In addition, both Clone B21 and N12 exhibit the same morphogenesis in Matrigel as the parent (Fig. 5B). We then determined whether the expanded EpCAM+CD49f+ cells could survive and engraft invivo. An ideal location for engraftment would be the native gallbladder. However, because there currently are no protocols that allow for the injection and maintenance of cells in the gallbladder, we attempted engraftment at an ectopic location. buy Apoptosis Compound Library Okumura et al.26 have reported the long-term engraftment of invitro explants of human gallbladder in the subcutaneous space of athymic nude mice. We injected the expanded EpCAM+CD49f+ cells mixed with

Matrigel into the subcutaneous neck region of immunodeficient mice. We observed the formation of cyst-like check details structures in the subcutaneous space 1 week postinjection (Fig. 6A). These cysts consisted of cells organized around a central lumen. Seven of seven (100%) mice injected formed cysts. However, engraftment was short term. Only 1 of 3 (33%) mice exhibited cyst formation at 2 weeks. Similar results were obtained with clonal cultures. We then isolated cells from cysts invivo 2 weeks postinjection and cultured them invitro to test their ability to reinitiate cultures with stem cell properties. Flow cytometry

analyses showed that cells isolated from cysts invivo were EpCAM+CD49f+. These cells reexpanded in vitro on feeder cells, forming colonies morphologically identical to parent and clonal cultures (data not shown), and remained EpCAM+CD49f+ (Fig. 6B). These data indicate that expanded EpCAM+CD49f+ cells survive and engraft invivo while retaining their proliferative ability in vitro. There is evidence indicating that intra- and extrahepatic bile duct cells develop separately.7 To date, there are no reports of the molecular differences—if any—between IHBD and gallbladder cells. We first screened primary IHBD cells with the same antibody panel used for primary gallbladder cells (Supporting Table 1). Most IHBD cells express EpCAM12 and we used EpCAM expression to separate IHBD cells from other

liver cells. Briefly, after liver perfusion of GFP+ mice, the high spin fraction was separated and used to isolate IHBD cells. Interestingly, we found differences in integrin expression, including CD49f GBA3 (Fig. 7A). Other notable markers that showed differences between IHBD and gallbladder cells were CD49e, CD81, CD54, CD26, and CD166 (Fig. 7A). We then determined whether expanded gallbladder cells and IHBD cells were different. IHBD cells are capable of expansion on LA7 feeders, and feeder cells select for EpCAM+ cells (Fig. 7B). In addition, IHBD cells form flat colonies similar to gallbladder cells. The phenotypic profiles of IHBD cells and gallbladder cells converged in culture, and we did not detect any differences using the foregoing panel of antibodies.