Tsui Yat-Ping, Mung Alan Kwan-Long, Chan Ying-Shing, Shum Daisy Kwok-Yan, Shea Graham Ka-Hon
School of Biomedical Sciences, The University of Hong Kong.
Department of Orthopaedics and Traumatology, The University of Hong Kong;
J Vis Exp. 2017 Jun 14(124):55794. doi: 10.3791/55794.
This manuscript describes a means to enrich for neural progenitors from the marrow stromal cell (MSC) population and thereafter to direct them to the mature Schwann cell fate. We subjected rat and human MSCs to transient hypoxic conditions (1% oxygen for 16 h) followed by expansion as neurospheres upon low-attachment substratum with epidermal growth factor (EGF)/basic fibroblast growth factor (bFGF) supplementation. Neurospheres were seeded onto poly-D-lysine/laminin-coated tissue culture plastic and cultured in a gliogenic cocktail containing β-Heregulin, bFGF, and platelet-derived growth factor (PDGF) to generate Schwann cell-like cells (SCLCs). SCLCs were directed to fate commitment via coculture for 2 weeks with purified dorsal root ganglia (DRG) neurons obtained from E14-15 pregnant Sprague Dawley rats. Mature Schwann cells demonstrate persistence in S100β/p75 expression and can form myelin segments. Cells generated in this manner have potential applications in autologous cell transplantation following spinal cord injury, as well as in disease modeling.
本手稿描述了一种从骨髓基质细胞（MSC）群体中富集神经祖细胞并随后将其定向分化为成熟施万细胞命运的方法。我们将大鼠和人类MSC置于短暂缺氧条件下（1%氧气，持续16小时），然后在补充表皮生长因子（EGF）/碱性成纤维细胞生长因子（bFGF）的低附着基质上作为神经球进行扩增。将神经球接种到聚-D-赖氨酸/层粘连蛋白包被的组织培养塑料上，并在含有β-调节素、bFGF和血小板衍生生长因子（PDGF）的成胶质细胞混合培养液中培养，以生成施万细胞样细胞（SCLC）。通过与从E14 - 15天怀孕的Sprague Dawley大鼠获得的纯化背根神经节（DRG）神经元共培养2周，将SCLC定向诱导分化。成熟的施万细胞在S100β/p75表达上持续存在，并能形成髓鞘节段。以这种方式产生的细胞在脊髓损伤后的自体细胞移植以及疾病建模中具有潜在应用。
