In order to unambiguously demonstrate that HSCs could give rise to both blood and hepatocytes, we decided to follow the progeny of a single prospectively isolated HSC after transplantation into lethally irradiated host mice. Single side population (SP) CD45+ cells from CD45.2 Rosa26 mice were transplanted into irradiated CD45.1 congenic recipients; hematopoietic and hepatic engraftment was analyzed in these primary hosts. Subsequently, HSC-derived BM cells from the primary hosts were transplanted into mutant secondary recipients for subsequent liver-engraftment analysis.
As previously reported (12
), approximately 25% of primary single-HSC recipients showed long-term and multilineage hematopoietic chimerism. Four mice with higher than 60% single-cell-derived blood engraftment were selected for liver-engraftment analysis. Two of these mice were treated with the hepatotoxin 3,5-dietoxycarbonyl-1,4-dihydrocollidine (DDC) (18
). Fourteen months after transplantation, liver samples were examined by X-gal staining. We detected donor cells displaying a characteristic hepatocyte-like morphology at frequencies of 1 in 300,000 and 1 in 150,000 for the noninjured and DDC-treated mice, respectively (Figure A). Donor hepatocyte-like cells were found primarily as isolated cells, and, in a few cases in the DDC-treated group, as clusters of two and, in one case, three cells (data not shown).
Figure 1 Hepatic differentiation after transplantation of a single HSC. Fourteen months after transplantation of a single β-gal+ HSC, random liver sections were analyzed by X-gal staining. (A) Cells with hepatocyte morphology (large cells with (more ...)
In order to more rigorously assess the hepatic potential of HSCs, BM cells derived from the single HSC were isolated by flow cytometry on the basis of their expression of the CD45.2 allele and transplanted into lethally irradiated recipients that were deficient in expression of fumarylacetoacetate hydrolase (FAH), an essential liver enzyme (19
mice can be maintained on the drug 2-(2-nitro-4-trifluoromethylbenzol)-1,3-cyclohexanedione (NTBC), and transplantation of whole BM (WBM) cells has previously been shown to rescue this deficiency (10
). In the absence of NTBC, normal hepatocytes possess a remarkable selective growth advantage and can repopulate a mutant liver. In order to maximize survival of BM-recipient mice, NTBC therapy was intermittently reinstituted and withdrawn. After verification of hematopoietic engraftment, 4–7 months after transplantation, the Fah–/–
transplant recipients were sacrificed and their livers analyzed histologically. Whole-mount X-gal staining of liver lobes for β-gal+
cells originally derived from the single HSC revealed extensive liver repopulation comprising about 40–60% of the hepatic parenchyma (Figure B). Hepatic engraftment, in agreement with previous reports (10
), appeared oligoclonal in nature, with approximately 200–400 β-gal+
nodules of repopulation per mouse liver, similar to the prevalence of repopulation clusters observed after WBM transplantation. Histochemical analysis of livers of transplanted mice revealed extensive areas of cells with hepatocyte morphology expressing both the hepatocyte-specific FAH enzyme and β-gal (Figure , C–E). Furthermore, serum biochemical measurements of liver function were taken. Transplanted mutant mice showed nearly wild-type levels of all functional indicators analyzed (Table ). These results strongly indicate that cells derived from a single HSC can produce, in addition to hematopoietic cells, fully functional hepatocytes and correct a metabolic disease.
Functional parameters of single-HSC_derived hepatocytes
Although these results indicate that cells derived from a single HSC can produce functional hepatocytes, it was unclear whether the HSC itself or its differentiated hematopoietic progeny fused with the host hepatocytes. In order to test whether mature hematopoietic cells of the lymphoid lineage could act as fusogenic partners for hepatocytes, we transplanted 1 ∞ 106
WBM cells from mice deficient in both recombinase-activating gene 2 (Rag-2) and the common cytokine receptor γ chain (γc) into Fah–/–
hosts. Absence of both Rag-2 and γc in mice results in the complete absence of circulating B, T, and NK cells, but normal numbers of myeloid and erythroid progeny (21
). Four weeks after BM transplantation, NTBC withdrawal was initiated in order to select for FAH+
hepatocytes. Transplanted mice were able to survive in the absence of NTBC and were maintained without the drug for more than 5 months. Immunohistochemical staining of liver sections from these mice revealed typical FAH+
nodules throughout the liver parenchyma (data not shown). The kinetics of repopulation and the number of FAH+
nodules per section (Table ) in these mice were no different from those in mice transplanted with normal BM. These data suggest that cells of the lymphocyte lineage are not required as hepatocyte fusion partners.
Contribution of different hematopoietic lineages to hepatocytes in the FAH model
Next we wanted to evaluate the hepatic contribution of mature hematopoietic cells of the myeloid lineage. In order to trace myeloid cells in vivo, we used Cre/lox recombination, wherein Cre recombinase, specifically expressed in myeloid cells under the endogenous regulatory sequences of the lysozyme-M (LysM) gene (22
), irreversibly unblocks expression of a β-gal reporter (R26R) (23
), thus marking all myeloid cells and their downstream progeny. The LysM locus in mice is exclusively active in hematopoietic cells of the myelomonocytic lineage, being expressed moderately in committed myeloid progenitors and expressed highly in mature macrophages and neutrophils. We transplanted 1 ∞ 106
BM cells from mice that were transgenic for both the LysM-Cre gene and the R26R gene (LysM-Cre/R26R), and that also carried the CD45.2 allele, into lethally irradiated CD45.1 Fah–/–
recipients. The degree of donor-derived hematopoietic chimerism in these hosts averaged 85% ± 2.9% in their peripheral blood. In order to verify the myeloid specificity of LysM-Cre expression, donor CD45.2 blood cells were analyzed for β-gal expression. Whereas only 2–8% of circulating B cells (B220+
) and 1–6% of T cells (CD3+
) expressed β-gal, more than 70% of granulocytic (Gr-1high
) and 80% of monocytic (Mac-1high
) cells were β-gal+
Figure 2 Myeloid-derived hepatocyte regeneration in Fah_/_ hosts. (A) Cytofluorimetric analysis of peripheral blood from FAH mutant mice transplanted with LysM-Cre/R26R BM. The flow profiles shown were previously gated on CD45.2+ cells in order to restrict (more ...)
Six months after transplantation, all Fah–/–
recipient mice were NTBC independent; thus, they were sacrificed and analyzed for donor-derived hepatic contribution by X-gal whole-mount staining of liver lobes. As a positive control, staining of livers of Fah–/–
mice transplanted with WBM from a ubiquitously expressed β-gal reporter (Rosa26 mice; ref. 24
) revealed widespread engraftment with a typical nodular morphology (Figure B). In these Rosa26 WBM recipients, we estimated the average number of β-gal+
nodules per liver to be 279 ± 38 (Table ). Analysis of livers of LysM-Cre/R26R WBM recipients also demonstrated extensive hepatic repopulation with β-gal+
myeloid-derived clusters (Figure B). The number of blue nodules was 247 ± 45 (n
= 4), similar to the control values. Further confirmation of the hepatic nature of the repopulation nodules was established by FAH immunohistochemistry (Figure C).
It was recently reported that the LysM promoter used in our experiments could also be active in a small number of pluripotent HSCs (25
). Thus, it could be possible that HSCs were still directly responsible for the generation of hepatocytes. In our study, only 3–8% of Lin–
SP cells from LysM-Cre/R26R mice were positive for β-gal activity (data not shown). If HSCs were solely responsible for the entire BM hepatogenic activity, then we would only expect 3–8% of the number of marrow-derived nodules to be lacZ+
in our lineage-tracing experiment. Clearly this was not the case; the same number of lacZ+
nodules was obtained whether the transplanted BM was derived from LysM-Cre/R26R or constitutive Rosa26 mice. Furthermore, in the recipients of LysM-Cre/R26R BM, costaining of liver sections for FAH and lacZ revealed that the majority of nodules are positive for both markers (data not shown), thus demonstrating that all hepatocytes are derived from LysM-expressing progenitors. Thus, although our experiments do not directly rule out that a few HSCs might be contributing to hepatocyte regeneration, they directly demonstrate that the great majority of this contribution is through a myeloid cell intermediate.
We designed an additional experiment to confirm the myeloid origin of the repopulating nodules. For this experiment we transplanted LysM BM into Fah–/–
recipients that also carried an allele for the R26R reporter. In this context, only hematopoietic cells actively expressing high levels of Cre at the time of spontaneous fusion with hepatocytes would activate expression of β-gal in Fah–/–
/R26R host nuclei. Since expression of Cre in HSCs seems to be oscillatory and transient and at much lower levels than in myeloid cells (25
), this experiment would more effectively rule out contribution by non-myelomonocytic progenitors. As expected, histochemical analysis of two livers of Fah–/–
/R26R recipients 5 months after transplantation revealed widespread repopulation by fused β-gal+
hepatic nodules (Figure B) that were also positive for FAH expression (Figure D). The number of nodules in these livers also correlated very well with that in mice transplanted with Rosa26 BM (Table ), indicating that the great majority of hepatic nodules in the FAH model arise from random fusion of myeloid cells.