Basic Biology II
$1 411 423
The approximately 25 feet of adult intestine is essential for nutrient absorption, secretion of hormones, and as a barrier to infection. Disorders of the intestine including inflammatory bowel disease, mesenteric ischemia, congenital syndromes and trauma, all significantly interfere with intestinal function and in severe cases result in “short-gut” syndromes and effective intestinal failure. Supportive measures are utilized including total parenteral nutrition, in which patients receive all of their nutrition intravenously, or even intestinal transplantation. One goal of the proposed work is to induce human embryonic stem (hES) cells to be reliably converted to intestinal stem cells in culture, for eventual therapy of these disorders. This will be accomplished by addition of hormones, of candidate genes, and by placing the hESC into a nutritive “niche” that promotes intestinal stem cell genesis and function. The generation of human intestinal stem cells from hESC will provide novel therapeutic approaches to the numerous conditions resulting in effective intestinal failure, for which currently available therapies are decidedly suboptimal. A second goal of the proposal is to generate and study colon cancer stem cells from the same intestinal cultures, to better understand the biology of colon cancer and to catalyze the rational development of therapies thereof. Previous studies of cancer stem cells have not allowed their study within a native environment, which will be performed here using our methodology. These studies will reveal temporal patterns of colon cancer progression, identify therapeutic/diagnostic targets, and validate an system for the evaluation of new colon cancer therapeutics.
Statement of Benefit to California:
The proposed research will develop new human embryonic stem cell-based technologies enabling the robust propagation of intestinal tissue its associated stem cells, and cancerous derivatives 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, congentital intestinal disorders and cancer.
EXECUTIVE SUMMARY The overall goal of this effort is to derive both normal and neoplastic intestinal stem cells (ISCs) from human pluripotent stem cells (hPSCs) using a novel, in vitro niche that is meant to reflect the cellular intestinal microenvironment. To achieve these ends, applicant proposes two aims. First, ISCs will be derived from embryonic stem cells (hESCs) and human adipose-derived induced pluripotent stem cells (hiPSCs) using rational reprogramming methods within an explant culture system. In Aim 2, a series of molecular manipulations will be performed in an effort to derive and subsequently characterize intestinal cancer stem cells (CSCs). The reviewers agreed that this proposal addresses a major unsolved problem and if successful, could have a significant impact on the field of regenerative medicine. The combination of an innovative explant culture approach with a strong mechanistic focus was thought to comprise an important first step towards achieving the numbers and types of cells that would be necessary for clinical breakthroughs. While they strongly acknowledged the creativity and compelling rationale, reviewers were somewhat less enthusiastic about the objectives of Aim 2. While some appreciated its novelty, most felt that the information generated from these studies would only incrementally advance our understanding of the processes that lead to oncogenesis. Despite a logical, well written and carefully designed research plan, the reviewers identified a number of issues that were thought to undermine the feasibility of this effort. Importantly, the preliminary finding that hESCs differentiate into non-intestinal epithelium within the explant environment raised questions about the authenticity of this model as an embryonic niche. One reviewer suggested that the explants might in fact be akin to a later or adult stage of development, thereby calling into question whether the appropriate signals would be in place to direct the intestinal differentiation of hPSCs. Beyond this fundamental uncertainty, some reviewers noted that certain necessary cell lines had not yet been produced, cloned or validated. It was not clear that if these strategies should fail whether the back up methods would result in equivalently useful reagents. Another reviewer commented that while the applicant had discussed potential pitfalls and alternative strategies, not all of these appeared realistic given the time-line of the primary approaches. While these weaknesses were significant, the reviewers were compelled to acknowledge the strengths represented by the applicant’s unique experience with the explant system as well as access to a valuable and useful antibody reagent. Reviewers lauded the achievements of the principal investigator (PI) as a highly acclaimed clinician scientist with an outstanding and productive track record. His/her accomplishments were fundamental in establishing the unique model that forms the basis of this investigation. The collaborators and research environment were also considered to be excellent. In summary, this proposal studies the derivation of ISCs from human pluripotent stem cells. Though the reviewers valued the PI’s expertise, productivity, and novel in vitro culture system, their enthusiasm diminished substantially over concerns about impact and feasibility.