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C. Leveau et al.
respective contributions of cells originating from ctrl or fsn LSK donors during hematopoietic reconstitution. As early as 2 weeks after transfer, the proportion of LSKfsn donor- derived leukocytes in the recipients’ blood was higher than that of LSKctrl. These differences persisted 14 weeks after transfer (Figure 4A). The proportions of cells originat- ing from donor LSK in the recipients’ organs, particularly the thymus and BM, were higher in Ctrl-LSKfsn mice than in Ctrl-LSKctrl mice (Figure 4B). In the spleen, the reconsti- tution advantage of LSKfsn donor-derived cells led to the expansion of neutrophil, eosinophil and monocyte lineag- es and to a lesser extent, T-cell lineages (Figure 4C). To fur- ther evaluate the effect of Ttc7a loss on long-term recon- stitution, total BM cells from primary recipient mice were transplanted into secondary recipients. The competitive advantage of Ttc7a-deficient LSK donor cells with regards to reconstitution was maintained and even enhanced upon secondary and tertiary transplantation (Figure 4D). Thus, our results show that a defect in Ttc7a improves the competitive fitness of HSC following transplantation.
Loss of Ttc7a increases the long-term self-renewal potential of hematopoietic stem cells
In view of the elevated proliferative capacity of Ttc7a- deficient hematopoietic cells, we next sought to assess the
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properties of HSC that could modify their reconstitution potential (i.e., quiescence and self-renewal capacity). To evaluate the impact of Ttc7a loss on the quiescence of reconstituting HSC, we measured bromodeoxyuridine (BrdU) incorporation in control and Ttc7a-deficient HSPC before and after BM transplantation. Upon 24 h of BrdU treatment, we observed similar percentages of BrdU-posi- tive (BrdU+) HSC, HPC-1 and HPC-2 cells purified from control and fsn mice (Online Supplementary Figure S4A). Similar results were obtained when comparing cell cycle progression in control and Ttc7a-deficient HSC upon transplantation of irradiated recipients (Online Supplementary Figure S4B, C). Altogether, these results sug- gest that the increased repopulation capacity of fsn HSC was not caused by a disturbed quiescent state.
We then looked at whether the long-term self-renewal ability of HSC was altered in this context. We therefore performed serial BM transplants from irradiated mice hav- ing received whole BM from 3-week old ctrl or fsn mice. Unexpectedly, Ttc7a-deficient cells successfully sustained BM reconstitution longer than ctrl cells did. The ctrl HSC sustained six rounds of transplantation (Figure 5A) but all the Ctrlctrl mice died during the seventh round of BM trans- fer (Figure 5A, B). In contrast, most Ctrlfsn mice survived 6 weeks after the seventh round and were able to undergo
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Figure 4. Ttc7a-deficient hematopoietic stem cells have a higher repopulation capacity. (A-C) Lethally irradiated CD45.1 mice were reconstituted with a mix of con- trol whole bone marrow (BM) and sorted LSK cells purified from ctrl (Ctrl-LSKctrl – black bars and lines) or Ttc7a-deficient mice (Ctrl-LSKfsn – red bars and lines) (mean ± standard error of mean) *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001 (two-tailed t-test). These data are representative of three independent experiments. Proportion of LSK donor-derived leukocytes in the blood over time (A), in lymphoid organs (15 weeks after transfer) (B) and in the spleen for the different types of leukocytes (C) (n=8). (D) BM cells from first and then second round recipient mice of each group were pulled and transplanted to secondary and tertiary ctrl CD45.1 recipients, respectively. The proportions of LSK donor-derived leukocytes of Ctrl-LSKctrl and Ctrl-LSKfsn mice were determined in each round (n=14 for control and n=16 for Ttc7a-reconstituted mice).
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haematologica | 2020; 105(1)