Stem Cell-Based Models of Human Amniogenesis and Primordial Germ Cell Specification

Abstract

Specification of primordial germ cells (PGCs), the embryonic precursors of sperm and eggs, is a critical step in human embryogenesis and reproductive health. PGCs transmit genetic and epigenetic information across generations and are the origin of new individuals and the driving force for genetic diversity and evolution. Germline defects underlie different human diseases, most notably infertility[1]. Therefore, understanding the development of human PGCs (hPGCs) has important implications for reproductive medicine and human development. Despite its fundamental and clinical importance, the development of PGCs in humans remains mysterious due to the scarcity and restricted availability of in vivo human embryo specimens and a lack of in vitro models. In this dissertation, I leverage the developmental potential and self-organizing property of human pluripotent stem cells (hPSCs) in conjunction with 2D and 3D bioengineering tools to generate an amniogenic gel-3D culture system for the derivation of human PGC-like cells (hPGCLCs). In the first section, I demonstrate an efficient method for hPGCLC derivation in a synthetic bioengineered environment mimicking the posterior end of the primate embryonic sac. Within a simple, homogeneous 3D Geltrex overlay condition, hPSCs cluster and undergo lumenogenesis and amniogenesis to form an embryonic-like sac, with hPGCLCs emerging spontaneously during this process. Our co-culture experiments suggest that amniotic ectoderm-like cells induce hPGCLC specification from hPSCs. Importantly, using this synthetic system, we, for the first time, reveal a critical role of ISL1, a very early amnion marker in hPGCLC specification using ISL1-knockout hPSC lines. Given its simplicity, high efficiency, and in vivo-like environment, our biomimetic method provides a novel and convenient strategy for the derivation of hPGCLCs and advancement of human embryology and reproductive biology. In the second section I present a novel amnion microtissue array platform that allows for quantitative phenotyping of lumenogenesis and amniogenesis of the epiblast and I demonstrate its potential application for embryonic toxicity profiling. Further, I describe a computer-assisted analysis pipeline that enhances the high-throughput potential of the system by automatically processing imaging data and quantifying morphological and biological features of amnion microtissues. Together, the findings in this dissertation will resonate with a significant number of stem cell biologists, developmental biologists, bioengineers, biomaterial scientists, and clinicians who seek to advance personalized iPSC therapies and reproductive and regenerative medicine.