Human umbilical cord mesenchymal stem cells and the treatment of spinal cord injury

Objective To review the recent studies about human umbilical cord mesenchymal stem cells （hUCMSCs） and advances in the treatment of spinal cord injury. Data sources Published articles （1983-2007） about hUCMSCs and spinal cord injury were selected using Medline. Study selection Articles selected were relevant to development of mesenchymal stem cells （MSCs） for transplantation in spinal cord injury therapy. Of 258 originally identified articles 51 were selected that specifically addressed the stated purpose. Results Recent work has revealed that hUCMSCs share most of the characteristics with MSCs derived from bone marrow and are more appropriate to transplantation for cell based therapies. Conclusions Human umbilical cord could be regarded as a source of MSCs for experimental and clinical needs. In addition, as a peculiar source of stem cells, hUCMSCs may play an important role in the treatment of spinal cord injury. Chin Med J 2009;122（2）：225-231     

Methods of isolation, expansion, differentiating induction and preservation of human umbilical cord mesenchymal stem cells

Objective  This literature review aims to summarize the methods of isolation, expansion, differentiation and preservation of human umbilical cord mesenchymal stem cells (hUCMSCs), for comprehensive understanding and practical use in preclinical research and clinical trials.

Data sources  All the literature reviewed was published over the last 10 years and is listed in PubMed and Chinese National Knowledge Infrastructure (CNKI). Studies were retrieved using the key word “human umbilical cord mesenchymal stem cells”.

Results  Explants culture and enzymatic digestion are two methods to isolate hUCMSCs from WJ and there are modifications to improve these methods. Culture conditions may affect the expansion and differentiating orientations of hUCMSCs. In addition, hUCMSCs can maintain their multi-potential effects after being properly frozen and thawed.

Conclusion  Considering their multi-potential, convenient and non-invasive accessibility, low immunogenicity and the reported therapeutic effects in several different preclinical animal models, hUCMSCs have immense scope in regeneration medicine as a substitute for MSCs derived from bone marrow or umbilical cord blood.

     

In Vitro Chondrogenic Phenotype Differentiation of Bone Marrow-derived Mesenchymal Stem Cells

In order to study the chondrogenic phenotype differentiation of adult sheep bone marrowderived mesenchymal stem cells (MSCs) in a defined medium as potential seed cells for cartilage tissue engineering, MSCs were isolated by density centrifugation with Percoll solution from bone marrow aspirated from sheep iliac crest. The third passage of MSCS were induced with H-DMEM containing TGF-β3 .IGF-I, Dexamethasone and VitC. The shape and ultrastructure of cells were observed, toluidine blue stain for GAG and immunohistochemistry for type II collagen were applied for chondrogenic phenotype identification. After 14 days of induction, MSCs changed from a spindlelike appearance to a polynal shape, a large amount of endoplasmic reticulum, Golgi complex and mitochondria were observed, and the differentiation of MSCs chondrogenic phenotype was verified by positive staining of toluidine blue and immunohistochemistry. MSCs derived from bone marrow can differentiate to chondrogenic phenotype when induced in vitro and can be used as optimal seed cells for cartilage tissue engineering.     

Differentiation character of adult mesenchymal stem cells and transfection of MSCs with lentiviral vectors

This study examined the differentiation character and pluripotency of mesenchymal stem cells (MSCs) under different conditions. Adult MSCs were initially isolated from the bone marrow of rats, cultured in vitro and identified by flow cytometry. After MSCs were transferred to osteogenic and adipogenic medium respectively, the morphological characterization of induced cells was observed. The expression of marker genes was detected by RT-PCR analysis. Then MSCs were transfected with lenti- viral vectors pGC-FU-Sox9-EGFP. Enhanced green fluorescence protein (EGFP) expression and trans- fection efficiency were determined by fluorescence microscopy. The results demonstrated that EGFP caused no effect on the multilineage potential of adult MSCs. Sox9 gene expression of high level was maintained stable in the transfected MSCs and induced MSCs to differentiate into chondrocytes. Ag- gracan was positive in chondrogenic lineages and the expression of aggracan and type Ⅱ collagenwas significantly increased during MSCs chondrogenic differentiation. It was concluded that Sox9 gene-modified adult MSCs may be promising candidate cells for further studies on tissue engineering. EGFP facilitates the research on MSCs physiological behavior and application in tissue engineering during differentiation both in vitro and in vivo.     

Human umbilical cord-derived endothelial progenitor cells promote growth cytokines-mediated neorevascularization in rat myocardial infarction

Background Cell-based vascular therapies of endothelial progenitor cells （EPCs） mediated neovascularization is still a novel but promising approach for the treatment of ischemic disease. The present study was designed to investigate the therapeutic potentials of human umbilical cord blood-derived EPCs （hUCB-EPCs） in rat with acute myocardial infarction. Methods Human umbilical cord blood （hUCB） mononuclear cells were isolated using density gradient centrifugation from the fresh human umbilical cord in healthy delivery woman, and cultured in M199 medium for 7 days. The EPCs were identified by double-positive staining with 1,1＇-dioctadecyl-3,3,3＇,3＇-tetramethylindocarbocyanine percholorate-labeled acetylated low-density lipoprotein （DiI-Ac-LDL） and fluorescein isothiocyanate-conjugated Ulex europaeus lectin （FITC-UEA-I）. The rat acute myocardial infarction model was established by the ligation of the left anterior descending artery. The hUCB-EPCs were intramyocardially injected into the peri-infarct area. Four weeks later, left ventricular function was assessed by a pressure-volume catheter. The average capillary density （CAD） was evaluated by anti-VIII immunohistochemistry staining to reflect the development of neovascularization at the peri-infarct area. The graft cells were identified by double immunofluorescence staining with human nuclear antigen （HNA） and CD31 antibody, representing human origin of EPCs and vascular endothelium, respectively. Expressions of cytokines, proliferating cell nuclear angigen （PCNA）, platelet endothelial cell adhesion molecule （PECAM） and vascular endothelial growth factor （VEGF） were detected to investigate the underlying mechanisms of cell differentiation and revascularization. Results The donor EPCs were detectable and integrated into the host myocardium as confirmed by double-positive immunofluorescence staining with HNA and CD31. And the anti-VIII staining demonstrated a higher degree of microvessel formation in EPCs transplanted rats, associated with a significant improvement of global heart function in terms of the increase of left ventricular end-systolic pressure （LVESP）, ＋dp/dtmax and -dp/dtmax as well as the decrease of LVEDP in rats with EPCs therapy comparing to the control rats （P〈0.05）. Moreover, the expression of the rat PCNA mRNA and PECAM were both enhanced in the EPCs group compared with that of the control group. Conclusions The human umbilical cord blood-derived EPCs could incorporate into new-born capillaries in rat myocardium, induce revascularization and improve the proliferation activity in the peri-infarct area, resulting in the improvement of global heart function. This may indicate a promising stem cell resource in cell-based therapy for ischaemic diseases.     

RhoA/ROCK signaling in chondrogenic differentiation of MSCs induced by TGF-β1

Objective： To investigate the role of RhoA/ROCK in the process of chondrogenic differentiation of MSCs in vitro induced by TGF-β1. Methods ： MSCs were isolated from rat bone marrow, cultured and passaged. The cultured MSCs at the 3rd passage were induced to differentiate into chondrocytes in induction medium containing TGF-β1, the expressions of RhoA and ROCKI/2 in the induced MSCs cells were detected by Western-blot and RT-PCR. Results： In parallel to chondrogenic marker gene expressions of collagen Ⅱand aggrecan, ROCK inhibition by Y27632 in these cells caused a significant decrease in mRNA level of collagen Ⅱ. Conclusion： The results suggest that RhoA/ROCK pathway may play an important and complex role in regulation of chondrogenic differentiation and gene expression.     

人脐带间充质干细胞在不同孔径聚碳酸酯膜上的生长及迁移

目的 观察人脐带间充质干细胞(hUCMSCs)在不同孔径Transwell插件底部聚碳酸酯膜上的生长、迁移情况,探讨对hUCMSCs进行非直接接触式共培养诱导分化时选择适宜膜孔径Transwell插件的依据.方法 体外分离、培养hUCMSCs,以丝裂霉素C处理后接种于常用的0.4、3.0和8.0μm三种膜孔径的6孔板Transwell插件中,培养7 d,观察、计数三种孔径插件底部贴壁细胞,计算迁移率,并在扫描电镜下观察细胞在多孔膜上的生长、迁移情况.结果 培养7 d后,0.4、3.0和8.0μm三种插件内贴壁hUCMSCs的迁移率分别为0、1.8%、8.0%,0.4 μm孔径迁移率与其他两组相比差异有统计学意义(P＜0.01).扫描电镜下观察到细胞在3.0和8.0 μm孔径聚碳酸酯膜底面生长以及穿越微孔的现象,而在0.4μm孑L径膜则未发现.结论 3.0和8.0 μm孔径聚碳酸酯膜允许hUCMSCs迁移穿越,而0.4 μm孔径聚碳酸酯膜则不允许,或可利用其正反面进行近距离非直接接触式共培养诱导hUCMSCs分化.

Abstract：

Objective To observe the growth and migration of human umbilical cord mesenchymal stem cells (hUCMSCs) on polycarbonate membrane with different pore sizes and explore the criteria of selecting optimal Transwell insert for indirect co-culture to induce the differentiation of hUCMSCs.Methods hUCMSCs were isolated in vitro and then expanded in culture medium.After the treatment of mitomycin C,the cells were seeded on porous membranes of 6-well-dish Transwell inserts with different pore sizes of 0.4,3.0 and 8.0 μm respectively.After culturing for 7 days,the cells were observed and counted on the bottom of each porous membrane.Then the calculation of migration ratio was performed.The growth and migration of hUCMSCs on porous membranes were also examined under scanning electron microscope (SEM).Results The migration ratios of hUCMSCs on membranes of 0.4,3.0 and 8.0 μm pore sizes were 0,1.8% and 8.0% respectively.The migration ratio of cells on 0.4 μm pore size membrane was statistically different from that of the other two pore size groups (P ＜ 0.01).Under SEM,a small portion of cells were growing on the bottoms of membranes and moving through the pores.But there was no cell movement through 0.4 μm pore size membrane.Conclusions hUCMSCs can migrate through the polycarbonate membranes of 3.0 μm and 8.0 μm pore sizes but not through the 0.4 μm one.Thus both sides of polycarbonate membrane of 0.4 μm pore size may be used for close indirect co-culture to induce the differentiation of hUCMSCs.     

Effect of the gap junction blocker 1-heptanol on chondrogenic differentiation of mouse bone marrow mesenchymal stem cells in vitro

Objective:To investigate the effect of the gap junction blocker 1-heptanol on the in vitro chondrogenic differentiation of mouse bone marrow mesenchymal stem cells(MSCs) following induction by GDF-5. Methods:MSCs were isolated from mouse bone marrow and cultured in vitro. After 3 passages cells were induced to undergo chondrogenic differentiation with recombinant human GDF-5(100 ng/ml), with or without 1-heptanol(2.5μmol/L). The effect of 1-heptanol on MSCs proliferation was investigated using the MTT assay...     

人脐带华通氏胶间充质干细胞的分离、培养、鉴定及冻存、复苏

目的：探讨从人脐带华通氏胶（Wharton’s jelly,WJ）中分离、培养、鉴定间充质干细胞（mesenchymal stemcells,MSCs）及其冻存、复苏的方法。方法：采用植块法分离、培养间充质干细胞,流式细胞仪检测P3代细胞免疫表型,鉴定其向成骨、成脂方向诱导分化的能力;将P1细胞冻存6个月后复苏,鉴定复苏后细胞的特性。结果：植块法容易从人脐带华通氏胶中获得间充质干细胞;组织块贴壁后6 d可见组织块周围细胞爬出,原代培养14~18 d细胞融合70%~80%;P3代细胞强烈表达CD73、CD90、CD105,不表达CD14、CD34、CD45、CD79a和HLA-DR;成骨诱导分化后10 d,可见明显钙结节;成脂诱导14 d,有明显的脂滴出现,油红O染色阳性。冻存再复苏细胞活力达80%,细胞免疫表型及成骨、成脂诱导显示与冻存前细胞呈相同的特性。结论：组织块培养法可从人脐带华通氏胶中分离、培养出纯度较高间充质干细胞,冻存、复苏不改变其特性。     

人脐血间充质干细胞体外分离培养与扩增实验研究

目的探讨脐血来源的人间充质干细胞（HMSCs）在体外分离培养与扩增的可行性。方法在无菌条件下收集正常足月胎儿的脐带血,经复合枸橼酸钠抗凝,用相对密度为1．077g／L的Ficoll淋巴细胞分离液分离脐血的单个核细胞,以30％胎牛血清进行培养和扩增,用流式细胞仪检测MSCs的表面标志。结果来源于人脐血的单个核细胞接种于特定培养基后,可产生贴壁细胞,主要表现为破骨样细胞和间充质样细胞,经传几代后,可得纯化扩增的人脐血MSCs。流式细胞仪检测结果显示,人脐血MSCs不表达GD14、CD19,强表达CD105、CD44。结论来源于人脐血的MSCs在体外可以分离培养、扩增,为MSCs的进一步研究奠定基础。     

Effectene介导钆喷酸葡胺标记人脐带间充质干细胞及体外磁共振成像研究

目的应用Effectene介导钆喷酸葡胺（Gd-DTPA）标记人脐带间充质干细胞（hUCMSCs）,研究磁标记干细胞的生物学特性,探讨Gd-DTPA标记干细胞体外磁共振成像（MRI）规律。方法组织块贴壁法分离纯化hUCMSCs,通过传代培养、扩增,鉴定细胞在体外的生物学特性。应用Effectene转染Gd-DTPA标记hUCMSCs,计数法检测Gd-DTPA标记干细胞的增殖能力,体外诱导其向成脂细胞和成骨细胞分化,观察Gd-DTPA影响hUCMSCs的生物学特性。应用1．5T临床应用型MRI系统,观察Gd-DTPA标记hUCMSCs的信号强度随细胞传代的变化规律,并探索MRI的最低细胞量。结果应用组织块贴壁法接种2周后获得原代细胞,细胞呈长梭形,漩涡样生长,传2代后细胞形态更为均匀一致。流式细胞仪检测第3代细胞高表达CD29、CD44、CD90和CD105,不表达CD31、CD45、CD40和HLA-DR。体外定向诱导能分化为成脂细胞和成骨细胞。Effectene能成功转染Gd-DTPA进入干细胞,检测标记后的干细胞增殖能力未受到影响,并能在体外诱导向成骨细胞和成脂细胞分化。体外MRI扫描Gd-DTPA标记的干细胞在T1WI呈现高信号,体外持续示踪时间约12d。结论应用组织块贴壁法能有效分离纯化hUCMSCs。应用Effectene转染Gd-DTPA标记hUCMSCs进行体外MRI示踪是可行的。     

特定微环境下诱导脐血间充质干细胞向神经细胞的分化

目的 在体外分离培养人脐血间充质干细胞(marrow mesenchymal stem cells,MSCs),探讨脐血间充质干细胞体外培养生长特性及向神经细胞分化的诱导条件.方法 在体外将脐血MSCs进行向神经细胞的定向诱导,观察脐血MSCs的形态变化,并在诱导后第5天用免疫荧光染色检测脐血MSCs中神经元细胞特异性的标志物;并与单纯低糖DMEM培养基培养的对照组脐血MSCs相比较.结果 诱导组中的脐血MSCs生长伸展,形成突起,呈放射状,并可互相形成连接,免疫荧光显示特异性烯醇化酶(neuronspecific enolase,NSE)阳性,表现出神经元细胞的特性.而对照组中的脐血MSCs未形成神经样形态结构,免疫荧光NSE阴性.结论 脐血MSCs可在体外经化学方法 定向诱导分化为神经细胞,可应用于神经修复及再生等领域.     

人脐血间充质干细胞体分离、培养及向脂肪细胞分化的实验研究

目的:探讨脐血间充质干细胞(UCB-MSCs)的分离培养及向脂肪细胞的分化潜能。方法:从足月儿脐血中获得间充质干细胞,体外培养传代,流式细胞检测细胞表面及抗原细胞周期;予10-6M地塞米松、100μg/ml1-甲基-3-异丁基-黄嘌呤(IBMX)、50μg/ml抗坏血酸的IMDM培养基,对MSCs进行诱导向脂肪细胞分化,油红-O染色染色鉴定。结果:成功建立了脐血间充质干细胞分离及培养扩增的方法;流式细胞仪检测结果显示,贴壁细胞均表达CD105、CD29和CD44,不表达造血细胞表型CD34、CD45和内皮细胞表型CD31;经脂肪培养液诱导后在细胞胞浆里可见颗粒状红色脂滴形成。结论:脐血间充质干细胞能进行体外分离培养,并能诱导分化为脂肪细胞。     

人脐带间充质干细胞分离培养方法的优化

目的：优化人脐带间充质干细胞 （ umbilical cord mesenchymal stem cells, UC - MSCs）分离培养的方法。方法：无菌条件下剔除剖腹产胎儿脐带动静脉,用牙剪子剪为约lmm。但彼此相连的组织块,得到贴壁细胞,采用半量换液进行原代及传代培养。用流式细胞仪检测其免疫表型。结果：成功从人脐带中分离纯化得到UC—MSCs,呈条形和成纤维细胞样的两种形态。UC—MSCs高度表达CD105、CD73．CD44、CDg0,极少数表达CIM5、CD34-CD14和HLA—DR。结论：建立出一种快捷经济的UC—MSCs体外分离培养方法。     

人脐带间充质干细胞裂解液在体外对食管癌EC9706细胞增殖与迁移的抑制作用

目的探讨人脐带间充质干细胞(hUCMSCs)裂解液在体外对食管癌EC9706细胞增殖、迁移的抑制作用。方法用组织块培养法体外培养hUCMSCs,流式细胞仪检测细胞表型;收集hUCMSCs,并用反复冻融法制备hUCMSCs裂解液;不同浓度hUCMSCs裂解液作用于食管癌EC9706细胞一定时间,采用四甲基偶氮唑盐(MTT)法检测细胞增殖状况,倒置显微镜观察细胞形态。通过Tranwell迁移实验观察hUCMSCs裂解液对食管癌EC9706细胞迁移能力的影响。结果人脐带组织块培养7¹2 d后可见贴壁的成纤维样细胞,流式细胞仪分析显示第5代细胞均表达CD29、CD90和CD166,不表达CD34、CD45和人类白细胞抗原-DR。MTT法显示加入hUCMSCs数等于或大于食管癌细胞数的裂解液时,与对照组比较明显抑制食管癌细胞的生长(P<0.05),且实验组各组抑制食管癌细胞增殖能力差异无统计学意义(P>0.05)。Tranwell迁移实验显示,实验组EC9706细胞的迁移较对照组明显受到抑制(P<0.05)。结论 hUCMSCs裂解液对食管癌细胞EC9706的增殖和迁移均有抑制作用。     

3种人类间充质干细胞体外增殖能力和成脂能力的比较

目的: 从人足月胎儿的脐带组织和胎盘组织中分离、培养间充质干细胞（MSCs）,观察人脐带组织MSCs（UC-MSCs）、胎盘组织MSCs（PL-MSCs）和胚胎组织MSCs（FT-MSCs）体外增殖能力和成脂分化潜能,为干细胞进一步应用于临床提供实验依据。方法: 无菌条件下获取人足月胎儿脐带组织和胎盘组织,通过Ⅰ型胶原酶酶解法分离出UC-MSCs和PL-MSCs,FT-MSCs由中科生物工程有限公司提供,取第3或4代MSCs进行检测。倒置显微镜观察细胞形态;活细胞计数法检测细胞增殖能力;流式细胞术测定UC-MSCs、PL-MSCs和FT-MSCs细胞周期并鉴定细胞表面标志物的表达阳性率。取3种组织来源的第3代MSCs进行成脂诱导,诱导18 d进行油红O染色,观察并比较3种不同组织来源MSCs的成脂能力。结果：从3种不同组织中均可获取梭形贴壁的MSCs,表面标记物CD44、CD73、CD90和CD105均呈阳性表达,CD14、CD34和CD45均为阴性表达;FT-MSCs增殖能力明显强于UC-MSCs和PL-MSCs（P<0.01）,FT-MSCs、UC-MSCs和PL-MSCs的增殖指数（PI）分别为(36.66±1.30)%、(18.23±1.10)%和(10.03±1.20)%,3种MSCs的PI比较差异均有统计学意义(P<0.01)。3种MSCs经过成脂诱导液诱导后,油红O染色结果均为阳性,但是UC-MSC s体外成脂能力(油红O染色阳性率)明显强于FT-MSCs和PL-MSCs(P<0.01)。结论：3种不同组织来源MSCs在形态和细胞表面标记物等方面相似,FT-MSCs具有更强的增殖能力和增殖活性,UC-MSCs具有更强的体外成脂潜能。