Functional analysis and molecular profiling of a rare mouse adult pancreas subpopulation with stem cell features.

The existence of stem cells in the adult mouse pancreas has not been documented despite evidence suggesting the presence of such a population. Putative progenitor cells have been isolated from the centroacinar component based on their high aldehyde dehydrogenase (Aldh) enzymatic activity but the lack of a specific molecular marker has not allowed their characterization. It has been shown previously in the lab that the mitochondrial aldehyde dehydrogenase 1b1 (Aldh1b1) gene is expressed in all embryonic pancreas progenitors and its expression in the adult mice persists only in rare elongated cells with centroacinar morphology. The number of Aldh1b1+ cells expanded significantly following pharmacological beta or acinar cell ablation. In this thesis I introduced Aldh1b1 as a novel exclusive marker for centroacinar cells. I established an Aldh1b1 lineage tracer line and used it to determine whether Aldh1b1+ cells expand and differentiate to other pancreatic cell types during pancreas homeostasis and following regeneration. I found that the Aldh1b1+ cell progeny expands over time and contributes to the endocrine lineage, as either alpha or beta cells as well as to the acinar and duct lineages under homeostatic conditions. These data suggested that Aldh1b1+ cells are mostly quiescent and self-renew in vivo. To define their molecular signature we performed single cell RNA sequencing. This revealed two distinct in vivo differentiation routes of these cells and suggested possibilities to isolate stem and committed progenitor cell populations using cell surface markers and maintain them or guide their differentiation using combinations of extracellular signals. Additionally, I found that adult pancreas Aldh+ cells can form self-renewing spheroids in vitro and this ability segregated exclusively with Aldh1b1 expressing cells. Spheroids can be passaged for at least 11 passages over 60 days; they retain Aldh1b1 expression and express the ductal marker Ck19 and progenitor markers such as Sox9 and Pdx1. To investigate the functional role of Aldh1b1 in the maintenance and expansion of adult pancreas progenitor cells I established an Aldh1b1 conditional knockout mouse line and subsequent experiments showed that Aldh1b1 activity is important for the expansion of adult pancreas progenitors in vitro. In summary, we have identified earlier unknown bona fide stem cells in the adult mouse pancreas, quantified their contribution during pancreas homeostasis and defined their molecular signature. Furthermore, we can isolate these stem cells and expand them as spheroids, thus establishing a system to study and manipulate them in vitro. The findings of this thesis open new research venues regarding pancreas regeneration and, quite possibly, pancreatic cancer development

Aldh1b1 expression defines progenitor cells in the adult pancreas and is required for Kras-induced pancreatic cancer.

The presence of progenitor or stem cells in the adult pancreas and their potential involvement in homeostasis and cancer development remain unresolved issues. Here, we show that mouse centroacinar cells can be identified and isolated by virtue of the mitochondrial enzyme Aldh1b1 that they uniquely express. These cells are necessary and sufficient for the formation of self-renewing adult pancreatic organoids in an Aldh1b1-dependent manner. Aldh1b1-expressing centroacinar cells are largely quiescent, self-renew, and, as shown by genetic lineage tracing, contribute to all 3 pancreatic lineages in the adult organ under homeostatic conditions. Single-cell RNA sequencing analysis of these cells identified a progenitor cell population, established its molecular signature, and determined distinct differentiation pathways to early progenitors. A distinct feature of these progenitor cells is the preferential expression of small GTPases, including Kras, suggesting that they might be susceptible to Kras-driven oncogenic transformation. This finding and the overexpression of Aldh1b1 in human and mouse pancreatic cancers, driven by activated Kras, prompted us to examine the involvement of Aldh1b1 in oncogenesis. We demonstrated genetically that ablation of Aldh1b1 completely abrogates tumor development in a mouse model of Kras(G12D)-induced pancreatic cancer