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Department of Immunology, Institute for Frontier Medical Sciences, Kyoto University, Sakyo-ku, Kyoto, Japan
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The problem with the MLP(MTB) assay, however, is that this method is unable to detect the erythroid potential of these progenitors. Therefore, it remained unclear whether the p-Multi represent the HSC themselves or the p-Multi also include progenitors restricted to myeloid, T, and B cell lineages (p-MTB). The earliest progenitors of M/E lineages have been designated as CFU-granulocyte/erythrocyte/macrophage/megakaryocyte (CFU-GEM-Meg), which represent the progenitors showing the broadest developmental capability in a CFU culture (CFU-C) assay. Because it was recently shown that common M/E progenitors, which are distinct from the HSCs, exist in murine bone marrow (13) and FL (14), now it is clear that the CFU-GEM-Meg represents both M/E progenitors and HSCs. In contrast, it remains to be clarified whether myeloid and erythroid potentials are always set together or only myeloid potential accompanies lymphoid branches.
In the present study, we devised new clonal assay systems capable of investigating the developmental potential of a single progenitor for generation of: 1) myeloid, erythroid, and B cell lineages (MLP(MEB) assay) and 2) myeloid, erythroid, T, and B cell lineages (MLP(METB) assay). Using these assays in combination with the CFU-C assay, we investigated the developmental potential of individual cells in various subpopulations of lineage marker-negative (Lin-) c-kit+ FL cells. The results indicated that the first lineage commitment step in hemopoiesis is the production of common myelolymphoid progenitors (CMLP or p-MTB) and common myeloerythroid progenitors (p-ME). These findings enabled us to illustrate the framework of the early stages of lineage commitment in fetal hemopoiesis.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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C57BL/6 (B6) mice were purchased from SLC (Shizuoka, Japan), and B6 Ly-5.1 mice were maintained in our animal facility. B6Ly-5.1 fetuses at 13 days postcoitum (dpc) were used as the progenitor source.
Growth factors
Recombinant human erythropoietin (Epo; Genzyme, Cambridge, MA), recombinant murine (rm) stem cell factor (SCF) (Genzyme), and rmIL-3 (Genzyme) were used.
Antibodies
The following Abs were used: anti-Ly-5.1 (A20-1.7; donated
by Y. Saga, Banyu Seiyaku, Tokyo, Japan); anti-c-kit
(ACK-2; donated by S.-I. Nishikawa, Kyoto University); anti-IL-7R
(A7R34, donated by S.-I. Nishikawa, Kyoto University); TER119 (TER)
(15) and anti-Fc
RII/III (FcR) (2.4G2; BD
PharMingen, San Diego, CA); FITC anti-Gr-1 (RA3-8C5; Caltag
Laboratories, San Francisco, CA); FITC anti-B220, PE anti-B220,
and allophycocyanin anti-B220 (RA-6B2; Caltag Laboratories); FITC
anti-Thy-1.2 (5a-8; Caltag Laboratories); PE anti-Sca-1
(E13-161.7; BD PharMingen); allophycocyanin anti-CD45
(30F11.1; BD PharMingen); PE anti-Mac-1 (M1/70; Caltag
Laboratories); and Red670-streptavidin (Life Technologies, Grand
Island, NY). TER was labeled with FITC. Anti-FcR and anti-IL-7R
were biotinylated. Anti-Ly-5.1 and anti-c-kit were
labeled with cyanine 5 (Cy5 labeling kit; Biological Detection Systems,
Pittsburgh, PA).
Staining and sorting of progenitors
Basic methods for surface staining of cells, flow cytometric analysis, and sorting of stained cells are as previously described (7). FL cells were four-color stained with anti-Lin, anti-Sca-1, anti-FcR, and anti-IL-7R, or with anti-Lin, anti-Sca-1, anti-CD45, and anti-FcR plus anti-IL-7R. Cells were sorted using a FACSVantage. Nonviable cells were excluded by forward and side scatter profiles.
Stromal cell lines and MLP(MEB) assay
Stromal cell lines PA6, OP9, and TSt-4 were used to investigate the generation of myeloid, erythroid, and B cells, respectively (16, 17, 18). The culture medium was RPMI 1640 supplemented with 5% FCS, sodium pyruvate (1 mM), nonessential amino acid solution (0.1 mM), 2-ME (5 x 10-5 M), streptomycin (100 µg/ml), and penicillin (100 µg/ml). Epo (2 U/ml) was added to the medium for coculture with OP9. In assaying the progenitor activity of a cell population, 50 sorted cells were cultured on stromal cell monolayers in a 24-well plate. Various days after culture, the floating cells were recovered and served for analysis with a flow cytometer.
In the clonal assay of progenitors, namely the MLP(MEB) assay, TSt-4 was monolayered in a 96-well plate, and culture medium was supplemented with Epo (2 U/ml) and G-CSF (10 ng/ml). Single cells were seeded into each well, and cell growth was judged with an inverted microscope. On the seventh day of culture, about half of the floating cells were harvested for flow cytometric analysis. The remaining cells in all wells were cultured for an additional 2 wk to confirm the progenitor activity observed on day 7.
High oxygen submersion (HOS) culture and MLP(METB) assay
The basic procedures for HOS culture of fetal thymus (FT) have been described previously (7). In brief, to prepare hemopoietic cell-depleted FT lobes, thymuses obtained from 15-dpc fetuses of B6 mice were cultured on polycarbonate filters (pore size 8 mm; Nuclepore, Pleasanton, CA) floating on culture medium containing deoxyguanosine (dGuo) (1.35 mM) for 6 days in a humidified atmosphere of 5% CO2 and 95% air. The lobes were washed, and single dGuo-treated lobes were placed into the wells of a 96-well V-bottom plate, to which the progenitors were added. Plates were centrifuged at 150 x g for 5 min at room temperature and placed into a plastic bag, and the air inside was exchanged with a gas mixture of 5% CO2, 70% O2, and 25% N2. The plastic bag was then incubated at 37°C. Culture period was 12–14 days.
The MLP(METB) assay is a modification of the MLP(MTB) assay, the procedure of which has been detailed elsewhere (7). Almost all procedures are the same as in the HOS culture. The differences are that a single cell is cultured together with a dGuo-treated FT lobe, and that the culture medium is supplemented with rmSCF (10 ng/ml), rmIL-3 (3 ng/ml), rmIL-7 (5 ng/ml), and Epo (2 U/ml) to support the growth of not only T cells, but also of myeloid, erythroid, and B cells. Cells grown outside the lobe were harvested by gentle pipetting on day 6, and used for flow cytometric analysis for erythroid and myeloid cells. The lobe was cultured for an additional 5 days, and the recovered cells from both inside and outside the lobe were analyzed for detection of T and B cells. Details of cell staining and flow cytometric analysis are as described previously (7).
CFU-C assay
FL cells were cultured in MEM (Life Technologies) containing 30% FCS, 1% methylcellulose, 1% fraction-V BSA (Sigma-Aldrich, St. Louis, MO), 2-ME (5 x 10-5 M), L-glutamine (1 mM), SCF (10 ng/ml), IL-3 (10 ng/ml), and Epo (2 U/ml). Numbers of colonies were counted on day 2 for CFU erythrocyte and on day 8 for all other colonies.
RT-PCR
mRNA was prepared from 104 cells, and cDNA
was synthesized with reverse transcriptase. cDNA equivalent to 500
cells were used for PCR. PCR was conducted as follows: denaturation at
94°C for 1 min, annealing at 53–65°C for 1 min, and elongation at
72°C for 2 min. The sequence of primers, annealing temperature, and
cycle numbers were as follows. Primers were for 
-actin,
5'-TCCTGTGGCATCCATGAAACT-3' and 5'-GAAGCACTTGCGGTGCACGAT-3'; for
Gata-1, 5'-TCCCAGTCCTTTCTTCTC-3' and
5'-ACAATTCCCACTACTGCTGC-3'; for Gata-2,
5'-ACACACCACCCGATACCCACCTAT-3' and 5'-GCCATGGCAGTCACCATGCT-3';
for EpoR, 5'-GGACACCTACTTGGTATTGG-3' and
5'-GACGTTGTAGGCTGGAGTCC-3'; for c-fms,
5'-CTGGAGAAGAAATATGTGCG-3' and 5'-TTCAGACCAAGCGAGAAGAT-3'; for
Gata-3, 5'-GGCCATTCGTACATGGAA-3' and
5'-GCCGTGGTGGATGGAC-3'; for Pax-5,
5'-TCCTCGGACCATCAGGACAG-3' and 5'-CCTGTTGATGGAGCTGACGC-3'; for
PU.1, 5'-AGATGCACGTCCTCGATACT-3' and
5'-TTGTGCTTGGACGAGAACTG-3'; for c-myb,
5'-AATATGGTCCGAAGCGTTGG-3' and 5'-CTCAGGGTCTTCGTCGTTAT-3';
for G-CSFR, 5'-TCATCACTCTGCCTCACTTG-3' and
5'-GAGACTACATCAGGGCCAAT-3'. Annealing temperature and cycle numbers
were 55°C/30 cycles for -actin; 58°C/35 cycles for
Gata-1; 65°C/40 cycles for Gata-2; 53°C/30
cycles for EpoR; 55°C/30 cycles for c-fms;
55°C/30 cycles for Gata-3; 58°C/35 cycles for
Pax5; 58°C/30 cycles for PU.1; 58°C/30 cycles
for c-myb; 55°C/35 cycles for
G-CSFR.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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FL cells from fetuses at 13 dpc were stained with various mAbs for
flow cytometric analysis. Approximately 8% of FL cells at this
gestational age are
Lin-c-kit+ (Fig. 1
A), and virtually all
Lin- cells are
c-kit+. A very small proportion of the
Lin-c-kit+ cells
expresses Sca-1 at high levels. This Sca-1 high-expressing cell
population was designated as Fr.1 (Fig. 1B). Our previous
study indicated that all multipotent progenitors (p-Multi) in FL, which
are capable of generating myeloid, T, and B cells, were exclusively
found in this Fr.1, and the Sca-1 negative to low positive
(Sca-1-) subpopulation represents the
progenitors at more advanced stages (9). It has been shown
that the expression of FcR begins at earlier stages in hemopoiesis
(7, 13, 19). We have previously shown that the expression
of IL-7R defines a subpopulation within the
Lin-c-kit+
population (10). Thus, to subdivide the
Lin-Sca-1- subpopulation,
we stained FL cells in four colors with anti-Lin, anti-Sca-1,
anti-FcR, and anti-IL-7R. It is shown in Fig. 1C
that the Lin-Sca-1-
subpopulation can be further subdivided into FcR high-expressing
(FcRhigh) IL-7R- (Fr.3)
and FcR-IL-7R+ (Fr.4)
subpopulations. Fr.4 is the population in which p-T or p-B is
enriched depending upon the gestational age (10). We have
also found that
Lin-Sca-1- cells contain
both CD45- cells and CD45+
cells. Lin-Sca-1- cells
can also be subdivided into
CD45+FcR-IL-7R-
(Fr.2),
CD45-FcR-IL-7R-
(Fr.5), and CD45+FcR/IL-7R+
(Fr.3 plus Fr.4) subpopulations, as shown in Fig. 1D.
Proportions of cells in Fr.1 to Fr.5 are indicated in Fig. 1E.
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Subpopulations of FL cells (Fr.1 to Fr.5 in Fig. 1
) were
cocultured at 50 cells/well on three different stromal cell lines, PA6,
OP9, and TSt-4, which efficiently support the generation of myeloid
cells, erythrocytes, and B cells, respectively (16, 17, 18). T
cell potential was investigated by culturing with a dGuo-treated FT
lobe. Various days after culture, cells were harvested, counted, and
stained with mAb to lineage markers for analysis with a flow cytometer.
Because the time course of cell generation differs among lineages (data
not shown), the peak cell numbers are shown in Fig. 2. Progenitors in Fr.1 generate cells of
all lineages examined, suggesting that this fraction contains the HSC
or a mixture of progenitors for all lineages. Progenitors in Fr.2
showed the potential to generate myeloid and erythroid cells, but did
not show any T or B cell generation. T and B cell generation was
observed when a large number (
103/well) of
Fr.2 cells are cultured (data not shown), but such T or B cell
progenitors may have been diluted out from the triplicate cultures of
50 cells in this experiment. These results indicated that Fr.2 does not
contain any HSCs, but contains M/E lineage-restricted progenitors or a
mixture of myeloid and erythroid progenitors. Fr.3 was shown to
exclusively contain myeloid lineage-restricted progenitors. As has been
reported (10), progenitors in Fr.4 are almost completely
restricted to the T or B cell lineage, although a marginal level of
macrophage potential has also been observed. Fr.5 contains only
erythrocyte precursors.
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). In Fr.2, the
proportions of CFU-GM, CFU-G, and CFU-M are much higher than in Fr.1,
and burst-forming unit erythroid appears in this fraction. CFU-GEM-Meg
are still seen in this fraction, while progenitors for the T and B cell
lineages are undetectable (Fig. 2), demonstrating the existence of M/E
progenitors that lack lymphoid potential. A large majority of colonies
in Fr.3 were CFU-M or CFU-G, although a small number of CFU-GM were
also seen. No CFU-GEM-Meg or erythroid colonies were found in this
fraction. Fr.4 contains a small number of CFU-M, conforming to the
results shown in Fig. 2. Fr.5 exclusively contains CFU
erythrocyte. These results confirmed the findings in Fig. 2 that
the lineage restriction progresses in parallel with the phenotypic
change.
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Progenitor activity of individual cells from Fr.1 (total 100
cells) was examined by culturing them in a 96-well plate with the
stromal cell line TSt-4 in the presence of G-CSF and Epo (Fig. 3A). Addition of Epo and G-CSF
to cocultures with stromal cell lines barely interfered with the B cell
potential of the progenitors (data not shown). These culture conditions
support the generation of myeloid, erythroid, and B lineage cells, and
this culture system was termed the MLP(MEB) assay. After 7 days of
culture, grown cells were harvested for microscopic observation and
flow cytometric analysis. Progenitors generating myeloid, erythroid,
and B cells (M/E/B type), which can be regarded as the HSCs, are
exclusively seen in Fr.1 (Fig. 3B, extreme
left panel). Fr.1 contains quite a large number of M/B type
progenitors. A large proportion of these M/B type progenitors should be
the p-Multi, as previously determined by the MLP(MTB) assay, rather
than bipotent p-MB, because p-MB are very rare, but p-Multi are
abundant in the Sca-1high population
(9) (see also Fig. 4).
Distinction of M/B type from M/E/B type may indicate that the
p-Multi detected by the MLP(MTB) assay are not necessarily the HSCs,
but include the p-MTB, which can be called the CMLP. M/E, M, and B type
progenitors were also detected in this population. However, B/E type
progenitors were not detected, suggesting that the lineage restriction
to a stage generating B cells and erythrocytes does not occur.
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B). The activity of
the M/E type progenitors for production of myeloid and erythroid cells
is comparable with that of the M/E/B type progenitors seen in Fr.1
(Fig. 3C). About half of the M/E type clones are comprised
of granulocytes, erythroid cells, macrophages, and megakaryocytes,
whereas the remaining half tend to lack megakaryocytes. An example of
Giemsa staining of the cells in a M/E type clone generated on a TSt-4
monolayer, which corresponds to a CFU-GEM-Meg in the CFU-C assay, is
shown in Fig. 3D, compared with cells in an M type clone,
which corresponds to a CFU-GM.
Expression of -globin, mb-1, and c-fms
in different types of clones was investigated by RT-PCR as the genes
specific for erythroid, B, and myeloid lineages, respectively (Fig. 3E). The results are completely in accordance with the
progenitor types, as determined by flow cytometric analysis (Fig. 3B). For example, the absence of lymphoid cells in M/E type
colonies from Fr.2 was confirmed by the failure to detect
mb-1 mRNA (Fig. 4E, lanes 4–6) that
is expressed in B lineage cells beginning at the early stages
(20). These results may have confirmed the presence of
common progenitors for M/E lineages that have no lymphoid
potential.
Fr.3 exclusively contains myeloid lineage-restricted progenitors (Fig. 3B). A large majority of these myeloid progenitors produce
only macrophages or granulocytes. The proportion of the
granulocyte/macrophage bipotent type progenitors in Fr.3 was comparable
with that of CFU-GM colonies detected by the CFU-C assay (Table I) in
this fraction (data not shown). The absence of B and erythroid lineage
cells in the myeloid colonies was confirmed by RT-PCR analysis (Fig. 3E, lanes 7 and 8). These results
establish the presence of macrophage/granulocyte type progenitors
without lymphoid and erythroid potentials; such progenitors may
subsequently be restricted to the macrophage or granulocyte lineages.
Fr.4, namely the
c-kit+Sca-1-IL-7R+
cells, has previously been shown to contain virtually
exclusively p-T and p-B using the MLP(MTB) assay, while a very
small number of macrophage progenitors was also present in this
fraction (10). This is confirmed by the data shown in the
extreme right panel of Fig. 3B.
Discrimination of CMLP from HSCs with the MLP(METB) assay
The developmental potential of individual progenitors in Fr.1 and
Fr.2 was further investigated using the MLP(METB) assay (Fig. 4A), which was also newly devised for the present study. The
procedure of this assay is basically the same as the MLP(MTB) assay,
which has been designed to examine the developmental potential of a
progenitor toward myeloid, T, and B cell lineages. In the MLP(METB)
assay, Epo is added to the culture to support erythropoiesis. Because
the MLP(METB) assay is able to determine the developmental potential of
individual progenitors toward the myeloid, erythroid, T, and B cell
lineages, it is formally possible to discriminate 15 different types of
progenitors, which are listed in Fig. 4C. Of the 15 types, 9
types were actually detected, which are p-METB, p-MTB, p-ME, p-MT,
p-MB, p-M, p-T, p-B, and p-E. Flow cytometric profiles of cells
generated from p-METB, p-MTB, p-ME, and p-E are shown in Fig. 4B. Profiles of cells derived from other types of
progenitors have been shown in previous studies (7, 9).
Six possible types, p-MET, p-MEB, p-ETB, p-ET, p-EB, and p-TB, have
never been detected, suggesting that such stages are absent in
FL.
The p-Multi, as determined by the previously used MLP(MTB) assay
(7, 9), was found to comprise at least two distinct
progenitor types, p-METB and p-MTB, with the latter in much larger
numbers. The p-METB may represent the HSCs themselves or the immediate
progeny of the HSCs retaining the same developmental potential as the
HSCs. The p-MTB may represent the earliest stage of differentiation
toward T and B cells, and thus can be named as the CMLP.
Furthermore, detection of p-ME with the MLP(METB)assay in Fr.2
confirmed the detection of M/E type progenitors in this fraction with
the MLP(MEB) assay (Fig. 3B). The inability of the p-MTB to
produce erythroid cells was confirmed by the failure to detect the
erythroid marker -globin using RT-PCR in the cells derived from the
p-MTB (data not shown).
Expression of lineage-associated genes in FL subpopulations
cDNA samples equivalent to 500 cells from subpopulations of FL
cells (indicated in Fig. 1) were used for PCR analysis to examine the
expression of transcription factors, which have been shown to play an
important role in hemopoiesis or lineage restriction (21, 22). The expression of receptors for lineage-specific growth
factors was also examined. As shown in Fig. 5, c-myb, PU.1,
Gata-1, Gata-2, and Gata-3 are
expressed at the earliest stage (Fr.1), and the expression levels of
these genes, except for Gata-3, does not seem to decline
until the Fr.2 stage. Expression of Gata-3 was the highest
at the Fr.4 stage, in which p-T and p-B are enriched (10).
Expression of Gata-1 and PU.1 in Fr.3 to Fr.5
seems reciprocal: Gata-1 is strongly expressed in Fr.5, but
hardly detectable in Fr.3 and Fr.4, whereas the reverse is true for
PU.1. B cell lineage-specific Pax-5 and erythroid
lineage-specific EpoR are exclusively expressed in Fr.4 and
Fr.5, respectively. c-fms and G-CSFR are
expressed from the earliest stage, and the expression continued onto
the stage of Fr.4. Because Fr.4 almost exclusively contains T and B
cell progenitors (4) (see also Fig. 2), and the cells in
this fraction barely respond to M-CSF or G-CSF (data not shown), the
expression of these genes could be related to remnant myeloid
potential.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B).
The p-METB is thought to be the HSC, because it is able to generate all
major lineage cells, and both p-METB and HSCs with long-term
reconstitution ability are exclusively found in Fr.1 (Fig. 4, and our
unpublished data). In contrast, in any in vitro clonal assay systems,
the detection of progenitors showing restricted potential may face the
problem as to whether the developmental potential of the progenitor is
fully induced in the culture condition used. The existence of p-ME may
be evident, because this type of progenitor was found in the population
in which no multipotent progenitors reside (Fr.2, Figs. 3B
and 4C). As for the discrimination of p-MTB from p-METB,
however, the possibility could not completely be ruled out that some of
p-METB are judged as p-MTB. Nevertheless, we regarded that most of the
progenitors that have expressed myeloid, T, and B cell potentials, but
not erythroid potential are really the p-MTB. This can be said because
the number of MEB type progenitors detected by using Epo-supplemented
OP9 stromal cells (data not shown), which is known to be the most
efficient culture system for induction of erythroid potential
(16), was comparable with the number of p-METB or MEB type
progenitor determined in the present study. The finding that the number
of HSCs (120 repopulating U/13 dpc FL) estimated by Ema and Nakauchi
(23) is comparable with that of p-METB (350/13 dpc FL,
see Fig. 4C), but not to the sum of p-METB and p-MTB, may
also support the discrimination of p-MTB from p-METB.
The existence of p-MTB was elucidated for the first time in the present
study, which can be termed the CMLP. It has recently been proposed that
the first major forking point in hemopoiesis in the bone marrow is the
production of M/E progenitors and CLP (4, 13). Because our
present findings in FL indicated that the CLP is not p-TB but p-MTB,
p-MTB rather than p-TB may be the key stage in branching toward the T
and B cell lineages in FL. During the past several years of
investigation on FL progenitors, we have repeatedly shown that the p-TB
type does not exist in FL. The present investigation with the MLP(METB)
assay strongly suggests that the absence of some possible types of
progenitor such as the p-TB is not exceptional. As seen in Fig. 4C, p-MET, p-MEB, p-ETB, p-ET, and p-EB types are also
undetected, implying that such stages do not exist. These results
further suggested that the erythroid potential is not maintained in the
lymphoid pathway, thus proposing that the first branch in hemopoiesis
may be between p-MTB and p-ME (Fig. 6). A
large proportion of p-MTB identified in our study was able to produce
both macrophages and granulocytes (data not shown). However, it is
probable that the granulocyte/macrophage double producer gives rise to
a macrophage producer along with the progress of differentiation,
because T/B/macrophage type progenitors have been reported
(6). The CMLP or p-MTB is exclusively found in Fr.1, in
which the HSCs belong, but not in Fr.4, in which p-T and p-B are
enriched (10). The phenotypic similarity between HSCs and
CMLP suggests that the CMLP is an immediate progeny of the
HSCs.
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), in which myeloid potential is only marginally
present (10). Similarly, p-B may be the immediate progeny
of p-MB. Although p-B scarcely generate DC (26 , and our
unpublished data), B cells retain an Ag-presenting function, and they
sometimes show a morphology indistinguishable from DC
(27). The findings that the myeloid potential accompanies all erythrocyte, T cell, and B cell branches may be compatible with the idea that the myeloid lineage represents the prototype of hemopoietic cells. The process of lineage commitment revealed in the present study could be the recapitulation of the phylogenic development of hemopoietic cells, in that all blood cells are thought to have evolved from macrophage-like phagocytic cells (28). It is likely that genes for molecules constructing erythrocytes, T cells, and B cells are controlled to be expressed after the expression of a basic program, which may lead to the macrophage or myeloid lineage. It remains unclear, however, whether myeloid differentiation is always accompanied with differentiation of erythrocytes, T cells, and B cells, or whether a myeloid-specific pathway independent of the other lineages exists.
It is highly probable that transcription factors are involved in
lineage restriction, although the key molecules, except for
Pax5 in the B cell lineage (29), have
not yet been identified. The results of RT-PCR analysis indicate that
the expression of many of the lineage-specific transcription factors
begins at the earliest stage, conforming to the previously proposed
idea that some of the lineage-specific genes are initiated to be
expressed at an uncommitted progenitor stage (30).
Reciprocal expression of PU.1 and Gata-1 between
Fr.3 and Fr.5, which represent p-M and p-E stages, respectively, seems
to be related to the shutting down of erythroid potential and myeloid
potential (Fig. 5). These observations are in complete agreement with
the previous findings that predominant expression of PU.1 or
Gata-1 promoted the commitment toward myeloid or erythroid
lineage, respectively (31, 32). To date, no candidate
genes have been found that could play a part in controlling the lineage
commitment between lymphoid and erythroid lineages.
The present findings indicate that hemopoiesis progresses through an
ordered restriction process rather than random processes. The ordered
restriction process has previously been proposed (33), and
recent molecular studies on hemopoiesis have suggested a hierarchy in
the lineage restriction process (21, 22). Although the
models proposed in these studies are different from each other and from
the model we propose (Fig. 6), they are based on the common concept
that hemopoiesis progresses according to a set program. Nevertheless,
lineage restriction in hemopoiesis is frequently referred to, without
any firm evidence, as a random process. This is usually called the
stochastic model rather than the random restriction model, the latter
more exactly expressing the meaning of this concept (34, 35). If hemopoiesis progresses through a random process, all 15
possible types of progenitors should be constantly detected with the
MLP(METB) assay. In the present study, however, of the 15 theoretical
types of progenitors, 6 are thought to be defective (Fig. 4), strongly
suggesting that the hemopoiesis progresses through an ordered
restriction process.
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Acknowledgments |
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Footnotes |
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2 M.L. and H.K. contributed equally to this paper.
3 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: Katsura{at}frontier.kyoto-u.ac.jp
4 Abbreviations used in this paper: HSC, hemopoietic stem cell; CFU-C, CFU culture; CFU-G, CFU granulocyte; CFU-GEM-Meg, CFU granulocyte/erythrocyte/macrophage/megakaryocyte; CFU-GM, CFU granulocyte-macrophage; CFU-M, CFU macrophage; CLP, common lymphoid progenitor; CMLP, common myelolymphoid progenitor; dGuo, deoxyguanosine; dpc, days postcoitum; E, erythroid; Epo, erythropoietin; FL, fetal liver; FT, fetal thymus; HOS, high oxygen submersion; Lin, lineage marker; M, myeloid; MLP, multilineage progenitor; rm, recombinant murine; SCF, stem cell factor.
Received for publication December 4, 2001. Accepted for publication July 24, 2002.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RIII defines distinct subpopulations of fetal liver B cell and myeloid precursors. Eur. J. Immunol. 25:2308.[Medline]
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 B. Giebel, T. Zhang, J. Beckmann, J. Spanholtz, P. Wernet, A. D. Ho, and M. Punzel Primitive human hematopoietic cells give rise to differentially specified daughter cells upon their initial cell division Blood, March 1, 2006; 107(5): 2146 - 2152. [Abstract] [Full Text] [PDF]
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D. L. Stachura, S. T. Chou, and M. J. Weiss Early block to erythromegakaryocytic development conferred by loss of transcription factor GATA-1 Blood, January 1, 2006; 107(1): 87 - 97. [Abstract] [Full Text] [PDF]
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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]
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A. Y. Lai, S. M. Lin, and M. Kondo Heterogeneity of Flt3-Expressing Multipotent Progenitors in Mouse Bone Marrow J. Immunol., October 15, 2005; 175(8): 5016 - 5023. [Abstract] [Full Text] [PDF]
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B. Larrivee, I. Pollet, and A. Karsan Activation of Vascular Endothelial Growth Factor Receptor-2 in Bone Marrow Leads to Accumulation of Myeloid Cells: Role of Granulocyte-Macrophage Colony-Stimulating Factor J. Immunol., September 1, 2005; 175(5): 3015 - 3024. [Abstract] [Full Text] [PDF]
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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]
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R. Haddad, P. Guardiola, B. Izac, C. Thibault, J. Radich, A.-L. Delezoide, C. Baillou, F. M. Lemoine, J. C. Gluckman, F. Pflumio, et al. Molecular characterization of early human T/NK and B-lymphoid progenitor cells in umbilical cord blood Blood, December 15, 2004; 104(13): 3918 - 3926. [Abstract] [Full Text] [PDF]
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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]
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