The translation of this technique to a model that lends itself to mechanistic studies because of the wide variety of investigational tools available in the mouse is an advance. The implantation of OUs on a biodegradable polymer into the omentum allows the generation of TESI in an anatomic location, containing a large amount of intestinal mucosa as well as the mesenchymal components of intestine, including muscle and nerve. Using appropriate markers, we confirmed that the engineered tissue recapitulates the morphology of the native intestine with a fully differentiated epithelium adjacent to an innervated muscularis. This engineered tissue also bears both Lgr5- or DcamKL-1-positive cells, two putative intestinal stem cell populations, as well as the mesenchymal component of the stem cell niche, the ISEMFs, in their accustomed location.
OUs are heterogeneous clusters of cells that can contain both epithelial and mesenchymal cells, and in particular, some OUs contain either Lgr5- or DcamKL-1-positive cells adjacent to the mesenchymal ISEMF. The mesenchymal epithelial proximity, conserved in the OUs, is also seen during the growth of the TESI. Lineage tracing, which was not possible in previous tissue-engineered intestine models, demonstrates that all the essential components of the engineered intestine, epithelium, muscularis, nerves, and part of the blood supply are derived from the implanted cells.
Immediately after implantation, the majority of the implanted cells die. This is probably multifactorial. Because the OUs are generated from full-thickness intestine, the differentiated epithelial cells that no longer have the capacity to proliferate likely undergo apoptosis. Indeed, 3 days after implantation, we were unable to identify any differentiated epithelial cells (data not shown). These data suggest that the engineered tissue regenerates only from the implanted undifferentiated progenitor/stem cells. It is also probable that during the engineering process, some cells lose their anchorage to the extracellular matrix or neighboring cells and therefore undergo anoikis. Moreover, before the tissue completes angiogenesis and/or vasculogenesis, the implanted cells survive by imbibition of nutrients. Therefore, the cells located in the middle of the polymer may not receive adequate nutrients and oxygen to survive. This is supported by our observation that the regeneration of the tissue is initiated by a few OUs located on the outside layer of the polymer, in proximity to the omentum.
As early as 7 days after implantation, a flat epithelium and rudimentary crypt structure develops from the OUs that survive. By 14 days, crypt-villus structures are observed with a flat epithelium at each end of the epithelium. This process of formation is reminiscent of a wound healing process rather than a developmental process. After injury of the native intestine, an undifferentiated flat epithelium, originating from the edge of the wound, covers the exposed mesenchyme. This is followed by the invagination of new crypts and finally by the formation of villi.30
It is interesting to note that the number of Lgr5-positive cells as well as the number of differentiated epithelial cells was overall higher than what is found in native ileum. Similar findings have been observed in regenerating intestine, suggesting that the engineered intestine is still regenerating 4 weeks after implantation.31
Further investigation of the molecular and cellular mechanisms of TESI formation may help to identify mechanisms of tissue repair and vice-versa.
It is known that the intestinal epithelial stem cells survive in a mesenchymal cell niche toward the base of the intestinal crypt. Existing in a subepithelial location from esophagus to large intestine, ISEMFs are key mesenchymal cells supporting the intestinal stem cell and a major source of instructive signals to the intestinal epithelium. Beyond fetal development, a balanced gut homeostasis and repair process depends on mesenchyme–epithelial cross talk.32–34
Evidence for the preservation of an intact, populated stem cell niche in fully formed TESI includes the demonstration of a differentiated epithelium at 4 weeks, allowing for a number of rounds of the customary epithelial regeneration that occurs every 3–7 days. Although the roles of Lgr5- or DcamKL-1-positive cells are still being defined, demonstration of the presence of both Lgr5- and DcamKL-1-positive cells in our tissue-engineered intestine reinforces our observation that engineered intestine is very similar to native intestine. Presence of those cells may be crucial for the normal homeostasis of the tissue for the lifetime of a patient in a clinical setting, and to exceed the current survival rates of nonautologous intestinal transplanted grafts. The presence of those putative stem cells also gives hope that the tissue would be able to repair itself in case of damage.
Recent experiments using isolated Lgr5-positive cells demonstrated that those cells were able to proliferate and form cyst-like structures with a differentiated epithelium in vitro
In order to achieve the development of crypt/villus structures from those isolated cells, the microenvironment of the cells was artificially recreated by culturing them in Matrigel with the addition of Wnt agonist R-spondin 1, EGF, and Noggin.18
However, this technique, although promising, has various limitations that will need to be addressed before translation to human therapy. First, the sorting process to purify single-cell populations is somewhat harsh and time-intensive. This allows the growth of only 6% of the cells, and their development was limited to a few crypt-villus structures. It may be difficult to scale up sorted single cells for autologous human therapy. Moreover, prolonged culture could result in transformation of the cells leading to either loss of function or tumorigenic potential.35
Finally, implantation of a stock cell line or nonautologous cultured cells might require immunosuppression. The generation of TESI with our multicellular approach required no in vitro
culture, limiting the associated risk of cell transformation. However, most importantly, maintaining the epithelial–mesenchymal relationship allows a more rapid and robust growth with large amount of mucosa and a higher success rate of mucosal generation. Indeed, we were able to generate a new intestinal mucosa demonstrating epithelial differentiation in 86% of our implants. At 6 weeks, the resulting tissues contained in average 3 times the initial implanted number of cells. Moreover, this approach also provides the mesenchymal components (i.e., muscularis, nerves, and blood supply) necessary for functional intestine that single-cell approaches currently lack.
Transition to the mouse model is a very exciting advance, as it will allow us to further investigate the molecular and cellular mechanisms involved during the growth of tissue-engineered intestine. A better understanding of those processes is crucial for a successful, safe, and optimized transition to human therapy.