Rolando Carrisoza-Gaytán, Abigail Daily, Kayla J Wolf, Samia Lasaad, Anna Cantalupo, Pooja Nair, Ronald Van Gaal, Per Uhlén, Jennifer A Lewis, Lisa M Satlin
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Comparison of cells in tubules microdissected from UB and CD organoids cultured for 34-35 days (d) or 62-65 d showed an: (i) increased [Ca<sup>2+</sup>]<sub>i</sub> response to basolateral application of the selective PIEZO1 agonist Yoda1 and (ii) decreased time to peak [Ca<sup>2+</sup>]<sub>i</sub> with advancing days in culture. Single cell analyses of the Yoda1-induced [Ca<sup>2+</sup>]<sub>i</sub> response revealed 7-15 mHz [Ca<sup>2+</sup>]<sub>i</sub> oscillations that were more prevalent with advancing days in culture and differentiation (CD vs. UB). Concurrent exposure to inhibitors of the sarcoplasmic/endoplasmic reticulum Ca<sup>2+</sup>-ATPase (SERCA) or the plasma membrane Ca<sup>2+</sup>-ATPase (PMCA) dampened the amplitude of the Yoda1-induced [Ca<sup>2+</sup>]<sub>i</sub> oscillations. Bulk RNA analysis and pathway enrichment analysis revealed broad changes in genes associated with Ca<sup>2+</sup> signaling, but not <i>PIEZO1</i>, with advancing days in culture and differentiation. These findings are consistent with a developmental increase in activity of PIEZO1 channels and/or maturation of associated pathways shaping Ca<sup>2+</sup> signaling dynamics in maturing UB and CD organoids. Decoding of [Ca<sup>2+</sup>]<sub>i</sub> oscillations may identify molecular mechanisms important in morphologic and functional differentiation of organoid tubules.</p>","PeriodicalId":7585,"journal":{"name":"American journal of physiology. 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Single cell analyses of the Yoda1-induced [Ca<sup>2+</sup>]<sub>i</sub> response revealed 7-15 mHz [Ca<sup>2+</sup>]<sub>i</sub> oscillations that were more prevalent with advancing days in culture and differentiation (CD vs. UB). Concurrent exposure to inhibitors of the sarcoplasmic/endoplasmic reticulum Ca<sup>2+</sup>-ATPase (SERCA) or the plasma membrane Ca<sup>2+</sup>-ATPase (PMCA) dampened the amplitude of the Yoda1-induced [Ca<sup>2+</sup>]<sub>i</sub> oscillations. Bulk RNA analysis and pathway enrichment analysis revealed broad changes in genes associated with Ca<sup>2+</sup> signaling, but not <i>PIEZO1</i>, with advancing days in culture and differentiation. These findings are consistent with a developmental increase in activity of PIEZO1 channels and/or maturation of associated pathways shaping Ca<sup>2+</sup> signaling dynamics in maturing UB and CD organoids. 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引用次数: 0
摘要
人类干细胞感知的机械信号通过离散的信号传导途径来调节发育表型和功能。从肾元谱系来源的类肾器官中分离的近端小管经历了基侧机械传感器PIEZO1和Ca2+信号转导途径的丰度和/或活性的发育增加(Carrisoza-Gaytán等)。AJP细胞物理,2023)。在这里,我们研究了人类ipsc衍生的输尿管芽(UB)和收集管(CD)类器官细胞是否在PIEZO1功能上表现出类似的发育增加。对培养34-35天(d)或62-65天的UB和CD类器官微解剖的细胞进行比较,发现:(i)选择性PIEZO1激动剂Yoda1的基底侧应用增加了[Ca2+]i的反应,(ii)随着培养天数的增加,[Ca2+]i达到峰值的时间减少。对yoda1诱导的[Ca2+]i响应的单细胞分析显示,随着培养和分化的进展,7-15 mHz [Ca2+]i振荡更为普遍(CD vs. UB)。同时暴露于肌浆/内质网Ca2+- atp酶(SERCA)或质膜Ca2+- atp酶(PMCA)抑制剂可抑制yoda1诱导的[Ca2+]i振荡的幅度。大量RNA分析和途径富集分析揭示了Ca2+信号相关基因的广泛变化,但不包括PIEZO1,随着培养和分化的推进。这些发现与在成熟的UB和CD类器官中形成Ca2+信号动力学的PIEZO1通道活性的发育增加和/或相关途径的成熟一致。解码[Ca2+]i振荡可以识别在类器官小管形态和功能分化中重要的分子机制。
Ca2+ signal dynamics in maturing ureteric bud (UB) and collecting duct (CD) derived organoid tubules.
Mechanical signals sensed by human stem cells are transduced via discrete signaling pathways to modulate developmental phenotype and function. Proximal tubules isolated from nephron lineage-derived kidney organoids undergo a developmental increase in abundance and/or activity of the basolateral mechanosensor PIEZO1 and Ca2+ signal transduction pathways (Carrisoza-Gaytán et al. AJP Cell Physiol, 2023). Here, we investigate whether human iPSC-derived ureteric bud (UB) and collecting duct (CD) organoid cells exhibit a similar developmental increase in PIEZO1 function. Comparison of cells in tubules microdissected from UB and CD organoids cultured for 34-35 days (d) or 62-65 d showed an: (i) increased [Ca2+]i response to basolateral application of the selective PIEZO1 agonist Yoda1 and (ii) decreased time to peak [Ca2+]i with advancing days in culture. Single cell analyses of the Yoda1-induced [Ca2+]i response revealed 7-15 mHz [Ca2+]i oscillations that were more prevalent with advancing days in culture and differentiation (CD vs. UB). Concurrent exposure to inhibitors of the sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA) or the plasma membrane Ca2+-ATPase (PMCA) dampened the amplitude of the Yoda1-induced [Ca2+]i oscillations. Bulk RNA analysis and pathway enrichment analysis revealed broad changes in genes associated with Ca2+ signaling, but not PIEZO1, with advancing days in culture and differentiation. These findings are consistent with a developmental increase in activity of PIEZO1 channels and/or maturation of associated pathways shaping Ca2+ signaling dynamics in maturing UB and CD organoids. Decoding of [Ca2+]i oscillations may identify molecular mechanisms important in morphologic and functional differentiation of organoid tubules.
期刊介绍:
The American Journal of Physiology-Cell Physiology is dedicated to innovative approaches to the study of cell and molecular physiology. Contributions that use cellular and molecular approaches to shed light on mechanisms of physiological control at higher levels of organization also appear regularly. Manuscripts dealing with the structure and function of cell membranes, contractile systems, cellular organelles, and membrane channels, transporters, and pumps are encouraged. Studies dealing with integrated regulation of cellular function, including mechanisms of signal transduction, development, gene expression, cell-to-cell interactions, and the cell physiology of pathophysiological states, are also eagerly sought. Interdisciplinary studies that apply the approaches of biochemistry, biophysics, molecular biology, morphology, and immunology to the determination of new principles in cell physiology are especially welcome.