hPSC-Derived Enteric Ganglioids Model Human ENS Development and Function

Abstract

The enteric nervous system (ENS) plays a central role in gut physiology and mediating the crosstalk between the gastrointestinal (GI) tract and other organs. The human ENS has remained elusive, highlighting the need for an in vitro modeling and mapping blueprint. Here we map out the developmental and functional features of the human ENS, by establishing robust and scalable 2D ENS cultures and 3D enteric ganglioids from human pluripotent stem cells (hPSCs). These models recapitulate the remarkable neuronal and glial diversity found in primary tissue and enable comprehensive molecular analyses that uncover functional and developmental relationships within these lineages. As a salient example of the power of this system, we performed in-depth characterization of enteric nitrergic neurons (NO neurons) which are implicated in a wide range of GI motility disorders. We conducted an unbiased screen and identified drug candidates that modulate the activity of NO neurons and demonstrated their potential in promoting motility in mouse colonic tissue ex vivo. We established a high-throughput strategy to define the developmental programs involved in NO neuron specification and discovered that PDGFR inhibition boosts the induction of NO neurons in enteric ganglioids. Transplantation of these ganglioids in the colon of NO neuron-deficient mice results in extensive tissue engraftment, providing a xenograft model for the study of human ENS in vivo and the development of cell-based therapies for neurodegenerative GI disorders. These studies provide a framework for deciphering fundamental features of the human ENS and designing effective strategies to treat enteric neuropathies.

Inhibition of muscarinic receptor signaling protects human enteric inhibitory neurons against platin chemotherapy toxicity

“…Next, we set out to determine if our hPSC-derived enteric neurons (henceforth referred to as “stage 1 2D enteric neurons” based on their differentiation stage and format) contain the same neuronal diversity as other published enteric neuron datasets. We compared the neurochemical composition of our dataset to hPSC-derived stage 1 and stage 2 ganglioid (3D) enteric neurons published by our group 21 , primary fetal human enteric neurons published by Teichmann and colleagues 40 , as well as primary adult human colonic enteric neurons published by Regev and colleagues 20 . For neurotransmitters, we utilized a two-step neurochemical identity-defining approach, that our group has reported previously, to identify all the cells that belong to six neurotransmitter identities: serotonergic, GABAergic, catecholaminergic, glutamatergic, cholinergic, and nitrergic 21 .…”

“…We compared the neurochemical composition of our dataset to hPSC-derived stage 1 and stage 2 ganglioid (3D) enteric neurons published by our group 21 , primary fetal human enteric neurons published by Teichmann and colleagues 40 , as well as primary adult human colonic enteric neurons published by Regev and colleagues 20 . For neurotransmitters, we utilized a two-step neurochemical identity-defining approach, that our group has reported previously, to identify all the cells that belong to six neurotransmitter identities: serotonergic, GABAergic, catecholaminergic, glutamatergic, cholinergic, and nitrergic 21 . We found that all six neurochemical identities are represented in our cultures, recapitulating the neurotransmitter diversity of other datasets (Figure S4F).…”

“…We found that all six neurochemical identities are represented in our cultures, recapitulating the neurotransmitter diversity of other datasets (Figure S4F). Furthermore, enteric neurons that produce multiple neurotransmitters have recently been characterized in some detail 21 . To determine if our stage 1 2D enteric neurons recapitulate the same neurotransmitter complexity as other enteric neuron tissues, we quantified the number of cells predicted to synthesize only one, two, three, or more neurotransmitters across all five datasets.…”

“…Similarly, cholinergic neurons, expressing the acetylcholine rate limiting enzyme CHAT , exist outside of the cholinergic neuron clusters (Figure S5C). To take a more holistic look at these two these populations, we identified all nitrergic neurons and cholinergic neurons using our two-step neurochemical identity-defining approach 21 , thereby binning all enteric neurons in our dataset into nitrergic, cholinergic, or other categories (Figure S5C). We then module scored the nitrergic, cholinergic, and other neurons based on their expression of the genes in the platin excitotoxicity transcriptional program (Supplementary Table 7).…”

“…Here, we utilized our established method of deriving enteric neurons from human pluripotent stem cells (hPSCs) [21][22][23] to build a robust in vitro model of platin-induced enteric neuropathy and uncover the mechanism of toxicity. Using this system, we determined the cellular and molecular hallmarks of platin-induced enteric neuropathy through comprehensive phenotypic and transcriptomic profiling.…”