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

人类性腺发育、功能和疾病的多能干细胞衍生类器官模型

基本信息

批准号：
10816755
负责人：
Philip W Jordan
金额：
$ 58.41万
依托单位：
HENRY M. JACKSON FDN FOR THE ADV MIL/MED
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-09-18 至 2026-08-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10816755
关键词：
3-Dimensional Biological Models Bioreactors Brain CRISPR/Cas technology Cell Death Cells Chicken Model Chickens Circulation Coculture Techniques Contraceptive methods DNA Sequence Alteration Data Development Diagnosis Diagnostic Diameter Diffusion Disease Disease model Drug Screening Drug Side Effects Embryo Embryonic Development Engineered Gene Engineering Enhancers Environment Evaluation FOXL2 gene Female Fertilization Foundations Gametogenesis Generations Genes Germ Cells Gonadal structure Human Immunodeficient Mouse In Vitro Incidence Infertility Investigation Knowledge Mammals Maps Meiosis Methods Microscopy Modeling Molecular Monkeys Mus Mutation Nutrient Organ Model Organoids Ovary Oxygen Patients Pattern Pluripotent Stem Cells Positioning Attribute Procedures Production Protocols documentation Publishing Quantitative Reverse Transcriptase PCR Regenerative Medicine Reproductive system Research Resolution SF1 Sexual Development Slice Somatic Cell Specific qualifier value Standardization Structure of primordial sex cell System Techniques Technology Testing Testis Tissues Toxic effect Toxicology Turner&apos s Syndrome WT1 gene Western Blotting Xenograft procedure assisted reproduction cell motility congenital anomaly diagnostic assay disease phenotype embryonic stem cell epigenome experimental study fetal gonad development human female human male human model human pluripotent stem cell male model organism mouse model mutant novel offspring organ growth postnatal pre-clinical reproductive reproductive development sex sex chromosome aneuploidy sex determination sex development disorder stem cells success tool transcription factor transcriptome transcriptomics

项目摘要

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.

项目摘要使用人类多能干细胞（HPSC）的三维器官技术是一个有价值的系统成功使用了十多年来，以研究器官发育和模型疾病。但是，标准建模人类性腺发展和研究差异和性疾病的原因的策略未建立发展（DSD）和不育。我们已经开发了一种新颖的，简单的基于生物反应器的器官系统，用于建模早期人为性腺使用HPSC的开发。但是，产生的性腺类器官尚未全面特征。在AIM 1中，我们将在不同阶段的不同阶段表征男性和女性人类性腺类器官发展。我们将利用将同时确定转录组的组合“ OMICS”方法和单细胞的表观基因组，并绘制其在性腺类器官中的位置。这些数据结合在一起使我们能够确定我们将针对人类的相应阶段进行评估的细胞子群（以及其他哺乳动物）高分辨率的胚胎和胎儿性腺发育。这些研究将奠定用于在胚胎发生过程中建模男性和雌性性腺发育的标准器官方案。 HPSC是设计多样性组织中解剖表型的理想模型系统。我们将使用我们的性腺器官模型以模拟DSD的示例。当所有先天性时，DSD的比率高达1：200至1：300 考虑异常。 DSD可能是由性染色体非整倍性或基因中的突变引起的睾丸或卵巢的性别确定和发展。在AIM 2中，我们将使用CRISPR-CAS9创建HPSC 该携带Sox9增强子突变是最近被发现引起男性和女性DSD的。突变体 Sox9增强子HSPC将用于在基于生物反应器的器官系统中创建类器官将评估开发，并将结果与AIM 1中从我们的研究中获得的结果进行比较。我们将扩展性腺器官研究以评估生殖细胞发育。迄今为止，人类原始细菌细胞样细胞（PGCLC）可以在体外成功地从HPSC中产生，我们在我们的体外概括了实验室。但是，减数分裂能力的细胞和受精的游戏的产生仍然是一个挑战。在AIM 3中，我们将检验以下假设：HPSC衍生的性腺类器官将是最佳 PGCLC进行殖民化，分化和获得能力进行的环境配子发生。我们将使用优化的标准程序和“ OMIC”方法来表征这些含PGCLC的类器官，并将其与从AIM 1获得的数据进行比较，以及使用的数据人类性腺。我们的研究目标的成功将导致建立睾丸和卵巢器官系统可用于诊断测定，毒性和药物筛查，避孕测试，疾病建模，作为以及辅助生殖。