Department of Developmental and Molecular Biology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA
* Author for correspondence (e-mail: graf{at}aecom.yu.edu)
Accepted 29 October 2004
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SUMMARY |
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Key words: Hematopoietic plasticity, Hepatocyte development, Endothelial cell development, Hematopoietic stem cells, Lineage tracing
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Introduction |
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Based on transplantation experiments into irradiated hosts, it has been
proposed that hematopoietic stem cells (HSCs) also possess developmental
plasticity and can trans-differentiate into cells of all three germ layers
(Graf, 2002;
Herzog et al., 2003
;
Wagers and Weissman, 2004
).
Two non-hematopoietic cell types for which this has been documented after
transplantation of single HSCs are hepatocytes and endothelial cells
(Camargo et al., 2004
;
Grant et al., 2002
).
Hepatocytes are the progeny of hepatoblasts, endodermal derivatives specified
in the mouse around midgestation (Zaret,
2002
), which co-localize with HSCs to the fetal liver
(Godin and Cumano, 2002
;
Suzuki et al., 2000
). In
FAH-deficient mice, a well-studied model of chronic liver failure
(Lagasse et al., 2000
), blood
to liver conversions have been shown to be mediated by fusion of macrophages
with hepatocytes (Camargo et al.,
2004
; Willenbring et al.,
2004
). However, direct trans-differentiation of HSCs into
hepatocytes has recently been reported for wild-type mice following liver
injury and HSC transplantation (Jang et
al., 2004
). Finally, after transplantation of human HSCs into
fetal sheep, as much as 20% of hepatocytes were reported to be of donor origin
(Almeida-Porada et al.,
2004
).
Endothelial cells, like hematopoietic cells, are of mesodermal origin. The
development of these two cell types, which share the expression of several
marker genes, is regulated by overlapping sets of transcription factors
(Bloor et al., 2002;
Ema et al., 2003
;
Oettgen, 2001
). Definitive
HSCs arise from vascular endothelial cells of the dorsal aorta
(de Bruijn et al., 2002
;
Jaffredo et al., 1998
;
Nishikawa et al., 1998
). A
common precursor of endothelial and hematopoietic cells, the hemangioblast,
has been identified in embryonic stem cell cultures
(Choi et al., 1998
), further
underscoring the close relationship of these two lineages. Recently, it has
been reported that fetal as well as adult HSCs transplanted into irradiated
hosts can generate endothelial cells and thus appear to function as
hemangioblasts (Bailey et al.,
2004
; Grant et al.,
2002
; Tamura et al.,
2002
).
These studies raise the possibility that hematopoietic contributions to
hepatocytes and endothelial cells occur as normal development processes. This
may happen particularly in the fetal liver where hematopoiesis, vasculogenesis
and hepatocyte differentiation coincide. Alternatively, hematopoietic
plasticity might also play a role in adults as a mechanism of tissue
maintenance (Blau et al.,
2001). To test these possibilities, we designed a mouse model
based on Cre/loxP technology in which essentially all hematopoietic cells,
including fetal and adult HSCs, are irreversibly labeled by YFP expression
('vav ancestry mice'). We analyzed the hepatocyte and endothelial cell
compartment of these mice in comparison with lysozyme ancestry mice, in which
most myelomonocytic cells and to a lesser degree all other adult hematopoietic
cell types, including HSCs, irreversibly express YFP
(Ye et al., 2003
).
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Materials and methods |
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Mice
PCR primers used to identify mice containing vav-Cre were:
5'-CCATGGCACCCAAGAAGAAG-3' (Vav1) and
5'-GCTTAGTTTTCCTGCAGCGG-3' (Vav2), which give a product of 2.6 kb
that spans from the start codon of Cre to the stop codon of YFP. PCR reactions
were carried out with 100 ng of tail DNA for 35 cycles (30 seconds
denaturation at 94°C, 60 seconds annealing at 60°C and 150 seconds
elongation at 72°C) and the products analyzed on a 1.5% agarose gel. Vav
ancestry mice were obtained by crossing vav-Cre transgenic mice with
ROSA26R-lacZ and ROSA26R-YFP reporter mice, respectively
(Soriano, 1999;
Srinivas et al., 2001
). The
phenotypes of the offspring were determined by examining ear clips under a
Nikon Eclipse E600 fluorescence microscope equipped with a filter to visualize
YFP (Chroma Technology, Rockingham, VT #41028). Mice with YFP+
cells of dendritic morphology were identified as `vav ancestry mice'; mice
with patches of YFP+ non-hematopoietic cells as `chimeric control
mice'; and mice with no YFP+ cells as negative control mice. To
obtain mice homozygous for the reporter gene, female vav ancestry mice were
crossed with ROSA26R males. The lysozyme ancestry mice used for this study
were homozygous for ROSA26R-YFP and either homozygous or heterozygous for
LysM-Cre. They were genotyped as described
(Ye et al., 2003
). Mice were
bred and maintained in accordance with guidelines from the Institute for
Animal Studies of the Albert Einstein College of Medicine.
Bone marrow reconstitution
Mononucleated bone marrow cells from vav ancestry mice were prepared by
sterile flushing of the tibia and femur bones as described before
(Ye et al., 2003). A sample of
these cells (2 x106) was injected into the tail vein of
non-transgenic littermates lethally irradiated with two doses of 600 rad in
the 24 hours before the transplantation. The degree of donor engraftment was
determined by flow cytometry of peripheral blood cells 4 weeks after
transplantation and at the time when the animals were sacrificed (all mice
used in this study had a donor contribution of at least 90% and 95% at these
two time points, respectively).
Liver injury
Adult mice were injected intraperitoneally with a single dose of carbon
tetrachloride (CCl4) solution (10 µl per 3 g bodyweight of a 30%
solution in plant oil, kindly provided by D. Shafritz). Blood plasma was
isolated 30 hours post injection and at the time of sacrifice. ALT levels were
determined by the Chemistry Laboratory of the Jacobi Medical Center.
FACS analysis of hematopoietic cells
Fetal livers were dissected from staged embryos and dissociated by gently
pressuring them through a 40 µm cell strainer. Cell suspensions were
stained in 4% FBS/PBS with biotinylated antibodies against the lineage markers
B220 (RA3-6B2, Pharmingen, San Diego, CA), CD3 (145-2C11), CD4 (L3T4), CD19
(1D3), Gr1 (RB6-8C5) and Ter119 (TER-119) and the stem cell markers Sca1 (D7,
PE-conjugated), and Kit (2B8, APC-conjugated) followed by Alexa Fluor
680®-conjugated streptavidin (Molecular Probes, Eugene, OR). Adult bone
marrow cells were harvested from hind limb bones and mature erythrocytes lysed
as described (Ye et al.,
2003). For the analysis of adult HSCs antibodies against Mac1
(M1/70) and CD34 (RAM34) were included in the lineage mix. Dead cells were
excluded by either DAPI (0.4 µg/ml) or propidium iodide (1 µg/ml)
staining. Analyses were performed with either an LSRII flow cytometer
(Becton-Dickinson) or a cell sorter (MoFlo-MLS, Cytomation, Fort Collins),
collecting 1,000,000-2,500,000 events per sample. Data analysis was done with
FlowJo (San Carlos, CA) software.
Preparation of frozen tissue sections
Mice were anesthetized and slowly perfused with 10 ml ice-cold PBS followed
by 15 ml 4% paraformaldehyde (PFA) using a 23 gauge butterfly needle inserted
into the left ventricle. The organs of interest were dissected out and fixed
in 15 ml 1.5% PFA in 30% sucrose/PBS for 1 hour at 4°C after which they
were sliced into 1-2 mm thick blocks and incubated for another 8-12 hours in
the fixation solution. The blocks were washed twice with 30% sucrose/PBS,
dried with tissue paper, embedded in OCT compound (Sakura, Torrance, CA) and
submersed in 2-methylbutane at -80°C. Sections (10 µm) were cut using a
cryostat (Leica CM1900), transferred onto pre-cleaned glass slides
(Superfrost/Plus, Fisher Scientific) and stored at -80°C.
Analysis of frozen sections
Immunofluorescence was performed using the M.O.M. basic kit (Vector Labs,
Burlingame, CA) following the manufacturer's instructions with the additional
inclusion of 0.3% Triton-100 in all solutions and of 3% BSA and 5% goat serum
in the blocking solution. For hepatocyte analysis, liver sections were stained
with a cocktail of APC-conjugated antibodies against the hematopoietic markers
CD45 (Pharmingen, 30-F11), F4/80 (Caltag, Burlingame, CA) and Mac1. DAPI (0.4
µg/ml) was included to visualize nuclei. Sections were screened for
YFP+ hepatocytes using a Nikon Eclipse E600 microscope equipped
with a 40 x objective and filters to visualize DAPI (Chroma #31000), YFP
(Chroma #41028), Alexa Fluor® 546 (Chroma #41002c) and APC (Chroma
#41013). Images were taken using a MagnaFire (Optronics, Goleta, CA) camera
and analyzed with Adobe Photoshop software. Hepatocytes were identified based
on cell morphology (brightfield), morphology of the nucleus (DAPI filter),
absence of hematopoietic markers (APC filter) and their characteristic
cytoplasmic autofluorescence (red filter). Slides containing YFP+
hepatocytes were stained with an antibody against mouse albumin (Accurate,
Westbury, NY) followed by an Alexa Fluor 546-conjugated secondary goat
anti-rabbit antibody (Molecular Probes). The number of hepatocytes in a
section was calculated as follows. First, the total area of the section was
determined using low magnification images (2 x objective) and Adobe
Photoshop. This area value was multiplied with the average number of
hepatocytes per high magnification field (40 x objective, n=20
images) and with 400 (the difference in area magnification between a 2 x
and a 40 x objective). Finally, the total number of hepatocytes was
adjusted by multiplication with a `fluorescence factor' between 0.7 and 0.9,
as most sections contained small areas in which YFP fluorescence was not
preserved.
For endothelial cell analysis, frozen sections were first stained with an antibody against CD45 (30-F11) followed by an Alexa Fluor 546-conjugated secondary goat anti-mouse antibody (Molecular Probes) and DAPI. After washing, the sections were stained with an APC-conjugated antibody against CD31 (MEC13.3). Images were taken as described above.
Analysis of endothelial cells by FACS
For the analysis of liver and kidney endothelial cells, mice were perfused
into the left ventricle using the solutions described previously for rat liver
perfusion (Neufeld, 1997) but
replacing collagenase with Liberase Blendzyme 3 (0.21 U/ml, Roche Diagnostics,
Indianapolis, IN). The dissected organs were incubated with the enzyme at
37°C for 45 minutes under gentle rotation. Cells were then dissociated
with a 25 ml pipette, filtered through a 40 µm cell strainer, large cells
removed by centrifugation at 30 g for 1 minute and the
supernatant centrifuged at 300 g for 5 minutes. The pellet was
washed twice with 4% FBS/PBS and then resuspended in the same solution. For
FACS analysis, the cells were incubated for 5 minutes with anti-mouse CD16/32
antibody (FcBlock, Pharmingen) to block unspecific binding and then stained
with antibodies against CD45, Ter119 (both PE-conjugated) and CD31
(APC-conjugated). FACS analyses were performed as described for hematopoietic
cells. Staining with DAPI showed the viability of the isolated endothelial
cells to be between 50%-85%.
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Results |
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Lysozyme ancestry mice are a complementary tool for hematopoietic lineage tracing
Because in vav ancestry mice all HSCs, committed progenitors and other
nucleated hematopoietic cells are YFP labeled, this model alone does not allow
drawing conclusions about the identity of the responsible cell type, in case
evidence for hematopoietic plasticity are found. Therefore, LysM-Cre mice
(Clausen et al., 1999) were
bred with ROSA26 reporter mice containing either YFP or lacZ. In the
resulting lysozyme ancestry mice, Cre expression from the endogenous lysozyme
M locus leads to the irreversible activation of ROSA26 driven YFP in about
90% of blood myelomonocytic cells (Ye
et al., 2003
) and essentially 100% of tissue macrophages (data not
shown). In addition, a varying subset of adult long-term reconstituting HSCs
and of all other hematopoietic cell types is reporter positive as a
consequence of myeloid linage priming in HSCs
(Ye et al., 2003
). The degree
of labeling of the LSK population ranges from
3% to
30%, depending
on the animal, and can be reliably deduced from the labeling index of
peripheral B cells (Ye et al.,
2003
). As macrophages are therefore the only cell type completely
labeled in both ancestry models (Fig.
1E), contributions of these cells to non-hematopoietic cells
should lead to the same labeling frequencies in both mouse models, while
contributions involving another hematopoietic cell type should be found less
frequently in lysozyme than in vav ancestry mice.
Concomitant identification of reporter positive hepatocytes, endothelial cells and hematopoietic cells in chimeric control mice
Chimeric control mice, expressing either YFP or lacZ, were used to
determine optimal conditions of tissue-preparation for reporter gene detection
in frozen sections. We found that direct YFP fluorescence yields the best
signal-to-noise ratio and no false-positive cells. In addition, it is most
conveniently combined with antibody staining to determine the identity of
reporter gene expressing cells (Fig.
2A,B; data not shown). Hepatocytes were identified as large
polygonal CD45-cells with prominent round nuclei. As shown in
Fig. 2C-H, YFP+
hepatocytes (broken white outline) could be clearly distinguished from
adjacent YFP- hepatocytes in liver sections from chimeric control
mice. Fig. 2C-H also shows that
YFP+ endothelial cells (white arrowheads) can be distinguished from
YFP+ hematopoietic cells (white arrows) by their strong expression
of CD31 and absence of CD45. In general, YFP+ cells showed strong
nuclear fluorescence (compare Fig.
2F,G). This feature facilitated the distinction between YFP
fluorescence and autofluorescence, which is largely cytoplasmic, especially in
hepatocytes. As negative controls, we analyzed ROSA26R-YFP (that lack the
vav-Cre transgene) and vav-Cre transgenic mice (that lack the floxed ROSA-YFP
allele). Liver sections encompassing more than 500,000 hepatocytes from these
mice revealed no evidence for spontaneous read-through of the stop cassette or
re-activation of the IRES-YFP element. Larger hepatic vessels, such as portal
veins, frequently exhibited a rim of extracellular autofluorescence that was
strongest in the blue/green part of the spectrum. This rim, which was also
seen in wild-type mice, most probably delineates the position of the basement
membrane as endothelial cells were found on its luminal side and tissue
hematopoietic cells on its abluminal side
(Fig. 2I-K). Together, these
pilot experiments show that YFP-positive hepatocytes, endothelial and
hematopoietic cells can be readily identified by the lineage tracing approach
taken.
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Liver damage leads to a moderate increase of hepatocyte labeling in vav ancestry mice
To address the issue of whether liver injury increases the frequency of
hematopoietic contributions to hepatocytes, two adult vav ancestry mice were
injected with carbon tetrachloride (CCl4) at a concentration that
induces necrosis in 60% of hepatocytes
(Yuan et al., 2003
). Thirty
hours after injection, we measured the blood plasma levels of alanine amino
transferase (ALT), an indicator for injury to the liver parenchyma. These were
determined to be 26 units per liter (U/I) for a control vav ancestry mouse
injected with oil and 4660 U/I and 7740 U/I for the injured mice. The
experimental mice were sacrificed 3 weeks after CCl4
administration, at which time they exhibited plasma ALT levels of 31 U/I and
18 U/I, respectively. Compared with uninjured mice, liver sections of the
injured mice revealed a two- to threefold increase in the frequency of
YFP+ hepatocyte clusters (
1 in 26,000 and
1 in 33,000,
respectively, Table 1) and a
slight increase in cluster size, with clusters containing up to five
YFP+ hepatocytes (in the plane of a 10 µm section). The cluster
size distribution compared with uninjured mice is shown in
Fig. 4.
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Discussion |
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Hematopoietic to hepatocyte contributions do not occur during early stages of development
The vast majority of LSK cells from E13.5 fetal liver of vav ancestry mice
already express YFP at maximum levels (Fig.
1D). This implies that vav-Cre expression begins shortly after the
emergence of the first HSCs, as there is an inherent delay between Cre
expression and reporter gene activation
(Prost et al., 2001). The
presence of YFP+ hematopoietic cells in E10.5 fetal liver shows
that yolk sac-derived progenitors, which home to the fetal liver before the
colonization of this organ by HSCs, are also labeled in vav ancestry mice.
Together, these observations suggest that even the earliest hematopoietic to
hepatocyte contributions should have been detected in the vav ancestry model.
The number of hepatocytes increases
100 fold between E13.5 and adulthood
(Anzai et al., 2003
;
Wang et al., 2002
), resulting
in a liver that is a mosaic of clonally derived colonies
(Kennedy et al., 1995
;
Shiojiri et al., 2000
), as can
also be seen in chimeric control mice (Fig.
2A). Therefore, early hematopoietic contributions should have led
to clusters of 100 or more YFP+ hepatocytes in adult vav ancestry
mice. The absence of such clusters therefore strongly argues against fetal
hematopoietic to hepatocyte contributions. This implies that the mouse fetal
liver environment, which supports the proliferation and differentiation of
hepatocytes and their precursors, does not trigger a hepatic potential in
hematopoietic cells. In addition, the observed prevalence of single
YFP+ hepatocytes suggests that few, if any, conversions occur
perinatally, as the liver weight increases 20-fold after birth
(Lau et al., 2003
). We
therefore conclude that hematopoietic plasticity plays no role in liver
development.
Macrophages are the source for the observed hematopoietic contributions to hepatocytes
A possible explanation for the absence of hematopoietic contributions in
the embryo is that the fetal liver lacks the cell types involved in the
process. Thus, there could be a requirement for mature hepatocytes, cells
whose differentiation is completed only around birth
(Zaret, 2002). Alternatively,
the relevant blood cell type might be the Kupffer cell, a specialized tissue
macrophage that develops postnatally
(Morris et al., 1991
). This
possibility is supported by the observation that vav and lysozyme ancestry
mice exhibit similar frequencies of labeled hepatocytes. Any significant
contribution of hematopoietic cells other than macrophages should have led to
a greater than 20-fold difference in the hepatocyte labeling index between vav
ancestry mice and the two lysozyme ancestry mice with a low HSC labeling index
studied (Table 1). It is also
unlikely that YFP+ HSCs of lysozyme ancestry mice are particularly
plastic, as we found no significant differences in the hepatocyte labeling
indices between mice with low and high HSC labeling
(Table 1). Finally, the
observations with the lck ancestry mouse rule out a contribution of T lineage
cells. The conclusion that macrophages contribute to hepatocytes during normal
development is in agreement with observations made after transplantation of
hematopoietic cells into FAH-deficient mice
(Camargo et al., 2004
;
Willenbring et al., 2004
). In
addition, the frequency of labeled hepatocyte clusters observed in our study,
650 per liver, falls within the range of hematopoietic derived colonies
in the FAH model (Camargo et al.,
2004
; Wang et al.,
2002
; Willenbring et al.,
2004
), suggesting that hepatic contributions occurring during
normal development are also mediated by cellular fusion.
The two- to threefold elevated number of YFP+ hepatocyte clusters observed after CCl4 injection might be best explained by an inflammatory response that leads to an increase in the number of activated liver macrophages and consequently to additional fusions with hepatocytes. The increased cluster size in response to liver injury shows that hepatocytes of hematopoietic origin can divide normally.
Hematopoietic stem cells do not function as hemangioblasts during mouse development
The absence of YFP+ endothelial cells in the liver strongly
indicates that hematopoietic cells do not contribute to the formation of
hepatic endothelial cells during development. Thus, although the first
vascular structures develop at E9.5 in the liver primordium
(Matsumoto et al., 2001), the
majority of endothelial cells are generated during subsequent organ growth, at
a time when most hematopoietic cells are already YFP labeled in vav ancestry
mice. Further support for the conclusion that definitive HSCs, which originate
from the endothelial cell layer of the dorsal aorta, have lost their
endothelial potential comes from the lack of YFP+ endothelial cells
in the heart, brain and lung of vav ancestry mice (data not shown). The
situation in the kidney is more complex as an endothelial subset contained in
the vasa recta is YFP labeled. Because no such cells were found in lysozyme
ancestry mice or after the transplantation of vav ancestry bone marrow, we do
not believe that they are of hematopoietic origin. However, we cannot entirely
rule out a contribution made by hematopoietic cells exclusively labeled in the
vav ancestry model and not present in adult bone marrow.
Our inability to detect hematopoietic to endothelial cell conversions was
unexpected based on reports describing such transitions after transplantation
of HSCs either with or without additional injury
(Bailey et al., 2004;
Grant et al., 2002
;
Sata et al., 2002
;
Tamura et al., 2002
). This
apparent discrepancy cannot simply be due to transplantation-associated
injuries as we could not detect YFP+ endothelial cells in mice
reconstituted with bone marrow cells from vav ancestry mice
(Fig. 6H;
Fig. 7D). A possible
explanation might be differences in the criteria used to identify endothelial
cells. For example, in one study (Bailey et
al., 2004
), endothelial cells were identified as expressing lower
levels of CD31 than hematopoietic cells, whereas we found the opposite to be
the case (Fig. 6C). In
addition, in studies where sections were not simultaneously stained for the
expression of hematopoietic and endothelial markers
(Gao et al., 2001
;
Grant et al., 2002
;
Sata et al., 2002
;
Tamura et al., 2002
),
hematopoietic cells might have been erroneously identified as endothelial
cells due to the close apposition of the two cell types (see, for example,
Fig. 2C-H,
Fig. 5). Finally, in reports
using GFP as a reporter (Bailey et al.,
2004
), endothelial cells might have been misidentified as being of
donor origin due to the autofluorescence of their basement membrane
(Fig. 2J,K). In this regard
chimeric control mice provided a unique tool to exclude artifacts and to
optimize conditions for the identification of different types of reporter
labeled cells. However, it is still possible that hematopoietic to endothelial
cell conversions occur in organs not analyzed in this study or can be induced,
for example when cells in the retina are injured and exposed to VEGF
(Grant et al., 2002
). Perhaps
injury-mediated re-vascularization is fundamentally different from normal
vasculogenesis and angiogenesis.
Another issue is the origin and function of endothelial progenitor cells,
which have been observed in the embryo
(Wei et al., 2004) and in the
circulation after bone marrow transplantation
(Asahara et al., 1999
). Our
data suggest that they are either not of hematopoietic origin or, if so, they
are not involved in the generation of endothelial cells during normal
ontogeny. Of note, CD45-Mac1-CD31- cultured
bone marrow stromal cells of vav ancestry mice are YFP- (M.S. and
T.G., unpublished). Whether these cells or other rare nonhematopoietic bone
marrow subsets have angiogenic potential remains to be determined.
In summary, our data indicate that once HSCs are specified, neither they nor their progeny become reprogrammed into hepatocytes or endothelial cells during fetal or early postnatal development. Furthermore, while macrophage to hepatocyte contributions can be detected as an ongoing process in the adult mouse, they do not constitute a significant source of cell replacement during normal tissue maintenance or regeneration. Our work thus supports the classical concept that germ layer specification sets developmental boundaries.
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ACKNOWLEDGMENTS |
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