Whole gut imaging allows quantification of all enteric neurons in the adult zebrafish intestine.

BACKGROUND: A fundamental understanding of the enteric nervous system in normal and diseased states is limited by the lack of standard measures of total enteric neuron number. The adult zebrafish is a useful model in this context as it is amenable to in toto imaging of the intestine. We leveraged this to develop a technique to image and quantify all enteric neurons within the adult zebrafish intestine and applied this method to assess the relationship between intestinal length and total enteric neuron number. METHODS: Dissected adult zebrafish intestines were immunostained in wholemount, optically cleared with refractive index-matched solution, and then imaged in tiles using light-sheet microscopy. Imaging software was used to stitch the tiles, and the full image underwent automated cell counting. Total enteric neuron number was assessed in relation to intestinal length using linear regression modeling. KEY RESULTS: Whole gut imaging of the adult zebrafish intestine permits the visualization of endogenous and immunohistochemistry-derived fluorescence throughout the intestine. While enteric neuron distribution is heterogeneous between intestinal segments, total enteric neuron number positively correlates with intestinal length. CONCLUSIONS & INFERENCES: Imaging of all enteric neurons within the adult vertebrate intestine is possible in models such as the zebrafish. In this study, we apply this to demonstrate a positive correlation between enteric neuron number and intestinal length. Quantifying total enteric numbers will facilitate future studies of enteric neuropathies and ENS structure in animal models and potentially in biopsied tissue samples.

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.

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.

Murine and human tissue-engineered esophagus form from sufficient stem/progenitor cells and do not require microdesigned biomaterials.

PURPOSE: Tissue-engineered esophagus (TEE) may serve as a therapeutic replacement for absent foregut. Most prior esophagus studies have favored microdesigned biomaterials and yielded epithelial growth alone. None have generated human TEE with mesenchymal components. We hypothesized that sufficient progenitor cells might only require basic support for successful generation of murine and human TEE. MATERIALS AND METHODS: Esophageal organoid units (EOUs) were isolated from murine or human esophagi and implanted on a polyglycolic acid/poly-l-lactic acid collagen-coated scaffold in adult allogeneic or immune-deficient mice. Alternatively, EOU were cultured for 10 days in vitro prior to implantation. RESULTS: TEE recapitulated all key components of native esophagus with an epithelium and subjacent muscularis. Differentiated suprabasal and proliferative basal layers of esophageal epithelium, muscle, and nerve were identified. Lineage tracing demonstrated that multiple EOU could contribute to the epithelium and mesenchyme of a single TEE. Cultured murine EOU grew as an expanding sphere of proliferative basal cells on a neuromuscular network that demonstrated spontaneous peristalsis in culture. Subsequently, cultured EOU generated TEE. CONCLUSIONS: TEE forms after transplantation of mouse and human organ-specific stem/progenitor cells in vivo on a relatively simple biodegradable scaffold. This is a first step toward future human therapies.