Basic Biology V
$1 169 495
We would have more effective treatment options for inflammatory diseases of the intestine if we could stimulate stem cells to regenerate the lining of the gut. In this project we are testing the role of a protein called ErbB4 in controlling the human intestinal stem cell environment. We have preliminary data showing that ErbB4 promotes the survival of cells called Paneth cells, which are in turn required for stem cell maintenance. Proposed studies will use cutting-edge intestinal culture systems to test (1) how ErbB4 regulates human stem cell numbers, types, and growth; (2) how regulation of Paneth cell development and survival is involved in ErbB4 effects on stem cells; and (3) the effects of ErbB4 on stem cell-driven intestinal regeneration after injury. The ultimate goal of these studies is to advance our understanding of the regulation of the human intestinal stem cell niche, with implications for future regenerative medicine therapies in this tissue.
Statement of Benefit to California:
Diseases in which the intestinal epithelium is damaged or reduced in surface area constitute major public health problems, nationwide and in California. Inflammatory bowel disease afflicts ~1.5 million American patients (with ~1/8 the American population, this translates to an estimated 187,500 patients in-state), necrotizing enterocolitis occurs in up to 10% of premature infants (and has a ~30% mortality rate), and short bowel syndrome affects 24.5/100,000 live births (>30% mortality). This project is focused on testing a mechanism of regulation of human intestinal stem cells by the growth factor receptor ErbB4, which could be useful to improve treatment and outcome in for these diseases. California will benefit from this project in several ways. First and foremost, many Californians are affected by these diseases, and improved medical care for IBD, NEC, and SBS would have a major impact both on quality of life and the cost of medical care. Second, the work will generate employment opportunities and spend supply costs within the state. Third, developments at the leading edge of the human intestinal regeneration field will attract top scientists and biotech, contributing to California’s society and economy. Finally, if successful this work will generate intellectual property that can be exploited and thus will also benefit the state’s economy.
This Fundamental Mechanisms proposal aims to test the role of a protein called ERBB4 in promoting the survival of Paneth cells within the human intestinal stem cell (ISC) niche. Paneth cells are hypothesized to be required for human ISC maintenance and thus intestinal regeneration. The proposed studies will use in vitro intestinal cell culture systems, known as intestinal organoids, to test how ERBB4 activity regulates expression of human ISC markers and proliferation of ISC. Additionally, these studies will test how ERBB4 signaling might regulate Paneth cell development and survival. Lastly, these studies will assess the effects of ERBB4 on stem cell-driven organoid regeneration after injury (dissociation), and on protection of the ISC from cytokine-induced cell death. Significance and Innovation - There are many intestinal disorders that might ultimately gain therapeutic benefit from a better understanding of the intestinal stem cell and its niche. - The study of complex intestinal organoid structures has increased physiological relevance relative to the study of monolayer cultures. - Though much has been learned recently using intestinal organoids from nonhuman vertebrate models, this approach is innovative in that it is studying the human system. Feasibility and Experimental Design - Reviewers questioned the rationale for focusing on ERBB4, since the preliminary data were not convincing that ERBB4 was the only or primary member of the ERBB protein family that might play a role in the ISC niche. - Reviewers felt that it would be challenging to obtain the human tissues as needed for generating organoids, since the tissue must be used quickly after harvest. - Since organoid cultures aren’t homogeneous, reviewers wondered how organoids at different stages of development would be separated for independent analysis. In addition, differences in human genetic background and pathology would be expected to contribute to heterogeneous outcomes, further complicating analysis. - Some doubts were expressed regarding the relative importance of Paneth cells in maintenance of the ISC niche. For example, it was noted that mice lacking Paneth cells have been generated, and they have relatively normal intestines. Principal Investigator (PI) and Research Team - The PI is a fairly new investigator, had a good publication record as a graduate student, and has been moderately productive since starting his/her own research lab. - The PI has assembled a qualified team with organoid culture expertise, although some concerns were expressed about the team’s ability to carry out specific applications such as viral transduction of organoids. Responsiveness to the RFA - The proposal is responsive to the RFA in that it uses primary human tissue in three-dimensional organoid structures to understand the mechanisms of maintenance of the ISC niche.