Interrogating Organ-Specific Niche Factors in the Developing Human Intestine to Inform Organ Engineering

Abstract

The human intestine is a remarkable organ that performs diverse and necessary functions for survival including nutrient uptake, hormone production, and host immunity. Given the prevalence and severity of gastrointestinal diseases in human health, there has been great interest in developing in vitro models to enable the study of human intestinal development, disease, and regeneration. To address this need, methods have been developed to generate complex three-dimensional in vitro intestinal cultures (organoids) from primary human donor tissue or human pluripotent stem cells (hPSCs). These organoid cultures partially recapitulate the cellular diversity and tissue organization of the native human intestine. Research is now focused on advancing existing systems towards more faithfully recapitulating the native organ and utilizing intestinal organoids for biological discovery. In this dissertation, I present an in-depth characterization of in vivo human intestinal development from 7–21 weeks post conception and of corresponding organoid models using single cell mRNA-sequencing (scRNA-seq).

One focus of this dissertation was to characterize the developing human intestinal stem cell (ISC) niche—a specialized microenvironment that supports ISC survival and function. This work examined the expression and localization of intestinal stem cell (ISC) niche factors inferred from murine studies, including Epidermal Growth Factor (EGF), WNTs, and R-spondins, to determine their relevance during human intestinal development. Both molecular and spatial characterization of the in vivo human intestine revealed that EGF, a prominent niche factor in mice, is expressed outside of the ISC crypt niche during human development. However, the EGF ligand family member Neuregulin 1 (NRG1) is expressed in a subepithelial PDGFRAHI/F3HI/DLL1HI mesenchymal population that lines the crypt-villus axis of the developing human intestine. Exogenously supplied NRG1, but not EGF, improved cellular diversity in human epithelial organoids by permitting differentiation of secretory lineages. This work illuminates the complexities of intestinal EGF/ERBB signaling and delineates key niche cells and signals of the developing intestine.

Another focus of this thesis was to investigate the cellularity of hPSC-derived human intestinal organoids (HIOs), as HIOs lack several cellular populations found in the native organ, including vasculature. scRNA-seq of HIOs identified a population of endogenous endothelial cells (ECs) present early in HIO differentiation that declines over time in culture. In this thesis, I developed a method to expand and maintain this endogenous population of ECs within HIOs. Given that ECs possess organ-specific gene expression, morphology, and function, we used bulk RNA-seq and scRNA-seq to interrogate the developing human intestine, lung, and kidney to identify organ-enriched EC gene signatures. By comparing these gene signatures and validated markers of HIO ECs, I found that HIO ECs grown in vitro share the highest similarity with native intestinal ECs relative to kidney and lung. Together, these data demonstrate that HIOs can co-differentiate a native EC population that is partially patterned with an intestine-specific EC transcriptional signature in vitro.

Collectively, this thesis provides new single cell resolution insights into in vivo human intestinal development and corresponding organoid model systems. Characterization of the native tissue helps define a more physiologically relevant niche environment to both improve fidelity of corresponding epithelial organoid model systems and deepen our understanding of human intestinal development. Additionally, a native population of ECs is documented and expanded within hPSC-derived human intestinal organoid models. This thesis advances human intestinal organoid models to more faithfully recapitulate the complexity of the native tissue.