rabbit ADSCs in tissue culture, in agreement with previously reported data for human ADSCs. 11 Furthermore, a similar number of proteins with > two unique peptides was identified in tissue culture and excised laryngeal ellipsoids at 12 weeks post-IML ADSC injection (tissue culture: 1,883 ± 279; rabbit larynges: 1,327 ± 204). Figure 2 shows results from one of two independent nLC-MS/MS runs, in which the number of unique peptides is plotted relative to the total number of identified proteins as well as the overlap between the two runs.…”

“…This facilitates collection of tissue sections following from slides that were previously subjected to routine protocols for fixation, paraffin embedding, and sectioning. A limited number of slides (10)(11)(12)(13)(14)(15) contained sufficient material to generate reproducible proteomics data in two independent runs. In the MACD+ADSC group, we identified CD9, CD44, fibronectin, and vimentin, which were previously recognized as ADSC biomarkers.…”

Discrete adipose-derived stem cell subpopulations may display differential functionality after in vitro expansion despite convergence to a common phenotype distribution

“…By the same token, it would be of interest to determine how significant is the association of CD146 with the regenerative potential in the clinical setting. Since, as mentioned above, this phenotype plays a role in revascularization processes, it can be envisaged that in conjunction with appropriate preconditioning regimen, such as hypoxia [8, 15, 16, 60–63], substantially improved therapeutic outcome would be achieved.…”

Chemical composition and the potential for proteomic transformation in cancer, hypoxia, and hyperosmotic stress

“…in hypoxic (1% O 2 ) vs normoxic (20% O 2 ) conditions. Source: Tables 1 and 2 of Riis et al (2016).…”