Neural Crest Cell Implantation Restores Enteric Nervous System Function and Alters the Gastrointestinal Transcriptome in Human Tissue-Engineered Small Intestine

Abstract

SummaryAcquired or congenital disruption in enteric nervous system (ENS) development or function can lead to significant mechanical dysmotility. ENS restoration through cellular transplantation may provide a cure for enteric neuropathies. We have previously generated human pluripotent stem cell (hPSC)-derived tissue-engineered small intestine (TESI) from human intestinal organoids (HIOs). However, HIO-TESI fails to develop an ENS. The purpose of our study is to restore ENS components derived exclusively from hPSCs in HIO-TESI. hPSC-derived enteric neural crest cell (ENCC) supplementation of HIO-TESI establishes submucosal and myenteric ganglia, repopulates various subclasses of neurons, and restores neuroepithelial connections and neuron-dependent contractility and relaxation in ENCC-HIO-TESI. RNA sequencing identified differentially expressed genes involved in neurogenesis, gliogenesis, gastrointestinal tract development, and differentiated epithelial cell types when ENS elements are restored during in vivo development of HIO-TESI. Our findings validate an effective approach to restoring hPSC-derived ENS components in HIO-TESI and may implicate their potential for the treatment of enteric neuropathies.

Enteric neural stem cell therapies for enteric neuropathies

“…Our recent work demonstrated that in vivo transplantation of ENSC into an nNOS‐deficient microenvironment led to the development of nNOS + neurons. Unexpectedly, we also observed significant increases in ICC numbers throughout the colon of these mice, results which appear consistent with observations of transcriptomic alteration after ENS incorporation within human intestinal organoid units . It is now clear that a much more detailed understanding of the effects that transplanted ENSC, be that gut or pluripotent‐derived, have on the host neuromusculature is required.…”

“…More recently, efforts have focused on tissue engineering approaches including the development of human intestinal organoids (HIOs) and subsequent implantation of pluripotent‐derived enteric NCCs (ENCC) . Here, using 2 differing derivation approaches, the incorporation of ES or iPSC‐derived enteric NCCs within HIOs led to the functional incorporation of neuronal and glial cell types within organoid units which have previously been shown to contain mesenchymal cell types but lack a neural crest component.…”

“…Here, using 2 differing derivation approaches, the incorporation of ES or iPSC‐derived enteric NCCs within HIOs led to the functional incorporation of neuronal and glial cell types within organoid units which have previously been shown to contain mesenchymal cell types but lack a neural crest component. In both of these studies, incorporation of ENCC led to the functional development of albeit rudimentary activity, such as ENS‐mediated contraction of HIOs and calcium activity within transplanted ENS cells derived from a human iPSC line expressing GCaMPf . These studies critically provide evidence that it is possible to form functional intestinal units in vitro, which may play a significant role in drug discovery and patient specific preclinical testing of cellular and compound based strategies.…”

Short‐term and long‐term human or mouse organoid units generate tissue‐engineered small intestine without added signalling molecules

“…In our hands, OUs maintained in culture successfully generated human TESI that appeared similar to TESI derived from direct reimplantation of OUs, as we have performed previously (Barthel et al., , b; Grant et al., ; Grikscheit et al., ; Levin et al., ; Sala et al., ; Schlieve et al., ; Speer et al., ; Spurrier & Grikscheit, ). Differentiated epithelial cell types were identified in the cultured OUs at both short‐ and long‐term time points (Figure ).…”

Isolation and characterisation of mouse intestinal mesoangioblasts

“…In regards to specific cellular components, the primary focus of recellularisation studies have been both the epithelial and vascular compartments as reported by Kitano et al, where intestinal organoids were used for the regeneration of a functional intestinal mucosa [8]. Less attention has been placed on the neuromuscular compartment with few studies reporting on the use of neural crest cells derived from induced pluripotent stem cells [11, 12]. Indeed, there have been no reported studies focussed on the muscular compartment in itself, highlighting the need for research into this under-represented, but critical, cell type for intestinal regeneration.…”