Dorrell C, Erker L, Schug J, Kopp JL, Canaday PS, Fox AJ, et al. Prospective isolation of a bipotential clonogenic liver progenitor cell in adult mice. Genes Dev 2011;25:1193-1203. (Reprinted with permission.)
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 CD45(-)/CD11b(-)/CD31(-)/MIC1-1C3(+)/CD133(+)/CD26(-), 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)-R26R(YFP) 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.
Shin S, Walton G, Aoki R, Brondell K, Schug J, Fox A, et al. Foxl1-Cre-marked adult hepatic progenitors have clonogenic and bilineage differentiation potential. Genes Dev 2011;25:1185-1192. (Reprinted with permission.)
Isolation of hepatic progenitor cells is a promising approach for cell replacement therapy of chronic liver disease. The winged helix transcription factor Foxl1 is a marker for progenitor cells and their descendants in the mouse liver in vivo. Here, we purify progenitor cells from Foxl1-Cre; RosaYFP mice and evaluate their proliferative and differentiation potential in vitro. Treatment of Foxl1-Cre; RosaYFP mice with a 3,5-diethoxycarbonyl-1,4-dihydrocollidine diet led to an increase of the percentage of YFP-labeled Foxl1(+) cells. Clonogenic assays demonstrated that up to 3.6% of Foxl1(+) cells had proliferative potential. Foxl1(+) cells differentiated into cholangiocytes and hepatocytes in vitro, depending on the culture condition employed. Microarray analyses indicated that Foxl1(+) cells express stem cell markers such as Prom1 as well as differentiation markers such as Ck19 and Hnf4a Thus, the Foxl1-Cre; RosaYFP model allows for easy isolation of adult hepatic progenitor cells that can be expanded and differentiated in culture.
The shortage of human donor livers, low engraftment rates, and poor survival of transplanted hepatocytes hamper the use of clinical and experimental hepatocyte transplantation. In healthy organs, liver progenitor cells (LPCs) are generally dormant (or slowly cycling) and are only present in low numbers in different niches of the liver.1, 2 When a liver gets injured and the regenerative capacity of mature hepatocytes and/or cholangiocytes is impaired, these LPCs become activated in humans as well as in animal models of liver disease3 and can replace dysfunctional or damaged parenchymal cells. Because of their high proliferative ability and differentiation potential toward hepatocytes and cholangiocytes, LPCs are considered as an attractive alternative source for cell therapy. However, their isolation remains challenging.4 So far, antibodies recognizing cell-surface proteins, such as cluster of differentiation (CD)133, epithelial cell adhesion molecule (EpCAM), CD49f, and CD24, were used to isolate putative adult LPCs mainly from injured mouse livers.5-9 The use of liver injury models increases the overall yield of LPCs, but gives a mix of dormant and activated LPCs, which complicates the characterization of these cells. An expected “gold standard” for the isolation of adult LPCs has, therefore, not yet been established.
Recently, two elegant reports have shed new light on the identity/biology of LPCs, giving new hope for their prospective use in liver cell replacement therapy.10, 11 First, the investigators describe two novel approaches for the successful isolation of bipotential LPCs from normal and DDC (3,5-diethoxycarbonyl-1,4-dihydrocollidine)-induced mouse livers. Second, they both demonstrate the progenitor features of these populations by clonally expanding and differentiating them to functional mature cells. Third, based on hierarchical clustering of gene-array data, they attempt to describe how LPCs react upon liver injury. From this, they conclude that the LPC response appears to be biphasic: primarily, a set of genes awakens the LPCs from a dormant state, whereas in a second phase, the expression of genes involved in metabolism and motility gets dramatically changed, which further favors the reconstitution of the liver mass.
The Dorrell article is unique in the sense that the investigators isolated LPCs from healthy livers and at several time points during liver injury using the monoclonal antibody, MIC1-1C3 (macrophage inhibitory cytokine-1-1C3), which is specific for duct cells.12 It allowed them to compare the gene-expression profile of dormant LPCs with activated LPCs.11 In another approach, Shin et al. circumvented the need for LPC-specific antibodies by using a transgenic mouse engineered to express yellow fluorescent protein (YFP) whenever the transcription factor, Foxl1 (Forkhead Box l1), was expressed (Foxl1Cre;Rosa YFP). Because Foxl1 is only expressed in activated LPCs,13 they could compare gene-array data from LPCs isolated at different time points during DDC treatment. LPCs were separated based on their MIC1-1C3 and YFP positivity from other nonparenchymal cells by flow cytometry for further analysis (Fig. 1A). Both reports are noteworthy because LPCs were isolated at different time points of liver injury and both demonstrate that isolated LPCs can be clonally expanded, even up to 15 passages using conditioned media from E14,5 liver cells.10, 11 It would now be a great advantage to identify those factors that allow the expansion of the progenitor cells. Furthermore, both studies unequivocally show that the isolated LPCs are bipotent by in vitro differentiation of a clonally expanded LPC (MIC1-1C3+ or Foxl1+) toward a cholangiocytic and hepatocytic cell type, refuting the existence of two unipotent LPCs.
Recently, Okabe et al. demonstrated that EpCAM (TROP1) is expressed both in cholangiocytes of healthy mouse livers and in oval cells (i.e., activated LPCs) when mice were fed with DDC containing chow. Its related protein, TROP2, is expressed exclusively in oval cells and not in the healthy liver.8 Foxl1, like TROP2, also appears to be an oval cell and not a dormant LPC marker. However, for Foxl1, it is tempting to address a function during LPC activation, because earlier studies using Foxl1−/− mice showed that Foxl1 promoted liver repair after bile-duct ligation-induced liver injury.14 Giving DDC chow to Foxl1−/− mice and determine whether they still exhibit a normal oval cell response is an obvious way to test this hypothesis. Though the MIC1-1C3 antibody can be used to identify dormant LPCs, it is not exclusive for the liver. Its expression in the pancreas11, 12 suggests that MIC1-1C3 might be a common marker for stem cells within endoderm-derived digestive organs, joining Sox915 and Sox17.16
Transcriptome profiling of dormant and activated LPCs undertaken by Shin et al. revealed drastic changes in gene expression of the isolated LPCs at different times of the injury. The wealth of data generated by these experiments need further analysis, but bioinformatic pathway analysis already allowed the investigators to identify processes regulated during LPC activation (illustrated in Fig. 1B). Importantly, although the isolation strategy of the LPCs was different, both studies identified similar relevant pathways. It is not surprising to find that LPCs overexpress drug metabolism and defense response genes because they have to survive several insults during the organism's life. It also makes sense to observe that, in their dormant state, LPCs have low expression of cell-cycle–related genes, compared to their counterpart during injury. Similarly, the overrepresentation of pathways involved in the remodeling of the LPC niche is conceivable because of the necessity of the progeny to be liberated from the niche. More advanced analysis of the data sets would certainly reveal new potential regulators of LPC activation or stemness.
We now look forward to reports that will use a combination of LPC cell-surface markers, such as EpCAM, Trop2, CD133, Dlk1, CD49f, and MIC1-1C3, to isolate LPCs from healthy and injured livers. Cell-surface markers combined with functional characteristics of LPCs, such as overexpression of pumps17 or aldehyde dehydrogenase activity,18 could further specify this population. Finally, the report by Shin et al. clearly has set the stage for many investigators to use transgenic mice for the isolation of LPCs. Good candidates for such studies are the already published reports on Sox9-Cre15 and CK19-Cre mice.19 Though the use of these mice provides the field with elegant tools to further characterize LPCs, we are still in need of strategies to isolate LPCs from human tissues to verify the findings obtained in mice.