Prospective isolation of a bipotential clonogenic liver progenitor cell in adult mice

Abstract

The molecular identification of adult hepatic stem/progenitor cells has been hampered by the lack of truly specific markers. To isolate putative adult liver progenitor cells, we used cell surface-marking antibodies, including MIC1-1C3, to isolate subpopulations of liver cells from normal adult mice or those undergoing an oval cell response and tested their capacity to form bilineage colonies in vitro. Robust clonogenic activity was found to be restricted to a subset of biliary duct cells antigenically defined as-, at a frequency of one of 34 or one of 25 in normal or oval cell injury livers, respectively. Gene expression analyses revealed that Sox9 was expressed exclusively in this subpopulation of normal liver cells and was highly enriched relative to other cell fractions in injured livers. In vivo lineage tracing using Sox9creER T2 -R26RYFP mice revealed that the cells that proliferate during progenitor-driven liver regeneration are progeny of Sox9-expressing precursors. A comprehensive array-based comparison of gene expression in progenitor-enriched and progenitor-depleted cells from both normal and DDC (3,5-diethoxycarbonyl-1,4-dihydrocollidine or diethyl1,4-dihydro-2,4,6-trimethyl-3,5-pyridinedicarboxylate)-treated livers revealed new potential regulators of liver progenitors.

Bone marrow injection stimulates hepatic ductular reactions in the absence of injury via macrophage-mediated TWEAK signaling

“…The clonal colonies when formed contain both hepatocellular and biliary markers, indicating that the isolated and purified HPCs are bipotential and selfrenewing. These markers include MIC1-1C3, identified by Dorrell et al following generation of antibodies to murine DRs (27) and more recently has been shown to select an enriched population of clonogenic HPCs (15). Our findings therefore highlight a therapeutic means by which these host DRs can be directly stimulated and we propose this pathway as a possible mechanism by which autologous BMC therapy may be beneficial in human liver disease.…”

“…The precise characterization of hepatic stem cells and HPCs remains contentious with numerous, often nonspecific markers used to identify this population, although the majority consensus is that HPCs exist within the biliary tree and DRs. Various cells of the DR have been demonstrated to be capable of hepatocellular regeneration (9,14,15,26). Following macrophage transfer we go on to show that accompanying the DR response identified histologically, there is an expansion of a defined Ter119 − /CD31 − /CD45 − /CD24 + /EpCAM + cell population in the healthy livers that contains HPCs.…”

“…Cytokeratins (CK) 7 and 19, EpCAM (Epithelial Cell Adhesion Molecule), Dlk1 (Delta Like Homolog), MIC1-1C3, and Sox9 [sex determining region Y (SRY) box] are all accepted, albeit unspecific, markers of DRs, and have been used to define essential mediators, including many cytokines, which control the DR response to experimental liver injury (5,(13)(14)(15). Among the most recently identified of these paracrine signals is TNF-like weak inducer of apoptosis (TWEAK), a cytokine of the TNF family, which directly stimulates proliferation of DRs in vivo and HPCs in vitro (16,17).…”

In vivo reprogramming of Sox9 + cells in the liver to insulin-secreting ducts

“…Moreover the experiments with the Sox9-CreER; mTmG mice show that they arise from cells labeled for Sox9 expression at the ductal plate stage of development. These are probably small bile ducts (22,23) but the cells of origin might also be hepatoblastlike progenitor cells, thought to be in the periportal region associated with small bile ducts (26,27) or perhaps they derive from the peribiliary glands, which are associated with larger bile ducts and are reported to contain cells expressing markers characteristic of embryonic endoderm (18,19,(28)(29)(30)(31)(32).…”

Flow Cytometric Sorting of Cells from Solid Tissue – Reagent Development and Application

“…Some methods will alter or remove antigens of interest rendering them unrecognizable. We have found, for example, that the antigen recognized by one of our in-house favorite antibodies (MIC1-1C3) is unusually sensitive to digestion with Pronase, a mixture of strong proteases (Dorrell et al, 2011a). It is very important to try to optimize your digestion protocol but bear in mind that, at the end of the day, if you can't get the cells into single-cell suspension then they can't be sorted and studied in isolation.…”