Yan Haoni, Abdulla Aynur, Wang Aiting, Ding Shuyu, Zhang Manlin, Zhang Yizhi, Zhuang Tsz Yui, Wu Leqi, Wang Yan, Ren Rongrong, Jiang Lai, Ding Xianting
Department of Anesthesiology and Surgical Intensive Care Unit, Xinhua Hospital, School of Medicine and School of Biomedical Engineering, Shanghai Jiao Tong University, Shanghai, 200030, P. R. China.
State Key Laboratory of Oncogenes and Related Genes, Institute for Personalized Medicine, Shanghai Jiao Tong University, Shanghai, 200030, P. R. China.
Adv Sci (Weinh). 2025 Jan;12(2):e2406509. doi: 10.1002/advs.202406509. Epub 2024 Nov 21.
Proteomic communications in neighboring microenvironments during early organ development is a dynamic process that continuously reshapes human embryonic stem cells (hESCs) developmental fate. Such dynamic proteomic alteration in the microenvironment consists of both freely secreted proteome and exosome-encapsulated proteome. Simultaneous monitoring of the time-lapse shift of both proteomes with live organoids remains technically challenging. Here, a continuous organoid secretion/encapsulation proteome tandem LC-MS/MS (COSEP-LCM) is introduced, which permits time-lapse monitoring of proteomic alterations both in free secretion form and in exosome encapsulated form at live organoids' microenvironment. Continuous growth of human cerebral organoids (COs) and free-secretion/exosome-encapsulation proteomics acquisition with COSEP-LCM for 60 days is demonstrated. SERPINF1, F5, and EFNB1 are initially enriched inside exosomes as encapsulated excretion and then gradually enriched outside exosomes as freely secreted excretion, while C3 is initially enriched outside exosomes as freely secreted excretion and gradually enriched inside exosomes as encapsulated excretion. Such dynamic excretion pattern paradigm shift may imply critical developmental strategy evolution during early human cerebral development. COSEP-LCM offers a platform technique for continuous inside/outside exosome proteomics co-analysis in live organoids' microenvironment.
早期器官发育过程中邻近微环境中的蛋白质组通讯是一个动态过程，不断重塑人类胚胎干细胞（hESCs）的发育命运。微环境中这种动态蛋白质组改变包括自由分泌的蛋白质组和外泌体包裹的蛋白质组。使用活类器官同时监测这两种蛋白质组的延时变化在技术上仍然具有挑战性。在此，我们介绍了一种连续类器官分泌/包裹蛋白质组串联液相色谱-质谱/质谱（COSEP-LCM）方法，它能够对活类器官微环境中自由分泌形式和外泌体包裹形式的蛋白质组变化进行延时监测。我们展示了人类脑类器官（COs）的连续生长以及使用COSEP-LCM进行60天的自由分泌/外泌体包裹蛋白质组学采集。丝氨酸蛋白酶抑制剂F1（SERPINF1）、凝血因子V（F5）和表皮生长因子受体结合蛋白1（EFNB1）最初作为包裹性排泄物在外泌体内富集，然后逐渐作为自由分泌排泄物在外泌体之外富集，而补体C3最初作为自由分泌排泄物在外泌体之外富集，随后逐渐作为包裹性排泄物在外泌体内富集。这种动态排泄模式的范式转变可能意味着人类早期脑发育过程中关键发育策略的演变。COSEP-LCM为在活类器官微环境中连续进行外泌体蛋白质组学的内外联合分析提供了一种平台技术。
