The roughly 25 feet of intestine in the adult human play numerous essential roles in daily life, such as nutrient absorption, secretion of hormones, and serving as a barrier to infection. Commensurate with these diverse roles, diseases of the intestine are a considerable source of human morbidity and mortality. Indeed, numerous pathologic conditions including inflammatory bowel diseases, mesenteric ischemia, congenital syndromes and trauma, with or without concomitant intestinal resection, all impair intestinal function to the extent that “short-gut” syndromes develop—resulting in effective intestinal failure. Current therapies rely on supportive measures such as total parenteral nutrition, in which patients receive all of their nutrition intravenously, or even intestinal transplantation.
The adult intestine is populated by specialized but highly active intestinal stem cells, which ideally could be harnessed for stem cell therapies of these disabling conditions. However, despite intensive research, no methods currently exist for identifying, isolating, and growing these intestinal stem cells for therapeutic purposes.
Our goal is to develop technologies enabling human embryonic stem (hES) cells to be reliably converted to intestinal cells in culture. Human ES cells can be readily grown in culture but represent a completely undifferentiated tabula rasa. Here, we propose studies to convert hES cells to intestinal stem cells and thence to mature intestinal cells. Towards this goal, we have developed the first methodology to induce intestinal cells to divide and expand as cultures, or “explants” outside of the body. This success has been reliant on the provision in our explants of a nutritive “niche”, a specialized area in which signals conductive to intestinal stem cell survival are highly concentrated. In this proposal, the hES cells will be placed in this niche of our explant culture, amidst signals that would promote their conversion from a naïve state into intestinal stem cells and their mature progeny. We will further refine these methods by coaxing hES cells along the first steps towards intestine prior to placing them in the explant niche, as well as by adding hormones to encourage growth of intestinal cells. The use of hES cells could greatly enhance the growth of our explant cultures, vastly expanding the yield of cultured intestine.
The therapeutic applications of this work are clear. The combination of ES cell technology along with our explant culture system holds considerable promise for the eventual generation of large quantities of intestinal stem cells, or even artificial intestine. Hopefully, these will yield effective therapies for the numerous conditions resulting in effective intestinal failure, for which currently available therapies are decidedly suboptimal.
The proposed research will develop new human embryonic stem cell-based technologies enabling the robust propagation of intestinal tissue and its associated stem cells outside of the human body, in laboratory culture. These studies have implications for the treatment of disabling conditions of the intestinal tract including inflammatory bowel disease, mesenteric ischemia and congentital intestinal disorders.
SYNOPSIS: This proposed research intends to develop the means for restorative intestinal regeneration by seeding human embryonic stem cells onto biopsied intestinal mucosum as a substratum and niche for intestinal progenitor cell propagation and differentiation. Aim 1 will determine whether a successful mouse intestinal explant-culture, which has been shown to recapitulate the intestinal stem cell niche, can induce human ESCs to differentiate into intestinal cells. Human ESCs prior to and after pre-differentiation to endoderm will be tested. Aim 2 will test whether R-spondin1-stimulation of Wnt signaling can enhance the incorporation, proliferation and differentiation of hESCs in the intestinal explants. Aim 3 will develop the methodology to prepare human intestinal explants from endoscopic biopsy that would allow a fully humanized system for ex vivo production of intestine for transplantation.
INNOVATION AND SIGNIFICANCE: Despite intensive investigation, it has not been possible to isolate and manipulate intestinal stem cells for possible restorative therapy. This proposal describes a very innovative and effective system that has the ability to maintain intestinal cell proliferation within the context of the mucosa. Therefore it has the potential to be an excellent natural substratum and niche for human ESCs to expand an intestinal epithelium with differentiating cells to restore the major cell types of the intestinal epithelium. Although many studies have addressed the generation of pancreatic endoderm and hepatic endoderm from ESC, few have attempted to induce differentiation from hESC to gastrointestinal epithelium. If this promising strategy proves feasible, obtaining insights in intestinal disorders and potentially, treatment of a number of intestinal diseases that constitute ‘short gut’ syndromes may become routine.
STRENGTHS: This is an excellent research plan based on a thorough understanding of the maintenance, propagation and differentiation of intestinal epithelium, the regulatory signaling of the stem cell niche, and strong preliminary results by an expert team of investigators. The preliminary experiments characterizing the mouse intestinal explants demonstrated a remarkable capacity of the explants for growth and maintenance and the proper response to Wnt-control of cell replenishment; this suggests the potential for appropriately hosting human ES cells. The planned experiments are a straightforward, logical and insightful approach to a therapeutic solution of a major and widespread health problem. The research plan is strengthened by the collaborative agreement with Dr. Julie Baker in Genetics at Stanford to provide cells with endoderm phenotype derived from human ES cells. The proposed research is a good opportunity to take advantage of this important advance for human stem cell biology of the gut.
WEAKNESSES: There are major technical hurdles not addressed by the preliminary results demonstrating the vigor of the mouse intestinal crypt culture. One concern is whether conditions can be found that promote the efficient incorporation of human embryonic stem cells into the epithelium in a manner that allows proper access to substratum, the Wnt-niche, and appropriate epithelial neighbors. Continuing the mouse ES cell experiments would have provided quick preliminary evidence relevant to the ability of ES cells to associate productively with the intestinal explants. The strength and inventiveness of the research plan and the significance of success to treatment of intestinal disease far outweigh the perceived weaknesses.
The proposal is very ambitious with Aims 1 and 2 more than sufficient for the 2-year term. It is more likely that Aim 1B will succeed rather than Aim 1A; this might need to be coupled to promoter selection method.
DISCUSSION: The mouse intestinal explant cell system (propsed to recapitualte the ESC niche) has been maintained for up to 70 days. The collaborator, Dr. Julie Baker, has recapiulated the Novocell protocol to differentiate hESC to endoderm phenotype. The applicant is exceeding well-funded. The proposal involves a massive amount of work. Discourage applicant from carrying out aim 3, accomplishment of aim 1 alone would be great. There was a question as to how gut explants were sterilized; response was to use a lot of antibiotics.