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The Journal of Immunology, 2001, 166: 3290-3296.
Copyright © 2001 by The American Association of Immunologists

Immature Multipotent Hemopoietic Progenitors Lacking Long-Term Bone Marrow-Reconstituting Activity in the Aorta-Gonad-Mesonephros Region of Murine Day 10 Fetuses1

Koichiro Ohmura*,{dagger}, Hiroshi Kawamoto*, Min Lu*, Tomokatsu Ikawa*, Shoichi Ozaki{dagger}, Kazuwa Nakao{dagger} and Yoshimoto Katsura2,*

* Department of Immunology, Institute for Frontier Medical Sciences, and {dagger} Department of Medicine and Clinical Science, Faculty of Medicine, Kyoto University, Kyoto, Japan


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Previous studies indicated that multipotent progenitors exist in early fetuses that do not contain long-term reconstituting (LTR) activity. However, it remained unclear whether these multipotent progenitors are committed to the hemopoietic lineage or are immature mesodermal cells or hemangioblasts. In this study, we have succeeded in enriching the multipotent progenitors that are capable of generating myeloid, T, and B cells in the LFA-1- subpopulation of TER-119-c-kit+CD45+ cells from the aorta-gonad-mesonephros (AGM) region of day 10 fetuses. We found that these day 10 AGM LFA-1- cells do not show the LTR activity, whereas day 11 AGM LFA-1- cells do have such an activity. These results strongly suggest that multipotent progenitors lacking LTR activity emerge as CD45+ hemopoietic progenitor cells in the AGM region on the 10th day of gestation, and such p-Multi mature into hemopoietic stem cells by acquiring LTR activity.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Hemopoietic stem cells (HSC)3 are defined as progenitors having multipotentiality and self-renewal capability, as well as the ability to repopulate all lineages of hemopoietic system over a long term (long-term reconstituting (LTR) activity) (1, 2, 3, 4, 5, 6, 7). In contrast, in early fetal hemopoietic tissues, it has been suggested that multipotent progenitors exist that lack LTR activity. For example, Godin et al. (8) reported that progenitors in paraaortic splanchnopleura from fetuses at 9.5 days postcoitus (dpc) are able to give rise to myeloid (M), B, and T cells, whereas LTR-HSC have not been found until 10.5 dpc (9, 10). Other research groups have also shown that the cells in both the yolk sac (YS) and embryonal body (EB) of 8–9 dpc fetuses can generate T and B lymphocytes (11, 12, 13, 14), but do not contain LTR activity (15). However, in these studies hemopoietic progenitors and immature mesodermal cells were not distinguished. This leaves open the possibility that such multipotential activity was due to immature mesodermal cells or hemangioblasts, which can differentiate into both hemopoietic and endothelial cells, rather than authentic hemopoietic progenitors.

Hemopoiesis is known to begin at 7.5 dpc in the extraembryonic mesoderm or YS, where erythropoiesis is mainly seen (16). After 9.5 dpc, hemopoietic clusters are visible in the EB (17). These clusters can be seen in the intraarterial sites of the dorsal aorta and the postumbilical arteries as well as the mesentery. Such hemopoietic tissues in the EB are called the aorta-gonad-mesonephros (AGM) region. Because no hemopoietic clusters are recognized by microscopic observation in the EB before 9.5 dpc (17), progenitors detected before 9.5 dpc can be regarded as immature mesodermal cells or hemangioblasts. In fact, lymphopoietic progenitors in 9.5 dpc fetuses are found mainly in vascular endothelial cadherin-positive CD45- cell populations, which can also generate endothelial cells (18). Only after 9.5 dpc does lymphopoietic activity become consistently detectable (11, 19). Previously, we have examined the developmental potential of CD45+ progenitors in the 10.0 dpc AGM region by a clonal assay named multilineage progenitor (MLP) assay (19, 20), which can determine the developmental capability of a single progenitor toward M, T, and B cell lineages. We found that in addition to progenitors restricted to M, T, or B cell lineage (p-M, p-T, and p-B, respectively), multipotent progenitors (p-Multi) generating M, T, and B cells were present in the 10.0 dpc AGM region (19). Intermediate types of bipotent progenitors capable of generating M and T cells (p-MT) or those capable of generating M and B cells (p-MB) were also found. In contrast, LTR-HSC have been reported to emerge first in the AGM region of 10.5 dpc fetuses (9). Thus, the question arises as to whether the p-Multi in the 10.0 dpc AGM region are distinct from LTR-HSC.

Recently, it has become possible to detect the LTR activity of as few as one to two stem cells by a bone marrow repopulation assay (21). Because ~10 p-Multi are present in the AGM region of a single 10.0 dpc fetus (19), it is possible to examine whether the AGM p-Multi have the LTR activity. In this study, we have examined the characteristics of hemopoietic lineage-committed progenitors by using the CD45+ population of AGM cells as a progenitor source. We found that p-Multi are highly enriched in TER-119(TER)-c-kit+CD45+LFA-1- population of 10.0 dpc AGM cells and showed that this cell population did not contain LTR-HSC. This study provides direct evidence that multipotent hemopoietic progenitors emerge ontogenically earlier than LTR-HSC.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice

Adult C57BL/6 (B6) mice were purchased from Japan SLC (Shizuoka, Japan), and B6Ly5.1 mice were maintained in our animal facility. 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, and the embryos with 30–35 pairs of somites were regarded as 10.0 dpc of gestation. The 11 and 12 dpc embryos used in this study include 11.0–11.5 and 12.0–12.5 dpc fetuses, respectively.

Monoclonal Abs

The following Abs were used: FITC-anti-Mac-1 and PE-anti-Mac-1 (M1/70; Caltag Laboratories, San Francisco, CA); FITC-anti-Gr-1 and PE-anti-Gr-1 (RB6-8C5; PharMingen, San Diego, CA); FITC-anti-B220, PE-anti-B220, and allophycocyanin-anti-B220 (RA3-6B2; Caltag); FITC-anti-Thy-1.2 and PE-anti-Thy-1.2 (5a-8; Caltag); FITC-anti-CD8 (YTS169.4; Caltag); FITC-anti-mouse IgM (Cappel, West Chester, PA); FITC-anti-TCR{gamma}{delta} (GL-3; Caltag); FITC-anti-CD44 (IM7.8.1; Caltag); FITC-anti-LFA-1 (121/7; Caltag); FITC-streptavidin (Caltag); PE-anti-CD45 (30F11.1; PharMingen); PE-anti-Sca-1 (E13-161.7; PharMingen); PE-TER (PharMingen); and Red670-streptavidin (Caltag). TER (22), anti-Ly5.1 (A20-1.7, donated by Y. Saga, Banyu Seiyaku, Tokyo, Japan), anti-Fc{gamma}RII/III (FcR) (2.4G2), and anti-CD34 (49E8; donated by H. Nakauchi, University of Tsukuba) were labeled with FITC in our laboratory. Anti-c-kit (ACK-2; donated by S.-I. Nishikawa, Kyoto University, Kyoto, Japan) and anti-Ly5.1 were conjugated with cyanine 5 (Cy5) using a labeling kit (Biological Detection Systems, Pittsburgh, PA).

Staining and sorting of progenitor cells

The basic methods for cell surface staining and analysis have been described previously (23). Single cell suspensions prepared from the AGM region, YS, and fetal liver (FL) of B6Ly5.1 fetuses by passage of the tissues through a 26-gauge needle were stained with various mAbs and were sorted using a FACSVantage. Sorted cells were reanalyzed to check their purity and were found to be >98% pure.

Cultures with stromal cell monolayer

The stromal cell line TSt-4 (19) was used to investigate the development of B and M cells. Single sorted AGM or FL cells were individually placed onto a confluent monolayer of TSt-4 cells in a 48-well plate (Costar, Cambridge, MA) using a micropipette under microscopic visualization. Medium changes were performed every 5 days. At day 14 of culture, all cells in each well were collected by trypsinization and analyzed with a flow cytometer. In some experiments, 2 U/ml of human erythropoietin (Epo; Genzyme, Cambridge, MA) and 10 ng/ml of human G-CSF (Genzyme) were added to the medium to promote the generation of erythroid cells and granulocytes. A portion of floating cells in each well was harvested on the 10th day of the culture and stained with a combination of mAbs for analysis with a flow cytometer. For Giemsa staining, cells were spun onto glass slides. Remaining cells were cultured for an additional 6 days and examined for generation of mature B cells.

MLP assay culture and analysis of cultured cells

The basic procedures for MLP assay culture have been described previously (20). Briefly, single sorted B6Ly5.1 cells were individually put into wells of a V-bottom 96-well plate (Costar) in which a deoxyguanosine (dGuo)-treated B6 fetal thymus (FT) lobe had been placed. A mixture of cytokines including 10 ng/ml of recombinant murine (rm) stem cell factor (Genzyme), 200 U/ml of rmIL-7 (donated by T. Sudo, Basic Research Laboratory, Toray, Kanagawa, Japan), and 1 ng/ml of rmGM-CSF (Life Technologies, Gaithersburg, MD), was added to the culture. Plates were centrifuged and placed 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. On day 5 of culture, half of the medium was replaced with medium containing 200 U/ml of IL-7 and 1 ng/ml of GM-CSF.

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-Ly5.1, whereas the other half was stained with FITC-anti-Mac-1, FITC-anti-Gr-1, PE-anti-B220, and Cy5-anti-Ly5.1. Surface phenotypes were analyzed with a FACSVantage by using CellQuest software (version 1.2.2). More detailed procedures for analysis have been described previously (19, 20).

RT-PCR

RNA isolation. mRNA was isolated from cultured cells (10,000 cells), TER+ bone marrow cells (3000 cells), and peripheral blood cells from 12 dpc fetuses (3000 cells) 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 RT 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 of Moloney murine leukemia virus reverse transcriptase (Life Technologies). Reaction samples were incubated at 37°C for 60 min, inactivated at 70°C for 5 min, and then chilled on ice.

PCR. cDNA was amplified by PCR using various sets of primers. Primers used were: {beta}-major globin sense, 5'-CTGACAGATGCTCTCTTGGG-3'; {beta}-major globin antisense, 5'-CACAACCCCAGAAACAGACA-3'; {beta}-H1 globin sense, 5'-AGTCCCCATGGAGTCAAAGA-3'; {beta}-H1 globin antisense, 5'-CTCAAGGAGACCTTTGCTCA-3'; {beta}-actin sense, 5'-TCCTGTGGCATCCATGAAACT-3'; {beta}-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, 0.5 M KCl, and 15 mM MgCl2), 0.16 µl of 25 mM dNTPs, 0.6 U of 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 {beta}-actin, 25 cycles for {beta}-major, and 30 cycles for {beta}-H1. Fifteen microliters of PCR product was electrophoresed through a 1.2% agarose gel and stained with ethidium bromide.

Reconstitution analysis

Eight- to 10-wk-old B6 recipient mice were whole body-irradiated (9.2 Gy) with a therapeutic 60Co {gamma}-ray source. Progenitor samples were injected i.v. via the tail vein together with 2 x 105 B6 bone marrow cells. Mice were housed in positive pressure cabinets.

Peripheral blood was periodically collected from the retro-orbital venous plexus of recipient mice to monitor reconstitution by donor-type progenitors. RBC in the samples were lysed by a 2-min incubation in 0.15 M ammonium chloride plus 0.01 M potassium bicarbonate. Cells were subsequently three-color stained either with FITC-Ly5.2, PE-Mac-1, and Cy5-Ly5.1 or with FITC-Thy-1, PE-B220, and Cy5-Ly5.1, and analyzed by flow cytometry. Mice were scored as positive when they had >1% of donor-derived cells of at least one lineage at 1 month after reconstitution. Six months after reconstitution, mice were scored as positive when >1% of cells were donor derived in all M, T, and B cell lineages.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Enrichment of p-Multi in 10.0 dpc AGM region

Cells from the 10.0 dpc AGM region were stained with various mAbs for flow cytometric analysis. The hemopoietic progenitor cells in the AGM region have been shown to express c-kit (10, 18), and TER is virtually the only lineage marker expressed by 10.0 dpc AGM cells (data not shown). Therefore, we investigated the expression of various surface markers, which have been reported to be useful for enriching immature cells (10, 21, 24, 25, 26, 27, 28, 29, 30), on the TER-c-kit+ population. As shown in Fig. 1GoA, 10.0 dpc AGM c-kit+ cells hardly express Sca-1 or Thy-1, whereas all c-kit+ cells are positive for CD44. LFA-1 and CD34 were found to clearly divide the c-kit+ AGM cells into two subpopulations, and expression levels of FcR and Mac-1 showed a broad distribution.



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  		FIGURE 1. Flow cytometric profiles of 10.0 dpc AGM cells. A, Cells from 10.0 dpc AGM region and 12 dpc FL were stained with a combination of TER, anti-c-kit, and various mAbs. Expression of different surface markers on TER-c-kit+ cells is shown. B, 10.0 dpc AGM cells were four-color stained with TER, anti-CD45, anti-c-kit, and anti-LFA-1, or with TER, anti-CD45, anti-c-kit, and anti-CD34. The numbers indicated in each panel represent the percentages of cells in the gated area or quadrants.

 
Our preliminary studies indicated that LFA-1 or CD34, but not Mac-1 or FcR, were useful for the enrichment of p-Multi. Expression profiles of these markers and c-kit on CD45+ 10.0 dpc AGM cells were investigated by four-color staining. c-kit vs LFA-1 and c-kit vs CD34 expression profiles of the TER-CD45+ population are shown in Fig. 1GoB. The cells in the LFA-1-, LFA-1+, CD34-, and CD34+ subpopulations of TER-c-kit+CD45+ cells were used as progenitor sources in the following experiments.

By culturing single cells on a stromal cell monolayer that supports the generation of M and B cells, progenitors can be classified into three types (31); M progenitors, B cell progenitors, and M/B bipotent progenitors. The clonal assay was performed on a monolayer of the stromal cell line TSt-4 (19). LFA-1-, LFA-1+, CD34-, and CD34+ subpopulations of the TER-c-kit+CD45+ cells were sorted, and the individual cells of each subpopulation were put on the stromal cell monolayer in a 96-well plate. After 14 days of culture, cells grown in each well were collected and analyzed for expression of B220 and Mac-1/Gr-1 by a flow cytometer. Numbers of M, B, and M/B progenitors are scored in Table IGo. M/B bipotent progenitors, a large proportion of which have T cell potential (Ref. 31 , and our unpublished data), are found to be enriched almost exclusively in CD34+ and LFA-1- subpopulations, and the frequency of M/B type progenitors was 2.4 times higher in the LFA-1- subpopulation than in CD34+ subpopulation. All the LFA-1- cells were also found to be CD34+ (data not shown). These results indicated that the LFA-1- population is the most useful for further characterization of the p-Multi.


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  		Table I. M and B cell generation from single progenitors of the 10 dpc AGM region on the stromal cell monolayera

 
Frequency of different types of progenitors in LFA-1- population detected by the MLP assay

The developmental potential of individual LFA-1- cells in the 10.0 dpc AGM region was investigated using the MLP assay. After 10 days of culture, cells inside and outside the FT lobe were harvested from each well and checked for the expression of M, T, and B cell-specific markers by flow cytometry. Six different types of progenitors, p-Multi, p-MT, p-MB, p-M, p-T, and p-B, were detected in this population. The flow cytometric profiles of the cells derived from these progenitors are not shown because they are similar to those previously shown (19, 20). Among 216 LFA-1- cells examined, 74 showed progenitor activity, and among them 23 were found to be p-Multi (Fig. 2GoA). These results indicate that the extent of enrichment attained here is ~8 times higher than that in the TER-c-kit+CD45+ population where the frequency of p-Multi was 8/582 (19). This also means that the p-Multi were ~400 times enriched relative to unfractionated AGM cells. The enrichment level of p-Multi in the LFA-1- population of AGM cells is comparable to that of c-kit+CD45+Sca-1high (Sca-1high) population of 12 dpc FL cells (Ref. 32 and Fig. 2GoC). This level of enrichment is also comparable to that of p-Multi detected as lympho-myeloid progenitors in the AA4.1+ cells of 10.0 dpc AGM region (33). In addition to p-Multi, bipotent progenitors p-MT and p-MB were also enriched in this population. p-TB type bipotent progenitors were not detected, confirming our previous findings with AGM cells (19), FL cells (20, 32, 34), and FT cells (35). Preliminary experiments indicated that no p-Multi were found in the LFA-1+ subpopulation (data not shown). Consistent with this is the finding that M/B type progenitors were very rare in LFA-1+ fraction as determined by coculture with a stromal cell line (Table IGo).



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  		FIGURE 2. The frequency and total number of different types of progenitors in LFA-1- population of the 10.0 dpc AGM region and YS, and in the Sca-1high population of 12 dpc FL. Single LFA-1- (TER-c-kit+CD45+LFA-1-) cells in the AGM region (A) or YS (B) from 10.0 dpc fetuses were cultured together with a dGuo-treated FT lobe in the medium containing stem cell factor, IL-7, and GM-CSF. In C, the same clonal culture of cells from the Sca-1high (c-kit+CD45+Sca-1high) population of 12 dpc FL was conducted. The numbers of examined cells are indicated in the upper part of each panel. In calculating the total numbers of progenitors, the numbers of cells in the AGM region, YS and FL were regarded as 3 x 104, 1 x 105, and 2 x 106, respectively. The percentage of LFA-1- cells in both the AGM region and YS as well as of Sca-1high cells in FL was regarded as 0.3%. The results are combined data from eight independent experiments.

 
The developmental potential of individual LFA-1- cells from 10.0 dpc YS was examined with the MLP assay (Fig. 2GoB). It is evident that the 10.0 dpc YS contain p-Multi, although the frequency, as well as the total number, of the p-Multi was much lower in the YS than in the AGM region. The results also indicated that a large majority of progenitors in the YS were p-M.

Lower growth potential of progenitors in the 10.0 dpc AGM region than those in 12 dpc FL

Mean numbers of cells generated from single p-M, p-T, p-B, or p-Multi of the 10.0 dpc AGM region and 12 dpc FL are shown in Fig. 3Go. In all cases, progenitors in the AGM region produced fewer cells than those in FL, a trend which is reproducible and also observed in cultures on stromal cell monolayers (see Fig. 4Go). These results suggest that there is a difference in the growth potential of progenitors derived from different sources.



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  		FIGURE 3. Difference in the number of cells generated from individual progenitors of the 10 dpc AGM region and 12 dpc FL. Single cells from the LFA-1- population of the 10.0 dpc AGM region and Sca-1+ population of 12 dpc FL were cultured under the MLP assay conditions. After 10 days of culture, cells were harvested from each well, and mean numbers and SEM of cells generated from different types of progenitors are shown. Numbers of clones used for the calculation of mean cell numbers for p-M, p-T, p-B, and p-Multi were 29, 6, 2, and 23, respectively, in AGM progenitors, and were 22, 4, 4, and 15, respectively, in FL progenitors.

 


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  		FIGURE 4. Characterization of cells generated in vitro from a single p-Multi in the 10.0 dpc AGM region and in 12 dpc FL. Single LFA-1- cells from the 10.0 dpc AGM region or single Sca-1high cells from 12 dpc FL were individually cultured for 16 days with a dGuo-treated FT lobe under MLP assay conditions (A) or cultured in 48-well plates on a monolayer of the stromal cell line TSt-4 in the presence of Epo and G-CSF for 10 days (B, D, and E). In C, cultivation of the wells used for B and D was continued for an additional 6 days. A, Representative profiles for CD4 vs CD8 and TCR{alpha}{beta} vs TCR{gamma}{delta} on T cells generated from an AGM p-Multi and an FL p-Multi. B, M and erythroid cells generated from a p-Multi in the AGM region and FL. These clones are the same as used for C and D. A large gating area was set in forward-side scatter to include all viable M, erythroid and B cells. C, B cells generated from a p-Multi in the AGM region and FL. A small gating area was set in forward-side scatter to exclude macrophages. D, Giemsa staining of cells generated from a p-Multi in AGM region and FL. Granulocytes, macrophages, and erythrocytes can be seen in both cases. E, RT-PCR analysis for the expression of {beta}-major and {beta}-H1 genes in clones of p-Multi from the 10.0 dpc AGM region and 12 dpc FL. Lanes 1, 2, 3, and 4 represent cells from a 10 dpc AGM LFA-1- progenitor, cells from a 12 dpc FL Sca-1high progenitor, TER+ cells in adult bone marrow, and unfractionated peripheral blood cells in 12 dpc fetuses, respectively. Average ± SD of cell recoveries from AGM p-Multi and FL p-Multi are 1.6 ± 1.2 x 104 (n = 5), 4.8 ± 1.9 x 104 (n = 5) in A, 2.5 ± 1.2 x 104 (n = 6), 8.0 ± 3.2 x 104 (n = 6) in B, and 5.0 ± 2.1 x 104 (n = 6), 12.0 ± 7.0 x 104 (n = 6) in C.

 
The T cell-generating capacity of p-Multi in 10.0 dpc AGM region and 12 dpc FL was characterized by extending the culture period of the MLP assay. Cells generated on the 16th day in MLP assay cultures of a single progenitor from the AGM region were compared with those derived from 12 dpc FL progenitors with respect to CD4 vs CD8 and TCR{alpha}{beta} vs TCR{gamma}{delta} expression (Fig. 4GoA). These progenitors are p-Multi because they were also capable of producing M and B cells (data not shown). As seen, AGM p-Multi produced much fewer mature T cells than the FL p-Multi, consistent with the data in Fig. 3Go.

Because the MLP assay conditions do not seem to be optimal for maturation and growth of M and B lineage cells, cultures with TSt-4 stromal cells were used to compare the M and B cell-producing potential of a p-Multi from the 10.0 dpc AGM region with that of a p-Multi from 12 dpc FL. Epo and G-CSF were added to the culture to support the generation and/or growth of erythroid cells and granulocytes. Individual cells from the 10.0 dpc AGM LFA-1- population or 12 dpc FL Sca-1high population were cultured in 48-well plates for 10 days, after which the cells were collected and analyzed by flow cytometry. Progenitors generating B, M, and erythroid cells were considered to be HSC because almost all clones showed T cell potential when a portion of the colony was cultured with a dGuo-treated FT lobe (data not shown). Hereafter, we will refer to these progenitors as p-Multi. Examples of FACS profiles and Giemsa staining micrographs of cells derived from an AGM p-Multi and a FL p-Multi are shown in Fig. 4Go, B and D. Erythroid cells (TER+B220-Mac-1-Gr-1-) and granulocytes (TER-B220-Mac-1+Gr-1+) are generated from both AGM and FL p-Multi. Cells displayed in the upper right quadrant of TER+Mac-1+Gr-1+ (Fig. 4GoB) are considered to be macrophages with nonspecific autofluorescence. The proportions of mature type M and erythroid cells generated from the AGM progenitor were much smaller than those derived from the FL progenitor. Generation of macrophages, granulocytes, and adult type erythrocytes was confirmed by Giemsa staining (Fig. 4GoD). RT-PCR for {beta}-globin genes was performed with mRNA collected from the clones that contained erythroid cells. The results showed that erythrocytes generated from both AGM and FL clones mainly produced adult-type hemoglobin, although they also express a faint band of fetal-type hemoglobin mRNA (Fig. 4GoE, lanes 1 and 2).

The remaining cells from these p-Multi clones were cultured for another 6 days under the same conditions, and then examined for expression of B cell markers. Representative flow cytometric profiles for B220 vs IgM are shown in Fig. 4GoC. The proportion of B220+ cells derived from an AGM progenitor was comparable to that derived from an FL progenitor. However, both the proportion of IgM+ cells and the expression level of IgM on B220+ cells derived from the AGM progenitor were much smaller than those from the FL progenitor. Expression of CD5 was not observed in any clones (data not shown). These results indicate that the p-Multi in the 10.0 dpc AGM region produced progeny less effectively than the FL p-Multi.

The p-Multi in the AGM region of 10.0 dpc fetuses lack LTR activity

Because p-Multi were found to be enriched in the LFA-1- population of the 10.0 dpc AGM region (Fig. 2Go), we examined the LTR activity of the LFA-1- cells. LFA-1- cells used here are CD45+ (Fig. 1GoB), and thus hemangioblasts and immature mesodermal cells are excluded from this population. One hundred AGM LFA-1- cells (including ~10% p-Multi) from 10.0 dpc B6Ly5.1 fetuses were transferred to lethally irradiated B6 adult mice together with 2 x 105 unfractionated B6 bone marrow cells. LTR activity of 300 LFA-1+ cells from the 10.0 dpc AGM region was also investigated. The numbers of these cells are almost equivalent to a total of LFA-1- and LFA-1+ cells in a fetus. Neither LFA-1- nor LFA-1+ cells from the 10.0 dpc AGM region showed any LTR activity (Table IIGo). Moreover, donor-derived cells were not detected at all, even 1 month after the transfer. This is in contrast to the results showing that only 50 Sca-1high cells from 12 dpc FL (containing ~10 p-Multi) are able to reconstitute hemopoiesis in two of five recipient mice, and a much higher level of reconstitution was attained with 100 Sca-1high FL cells. These results indicate that the p-Multi in 10.0 dpc AGM region lack LTR activity. We refer to the p-Multi devoid of LTR activity as pre-HSC.


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  		Table II. LTR activity of 10.0 dpc AGM progenitors

 
It has been shown that LTR-HSC are present in the AGM region of 11 dpc fetuses (9, 10). Because our preliminary experiments indicated that p-Multi are enriched in the LFA-1- population of 11 dpc AGM cells, we examined the LTR activity of LFA-1- cells from the 11 dpc AGM region (Table IIIGo). One hundred LFA-1- and 3000 LFA-1+ 11 dpc AGM cells were i.v. transferred to the lethally irradiated mice together with 2 x 105 unfractionated recipient type bone marrow cells. The number of LFA-1- and LFA-1+ cells used here is equivalent to half of the LFA-1- cells and four times that of the LFA-1+ cells in a fetus, respectively. We similarly transferred 300 FL LFA-1- cells and 15,000 FL LFA-1+ cells from 11 dpc fetuses, equivalent to 0.8 and 1.5 fetuses, respectively. LFA-1- but not LFA-1+ cells from the AGM region as well as FL of 11 dpc fetuses are positive for LTR activity. These results suggest that the AGM p-Multi seen in 10.0 dpc fetuses have matured from pre-HSC to HSC in a single day, or that LTR-HSC have newly emerged in the AGM region from 10.0 to 11 dpc.


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  		Table III. LTR activity of progenitors in AGM region and FL of 11 dpc fetuses

 
We have examined the expression of various surface molecules on LFA-1- cells from 10 dpc AGM and 11 dpc AGM/FL, and found that all of them were CD44+FcR-CD34+Thy-1- (data not shown). The only phenotypic difference between 10 and 11 dpc LFA-1- cells was that LFA-1- cells from 11 dpc AGM and FL begin to express Sca-1, although at a low level (Fig. 5Go). These findings suggest that initiation of Sca-1 expression on LFA-1- cells correlates with the acquisition of LTR activity.



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  		FIGURE 5. Sca-1 expression on the cells from 11 dpc AGM region. Cells were stained in four colors with TER, anti-c-kit, anti-Sca-1, and anti-LFA-1. Expression of Sca-1 vs LFA-1 on TER-c-kit+ cells is shown. The numbers in the panels indicate the percentage of cells in each quadrant.

 

   Discussion
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 Abstract
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 Materials and Methods
 Results
 Discussion
References
 
In this study, we succeeded in enriching p-Multi in the TER-c-kit+CD45+LFA-1- population of the 10.0 dpc AGM region and showed that these AGM p-Multi lack LTR activity. These results, together with our previous findings (18, 19), suggest that the multipotent hemopoietic progenitor branches from a hemangioblast before acquiring LTR activity.

As shown in our previous paper (32), p-Multi in 12 dpc FL were enriched in the Sca-1high subpopulation of CD45+c-kit+ cells. However, the Sca-1 marker cannot be used to enrich AGM p-Multi, because 10.0 dpc AGM cells hardly express this molecule on their surface (Fig. 1GoA). In this study, we found that the AGM p-Multi are enriched in the LFA-1- subpopulation of CD45+c-kit+ AGM cells. The absence of LFA-1 molecules on p-Multi is consistent with the previous reports in human bone marrow that the LFA-1- subpopulation of CD34+ cells are more primitive than the LFA-1+ subpopulation, as determined by long-term bone marrow culture and colony assay (28, 29).

Enrichment of the p-Multi in the LFA-1- population at a high level enabled us to investigate the LTR activity of AGM p-Multi more precisely. As shown in Table IIGo, 100 or 150 LFA-1- cells, which include ~10% p-Multi, failed to reconstitute hemopoiesis in irradiated adult recipients. None of the 14 recipients examined in these experiments show reconstitution with LFA-1- cells. This means that no LTR-HSC were found among a total of 1750 LFA-1- cells from 10.0 dpc fetuses, which include ~190 p-Multi, a number equivalent to 19 fetuses. This is in contrast with the fact that 50 or 100 Sca-1high cells from 12 dpc FL, including 10 and 20 p-Multi, respectively, showed LTR activity. Because the LTR activity of LFA-1- cells from the 11 dpc AGM region was comparable to that of Sca-1high FL cells from 12 dpc fetuses (Tables IIGo and IIIGo), it is likely that the p-Multi in the 10.0 dpc AGM region acquire LTR activity within 1 day, or that the 10.0 dpc p-Multi are replaced with new LTR-HSC type p-Multi during this period. Inducible potential for LTR-HSC has been reported by two research groups. Yoder and his colleagues (15, 36, 37) showed that the progenitors in the 9.0 dpc YS acquired LTR activity upon transfer into the myeloabrated neonatal liver, whereas Medvinsky and Dzierzak (38) showed that LTR-HSC were induced by organ culture of 10 dpc AGM region for a few days. However, it was unclear whether the LTR-HSC detected in these experiments were derived from immature mesodermal cells or pre-HSC. In contrast, we have obtained preliminary data that suggested that CD45+ pre-HSC matured into LTR-HSC. We cultured 200 or 400 LFA-1- 10.0 dpc AGM cells in a well of a six-well plate with OP9 (39, 40) stromal cells for 3 days, and cells from each well were transferred into a lethally irradiated mouse together with the recipient-type bone marrow cells. All lineages in 2 of 12 and 2 of 6 recipient mice, respectively, in these experiments were repopulated with the donor-derived hemopoietic cells for >6 mo. More detailed investigation may be required to determine whether LTR-HSC are derived from pre-HSC type p-Multi because our data does not completely rule out the possibility that a very small number of LTR-HSC included in the primary culture multiplied during the coculture with stromal cells.

The failure of p-Multi in the AGM region of 10.0 dpc fetuses to show LTR activity may be ascribed either to the insufficiency of self-renewal capacity or the inability of the AGM cells to adapt to the adult bone marrow environment. Our present and previous (19) investigations indicated that not only p-Multi but also unipotent type progenitors produced fewer progeny cells in MLP assay culture. The same tendency was also seen in cultures with stromal cells in the presence of cytokines (Fig. 4Go). These results may indicate that the self-renewal capacity of AGM progenitors is lower than that of FL progenitors. In contrast, the finding that AGM p-Multi failed to show any reconstitution potential even 1 month after cell transfer (Table IIGo) suggests a deficiency of adaptation, e.g., an inability to home to the bone marrow or cooperate with bone marrow environment for hemopoiesis.

p-Multi were also found in the 10.0 dpc YS, although the number was much smaller than that found in the AGM region (Fig. 2Go). Because the YS p-Multi, like the AGM p-Multi, failed to show any LTR activity (data not shown), they were considered to be pre-HSC. Although it is still controversial whether such YS p-Multi are merely immigrants from the AGM region or are generated in the YS, we propose that the p-Multi in the YS originate in the YS itself. This is based on our previous findings that the c-kit+ vascular endothelial cadherin-positive hemangioblast type cells are present in the YS as well as the AGM region, and they were able to generate not only erythromyeloid cells but also T and B lineage cells (18), suggesting that the YS-derived hemangioblasts contribute to the definitive hemopoiesis. Thus, it is possible that the pre-HSC found in the 10.0 dpc AGM region are the immigrants from the YS. This could be clarified if pre-HSC are detected before the appearance of LTR-HSC in the organ culture of the AGM region removed before circulation begins.

Yoder et al. (36) reported that the precursors capable of becoming LTR-HSC are far more abundant in the YS than in the AGM region at 9.0 dpc of gestation. Their result does not seem to conform to the present findings that the total number of p-Multi in 10.0 dpc YS is much smaller than that in the 10.0 dpc AGM region. However, the discrepancy can be explained if the 9.0 dpc YS contain plenty of CD45- hemangioblasts or immature mesodermal cells that can become LTR-HSC because sorted CD45+ cells were used in our experiments, whereas CD34+ YS cells, which contain both CD45+ and CD45- cells, were transferred in their experiments.

This study shows that LFA-1- p-Multi in the 10.0 dpc AGM region can be categorized as pre-HSC, which do not have LTR activity, thus revealing a novel developmental stage of hemogenesis during mouse ontogeny. Clarification of how pre-HSC acquire LTR potential will provide a clue to elucidating the mechanism of development and self-renewal of the HSC.


   Acknowledgments
 
We are indebted to Dr. Stuart Berzins (Joslin Diabetes Center, Harvard Medical School, Boston, MA) for critical reading of the manuscript, and to Y. Takaoki for secretarial assistance.


   Footnotes
 
1 This study was partially supported by grants from the Ministry of Education, Science, Sports and Culture, Japan. Back

2 Address correspondence and reprint requests to Dr. Yoshimoto Katsura, Department of Immunology, Institute for Frontier Medical Sciences, Kyoto University, Kyoto, Japan. Back

3 Abbreviations used in this paper: HSC, hemopoietic stem cell(s); AGM, aorta-gonad-mesonephros; M, myeloid; dGuo, deoxyguanosine; dpc, days postcoitus; EB, embryonal body; FL, fetal liver; FT, fetal thymus; LTR, long-term reconstituting; MLP, multilineage progenitor; p-B, B cell lineage committed progenitor; p-M, M lineage committed progenitor; p-Multi, multipotent progenitor(s); p-MB, bipotent progenitors capable of generating M and B cells; p-MT, bipotent progenitors capable of generating M and T cells; p-T, T cell lineage committed progenitor; YS, yolk sac; B6, C57BL/6; rm, recombinant murine. Back

Received for publication September 18, 2000. Accepted for publication December 28, 2000.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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