Department of Anatomy and Neurobiology, Medical College of Virginia Campus, Virginia Commonwealth University, Richmond, VA 23298, USA.
Department of Biomedical Engineering, College of Engineering, Virginia Commonwealth University, Richmond, VA 23284, USA.
J Neurosci Methods. 2024 Jun;406:110114. doi: 10.1016/j.jneumeth.2024.110114. Epub 2024 Mar 24.
Induced pluripotent stem cells (iPSCs) derived neural stem cells (NSCs) provide a potential for autologous neural transplantation therapy following neurological insults. Thus far, in preclinical studies the donor iPSCs-NSCs are mostly of human or mouse origin with concerns centering around graft rejection when applied to rat brain injury models. For better survival and integration of transplanted cells in the injured brain in rat models, use of rat-iPSC-NSCs and in combination with biomaterials is of advantageous. Herein, we report a detailed method in generating rat iPSCs with improved reprogramming efficiency and differentiation into neurons.
Rat fibroblasts were reprogrammed into iPSCs with polybrene and EF1α-STEMCCA-LoxP lentivirus vector. Pluripotency characterization, differentiation into neuronal linage cells were assessed with RT-qPCR, Western blotting, immunostaining and patch-clamp methods. Cells were cultured in a custom-designed integrin array system as well as in a hydrogel-based 3D condition.
We describe a thorough method for the generation of rat-iPSC-NSCs, and identify integrin αβ as a substrate for the optimal growth of rat-iPSC-NSCs. Furthermore, with hydrogel as the supporting biomaterial in the 3-D culture, when combined with integrin αβ binding peptide, it forms a conducive environment for optimal growth and differentiation of iPSC-NSCs into mature neurons.
Published studies about rat-iPSC-NSCs are rare. This study provides a detailed protocol for the generation of rat iPSC-NSCs and optimal growth conditions for neuronal differentiation. Our method is useable for studies to assess the utility of rat iPSC-NSCs for neural transplantation in rat brain injury models.
诱导多能干细胞（iPSC）衍生的神经干细胞（NSC）为神经损伤后的自体神经移植治疗提供了可能。迄今为止，在临床前研究中，供体 iPSC-NSC 主要来源于人类或小鼠，当应用于大鼠脑损伤模型时，主要关注移植物排斥问题。为了提高大鼠模型中移植细胞的存活率和整合性，使用大鼠 iPSC-NSC 并结合生物材料是有利的。在此，我们报告了一种详细的方法，可提高大鼠 iPSC 的重编程效率，并使其分化为神经元。
用多柔比星和 EF1α-STEMCCA-LoxP 慢病毒载体将大鼠成纤维细胞重编程为 iPSC。通过 RT-qPCR、Western blot、免疫染色和膜片钳方法评估多能性特征和向神经元谱系细胞的分化。将细胞在定制的整合素阵列系统以及基于水凝胶的 3D 条件下进行培养。
我们描述了一种生成大鼠 iPSC-NSC 的全面方法，并确定整合素 αβ 是大鼠 iPSC-NSC 最佳生长的底物。此外，在 3D 培养中用水凝胶作为支撑生物材料，与整合素 αβ 结合肽结合时，形成有利于 iPSC-NSC 向成熟神经元最佳生长和分化的环境。
关于大鼠 iPSC-NSC 的已发表研究很少。本研究提供了一种详细的大鼠 iPSC-NSC 生成方案和神经元分化的最佳生长条件。我们的方法可用于评估大鼠 iPSC-NSC 在大鼠脑损伤模型中的神经移植应用价值的研究。
