Data_Sheet_1_Substrate-bound and soluble domains of tenascin-C regulate differentiation, proliferation and migration of neural stem and progenitor cells.pdf

IntroductionThe lack of regenerative capacity of the central nervous system is one of the major challenges nowadays. The knowledge of guidance cues that trigger differentiation, proliferation, and migration of neural stem and progenitor cells is one key element in regenerative medicine. The extracellular matrix protein tenascin-C (Tnc) is a promising candidate to regulate cell fate due to its expression in the developing central nervous system and in the adult neural stem cell niches. Of special interest are the alternatively spliced fibronectin type III (FnIII) domains of Tnc whose combinatorial diversity could theoretically generate up to 64 isoforms in the mouse. A total of 27 isoforms have already been discovered in the developing brain, among others the domain combinations A1D, CD, and A124BCD.MethodsIn the present study, these domains as well as the combination of the constitutively expressed FnIII domains 7 and 8 (78) were expressed in Chinese hamster ovary cells as pseudo-antibodies fused to the Fc-fragment of a human immunoglobulin G antibody. The fusion proteins were presented to primary mouse neural stem/progenitor cells (NSPCs) grown as neurospheres, either as coated culture substrates or as soluble additives in vitro. The influence of the domains on the differentiation, proliferation and migration of NSPCs was analyzed.ResultsWe observed that the domain combination A124BCD promoted the differentiation of neurons and oligodendrocytes, whereas the domain A1D supported astrocyte differentiation. The constitutively expressed domain 78 had a proliferation and migration stimulating impact. Moreover, most effects were seen only in one of the presentation modes but not in both, suggesting different effects of the Tnc domains in two- and three-dimensional cultures.DiscussionThis knowledge about the different effect of the Tnc domains might be used to create artificial three-dimensional environments for cell transplantation. Hydrogels spiked with Tnc-domains might represent a promising tool in regenerative medicine.

引言中枢神经系统的再生能力不足是目前面临的主要挑战之一。了解触发神经干和祖细胞分化、增殖及迁移的导向信号是再生医学的关键要素。细胞外基质蛋白层粘连蛋白-C（Tenascin-C，简称Tnc）因其在中枢神经系统的发育阶段及成年神经干细胞微环境中表达，成为调控细胞命运的有望候选者。特别值得关注的是Tnc中可变剪接的纤连蛋白类型III（FnIII）结构域，其组合多样性理论上能在小鼠中产生高达64种异构体。在发育中的大脑中已发现27种异构体，其中A1D、CD和A124BCD等结构域组合尤为突出。

方法在本研究中，我们将这些结构域以及组成型表达的FnIII结构域7和8（78）在中华仓鼠卵巢细胞中表达，作为与人类免疫球蛋白G抗体Fc片段融合的伪抗体。这些融合蛋白被展示给作为神经球培养的原始小鼠神经干细胞/祖细胞（NSPCs），无论是作为涂层培养底物还是在体外作为可溶性添加剂。分析了这些结构域对NSPCs的分化、增殖和迁移的影响。

结果我们观察到，结构域组合A124BCD促进了神经元和少突胶质细胞的分化，而结构域A1D支持星形胶质细胞的分化。组成型表达的领域78具有增殖和迁移刺激作用。此外，大多数影响仅在一种展示模式下观察到，而不是两种方式都观察到，这表明Tnc结构域在二维和三维培养中具有不同的作用。

讨论关于Tnc结构域不同作用的知识可能被用于创建人工三维环境以供细胞移植使用。富含Tnc结构域的水凝胶可能在再生医学中成为一种有希望的工具。