Intestinal homeostasis is maintained by the robust activity of intestinal stem cells (ISC) which allow complete regeneration of the intestinal epithelium every 5-7 days. Recent functional studies have established the existence of two types of ISC, a crypt base columnar (CBC) cell expressing LGR5 and prominin-1, and a distinct ISC population expressing Bmi1, located higher in the crypt at approximately the +4 position and restricted to the small intestine. Despite potent stem cell attributes of the Bmi1+ cells in lineage tracing studies, their regulation, relationship to LGR5+ cells and transcriptome have remained poorly defined. The overall goal of this application is the analysis of the Bmi1+ lineage in vivo and in vitro, using recently developed Bmi1-CreER knock-in mice, our robust methodology for small intestinal culture, and R-Spondin1 and Dkk1 adenoviruses allowing gain- and loss-of-function Wnt manipulation in vivo. Accordingly, Aim 1 will investigate the regulation and functional relevance of the Bmi1+ lineage during intestinal regeneration. The number and fate of Bmi1+ cells will be examined during regeneration in response to radiation or R-spondin1, both in vivo and in vivo using the Bmi1-CreER mouse or cultures derived thereof. Importantly, the functional contribution of the Bmi1+ ISC to intestinal regeneration after radiation or R-spondin treatment will be assessed by diphtheria toxin-mediated lineage ablation.
Aim 2 will address the important question of relationships between the Bmi1+ and LGR5+ ISC lineages. Fate mapping of the Bmi1 lineage will be performed in vivo and in vitro to formally demonstrate if Bmi1+ cells or their progeny can express LGR5. Culture of isolated Bmi1+ cells from Bmi1-CreER mice will be performed both within and without an ISC niche to explore if Bmi1+ cells can give rise to LGR5+ cells in vitro.
In Aim 3, transcriptional profiling of Bmi1+ cells will be performed and compared to the published LGR5+ transcriptome and target validation performed exploiting in vitro intestinal culture. Finally, Aim 4 will explore the ex vivo expansion and transplantation of Bmi1+ ISC. Our ISC niche-dependent intestinal culture system, as well as niche-free systems will be used to expand Bmi1+ cells ex vivo, followed by single cell or population transplantation in vivo. Questions of plasticity will also be addressed with introduction of small intestine Bmi1+ cells into the colon both in vitro and in vivo.
The intestine possesses highly active stem cell populations with therapeutic relevance to diverse conditions including inflammatory bowel diseases, metabolic disorders and cancer. Here, the Bmi1+ intestinal stem cell population will be investigated with regards to regenerative potential both in vivo and in vitro.
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