Recording History: A Lineage Tracing Molecular Recorder for Human Induced Pluripotent Stem Cell Gastruloids

Abstract

Mammalian development is a complex process with poorly defined differentiation pathways beyond some of the earliest embryonic stages. Technological advancements in genome engineering and single cell genomics have revitalized the subfield of lineage tracing due to the increased ability of such models to map developmental trajectories. However, studies of human development are limited due to ethical guidelines and available technologies that can be implemented within a human system. While progress has been made in the field of lineage tracing, many challenges persist delaying implementation for human developmental studies. Here I develop modifications for a Cas9-based lineage tracing technology, based on Chan, Smith et al.’s molecular recorder, to be created in a human induced pluripotent stem cell (hiPSC) gastruloid. The study has two arms focusing on the creation of the tracer cell line and gastruloid generation, respectively. I have worked on the integration of a Cas9 system into hiPSCs that edits at predesigned target sites to introduce stochastic indel mutations. Through nucleofection I have performed proof of principle experimentation for the knockout of TP53, which poses a survival barrier for a tracer hiPSC line. The gastruloid generation protocol utilizes chemically induced aggregation and differentiation to simulate embryogenesis. This will create a promising platform to study developmental trajectories in human development previously limited in research and highlight genes of interest to be examined for future studies.