Human and mouse tissue-engineered small intestine both demonstrate digestive and absorptive function

Abstract

Short bowel syndrome (SBS) is a devastating condition in which insufficient small intestinal surface area results in malnutrition and dependence on intravenous parenteral nutrition. There is an increasing incidence of SBS, particularly in premature babies and newborns with congenital intestinal anomalies. Tissue-engineered small intestine (TESI) offers a therapeutic alternative to the current standard treatment, intestinal transplantation, and has the potential to solve its biggest challenges, namely donor shortage and life-long immunosuppression. We have previously demonstrated that TESI can be generated from mouse and human small intestine and histologically replicates key components of native intestine. We hypothesized that TESI also recapitulates native small intestine function. Organoid units were generated from mouse or human donor intestine and implanted into genetically identical or immunodeficient host mice. After 4 wk, TESI was harvested and either fixed and paraffin embedded or immediately subjected to assays to illustrate function. We demonstrated that both mouse and human tissue-engineered small intestine grew into an appropriately polarized sphere of intact epithelium facing a lumen, contiguous with supporting mesenchyme, muscle, and stem/progenitor cells. The epithelium demonstrated major ultrastructural components, including tight junctions and microvilli, transporters, and functional brush-border and digestive enzymes. This study demonstrates that tissue-engineered small intestine possesses a well-differentiated epithelium with intact ion transporters/channels, functional brush-border enzymes, and similar ultrastructural components to native tissue, including progenitor cells, whether derived from mouse or human cells.

Keywords: intestinal failure; intestinal stem cell; regenerative medicine; short bowel syndrome; tissue engineering.

Fig. 1.

Tissue-engineered small intestine (TESI) recapitulates the ultrastructure of native intestine. Transmission electron microscopy (TEM) on a low-power field shows a crypt-like structure in both human and mouse-derived TESI. Within these crypts, dense vesicles within supporting Paneth cells are noted flanking characteristic triangular-shaped crypt-base columnar cells (CBCs), an intestinal epithelial progenitor cell. There are goblet cells containing mucus and absorptive polarized enterocytes bearing apical microvilli facing the luminal compartment. Enteroendocrine cells are notable for smaller cytoplasmic granules containing secretory products than the larger vesicles demonstrated in the Paneth cells. TESI epithelial cells exhibited intact tight junctions bridging complex intercalated intercellular spaces (arrows). Scale bars as indicated.

Fig. 2.

Goblet cells are abundant in TESI. Goblet cells are identified by Alcian blue staining throughout the epithelium of human TESI (hTESI) (B) and mouse TESI (mTESI) (D). Goblet cell location and appearance are similar to native human ileum (A) and mouse jejunum (C) controls. Scale bar = 50 μm.

Fig. 3.

Cytoskeletal elements are intact in TESI. A: punctate areas at the apical junction between epithelial cells demonstrate immunofluorescent staining for zonula occludens (ZO)-1. B: actin localizes to cell membranes. Blue indicates nuclear Hoechst dye. Scale bars as indicated.

Fig. 4.

Presence of Cdc42 and Na⁺/K⁺ ATPase demonstrate TESI polarity. Cdc42 (red) localizes faintly to the brush border and primarily to secretory cells in TESI epithelium as in controls. Na⁺/K⁺ATPase (green) localizes to the basolateral membranes throughout. Blue = nuclear Hoechst dye. Scale bar = 50 μm.

Fig. 5.

Cdc42 colocalizes with secretory cell types in TESI. Overlapping Cdc42 stains with mucin 2 (Muc2) and lysozyme in mTESI (top and top, middle) and hTESI (bottom, middle and bottom) demonstrate colocalization with secretory cell types. Cdc42 and lysozyme colocalization is noted in fewer cells in mTESI. Scale bar = 50 μm.

Fig. 6.

Major intestinal isoforms of the sodium/hydrogen exchanger (NHE), including NHE1 and NHE3 are present on the epithelial membrane of TESI. NHE1 (A) stains both apical and basolateral membranes, whereas NHE3 (B) stains the apical membrane more specifically. Blue = nuclear Hoechst dye. Scale bar = 50 μm.

Fig. 7.

Water channels and chloride transporter controlling transcellular water transport are present in TESI. A: aquaporin shows apical specificity in TESI as in native intestine. B: cystic fibrosis transmembrane conductance regulator (CFTR) localizes to the brush border in TESI and controls. Blue = nuclear Hoechst dye. Scale bar = 50 μm.

Fig. 8.

TESI possesses membrane components necessary for complex sugar breakdown and absorption. A: immunohistochemistry demonstrates strong apical staining for the sodium glucose transporter (SGLT-1) in mouse native jejunum and slightly less intense but properly localized staining in mTESI. B: dimeric brush-border enzyme sucrase isomaltase also showed slightly less intense staining in hTESI and mTESI compared with controls. Blue = nuclear Hoechst dye. Scale bar = 50 μm.

Fig. 9.

Functional brush-border enzymes are present in TESI epithelium. A: intestinal alkaline phosphatase (IAP) is active at the brush border of TESI and control intestine, as indicated by the red fluorescent byproduct of a completed enzymatic reaction carried out by IAP. Blue = nuclear Hoechst dye. Scale bar = 50 μm. B: functional membrane disaccharidases are present on the brush border of TESI. The amount of glucose released after hydrolysis of the complex sugars sucrose and maltose is shown. Sucrase was measured following incubation with TESI homogenate (B2). Homogenized adult C57BL/6 jejunum (JEJ) and human ileum (ILE) served as positive controls. Control tissue homogenate incubated without added complex sugar served as negative control (blank). 3 mTESI and 3 hTESI were studied (sequentially numbered TESI represent individual TESI samples). Both maltase (B1) and sucrase (B2) were active in mTESI and hTESI although only at a fraction of the activity of native mouse and human intestine. Maltase activity was higher than sucrase in both hTESI and mTESI.

Fig. 10.

Rapidly dividing label-retaining cells are present in TESI. Ki-67 localizes rapidly dividing cells in hTESI epithelium and less so in mesenchyme. These cells appear to be concentrated in the forming crypt-like structure. Ki-67 more reliably stains crypt structures in mTESI, with interspersed mesenchymal cells. Human ileum and mouse jejunum controls demonstrate that Ki-67 is localized in crypt epithelium. Scale bar = 50 μm.

Fig. 11.

Apical proteins gain expression at different time points in TESI. At week 2, there is no apical staining of sucrase isomaltase in TESI. By week 4, it is strongly expressed. Aquaporin 7 stained strongly at the apical membrane of TESI by week 2 and remained present at week 4. Blue = nuclear Hoechst dye. Scale bar = 20 μm.