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Fas-L is a TNF family member known to trigger cell death. It has recently become evident that Fas-L can transduce also non-apoptotic signals. Mesenchymal stem cells (MSCs) are multipotent cells that are derived from various adult tissues. Although MSCs from different tissues display common properties they also display tissue-specific characteristics. Previous works have demonstrated massive apoptosis following Fas-L treatment of bone marrow-derived MSCs both in vitro and following their administration in vivo. We therefore set to examine Fas-L-induced responses in adipose-derived stem cells (ASCs). Human ASCs were isolated from lipoaspirates and their reactivity to Fas-L treatment was examined. ASCs responded to Fas-L by simultaneous apoptosis and proliferation, which yielded a net doubling of cell quantities and a phenotypic shift, including reduced expression of CD105 and increased expression of CD73, in association with increased bone differentiation potential. Treatment of freshly isolated ASCs led to an increase in large colony forming unit fibroblasts, likely produced by early stem cell progenitor cells. Fas-L-induced apoptosis and proliferation signaling were found to be independent as caspase inhibition attenuated Fas-L-induced apoptosis without impacting proliferation, whereas inhibition of PI3K and MEK, but not of JNK, attenuated Fas-L-dependent proliferation, but not apoptosis. Thus, Fas-L signaling in ASCs leads to their expansion and phenotypic shift toward a more potent stem cell state. We speculate that these reactions ensure the survival of ASC progenitor cells encountering Fas-L-enriched environments during tissue damage and inflammation and may also enhance ASC survival following their administration in vivo.

Background: Little is known about the potentially unfavorable effects of mesenchymal stromal cell (MSC) activation on the heart. MSCs can respond to tissue injury by anti- or proinflammatory activation. We aimed to study the potential negative interaction between left ventricular dysfunction (LVD) and MSC activation., Methods: We isolated MSCs from cardiac and subcutaneous fat tissues of mice with LVD 28 days after myocardial infarction or sham operation. To evaluate the effect of LVD on MSCs, we characterized cardiac MSCs and subcutaneous MSCs in vitro. Subsequently, we injected MSCs or saline into the infarcted myocardium of mice and evaluated LV remodeling and function 28 days after myocardial infarction. To test the hypothesis that toll-like receptor 4 ( TLR4 ) mediates proinflammatory polarization of MSCs, we characterized cardiac MSCs from TLR4 -/- and wild-type (WT) mice after inflammatory stimulation in vitro. Next, we transplanted cardiac MSCs from TLR4 -/- and WT male mice into the infarcted myocardium of female WT mice and evaluated infarct size, MSC retention, inflammation, remodeling, and function after 7 days., Results: LVD switched cardiac MSCs toward an inflammatory phenotype, with increased secretion of inflammatory cytokines as well as chemokines. The effect of LVD on subcutaneous MSCs was less remarkable. Although transplantation of cardiac MSCs and subcutaneous MSCs from LVD and sham hearts did not improve LV remodeling and function, cardiac MSCs from LVD exacerbated anterior wall thinning 28 days after myocardial infarction. The inflammatory polarization of cardiac MSCs by LVD was mediated by TLR4 , as we found less secretion of inflammatory cytokines and higher secretion of anti-inflammatory cytokines from activated cardiac MSCs of TLR4 -deficient mice, compared with WT cardiac MSCs. Significantly, TLR4 deficiency preserved the expression of CD47 (don't eat me signal) on cardiac MSCs after both TLR4 stimulation in vitro and transplantation into the infarcted heart. Compared with WT cardiac MSCs and saline, TLR4 -/- cardiac MSCs survived in the cardiac tissue and maintained their reparative properties, reduced infarct size, increased scar thickness, and attenuated LV dilatation 7 days after myocardial infarction., Conclusions: The environment of the failing and infarcted myocardium drives resident and transplanted MSCs toward a proinflammatory phenotype and restricts their survival and reparative effects in a mechanism mediated by TLR4 ., (© 2017 American Heart Association, Inc.)

Background: Adipose tissue-derived mesenchymal stem cells (ASCs) can be isolated from subcutaneous fat harvested by tissue resection or liposuction., Objectives: The authors compared ASCs isolated by tissue resection or power-assisted liposuction (PAL) to determine whether either surgical procedure yielded ASCs with improved purity and competence that was preserved for several passages., Methods: For this experimental study, ASCs were isolated from fat harvested by tissue resection or PAL from six patients who underwent abdominoplasty. ASCs were counted to determine cell yields, and viabilities were assessed with an amine-reactive dye and by fluorescence-activated cell sorting (FACS). Cell phenotypes were determined by immunostaining and FACS, and doubling times were calculated. Senescence ratios of the cells were detected by gene profiling and by assaying β-galactosidase activity. Multipotency was evaluated by induced differentiation analyses., Results: No significant differences were observed in cell numbers or viabilities of ASCs isolated following either surgical method of fat harvesting. Both populations of cultured ASCs expressed markers of mesenchymal stem cells and preserved this expression pattern through the third passage. PAL and tissue resection yielded ASCs with similar division rates, similar senescence ratios into the fourth passage, and similar capacities to differentiate into osteocytes or adipocytes., Conclusions: Fat harvested by PAL or tissue resection yielded uniform cultures of ASCs with high division rates, low senescence ratios, and multipotency preserved into passages 3 and 4. Because PAL is less invasive, it may be preferable for the isolation of ASCs., (© 2015 The American Society for Aesthetic Plastic Surgery, Inc. Reprints and permission: journals.permissions@oup.com.)

Mesenchymal stromal cells (MSCs) are multipotent and can be derived from different adult tissues including fat. Our repeated attempts to produce long-term proliferative cultures of rat abdominal adipose stem cells (aASCs) under normal oxygen concentration (21%) were unsuccessful. We set to examine the events controlling this cytostasis of aASCs and found that it resulted from overproduction of reactive oxygen species (ROS) that led to apoptosis. ROS overproduction in aASCs was accompanied by increased expression of NOX1 but not of NOX2 or NOX4. NOX family members are an important source of intracellular ROS pointing to NOX1 involvement in ROS accumulation. This was verified when aASCs that were grown under 3% oxygen conditions expanded long term, displaying reduced NOX1 expression and decreased ROS accumulation. NOX1 involvement in aASC cytostasis was reaffirmed when cells that were expanded under normoxic conditions in the presence of a specific NOX1 inhibitor, ML171, demonstrated reduced ROS accumulation, reduced apoptosis and long-term expansion. aASC expansion arrest was accompanied also by a weak fat differentiation and migratory potential, which was enhanced by NOX1 inhibition. This suggests an inhibitory role for NOX1-induced ROS overproduction on aASCs, their fat differentiation and migratory potential. In contrast to aASCs, similar cells produced from subcutaneous fat were easily expanded in normoxic cultures, exhibiting low ROS concentrations, a low number of apoptotic cells and improved fat differentiation and migration. Taken together, our results show, for the first time, that NOX1-induced ROS accumulation halts ASC expansion and reduces their differentiation and migratory potential under normoxic conditions. Importantly, this phenotype comprises a tissue-specific signature as it was evident in aASCs but not in subcutaneous ASCs. NOX-induced ROS accumulation and cytokine production by fat are part of the metabolic syndrome. The similarity of this phenomenon to aASC phenotype may indicate that they arise from similar molecular mechanisms.

Introduction: Mesenchymal stem cells (MSCs) are multipotent and have been derived from various tissues. Although MSCs share many basic features, they often display subtle tissue specific differences. We previously demonstrated that bone marrow (BM) MSCs frequently become polyploid in culture. This tendency was mediated by a reduction in the expression of H19 long non-coding RNA during the transition from a diploid to a polyploid state., Methods: MSCs were derived from both BM and adipose tissue of mice and expanded under normoxic and hypoxic culture conditions. Cells were stained by propidium iodide and their ploidy was evaluated by FACS. Gene expression of independent MSC preparations was compared by quantitative real time PCR and protein expression levels by Western blot analysis. p53 silencing in MSCs was performed by a specific small hairpin RNA (shRNA)., Results: We set to examine whether genomic instability is common to MSCs originating from different tissues. It is demonstrated that adipose derived MSCs (ASCs) tend to remain diploid during culture while a vast majority of BM MSCs become polyploid. The diploid phenotype of ASCs is correlated with reduced H19 expression compared to BM MSCs. Under hypoxic conditions (3% oxygen) both ASCs and BM MSCs demonstrate increased RNA expression of H19 and Vascular endothelial growth factor A. Importantly, ASC gene expression is significantly less variable than BM MSCs under both oxygen conditions, indicating to their superior homogeneity. Gene expression analysis revealed that p53 target genes, often induced by DNA damage, are up-regulated in ASCs under basal conditions. However, p53 activation following treatment with DNA damaging agents was strongly elevated in BM MSCs compared to ASCs. We found that p53 is involved in maintaining the stable diploid state of ASCs as p53 shRNA induced ploidy changes in ASCs but not in BM MSCs., Conclusions: The increased genomic stability of murine ASCs together with their lower H19 expression and relative homogeneity suggest a tissue specific higher stability of ASCs compared to BM MSCs, possibly due to higher activity of p53. The tissue specific differences between MSCs from a different tissue source may have important consequences on the use of various MSCs both in vitro and in vivo.

Background: Human mesenchymal stromal cells (hMSCs) from adipose cardiac tissue have attracted considerable interest in regard to cell-based therapies. We aimed to test the hypothesis that hMSCs from the heart and epicardial fat would be better cells for infarct repair., Methods and Results: We isolated and grew hMSCs from patients with ischemic heart disease from 4 locations: epicardial fat, pericardial fat, subcutaneous fat, and the right atrium. Significantly, hMSCs from the right atrium and epicardial fat secreted the highest amounts of trophic and inflammatory cytokines, while hMSCs from pericardial and subcutaneous fat secreted the lowest. Relative expression of inflammation- and fibrosis-related genes was considerably higher in hMSCs from the right atrium and epicardial fat than in subcutaneous fat hMSCs. To determine the functional effects of hMSCs, we allocated rats to hMSC transplantation 7 days after myocardial infarction. Atrial hMSCs induced greatest infarct vascularization as well as highest inflammation score 27 days after transplantation. Surprisingly, cardiac dysfunction was worst after transplantation of hMSCs from atrium and epicardial fat and minimal after transplantation of hMSCs from subcutaneous fat. These findings were confirmed by using hMSC transplantation in immunocompromised mice after myocardial infarction. Notably, there was a correlation between tumor necrosis factor-α secretion from hMSCs and posttransplantation left ventricular remodeling and dysfunction., Conclusions: Because of their proinflammatory properties, hMSCs from the right atrium and epicardial fat of cardiac patients could impair heart function after myocardial infarction. Our findings might be relevant to autologous mesenchymal stromal cell therapy and development and progression of ischemic heart disease.

Background: Mesenchymal stromal cells (MSCs) improve tissue repair but their mechanism of action is not fully understood. We aimed to test the hypothesis that MSCs may act via macrophages, and that specifically, human cardiac adipose tissue-derived mesenchymal stromal cells (AT-MSCs) can polarize human macrophages into a reparative, anti-inflammatory (M2) phenotype. Methods and, Results: We isolated and grew AT-MSCs from human cardiac adipose tissue obtained during cardiac surgery. Macrophages were grown from CD14(+) monocytes from healthy donor blood and then cocultured with AT-MSCs, with and without transwell membrane, for 1 to 14 days. In response to AT-MSCs, macrophages acquired a star-shaped morphology, typical of alternatively activated phenotype (M2), and increased the expression of M2 markers CD206(+), CD163(+), and CD16(+) by 1.5- and 9-fold. Significantly, AT-MSCs modified macrophage cytokine secretion and increased the secretion of anti-inflammatory and angiogenic cytokines: interleukin (IL)-10 (9-fold) and vascular endothelial growth factors (3-fold). Moreover, AT-MSCs decreased macrophage secretion of inflammatory cytokines such as IL-1α (2-fold), tumor necrosis factor α (1.5-fold), IL-17 (3-fold), and interferon gamma (2-fold). Remarkably, the interaction between AT-MSCs and macrophages was bidirectional and macrophages enhanced AT-MSC secretion of typical M2 inducers IL-4 and IL-13. Notably, AT-MSCs decreased macrophage phagocytic capacity. Finally, IL-6 mediates the M2 polarization effect of AT-MSCs on macrophages, by increasing M2-associated cytokines, IL-10 and IL-13., Conclusions: Human cardiac AT-MSCs can polarize human macrophages into anti-inflammatory phenotype. Our findings suggest a new mechanism of action of AT-MSCs that could be relevant to the pathogenesis and treatment of myocardial infarction, atherosclerosis, and various cardiovascular diseases.

Mesenchymal stromal cells (MSC) are used extensively in clinical trials; however, the possibility that MSCs have a potential for malignant transformation was raised. We examined the genomic stability versus the tumor-forming capacity of multiple mouse MSCs. Murine MSCs have been shown to be less stable and more prone to malignant transformation than their human counterparts. A large series of independently isolated MSC populations exhibited low tumorigenic potential under syngeneic conditions, which increased in immunocompromised animals. Unexpectedly, higher ploidy correlated with reduced tumor-forming capacity. Furthermore, in both cultured MSCs and primary hepatocytes, polyploidization was associated with a dramatic decrease in the expression of the long noncoding RNA H19. Direct knockdown of H19 expression in diploid cells resulted in acquisition of polyploid cell traits. Moreover, artificial tetraploidization of diploid cancer cells led to a reduction of H19 levels, as well as to an attenuation of the tumorigenic potential. Polyploidy might therefore serve as a protective mechanism aimed at reducing malignant transformation through the involvement of the H19 regulatory long noncoding RNA.