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。     

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。     

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.     

大鼠骨髓间充质干细胞向肝样细胞的定向诱导分化

目的 探讨肝细胞生长因子(Hepatocyte growth factor,HGF)和碱性成纤维细胞生长因子(Basic fibroblast growthfactor,bFGF)诱导大鼠骨髓间充质干细胞(Bone marrow mesenchymal stem cells,BM-MSCs)分化为肝样细胞的可行性。方法取SD大鼠股骨骨髓,直接贴壁法分离纯化BM-MSCs,并体外传代,流式细胞术和成骨诱导对其进行鉴定。取第3代BM-MSCs,分为2组:实验组用HGF(20 ng/ml)和bFGF(10 ng/ml)进行诱导,阴性对照组不加诱导剂,倒置显微镜下观察细胞形态变化;RT-PCR法检测诱导后细胞甲胎蛋白(Alpha fetoprotein,AFP)和白蛋白(Albumin,ALB)基因mRNA的转录水平;免疫细胞化学染色法检测诱导后细胞的AFP和ALB蛋白的表达。结果第3代BM-MSCs表型标志和功能特性均符合MSCs的特点。BM-MSCs经HGF和bFGF诱导后呈肝样细胞形态。实验组细胞可检测出AFP和ALB基因mRNA的表达。实验组细胞诱导后第7天,AFP蛋白开始表达,第14天时表达降低,第21天时不表达;ALB于诱导后第14天出现表达,并随诱导时间的延长表达逐渐增加。结论 HGF和bFGF具有体外诱导BM-MSCs向肝样细胞分化的作用。     

骨髓间充质干细胞对大鼠胰腺损伤组织的修复作用

目的观察骨髓间充质干细胞(MSCs)对大鼠胰腺损伤组织的修复作用。方法应用贴壁法分离、纯化、扩增大鼠MSCs,经流式细胞仪检测其细胞周期及表面标志后,用DAPI标记,经尾静脉注入胰腺损伤模型大鼠体内,15d后,在激光共聚焦显微镜下观察MSCs在大鼠胰腺组织的定位,组织病理切片观察胰腺损伤组织的病理改变,PCR检测胰腺损伤区组织的Sry基因。结果体外纯化、扩增、富集的MSCs经流式细胞仪检测,86.67%的细胞处于G0/G1期,细胞表面CD34呈阴性表达,CD44呈阳性表达。DAPI标记的MSCs移植治疗15d后,激光共聚焦显微镜下可见,MSCs在损伤的胰腺组织中多见,在正常胰腺组织中偶见;组织病理切片可见损伤的胰腺组织结构开始恢复,胰岛再建;PCR结果显示,治疗组胰腺组织可扩增出Sry基因。结论MSCs对大鼠胰腺损伤组织可能具有修复作用。     

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。     

骨髓间充质干细胞对胰腺损伤模型大鼠血清生化指标的影响

目的观察大鼠骨髓间充质干细胞(MSCs)对胰腺损伤模型大鼠血清生化指标的影响。方法胰腺结扎建立大鼠胰腺损伤模型;体外分离纯化及培养大鼠的MSCs,流式细胞术检测细胞周期及细胞表面标志,经尾静脉回输治疗胰腺损伤,逐日观察,并于回输后24h、48h、7d和15d,分别采血,分离血清,检测血淀粉酶与脂肪酶含量。结果分离纯化的MSCs细胞周期中,86.67%处于G0/G1期,表达MSCs表面标志CD44,不表达造血干细胞表面标志CD34。治疗组在回输MSCs15d后,肉眼可见坏死的胰腺组织外观基本恢复正常,24h、48h和7d,血清淀粉酶含量均低于模型组,且差异有统计学意义,在15d时接近于模型组,差异无统计学意义;在24h、48h、7d和15d时,治疗组血清中脂肪酶含量均低于模型组,且差异有统计学意义;24h、48h、7d和15d,治疗组血清中血淀粉酶和脂肪酶含量仍高于正常对照组,且差异有统计学意义。结论骨髓间充质干细胞对胰腺组织损伤的模型大鼠具有治疗作用。     

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。     

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。     

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。     

Potential Spermatogenesis Recovery with Bone Marrow Mesenchymal Stem Cells in an Azoospermic Rat Model

Non-obstructive azoospermia is the most challenging type of male infertility. Stem cell based therapy provides the potential to enhance the recovery of spermatogenesis following cancer therapy. Bone marrow-derived mesenchymal stem cells (BMSCs) possess the potential to differentiate or trans-differentiate into multi-lineage cells, secrete paracrine factors to recruit the resident stem cells to participate in tissue regeneration, or fuse with the local cells in the affected region. In this study, we tested whether spermatogenically-induced BMSCs can restore spermatogenesis after administration of an anticancer drug. Allogeneic BMSCs were co-cultured in conditioned media derived from cultured testicular Sertoli cells in vitro, and then induced stem cells were transplanted into the seminiferous tubules of a busulfan-induced azoospermatic rat model for 8 weeks. The in vitro induced BMSCs exhibited specific spermatogonic gene and protein markers, and after implantation the donor cells survived and located at the basement membranes of the recipient seminiferous tubules, in accordance with what are considered the unique biological characteristics of spermatogenic stem cells. Molecular markers of spermatogonial stem cells and spermatogonia (Vasa, Stella, SMAD1, Dazl, GCNF, HSP90α, integrinβ1, and c-kit) were expressed in the recipient testis tissue. No tumor mass, immune response, or inflammatory reaction developed. In conclusion, BMSCs might provide the potential to trans-differentiate into spermatogenic-like-cells, enhancing endogenous fertility recovery. The present study indicates that BMSCs might offer alternative treatment for the patients with azoospermatic infertility after cancer chemotherapy.     

Addition of Adipose-Derived Stem Cells to Mesenchymal Stem Cell Sheets Improves Bone Formation at an Ectopic Site

To determine the effect of adipose-derived stem cells (ADSCs) added to bone marrow-derived mesenchymal stem cell (MSC) sheets on bone formation at an ectopic site. We isolated MSCs and ADSCs from the same rabbits. We then prepared MSC sheets for implantation with or without ADSCs subcutaneously in the backs of severe combined immunodeficiency (SCID) mice. We assessed bone formation at eight weeks after implantation by micro-computed tomography and histological analysis. In osteogenic medium, MSCs grew to form multilayer sheets containing many calcium nodules. MSC sheets without ADSCs formed bone-like tissue; although neo-bone and cartilage-like tissues were sparse and unevenly distributed by eight weeks after implantation. In comparison, MSC sheets with ADSCs promoted better bone regeneration as evidenced by the greater density of bone, increased mineral deposition, obvious formation of blood vessels, large number of interconnected ossified trabeculae and woven bone structures, and greater bone volume/total volume within the composite constructs. Our results indicate that although sheets of only MSCs have the potential to form tissue engineered bone at an ectopic site, the addition of ADSCs can significantly increase the osteogenic potential of MSC sheets. Thus, the combination of MSC sheets with ADSCs may be regarded as a promising therapeutic strategy to stimulate bone regeneration.     

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.     

MicroRNAs Modulate Signaling Pathways in Osteogenic Differentiation of Mesenchymal Stem Cells

Mesenchymal stem cells (MSCs) have been identified in many adult tissues and they have been closely studied in recent years, especially in view of their potential use for treating diseases and damaged tissues and organs. MSCs are capable of self-replication and differentiation into osteoblasts and are considered an important source of cells in tissue engineering for bone regeneration. Several epigenetic factors are believed to play a role in the osteogenic differentiation of MSCs, including microRNAs (miRNAs). MiRNAs are small, single-stranded, non-coding RNAs of approximately 22 nucleotides that are able to regulate cell proliferation, differentiation and apoptosis by binding the 3′ untranslated region (3′-UTR) of target mRNAs, which can be subsequently degraded or translationally silenced. MiRNAs control gene expression in osteogenic differentiation by regulating two crucial signaling cascades in osteogenesis: the transforming growth factor-beta (TGF-β)/bone morphogenic protein (BMP) and the Wingless/Int-1(Wnt)/β-catenin signaling pathways. This review provides an overview of the miRNAs involved in osteogenic differentiation and how these miRNAs could regulate the expression of target genes.     

Receptor-Targeted,Magneto-Mechanical Stimulation of Osteogenic Differentiation of Human Bone Marrow-Derived Mesenchymal Stem Cells

Mechanical cues are employed to promote stem cell differentiation and functional tissue formation in tissue engineering and regenerative medicine. We have developed a Magnetic Force Bioreactor (MFB) that delivers highly targeted local forces to cells at a pico-newton level, utilizing magnetic micro- and nano-particles to target cell surface receptors. In this study, we investigated the effects of magnetically targeting and actuating specific two mechanical-sensitive cell membrane receptors—platelet-derived growth factor receptor α (PDGFRα) and integrin α_νβ₃. It was found that a higher mineral-to-matrix ratio was obtained after three weeks of magneto-mechanical stimulation coupled with osteogenic medium culture by initially targeting PDGFRα compared with targeting integrin α_νβ₃ and non-treated controls. Moreover, different initiation sites caused a differentiated response profile when using a 2-day-lagged magneto-mechanical stimulation over culture periods of 7 and 12 days). However, both resulted in statistically higher osteogenic marker genes expression compared with immediate magneto-mechanical stimulation. These results provide insights into important parameters for designing appropriate protocols for ex vivo induced bone formation via magneto-mechanical actuation.