Funding opportunities

Differentiation of Human Embryonic Stem Cells to Intestinal Fates

Funding Type: 
SEED Grant
Grant Number: 
Principle Investigator: 
Funds requested: 
$578 943
Funding Recommendations: 
Grant approved: 
Public Abstract: 
Statement of Benefit to California: 
Review Summary: 
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.

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