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

BACKGROUND: Complex 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. METHOD: Using 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. RESULTS: Upon 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 SP2(CD73+90+105+34-146+271-) outperformed others in terms of wound healing. CONCLUSIONS: Our 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 MATERIAL: The online version of this article (doi:10.1186/s13287-016-0435-8) contains supplementary material, which is available to authorized users

Effect of unaccustomed eccentric exercise on proprioception of the knee in weight and non-weight bearing tasks

The study investigates the effects of eccentric exercise of the quadriceps on proprioception of the knee in weight and non-weight bearing tasks. Proprioception of the exercised leg was assessed at 120 and 150 of knee extension in 15 healthy adults (age 25.0 ± 3.6 yrs) before, immediately after, and 24 h following eccentric exercise of the quadriceps. Three tests of proprioception were performed: 1. matching the position of the exercised leg (right leg) to the reference leg (left leg) in sitting (non-weight bearing matching task); 2. repositioning the exercised leg after active movement in sitting (non-weight bearing repositioning task); 3. repositioning the exercised leg after active movement in standing (weight bearing task). Maximum knee extension force was reduced by 77.0 ± 12.3 % immediately after the exercise, and by 82.7 ± 16.2% 24 h post exercise, with respect to baseline (P < 0.001). The absolute error in the non-weight bearing matching task at 120 of knee extension was greater immediately following eccentric exercise (12.3 ± 5.6, P < 0.001) and 24 h after exercise (8.1 ± 4.5, P < 0.05) compared to baseline (5.8 ± 2.7). Similarly, the absolute error in the non-weight bearing repositioning task at 120 was greater both immediately (5.9 ± 3.1 , P < 0.01) and 24 h post exercise (5.2 ± 3.0 , P < 0.05) compared to baseline (4.5 ± 2.6 ). Therefore, in both non-weight bearing tasks, the subjects matched the position of their leg after eccentric exercise by adopting a more extended knee position of the exercised limb. Furthermore, the subjects showed higher variability in their performance immediately post exercise (P < 0.05, compared to baseline) but not 24 h after. In contrast, eccentric exercise did not affect the repositioning errors in the weight bearing task. In conclusion, eccentric exercise of the quadriceps impairs proprioception of the knee both immediately after and 24 h post exercise, but only in non-weight bearing tasks

Activation of protease-activated receptor 2 induces VEGF independently of HIF-1.

BACKGROUND: Human 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 FINDINGS: Immunohistochemistry 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. CONCLUSION: In hASCs trypsin and hypoxia induce VEGF expression through separate pathways

Stabilisation of HIF-1 in hASCs after trypsin and 1% oxygen exposure alone or in combination.

<p>(<b>A</b>) HIF-1 activation/stabilization during 6 hours culture after trypsin exposure in combination with 24 hours in hypoxic/normoxic conditions was analysed by ELISA. All cells were harvested <i>in situ</i>. Values are represented as the mean and SEM (n = 12). Asterisks denote statistical difference between this and all other groups (p<0.05). (<b>B</b>) Analysis of HIF-1α induction at 4 and 12 hours following 5 min trypsin exposure was done by immunoblotting. All cells were harvested <i>in situ</i>. HIF-1α positive controls are ASCs subjected to 48 hours of 1% oxygen.</p

Trypsin-activated PAR2 intracellular signaling in hASCs.

<p>(<b>A</b>) Schematic rendition of signal-transduction pathways linking PAR2 and <i>VEGF</i>. (<b>B</b>) The effect of specific kinase inhibitors on suppressing trypsin-induced <i>VEGF</i> activation after 5 min trypsin exposure was assessed by real-time RT-PCR (n = 6). Expression levels were normalized to the levels induced by trypsin (Ctrl). (<b>C</b>) The effect of PI3K and Mek inhibitors on phosphorylation of Akt and Erk1/2, respectively, as a result of 5 min trypsin exposure was determined by immunoblotting. PI3K and Mek inhibitors were added 2 hours prior to trypsin exposure. Cells after a 4-day culture at 20% oxygen were used as controls (Ctrl). Representative data obtained from ASC12 cells are presented. Values are represented as the mean and SEM. Abbreviations: PAR2, protease-activated receptor 2; VEGF, vascular endothelial growth factor; Ctrl, control.</p