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.
