Page 179 - 2021_06-Haematologica-web

BMPR2 regulates the self-renewal of adult HSCs
SLAM populations, including the LT-HSC (LSK- CD150+CD48-) (Figure 3E and F).
Deletion of BMPR-II results in compromised self-renewal capacity and altered long-term hematopoietic stem cell-quality
In order to assay the self-renewal ability of BMPR-II deficient HSC, secondary and tertiary BM transplanta- tions were performed. We transplanted a fixed number of cells from primary recipients to lethally irradiated second- ary recipients. Similarly, BM from secondary recipients was transplanted to lethally irradiated tertiary recipients. The overall donor contribution of BMPR-II-/- HSC dropped dramatically upon secondary transplantation, as compared to WT cells, which exhibited stable reconstitu- tion across consecutive transplantations (Figure 3G). Upon tertiary transplantation, BMPR-II-/- cells dropped further, indicating a severely compromised ability to self- renew under stressed conditions (Figure 3G). BMPR-II+/- BM cells displayed sustained donor contribution in sec- ondary recipients, but appeared to drop upon tertiary transplantation, although not significantly so (Figure 3G). Furthermore, quantification of LT-HSC revealed decreas- ing numbers of BMPR-II-/- derived cells across consecutive transplantations and in tertiary recipients the contribu- tion to LT-HSC was essentially nonexistent (Figure 3H). These data show that BMPR-II-mediated signaling is essential for self-renewal of LT-HSC in vivo.
In agreement with the in vivo transplantation data stat- ed above is the in vitro serial replating assay which shows a significant decrease in BMPR-II-/- colony number after three platings (Online Supplementary Figure S2E).
In order to verify that the observed defect in regenera- tive capacity was caused by a qualitative defect of HSC, we transplanted ten sorted BMPR-II-/- or WT LT-HSC in conjunction with congenic WT support BM cells (Figure 3I). In agreement with previous transplantations, overall donor contribution of BMPR-II-/- LT-HSC was significant- ly reduced at 16 weeks post-transplantation in PB (Figure 3J) and the lineage distribution was unaffected (Figure 3K). Furthermore, the LSK compartment in BM was sig- nificantly reduced, as was the CD150-CD48- and CD150- CD48+ subset of LSK cells (Figure 3L). The LT-HSC showed a similar reduction, though it did not reach sig- nificance (P=0.09) (Figure 3L).
Loss of BMPR-II causes transcriptional cell cycle perturbation but has little or no effect on cell cycle and apoptosis in long-term hematopoietic stem cells
In order to investigate the biological properties of BMPR-II-/- primitive hematopoietic cells, we analyzed apoptosis and cell cycle parameters of BM cells from BMPR-II-/- and WT mice by flow cytometry. The fraction of apoptotic (AnnexinV+) cells within LSK/LSK-SLAM populations did not differ between BMPR-II-/- and WT BM (Figure 4A and B). Cell cycle distribution, analyzed using Ki67 and DAPI, was mostly unaltered in all hematopoietic populations tested between BMPR-II-/- and controls (Figure 4C). We observed a slight decrease in qui- escent G0-phase LT-HSC and a slight increase in LT-HSC in G1-phase, though these differences did not reach sig- nificance (Figure 4D). Similar results were seen in other primitive hematopoietic populations, with a significant decrease of cells in G0 in the CD150-CD48- and CD150-
CD48+ subsets of LSK cells (Online Supplementary Figure S3A to C). In contrast to the lack of significant cell cycle perturbation in LT-HSC was the observed enrichment in gene sets pertaining to cell cycling (Online Supplementary Figure S4A). When the hematopoietic system was put under stress following in vivo treatment with 5-fluo- rouracil, the blood, BM, and spleen were mostly unaffect- ed. Even though white blood cells and splenic LT-HSC were reduced, this was not significant (Online Supplementary Figure S5A to D). Furthermore, the prolifer- ative capacity of BMPR-II-/- c-kit+ BM cells in vitro was nor- mal when assayed under serum-free conditions in the presence of SCF, IL-3, and IL-6 (Figure 4E).
Homing is unaffected by deletion of BMPR-II
As BMP signaling has been linked to HSC homing via maintenance of ITGA4 expression during ex vivo culture, we investigated if loss of BMPR-II resulted in a homing defect.20 We transplanted unfractionated BMPR-II-/- and WT BM cells, with or without competitor cells, to lethally irradiated recipients. Following 20 hours, BM was ana- lyzed by flow cytometry. The donor contribution to Lin- Sca1+CD150+ cells as well as to the overall Lin- population was not significantly altered between BMPR-II-/- and WT cells (Figure 4F; Online Supplementary Figure S6A). Donor contribution following competitive transplantation was also not significantly altered (Online Supplementary Figure S6B to C). Likewise, the expression of ITGA4 (CD49d) was unaltered between BMPR-II-/- and control LT-HSC, indicating that BMP signaling does not regulate ITGA4
expression in vivo (Online Supplementary Figure S6D).
Reduced phosphorylation of SMAD1 upon BMPR-II deletion
In order to investigate the SMAD signaling status of BMPR-II-/- hematopoietic cells, we performed western blots of purified c-kit+ cells incubated with/without BMP4 in vitro. As expected, BMPR-II-/- cells exhibited significant- ly reduced phosphorylated SMAD1/5, both in the pres- ence and absence of BMP4 stimulation (Figure 5A). WT cells exhibited robust levels of phosphorylated SMAD1/5, but the level was not further increased upon BMP4 exposure, suggesting already saturated levels. These data confirm that deletion of BMPR-II translates into a functional reduction of SMAD signaling.
Limited SMAD-dependent transcriptional activity in hematopoietic populations
Although SMAD1/5-mediated BMP signaling is dispen- sable for HSC function, transcriptional activity of SMAD downstream of BMP has not been characterized in detail in hematopoietic cells. Using a BRE-GFP reporter mouse, a well-established model for gauging in vivo transcriptional activity of SMAD1/5/8,26,32-34 cells responding transcription- ally to BMP through SMAD were measured by green fluo- rescent protein (GFP), allowing in vivo analysis. BRE-GFP BM cells displayed limited activation of the SMAD path- way, with the highest proportion of GFP+ cells reaching only 2.79 % on average (LSK CD150-CD48- population) (Figure 5B). In order to investigate whether hematopoietic cells could respond to BMP signaling via the SMAD path- way, BRE-GFP cells were stimulated in vitro for 16 hours with/without BMP4. No significant difference in GFP+ cells was found in any BM population (Figure 5C).
haematologica | 2021; 106(8)
2207