Generation of GABAergic striatal neurons by a novel iPSC differentiation protocol enabling scalability and cryopreservation of progenitor cells
Generation of GABAergic striatal neurons by a novel iPSC differentiation protocol enabling scalability and cryopreservation of progenitor cells
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DOI：
10.1007/s10616-020-00406-7
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发表时间：
2020-06-09
期刊：
影响因子：
2.2
通讯作者：
Malakhova, Anastasia A.
中科院分区：
文献类型：
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作者：
Grigor'eva, Elena, V;Malankhanova, Tuyana B.;Malakhova, Anastasia A.
细胞模型是研究遗传性人类神经退行性疾病的有前途的工具。多能干细胞的神经元衍生物提供了研究神经变性过程的不同阶段的机会。因此，相关细胞类型的简单和大规模生产是需要克服的关键障碍。在这项工作中，我们提出了一种替代方案，使iPSC分化为GABA能中型棘神经元（MSN）。第一阶段涉及通过SB431542和LDN193189处理的双SMAD信号传导抑制，这导致神经外胚层细胞的产生。此外，我们使用bFGF作为神经元存活因子和dorsomorphin来抑制BMP信号传导。dorsomorphin和SB 431542的联合处理显著增强了神经元诱导，这通过端脑特异性标记物SOX 1和OX 2以及前脑标记物PAX 6的表达增加来证实。下一阶段涉及活跃增殖的MSN祖细胞的衍生。在这个阶段，我们的方案的一个重要特征是能够以高密度进行前体细胞的长期培养而不损失表型特性。此外，该方案能够实现MSN祖细胞的多个扩增步骤（> 180天培养）和冷冻保存。因此，这种方法可以快速生产大量与基础研究、大规模药物筛选和毒理学研究相关的神经元。
Cell models are promising tools for studying hereditary human neurodegenerative diseases. Neuronal derivatives of pluripotent stem cells provide the opportunity to investigate different stages of the neurodegeneration process. Therefore, easy and large-scale production of relevant cell types is a crucial barrier to overcome. In this work, we present an alternative protocol for iPSC differentiation into GABAergic medium spiny neurons (MSNs). The first stage involved dual-SMAD signalling inhibition through treatment with SB431542 and LDN193189, which results in the generation of neuroectodermal cells. Moreover, we used bFGF as a neuronal survival factor and dorsomorphin to inhibit BMP signalling. The combined treatment of dorsomorphin and SB431542 significantly enhanced neuronal induction, which was confirmed by the increased expression of the telencephalic-specific markersSOX1andOTX2as well as the forebrain markerPAX6. The next stage involved the derivation of actively proliferating MSN progenitor cells. An important feature of our protocol at this stage is the ability to perform prolonged cultivation of precursor cells at a high density without losing phenotypic properties. Moreover, the protocol enables multiple expansion steps (> 180 days cultivation) and cryopreservation of MSN progenitors. Therefore, this method allows quick production of a large number of neurons that are relevant for basic research, large-scale drug screening, and toxicological studies.