«3. Medizinische Klinik und Poliklinik – Hämatologische Forschung Cks1 is a critical regulator of hematopoietic stem cell cycling, quiescence and ...»
WT and Cks1-/- donor cells express the CD45.2 surface marker. Helper spleen cells were derived from CD45.1 mice and recipients express both CD45.1 and CD45.2. Bone marrow cells (2x105 per recipient mouse) from WT and Cks1deficient mice were transplanted together with helper spleen cells (4x105 per recipient mouse) in lethally irradiated (9Gy) recipients (Fig. 16A). The analysis of the primary recipients did not show significant changes in the engraftment of Cks1-/- donor cells compared to WT donor cells in blood, bone marrow, spleen and lymph nodes (LN) (Fig. 16B, C). The absolute numbers of donor-derived Lin-, MPP, LSK and CD150+ LSK cells were comparable in both groups except for few individual mice (Fig. 16D,E). These analysis demonstrate that Cks1deficient BM cells can reconstitute the hematopoiesis in irradiated mice.
Figure 16: Primary BM Transplantations. Cks1-/- BM cells are able to reconstitute recipient mice.
(A) Scheme of the transplantation model: 2x105 donor BM cells were transplanted in lethally irradiated recipients together with 4x105 spleen helper cells. (B) Blood engraftment at week 5, 10 and 16 after transplantation of 2x105 WT or Cks1-/- BM cells. (C) Engraftment in different organs: BM, spleen and LN respectively. (D) Representative dot blots from the analysis of donor CD45.2+ LSK and MPP cells in the bone marrow of recipient mice 16 weeks after transplantation. (E) Absolute numbers of donor BM, MPP, LSK and CD150+ LSK on week 16 after transplantation (recipients of Cks1+/+ cells: n=8, recipients of Cks1-/- cells: n=11; 2 independent experiments).
4.2.10. Secondary bone marrow transplantations: increased engraftment of Cks1-/- cells To test whether absence of Cks1 affects the self-renewal capability of HSC, secondary transplantations were performed (Fig. 17A). For this purpose 1x106 BM cells from the primary recipients of WT or Cks1-/- BM cells (week 16 after transplantation) together with 4x105 WT helper spleen cells were transplanted in lethally irradiated recipients. Surprisingly, five weeks after the secondary transplantation the percentage of Cks1-/- donor cells in the blood of the recipients was increased and at week 10 and 22 the donor cells engraftment of the Cks1-/- secondary recipients was significantly higher as compared to the WT secondary recipients (Fig.17B). Flow cytometry analysis of the recipient mice at week 22 post transplantation also revealed a significantly higher percentage of donor Cks1-/- cells in the bone marrow (Fig. 17C). There were no significant differences in the spleen engraftment (Fig. 17C). Most importantly, the absolute cell numbers of Cks1-/- donor BM cells, as well as the single populations: MPP, LSK and CD150+ LSK, was significantly increased compared to WT recipient controls (Fig. 17D, E). These results suggest that Cks1 is a crucial regulator in the LT-HSC.
Figure 17: Secondary BM Transplantations. Increased engraftment of Cks1-/- cells (A) Scheme of the transplantation model: 1x106 primary recipient delivered BM cells were transplanted in lethally irradiated secondary recipients together with 4x105 helper spleen cells.
(B) Blood engraftment at week 5, 10 and 22 after secondary transplantation of 1x106 BM cells from primary recipients of WT or Cks1-/- BM cells. (C) Engraftment in BM and spleen of the secondary recipients. (D) Representative dot blots from the analysis of CD45.2+ donor LSK and MPP cells in the bone marrow of the secondary recipients 22 weeks after transplantation. (E) Absolute number of donor BM, MPP, LSK and CD150+ LSK at week 22 after secondary transplantation (recipients of Cks1+/+ cells: n=8, recipients of Cks1-/- cells: n=8; 2 independent experiments).
11. Loss of Cks1 results in increased LSK frequency and increased replating capability after cultivating in vitro The secondary BM transplantation experiments suggest that lack of Cks1 result in the prolonged ability to maintain the HSC. To further test this possibility, an in vitro cell culture experiment was performed, in which FACS-sorted HSC (CD150+ and CD150- LSK) from WT and Cks1-/- mice were activated to proliferate using the cytokines SCF, TPO and Flt3L and the frequency of the remaining LSK was analyzed 48 hours after cultivation. Flow cytometric analysis revealed that culture of Cks1-/- CD150+ and CD150- LSK cells contained a larger proportion of cells maintaining the HSC phenotype as compared to WT cells (Fig 18A). Next, an in vitro surrogate assay for selfrenewal again using FACS-sorted CD150+ and CD150- LSK from WT and Cks1-/- was performed. The LSK were plated into growth factor-supplemented methyl cellulose. After initial colony formation, cells were recovered from the methyl cellulose and plated at equal numbers for several passages (Fig 18B).
As expected, WT HSC lost the ability to form colonies after 3 passages (Fig 18C and ). In contrast, Cks1-/- HSC cells, which formed fewer colonies after the first plating, continued to form detectable numbers of colonies until up to the sixth round of replating as opposed to WT HSC (Fig 18C).
Taken together, the in vitro and in vivo experiments show that Cks1 expression is a decisive factor for the maintenance of HSC dormancy.
Figure 18: Loss of Cks1 in LSK results in increased replating capability and increased LSK frequency after cultivating in vitro.
(A) On the left: frequency of remaining CD150+ and CD150- LSK 48 hours after cultivation in medium with supplemented SCF, TPO and Flt3L analyzed by FACS (2 independent experiments); on the right: representative FACS analysis of the experiment. (B) Scheme of methylcellulose replating experiment: sorted CD150+ und CD150- WT and Cks1-/- LSK were cultivated in methylcellulose for 12 days. Colonies were counted, the cells were homogenized and equal numbers of cells were replated. This procedure was repeated until no colonies were detected. (C) Colony numbers in the replating experiment (2 independent experiments).
4.2.12. Transplantations of CD150+ LSK cells: loss of Cks1 leads to accumulation of LT-HSC The serial BM transplantations (Fig. 17) suggest that Cks1-/- donor HSC possess an enhanced ability to self-renew. Though, since the percentage of CD34- LSK as well as the percentage of CD150+CD34- LSK of absolute cell number at steady state was significantly increased in the Cks1-/- mice (Fig. 9B), the Cks1-/- transplant contained more CD150+ LSK. Calculated 22,17 Cks1-/CD150+ LSK vs. 16,82 WT CD150+ LSK were transplanted per recipient mice in the primary transplantation. Also, the frequency of these early subpopulations, which are responsible for the secondary engraftment, was already slightly increased (not significant) at week 16 after primary transplantation (Fig. 16E). To avoid aberration and transplantation of unequal HSC numbers and to test the quality of LT-HSC lacking Cks1 in vivo, serial transplantations with sorted CD150+ LSK were performed. The bone marrow from 2 WT or 2 Cks1-/- was pooled, depleted for lineage-committed cells and stained with cell surface antibodies. CD150+ LSK cells were sorted and 300 CD150+ LSK were transplanted together with 1x105 recipient-derived BM and 5x105 recipient-derived helper spleen cells into lethally irradiated CD45.1xCD45.2 recipients (Fig. 19A, B). The CD150+ LSK cells from both genotypes were able to engraft and repopulate the blood system of the recipient mice. Strikingly, the engraftment in the recipients of Cks1-/- delivered CD150+ LSK was significantly decreased in blood, BM, spleen and LN (Fig. 19C, D).
This finding is consistent with the slower cycling of cells lacking Cks1 (Fig. 12C and 15D). Different organs of the recipient mice were also analyzed for the donor delivered mature populations. Interestingly, a similar effect as after chemo-ablative stress (Fig. 14D) was observed. The fraction of the Cks1-/CD150+ LSK derived B cells was significantly higher in the blood, bone marrow and the lymph node. On the other hand, the fraction of T cells was significantly decreased in the spleen and lymph nodes (Fig. 19E).
Figure 19: Transplantations of CD150+ LSK cells. Decreased engraftment of Cks1deficient cells and increase in the Cks1-/- donor-delivered B cell population.
(A) Scheme of the transplantation experiment: 300 sorted CD150+ LSK from WT and Cks1-/mice were transplanted in lethally irradiated recipients together with 5x105 helper WT spleen cells and 1x105 helper WT BM cells. (B) Sorting strategy for the transplantation of 300 CD150+ LSK, gated on Lin- cells. (C) On the left: representative dot blots from the blood FACS analysis of recipient mice. On the right: Engraftment (% CD45.2+ cells) in the peripheral blood of the recipients 4, 10 and 16 weeks after transplantation. (D) Engraftment in BM, spleen and LN 16 weeks after transplantation. (E) Frequency of donor-derived B cells (B), T cells (T) and granulocytes (G) in the indicated tissues (recipients of Cks1+/+ cells: n=8, recipients of Cks1-/cells: n=11; 2 independent experiments).
RESULTS Further flow cytometric analysis of the donor-derived early hematopoietic subsets in the sacrificed recipients at week 16 after transplantation revealed a significant decrease in the MPP population within the lineage negative subset in the recipients of Cks1-/- HSC (Fig. 20A, B). At the same time, the fraction of recovered Cks1-/- donor-derived CD150+ LSK was increased (Fig. 20A, B).
Furthermore, the frequency of the Cks1-/- donor-derived CD150+ cells within the donor LSK population was increased (Fig. 20C). The decrease in engraftment capacity of the LT-HSC lacking Cks1 and the accumulation of these cells 16 weeks after transplantation confirmed the suggestion, that Cks1 deficiency alters the function of the early hematopoietic cells probably through maintaining quiescence and thus prohibiting cell cycle entry and proliferation.
Figure 20: Transplantations of CD150+ LSK cells. Loss of Cks1 leads to decreased engraftment and accumulation of CD150+ LSK.
(A) Representative FACS analysis 16 week after transplantation of WT respectively Cks1-/CD45.2+ CD150+ LSK in CD45.1xCD45.2 recipients, gated on Lin- donor cells. (B) Relative number of MPP, LSK and CD150+ LSK within the donor Lin- population 16 weeks after transplantation of 300 CD150+ LSK. (C) Relative number of CD150+ LSK within the donor LSK population (recipients of Cks1+/+ cells: n=8, recipients of Cks1-/- cells: n=11; 2 independent experiments).
4.2.13. Secondary transplantations of CD150+ LSK cells: Cks1-/- HSC proliferate slower HSC self-renewal is experimentally defined as the capacity for long-term reconstitution of all blood lineages upon transplantation into recipient . To look further into the self-renewing potential and persistence of WT vs. Cks1-/CD150+ LSK, serial transplantations were performed. 300 donor-derived CD150+ LSK from the primary recipients were transplanted together with 1x105 recipient-derived BM and 5x105 recipient-derived helper spleen cells into lethally irradiated CD45.1xCD45.2 secondary recipients (Fig. 21A, B). Similar effects as after the primary transplantations were observed. The Cks1-/- cells engraftment was decreased compared to the control WT cells but the difference was not significant (Fig. 21C, D). Again, more B cells and partly less T cells arose from donor delivered Cks1-/- CD150+ LSK (Fig. 21E). Interestingly, in contrast to the primary CD150+ LSK transplantation, also the myeloid fraction was affected and Cks1-/- donor-delivered granulocyte population were decreased in the blood and BM of the secondary recipients (Fig. 21E). The analysis of the secondary recipients at week 16 post transplantation also revealed similar to the primary transplantations results for the donor delivered early hematopoietic populations. The frequency of the Cks1-/- donor-delivered MPP in the Lin- subset was decreased and the one of LSK was increased (Fig.
22A, B). To further look in the proliferation of the donor cells the secondary recipients were intraperitoneally injected with BrdU 12 hours before sacrifice. As expected, the BrdU incorporation in Cks1-/- donor-derived LSK populations was decreased (Fig. 22A, C), proving the assumption that these cells divide slower, thus possess reduced engraftment capacity.
Figure 21: Secondary transplantations of CD150+ LSK cells: Cks1 controls lineage differentiation.
(A) Scheme of the experiment: 300 donor-derived CD150+ LSK were FACS-sorted from primary recipients of WT and Cks1-/- CD150+ LSK cells 16 weeks after transplantation and were secondary transplanted in lethally irradiated WT recipients together with 5x105 WT helper spleen cells and 1x105 WT helper BM cells. 12 hours before the analysis of the recipient mice, BrdU was injected i.p. (B) Sorting strategy for the secondary transplantation of 300 donorderived CD150+ LSK, gated on Lin- cells. (C) Engraftment (% CD45.2+ cells) in the peripheral blood of the recipients 4, 10 and 16 weeks after transplantation. (D) Engraftment in BM, spleen and LN. (E) Donor-derived B cell (B), T cell (T) and granulocyte (G) populations (recipients of Cks1+/+ cells: n=8, recipients of Cks1-/- cells: n=9; 2 independent experiments).
RESULTS Figure 22: Secondary transplantations of CD150+ LSK cells: Cks1-/- HSC proliferate slower.
(A) Representative FACS analysis 16 weeks after secondary transplantation of 300 donorderived CD45.2+ CD150+ LSK in CD45.1xCD45.2 recipients, gated on Lin- donor cells. Lower panel: BrdU incorporation in the donor-delivered LSK fractions. (B) Relative number of MPP and LSK within the donor Lin- population 16 weeks after secondary transplantation of 300 CD150+ LSK. (C) Percentage of BrdU+ MPP and LSK cells after the secondary transplantation (recipients of Cks1+/+ cells: n=8, recipients of Cks1-/- cells: n=9; 2 independent experiments).