Interrogating Stem Cell Niche Factors in the Developing Human Gut to Advance in Vitro Models

Abstract

FFor centuries, scientists have been fascinated by how complex organisms like humans develop from a single cell. Yet historically the understanding of our own development has been hampered by ethical and technical restrictions. To address these limitations, scientists developed 3D cell models, known as organoids, to elucidate the cellular mechanisms driving development. However, current organoid models are limited by their ability to replicate the complex cell type make up, organization, and function of entire organs. Of interest to this work, organoid models of the human intestine lack spatial organization and many specialized cell types important for intestinal physiology. In this dissertation, I leverage single cell RNA sequencing (scRNA-seq) to characterize the developing human intestine. I identify Epiregulin (EREG) as a crucial developmental cue unique to the developing human intestine. I then build on this finding by investigating the potential for EREG to improve the two most common 3D organoid models of the human gut, enteroids and human intestinal organoids (HIOs). Chapter 2 focuses on understanding important stem cell niche cues in the intestinal epithelium. Using scRNA-seq, I interrogate the cell signaling environment within the developing human intestine to identify niche cues important for epithelial development and homeostasis. I identify an Epidermal Growth Factor (EGF) family member, Epiregulin (EREG), as robustly expressed in the developing human crypt. My results demonstrate that EREG can replace EGF in vitro, and EREG leads to spatially resolved enteroids. EREG-grown enteroids recapitulate many aspects of the human intestine that are not captured by previous enteroid models. This includes organoid budding, proliferative crypt domains and a differentiated villus-like central lumen. Utilizing multi-omic profiling of native crypts, EGF-grown, and EREG-grown enteroids I show that EGF-enteroids have an altered chromatin landscape that is influenced by concentration and leads to ectopic expression of stomach genes. This contrasts with EREG-grown enteroids, which retain intestinal markers in culture. These findings mark a significant advancement in the field by enabling better interrogation of stem cell function, spatial organization, cellular differentiation, and disease modeling. Chapter 3 interrogates a role for EREG in the development of the human intestine using a complex HIO model derived from human pluripotent stem cells. HIOs possess both epithelial and mesenchymal lineages in vitro but have historically lacked important cell types such as neurons, endothelial cells, immune cells, and smooth muscle. Leveraging the findings from Chapter 2, I developed an in vitro method to produce HIOs with epithelium, mesenchyme, enteric neuroglial populations, endothelial cells, and organized smooth muscle in a single differentiation. When transplanted into a murine host, these populations expand and organize to support organoid maturation. These EREG HIOs demonstrate enteric nervous system function, with HIOs undergoing peristalsis-like contractions and by utilizing in vitro microfluidic devices to model vascular-like flow, I show that EREG-HIOs form functional vasculature. I build on this finding by showing that transplantation of EREG-HIOs into a mouse stimulates endothelial cells to anastomose with host vasculature. Collectively, I report an in vitro hPSC-derived model of the human gut that simultaneously co-differentiates epithelial, stromal, endothelial, neural, and organized muscle populations. Taken together this work illustrates the power of benchmarking the native human organ at single cell resolution and demonstrates how principles of development can be used to improve in vitro models of the human intestine.