Pluripotent Stem Cell-derived Liver Organoids as a Patient-based New Model for Deliverable Assessment of GH Insensitivity Syndrome

项目摘要 循环胰岛素样生长因子（IGF）-I，对人类发育，生长和发育的所有阶段都至关重要。 IGF-I主要由肝脏产生，出生后肝脏产生IGF-I的比例很高。 依赖于垂体分泌的生长激素（GH）。肝脏人GH-IGF-I轴对于 正常的人类生长突出了GH下游的多种遗传缺陷，在儿童中， 严重生长障碍（身高低于平均值-3至-10标准差）、GH不敏感（GHI）和 IGF-I缺乏症（IGFD）。STAT 5 B（信号转导和激活因子）罕见突变的鉴定 转录5 B）在儿童GHI和IGFD，连同超过80个报告的GHR（GH受体） 受影响儿童的突变，坚定地确立了GHR-STAT 5 B途径对GH- IGF-I产生的诱导调节。然而，令人不安的是，>60%的GHI，特发性矮小儿童， 没有携带GHR或STAT 5 B缺陷，但IGF-I缺乏，表明对IGF-I的理解仍然有限。 肝GHR-STAT 5 B-IGF-I生长轴和目前模型系统显然是不够的。 我们建议建立一个新的基于患者的GHI和IGFD可交付评估模型， 基于新兴的人类诱导多能干细胞（iPSC）衍生的类器官（“迷你器官”）， 为疾病调查和精准医疗提供有前景的“活的复制品”模型。我们假设人类 对GH提示的肝反应可以在患者iPSC衍生的肝类器官中重现。这个人类 的模型提供了独特的机会来探测人类肝脏GHR-STAT 5 B依赖性和非依赖性 患者特异性类器官中的基因调控机制，并跟踪发育中的 肝脏类器官激素线索，平行于子宫内和产后发育过程。具体 目标将利用我们独特的一组特征化的患者原代细胞来评估分子特征， iPSC衍生的类器官对GH线索的敏感性和功能性，以建立第一个可行的人类 基于人肝GH-IGF-I生长轴机制研究的模型。具体目标将 (1)评估人iPSC衍生的肝类器官中GHR-STAT 5 B-IGF-I轴的发育表达 模型;（2）描述肝脏STAT 5 B依赖性和非依赖性GH调节基因表达， 人iPSC衍生的肝类器官。在这些研究结束时，我们将建立和 证明了患者特异性的iPSC衍生的肝类器官系统用于评估功能性肝细胞的效用。 遗传缺陷导致GHI和IGFD。获得的新的机械见解包括改进的 了解人类肝脏GHR-STAT 5 B介导的IGF-I表达调节， 鉴定人GH反应性的新标记物。从长远来看，这些新的基于患者的模型 为未来的个性化治疗测试提供平台，以改善患者管理和护理 受影响的儿童，并为未来评估肝脏对其他激素线索的反应。

PROJECT SUMMARY Circulating insulin-like growth factor (IGF)-I, critical for all phases of human development, growth, and maturation, is produced predominantly by the liver, with post-natal hepatic production of IGF-I highly dependent on pituitary-secreted growth hormone (GH). The importance of the hepatic human GH-IGF-I axis for normal human growth is highlighted by the multiple genetic defects downstream of GH, in children who present with severe growth failure (height -3 to -10 standard deviation below the mean), GH insensitivity (GHI) and IGF-I deficiency (IGFD). The identification of rare mutations of STAT5B (signal transducer and activator of transcription 5B) in children with GHI and IGFD, together with the more than 80 reported GHR (GH receptor) mutations in affected children, firmly established the critical importance of the GHR-STAT5B pathway for GH- induced regulation of IGF-I production. However, a disturbing >60% of GHI, idiopathic short statured children, do not carry GHR or STAT5B defects, yet are IGF-I deficient, indicating still limited understanding of the hepatic GHR-STAT5B-IGF-I growth axis and that current model systems are clearly insufficient. We propose establishing a new patient-based model for deliverable assessment of GHI and IGFD, based on emerging human induced-pluripotent stem cells (iPSC)-derived organoids (“mini-organs”), which are promising “living replica” models for disease investigation and precision medicine. We hypothesize that human hepatic response to GH cues can be recapitulated in patient iPSC-derived liver organoids. This human- based model offers unique opportunities to probe human hepatic GHR-STAT5B dependent and independent mechanisms of gene regulation in patient-specific organoids and to follow responsiveness of the developing liver organoid to hormonal cues, paralleling in utero and post-natal developmental processes. The Specific Aims will leverage our unique panel of characterized, patient primary cells to assess molecular signatures, sensitivity, and functionality of iPSC-derived organoids to GH cues, towards establishing the first viable human based models for mechanistic investigation of the human hepatic GH-IGF-I growth axis. The Specific Aims will (1) assess the developmental expression of the GHR-STAT5B-IGF-I axis in human iPSC-derived liver organoid model; and (2) delineate hepatic STAT5B-dependent and independent GH-regulated gene expression in human iPSC-derived liver organoids. At the conclusion of these studies we will have established and demonstrated the utility of a patient-specific, iPSC-derived liver organoid system for evaluating functional disruptions by genetic defects leading to GHI and IGFD. New mechanistic insights gained include improved understanding of human, hepatic GHR-STAT5B mediated regulation of IGF-I expression, with potential for identifying new markers of human GH responsiveness. In the long term, these novel patient-based models provide a platform for future personalized therapeutic testing, towards improving patient management and care of affected children, and for future evaluation of liver responsiveness to other hormonal cues.