Establishment of experimental paradigm for generation of functioning enteric neurons from human pluripotent stem cells using single cell transcriptomics and human innervated colonic organoids

Abstract

Generation of fully-functioning cells from human pluripotent stem cells (hPSCs) remains challenging. In this study, we performed single-cell RNA sequencing (scRNA-seq) to systematically analyze the lineage commitment process after the hPSCs exist their pluripotent state and the timing of various differentiation cues underlying the generation of neural crest (NC) and their subsequent neuronal lineage differentiation and established a hPSC-derived innervated colonic organoid (HCOs) model for appraising the function of hPSC-derived enteric neurons. scRNA-seq analysis revealed five main clusters of cells from the pool of hPSC-derived NC, each cluster of cells exhibited unique expression pattern resembling NCs at different developmental stages in vivo. In particular, we found that HEDGEHOG (HH) pathway is activated in the post-migratory NC-like cells. Using chemical- and gene-targeting-mediated modulation of HH signaling, we further defined the critical treatment window for HH to increase the NC-yield from hPSC. By profiling the single cell transcriptomes of hiPSC-derived NC cells and their neuronal progenies, we further delineated how HH alters the topology of the neuronal differentiation path of NC and primes NC toward the neurogenic lineage. Subsequent in vitro differentiation assay further indicated that activation of HH signaling during the hPSC-to-NC transition can greatly improve the subsequent neuronal lineage differentiation of NC. More importantly, we established a differentiation protocol for the generation of a human innervated HCOs model and used them for assessing the functional competency of hPSC-derived enteric neurons. Our innervated HCOs model contained defined crypts, colonic epithelium, various types of colon cell (e.g. Goblet- and endocrine-like cells) as well as functional enteric nervous system. With our innervated HCOs model, we further demonstrated that activation of HH during NC induction can greatly improve the functional competency and the neuromuscular coupling of hPSC-derived enteric neurons with high reproducibility across hPSC lines. In summary, we established an experimental paradigm to systematically optimize the differentiation protocol for the generation of functioning NC in a systematic way.