GSBS Dissertations and Theses

ORCID ID

0000-0002-3150-8400

Publication Date

2019-02-12

Document Type

Doctoral Dissertation

Academic Program

Interdisciplinary Graduate Program

Department

Program in Molecular Medicine

First Thesis Advisor

Rene Maehr

Keywords

dCas9-KRAB, human pluripotent stem cells, CRISPR, single-cell RNA-sequencing, thymus, development, endoderm, CRISPR-effector

Abstract

The definitive endoderm gives rise to several specialized organs, including the thymus. Improper development of the definite endoderm or its derivatives can lead to human disease; in the case of the thymus, immunodeficiency or autoimmune disorders. Human pluripotent stem cells (hPSCs) have emerged as a system to model human development, as study of their differentiation allows for elucidation of the molecular basis of cell fate decisions, under both healthy and impaired conditions. Here, we first developed a CRISPR-effector system to control endogenous gene expression in hPSCs, a novel approach to manipulating hPSC state. Next, the human-specific, loss-of-function phenotypes of candidate transcription factors driving hPSC-to-definitive endoderm differentiation were analyzed through combined CRISPR-perturbation and single-cell RNA-sequencing. This analysis revealed the importance of TGFβ mediators in human definitive endoderm differentiation as well as identified an unappreciated role for FOXA2 in human foregut development. Finally, as the differentiation of definitive endoderm to thymic epithelial progenitors (TEPs) is of particular interest, a single-cell transcriptomic atlas of murine thymus development was generated in anticipation of identifying factors driving later stages of TEP differentiation. Taken together, this dissertation establishes a CRISPR-effector system to interrogate gene and regulatory element function in hPSC differentiation strategies, details the role of specific transcription factors in human endoderm differentiation, and sets the groundwork for future investigations to characterize hPSC-derived TEPs and the factors driving their differentiation.

DOI

10.13028/js5y-5g70

Rights and Permissions

Copyright is held by the author, with all rights reserved.

Available for download on Saturday, March 20, 2021

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