骨髓基质干细胞在体外向软骨细胞分化

目的观察骨髓基质干细胞(B M SC s)在体外能否分化为软骨细胞。方法利用高密度细胞球培养体系在含转化生长因子-β1(TG F-β1)的培养基中培养B M SC s21d,用免疫组化甲苯胺蓝染色方法分析培养的B M SC s球中蛋白多糖(软骨细胞分泌的主要基质成份)的表达、用免疫组化和R T-P C R方法分析Ⅱ型胶原(软骨细胞特异分泌的主要胶原蛋白)的表达来评估B M SC s是否分化为软骨细胞。结果TG F-β1作用的B M SC s表达了Ⅱ型胶原和蛋白多糖。结论B M SC s在体外特定的培养条件下可分化为软骨细胞,从而可能成为临床上治疗创伤或骨关节炎所致的软骨缺损所需的合适的自体来源的种子细胞。     

体外负压培养对骨髓间充质干细胞成骨活性的影响

目的：探讨体外负压培养对骨髓间充质干细胞（bone marrow derived stroma cells,BMSCs）成骨活性的影响。方法：取第3代BMSCs分为实验组和对照组,实验组进行间歇性负压培养,设置压力为17kPa,每次30min,每日4次,干预2周;对照组于普通CO2培养箱中常规培养。倒置显微镜下观察细胞形态,检测ALP活性,免疫组织化学检测Ⅰ型胶原的表达,RT-PCR检测2周后以及终止负压后1、2、3d骨保护素（osteoprotegerin,OPG）mRNA和骨保护素配体（osteoprotegerin ligand,OPGL）mRNA表达水平。结果：诱导2周后,BMSCs呈现出显著的成骨细胞特性,与对照组比较,ALP活性显著增加,Ⅰ型胶原表达阳性,实验组细胞OPG mRNA表达水平显著提高,OPGL mRNA表达水平显著降低,且差异均有统计学意义（P〈0.05）。负压终止后3d,两组细胞OPG mRNA和OPGL mRNA表达水平差异无统计学意义（P〉0.05）。结论：体外负压培养可以提高BMSCs成骨活性。     

表达HGF的骨髓间充质干细胞促进小移植肝早期肝再生

目的 建立大鼠小体积肝移植模型,输注表达人肝细胞生长因子(human hepatocyte growth factor,hHGF)的骨髓间充质干细胞(mesenchymal stem cells,MSCs),研究其在移植早期对小移植肝促再生作用.方法 将已建立的表达hHGF和绿色荧光蛋白(green fluorescence protein,GFP)的MSCs,分别命名为HGF/MSCs,GFP/Mscs.建立大鼠30%肝移植模型.受体分为4组,实验组输注5×106HGF/MSCs;对照组则分别输注相同体积的生理盐水(PS),5×106 GFP/MSCs或1.0×109 pfu含hHGF的重组腺病毒液(Ad-HGF).分别于术后1,3,5,7 d各组随机抽取5只大鼠处死.取血检测血清ALT和hHGF.记录移植物湿重.取肝组织检测hHGF、c-met表达,以及肝细胞凋亡和增殖活性.另每组15只,分组同上,用于观察生存期.结果 PS组大鼠7 d生存率33.3%;组织学及血清学检查示术后肝脏损伤重,汇管区单核细胞浸润多;而实验组大鼠7 d生存率为73.3%.肝脏损伤轻,炎性细胞浸润少;实验组移植肝再生较PS组明显增加.结论 大鼠部分肝移植后,输注HGF/MSCs能够保护小体积移植肝,促进小移植肝再生,提高7 d生存率.     

Microenvironment and stem properties of bone marrow-derived mesenchymal cells

Adult stem cells are self-renewing, pluripotent, and able to repopulate the tissue in which they reside. Cells endowed with these properties have been isolated from several tissues and an increasing number of reports provide evidence of their ability, following transplantation, to engraft host tissues other than those of their origin. In this setting, interest in the well-documented capacity of bone marrow stromal cells to undergo multilineage differentiation is growing. Neural and cardiomyogenic lineages have recently been proposed as additional differentiative pathways of these cells. However, culture conditions and inductive molecules can alter the behavior of bone marrow stromal cells and the microenvironment is critical for proper in vivo delivery. The maintenance of their stem properties and the possibility of reprogramming their commitment is a field of primary interest given the potential use of these cells in regenerative medicine. We discuss here how the microenvironmental cues, and the growth factors that physiologically govern commitment and subsequent differentiation, influence the properties of bone marrow stromal cells and modulate their engraftment into host tissues.     

骨髓源性肝干细胞的确认及定向分化的实验研究

目的 确定骨髓源性肝干细胞的表面标记，体外培养诱导其分化为肝细胞。方法 采用流式细胞仪检测多种大鼠肝损伤模型骨髓中干细胞标记的细胞群体的数量变化，寻找可能的肝干细胞，免疫磁珠分离各骨髓干细胞标记的细胞群体，进行体外培养诱导分化，观察细胞形态的变化，免疫组化技术检测清蛋白(白蛋白)，AFP，CK8／18等肝细胞标记的表达。结果 各模型大鼠骨髓内β2微球蛋白阴性(β2m^-)细胞显著增高，体外培养经诱导后呈多角形细胞表现，清蛋白，AFP，CK8／18表达阳性。其他干细胞类型细胞数量变化小，体外培养未见肝细胞样变化。结论 β2m^-细胞的数量随肝损伤而变化，在体外具有向肝细胞分化的能力，可能成为肝干细胞的标记物。     

共培养系统下软骨细胞对骨髓间质干细胞向软骨细胞分化的诱导作用

 目的观察共培养系统下正常软骨细胞和关节炎软骨细胞对骨髓间质干细胞(bone marrow mesenchymal stem cells,BMSCs)向软骨细胞分化的促进作用。方法分离新西兰兔 BMSCs及正常软骨细胞。制作兔膝关节炎模型,提取兔关节炎软骨细胞。将 BMSCs与低熔点琼脂糖复合成凝胶块,构建软骨细胞-BMCSs共培养系统,分为正常软骨细胞 P0-BMSCs组、正常软骨细胞 P3-BMSCs组、关节炎软骨细胞 P0-BMSCs组、关节炎软骨细胞 P3-BMSCs组及 BMSCs组(对照组)。在 3、7、14天取材行实时定量 PCR、糖胺聚糖含量、细胞活性检测及组织切片观察。结果(1)正常软骨细胞 P0-BMSCs组的 II 型胶原基因表达增强,在 3、7、14天分别为对照组的 5.1、7.2、11.2倍; 正常软骨细胞 P3-BMSCs组 I 、 II 型胶原及蛋白聚糖基因表达均未见增强; 关节炎软骨细胞 P0-BMSCs组蛋白聚糖基因表达增强,在 14天为对照组的 7.8倍; 关节炎软骨细胞 P3-BMSCs组 I 型胶原基因表达水平在三个时间点均低于对照组。(2)正常软骨细胞 P0-BMSCs组糖胺聚糖含量为对照组的 2.59倍。除关节炎软骨细胞 P0-BMSCs组外,其余三组与对照组比较差异均有统计学意义。阿新蓝染色各组均为阳性,正常软骨细胞 P0-BMSCs组的细胞及细胞外基质蓝染最深。结论兔正常 P0软骨细胞与兔关节炎 P0软骨细胞能够有效促进 BMSCs向软骨细胞分化,正常 P3软骨细胞的促分化作用微弱,而关节炎 P3软骨细胞不具有促分化作用。     

Small-diameter blood vessels engineered with bone marrow-derived cells

OBJECTIVE: The objective of this study is to investigate if bone marrow-derived cells (BMCs) regenerate vascular tissues and improve patency in tissue-engineered small-diameter (internal diameter = 3 mm) vascular grafts. SUMMARY BACKGROUND DATA: BMCs have demonstrated the ability to differentiate into endothelial-like cells and vascular smooth muscle-like cells and may offer an alternative cell source for vascular tissue engineering. Thus, we tissue-engineered small-diameter vascular grafts with BMCs and decellularized arteries. METHODS: Canine BMCs were differentiated in vitro into smooth muscle alpha-actin/smooth muscle myosin heavy-chain-positive cells and von Willebrand factor/CD31-positive cells and seeded onto decellularized canine carotid arteries (internal diameter = 3 mm). The seeded grafts were implanted in cell donor dogs. The vascular-tissue regeneration and graft patency were investigated with immunohistochemistry and angiography, respectively. RESULTS: The vascular grafts seeded with BMCs remained patent for up to 8 weeks in the canine carotid artery interposition model, whereas nonseeded grafts occluded within 2 weeks. Within 8 weeks after implantation, the vascular grafts showed regeneration of the 3 elements of artery (endothelium, media, and adventitia). BMCs labeled with a fluorescent dye prior to implantation were detected in the retrieved vascular grafts, indicating that the BMCs participated in the vascular tissue regeneration. CONCLUSIONS: Here we show that BMCs have the potential to regenerate vascular tissues and improve patency in tissue-engineered small-diameter vascular grafts. This is the first report of a small-diameter neovessel engineered with BMCs as a cell source.     

Th1 polarization in murine IgA nephropathy directed by bone marrow-derived cells

IgA nephropathy is the most common form of progressive glomerulonephritis although the pathophysiology of this nephropathy is unclear. The ddY mouse is a spontaneous animal model with variable incidence and extent of glomerular injury mimicking human IgA nephropathy. Here, we transplanted bone marrow cells from 20-week-old ddY mice with beginning or quiescent IgA nephropathy into irradiated similar ddY mice, C57Bl/6 (Th1 prone) mice, or BALB/c (Th2 prone) mice. Serum IgA/IgG complex and Th1/Th2 polarization of spleen cells was determined by enzyme-linked immunosorbent assay and confirmed by fluorescent cytometric analysis. The ddY mice with commencing IgA nephropathy demonstrated strong polarization toward Th1, while those with quiescent disease were Th2 polarized. Serum levels of IgA/IgG2a immune complex significantly correlated with the severity of the glomerular lesions. Bone marrow taken from mice with commencing IgA nephropathy conferred IgA nephropathy with Th1 polarization in recipient-quiescent mice, while transplantation from the quiescent mice ablated glomerular injury and mesangial IgA/IgG deposition in those commencing IgA disease. However, adoptive transfer of CD4(+) T cells from those whose disease began failed to induce any IgA deposition or renal injury. Our study suggests that bone marrow cells, presuming IgA producing cells, may initiate this disease. Th1 cells may be involved in the pathophysiology of the disease after glomerular IgA deposition.     

Characterization of bone marrow-derived mesenchymal stem cells in aging

Adult mesenchymal stem cells are a resource for autologous and allogeneic cell therapies for immune-modulation and regenerative medicine. However, patients most in need of such therapies are often of advanced age. Therefore, the effects of the aged milieu on these cells and their intrinsic aging in vivo are important considerations. Furthermore, these cells may require expansion in vitro before use as well as for future research. Their aging in vitro is thus also an important consideration. Here, we focus on bone marrow mesenchymal stem cells (BMSCs), which are unique compared to other stem cells due to their support of hematopoietic cells in addition to contributing to bone formation. BMSCs may be sensitive to age-related diseases and could perpetuate degenerative diseases in which bone remodeling is a contributory factor. Here, we review (1) the characterization of BMSCs, (2) the characterization of in vivo-aged BMSCs, (3) the characterization of in vitro-aged BMSCs, and (4) potential approaches to optimize the performance of aged BMSCs. This article is part of a Special Issue entitled “Stem Cells and Bone”.     

Immunotherapy of NOD mice with bone marrow-derived dendritic cells

We evaluated two bone marrow-derived dendritic cell (DC) populations from NOD mice, the murine model for type 1 human diabetes. DCs derived from GM-CSF [granulocyte/macrophage colony-stimulating factor] + interleukin (IL)-4 cultures expressed high levels of major histocompatibility complex (MHC) class II, CD40, CD80, and CD86 molecules and were efficient stimulators of naive allogeneic T-cells. In contrast, DCs derived from GM-CSF cultures had low levels of MHC class II costimulation/activation molecules, were able to take up mannosylated bovine serum albumin more efficiently than GM + IL-4 DCs, and were poor T-cell stimulators. The two DC populations migrated to the spleen and pancreas after intravenous injection. To determine the ability of the two DC populations to modulate diabetes development, DCs were pulsed with a mixture of three islet antigen-derived peptides or with medium before injection into prediabetic NOD mice. Despite phenotypic and functional differences in vitro, both populations prevented in vivo diabetes development. Pulsing of the DCs with peptide in vitro did not significantly improve the ability of DCs to prevent disease, which suggests that DCs may process and present antigen to T-cells in vivo. In addition, we detected GAD65 peptide-specific IgG1 antibody responses in DC-treated mice. Overall, these results suggest that a Th2 response was generated in DC-treated mice. This response was optimal when using GM + IL-4 DCs, which suggests that the balance between regulatory Th2 and effector Th1 cells may have been altered in these mice.     

Effects of bone marrow-derived mesenchymal stem cells on glomerular podocyte injured by lipopolysaccharide

Objective To observe the effects of bone marrow-derived mesenchymal stem cells (BMSC) on glomerular podocyte injured by lipopolysaccharide (LPS) and the expression of related protein. Methods Podocytes are divided into control group, BMSC group, LPS group and LPS plus BMSC group. After 24 hours of intervention, observing each experimental group podocyte form under inverted phase contrast microscope;detecting the expressions of mRNA and protein of nephrin, CD2AP, synaptopodin, and TRPC6 by RT-PCR and Western-blot. Results Compared with control group, expressions of nephrin, CD2AP, and synaptopodin in LPS group decreased (P＜0.05) while that of TRPC6 increased (P＜0.05); compared with LPS group, expressions of nephrin, CD2AP, and synaptopodin in LPS+MSC group increased (P＜0.05) while that of TRPC6 decreased (P＜0.05). Conclusion BMSC may relieve LPS-induced podocyte injury.     

Propagation of adult rat bone marrow-derived hepatocyte-like cells by serial passages in vitro

Previously, we found that hepatocyte growth factor receptor (c-Met)-and alpha-fetoprotein (AFP)-expressing cells were present in adult rat bone marrow, and that these cells also expressed hematopoietic stem cell markers, such as CD34, Thy-1, and c-Kit. When bone marrow cells were cultured in a hepatocyte growth medium (HGM) with HGF and EGF, colonies composed of polygonal cells resembling mature hepatocytes appeared by 2 weeks and grew very slowly because of overgrowth of stromal cells. At days 34-41, 2-mm2 sheets of hepatocyte-like cells were cut out of their colonies by scratching with an injection needle under observation with a phase contrast microscope, transferred into wells of 24-well plates, and cultured in the HGM medium in the presence or absence of HGF and EGF. When cells reached confluence, cells were detached with trypsin and EDTA and transferred step by step into bigger culture vessels. Thus, hepatocyte-like cells were expanded 1000-fold during less than 4 months. These cells were immunocytochemically stained for albumin and also for AFP and the hematopoietic stem cell markers described above, showing characteristics of oval cells. By RT-PCR, we detected mRNAs of tryptophan-2,3-dioxygenase and tyrosine aminotransferase, markers of hepatocytes at a terminal differentiation stage. The present culture system may be useful for supply of hepatocyte resources for cell transplantation therapy.     

利塞膦酸盐对BMSCs诱导的成骨细胞在脱钙骨支架上生物学行为的影响

目的:观察不同浓度利塞膦酸盐对脱钙骨支架中成骨细胞活性的影响.方法:选用含不同浓度利塞膦酸钠的培养液(10～-7、10-810-9,10-10 mol/L)对BMSCs诱导的成骨细胞与脱钙骨支架复合物进行培养,通过MTT,上清液碱性磷酸酶活性及Ⅰ型前胶原羧端肽、骨钙素检测对比观察不同浓度利塞膦酸钠对成骨细胞的作用效果.结果:MTT显示不同浓度利塞膦酸钠组细胞均随培养时间的增加而明显增殖,相同培养时间各不同浓度的利塞膦酸钠组的细胞增殖能力均高于对照纽(P＜0.05),且在10-10～10-7mol/L浓度范围内呈逐渐增加趋势.各不同浓度的利塞膦酸钠组的上清液ALP活性表达、Ⅰ型前胶原羧端肽表达、骨钙素表达均随培养时间的增加而明显增加,相同培养时间各组数值均高于对照组(P＜0.05),且在10-10～10-7 mol/L浓度范围内呈逐渐增加趋势.结论:利塞膦酸钠对成骨细胞在支架材料中的粘附、生长、增殖及骨化作用均有良好的促进作用.     

Effects of transforming growth factor beta on cells derived from bone and callus of patients with osteogenesis imperfecta

We studied the influence of transforming growth factor beta (TGF-β) on cultured bone cells derived from two patients with osteogenesis imperfecta (OI) and from human controls. Additionally, cells from a hyperplastic callus that had developed spontaneously at the femur of the patient in Case 1 and cells from a normal fracture callus were included in the study. TGF-β increased the synthesis of total protein and collagen of all cells without changing the pattern of interstitial collagens. Proliferation was stimulated by TGF-β in the OI bone cells from Case 1, in cells from the central part of the hyperplastic callus, and in cells from the fracture callus. In Case 2, proliferation of bone cells was decreased by low concentrations of TGF-β. Alkaline phosphatase (AP) activity was enhanced by TGF-β in normal human bone cells, not affected in bone cells from the patient in Case 2 or in cells from the central part of the hyperplastic callus, and inhibited in bone cells and cells from the peripheral part of the hyperplastic callus of Case 1 and in cells from the fracture callus. We conclude that TGF-β has common and specific effects on cultured human cells derived from different types of skeletal tissues. Simultaneous stimulation of collagen synthesis and AP activity by TGF-β was restricted to normal human bone cells and might reflect their mature state of osteoblastic differentiation. Cells derived from bone of both patients with OI, from the hyperplastic callus, and from the fracture callus showed a different response pattern to TGF-β. The difference in response to TGF-β of bone cells from the two patients with OI might be related to the development of a hyperplastic callus in one of them (Case 1). This study indicates that the effects of TGF-β on cells isolated from human skeletal tissues depend on their metabolic state and their stage of maturation and might be correlated with the formation of a hyperplastic callus in OI.     

骨髓源性肝干细胞定向分化及脾内移植研究

目的　寻找骨髓源性肝干细胞的表面标记 ,进行定向分化及脾内移植研究。方法　流式细胞仪检测淤胆大鼠骨髓中干细胞群体的数量变化 ,寻找肝干细胞标记 ,免疫磁珠分离 ,进行体外诱导分化和肝再生模型的脾内移植 ,观察细胞形态的变化 ,免疫组织化学和免疫荧光技术检测白蛋白、AFP、CK8/18等肝细胞标记的表达。结果　淤胆鼠 β2微球蛋白阴性 (β2 m-)细胞数量较对照组数量明显增高 ,分别为 (6.17± 2 .70 ) %和 (0 .79± 0 .61) % (P <0 .0 1) ,β2 m-细胞体外培养及脾内移植均可出现肝细胞样细胞 ,白蛋白、AFP、CK8/18表达阳性。结论　β2m 细胞在体内外具有向肝细胞分化的能力 ,脾内移植是肝干细胞移植可供选择的部位之一。