Parasympathetic neurons derived from human pluripotent stem cells model human diseases and development. Parasympathetic neurons derived from human pluripotent stem cells model human diseases and development

Parasympathetic neurons (parasymNs) belong to the autonomic nervous system (ANS) and are critical for unconscious body responses, including rest-and-digest and calming the body. ParasymN dysfunction has been found involved in neural diseases such as autonomic neuropathy and neurological autoimmune disease; and parasymN innervation is important for organ development. However, human parasymN function and dysfunction is vastly understudied, due to the lack of a model system. Human pluripotent stem cell (hPSC)-derived neurons can fill this void and serve for disease modeling, drug screening, and transplantation therapy. Here, we developed a differentiation paradigm detailing for the first time, the derivation of functional human parasymNs from Schwann cell progenitors (SCP). We employ these neurons (i) to assess human ANS development paradigms, (ii) to model neuropathy in the genetic disorder Familial Dysautonomia (FD), (iii) to show parasymN dysfunction during SARS-CoV-2 infection, (iv) to model the autoimmune disease Sjörgen’s syndrome and, (v) to show that parasymN innervation of white adipocytes during development and help mature the tissue. Our model system will become instrumental for future disease mechanistic and drug discovery studies as well as for human developmental studies. Overall design: To understand the development of parasympathetic neurons, their function, and their missregulation in diseases, we developed a protocol to differentiate parasympathetic neurons from human pluripotent stem cells. To characterize their transcriptome at different developmental stages, we collected RNA on different days throuhgout the differentiation (day 10, 14, 16, and 30) from three biological replicates (independent differentiations). We also collected RNA from parasympathetic neurons (day 30) differentiated from iPSCs dervied from one Familial dysautonomia patient (S2). We performed RNAseq of each sample. To characterize their cellular composition, we collected single cell RNA data on day 30 from two independent differentiations (biological replicates).

副交感神经元（parasympathetic neurons, parasymNs）隶属于自主神经系统（autonomic nervous system, ANS），对机体无意识生理应答至关重要，涵盖“休息-消化”状态调控与机体舒缓功能。研究表明，副交感神经元功能异常与自主神经病、神经性自身免疫病等多种神经疾病密切相关，且副交感神经支配对器官发育具有关键意义。然而，由于缺乏适配的模型系统，目前针对人类副交感神经元的功能与功能异常的研究仍极度匮乏。人类多能干细胞（human pluripotent stem cell, hPSC）诱导分化的神经元可填补这一研究空白，可用于疾病建模、药物筛选与移植治疗。

本研究首次构建了一套分化范式，实现了从雪旺细胞前体（Schwann cell progenitors, SCP）向具有功能的人类副交感神经元的定向诱导分化。我们利用该神经元模型开展了五项研究：其一，解析人类自主神经系统的发育范式；其二，针对遗传性疾病家族性自主神经失调症（Familial Dysautonomia, FD）构建神经病变模型；其三，揭示严重急性呼吸综合征冠状病毒2（SARS-CoV-2）感染过程中的副交感神经元功能异常；其四，建立干燥综合征（Sjögren’s syndrome）的自身免疫病研究模型；其五，证实发育过程中白色脂肪组织的副交感神经支配可促进该组织成熟。本模型系统将为后续疾病机制研究、药物发现以及人类发育生物学研究提供重要工具。

整体实验设计：为阐明副交感神经元的发育过程、生理功能及其在疾病中的异常调控机制，我们建立了一套从人类多能干细胞诱导分化副交感神经元的标准化实验方案。为表征其不同发育阶段的转录组特征，我们在分化进程的多个时间节点（第10、14、16和30天）收集了3份生物学重复样本（独立分化批次）的总RNA。此外，我们还收集了1份来自家族性自主神经失调症患者（S2）的诱导多能干细胞（induced pluripotent stem cells, iPSCs）诱导分化得到的第30天副交感神经元的总RNA。对所有样本均进行了RNA测序（RNAseq）。为表征其细胞组成特征，我们从2份独立分化批次的第30天样本中获取了单细胞RNA测序数据。