Identification and expansion of a unique stem cell population from adult mouse gallbladder

Abstract

The identification of resident stem cells in the mouse gallbladder is to date, unexplored. In addition, the relationship between adult gallbladder stem cells and intrahepatic bile duct (IHBD) cells is not well understood. The goal of this study was to isolate stem cells from an adult mouse gallbladder and determine if they were unique compared to IHBD cells. By limiting dilution analyses and index sorts, we found that an EpCAM+CD49fhi sub-population from primary gallbladder is enriched in colony forming cells compared to EpCAM+CD49flo cells. EpCAM+CD49fhi cells expressed CD29, CD133 and Sca1 but were negative for lineage markers CD31, CD45 and F4/80. Using a novel feeder cell culture system, we observed long-term (>passage 20) and clonal expansion of the EpCAM+CD49fhi cells in vitro. In a matrigel differentiation assay, EpCAM+CD49f+ cells expanding in vitro underwent organotypic morphogenesis forming ductular structures and cysts. These structures are similar to, and recapitulate a transport function of primary gallbladder. EpCAM+CD49f+ cells also engraft into the subcutaneous space of recipient mice. We compared primary gallbladder and IHBD cells by flow cytometry and found phenotypic differences in expression of CD49f, CD49e, CD81, CD26, CD54 and CD166. In addition, oligonucleotide microarrays showed that the expanded EpCAM+CD49f+ gallbladder cells and IHBD cells exhibit differences related to lipid and drug metabolism. Notable genes that were different are cytochrome P450, glutathione-S-transferase, Indian hedgehog and solute carrier family genes. Conclusion we have isolated an epithelial cell population from primary mouse gallbladder with stem cell characteristics and found it to be unique compared to IHBD cells.

Identification of a candidate stem cell in human gallbladder

“…Recently, Spence et al (8) by using a PDX1-Cre transgenic mouse showed that hepatocytes and IHBD cells descend from Pdx1− cells while the EHBD cells including the ventral pancreas derive from Pdx1+ cells. These data were corroborated by a study in our lab, where we found that adult mouse gallbladder stem cells have a distinct phenotypic and expression profile compared to adult mouse IHBD cells (9). However, the differences between human IHBD and EHBD cells have not yet been explored.…”

“…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 (9). 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.…”

“…Gallbladder and liver samples were incubated with EBSS/10mM EGTA/1% HEPES for 15min at 37C and treated with 1 mg/ml CollagenaseII (Invitrogen, CA) +1mg/ml Hyaluronidase (Sigma) + 100 μg/ml of DNaseI (Roche, IN) for 1–1.5 hrs followed by 0.25%Trypsin /0.1%EDTA (Fisher Scientific, MA) for 30 min to obtain a cell suspension. Cell suspensions were plated on irradiated rat feeder cells as described previously (9). …”

“…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 (9). In this report, we adapt these assays to the study of human cells.…”

Novel surface markers directed against adult human gallbladder

“…The extrahepatic biliary tree (comprised of extrahepatic bile ducts, common duct, cystic duct and gallbladder) shares a common progenitor with the ventral pancreas during embryonic development [1]. In the adult, putative facultative stem/progenitor cells are postulated to exist in injured liver [2–6], pancreas [7], and the extrahepatic biliary system [8–12]. …”