Little is known about the resident stem cells in gallbladder and the relationship between the stem cells of the hepato-biliary system. Our goal here was to identify and characterize stem cells in the adult mouse gallbladder. We found that an EpCAM+CD49ffhi sub-population from primary mouse gallbladder can expand from single cells, and exhibits morphogenesis in organotypic culture in vitro. Both parent and clonal cultures were capable of survival and short-term morphogenesis in an adapted in vivo assay. We therefore concluded that EpCAM+CD49ffhi gallbladder cells satisfy the stem cell criteria of clonogenic self-renewal and lineage commitment and represent a gallbladder stem cell population. Last we determined that gallbladder stem cells and IHBD cells expanded in vitro have distinct transcriptomes, suggesting that the cells of the IHBD and EHBD systems are different.
This study is the first to describe the identification and prospective isolation of stem cells from an uninjured mouse gallbladder. Previous reports of stem cells in the EHBD system have focused on injury models (28
) or disease conditions such as biliary atresia (29
). Furthermore, these studies do not distinguish epithelial from non-epithelial cells in their isolation protocols. We used EpCAM to isolate gallbladder epithelial cells thereby preventing contamination by non-epithelial cells. This is especially important as we detect EpCAM-CD49f+ cells in primary gallbladder by both immunohistochemistry and flow cytometry. The isolation and subsequent expansion of EpCAM+CD49f+ cells thereby allows for the definitive identification of resident epithelial stem cells.
We confirmed CD49f as a gallbladder stem cell marker by LDAs and index sorts from primary gallbladder. EpCAM+CD49fhi
cells have a significantly higher CFU readout relative to EpCAM+CD49flo
cells. The low enrichment in CFU readout indicates that additional markers are required to further purify stem cells such that single cells can be isolated and expanded (31
). Therefore, expression of EpCAM and CD49f enriches, but does not select for stem cells. All gallbladder epithelial cells expanded in vitro
were EpCAM+CD49f+. However, these cells exhibited morphological heterogeneity at first expansion, forming flat and glandular colonies. Interestingly, none of the glandular colonies and only a fraction of the flat colonies were capable of serial passage. It appears that the EpCAM+CD49fhi
population in primary gallbladder is itself heterogeneous with only a sub-population of cells capable of self-renewal. We could not identify any additional markers to select for this specific sub-population directly from primary tissue and therefore characterized the stemness of the EpCAM+CD49f+ cells expanded past p0. We determined that the expanded EpCAM+CD49f+ cells can self-renew clonogenically. However, defined protocols for gallbladder epithelial cell differentiation do not exist. In the past, researchers have used collagen gel sandwich culture to observe cyst morphogenesis with rabbit gallbladder epithelial cells (32
). The collagen gel is supplemented with exogenous growth factors such as EGF and TGFβ1. We postulate that our 3D culture system is similar to collagen gel culture in that matrigel is an appropriate growth factor containing extracellular matrix that supports morphogenesis. Cyst formation in our culture is similar in morphology and ultrastructure to that observed before (32
). We also observed dye transport that is reminiscent of a transport function of the gallbladder. In addition, we observed similar morphogenesis in vivo
after transplantation. We chose an ectopic location, as engraftment in the native gallbladder would be technically challenging and the subcutaneous space has been shown to engraft human gallbladder cells (26
). Lee et al. (34
) have shown that gallbladder cells can engraft into the native liver of SCID mice. However, engraftment was significant only with tremendous injury to the liver (retrorsine and partial hepatectomy or carbon tetrachloride treatment) and required very large numbers of cells. For these reasons, we concluded that subcutaneous rather than liver engraftment would be a more apt in vivo
assay. In our hands, the EpCAM+CD49f+ cells only engraft in the short term (2 weeks post transplantation). This short-term engraftment might be due to a lack of growth stimulus in the recipient. Also under physiological conditions, the rate of cell proliferation in the gallbladder epithelium is low (35
). Future studies will determine if long-term engraftment of EpCAM+CD49f+ cells is possible. For now, these data conclusively show that parent and clonal EpCAM+CD49f+ cells can organize into organotypic structures that mimic the morphology, ultrastructure and function of the native gallbladder, both in vitro
and in vivo
Spence et al. (7
) have recently showed that IHBD and EHBD cells develop from separate precursors. However, there are no reports describing their similarities or differences in the adult. We found that expression of CD49f, CD49e, CD81, CD26, CD54 and CD166 was different between primary IHBD and gallbladder cells. The goal of our experiment was to evaluate the differences if any between gallbladder stem cells and IHBD cells. Expanded EpCAM+CD49f+ gallbladder cells (>p0) represent a purer stem cell population than primary EpCAM+CD49fhi
cells. The latter forms both flat and glandular colonies and only a fraction of the flat can self-renew. Therefore, we ran microarray analyses on expanded EpCAM+CD49f+ cells (>p1) and expanded IHBD cells. The major groups of differentially expressed genes were cytochrome P450 genes, glutathione-S-transferase and the solute carrier family genes. Also interferon (IFN)-inducible protein 27 was differentially expressed between gallbladder and IHBD cells. Interestingly, expression of CD54 is known to be immunologically mediated (36
). The immunologic properties of bile duct cells have long been considered. They are the primary site of damage in inflammatory diseases such as primary biliary cirrhosis (37
) and biliary atresia (9
) and in liver allograft rejection (38
). The differential expression of an IFN-inducible protein and CD54 indicates that the immunologic properties of IHBD and gallbladder cells could be different. Studies of IHBD cells are hindered by a technical inability to isolate and expand them from primary tissue (2
). We circumvented this hurdle by using LA7 feeder cells that allow for a robust expansion of IHBD epithelial (EpCAM+) cells. This expansion assay along with the 3D matrigel assay could serve as interchangeable and technically easy tools to study bile duct cells. In all, the complete elucidation of the differences between the IHBD and gallbladder cells belongs to another study, as does the evaluation of the stem cell characteristics of the expanded IHBD cells. The focus of this manuscript was to isolate and characterize gallbladder stem cells.
We postulate that this study will have important clinical significance. Gallbladder stem cells could be used to treat biliary atresia as has been noted with hepatic progenitor cells (40
). These cells could also be reprogrammed into hepatocytes or endocrine cells. There have been recent reports of the differentiation of gallbladder epithelial cells into hepatocytes (34
) and ectopic endocrine cells have been observed in the EHBD cells of Hes1−/−
deficient mice (42
). Gallbladder stem cells might be capable of such plasticity, which along with the ready availability of donor tissue would make them an attractive candidate for cell-based therapy.