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BMPR2 regulates the self-renewal of adult HSCs
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Figure 1. Expression of bone morphogenetic protein receptors in primitive hematopoietic subsets. (A) Expression of bone morphogenetic protein (BMP) receptor BMPR-II relative to HPRT in indicated subsets of bone mar- row (BM) hematopoietic cell populations (n=3-4). LT-HSC: long term-hematopoietic stem cells; ST-HSC: short term- HSC; LMPP: lymphoid-primed multipotent progenitor; GMP: granulocyte-macrophage progenitor; MkP: megakaryocyte progenitor; PreGM: pre-granulocyte- macrophage progenitor; PreMegE: pre-megakaryocyte-ery- throid progenitor; PreCFU-E: pre-colony-forming unit-ery- throid progenitor; CFU-E: colony-forming unit-erythroid pro- genitor. (B) Expression of type-I receptors, ALK2, ALK3, and ALK6 in indicated hematopoietic cell populations (n=3). **P<0.01 in comparison to all other populations except for LMPP (A); *P<0.05; ns:not significant; ALK6 data was not statistically tested (B). MEF: mouse embryon- ic fibroblasts.
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full role of BMP in adult hematopoiesis. We conditionally deleted BMPR-II in hematopoietic cells, employing the Cre/loxP system with the Vav-Cre driver strain.2728 Efficient deletion of exon 4-5 of the BMPR-II gene in hematopoietic cells was confirmed by PCR analysis of individual colonies from BM, reaching 98.85% efficiency (n=160 alleles; Online Supplementary Figure S1A). Recombination at the BMPR-II locus resulted in efficient reduction of BMPR-II mRNA in purified LT-HSCs (Online Supplementary Figure S1B and C). Vav-Cre mediated dele- tion in mice homozygous for floxed BMPR-II alleles (BMPR-IIfl/fl;Vav-Cre, hereafter referred to as BMPR-II-/-) did not result in embryonic lethality although the Vav promoter is active from embryonic day (E) 10.5,30 indicat- ing that BMPR-II signaling is not endogenously required in HSC for normal development after E10.5. All PB parameters were normal in adult BMPR-II-/- and BMPR- II+/- mice at steady state compared to WT littermates (Figure 2A and B). Similarly, B/T/myeloid lineage distribu- tion and number of cells in the BM of mutant mice were unaltered compared to WT littermates (Figure 2C and data not shown). Megakaryocytic lineage distribution and progenitor populations were also unaltered (Online Supplementary Figure S2A and B). In order to further ana- lyze HSPC lacking BMPR-II, we performed flow cytome- try analyses on BM from BMPR-II-/-, BMPR-II+/-, and WT littermate mice. Interestingly, BMPR-II-/- mice had signifi- cantly fewer LSK cells in the BM as compared to WT mice (Figure 2D to E). Further analyses by SLAM markers did not reveal significant differences in more primitive sub-
sets of LSK cells, such as LT-HSC (Figure 2D to E). Similarly, when assessing HSC phenotypic aging by CD41 expression31 we saw no significant differences between WT and BMPR-II-/- LT-HSC (Online Supplementary Figure S2C). However, in agreement with the reduced number of LSK cells, the colony forming capacity of BM cells from BMPR-II-/- mice was significant- ly reduced compared to that of WT littermates (Figure 2F; Online Supplementary Figure 2D), suggesting that primitive hematopoiesis might be altered in BMPR-II-/- mice.
BMPR-II deficient hematopoietic stem cells exhibit reduced regenerative potential upon transplantation
In order to test the regenerative capacity of BMPR-II deficient HSC, we transplanted unfractionated BM cells from BMPR-II-/-, BMPR-II+/-, and WT mice at a 1:1 ratio with congenic WT competitor cells to lethally irradiated recipients (Figure 3A). BMPR-II-/- BM cells exhibited sig- nificantly reduced reconstitution capacity in PB short term at 4 weeks (data not shown) and a similar, though non-significant, reduction in PB long term at 16 weeks post-transplantation (Figure 3A to C). Deficiency of BMPR-II did not affect lineage distribution, though a slight decrease in donor contribution to myeloid cells could be observed (Figure 3D). In order to further investi- gate the ability of BMPR-II-/- cells to contribute to primi- tive hematopoietic cells, we quantified the number of donor-derived LSK-SLAM cells in BM. Interestingly, BMPR-II-/- cells exhibited a significantly reduced contribu- tion to the entire LSK compartment including all LSK-
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