Department of Cell Biology and Genetics, Erasmus University Medical Center, PO Box 1738, 3000 DR, Rotterdam, The Netherlands
* Author for correspondence (e-mail: e.dzierzak{at}erasmusmc.nl)
Accepted 8 December 2004
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SUMMARY |
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Key words: Mesenchyme, Hematopoiesis, AGM, Development, Embryo
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Introduction |
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During mouse ontogeny, the first adult-type HSCs are generated at E10.5 in
the aorta-gonad-mesonephros (AGM) region
(Medvinsky and Dzierzak, 1996;
Muller et al., 1994
).
Initially, HSCs are restricted to the regions of major vasculature: the
vitelline and umbilical arteries and the dorsal aorta
(de Bruijn et al., 2000
). One
day after their emergence in the AGM region, HSCs are also found in the other
subregion of the AGM, the urogenital ridges, and also in the yolk sac (YS) and
fetal liver (de Bruijn et al.,
2000
; Medvinsky and Dzierzak,
1996
; Muller et al.,
1994
). Between E10.5 and E12.0, HSCs amplify within the AGM region
and, later, these cells are thought to migrate and colonize the fetal liver,
where they undergo extensive expansion to the numbers found in the adult
(Kumaravelu et al., 2002
).
Finally, just before birth, HSCs are thought to migrate to the BM, which
remains the main site of hematopoiesis throughout adult life
(Cumano and Godin, 2001
). Thus,
HSC supportive microenvironments, presumably of mesenchymal origin, exist in
several anatomical sites during ontogeny, the AGM, fetal liver and BM.
Recently, an early mesodermal precursor has been identified in the E9.5
mouse dorsal aorta. When transplanted into chick embryos, these cells
participated in the development of vasculature and were integrated into blood,
bone, cartilage and muscle (Minasi et al.,
2002). Moreover, clonal progeny from an aorta cell line was able
to differentiate in vitro into cells of hematopoietic, osteogenic, adipogenic
and myogenic lineages (Minasi et al.,
2002
). Other reports demonstrate that cells from mouse embryonic
dorsal aorta and fetal liver can differentiate in vivo along the myogenic
lineage (De Angelis et al.,
1999
; Fukada et al.,
2002
). Hence, during midgestation, precursor cells for several
mesenchymal lineages can be found within hematopoietic sites, but it is
unknown whether there is a specific localization of such precursors to these
sites. Moreover, the results of Blazsek et al. suggest that, during
development, acquisition of hematopoietic competence by the BM is correlated
with the emergence of cell aggregates (hematon units) composed of mesenchymal,
endothelial and hematopoietic cells, as well as hematopoietic progenitors
(Blazsek et al., 2000
). These
studies raise a number of questions. Where do mesenchymal stem/progenitor
cells first emerge in the embryo? How are they distributed during ontogeny? Is
their temporal and spatial distribution pattern effectively correlated with
hematopoietic territories? And finally, is the presence of MSCs dependent on
HSC activity?
We carried out a comprehensive anatomical mapping and frequency analysis of mesenchymal progenitors during ontogeny. The data presented here show that AGM, fetal liver, and neonatal and adult BM harbor stem/progenitor cells for several mesenchymal lineages. The presence of these cells in circulating blood suggests that at least part of these progenitors can migrate between tissues. Moreover, the progressive increase in numbers of mesenchymal progenitor/stem cells throughout development indicates that these hematopoietic sites provide a microenvironment that supports the generation, maintenance and/or proliferation of mesenchymal stem/progenitor cells. Additionally, in E11 Runx1 deficient embryos, the presence, differentiation potential and frequency of these cells is not dependent upon HSC activity. Thus, our results show that mesenchymal progenitor/stem cells develop within the major hematopoietic sites throughout ontogeny, raising the possibility of a parallel and coordinate development of both hematopoietic and mesenchymal systems.
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Materials and methods |
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Tissues and cell preparations
YS, head, somites, limb buds, heart, liver, umbilical and vitelline
arteries, and AGM region were dissected from E11 mouse embryos
(Fig. 1A). Some AGMs were
longitudinally sub-dissected, separating the dorsal aorta with its surrounding
mesenchyme (AoM) from the gonads and mesonephros (urogenital ridges; UGRs)
(Fig. 1B). All tissues were
incubated for one hour at 37°C with 0.125% collagenase in phosphate
buffered saline solution (PBS) containing 10% fetal calf serum (FCS) and
penicillin/streptomycin (P/S). Primary cells were then dispersed, washed,
resuspended in PBS with 10% FCS and P/S, and used in differentiation assays
and frequency analysis. Hematopoietic tissues at other time points were
isolated and tested: E12 YS, AGM and liver; E14 liver; neonatal (1 week old)
and adult (12-14 weeks old) BM. After collagenase digestion, E14 liver cells
were submitted to a ficoll gradient to deplete erythrocytes. Femora were
dissected from surrounding tissues and BM cells flushed using a 25G needle.
Cells were dispersed, resuspended in PBS with 10% FCS and P/S, passed through
a nylon mesh filter. Blood was obtained at E11, E12, E14 and E17. Embryos were
isolated with yolk sac intact, washed and placed in a culture dish containing
PBS+10% FCS. Under the dissection microscope, the vitelline and umbilical
arteries were cut at the base of the yolk sac allowing the blood to flow
freely into the medium. The medium was collected and centrifuged, nucleated
cells counts performed, and blood cells directly cultured.
|
Adipogenic differentiation
Primary cells from the several tissues were cultured for 2 to 3 days in
DMEM with 10% FCS and P/S, and then treated in adipogenic medium consisting of
DMEM, 1% FCS, P/S, 10-7 M dexamethasone and 100 ng/ml insulin.
Seven to 10 days after stimulation, the cultures were evaluated based on
morphology and Pparg gene expression.
Chondrogenic differentiation
A micro-mass culture system was used
(Dennis et al., 1999). Primary
cells from the several tissues were placed in polypropylene tubes, centrifuged
to form a micro-mass and cultured for 21 days in DMEM containing
ITS+ (insulin-transferrin-selenium), P/S, 0.1 mM L-ascorbic acid
2-phosphate, 10-9 M dexamethasone and 20 ng/ml TGFß1. After 21
days, tissue aggregates were frozen, sectioned and either stained with
Toluidine Blue (which detects proteoglycans in the extracellular matrix), or
fixed and subsequently stained with a collagen type II specific antibody
(CIIC1, Developmental Studies Hybridoma Bank, Iowa). Secondary antibody,
anti-mouse immunoglobulin-HRP (Dako, Denmark) and a DAB substrate for
peroxidase (Dako, USA) were used.
Frequency analysis
To estimate the frequency of osteogenic, adipogenic and chondrogenic
progenitors present in the several hematopoietic tissues throughout
development, primary cells of each tissue were tested in colony forming unit
(CFU) assays (Friedenstein et al.,
1974; Owen et al.,
1987
; Sekiya et al.,
2002
; Simmons and Torok-Storb,
1991
) in association with the above mentioned differentiation
assays. To determine the frequency of osteogenic and adipogenic progenitors,
the minimum cell-seeding density at which cells grow in distinct colonies was
determined for each tissue type (Table
1). With regard to the chondrogenic lineage, the minimum cell
input for the formation of a tissue aggregate (or pellet) in micro-mass
cultures was also determined (Table
1).
|
Adipogenic progenitors
After 7 to 10 days of treatment, frequency was taken as the number of
adipogenic colonies (Sekiya et al.,
2002) divided by the input cell number.
Chondrogenic progenitors
After 21 days of culture in chondrogenic differentiation medium, frequency
was determined by the number of distinct cartilage foci
(Fig. 2E) divided by the input
cell number. In this calculation, we assume that each foci in the micromass
cultures would develop from at least one chondrogenic progenitor. Results from
experiments with mixes of phenotypically marked cells indicate that
approximately 80% of foci are most likely clonally-derived (S.C.M.,
unpublished). Thus, our indicated values may represent a slight
underestimation of chondrogenic progenitors.
|
Gene expression
Reverse-transcriptase polymerase chain reaction (RT-PCR) was used to
determine the expression of osteocalcin and Pparg in both freshly
isolated cells and cultures stimulated towards differentiation. Target genes
were amplified by PCR with the following primers:
mouse Cbfa1, 5'-ACCACAGAACCACAAGTGCGG-3' and 5'-CTGAAGAGGCTGTTTGACGC-3';
mouse osteopontin, 5'-CTATAGCCACATGGCCGG-3' and 5'-GAGGTCCTCATCTGTGGC-3';
mouse osteocalcin, 5'-CTGACCTCACAGATCCCAAGC-3' and 5'-CTGTGACATCCATACTTGCAG-3';
mouse Pparg, 5'-GACCTCTCCGTGATGGAA-3' and 5'-GCTGGTCGATATCACTGG-3'; and
the house-keeping gene mouse ß actin, 5'-CCTGAACCCTAAGGCCAACCG-3' and 5'-GCTCATAGCTCTTCTCCAGGG-3'.
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Results |
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Cells from YS, vitelline and umbilical arteries, head, heart and liver did
not contain detectable progenitors for any of the evaluated lineages, even
when cultured at high-cell densities. Although somites and limb buds contained
cells able to form cartilage in vitro, this is due to the already
differentiating prechondrogenic mass that constitutes these tissues
(Cheah et al., 1991) and is not
attributable to mesenchymal stem/progenitor cells. Thus, mesenchymal
stem/progenitor cells are exclusively localized in the AGM region of the E11
embryo.
Sublocation of mesenchymal stem/progenitor cells within the E11 AGM
To more specifically localize mesenchymal stem/progenitor cells within the
AGM, the dorsal aorta with its surrounding mesenchyme (AoM) and the urogenital
ridges (UGRs) (Fig. 1B) were
subdissected and analyzed. Differentiation cultures revealed that progenitors
with osteogenic potential, as determined by ALP activity, were present
throughout the entire AGM region. In the whole AGM region, osteogenic
precursors were present at a frequency of 1.93±0.40/104
cells [Fig. 3A; there was no
contribution of ALP-positive primordial germ cells in the AGM region to this
frequency (see figure legend)]. The UGRs had a slightly higher frequency of
these cells (2.27±0.25/104 cells), and the AoM, a 2-fold
lower frequency (1.07±0.40/104 cells). The absolute number
of osteogenic progenitors was also higher in the UGRs than in the AoM
(Table 3). Because the number
of osteogenic precursors in the whole AGM region was higher than the sum of
these cells in the AoM and UGRs, either a loss of osteogenic progenitor cells
is incurred during subdissection, or cells of both subregions are required for
optimal differentiation potential to be revealed.
|
|
Chondrogenic progenitors were present at a lower frequency in the AGM (0.85±0.22/104 cells; Fig. 3C) than either osteogenic or adipogenic progenitors. Such progenitors were located both in the UGRs and AoM at frequencies of 0.70±0.18/104 and 0.48±0.17/104 cells, respectively. Again, the AoM appeared to contain fewer progenitors than the UGRs, although this difference was not statistically significant (P=0.06).
In summary, cultures from both AoM and UGRs revealed that osteogenic and chondrogenic progenitor cells are distributed throughout the entire AGM region, whereas precursor cells for the adipogenic lineage are exclusively located in the UGRs. Moreover, both the frequency and absolute number of adipogenic progenitors in the UGRs (Table 3) are substantially higher than the frequency and absolute number of progenitors for the other two lineages, indicating that this site provides a particularly favorable microenvironment for the adipogenic differentiation. These findings suggest that at this developmental stage, the mesenchymal progenitors found in the AoM have a more restricted potential than the progenitors detected in the UGRs, or that adipogenic cells arise from a different progenitor than osteogenic and chondrogenic cells.
Mesenchymal stem/progenitor cells progressively increase in number in hematopoietic sites during development
The finding that mesenchymal progenitors are exclusively localized in the
E11 AGM region prompted us to examine other hematopoietic sites for such
activity. AGM, YS, liver and BM cells were isolated and tested at several
developmental stages. At E12 the AGM region continued to harbor mesenchymal
progenitors with potential for osteogenic, adipogenic and chondrogenic
lineages, while no detectable progenitors were found in the highly
hematopoietic E12 YS. Mesenchymal progenitors were present in the E14 liver,
neonatal BM and adult BM and were capable of differentiating into all three
lineages. Thus, mesenchymal cells, similar to those previously found in adult
BM (Kuznetsov et al., 1997;
Pereira et al., 1998
;
Pittenger et al., 1999
;
Prockop, 1997
), are present in
most primary hematopoietic tissues throughout development. The frequency and
absolute number of osteogenic, adipogenic and chondrogenic progenitors was
determined in each of these hematopoietic sites and the results are shown in
Fig. 4.
|
Like the osteogenic progenitor frequency, the frequency of adipogenic progenitors in the AGM did not vary significantly from E11 to E12 (Fig. 4B). Nonetheless, the absolute number of these cells per AGM did increase slightly (P=0.1). In the liver, these progenitors were detected only beginning at E14 and, although their frequency in this tissue was very low when compared with the E11/12 AGM, the absolute number of adipogenic progenitors per tissue was higher (244 in E14 liver as compared with 124-183 in the E11/12 AGM). Both in neonatal and adult BM, the frequency of adipogenic progenitors decreased more than 10-fold when compared with the progenitor frequency in the E14 liver. The absolute number of these progenitors decreased in the neonatal BM to 72±25, whereas this number increased to 191±53 in the adult stage. Thus, throughout development the number of adipogenic progenitors is variable, between 100-250 progenitors per tissue.
The frequency of chondrogenic progenitors in the AGM region did not statistically vary from E11 to E12, but a developmentally progressive decrease in the frequency was observed in the E14 liver (P=0.01, when compared with E11/12 AGM), and in neonatal and adult BM (P<0.001, when compared with E14 liver). However, a significant (P=0.007) increase in the absolute number of chondrogenic progenitors was observed in E14 liver (143±37) when compared with E12 AGM (34±7). This increased number of progenitors persisted in the adult BM (Fig. 4C).
In summary, spatial quantification of mesenchymal progenitors during development demonstrates that although progenitor frequency decreases, the absolute numbers of progenitors increases to a plateau level. Osteogenic progenitors predominate in adult BM, while adipogenic progenitors predominate in the AGM. Taken together, these results suggest a developmentally progressive amplification of mesenchymal progenitors in each of the hematopoietic sites, AGM, liver and BM.
Mesenchymal progenitors are present in embryonic blood
Although there is a progressive appearance and increase in mesenchymal
progenitor activity in the liver and BM during development, it is undetermined
whether these tissues are seeded through the circulation with progenitors
arising from, for example, the AGM region. It is generally accepted that for
the hematopoietic system, hematopoietic cells (erythroblasts) arising in the
YS, migrate and colonize the liver rudiment at late E9
(Houssaint, 1981;
Johnson and Jones, 1973
). To
investigate the possibility that migration of mesenchymal progenitors may
occur between tissues, embryonic blood was tested for the presence of such
cells at E11, E12, E14 and E17. At E11 and E17, even with high cell seeding
densities, no progenitors for any of the three mesenchymal lineages could be
detected in circulating blood. However, both at E12 and E14, stem/progenitor
cells for the osteogenic, adipogenic and chondrogenic lineages were detected
in circulating blood (Fig. 5).
The frequency of these cells in blood both at E12 and E14 was extremely low
(on average 30-500 times lower than progenitor frequency in E12 AGM and E14
liver). These results suggest that at least some mesenchymal stem/progenitor
cells may relocate to the different hematopoietic sites through the
circulation.
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Discussion |
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Rather than being localized in the aorta with mesangioblasts, we found
mesenchymal stem/progenitor cells for osteogenic, adipogenic and chondrogenic
lineages localized to the UGRs. We also found mesenchymal progenitor cells
localized to the aorta, but these had a more restricted potential (osteogenic
and chondrogenic). AGM subregional localization differences are also known for
HSCs. At E11, HSCs localize to the aorta, but by E12, they can be found both
in the aorta and UGRs (de Bruijn et al.,
2000). Moreover, other investigators have demonstrated that cells
readily migrate from the mesonephros to the gonads at E10/E11
(Martineau et al., 1997
;
Perez-Aparicio et al., 1998
).
Thus, as development proceeds during the crucial period from E9 to E11, there
appears to be a widespread and ongoing spatial reorganization and/or
maturation/differentiation of several stem cells and progenitors in the AGM
region, and this most likely includes mesenchymal stem/progenitor cells.
We have shown that at E12 the AGM continues to harbor mesenchymal
stem/progenitor cells, and, thereafter, we found such cells in the E14 liver,
and later in the neonatal and adult BM. In all these locations,
stem/progenitor cells possessed similar potentials, being able to
differentiate into cells of the osteogenic, adipogenic and chondrogenic
lineages. Their frequency sequentially decreased from site to site during
development. However, the absolute number of mesenchymal stem/progenitor cells
in these tissues increased with developmental time to reach relative plateau
levels. Hence, our results demonstrate that AGM, liver and BM are excellent
environments for the maintenance and expansion of mesenchymal stem/progenitor
cells. Although, the increases in mesenchymal stem/progenitor cells parallel
the increase in HSC numbers in these tissues throughout ontogeny
(Kumaravelu et al., 2002;
Morrison et al., 1995
), a
possible developmental relationship between mesenchymal and hematopoietic
cells leading to a parallel and coordinate development of these systems still
needs to be proven.
As shown in Table 1, the plating densities at which mesenchymal progenitors could be detected increased from site to site during ontogeny, implicating a decrease in the frequency of detectable mesenchymal progenitors throughout development. This decrease reflects either a `real' decrease in the incidence of mesenchymal progenitors, or it could be related to the cells constituting the microenvironment that would differentially affect the in vitro proliferation and differentiation capacity of the progenitor cells. Because we could detect an increase in the absolute numbers of these progenitors from hematopoietic site to hematopoietic site during development, the microenvironments of these organs seem to be supportive of mesenchymal progenitor expansion and differentiation, and, therefore, the decrease in frequency is most likely due to the large increase in cells of other lineages in these tissue rudiments.
Thus, we have shown that during development: (1) mesenchymal stem/progenitor cells are present in the major hematopoietic sites; (2) hematopoietic organs provide a good microenvironment for the maintenance and expansion of these cells; and (3) a small part of the mesenchymal progenitor population is in the circulation. These findings provide new information supporting the notion that the origins and formation of the hematopoietic supportive microenvironment and the hematopoietic system closely parallel each other.
It should be mentioned that our studies were not carried out at a clonal level and thus it is difficult to discern between true MSCs and mesenchymal progenitors within the evaluated population. Nevertheless, because in each evaluated site the frequency and number of precursor cells varied from lineage to lineage, it is likely that the analyzed cell population actually consists of both true stem cells and progenitors at several stages of differentiation.
Mesenchymal-derived stromal cells are known to be essential in regulating
the balance between HSC self-renewal and differentiation from the earliest
stages in the AGM to the adult BM. Stromal cell lines with hematopoietic
supportive ability have been isolated from the AGM region and its associated
subregions (Oostendorp et al.,
2002a; Oostendorp et al.,
2002b
), and indeed, one of the most supportive stromal clones is
derived from the UGRs. Highly supportive stroma cells have also been isolated
from the fetal liver (Moore et al.,
1997
) and the adult BM. Moreover, these stromal clones, including
AGM-derived clones, have the potential to differentiate to most of the
mesenchymal lineages in appropriate culture conditions (C. Durand, E. Haak and
E.D., unpublished). This, taken together with the fact that mesenchymal stem
cells from BM enhance engraftment of HSCs after transplantation
(Almeida-Porada et al., 2000
),
suggests that both HSCs and mesenchymal-derived cells work in concert with
each other.
While it is clear that HSCs require the presence of stromal cells for their maintenance and differentiation, it was unclear whether the reverse was true. Our results in Runx1 deficient embryos, demonstrate that the presence, differentiation potential and frequency of mesenchymal progenitor/stem cells in E11 AGM is not dependent on HSC activity. Although Runx1 deficiency leads to the arrest of HSC emergence and/or function, it is uncertain whether this transcription factor also acts within mesenchymal lineage cells to affect HSC support. We have been able to isolate hematopoietic supportive stromal cells from Runx1-/- E11 AGMs, strongly supporting the notion that the Runx1 transcription factor does not play a crucial role in the generation of this mesenchymal stem/progenitor cell compartment (E. Haak and K. Harvey, unpublished).
In contrast to our findings that mesenchymal stem/progenitor cells are
localized to the AGM and other major (highly vascularized) hematopoietic
tissues throughout development, we found no mesenchymal stem/progenitor cells
in the highly vascularized midgestation YS. This may not be surprising as the
YS is a simple two-layered structure and it becomes extinct in late gestation.
We also found site-specific variations in differentiation potential in the
other major hematopoietic sites. For example, whereas osteogenic and
chondrogenic progenitors were both located in the AoM and UGR sub regions,
adipogenic progenitors were only present in the UGR part of the AGM. Thus, it
appears that specific tissues limit differentiation potential, influence the
mesenchymal lineage hierarchy and/or are unable to maintain mesenchymal
stem/progenitor cells. Some studies examining the mesenchymal differentiation
hierarchy have shown that the adipogenic lineage diverges and becomes
independent earlier than the osteogenic and chondrogenic lineages
(Banfi et al., 2000). Hence,
future studies should focus on the mesenchymal lineage hierarchy in the
different ontogenic sites to determine the mechanisms by which mesenchymal
differentiation occurs.
In conclusion, mesenchymal stem/progenitor cells were detected in the major hematopoietic sites during mouse development: in the AGM and liver at midgestational stages, and later in neonatal and adult BM. These cells are able to amplify within the hematopoietic sites. At E11, the Runx1 HSC deficiency does not affect the presence, differentiation potential or frequency of mesenchymal progenitors. As mesenchymal stem/progenitor cells are found in the midgestation circulation, lineage tracing through development will be our future focus, so as to determine whether indeed, mesenchymal stem/progenitor cells migrate and colonize the major hematopoietic tissues to supply the hematopoietic supportive microenvironment.
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ACKNOWLEDGMENTS |
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