FLT3/FLK2, a member of the receptor tyrosine kinase family, plays a critical role in maintenance of hematopoietic homeostasis, and the constitutively active form of the FLT3 mutation is one of the most common genetic abnormalities in acute myelogenous leukemia. In murine hematopoiesis, Flt3 is not expressed in self-renewing hematopoietic stem cells, but its expression is restricted to the multipotent and the lymphoid progenitor stages at which cells are incapable of self-renewal. We extensively analyzed the expression of Flt3 in human (h) hematopoiesis. Strikingly, in both the bone marrow and the cord blood, the human hematopoietic stem cell population capable of long-term reconstitution in xenogeneic hosts uniformly expressed Flt3. Furthermore, human Flt3 is expressed not only in early lymphoid progenitors, but also in progenitors continuously along the granulocyte/macrophage pathway, including the common myeloid progenitor and the granulocyte/macrophage progenitor. We further found that human Flt3 signaling prevents stem and progenitors from spontaneous apoptotic cell death at least through up-regulating Mcl-1, an indispensable survival factor for hematopoiesis. Thus, the distribution of Flt3 expression is considerably different in human and mouse hematopoiesis, and human FLT3 signaling might play an important role in cell survival, especially at stem and progenitor cells that are critical cellular targets for acute myelogenous leukemia transformation.

Hematopoiesis is one of the most intensely studied stem cell systems where hematopoietic stem cells (HSCs)3 self-renew, generate a variety of lineage-restricted progenitors, and continuously supply all types of mature blood cells. The technical advances of the multicolor FACS and the use of mAbs have enabled the prospective isolation of hematopoietic stem and progenitor cells according to the surface marker expression. In mice, multipotent hematopoietic activity resides in a small fraction of bone marrow (BM) cells lacking the expression of lineage-associated surface marker (Lin) but expressing high levels of Sca-1 and c-Kit (1, 2). Within the c-Kit+LinSca-1+ (KLS) fraction, the most primitive self-renewing HSCs with long-term reconstituting activity (LT-HSCs) do not express murine (m) CD34, but they do express mCD38 and a low level of mCD90 (Thy1), whereas mCD34+, mCD38, or mThy1 KLS cells are short-term HSCs (ST-HSCs) or multipotent progenitors that do not self-renew (3, 4, 5). Downstream of the mCD34+ ST-HSC stage, common lymphoid progenitors (CLPs) (6) and common myeloid progenitors (CMPs) (7) that can differentiate into all lymphoid cells and myelo-erythroid cells, respectively, have been purified. CMPs differentiate into granulocyte/macrophage progenitors (GMPs) and megakaryocyte/erythrocyte progenitors (MEPs), both of which are also prospectively isolatable by FACS (7).

Interestingly, the expression pattern of these surface markers in early stem and progenitor populations are considerably different in human (h) hematopoiesis. In humans, LT-HSCs express hCD34 (8). The hLT-HSC resides in the hCD34+hCD38 (9, 10) or the hCD34+hCD90+ (11, 12, 13) fractions in both human BM and cord blood (CB). It is still unclear what percent of hCD34+hCD38 or hCD34+hCD90+ cells are LT-HSCs in human hematopoiesis. The human counterpart for mCMPs, mGMPs, mMEPs, or mCLPs is also isolatable in the BM and the CB within the hCD34+hCD38+ progenitor fraction (14, 15). It has thus been suggested that, despite the difference in the expression patterns of key Ags in human and mouse hematopoiesis, lineage commitment processes from HSCs to mature blood cells might be generally preserved in both species. For example, the existence of prospectively isolatable CMPs and CLPs suggests that lineage commitment from HSCs involves myeloid vs lymphoid bifurcation in both mouse and human.

Recently, two independent groups have reported that in murine hematopoiesis, Flt3/Flk2, a tyrosine kinase receptor, is expressed in ST-HSCs but not in LT-HSCs. One group showed that mCD34 KLS cells (LT-HSCs) are mFlt3 (16), and the other showed that only the mFlt3 fraction of mCD90low KLS cells possesses LT-HSC activity (17). Each group further studied the detailed differentiation activity of mFlt3+ KLS cells, but drew different conclusions. Adolfsson et al. (18) reported that the mFlt3+mCD34+ KLS population maintains the granulocyte/macrophage (GM) and the T/B lymphoid, but not the megakaryocyte/erythrocyte (MegE) potential, if any. This result suggests that, in addition to the lymphoid vs myeloid developmental pathway represented by CLPs and CMPs, respectively, there is a critical stage common to GM, T, and B lymphoid cells. The other group, however, showed that mFlt3+mCD90 KLS cells are multipotent, thus claiming that the stage common to GM/lymphoid lineages proposed by Adolfsson et al. (18) does not constitute a major pathway for hematopoietic development (19). In contrast, downstream of the mST-HSC stage, there is a general agreement that mFlt3 is expressed in progenitors with lymphoid potential, such as the majority of CLPs and a minor fraction of CMPs, that retain a weak B cell potential (20), whereas it is down-regulated in late myeloid stages, such as GMPs and MEPs (20, 21). The Flt3 ligand (FL) is required for development of CLPs from mFlt3+ KLS cells, whereas mFlt3 is dispensable for HSC maintenance and myeloid development (22). These results suggest that in mouse hematopoiesis, Flt3 signaling plays an important role in lymphoid, but not in HSC or myeloid, development.

The precise expression and the role of hFlt3 in human hematopoiesis, however, remain unclear. Around 40–80% of hCD34+ BM and CB cells express hFlt3 (23, 24). Although a fraction of both the hFlt3+ and the hFlt3 populations gave rise to multilineage “mixed” colonies containing all myelo-erythroid components, the hFlt3+hCD34+ and hFlt3hCD34+ populations predominantly formed GM and erythroid colonies, respectively (23, 24, 25). It has also been shown that cells with NOD/SCID reconstitution activity reside in the hCD34+hFlt3+ fraction (24). These data collectively suggest that LT-HSCs and GMPs may reside mainly in the hFlt3+hCD34+ fraction, whereas MEPs may be contained in the hFlt3hCD34+ fraction. Therefore, the expression pattern of Flt3 could be quite different in mouse and human hematopoiesis. Flt3 expression has also been implicated in development of human acute myelogeneous leukemia (AML). Flt3 is expressed in leukemic blasts in most cases with AML (26, 27). Furthermore, FLT3 is one of the most frequently mutated genes in AML (28, 29), and the FLT3 mutants transduce the constitutively active FLT3 signaling, that could be the cause of poor prognosis in AML with FLT3 mutations (30, 31, 32).

In this study, we extensively analyzed the expression and function of hFlt3 in steady-state human BM and CB hematopoiesis. Interestingly, hFlt3 was expressed in the entire human BM and CB HSC population, and purified hFlt3+ HSCs could reconstitute multilineage cells for a long-term in our xenogeneic transplantation system (33). Therefore, unlike mouse hematopoiesis, the negative expression of Flt3 does not mark LT-HSCs in human. Furthermore, in striking contrast to mouse hematopoiesis where mFlt3 is expressed in CLPs but not GMPs (20, 21), hFlt3 was expressed in GMPs as well as in CLPs at a high level. The hFlt3 signaling did not affect the lineage fate decision of hHSCs, but supported cell survival of hFlt3+ stem and progenitor cells, at least through the up-regulation of Mcl-1, a survival promoting Bcl-2 homologue (34). These data collectively suggest that Flt3 signaling plays a critical role in maintenance of self-renewing LT-HSCs, and of GM and lymphoid progenitors in human hematopoiesis.

Fresh human steady-state BM and CB samples were collected from healthy adults and newborns after normal deliveries. Informed consent was obtained from all subjects. The Institutional Review Board of each institution participating in this project approved all research on human subjects.

The BM and CB mononuclear cells were prepared by gradient centrifugation and the CD34+ cells were enriched from mononuclear cells by using the Indirect CD34 MicroBead kit (Miltenyi Biotec) as described previously (14). For the analyses and sorting of myeloid progenitors, cells were stained with a Cy5-PE- or PC5-conjugated lineage mixture, including anti-hCD3 (HIT3a), hCD4 (RPA-T4), hCD7, hCD8 (RPA-T8), hCD10 (HI10a), hCD19 (HIB19), hCD20 (2H7), hCD11b (ICFR44), hCD14 (RMO52), hCD56 (NKH-1), and hGPA (GA-R2), FITC-conjugated anti-hCD34 (8G12), or anti-hCD45RA (HI100), PE-conjugated anti-hFlt3 (CD135) (4G8), or anti-hCD123 (6H6), allophycocyanin-conjugated anti-hCD34 (8G12), or anti-hCD38 (HIT2), Pacific Blue-conjugated anti-hCD45RA (HI100), and biotinylated anti-hCD38 (HIT2), or anti-hCD123 (9F5). The lymphoid progenitors were stained with the same lineage mixture except for the omission of anti-hCD10 and hCD19 followed by FITC-conjugated anti-hCD10 (SS2/36), PE-Cy7-conjugated anti-hCD19 (SJ25C1), and anti-hFlt3, hCD34, hCD38, and hCD45RA as described above. For additional analyses, PE-Cy7-conjugated anti-hCD34 (8G12), FITC-conjugated anti-hCD90 (5E10), PE-conjugated anti- hCD117 (YB5.B8), biotinylated anti-hFlt3 (BV10A4H2), and PE-conjugated anti-hCD127 (R34.34) mAbs were used. Streptavidin-conjugated allophycocyanin-Cy7 or PE-Cy7 was used for visualization of the biotinylated Abs (BD Pharmingen). The dead cells were excluded by propidium iodide (PI) staining. Appropriate isotype-matched, irrelevant control mAbs were used to determine the level of background staining. The cells were sorted and analyzed by FACS Aria (BD Biosciences). The sorted cells were subjected to an additional round of sorting using the same gate to eliminate contaminating cells and doublets. For single-cell assays, the re-sort was performed by using an automatic cell-deposition unit system (BD Biosciences).

Clonogenic CFU assays were performed using a methylcellulose culture system that was set up to detect all possible outcomes of myeloid differentiation as reported previously (14, 35). For myeloid colony formation, cells were cultured in IMDM-based methylcellulose medium (Methocult H4100; StemCell Technologies) supplemented with 20% FCS, 1% BSA, 2 mM l-glutamine, 50 μM 2-ME, and antibiotics in the presence of human cytokines such as IL-3 (20 ng/ml), stem cell factor (SCF) (20 ng/ml), FL (20 ng/ml), IL-11 (10 ng/ml), thrombopoietin (Tpo) (50 ng/ml), erythropoietin (Epo) (4 U/ml), and GM-CSF (50 ng/ml). All cytokines were obtained from R&D Systems. Colony numbers were enumerated on day 14 of culture. For the short-term liquid cell culture, cells were cultured in IMDM with 10% FCS in the presence of the cytokines described above. All of the cultures were incubated at 37°C in a humidified chamber under 5% CO2.

To exclude the unexpected effects of FCS and to evaluate the effects of cytokine stimulation precisely, the cells were prepared in the FCS-free condition. The anti-apoptotic effect of FL and SCF was evaluated after 24 h serum-free liquid culture, using Annexin V and PI staining (BD Pharmingen). The sorted cells were cultured in the serum-free medium (STEMPRO-34 SFM; Invitrogen) with or without FL (20 ng/ml) and/or SCF (20 ng/ml) for 24 h. The living cells were defined as Annexin V/PI among the live-gated cells (as shown in Fig. 5B). For the cytokine stimulation assays, cells were sorted in the IMDM and then the cytokines were added.

The NOD.Cg-PrkdcscidIL-2rgtmlWjl/Sz (NOD/SCID/IL2rγnull) mice were developed at The Jackson Laboratory. The NOD/SCID/IL2rγnull strain was established by backcrossing a complete null mutation at γc locus (36) onto the NOD.Cg-Prkdcscid strain. The establishment of this mouse line has been reported elsewhere (37). For the reconstitution assays, the sorted cells were transplanted into irradiated (100cGy) NOD/SCID/IL2rγnull newborns via a facial vein within 48 h of birth. To confirm the long-term reconstitution by hHSCs, the chimerism of circulating human blood cells were analyzed until at least 24 wk after transplantation, as previously reported (33). In addition to the Abs described above, the following mAbs were used: allophycocyanin-conjugated anti-hCD45 (J33), PE-Cy7-conjugated anti-hCD123 (6H6), FITC-conjugated anti-hCD33 (HIM3–4) or hCD14 (M5E2), and PE-conjugated anti-hCD41 (VIPL3), hCD56 (B159), anti-hGlycophorin A (GPA) (GA-R2), or anti-hCD3 (HIT3a).

To examine the gene expression profile of each population, RNA was isolated from 2,000-sorted cells using Isogen reagent (Nippon gene) according to the manufacturer's instructions. The total RNA was reverse transcribed to cDNA using a TaKaRa RNA PCR kit (Takara Shuzo). The mRNA levels were quantified in triplicate using a real-time PCR (7500 Real-Time PCR system; Applied Biosystems). hβ2-microglobulin mRNA was separately amplified in the same plate to be used for internal control. The primer and probes were designed by Primer Express software (Applied Biosystems).

The hCD34+Lin population was divided into hCD38+ and hCD38 populations (Fig. 1, A and B). It has been shown that HSCs with long-term reconstitution activity reside in the hCD38 fraction within the hCD34+ BM and CB populations (9, 10). As shown in Fig. 1 A, in the BM, hCD38 cells constituted only ∼5% of the LinhCD34+ population. This population uniformly expressed hFlt3 at a low level. More than 60% of the hCD34+hCD38 BM cells also expressed hCD90, another critical marker for hHSCs (11, 12, 13), whereas the hCD34+hCD38+Lin fraction was constituted of hCD90 lineage-committed progenitors.

FIGURE 1.

A flow cytometric analyses of human early hematopoietic populations in the BM and the CB. In hCD34+hCD38 immature BM (A) and CB (B) cells, hFlt3 was expressed at a low level in both hCD90 (Thy1) positive and negative fractions. In contrast, the hCD34+hCD38+ BM and CB progenitor populations did not express hCD90, and hFlt3 was expressed in only a fraction of these populations. C, hHSCs and myeloid progenitors expressed c-Kit at high levels, and CLPs at a low level. Representative data of independent five experiments are shown here.

FIGURE 1.

A flow cytometric analyses of human early hematopoietic populations in the BM and the CB. In hCD34+hCD38 immature BM (A) and CB (B) cells, hFlt3 was expressed at a low level in both hCD90 (Thy1) positive and negative fractions. In contrast, the hCD34+hCD38+ BM and CB progenitor populations did not express hCD90, and hFlt3 was expressed in only a fraction of these populations. C, hHSCs and myeloid progenitors expressed c-Kit at high levels, and CLPs at a low level. Representative data of independent five experiments are shown here.

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In the CB, only ∼30% of hCD34+hCD38 cells expressed hCD90 (Fig. 1 B). In the NOD/SCID/IL2rγnull newborn system, the hCD34+hCD38hCD90+ population was highly enriched for HSCs capable of long-term reconstitution as compared with the hCD34+hCD38hCD90 CB fraction (F. Ishikawa, unpublished data). The vast majority of hCD34+hCD38 cells expressed hFlt3 at a low level as previously reported (38). Furthermore, the hCD34+hCD38hCD90+ CB population expressed hFlt3.

These data clearly show that hFlt3 is expressed in all cells with the hHSC phenotype in both the BM and the CB, and suggest that Flt3 expression does not discriminate ST-HSCs from LT-HSCs in human as it does in mouse (16, 17). In contrast, the BM and the CB hCD34+hCD38+ progenitor fraction expressed negative to high levels of hFlt3. We thus further subfractionated the hCD34+hCD38+ population to evaluate the hFlt3 expression in a variety of lineage-restricted progenitors.

In mouse hematopoiesis, the expression of mFlt3 is associated with early lymphoid progenitor activities; it is expressed in the majority of CLPs, and in the minority of CMPs with weak B cell potential (20), but not in MEPs or GMPs (20) (21). Fig. 2 shows the expression of hFlt3 in the myeloid and lymphoid progenitor populations. According to the phenotypic definition of human myeloid and lymphoid progenitors (14, 15, 39, 40), hCD34+hCD38+ cells were subfractionated into myeloid and lymphoid progenitors, including the hCD45RAhCD123 (IL-3Rα)low CMP, the hCD45RAhCD123 MEP, the hCD45RA+hCD123low GMP, the hCD10+hCD19 CLP, and the hCD10+hCD19+proB populations. Interestingly, in both the human BM and CB, ∼70–80% of CMPs expressed hFlt3, whose level was progressively up-regulated at the GMP stage. In contrast, hFlt3 expression was completely shut down in MEPs. In the lymphoid lineage, the hCD34+hCD38+hCD10+ CLP (15) strongly expressed hFlt3, whereas hFlt3 was down-regulated in the proB cells. The expression level of hFlt3 in GMPs and CLPs appears to be higher than that in hCD34+hCD38hCD90+ HSCs (Fig. 2). We also tested the level of hFlt3 transcripts in purified hBM HSCs and progenitor populations (Fig. 3,A). The pattern of hFlt3 mRNA expression was generally consistent with that in hFlt3 protein, as evaluated by using anti-hFlt3 Abs on FACS (Figs. 1 and 2). Consistent with a previous report (41), MEPs and hFlt3 CMPs had the lowest levels, GMPs and CLPs had the highest levels, and the hCD34+hCD38 HSC population had a medium level of hFlt3 mRNA. Collectively, functional hLT-HSCs express hFlt3 mRNA and surface protein, and the distribution of Flt3 is quite different between human and mouse in early hematopoiesis.

FIGURE 2.

The expression patterns of hFlt3 are similar in early human BM (A) and CB (B) hematopoiesis. In the myeloid pathway in both the BM and CB, hFlt3 was up-regulated into the GM pathway, but was down-regulated in the MegE pathway; GMPs expressed hFlt3 at a high level, whereas MEPs did not express hFlt3. CMPs contained both hFlt3+ and hFlt3 fractions. In the lymphoid pathway, CLPs expressed hFlt3 at a high level in BM (A) and a low level in CB (B), whereas hCD10+hCD19+ proB cells did not express hFlt3 in either the BM or the CB. Representative data of independent five experiments are shown here.

FIGURE 2.

The expression patterns of hFlt3 are similar in early human BM (A) and CB (B) hematopoiesis. In the myeloid pathway in both the BM and CB, hFlt3 was up-regulated into the GM pathway, but was down-regulated in the MegE pathway; GMPs expressed hFlt3 at a high level, whereas MEPs did not express hFlt3. CMPs contained both hFlt3+ and hFlt3 fractions. In the lymphoid pathway, CLPs expressed hFlt3 at a high level in BM (A) and a low level in CB (B), whereas hCD10+hCD19+ proB cells did not express hFlt3 in either the BM or the CB. Representative data of independent five experiments are shown here.

Close modal
FIGURE 3.

Long-term reconstitution potential of hFlt3+hCD34+hCD38hCD90+Lin cells in NOD/SCID/IL2rγnull newborn mice. A, Analyses of the relative expression levels of hFlt3 transcript by real-time PCR. Each bar shows the n-fold difference of the level of hFlt3 mRNA in comparison to that of the whole CMP. The mean value and SD of BM samples from three independent normal donors are shown. Note that the levels of hFlt3 transcripts are well correlated with those of surface hFlt3 expression determined by FACS (Fig. 2 A). B, The long-term and multilineage reconstitution of human cells in mice injected with 1 × 103 hFlt3+hCD34+hCD38Lin CB cells 4 (upper panels) or 6 (lower panels) mo after transplantation. Representative two results out of five experiments are shown. C, Multilineage reconstitution 6 (upper panels) and 15 wk (lower panels) after i.v. injection of 5 × 103 hFlt3+hCD34+hCD38hCD90+Lin BM HSCs into NOD/SCID/IL2rγnull newborns. Donor-derived viable human cells were evaluated as hCD45+PI cells. hCD33+ granulocytes, hCD14+ monocytes, hCD41+ megakaryocytes, hCD19+ B cells, hCD3+ T cells, and hCD56+ NK cells were detected in the BM of recipient mice. D, Stem and progenitor analyses of BM from mice reconstituted with hFlt3+ HSCs. The BM contained hFlt3+hCD34+hCD38 HSCs, and all types of myeloid progenitors within the hCD34+hCD38+ population, including hCD45RAhCD123low CMPs, hCD45RA+hCD123low GMPs, and hCD45RAhCD123 MEPs. The expression patterns of hFlt3 in each population were identical with those of freshly isolated stem and progenitor cells. A representative experiment by using BM samples from three independent normal donors is shown.

FIGURE 3.

Long-term reconstitution potential of hFlt3+hCD34+hCD38hCD90+Lin cells in NOD/SCID/IL2rγnull newborn mice. A, Analyses of the relative expression levels of hFlt3 transcript by real-time PCR. Each bar shows the n-fold difference of the level of hFlt3 mRNA in comparison to that of the whole CMP. The mean value and SD of BM samples from three independent normal donors are shown. Note that the levels of hFlt3 transcripts are well correlated with those of surface hFlt3 expression determined by FACS (Fig. 2 A). B, The long-term and multilineage reconstitution of human cells in mice injected with 1 × 103 hFlt3+hCD34+hCD38Lin CB cells 4 (upper panels) or 6 (lower panels) mo after transplantation. Representative two results out of five experiments are shown. C, Multilineage reconstitution 6 (upper panels) and 15 wk (lower panels) after i.v. injection of 5 × 103 hFlt3+hCD34+hCD38hCD90+Lin BM HSCs into NOD/SCID/IL2rγnull newborns. Donor-derived viable human cells were evaluated as hCD45+PI cells. hCD33+ granulocytes, hCD14+ monocytes, hCD41+ megakaryocytes, hCD19+ B cells, hCD3+ T cells, and hCD56+ NK cells were detected in the BM of recipient mice. D, Stem and progenitor analyses of BM from mice reconstituted with hFlt3+ HSCs. The BM contained hFlt3+hCD34+hCD38 HSCs, and all types of myeloid progenitors within the hCD34+hCD38+ population, including hCD45RAhCD123low CMPs, hCD45RA+hCD123low GMPs, and hCD45RAhCD123 MEPs. The expression patterns of hFlt3 in each population were identical with those of freshly isolated stem and progenitor cells. A representative experiment by using BM samples from three independent normal donors is shown.

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In contrast, c-Kit was expressed at high levels in human HSCs and myelo-erythroid progenitors, while at a low level in CLPs (Fig. 1 C). The expression pattern of c-Kit in human hematopoietic stem and progenitor cells is generally consistent with that in mouse hematopoiesis (4, 6, 7), suggesting that the c-Kit expression program is preserved in mouse and human hematopoiesis.

In the NOD/SCID/IL2rγnull newborn system, hCD34+hCD38 BM and CB cells are capable of reconstitution of all hematopoietic lineages for a long term (33). The entire hCD34+hCD38 BM population expressed hFlt3 (Fig. 1,A), suggesting that functional hBM HSCs possess hFlt3 on their surface. In contrast, hCD34+hCD38 CB cells contained some hCD90 cells that did not express hFlt3. To formally test whether Flt3-expressing hCD34+hCD38 CB cells possess LT-HSC activity, we transplanted hFlt3+hCD34+hCD38hCD90+ CB cells in to NOD/SCID/IL2rγnull newborns. As shown in Fig. 3 B, NOD/SCID/IL2rγnull mice transplanted with 1 × 103 hFlt3+hCD34+hCD38hCD90+ CB cells reconstituted all hematolymphoid lineages for >6 mo, indicating that hFlt3 is expressed in functional hHSCs in CB as well as in BM.

Fig. 3,C shows the phenotypic analysis of human progeny from 5 × 103 hFlt3+hCD34+hCD38hCD90+ BM cells 6 (upper panels) or 15 wk (lower panels) after transplantation into NOD/SCID/IL2rγnull newborns (33). hFlt3+hCD34+hCD38hCD90+ BM cells differentiated into all hematopoietic lineage cells, including hCD33+ granulocytes, hCD14+ monocytes, hCD41+ megakaryocytes, hCD19+ B cells, hCD3+ T cells, hCD56+ NK cells (Fig. 3,C), and hGPA+ erythrocytes (not shown). Furthermore, transplanted hFlt3+hCD34+hCD38 HSCs purified from primary recipients developed secondary hFlt3+ HSCs and hFlt3−/+ CMPs, hFlt3 MEPs, and hFlt3+ GMPs recapitulating normal human hematopoietic development. Thus, the hCD34+hCD38hCD90+ BM population contains cells with long-term SCID reconstitution potential as reported (33, 42), and all cells within this population express hFlt3 on their surface (Fig. 3 D).

Fig. 4,A shows the differentiation potential of purified BM progenitors in vitro in the presence of the myeloid cytokine mixture containing SCF, FL, IL-3, IL-11, Tpo, Epo, and GM-CSF. hFlt3+ CMPs formed a variety of myelo-erythroid colonies including clonogenic CFU-granulocyte/erythroid/macrophage/megakaryocyte (CFU-GEMM), whereas hFlt3 CMPs did not form CFU-GEMM, but preferentially differentiated into the MegE lineage. Since GMPs (hFlt3+) and MEPs (hFlt3) exclusively gave rise to GM- and MegE-related colonies, respectively, hFlt3 expression could be associated with GM lineage development. These results suggested that hFlt3+ CMPs might differentiate into MEPs via hFlt3 CMPs. We thus directly tested the lineage relationship of these purified myelo-erythroid progenitor populations (Fig. 4,B). hFlt3+ and hFlt3 CMPs were purified and cultured in vitro. Then, 72 h after the initiation of culture, hFlt3+ CMPs gave rise to hFlt3 CMPs, hFlt3+ GMPs and hFlt3 MEPs, whereas hFlt3 CMPs did not up-regulate hFlt3, differentiating only into hFlt3 MEPs. Such phenotypically defined secondary myeloid progenitors displayed differentiation activity consistent with their phenotypic definition (Fig. 4 C). These data suggest that multipotent hFlt3+ CMPs can differentiate into both GMPs and MEPs, whereas hFlt3 CMPs represent a transitional stage into MEPs.

FIGURE 4.

The lineage potential and the relationship of myeloid progenitor populations. A, Clonogenic colony formation of purified populations on methylcellulose in the presence of cytokine mixture. The hCD34+hCD38 HSCs and hFlt3+CMPs gave rise to various myeloid colonies including CFU-GEMM, whereas GMPs and MEPs formed exclusively GM and MegE lineage-related colonies, respectively. In contrast, hFlt3CMPs predominantly gave rise to MegE lineage-related colonies but failed to form CFU-GEMM. The mean value of eight independent experiments is shown. CFU-M: CFU-macrophage, CFU-G: CFU-granulocyte, CFU-GM: CFU-granulocyte/macrophage, CFU-MegE: CFU-megakaryocyte/erythroid, CFU-Meg: CFU-megakaryocyte, and BFU-E: burst-forming units-erythroid. B, The lineal relationship between hFlt3+CMPs and hFlt3CMPs. After 72 h of culturing, hFlt3+CMPs gave rise to hFlt3CMPs, GMPs, and MEPs. In contrast, hFlt3CMPs differentiated into only MEPs, thus suggesting hFlt3CMP to be a transitional intermediate population from hFlt3+CMPs to hFlt3MEPs. C, The colony formation activity of phenotypically defined secondary CMPs, GMPs, and MEPs purified from the primary culture of hFlt3+CMPs or hFlt3CMPs. Each population displayed the colony formation activity consistent with their phenotypic definition. The mean value of four independent experiments is shown.

FIGURE 4.

The lineage potential and the relationship of myeloid progenitor populations. A, Clonogenic colony formation of purified populations on methylcellulose in the presence of cytokine mixture. The hCD34+hCD38 HSCs and hFlt3+CMPs gave rise to various myeloid colonies including CFU-GEMM, whereas GMPs and MEPs formed exclusively GM and MegE lineage-related colonies, respectively. In contrast, hFlt3CMPs predominantly gave rise to MegE lineage-related colonies but failed to form CFU-GEMM. The mean value of eight independent experiments is shown. CFU-M: CFU-macrophage, CFU-G: CFU-granulocyte, CFU-GM: CFU-granulocyte/macrophage, CFU-MegE: CFU-megakaryocyte/erythroid, CFU-Meg: CFU-megakaryocyte, and BFU-E: burst-forming units-erythroid. B, The lineal relationship between hFlt3+CMPs and hFlt3CMPs. After 72 h of culturing, hFlt3+CMPs gave rise to hFlt3CMPs, GMPs, and MEPs. In contrast, hFlt3CMPs differentiated into only MEPs, thus suggesting hFlt3CMP to be a transitional intermediate population from hFlt3+CMPs to hFlt3MEPs. C, The colony formation activity of phenotypically defined secondary CMPs, GMPs, and MEPs purified from the primary culture of hFlt3+CMPs or hFlt3CMPs. Each population displayed the colony formation activity consistent with their phenotypic definition. The mean value of four independent experiments is shown.

Close modal

We wished to elucidate the role of Flt3 signaling in human hematopoiesis. We first tested the effect of Flt3 signaling on the differentiation of HSCs, CMPs, and GMPs. Purified hFlt3+ HSCs, CMPs, and GMPs were cultured in methylcellulose in the presence of the myeloid cytokine mixture, with or without hFL. As shown in Fig. 5,A, the addition of FL in the culture did not affect the percentage of GM, MegE, or mix colonies in any of these populations. Interestingly, however, the colony numbers significantly increased in all cases when FL was added to the culture. This effect was dose-dependent, and the stimulatory activity of FL reached its peak at a concentration of 5 ng/ml (not shown). The plating efficiencies of hFlt3+ HSCs, CMPs, and GMPs cultured with the cytokine mixture containing FL (20 ng/ml) were significantly higher than those cultured without FL, suggesting that FL signaling may enhance the viability of cells (Fig. 5,A). We then directly tested the viability of HSCs, CMPs, and GMPs 24 h after the initiation of culture in serum-free media, with or without FL. The live, apoptotic, and dead cells after culture were enumerated by the Annexin/PI staining (43). In this staining, live cells are Annexin/PI, whereas Annexin+/PI and Annexin+/PI+ cells are apoptotic and dead cells, respectively (Fig. 5,B). Without FL, a considerable proportion of purified HSCs, CMPs, and GMPs rapidly became Annexin+/PI and Annexin+/PI+ cells undergoing apoptotic cell death. The addition of FL significantly blocked apoptotic cell death in all of these populations, indicating that FL plays a critical role in human hematopoietic stem and progenitor cell survival (Fig. 5 B). These data strongly suggest that Flt3 signaling does not instruct hematopoietic lineage commitment in hFlt3-expressing myeloid progenitors, but it does promote their survival.

FIGURE 5.

Effect of FL and SCF on the survival of purified progenitors. A, The effect of additional FL on colony formation of purified progenitors in methylcellulose in the presence of SCF, IL-3, IL-11, GM-CSF, Epo, and Tpo. Results from five independent experiments are shown here. Note that colony numbers are increased by the addition of FL into cultures in all hFlt3-expressing subsets including HSCs, CMPs, and GMPs but not in hFlt3 MEPs. B, An evaluation of apoptotic cell death in cultures of stem and progenitor cells. HSCs, hFlt3+ CMPs, and GMPs were cultured in the serum-free media, with or without FL, and analyzed at 12, 18, 24, 30, 48, and 72 h after initiation of culture. A representative data obtained after 24-h culture is shown. C, Anti-apoptotic effects of FL and/or SCF on HSCs and Flt3+ CMPs. Annexin PI live cells were enumerated after 24-h culture in a serum-free media. Each graph shows n-fold differences in the percentage of live cells relative to the ones without cytokine. Each bar represents the mean value and the SD of five independent samples.

FIGURE 5.

Effect of FL and SCF on the survival of purified progenitors. A, The effect of additional FL on colony formation of purified progenitors in methylcellulose in the presence of SCF, IL-3, IL-11, GM-CSF, Epo, and Tpo. Results from five independent experiments are shown here. Note that colony numbers are increased by the addition of FL into cultures in all hFlt3-expressing subsets including HSCs, CMPs, and GMPs but not in hFlt3 MEPs. B, An evaluation of apoptotic cell death in cultures of stem and progenitor cells. HSCs, hFlt3+ CMPs, and GMPs were cultured in the serum-free media, with or without FL, and analyzed at 12, 18, 24, 30, 48, and 72 h after initiation of culture. A representative data obtained after 24-h culture is shown. C, Anti-apoptotic effects of FL and/or SCF on HSCs and Flt3+ CMPs. Annexin PI live cells were enumerated after 24-h culture in a serum-free media. Each graph shows n-fold differences in the percentage of live cells relative to the ones without cytokine. Each bar represents the mean value and the SD of five independent samples.

Close modal

SCF, the ligand for c-Kit, has also been shown to play a critical role in the maintenance of survival in early hematopoiesis. Both c-Kit and Flt3 belong to the class III receptor tyrosine kinase (RTK) family, sharing their major signaling cascade (44). Human HSCs, CMPs, and GMPs expressed both c-Kit and Flt3 at the single cell level (Fig. 1). Thus, we tested the anti-apoptotic effect of SCF in this system. As shown in Fig. 5 C, in all HSC, CMP, and GMP populations, SCF also displayed anti-apoptotic effects whose impact on cell survival is similar to that of FL. Furthermore, in HSCs and CMPs, the combination of FL and SCF further increased percentages of live cells as compared with those in the presence of either FL or SCF alone, suggesting that SCF and FL signals collaborate to maintain cell survival of HSCs and CMPs.

The question: is the mechanism of cell survival enhancement by signaling of RTKs, such as Flt3 and c-Kit? We have shown that in murine hematopoiesis, Mcl-1, a Bcl-2 homologue, is indispensable for hematopoietic stem and progenitor cell survival, and that c-Kit signaling is one of the most critical inducers for Mcl-1 expression in mHSCs (45). We therefore hypothesized that Flt3, as well as c-Kit, signaling may up-regulate Mcl-1 to maintain cell survival in human hematopoiesis as well.

Fig. 6 A shows the distribution of the transcripts of Bcl-2 family molecules including Mcl-1, Bcl-2, and Bcl-xL in human stem and progenitor cells. Mcl-1 is expressed at the highest level in HSCs. CMPs and CLPs expressed similar levels of Mcl-1, and MEPs expressed Mcl-1 at the lowest level. This expression pattern of Mcl-1 transcript in human hematopoiesis is consistent with that in murine hematopoiesis (45). In contrast, Bcl-2 was highly expressed in GMPs and CLPs, whereas Bcl-xL was expressed in MEPs at the highest level.

FIGURE 6.

A, Quantitative RT-PCR assays for human anti-apoptotic genes such as Mcl-1, Bcl-2, and Bcl-xL in purified HSCs and each progenitor population. Each bar represents an n-fold difference in the amount of anti-apoptotic gene expression relative to that in Flt3+ CMPs. Note that Mcl-1 expression level is highest in HSCs, whereas Bcl-2 and Bcl-xL expression is most pronounced in GMPs and MEPs, respectively. B, Changes in anti-apoptotic gene expression in each progenitor after incubation with FL and/or SCF. Significant up-regulation of Mcl-1 mRNA was seen in HSCs, Flt3+ CMPs, GMPs, and CLPs after incubation with FL and/or SCF. Each bar represents the mean value and the SD of sixe independent samples.

FIGURE 6.

A, Quantitative RT-PCR assays for human anti-apoptotic genes such as Mcl-1, Bcl-2, and Bcl-xL in purified HSCs and each progenitor population. Each bar represents an n-fold difference in the amount of anti-apoptotic gene expression relative to that in Flt3+ CMPs. Note that Mcl-1 expression level is highest in HSCs, whereas Bcl-2 and Bcl-xL expression is most pronounced in GMPs and MEPs, respectively. B, Changes in anti-apoptotic gene expression in each progenitor after incubation with FL and/or SCF. Significant up-regulation of Mcl-1 mRNA was seen in HSCs, Flt3+ CMPs, GMPs, and CLPs after incubation with FL and/or SCF. Each bar represents the mean value and the SD of sixe independent samples.

Close modal

Purified stem and progenitor populations were incubated with FL and/or SCF in serum-free media. Both FL and SCF dramatically up-regulated the expression of Mcl-1 in a dose-dependent manner, and it reached its peak 30 min after initiation of culture at a concentration of 5 ng/ml (data not shown). Fig. 6 B shows the relative expression level of Mcl-1, Bcl-2, and Bcl-xL in the presence of 20 ng/ml FL and/or SCF. We found that both FL and SCF significantly up-regulated the expression of Mcl-1, but not of Bcl-2 or Bcl-xL, in HSCs, CMPs, and GMPs. These data collectively suggest that one of the important functions of these class III RTKs is to specifically activate Mcl-1 expression. Interestingly, in HSCs, FL and SCF displayed an additive effect on the up-regulation of Mcl-1. Therefore, Flt3 and c-Kit signaling collaborate to protect Flt3+ HSCs and early myeloid progenitors from apoptotic cell death, presumably through activating anti-apoptotic Mcl-1 transcription. In CLPs, however, FL activated not only Mcl-1 but also Bcl-2 transcription.

In this study, by using a multicolor FACS and a highly efficient xenograft system, we provide evidence that the distribution of Flt3 RTK is quite different in human and mouse hematopoiesis. First, although mouse LT-HSCs do not express mFlt3, the HSC-enriched hCD34+hCD38hLin population, that can reconstitute human hematopoiesis for a long-term in our xenogenic mouse model, uniformly expresses hFlt3 in both BM and CB. It is still unclear whether SCID-repopulating cells directly correspond to hLT-HSCs. However, because the hCD34+hCD38+hLin cells never reconstituted in xenogenic hosts for a long-term in our and others' experiments (42), it is highly likely that hCD34+hCD38hLin population is highly enriched for hLT-HSCs. Therefore, it is suggested that the negative expression of hFlt3 does not mark LT-HSCs in human, while mFlt3 does in mouse (16, 17). Second, in contrast to mouse hematopoiesis, where mFlt3 expression is restricted within progenitor populations of lymphoid potential including CLPs and a minority of CMPs that can differentiate into B cells (20), hFlt3 is expressed in human CMPs and GMPs, as well as in CLPs. The Flt3 expression is suppressed after cells are committed into the MegE lineage in both human and mouse. The distribution of Flt3 in mouse and human hematopoiesis is schematized in Fig. 7. The significant difference of Flt3 distribution in human and mouse hematopoiesis suggests that the critical role of Flt3 signaling in hematopoietic development could also be different between these species.

FIGURE 7.

Proposed differential expression of human and mouse Flt3 in steady-state hematopoiesis. Cellular morphology of directly sorted each progenitors (May-Giemsa ×1000) is shown here. In human, the most primitive LT-HSC expressed hFlt3 at a low level and its expression is up-regulated at the early GM and the lymphoid progenitor stages, while it is down-regulated in MEPs. In contrast, the mouse LT-HSC lacks mFlt3 expression, and mFlt3 is expressed in cells primed to the lymphoid pathway, including CLPs and a fraction of CMPs.

FIGURE 7.

Proposed differential expression of human and mouse Flt3 in steady-state hematopoiesis. Cellular morphology of directly sorted each progenitors (May-Giemsa ×1000) is shown here. In human, the most primitive LT-HSC expressed hFlt3 at a low level and its expression is up-regulated at the early GM and the lymphoid progenitor stages, while it is down-regulated in MEPs. In contrast, the mouse LT-HSC lacks mFlt3 expression, and mFlt3 is expressed in cells primed to the lymphoid pathway, including CLPs and a fraction of CMPs.

Close modal

We further found that the important function of hFlt3 should include the maintenance of cell survival via the up-regulation of anti-apoptotic Mcl-1 in early hematopoiesis. Previous studies have demonstrated that FL can support in vitro survival of human long-term culture-initiating cells (24, 46, 47). MCL-1 is a non-redundant anti-apoptotic protein, at least in mouse hematopoiesis, because the removal of Mcl-1 from hematopoietic cells in a conditional knockout system caused fatal hematopoietic failure, and because in vitro disruption of Mcl-1 in mouse HSCs, CMPs, or CLPs rapidly induced their apoptotic cell death (45). The expression level of Mcl-1 was the highest at the HSC stage and gradually declined as HSCs differentiate into myeloid and lymphoid progenitors in mouse hematopoiesis (45). The pattern of Mcl-1 distribution is well preserved in human hematopoiesis (Fig. 6,A), suggesting that Mcl-1 might also be essential for hHSC survival. In mouse HSCs, Mcl-1 is up-regulated by signals from cytokines including SCF, IL-6, and IL-11, and SCF exerts the most potent effect on the up-regulation of Mcl-1(45). In contrast to mouse LT-HSCs that express c-Kit but not Flt3, functional hLT-HSCs coexpress c-Kit and Flt3 (Fig. 1), and importantly, FL as well as SCF are potent inducers for Mcl-1 transcription (Fig. 6). The fact that FL and SCF activated only Mcl-1, but not Bcl-2 or Bcl-xL, in turn suggests that Mcl-1 might be the most critical survival factor controlled by exogenous cytokine signals at the HSC stage. Although it remains unclear whether hFlt3 and/or c-Kit signaling is absolutely required for hHSC survival, our data suggest that, to maintain the Mcl-1 level in hHSCs, the Flt3/FL system could work as an alternative to the SCF/c-Kit system. This is of interest because the SCF/c-Kit system is non-redundant in mouse hematopoiesis (48), where mouse LT-HSCs express only c-Kit, but not Flt3.

The anti-apoptotic effect of hFlt3 signaling was also seen in hFlt3-expressing myeloid progenitor populations. The incubation of CMPs and GMPs with FL significantly prevented their apoptotic cell death in vitro, and FL, as well as SCF, rapidly activated the Mcl-1 transcription in these progenitors. Interestingly, in CLPs, FL activated not only Mcl-1 but also Bcl-2. In lymphopoiesis, Bcl-2 (49, 50), as well as Mcl-1 (51), is critical. FL may collaborate with IL-7 to maintain lymphoid cell survival by up-regulating both Bcl-2 and Mcl-1. Collectively, in humans, Flt3 signaling might support cell survival in early hematopoietic stages with only the exception of the MegE lineage developmental pathway.

Our data also provides an important insight into pathogenesis of AML with FLT3 mutations. A total of 15–35% of AML patients have either internal tandem duplications (ITDs) in the juxtamembrane domain or mutations in the activating loop of FLT3 (28, 29), resulting in ligand-independent constitutive signal activation. The FLT3 mutations are rarely found in acute lymphoblastic leukemia (28, 29). The etiologic link of FLT3 mutations with AML does not fit the lymphoid-only expression pattern of Flt3 in mouse hematopoiesis. In mouse models, however, the ectopic expression of FLT3-ITDs in the bone marrow promotes development of myeloproliferative disorders, but these mutations themselves do not cause leukemia (52). We have found that AML cells with FLT3-ITD mutations possess extremely high levels of Mcl-1, and transduction of FLT3-ITD into normal HSCs induces rapid up-regulation of Mcl-1 of up to >10-fold higher levels (G. Yoshimoto and K. Akashi, manuscript in preparation). Because the expression of FLT3 mutations should occur in concert with that of normal Flt3, our data suggest that once FLT3 mutations are acquired in human hematopoiesis, abnormal survival-promoting signals of Mcl-1 should be expressed in LT-HSCs, and is progressively up-regulated in GMPs. It has been shown that both LT-HSCs and GMPs are the critical cellular target for leukemic transformation. The reinforced survival of CMPs/GMPs by blocking two independent apoptotic pathways (53), or the enforced expression of bcr-abl together with survival-promoting Bcl-2 at the GMP stage (54), results in AML development in mouse models. In human bcr-abl-positive chronic myelogeneous leukemia, GMPs could be the target for blastic transformation by acquisition of β-catenin signaling (55). GMPs can also be converted into leukemic stem cells simply by transducing leukemia fusion genes, such as MLL-ENL (56) or MOZ-TIF2 (57). Thus, these data collectively suggest that the acquisition of FLT3 mutations in human hematopoiesis might induce the reinforced survival of cells at the HSC and myeloid progenitor stages, where FLT3 mutations might collaborate with other genetic abnormalities to achieve full AML transformation.

In conclusion, our data show that the distribution of Flt3 is quite different in mouse and human hematopoeisis. hFlt3 targets LT-HSCs and myeloid progenitors except for MEPs. Flt3 signaling might support cell survival in early hematopoiesis including the HSC and the myeloid progenitor stages through up-regulation of Mcl-1. This is a striking example that the expression pattern of key molecules could be significantly different between human and mouse. Accordingly, special considerations are required in using mouse models to understand the role of Flt3 and FLT3 mutations in human hematopoiesis.

The authors have no financial conflict of interest.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1

This work was supported in part by a Grant-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology in Japan (19659248 to T.M., and 17109010 and 17047029 to K.A.) and the Mochida Memorial Foundation for Medical and Pharmaceutical Research (to T.M.).

3

Abbreviations used in this paper: HSC, hematopoietic stem cell; AML, acute myelogenous leukemia; BM, bone marrow; KLS, c-Kit+LinSca-1+; LT-HSC, HSC with long-term reconstituting activity; ST-HSC, short-term HCS; m, murine; h, human; CLP, common lymphoid progenitor; CMP, common myeloid progenitor; GM, granulocyte/macrophage; GMP, GM progenitor; MEP, megakaryocyte/erythrocyte progenitor; CB, cord blood; MegE, megakaryocyte/erythrocyte; FL, Flt3 ligand; PI, propidium iodide; SCF, stem cell factor; Tpo, thrombopoietin; Epo, erythropoietin; CFU-GEMM, CFU-granulocyte/erythroid/macrophage/megakaryocyte; RTK, receptor tyrosine kinase; ITD, internal tandem duplication.

1
Ikuta, K., I. L. Weissman.
1992
. Evidence that hematopoietic stem cells express mouse c-kit but do not depend on steel factor for their generation.
Proc. Natl. Acad. Sci. USA
89
:
1502
-1506.
2
Spangrude, G. J., S. Heimfeld, I. L. Weissman.
1988
. Purification and characterization of mouse hematopoietic stem cells.
Science
241
:
58
-62.
3
Morrison, S. J., I. L. Weissman.
1994
. The long-term repopulating subset of hematopoietic stem cells is deterministic and isolatable by phenotype.
Immunity
1
:
661
-673.
4
Osawa, M., K. Hanada, H. Hamada, H. Nakauchi.
1996
. Long-term lymphohematopoietic reconstitution by a single CD34-low/negative hematopoietic stem cell.
Science
273
:
242
-245.
5
Randall, T. D., F. E. Lund, M. C. Howard, I. L. Weissman.
1996
. Expression of murine CD38 defines a population of long-term reconstituting hematopoietic stem cells.
Blood
87
:
4057
-4067.
6
Kondo, M., I. L. Weissman, K. Akashi.
1997
. Identification of clonogenic common lymphoid progenitors in mouse bone marrow.
Cell
91
:
661
-672.
7
Akashi, K., D. Traver, T. Miyamoto, I. L. Weissman.
2000
. A clonogenic common myeloid progenitor that gives rise to all myeloid lineages.
Nature
404
:
193
-197.
8
Okuno, Y., H. Iwasaki, C. S. Huettner, H. S. Radomska, D. A. Gonzalez, D. G. Tenen, K. Akashi.
2002
. Differential regulation of the human and murine CD34 genes in hematopoietic stem cells.
Proc. Natl. Acad. Sci. USA
99
:
6246
-6251.
9
Terstappen, L. W., S. Huang, M. Safford, P. M. Lansdorp, M. R. Loken.
1991
. Sequential generations of hematopoietic colonies derived from single nonlineage-committed CD34+CD38 progenitor cells.
Blood
77
:
1218
-1227.
10
Ishikawa, F., A. G. Livingston, H. Minamiguchi, J. R. Wingard, M. Ogawa.
2003
. Human cord blood long-term engrafting cells are CD34+ CD38.
Leukemia
17
:
960
-964.
11
Craig, W., R. Kay, R. L. Cutler, P. M. Lansdorp.
1993
. Expression of Thy-1 on human hematopoietic progenitor cells.
J. Exp. Med.
177
:
1331
-1342.
12
Murray, L., B. Chen, A. Galy, S. Chen, R. Tushinski, N. Uchida, R. Negrin, G. Tricot, S. Jagannath, D. Vesole, et al
1995
. Enrichment of human hematopoietic stem cell activity in the CD34+Thy-1+Lin subpopulation from mobilized peripheral blood.
Blood
85
:
368
-378.
13
Baum, C. M., I. L. Weissman, A. S. Tsukamoto, A. M. Buckle, B. Peault.
1992
. Isolation of a candidate human hematopoietic stem-cell population.
Proc. Natl. Acad. Sci. USA
89
:
2804
-2808.
14
Manz, M. G., T. Miyamoto, K. Akashi, I. L. Weissman.
2002
. Prospective isolation of human clonogenic common myeloid progenitors.
Proc. Natl. Acad. Sci. USA
99
:
11872
-11877.
15
Galy, A., M. Travis, D. Cen, B. Chen.
1995
. Human T, B, natural killer, and dendritic cells arise from a common bone marrow progenitor cell subset.
Immunity
3
:
459
-473.
16
Adolfsson, J., O. J. Borge, D. Bryder, K. Theilgaard-Monch, I. Astrand-Grundstrom, E. Sitnicka, Y. Sasaki, S. E. Jacobsen.
2001
. Upregulation of Flt3 expression within the bone marrow Lin()Sca1(+)c-kit(+) stem cell compartment is accompanied by loss of self-renewal capacity.
Immunity
15
:
659
-669.
17
Christensen, J. L., I. L. Weissman.
2001
. Flk-2 is a marker in hematopoietic stem cell differentiation: a simple method to isolate long-term stem cells.
Proc. Natl. Acad. Sci. USA
98
:
14541
-14546.
18
Adolfsson, J., R. Mansson, N. Buza-Vidas, A. Hultquist, K. Liuba, C. T. Jensen, D. Bryder, L. Yang, O. J. Borge, L. A. Thoren, et al
2005
. Identification of Flt3+ lympho-myeloid stem cells lacking erythro-megakaryocytic potential a revised road map for adult blood lineage commitment.
Cell
121
:
295
-306.
19
Forsberg, E. C., T. Serwold, S. Kogan, I. L. Weissman, E. Passegue.
2006
. New evidence supporting megakaryocyte-erythrocyte potential of flk2/flt3+ multipotent hematopoietic progenitors.
Cell
126
:
415
-426.
20
D'Amico, A., L. Wu.
2003
. The early progenitors of mouse dendritic cells and plasmacytoid predendritic cells are within the bone marrow hemopoietic precursors expressing Flt3.
J. Exp. Med.
198
:
293
-303.
21
Karsunky, H., M. Merad, A. Cozzio, I. L. Weissman, M. G. Manz.
2003
. Flt3 ligand regulates dendritic cell development from Flt3+ lymphoid and myeloid-committed progenitors to Flt3+ dendritic cells in vivo.
J. Exp. Med.
198
:
305
-313.
22
Sitnicka, E., D. Bryder, K. Theilgaard-Monch, N. Buza-Vidas, J. Adolfsson, S. E. Jacobsen.
2002
. Key role of flt3 ligand in regulation of the common lymphoid progenitor but not in maintenance of the hematopoietic stem cell pool.
Immunity
17
:
463
-472.
23
Rappold, I., B. L. Ziegler, I. Kohler, S. Marchetto, O. Rosnet, D. Birnbaum, P. J. Simmons, A. C. Zannettino, B. Hill, S. Neu, et al
1997
. Functional and phenotypic characterization of cord blood and bone marrow subsets expressing FLT3 (CD135) receptor tyrosine kinase.
Blood
90
:
111
-125.
24
Sitnicka, E., N. Buza-Vidas, S. Larsson, J. M. Nygren, K. Liuba, S. E. Jacobsen.
2003
. Human CD34+ hematopoietic stem cells capable of multilineage engrafting NOD/SCID mice express flt3: distinct flt3 and c-kit expression and response patterns on mouse and candidate human hematopoietic stem cells.
Blood
102
:
881
-886.
25
Gotze, K. S., M. Ramirez, K. Tabor, D. Small, W. Matthews, C. I. Civin.
1998
. Flt3high and Flt3low CD34+ progenitor cells isolated from human bone marrow are functionally distinct.
Blood
91
:
1947
-1958.
26
Carow, C. E., M. Levenstein, S. H. Kaufmann, J. Chen, S. Amin, P. Rockwell, L. Witte, M. J. Borowitz, C. I. Civin, D. Small.
1996
. Expression of the hematopoietic growth factor receptor FLT3 (STK-1/Flk2) in human leukemias.
Blood
87
:
1089
-1096.
27
Rosnet, O., H. J. Buhring, S. Marchetto, I. Rappold, C. Lavagna, D. Sainty, C. Arnoulet, C. Chabannon, L. Kanz, C. Hannum, D. Birnbaum.
1996
. Human FLT3/FLK2 receptor tyrosine kinase is expressed at the surface of normal and malignant hematopoietic cells.
Leukemia
10
:
238
-248.
28
Stirewalt, D. L., J. P. Radich.
2003
. The role of FLT3 in haematopoietic malignancies.
Nat. Rev. Cancer
3
:
650
-665.
29
Gilliland, D. G., J. D. Griffin.
2002
. The roles of FLT3 in hematopoiesis and leukemia.
Blood
100
:
1532
-1542.
30
Kiyoi, H., T. Naoe, Y. Nakano, S. Yokota, S. Minami, S. Miyawaki, N. Asou, K. Kuriyama, I. Jinnai, C. Shimazaki, et al
1999
. Prognostic implication of FLT3 and N-RAS gene mutations in acute myeloid leukemia.
Blood
93
:
3074
-3080.
31
Schnittger, S., C. Schoch, M. Dugas, W. Kern, P. Staib, C. Wuchter, H. Loffler, C. M. Sauerland, H. Serve, T. Buchner, et al
2002
. Analysis of FLT3 length mutations in 1003 patients with acute myeloid leukemia: correlation to cytogenetics, FAB subtype, and prognosis in the AMLCG study and usefulness as a marker for the detection of minimal residual disease.
Blood
100
:
59
-66.
32
Thiede, C., C. Steudel, B. Mohr, M. Schaich, U. Schakel, U. Platzbecker, M. Wermke, M. Bornhauser, M. Ritter, A. Neubauer, et al
2002
. Analysis of FLT3-activating mutations in 979 patients with acute myelogenous leukemia: association with FAB subtypes and identification of subgroups with poor prognosis.
Blood
99
:
4326
-4335.
33
Ishikawa, F., M. Yasukawa, B. Lyons, S. Yoshida, T. Miyamoto, G. Yoshimoto, T. Watanabe, K. Akashi, L. D. Shultz, M. Harada.
2005
. Development of functional human blood and immune systems in NOD/SCID/IL2 receptor γ chain(null) mice.
Blood
106
:
1565
-1573.
34
Kozopas, K. M., T. Yang, H. L. Buchan, P. Zhou, R. W. Craig.
1993
. MCL1, a gene expressed in programmed myeloid cell differentiation, has sequence similarity to BCL2.
Proc. Natl. Acad. Sci. USA
90
:
3516
-3520.
35
Miyamoto, T., I. L. Weissman, K. Akashi.
2000
. AML1/ETO-expressing nonleukemic stem cells in acute myelogenous leukemia with 8;21 chromosomal translocation.
Proc. Natl. Acad. Sci. USA
97
:
7521
-7526.
36
Cao, X., E. W. Shores, J. Hu-Li, M. R. Anver, B. L. Kelsall, S. M. Russell, J. Drago, M. Noguchi, A. Grinberg, E. T. Bloom, et al
1995
. Defective lymphoid development in mice lacking expression of the common cytokine receptor γ chain.
Immunity
2
:
223
-238.
37
Shultz, L. D., B. L. Lyons, L. M. Burzenski, B. Gott, X. Chen, S. Chaleff, M. Kotb, S. D. Gillies, M. King, J. Mangada, D. L. Greiner, R. Handgretinger.
2005
. Human lymphoid and myeloid cell development in NOD/LtSz-scid IL2R γ null mice engrafted with mobilized human hemopoietic stem cells.
J. Immunol.
174
:
6477
-6489.
38
Ebihara, Y., M. Wada, T. Ueda, M. J. Xu, A. Manabe, R. Tanaka, M. Ito, H. Mugishima, S. Asano, T. Nakahata, K. Tsuji.
2002
. Reconstitution of human haematopoiesis in non-obese diabetic/severe combined immunodeficient mice by clonal cells expanded from single CD34+CD38 cells expressing Flk2/Flt3.
Br. J. Haematol.
119
:
525
-534.
39
Ryan, D. H., B. L. Nuccie, I. Ritterman, J. L. Liesveld, C. N. Abboud, R. A. Insel.
1997
. Expression of interleukin-7 receptor by lineage-negative human bone marrow progenitors with enhanced lymphoid proliferative potential and B-lineage differentiation capacity.
Blood
89
:
929
-940.
40
LeBien, T. W..
2000
. Fates of human B-cell precursors.
Blood
96
:
9
-23.
41
Chicha, L., D. Jarrossay, M. G. Manz.
2004
. Clonal type I interferon-producing and dendritic cell precursors are contained in both human lymphoid and myeloid progenitor populations.
J. Exp. Med.
200
:
1519
-1524.
42
Majeti, R., C. Y. Park, I. L. Weissman.
2007
. Identification of a hierarchy of multipotent hematopoietic progenitors in human cord blood.
Cell Stem Cell.
1
:
635
-645.
43
van Engeland, M., L. J. Nieland, F. C. Ramaekers, B. Schutte, C. P. Reutelingsperger.
1998
. Annexin V-affinity assay: a review on an apoptosis detection system based on phosphatidylserine exposure.
Cytometry
31
:
1
-9.
44
Gilliland, D. G., J. D. Griffin.
2002
. Role of FLT3 in leukemia.
Curr. Opin. Hematol.
9
:
274
-281.
45
Opferman, J. T., H. Iwasaki, C. C. Ong, H. Suh, S. Mizuno, K. Akashi, S. J. Korsmeyer.
2005
. Obligate role of anti-apoptotic MCL-1 in the survival of hematopoietic stem cells.
Science
307
:
1101
-1104.
46
Shah, A. J., E. M. Smogorzewska, C. Hannum, G. M. Crooks.
1996
. Flt3 ligand induces proliferation of quiescent human bone marrow CD34+CD38 cells and maintains progenitor cells in vitro.
Blood
87
:
3563
-3570.
47
Gabbianelli, M., E. Pelosi, E. Montesoro, M. Valtieri, L. Luchetti, P. Samoggia, L. Vitelli, T. Barberi, U. Testa, S. Lyman, et al
1995
. Multi-level effects of flt3 ligand on human hematopoiesis: expansion of putative stem cells and proliferation of granulomonocytic progenitors/monocytic precursors.
Blood
86
:
1661
-1670.
48
Witte, O. N..
1990
. Steel locus defines new multipotent growth factor.
Cell
63
:
5
-6.
49
Nakayama, K., K. Nakayama, I. Negishi, K. Kuida, H. Sawa, D. Y. Loh.
1994
. Targeted disruption of Bcl-2 α β in mice: occurrence of gray hair, polycystic kidney disease, and lymphocytopenia.
Proc. Natl. Acad. Sci. USA
91
:
3700
-3704.
50
Akashi, K., M. Kondo, U. von Freeden-Jeffry, R. Murray, I. L. Weissman.
1997
. Bcl-2 rescues T lymphopoiesis in interleukin-7 receptor-deficient mice.
Cell
89
:
1033
-1041.
51
Opferman, J. T., A. Letai, C. Beard, M. D. Sorcinelli, C. C. Ong, S. J. Korsmeyer.
2003
. Development and maintenance of B and T lymphocytes requires antiapoptotic MCL-1.
Nature
426
:
671
-676.
52
Kelly, L. M., Q. Liu, J. L. Kutok, I. R. Williams, C. L. Boulton, D. G. Gilliland.
2002
. FLT3 internal tandem duplication mutations associated with human acute myeloid leukemias induce myeloproliferative disease in a murine bone marrow transplant model.
Blood
99
:
310
-318.
53
Traver, D., K. Akashi, I. L. Weissman, E. Lagasse.
1998
. Mice defective in two apoptosis pathways in the myeloid lineage develop acute myeloblastic leukemia.
Immunity
9
:
47
-57.
54
Jaiswal, S., D. Traver, T. Miyamoto, K. Akashi, E. Lagasse, I. L. Weissman.
2003
. Expression of BCR/ABL and BCL-2 in myeloid progenitors leads to myeloid leukemias.
Proc. Natl. Acad. Sci. USA
100
:
10002
-10007.
55
Jamieson, C. H., L. E. Ailles, S. J. Dylla, M. Muijtjens, C. Jones, J. L. Zehnder, J. Gotlib, K. Li, M. G. Manz, A. Keating, et al
2004
. Granulocyte-macrophage progenitors as candidate leukemic stem cells in blast-crisis CML.
N. Engl. J. Med.
351
:
657
-667.
56
Cozzio, A., E. Passegue, P. M. Ayton, H. Karsunky, M. L. Cleary, I. L. Weissman.
2003
. Similar MLL-associated leukemias arising from self-renewing stem cells and short-lived myeloid progenitors.
Genes Dev.
17
:
3029
-3035.
57
Huntly, B. J., H. Shigematsu, K. Deguchi, B. H. Lee, S. Mizuno, N. Duclos, R. Rowan, S. Amaral, D. Curley, I. R. Williams, et al
2004
. MOZ-TIF2, but not BCR-ABL, confers properties of leukemic stem cells to committed murine hematopoietic progenitors.
Cancer Cell.
6
:
587
-596.