| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
*
Department of Immunology, Institute for Frontier Medical Sciences, and
Department of Medicine and Clinical Science, Graduate School of Medicine, Kyoto University, Sakyo-ku, Kyoto, Japan
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
It is still controversial when in the fetus the progenitors capable of generating lymphocytes emerge, despite extensive investigations having been undertaken on the differentiational potential of early progenitors in the EB. Ogawa and colleagues (12) have previously shown that B cell progenitors emerge in the 9.6-dpc EB by culturing whole embryo cells on a monolayer of a bone marrow (BM)-derived stromal cell line. On the other hand, Cumano et al. (3) showed that progenitors in 8.5-dpc (10–12 somite stage) P-Sp generated B cells when they were cultured on a stromal cell monolayer in the presence of cytokines, including SCF and IL-7, and moreover that cells derived from a single 9.5-dpc P-Sp cell precultured on the stromal cell layer for 14 days showed T cell progenitor activity when transferred into a deoxyguanosine (dGuo)-treated fetal thymus (FT) lobe (13). Recently, we found that the progenitors in the caudal half of 9.5-dpc EB were able to generate both T and B cells (14), whereas 8.5-dpc (5–10 somite stage) embryos did not contain T or B cell progenitors. Thus, a dramatic change of the characteristics of progenitors, such as commitment to T or B cell lineage, has occurred at about 9.5 dpc.
The presence of B cell lineage-committed progenitors (p-B) has been elucidated in the FL at 12 dpc by the group of Cumano (15, 16). In their experimental system, however, T cell lineage-committed progenitors (p-T) cannot be detected, because p-T do not differentiate on a stromal cell monolayer. Nor can it be clarified whether the progenitors generating only B cells, only myeloid cells, or both B and myeloid cells on the stromal cell layer retained T cell-generating ability. To broaden the area of analysis, we have recently established a clonal assay system, named multilineage progenitor (MLP) assay, which is effective in determining the developmental potential of individual progenitors toward T, B, and myeloid cell lineages (17). By using this assay system, we have shown the presence of various types of progenitors, including multipotent progenitors (p-Multi), p-T, p-B, and myeloid lineage-committed progenitors (p-M) in 12-dpc FL (17, 18). We have also shown that it is p-T, but not p-Multi, that migrate to the thymus to produce T cells (18, 19). However, since the thymic lymphoid cells appear at 11 dpc (20), it is probable that the first p-T migrating into the FT may have emerged somewhere other than FL before 11 dpc. In the present study, we show that lineage-committed progenitors as well as p-Multi are present in the AGM region of 10-dpc fetuses.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Adult C57BL/6 (B6) mice were purchased from Japan SLC (Shizuoka, Japan). B6Ly 5.1 mice were maintained in our animal facility. (B6 x B6Ly 5.1)F1 fetuses were obtained by crossing the male B6Ly 5.1 mice with female B6 mice. Embryos at various stages of gestation were obtained from time-mated pregnant mice. The date of finding the vaginal plug was designated as 0 dpc. Precise embryonic stages were determined by counting the pairs of somites. Embryos with 24–29 pairs of somites were regarded as 9.5 dpc, and those with 30–35 pairs of somites were regarded as 10.0 dpc of gestation.
Organs and cell suspension
AGM regions, fetal blood (FB), FL, and FT were obtained from B6Ly 5.1 fetal mice or (B6 x B6Ly 5.1)F1 fetuses. Single cell suspensions of the AGM cells were prepared by passage of tissues containing AGM region through a 26-gauge needle. FB was collected by cutting the umbilical cord without damaging the embryo. Embryos were left bleeding in the medium. To get rid of maternal blood, the uterus and embryos had been extensively washed.
Monoclonal Abs
The following Abs were used: FITC anti-Mac-1 (M1/70; Caltag,
San Francisco, CA), FITC anti-Gr-1 (RA3-8C5; PharMingen, San Diego,
CA), FITC anti-B220 (RA3-6B2; Caltag), FITC anti-Thy-1.2 (5a-8;
Caltag), FITC anti-CD8 (YTS169.4; Caltag), FITC anti-mouse IgM
(Cappel, West Chester, PA), FITC anti-TCR
(GL-3; Caltag),
FITC anti-CD3
(145-2C11; PharMingen), FITC anti-Vß6
(RR4-7; PharMingen), FITC anti-Vß8 (KJ16; Caltag), FITC
anti-V3 (536; PharMingen), PE anti-B220 (RA3-6B2; Caltag),
PE anti-CD45 (30F11.1; PharMingen), PE anti-Thy-1.2 (5a-8;
Caltag), PE anti-CD4 (GK1.5; Caltag), PE anti-TCR
ß
(H57-597; Caltag), PE anti-Sca-1 (E13-161.7; PharMingen).
Anti-TER119 (TER) (established by Dr. T. Kina in our laboratory) and
anti-Ly-5.1 (A20-1.7, donated by Dr. Y. Saga, Banyu Seiyaku, Tokyo,
Japan) were labeled with FITC in our laboratory. Anti-c-kit
(ACK-2, donated by Dr. S.-I. Nishikawa, Kyoto University, Kyoto, Japan)
and anti-Ly-5.1 were conjugated with cyanine 5 (Cy5) using a
labeling kit (Biological Detection Systems, Pittsburgh, PA). Anti-TER,
anti-Mac-1, anti-Gr-1, anti-B220, and anti-Thy-1.2 were
used as lineage markers (Lin).
High oxygen submersion (HOS) organ culture and MLP assay culture
The basic procedures for HOS culture have been described previously (21). Briefly, FT obtained from 15-dpc fetuses of B6 mice were treated with dGuo. The lobes were washed, and single dGuo-treated lobes were placed into wells of a V-bottom 96-well plate (Costar, Cambridge, MA), into which a suspension of progenitor cells was added. The plates were sealed in a plastic bag (Ohmi Oder Air Service, Hikone, Japan) and the air inside was replaced by a gas mixture (70% O2, 25% N2, and 5% CO2). The plastic bag was incubated at 37°C. RPMI 1640 medium supplemented with 10% FCS, L-glutamine (2 mM), sodium pyruvate (1 mM), sodium bicarbonate (2 mg/ml), nonessential amino acid solution (0.1 mM), 2-ME (5 x 10-5 M), streptomycin (100 µg/ml), and penicillin (100 U/ml) was used as complete medium. Medium change was performed every 4 to 5 days.
The MLP assay culture has been designed as a modification of the HOS culture (17). Cells sorted with a flow cytometer were suspended in a 9-cm plastic dish (INA · OPTICA, Osaka, Japan) at the concentration of about 100 cells/ml. A single cell was picked up using a micropipette with a long fine tip (QSP, Petalurna, CA) under direct microscopic visualization, and was put into each well of a V-bottom 96-well plate in which a dGuo-treated FT lobe had been placed in complete medium. A mixture of cytokines including 10 ng/ml murine rSCF (Genzyme, Cambridge, MA), 200 U/ml murine IL-7 (donated by Dr. T. Sudo, Basic Research Laboratory, Toray, Kanagawa, Japan), and 1 ng/ml murine GM-CSF (Life Technologies, Gaithersburg, MD) was added to the culture to promote the growth of B and myeloid cells. The difference of the cytokine mixture used here from that used for the MLP assay for FL progenitors (17) is that GM-CSF was used in place of IL-3. This change is due to the fact that most progenitors in the AGM region tend to become blastic and do not differentiate in the presence of IL-3 (our unpublished data). Plates were centrifuged, placed in a plastic bag, and cultured under HOS conditions at 37°C. Half of the medium was replaced with complete medium containing 200 U/ml IL-7 and 1 ng/ml GM-CSF on the fifth day.
Cocultivation of progenitors with a stromal cell line
The stromal cell line TSt-4 was used to investigate the development of B and myeloid cells, as described previously (22). To a confluent monolayer of TSt-4 cells in a six-well plate (Costar), FL or AGM cells were added. The culture medium is the same complete medium as used in HOS cultures, except that the concentration of FCS is 5%.
Cell sorting and analysis
The basic methods for cell surface staining and analysis have been described previously (23). Cells obtained from the AGM region, FB, FL, FT, and BM were stained with various mAbs and were sorted using a FACS Vantage. The sorted cells were reanalyzed to check their purity, and were found to be >98% pure.
In the MLP assay, cells generated inside and outside the FT lobe were harvested from each well 10 days after cultivation, and single cell suspensions were made. Half of the cell sample was stained with FITC anti-B220, PE anti-Thy-1.2, and Cy5 anti-Ly-5.1, whereas the other half was stained with FITC anti-Mac-1, FITC anti-Gr-1, PE anti-B220, and Cy5 anti- Ly 5.1. In some experiments, flow-cytometric analysis was performed by using half of each sample, while the remaining half was used for PCR analysis. Surface phenotypes were analyzed with a FACS Vantage by using CELL Quest software (version 1.2.2). Cells showing the phenotype of Ly 5.1+Thy-1+B220- or Ly-5.1+Thy-1-B220+ were tentatively regarded as donor-derived T or B cells, respectively. These populations were then checked for forward-side scatter and expression of Mac-1/Gr-1, and the cells falling within the lymphocyte area and expressing neither Mac-1 nor Gr-1 were finally considered to be either donor-derived T or B cells. Ly 5.1+Thy-1-B220-Mac-1+Gr-1+ cells were judged as donor-derived myeloid cells. Although this population contains cells with low to middle levels of staining for B220 or Thy-1, such a staining was regarded as nonspecific because these Mac-1+/Gr-1+ cells were found to fall in the area of macrophages in forward-side scatter analysis.
RT-PCR
RNA isolation. mRNA was isolated from TER-c-kit+Ly 5.1+ cells (3000 cells) in 10-dpc AGM region and FB of (B6 x B6Ly 5.1)F1 fetuses, Lin-c-kit+CD45+ cells in 12-dpc FL and FT cells (3000 cells each) of B6 fetuses, and c-kit+CD45+Sca-1high cells in 12-dpc FL cells (3000 cells) of B6 fetuses by using a QuickPrep Micro mRNA Purification Kit (Pharmacia, Little Chalfont, U.K.).
Reverse transcription. A mixture of mRNA solution and 5 µl of 0.04 µg/µl oligo(dT) primers (Life Technologies) was incubated at 65°C for 5 min. Samples were placed on ice and the following reagents were added: 4 µl of 5x reverse-transcription buffer (0.25 M Tris-HCl, pH 8.3, 0.375 M KCl, and 15 mM MgCl2), 2 µl of 0.1 M DTT, 0.4 µl of 25 mM dNTPs, and 100 U M-MLV reverse transcriptase (Life Technologies). The reaction samples were incubated at 37°C for 60 min and inactivated at 70°C for 5 min, then chilled on ice.
PCR. cDNA was amplified by PCR using various sets of primers. Primers used: TCF-1 sense, 5'-CCAGCTTTCTCCACTCTACG-3'; TCF-1 antisense, 5'-TCAAGGATGGGTGGGTGAAC-3'; GATA-3 sense, 5'-TCGGCCATTCGTACATGGAA-3'; GATA-3 antisense, 5'-GAGAGCCGTGGTGGATGGAC-3'; mb-1 sense, 5'-ATCATCTTGCTGTTCTGTGC-3'; mb-1 antisense, 5'-ACACTAACGAGGATGCTGTA-3'; c-fms sense, 5'-CTGGAGAAGAAATATGTGCG-3'; c-fms antisense, 5'-TTCAGACCAAGCGAGAAGAT-3'; IL-7R sense, 5'-AAGGATGTGGTGAATGCAGG-3'; IL-7R antisense, 5'-ACAATAGGACAGGTTCATGG-3'; ß-actin sense, 5'-TCCTGTGGCATCCATGAAACT-3'; ß-actin antisense, 5'-GAAGCACTTGCGGTGCACGAT-3'. The reaction volume was 20 µl containing 2 µl of cDNA sample, 2 µl of 10x PCR buffer (0.1 M Tris-HCl, pH 9, 0.5 M KCl, and 15 mM MgCl2), 0.16 µl of 25 mM dNTPs, 0.6 U Taq polymerase (Pharmacia), and 0.4 µl of each primer (10 mM). After incubation for 5 min at 94°C, PCR amplification was performed using the Thermal-Cycler (TAKARA, Otsu, Japan). Cycling times and temperatures were as follows: denaturation at 94°C for 1 min, annealing at 55°C for 1 min, and elongation at 72°C for 2 min. Amplification was performed for 20 cycles for ß-actin and 30 cycles for all other genes. Fifteen microliters of PCR product were electrophoresed through 1.2% agarose or 5% polyacrylamide gel and stained with ethidium bromide.
PCR analysis of TCR gene rearrangement
After 10 days of MLP assay culture, half of each sample was used
for flow-cytometric analysis, and the remaining half was subjected to
PCR analysis. Cells (3000 cells) were resuspended in 20 µl of 1x PCR
buffer (10 mM Tris-HCl, pH 9, 50 mM KCl, and 1.5 mM
MgCl2) including 0.45% Nonidet P-40, 0.45%
Tween 20, and 1.2 µg proteinase K (Sigma, St. Louis, MD), and
incubated at 55°C for 1 h, then 95°C for 10 min. Samples of
these disrupted cells were used as templates for PCR amplification.
Primers were: Dß2, 5'-GCACCTGTGGGGAAGAAACT-3';
Jß2.6, 5'-TGAGAGCTGTCTCCTACTATCGATT-3';
V4, 5'-AGTGTTCAGAAGCCCGATGCA-3';
J1, 5'-AGAGGGAATTACTATGAGCT-3'. The reaction
volume was 20 µl containing 5 µl of the cell extract (equivalent to
750 cells), 1.5 µl of 10x PCR buffer, 0.16 µl of 25 mM dNTPs, 0.4
µl of each primer (10 mM), and 0.6 U Taq polymerase.
Thermocycling conditions were as follows: 5 min at 94°C, followed by
35 cycles of 1 min at 94°C, 1 min at 60°C for D-Jß rearrangement
or at 55°C for V-J rearrangement, 2 min at 72°C, and 10 min at
72°C. Amplified DNA products were applied to a 1.2% agarose gel,
electrophoresed, and stained with ethidium bromide.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
We first investigated when progenitors capable of generating T cells emerge in the AGM region. Unfractionated cells from the AGM region of 9.5-dpc fetuses (4–10 x 104 cells; equivalent to 2–5 fetuses) or those of 10.0-dpc fetuses (3 x 104 cells; equivalent to 1 fetus) were cultured together with a dGuo-treated FT lobe, and the cells generated in the wells were assayed with a flow cytometer. It was found that T cell generation was always observed in cultures of 10.0-dpc AGM cells, whereas in cultures of 9.5-dpc AGM cells, T cell generation was seen only in two of seven experiments (data not shown). These results suggested that progenitors capable of generating T cells emerged between 9.5 and 10.0 dpc.
We investigated the time course of T cell generation from
10.0-dpc AGM progenitors. Unfractionated AGM cells (3 x
104 cells per well), and as controls 12-dpc FL cells (3 x
103 cells per well) and
Lin-c-kit+ adult BM
cells (2 x 103 cells per well), were
cultured together with a dGuo-treated FT lobe. The number of cells used
in each group is set in order that the numbers of T cell progenitors
included in these groups are comparable. Cells grown in each well were
harvested at different days, enumerated, and analyzed by
flow-cytometric analysis. Numbers of Thy-1+ cells
per well are plotted in Fig. 1
A, and CD4 vs CD8 and
TCRß vs TCR expression profiles of cells generated in these
cultures are shown in Fig. 1B. T cell generation from AGM
progenitors was much more rapid than that from BM progenitors (Fig. 1A), although it was slower by about 2 days than that from
12-dpc FL progenitors. The time course of T cell generation from AGM
progenitors seems to conform to the profile seen in ontogenic T cell
development in FT (18, 24). It was also found that the
proportions of Vß6+,
Vß8+, or V3+ cells
among T cells generated from the 10.0-dpc AGM progenitors were
comparable to those among T cells generated from 12-dpc FL progenitors
(Table I). From these results, the T cell
progenitors in the AGM region may have been qualified as those
migrating into FT.
|
|
We next examined the ability of AGM progenitors to generate B and myeloid cells. When unfractionated cells from 9.5-dpc AGM region (1 x 105 cells) were cultured on a monolayer of the stromal cell line TSt-4, which supports the development of both B and myeloid cells, myeloid cell generation was constantly observed, whereas B cell generation was only occasionally seen (data not shown) like the T cell generation in FT organ cultures (see preceding section). On the other hand, 10.0-dpc AGM cells (3 x 104 unfractionated cells) constantly produced B and myeloid cells. In the following experiments, we used 10.0-dpc fetuses as the source of AGM progenitors.
AGM cells (3 x 104 unfractionated cells) from 10.0-dpc
fetuses, and for comparison 12-dpc FL cells (3 x 103
unfractionated cells), were cultured on a monolayer of TSt-4, and
floating cells were harvested on days 5, 10, and 14. The recovered
cells were assayed for expression of B220, Mac-1/Gr-1, and surface IgM
(Fig. 2). At an early phase (day 5), the
culture of AGM progenitors was predominated by myeloid cells. This is
in contrast with the fact that the cultures of FL progenitors were
dominated by B cells. B cell growth from AGM progenitors became
prominent on day 10 of culture (data not shown), and on day 14 the
number of B cells became comparable with that in cultures of FL
progenitors. The B cells derived from 10.0-dpc AGM progenitors express
surface IgM within 14 days after initiation of the culture. It was also
found that these B cells were negative for surface CD5 (data not
shown). The results in this and the preceding section indicate that T
cell progenitors emerge nearly at the same gestational age as B cell
progenitors.
|
AGM and FB cells from 10.0-dpc fetuses, and also FL and FT cells
from 12-dpc fetuses, were stained with various mAb and analyzed by flow
cytometry. Flow-cytometric profiles of AGM, FB, and FL cells are shown
in Fig. 3A. Previous
investigations indicated that progenitors are enriched in the
Sca-1+ population in FL and FB of 12–13-dpc
fetuses (18, 25), but no Sca-1-expressing cells are found
in the AGM region or FB of 10.0-dpc fetuses (Fig. 3A). On
the other hand, it seemed probable that hemopoietic progenitors are
enriched in the
TER-c-kit+CD45+
population, because the
TER-c-kit+CD45-
population contains angioblasts (26) and probably also
primordial germ cell progenitors (27).
|
B). In contrast, the bands
representing the products of mb-1, c-fms, and IL-7R in the
Sca-1high cells of 12-dpc FL were faint or almost
invisible. These findings strongly suggested that in the AGM region,
various types of committed progenitors are present in the
c-kit+CD45+
population. The failure to detect any TCF-1 transcripts except for FT
may indicate that T cell differentiation has not yet begun in the AGM
region nor in the FB. Identification of lineage-committed as well as multipotent progenitors in the AGM region
In the present study, we succeeded in applying the MLP assay
system to investigate the developmental potential of individual
progenitors in the AGM region. This was attained by changing the
combination of cytokines added to the HOS culture (see Materials
and Methods). Single cells of the
TER-c-kit+CD45+ AGM population
obtained from 10.0-dpc fetuses of B6Ly-5.1 or (B6 x B6Ly
5.1)F1 mice were cultured together with a
dGuo-treated lobe in the presence of SCF, GM-CSF, and IL-7 (Fig. 4A). On the tenth day of
culture, cells were harvested from each well, stained with various mAb,
and flow cytometrically analyzed.
|
. It was found that p-T in the
AGM region comprise two types, p-T1 and
p-T2 (Fig. 4B).
p-T2 are similar to the p-T in FL, because this
type of p-T can generate a large number (>20,000 cells) of
Thy-1+ cells, and a portion of these cells
expresses CD4 and/or CD8 (Fig. 4B, extreme right
panels) as well as TCR (not shown) on their surface. The other
type (p-T1), which comprises the majority of p-T
in the AGM region, generates only 500-3000 Thy-1+
cells, and these Thy-1+ cells do not express
other T cell markers, nor the NK cell marker NK 1.1 (not shown), even
after an extended culture period. Removal of GM-CSF from the culture
medium did not influence the phenotype of Thy-1+
cells derived from AGM p-T1 (data not shown).
It was also found that two types of p-Multi
(p-Multi1 and p-Multi2)
exist in the AGM region (Fig. 4C).
p-Multi1-type progenitors generate a small number
(<5000 cells) of
Thy-1+CD4-CD8-
cells by day 10 of culture, whereas p-Multi2-type
progenitors are capable of generating a large number of T cells
expressing CD4 and/or CD8 within 10 days of culture.
p-Multi2-type progenitors have never been
detected in 12-dpc FL (17, 18 , and our unpublished data)
or in the 11-dpc AGM region (not shown). Because a large majority of
p-T in the AGM region are p-T1 type, it is likely
that the rather rapid generation of mature T cells from unfractionated
AGM cells seen in Fig. 1 is at least partially contributed by the
p-Multi2.
We next investigated whether the Thy-1+ cells
generated from these p-T1 are authentic T cells.
DNA was extracted from half of Thy-1+ cells
derived from two p-T1 and one
p-T2, and served for PCR analysis to determine
the Dß2-Jß2 and V3/V4-J1 rearrangements. T cells derived
from a p-Multi1 of the AGM region were also
investigated. As positive and negative controls for PCR, 16-dpc FT
cells and cells derived from a p-M, respectively, were used. Results
are shown in Fig. 5. FT cells from 16-dpc
fetuses show six D-Jß-rearranged bands and a germline band (1.8 kb)
as well as V-J-rearranged bands (V4J1 and V3J1). These
six D-Jß- and two V-J-rearranged bands were observed in all
samples examined in this study, including the case of 2
p-T1, indicating that cells generated from the
p-T1 are surely T cells, and that both
p-T1 and p-T2 extensively
proliferated before rearrangement of TCR genes.
|
Fig. 6 scores the numbers of
different types of progenitors found in 582
TER-c-kit+CD45+
AGM cells, and 200
TER-c-kit+CD45+
FB cells from 10.0-dpc fetuses. Progenitors detected among 234
Lin-c-kit+CD45+
FL cells from 12-dpc fetuses are also shown. Numbers of progenitors per
AGM region, FB, or FL of one fetus can be read on the bottom scale. The
proportions of six different types of progenitors in the AGM region are
not so much different from those in FL. However, the numbers of
progenitors in the AGM region were much smaller than those in FL. For
example, the number of p-Multi in the AGM region was only 8, whereas
about 2000 p-Multi are present in FL. Such a large difference, however,
simply reflects the difference in cell numbers between the 10.0-dpc AGM
region and 12-dpc FL. Although not indicated in Fig. 6, among 4 p-T in
the AGM region, 3 were p-T1 type and 1 was
p-T2 type, and among 8 p-Multi in the AGM region,
6 were p-Multi1 type and 2 were
p-Multi2 type.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Characteristics of the progenitors in AGM region are slightly different from those in FL. For example, AGM progenitors do not express Sca-1, which is the marker of the earliest progenitors in FL (18, 25). GM-CSF, but not IL-3, was effective in supporting the differentiation of AGM progenitors toward myeloid lineage, although FL progenitors respond to both IL-3 and GM-CSF (17). With IL-3, AGM progenitors tend to become blastoid without generating T, B, or myeloid cells. These differences may reflect the difference in developmental stage of progenitors obtained at different embryonic ages as well as the in vivo environment they belonged to. Of interest is the fact that the same six types of progenitors as found in FL (17, 18) were identified in the AGM region, regardless of such differences in surface phenotype and cytokine responsiveness. This may indicate that hemopoietic stem cells or multipotent progenitors begin lineage commitment in the AGM region before completing the maturational steps of the multipotent progenitor itself. Conforming to the finding in FL (17, 18), all bipotent progenitors detected in the AGM region were p-MT and p-MB types, and p-TB-type progenitors were not detected. These results strongly suggested that commitment to p-T and p-B in the AGM region occurs mainly through bipotent p-MT and p-MB progenitors, respectively. Although p-TB-type progenitors have been reported to be present in the Lin-c-kitlowIL-7R+ population of adult BM (33), a recent report from the same research group documented that the p-TB-type progenitors may not exist in FL (34). Our present study strongly suggested that the process of hemopoietic cell differentiation in the AGM region is similar to that in FL, but not BM.
We have previously reported the existence of p-T in 12-dpc FL
(17, 18). The present work is the first to identify p-T in
10-dpc fetuses, at the gestational age when the thymus has not yet
developed. We consider that the progenitors detected as p-T are T cell
lineage restricted in vivo, because of the following reasons. First,
the MLP assay is effective in discriminating p-T from p-Multi in FL, as
detailed in our previous study (18, 19). Second, it was
found that the AGM p-T are self reproductive or proliferate before the
rearrangement of TCRß gene without producing other lineage cells
(Fig. 5). Such a self-reproducing capability may also indicate that the
lineage commitment is a very early event in hemopoiesis. Third, RT-PCR
analysis indicated that the transcription factor GATA-3 that is shown
to be specific to T cell lineage (28) was expressed in the
c-kit+ AGM cells (Fig. 3B). The
expression of IL-7R that is essential to early differentiation of T and
B lineage cells is also seen in the c-kit+
AGM cells (Fig. 3B) as in c-kit+
FL cells, in which a large number of p-T were present
(18).
It was also found that unipotent progenitors are heterogeneous in their
differentiational potential. For example, p-T can be divided into
p-T1 and p-T2 (Fig. 4B). Among a total of 19 p-T to date detected in the AGM
region, 17 were p-T1 type and 2 were
p-T2 type. In contrast, only 1 p-T detected in
the 10.0-dpc FB was p-T2 type. In 11-dpc AGM region, 2 of 7 p-T
were p-T2 type (data not shown), indicating that
the proportion of p-T2 increases with the fetal
age. Such p-T1-type progenitors, however, have
not ever been observed in 12-dpc FL (data not shown). It has not yet
been clarified, however, whether p-T1 are simply
the immature type of p-T that could mature into
p-T2 if maintained in the environment of the AGM
region, or whether they are the progenitors of a different type of T
cells, such as those developing extrathymically. On the other hand, it
is improbable that p-T1 is restricted to the NK
lineage, because TCR gene rearrangement is seen in the progeny of
p-T1 (Fig. 5).
It was surprising that p-Multi2-type progenitors
were discovered in the AGM region, which generate T cells as rapidly as
p-T2 (Fig. 4). It is highly probable that
p-T2-type progenitors are derived from such
p-Multi2. More intensive investigation, however,
is required to clarify the maturational steps of hemopoietic stem cells
themselves as well as the mechanism of lineage commitment.
The number of progenitors capable of generating B cells, which is the
total number of p-Multi, p-MB, and p-B in the AGM region of a 10.0-dpc
fetus (30–35 somite stage) detected by our MLP assay, was 10 (Fig. 6).
This value is equivalent to the number of the B cell progenitors (15
cells) in a 9.5-dpc P-Sp (25 somite stage) previously estimated by
culturing single cells on the stromal cell monolayer (13),
indicating that the seeding efficiency in the MLP assay cultures is
comparable to that in cultures on stromal cells. On the other hand, the
MLP assay is much more efficient in detecting progenitors than the
CFU-S assay. It has been shown that day 8 CFU-S and day 11 CFU-S
observed in the 10-dpc AGM region were only 0.8 and 1.2, respectively
(1, 35), and a large proportion of these CFU-S was
erythroid progenitors (35). With the MLP assay, the total
number of progenitors capable of generating myeloid cells (the sum of
p-Multi, p-MT, p-MB, and p-M) was 38.
The numbers of progenitors in the 10-dpc AGM region are much smaller than those in 12-dpc FL, the numbers of p-Multi, p-T, p-B and p-M in the AGM region being 1/250, 1/1000, 1/400, and 1/200 of those in 12-dpc FL, respectively. If all FL progenitors are derived from the AGM region, extensive proliferation and/or self-reproduction of p-Multi, as well as delivery of committed progenitors should have occurred either or both in the AGM region and FL. Immigration from the YS may also contribute to the definitive hemopoiesis in FL (10, 11).
Generation of T and B cells from 9.5-dpc AGM cells was observed only in two of seven experiments, despite the fact that a large number of cells, equivalent to two to five fetuses, were seeded in each culture. On the other hand, 10.0-dpc AGM progenitors always generated T and B cells. Garcia-Porrero et al. (36) showed by microscopic observation that the formation of the hemopoietic clusters on the vascular endothelial cell layer of the AGM region starts at 9.5 dpc. Our recent work further suggested that the main lymphoid progenitors in 9.5-dpc fetuses are the vascular endothelial cadherin-positive cells (14). These results strongly suggest that before 9.5 dpc, the progenitors generating lymphocytes are the hemangioblasts that can give rise to both hemopoietic cells and vascular endothelial cells, and that multipotent progenitors restricted to hemopoietic lineages emerge in the AGM region at about 9.5 dpc. Unstable generation of T and B cells from 9.5-dpc AGM progenitors may be attributable to incomplete commitment of the hemangioblast to the hemopoietic lineage. On the other hand, conflicting results about the generation of T and B cells from the progenitors in 8.5–9.5-dpc fetuses in other papers (12, 13, 14, 37, 38) could be due to a difference in cell preparation. Vigorous pipetting or enzymatic cell separation may have released a portion of the hemangioblasts from the endothelial cell layer.
Another important finding is that a large majority of hemopoietic
progenitors in 10.0-dpc FB are p-M, and the number of p-M in 10.0-dpc
FB reached nearly 1/5 of that in 12-dpc FB (18), despite
the fact that the total number of all types of progenitors in FB was
1/250 of that in 12-dpc FB (Fig. 6). Our preliminary experiments showed
that at least 1/5 of the p-M in the AGM region can also generate
erythrocytes. Circulation of a relatively large number of p-M in
10.0-dpc fetuses may reflect the preferential requirement of phagocytes
and erythrocytes during the earlier developmental stages of the
embryo.
Because the p-Multi are capable of generating T, B, and myeloid cells, it is probable that at least a portion of the p-Multi represents the hemopoietic stem cells. Although we have not yet completed establishing an MLP assay that covers the erythroid lineage, preliminary experiments indicated that only a small portion of p-Multi in the AGM region generates erythrocytes, thus suggesting that a large majority of p-Multi are restricted to myeloid, T, and B cell lineages (p-MTB). The presence of such p-MTB-type progenitors has also been reported recently by Lacaud et al. (39). On the other hand, we do not have any evidence to regard the AGM p-Multi that are able to generate erythrocytes as the conventional stem cells, because LTR-HSC activity has not been detected in 10-dpc AGM cells (5, 27). If the p-Multi in the AGM region do not have LTR-HSC activity, the AGM p-Multi could represent a primitive type multipotent progenitor giving rise to the stem cells. Studies are in progress to purify the p-Multi in 10.0-dpc AGM region and examine the LTR-HSC activity.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Yoshimoto Katsura, Department of Immunology, Institute for Frontier Medical Sciences, Kyoto University, Sakyo-ku, Kyoto 606-8507, Japan. E-mail address:
3 Abbreviations used in this paper: EB, embryo body; AGM, aorta-gonad-mesonephros; BM, bone marrow; dGuo, deoxyguanosine; dpc, days postcoitum; FB, fetal blood; FL, fetal liver; FT, fetal thymus; HOS, high oxygen submersion; HSC, hemopoietic stem cell; Lin, lineage markers; LTR, long-term repopulating; MLP, multilineage progenitor; p-B, progenitors committed to B cell lineage; p-M, progenitors committed to myeloid lineage; p-MB, bipotent progenitors capable of generating myeloid and B cells; p-MT, bipotent progenitors capable of generating myeloid and T cells; p-Multi, multipotent progenitors; P-Sp, paraaortic splanchnopleura; p-T, progenitors committed to T cell lineage; SCF, stem cell factor; YS, yolk sac.
Received for publication June 1, 1999. Accepted for publication August 18, 1999.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
ß-bearing T cells in the submersion organ culture of murine fetal thymus at high oxygen concentrations. Eur. J. Immunol. 23:200.[Medline]
and T cell receptor enhancers. J. Exp. Med. 173:1133.This article has been cited by other articles:
|
|
 |
|
  T. Yamashita, S. Freigang, C. Eberle, J. Pattison, S. Gupta, C. Napoli, and W. Palinski Maternal Immunization Programs Postnatal Immune Responses and Reduces Atherosclerosis in Offspring Circ. Res., September 29, 2006; 99(7): E51 - E64. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Lu, R. Tayu, T. Ikawa, K. Masuda, I. Matsumoto, H. Mugishima, H. Kawamoto, and Y. Katsura The Earliest Thymic Progenitors in Adults Are Restricted to T, NK, and Dendritic Cell Lineage and Have a Potential to Form More Diverse TCR{beta} Chains than Fetal Progenitors J. Immunol., November 1, 2005; 175(9): 5848 - 5856. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. C. WEISEL and M. A. S. MOORE Genetic and Functional Characterization of Isolated Stromal Cell Lines from the Aorta-Gonado-Mesonephros Region Ann. N.Y. Acad. Sci., June 1, 2005; 1044(1): 51 - 59. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Masuda, M. Itoi, T. Amagai, N. Minato, Y. Katsura, and H. Kawamoto Thymic Anlage Is Colonized by Progenitors Restricted to T, NK, and Dendritic Cell Lineages J. Immunol., March 1, 2005; 174(5): 2525 - 2532. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Ikawa, K. Masuda, M. Lu, N. Minato, Y. Katsura, and H. Kawamoto Identification of the earliest prethymic T-cell progenitors in murine fetal blood Blood, January 15, 2004; 103(2): 530 - 537. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 I. Nobuhisa, M. Takizawa, S. Takaki, H. Inoue, K. Okita, M. Ueno, K. Takatsu, and T. Taga Regulation of Hematopoietic Development in the Aorta-Gonad-Mesonephros Region Mediated by Lnk Adaptor Protein Mol. Cell. Biol., December 1, 2003; 23(23): 8486 - 8494. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Q. Shen, M. Lu, T. Ikawa, K. Masuda, K. Ohmura, N. Minato, Y. Katsura, and H. Kawamoto T/NK Bipotent Progenitors in the Thymus Retain the Potential to Generate Dendritic Cells J. Immunol., October 1, 2003; 171(7): 3401 - 3406. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Lu, H. Kawamoto, Y. Katsube, T. Ikawa, and Y. Katsura The Common Myelolymphoid Progenitor: A Key Intermediate Stage in Hemopoiesis Generating T and B Cells J. Immunol., October 1, 2002; 169(7): 3519 - 3525. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. A. Martinez-M., S. Minguet, P. Gonzalo, P. G. Soro, B. de Andres, A. Izcue, M. A. R. Marcos, and M.-L. Gaspar Long-lived polyclonal B-cell lines derived from midgestation mouse embryo lymphohematopoietic progenitors reconstitute adult immunodeficient mice Blood, September 15, 2001; 98(6): 1862 - 1871. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Itoi, H. Kawamoto, Y. Katsura, and T. Amagai Two distinct steps of immigration of hematopoietic progenitors into the early thymus anlage Int. Immunol., September 1, 2001; 13(9): 1203 - 1211. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Ohmura, H. Kawamoto, M. Lu, T. Ikawa, S. Ozaki, K. Nakao, and Y. Katsura Immature Multipotent Hemopoietic Progenitors Lacking Long-Term Bone Marrow-Reconstituting Activity in the Aorta-Gonad-Mesonephros Region of Murine Day 10 Fetuses J. Immunol., March 1, 2001; 166(5): 3290 - 3296. [Abstract] [Full Text] [PDF]
|
|
|
