Uncovering the Regulators of Enteric Neurogenesis and Diversification Using HPSC-Derived Models

Abstract

Neurogenesis in the enteric nervous system (ENS) is a highly dynamic and complex process that remains only partially understood. Enteric neural crest cells (ENCCs) primarily emerge from the vagal neural tube to enter the foregut and migrate caudally to colonize the entire gut. During this process, enteric progenitors must undergo differentiation stages that direct the emergence of diverse neuronal and glial subtypes, establishing ganglia essential for gastrointestinal (GI) function. The molecular regulatory programs that control neurogenesis, diversification, and maturation of enteric neurons have not been well described due to the transient nature and inaccessibility of progenitor and immature neuron cell types in the developing gut, particularly in humans. In this study, we utilized our human pluripotent stem cell (hPSC) differentiation system to overcome these limitations and characterize neurogenesis and the diversification of the enteric nervous system (ENS) over time. Through time-course analysis spanning approximately 100 days, we tracked the induction dynamics of various progenitor and neuronal populations using single-cell transcriptomics. To identify pathways involved in neurogenesis, we conducted a small molecule screen of nearly 1700 small molecular inhibitors and discovered factors that accelerate neurogenesis in hPSC-derived ENS cultures. The extent of neurogenesis was quantified by an increase in gene expression of neurotransmitter processing associated genes and synaptic proteins. Our single-cell transcriptomics analysis, along with the screen results, highlight pathways critical in the temporal regulation of neurogenesis in the ENS. The insights gained from this approach contribute to a deeper understanding of the molecular mechanisms underlying ENS development and neurogenesis and establish improved and accelerated enteric neuron differentiation strategies for drug testing and cell therapy applications.