1 Institute for Stem Cell Research, University of Edinburgh, West Main's Road,
King's Buildings, Edinburgh EH9 3JQ, UK
2 Statistics and Modelling Science Department, University of Strathclyde,
Livingston Tower, 26 Richmond Street, Glasgow G1 1XH, UK
3 John Hughes Bennett Laboratory, Department of Oncology, University of
Edinburgh, Western General Hospital, Crewe Road, Edinburgh EH4 2XU, UK
* Author for correspondence (e-mail: a.medvinsky{at}ed.ac.uk)
Accepted 18 July 2002
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SUMMARY |
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Key words: Haematopoietic stem cells, AGM region, Yolk sac, Liver, Mouse
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INTRODUCTION |
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The above data does not, however, rule out a possibility that at later
stages the YS is involved in the independent production or expansion of
definitive HSCs. Although early YS cells are unable to repopulate adult
irradiated recipients upon direct transplantation, when transplanted into the
embryo they can contribute to adult haematopoiesis
(Weissman et al., 1978;
Toles et al., 1989
). Analogous
results have been achieved by transplantation of YS cells into newborn
recipients (Yoder and Hiatt,
1997
; Yoder et al.,
1997
). In addition, by day 8 p.c. both the P-Sp and the YS contain
cells that can mature into definitive HSCs by co-culture with an AGM-derived
stromal cell line (Matsuoka et al.,
2001
). Thus at least from day 8 p.c. the YS contains cells
(pre-definitive HSCs), which are capable of development into definitive HSCs
upon maturation in an embryonic or newborn microenvironment
(Medvinsky and Dzierzak,
1999
). However, it remains unclear if and when during normal
embryo development these cells mature into definitive HSCs and whether they
have to migrate to an inductive AGM microenvironment in order to do so.
In our previous papers we focused on the initiation of definitive HSC
production in the mouse embryo (Muller et
al., 1994; Medvinsky and
Dzierzak, 1996
). Here we explore the subsequent stage of HSC
development from day 11 until day 13 when the number of definitive HSCs
increases in the embryo. From day 11 p.c. onwards the number of HSC/RUs
increases dramatically in the liver
(Morrison et al., 1995
;
Ema and Nakauchi, 2000
) but
little is known about their distribution in the rest of the embryo, the routes
of their migration and the mechanisms underlying their expansion in the liver.
Various types of multipotent, pluripotent and bi-potent myeloid and lymphoid
progenitors have been identified during embryogenesis in the YS, AGM region,
liver, thymic and splenic rudiments (Moore
and Metcalf, 1970
; Velardi and
Cooper, 1984
; Johnson and
Barker, 1985
; Wong et al.,
1986
; Eren et al.,
1987
; Liu and Auerbach,
1991
; Cumano et al.,
1992
; Morrison et al.,
1995
; Ema et al.,
1998
; Kawamoto et al.,
1998
; Nishikawa et al.,
1998
; Liu et al.,
1999
; Ohmura et al.,
2001
; Palis et al.,
2001
; Traver et al.,
2001
; Douagi et al.,
2002
) and different types of progenitor cells are disseminated via
the circulation (Moore and Metcalf,
1970
; Johnson and Barker,
1985
; Rodewald et al.,
1994
; Delassus and Cumano,
1996
). However, as the links within the haematopoietic hierarchy
and between tissues are unclear, it is difficult to resolve an entire
anatomical picture of the development of definitive HSCs. Although data from
some publications has revealed fragments of it
(Moore and Metcalf, 1970
;
Ikuta et al., 1990
;
Morrison et al., 1995
;
Berger and Sturm, 1996
;
Sanchez et al., 1996
;
de Bruijn et al., 2000
;
Ema and Nakauchi, 2000
;
Hsu et al., 2000
;
de Bruijn et al., 2002
;
North et al., 2002
), a
comprehensive quantitative anatomical map of HSC/RUs development during
embryogenesis has not been produced. Here, we have attempted to create a
temporal and spatial map of development of definitive HSC/RUs in the 11-13
d.p.c. mouse embryo. To this end, the number of HSC/RUs in different embryonic
tissues has been estimated using a limiting dilution method
(Szilvassy et al., 1990
). In
addition, the potential of the AGM region and the YS to produce and/or
maintain definitive HSC/RUs has been assessed using an organ culture approach
(Medvinsky and Dzierzak,
1996
).
We have found that expansion of the pool of HSC/RUs within the liver occurs
concurrently with increasing numbers of HSC/RUs in the circulation. In
addition to the previously reported activity of the AGM region during day
10-11 p.c. (Medvinsky and Dzierzak,
1996; de Bruijn et al.,
2000
), we report here that a day later, at 12 d.p.c., the YS
becomes competent to generate (and/or expand) definitive HSC/RUs. This finding
suggests that both the AGM region and the YS produce HSCs that colonise the
developing liver in two subsequent waves, the peaks of which fall on days
10-11 p.c. and day 12 p.c. respectively.
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MATERIALS AND METHODS |
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Organ culture
Tissues were cultured in myelo-cult medium (Stem Cell Technology)
supplemented with 10-6 M hydrocortisone hemi-succinate (Sigma) on
Durapore 0.65 µm filters (Millipore) supported by stainless steel stands
(5% CO2 in air) at the gas-liquid interface as previously described
(Medvinsky and Dzierzak,
1996). 12 d.p.c. foetal liver was explanted in small pieces
comparable in size to the AGM region. Cultures were set up for 3-5 days and
7-9 days.
Analysis of donor contribution into recipient haematopoietic
system
The contribution of donor cells was assessed 6-8 weeks and 3.5-5 months
after transplantation as described previously
(Medvinsky and Dzierzak,
1996). Briefly, the percentage of male donor cells in the
haematopoietic system of female recipients was assessed by comparison with
standards of serially diluted male in female DNA (0.1%, 1%, 10%, 100%). Both
test DNA and standards were amplified by PCR using primers specific for male
Y2B and mouse myogenin sequences. As previously, we have restricted our
analysis to those cells (definitive HSCs), which upon transplantation,
contributed at a level of 10% or higher in the haematopoietic system of
irradiated recipients (Muller et al.,
1994
; Medvinsky and Dzierzak,
1996
).
Transplantations of day 12 and 13 tissues in some cases were carried out
using Ly5.1 embryos and multilineage contribution was assessed in recipient
mice by assessing co-expression of the Ly5.1 marker and lineage specific
markers by antibody staining and subsequent analysis on FACSCalibur
(Beckton-Dickinson). For this purpose biotinylated anti-Mac-1, anti-B220
(secondary stained with Strepdavidin-PE; Sigma), PE-conjugated anti-CD3
and FITC-conjugated anti-Ly5.1 antibodies (Pharmingen) were used. In some
cases the bone marrow of reconstituted mice was transplanted into secondary
recipients.
Quantitation of HSC/RUs by limiting dilution analysis
Upon transplantation, one definitive HSC is sufficient to differentiate
into all lymphoid and myeloid cell types and to contribute over several months
at a high level to the haematopoietic system of an irradiated recipient
(Lemischka, 1992;
Morrison et al., 1997
). In
order to estimate the number of HSCs/RU in various organs we have adopted a
limiting dilution method by transplanting low numbers of HSCs (several
dilutions) into irradiated recipients
(Szilvassy et al., 1990
). Test
cells were co-transplanted intravenously with 2x104 bone
marrow cells to ensure short-term survival of the recipient. The number of
HSCs in tested tissues was estimated by Poisson statistics based on the
proportion of non-repopulated recipients in long-term (longer than 3.5 months)
repopulating experiments. Serial dilutions are expressed in embryo equivalents
(e.e.). A minimum of 2 and maximum of 12 different dilutions were used for
each tissue. For each dilution between 4 and 21 recipients were transplanted
in a minimum of two independent replicate experiments. The final numbers of
HSCs were estimated by the maximum likelihood method using Genstat 5 package
and expressed as the most probable numbers (MPN)
(GenStat 5 Release 3 Reference Manual,
1993
). The asymmetric error range in parentheses next to MPN and
also showing on the graphs is typical for this kind of analysis which involves
confidence interval estimation of the Poisson mean.
Our calculations are based on the assumption that HSCs from different
tissues and at different stages of development have an equal seeding
efficiency. However, this may not be the case and therefore we, as others
(Ema and Nakauchi, 2000),
introduce an operational term `repopulating unit' (RU), which is not
necessarily related to a single cell and appears next to HSC abbreviation in
the text.
In separate experiments we have found that the 2x104
carrier bone marrow cells injected per recipient contain on average 2 (1.6,
2.6) long-term repopulating HSC/RUs, which is similar to numbers reported
by some other groups (Abkowitz et al.,
2000). This may explain why when other researchers transplanted 10
times more bone marrow carrier cells (2x105) (about 20
HSC/RUs) along with day 11 embryonic liver no liver contribution was detected
in recipient mice (Ema and Nakauchi,
2000
), as the donor HSCs may have been out competed by an excess
of HSCs in the carrier bone marrow.
Assessment of content of circulating HSC/RUs in various embryonic
tissues
The number of definitive HSC/RUs in the circulation was assessed in
transplantion experiments as described above. The number of HSC/RUs in the
circulation quoted in Fig. 1A
and table 1 represent only a
proportion of all circulating HSC/RU i.e. those that were released upon
separation of the YS and the embryo body. The relative proportion of
circulatory blood cells in various embryonic tissues was estimated by a
comparison of the numbers of red blood cells in the tissues with those in the
circulation. To this end haemoglobinized cells from the circulation and cells
obtained after trypsinization of dissected tissues were stained with
0-Dianisidine and counted under the microscope
(Iuchi and Yamamoto, 1983) as
a measure of the contamination of tissues with embryonic blood. The likely
contribution of circulating HSC/RUs to the number of HSC/RUs recovered from
different embryonic tissues was calculated from the following formula:
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RESULTS |
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Tissue distribution of definitive HSC/RUs within 12 d.p.c.
embryo
On day 12 p.c. both the AGM region and the YS contain approximately two to
three HSC/RUs each, which is higher than on day 11 p.c.
(Fig. 1A). At this time the
embryonic circulation also contains about three HSC/RUs. The significant
numbers of HSC/RUs in the circulation suggests intensive trafficking of
HSC/RUs within the embryo. By day 12 p.c. the number of HSC/RUs in the
embryonic liver reaches 53 (43,69) thus increasing approximately
50-fold from day 11 p.c. Multilineage contribution to recipient mice was
confirmed by analysis of selected mice
(Fig. 2).
|
Estimates of possible HSC/RUs numbers derived from the circulation in these organs showed that HSC/RUs numbers within both the AGM region and the YS are significantly above the numbers of HSC/RUs attributable to circulating blood present in these tissues (Table 1). To directly test the number of circulating HSC/RUs contained within the AGM region we flushed out 12 d.p.c. dorsal aorta (Table 2). As expected the flushed out samples of embryonic blood contained fewer HSC/RUs than remained in the dorsal aorta [0.3 (0, 1.7) and 1.6 (1.2, 2.4) respectively]. Therefore, either both the AGM region and the YS on day 12 p.c. are involved in the specific production of HSC/RUs, or circulating HSC/RUs have been selectively retained in these tissues. This issue has been more closely examined in organ culture experiments described below.
|
In contrast to day 11 p.c., on day 12 p.c. HSC/RUs were consistently detected in the body of the embryo. The number of HSC/RUs in the body of the 12 d.p.c. embryo without the liver was estimated to be 12.1 (8.1, 19.3). When in addition to the liver, the AGM region and blood were also removed the total number of HSC/RUs in all remaining tissues was 5.8 (4.4, 8.2) (Table 1). From the amount of blood in body transplants (Table 1) we estimate that about 1.6 HSC/RUs in the embryo body belong to the pool of circulating HSC/RUs. Therefore, it may be that apart from the AGM region and the YS a few HSC/RUs are harboured in other tissues of the body. Amongst individually tested tissues (thymus, spleen, lung, forelimbs, heart and head) transplanted separately, the lungs consistently reconstituted irradiated recipients. They contained 0.4 (0.2, 0.6) HSC/RUs, which is above the expected 0.06 HSC/RUs that would be brought there by the circulation (Table 1). Forelimbs also contained about 0.5 (0.3, 0.9) HSCs. Some untested tissues may contain solitary HSC/RUs as well.
Tissue distribution of definitive HSC/RUs in 13 d.p.c. embryo
By day 13 p.c. of development the number of circulating HSC/RUs in the
embryonic vasculature remains high 5.9 (4.7, 7.7) and the number of
HSC/RUs in the liver continues to grow, reaching 260 (212, 320)
(Fig. 1). The number of HSC/RUs
decreases in both the AGM region and the YS to 0.8 (0.6, 1.2) and
0.8 (0.6, 1.2) HSC/RU per tissue respectively
(Fig. 1,
Table 1). The total number of
HSC/RUs in the body outside the liver remains stable compared to 12 d.p.c.
body (Table 1) and HSC/RUs are
no longer detectable in the lungs. Forelimb transplants reconstituted three
out of five recipient mice in two dilutions. Since the amount of blood in
distal limbs was extremely low, freely circulating HSC/RUs are unlikely to
account for this (Table 1).
HSC/RUs found in 12-13 d.p.c. limbs may reflect early colonisation of
developing long bones with HSC/RUs. Further analysis of the limbs of 14 d.p.c.
embryos will be required to test the reliability of this conclusion.
Analysis of the HSC potential of 11 d.p.c. embryonic tissues using an
organ culture approach
In the day 11 p.c. embryo five sites contain HSC/Rus: the AGM region, the
YS, the liver, blood and umbilical vessels
(Muller et al., 1994;
Medvinsky and Dzierzak, 1996
;
de Bruijn et al., 2000
). It has
been shown using an organ culture approach that the AGM region is the only one
of these tissues capable of expanding or generating HSC/RUs at this age
(Medvinsky and Dzierzak, 1996
;
de Bruijn et al., 2000
). Here
we have quantitatively reassessed the potential of 11 d.p.c. embryonic tissues
to produce HSC/RUs, by quantifying the number of HSC/RUs that are produced and
maintained in organ culture. After 3-5 days in culture, the number of HSC/RUs
in 11 d.p.c. AGM region increased from 0.9 (0.7, 1.1) to 12 (10.0,
17.6) (P<0.05) (Fig.
3A) followed by a drop in the numbers of HSC/RUs after 7-9 days in
culture.
|
As was shown previously, 11 d.p.c. YS is incapable of expanding the initial numbers of explanted HSC/RUs. Each explant of 11 d.p.c. YS contains approximately one HSC before and after 3 days in culture (Fig. 3A). However, in contrast to the AGM region, 11 d.p.c. YS explants are not able to maintain HSC/RUs for a longer time in culture. Similarly, in 11 d.p.c. liver about 1 HSC can be detected after 3 but not after 7 days in culture.
Analysis of HSC potential of 12 d.p.c. embryonic tissues by organ
culture
When 12 d.p.c. AGM region was tested, we found that after 3-5 days in vitro
it contained the same number of HSC/RUs as it initially contained in the
embryo (Fig. 3B). A slight
increase in the numbers of HSC/RUs was observed after 7-9 days in culture. 12
d.p.c. AGMs are larger than 11 d.p.c. AGMs and therefore culture conditions
could be suboptimal. To optimise the culture we reduced the size of the
explants by subdissection of 12 d.p.c. AGMs and found no signs of HSC
expansion in these cultures either (data not shown). In addition, we found
that the microenvironment of the 12 d.p.c. AGM is highly supportive of
long-term (up to 4 weeks) maintenance of HSC/RUs (Kumaravelu et al.,
unpublished observation). Thus, we infer that the ability of the AGM region to
expand (and/or generate) definitive HSC/RUs is significantly attenuated on day
12 p.c., as compared to 11 d.p.c., concurrent with progressive specification
of the AGM region into gonads and mesonephric derivatives.
In contrast to 11 d.p.c. YS, 12 d.p.c. YS explants acquire the capacity to increase the numbers of HSC/RUs during culture. Before culture 12 d.p.c. YS contains 1.8 (1.4, 2.4) HSC/RUs but after 3 days in culture it contains 6.8 (5.0, 9.8) HSC/RUs per explant (P<0.05) (Fig. 3B). However, like the 11 d.p.c. YS and in contrast to the AGM region, 12 d.p.c. YS was not able to maintain HSC/RUs in long-term cultures; after 7 days in culture the number of HSC/RUs dropped to less than 1, being 0.6 (0.4, 1.2) HSC/RUs per YS. This may possibly reflect the transitory nature of haematopoietic activity in the YS in vivo.
Explants of 12 d.p.c., foetal liver were not able to maintain the initial number of HSC/RUs in culture, which could be explained either by suboptimal culture conditions for this tissue or by immaturity of day 12 liver microenvironment (Fig. 3C).
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DISCUSSION |
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Early development of definitive HSC/RUs in the mouse embryo involves at
least two key stages; (i) initiation of definitive HSC/RUs (late day 10-early
day 11 p.c.) and (ii) expansion of the pool of definitive HSC/RUs. In
addition, some tissues may be involved in the maintenance of HSC/RUs. Since
HSCs may rapidly change their location during embryogenesis, detection of HSCs
in tissues does not identify whether these tissues are capable of generating,
expanding, or maintaining them or whether these tissues merely transiently
contain them. In order to try and distinguish between these possibilities, we
and others previously developed an organ culture approach which enables
isolated tissues to be individually tested to reveal their HSC activity
(Medvinsky and Dzierzak, 1996;
Cumano et al., 2001
). For
example, the number of HSC/RUs within the AGM region on days 10-11 p.c. is no
higher than within the YS and the liver. However, in contrast to other
embryonic tissues at this age, the AGM region is capable of autonomously
initiating and expanding HSC/RUs in vitro suggesting that the initial pool of
definitive HSC/RUs is generated within the AGM region and these then colonise
the liver (Morrison et al.,
1995
; Medvinsky and Dzierzak,
1996
; Ema and Nakauchi,
2000
).
Here, using an improved protocol we have been able to detect rare HSC/RUs
in day 11 circulation (compare with our previous report)
(Muller et al., 1994). This
reveals a previously proposed, but never experimentally shown, route by which
11 d.p.c. AGM derived HSCs can colonise the liver. During days 12-13 p.c. the
number of HSC/RUs in the circulation rises (approximately 6 HSC/RUs were
detected on day 13 p.c.) consistent with the more intense colonisation of the
foetal liver with HSCs from extra-hepatic sources during this period. It is
worth bearing in mind that HSC/RUs present in the circulation at the moment of
embryo dissection are likely to represent only a small proportion of total
HSC/RUs present in the circulation over the entire day of gestation.
Our present calculations indicate that the total number of HSC/RUs within
the developing embryo increases dramatically from about 3 (day 11 p.c.) to 66
(day 12 p.c.) mainly due to accumulation of HSC/RUs in the liver
(Table 3). This is in
accordance with previously published numbers of HSC/RUs in day 12 liver
(Ema and Nakauchi, 2000).
Owing to the length of the cell cycle it is unlikely that such an increase in
HSC number occurs entirely from amplification of a few HSC/RUs that initially
colonized the liver. If the possibility of de novo/primary formation of
HSC/RUs in the liver is ruled out then this increase must be the result of a
massive immigration of HSC/RUs from extra-liver source(s). Our quantitative
data are in accord with this hypothesis as explained below.
|
In the 11 d.p.c. embryo the number of HSC/RUs within the liver is low and can be easily explained by colonisation from the AGM region. In fact, one 11 d.p.c. AGM region can produce as many as twelve HSC/RUs after 3 days in culture; and we assume that in vivo this process may be significantly more efficient. By 12 d.p.c. the HSC productivity of the AGM region decreases as assessed by the organ culture test. However measurement of HSC/RUs numbers in uncultured AGMs showed that the number of HSC/RUs in the AGM region is still higher than would be expected in a non-haematopoietic tissue. Conversely, by 12 d.p.c. the YS showed noticeable HSC productivity in vitro. It was capable of expanding the number of HSC/RUs from about 1.8 to 6.8 during a 3-day culture period.
Thus, at early stages of liver colonisation the high cumulative HSC
productivity of the AGM region and the YS may provide the liver with a large
part of the `ready-to-use' pool of HSCs
(Fig. 4). It is interesting
that in the same culture conditions the liver itself was unable to increase or
even maintain the initial number of HSCs. This suggests either that by day 12
p.c. the liver is not yet competent to expand HSCs or that the culture
conditions used are not fully adequate. It is important to note in relation to
this that a cell line derived from day 14 foetal liver is capable of
maintaining HSCs over a period of 1 month
(Moore et al., 1997).
|
The generation/expansion of definitive HSCs/RUs in 12 p.c. YS culture is
likely related to previous observations that from 8-10 d.p.c. the YS contains
immature cells that when placed into an embryonic or newborn environment
become capable of contributing into adult haematopoiesis
(Weissman et al., 1978;
Toles et al., 1989
;
Yoder and Hiatt, 1997
;
Yoder et al., 1997
;
Matsuoka et al., 2001
). Our
data suggest that these early YS cells may not necessarily need processing in
the AGM region to become functional definitive HSCs but can mature in situ on
day 12 p.c. The controversy over the origin of HSCs has always centred on the
issue of whether the P-Sp/AGM region or the YS is the initial site of their
generation. However, these data indicate that definitive HSCs may develop
independently and asynchronously in these two different sites of the mouse
embryo suggesting that the argument over the first source of HSCs is not
relevant. However, at present, the possibility of cross-seeding of the YS and
the AGM region with definitive HSCs and/or their ancestor cells cannot be
excluded. Lineage tracing of YS and AGM haematopoiesis from early stages of
development is required to finally resolve this issue.
In summary, we have carried out a comprehensive anatomical mapping of the
development of definitive HSC/RUs in the mouse embryo from 11-13 d.p.c. during
which time the embryonic liver becomes colonised. We have shown that
increasing numbers of HSC/RUs in the liver is accompanied by the appearance of
growing numbers of HSC/RUs in the embryonic blood. The data presented here
suggests that in addition to early waves of colonisation with committed and
multipotent haematopoietic progenitors
(Moore and Metcalf, 1970;
Johnson and Barker, 1985
;
Dzierzak and Medvinsky, 1995
)
the liver is colonised by two consecutive waves of definitive HSC/RUs. The
initial wave of HSC/RUs arrives from the AGM region on day 10 p.c., reaches a
maximum by day 11 p.c. and disappears by day 13 p.c. On day 12 p.c. when AGM
activity is decreasing, the second wave of colonisation arrives from the YS.
This wave marks the embryonic stage when early YS cells mature into definitive
HSC/RUs.
A visual demonstration of development of HSC/RUs in 10-12 d.p.c. mouse embryo as it is viewed by the authors is presented as a movie and accompanies the Web version of the article (http://dev.biologists.org/supplemental/).
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ACKNOWLEDGMENTS |
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![]() |
Footnotes |
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