P. H. Dedhia1, F. K. Noto3, L. D. Shea3, J. R. Spence2 1University Of Michigan,Department Of Surgery,Ann Arbor, MI, USA 2University Of Michigan,Department Of Internal Medicine,Ann Arbor, MI, USA 3University Of Michigan,Department Of Biomedical Engineering,Ann Arbor, MI, USA
Introduction: Short bowel syndrome secondary to bowel resection for congenital anomalies, infection or inflammation, or intestinal ischemia can have devastating metabolic consequences. Supportive treatment such as parenteral nutrition is costly and has associated morbidity. Surgical intervention including bowel lengthening procedures and small bowel transplantation has met with varied success. Accordingly, alternative approaches to create tissue-engineered intestine offer novel, potentially curative therapeutic strategies to treat short bowel syndrome. Human enteroids, which are primary 3-dimensional tissue that can be generated from an endoscopic biopsy, recapitulate the complexities of the intestinal epithelium histologically and can be expanded exponentially in vitro. Consequently, enteroids can provide a limitless source of autologous intestinal epithelium. To date, successful transplantation of murine enteroids has required induction of colitis. In contrast, human enteroids have not been successfully transplanted into immunocompromised mice, likely due to inadequate supportive structures. Poly (lactide-co-glycolide) (PLG) scaffolds have been shown to provide structural support for human cells. We thus hypothesized that placement of human enteroids on PLG scaffolds embedded with growth factors would permit long-term survival of enteroids post-transplant.
Methods: We generated enteroids from human small intestinal tissue by isolating intestinal epithelium from associated structures using dispase. After expanding these enteroids in vitro, we embedded enteroid fragments and the growth factors, EGF, Noggin, and R-spondin, onto PLG scaffolds. Enteroid-laden scaffolds were then grown in vitro for 1 week and assessed for survival by calcein and ethidium homodimer staining. Enteroid-laden scaffolds were also transplanted into the epididymal fat pads of immunocompromised NSG mice and harvested at 1 and 4 weeks.
Results: Our data demonstrated that enteroids grown on PLG scaffolds were able to survive for 1 week in vitro as indicated by positive calcein staining. When enteroid-containing scaffolds were transplanted into murine epididymal fat pads, all explants demonstrated regions of cells that stained positive for human mitochondrial antigen, Cdx2, and E-cadherin after both, 1 and 4 weeks post-transplant (n = 5, and n= 4, respectively). Together, these data indicate that human enteroids persist and retain their intestinal identity when set in scaffolds and transplanted into mice.
Conclusion: This is the first report demonstrating survival of human epithelial enteroids in a transplant model. As such, these studies have important implications for short bowel syndrome, because enteroid-laden scaffolds are easily generated from autologous tissue. Furthermore, scaffolds can be designed to resemble gut tubes such that future studies may examine the ability of scaffolds to be incorporated into the small intestine.