Table_4_Secretome Analysis Performed During in vitro Cardiac Differentiation: Discovering the Cardiac Microenvironment.XLSX

Human pluripotent stem cells are an important tool for the study of developmental processes, such as cardiomyogenic differentiation. Despite the advances made in this field, the molecular and cellular signals involved in the commitment of embryonic stem cells to the cardiac phenotype are still under investigation. Therefore, this study focuses on identifying the extracellular signals involved in in vitro cardiac differentiation of human embryonic stem cells. Using a three-dimensional cardiomyogenic differentiation protocol, the conditioned medium and the extracellular matrix (ECM) of embryoid body cultures were collected and characterized at four specific time points. Mass spectrometry (MS) and antibody array analysis of the secretome identified a number of secreted proteins related to signaling pathways, such as Wnt and TGFβ, as well as many ECM proteins. When comparing the proteins identified at selected time points, our data pointed out protein interactions and biological process related to cardiac differentiation. Interestingly, the great changes in secretome profile occurred during the cardiac progenitor specification. The secretome results were also compared with our previous RNAseq data, indicating that the secreted proteins undergo some level of gene regulation. During cardiac commitment it was observed an increase in complexity of the ECM, and some proteins as IGFBP7, FN1, HSPG2, as well as other members of the basal lamina could be highlighted. Thus, these findings contribute valuable information about essential microenvironmental signals working on cardiomyogenic differentiation that may be used in future strategies for cardiac differentiation, cardiomyocyte maturation, and in advances for future acellular therapies.

人类多能干细胞（human pluripotent stem cells）是研究发育过程（例如心肌细胞分化）的重要工具。尽管该领域已取得诸多进展，但胚胎干细胞向心肌表型定型过程中涉及的分子与细胞信号通路仍有待深入研究。因此，本研究旨在鉴定人类胚胎干细胞体外心肌分化过程中相关的胞外信号。本研究采用三维心肌分化方案，在四个特定时间点收集拟胚体培养物的条件培养基与细胞外基质（extracellular matrix, ECM）并对其进行表征。通过对分泌组进行质谱（mass spectrometry, MS）分析与抗体芯片分析，本研究鉴定出多个与Wnt、TGFβ等信号通路相关的分泌蛋白，以及大量ECM蛋白。通过对比不同时间点鉴定出的蛋白，本研究数据揭示了与心肌分化相关的蛋白互作网络与生物学过程。值得注意的是，分泌组谱的显著变化发生在心肌祖细胞定型阶段。本研究还将分泌组结果与此前的RNA测序（RNAseq）数据进行比对，结果表明分泌蛋白存在一定程度的基因表达调控。在心肌细胞定型过程中，ECM的复杂性显著提升，IGFBP7、FN1、HSPG2等蛋白以及基膜的其他成员均表现出显著差异。综上，本研究结果为心肌分化过程中发挥关键作用的微环境信号提供了宝贵信息，这些信号有望应用于未来的心肌分化、心肌细胞成熟策略以及无细胞治疗相关研究进展中。