Conditional c-myb knockout in adult hematopoietic stem cells leads to loss of self-renewal due to impaired proliferation and accelerated differentiation

Disruption of c-myb Gene Impairs Adult Hematopoiesis in the BM.

The BM cellularity of the myb^f/f/MxCre (KO) mice was significantly reduced to 41% of litter-mate controls after pIpC administration to induce disruption of the c-myb gene (Fig. 1A). There were no notable abnormalities among myb^+/+, myb^f/+, myb^f/f, myb^+/+/MxCre, and myb^f/+/MxCre mice in the hematopoietic compartment. DNA analysis of total BM cells indicated that the c-myb floxed alleles were partially deleted (Fig. 1B). In addition, we observed a tight correlation between the levels of decreased BM cellularity and deletion efficiency of the c-myb floxed allele. The cellular number and percentages of neutrophils and B lymphoid cells in the pIpC-induced KO mice were also concomitantly reduced compared with the control mice, as determined by flow cytometric analysis (Fig. 1 C and D). Although the percentages of erythroid, monocytic, and megakaryocytic cells were increased in the pIpC-induced KO mice, the absolute number of these cells indicated a significant decrease by 57% for erythroid cells and 34% for monocytes and a nonsignificant reduction of 28% for megakaryocytes (Fig. 1 C and D). Together these data indicate that c-myb is required for BM hematopoiesis of the adult mice.

Deletion of the c-myb Gene Results in a Dramatic Reduction of HSCs.

Consistent with published reports, c-myb is expressed in LT-HSCs and ST-HSCs (Fig. 1E). In addition, c-myb is also expressed in MPPs (Fig. 1E). The percentages of cells in the myeloid progenitor compartment (Lin⁻c-Kit⁺Sca-1⁻, LKS⁻) and the stem cell compartment (Lin⁻c-Kit⁺Sca-1⁺, LKS⁺) of the pIpC-induced KO mice were dramatically decreased by 53% and 96%, respectively, compared with those of controls (Fig. 1 F and G). Further fractionation of the LKS⁺ by the Thy1 and CD135/Flt3 surface markers indicated that the numbers of LT-HSCs (LKS⁺Thy^loFlt3⁻), ST-HSCs (LKS⁺Thy1^loFlt3⁺), and MPPs (LKS⁺Thy1⁻Flt3⁺) were also greatly reduced by 96%, 94%, and 88%, respectively, in the pIpC-induced KO mice compared with controls (Fig. 1 F and G). Likewise, fractionation of the LKS⁺ into LT-HSCs, ST-HSCs, and MPPs by CD34 and Flt3 antigens produced the same dramatic decreases in cell numbers. Furthermore, staining for LT-HSCs and MPPs with the surface markers CD150/CD48/CD244 (14) along with lineage markers and c-Kit resulted in decreases of 94% for LT-HSCs and 87% for MPPs [supporting information (SI) Fig. S1]. These results demonstrate that c-myb is required for the maintenance of adult HSCs.

HSCs Have an Intrinsic Requirement for c-myb for Their Maintenance.

As c-myb has been reported to be expressed in BM stromal cells (15), we performed syngeneic competitive BM transplantation (BMT) as previously described (16) to determine the nature of the requirement of c-myb in HSCs. Total blood cells obtained via retro-orbital bleeding before pIpC administration indicated that the chimeric mice were reconstituted closely to the desired ratio of 2 to 1, with 2 parts CD45.2 experimental (myb^f/f or KO) cells to 1 part CD45.1 competitive cells (Fig. S2).

After pIpC administration, the ratios of CD45.2 to CD45.1 BM cells for all lineages examined in the pIpC-induced KO mice were inverted compared with those of the control chimeric mice: more CD45.1+ competitor cells than CD45.2+ pIpC-induced KO cells were present (Fig. 2 A and B). The CD45.2+ BM cells of erythroid, neutrophilic, monocytic, megakaryocytic, B lymphoid, and T lymphoid cells were dramatically decreased by 43% to 83% in the pIpC-induced chimeric KO mice compared with controls (Fig. 2B). Surprisingly, the decrease in BM CD45.2+ megakaryocytes of the pIpC-treated KO chimeric mice was significant compared with the nonsignificant decrease in the pIpC-induced KO mice (Figs. 1C and 2 A and B). In addition, the pIpC-induced chimeric KO CD45.2+ myeloid progenitor and stem cell compartments were severely diminished to 15% and 17%, respectively, of those of control CD45.2+ cells (Fig. 2 D and E). Concurrently, CD45.2+ LT-HSCs, ST-HSCs and MPPs in the pIpC-induced chimeric KO mice were all severely depleted to less than 20% of those in the control mice (Fig. 2 D and E). When myb^f/+/MxCre cells were used as donor cells to produce the mixed chimeras, heterozygous cells in blood, BM, and spleen behaved identically to myb^f/f control cells before and after pIpC induction. These competitive BMT experiments indicated that HSCs have an intrinsic requirement for c-myb for their maintenance.

c-myb Is Required for Self-Renewal of Adult HSCs.

To test rigorously whether c-myb is required for self-renewal of adult HSCs, serial transplantation was performed. Total BM cells from 3 primary transplants were isolated and pooled from each of the 2 experimental groups (Fig. 3A). Cells (1.5 × 10⁶) were transplanted into the lethally irradiated secondary recipient mice. The secondary transplants were examined at 10 weeks after reconstitution. The inverted ratio of CD45.2 to CD45.1 cells of various BM lineages as well as HSCs in the secondary KO chimeras were maintained from the primary pIpC-induced KO chimeric mice (Fig. 3D). These results were consistent with DNA analysis from BM of the secondary KO transplants, which indicated a nearly complete loss of the mutant alleles (Fig. 3C). Likewise, the reconstituted ratio of CD45.2 to CD45.1 cells in the secondary control chimeras was similar to that of the primary control transplants (Figs. 2 and 3). The data gathered from the secondary transplants demonstrated that c-myb is required for self-renewal of adult HSCs.

Disruption of c-myb Leads to Impaired Proliferation and Accelerated Differentiation of HSCs.

To understand the functions of c-myb in HSC development, we analyzed the proliferative, survival, and differentiation capacities of purified LT-HSCs, ST-HSCs, and MPPs via BrdU incorporation, caspase staining, and lineage marker expression, respectively. Because initial studies on sorted LKS⁺ and LKS⁺CD34- and LKS⁺Thy^lo HSCs showed no noticeable changes in BrdU incorporation or poly-caspase staining between IFN-induced purified KO and control cells at 24 h following IFN treatment but revealed alterations at 48 h, we performed all our functional studies on purified LT-HSCs, ST-HSCs, and MPPs at the 48 h time point.

At 48 h following IFN treatment, there were no differences in poly-caspase staining among IFN-induced KO LT-HSCs, ST-HSCs, and MPPs compared with controls (Fig. S3). However, despite the incomplete deletion of the c-myb floxed allele (Fig. S4A), BrdU incorporation was statistically significantly reduced, although modestly, in IFN-treated KO LT-HSCs, ST-HSCs, and MPPs (Fig. 4C). These studies indicate that c-myb is required for the proliferative capacity of HSCs.

Interestingly, the number of CD11b+ and CD41+ IFN-induced KO purified LT-HSCs were increased by twice as much as controls, indicating that disruption of c-myb accelerates differentiation (Fig. 4A). Surprisingly, there exists an approximately 20% atypical population in the IFN-induced KO LT-HSCs that expressed both CD11b and CD41 antigens on their cell surface, as opposed to less than 1.3% in the controls (Fig. 4A and Fig. S4), signifying that loss of c-myb also leads to aberrant development. In contrast, there were either no or subtle changes in the percentages of IFN-induced KO LT-HSCs that expressed Gr-1 or both Gr-1 and CD11b compared with controls (Fig. S4). Furthermore, forward scatter/side scatter morphological gating, which specifies size and granularity of the cells, was increased in the IFN-induced KO LT-HSCs, which is consistent with the increased expression of lineage markers and indicates accelerated differentiation (Fig. S3). In addition, surface expression of c-Kit was down-regulated in IFN-induced KO LT-HSCs but not in the controls (Fig. 4D). These results demonstrated that disruption of c-myb gene accelerates differentiation of LT-HSCs.

Similar to the IFN-induced KO LT-HSCs, IFN-induced KO ST-HSCs and MPPs had no differences in poly-caspase staining, slightly decreased BrdU incorporation and c-Kit expression, and caused increases in CD11b⁺, CD41⁺ and CD11b⁺CD41⁺ populations and greater forward/side scatter compared with controls (Fig. 4 B–D and Fig. S3 and Fig. S4). In contrast to IFN-induced KO LT-HSCs, IFN-induced KO ST-HSCs and MPPs displayed an approximate fourfold increase in the CD11b⁺Gr-1⁺ population and a distinct four- to fivefold decrease in the CD11b⁻Gr-1⁺ cells compared with controls (Fig. S4). Results obtained using LT-HSCs, ST-HSCs, and MPPs, which were sorted by surface expression of CD34 and Flt3, showed the same impaired proliferation and altered differentiation patterns as described earlier. Furthermore, IFN-induced purified KO LKS+ cells exhibited the same defective phenotypes as IFN-induced KO LT-HSCs, ST-HSCs, and MPPs. Therefore, c-myb regulates the proliferation and differentiation programs of LT-HSCs, ST-HSCs, and MPPs.

Disruption of c-myb Leads to Increased Presence of Aberrant and Differentiated HSCs in Mice.

To determine whether the results seen in vitro with sorted HSCs were artifacts or represented a naturally occurring phenomenon of the disrupted c-myb gene, we re-examined the BM of the pIpC-induced KO mice. The LKS⁺ compartment of the pIpC-induced KO mice exhibited decreased levels of c-Kit expression and increased levels of CD11b⁺CD41⁺ cells compared with control cells (Fig. 4E). These results from the pIpC-induced KO mice were consistent with the in vitro experiments using sorted HSCs and confirmed that c-myb regulates the differentiation of HSCs.

Disruption of c-myb Results in Loss of Growth on Methylcellulose and Multi-Lineage Differentiation.

It has recently been shown that knockdown c-myb LT-HSCs, ST-HSCs, and MPPs could form colonies on cytokine-containing methylcellulose for assessing multi-lineage differentiation (11). Hematopoietic colonies in IFN-induced, purified KO LT-HSCs and MPPs were significantly reduced compared with those of nontreated myb^f/f/MxCre and myb^f/f controls (Fig. 5A). In fact, colony formation using IFN-induced purified KO LKS⁺ cells was profoundly diminished by greater than 80% (Fig. 5A). Analysis of genomic DNA, extracted from the remaining colonies that did grow on the plates of any of the aforementioned sorted cells, revealed presence of only the c-myb floxed allele (Fig. 5B), indicating that cells with a disrupted c-myb gene will not grow and form multi-lineage colonies. These results demonstrated that c-myb is critical for HSC growth and multi-lineage differentiation.

Disruption of c-myb Results in Altered Gene Expression in HSCs.

To determine the molecular mechanism by which c-myb functions in HSCs, we first examined changes in the abundance of 5 genes (gfi-1, cxcr4, cebpa, flt3, bcl2) that were shown by microarray analysis of purified Lin⁻c-Kit⁺ BM cells to be altered when c-myb expression was lost (Table S1). These down-regulated genes were confirmed by semiquantitative RT-PCR and Northern blot analysis (Fig. S5). In addition, microarray analysis of purified LKS⁺ cells also demonstrated reduced expression of these genes.

We therefore investigated the expression levels of the aforementioned genes, as well as c-myc, in purified LT-HSCs and ST-HSCs. Cxcr4, c-myc, and bcl2 were decreased in IFN-induced KO LT-HSCs compared with those from controls (Fig. 5C). In IFN-induced KO ST-HSCs, cxcr4, gfi-1, c-myc, bcl2, and flt3 were reduced compared with those from controls (Fig. 5D). These studies indicate that these genes play a role in c-myb-regulated development of LT-HSCs and ST-HSCs.

Mice and Genotyping.

The construction of a conditional c-myb floxed mouse was previously reported (9). The c-myb mutant mice were backcrossed for at least 6 to 10 generations to C57BL/6 mice. The mutant mice were genotyped by using a 3-primer PCR amplification method: mybG2e 5′-ATT CCA GTG GTT CTT GAT AGC ATT ATC-3′; mybG11e, 5′-GCC GCT AAG CCA CAA TGG AAG GGC-3′; mybG19e, 5′-CCT TGA CTC TGA GTA AGA AAG TAA AC-3′.

In Vivo and in Vitro Deletion of the c-myb floxed Allele.

For in vivo disruption of the c-myb floxed allele, the myb^f/f/MxCre and control mice were given 250 μL of 2-mg/mL pIpC (P-1530; Sigma) by i.p. injection every other day for a total of 7 to 9 injections and analyzed 1 or 2 d after the last injection. pIpC was dissolved in sterile PBS solution by heating at 56 °C for 30 min and then stored in frozen aliquot at −20 °C. For injections, defrosted pIpC solution was heated at 56 °C for 8 min and allowed to cool at room temperature.

For in vitro deletion, 2 × 10⁴ units of IFN-α (R&D Systems) per mL SCF/IL3/IL6 cytokine-containing DMEM as described by Pear et al. (28) and listed in the legend for Fig. S4. For cell culture, regardless of the purified cell number, a minimum of 200 μL medium was used with a maximum concentration of 1 × 10⁶ cells/mL.

Mixed BM Chimeras.

For the competitive BMT experiments, 1 × 10⁶ unfractionated myb^f/f/MxCre or control BM cells and 0.5 × 10⁶ unfractionated competitor B6-CD45.1 BM cells were injected intravenously into the tail veins of lethally irradiated C57BL6/J mice (1,100 rad). FACS analysis was used to monitor for reconstitution at 10 weeks to 4 months post-transplantation via retro-orbital bleeding, and shortly afterward mice were given pIpC injections. For the second transplant, 1.5 × 10⁶ unfractionated BM cells from the pIpC-induced first transplant mice were used as donor cells. Mice were analyzed at 10 weeks post-transplantation.

Colony Assay on Methylcellulose.

See SI Materials and Methods for a detailed description of the colony assay on methylcellulose.

Flow Cytometry, Sorting, and Antibodies.

See SI Materials and Methods for a detailed description of flow cytometry, sorting, and antibodies.

In Vitro Functional Assay.

At the indicated time following IFNα treatment, purified cells were pulsed with BrdU (BD Biosciences) in fresh cytokine medium for 2 h at 37 °C in a tissue culture incubator under humidified conditions with 5% CO₂. Then the indicated fluorochrome inhibitor of caspases for detecting poly-caspase activity (V35117 FLICA kit; Invitrogen) was then added, and cells were returned to the incubator for an additional 1 h. After the incubation, purified cells were washed, stained with surface antibodies, and then fixed and permeabilized for anti-BrdU staining as specified by the manufacturer (BD Biosciences).

Semiquantitative RT-PCR Determination of mRNA Levels.

See Table S2 and SI Materials and Methods for a detailed discussion of semiquantitative RT-PCR determination of mRNA levels.

Statistical Analysis.

Data are expressed as mean ± SEM. Comparisons were analyzed by using Student 2-tailed paired or unpaired (with equal variance) t tests. Differences were considered significant at P < 0.05.