Isolation and characterization of exosomes from adipose tissue-derived mesenchymal stem cells

Mesenchymal stem cells (MSCs) are the subject of intense research as they are a potential therapeutic tool for several clinical applications. The new MSCs action models are focused on the use of MSC-derived secretome which contains several growth factors, cytokines, microRNAs, and extracellular vesicles such as exosomes. Exosomes have recently emerged as a component with great potential involved as mediators in cellular communication. The isolation and identification of exosomes has made it possible for them to be used in cell-free therapies. The purposes of this study are: (i) to detect exosomes released into adipose-derived MSC conditioned cell culture medium, (ii) to identify exosome morphology, and (iii) to carry out a complete characterization of said exosomes. Moreover, it is aimed at determining which method for exosome isolation would be best to use. Precipitation has been identified as a highly useful method of exosome isolation since it provides higher efficiency and purity values than other methods. A broad characterization of the exosomes present in the MSC-conditioned medium was also carried out. This work fills a gap in the existing literature on bioactive molecules which have attracted a great deal of interest due to their potential use in cellular therapies.     

脂肪组织来源的干细胞的研究进展

来自中胚层的脂肪组织与骨髓组织一样含有大量能自我更新和多向系分化潜能的细胞群称为脂肪干细胞。其取材方便，来源丰富，可在体外稳定增殖传代。并与骨髓间充质干细胞有相似的多向分化表面标志CD105、STRO-1以及CD166受体。研究发现它具有多向系分化潜能，除可以分化为问充质来源的脂肪、骨，软骨、脂肪以及骨骼肌、心肌等细胞，也可诱导分化为来源于外胚层的神经细胞以及具有功能性的血管内皮细胞，用以修复骨、软骨、心肌、骨骼肌、血管以及神经等组织。其具有造血支持作用以及可被逆转录病毒、腺病毒以及慢病毒较高的转染效率等优点，可以作为基因转移良好的靶细胞。因而，脂肪来源的干细胞其有望成为组织工程、细胞治疗以及基因转染良好的种子细胞。     

Adipogenesis induced by human adipose tissue-derived stem cells

Adipose tissue-derived stem cells (ASCs), including preadipocytes, may play an important role in de novo adipogenesis and are expected to be a useful external source of cells for adipose tissue engineering. In this study, we examined in vivo adipogenesis up to 24 weeks after implantation, induced by human ASCs that were isolated from adipose tissues and expanded in vitro. ASCs proliferated in vitro in the presence of basic fibroblast growth factor (bFGF), and the number of cells increased by more than 1000-fold at the fourth passage. The ability to differentiate into mature adipocytes was maintained up to the third passage. We incorporated designated numbers of third-passage-expanded cells into a type I collagen scaffold and implanted them into the back of nude mice with or without controlled-release bFGF. After the implantation of 2 x 10(6) ASCs with controlled-release bFGF, the greatest cross-sectional surface area of adipose tissue in the scaffold was 1.19 mm(2) at 12 weeks and 2.14 mm(2) at 24 weeks. About 2 x 10(6) ASCs with controlled-release bFGF was the best condition for total adipogenesis. Immunohistochemical analysis with antihuman vimentin antibody showed that the area of human-origin adipose tissue was maximum in the group with 8 x 10(6) ASCs incorporated in a scaffold at both 12 and 24 weeks. The amount of human-origin adipose tissue increased in all groups with implanted ASCs from 12 to 24 weeks. Only trace of human-origin adipose tissue was observed in other groups implanted ASCs. Our results show that human ASCs not only function as progenitor cells for in vivo adipogenesis, but also induce de novo adipogenesis for long period.     

脂肪组织源性干细胞的生物学特性与多向分化潜能

回顾脂肪组织源性干细胞的生物学特性与多向分化潜能研究新进展。源于中胚层的脂肪组织源性干细胞具有自我更新及多向分化潜能，在体外适当的诱导条件下可向脂肪细胞、软骨细胞、成骨细胞、骨骼肌、心肌细胞、内皮细胞及神经细胞分化，在组织工程及基因治疗中具有良好的应用前景。对脂肪组织源性干细胞生物学特性及多向分化潜能的研究．将为脂肪组织源性干细胞成为现代组织工程学研究的理想种子细胞并最终用于以细胞为基础的临床治疗提供前提和保障。     

纯化取材脂肪提高实验中干细胞培养的纯度

背景：在以往脂肪干细胞原代培养的文献中大多只是剔除脂肪表面的血管组织,而对于脂肪内部的血管,尤其是血管周围淋巴结的剔除很少有文献报道。 目的：探讨通过脂肪纯化来提高培养干细胞纯度的可行性。 方法：将取材自同一大鼠腹股沟处的脂肪组织分成两份,一份给予纯化处理,剔除表面的血管、皮肤和肌肉组织的同时一并剔除组织内血管及其周围结节样组织。而另一份仅剔除表面血管及可能黏附的皮肤和肌肉组织,分别对两份脂肪行干细胞原代培养。 结果与结论：经苏木精-伊红染色显微镜下观察,证实血管周围结节样组织为淋巴结。纯化脂肪组培养的细胞形态一致、呈长梭形。脂肪干细胞相关性抗原CD90的免疫荧光和流式细胞仪检测结果均证明纯化脂肪组培养的细胞纯度较未纯化组高。实验结果提示通过纯化取材可以提高原代培养的脂肪干细胞纯度。     

成人脂肪源间充质干细胞在小鼠体内具有血液血管干细胞特性

目的:观察成人脂肪源Flk1+ CD31- CD34-细胞在小鼠体内是否具有血液血管干细胞的特性。方法:将男性成人脂肪源Flk1+ CD31- CD34-细胞经尾静脉输入6-8周龄、接受亚致死剂量射线照射的非肥胖型糖尿病及严重联合免疫缺陷(NOD/SCID)小鼠体内(每只输入剂量为105,悬于0.4m LRPMI-1640培养基中),对照组接受同等剂量的RPMI-1640培养基中。2个月后处死小鼠。用聚合酶链式反应、流式细胞术、三色荧光法及荧光原位杂交法检测脂肪源Flk1+ CD31- CD34-细胞在实验小鼠骨髓及消化系统内的分化情况。结果:脂肪源Flk1+ CD31- CD34-细胞在单细胞水平上在NOD/SCID小鼠体内可分化为内皮细胞和造血细胞。结论:脂肪源Flk1+ CD31- CD34-细胞在体内具有血液血管干细胞的特性,这类细胞有可能作为种子细胞用以治疗血液和血管性疾病。     

差速贴壁法分离培养脂肪源间充质干细胞

目的:探讨差速贴壁法从大鼠腹股沟脂肪垫分离纯化脂肪源间充质干细胞(adipose tissue-derived mesenchymal stem cells,ADMSCs)的可行性。方法:采用差速贴壁培养法分离ADMSCs,并与普通培养法得到的ADMSCs进行表面分子CD44阳性率对比。在第2代ADMSCs中加入条件培养基进行诱导,根据条件培养基的不同分成3组:①成骨诱导组:加入成骨培养基;②成脂诱导组:加入成脂培养基;③对照组:仅加入基础培养基。成骨诱导组和对照组进行碱性磷酸酶(ALP)检测,成脂诱导组和对照组进行油红O染色检测。结果:差速贴壁培养法获得CD44阳性率更高的ADMSCs。成骨诱导组的ALP大大高于对照组,成脂诱导组油红O染色阳性,对照组油红O染色均为阴性。结论:差速贴壁培养法从大鼠腹股沟脂肪垫中分离得到高纯度ADMSCs。     

Soluble factors-mediated immunomodulatory effects of canine adipose tissue-derived mesenchymal stem cells

Adipose tissue-derived mesenchymal stem cells (AD-MSCs), which can differentiate into several lineages, have immunomodulatory properties similar to those of bone marrow-derived MSCs. However, the specific mechanism by which the immunomodulatory effect of MSCs occurs is not clear. In this study, we isolated canine AD-MSCs (cAD-MSCs) and induced their development into adipocyte, osteocyte, and neuron-like cells. We then investigated their phenotype and cytokine expression to determine whether they were able to exert an immunomodulatory effect and what the underlying mechanisms of this effect were. cAD-MSCs expressed CD44, CD90, and MHC class I and were also partially positive for the expression of CD34; however, they did not express CD14 and CD45. In addition, they expressed the mRNA of transforming growth factor beta (TGF-beta), IL-6, IL-8, CCL2, CCL5, vascular endothelial growth factor, hepatocyte growth factor (HGF), tissue inhibitor metalloproteinase-1/2, and cyclooxygenase-2 but not that of IL-10. Further, leukocyte proliferation induced by mitogens was suppressed when they were cocultured with irradiated cAD-MSCs, as well as with culture supernatants of cAD-MSCs alone. Moreover, TNF-alpha production significantly decreased, whereas TGF-beta, IL-6, and interferon-gamma production significantly increased in cAD-MSCs that were cocultured with leukocytes. Finally, immonomodulatory factors of MSCs, such as TGF-beta, HGF, prostaglandin E2 (PGE2), and indoleamine 2, 3 dioxygenase (IDO), increased significantly in cAD-MSCs that were cocultured with leukocytes; however, the production of PGE2 and IDO showed different kinetics, and leukocyte proliferation was effectively restored by PGE2 and IDO inhibitors. Taken together, these results indicate that the immunomodulatory effects of cAD-MSCs are associated with soluble factors (TGF-beta, HGF, PGE2, and IDO). Therefore, it is suggested that cAD-MSCs have a potential therapeutic use in the treatment of immune-mediated disease.     

In vitro protection of adipose tissue-derived mesenchymal stem cells by erythropoietin

Mobilization of stem cells and their differentiation into cardiomyocytes are known to have protective effects after myocardial infarction. The integrity of transplanted mesenchymal stem cells for cardiac regeneration is dependent on cell–cell or cell–matrix interaction, which is adversely affected by reactive oxygen species in an ischemic environment. Treatment with erythropoietin was shown to protect human adipose tissue derived mesenchymal stem cells in an ischemic injury in vitro model. The analyses indicated that expression of erythropoietin receptors played a pivotal role in erythropoietin mediated cell survival. In this study, the anti-apoptotic effect of erythropoietin on stem cells was analyzed in apoptosis-induced human mesenchymal stem cells. Apoptosis was induced in cultured adult human adipose tissue derived mesenchymal stem cells by hydrogen peroxide. A group of cultured cells was also treated with recombinant human erythropoietin in a concentration of 50 ng mL⁻¹. The degree of apoptosis was analyzed by flow-cytometry and immunohistochemical staining for Caspase 3. The average percentages of apoptotic cells were significantly higher in H₂O₂-induced stem cells than in cells co-cultured with erythropoietin (63.03 ± 4.96% vs 29 ± 3.41%, p < 0.01). We conclude that preconditioning with erythropoietin suppresses apoptosis of mesenchymal stem cells and enhances their survival.     

神经型钙黏蛋白对小鼠脂肪间充质干细胞的成神经作用

目的:探讨神经型钙黏蛋白(N-cadherin)在小鼠脂肪间充质干细胞(ADMSCs)成神经分化中的作用。方法;用差速培养法获取小鼠ADMSCs,传至第5代,经成神经诱导液诱导ADMSCs 7 d后,免疫荧光检测胶质纤维酸性蛋白(GFAP)、神经元特异性烯醇化酶(NSE)和微管相关蛋白2(MAP2)的表达,半定量PCR检测细胞中N-cadherin mRNA的表达;采用N-cadherin对细胞进行基因修饰,免疫荧光检测神经丝蛋白(NF)和GFAP的表达。结果:ADMSCs经成神经诱导7 d后表达GFAP、NSE和MAP2,半定量PCR结果显示,与对照组相比成神经诱导后的细胞表达N-cadherin显著增高。转染N-cadherin的N-cadherin细胞培养24 h后发出细长的突起,与相邻细胞之间形成网状连接。免疫荧光结果显示,转染后的细胞NF和GFAP表达阳性。结论:ADMSCs在体外多种作用下,具有向神经细胞分化的潜能。N-cadherin转染后能改变ADMSCs的形态,并且在ADMSCs向神经分化中发挥一定的作用。     

脂肪组织来源干细胞构建皮肤复合组织修复创面缺损

BACKGROUND: There is no clear understanding on the effects of subcutaneous fat and stem cells on wound healing. OBJECTIVE: To explore the therapeutic effects of skin composite prepared with adipose tissue-derived stem cells on skin defects. METHODS: Epidermal cells, fibroblasts, adipose tissue-derived stem cells as seed cells and bovine collagen gel as a scaffold were used to build a complex with a variety of cells. A 6-mm diameter circular skin defect was made on the both sides of the rat back. The right side as experimental side was implanted with an 8-mm diameter multilayer skin composite, and the left side (control side) was only treated with a simple dressing. RESULTS AND CONCLUSION: For the constructed multi-layer skin composite, the epidermal layer was continuously merged into the multi-layer, the fibroblasts evenly distributed in the corium layer, and lipid droplets existed in the fat layer in which the cells distributed uniformly. Cell aggregation was obviously observed at the junction of different layers. In the experimental side, the rate of wound healing, granulation tissue thickness, the thickness of dermis and the capillary density were significantly higher than those in the control side. Taken together, we can construct multilayer skin composites with a variety of cells as seed cells, such as epidermal cells, fibroblasts and adipose tissue-derived stem cells, and bovine collagen gel as a scaffold, which promote wound healing and increase the thickness of dermis.        

The growth kinetic,differentiation properties,karyotyping, and characterization of adipose tissue-derived stem cells in hamster

Multilineage properties of mesenchymal stem cells (MSCs) cause their contribution in repair of different tissue. Adipose tissue is considered a good source of MSCs because of its abundance and easy access. The aims of this study were to characterize and determine the differentiation properties and growth kinetics of adipose tissue-derived stem cells (AT-SCs) from Syrian hamster. Fat tissue was isolated from the abdominal and pelvic regions of the hamster and by using type I collagenase, and cells were isolated and cultured. AT-SCs were characterized by morphology, cell surface markers, and adipogenic and osteogenic differentiations. AT-SCs were transferred to 24 well plates, and the growth kinetic was assessed by evaluation of the population doubling time (PDT). To show the chromosome stability of the AT-SCs, karyotyping was done. Hamsters’ AT-SCs showed fibroblast like morphology and expressed CD73 and CD29 but not CD45. AT-SCs could be differentiated to adipocytes and osteoblasts verified by Alizarin Red and Oil Red O staining methods. The PDT of the passages 2 and 3 of the AT-SCs were 51.6 and 80.6 h, respectively. The karyotyping was normal until passage 4. Hamsters’ AT-SCs showed mesenchymal properties denoting to these cells as a good source for the cell transplantation purposes in experimental and preclinical studies in hamster as an animal model.     

Chondrogenic potential of bone marrow- and adipose tissue-derived adult human mesenchymal stem cells

Regarding cartilage repair, tissue engineering is currently focusing on the use of adult mesenchymal stem cells (MSC) as an alternative to autologous chondrocytes. The potential of stem cells from various tissues to differentiate towards the chondrogenic phenotype has been investigated and it appears that the most common and studied sources are bone marrow (BM) and adipose tissue (AT) for historical and easy access reasons. In addition to three dimensional environment, the presence of member(s) of the transforming growth factor (TGF-β family and low oxygen tension have been reported to promote the in vitro differentiation of MSCs. Our work aimed at characterizing and comparing the degree of chondrogenic differentiation of MSCs isolated from BM and AT cultured in the same conditions. We also further aimed at and at determining whether hypoxia (2% oxygen) could affect the chondrogenic potential of AT-MSCs. Cells were first expanded in the presence of FGF-2, then harvested and centrifuged to allow formation of cell pellets, which were cultured in the presence of TGF-β3 and/or Bone Morphogenetic Protein-2 (BMP-2) and with 2 or 20% oxygen tension, for 24 days. Markers of the chondrocyte (COL2A1, AGC1, Sox9) and hypertrophic chondrocyte (COL10A1, MMP-13) were monitored by real-time PCR and/or by immunohistological staining. Our data show that BMP-2/TGF-β3 combination is the best culture condition to induce the chondrocyte phenotype in pellet cultures of BM and AT-MSCs. Particularly, a switch in the expression of the pre-chondrogenic type IIA form to the cartilage-specific type IIB form of COL2A1 was observed. A parallel increase in gene expression of COL10A1 and MMP-13 was also recorded. However when AT-MSCs were cultured in hypoxia, the expression of markers of hypertrophic chondrocytes decreased when BMP-2/TGF-β3 were present in the medium. Thus it seems that hypoxia participates to the control of AT-MSCs chondrogenesis. Altogether, these cellular model systems will help us to investigate further the potential of different adult stem cells for cartilage engineering.     

Electrospinning adipose tissue-derived extracellular matrix for adipose stem cell culture

Basement membrane-rich extracellular matrices, particularly murine sarcoma-derived Matrigel, play important roles in regenerative medicine research, exhibiting marked cellular responses in vitro and in vivo, although with limited clinical applications. We find that a human-derived matrix from lipoaspirate fat, a tissue rich in basement membrane components, can be fabricated by electrospinning and used to support cell culture. We describe practical applications and purification of extracellular matrix (ECM) from adipose tissue (At-ECM) and its use in electrospinning scaffolds and adipose stem cell (ASC) culture. The matrix composition of this purified and electrospun At-ECM was assessed histochemically for basement membrane, connective tissue, collagen, elastic fibers/elastin, glycoprotein, and proteoglycans. Each histochemical stain was positive in fat tissue, purified At-ECM, and electrospun At-ECM, and to some extent positive in a 10:90 blend with polydioxanone (PDO). We also show that electrospun At-ECM, alone and blended with PDO, supports ASC attachment and growth, suggesting that electrospun At-ECM scaffolds support ASC cultivation. These studies show that At-ECM can be isolated and electrospun as a basement membrane-rich tissue engineering matrix capable of supporting stem cells, providing the groundwork for an array of future regenerative medicine advances.