Immunomodulatory effect of human adipose tissue-derived adult stem cells: comparison with bone marrow mesenchymal stem cells

Like mesenchymal stem cells from bone marrow (BM-MSCs), adipose tissue-derived adult stem cells (ADAS cells) can differentiate into several lineages and present therapeutical potential for repairing damaged tissues. The use of allogenic stem cells can enlarge their therapeutical interest, provided that the grafted cells could be tolerated. We investigate here, for the first time, the immunosuppressive properties of ADAS cells compared with the well-characterized immunosuppressive properties of BM-MSCs. ADAS cells did not provoke in vitro alloreactivity of incompatible lymphocytes and, moreover, suppressed mixed lymphocyte reaction (MLR) and lymphocyte proliferative response to mitogens. The impairment of inhibition when ADAS cells and BM-MSCs were separated from lymphocytes by a permeable membrane suggests that cell contact is required for a full inhibitory effect. Hepatocyte growth factor is secreted by both stem cells but, similar to interleukin-10 and transforming growth factor-beta (TGF-beta), the levels of which were undetectable in supernatants of MLR inhibited by ADAS cells or BM-MSCs, it did not seem implicated in the stem cell suppressive effect. These findings support that ADAS cells share immunosuppressive properties with BM-MSCs. Therefore, ADAS cell-based reconstructive therapy could employ allogenic cells and because of their immunosuppressive properties, ADAS cells could be an alternative source to BM-MSCs to treat allogenic conflicts.     

Interaction of human adipose tissue-derived mesenchymal stromal cells with breast cancer cells

Human adipose tissue was shown to be a very attractive source of mesenchymal stromal cells that have a wide scale of potential applications in reconstructive plastic surgery and regenerative medicine. However, these cells were described to have profound effects on biological behaviour of tumour cells. The aim of this study was to analyze the influence of adipose tissue-derived human mesenchymal stromal cells (AT-MSC) on the proliferation of breast cancer cells. We have tested proliferation of three different human breast cancer cell lines under the influence of AT-MSC derived soluble factors as well as in the direct cocultures. These data were supplemented with the expression analysis of cytokines and their cognate receptors on the target cells. We have observed stimulation of proliferation in breast cancer cells MDA-MB-361, T47D and EGFP-MCF7. AT-MSC were found to secrete wide scale of cytokines, chemokines and growth factors, thus we concluded that this pro-proliferative effect was a result of their synergistic action. These data bring out a need to evaluate whether primary breast tumour derived human cells would respond to these type of stimuli in a similar manner in order to exclude any potential clinical risk related to the application of human mesenchymal stromal cells under the context of patient with history of breast cancer malignancy.     

TSH-induced gene expression involves regulation of self-renewal and differentiation-related genes in human bone marrow-derived mesenchymal stem cells

Bone marrow-derived mesenchymal stem cells are pluripotent cells that are capable of differentiating into a variety of cell types including neuronal cells, osteoblasts, chondrocytes, myocytes, and adipocytes. Despite recent advances in stem cell biology, neuroendocrine relations, particularly TSH interactions remain elusive. In this study, we investigated expression and biological consequence of TSH receptor (TSHR) interactions in mesenchymal stem cells of cultured human bone marrow. To the best of our knowledge, we demonstrated for the first time that human bone marrow-derived mesenchymal stem cells expressed a functional thyrotropin receptor that was capable of transducing signals through cAMP. We extended this study to explore possible pathways that could be associated directly or indirectly with the TSHR function in mesenchymal stem cells. Expression of 80 genes was studied by real-time PCR array profiles. Our investigation indicated involvements of interactions between TSH and its receptor in novel regulatory pathways, which could be the important mediators of self-renewal, maintenance, development, and differentiation in bone marrow-derived mesenchymal stem cells. TSH enhanced differentiation to the chondrogenic cell lineage; however, further work is required to determine whether osteoblastic differentiation is also promoted. Our results presented in this study have opened an era of regulatory events associated with novel neuroendocrine interactions of hypothalamic-pituitary axis in mesenchymal stem cell biology and differentiation.     

Efficacy and safety of human adipose tissue-derived mesenchymal stem cells for supporting hematopoiesis

We have demonstrated that adipose tissue-derived mesenchymal stem cells (ADSCs) from mice are capable of reconstituting the hematopoietic microenvironment, and facilitate hematopoiesis more effectively than bone marrow-derived mesenchymal stem cells (BMSCs) in mouse. The ready accessibility of fat tissue rich in MSCs and the higher hematopoiesis-supporting capacities of ADSCs suggest that ADSCs might represent a new therapeutic modality for the regeneration of impaired hematopoiesis. As a further step towards their use in clinical practice, we established human BMSCs and ADSCs from healthy volunteers of similar age, and compared their proliferation capacities, hematopoiesis-supporting properties, and safety. In vitro cell proliferation studies revealed that ADSCs have a higher population doubling number than BMSCs. In vitro co-culture assays showed that ADSCs not only support human CD34(+) peripheral blood stem cells (PBSCs), but also yield significantly more non-adherent hematic cells than BMSCs. In vitro progenitor assays revealed that ADSCs promote a higher frequency of early progenitors than do BMSCs. Interestingly, BM cellularity in irradiated mice that had received ADSCs tended to be higher than that of mice treated with BMSCs. When MSCs were injected into the BM cavity of tibiae, we observed no evidence of MSC-induced toxicity either during or after treatment. In addition, no microscopic abnormalities were observed in the bone marrow and major organs.     

Effects of human placental serum on proliferation and morphology of human adipose tissue-derived stem cells

Media used for tissue culture may have significant effects on the growth and morphology of the adipose tissue-derived stem cells (ADSCs). As fetal bovine serum (FBS) may induce an immunological reaction and health risks, this study was designed to evaluate and compare the effects of human placental serum (HPS) on the proliferation and morphology of hADSCs. We cultured hADSCs for at least three passages in four different culture media containing either FBS, HPS, autologous serum (AS) or human allogeneic serum (HAS). Morphological and immunophenotypic characteristics, as well as proliferation rates of the hADSCs were determined. The rates of proliferation of hADSCs seemed as follows: AS≥HPS>HAS>FBS. Morphologically, hADSCs isolated and expanded in medium containing HPS were similar to those grown in medium containing AS, whereas the morphology of cells cultured in human sera was different in comparison with FBS-ADSCs cultures. The immunophenotypic markers of hADSCs grown up in medium containing placental serum such as CD44+, CD90+ and CD105+, were similar to hADSCs grown up in media containing other sera. These results indicate that medium enriched with HPS provided a better microenvironment for hADSCs in comparison with medium enriched with commercially available FBS, and other human sera.     

Hepatogenic differentiation of human mesenchymal stem cells from adipose tissue in comparison with bone marrow mesenchymal stem cells

INTRODUCTIONMost liver diseases lead to hepatocyte dysfunction with the possibility of eventual organ failure. The replacement of diseased hepatocytes and the stimulation of endogenous or exogenous regeneration by stem cells are the main aims of liver-dir…     

人脂肪间充质干细胞体外分离、培养的方法研究

目的：探讨人脂肪间充质干细胞体外分离及培养方法。方法用消化离心的方法获得人脂肪间充质干细胞,以1×104/cm2接种于体积分数为0．1％胎牛血清的LG-DMEM培养基中,并进行细胞形态学观察,绘制细胞生长曲线,流式细胞仪检测细胞表面抗原,行成脂及成骨诱导检测其多向分化潜能。结果获取的脂肪间充质干细胞大小较为均匀,呈梭形的成纤维细胞样,细胞增殖良好;细胞生长曲线测定表明接种后第4天细胞进入指数增长期,第8天后生长进入平台期,体外能长期培养存活,并保持不分化状态;流式细胞仪检测表明细胞高表达 CD13、CD73,低表达 CD34、CD45及 HLA-DR;能诱导成脂及成骨细胞。结论利用消化离心法在体外能分离得到脂肪间充质干细胞,细胞生长良好,可作为组织工程的种子细胞。     

体外诱导入骨髓间充质干细胞向肝系细胞分化过程中的基因表达谱变化

目的 应用微阵列分析骨髓间充质干细胞向肝系细胞分化的基因表达变化.方法 收集健康志愿者胸骨骨髓,分离间充质干细胞体外培养,应用HGF+ FGF4诱导分化并鉴定分化细胞;利用微阵列技术筛选骨髓间充质干细胞向肝系细胞分化的有关基因.结果 121条基因表达发生变化,其中89条上调,32条下调.结论 相关基因涉及信号转导、能量代谢、基因转录、蛋白质翻译与合成等.SGK、ApoB、LDLR和CETP等可能在其分化过程中有非常重要的意义.     

Prostacyclin improves transcoronary myocardial delivery of adipose tissue-derived stromal cells.

AIMS: Heart transplantation of adipose tissue-derived stromal cells (ADSCs) is under evaluation as a therapy for cardiac repair. Prostacyclin (PGI2), a vasodilator with additional effects on platelet aggregation and blood cell adhesion, exerts cardioprotection and might favour the myocardial delivery of ADSCs. We investigated the engraftment and influence on cardiac function of the transcoronary delivery of ADSCs and the effects of PGI2 compared with nitroglycerin (NTG) and adenosine (Ado) in isolated-perfused mouse hearts. METHODS AND RESULTS: Infusion of ADSCs at <1 x 10(6) cells/mL caused no significant changes in contractility and rhythm, whereas higher cell doses caused cardiac dysfunction. Perfusion with PGI2, NTG, and Ado concentration-dependently increased coronary flow (CF). Perfusion with PGI2, at variance from NTG and Ado, increased ADSC delivery and entrance into the myocardial interstitium without affecting ventricular or metabolic functions and CF (engrafted ADSCs, as percentage of control, at doses producing 50% of maximum vasodilation: PGI2: 220+/-12, P < 0.001; NTG: 110+/-8, P = N.S.; Ado: 80+/-5, P = N.S.). CONCLUSION: PGI2 safely increases myocardial delivery of ADSCs, by mechanisms independent of its vasodilatory properties, with a potential for its use in cell therapy for cardiac repair.     

Unsorted human adipose tissue-derived stem cells promote angiogenesis and myogenesis in murine ischemic hindlimb model

We examined the protective effect of unsorted human adipose tissue-derived stem cells (hADSCs) with a short-term culture in endothelial differentiation medium on tissue repair after ischemic injury. hADSCs were isolated from human subcutaneous adipose tissue and cultured in vitro in endothelial differentiation medium for 2 wks before transplantation. Cultured hADSCs showed a typical mesenchymal stromal cell-like phenotype, positive for endothelial-specific markers including VE-cadherin, Flt-1, eNOS, and vWF but not CD31. Two hours after ligation of the femoral artery and vein, mice were injected with the unselected hADSCs locally near the surgery site and tested for tissue perfusion and repair. Tissue perfusion rates of the ischemic limbs were significantly higher in the group treated with hADSCs compared with those of the control mice as early as post-operative day 3 (median 195.3%/min; interquartile range, 82.0–321.1 vs. median 47.1%/min; interquartile range, 18.0–58.7; p = 0.001 by Friedman two-way analysis). Subsequently, the mice treated with hADSC showed better prognosis at 4 wks after surgery, and the histological analysis revealed increased vascular density and reduced muscle atrophy in the hADSC-transplanted limbs. Moreover, hADSC-treated muscle contained differentiated myocytes positive for human NF-κB and myogenin antigen. These results collectively indicate that unsorted hADSCs after a 2-wk-in vitro culture have a therapeutic potential in ischemic tissue injury via inducing both angiogenesis and myogenesis.     

Heterogeneity among human bone marrow-derived mesenchymal stem cells and neural progenitor cells

BACKGROUND AND OBJECTIVES: Mesenchymal stem cells (MSC) and neural progenitor cells (NPC) are pluripotent cells. The former can give rise to myocytes, chondrocytes, adipocytes, and osteogenic cells, while the latter can give rise to astrocytes, neurons, and oligodendrocytes. The aim of this study was to analyze and compare the antigen expression patterns of MSC and NPC. DESIGN AND METHODS: Human bone marrow-derived MSC and NPC were analyzed by flow cytometry and immunocytochemistry using a variety of unique monoclonal antibodies (57D2, W4A5, W8B2) generated in our laboratory. In addition, the expression profile of CD antigens and intracellular differentiation markers was analyzed. RESULTS. We show for the first time that CD10+, CD13+, CD61+, CD90+, CD105 (endoglin)+, CD45-, CD34-, and CD133- MSC also expressed CD109, CD140b (PDGF-RB), CD164, and CD172a (SIRPa). In addition, we found heterogeneity of MSC as demonstrated by the preferential expression of nestin and W8B2 antigen on distinct MSC subpopulations. Morphologically, these populations comprised small single cells and larger cells with polygonal appearance. NPC expressed high levels of CD56, CD90 and nestin and moderate levels of CD15, W4A5, and 57D2 antigens. In contrast, CD133 and CD172 were found only on NPC subpopulations. INTERPRETATION AND CONCLUSIONS: Our data demonstrate nestin expression in most NPC as well as in immature MSC subpopulations. MSC and NPC subpopulations can now be distinguished using our novel antibodies W8B2, 57D2, and W4A5.     

Gene expression profiling suggests a pathological role of human bone marrow-derived mesenchymal stem cells in aging-related skeletal diseases

Aging is associated with bone loss and degenerative joint diseases, in which the aging of bone marrow-derived mesenchymal stem cell (bmMSC)[1] may play an important role. In this study, we analyzed the gene expression profiles of bmMSC from 14 donors between 36 and 74 years old, and obtained age-associated genes (in the background of osteoarthritis) and osteoarthritis-associated genes (in the background of old age). Pathway analysis of these genes suggests that alterations in glycobiology might play an important role in the aging of human bmMSC. On the other hand, antigen presentation and signaling of immune cells were the top pathways enriched by osteoarthritis-associated genes, suggesting that alteration in immunology of bmMSC might be involved in the pathogenesis of osteoarthritis. Most intriguingly, we found significant age-associated differential expression of HEXA, HEXB, CTSK, SULF1, ADAMTS5, SPP1, COL8A2, GPNMB, TNFAIP6, and RPL29; those genes have been implicated in the bone loss and the pathology of osteoporosis and osteoarthritis in aging. Collectively, our results suggest a pathological role of bmMSC in aging-related skeletal diseases, and suggest the possibility that alteration in the immunology of bmMSC might also play an important role in the etiology of adult-onset osteoarthritis.     

shR-377联合细胞因子诱导人脂肪间充质干细胞向神经干细胞定向分化

目的探索sh R-377联合细胞因子诱导人脂肪间充质干细胞(HADMSCs)向神经干细胞(NSCs)定向分化。方法采用吸脂术获取健康青年人腹部大网膜脂肪组织,胶原酶消化法分离HADMSCs,行表型鉴定和细胞周期检测。sh R-377转染第三代HADMSCs,48 h后流式检测nestin表达率,72 h后行RT-q PCR和免疫细胞化学测定nestin和musashi,并对转化后细胞增殖培养分析。结果流式结果显示HADMSCs表面标志CD45及CD117表达呈阴性,CD29及CD44呈阳性,诱导24 h后nestin表达率为(64.6±3.2%);RT-q PCR结果表明诱导72 h后神经干样细胞球nestin、musashi表达阳性,免疫细胞化学染色可见nestin染色阳性。神经干样细胞球增殖培养7 d可见多个克隆球。神经干样细胞球进一步分化培养,可见到很明显的细长突触,双极或多极神经元样形态。结论 sh R-377联合细胞因子的诱导方法可以使HADMSCs定向分化为NSCs,诱导时间约48 h。转化时间和转化率比传统方法有较大提高,为体外大量获得NSCs提供了实验依据。