Modeling Chromosomal Mosaicism During Early Human Embryogenesis on Microraft Platform

Abstract

Aneuploidy and chromosomal mosaicism in human embryos complicate predicting pregnancy outcomes already plagued with frequent pregnancy losses. Given the rapid increase of assisted reproductive technology (ART) treatments, there is a critical need to better assess the developmental potential of embryo candidates, especially in patients with poor prognoses. Emerging evidence suggests that human mosaic embryos can selectively eliminate aneuploid cells for healthy development; however, the mechanisms mediating this embryonic self-correction have yet to be systematically studied in humans. Because both technical and ethical limitations restrict studying cellular processes during early human embryogenesis, I will develop a novel platform to quantitatively screen and sort gastruloids—an in vitro multicellular model recapitulating cell fate and signaling during gastrulation—comprised of euploid and aneuploid human pluripotent stem cells (hPSCs). Analyzing single gastruloids is challenging by most current technologies, which only allow for low-throughput sorting or bulk analyses. Thus, new tools are required to systematically study the heterogeneity among gastruloids undergoing dramatic changes during self-organization. The project was divided into three Aims. Firstly, I developed an automated system to perform image-based screens of single gastruloids by isolating individual colonies for downstream analyses. Secondly, the emergence of gastruloid heterogeneity derived from multiple aliquots of a single cell line was quantified by coupling both phenotypic and transcriptomic information. Low-dimensional latent representations were created from imaging time-series using deep learning (DL) of developing gastruloids derived from hPSCs expressing a SOX2-mCitrine reporter to track SOX2 dynamics. Among single gastruloids across multiple experimental batches, simple-to-complex variability in patterning behavior were assessed. Endpoint RNA-sequencing (RNA-seq) data from individually isolated gastruloids also revealed distinct clusters associated with batch and subclusters within batches. Thirdly, I assessed the phenotypic and transcriptomic differences between euploid and mosaic (derived from 50% euploid and 50% aneuploid cells) gastruloids to demonstrate the potential of this platform for studying chromosomal mosaicism during early human embryogenesis. The platform will enable future research to elucidate mechanisms for aneuploidy depletion critical for overcoming error-prone development and to improve reproductive treatments for an increasing number of patients.