Distinction Between Hematopoietic and Hepatopoietic Progenitor Cells.
The fetal liver at ED12.5 provides an ideal opportunity to study common and distinct markers of HSC versus FLEC, because both of these cell types are present at this time during liver development. We studied antigens known to be expressed by HSC that have also been reported to be expressed on FLEC, such as CD343, 4 and c-Kit,3, 6 or on adult liver oval cells, such as CD45, CD34, Sca-1, and c-Kit.7–10, 12–14 Of these markers, only Sca-1 was clearly detected in both the hematopoietic and epithelial cell fractions. Sca-1 is a frequently used marker for enrichment and characterization of murine HSC.34 It has been shown to be required for the regulation of HSC self-renewal and for development of committed progenitor cells.35 In previous studies, Sca-1 has also been described in nonhematopoietic progenitor cell compartments, such as mammary gland epithelial progenitor cells,36 adult cardiac progenitors,37 and lung epithelial–specific progenitors,38 so that its expression in FLEC is not surprising.
C-Kit, the classical marker for HSC, was not expressed by ED12.5 FLEC, although a low percentage of c-Kit+ epithelial cells cannot be excluded. Our results are consistent with those of Suzuki et al., who reported that endodermal stem cells in the mouse fetal liver are c-Kit- at ED13.5,18, 19 and Tanimizu et al.,24 who reported that Dlk+ FLEC isolated from ED14.5 murine fetal liver are c-Kit−. However, hematopoietic markers CD34 and CD45 were not expressed on mouse ED12.5 FLEC, in contrast to their expression on mouse oval cells.14
The percentage of epithelial cells, as defined by FACS analysis after staining for AFP or ALB, was approximately 2.5% of total cells, a percentage similar to that originally described by Sigal et al.16 in ED15 rat fetal liver. On cytospins, all AFP+ cells were also PanCK+ and vice versa. Using FACS analysis and double-immunostaining for the hepatic epithelial-specific cytoplasmic markers AFP and ALB and surface markers Liv2, E-cadherin and Dlk provided a definitive approach to characterize epithelial cell populations in the fetal liver and proved to be useful in separating these cells from HSC.
Liv2 has been identified as a specific marker for epithelial cells in the ED9.5 to ED12.5 murine fetal liver by Watanabe et al.22 and is not expressed in other tissues. Terai et al.39 used Liv2 to show transdifferentiation of transplanted bone marrow cells into hepatocytes, but the physiological role of Liv2 has not yet been determined. In our study, we demonstrated by double-labeling immunocytochemistry on cytospins, as well as in FACS analysis, that Liv2 is a highly specific surface marker for AFP+/ALB+ FLEC.
E-cadherin has been used previously by Nitou et al.23 as a selection marker for epithelial cells in the murine fetal liver, obtaining 95% purity by microbead technology. Unfortunately however, these investigators did not perform liver repopulation studies to demonstrate the in vivo potential of their enriched FLEC. In the fetal liver, staining for E-cadherin detected all AFP+ cells, and using E-cadherin in FACS sorting, we obtained up to 95% purity and a viability >90% by trypan blue dye exclusion. These cells were viable in cell culture and yielded high levels of repopulation after their transplantation.
Dlk is expressed from ED10.5 to ED18.5 in the murine fetal liver and disappears in neonatal and adult liver. Tanimizu et al.24 used Dlk to isolate epithelial cells from ED14.5 murine fetal liver by Automacs and FACS sorting, and Dlk was recently described as a marker for a subset of oval cells in hepatocytic differentiation in the 2-AAF/PH and retrorsine/PH rat models.40, 41 However, Dlk is not specific for epithelial lineages, as it is found at ED13.5 also in pituitary, lung, vertebra, and tongue.42 Dlk-null mice show no apparent defects in liver formation or hematopoiesis.43 We detected Dlk by double-label immunocytochemistry on cytospins and in FACS analysis, showing that ALB+/AFP+ cells are Dlk+ and vice versa, and demonstrating that Dlk is specific for epithelial cells in the mouse fetal liver.
The hallmark feature of a progenitor or stem cell is its ability to repopulate an organ or tissue with functionally differentiated progeny. Up to now, the in vivo differentiation potential of immuno-enriched murine fetal liver cells has been difficult to show. Suzuki et al.18 demonstrated small hepatocytic clusters after transplantation of sorted Ter119−/CD45−/c-Kit−/CD49f+/CD29+ cells in retrorsine/CCl4-treated recipients,18 and Tanimizu et al.24 reported scattered GFP+/ALB+ cells after transplantation of Dlk+ cells into the Jo1/Fas model.24 Crude EGFP-marked murine fetal liver cells also showed clusters after transplantation into uPA transgenic mice, but repopulation was reported to be only 1% to 2%.44 In the current study, using the DPPIV cell transplantation model, we obtained up to 80% repopulation 4 months after transplantation of crude mouse fetal liver cells, proving engraftment, differentiated hepatocyte function, and replacement of whole liver lobules. In addition, we show large hepatocytic clusters after transplantation of purified ALB+/AFP+ murine FLEC, demonstrating their high proliferative potential and repopulation capacity in vivo. We also have demonstrated the hepatocytic phenotype of the repopulating cells by 4 different methods: enzyme histochemistry for DPPIV, immunohistochemistry for ALB, and enzyme histochemistry for G6P and glycogen. Suzuki et al.19 detected bile ducts after transplantation of cultured cells in the DAPM model, and Strick-Merchand et al.45 showed donor-derived bile ducts after transplantation of fetal liver epithelial cell lines into uPA transgenic mice. However, the retrorsine/CCl4 liver injury model favors hepatocytic proliferation and differentiation, and in the present study, we had only limited evidence for donor-derived bile ducts.
We also observed extensive repopulation of the recipient's liver sinusoids by transplanted endothelial cells, as determined by double-staining with DPPIV and CD31 (PECAM-1). The striking relation between the repopulation by endothelial and hepatocytic cells suggests a possible synergy, cooperation, or at least common mechanisms during engraftment between epithelial and endothelial cells. Hoppo et al.21 identified a mesenchymal cell fraction in the murine fetal liver that facilitated the in vitro culture of isolated epithelial cells, and this or a related cell fraction might also facilitate the engraftment of isolated epithelial cells after transplantation.
In conclusion, we characterized a population of ED12.5 murine FLEC that is essentially homogeneous for all analyzed markers (AFP, ALB, PanCK, Liv2, E-cadherin, Dlk, Sca-1), can be sorted with specific surface markers to high purity (95%), and can differentiate in vivo into mature, functional hepatocytes with high liver repopulation capacity. This cell population is clearly distinct from the abundant hematopoietic populations present at this stage of liver development. The in vivo properties of ED12.5 FLEC suggest that they will be particularly advantageous for liver repopulation studies in a wide variety of pathophysiologic states and in mouse models of human liver diseases.