Experimental and Therapeutic Opportunities for Stem Cells in Multiple Sclerosis

Multiple Sclerosis (MS) is an inflammatory demyelinating neurodegenerative disorder of the brain and spinal cord that causes significant disability in young adults. Although the precise aetiopathogenesis of MS remains unresolved, its pathological hallmarks include inflammation, demyelination, axonal injury (acute and chronic), astrogliosis and variable remyelination. Despite major recent advances in therapeutics for the early stage of the disease there are currently no disease modifying treatments for the progressive stage of disease, whose pathological substrate is axonal degeneration. This represents the great and unmet clinical need in MS. Against this background, human stem cells offer promise both to improve understanding of disease mechanism(s) through in-vitro modeling as well as potentially direct use to supplement and promote remyelination, an endogenous reparative process where entire myelin sheaths are restored to demyelinated axons. Conceptually, stem cells can act directly to myelinate axons or indirectly through different mechanisms to promote endogenous repair; importantly these two mechanisms of action are not mutually exclusive. We propose that discovery of novel methods to invoke or enhance remyelination in MS may be the most effective therapeutic strategy to limit axonal damage and instigate restoration of structure and function in this debilitating condition. Human stem cell derived neurons and glia, including patient specific cells derived through reprogramming, provide an unprecedented experimental system to model MS “in a dish” as well as enable high-throughput drug discovery. Finally, we speculate upon the potential role for stem cell based therapies in MS.     

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。     

Signaling Involved in Hair Follicle Morphogenesis and Development

Hair follicle morphogenesis depends on Wnt, Shh, Notch, BMP and other signaling pathways interplay between epithelial and mesenchymal cells. The Wnt pathway plays an essential role during hair follicle induction, Shh is involved in morphogenesis and late stage differentiation, Notch signaling determines stem cell fate while BMP is involved in cellular differentiation. The Wnt pathway is considered to be the master regulator during hair follicle morphogenesis. Wnt signaling proceeds through EDA/EDAR/NF-κB signaling. NF-κB regulates the Wnt pathway and acts as a signal mediator by upregulating the expression of Shh ligand. Signal crosstalk between epithelial and mesenchymal cells takes place mainly through primary cilia. Primary cilia formation is initiated with epithelial laminin-511 interaction with dermal β-1 integrin, which also upregulates expression of downstream effectors of Shh pathway in dermal lineage. PDGF signal transduction essential for crosstalk is mediated through epithelial PDGF-A and PDGFRα expressed on the primary cilia. Dermal Shh and PDGF signaling up-regulates dermal noggin expression; noggin is a potent inhibitor of BMP signaling which helps in counteracting BMP mediated β-catenin inhibition. This interplay of signaling between the epithelial and dermal lineage helps in epithelial Shh signal amplification. The dermal Wnt pathway helps in upregulation of epithelial Notch expression. Dysregulation of these pathways leads to certain abnormalities and in some cases even tumor outgrowth.     

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。     

Human Mesenchymal Stem Cells Modulate Inflammatory Cytokines after Spinal Cord Injury in Rat

Transplantation of mesenchymal stem cells (MSC) improves functional recovery in experimental models of spinal cord injury (SCI); however, the mechanisms underlying this effect are not completely understood. We investigated the effect of intrathecal implantation of human MSC on functional recovery, astrogliosis and levels of inflammatory cytokines in rats using balloon-induced spinal cord compression lesions. Transplanted cells did not survive at the lesion site of the spinal cord; however, functional recovery was enhanced in the MSC-treated group as was confirmed by the Basso, Beattie, and Bresnahan (BBB) and the flat beam test. Morphometric analysis showed a significantly higher amount of remaining white matter in the cranial part of the lesioned spinal cords. Immunohistochemical analysis of the lesions indicated the rearrangement of the glial scar in MSC-treated animals. Real-time PCR analysis revealed an increased expression of Irf5, Mrc1, Fgf2, Gap43 and Gfap. Transplantation of MSCs into a lesioned spinal cord reduced TNFα, IL-4, IL-1β, IL-2, IL-6 and IL-12 and increased the levels of MIP-1α and RANTES when compared to saline-treated controls. Intrathecal implantation of MSCs reduces the inflammatory reaction and apoptosis, improves functional recovery and modulates glial scar formation after SCI, regardless of cell survival. Therefore, repeated applications may prolong the beneficial effects induced by MSC application.     

调节性树突状细胞在免疫相关性疾病中的研究进展

树突状细胞(Dendritic cell,DC)是机体免疫系统中一组形态和功能异质性的专职抗原提呈细胞,其既能启动初始免疫应答,也能负向调节免疫反应。具有负向调节免疫应答功能的树突状细胞称为调节性DC(Regulatory DC,DCreg)。DCreg在某些感染性疾病、自身免疫性疾病、肿瘤等的发生发展过程中起重要作用,也有望在临床中成为重要的治疗工具。DCreg的分化与细胞所处的微环境有关,结合微环境研究DCreg才能客观地反应机体免疫功能的真实状态。本文就DCreg分化的微环境、血管活性肠肽(Vasoactive intestinal peptide,VIP)诱导的DCreg(DCVIP)以及部分DCreg在免疫相关性疾病中的临床意义等方面的最新研究进展作一综述。     

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。