Adipose tissue-derived stem cells as a therapeutic tool for cardiovascular disease

Adipose tissue-deried stem cells( ADSCs) are adult stem cells that can be easily harvested from subcutaneous adipose tissue. Many studies have demonstrated that ADSCs differentiate into vascular endothelial cells(VECs), vascular smooth muscle cells(VSMCs), and cardiomyocytes in vitro and in vivo. However, ADSCs may fuse with tissue-resident cells and obtain the corresponding characteristics of those cells. If fusion occurs, ADSCs may express markers of VECs, VSMCs, and cardiomyocytes without direct differentiation into these cell types. ADSCs also produce a variety of paracrine factors such as vascular endothelial growth factor, hepatocyte growth factor, and insulin-like growth factor-1 that have proangiogenic and/or antiapoptotic activities. Thus, ADSCs have the potential to regenerate the cardiovascular system via direct differentiation into VECs, VSMCs, and cardiomyocytes, fusion with tissueresident cells, and the production of paracrine factors. Numerous animal studies have demonstrated the efficacy of ADSC implantation in the treatment of acute myocardial infarction(AMI), ischemic cardiomyopathy(ICM), dilated cardiomyopathy, hindlimb ischemia, and stroke. Clinical studies regarding the use of autologous ADSCs for treating patients with AMI and ICM have recently been initiated. ADSC implantation has been reported as safe and effective so far. Therefore, ADSCs appear to be useful for the treatment of cardiovascular disease. However, the tumorigenic potential of ADSCs requires careful evaluation before their safe clinical application.

High-Fat Diets-Induced Metabolic Alterations Alter the Differentiation Potential of Adipose Tissue-Derived Stem Cells

Background: Adipose tissue-derived stem cells (ASC) possess the ability to differentiate into adipocytes or endothelial cells to help in the adipogenesis, vasculogenesis and vascular repair. This study aims at determining the impact of high-fat diets (HFD)-induced type 2 diabetes (T2D) on the differentiation potential of ASC. Results: C57BL/6J male mice were fed a vegetal (VD) or an animal (AD) HFD. Isolation of ACS from mice showing different levels of metabolic alterations reveals that advanced T2D did not affect the number of cells per gram of tissue. Rather, a higher proportion of inflammatory CD36+ cells was identified in HFD fed mice. Despite a marked decreased expression of adipogenic genes (aP2, C/EBPα and PPARγ2), ASC from HFD groups had a higher adipogenic potential and a lower endothelial differentiation potential in vitro compared to control. ASC from the VD group had enhanced cyclin B1 expression and had more adipogenic potential compared to AD group. Conclusion: Our results demonstrate that the metabolic modifications, linked to the nature of fatty acids in diets, modulate the differentiation potential of ASC with increased adipogenesis to the detriment of the endothelial pathway. Results highlight the importance of evaluating the ASC differentiation behavior in a context of autologous cell-based therapy for the repair of vascular tissues in diabetic patients.

Optimization of adipose tissue-derived mesenchymal stromal cells transplantation for bone marrow repopulation following irradiation

BACKGROUND Bone marrow(BM)suppression is one of the most common side effects of radiotherapy and the primary cause of death following exposure to irradiation.Despite concerted efforts,there is no definitive treatment method available.Recent studies have reported using mesenchymal stromal cells(MSCs),but their therapeutic effects are contested.AIM We administered and examined the effects of various amounts of adipose-derived MSCs(ADSCs)in mice with radiation-induced BM suppression.METHODS Mice were divided into three groups:Normal control group,irradiated(RT)group,and stem cell-treated group following whole-body irradiation(WBI).Mouse ADSCs(mADSCs)were transplanted into the peritoneal cavity either once or three times at 5×10^(5) cells/200μL.The white blood cell count and the levels of,plasma cytokines,BM mRNA,and BM surface markers were compared between the three groups.Human BM-derived CD34+hematopoietic progenitor cells were co-cultured with human ADSCs(hADSCs)or incubated in the presence of hADSCs conditioned media to investigate the effect on human cells in vitro.RESULTS The survival rate of mice that received one transplant of mADSCs was higher than that of mice that received three transplants.Multiple transplantations of ADSCs delayed the repopulation of BM hematopoietic stem cells.Anti-inflammatory effects and M2 polarization by intraperitoneal ADSCs might suppress erythropoiesis and induce myelopoiesis in sub-lethally RT mice.CONCLUSION The results suggested that an optimal amount of MSCs could improve survival rates post-WBI.

Effects of macrophages on the osteogenic differentiation of adipose tissue-derived stem cells in two-dimensional and three-dimensional cocultures

BACKGROUND Fracture is one of the most pervasive injuries in the musculoskeletal system,and there is a complex interaction between macrophages and adipose tissue-derived stem cells(ADSCs)in fracture healing.However,two-dimensional(2D)coculture of macrophages and ADSCs can not accurately mimic the in vivo cell microenvironment.AIM To establish both 2D and 3D osteogenic coculture models to investigate the interaction between macrophages and ADSCs.METHODS After obtaining ADSCs from surgery and inducing differentiation of the THP1 cell line,we established 2D and 3D osteogenic coculture models.To assess the level of osteogenic differentiation,we used alizarin red staining and measured the relative expression levels of osteogenic differentiation markers osteocalcin,Runt-related transcription factor 2,and alkaline phosphatase through polymerase chain reaction.Verification was conducted by analyzing the expression changes of N-cadherin and the activation of the Wnt/β-catenin signaling pathway using western blotting.RESULTS In this study,it was discovered that macrophages in 3D culture inhibited osteogenic differentiation of ADSCs,contrary to the effect in 2D culture.This observation confirmed the significance of intricate intercellular connections in the 3D culture environment.Additionally,the 3D culture group exhibited significantly higher N-cadherin expression and showed reducedβ-catenin and Wnt1 protein levels compared to the 2D culture group.CONCLUSION Macrophages promoted ADSC osteogenic differentiation in 2D culture conditions but inhibited it in 3D culture.The 3D culture environment might inhibit the Wnt/β-catenin signaling pathway by upregulating N-cadherin expression,ultimately hindering the osteogenic differentiation of ADSCs.By investigating the process of osteogenesis in ADSCs,this study provides novel ideas for exploring 3D osteogenesis in ADSCs,fracture repair,and other bone trauma repair.

Outcomes of combined mitochondria and mesenchymal stem cellsderived exosome therapy in rat acute respiratory distress syndrome and sepsis

BACKGROUND The treatment of acute respiratory distress syndrome(ARDS)complicated by sepsis syndrome(SS)remains challenging.AIM To investigate whether combined adipose-derived mesenchymal-stem-cells(ADMSCs)-derived exosome(EXAD)and exogenous mitochondria(mitoEx)protect the lung from ARDS complicated by SS.METHODS In vitro study,including L2 cells treated with lipopolysaccharide(LPS)and in vivo study including male-adult-SD rats categorized into groups 1(sham-operated-control),2(ARDS-SS),3(ARDS-SS+EXAD),4(ARDS-SS+mitoEx),and 5(ARDS-SS+EXAD+mitoEx),were included in the present study.RESULTS In vitro study showed an abundance of mitoEx found in recipient-L2 cells,resulting in significantly higher mitochondrial-cytochrome-C,adenosine triphosphate and relative mitochondrial DNA levels(P<0.001).The protein levels of inflammation[interleukin(IL)-1β/tumor necrosis factor(TNF)-α/nuclear factor-κB/toll-like receptor(TLR)-4/matrix-metalloproteinase(MMP)-9/oxidative-stress(NOX-1/NOX-2)/apoptosis(cleaved-caspase3/cleaved-poly(ADP-ribose)polymerase)]were significantly attenuated in lipopolysaccharide(LPS)-treated L2 cells with EXAD treatment than without EXAD treatment,whereas the protein expressions of cellular junctions[occluding/β-catenin/zonula occludens(ZO)-1/E-cadherin]exhibited an opposite pattern of inflam-mation(all P<0.001).Animals were euthanized by 72 h post-48 h-ARDS induction,and lung tissues were harvested.By 72 h,flow cytometric analysis of bronchoalveolar lavage fluid demonstrated that the levels of inflam-matory cells(Ly6G+/CD14+/CD68+/CD11b/c+/myeloperoxidase+)and albumin were lowest in group 1,highest in group 2,and significantly higher in groups 3 and 4 than in group 5(all P<0.0001),whereas arterial oxygen-saturation(SaO2%)displayed an opposite pattern of albumin among the groups.Histopathological findings of lung injury/fibrosis area and inflammatory/DNA-damaged markers(CD68+/γ-H2AX)displayed an identical pattern of SaO2%among the groups(all P<0.0001).The protein expressions of inflammatory(TLR-4/MMP-9/IL-1β/TNF-α)/oxidative stress(NOX-1/NOX-2/p22phox/oxidized protein)/mitochondrial-damaged(cytosolic-cytochrome-C/dynamin-related protein 1)/autophagic(beclin-1/Atg-5/ratio of LC3B-II/LC3B-I)biomarkers exhibited a similar manner,whereas antioxidants[nuclear respiratory factor(Nrf)-1/Nrf-2]/cellular junctions(ZO-1/E-cadherin)/mitochondrial electron transport chain(complex I-V)exhibited an opposite manner of albumin among the groups(all P<0.0001).CONCLUSION Combined EXAD-mitoEx therapy was better than merely one for protecting the lung against ARDS-SS induced injury.

Stem cells： novel players in the treatment of erectile dysfunction

Stem cells are defined by their capacity for both self-renewal and directed differentiation; thus, they represent great promise for regenerative medicine. Historically, stem cells have been categorized as either embryonic stem cells （ESCs） or adult stem cells （ASCs）. It was previously believed that only ESCs hold the ability to differentiate into any cell type, whereas ASCs have the capacity to give rise only to cells of a given germ layer. More recently, however, numerous studies demonstrated the ability of ASCs to differentiate into cell types beyond their tissue origin. The aim of this review was to summarize contemporary evidence regarding stem cell availability, differentiation, and more specifically, the potential of these cells in the diagnosis and treatment of erectile dysfunction （ED） in both animal models and human research. We performed a search on PubMed for articles related to definition, iocalisation and circulation of stem cells as well as the application of stem cells in both diagnosis and treatment of ED. Strong evidence supports the concept that stem cell therapy is potentially the next therapeutic approach for ED. To date, a large spectrum of stem cells, including bone marrow mesenchymal stem cells, adipose tissue-derived stem cells and muscle-derived stem cells, have been investigated for neural, vascular, endothelial or smooth muscle regeneration in animal models for ED. In addition, several subtypes of ASCs are localized in the penis, and circulating endogenous stem cells can be employed to predict the outcome of ED and ED-related cardiovascular diseases.

Unmodified autologous stem cells at point of care for chronic myocardial infarction

BACKGROUND Numerous studies investigated cell-based therapies for myocardial infarction(MI).The conflicting results of these studies have established the need for developing innovative approaches for applying cell-based therapy for MI.Experimental studies on animal models demonstrated the potential of fresh,uncultured,unmodified,autologous adipose-derived regenerative cells(UAADRCs)for treating acute MI.In contrast,studies on the treatment of chronic MI(CMI;>4 wk post-MI)with UA-ADRCs have not been published so far.Among several methods for delivering cells to the myocardium,retrograde delivery into a temporarily blocked coronary vein has recently been demonstrated as an effective option.AIM To test the hypothesis that in experimentally-induced chronic myocardial infarction(CMI;>4 wk post-MI)in pigs,retrograde delivery of fresh,uncultured,unmodified,autologous adipose-derived regenerative cells(UA-ADRCs)into a temporarily blocked coronary vein improves cardiac function and structure.METHODS The left anterior descending(LAD)coronary artery of pigs was blocked for 180 min at time point T0.Then,either 18×106 UA-ADRCs prepared at“point of care”or saline as control were retrogradely delivered via an over-the-wire balloon catheter placed in the temporarily blocked LAD vein 4 wk after T0(T1).Effects of cells or saline were assessed by cardiac magnetic resonance(CMR)imaging,late gadolinium enhancement CMR imaging,and post mortem histologic analysis 10 wk after T0(T2).RESULTS Unlike the delivery of saline,delivery of UA-ADRCs demonstrated statistically significant improvements in cardiac function and structure at T2 compared to T1(all values given as mean±SE):Increased mean LVEF(UA-ADRCs group:34.3%±2.9%at T1 vs 40.4±2.6%at T2,P=0.037;saline group:37.8%±2.6%at T1 vs 36.2%±2.4%at T2,P>0.999),increased mean cardiac output(UA-ADRCs group:2.7±0.2 L/min at T1 vs 3.8±0.2 L/min at T2,P=0.002;saline group:3.4±0.3 L/min at T1 vs 3.6±0.3 L/min at T2,P=0.798),increased mean mass of the left ventricle(UA-ADRCs group:55.3±5.0 g at T1 vs 71.3±4.5 g at T2,P<0.001;saline group:63.2±3.4 g at T1 vs 68.4±4.0 g at T2,P=0.321)and reduced mean relative amount of scar volume of the left ventricular wall(UA-ADRCs group:20.9%±2.3%at T1 vs 16.6%±1.2%at T2,P=0.042;saline group:17.6%±1.4%at T1 vs 22.7%±1.8%at T2,P=0.022).CONCLUSION Retrograde cell delivery of UA-ADRCs in a porcine model for the study of CMI significantly improved myocardial function,increased myocardial mass and reduced the formation of scar tissue.

Over-expression of VEGF165 in the adipose tissue-derived stem cells via the lentiviral vector

Background Many researchers studied the possibility of using stem cells as gene therapeutic vector. But few related reports on the adipose tissue-derived stem cells （ADSCs） are available. Therefore we intended to construct a lentiviral VEGF165 expression vector and then infect the ADSCs to produce therapeutic seed cells.Methods EHS1001-68950485313912 clone was mutated by PCR method to produce consensus fragment of VEGF165 transcript （NM_001025368）. Lentivirus was enveloped with pGC-FU, pHelper 1.0 and pHelper 2.0 plasmids in 293T cells.And then the ADSCs （multiplicity of infection=20） were transfected with the vectors after titer determination. Stable expression of VEGF165 in ADSCs was confirmed by immunofluorescence staining, enzyme-linked immunosorbent assay （ELISA） and Western blotting analysis.Results DNA sequencing and 293T transfection verified VEGF165 was linked to the GFP fused vector. The virus titer is up to 2x10a determined by quantitative PCR. VEGF165 transduced cells could show green fluorescence confirmed by immunofluorescence staining （almost 95%）. ELISA analyses could detect out the density of VEGF was 850.86-1202.13pg/ml （mean （923.00±31.22） pg/ml） in the supernatant of VEGF16s-transduced cells but not detected in the GFP-transduced cells （P 〈0.001） and the Western blotting analyses also confirmed VEGF165 expression in VEGF165-transduced cells.Conclusions The VEGF165 over-expression ADSCs were obtained and may be used as a cell therapeutic tool and may be applied for vascular regeneration, especially in the treatment of erectile dysfunction.

Rho-associated coiled kinase inhibitor Y-27632 promotes neuronal-like differentiation of adult human adipose tissue-derived stem cells

Background Y-27632 is a specific inhibitor of Rho-associated coiled kinase （ROCK） and has been shown to promote the survival and induce the differentiation of a variety of cells types. However, the effects of Y-27632 on adult human adipose tissue-derived stem cells （ADSCs） are unclear. This study aimed to investigate the effects of Y-27632 on the neuronal-like differentiation of ADSCs. Methods ADSCs were isolated from women undergoing plastic surgery and cultured. ADSCs were treated with different doses of Y-27632 and observed morphological changes under microscope. The expression of nestin, neuron specific enolase （NSE） and microtubule-associated protein-2 （MAP-2） in ADSCs treated with Y-27632 was detected by immunocytochemistry and Western blotting analysis. Results Y-27632 had the potency to induce neuronal-like differentiation in ADSCs in a dose-dependent manner. Moreover, the differentiation induced by Y-27632 was recovered upon drug withdraw. ADSCs treated with Y-27632 expressed neuronal markers such as NSE, MAP-2 and nestin while untreated ADSCs did not express these markers. Conclusion Selective ROCK inhibitor Y-27632 could potentiate the neuronal-like differentiation of ADSCs, suggesting that Y-27632 could be utilized to induce the differentiation of ADSCs to neurons and facilitate the clinical application of ADSCs in tissue engineering.

Chemically induced hypoxia by dimethyloxalylglycine(DMOG)-loaded nanoporous silica nanoparticles supports endothelial tube formation by sustained VEGF release from adipose tissue-derived stem cells

Inadequate vascularization leading to insufficient oxygen and nutrient supply in deeper layers of bioartificial tissues remains a limitation in current tissue engineering approaches to which prevascularization offers a promising solution.Hypoxia triggering pre-vascularization by enhanced vascular endothelial growth factor(VEGF)expression can be induced chemically by dimethyloxalylglycine(DMOG).Nanoporous silica nanoparticles(NPSNPs,or mesoporous silica nanoparticles,MSNs)enable sustained delivery of molecules and potentially release DMOG allowing a durable capillarization of a construct.Here we evaluated the effects of soluble DMOG and DMOG-loaded NPSNPs on VEGF secretion of adipose tissue-derived stem cells(ASC)and on tube formation by human umbilical vein endothelial cells(HUVEC)-ASC co-cultures.Repeated doses of 100 mM and 500 mM soluble DMOG on ASC resulted in 3-to 7-fold increased VEGF levels on day 9(P<0.0001).Same doses of DMOG-NPSNPs enhanced VEGF secretion 7.7-fold(P<0.0001)which could be maintained until day 12 with 500 mM DMOG-NPSNPs.In fibrin-based tube formation assays,100 mM DMOG-NPSNPs had inhibitory effects whereas 50 mM significantly increased tube length,area and number of junctions transiently for 4 days.Thus,DMOG-NPSNPs supported endothelial tube formation by upregulated VEGF secretion from ASC and thus display a promising tool for prevascularization of tissue-engineered constructs.Further studies will evaluate their effect in hydrogels under perfusion.

Transplantation of neural progenitor cells differentiated from adipose tissue-derived stem cells for treatment of sciatic nerve injury

Objectives: Currently, the clinical repair of sciatic nerve injury remains difficult.Previous studies have confirmed that transplantation of adipose tissue-derived stem cells promotes nerve regeneration and restoration at peripheral nerve injury sites. Methods: In this study, adipose tissue-derived stem cells were induced to differentiate into neural progenitor cells, transfected with a green fluorescent protein-containing lentivirus, and then transplanted into the lesions of rats with sciatic nerve compression injury. Results: Fluorescence microscopy revealed that the transplanted cells survived,migrated, and differentiated in rats. At two weeks post-operation, a large number of transplanted cells had migrated to the injured lesions; at six weeks post-operation, transplanted cells were visible around the injured nerve and several cells were observed to express a Schwann cell marker. Sciatic function index and electrophysiological outcomes of the transplantation group were better than those of the control group. Cell transplantation promoted the recovery of motor nerve conduction velocity and compound muscle action potential amplitude, and reduced gastrocnemius muscle atrophy.Conclusions: Our experimental findings indicate that neural progenitor cells,differentiated from adipose tissue-derived stem cells, are potential seed stem cells that can be transplanted into lesions to treat sciatic nerve injury. This provides a theoretical basis for their use in clinical applications.

Recent advances in andrology-related stem cell research

Stem cells hold great promise for regenerative medicine because of their ability to self-renew and to differentiate into various cell types. Although embryonic stem cells （BSC） have greater differentiation potential than adult stem cells, the former is lagging in reaching clinical applications because of ethical concerns and governmental restrictions. Bone marrow stem cells （BMSC） are the best-studied adult stem cells （ASC） and have the potential to treat a wide variety of diseases, including erectile dysfunction （ED） and male infertility. More recently discovered adipose tissuederived stem cells （ADSC） are virtually identical to bone marrow stem cells in differentiation and therapeutic potential, but are easier and safer to obtain, can be harvested in larger quantities, and have the associated benefit of reducing obesity. Therefore, ADSC appear to be a better choice for future clinical applications. We have previously shown that ESC could restore the erectile function of neurogenic ED in rats, and we now have evidence that ADSC could do so as well. We are also investigating whether ADSC can differentiate into Leydig, Sertoli and male germ cells. The eventual goal is to use ADSC to treat male infertility and testosterone deficiency. （Asian JAndrol 2008 Mar; 10： 171-175）

Stem cell therapy for erectile dysfunction

Erectile dysfunction(ED)is an important health problem that has commonly been clinically treated using phosphodiesterase type 5 inhibitors(PDE5Is).However,PDE5Is are less effective when the structure of the cavernous body has been severely injured,and thus regeneration is required.Stem cell therapy has been investigated as a possible means for regenerating the injured cavernous body.Stem cells are classified into embryonic stem cells and adult stem cells(ASCs),and the intracavernous injection of ASCs has been explored as a therapy in animal ED models.Bone marrowderived mesenchymal stem cells and adipose tissuederived stem cells are major sources of ASCs used for the treatment of ED,and accumulated evidence now suggests that ASCs are useful in the restoration of erectile function and the regeneration of the cavernous body.However,the mechanisms by which ASCs recover erectile function remain controversial.Some studies indicated that ASCs were differentiated into the vascular endothelial cells,vascular smooth muscle cells,and nerve cells that originally resided in the cavernous body,whereas other studies have suggested that ASCs improved erectile function via the secretion of anti-apoptotic and/or proangiogenic cytokines ratherthan differentiation into other cell types.In this paper,we reviewed the characteristics of stem cells used for the treatment of ED,and the possible mechanisms by which these cells exert their effects.We also discussed the problems to be solved before implementation in the clinical setting.

Metformin promotes angiogenesis by enhancing VEGFa secretion by adipose-derived stem cells via the autophagy pathway

Human adipose tissue-derived stem cell(ADSC)derivatives are cell-free,with low immunogenicity and no potential tumourigenicity,making them ideal for aiding wound healing.However,variable quality has impeded their clinical application.Metformin(MET)is a 5′adenosine monophosphate-activated protein kinase activator associated with autophagic activation.In this study,we assessed the potential applicability and underlying mechanisms of MET-treated ADSC derivatives in enhancing angiogenesis.We employed various scientific techniques to evaluate the influence of MET on ADSC,assess angiogenesis and autophagy in MET-treated ADSC in vitro,and examine whether MET-treated ADSC increase angiogenesis.We found that low MET concentrations exerted no appreciable effect on ADSC proliferation.However,MET was observed to enhance the angiogenic capacity and autophagy of ADSC.MET-induced autophagy was associated with increased vascular endothelial growth factor A production and release,which contributed to promoting the therapeutic efficacy of ADSC.In vivo experiments confirmed that in contrast to untreated ADSC,MET-treated ADSC promoted angiogenesis.Our findings thus indicate that the application of MET-treated ADSC would be an effective approach to accelerate wound healing by promoting angiogenesis at wound sites.

The CD133/1⁺cell subset from human subcutaneous adult fat retains hemogenic potential

Research has shown that cells from adult fat tissue can effect long-term blood reconstitution. Fat-derived multipotentiality was ascribed to CD34+ perivascular populations from its prominent microvasculature, that represent mostly non-hemogenic, mesenchymal cells, although this tissue contains a CD34+45+ subset committed to a hemogenic fate. Here, in order to analyze cell subsets presenting hemogenic capabilities within fat, CD133/1+ and pericytes, the latter defined by CD140b (PDGFRb, Platelet-Derived Growth Factor Receptor Beta) expression, were immunomagnetically selected from stromal-vascular fractions (SVF). In Vitro Colony Forming Unit (CFU) assays were negative for CD140b+ pericytes and positive for CD133/1+ cells when a prolonged CFU assay was performed, revealing fat as another store of primitive progenitors that retain hemogenic potential.

人脂肪间充质干细胞向视网膜色素上皮样细胞的诱导分化及其在体应用研究

背景 目前,干细胞疗法治疗视网膜变性疾病是眼科研究的热点之一.研究已证实骨髓间充质干细胞（MSCs）可以体外诱导分化为视网膜色素上皮（RPE）样细胞,但由于取材困难,临床应用受到了一定的限制.人脂肪间充质干细胞（ADSCs）与MSCs有类似的特性且容易获取,但人ADSCs能够诱导分化为RPE细胞的研究尚不多见. 目的 评估人ADSCs向RPE样细胞诱导分化的可行性及其在体应用的安全性.方法 将体外培养的第3代人ADSCs接种于6孔板进行培养,12h后于实验组培养液中加入100 ng/ml表皮生长因子（EGF）、50 μmol/L牛磺酸和5×10-7 mol/L视黄酸进行诱导,常规培养的细胞作为对照组.采用RPE细胞标志物Pan细胞角蛋白（Pan-CK）单克隆抗体进行免疫荧光检测,对诱导的细胞进行鉴定,采用细胞膜示踪剂PKH26标记法示踪诱导细胞,将诱导后的RPE样细胞悬液lμl注入6只BALB/c裸鼠的右眼玻璃体腔,另6只裸鼠玻璃体内注射等容量PBS.于注射后1个月摘取实验动物右眼眼球,各组中3只眼球用于组织病理学检查,在光学显微镜下观察玻璃体视网膜的形态学改变,另3只眼球在透射电子显微镜下观察玻璃体视网膜超微结构的改变,评估诱导细胞在体应用的安全性. 结果 体外培养的第3代人ADSCs呈细长多角形,生长良好.对照组细胞Pan-CK表达缺失,而诱导细胞的细胞膜Pan-CK表达阳性,呈红色荧光.诱导的细胞经PKH26标记后细胞膜呈红色荧光.诱导的细胞悬液于裸鼠玻璃体内注射后1个月,光学显微镜下可见诱导细胞位于视网膜表面,视网膜各层组织结构排列清晰;透射电子显微镜下可见视网膜神经节细胞（RGCs）细胞膜完整,线粒体结构正常,染色质均匀. 结论 人ADSCs体外诱导后可分化为RPE样细胞,PKH26可对诱导后细胞进行细胞示踪,分化的细胞玻璃体腔注射后短期内未发现视网膜的不良反应。

温度敏感性材料培养的脂肪源性干细胞作为眼表重建种子细胞的可行性研究

背景 组织工程角膜学的发展为角膜盲的治疗提供了新的选择,但目前暂未发现培养方法简单、可行性好的角膜上皮种子细胞和理想的支架材料.研究表明,脂肪源性干细胞（ADSCs）具有自我更新能力同时也具有类上皮的特质,而温度敏感性材料（TRSs）作为支架进行干细胞培养也为细胞层片技术的进步提供了技术支撑. 目的 研究兔ADSCs在TRSs上的培养特性,并与经典的口腔黏膜上皮细胞（OMECs）特性进行比较,探讨ADSCs作为眼表重建种子细胞的可行性.方法 异丙基丙烯酰胺溶于二丙醇后均匀涂于直径35 mm的聚苯乙烯培养皿表面,利用电子束照射制备TRSs,兔颈背部皮下脂肪组织2～3g经消化培养后获得ADSCs,同时取出兔口腔黏膜组织进行消化培养,获得OMECs.将2种干细胞均接种于TRSs上继续培养,比较2种细胞形态、生长速度、脱附时间和成活细胞的总计数,将ADSCs层片和OMECs层片行组织病理学检查,观察2种细胞的形态特征;采用免疫组织化学法检测2种细胞中干细胞标志物和上皮细胞标志物的表达;应用扫描电子显微镜检查2种细胞层片的表面超微结构.结果 自制TRSs透明性和光滑度与普通培养皿接近,任意观察的5个样品中有4个水接触角＞10°,成功率为80％.TRSs上培养的ADSCs呈长梭形,OMECs呈不规则圆形.ADSCs生长周期在TRSs上为12～ 14 d,脱附时间为（46.0±9.6） min,细胞总计数为（7.9±1.1）×105/片,而TRSs培养的OMECs生长周期为14～16 d,脱附时间为（91.9±10.9） min,细胞计数为（45.8±26.5） ×105/片,2种细胞间层片脱附时间和细胞计数的差异均有统计学意义（P=0.002、0.028）.组织病理学检查显示,TRSs上的ADSCs呈1～3层排列,而OMECs呈4～5层覆层结构.免疫组织化学染色显示,ADSCs和OMECs细胞层片细胞角蛋白12（CK12）及干细胞标志物p63、ATP结合转运蛋白G超家族成员2（ABCG2）均呈阳性表达.扫描电子显微镜下观察可见ADSCs和OMECs表面均有致密的上皮微绒毛结构,细胞间连接紧密.结论 自制的TRSs可作为脂肪干细胞的培养支架,ADSCs取材广泛,TRSs上培养的ADSCs层片细胞活力好,可操作性强,可作为眼表重建新的种子来源。

体外培养脂肪干细胞向心肌细胞诱导的实验研究

目的:研究诱导体外培养脂肪干细胞(ADSCs)向心肌细胞分化的方法。方法:培养原代脂肪干细胞经CD34、CD44、CD105抗体标记鉴定后,进行5-aza诱导培养。通过鉴定心肌细胞特异性抗原横纹肌肌动蛋白及心肌特异性肌钙蛋白T检测细胞分化情况。结果:ADSCs高表达间充质干细胞特异抗原CD44、CD105,不表达CD34;表达HLA-I抗原,不表达HLA-抗原。经5-aza诱导后,细胞形态变为类似于心肌样细胞。免疫细胞化学检测横纹肌肌动蛋白及心肌特异性肌钙蛋白T呈阳性,流式细胞仪显示心肌细胞特异性抗原阳性细胞为94%。结论:脂肪干细胞具有向心肌细胞分化的可能,在组织工程学及干细胞移植领域有着良好的应用前景。

脂肪干细胞体外快速扩增实验研究

目的:探索在旋转生物反应器内应用微载体技术快速扩增兔脂肪干细胞的方法。方法:比较应用微载体结合旋转生物反应器(RCSS)进行动态培养与平皿静态培养两种方法中,脂肪干细胞生长曲线、倍增时间和生长周期的情况。结果:实验组RCSS法培养中脂肪干细胞密度可达最初接种的19倍左右,而对照组平皿静态培养仅为6倍余。实验组倍增时间短于对照组,且细胞周期分析显示G2-M期+S期细胞比例高于对照组。结论:利用微载体细胞培养技术可快速地在体外扩增脂肪干细胞。

Current Status of ADSCs-Enriched Fat Grafts in Plastic Surgery

Autologous fat grafting is an increasingly popular technique in plastic surgery for volume augmentation and rejuvenation.However,the unpredictability of long-term volume retention limits its clinical application.Various animal studies have documented the positive effects of adipose tissue-derived stem cells(ADSCs)on the acceleration of lipofilling.However,the results have been inconsistent,and there is an insufficient number of high-quality clinical studies to formulate evidence-based recommendations for ADSC-enriched fat grafts.Moreover,related technical standards,such as the final count of harvested ADSCs and the enrichment ratio,have not yet been established.This systematic review included all clinical trials on ADSC-enriched fat grafts in plastic surgery from PubMed in the past 10 years,as well as all registered clinical trials on ClinicalTrials.Gov.To examine the current landscape of ADSCs harvest,we summarize the current applications of ADSCs in the field of plastic surgery and discuss the current barriers to universal clinical use.