A Systems-Level Characterization of the Differentiation of Human Embryonic Stem Cells into Mesenchymal Stem Cells

Mesenchymal stem/stromal cells (MSCs) are self-renewing multipotent cells with regenerative, secretory and immunomodulatory capabilities that are beneficial for the treatment of various diseases. To avoid the issues that come with using tissue-derived MSCs in therapy, MSCs may be generated by the differentiation of human embryonic stems cells (hESCs) in culture. However, the changes that occur during the differentiation process have not been comprehensively characterized. Here, we combined transcriptome, proteome and phosphoproteome profiling to perform an in-depth, multi-omics study of the hESCs-to-MSCs differentiation process. Based on RNA-to-protein correlation, we determined a set of high confidence genes that are important to differentiation. Among the earliest and strongest induced proteins with extensive differential phosphorylation was AHNAK, which we hypothesized to be a defining factor in MSC biology. We observed two distinct expression waves of developmental HOX genes and an AGO2-to-AGO3 switch in gene silencing. Exploring the kinetic of non-coding ORFs during differentiation, we mapped new functions to well annotated long non-coding RNAs (CARMN, MALAT, NEAT1, LINC00152) as well as new candidates which we identified to be important to the differentiation process. Phosphoproteome analysis revealed ESC and MSC-specific phosphorylation motifs with PAK2 and RAF1 as top predicted upstream kinases in MSCs. Our data represent a rich systems-level resource on ESC-to-MSC differentiation that will be useful for the study of stem cell biology.

间充质干/基质细胞（Mesenchymal stem/stromal cells, MSCs）是一类具有自我更新能力的多潜能细胞，具备再生、分泌及免疫调节功能，在多种疾病的治疗中具有潜在应用价值。为解决临床治疗中应用组织源性MSCs所面临的问题，可通过体外诱导人类胚胎干细胞（human embryonic stems cells, hESCs）定向分化获得MSCs。然而，该分化过程中发生的细胞分子变化尚未得到全面系统的表征。本研究整合转录组、蛋白质组及磷酸化蛋白质组谱分析技术，对hESCs向MSCs的分化进程开展了深入的多组学研究。基于RNA与蛋白质的相关性分析，我们筛选得到一批对分化进程至关重要的高置信度基因。在早期即被显著诱导且存在广泛差异磷酸化的蛋白质中，AHNAK是其中的关键成员之一，我们推测其为MSCs生物学特性的核心决定因子。研究中我们观察到发育同源盒（HOX）基因呈现两个截然不同的表达波峰，以及基因沉默通路中AGO2向AGO3的表达转换现象。通过探究分化进程中非编码开放阅读框（ORFs）的动力学特征，我们为已注释的长链非编码RNA（CARMN、MALAT、NEAT1、LINC00152）赋予了全新的功能，并鉴定出一批在分化过程中发挥关键调控作用的新型候选非编码RNA。磷酸化蛋白质组分析还揭示了胚胎干细胞（embryonic stem cells, ESC）与MSCs特异性的磷酸化基序，并预测PAK2与RAF1为MSCs中最主要的上游调控激酶。本研究产生的数据集为hESCs向MSCs的分化过程提供了丰富的系统级研究资源，将为干细胞生物学领域的相关研究提供重要的理论与数据支撑。