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Niche recycling through division-independent egress of hematopoietic stem cells.

Bhattacharya D, Czechowicz A, Ooi AG, Rossi DJ, Bryder D, Weissman IL - J. Exp. Med. (2009)

Bottom Line: Hematopoietic stem cells (HSCs) are thought to reside in discrete niches through stable adhesion, yet previous studies have suggested that host HSCs can be replaced by transplanted donor HSCs, even in the absence of cytoreductive conditioning.Bromodeoxyuridine (BrdU) feeding experiments demonstrated that HSCs in the peripheral blood incorporate BrdU at the same rate as do HSCs in the bone marrow, suggesting that egress from the bone marrow to the blood can occur without cell division and can leave behind vacant HSC niches.These data provide insight as to how HSC replacement can occur despite the residence of endogenous HSCs in niches, and suggest therapeutic interventions that capitalize upon physiological HSC egress.

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Affiliation: Institute of Stem Cell Biology and Regenerative Medicine Stanford University School of Medicine Stanford, CA 94305, USA. deeptab@wustl.edu

ABSTRACT
Hematopoietic stem cells (HSCs) are thought to reside in discrete niches through stable adhesion, yet previous studies have suggested that host HSCs can be replaced by transplanted donor HSCs, even in the absence of cytoreductive conditioning. To explain this apparent paradox, we calculated, through cell surface phenotyping and transplantation of unfractionated blood, that approximately 1-5% of the total pool of HSCs enters into the circulation each day. Bromodeoxyuridine (BrdU) feeding experiments demonstrated that HSCs in the peripheral blood incorporate BrdU at the same rate as do HSCs in the bone marrow, suggesting that egress from the bone marrow to the blood can occur without cell division and can leave behind vacant HSC niches. Consistent with this, repetitive daily transplantations of small numbers of HSCs administered as new niches became available over the course of 7 d led to significantly higher levels of engraftment than did large, single-bolus transplantations of the same total number of HSCs. These data provide insight as to how HSC replacement can occur despite the residence of endogenous HSCs in niches, and suggest therapeutic interventions that capitalize upon physiological HSC egress.

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HSCs can egress into the peripheral blood without dividing. Mice were fed BrdU in the drinking water for 3 or 6 d (experiment 1), 9 d (experiment 2), or 12 d (experiment 3), and the percentage of peripheral blood HSCs that had incorporated BrdU was quantified. Control mice were not fed BrdU, but bone marrow HSCs were isolated as from experimental groups. HSCs were identified as described in Fig. 3 A, and myeloid progenitors (MP) were identified as lineage− c-kit+ Sca-1− cells. Peripheral blood was pooled from 20 mice for each experiment, and 2 independent experiments were performed for each time point.
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fig4: HSCs can egress into the peripheral blood without dividing. Mice were fed BrdU in the drinking water for 3 or 6 d (experiment 1), 9 d (experiment 2), or 12 d (experiment 3), and the percentage of peripheral blood HSCs that had incorporated BrdU was quantified. Control mice were not fed BrdU, but bone marrow HSCs were isolated as from experimental groups. HSCs were identified as described in Fig. 3 A, and myeloid progenitors (MP) were identified as lineage− c-kit+ Sca-1− cells. Peripheral blood was pooled from 20 mice for each experiment, and 2 independent experiments were performed for each time point.

Mentions: If circulating HSCs egress from the bone marrow in a division-dependent manner (Fig. 1 A), after an appropriate period of BrdU feeding, this model of HSC egress would predict that all peripheral blood HSCs would have incorporated BrdU, whereas only a fraction of the bone marrow HSCs would have incorporated BrdU. More specifically, HSCs destined for peripheral blood would incorporate BrdU at more rapid rates than would the total pool of bone marrow HSCs. In contrast, the division-independent model (Fig. 1 B) would predict that after an appropriate labeling period, not all blood HSCs would have incorporated BrdU, and instead, the extent of BrdU incorporation would be similar to that of bone marrow HSCs. To test these models, 20 mice were fed BrdU in their drinking water for 3 d, and the levels of BrdU incorporation in pooled peripheral blood and bone marrow HSCs were quantified. Only ∼9% of both blood and bone marrow HSCs had incorporated BrdU, suggesting that cellular division is not a requirement for HSC egress into the bloodstream (Fig. 4, experiment 1). Even after a longer BrdU feeding period of 6 d in which HSCs from 20 mice were pooled and analyzed, only 18% of peripheral blood and bone marrow HSCs had incorporated BrdU (Fig. 4, experiment 1). These experiments were repeated with similar results. After 9 and 12 d of BrdU feeding, the frequency of BrdU+ HSCs found in the peripheral blood had still not exceeded that in the bone marrow (Fig. 4, experiments 2 and 3). Interestingly, the percentage of HSCs in the bone marrow that had incorporated BrdU did not change between 9 and 12 d of feeding, which is consistent with the proposal that distinct proliferative and relatively nonproliferative HSC populations exist (Nygren and Bryder, 2008; Wilson et al., 2008). It is possible that the actual levels of proliferation may be even less than these values indicate, as BrdU itself has been suggested to induce the proliferation of HSCs (Nygren and Bryder, 2008; Wilson et al., 2008). Our levels of BrdU incorporation in bone marrow HSCs are likely lower than that reported by two recent studies because of the lower dose of BrdU administered in this study (Kiel et al., 2007b; Wilson et al., 2008). This lower BrdU dose was still sufficient for distinguishing differences in proliferative rates, as highly proliferative myeloid progenitors had incorporated more BrdU than had HSCs at all time points (Fig. 4). Thus, given the purity of these peripheral blood HSCs as shown by the clonal assays and in vivo reconstitutions (Fig. 3), it is clear from both experiments that not all peripheral blood HSCs incorporated BrdU, strengthening the interpretation that HSC intravasation need not necessarily be accompanied by cellular division.


Niche recycling through division-independent egress of hematopoietic stem cells.

Bhattacharya D, Czechowicz A, Ooi AG, Rossi DJ, Bryder D, Weissman IL - J. Exp. Med. (2009)

HSCs can egress into the peripheral blood without dividing. Mice were fed BrdU in the drinking water for 3 or 6 d (experiment 1), 9 d (experiment 2), or 12 d (experiment 3), and the percentage of peripheral blood HSCs that had incorporated BrdU was quantified. Control mice were not fed BrdU, but bone marrow HSCs were isolated as from experimental groups. HSCs were identified as described in Fig. 3 A, and myeloid progenitors (MP) were identified as lineage− c-kit+ Sca-1− cells. Peripheral blood was pooled from 20 mice for each experiment, and 2 independent experiments were performed for each time point.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2806613&req=5

fig4: HSCs can egress into the peripheral blood without dividing. Mice were fed BrdU in the drinking water for 3 or 6 d (experiment 1), 9 d (experiment 2), or 12 d (experiment 3), and the percentage of peripheral blood HSCs that had incorporated BrdU was quantified. Control mice were not fed BrdU, but bone marrow HSCs were isolated as from experimental groups. HSCs were identified as described in Fig. 3 A, and myeloid progenitors (MP) were identified as lineage− c-kit+ Sca-1− cells. Peripheral blood was pooled from 20 mice for each experiment, and 2 independent experiments were performed for each time point.
Mentions: If circulating HSCs egress from the bone marrow in a division-dependent manner (Fig. 1 A), after an appropriate period of BrdU feeding, this model of HSC egress would predict that all peripheral blood HSCs would have incorporated BrdU, whereas only a fraction of the bone marrow HSCs would have incorporated BrdU. More specifically, HSCs destined for peripheral blood would incorporate BrdU at more rapid rates than would the total pool of bone marrow HSCs. In contrast, the division-independent model (Fig. 1 B) would predict that after an appropriate labeling period, not all blood HSCs would have incorporated BrdU, and instead, the extent of BrdU incorporation would be similar to that of bone marrow HSCs. To test these models, 20 mice were fed BrdU in their drinking water for 3 d, and the levels of BrdU incorporation in pooled peripheral blood and bone marrow HSCs were quantified. Only ∼9% of both blood and bone marrow HSCs had incorporated BrdU, suggesting that cellular division is not a requirement for HSC egress into the bloodstream (Fig. 4, experiment 1). Even after a longer BrdU feeding period of 6 d in which HSCs from 20 mice were pooled and analyzed, only 18% of peripheral blood and bone marrow HSCs had incorporated BrdU (Fig. 4, experiment 1). These experiments were repeated with similar results. After 9 and 12 d of BrdU feeding, the frequency of BrdU+ HSCs found in the peripheral blood had still not exceeded that in the bone marrow (Fig. 4, experiments 2 and 3). Interestingly, the percentage of HSCs in the bone marrow that had incorporated BrdU did not change between 9 and 12 d of feeding, which is consistent with the proposal that distinct proliferative and relatively nonproliferative HSC populations exist (Nygren and Bryder, 2008; Wilson et al., 2008). It is possible that the actual levels of proliferation may be even less than these values indicate, as BrdU itself has been suggested to induce the proliferation of HSCs (Nygren and Bryder, 2008; Wilson et al., 2008). Our levels of BrdU incorporation in bone marrow HSCs are likely lower than that reported by two recent studies because of the lower dose of BrdU administered in this study (Kiel et al., 2007b; Wilson et al., 2008). This lower BrdU dose was still sufficient for distinguishing differences in proliferative rates, as highly proliferative myeloid progenitors had incorporated more BrdU than had HSCs at all time points (Fig. 4). Thus, given the purity of these peripheral blood HSCs as shown by the clonal assays and in vivo reconstitutions (Fig. 3), it is clear from both experiments that not all peripheral blood HSCs incorporated BrdU, strengthening the interpretation that HSC intravasation need not necessarily be accompanied by cellular division.

Bottom Line: Hematopoietic stem cells (HSCs) are thought to reside in discrete niches through stable adhesion, yet previous studies have suggested that host HSCs can be replaced by transplanted donor HSCs, even in the absence of cytoreductive conditioning.Bromodeoxyuridine (BrdU) feeding experiments demonstrated that HSCs in the peripheral blood incorporate BrdU at the same rate as do HSCs in the bone marrow, suggesting that egress from the bone marrow to the blood can occur without cell division and can leave behind vacant HSC niches.These data provide insight as to how HSC replacement can occur despite the residence of endogenous HSCs in niches, and suggest therapeutic interventions that capitalize upon physiological HSC egress.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Stem Cell Biology and Regenerative Medicine Stanford University School of Medicine Stanford, CA 94305, USA. deeptab@wustl.edu

ABSTRACT
Hematopoietic stem cells (HSCs) are thought to reside in discrete niches through stable adhesion, yet previous studies have suggested that host HSCs can be replaced by transplanted donor HSCs, even in the absence of cytoreductive conditioning. To explain this apparent paradox, we calculated, through cell surface phenotyping and transplantation of unfractionated blood, that approximately 1-5% of the total pool of HSCs enters into the circulation each day. Bromodeoxyuridine (BrdU) feeding experiments demonstrated that HSCs in the peripheral blood incorporate BrdU at the same rate as do HSCs in the bone marrow, suggesting that egress from the bone marrow to the blood can occur without cell division and can leave behind vacant HSC niches. Consistent with this, repetitive daily transplantations of small numbers of HSCs administered as new niches became available over the course of 7 d led to significantly higher levels of engraftment than did large, single-bolus transplantations of the same total number of HSCs. These data provide insight as to how HSC replacement can occur despite the residence of endogenous HSCs in niches, and suggest therapeutic interventions that capitalize upon physiological HSC egress.

Show MeSH
Related in: MedlinePlus