The goals of this study were

The goals of this study were to evaluate if human fetal gallbladder contains a clonogenic candidate stem cell population and compare its phenotypic and expression profiles to those of fetal IHBD cells. The evaluation of human fetal gallbladder stem buy SM-164 would have important ramifications for the study of congenital diseases such as biliary atresia (Bassett and Murray, 2008) and gallbladder carcinoma, the most common malignancy of the bile duct system (Miller and Jarnagin, 2008), because of the importance of stem cells in development, tissue regeneration and cancer (Lagasse, 2008). In addition, a comparison of human fetal gallbladder and IHBD cells would further elucidate the ontogeny of bile duct cells and represent the first time that the developmental differences between the human IHBD and EHBD cells have been explored. Stem cells are defined by their ability for single cell self-renewal and lineage commitment (Wagers and Weissman, 2004). We have previously used colony forming assays along with single cell and morphogenesis assays to characterize a resident stem cell population in adult mouse gallbladder (Manohar et al., 2011). In this report, we adapt these assays to the study of human cells. We identify an EpCAM+CD13+CD44+ epithelial subpopulation from primary human fetal gallbladder that can expand in vitro through seven passages, exhibits single-cell self-renewal and engrafts in the subcutaneous space of immunodeficient mice. Last, we found that expanded human IHBD cells and gallbladder cells had distinct phenotypic and expression profiles with many of the predicted functional differences between both cell types mirroring those from our previous report (Manohar et al., 2011). To our knowledge, this is the first report to prospectively isolate a clonogenic epithelial population from human fetal gallbladder and evaluate its genealogy relative to IHBD cells.
Methods
Results
Discussion Here we reported a novel culture system that selects for human gallbladder epithelial cells and supports single cell self-renewal. In a previous study, Kobayashi et al. (1991) used human gallbladder myofibroblasts as feeders to culture gallbladder cells. However, their culture system does not select for epithelial cells and clonal expansion was not reported. Our use of the LA7 feeder cell culture system and an epithelial cell marker (EpCAM) to definitively separate epithelial from mesenchymal and hematopoietic cells, allows for the identification of resident clonogenic cells from gallbladder epithelium. This is especially important as we identified EpCAM−CD13+ and EpCAM−CD44+ cells in primary gallbladder (data not shown). A key characteristic of stem cells is their ability to grow from single cells and differentiate into lineage-committed progeny. And for some stem cell populations clonogenicity in vitro can be used as a surrogate assay for the identification of their in vivo counterparts. Combined expression of EpCAM, CD13 and CD44 highlighted specific heterogeneity on primary gallbladder epithelial cells, allowing for their fractionation into three distinct populations. We found that EpCAM+CD44+CD13+ cells are the most enriched in colony forming ability, exhibit single cell self-renewal and morphogenesis and give rise to cultures in vitro that survived and engrafted in the subcutaneous space of immunodeficient mice. These cells are candidates to represent bona fide gall bladder stem cells in vivo. As there are no specific markers of gallbladder differentiation, we could only evaluate morphogenesis, but not specific differentiation of gallbladder cells. Our in vitro morphogenesis experiments were supported by in vivo experiments, where we observed short-term (≤2weeks) engraftment of expanded gallbladder cells. Importantly, in our previous work with adult mouse gallbladder cells, we also only observed short-term engraftment of the cells (Manohar et al., 2011). This could be for lack of growth stimulus from the recipient animal. Moreover, the rate of self-renewal of gallbladder cells in vivo is known to be low (Lamote and Willems, 1997). We concluded that as the primary EpCAM+CD44+CD13+ cells exhibited clonal self-renewal, expansion in vitro and engraftment in vivo, they represent the most clonogenic cell population from fetal gallbladder identified to date.