Evaluating the Effect of Hypoxia on Adipose-Derived Mesenchymal Stromal Cells In Vivo: A Systematic Review.

Objective To investigate the effects of adenovirus-mediated co-transfection of bone morphogenetic protein-2 (BMP-2) gene and bone morphogenetic protein-7 (BMP-7) gene on osteogenic differentiation of adipose-derived mesenchymal stromal cells (ADSCs).Methods The expression adenovirus vectors with green fluorescent protein gene carrying BMP-2 and adenovirus vectors with red fluorescent protein gene carrying BMP-7 were packaged and produced respectively.Rat adipose-derived stromal cells were isolated and cultured in vitro,and were transfected with Ad-BMP-2 (Ad-BMp-2 group),Ad-BMP-7 (Ad-BMP-7 group),co-transfected with Ad-BMP-2 and Ad-BMP-7 (Ad-BMP-2 ± Ad-BMP-7 group),or transfected with no virus (control group).The expression of osteocalcin (OCN) and collagen Ⅰ protein at 14th day after transfection was detected by using Western blotting in each group.Alkaline phosphatase (ALP) activity in ADSCs was measured.Results The expression of OCN and collagen Ⅰ protein in cotransfected group was significantly higher than that in the other groups (P ＜ 0.05).The ALP activity in cotransfected group was significantly stronger than that in the other groups (P ＜ 0.01).Conclusion Adenovirus-mediated co-transfection of BMP-2 gene and BMP-7 gene may promote osteogenic differentiation of adipose-derived ADSCs. Key words: Bone morphogenetic protein; Adenovirus; Adipose-derived stromal cells; Osteogenic differentiation

Mesenchymal stromal cells (MSCs) have been used in the treatment of Crohn's disease (CD) because of the immunomodulatory ability. The aim of this study was to investigate the therapeutic effect of adipose-derived MSCs (AD-MSCs) and to compare the therapeutic effect of AD-MSCs with that of bone marrow MSCs (BM-MSCs) in a murine model of CD. Murine colitis model of CD was created by trinitrobenzene sulfonic acid (TNBS). Twelve hours after treatment with TNBS, the mouse model was injected with MSCs intraperitoneally. Real-time polymerase chain reaction and immunohistochemistry staining were used to measure the expression levels of inflammatory cytokines in colonic tissues to investigate the therapeutic effect of AD-MSCs. The ten-day survival was recorded after infusion of MSCs. Intraperitoneal injection of MSCs alleviated the clinical and histopathologic severity of intestinal inflammation, and improved the survival of the TNBS-induced mouse model of CD. AD-MSCs could effectively increase the expression of interleukin-10 and reduce the secretion of pro-inflammatory cytokines including tumor necrosis factor-α, interleukin-12, and vascular endothelial growth factor. The mucosal injury was repaired by AD-MSCs. These effects were comparable between AD-MSCs and BM-MSCs. The therapeutic effect appears similar between AD-MSCs and BM-MSCs in treating CD. AD-MSCs may be a potential alternative of cell-based therapy for CD.

Prevention of skin flap necrosis by use of adipose-derived stromal cells with light-emitting diode phototherapy

Adipose-derived mesenchymal stromal cells primed by macrophages decrease inflammation and multiple organ lesions improving mice survival after cecal ligation and puncure-induced sepsis

Adipose-derived mesenchymal stromal cells (AD-MSCs) have been extensively studied in recent years. Their attractiveness is due to the ease of obtaining clinical material (fat tissue, lipoaspirate) and the relatively large number of AD-MSCs present in adipose tissue. In addition, AD-MSCs possess a high regenerative potential and immunomodulatory activities. Therefore, AD-MSCs have great potential in stem cell-based therapies in wound healing as well as in orthopedic, cardiovascular, or autoimmune diseases. There are many ongoing clinical trials on AD-MSC and in many cases their effectiveness has been proven. In this article, we present current knowledge about AD-MSCs based on our experience and other authors. We also demonstrate the application of AD-MSCs in selected pre-clinical models and clinical studies. Adipose-derived stromal cells can also be the pillar of the next generation of stem cells that will be chemically or genetically modified. Despite much research on these cells, there are still important and interesting areas to explore.

Objective To investigate the effect and related mechanism of apolipoprotein A5 (ApoA5) on adipogenic differentiation of human adipose-derived mesenchymal stem cells (AMSC). Methods Subcutaneous adipose tissue was obtained from 40 patients undergoing abdominal surgery at our hospital from February to July 2015. After induction of human AMSC by collagenase digestion, the adipose tissue was induced to differentiate into mature adipocytes and treated with ApoA5 at 600 and 1 200 ng/ml, respectively (ApoA5 intervention groups). Cells treated without ApoA5 protein were used as control group. The cells were harvested on the 7th and 14th day of differentiation, and the following assays were performed: (1) the effect of ApoA5 on TG content was measured by a TG assay kit; (2) RT-qPCR assay was used to detect the effect of ApoA5 on aP2 and FAS mRNA expression; (3) the effect of ApoA5 on the expression of CIDEC mRNA and protein was detected by RT-qPCR and Western blot; (4) the effect of ApoA5 on the expression of C/EBPβ mRNA and protein was detected by RT-qPCR and Western blot; (5) using lentiviral transfection technique, we overexpressed the gene of CIDEC in AMSC and cells were divided into lentiviral negative control group, lentiviral over-expressed CIDEC group and lentiviral over-expressed CIDEC+ApoA5 intervention group (the ApoA5 intervention concentration was 1 200 ng/ml). Thereby, we examined the effect of ApoA5 on the above indicators in adipogenic differentiation of AMSC in the case of CIDEC overexpression. Results (1) Effect of ApoA5 on TG content in AMSC: on the 7th and 14th day after the intervention, the TG levels were lower in ApoA5 600 and 1 200 ng/ml group AMSC than those in the control group (all P 0.05). (6) The effect of ApoA5 on the expression of aP2 and FAS mRNA in AMSC after CIDEC overexpression: on the 7th day after intervention, the expression levels of aP2 and FAS mRNA in AMSC were higher in the lentivirus over-expressed CIDEC group than in the lentivirus negative control group (both P 0.05). (7) The effect of ApoA5 on the expression of C/EBPβ mRNA and protein in AMSC after CIDEC overexpression: on the 7th day after intervention, the mRNA and relative protein expressions of C/EBPβ in AMSC were higher in lentivirus-overexpressed CIDEC group than in lentivirus negative control group (both P 0.05). Conclusions ApoA5 can inhibit the adipogenic differentiation of AMSC,and this effect may be mediated by down-regulating the expression of CIDEC. Furthermore, our results indicate that CIDEC could be considered as a key factor in adipogenic differentiation. Key words: Apolipoproteins A; Adipogenesis; Adipose-derived mesenchymal stem cells

BackgroundThe early but not the late phase of experimental inflammatory osteoarthritis (CiOA) is characterised by strongly elevated levels of S100A8/A91 and interleukin-1 beta (IL-1β).1,2 Adipose-derived mesenchymal stromal cells (ASCs) exhibit...

Local application of adipose-derived mesenchymal stromal cells in experimental inflammatory OA results in interleukin-1β-mediated attraction of PMNs and reduced S100A8/A9 release

Background: Injection of adipose-derived mesenchymal stromal cells (ASCs) into murine knee joints after induction of inflammatory collagenase-induced osteoarthritis (CiOA) reduces development of joint pathology. This protection is only achieved when ASCs are applied in early CiOA, which is characterized by synovitis and high S100A8/A9 and IL-1β levels, suggesting that inflammation is a prerequisite for the protective effect of ASCs. Our objective was to gain more insight into the interplay between synovitis and ASC-mediated amelioration of CiOA pathology.Methods: CiOA was induced by intra-articular collagenase injection. Knee joint sections were stained with hematoxylin/eosin and immunolocalization of polymorphonuclear cells (PMNs) and ASCs was performed using antibodies for NIMP-R14 and CD271, respectively. Chemokine expression induced by IL-1β or S100A8/A9 was assessed with qPCR and Luminex. ASC-PMN co-cultures were analyzed microscopically and with Luminex for inflammatory mediators. Migration of PMNs through transwell membranes toward conditioned medium of non-stimulated ASCs (ASCNS-CM) or IL-1β-stimulated ASCs (ASCIL-1β-CM) was examined using flow cytometry. Phagocytic capacity of PMNs was measured with labeled zymosan particles.Results: Intra-articular saline injection on day 7 of CiOA increased synovitis after 6 h, characterized by PMNs scattered throughout the joint cavity and the synovium. ASC injection resulted in comparable numbers of PMNs which clustered around ASCs in close interaction with the synovial lining. IL-1β-stimulation of ASCs in vitro strongly increased expression of PMN-attracting chemokines CXCL5, CXCL7, and KC, whereas S100A8/A9-stimulation did not. In agreement, the number of clustered PMNs per ASC was significantly increased after 6 h of co-culturing with IL-1β-stimulated ASCs. Also migration of PMNs toward ASCIL-1β-CM was significantly enhanced (287%) when compared to ASCNS-CM. Interestingly, association of PMNs with ASCs significantly diminished KC protein release by ASCs (69% lower after 24 h), accompanied by reduced release of S100A8/A9 protein by the PMNs. Moreover, phagocytic capacity of PMNs was strongly enhanced after priming with ASCIL-1β-CM.Conclusions: Local application of ASCs in inflamed CiOA knee joints results in clustering of attracted PMNs with ASCs in the synovium, which is likely mediated by IL-1β-induced up-regulation of chemokine release by ASCs. This results in enhanced phagocytic capacity of PMNs, enabling the clearance of debris to attenuate synovitis.

BackgroundInjection of adipose-derived mesenchymal stromal cells (ASCs) into knee joints after induction of experimental inflammatory osteoarthritis (CiOA) reduces development of joint pathology.1 This protection is only achieved when ASCs are...

BackgroundRecent studies have shown that mild synovitis in early phases of osteoarthritis (OA) is conducive to development of joint damage. OA synovitis is characterized by elevated levels of pro-inflammatory factors...

IntroductionIntra-articular injection of adipose-derived mesenchymal stromal cells (ASCs) and/or platelet-rich plasma (PRP) have been reported to independently and synergistically improve healing of osteochondral lesions in animal models. However, their independent and combined effects when localized to an osteochondral lesion by encapsulation within a photocrosslinkable methacrylated gelatin hydrogel (GelMA) have not been explored. Herein we investigated a unique combination of allogeneic ASCs and PRP embedded in GelMA as a single-stage treatment for osteochondral regeneration in a rabbit model.MethodsThirty mature rabbits were divided into six experimental groups: (1) Sham; (2) Defect; (3) GelMA; (4) GelMA + ASCs; (5) GelMA + PRP; and (6) GelMA + ASCs + PRP.At 12 weeks following surgical repair, osteochondral regeneration was assessed on the basis of gross appearance, biomechanical properties, histological and immunohistochemical characteristics, and subchondral bone volume.ResultsIn terms of mechanical property reflecting the ability of neotissue to bear stress, PRP only group were significantly lower than the Sham group (p = 0.0098). On the other hand, ASCs only and ASCs combined with PRP groups did not exhibit significantly difference, which suggesting that incorporation of ASCs assists in restoring the ability of the neotissue to bear stresses similarly to native tissue (p = 0.346, p = 0.40, respectively). Safranin O in ASCs combined with PRP group was significantly higher than the Defect and GelMA only groups (p = 0.0009, p = 0.0017, respectively). Additionally, ASCs only and ASCs combined with PRP groups presented especially strong staining for collagen type II. Surprisingly, PRP only and PRP + ASCs groups tended to exhibit higher collagen type I and collagen type X staining compared to ASCs only group, suggesting a potential PRP-mediated hypertrophic effect.ConclusionRegeneration of a focal osteochondral defect in a rabbit model was improved by a single-stage treatment of a photocrosslinked hydrogel containing allogenic ASCs and autologous PRP, with the combination of ASCs and PRP producing superior benefit than either alone. No experimental construct fully restored all properties of the native, healthy osteochondral unit, which may require longer follow-up or further modification of PRP and/or ASCs characteristics.

Introduction: Adipose-derived multipotent mesenchymal stromal cells (ADSCs) are widely used for cell therapy, in particular for the treatment of diseases of the nervous system. An important issue is to predict the effectiveness and safety of such cell transplants, considering disorders of adipose tissue under age-related dysfunction of sex hormones production. The study aimed to investigate the ultrastructural characteristics of 3D spheroids formed by ADSCs of ovariectomized mice of different ages compared to age-matched controls.Methods: ADSCs were obtained from female CBA/Ca mice randomly divided into four groups: CtrlY—control young (2 months) mice, CtrlO—control old (14 months) mice, OVxY—ovariectomized young mice, and OVxO—ovariectomized old mice of the same age. 3D spheroids were formed by micromass technique for 12–14 days and their ultrastructural characteristics were estimated by transmission electron microscopy.Results and Discussion: The electron microscopy analysis of spheroids from CtrlY animals revealed that ADSCs formed a culture of more or less homogeneous in size multicellular structures. The cytoplasm of these ADSCs had a granular appearance due to being rich in free ribosomes and polysomes, indicating active protein synthesis. Extended electron-dense mitochondria with a regular cristae structure and a predominant condensed matrix were observed in ADSCs from CtrlY group, which could indicate high respiratory activity. At the same time, ADSCs from CtrlO group formed a culture of heterogeneous in size spheroids. In ADSCs from CtrlO group, the mitochondrial population was heterogeneous, a significant part was represented by more round structures. This may indicate an increase in mitochondrial fission and/or an impairment of the fusion. Significantly fewer polysomes were observed in the cytoplasm of ADSCs from CtrlO group, indicating low protein synthetic activity. The cytoplasm of ADSCs in spheroids from old mice had significantly increased amounts of lipid droplets compared to cells obtained from young animals. Also, an increase in the number of lipid droplets in the cytoplasm of ADSCs was observed in both the group of young and old ovariectomized mice compared with control animals of the same age. Together, our data indicate the negative impact of aging on the ultrastructural characteristics of 3D spheroids formed by ADSCs. Our findings are particularly promising in the context of potential therapeutic applications of ADSCs for the treatment of diseases of the nervous system.

Adipose-derived mesenchymal stem cells (ADSCs), which tended to neurogenically differentiate spontaneously after achieving high confluence, were observed. Human ADSCs reaching 80% confluence were cultured in DMEM without an inducing factor for 24 h and then maintained in DMEM plus 1% FBS medium for 7 days. The neurogenic, adipogenic, and osteogenic genes of the factor-induced and confluence-initiated differentiation of the ADSCs and bone marrow-derived mesenchymal stem cells (BMSCs) at passages 3 to 5 were determined and compared using RT-qPCR, and the neurogenic differentiation was confirmed using immunofluorescent staining. In vitro tests revealed that the RNA and protein expression of neuronal markers, including class III β-tubulin (TUBB3), microtubule-associated protein 2 (MAP2), neurofilament medium polypeptide (NEFM), neurofilament heavy polypeptide (NEFH), and neurofilament light polypeptide (NEFL), had been enhanced in the confluence-initiated differentiation of the ADSCs. In addition, the expressions of neurotrophins, such as the nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and glial cell-derived neurotrophic factor (GDNF), were also elevated in the confluence-initiated differentiation of the ADSCs. However, the confluent ADSCs did not show a tendency toward spontaneous adipogenic and osteogenic differentiation. Moreover, compared with the confluent ADSCs, the tendency of spontaneous neurogenic, adipogenic, and osteogenic differentiation of the confluent human bone marrow mesenchymal stem cells (BMSCs) was not observed. The results indicated that ADSCs had the potential to spontaneously differentiate into neuron-like cells during the confluent culture period; however, this tendency was not observed in BMSCs.

Objective To investigate the effectiveness of human insulin-like growth factor-I （hIGF- I ） in transfecting adipose-derived mesenchymal stem cells （AdMSCs） and the expression of the transfected genes and find the possibility of differentiation of ADSCs to chondrocytes after hIGF- I transfection. Methods AdMSCs were extracted from the fat tissue of the back of rabbits＇ neck and cultured in vitro by monolayer. Then, the recombinant eukaryotic expression plasmid pcDNA3.1 ＋ hIGF- I was transfected into AdMSCs by Lipofectamine 2000. The growth of the ADSCs was observed by inverted microscope at 4-72 hours after transfection, at the end of passage and after stable transfection respectively, and the transfection efficiency was calculated. The hIGF- I concentration in the supernatant was detected by enzyme-linked immunosorbent assay （ELISA）, the expression of collagen-Ⅱ by immunohistochemistry, the expression of hIGF- I mRNA by reverse transcription polymerase chain reaction （ RT- PCR）, the expression of hIGF- I protein by Western blot and the cell cycle by flow cytometry. Results AdMSCs were shaped long shuttle and adhered to the disk at the 4th hour. AdMSCs were increased and distributed in cluster at 24th hour. After transfection, AdMSCs were proliferated actively, with shortened doubling generation time. The concentration of hIGF- I in the supernatant increased slowly after transfec- tion and reached peak at 32.45 ng/ml at the 72nd hour. The hIGF- I concentration remained at 28.36 ng/ml after stable transfeetion. Immunohistoehemistry affirmed a large number of collagen-Ⅱ in the endochylema and positive expression of hIGF- I mRNA and hIGF- I protein. The percentage of AdMSCs at stage S increased after transfection, which was significantly higher than that of AdMSCs without transfection, with statistical difference （ P 〈 0.05）. Conclusion After stable transfection, AdMSCs can secrete high concentration of hIGF- I that can promote cell proliferation and differentiation of AdMSCs to chondrocytes. Key words: Insulin-like growth factor- I ; Mesenchymal stem cells; Transfection; Cell cycle ; Chondrocyte