Human and Murine Tissue-Engineered Colon Exhibit Diverse Neuronal Subtypes and Can Be Populated by Enteric Nervous System Progenitor Cells When Donor Colon Is Aganglionic

Abstract

Purpose: Tissue-engineered colon (TEC) might potentially replace absent or injured large intestine, but the enteric nervous system (ENS), a key component, has not been investigated. In various enteric neuropathic diseases in which the TEC is derived from aganglionic donor colon, the resulting construct might also be aganglionic, limiting tissue engineering applications in conditions such as Hirschsprung disease (HD). We hypothesized that TEC might contain a diverse population of enteric neuronal subtypes, and that aganglionic TEC can be populated by neurons and glia when supplemented with ENS progenitor cells in the form of neurospheres. Materials and Methods: Human and murine organoid units (OU) and multicellular clusters containing epithelium and mesenchyme were isolated from both mouse and human donor tissues, including from normally innervated and aganglionic colon. The OU were seeded onto a biodegradable scaffold and implanted within a host mouse, resulting in the growth of TEC. Aganglionic murine and human OU were supplemented with cultured neurospheres to populate the absent ENS not provided by the OU to rescue the HD phenotype. Results: TEC demonstrated abundant smooth muscle and clusters of neurons and glia beneath the epithelium and deeper within the mesenchyme. Motor and afferent neuronal subtypes were identified in TEC. Aganglionic OU formed TEC with absent neural elements, but neurons and glia were abundant when aganglionic OU were supplemented with ENS progenitor cells. Conclusion: Murine and human TEC contain key components of the ENS that were not previously identified, including glia, neurons, and fundamental neuronal subtypes. TEC derived from aganglionic colon can be populated with neurons and glia when supplemented with neurospheres. Combining tissue engineering and cellular replacement therapies represents a new strategy for treating enteric neuropathies, particularly HD.

The Contributions of Human Mini-Intestines to the Study of Intestinal Physiology and Pathophysiology

“…Such models are being considered for use in transplantation for short gut syndrome (132, 133). An advantage is that the patient can be used as the source from which to grow the enteroids, minimizing the risk of rejection and the need for long-term immunosuppression.…”

Drug Discovery via Human-Derived Stem Cell Organoids

“…First, the limited number of cell types in intestinal epithelial organoids and the absence of the immune, nervous, and vascular system in cultured intestinal organoids result in drug effects that are different from those in vivo ( Fuller et al, 2012 ; Jabaji et al, 2013 ; Grun et al, 2015 ). Additionally, although the main cell types of epithelial cells that are generated in organoids are similarly diverse in comparison with those found in vivo , the 3D organization cannot be spatially and structurally similar to an in vivo murine intestine or consistent with other organoids ( Onuma et al, 2013 ; Wieck et al, 2015 ). Third, the microenvironment and hormone levels, pH, and the health status of the human body as well as epigenetic factors cannot be fully reproduced in an ex vivo organoid ( Lancaster and Knoblich, 2014 ; Dekkers et al, 2015 ; Wroblewski et al, 2015 ).…”

“…The widespread implementation of organoid technologies provides a more physiologically relevant platform for high-throughput screening during drug discovery ( Eglen and Randle, 2015 ; Walsh et al, 2016 ). An organoid means ex vivo three-dimensional (3D) tissue originating from organ stem cells, embryonic stem cells (ESCs), or induced pluripotent stem cells (iPSCs), with structure and function similar to those of the original organ to some degree ( McCracken et al, 2011 ; Fuller et al, 2012 ; Lancaster and Knoblich, 2014 ; Ordonez-Moran et al, 2015 ; Tamminen et al, 2015 ; Wieck et al, 2015 ). So far, various organoid systems have been successfully established from a specific organ and could be expanded infinitely ( Sato et al, 2011 ; Hisha et al, 2013 ; Mahe et al, 2013 ; Takebe et al, 2013 ; Gehart and Clevers, 2015 ; Rookmaaker et al, 2015 ; Xinaris et al, 2015 ; Yin et al, 2016 ).…”

Generation of an artificial intestine for the management of short bowel syndrome

“…Further, Wieck et al . demonstrated the ability of cultured murine enteric neurospheres to form enteric nervous system components when co-seeded with organoids isolated from mice with aganglionic colon and implanted in an omental model [52]. While not performed in the small intestine, this study is important as a first step towards engineering an intact enteric nervous system in artificial intestine.…”

“…This study, along with Wieck et al . [52] are the first studies that have begun to perform co-cultures and experiments with IECs and other intestinal components.…”