Administration of mesenchymal stem cells and ziprasidone enhanced amelioration of ischemic brain damage in rats

Ziprasidone is a benzisothiazolyl piperazine derivative that was developed from the chemically related antipsychotic drug tiospirone, and it improves neurological functions of the ischemic brain and is effective in treatment of schizophrenia. Mesenchymal stem cells (MSCs) are considered as a leading candidate for neurological regenerative therapy because of their neural differentiation properties in damaged brain. We investigated whether the transplantation of neural progenitor cells (NPCs) derived from adipose mesenchymal stem cells combined with ziprasidone enhances neuroprotective effects in an animal model of focal cerebral ischemia. In combination therapy groups, significant reduction of infarct volume and improvement of neurological functions were observed at 3 days after middle cerebral artery occlusion (MCAO) compared with monotherapy. Co-administration of ziprasidone and NPCs enhanced the anti-apoptotic effect and reduced the number of terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive apoptotic cells compared with the NPCs alone group at 7 days after MCAO. Ziprasidone or the combination of ziprasidone and NPCs induced the expression of endogenous neurotrophic factor gene brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), and glial cell-derived neurotrophic factor (GDNF). The immunohistochemical investigation revealed that the ziprasidone and NPCs attenuated the increased intensity of microglial marker (Iba-1) in the infarcted cortical area. Moreover, the number of transplanted NPCs on day 7 with combination therapy was significantly higher than with NPCs alone. These effects might be responsible for improved functional behavior and increased survival of NPCs. Our finding indicates that combination therapy of ziprasidone and NPCs enhances neuroprotection against ischemic brain injury.     

SUMO‐modified bone marrow mesenchymal stem cells promoted the repair of articular cartilage in rats

Articular cartilage damage can lead to joint deformity, pain, and severe dysfunction. However, due to the lack of blood vessels and nerves in articular cartilage, the self‐healing capacity of damaged cartilage is limited. In this study, we overexpressed small ubiquitin‐like modifier (SUMO)1, SUMO2/3, and SUMO1/2/3 in bone marrow mesenchymal stem cells (BMSCs). Then, these cells were inoculated on surfaces of different hardness, and their differentiation into chondrocytes, hypoxic tolerance ability, and inflammatory response was detected. Finally, BMSCs were transplanted into the injured knee joint cavity of the rats, and the repair was evaluated. We found that BMSCs overexpressing SUMO1 were more likely to differentiate into articular cartilage along with the hardness of the surface, while BMSCs overexpressing SUMO2/3 could reduce inflammation response and improve the damaged cartilage microenvironment. In the rat model, BMSCs overexpressing SUMO1/2/3 transplanted on injured articular cartilage surface showed better survival, less inflammatory response, and improved tissue repair capability. In conclusion, BMSCs overexpressing SUMO are more tolerant to hypoxia conditions, and have stronger repair ability for damaged chondrocytes in vitro and for articular cartilage injury model in rats, and are excellent seed cells for repairing articular cartilage.     

川芎嗪体外诱导小鼠骨髓间充质干细胞分化为神经元样细胞的研究

为了探讨川芎嗪体外诱导小鼠骨髓间质干细胞（BMSCs）分化为神经元样细胞的作用,以小鼠骨髓间充质干细胞为研究对象,实验分为空白对照组、β-巯基乙醇（BME）阳性对照组和川芎嗪诱导组。采用荧光免疫化学和Western blot方法,分别检测神经干细胞巢蛋白（nestin）和经元特异性烯醇化酶（NSE）的表达;RT-PCR检测诱导不同时间对神经细胞相关基因Nestin、NSE、β-微管蛋白III（β-Tubulin III）和核受体相关因子-1（Nurr1）mRNA表达的影响。结果显示川芎嗪诱导间充质干细胞24 h后,细胞形态发生显著改变,细胞突起形成且数目不等,形成神经元样细胞。细胞死亡率低于β-巯基乙醇诱导组。免疫荧光化学法和western blot结果显示：川芎嗪诱导后的细胞nes-tin和NSE蛋白表达呈阳性,且表达丰度显著高于β-巯基乙醇诱导组。川芎嗪作用不同时间的BMSCs表达神经细胞相关基因Nestin、β-Tubulin III、NSE和Nurrl。结果表明川芎嗪能定向诱导小鼠骨髓间充质干细胞分化为神经元样细胞,是较理想的诱导剂。     

CD200 expression in human cultured bone marrow mesenchymal stem cells is induced by pro‐osteogenic and pro‐inflammatory cues

Similar to other adult tissue stem/progenitor cells, bone marrow mesenchymal stem/stromal cells (BM MSCs) exhibit heterogeneity at the phenotypic level and in terms of proliferation and differentiation potential. In this study such a heterogeneity was reflected by the CD200 protein. We thus characterized CD200^pos cells sorted from whole BM MSC cultures and we investigated the molecular mechanisms regulating CD200 expression. After sorting, measurement of lineage markers showed that the osteoblastic genes RUNX2 and DLX5 were up‐regulated in CD200^pos cells compared to CD200^neg fraction. At the functional level, CD200^pos cells were prone to mineralize the extra‐cellular matrix in vitro after sole addition of phosphates. In addition, osteogenic cues generated by bone morphogenetic protein 4 (BMP4) or BMP7 strongly induced CD200 expression. These data suggest that CD200 expression is related to commitment/differentiation towards the osteoblastic lineage. Immunohistochemistry of trephine bone marrow biopsies further corroborates the osteoblastic fate of CD200^pos cells. However, when dexamethasone was used to direct osteogenic differentiation in vitro, CD200 was consistently down‐regulated. As dexamethasone has anti‐inflammatory properties, we assessed the effects of different immunological stimuli on CD200 expression. The pro‐inflammatory cytokines interleukin‐1β and tumour necrosis factor‐α increased CD200 membrane expression but down‐regulated osteoblastic gene expression suggesting an additional regulatory pathway of CD200 expression. Surprisingly, whatever the context, i.e. pro‐inflammatory or pro‐osteogenic, CD200 expression was down‐regulated when nuclear‐factor (NF)‐κB was inhibited by chemical or adenoviral agents. In conclusion, CD200 expression by cultured BM MSCs can be induced by both osteogenic and pro‐inflammatory cytokines through the same pathway: NF‐κB.     

Comparison of gingiva‐derived and bone marrow mesenchymal stem cells for osteogenesis

Presently, bone marrow is considered as a prime source of mesenchymal stem cells; however, there are some drawbacks and limitations. Compared with other mesenchymal stem cell (MSC) sources, gingiva‐derived mesenchymal stem cells (GMSCs) are abundant and easy to obtain through minimally invasive cell isolation techniques. In this study, MSCs derived from gingiva and bone marrow were isolated and cultured from mice. GMSCs were characterized by osteogenic, adipogenic and chondrogenic differentiation, and flow cytometry. Compared with bone marrow MSCs (BMSCs), the proliferation capacity was judged by CCK‐8 proliferation assay. Osteogenic differentiation was assessed by ALP staining, ALP assay and Alizarin red staining. RT‐qPCR was performed for ALP, OCN, OSX and Runx2. The results indicated that GMSCs showed higher proliferative capacity than BMSCs. GMSCs turned more positive for ALP and formed a more number of mineralized nodules than BMSCs after osteogenic induction. RT‐qPCR revealed that the expression of ALP, OCN, OSX and Runx2 was significantly increased in the GMSCs compared with that in BMSCs. Moreover, it was found that the number of CD90‐positive cells in GMSCs elevated more than that of BMSCs during osteogenic induction. Taking these results together, it was indicated that GMSCs might be a promising source in the future bone tissue engineering.     

Bone marrow and adipose mesenchymal stem cells attenuate cardiac fibrosis induced by methotrexate in rats

Mesenchymal stem cells (MSCs) are an ideal adult stem cell with capacity for self‐renewal and differentiation with an extensive tissue distribution. The present study evaluates the therapeutic effects of bone marrow mesenchymal stem cells (BM‐MSCs) or adipose‐derived mesenchymal stem cells (AD‐MSCs) against the development of methotrexate (MTX)‐induced cardiac fibrosis versus dexamethasone (DEX). Rats were allocated into five groups; group 1, received normal saline orally; group 2, received MTX (14 mg/kg/week for 2 weeks); groups 3 and 4, treated once with 2 × 10 ⁶ cells of MTX + BM‐MSCs and MTX + AD‐MSCs, respectively; and group 5, MTX + DEX (0.5 mg/kg, for 7 days, P.O.). MTX induced cardiac fibrosis as marked changes in oxidative biomarkers and elevation of triglyceride, cholesterol, aspartate aminotransferase, gamma‐glutamyl transferase, creatine kinase, and caspase‐3, as well as deposited collagen. These injurious effects were antagonized after treatment with MSCs. So, MSCs possessed antioxidant, antiapoptotic, as well antifibrotic effects, which will perhaps initiate them as notable prospective for the treatment of cardiac fibrosis.     

Persistent DNA damage‐induced premature senescence alters the functional features of human bone marrow mesenchymal stem cells

Human mesenchymal stem cells (hMSCs) are adult multipotent stem cells located in various tissues, including the bone marrow. In contrast to terminally differentiated somatic cells, adult stem cells must persist and function throughout life to ensure tissue homeostasis and repair. For this reason, they must be equipped with DNA damage responses able to maintain genomic integrity while ensuring their lifelong persistence. Evaluation of hMSC response to genotoxic insults is of great interest considering both their therapeutic potential and their physiological functions. This study aimed to investigate the response of human bone marrow MSCs to the genotoxic agent Actinomycin D (ActD), a well‐known anti‐tumour drug. We report that hMSCs react by undergoing premature senescence driven by a persistent DNA damage response activation, as hallmarked by inhibition of DNA synthesis, p21 and p16 protein expression, marked Senescent Associated β‐galactosidase activity and enlarged γH2AX foci co‐localizing with 53BP1 protein. Senescent hMSCs overexpress several senescence‐associated secretory phenotype (SASP) genes and promote motility of lung tumour and osteosarcoma cell lines in vitro. Our findings disclose a multifaceted consequence of ActD treatment on hMSCs that on the one hand helps to preserve this stem cell pool and prevents damaged cells from undergoing neoplastic transformation, and on the other hand alters their functional effects on the surrounding tissue microenvironment in a way that might worsen their tumour‐promoting behaviour.     

Reconstruction of tissue‐engineered bone with bone marrow mesenchymal stem cells and partially deproteinized bone in vitro

We have explored the optimal seeding density and timing for transplantation of the tissue‐engineered bone with BMMSCs (bone marrow mesenchymal stem cells) and PDPB (partially deproteinized bone) in vitro. Rabbit BMMSCs of different densities were seeded into PDPB generated from fresh pig vertebrates to reconstruct tissue‐engineered bone in vitro. Adhesion and proliferation of BMMSCs were analysed by MTT [3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl‐2H‐tetrazolium bromide] assay from which growth curves of BMMSCs on the PDPB materials were generated. The data show that BMMSCs began to adhere to PDPB after 24 h of primary culture, all groups reaching peak growth on the 6th day, after which the value of A decreased gradually and reached a plateau phase. The optimal BMMSCs seeding density of 5×10⁶/ml achieved an excellent adhesion and proliferation activity on PDPB. In summary, the best cell seeding density of constructing tissue‐engineered bone with BMMSCs in vitro is 5×10⁶/ml, the optimal timing to transplant is the 6th day.     

Combination therapy with bone marrow stromal cells and FK506 enhanced amelioration of ischemic brain damage in rats

AimsWe previously reported that cysteinyl leukotriene receptor 2 (CysLT₂) mediates ischemic astrocyte injury, and leukotriene D₄-activated CysLT₂ receptor up-regulates the water channel aquaporin 4 (AQP4). Here we investigated the mechanism underlying CysLT₂ receptor-mediated ischemic astrocyte injury induced by 4-h oxygen-glucose deprivation and 24-h recovery (OGD/R).Main methodsPrimary cultures of rat astrocytes were treated by OGD/R to construct the cell injury model. AQP4 expression was inhibited by small interfering RNA (siRNA). The expressions of AQP4 and CysLTs receptors, and the MAPK signaling pathway were determined.Key findingsOGD/R induced astrocyte injury, and increased expression of the CysLT₂ (but not CysLT₁) receptor and AQP4. OGD/R-induced cell injury and AQP4 up-regulation were inhibited by a CysLT₂ receptor antagonist (Bay cysLT2) and a non-selective CysLT receptor antagonist (Bay u9773), but not by a CysLT₁ receptor antagonist (montelukast). Knockdown of AQP4 by siRNA attenuated OGD/R injury. Furthermore, OGD/R increased phosphorylation of ERK1/2 and p38, whose inhibitors relieved the cell injury and AQP4 up-regulation.SignificanceThe CysLT₂ receptor mediates AQP4 up-regulation in astrocytes, and up-regulated AQP4 leads to OGD/R-induced injury, which results from activation of the ERK1/2 and p38 MAPK pathways.     

人骨髓间充质干细胞在成年大鼠脑内的迁移及分化

骨髓间充质干细胞 (mesenchymalstemcells,MSCs)是目前备受关注的一类具有多向分化潜能的组织干细胞 ,体外可以分化为骨、软骨、脂肪等多种细胞。因此 ,MSCs是细胞治疗和基因治疗的种子细胞之一。为了探索MSCs的迁移和分化趋势 ,为帕金森病 (Parkinsondisease,PD)的干细胞治疗提供理论和实验依据 ,本实验将体外扩增并转染增强型绿色荧光蛋白 (enhancedgreenfluorescentprotein ,EGFP)的人骨髓MSCs注入PD大鼠脑内纹状体 ,观察了人骨髓MSCs在大鼠脑内的存活、迁移、分化以及注射MSCs前后大鼠的行为变化。结果表明 ,人骨髓MSCs在大鼠脑内可存活较长时间 ( 10周以上 ) ;随着时间的延长 ,MSCs迁移范围扩大 ,分布于纹状体、胼胝体、皮质以及脑内血管壁 ;免疫组化法检测证实MSCs在大鼠脑内表达人神经丝蛋白 (neurofilament,NF)、神经元特异性烯醇化酶 (neuron specificeno lase,NSE)以及胶质原纤维酸性蛋白 ( glialfibrillaryacidprotein ,GFAP) ;PD大鼠的异常行为有所缓解 ,转圈数由 8 86±2 0 9r/min下降到 4 87± 2 0 6r/min ,统计学分析P <0 0 5为差异显著。以上观察结果表明 ,骨髓MSCs有望成为治疗PD的种子细胞     

Microtubule-interacting drugs induce moderate and reversible damage to human bone marrow mesenchymal stem cells

Objectives:  This study aimed to investigate molecular and cellular changes induced in human bone marrow mesenchymal stem cells (hMSCs) after treatment with microtubule-interacting agents and to estimate damage to the bone marrow microenvironment caused by chemotherapy.
Materials and methods:  Using an in vitro hMSC culture system and biochemical and morphological approaches, we studied the effect of nocodazole and taxol® on microtubule and nuclear envelope organization, tubulin and p53 synthesis, cell cycle progression and proliferation and death of hMSCs isolated from healthy donors.
Results and conclusions:  Both nocodazole and taxol reduced hMSC proliferation and induced changes in the microtubular network and nuclear envelope morphology and organization. However, they exhibited only a moderate effect on cell death and partial arrest of hMSCs at G₂ but not at M phase of the cell cycle. Both agents induced expression of p53, exclusively localized in abnormally shaped nuclei, while taxol, but not nocodazole, increased synthesis of β-tubulin isoforms. Cell growth rates and microtubule and nuclear envelope organization gradually normalized after transfer, in drug-free medium. Our data indicate that microtubule-interacting drugs reversibly inhibit proliferation of hMSCs; additionally, their cytotoxic action and effect on microtubule and nuclear envelope organization are moderate and reversible. We conclude that alterations in human bone marrow cells of patients under taxol chemotherapy are transient and reversible.     

Polyethylenimine‐mediated gene delivery into human bone marrow mesenchymal stem cells from patients

Transplantation of mesenchymal stem cells (MSCs) derived from adult bone marrow has been proposed as a potential therapeutic approach for post‐infarction left ventricular (LV) dysfunction. However, age‐related functional decline of stem cells has restricted their clinical benefits after transplantation into the infarcted myocardium. The limitations imposed on patient cells could be addressed by genetic modification of stem cells. This study was designed to improve our understanding of genetic modification of human bone marrow derived mesenchymal stem cells (hMSCs) by polyethylenimine (PEI, branched with Mw 25 kD), one of non‐viral vectors that show promise in stem cell genetic modification, in the context of cardiac regeneration for patients. We optimized the PEI‐mediated reporter gene transfection into hMSCs, evaluated whether transfection efficiency is associated with gender or age of the cell donors, analysed the influence of cell cycle on transfection and investigated the transfer of therapeutic vascular endothelial growth factor gene (VEGF). hMSCs were isolated from patients with cardiovascular disease aged from 41 to 85 years. Optimization of gene delivery to hMSCs was carried out based on the particle size of the PEI/DNA complexes, N/P ratio of complexes, DNA dosage and cell viability. The highest efficiency with the cell viability near 60% was achieved at N/P ratio 2 and 6.0 μg DNA/cm². The average transfection efficiency for all tested samples, middle‐age group (<65 years), old‐age group (>65 years), female group and male group was 4.32%, 3.85%, 4.52%, 4.14% and 4.38%, respectively. The transfection efficiency did not show any correlation either with the age or the gender of the donors. Statistically, there were two subpopulations in the donors; and transfection efficiency in each subpopulation was linearly related to the cell percentage in S phase. No significant phenotypic differences were observed between these two subpopulations. Furthermore, PEI‐mediated therapeutic gene VEGF transfer could significantly enhance the expression level.     

Polyphenols suppress hydrogen peroxide‐induced oxidative stress in human bone‐marrow derived mesenchymal stem cells

Human mesenchymal stem cells (hMSCs) are considered a highly promising candidate cell type for cell‐based tissue engineering and regeneration because of their self‐renewal and multi‐lineage differentiation characteristics. Increased levels of reactive oxygen/nitrogen species (ROS/RNS) are associated with tissue injury and inflammation, impact a number of cellular processes, including cell adhesion, migration, and proliferation, and have been linked to cellular senescence in MSCs, potentially compromising their activities. Naturally occurring polyphenolic compounds (polyphenols), epigallocatechin‐3‐gallate (EGCG), and curcumin, block ROS/RNS and are potent inflammation‐modulating agents. However, their potential protective effects against oxidative stress in hMSCs have not been examined. In this study, we carried out a systematic analysis of the effects of polyphenols on hMSCs in their response to oxidative stress in the form of treatment with H₂O₂ and S‐nitroso‐N‐acetylpenicillamine (SNAP), respectively. Parameters measured included colony forming activity, apoptosis, and the levels of antioxidant enzymes and free reactive species. We found that polyphenols reversed H₂O₂‐induced loss of colony forming activity in hMSCs. In a dose‐dependent manner, polyphenols inhibited increased levels of ROS and NO, produced by H₂O₂ or SNAP, respectively, in MSCs. Notably, polyphenols rapidly and almost completely blocked H₂O₂‐induced ROS in the absence of significant direct effect on H₂O₂ itself. Polyphenols also protected the antioxidant enzymes and reduced apoptotic cell death caused by H₂O₂ exposure. Taken together, these findings demonstrate that EGCG and curcumin are capable of suppressing inducible oxidative stress in hMSCs, and suggest a possible new approach to maintain MSC viability and potency for clinical application. J. Cell. Biochem. 114: 1163–1173, 2013. © 2012 Wiley Periodicals, Inc.     

重组杆状病毒转导恒河猴骨髓间充质干细胞

[目的]研究重组杆状病毒(Bac-CMV-EGFP)能否能有效转导恒河猴骨髓间充质干细胞(rhesus Bone marrow-derived Mesenchymal Stem Cells,rBMSCs),及杆状病毒转导后对细胞活力,增殖及分化能力的影响.[方法]体外原代培养rBMSCs,不同剂量的杆状病毒转导3代以后的细胞,并用流式细胞仪分别检测其转导效率.在较高的杆状病毒转导效率下,检测rBMSCs细胞活力,增殖及分化能力,并与正常对照组细胞进行比较.[结果]杆状病毒在感染指数(Multiplicity Of Infection,MOI)为300v.g/cell,孵育温度为25度,孵育时间为4h的转导条件下,对rBMSCs转导效率可达80%左右.进一步检测后发现,高效转导杆状病毒后的rBMSCs的细胞活力,增殖及分化能力与未转导病毒细胞组无明显变化.[结论]重组杆状病毒可安全有效地基因修饰rBMSCs,且不影响其生物特性,为今后的体内基因治疗灵长类动物模型试验奠定了基础.     

人骨髓间充质干细胞向血管内皮细胞诱导分化的研究

目的:分析人骨髓间充质干细胞(hMSCs)和脐静脉内皮细胞(hUVECs)的基因表达差异,探讨体外基因转染诱导内皮分化的可行性以及作为血管组织工程种子细胞来源的应用前景。方法:分别从人骨髓和脐静脉分离间充质干细胞(hMSCs)和内皮细胞(hUVECs),扩增培养后进行流式细胞仪、免疫细胞化学,免疫荧光鉴定和超微结构观察。通过BiostarH-40S表达谱芯片分析,选择两者的差异表达基因,导入hMSCs,经RT-PCR、ELISA鉴定该基因的转染和表达,并分析hMSCs的内皮分化程度。结果:hMSCs表达内皮细胞的多种特异性mRNA,经VEGFl65基因瞬时转染后RT-PCR有明显条带,ELISA定量检测VEGF165蛋白表达为(707.9±11.3)ng/L,同时CD44表达明显下调38.80%,CD31则明显上调达56.82%,FI-1,FVⅢAg和CD34的表达也有不同程度升高。结论:hMSCs具有内皮分化潜能,体外基因转染诱导hMSCs产生功能性内皮细胞和组织工程化血管具有广阔前景。