Mori Hideki, Hara Masayuki
Department of Biological Science, Graduate School of Science, Osaka Prefecture University, 1-2 Gakuen-cho, Naka-ku, Sakai, Osaka 599-8570, Japan.
Department of Biological Science, Graduate School of Science, Osaka Prefecture University, 1-2 Gakuen-cho, Naka-ku, Sakai, Osaka 599-8570, Japan.
J Biosci Bioeng. 2016 May;121(5):584-90. doi: 10.1016/j.jbiosc.2015.09.010. Epub 2015 Oct 21.
Neural stem/progenitor cells (NSPCs) in the central nervous system (CNS) have the capacity to self-renew by proliferation and are multipotent, giving rise to neurons, astrocytes, and oligodendrocytes. NSPCs can be amplified in neurosphere suspension cultures for cell transplantation therapy to treat CNS diseases as well as for in vitro pharmacological/toxicological assays; however, these suspension cultures have certain limitations, including the inconvenience of changing the culture medium as well as difficulty of live imaging. In the present study, we prepared a gamma-crosslinked poly(vinyl alcohol) (PVA) hydrogel and assessed its suitability as a substrate for adherent NSPC cultures. Differentiation was determined by evaluating the expression of the markers nestin (progenitors), βIII tubulin (neurons), and glial fibrillary acidic protein and S100β (glia) by immunocytochemistry and quantitative reverse transcriptase PCR. The levels of the marker genes were similar between the two types of culture; although some variability was observed, there were no fold differences in expression. NSPCs adhered to the PVA gel as clusters and grew without differentiating into neurons and glia. The proliferation rate of cells grown on the soft PVA gel [3.75-7.5% (w/v) PVA] was approximately 70% of that of neurospheres in suspension. We conclude that gamma-crosslinked PVA hydrogels can function as a novel scaffold for maintaining adherent NSPCs in an undifferentiated state.
中枢神经系统（CNS）中的神经干细胞/祖细胞（NSPCs）具有通过增殖进行自我更新的能力，并且具有多能性，可分化为神经元、星形胶质细胞和少突胶质细胞。NSPCs可在神经球悬浮培养物中扩增，用于细胞移植治疗中枢神经系统疾病以及进行体外药理/毒理学分析；然而，这些悬浮培养存在一定局限性，包括更换培养基不便以及活体成像困难。在本研究中，我们制备了一种γ-交联聚乙烯醇（PVA）水凝胶，并评估了其作为贴壁NSPC培养底物的适用性。通过免疫细胞化学和定量逆转录聚合酶链反应评估巢蛋白（祖细胞）、βIII微管蛋白（神经元）、胶质纤维酸性蛋白和S100β（神经胶质）标志物的表达，以确定分化情况。两种培养类型之间标志物基因的水平相似；尽管观察到一些变异性，但表达上没有倍数差异。NSPCs以簇状附着在PVA凝胶上生长，且未分化为神经元和神经胶质。在柔软的PVA凝胶[3.75 - 7.5%（w/v）PVA]上生长的细胞增殖率约为悬浮神经球的70%。我们得出结论，γ-交联PVA水凝胶可作为一种新型支架，用于维持贴壁NSPCs处于未分化状态。
