Data_Sheet_1_A defined method for differentiating human iPSCs into midbrain dopaminergic progenitors that safely restore motor deficits in Parkinson’s disease.docx

BackgroundParkinson’s disease (PD) is a progressive neurodegenerative condition that primarily affects motor functions; it is caused by the loss of midbrain dopaminergic (mDA) neurons. The therapeutic effects of transplanting human-induced pluripotent stem cell (iPSC)-derived mDA neural progenitor cells in animal PD models are known and are being evaluated in an ongoing clinical trial. However, However, improvements in the safety and efficiency of differentiation-inducing methods are crucial for providing a larger scale of cell therapy studies. This study aimed to investigate the usefulness of dopaminergic progenitor cells derived from human iPSCs by our previously reported method, which promotes differentiation and neuronal maturation by treating iPSCs with three inhibitors at the start of induction.
MethodsHealthy subject-derived iPS cells were induced into mDA progenitor cells by the CTraS-mediated method we previously reported, and their proprieties and dopaminergic differentiation efficiency were examined in vitro. Then, the induced mDA progenitors were transplanted into 6-hydroxydopamine-lesioned PD model mice, and their efficacy in improving motor function, cell viability, and differentiation ability in vivo was evaluated for 16 weeks.
ResultsApproximately ≥80% of cells induced by this method without sorting expressed mDA progenitor markers and differentiated primarily into A9 dopaminergic neurons in vitro. After transplantation in 6-hydroxydopamine-lesioned PD model mice, more than 90% of the engrafted cells differentiated into the lineage of mDA neurons, and approximately 15% developed into mature mDA neurons without tumour formation. The grafted PD model mice also demonstrated significantly improved motor functions.
ConclusionThis study suggests that the differentiation protocol for the preparation of mDA progenitors is a promising option for cell therapy in patients with PD.

背景 帕金森病（Parkinson’s disease, PD）是一种进行性神经退行性疾病，主要累及运动功能，其致病机制为中脑多巴胺能（midbrain dopaminergic, mDA）神经元丢失。将人诱导多能干细胞（induced pluripotent stem cell, iPSC）诱导分化得到的mDA神经前体细胞移植到动物PD模型中，已被证实具有治疗效果，相关疗法目前正处于临床试验阶段。然而，优化诱导分化方法的安全性与效率，对于开展更大规模的细胞治疗研究至关重要。本研究旨在评估我们此前报道的一种诱导方法制备的人iPSC来源多巴胺能前体细胞的应用价值：该方法在诱导初期通过三种抑制剂处理，以促进细胞分化与神经元成熟。

方法 本研究采用我们此前报道的CTraS介导方法，将健康受试者来源的iPS细胞诱导分化为mDA前体细胞，并在体外检测了细胞特性与多巴胺能分化效率。随后，将诱导得到的mDA前体细胞移植到6-羟基多巴胺损伤的PD模型小鼠体内，连续16周评估其在改善运动功能、细胞存活能力以及体内分化能力方面的疗效。

结果 采用该方法诱导得到的未分选细胞中，约≥80%表达mDA前体细胞标志物，且在体外主要分化为A9型多巴胺能神经元。将其移植到6-羟基多巴胺损伤的PD模型小鼠体内后，超过90%的移植细胞分化为mDA神经元谱系，其中约15%发育为成熟mDA神经元，且未形成肿瘤。接受移植的PD模型小鼠的运动功能也得到了显著改善。

结论 本研究表明，本研究所用的mDA前体细胞制备分化方案，有望成为帕金森病患者细胞治疗的潜在选择方案。