J. N. Rea1,2, C. R. Schlieve1,2, K. L. Fowler1,2, S. Huang5,6, J. R. Spence4,5,6, T. C. Grikscheit1,2,3 1Children’s Hospital Los Angeles,Developmental Biology And Regenerative Medicine Program, The Saban Research Institute,Los Angeles, CA, USA 2Children’s Hospital Los Angeles,Department Of Surgery, Division Of Pediatric Surgery,Los Angeles, CA, USA 3University Of Southern California,Keck Medical School,Los Angeles, CA, USA 4University Of Michigan,Department Of Internal Medicine,Ann Arbor, MI, USA 5University Of Michigan,Department Of Cell And Developmental Biology,Ann Arbor, MI, USA 6University Of Michigan,Department Of Biomedical Engineering,Ann Arbor, MI, USA
Introduction:
Intestinal villus formation increases the epithelial surface area of the gut for nutrient absorption and in several species the previllus epithelial layer is flat and pseudostratified with alteration of the nuclear position according to cell cycle. In the mouse, the epithelial cell shape changes from an average height around 15 µm to 50 µm around E14.5, and human villification follows a similar program1. A grading system 1-4 was developed to evaluate villification in tissue-engineered small intestine (TESI), which is a potential therapy for intestinal loss. Grade 2 TESI was defined as previllus monolayer epithelium without crypt invaginations. We hypothesized that villus morphogenesis might replicate human and mouse programs with changes in the epithelial thickness.
Methods:
Human postnatal TESI (hTESI), human fetal TESI (hfTESI), human intestinal organoid TESI (HIO-TESI), and mouse postnatal TESI (mTESI) were generated as previously described. Two types of Grade 2 epithelium were identified, absence (Grade 2a) or presence (Grade 2b) of secretory cells. Therefore H&E staining of Grade 2a (n = 6) and Grade 2b (n = 6) samples of each TESI preparation were captured by bright field microscopy. Eight random measurements of epithelial cell height and width were recorded for each sample (ImageJ) and analyzed by two-way ANOVA after outlier analysis for each TESI preparation (Prism software).
Results:
Epithelial cell height was lower in the absence of secretory cells when TESI was derived from human fetal tissue or induced pluripotent stem cells (hfTESI and HIO-TESI) but in postnatally-derived hTESI and mTESI, there was no difference in the epithelial cell height between the groups. Comparable to the known changes in epithelial thickness for mouse villus development (15->50 µm), both hfTESI (Grade 2a: 18.86 µm vs Grade 2b: 38.85 µm, p = 0.0079) and HIO-TESI (Grade 2a: 13.82 µm vs Grade 2b: 31.26 µm, p = 0.0231) exhibit marked increases in thickness when secretory cells are present. As in murine/human development, there were no changes in epithelial cell width in any of the TESI preparations.
Conclusion:
Secretory cells are thought to perform important stem/progenitor cell niche functions and develop contemporaneously with crypt formation. Further investigation of the process of villus morphogenesis in TESI may indicate points for intervention to improve TESI villus formation and therefore, perhaps, function.
