Generation and optimization of highly pure motor neurons from human induced pluripotent stem cells via lentiviral delivery of transcription factors
Generation and optimization of highly pure motor neurons from human induced pluripotent stem cells via lentiviral delivery of transcription factors
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DOI：
10.1152/ajpcell.00279.2020
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发表时间：
2020-10-01
影响因子：
5.5
通讯作者：
Ding, Baojin
中科院分区：
文献类型：
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作者：
Sepehrimanesh, Masood;Ding, Baojin
从人诱导多能干细胞（hiPSC）产生神经元克服了人脑样品的有限获取，极大地促进了神经系统疾病研究的进展。然而，它仍然是一个挑战，以产生一个特定的神经元亚型与高纯度和产量，以确定发病神经元的发病机制，使用生化方法。运动神经元（MN）是负责自主运动和意志运动的专门神经元亚型。MN中的功能障碍涉及多种运动疾病，例如肌萎缩性侧索硬化症（ALS）。在这项研究中，我们通过慢病毒递送转录因子从人iPSC产生功能性MN。此外，我们通过使用不同的转录因子组合优化了诱导条件，发现表达三种因子[神经生成素-2（NGN 2）、胰岛素基因增强子1（ISL 1）和LIM/同源框3（LHX 3）]的单个慢病毒载体对于诱导iPSC衍生的MN（iPSC-MN）是必要的和足够的。这些MN强烈表达一般神经元标记物[微管相关蛋白2（MAP 2），神经丝蛋白（SMI-32）和微管蛋白β-3 III类（TUBB 3）]和MN特异性标记物[HB 9和胆碱乙酰转移酶（ChAT）]，并在3周内显示电成熟和动作电位放电。这种方法显着提高了神经元的存活率，产量和纯度，使其成为可行的，以获得丰富的材料，在建模运动疾病的生化研究。
Generation of neurons from human induced pluripotent stem cells (hiPSCs) overcomes the limited access to human brain samples and greatly facilitates the progress of research in neurological diseases. However, it is still a challenge to generate a particular neuronal subtype with high purity and yield for determining the pathogenesis of diseased neurons using biochemical approaches. Motor neurons (MNs) are a specialized neuronal subtype responsible for governing both autonomic and volitional movement. Dysfunctions in MNs are implicated in a variety of movement diseases, such as amyotrophic lateral sclerosis (ALS). In this study, we generated functional MNs from human iPSCs via lentiviral delivery of transcription factors. Moreover, we optimized induction conditions by using different combinations of transcription factors and found that a single lentiviral vector expressing three factors [neurogenin-2 (NGN2), insulin gene enhancer 1 (ISL1), and LIM/homeobox 3 (LHX3)] is necessary and sufficient to induce iPSC-derived MNs (iPSC-MNs). These MNs robustly expressed general neuron markers [microtubule-associated protein 2 (MAP2), neurofilament protein (SMI-32), and tubulin beta-3 class III (TUBB3)] and MN-specific markers [HB9 and choline acetyltransferase (ChAT)] and showed electrical maturation and firing of action potentials within 3 wk. This approach significantly improved the neuronal survival, yield, and purity, making it feasible to obtain abundant materials for biochemical studies in modeling movement diseases.