神经干细胞的分化影响其对肝细胞生长因子的趋化性迁移

神经干细胞（neural stem cells,NSCs）的定向迁移对神经系统发育和损伤后修复至关重要,但NSCs的定向迁移与NSCs的分化之间的关系鲜有研究。该研究以此为切入点,以肝细胞生长因子（hepatocyte growth factor,HGF）为趋化因子,神经干细胞系C17．2为研究对象,首先,建立了不同分化阶段的NSCs（分别分化0,12,24,72h）的分化模型;其次,运用Boyden chamber和Dunn chamber研究了不同分化状态下的NSCs对HGF的趋化性迁移。Boyden chamber结果显示：下室加入HGF后,分化12,24h的NSCs迁移至膜下方的细胞数目显著高于分化0,72h的NSCs;Dunn chamber结果显示：分化12,24h的NSCs迁移效率显著高于分化0,72h的NSCs。这些结果表明,NSCs的分化影响其对HGF的趋化性迁移,为在临床上更有效地利用NSCs治疗各种神经系统退行性疾病提供了理论依据。     

干细胞因子诱导神经干细胞定向迁移

神经干细胞(neural stem cells,NSCs)能够定向追踪胶质瘤,然而其内在作用机制及影响因素尚不明了。本实验运用Boyden chamber及Dunn chamber来研究干细胞因子(stem cell factor,SCF)诱导C17.2神经干细胞的趋化性迁移。Boyden chamber结果显示,下室加入SCF后迁移至膜下方的C17.2细胞数显著多于对照组;Dunn chamber结果显示,仅在外槽加入SCF的C17.2细胞迁移速率及迁移效率显著高于内外槽均加入SCF的细胞,单个细胞迁移轨迹分析表明仅在外槽加入SCF后细胞朝着浓度梯度方向迁移而内外槽均加入SCF后细胞的迁移则无规律。这些结果表明,SCF能够诱导C17.2细胞的定向迁移,为进一步了解神经干细胞定向追踪胶质瘤提供了理论依据。     

Tie-2单克隆抗体鉴定人骨髓间充质干细胞体外定向诱导分化的内皮细胞

目的:体外扩增和定向诱导成人骨髓间充质干细胞(MSCs)向内皮细胞分化,并探讨其可行性和条件.方法:利用Percoll(1 073 g/L)从正常成人骨髓中分离MSCs,用含10?S的LG-DMEM培养基进行纯化和扩增培养,流式细胞仪分析鉴定MSCs的纯度.用含VEGF(10μg/L)的HGDMEM培养基诱导MSCs向内皮细胞定向分化,Tie-2单克隆抗体的免疫组化法和透射电镜(TEM)鉴定其细胞的性质.结果:5.0×105个MSCs在体外扩增15代后,获得了8.0×1012个MSCs,扩增了约1.6×107倍.加入诱导培养体系培养14～21 d,光镜下可观察到内皮细胞呈典型的"鹅卵石"样:90%的细胞Tie-2免疫组化呈阳性反应;TEM下可观察到胞浆内有Weible-palade小体.结论:成人骨髓MSCs在体外具有定向诱导分化为内皮细胞的潜能,为构建心脏组织工程瓣种子细胞的来源提供了可能性.     

TGF-β1诱导马鹿茸MSCs软骨分化及c-myc基因的表达

鹿茸间充质干细胞(MSCs)是维持茸再生与骨化的重要组织,旨在研究鹿茸MSCs的软骨分化及原癌基因c-myc对该过程的调控作用。利用成年塔里木马鹿生长60 d的鹿茸第2代间充质干细胞(MSCs,P2),通过TGF-β1(10 ng/m L浓度)刺激,诱导塔里木马鹿茸间充质干细胞向软骨分化,采用免疫组化和阿利新蓝染色鉴定诱导结果,并通过q PCR方法检测软骨分化过程中c-myc基因的表达变化。结果显示,MSCs在诱导后的第9天开始出现细胞形态变化,由梭形向多角形转变,原来菊花状的分布逐渐向铺路石状变化,至14 d可观察到软骨陷窝,21 d软骨细胞基质明显,并开始出现细胞凋亡。非诱导组28 d细胞出现凋亡,细胞内发现空泡。35 d两组细胞凋亡明显,细胞折光性变差,间隙变大。阿利新蓝染色鉴定,诱导至第14天细胞基质中开始出现大量阳性染色。免疫组化实验检测,诱导至21 d的细胞基质中出现棕色Col II阳性反应物,随培养时间增加颜色加深,并集中分布在细胞及其周围基质中。在软骨分化进程中,第7、14、21和28天,诱导组c-myc基因表达与非诱导组相比显著下调(P0.05),但诱导至35 d,诱导组c-myc表达与非诱导组相比无显著差异(P0.05)。在TGF-β1刺激下,塔里木马鹿茸MSCs可以分化成软骨,原癌基因c-myc下调表达诱导鹿茸MSCs进入凋亡状态并分化为软骨细胞。     

隔离器内残留过氧化氢去污剂对MSCs和NK两种细胞增殖及生物学特性的影响研究

该文研究了特定过氧化氢去污剂残留量对间充质干细胞(mesenchymal stem cells,MSCs)和自然杀伤细胞(natural killer cell,NK)的增殖及生物学特性的影响。采用低浓度过氧化氢去污剂建立特定残留量进行MSCs和NK细胞培养,应用细胞计数法分析细胞增殖能力,流式细胞术检测细胞表面标记,并采用诱导分化培养基定向诱导MSCs成骨、成脂、成软骨分化。结果显示,MSCs和NK细胞在3～5 mg/m³浓度的过氧化氢环境下形态正常且增殖活跃。流式细胞仪检测结果显示,MSCs的CD73、CD105、CD90均呈高表达,CD45、CD34、CD14、CD79a、HLA-DR为阴性表达;NK细胞表达CD56⁺和CD3~–的百分比超过70%。诱导后,MSCs具有成骨、成脂、成软骨分化的潜能。总之,MSCs和NK细胞在含有低浓度的残留过氧化氢去污剂的培养环境中生长良好,与未含有过氧化氢去污剂的培养环境比较,增殖能力和分化潜能均无显著差异。     

SDF-1/CXCR4 轴在间充质干细胞归巢机制中的研究进展

间充质干细胞(Mesenchymal Stem Cells,MSCs)是一种具有多向分化潜能的成体干细胞,其具有分泌营养物质和调节炎症反应的能力,虽然间充质干细胞在组织修复、重塑和免疫调节方面已得到临床运用,但MSCs趋化和归巢的机制仍不清楚。基质细胞衍生因子-1(stromal cell-derived factor 1,SDF-1)和其趋化因子受体4(C-X-C chemokine receptor 4,CXCR4)在介导MSCs的分化、迁移和归巢中起着至关重要的作用,若能深入探讨、明确其在归巢中的作用,期望给间充质干细胞在临床的应用开辟新的应用前景。     

间充质干细胞体外分化为多巴胺能神经元的研究进展

骨髓间充质干细胞（MSCs）有来源广泛、易于分离培养、不易引起免疫排斥等特点,使其成为细胞治疗和基因治疗的种子细胞,具有广泛的科研和临床应用价值。骨髓MSCs具有多向分化潜能,在特定条件下能诱导分化成神经元甚至是更为特异的多巴胺能神经元,为帕金森病进行细胞移植疗法提供了理想的细胞来源。本文就近年来体外诱导MSCs向多巴胺能神经元定向分化所涉及到的常用诱导因素和诱导方法及途径予以综述。     

VEGF通过调节黏着斑促进间充质干细胞的黏附及铺展

间充质干细胞（mesenchymalstemcells,MSCs）具有多向分化潜能并能在体外趋化剂或细胞因子的作用下进行定向迁移,体内移植后可趋向迁移至脑瘤病灶区。细胞黏附是细胞迁移的首要条件,了解细胞黏附及其调控有助于细胞迁移机制的研究。细胞黏附及铺展涉及到黏着斑（f0－caladhesions,FAs）的动态变化以及细胞骨架的重排。细胞铺展面积在黏附过程中逐渐增大,黏附初期形成的小的黏着复合物逐渐成熟,聚集在一起形成较大的FAs。肌动蛋白（F—actin）聚集形成的螺线圈样微丝结构逐渐被应力纤维代替,细胞也由圆形变为具有极性的梭形或多角形。黏着斑激酶（focal adhesion kinase,FAK）和桩蛋白（paxillin）具有调节FAs聚合及骨架重排的作用,其中,Y397－FAK和Y31／Y118－paxillin的磷酸化活性在细胞铺展过程中不断变化。FAs组装时,Y397－FAK的磷酸化活性升高;FAs成熟后,Y397．FAK的磷酸化活性下降。活化的FAK能够磷酸4LY31／Y118-paxillin,激活paxillin参与调节细胞骨架的形成和排列。血管内皮生长因子（vascular endothelial growthfactor,VEGF）诱导~SMSCs黏附过程中,细胞面积变大,完全铺展的时间缩短,黏着斑及细胞骨架的形成均提前。另外,VEGF诱导的细胞铺展过程中形成的FAs形态细长,数量较多。该研究表明,VEGF通过调节黏着斑和细胞骨架促L~MSCs的黏附与铺展,提示vEGF可以通过调节黏着斑进而调控MSCs的定向迁移,为细胞迁移行为的研究提供理论基础。     

脐血间充质干细胞诱导向神经元样细胞分化的实验研究

目的:研究脐血间充质干细胞生物学特性及向神经元样细胞分化的潜能。方法:采用密度梯度离心结合贴壁培养法自脐血中分离间充质干细胞,观察细胞生长情况,描绘生长曲线,流式细胞仪检测细胞表面标志物,分别向成骨细胞、脂肪细胞、神经元样细胞进行诱导分化,通过茜素红染色、油红O染色检测脐血间充质干细胞成骨、成脂肪细胞诱导分化能力,而以免疫组织化学检测诱导后细胞表面神经标志物的表达。结果:纯化的脐血间充质干细胞贴壁生长,呈均一梭形,生长曲线呈S型,并以P3代增殖能力最强,细胞表面不表达或弱表达CD34、CD35、CD106,高表达CD29、CD44、CD105。成骨诱导2周后,可检测到钙化基质的形成,成脂肪诱导3周后,可检测到脂滴的形成。向神经元样细胞诱导分化后,可观察到典型的神经元样形态改变,且NSE、NF、GFAP阳性表达。结论:分离纯化的脐血间充质干细胞具有较强的增殖能力与分化潜能,并在体外诱导条件下可以向神经元样细胞定向分化。     

人脐血间充质干细胞诱导分化为神经细胞的研究

目的探讨体外诱导人脐血间充质干细胞(MSCs)向神经细胞分化的条件,为治疗中枢神经系统损伤提供实用的干细胞来源。方法体外分离、纯化、扩增脐血MSCs,流式细胞仪检测细胞表面标志。采用脑源性神经营养因子BDNF 10ng/ml 维甲酸RA0.5μM 碱性成纤维生长因子bFGF 20ng/ml协同诱导脐血MSCs定向分化。免疫荧光染色检测诱导后细胞的星形胶质细胞特异标志GFAP及神经元特异标志MAP2的表达情况。建立大鼠脊髓横断损伤模型,将BrdU标记的诱导后的细胞移植入损伤的脊髓中,采用BBB运动功能评分标准在术后24h及1、2、3、4、5周各时间点对大鼠进行运动功能评分。用组织学和免疫组化方法检测移植到大鼠脊髓中的BrdU阳性细胞的存活、迁移、分化情况。结果脐血MSCs体外培养三代后,细胞表面CD11b、CD34、CD45和CD44表达阴性。诱导分化7d后,大部分细胞的形态类似神经元,免疫荧光染色检测MAP2阳性细胞占大多数,明显多于GFAP阳性细胞。5周后,细胞移植组大鼠的后肢运动功能恢复情况较对照组好。免疫组织化学结果显示植入的细胞可长时间在宿主脊髓中存活,并向损伤处两端迁移。结论人脐血MSCs于体外在特定的条件下可以诱导分化为神经元样细胞。移植脐血MSCs诱导后的神经细胞可在损伤的脊髓中存活、迁移,并能促进脊髓损伤后行为和功能恢复。     

Neural differentiation of mesenchymal stem cells influences chemotactic responses to HGF

Recently, mesenchymal stem cells (MSCs) have been extensively used for cell‐based therapies in neuronal degenerative disease. Although much effort has been devoted to the delineation of factors involved in the migration of MSCs, the relationship between the chemotactic responses and the differentiation status of these cells remains elusive. Here, we report that MSCs in varying neural differentiation states display different chemotactic responses to hepatocyte growth factor (HGF): first, the number of chemotaxing MSCs and the optimal concentrations of HGF that induced the peak migration varied greatly; second, time‐lapse video analysis showed that MSCs in certain differentiation state migrated more efficiently toward HGF; third, the phosphorylation levels of Akt, ERK1/2, SAPK/JNK, and p38MAPK were closely related to the differentiation levels of MSCs subjected to HGF; and finally, although inhibition of ERK1/2 signaling significantly attenuated HGF‐stimulated transfilter migration of both undifferentiated and differentiating MSCs, abolishment of PI3K/Akt, p38MAPK, or SAPK/JNK signaling only decreased the number of migrated cells in certain differentiation state(s). Blocking of PI3K/Akt or MAPK signaling impaired the migration efficiency and/or speed, the extent of which depends on the cell differentiation states. Meanwhile, F‐actin rearrangement, which is essential for MSCs chemotaxis, was induced by HGF, and the time points of cytoskeletal reorganization were different among these cells. Collectively, these results demonstrate that neural differentiation of MSCs influences their chemotactic responses to HGF: MSCs in varying differentiation states possess different migratory capacities, thereby shedding light on optimization of the therapeutic potential of MSCs to be employed for neural regeneration after injury. J. Cell. Physiol. 228: 149–162, 2013. © 2012 Wiley Periodicals, Inc.     

Differentiation-dependent secretion of proangiogenic factors by mesenchymal stem cells

Mesenchymal stem cells (MSCs) are a promising cell population for cell-based bone repair due to their proliferative potential, ability to differentiate into bone-forming osteoblasts, and their secretion of potent trophic factors that stimulate angiogenesis and neovascularization. To promote bone healing, autogenous or allogeneic MSCs are transplanted into bone defects after differentiation to varying degrees down the osteogenic lineage. However, the contribution of the stage of osteogenic differentiation upon angiogenic factor secretion is unclear. We hypothesized that the proangiogenic potential of MSCs was dependent upon their stage of osteogenic differentiation. After 7 days of culture, we observed the greatest osteogenic differentiation of MSCs when cells were cultured with dexamethasone (OM+). Conversely, VEGF protein secretion and upregulation of angiogenic genes were greatest in MSCs cultured in growth media (GM). Using conditioned media from MSCs in each culture condition, GM-conditioned media maximized proliferation and enhanced chemotactic migration and tubule formation of endothelial colony forming cells (ECFCs). The addition of a neutralizing VEGF(165/121) antibody to conditioned media attenuated ECFC proliferation and chemotactic migration. ECFCs seeded on microcarrier beads and co-cultured with MSCs previously cultured in GM in a fibrin gel exhibited superior sprouting compared to MSCs previously cultured in OM+. These results confirm that MSCs induced farther down the osteogenic lineage possess reduced proangiogenic potential, thereby providing important findings for consideration when using MSCs for bone repair.     

Dynamics of focal adhesions and reorganization of F‐actin in VEGF‐stimulated NSCs under varying differentiation states

Precise migration of neural stem/progenitor cells (NSCs) is crucially important for neurogenesis and repair in the nervous system. However, the detailed mechanisms are not clear. Our previous results showed that NSCs in varying differentiation states possess different migratory ability to vascular endothelial growth factor (VEGF). In this study, we demonstrate the different dynamics of focal adhesions (FAs) and reorganization of F‐actin in NSCs during spreading and migration stimulated by VEGF. We found that the migrating NSCs of 0.5 and 1 day differentiation possess more FAs at leading edge than cells of other states. Moreover, the phosphorylation of focal adhesion kinase (FAK) and paxillin in NSCs correlates closely with their differentiation states. VEGF promotes FA formation with broad lamellipodium generation at the leading edge in chemotaxing cells of 0, 0.5, and 1 day differentiation, but not in cells of 3 days differentiation. Furthermore, cells of 1 day differentiation show a maximal asymmetry of FAs between lamella and cell rear, orchestrating cell polarization and directional migration. Time‐lapse video analysis shows that the disassembly of FAs and the cell tail detachment in NSCs of 1 day differentiation are more rapid, along with the concurrent enlarged size of FAs at the leading edge, leading to the most effective chemotactic response to VEGF. Collectively, these results indicate that the dynamics of FAs and reorganization of F‐actin in NSCs that undergo directional migration correlate closely with their differentiation states, contributing to the different chemotactic responses of these cells to VEGF. J. Cell. Biochem. 114: 1744–1759, 2013. © 2013 Wiley Periodicals, Inc.     

Assays for in vitro monitoring of human airway smooth muscle (ASM) and human pulmonary arterial vascular smooth muscle (VSM) cell migration

Migration of human pulmonary vascular smooth muscle (VSM) cells contributes to vascular remodeling in pulmonary arterial hypertension and atherosclerosis. Evidence also indicates that, in part, migration of airway smooth muscle (ASM) cells may contribute to airway remodeling associated with asthma. Here we describe migration of VSM and ASM cells in vitro using Transwell or Boyden chamber assays. Because dissecting signaling mechanisms regulating cell migration requires molecular approaches, our protocol also describes how to assess migration of transfected VSM and ASM cells. Transwell or Boyden chamber assays can be completed in approximately 8 h and include plating of serum-deprived VSM or ASM cell suspension on membrane precoated with collagen, migration of cells toward chemotactic gradient and visual (Transwell) or digital (Boyden chamber) analysis of membrane. Although the Transwell assay is easy, the Boyden chamber assay requires hands-on experience; however, both assays are reliable cell-based approaches providing valuable information on how chemotactic and inflammatory factors modulate VSM and ASM migration.     

Cellular changes in bone marrow of malaria-infected mice. III. Chemotaxis of granulocytes

The number of bone marrow cells and their chemotactic activity was studied during malaria infection. Two days after infection of Balb/c mice with Plasmodium berghei, an increase in granulocyte number was observed in the blood. A modified Boyden chamber chemotaxis assay was employed to investigate the mechanism of granulocyte accumulation in the blood. Bone marrow cells from normal mice, from mice during a primary lethal infection and from immune mice after challenge were compared. The complement factor C5a showed chemotactic activity for bone marrow cells; a significant decrease of chemotaxis was only observed after 6 days of primary infection. Extracts of spleen, liver and infected erythrocytes lacked chemotactic activity, or caused inhibition of cell migration. Serum from mice with a 2-day primary infection contained chemotactic activity. The active component was heat labile, protease sensitive and had an estimated molecular weight of 250,000.     

The effect of cell sedimentation on measuring chondrocyte population migration using a Boyden chamber

The Boyden chamber assay provides a convenient method of assessing cell migration and measuring cell motility coefficients at the population level. Previous models of this assay completely ignore cell sedimentation in the suspension, assuming that all cells have already settled on the filter surface before commencing migration within the filter. However, ignoring cell sedimentation could lead to poor data interpretation because the time required for cells to settle through the suspension is close to the incubation period of only a few hours. This study models the Boyden chamber assay by incorporating the cell settling process to account for the cells remaining in the upper well when other cells migrate in the filter. The simulations in this study elucidate the experiments in the literature that test the haptotactic and chemotactic responses of rabbit chondrocytes to type II collagen. This study determines the cell population random motility, as well as the haptotaxis and chemotaxis coefficients, by fitting the experimental data. Results show that the chemotactic motility coefficient is 100 times greater than the haptotactic coefficient, and the equilibrium collagen-receptor dissociation constant is about 10-fold the haptotactic counterpart. Diffusion causes the soluble collagen gradients in the chemotactic case to decline over time, while the coated collagen gradients in the haptotactic assay are likely to remain fixed. As a result, the chemotactic case exhibits a lower number of migrated cells than the haptotactic assay. This study also demonstrates the influences of the dimensionless parameters that control cell behavior in the Boyden assay, providing a reference for future experiment designs.     

IGF-I and IGF-II stimulate directed cell migration of bone-marrow-derived human mesenchymal progenitor cells

Insulin-like growth factors (IGFs) are known to be key regulators of bone growth, remodeling, and repair. Since all these processes depend on the recruitment of cells with the potential to be committed to the osteoblastic lineage, we studied possible effects of IGF-I and -II on migration of human mesenchymal progenitor cells (MPC) using a modified Boyden chamber assay. The results were compared to those of primary osteoblasts and in vitro-osteogenic-differentiated MPC. IGF-I and -II stimulated cell migration of all these cell populations in a dose-dependent manner from 1 to 100 ng/mL. The maximal chemotactic index (CI) was 4-5 for MPC and primary osteoblasts and about 3 for in vitro-differentiated MPC. Checkerboard analysis revealed that IGFs stimulated true directed cell migration (chemotaxis) and not simply chemokinesis. Addition of an antibody against the type I IGF receptor (αIR3) completely abolished (MPC) or markedly reduced (primary osteoblasts) the chemotactic effects of each of the IGFs. IGFBP-3 itself had no direct effect, while IGFBP-5 stimulated MPC migration at concentrations of 80 and 160 ng/mL. Parallel application of IGFBP-3 had borderline inhibitory effects while the addition of 40 ng/mL of IGFBP-5 enhanced the chemotactic effect of IGF-I on MPC. In conclusion, our results show that IGF-I and -II are chemotactic factors for MPC and indicate that IGFBP-5 both modulates the IGF-I effect and directly stimulates migration of human mesenchymal progenitor cells.     

Chemotaxis and chemokinesis of rat polymorphonuclear leukocytes in response to 4-hydroxy-2-tetradecenal and 4-hydroxy-2-nonenal

Since a number of experimental evidences suggests that some lipoperoxidation products can affect leukocyte migration "in vitro", we have investigated the chemotactic and chemokinetic properties of two of these products (4-hydroxy-2,3-trans-tetradecenal and 4-hydroxy-2,3-trans-nonenal) using rat neutrophils. The cells were obtained from the pleural cavity after injection of 1.0 ml isologous serum. The granulocytes were suspended in Hanks' plus BSA 2% and the motility determined by means of a modified Boyden chamber. For evaluating the chemotactic properties, the aldehyde were added into the lower compartment, while for detecting the chemokinetic power, the compounds were placed in both the compartments. Our results show that both the chemicals (in a range between nano- and micromolar concentrations) are able to exert -at different degree- a chemotactic activity. In this connection, the more active aldehyde appeared to be the tetradecenal. On the contrary, the same compounds seem uneffective in stimulating the random migration of polymorphonuclear cells.     

An improved assay to quantitate the invasiveness of cells in modified Boyden chambers

The most commonly used means of assessing the invasiveness of cultured cells is the Boyden chamber assay, which requires that cells lyse Matrigel™, followed by migration through pores in a filter in response to a chemotactic gradient. This report describes a simple method, which greatly increases the speed and accuracy by which Boyden chamber assays can be analyzed, and permits the concurrent analysis of distinct cell subpopulations within specimens containing multiple-cell types.