Pluripotent stem cell-derived organoid model of human gonad development, functions, and disorders

项目摘要 利用人多能干细胞（hPSC）的三维类器官技术是一种有价值的系统 十多年来，它被成功地用于研究器官发育和疾病模型。但标 模拟人类性腺发育和研究性别差异和性别障碍原因的策略 发育（DSD）和不孕症尚未确定。 我们开发了一种新颖、简单、基于生物反应器的类器官系统，用于模拟早期人类性腺 使用hPSC的开发。然而，产生的性腺类器官还没有被全面地 表征了在目标1中，我们将表征处于不同发育阶段的男性和女性人类性腺类器官。 发展我们将利用组合的“组学”方法，同时确定转录组， 和单细胞的表观基因组，并绘制它们在性腺类器官内的位置。这些数据将 使我们能够识别细胞亚群，我们将针对人类（和其他人）的相应阶段进行评估。 哺乳动物）胚胎和胎儿性腺发育的高分辨率。这些研究将为 用于在胚胎发生期间对雄性和雌性性腺发育进行建模的标准化类器官方案。 hPSC是在不同组织中工程化疾病表型的完美模型系统。我们会用我们的生殖腺 类器官模型来模拟DSD的实例。当所有的先天性发育不良都发生时，其发生率高达1：200至1：300。 考虑了异常情况。DSD可以由性染色体非整倍性或基因突变引起， 性别决定和睾丸或卵巢的发育。在目标2中，我们将使用CRISPR-Cas9来创建hPSC 携带SOX 9增强子突变，这些突变最近被发现会导致男性和女性的DSD。突变体 SOX 9增强子hSPC将用于在我们的基于生物反应器的类器官系统中产生类器官，性腺 我们会评估发展情况，并将结果与目标1的研究结果作比较。 我们将扩大我们的性腺类器官的研究，以评估生殖细胞的发展。迄今为止，人类原始生殖细胞 细胞样细胞（PGCLC）可以在体外成功地从hPSC中产生，我们已经在我们的研究中概括了这一点。 实验室然而，减数分裂感受态细胞和受精感受态配子的产生仍然是一个问题。 挑战.在目标3中，我们将检验hPSC衍生的性腺类器官将作为最佳的生殖系统的假设。 PGCLC经历定殖、分化并获得经历 配子发生我们将使用优化的标准程序和“组学”方法来表征这些 将含有PGCLC的类器官与从Aim 1获得的数据以及使用PGCLC获得的数据进行比较。 人类胚胎性腺 我们的研究目标的成功将导致睾丸和卵巢类器官系统的建立 可用于诊断分析、毒性和药物筛选、避孕测试、疾病建模， 以及辅助生殖。

Project Summary Three-dimensional organoid technology, utilizing human pluripotent stem cells (hPSCs), is a valuable system successfully utilized for over a decade to investigate organ development and model diseases. However, standard strategies for modeling human gonad development and studying causes of differences and disorders of sex development (DSD) and infertility are not established. We have developed a novel, simple, bioreactor-based organoid system for modeling early human gonad development using hPSCs. However, generated gonad organoids have not been comprehensively characterized. In Aim 1 we will characterize the male and female human gonad organoids at different stages of development. We will utilize combined “omics” approaches that will concomitantly determine the transcriptome and epigenome of single cells and map their position within the gonad organoid. Combined, these data will enable us to identify cell sub-populations that we will evaluate against corresponding stages of human (and other mammals) embryonic and fetal gonad development at high resolution. These studies will lay the foundation of standardized organoid protocols for modeling male and female gonad development during embryogenesis. hPSCs are a perfect model system to engineer diseased phenotypes in diverse tissues. We will use our gonad organoid models to mimic examples of DSD. DSD occur at a ratio as high as 1:200 to 1:300 when all congenital anomalies are considered. DSD can arise from sex chromosome aneuploidy or mutations in genes required for sex determination and development of the testis or ovary. In Aim 2, we will use CRISPR-Cas9 to create hPSCs that harbor SOX9 enhancer mutations that were recently discovered to cause DSD in males and females. Mutant SOX9 enhancer hSPCs will be used to create organoids in our bioreactor-based organoid system, gonad development will be assessed, and results will be compared to those obtained from our studies in Aim 1. We will extend our gonad organoid studies to assess germ cell development. To date, human primordial germ cell-like cells (PGCLCs) can be successfully generated from hPSCs in vitro, which we have recapitulated in our lab. However, the production of meiosis-competent cells and fertilization-competent gametes remains a challenge. In Aim 3, we will test the hypothesis that hPSC-derived gonad organoids will serve as an optimal environment for PGCLCs to undergo colonization, differentiation, and gain the capacity to undergo gametogenesis. We will use optimized standard procedures and “omics” approaches to characterize these PGCLC-containing organoids and compare them to data obtained from Aim 1, as well as data obtained using human embryonic gonads. The success of our research objectives will lead to the establishment of testis and ovary organoid systems that can be used for diagnostic assays, toxicity and drug screening, contraception testing, disease modeling, as well as assisted reproduction.