Friday, May 9, 2025
5/9/2025
Modeling Chromosomal Mosaicism During Early Human Embryogenesis on Microraft Array Platform - PROJECT SUMMARY/ABSTRACT Aneuploidy and chromosomal mosaicism in human embryos complicate predicting pregnancy outcomes already plagued with frequent pregnancy losses. Given the rapid increase of fertility treatments such as 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. Both recent clinical and in vitro studies suggest 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. Here, I propose to investigate the role of autophagy, which regulates both cell survival and death, in aneuploidy depletion. Although previous work in mouse and human preimplantation models demonstrated autophagy upregulation in eliminated cell types, our preliminary data in a human gastrulation model revealed that autophagy instead aids in cell survival. 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 mosaic gastruloids—an in vitro multicellular model recapitulating cell fate and signaling during gastrulation—comprised of euploid and aneuploid human embryonic stem cells (hESCs). Analyzing single gastruloids is challenging by most current technologies like gene expression assays, 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 is divided into two main Aims. I will use an automated microscope system that I have developed to perform image-based phenotypic screens of single gastruloids using live reporters and a custom computational pipeline. Firstly, I will generate a mixed population of mosaic gastruloids and identify distinct phenotypes based on patterning behavior via clustering. Secondly, I will investigate autophagy-dependent aneuploidy depletion within the mosaic gastruloids by measuring autophagy markers (LC3B and p62) and p53- responsive genes (p53, p21, cyclin G1, bcl-2). The gastruloids will be treated with bafilomycin A1 or rapamycin to inhibit or enhance autophagy, respectively. I hypothesize that phenotypic abnormalities in mosaic human embryos are dependent on the degree of aneuploidy present, and that autophagy can prevent the elimination of aneuploid cells. The proposed project will enable future research to delineate other embryo self-correction mechanisms critical in overcoming error-prone development and to improve fertility treatments for an increasing number of patients.
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