¹ StemCells, 525 Del Rey Avenue, Suite C, Sunnyvale, California 94085, USA
² Department of Molecular and Medical Genetics, Oregon Health Sciences University, 3181 S.W. Sam Jackson Park Road, L103, Portland, Oregon 97201, USA
³ Department of Pathology, Texas Children's Hospital, Baylor College of Medicine, Houston, Texas 77030, USA
⁴ Department of Pathology and Developmental Biology, Stanford University School of Medicine, Stanford, California 94305, USA

Correspondence should be addressed to E Lagasse.
E-mail: elagasse@stemcell.net

The characterization of hepatic progenitor cells is of great scientific and clinical interest. Here we report that
intravenous injection of adult bone marrow cells in the FAH-/- mouse, an animal model of tyrosinemia type I, rescued the mouse and restored the biochemical function of its liver. Moreover, within bone marrow, only rigorously purified hematopoietic stem cells gave rise to donor-derived hematopoietic and hepatic regeneration. This result seems to contradict the conventional assumptions of the germ layer origins of tissues such as the liver, and raises the question of whether the cells of the hematopoietic stem cell phenotype are pluripotent
hematopoietic cells that retain the ability to transdifferentiate, or whether they are more primitive multipotent cells.

Bone Marrow Cells Can Cure a Metabolic Liver Disease:

To determine whether bone marrow contains cells that correct liver disease, we used bone marrow transplantation in the fumarylacetoacetate hydrolase (FAH)-deficient mouse, an animal model of fatal hereditary tyrosinemia type I [7]. Mutant mice have progressive liver failure and renal tubular damage unless treated with
2-(2-nitro-4-trifluoro-methylbenzyol)-1,3-cyclohexanedione (NTBC) [8]. We used FAH-/-/129SvJ mice as recipients for the engraftment by bone marrow cells, because this model shows a strong growth advantage of wild-type hepatocytes to repopulate mutant liver [9]. In the first experiment, we lethally irradiated nine female FAH mutants and transplanted each with 1 x 10 ⁶ unfractionated bone marrow cells from male ROSA26/129SvJ mice, wild-type for FAH and transgenic for the Escherichia coli lacZ gene [10]. Three weeks after transplantation,
we discontinued NTBC to allow selection of liver repopulating cells. Four of the nine mice survived the selection period and seemed clinically healthy. At 7 months after transplantation, we killed the mice and analyzed them. b-galactosidase staining of the liver showed donor-derived cells (about 30–50% of the livermass; Fig. 1a) in all four mice. 

Figure 1: Liver histology of the FAH-/- mice 7 months after bone marrow transplantation. FAH-/- mice were transplanted with 1 x 10 ⁶ bone marrow cells from ROSA26/129SvJ mice.

a, Repopulating nodules are detected in the liver with X-gal [10] (blue staining).
b, FAH staining of the nodule. The dark red areas are FAH-positive hepatocytes and are adjacent to an FAH-negative area.
c, Glutamine synthetase staining, showing a completely repopulated region. Original magnification, x 200. Glutamine synthetase staining (red) is limited to zone III hepatocytes surrounding the central vein (CV), whereas zone I cells next to the portal triad (PT) do not express this enzyme.

Hematopoietic Stem Cells Can Differentiate Into Hepatocytes:

To address the issue of the identity of the bone marrow cells that have the potential to generate hepatocytes, we determined whether highly purified and prospectively isolated hematopoietic stem cells (HSCs) could give rise to hepatocytes. HSCs have been rigorously and directly identified [11]; in the BA mouse strain, HSCs represent a rare population of 0.01–0.05% of whole bone marrow that can be reproducibly isolated using a combination of 13 distinct cell surface markers [12-15]. The functional properties of HSCs and their subsets have been established by transplantation into lethally irradiated host mice in conditions in which the progeny of a single stem cell can be identified [16-18]. HSCs are capable of long-term, multi-lineage reconstitution and radioprotection of a lethally irradiated host with an enrichment that mirrors their representation in bone marrow by several thousand-fold.

We isolated HSCs from the bone marrow of normal adult male ROSA26/BA mice (2 months old) by fluorescence-activated cell sorting (FACS) ( Fig. 2). 

Figure 2: Isolation and CD45 expression of mouse HSCs.

a, Phenotypic analysis of adult bone marrow cells and the restricted gates used to sort HSCs.
b, Analysis of the sorted KTLS HSCs. HSCs were sorted a second time directly into an Eppendorf tube for transplantation.
c, CD45 analysis of the sorted HSCs.
Left (b and c), CD45 expression on HSCs.

Most of the mice were engrafted at 2 months with 10–1,000 KTLS HSCs, and the reconstitution level was proportional to the number of KTLS HSCs injected. During the next 4 months, we applied positive selection of the engrafted cells twice to the FAH-/- mutant liver by removing NTBC from the drinking water and restarting the drug when total body weight decreased by more than 30%. We killed surviving mice after the second selection (6 months after HSC transplantation) and analyzed bone marrow, blood and spleen as single-cell suspensions by FACS for donor-specific multilineage reconstitution (B, T and myeloid lineages) of the hematopoietic system (Table 2). This analysis confirmed that the hematopoietic systems of all surviving host FAH-/- mice were engrafted long-term with donor male ROSA26 HSCs. In these experiments, we engrafted HSCs across minor histocompatibility barriers (C57Bl into 129SvJ) [21], which would explain the slightly lower (but not out of range) level of engraftment here compared with the level expected for congenic HSC reconstitution.

We monitored the degree of hepatic engraftment achieved by several criteria. We fixed and stained for b-galactosidase activity the whole median lobe of the liver of most experimental mice to detect any macroscopic nodules. For the rest of the liver, we analyzed serial sections for donor-derived hepatocytes by the presence of b-galactosidase-positive cells using histochemical staining, by the expression of FAH enzyme within the hepatocytes using immunostaining, and by the appearance of male donor cells using fluorescent in situ hybridization of Y chromosome sequences. We detected nodules of b-galactosidase-positive activity in the livers of mice injected with 50–1,000 KTLS HSCs (Fig. 3a). 

Figure 3: Liver histology of FAH-/- mice 6 months after KTLS HSC transplantation.

a, Liver lobe from an FAH-/- mouse with 50 (left) and 1,000 (right) transplanted ROSA26 KTLS HSCs. Repopulating b-galactosidase-positive nodules are shown in blue with X-gal.
b, Transplantation of 1,000 HSCs. Histology of the repopulated liver nodules, shown with staining for X-gal. Left and right, low- and high-power magnification, respectively, of two different nodules. Top, with hematoxylin; bottom, no counterstain.
c, Transplantation of 50 HSCs. Serial section of a liver nodule with staining for X-gal (left) and FAH (right).
d, Transplantation of 1,000 HSCs. Detection of Y-chromosome-positive nuclei in the liver. Serial section of a liver nodule stained for X-gal (top row, left), FAH (top row, right) and fluorescent in situ hybridization (middle row; yellow arrows indicate positive signals in the nuclei of cells). Bottom row, Cell-drop preparation from another mouse liver, showing a Y-chromosome-positive bi-nucleated hepatocyte (left) and non-parenchymal mononucleated cells (right).
e, Primary culture of hepatocytes 2 weeks on mouse fibroblast (STO) feeder cells from a mouse transplanted with 1,000 HSCs. Co-detection of hepatocytes positive for albumin (Alb; left) and X-gal (right).

For our final approach to analyze the hepatic nature of the repopulating nodules derived from HSCs, we compared the expression of hepatocyte and hematopoietic markers in the regenerating nodules that originated from transplantation of hepatocytes [23] and from the transplantation of extrahepatic cells (bone marrow cells and purified HSCs) (Fig. 4). 

Figure 4: Immunofluorescent staining of regenerating hepatic nodules in the FAH-/- mice.

Liver samples were collected 2 months after transplantation of 1 x 10 ⁴ hepatocytes a,
and 6 months after transplantation of 1 x 10 ³ purified HSCs b,
or 1 x 10 ⁶ bone marrow cells c,
Serial sections of frozen liver were stained for b-galactosidase activity (detected as blue with X-gal), fluorescein isothiocyanate (FITC)-conjugated dipeptidyl peptidase IV (CD26), phycoerythrin-conjugated (PE) albumin, FITC-conjugated E-cadherin and PE-conjugated hematopoietic markers (CD45, CD2, CD3, CD4, CD5, CD8, NK1.1, B220, Ter119, GR-1 and Mac-1). Nuclei were counterstained with Hoechst.

HSC-derived hepatocytes expressed the same hepatocyte markers as bone marrow-derived hepatocytes and transplanted hepatocytes. The fact that HSC-derived hepatocytes clustered and created a regional replacement of the diseased parenchyma, as did bone marrow-derived hepatocytes and transplanted hepatocytes, indicates functional involvement of these hepatocytes and the potential utility HSCs might have in liver cell therapy.

HSCs are the Only Bone Marrow Cells to Give Rise to Hepatocytes:

In a second set of experiments, we tested whether HSCs, as defined by the markers c-kit ^high, Lin ^- or Sca-1 ⁺, are the only cells in the adult bone marrow that contain the hepatic progenitors. To avoid excluding any cell populations, we divided bone marrow among c-kit ⁺ or c-kit ^- pools, Lin ⁺ or Lin ^- pools and Sca-1 ⁺ or Sca-1 ^- pools using flow cytometry (data not shown). If hepatic progenitors were expressing these antigens uniformly, hepatic engraftment would be enriched in one fraction and correspondingly depleted in the other. We injected each ROSA26 bone marrow subpopulation intravenously into lethally irradiated FAH-/- mice along with 2 x 10 ⁵ FAH-/- congenic adult female bone marrow cells as a radioprotective dose (Table 3).

After two cycles of selection, only one mouse in each group survived and was analyzed for hematopoietic and hepatic engraftment. To assess hematopoietic reconstitution, we analyzed blood, spleen and bone marrow cells for donor cells. For the determination of liver engraftment, we counted macroscopic hepatic nodules for each mouse analyzed. In addition, we screened 25 serial sections for b-galactosidase-positive donor hepatocytes and confirmed the engraftment by FAH staining. As has been reported, c-kit ^- cells (representing 92.3% of whole bone marrow), Lin ⁺ cells (representing 93.4% of whole bone marrow) and Sca-1 ^- cells (representing 95.8% of whole bone marrow) did not contribute much to long-term multi-lineage hematolymphoid reconstitution [14] (Table 3). Similarly, these populations showed no enrichment in hepatocyte engraftment (Table 3). Both long-term multi-lineage hematopoietic reconstitution and hepatocyte engraftment were the property of cells expressing the markers c-kit ⁺ (7.7% of whole bone marrow), Lin ^- (6.6% of whole bone marrow) and Sca-1 ⁺ (4.2% of whole bone marrow). Therefore, c-kit ^- Lin⁺ Sca-1 ^- cells, which represent 99.9% of the bone marrow, did not have much hematopoietic or hepatic-reconstituting activity. Hepatocyte repopulation was only seen concurrently with long-term multi-lineage hematopoietic reconstitution.

Discussion:

Here we have demonstrated that highly purified HSCs have hepatic as well as hematopoietic reconstitution activity. As few as 50 adult HSCs injected intravenously have the capacity to reconstitute hematopoiesis and give rise to hepatocytes, with intravenous injection of HSCs having about a 10% seeding efficiency in the bone marrow microenvironment [16, 25]. But it is not evident whether the HSCs themselves or their progeny seed the liver. HSCs have been found at low levels in adult mouse liver [26]; perhaps this specific population is involved in hepatic homeostasis. In bone marrow, only the purified KTLS HSC population has the capacity to give rise to hepatocytes.

Bone marrow populations or bone marrow cells enriched for HSCs have the potential to give rise to muscle [27, 28]. In addition, bone marrow stromal cells can differentiate into astrocytes [29] and bone marrow is also a source of primitive mesenchymal cells [30]; it will be necessary to test whether these muscle cells, primitive mesenchymal cells and astrocytes are also derived from HSCs. If resident bone marrow HSCs can form a variety of cell types, they may be more multipotent than the phrase 'pluripotent hematopoietic stem cell' indicates [31]. Studies with rodents indicate that brain cells can turn into blood [32] and that skeletal muscle contains cells that can become hematopoietic [33]. These findings indicate that HSCs could have been itinerant or multipotent cells and might be present in a variety of tissues. The results of classical studies of embryogenesis and its genetic control could be interpreted as showing the stepwise maturation of groups of cells into more and more defined cell fates; for example, the primitive gut endoderm region contains cells that bud off to develop the primitive liver and pancreas [34]. It is also possible that not all the cells on fixed sections are actually progenitors for such examples of organogenesis. What if most of the cells are in fact mainly suitable microenvironments for itinerant stem cells to
invade, in which they respond by providing tissue-specific differentiation? If they are indeed derived from rare stem cells, whether heterogenous or homogenous as to phenotype and function, then the long-held view that the germ layer (mesoderm, endoderm, ectoderm) origin of mammalian organs is known would be in doubt. Each tissue-specific stem cell must be identified, purified and demonstrated to determine its full developmental potential, as well as its germ layer origin.

Finally, two recent studies on human patients [35, 36] have reported that hepatocytes were derived from bone marrow cells, which confirms the previous studies in rodent models. Those two studies did not distinguish whether HSCs, mesenchymal stem cells or as-yet-unknown progenitors residing in the bone marrow were responsible for the liver engraftment. Here, we have shown that HSCs and only HSCs can give rise to hepatocytes. These studies in the mouse provide pre-clinical evidence that perhaps purified human HSCs [37] may similarly be a source of hepatocytes for therapeutic liver repopulation in the appropriate conditions.

In our studies of HSCs and in previous studies using mature hepatocytes [38], the functional and clinical reconstitution of the FAH-/- hosts after termination of NTBC was better with hepatocytes. We do not know whether this difference was qualitative or quantitative. In addition, we still must demonstrate that purified HSCs can rescue the FAH-/- mouse from its lethal metabolic disease. Therefore, we need to explore further the full potential of HSCs for treating liver disorders and the biological function HSCs might have in endogenous repair of liver injury. Nonetheless, the treatment of liver diseases with hematopoietic stem cells may have considerable advantages over the use of hepatocytes themselves. It could provide a relatively easy source of stem cells for liver therapy: HSCs can be obtained from living individuals using a moderately invasive procedure, in contrast to obtaining hepatocytes from cadaveric donors, as with whole-organ collections [39]. Millions of potential donors are
already enlisted in national and international bone marrow donor registries [40, 41]. Moreover, by simultaneously repopulating the hematopoietic and the hepatic systems, the tissue rejection that plagues transplant recipients might be overcome without the lifelong use of immunosuppressive medication. The recent advances in the field of bone marrow transplantation with the use of 'mini-transplants' [42] or hematopoietic chimerism and donor-specific tolerance without irradiation [43] make the application of transplanted HSCs much more probable for a variety of disorders, including liver disease.

Staining of HSCs:

Cells were stained as described [12]. For KTLS cells isolated from 2-month-old ROSA26/B mice (C57Bl/Ka-Thy1.1), the bone marrow cells were incubated with a biotinylated monoclonal antibody specific for Sca-1 (PharMingen, San Diego, California), then positively selected using the magnetic-activated cell separation magnetic bead system (Miltenyl Biotec, Auburn, California). The positively selected cells were stained with phycoerythrin-conjugated lineage markers (PharMingen, San Diego, California), which included RA3-6B2 (B220) for the B-lineage marker; RM2-5 (CD2), GK1.5 (CD4), 53-7.3 (CD5), 53.6.7 (CD8) and 145-2C11 (CD3) for T-cell markers; RB6-8C5 (GR-1) and M1/70 (CD11b, Mac-1) for myeloid markers; PK136 (NK1.1) for natural killer cells; and Ter119 for erythrocytes. The positively selected cells were also stained with fluorescein-conjugated
19XE5 (Thy1.1), allophycocyanin-conjugated 2B8 (c-kit) (PharMingen, San Diego, California) and streptavidin–Cy7APC (PharMingen, San Diego, California). After the final wash, cells were resuspended in a PBS/FCS buffer that contained 1 mg/ml propidium iodide to discriminate between viable and nonviable cells.

Purification of HSCs:

Isolation of HSCs was accomplished using a fluorescence-activated cell sorter (FACSTM Becton Dickinson Immunocytometry Systems, San Jose, California). The FACSVantage SE is configured with argon, krypton and
helium–neon ion lasers. Data parameters were collected in the list mode data file and were analyzed by the software program Flowjo ( http://www.Treestar.com). Pure populations of sorted HSCs were resorted directly into Eppendorf tubes by an automated cell deposition unit using counter mode.

Transplantation procedure:

The FAH-/- recipient mice were lethally irradiated with a total dose of 1,200 rads in a split dose with a 3-hour interval. Then, 1 d later, cells were injected intravenously into the retroorbital plexuses of anesthetized mice
using insulin syringes (Becton Dickinson, Franklin Lakes, New Jersey). Cells were injected at a dose of 100 ul per mouse. The protocol for transplantation of hepatocytes has been described [23].

Histology and Immunohistology:

Liver was embedded in optimum cutting temperature compound and frozen in isopentane. Serial sections 10 and 5 um in thickness, were stained histochemically for b-galactosidase and immunohistochemically with the polyclonal rabbit antibody against FAH (a gift from R.M. Tanguay), albumin (Accurate Chemical, Westbury, New York), dipeptidyl peptidase IV/CD26 (PharMingen, San Diego, California) or E-cadherin (Zymed, South San Francisco, California). For some samples, whole lobes of the liver were fixed in 4% paraformaldehyde at 4° C overnight and stained for b-galactosidase.

Detection of b-galactosidase:

FDG (fluorescein di-—D-galactopyranoside; Molecular Probes) was used as the fluorogenic substrate to detect b-galactosidase by flow cytometry [44]. X-gal was used as the substrate to detect b-galactosidase in sections [45].

Fluorescent in situ hybridization:

Cryostat sections 5 um in thickness were fixed three times, 10 min each, in Carnoy's fixative. The sections were then pre-treated at 37 °C for 30 min in preheated 2x SSC buffer, pH 7.0. Serial ethanol dehydration was done (1.5 min each), and the slides were air-dried at room temperature. Sections were denatured at 65 °C for 2 min in
preheated 70% formamide and 2x SSC buffer, pH 7.0, and were then 'quenched' with ice-cold 70% ethanol for 1.5 min. Serial ethanol dehydration was done again. The mouse Y chromosome probe labeled with fluorescein isothiocyanate (STAR*FISH; Cambio, Cambridge, England) was denatured at 65 °C for 10 min and applied to the sections at 45 °C. The sections were coverslipped and sealed with rubber cement for incubation overnight in a hydrated slide box at 42 °C. The next day, the coverslips were carefully removed in preheated 2x SSC buffer, pH 7.0, at 45 °C. The sections were washed twice in preheated 50% formamide in 2x SSC buffer for 5 min each at 45 °C and were then gently washed twice in preheated 0.1 SSC buffer for 5 min each at 45 °C. Sections were counterstained with Hoechst. For fluorescent in situ hybridization on cell-drop preparations, slides were treated
using the Cambio protocol (www.cambio.co.uk).

Number of hepatocytes estimated per nodule:

To determine the number of hepatocytes per nodule, we consider a nodule as a large sphere and a single hepatocyte a small sphere. Mathematically, the volume of a sphere is calculated by or defined as 4/3pr³. The number of hepatocytes estimated per nodule = 4/3pr³ (r=radius of the nodule)/4/3pr³ (r=radius of
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We thank L. Jerabek for help with the mice, A. Tsukamoto for review of the manuscript, M. Masek for optimizing staining protocols and M. Ferraz for animal care. This work was supported in part by the National Institutes of Health (I.L.W and M.G.) and the American Liver Foundation (X.W).

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