D. M. Schwartz1, N. Nagy1, L. S. Cheng1,2, K. Kitano1, H. C. Ott1, A. M. Goldstein1 1Massachusetts General Hospital,Boston, MA, USA 2University Of California – San Francisco,San Francisco, CA, USA
Introduction:
Patients with short bowel syndrome suffer extensive morbidity from reliance on parenteral nutrition. Small bowel transplant is unfortunately complicated by frequent graft failure. Intestinal tissue engineering aims to use patient-specific cells to create bioengineered small bowel. Regenerated intestine must have peristaltic function, and isolated enteric neural progenitor cells and smooth muscle cells are promising sources for creating neo-innervated and contractile bowel. We hypothesized that decellularized intestine, composed of only native extracellular matrix, could function as a permissive environment to support growth and development of these cell types.
Methods:
Intestinal scaffolds were generated from Sprague-Dawley rats using perfusion decellularization of whole jejunal segments. Colonic smooth muscle cells (SMCs) were isolated from 7-week old C57BL/6 mice, passaged in culture, and suspension-seeded onto the serosa of scaffolds (n=6). Neural stem cells were isolated from the intestine of 3-week old Actb-DsRed mice and propagated in culture as floating neurospheres. Secondary neurospheres were transplanted to the submucosa of decellularized scaffolds and cultured for 6 days (n=3). EdU was added as a marker of cell proliferation. To examine cell interactions, neurospheres were transplanted to the submucosa of scaffolds on which SMCs had been cultured for 3 weeks. This SMC and neurosphere co-culture was sustained for an additional week (n=2). All scaffolds were fixed, processed for immunohistochemistry, and analyzed for cell survival, migration, proliferation and maturation.
Results:
Smooth muscle cells transplanted onto decellularized intestinal segments survived in the host environment and organized into a four-cell-thick layer. The recellularized SMCs maintained immunoreactivity to calponin, a marker of differentiated smooth muscle. Enteric neural stem cells transplanted to the scaffolds survived and migrated an average maximal distance of 642 μm without additional stimulus. These cells maintained immunoreactivity to the neural crest lineage marker p75, and formed plexuses containing differentiated neurons, as evidenced by expression of PGP9.5 and NOS. A subpopulation of cells differentiated into enteric glia, expressing S100 and GFAP. Neural stem cells continued to proliferate throughout the culture period. In scaffolds co-cultured with SMC and neurospheres, the neural stem cells migrated along the smooth muscle layer and both cell types maintained their identities while establishing physical contact with each other.
Conclusion:
Decellularized intestinal scaffolds support the growth, differentiation, and proliferation of both neural stem cells and enteric smooth muscle cells in co-culture. These results support the achievability of regenerating an innervated muscularis externa, an essential step towards the ultimate goal of engineering small bowel with peristaltic function on a platform of decellularized extracellular matrix.