Hand and face vascularized composite allotransplantation (VCA) is an evolving and challenging field with great opportunities. During VCA, massive surgical damage is inflicted on both donor and recipient tissues, which may contribute to the high VCA rejection rates. To segregate between the damage-induced and rejection phase of post-VCA responses, we compared responses occurring up to 5 days following syngeneic versus allogeneic vascularized groin flap transplantations, culminating in transplant acceptance or rejection, respectively. The immune response elicited upon transplantation of a syngeneic versus allogeneic vascularized groin flap was compared at Post-operative days 2 or 5 by histology, immunohistochemistry and by broad-scope gene and protein analyses using quantitative real-time PCR and Multiplex respectively. Immune cell infiltration began at the donor-recipient interface and paralleled expression of a large group of wound healing-associated genes in both allografts and syngrafts. By day 5 post-transplantation, cell infiltration spread over the entire allograft but remained confined to the wound site in the syngraft. This shift correlated with upregulation of IL-18, INFg, CXCL9, 10 and 11, CCL2, CCL5, CX3CL1 and IL-10 in the allograft only, suggesting their role in the induction of the anti-alloantigen adaptive immune response. High resemblance between the cues governing VCA and solid organ rejection was observed. Despite this high resemblance we describe also, for the first time, a damage induced inflammatory component in VCA rejection as immune cell infiltration into the graft initiated at the surgical damage site spreading to the entire allograft only at late stage rejection. We speculate that the highly inflammatory setting created by the unique surgical damage during VCA may enhance acute allograft rejection.

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: Adipose-derived stem cell (ASC) expansion under atmospheric oxygen levels (21%) was previously shown to cause increased reactive oxygen species (ROS) accumulation and genetic instability compared to cells cultured under physiological oxygen levels (2–8%). However, since culture under physiological oxygen levels is costly and complicated, a simpler method to reduce ROS accumulation is desirable. The current study aimed to determine whether lower culture temperature can reduce ROS production in ASCs without impairing their culture expansion. Methods: Proliferation, differentiation, ROS accumulation, and gene expression were compared between ASC cultures at 35 °C and 37 °C. ASCs isolated either from rat fat depots or from human lipoaspirates were examined in the study. Results: Rat visceral ASCs (vASCs) cultured at 35 °C demonstrated reduced ROS production and apoptosis and enhanced expansion and adipogenic differentiation compared to vASCs cultured at 37 °C. Similarly, the culture of human ASCs (hASCs) at 35 °C led to reduced ROS accumulation and apoptosis, with no effect on the proliferation rate, compared to hASCs cultured at 37 °C. Comparison of gene expression profiles of 35 °C versus 37 °C vASCs uncovered the development of a pro-inflammatory phenotype in 37 °C vASCs in correlation with culture temperature and ROS overproduction. This correlation was reaffirmed in both hASCs and subcutaneous rat ASCs. Conclusions: This is the first evidence of the effect of culture temperature on ASC growth and differentiation properties. Reduced temperatures may result in superior ASC cultures with enhanced expansion capacities in vitro and effectiveness in vivo.

Adipose-derived stem cells are derived from the nonfat component of adipose tissue termed the stromal vascular fraction (SVF). The use of freshly isolated autologous SVF cells as an alternative to adult stem cells is becoming more common. Repeated SVF administration for improved clinical outcomes is complicated by the need for repeated liposuction. This can be overcome by cryopreservation of SVF cells. The current study aimed to assess whether SVF cells retain their stem cell potency during cryopreservation. SVF cells isolated from lipoaspirates (donor age: 46.1 ± 11.7 y; body mass index: 29.3 ± 4.8 kg/m2) were analyzed either immediately after isolation or following cryopreservation at −196°C. Analyses included assessment of nucleated cell counts by methylene blue staining, colony-forming unit fibroblast counts, surface marker expression using a flow cytometric panel (CD45, CD34, CD31, CD73, CD29, and CD105), expansion in culture, and differentiation to fat and bone. While cryopreservation reduced the number of viable SVF cells, stem cell potency was preserved, as demonstrated by no significant difference in the proliferation, surface marker expression in culture, bone and fat differentiation capacity, and the number of colony-forming unit fibroblasts in culture, in cryopreserved versus fresh SVF cells. Importantly, reduced cell counts of cryopreserved cells were due, mainly, to a reduction in hematopoietic CD45+ cells, which was accompanied by increased proportions of CD45−CD34+CD31− stem cell progenitor cells compared to fresh SVF cells. Cryopreservation of SVF cells did not affect their in vitro stem cell potency and may therefore enable repeated SVF cell administrations, without the need for repeated liposuction.

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.