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 invitro 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 subpopulation of cells capable of self-renewal. We could not identify PLX3397 concentration any additional markers to select for this specific subpopulation 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, 33 The collagen gel is supplemented with exogenous growth factors, such as epidermal growth factor (EGF) and transforming growth factor (TGF)β1. We postulate Saracatinib 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 was similar in morphology and ultrastructure to that observed

before.32, 33 We also observed dye transport reminiscent of a transport function of the gallbladder. In addition, we observed similar morphogenesis in vivo after transplantation. We chose an ectopic location, because 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 MCE native liver of severe combined immunodeficiency mice. However, engraftment was significant only with tremendous injury to the liver (e.g., 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 invivo assay. In our hands, the EpCAM+CD49f+ cells only engraft in the short term (2 weeks post-transplantation). This short-term engraftment might be the result of a lack of growth stimulus in the recipient. Also, under physiological conditions, the rate of cell proliferation in the gallbladder epithelium is low.