Single cell RNA sequencing of in vitro differentiation NMP protocols. Single cell RNA sequencing of in vitro differentiation NMP protocols

The mammalian embryos Caudal Lateral Epiblast (CLE) harbours bipotent progenitors, called Neural Mesodermal Progenitors (NMPs), that contribute to the spinal cord and the paraxial mesoderm throughout axial elongation. Here we performed a single cell analysis of different in vitro NMPs populations produced either from embryonic stem cells (ESCs) or epiblast stem cells (EpiSCs) and compared them to E8.25 CLE mouse embryos. In our analysis of this region our findings challenge the notion that NMPs can be defined by the exclusive coexpression of Sox2 and T at mRNA level. We analyse the in vitro NMP-like populations using a purpose-built Support Vector Machine (SVM) based on the embryo CLE and use it as a classification model to compare the in vivo and in vitro populations. Our results show that NMP differentiation from ESCs, leads to heterogeneous progenitor populations with few NMP-like cells, as defined by the SVM algorithm, whereas starting with EpiSCs, yields a high proportion of cells with the embryo NMP signature. We find that the population from which the Epi-NMPs are derived in culture contains a node-like population, which leads to suggest that this population probably maintains the expression of T in vitro and thereby a source of NMPs. In conclusion, differentiation of EpiSCs into NMPs reproduces events in vivo and suggests a sequence of events for the emergence of the NMPs population. Overall design: We performed single cell RNA sequencing (RNA-seq) for different in vitro NMPs populations produced either from ESCs or EpiSCs: ES-NMP, Epi-CE as well as of the T expressing cells from the Epi-CE population (Epi-CE-T) and Epi-NMP. 10x Genomics single cell transcriptomic service was used to sequence our 4 samples.

哺乳动物胚胎尾侧上胚层（Caudal Lateral Epiblast, CLE）蕴藏着一类双潜能祖细胞——神经中胚层祖细胞（Neural Mesodermal Progenitors, NMPs），其在轴向伸长过程中可分化为脊髓与轴旁中胚层。本研究对由胚胎干细胞（Embryonic Stem Cells, ESCs）或上胚层干细胞（Epiblast Stem Cells, EpiSCs）诱导生成的多类体外NMP群体开展单细胞分析，并将其与E8.25期小鼠胚胎的CLE进行对照。针对该区域的分析结果，对“以Sox2与T在mRNA水平的排他性共表达作为NMPs定义标准”这一观点提出了质疑。研究人员基于胚胎CLE构建了专用的支持向量机（Support Vector Machine, SVM）分类模型，用以分析体外NMP样群体，并借助该模型对比体内与体外的细胞群体。研究结果显示，由胚胎干细胞诱导分化得到的NMPs会形成异质性祖细胞群体，其中符合SVM算法定义的NMP样细胞占比极低；而以上胚层干细胞为起始材料诱导得到的NMPs，则有高比例的细胞呈现胚胎NMP的特征转录谱。我们发现，培养体系中用于生成Epi-NMPs的初始群体含有原结样细胞群，据此推测该群体可在体外持续维持T基因的表达，进而成为NMPs的来源。综上，以上胚层干细胞诱导分化为NMPs的过程可重现体内相关发育事件，为揭示NMPs群体的形成时序提供了理论依据。整体实验设计：本研究对多类体外NMP群体开展了单细胞RNA测序（single cell RNA sequencing, RNA-seq），包括由ESCs诱导得到的ES-NMP、由EpiSCs诱导得到的Epi-CE群体，以及Epi-CE群体中表达T的细胞亚群（Epi-CE-T）与Epi-NMP。本次测序采用10x Genomics单细胞转录组测序服务，共完成4个样本的测序。