Small molecule screen reveals pathways that regulate C4 secretion in stem-cell derived astrocytes.

In the brain, the complement system plays a crucial role in the immune response and in synaptic elimination during normal development and disease. Here, we sought to identify pathways that modulate the production of complement component 4 (C4), recently associated with an increased risk of schizophrenia. To design a disease-relevant assay, we first developed a rapid and robust 3D protocol capable of producing large numbers of astrocytes from pluripotent cells. Transcriptional profiling of these astrocytes confirmed the homogeneity of this population of dorsal fetal-like astrocytes. Using a novel ELISA-based small molecule screen, we identified epigenetic regulators, as well as inhibitors of intracellular signaling pathways, able to modulate C4 secretion from astrocytes. We then built a Connectivity Map to predict and validate additional key regulatory pathways, including one involving c-Jun-kinase (JNK). This work provides a foundation for developing therapies for CNS diseases involving the complement cascade. Overall design: Biological replicates of astrocytes differentited from human stem-cells (IPSCs, 1016A, n=3 ,ESCs, Hues8 n=3 and H1 n=2) were analyzed using scRNAseq

在大脑中，补体系统（complement system）在免疫应答以及正常发育和疾病进程中的突触清除过程中发挥关键作用。本研究旨在鉴定调控补体成分4（complement component 4, C4）表达的通路——该成分近期被发现与精神分裂症发病风险升高相关。
为设计与疾病相关的实验检测体系，我们首先开发了一种快速且稳定的3D培养方案，可从多能干细胞中获取大量星形胶质细胞（astrocytes）。对这些星形胶质细胞的转录组分析证实，该细胞群体为均一的背侧胎儿样星形胶质细胞。
利用基于酶联免疫吸附实验（enzyme-linked immunosorbent assay, ELISA）的新型小分子筛选模型，我们鉴定出了能够调控星形胶质细胞分泌C4的表观遗传调控因子，以及细胞内信号通路抑制剂。随后我们构建了连接图谱（Connectivity Map），以预测并验证其他关键调控通路，其中包括涉及c-Jun激酶（c-Jun-kinase, JNK）的通路。
本研究为开发针对补体级联反应相关中枢神经系统（central nervous system, CNS）疾病的治疗手段奠定了基础。
实验设计概述：对源自人类干细胞的分化星形胶质细胞的生物学重复样本进行了分析，样本包括诱导多能干细胞（induced pluripotent stem cells, IPSCs）株1016A（n=3）、胚胎干细胞（embryonic stem cells, ESCs）株Hues8（n=3）及H1（n=2），并采用单细胞RNA测序（single-cell RNA sequencing, scRNAseq）完成检测。