Department of Chemical and Biomedical Engineering, Cleveland State University, Cleveland, OH 44115, USA.
Biomaterials. 2013 Aug;34(25):5995-6007. doi: 10.1016/j.biomaterials.2013.04.042. Epub 2013 May 18.
The onset of neurodegenerative disorders is characterized by the progressive dysfunction and loss of subpopulations of specialized cells within specific regions of the central nervous system (CNS). Since CNS has a limited ability for self-repair and regeneration under such conditions, clinical transplantation of stem cells has been explored as an alternative. Although embryonic stem cells (ESCs) offer a promising therapeutic platform to treat a variety of neurodegenerative disorders, the niche microenvironment, which could regulate their differentiation into specialized lineages on demand, needs to be optimized for successful clinical transplantation. Here, we evaluated the synergistic role of matrix microenvironment (type, architecture, composition, stiffness) and signaling molecules (type, dosage) on murine ESC differentiation into specific neural and glial lineages. ESCs were cultured as embryoid bodies on either 2D substrates or within 3D scaffolds, in the presence or absence of retinoic acid (RA) and sonic hedgehog (Shh). Results showed that ESCs maintained their stemness even after 4 days in the absence of exogenous signaling molecules, as evidenced by Oct-4 staining. RA at 1 μM dosage was deemed optimal for neural differentiation and neurite outgrowth on collagen-1 coated substrates. Significant neural differentiation with robust neurite outgrowth and branching was evident only on collagen-1 coated 2D substrates and within 3D matrigel scaffolds, in the presence of 1 μM RA. Blocking α6 or β1 integrin subunits on differentiating cells inhibited matrigel-induced effects on neural differentiation and neurite outgrowth. Hydrogel concentration strongly regulated formation of neural and astrocyte lineages in 1 μM RA additive cultures. When RA and Shh were provided, either alone or together, 3D collagen-1 scaffolds enhanced significant motor neuron formation, while 3D matrigel stimulated dopaminergic neuron differentiation. These results suggest a synergistic role of microenvironmental cues for ESC differentiation and maturation, with potential applications in cell transplantation therapy.
神经退行性疾病的发病机制以中枢神经系统（CNS）特定区域的特定细胞亚群的进行性功能障碍和丧失为特征。由于中枢神经系统在这种情况下自我修复和再生的能力有限，因此已经探索了临床干细胞移植作为替代方法。虽然胚胎干细胞（ESCs）为治疗各种神经退行性疾病提供了有前途的治疗平台，但需要优化龛微环境，使其能够按需调节它们分化为特定谱系。在这里，我们评估了基质微环境（类型、结构、组成、硬度）和信号分子（类型、剂量）对鼠 ESC 分化为特定神经和神经胶质谱系的协同作用。在存在或不存在维甲酸（RA）和 sonic hedgehog（Shh）的情况下，将 ESC 培养为类胚体，在 2D 底物或 3D 支架上。结果表明，即使在没有外源性信号分子的情况下，ESC 也能维持其干性，这一点可以通过 Oct-4 染色来证明。1 μM 剂量的 RA 被认为是神经分化和胶原-1 包被基底上神经突生长的最佳选择。仅在胶原-1 包被的 2D 底物和存在 1 μM RA 的 3D matrigel 支架上，才能观察到明显的神经分化和强壮的神经突生长和分支。在分化细胞上阻断 α6 或 β1 整合素亚基可抑制 matrigel 对神经分化和神经突生长的影响。在 1 μM RA 添加剂培养物中，水凝胶浓度强烈调节神经和星形胶质细胞谱系的形成。当单独或一起提供 RA 和 Shh 时，3D 胶原-1 支架增强了显著的运动神经元形成，而 3D matrigel 刺激多巴胺能神经元分化。这些结果表明微环境线索对 ESC 分化和成熟具有协同作用，在细胞移植治疗中有潜在应用。
