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Blood, Vol. 95 No. 7 (April 1), 2000:
pp. 2284-2288
HEMATOPOIESIS
Expansion of hematopoietic stem cells in the developing liver of a
mouse embryo
Hideo Ema and
Hiromitsu Nakauchi
From the Department of Immunology, Institute of Basic Medical
Sciences, University of Tsukuba and CREST (JST), Tsukuba, Japan.
 |
Abstract |
The activity of hematopoietic stem cells in the developing liver of
a C57BL/6 mouse embryo was quantified by a competitive repopulation
assay. Different doses of fetal liver cells at days 11 to 18 of
gestation were transplanted into irradiated mice together with
2 × 105 adult bone marrow cells. A long-term
repopulation in myeloid-, B-cell, and T-cell lineage by fetal liver
cells was evaluated at 20 weeks after transplantation. At day 12 of
gestation multilineage repopulating activity was first detected in the
liver as 50 repopulating units (RU) per liver. The number of RU per
liver increased 10-fold and 33-fold by day 14 and day 16 of gestation,
and decreased thereafter, suggesting a single wave of stem cell
development in the fetal liver. A limiting dilution analysis revealed
that the frequency of competitive repopulating units (CRU) in fetal
liver cells at day 12 of gestation was similar to that at day 16 of
gestation. Because of an increase of total fetal liver cell number, the
absolute number of CRU per liver from days 12 to 16 of gestation
increased 38-fold. Hence, the mean activity of stem cells (MAS) that is given by RU per CRU remained constant from days 12 to 16 of gestation. From these data we conclude that hematopoietic stem cells expand in the
fetal liver maintaining their level of repopulating potential.
(Blood. 2000;95:2284-2288)
© 2000 by The American Society of Hematology.
| |
Introduction |
Recent studies on the development of hematopoietic
system have revealed the initiation sites of hematopoiesis and proposed a migration of stem cells during development of the mouse
embryo.1-5 Cells with long-term marrow repopulating
activity have been detected in the yolk sac (YS) and the
aorta-gonad-mesonephros (AGM) region at days 9 to 10 of gestation for
the first time in an embryonic life of mice.6,7
Hematopoietic stem cells are believed to migrate into the liver around
day 11, and subsequently into the bone marrow and spleen, whereas the
fetal liver remains as a main organ of definitive
hematopoiesis during the embryonic period.
Myeloid and B-cell precursors sequentially increase in the fetal liver
from mid-gestation to birth.1,8-10 However, the number of
T-cell precursor cells peaks at day 13 of gestation.11 It is generally believed that hematopoietic stem cells also expand in the
fetal liver, but the extent of their expansion and the kinetics of stem
cell development remain poorly understood. It has been shown that fetal
liver hematopoietic stem cells have a greater proliferative capacity
than do adult bone marrow stem cells.12-14 It has also been
reported that the frequency of hematopoietic stem cells in day 14-fetal
liver cells is comparable to that in adult bone marrow
cells.15 Fetal liver hematopoietic stem cells have been
characterized in comparison with adult bone marrow stem cells. However,
fetal liver stem cells at different time points of development have
never been compared. Herein, we measured a long-term multilineage
repopulating ability of the hematopoietic stem cells, which is referred
to as stem cell activity, in the fetal liver at successive stages of
gestation using 2 previously defined units.
We measured the numbers of repopulating units (RU)16 and
competitive repopulating units (CRU)17 in fetal liver
cells, based on a competitive repopulation assay,12 because
they are complementary. The activity of repopulating cells is given by RU, whereas the number of these cells is given by CRU. Given both numbers of RU and CRU, a mean activity of stem cell (MAS = RU/CRU) was introduced to compare the repopulating ability of individual CRU on
average between different cells examined. We demonstrate the total RU
per liver increased until day 16 of gestation and decreased thereafter.
The total CRU per liver also increased from day 12 to 16 of gestation,
along with an increase of fetal liver cell number. Because MAS was
similar at days 12 and 16 of gestation, we conclude that there is an
expansion of hematopoietic stem cells in the fetal liver.
| |
Materials and methods |
Mice
C57BL/6 mice (B6-Ly5.2) and their congenic strain (B6-Ly5.1) were
maintained in our animal facility. B6-F1 embryos were obtained from
mating pairs of B6-Ly5.1 males and B6-Ly5.2 females. The day of a
vaginal plug observed was designated as day 0 of gestation. Eight- to
10-week-old female mice were used as recipients in transplantation experiments.
Cells
Fetal liver was isolated from embryos at days 11 to 18 of gestation
under a dissecting microscope. Cell suspension was prepared in Hank's
balanced salt solution (HBSS, Life Technologies, Rockville, MD)
containing 2% fetal calf serum (FCS) by repeated flushing through
needles of 18 to 27 gauge. The cells were passed through a nylon mesh
with pore size of 70 µm (Falcon 2350, Becton Dickinson Labware,
Franklin Lakes, NJ). Bone marrow competitor cells of 8- to 10-week-old
B6-Ly5.1 mice were suspended in HBSS containing 2% FCS with an
18-gauge needle and passed through a nylon mesh. Viability of cells was
verified with the trypan blue dye exclusion.
Competitive repopulation assay
We performed population-type and limiting dilution-type assays, both
based on a competitive repopulation assay12 to which the
Ly5 system was adapted. A population-type assay was used to compare
repopulating activity between fetal liver and bone marrow cells.18 A limiting dilution analysis was used to estimate
a frequency of competitive repopulating units (CRU) in fetal liver cells as described.17 In both assays, 3 different numbers
of fetal liver cells (B6-F1) were mixed with
2 × 105 bone marrow cells (B6-Ly5.1). A cell
mixture in 200 µL of HBSS was injected into a group of B6-Ly5.2 mice
irradiated at a single dose of 9.5 Gy. Five or more recipients per
group were used for a population-type assay. Ten or more recipients per
group were used for a limiting dilution-type assay.
Analysis of the recipients
At 4 and 20 weeks after transplantation, peripheral blood cells of
the recipients were obtained by retro-orbital bleeding and stained with
fluorescence isothiocyanate (FITC)-conjugated anti-Ly5.1 (A20) and
biotinylated anti-Ly5.2 (104), followed by addition of
streptavidin-allophycocyanin (SA-APC). The cells were simultaneously
stained with PE-conjugated anti-B220 (RA3-6B2) or a mixture of
phycoerythrin (PE)-conjugated anti-Mac-1 (M1/70) and Gr-1 (RB6-8C5), or
a mixture of PE-conjugated anti-CD4 (GK1.4) and CD8 (53-6.7)
antibodies. All antibodies and reagents were purchased from PharMingen,
San Diego, CA. Multicolor analysis and cell sorting were performed on a
dual laser FACS Vantage (Becton Dickinson, San Jose, CA).
In a population-type assay, contribution of fetal liver-derived cells
against that of bone marrow competitor-derived cells was expressed by
the test donor-derived cells/competitor-derived cells (T/C) ratio. The
T/C ratio was defined as percentage of test donor-derived cells
(Ly5.1/Ly5.2-double positive F1 cells) divided by percentage of
competitor-derived cells (Ly5.1 cells) on FACS analysis of the
peripheral blood as demonstrated in Figure 1. Because Ly5.2 positive cells were gated
out from the calculation, T/C ratio calculated was not influence by the
residual host cells. Repopulating units (RU) was calculated using
Harrison's method16 as follows:
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| Fig 1.
Reconstitution analysis of the mice transplanted with day
12-fetal liver cells at 20 weeks after transplantation.
The representative FACS profile of peripheral blood analysis is shown.
The reconstitution in tri-lineage: myeloid, B cell, and T cell was a
criterion for engraftment. T: test donor cells (fetal liver cells
derived from F1 mouse), C: competitor cells (bone marrow cells from
B6-Ly5.1), and R: residual host cells from B6-Ly5.2. T/C ratio: a ratio
of (% test donor-derived cells)/ (% competitor-derived cells). In
this case, T = 45.0%, C = 44.0%, R = 1.0%, and T/C
ratio = 1.02.
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RU equals the percentage of donor cells times the number of competitor
cells times 10 5 divided by 100 minus percentage of donor
cells. By definition each RU represents the repopulating activity of
1 × 105 bone marrow cells.16 In this
study, the number of bone marrow competitor cells was fixed as
2 × 105 cells. T/C ratio defined above was applied
to Harrison's formula as follows: RU = T/C ratio × 2.
Limiting dilution analysis was performed as described.17 A
recipient mouse was regarded as positive when the test donor-derived cells consisted of more than 1% of test donor and competitor-derived cells in the peripheral blood. The frequency of repopulating cells (CRU) was estimated on the basis of Poisson statistics as
described.19-21 Reconstitution in myeloid-, T-cell, and
B-cell lineage was verified by detecting the cells positive for
Mac-1/Gr-1, B220 and CD4/CD8 within the gate of F1-test donor
cells in both population and limiting dilution -type assays
(see Figure 1). To compare the repopulating potentials
between different sources of cells, we defined the mean
activity of stem cells (MAS) as follows: MAS = RU/CRU.
| |
Results |
Measure of test donor-derived cells/competitor-derived cells
ratio
Fetal liver cells were obtained from embryos of B6 mice from day 11 to day 18 of gestation, and subjected to a population-type competitive
repopulation assay.18 At day 11 of gestation, the total
liver cells that contained 2 × 105 cells per embryo
on average were transplanted into lethally irradiated mice together
with 2 × 105 adult bone marrow cells.
At days 12 to 18 of gestation, 3 different numbers of fetal liver cells
were transplanted. At 4 and 20 weeks after transplantation, peripheral
blood cells were analyzed to evaluate contributions of fetal liver
cells (test donor) and bone marrow cells (competitor) to host
peripheral blood chimerism. Only myeloid lineage was taken into account
for hematopoietic reconstitution at 4 weeks after transplantation,
whereas the reconstitution in all myeloid, B-cell, and T-cell lineage
was a criterion for reconstitution at 20 weeks after transplantation.
As demonstrated in Figure 1, the cells derived from the fetal liver
expressed both the Ly5.1 and Ly5.2 antigens (F1-type cells). Bone
marrow cells used as competitor cells expressed only the Ly5.1 antigen (Ly5.1 cells). Therefore, in B6-Ly5.2 recipients, fetal liver-derived F1-type cells were distinguished from competitor-derived Ly5.1 cells
and residual host-derived Ly5.2 cells.
We obtained the T/C ratio (% test donor-derived cells/%
competitor-derived cells) to calculate RU.16 Under the
irradiation condition used, remaining host cells could be detected in
almost all recipient mice even 20 weeks after transplantation at a
frequency of 6.5% ± 4.4% (n = 12). Use of Ly5.1, Ly5.2, and
F1 cells has allowed more accurate estimation of stem cell activity by
removing the influence of remaining host cells from the analysis. The
T/C ratios for fetal liver cells at different days of gestation were listed in Table 1. In every reconstituted mouse,
fetal liver-derived cells were detected in all myeloid-, B-, and T-cell
lineages at 20 weeks after transplantation (data not shown).
In 2 independent experiments, fetal liver cells of gestation day 11 were transplanted into a total of 20 mice. Whole fetal liver cells from
an embryo were transplanted into a lethally irradiated adult recipient.
However, no mice showed a detectable level of reconstitution by test
donor cells for an observation period of 38 weeks. Transplantation of
gestation day 10-liver cells also resulted in no reconstitution (data
not shown). These data indicated that fetal liver, before day 12 of
gestation, did not contain hematopoietic stem cells capable of
competing against 2 × 105 adult bone marrow cells,
at least in an adult hematopoietic environment.
Developmental wave of hematopoietic stem cell activity in the
fetal liver
RU has been used to indicate the repopulating potential in a cell
population.16 On the basis of the data shown in Table 1, RU
was calculated by multiplying the mean T/C ratio by a factor of 2. After calculating RU per 1 × 105 cells for each
dose, the mean ± SD of the data for each gestation day was
obtained and shown in Table 2. The total
number of RU per liver was obtained by multiplying the number of RU per
1 × 105 by the total number of liver cells divided
by 105. Data in Table 2 are graphically demonstrated in
Figure 2.
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| Fig 2.
Development of stem cell activity in the fetal liver.
Graphical presentation of the data in Table 2. The mean of total
RU/liver at 4 and 20 weeks after transplantation are shown.
|
|
Repopulating activity in the liver was first detected at day 12 of
gestation. The RU for 4 and 20 weeks after transplantation were 13 and
50 per liver, respectively. The total RU per liver at 4 weeks after
transplantation gradually increased with day of gestation and reached a
plateau by day 16. In contrast, RU obtained 20 weeks after
transplantation showed a drastic increase with a peak at day 16 of
gestation and decreased thereafter suggesting a single wave of stem
cell development. The total number of RU per liver at day 14 and 16 of
gestation was 10- and 33-fold higher than that of RU at day 12 of gestation.
Number of hematopoietic stem cells in the fetal liver at days 12 and 16 of gestation
To gain further insight into the mechanism of an increase in the
total stem cell activity of the fetal liver, a limiting dilution-type assay was performed with fetal liver cells at days 12 and 16 of gestation. Three different numbers of fetal liver cells for each gestation day were mixed with 2 × 105 bone marrow
cells, and transplanted into irradiated adult recipients. Peripheral
blood cells of the recipients were analyzed 4 and 20 weeks after
transplantation to examine whether there was a significant contribution
by the fetal liver-derived cells (more than 1% chimerism). Figure
3 shows the result of in vivo limiting
dilution analysis. The frequency of CRU was determined according to
Poisson statistics as demonstrated in Figure 3. The frequency of CRU in
day 12 fetal liver was similar to that in day 16 fetal liver both at 4 and 20 weeks after transplantation (Table
3). The absolute numbers of CRU in the
liver at 4 and 20 weeks after transplantation showed 29-fold and
38-fold increases from days 12 to 16 of gestation.
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| Fig 3.
In vivo limiting dilution analysis of repopulating cells.
A group of irradiated mice were transplanted with varying numbers of
fetal liver cells at day 12 (A) and day 16 (B) of gestation. At 4 and
20 weeks after transplantation peripheral blood cells were analyzed for
the presence of fetal liver-derived cells. The number of recipients
that survived until the time of analysis were 6 to 13 mice and 8 to 13 mice per group for day 12 and day 16 fetal liver cell transplantation.
Mice that did not contain more than 1% of fetal liver-derived cells in
a total of fetal liver- and competitor-derived cells were considered to
be negative.
|
|
To estimate repopulating activity of a stem cell, we introduced a novel
unit, mean activity of stem cell (MAS), which can be obtained by
calculating the number of RU divided by CRU for the same number of
cells. MAS indicates the average repopulating ability of individual
stem cell. MAS of day 12 fetal liver cells was similar to that of day
16 fetal liver cells at the same time point after transplantation
(Table 3).
| |
Discussion |
The hematopoietic stem cell activity in the fetal liver at
successive stages of development was measured in a quantitative manner
using unfractionated fetal liver cells. Both total RU16 and
CRU17 per liver were taken into account for evaluation of this activity. Given the numbers of RU and CRU, the mean activity of
stem cell (MAS = RU/CRU) is proposed to compare the repopulating ability of individual CRU on average between different sources of
cells. In all experiments, 2 × 105 bone marrow
cells of 8- to 10-week-old B6 mice were used as competitor cells to
ensure the survival of the recipients and to obtain a constant
repopulating ability to compete. In addition, by means of Ly5.1-,
Ly5.2-, and F1-type cells, a competition between test donor and
competitor cells was assessed more accurately in different recipients
regardless of a variation in the amount of residual host cells on
analysis. The Ly5.1 and Ly5.2-negative cells, such as red blood cells
contaminated in samples, were also excluded from the analysis.
The total stem cell activity per liver from day 12 of gestation to the
day before birth was estimated (Table 2 and Figure 2). Stem cell
activity in day 11 fetal liver has been described.6 However, we could not detect it in our assay system. The number of
cells transplanted may not be sufficient to repopulate bone marrow
against 2 × 105 bone marrow competitor cells. It is
conceivable that the level of stem cell activity in day 11 fetal liver
is much lower than that in day 12.22,23
RU at 4 weeks after transplantation successively increased along with
day of gestation. This kinetics resembles that of in vivo and in vitro
colony-forming cells in the fetal liver.1,8 It is assumed
that myeloid lineage-committed precursor cells were mostly responsible
for repopulating activity at 4 weeks after transplantation. On the
other hand, RU at 20 weeks after transplantation exhibited an increase
in the fetal liver until day 16 of gestation during the embryonic life.
We interpreted that the number of RU at 4 and 20 weeks after
transplantation represented the short- and long-term repopulating
abilities of a cell population.24 A 32- and 33-fold
increase was observed for short- and long-term repopulating abilities
from days 12 to 16 of gestation. Because the frequencies of CRU
remained rather constant during this period, the total numbers of
short- and long-term CRU per liver showed a 29- and 38-fold increase,
which corresponded well with increases in the total RU. Thus, an
increase in CRU mostly accounts for increase in RU in this study.
The observation that RU at 20 weeks is higher than that at 4 weeks
after transplantation at all gestation days examined supports the
notion that stem cells in fetal liver have a higher intrinsic capacity
for self-renewal than do those in bone marrow.12 These data
indicate that RU at 4 weeks after transplantation may not reflect
actual stem cell activity in the case of fetal liver stem cells. These
findings may reflect a unique property of fetal liver-derived stem
cells in that they do not efficiently generate as many myeloid precursor cells in a short-term as do bone marrow stem cells. However,
they are able to repopulate more intensely in multilineage later on.
The MAS at short- and long-term remained similar between days 12 and 16 of gestation. On average, therefore, the repopulating ability of each
CRU did not change from days 12 to 16. It is concluded that major part
of hematopoietic precursor and stem cell expansion takes place in the
fetal liver by day 16 of gestation.
It has been shown that the hematopoietic activity in the AGM region and
yolk sac is maximal at day 11 of gestation, and rapidly ceases
thereafter.3,23,25 Our results support the notion that
hematopoietic stem cells migrate into the liver mostly between days 11 and 12. An increase in the number of hematopoietic stem cells up to
38-fold between days 12 and 16 is best explained by self-renewal of
these cells in the fetal liver, because no other active site
for hematopoiesis in this period has been described. Furthermore, in utero transplantation of fetal liver cells suggested self-renewal of hematopoietic stem cells in the liver.26
However, there still remains a possibility that prestem cells migrating from the AGM region and yolk sac give rise to stem cells in the liver
in their differentiation process. These prestem cells may be detectable
in the conditioned newborn recipients, but not in the irradiated adult
mice.7
RU at 20 weeks after transplantation showed a peak at day 16 of
gestation. The decline in RU after day 16 may suggest a massive exit of
stem cells from the liver to either spleen or bone marrow. A less
likely alternative is an abrupt reduction in self-renewal of
hematopoietic stem cells, due to the deterioration of hematopoietic microenvironment. This deterioration may also induce a rapid decrease of MAS or apoptosis of hematopoietic stem cells after day 17 of gestation.
| |
Acknowledgments |
We thank K. Shimada for helpful discussion, K. Fujii for advice in
statistical analysis, and M. Onodera and A. Shibuya for critical
reading of the manuscript.
| |
Footnotes |
Submitted August 26, 1999; accepted December 10, 1999.
Supported by grants from CREST of Japan Science and Technology
Corporation, the Ministry of Education, Science, Sports and Culture in
Japan, the Agency for Science and Technology, and the Japan Society for
the Promotion of Science JSPS-RFTF96I00202.
Reprints: Hiromitsu Nakauchi, Department of Immunology,
Institute of Basic Medical Sciences, University of Tsukuba and CREST
(JST), 1-1-1 Tennodai, Tsukuba, 305-8575 Japan; e-mail address:
nakauchi{at}md.tsukuba.ac.jp.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
| |
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Top
Abstract
Introduction