Laboratory of Developmental Biology, Faculty of Biochemistry and Molecular Medicine, University of Oulu, 90220, Oulu, Finland.
Infotech Oulu, University of Oulu, 90014, Oulu, Finland.
Cell Commun Signal. 2023 Dec 18;21(1):358. doi: 10.1186/s12964-023-01374-z.
During kidney organogenesis, metanephric mesenchyme (MM) and ureteric bud (UB) interact reciprocally to form nephrons. Signaling stimuli involved in these interactions include Wnts, growth factors and nano/micro particles. How UB and MM are interacting is not completely understood. Our study investigated the signaling and communication via extracellular vesicles (EVs) during nephrogenesis. Embryonic day (E) 11.5 mouse kidney UB and MM produce very low number of primary cells that have limited ability for proliferation in culture. Such limitations obstruct studying the role of EVs in induction of nephrogenesis. These issues necessitate to generate a nephrogenesis model allowing to study the comprehensive role of EVs during nephrogenesis.
Our study generated a UB derived cell line-based in vitro flexible model of nephrogenesis allowing expandable cell culturing, in addition to performing characterization, tracking and blocking of EVs. UB cell line aggregation with E11.5 MM cells induced the formation of segmented nephrons. Most efficient nephrogenesis was obtained by the co-culturing of 30,000 cells of UB cell line with 50,000 MM cells. Results revealed that both the UB and the MM secrete EVs during nephrogenesis. UB cell line derived EVs were characterized by their size, morphology and expression of markers (CD63, TSG101, CD9 and CD81). Furthermore, proteomics data of UB cell line-derived EVs revealed large number of proteins involved in nephrogenesis-related signaling pathways. Palmitoylated GFP-tagged EVs from UB cell line were found in the nephron formation zone in the developing kidney organoid. UB cell line derived EVs did not induce nephrogenesis in MM cells but significantly contributed to the survival and nephrogenesis-competency of MM cells. The secretion of EVs was continuously inhibited during the ongoing nephrogenesis by the knockdown of RalA and RalB gene expression using short hairpin RNAs. This inhibition partially impaired the ability of UB cell line to induce nephrogenesis. Moreover, impaired nephrogenesis was partially rescued by the addition of EVs.
Our study established a novel in vitro flexible model of nephrogenesis that solved the limitations of primary embryonic kidney cells and mouse embryonic stem cell kidney organoids for the EV research. EVs were found to be an integral part of nephrogenesis process. Video Abstract.
在肾脏器官发生过程中，后肾间充质（MM）和输尿管芽（UB）相互作用形成肾单位。参与这些相互作用的信号刺激物包括 Wnts、生长因子和纳米/微颗粒。UB 和 MM 是如何相互作用的尚不完全清楚。我们的研究调查了肾发生过程中外泌体（EVs）的信号和通讯。胚胎第 11.5 天（E）的小鼠肾脏 UB 和 MM 产生的初级细胞数量非常少，在培养中增殖能力有限。这些局限性阻碍了对 EVs 在诱导肾发生中的作用的研究。这些问题需要生成一个允许研究 EVs 在肾发生过程中的全面作用的肾发生模型。
我们的研究生成了一种基于 UB 衍生细胞系的体外灵活的肾发生模型，允许可扩展的细胞培养，此外还进行了 EVs 的表征、跟踪和阻断。UB 细胞系与 E11.5 MM 细胞的聚集诱导了分段肾单位的形成。通过共培养 30000 个 UB 细胞系细胞和 50000 个 MM 细胞获得了最有效的肾发生。结果表明，UB 和 MM 在肾发生过程中都分泌 EVs。UB 细胞系衍生的 EVs 的特征在于其大小、形态和标记物（CD63、TSG101、CD9 和 CD81）的表达。此外，UB 细胞系衍生的 EVs 的蛋白质组学数据显示了大量参与肾发生相关信号通路的蛋白质。在发育中的肾脏类器官的肾单位形成区发现了来自 UB 细胞系的棕榈酰化 GFP 标记的 EVs。UB 细胞系衍生的 EVs 不会诱导 MM 细胞中的肾发生，但会显著促进 MM 细胞的存活和肾发生能力。通过短发夹 RNA 敲低 RalA 和 RalB 基因表达，在持续进行的肾发生过程中持续抑制 EVs 的分泌。这种抑制部分削弱了 UB 细胞系诱导肾发生的能力。此外，通过添加 EVs 部分挽救了受损的肾发生。
我们的研究建立了一种新的体外灵活的肾发生模型，解决了原代胚胎肾细胞和小鼠胚胎干细胞肾类器官在 EV 研究中的局限性。发现 EVs 是肾发生过程的一个组成部分。
