Differentiation of human adipose-derived stem cells into neuron-like cells by Radix Angelicae Sinensis

Human adipose tissues are an ideal source of stem cells. It is important to find inducers that can safely and effectively differentiate stem cells into functional neurons for clinical use. In this study, we investigate the use of Radix Angelicae Sinensis as an inducer of neuronal differentiation. Primary human adipose-derived stem cells were obtained from adult subcutaneous fatty tissue, then pre-induced with 10% Radix Angelicae Sinensis injection for 24 hours, and incubated in serum-free Dulbecco＇s modified Eagle＇s medium/Nutrient Mixture F-12 containing 40% Radix Angelicae Sinensis to induce its differentiation into neuron-like cells. Butylated hydroxyanisole, a common in- ducer for neuronal differentiation, was used as the control. After human adipose-derived stem cells differentiated into neuron-like cells under the induction of Radix Angelicae Sinensis for 24 hours, the positive expression of neuron-specific enolase was lower than that of the butylated hydroxyani- sole-induced group, and the expression of glial fibrillary acidic protein was negative. Alter they were induced for 48 hours, the positive expression of neuron specific enolase in human adipose-derived stem cells was significantly higher than that of the butylated hydroxyanisole-induced group. Our experimental findings indicate that Radix Angelicae Sinensis can induce human adipose-derived stem cell differentiation into neuron-like cells and produce less cytotoxicity.

In vivo tracking of human adipose-derived stem cells labeled with ferumoxytol in rats with middle cerebral artery occlusion by magnetic resonance imaging

Ferumoxytol, an iron replacement product, is a new type of superparamagnetic iron oxide ap- proved by the US Food and Drug Administration. Herein, we assessed the feasibility of tracking transplanted human adipose-derived stem cells labeled with ferumoxytol in middle cerebral artery occlusion-injured rats by 3.0 T MRI in vivo. 1 × 104 human adipose-derived stem cells labeled with ferumoxytol-heparin-protamine were transplanted into the brains of rats with middle cerebral artery occlusion. Neurologic impairment was scored at 1, 7, 14, and 28 days after transplantation. T2-weighted imaging and enhanced susceptibility-weighted angiography were used to observe transplanted cells. Results of imaging tests were compared with results of Prussian blue staining. The modified neurologic impairment scores were significantly lower in rats transplanted with cells at all time points except I day post-transplantation compared with rats without transplantation. Regions with hypointense signals on T2-weighted and enhanced susceptibility-weighted angiography images corresponded with areas stained by Prussian blue, suggesting the presence of superparamagnetic iron oxide particles within the engrafted cells. Enhanced susceptibility-weighted angiography image exhibited better sensitivity and contrast in tracing ferumoxytol-heparin-protamine-labeled human adipose-derived stem ceils compared with T2-weighted imaging in routine MRI.

circ_0003204 regulates the osteogenic differentiation of human adipose-derived stem cells via miR-370-3p/HDAC4 axis

Human adipose-derived stem cells(hASCs)are a promising cell type for bone tissue regeneration.Circular RNAs(circRNAs)have been shown to play a critical role in regulating various cell differentiation and involve in mesenchymal stem cell osteogenesis.However,how circRNAs regulate hASCs in osteogenesis is still unclear.Herein,we found circ_0003204 was significantly downregulated during osteogenic differentiation of hASCs.Knockdown of circ_0003204 by si RNA or overexpression by lentivirus confirmed circ_0003204 could negatively regulate the osteogenic differentiation of hASCs.We performed dual-luciferase reporting assay and rescue experiments to verify circ_0003204 regulated osteogenic differentiation via sponging miR-370-3p.We predicted and confirmed that miR-370-3p had targets in the 3′-UTR of HDAC4 m RNA.The following rescue experiments indicated that circ_0003204 regulated the osteogenic differentiation of hASCs via miR-370-3p/HDAC4 axis.Subsequent in vivo experiments showed the silencing of circ_0003204 increased the bone formation and promoted the expression of osteogenic-related proteins in a mouse bone defect model,while overexpression of circ_0003204 inhibited bone defect repair.Our findings indicated that circ_0003204 might be a promising target to promote the efficacy of hASCs in repairing bone defects.

Tumor necrosis factor-αinhibition restores matrix formation by human adipose-derived stem cells in the late stage of chondrogenic differentiation

BACKGROUND Cartilage tissue engineering is a promising strategy for treating cartilage damage.Matrix formation by adipose-derived stem cells(ADSCs),which are one type of seed cell used for cartilage tissue engineering,decreases in the late stage of induced chondrogenic differentiation in vitro,which seriously limits research on ADSCs and their application.AIM To improve the chondrogenic differentiation efficiency of ADSCs in vitro,and optimize the existing chondrogenic induction protocol.METHODS Tumor necrosis factor-alpha(TNF-α)inhibitor was added to chondrogenic culture medium,and then Western blotting,enzyme linked immunosorbent assay,immunofluorescence and toluidine blue staining were used to detect the cartilage matrix secretion and the expression of key proteins of nuclear factor kappa-B(NF-κB)signaling pathway.RESULTS In this study,we found that the levels of TNF-αand matrix metalloproteinase 3 were increased during the chondrogenic differentiation of ADSCs.TNF-αthen bound to its receptor and activated the NF-κB pathway,leading to a decrease in cartilage matrix synthesis and secretion.Blocking TNF-αwith its inhibitors etanercept(1μg/mL)or infliximab(10μg/mL)significantly restored matrix formation.CONCLUSION Therefore,this study developed a combination of ADSC therapy and targeted anti-inflammatory drugs to optimize the chondrogenesis of ADSCs,and this approach could be very beneficial for translating ADSC-based approaches to treat cartilage damage.

Isolation and identification of adipose-derived stromal/stem cells from breast cancer patients after exposure neoadjuvant chemotherapy

BACKGROUND With recent research advances,adipose-derived stromal/stem cells(ASCs)have been demonstrated to facilitate the survival of fat grafts and thus are increasingly used for reconstructive procedures following surgery for breast cancer.Unfortunately,in patients,following radiation and chemotherapy for breast cancer suggest that these cancer treatment therapies may limit stem cell cellular functions important for soft tissue wound healing.For clinical translation to patients that have undergone cancer treatment,it is necessary to understand the effects of these therapies on the ASC's ability to improve fat graft survival in clinical practice.AIM To investigate whether the impact on ASCs function capacity and recovery in cancer patients may be due to the chemotherapy.METHODS ASCs were isolated from the cancerous side and noncancerous side of the breast from the same patients with receiving neoadjuvant chemotherapy(NAC)or notreceiving NAC.ASCs were in vitro treated with 5-fluorouracil(5-FU),doxorubicin(DXR),and cyclophosphamide(Cytoxan)at various concentrations.The stem cells yield,cell viability,and proliferation rates were measured by growth curves and MTT assays.Differentiation capacity for adipogenesis was determined by qPCR analysis of the specific gene markers and histological staining.RESULTS No significant differences were observed between the yield of ASCs in patients receiving NAC treatment and not-receiving NAC.ASCs yield from the cancerous side of the breast showed lower than the noncancerous side of the breast in both patients receiving NAC and not-receiving NAC.The proliferation rates of ASCs from patients didn’t differ much before and after NAC upon in vitro culture,and these cells appeared to retain the capacity to acquire adipocyte traits simile to the ASCs from patients not-receiving NAC.After cessation and washout of the drugs for another a week of culturing,ASCs showed a slow recovery of cell growth capacity in 5-FU-treated groups but was not observed in ASCs treated with DXR groups.CONCLUSION Neoadjuvant therapies do not affect the functioning capacity of ASCs.ASCs may hold great potential to serve as a cell source for fat grafting and reconstruction in patients undergoing chemotherapy.

Ultrastructure of neuronal-like cells differentiated from adult adipose-derived stromal cells

β-mercaptoethanol induces in vitro adult adipose-derived stromal cells （ADSCs） to differentiate into neurons. However, the ultrastructural features of the differentiated neuronal-like cells remain unknown. In the present study, inverted phase contrast microscopy was utilized to observe β-mercaptoethanol-induced differentiation of neuronal-like cells from human ADSCs, and immunocytochemistry and real-time polymerase chain reaction were employed to detect expression of a neural stem cells marker （nestin）, a neuronal marker （neuron-specific enolase）, and a glial marker （glial fibrillary acidic protein）. In addition, ultrastructure of neuronal-like cells was observed by transmission election microscopy. Results revealed highest expression rate of nestin and neuron-specific enolase at 3 and 5 hours following induced differentiation; cells in the 5-hour induction group exhibited a neuronal-specific structure, i.e., Nissl bodies. However, when induction solution was replaced by complete culture medium after 8-hour induction, the differentiated cells reverted to the fibroblast-like morphology from day 1. These results demonstrate that β-mercaptoethanol-induced ADSCs induced differentiation into neural stem cells, followed by morphology of neuronal-like cells. However, this differentiation state was not stable.

Human adipose tissue-derived stem cells in breast reconstruction following surgery for cancer: A controversial issue

Breast cancer is the most common cancer in women. Patients, in particular young women, after surgical removal of the tumor have a poorer quality of life and psychological problems. Plastic surgery procedures for breast reconstruction, including autologous fat grafting, concur to reduce cosmetic and psychological problems. The maintenance of the transplanted fat is partially due to the presence of resident adipose derived-stem cells (ASCs). The latter can be isolated by digestion and centrifugation from the stromal vascular fraction (SVF) of subcutaneous adipose tissue. Intraoperatory SVF/ASC enrichment has been proposed to stabilize and optimalize autologous fat engraftment for breast reconstructive surgery after mastectomy, but the safety of these procedures is still uncertain. Although the literature offers contrasting opinions concerning the effects of ASCs on cancer growth according to the tumor type, at the present time ASC implementation for regenerative medicine therapies should be carefully considered in patients previously treated for breast cancer. At the present, reconstructive therapy utilizing ASC-enriched fat grafting should be postponed until there is no evidence of active disease.

Biological character of human adipose-derived adult stem cells and influence of donor age on cell replication in culture

To investigate the biological character of human adipose-derived adult stem cells (hADAS cells) when cultured in vitro and the relationship between hADAS cell’s replication activity and the donor’s age factor, and to assess the stem cells as a new source for tissue engineering. hADAS cells are isolated from human adipose tissue of different age groups (from adolescents to olds: <20 years old, 21―40 years old, 41―60 years old and >61 years old groups). The protein markers (CD29, CD34, CD44, CD45, CD49d, HLA-DR, CD106) of hADAS cells were detected by flow cytometry (FCM) to identify the stem cell, and the cell cycle was examined for P20 hADAS cells to evaluate the safety of the subculture in vitro. The generative activity of hADAS cells in different age groups was also examined by MTT method. The formula “ log2T D = t logN t ? logN 0” was used to get the time doubling (TD) of the cells. The results showed that the cells kept heredity stabilization by chromosome analysis for at least 20 passages. The TD of these cells increased progressively by ageing, and the TD of the <20 years old group was lower than that of the >61 years old group (statistical analysis of variance (ANOVA), P=0.002, P<0.05). These find- ings suggested that a higher level of hADAS cells replication activity was found in the younger dona- tors, and they represent novel and valuable seed cells for studies of tissue engineering.

Conversion of Adipose-Derived Stem Cells into Sweat Gland-Like Cells: An In Vitro Phenotypic Study

Objective:This study was performed to explore the conversion of adipose-derived stem cells(ADSCs)into sweat gland-like cells for the purpose of sweat gland regeneration.Methods:ADSCs and human sweat gland(hSG)cells were isolated,cultured,and identified.The ADSCs were then cultured in combination with epidermal growth factor and/or cocultured with hSG cells in a Transwell coculturing system to transform the ADSCs into hSG-like cells.Phenotypic changes of the ADSCs were examined by morphological observation and immunocytochemical analysis of specific markers.Results:The ADSCs showed sweat gland-like morphologic changes and expressed sweat gland markers(cytokeratins 7,14,and 18).Conclusion:These findings revealed that ADSCs can differentiate into hSG-like cells after coculture in a Transwell system and that epidermal growth factor can enhance the efficiency of differentiation.ADSCs may serve as a potential source of cells for sweat gland regeneration.

Human adipose derived stem cells are superior to human osteoblasts (HOB) in bone tissue engineering on a collagen-fibroin-ELR blend

The ultrastructure of the bone provides a unique mechanical strength against compressive, torsional andtensional stresses. An elastin-like recombinamer (ELR) with a nucleation sequence for hydroxyapatitewas incorporated into films prepared from a collagen-silk fibroin blend carrying microchannel patternsto stimulate anisotropic osteogenesis. SEM and fluorescence microscopy showed the alignment ofadipose-derived stem cells (ADSCs) and the human osteoblasts (HOBs) on the ridges and in the groovesof microchannel patterned collagen-fibroin-ELR blend films. The Young's modulus and the ultimatetensile strength (UTS) of untreated films were 0.58 ± 0.13 MPa and 0.18 ± 0.05 MPa, respectively. After 28days of cell culture, ADSC seeded film had a Young's modulus of 1.21 ± 0.42 MPa and UTS of0.32 ± 0.15 MPa which were about 3 fold higher than HOB seeded films. The difference in Young'smodulus was statistically significant (p: 0.02). ADSCs attached, proliferated and mineralized better thanthe HOBs. In the light of these results, ADSCs served as a better cell source than HOBs for bone tissueengineering of collagen-fibroin-ELR based constructs used in this study. We have thus shown theenhancement in the tensile mechanical properties of the bone tissue engineered scaffolds by usingADSCs.

Enzymatic and non-enzymatic isolationsystems for adipose tissue-derived cells:current state of the art

In the past decade, adipose tissue became a highly interesting source of adult stem cells for plastic surgery andregenerative medicine. The isolated stromal vascular fraction (SVF) is a heterogeneous cell population including theadipose-derived stromal/stem cells (ASC), which showed regenerative potential in several clinical studies and trials.SVF should be provided in a safe and reproducible manner in accordance with current good manufacturing practices(cGMP). To ensure highest possible safety for patients, a precisely defined procedure with a high-quality control isrequired. Hence, an increasing number of adipose tissue-derived cell isolation systems have been developed.These systems aim for a closed, sterile, and safe isolation process limiting donor variations, risk for contaminations,and unpredictability of the cell material. To isolate SVF from adipose tissue, enzymes such as collagenase are used.Alternatively, in order to avoid enzymes, isolation systems using physical forces are available. Here, we provide anoverview of known existing enzymatic and non-enzymatic adipose tissue-derived cell isolation systems, which arepatented, published, or already on the market.

人脂肪干细胞体外向成骨细胞定向诱导分化的实验研究

目的:探讨人脂肪干细胞(hASCs)的分离方法、体外增殖能力及定向诱导分化为成骨细胞的潜能。方法:从脂肪抽吸物中分离、培养具有多向分化潜能的脂肪干细胞,比较原代(P0)及传代第1、2、5代(P1、P2、P5)细胞的生长曲线。实验组取P2细胞,应用成骨诱导液向成骨细胞定向诱导分化,并以未诱导组为对照组,利用ALP染色、von Kossa染色、免疫荧光检测、RT-PCR等方法对细胞成骨潜能进行评价。结果:传代细胞较原代细胞增殖速度快。P1、P2、P5均具有一些共同特征,传代培养的潜伏期约为24h,传代培养细胞的对数增殖期约为2～3d,接种后第4～5天进入平台期;细胞诱导14d后,实验组ALP染色呈阳性反应,对照组阴性;von Kossa染色,实验组出现深棕色结节状沉积,且随诱导时间增加而加深,对照组无结节状沉积出现;免疫荧光检测,实验组和对照组均表达Ⅰ型胶原,实验组骨钙素(OCN)、骨桥蛋白(OPN)检测为阳性,对照组为阴性或弱阳性;RT-PCR检测表明,实验组诱导14d时有ALP、Osteopontin表达,对照组阴性;实验组、对照组及成骨细胞均有I型胶原阳性表达。结论:hASCs在体外培养条件下生长良好,P2细胞在诱导培养下可向成骨细胞分化,为以hASCs为种子细胞构建组织工程骨奠定了基础。

3种成软骨诱导培养基对单层培养和微球培养的人脂肪干细胞成软骨效果比较

目的比较3种成软骨诱导培养基对单层培养和微球培养的人脂肪干细胞(human adipose mesenchymal stem cells,hASCs)体外成软骨分化的效果。方法从人脂肪组织中提取hASCs,分别采用单层培养和微球培养,加入3种不同成分的成软骨诱导培养基,A组:胎牛血清(FBS)+10 ng/ml转化生长因子3(TGF-β3);B组:胰岛素-转铁蛋白-亚硒酸钠(ITS)+1 ng/ml TGF-β3;C组:ITS+10 ng/mL TGF-β3。3周后提取单层培养的细胞蛋白进行成软骨相关标志物检测,并对细胞微球进行阿尔新蓝、甲苯胺蓝、天狼星红染色,比较成软骨效果。结果3周后单层培养体系C组的软骨标志物表达最高,微球培养阿尔新蓝、甲苯胺蓝、天狼星红染色C组着色最深。结论作为培养基添加物,ITS在诱导hASCs成软骨分化时的效果优于FBS,而作为刺激hASCs成软骨的重要生长因子TGF-β3,10 ng/ml的浓度比1 ng/ml作用更强。

Stem cell-derived hepatocyte therapy using versatile biomimetic nanozyme incorporated nanofiber-reinforced decellularized extracellular matrix hydrogels for the treatment of acute liver failure

Reactive oxygen species(ROS)-associated oxidative stress,inflammation storm,and massive hepatocyte necrosis are the typical manifestations of acute liver failure(ALF),therefore specific therapeutic interventions are essential for the devastating disease.Here,we developed a platform consisting of versatile biomimetic copper oxide nanozymes(Cu NZs)-loaded PLGA nanofibers(Cu NZs@PLGA nanofibers)and decellularized extracellular matrix(dECM)hydrogels for delivery of human adipose-derived mesenchymal stem/stromal cells-derived hepatocyte-like cells(hADMSCs-derived HLCs)(HLCs/Cu NZs@fiber/dECM).Cu NZs@PLGA nanofibers could conspicuously scavenge excessive ROS at the early stage of ALF,and reduce the massive accumulation of pro-inflammatory cytokines,herein efficiently preventing the deterioration of hepatocytes necrosis.Moreover,Cu NZs@PLGA nanofibers also exhibited a cytoprotection effect on the transplanted HLCs.Meanwhile,HLCs with hepatic-specific biofunctions and anti-inflammatory activity acted as a promising alternative cell source for ALF therapy.The dECM hydrogels further provided the desirable 3D environment and favorably improved the hepatic functions of HLCs.In addition,the pro-angiogenesis activity of Cu NZs@PLGA nanofibers also facilitated the integration of the whole implant with the host liver.Hence,HLCs/Cu NZs@fiber/dECM performed excellent synergistic therapeutic efficacy on ALF mice.This strategy using Cu NZs@PLGA nanofiber-reinforced dECM hydrogels for HLCs in situ delivery is a promising approach for ALF therapy and shows great potential for clinical translation.

Encapsulated three-dimensional bioprinted structure seeded with urothelial cells:a new construction technique for tissue-engineered urinary tract patch

Background:Traditional tissue engineering methods to fabricate urinary tract patch have some drawbacks such as compromised cell viability and uneven cell distribution within scaffold.In this study,we combined three-dimensional(3D)bioprinting and tissue engineering method to form a tissue-engineered urinary tract patch,which could be employed for the application on Beagles urinary tract defect mode to verify its effectiveness on urinary tract reconstruction.Methods:Human adipose-derived stem cells(hADSCs)were dropped into smooth muscle differentiation medium to generate induced microtissues(ID-MTs),flow cytometry was utilized to detect the positive percentage for CD44,CD105,CD45,and CD34 of hADSCs.Expression of vascular endothelial growth factor A(VEGFA)and tumor necrosis factor-stimulated gene-6(TSG-6)in hADSCs and MTs were identified by Western blotting.Then the ID-MTs were employed for 3D bioprinting.The bioprinted structure was encapsulated by transplantation into the subcutaneous tissue of nude mice for 1 week.After retrieval of the encapsulated structure,hematoxylin and eosin and Masson’s trichrome staining were performed to demonstrate the morphology and reveal collagen and smooth muscle fibers,integral optical density(IOD)and area of interest were calculated for further semiquantitative analysis.Immunofluorescent double staining of CD31 andα-smooth muscle actin(α-SMA)were used to reveal vascularization of the encapsulated structure.Immunohistochemistry was performed to evaluate the expression of interleukin-2(IL-2),α-SMA,and smoothelin of the MTs in the implanted structure.Afterward,the encapsulated structure was seeded with human urothelial cells.Immunofluorescent staining of cytokeratins AE1/AE3 was applied to inspect the morphology of seeded encapsulated structure.Results:The semi-quantitative assay showed that the relative protein expression of VEGFA was 0.355±0.038 in the hADSCs vs.0.649±0.150 in the MTs(t=3.291,P=0.030),while TSG-6 expression was 0.492±0.092 in the hADSCs vs.1.256±0.401 in the MTs(t=3.216,P=0.032).The semi-quantitative analysis showed that the mean IOD of IL-2 in the MT group was 7.67±1.26,while 12.6±4.79 in the hADSCs group,but semi-quantitative analysis showed that there was no statistical significance in the difference between the two groups(t=1.724,P=0.16).The semi-quantitative analysis showed that IOD was 71.7±14.2 in non-induced MTs(NI-MTs)vs.35.7±11.4 in ID-MTs for collagen fibers(t=3.428,P=0.027)and 12.8±1.9 in NI-MTs vs.30.6±8.9 in ID-MTs for smooth muscle fibers(t=3.369,P=0.028);furthermore,the mean IOD was 0.0613±0.0172 in ID-MTs vs.0.0017±0.0009 in NI-MTs forα-SMA(t=5.994,P=0.027),while 0.0355±0.0128 in ID-MTs vs.0.0035±0.0022 in NI-MTs for smoothelin(t=4.268,P=0.013),which indicate that 3D bioprinted structure containing ID-MTs could mimic the smooth muscle layer of native urinary tract.After encapsulation of the urinary tract patch for additional cell adhesion,urothelial cells were seeded onto the encapsulated structures,and a monolayer urothelial cell was observed.Conclusion:Through 3D bioprinting and tissue engineering methods,we provided a promising way to fabricate tissue-engineered urinary tract patch for further investigation.

Evaluation of Novel 3D Architectures Based on Knitting Technologies for Engineering Biological Tissues

Textile-based technologies are considered as potential routes for the production of 3D porous architectures for tissue engineering( TE) applications. We describe the use of two polymers,namely polybutylene succinate( PBS) and silk fibroin(SF) to produce fiber-based finely tuned porous architectures by weft and warp knittings. The obtained knitted constructs are described in terms of their morphology, mechanical properties,swelling ability,degradation behaviour,and cytotoxicity. Each type of polymer fibers allows for the processing of a very reproducible intra-architectural scaffold geometry,with distinct characteristics in terms of the surface physicochemistry,mechanical performance,and degradation capability,which has an impact on the resulting cell behaviour at the surface of the respective biotextiles. Preliminary cytotoxicity screening shows that both materials can support cell adhesion and proliferation. Furthermore, different surface modifications were performed( acid /alkaline treatment, UV radiation,and plasma) for modulating cell behavior. An increase of cell-material interactions were observed,indicating the important role of materials surface in the first hours of culturing. Human adipose-derived stem cells( hASCs) became an emerging possibility for regenerative medicine and tissue replacement therapies. The potential of the recently developed silk-based biotextile structures to promote hASCs adhesion,proliferation,and differentiation is also evaluated. The obtained results validate the developed constructs as viable matrices for TE applications. Given the processing efficacy and versatility of the knitting technology, and the interesting structural and surface properties of the proposed polymer fibers,it is foreseen that our developed systems can be attractive for the functional engineering of tissues such as bone,skin,ligaments or cartilage and also for develop more complex systems for further industrialization of TE products.

重组人碱性成纤维细胞生长因子对人脂肪干细胞分化为脂肪细胞的影响

目的探讨不同浓度重组人碱性成纤维细胞生长因子（recombinant human basic fibroblast growth factor，rh-bFGF）对体外培养的人脂肪来源干细胞（human adipose-derived stem cells，hASCs）诱导分化为脂肪细胞的影响，通过实验寻找促进hASCs向脂肪细胞转化的最佳浓度。方法取健康脂肪抽吸者脂肪混悬液进行分离提取hASCs，进行hASCs的培养、鉴定和成脂诱导分化，并在成脂诱导的细胞中分别加入0ng／ml rh-bFGF作为对照组，加入10、20、40ng／ml外源性rh—bFGF作为实验组。MTT比色法检测成脂细胞的增殖速率，油红O染色定性分析新形成脂肪细胞的时间，利用Western印迹法检测成熟脂滴表面标记蛋白CIDEC的表达。结果添加40ng／ml rh-bFGF的脂滴形成平均时间为（11．5±1．9）h，检测细胞增殖的吸光度值平均为0．52±0．10，10、20、40ng／ml rh-bFGF对细胞的增殖均有促进作用，尤以40ng／ml浓度最为明显，40ng／ml rh-bFGF成熟脂滴表面蛋白CIDEC的表达量高于其他组。结论在hASCs成脂诱导剂中添加rh-bFGF能有效促进成脂细胞的增殖速率，加速hASCs向脂肪细胞的分化，其中以40ng／ml rh-bFGF为最佳浓度。

LIF基因慢病毒载体构建及其在人脂肪间充质干细胞的表达

目的构建LIF慢病毒载体,转染人脂肪间充质干细胞,以此获得高表达LIF的人脂肪间充质干细胞,为LIF在人脂肪间充质干细胞介导的免疫调节效应机制研究中奠定基础。方法以PCR扩增得到LIF基因序列,采用质粒重组技术获得载有LIF基因的重组质粒,然后用重组质粒包装慢病毒。用胶原酶消化法分离培养人脂肪间充质干细胞,然后将慢病毒转染人脂肪间充质干细胞,通过荧光显微镜观察绿色荧光蛋白的表达,采用流式细胞技术检测慢病毒转染率,RT-PCR及Western blot技术检测LIF的表达水平,最后经SPSS 13.0软件对数据进行统计学分析,计量资料均以(x±s)表示,组间均数比较采用成对样本t检验,以P<0.05为差异有统计学意义。结果通过PCR技术扩增得到LIF基因序列,并且成功构建带有LIF基因的重组质粒,重组质粒经限制性内切酶双酶切鉴定,得到大小为608 bp左右的条带,大小与LIF基因序列匹配,通过重组质粒转染293T细胞后获得慢病毒颗粒,滴度检测达1×108TU/ml。成功分离得到纯度高的h ASCs,慢病毒转染h ASCs后,流式细胞仪检测慢病毒转染率可达90%。RT-PCR及Western blot结果经统计学分析提示,转染后LIF的表达水平明显升高。结论携带人LIF基因的慢病毒载体可以有效转染h ASCs,并且表达LIF。

Development of composite functional tissue sheets using hiPSC-CMs and hADSCs to improve the cardiac function after myocardial infarction

Human-induced pluripotent stem cell-derived cardiomyocytes(hiPSC-CMs)have been widely used in therapy of ischemic heart disease.However,there are still remaining issues that limit the therapeutic efficacy,such as immune rejection and low retention of hiPSC-CMs.Human adipose mesenchymal stromal cells(hADSCs)have been reported to be able to regulate the immune response,promote angiogenesis and promote the maturation of hiPSC-CMs.In this study,we co-cultured these two types of cells on fiber scaffold made of biodegradable poly(D,L-lactic-co-glycolic acid)(PLGA)polymer for several days to develop a composited 3D cardiac tissue sheet.As expected,the cells formed 231.00±15.14μm thickness tissue,with improved organization,alignment,ECM condition,contractile ability,and paracrine function compared to culture hiPSC-CMs only on PLGA fiber.Furthermore,the composited 3D cardiac tissue sheet significantly promoted the engraftment and survival after transplantation.The composited 3D cardiac tissue sheet also increased cardiac function,attenuated ventricular remodeling,decreased fibrosis,and enhanced angiogenesis in rat myocardial infarction model,indicating that this strategy wound be a promising therapeutic option in the clinical scenario.