骨髓间充质干细胞向视网膜神经节样细胞的分化

目的探讨乳鼠视网膜细胞条件分化液诱导骨髓间充质干细胞(BMSCs)的神经分化情况,以期为视网膜退行性疾病提供治疗方案。方法体外分离培养Wistar大鼠乳鼠BMSCs,观察BMSCs的增殖情况并进行鉴定;制备乳鼠视网膜细胞条件分化液,以其诱导BMSCs,观察BMSCs的神经分化情况,并行免疫组化鉴定。结果体外培养获得了较纯的BMSCs;在乳鼠视网膜细胞条件分化液的环境中,诱导后72h,BMSCs胞体收缩成锥形或球形,细胞突起变细、变长,呈神经细胞的典型形态;免疫组化结果显示,部分细胞呈神经元特异性烯醇化酶(NSE)、巢蛋白(nestin)和Thy1.1阳性反应。结论乳鼠视网膜细胞条件分化液可诱导BMSCs分化成视网膜神经节样细胞。     

bFGF促增殖后骨髓间充质干细胞向多巴胺能神经元样细胞的分化

目的探讨碱性成纤维细胞生长因子(Basic fibroblast growth factor,bFGF)体外作用于骨髓间充质干细胞(Mesen-chymal stem cells,MSCs)后,诱导其向神经元样细胞和多巴胺能神经元样细胞定向分化的情况。方法从鼠骨髓中获得MSCs,培养传代,用MTT法检测bFGF对骨髓MSCs生长的影响。10 ng/ml bFGF作用2 d后,通过IBMX、细胞因子bFGF、GDNFI、L-1β、中脑神经胶质细胞条件培养基和中脑神经细胞膜碎片等分组联合诱导骨髓MSCs向神经元样细胞、多巴胺能神经元样细胞分化,免疫细胞化学方法鉴定特异标志NSE、MAP-2a,b和TH的表达。结果在一定范围内,bFGF对骨髓MSCs具有明显的促增殖作用。分化的神经元样细胞表达NSE、MAP-2a,b和TH,联合应用GDNFI、L-1β、中脑条件培养基和中脑神经细胞膜碎片诱导7 d后,NSE阳性率为(27.774±2.747)%,MAP-2a,b为(28.006±3.080)%,TH为(3.098±0.352)%。结论体外骨髓MSCs被诱导分化成神经元样细胞和多巴胺能神经元样细胞,为帕金森等中枢神经系统疾病的细胞移植治疗奠定了基础。     

大鼠骨髓间充质干细胞向成骨与成脂分化过程中相关基因的表达变化

目的探讨大鼠骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs)向成骨与成脂分化过程中相关基因表达的变化。方法采用全贴壁法分离培养大鼠BMSCs,并观察其形态学特征的变化,MTT法检测其生长状况,并绘制生长曲线。分别采用成骨和成脂诱导剂对第4代BMSCs进行诱导分化,应用碱性磷酸酶试剂盒、茜素红和油红O染色液检测其ALP活性、成骨和成脂分化能力;RT-QPCR检测诱导0、7、14和21 d的成骨分化相关基因Runt相关转录因子2(Runx2)、骨钙素(osteocalcin,OCN)、碱性磷酸酶(ALP)及成脂分化相关基因过氧化物酶体增殖物激活受体γ(peroxidase proliferator activated receptor gamma,PPARγ)和脂肪酸结合蛋白(FABP4)的表达变化。结果全骨髓贴壁法能成功分离培养BMSCs,传代细胞生长增殖迅速,以长梭形细胞生长为主,细胞生长曲线呈S形。第4代BMSCs分别经成骨和成脂诱导剂诱导后,ALP、茜素红和油红O染色均呈阳性;诱导7、14和21 d后,Runx2、OCN、ALP、PPARγ和FABP4基因mRNA的表达量均显著高于0 d(P0.05);成骨分化过程中,Runx2和ALP在第7天时表达量最高,之后呈下降趋势,OCN的表达量呈稳定上升趋势;成脂分化过程中,PPARγ在第7天时表达量最高,FABP4始终高表达。结论 BMSCs具有易于体外分离培养、扩增和经诱导后具有多向分化潜能等特点,成骨和成脂分化相关基因的表达量随诱导时间延长而变化,呈明显的时序性表达差异,提示分别在成骨与成脂分化过程中起重要调控作用,为BMSCs在骨、细胞和基因等工程中的机制研究提供了实验依据。     

微囊化共培养诱导骨髓间充质干细胞体外定向成骨分化研究

为了模拟体内成骨微环境,为骨组织工程提供一种调控干细胞体外向成骨细胞定向分化的共培养新方法,SD大鼠骨髓间充质干细胞和包埋在海藻酸钠-聚赖氨酸-海藻酸钠(alginate-poly-lysine-alginate,APA)微胶囊中的SD大鼠成骨细胞进行体外共培养。共培养过程中,通过碱性磷酸酶(ALP)定量、定性分析以及钙化结节(von Kossa)染色等手段来评价骨髓间充质干细胞向成骨细胞定向分化。结果表明在体外微囊化共培养过程中,被诱导细胞的胞内ALP酶活性逐渐高于对照组的干细胞,接近于成骨细胞;ALP以及von Kossa定性染色证实被诱导细胞具有较高的ALP活性以及具有分泌钙基质的能力。微囊化成骨细胞和外部干细胞的共培养体系较好地模拟了体内干细胞向成骨细胞转化的成骨微环境,促进了干细胞向成骨细胞的体外定向分化;微胶囊膜将成骨细胞和干细胞进行了隔离,避免了两者的直接接触和可能的细胞交叉污染混合,同时利于分离目的细胞,这种微囊化共培养体系为骨组织工程提供了一种安全调控干细胞体外成骨定向分化的工程化新方法。     

体外诱导微环境对骨髓间充质干细胞成神经分化的影响及分化后的自发逆转现象

目的体外定向诱导大鼠骨髓间充质干细胞(Mesenchymal stem cells,MSCs)成神经分化,并探讨诱导微环境对其分化的影响及分化后的自发逆转现象。方法体外分离培养大鼠MSCs,流式细胞仪检测细胞表面标志。采用改良神经元诱导液[Modified neuronal induction media(MNM)]定向诱导MSCs,免疫荧光检测神经细胞表面标志。观察胎牛血清(FBS)浓度、细胞密度、MNM剂量、新鲜与使用过的MNM等不同诱导微环境对MSCs成神经分化的影响。结果 MSCs经MNM诱导后,6h即可见尼氏体,表达神经元特异性表面标志神经元特异性烯醇化酶(NSE)、巢蛋白(Nestin)和微管相关蛋白-2(MAP-2)。随着诱导微环境的改变,MSCs成神经分化率及神经元表面标志表达亦发生改变,且分化后的神经元样细胞可自发逆转。结论 MSCs能够在MNM微环境中定向成神经分化,但诱导微环境的改变可以从量和质两个层面影响MSCs定向分化。     

骨髓间充质干细胞在脊髓损伤模型大鼠体内的转化

目的观察静脉移植骨髓间充质干细胞(MSCs)在脊髓损伤模型大鼠体内向神经样细胞转化的情况。方法体外扩增培养MSCs,流式细胞仪检测表面标志CD34和CD44的表达;采用击打法制备大鼠脊髓损伤模型;Brdu标记的MSCs经尾静脉移植入脊髓损伤模型大鼠体内,输注后21d,取脊髓组织进行免疫组化染色,检测神经元特异性烯醇化酶(NSE)的表达。结果体外扩增的MSCs经流式细胞仪检测不表达CD34,表达CD44;激光共聚焦显微镜下观察可见,移植Brdu标记的MSCs组在大鼠损伤的脊髓组织中可同时表达Brdu和NSE。结论移植的MSCs可迁移到损伤的脊髓组织,并可转化为神经样细胞。     

大鼠骨髓间充质干细胞的体外分离培养与鉴定

目的建立大鼠骨髓间充质干细胞(Mesenchymal stem cells,MSCs)体外分离培养及鉴定的方法 ,为MSCs的系列研究奠定基础。方法采用全骨髓直接贴壁筛选法分离培养MSCs并传代,倒置相差显微镜下观察细胞形态,以MTT法检测细胞增殖水平并绘制生长曲线。取第3代MSCs,流式细胞术检测细胞周期和细胞表型,应用成骨细胞诱导液和脂肪样细胞诱导液诱导MSCs定向分化,鉴定其分化能力。结果全骨髓细胞培养5d,镜下可见贴壁细胞增殖明显,细胞形态较均一,大部分呈梭形,7d左右可传代,经2～3次传代后细胞呈单一梭形的成纤维样细胞,即MSCs;细胞生长曲线呈S形;经流式细胞仪检测,MSCs细胞76.01%处于G0/G1期,7.13%处于G2/M期,16.86%处于S期;MSCs表面不表达CD34;在特定诱导液作用下,MSCs可分别向成骨样细胞及脂肪样细胞分化。结论已成功建立了分离培养及鉴定MSCs的方法 ,可用来评价体外培养的MSCs。     

细胞传代对骨髓间充质干细胞成软骨细胞分化的影响

目的 观察在成软骨诱导培养条件下,细胞传代对骨髓间充质干细胞(MSCs)体外成软骨能力的影响.方法 不同代MSCs成软骨诱导后,观察细胞生物学特性以及通过免疫荧光,RT-PCR测定特异性软骨细胞外基质aggrecan的表达情况.结果 经成软骨诱导后,第2、4代MSCs表达aggrecan明显较第6、8代细胞高.结论 MSCs很可能由多种形态功能接近,分化潜能有略有差异的细胞组成;在成软骨诱导培养条件下,对此传代后成软骨能力减弱.     

去上皮羊膜及其浸提液体外诱导骨髓间充质干细胞的分化

目的观察去上皮羊膜及其浸提液体外诱导骨髓间充质干细胞(Bone marrow mesenchymal stem cells,BMSCs)向上皮细胞的分化,并探讨其机制。方法从胎儿四肢长骨分离BMSCs,扩增后采用流式细胞术分析第3代(P3)细胞表面抗原(CD29、CD34、CD71和HLA-DR)的表达,并用4,6-乙酰基-2-苯基吲哚(DAPI)标记第4代BMSCs(P4-BMSCs)。机械法去除正常胎盘羊膜上皮,制成去上皮羊膜,并制备去上皮羊膜浸提液。将DAPI标记的BMSCs接种于羊膜上,设加或不加表皮细胞生长因子(Epidermal growth factor,EGF)、类胰岛素1号生长因子(Insulin-like growth factor 1,IGF-1)、羊膜浸提液诱导组及细胞爬片对照组,体外诱导培养后,采用免疫荧光组织(细胞)化学染色学法检测各组细胞角蛋白(Cytokeratin,CK)、EGF-R和IGF-1-R的表达,并于诱导后第10天计算CK阳性细胞率。结果原代BMSCs呈典型旋涡状生长,P3细胞表达CD29和CD71,不表达CD34和HLA-DR。羊膜组和细胞爬片组BMSCs在加入EGF或IGF-1诱导后,表达EGF-R和IGF-1-R的时间较未加生长因子的对照组提前2～4 d,表达CK的时间提前2～6 d,单用羊膜组或羊膜浸提液组的表达时间差异无统计学意义(P>0.05);诱导第10天,单用羊膜或羊膜浸提液诱导组的CK阳性细胞表达率明显高于细胞爬片对照组(P<0.05);羊膜与EGF、IGF-1联合诱导组高于单用羊膜组(P<0.05);EGF诱导组高于IGF-1诱导组(P<0.05)。结论羊膜及羊膜浸提液、外源性EGF和IGF-1在体外均可诱导BMSCs向上皮细胞分化,羊膜可能主要通过其所含的细胞因子诱导BMSCs向上皮分化。     

白藜芦醇体外诱导大鼠骨髓基质细胞向神经元样细胞的分化

目的研究白藜芦醇体外诱导大鼠骨髓基质细胞(Marrow stroma cells,MSCs)向神经元样细胞的分化。方法采用全骨髓贴壁法分离培养MSCs,取第3代MSCs分为白藜芦醇诱导组、对照组和正常组,白藜芦醇诱导组先加入预诱导液(含10μg/L bFGF的DMEM/F12培养基)培养24 h,再更换诱导液(含15μmol/L白藜芦醇的DMEM/F12培养基)诱导6 h,然后更换维持液(含15μmol/L白藜芦醇,10μg/L bFGF,2%B27的DMEM/F12培养基),继续培养至72 h;对照组预诱导与白藜芦醇诱导组相同,诱导时仅加入DMEM/F12培养基诱导6 h,再更换维持液(含10μg/L bFGF,2%B27的DMEM/F12培养基),继续培养至72 h,倒置显微镜下观察诱导分化后MSCs形态;各组分别于诱导前及诱导后2、6、24、72 h采用间接免疫荧光法、Western blot法和RT-PCR法检测nestin、NSE蛋白及mRNA表达。结果白藜芦醇诱导后细胞胞体收缩,伸出长突起,类似神经元,间接免疫荧光染色显示诱导后细胞nestin和NSE阳性,对照组未见阳性细胞。白藜芦醇诱导组nestin、NSE蛋白及mRNA表达较对照组明显升高,诱导后2 h,nestin蛋白及mRNA表达达最高(P<0.01),之后逐渐下降;而NSE蛋白及mRNA表达逐渐升高,诱导后72 h达最高(P<0.01),对照组则无明显变化。结论白藜芦醇在体外可诱导大鼠骨髓基质细胞分化为神经元样细胞,为白藜芦醇在干细胞移植领域的应用提供了实验依据。     

Bone Marrow-Derived Vasculogenic Mesenchymal Stem Cells Enhance In Vitro Angiogenic Sprouting of Human Umbilical Vein Endothelial Cells

Vasculogenic properties of bone marrow-derived mesenchymal stem cells (MSCs) have been reported, but it is still unclear whether the vasculogenic properties are restricted to some populations of MSCs or whether the entire population of MSCs has these properties. We cultured two different populations of MSCs in different culture media and their vasculogenic properties were evaluated using In vitro spheroid sprouting assay. Neither population of MSCs expressed markers of endothelial progenitor cells (EPCs), but they were different in the profiling of angiogenic factor expression as well as vasculogenic properties. One population of MSCs expressed basic fibroblast growth factor (bFGF) and another expressed hepatocyte growth factor (HGF). MSCs expressing HGF exhibited In vitro angiogenic sprouting capacity in response to bFGF derived from other MSCs as well as to their autocrine HGF. The vasculogenic mesenchymal stem cells (vMSCs) derived from the bone marrow also enhanced In vitro angiogenic sprouting capacity of human umbilical vein endothelial cells (HUVECs) in an HGF-dependent manner. These results suggest that MSCs exhibit different vasculogenic properties, and vMSCs that are different from EPCs may contribute to neovascularization and could be a promising cellular therapy for cardiovascular diseases.     

骨髓间充质干细胞对海藻酸钙胶珠内神经干细胞增殖与分化的作用

神经干细胞(neural stem cells,NSCs)移植治疗神经损伤被认为是具有潜在应用价值的手段,但其来源困难;骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs)以其所具有的诸多优点,为神经损伤的治疗提供了一个新的思路。而BMSCs是否是通过作用于内源性的NSCs来促进神经修复,仍存在着争议。今采用海藻酸钙胶珠将NSCs包囊培养至一定大小的神经球后,再与BMSCs进行共培养,考察BMSCs对生长在海藻酸钙胶珠内的NSCs增殖与分化的作用,探讨BMSCs移植治疗神经疾病与损伤的作用机理。共培养过程中观察神经球结构的变化;共培养结束后计算NSCs的增殖倍数,对增殖条件下共培养的NSCs表型和多向分化潜能进行免疫荧光染色鉴定;对分化条件下共培养的NSCs向不同神经细胞分化的能力进行流式细胞仪检测。结果表明,BMSCs可使生长于支架内的NSCs迁出细胞球,对NSCs的增殖没有明显影响;但能够明显影响NSCs的分化,使其向少突胶质细胞分化的能力增加3倍,向星形胶质细胞分化的能力减弱1倍,而向神经元细胞分化的能力没有明显变化。BMSCs有可能是通过分泌某些因子增加了NSCs迁移及向少突胶质细胞分化的能力,从而促进神经损伤的修复。     

A Single-Cell Raman Spectroscopy Analysis of Bone Marrow Mesenchymal Stem/Stromal Cells to Identify Inter-Individual Diversity

The heterogeneity of stem cells represents the main challenge in regenerative medicine development. This issue is particularly pronounced when it comes to the use of primary mesenchymal stem/stromal cells (MSCs) due to a lack of identification markers. Considering the need for additional approaches in MSCs characterization, we applied Raman spectroscopy to investigate inter-individual differences between bone marrow MSCs (BM-MSCs). Based on standard biological tests, BM-MSCs of analyzed donors fulfill all conditions for their characterization, while no donor-related specifics were observed in terms of BM-MSCs morphology, phenotype, multilineage differentiation potential, colony-forming capacity, expression of pluripotency-associated markers or proliferative capacity. However, examination of BM-MSCs at a single-cell level by Raman spectroscopy revealed that despite similar biochemical background, fine differences in the Raman spectra of BM-MSCs of each donor can be detected. After extensive principal component analysis (PCA) of Raman spectra, our study revealed the possibility of this method to diversify BM-MSCs populations, whereby the grouping of cell populations was most prominent when cell populations were analyzed in pairs. These results indicate that Raman spectroscopy, as a label-free assay, could have a huge potential in understanding stem cell heterogeneity and sorting cell populations with a similar biochemical background that can be significant for the development of personalized therapy approaches.     

Radiation-Induced Osteocyte Senescence Alters Bone Marrow Mesenchymal Stem Cell Differentiation Potential via Paracrine Signaling

Cellular senescence and its senescence-associated secretory phenotype (SASP) are widely regarded as promising therapeutic targets for aging-related diseases, such as osteoporosis. However, the expression pattern of cellular senescence and multiple SASP secretion remains unclear, thus leaving a large gap in the knowledge for a desirable intervention targeting cellular senescence. Therefore, there is a critical need to understand the molecular mechanism of SASP secretion in the bone microenvironment that can ameliorate aging-related degenerative pathologies including osteoporosis. In this study, osteocyte-like cells (MLO-Y4) were induced to cellular senescence by 2 Gy γ-rays; then, senescence phenotype changes and adverse effects of SASP on bone marrow mesenchymal stem cell (BMSC) differentiation potential were investigated. The results revealed that 2 Gy irradiation could hinder cell viability, shorten cell dendrites, and induce cellular senescence, as evidenced by the higher expression of senescence markers p16 and p21 and the elevated formation of senescence-associated heterochromatin foci (SAHF), which was accompanied by the enhanced secretion of SASP markers such as IL-1α, IL-6, MMP-3, IGFBP-6, resistin, and adiponectin. When 0.8 μM JAK1 inhibitors were added to block SASP secretion, the higher expression of SASP was blunted, but the inhibition in osteogenic and adipogenic differentiation potential of BMSCs co-cultured with irradiated MLO-Y4 cell conditioned medium (CM- 2 Gy) was alleviated. These results suggest that senescent osteocytes can perturb BMSCs’ differential potential via the paracrine signaling of SASP, which was also demonstrated by in vivo experiments. In conclusion, we identified the SASP factor partially responsible for the degenerative differentiation of BMSCs, which allowed us to hypothesize that senescent osteocytes and their SASPs may contribute to radiation-induced bone loss.     

大鼠骨髓间充质干细胞体外向血管平滑肌样细胞诱导分化过程中Periostin的表达及其作用

目的探讨血小板源性生长因子(Platelet derived growth factor,PDGF)-BB诱导骨髓间充质干细胞(Bone marrow-derived mesenchymal stem cells,BMSCs)向血管平滑肌样细胞(Vascular smooth muscle-like cells,VSMLCs)分化过程中Periostin的表达及其在促VSMLCs分化中的作用。方法采用全骨髓贴壁培养法分离和培养大鼠BMSCs,取第2代BMSCs分为2组:诱导Ⅰ组(用50 ng/ml PDGF-BB单独向VSMLCs诱导)和诱导Ⅱ组(加入地塞米松1μmol/L、胰岛素1μmol/L、吲哚美辛1μmol/L、3-异丁基-1甲基黄嘌呤0.5 mmol/L,向脂肪样细胞诱导),以大鼠胸大动脉平滑肌细胞作为阳性对照。分别于诱导后7 d和14 d,采用RT-PCR检测细胞中平滑肌肌动蛋白(SMα-actin)、平滑肌肌球蛋白重链(SM MHC)、平滑肌肌钙结合蛋白(SM Calponin)和Periostin mRNA的转录水平,Western blot检测Periostin蛋白的表达水平。结果诱导Ⅰ组细胞的SMα-actin、SM MHC、SMCalponin和Periostin基因mRNA及Periostin蛋白的表达水平14 d比7 d显著增强,且差异有统计学意义(P<0.05);14 d与阳性细胞相比,差异无统计学意义(P>0.05);未诱导组及诱导Ⅱ组在14 d均无表达。结论 PDGF-BB(50 ng/ml)能够单独诱导BMSCs向VSMCs分化,Periostin在此过程中起重要作用。     

Density-Dependent Differentiation of Tonsil-Derived Mesenchymal Stem Cells into Parathyroid-Hormone-Releasing Cells

Mesenchymal stem cells (MSCs) can differentiate into endoderm lineages, especially parathyroid-hormone (PTH)-releasing cells. We have previously reported that tonsil-derived MSC (T-MSC) can differentiate into PTH-releasing cells (T-MSC-PTHCs), which restored the parathyroid functions in parathyroidectomy (PTX) rats. In this study, we demonstrate quality optimization by standardizing the differentiation rate for a better clinical application of T-MSC-PTHCs to overcome donor-dependent variation of T-MSCs. Quantitation results of PTH mRNA copy number in the differentiated cells and the PTH concentration in the conditioned medium confirmed that the differentiation efficiency largely varied depending on the cells from each donor. In addition, the differentiation rate of the cells from all the donors greatly improved when differentiation was started at a high cell density (100% confluence). The large-scale expression profiling of T-MSC-PTHCs by RNA sequencing indicated that those genes involved in exiting the differentiation and the cell cycle were the major pathways for the differentiation of T-MSC-PTHCs. Furthermore, the implantation of the T-MSC-PTHCs, which were differentiated at a high cell density embedded in hyaluronic acid, resulted in a higher serum PTH in the PTX model. This standardized efficiency of differentiation into PTHC was achieved by initiating differentiation at a high cell density. Our findings provide a potential solution to overcome the limitations due to donor-dependent variation by establishing a standardized differentiation protocol for the clinical application of T-MSC therapy in treating hypoparathyroidism.     

Proteomic Comparison of Bone Marrow Derived Osteoblasts and Mesenchymal Stem Cells

Mesenchymal stem cells (MSCs) can differentiate into osteoblasts, and therapeutic targeting of these cells is considered both for malignant and non-malignant diseases. We analyzed global proteomic profiles for osteoblasts derived from ten and MSCs from six healthy individuals, and we quantified 5465 proteins for the osteoblasts and 5420 proteins for the MSCs. There was a large overlap in the profiles for the two cell types; 156 proteins were quantified only in osteoblasts and 111 proteins only for the MSCs. The osteoblast-specific proteins included several extracellular matrix proteins and a network including 27 proteins that influence intracellular signaling (Wnt/Notch/Bone morphogenic protein pathways) and bone mineralization. The osteoblasts and MSCs showed only minor age- and sex-dependent proteomic differences. Finally, the osteoblast and MSC proteomic profiles were altered by ex vivo culture in serum-free media. We conclude that although the proteomic profiles of osteoblasts and MSCs show many similarities, we identified several osteoblast-specific extracellular matrix proteins and an osteoblast-specific intracellular signaling network. Therapeutic targeting of these proteins will possibly have minor effects on MSCs. Furthermore, the use of ex vivo cultured osteoblasts/MSCs in clinical medicine will require careful standardization of the ex vivo handling of the cells.