SCL regulates the Quiescence and the Long-Term Competence of Hematopoietic Stem Cells.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2520-2520
Author(s):  
Julie Lacombe ◽  
Sabine Herblot ◽  
Shanti Rojas-Sutterlin ◽  
André Haman ◽  
Stephane Barakat ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Hematopoietic Stem Cells ◽  
Gene Dosage ◽  
Critical Role ◽  
Bhlh Transcription Factor ◽  
Hematopoietic Stem

Abstract Abstract 2520 Poster Board II-497 The life-long production of blood cells depends on the regenerative capacity of a rare bone marrow population, the hematopoietic stem cells (HSCs). In the adult, the majority of HSCs are quiescent while a large proportion of progenitors are more cycling. The state of quiescence in HSCs is reversible and these cells can be triggered into cycle by chemotoxic injuries, exposure to cytokines in vitro, as well as transplantation in vivo. SCL/TAL1 is a bHLH transcription factor that has a critical role in generating HSCs during development. However, the role of SCL in adult HSCs is still a matter of debate. In the present study, we took several approaches to address this question. Scl expression was monitored by quantitative PCR analysis in a population that contains adult long-term reconstituting HSCs (LT-HSCs) at a frequency of 20–50%: Kit+Sca+Lin-CD150+CD48-. RT-PCR results were confirmed by β-galactosidase staining of these cells in Scl-LacZ mice. We show that Scl is highly expressed in LT-HSC and that its expression correlates with quiescence, i.e. Scl levels decrease when LT-HSCs exit the G0 state. In order to assess stem cell function, we performed several transplantation assays with adult bone marrow cells in which SCL protein levels were decreased at least two-fold by gene targeting or by RNA interference. 1) The mean stem cell activity of HSCs transplanted at ∼1 CRU was two-fold decreased in Scl heterozygous (Scl+/−) mice. 2) In competitive transplantation, the contribution of Scl+/− cells to primitive populations as well mature cells in the bone marrow was significantly decreased 8 months after transplantation. 3) In secondary transplantation assays, Scl+/− HSCs were severely impaired in their ability to reconstitute secondary recipient in stem cells and progenitor populations and in almost all mature lineages. 4) Reconstitution of the stem cell pool by adult HSCs expressing Scl-directed shRNAs was significantly decreased compared to controls. We therefore conclude that SCL levels regulate HSC long term competence. Since Scl levels decrease when LT-HSCs exit the G0 state, we addressed the question whether the cell cycle state of LT-HSCs is sensitive to Scl gene dosage. We stained bone marrow cell populations with Hoechst and Pyronin Y. At steady state, percentage LT-HSCs in G1 fraction appears to be significantly increased in mice lacking one allele of Scl. Furthermore, a three-fold increase in G1 fraction was also observed when cells were infected with Scl-directed shRNA, suggesting that a decrease in Scl levels facilitates G0-G1 transition. At the molecular level, we show by chromatin immunoprecipitation that SCL occupies the Cdkn1a and Id1 loci. Furthermore, in purified Kit+Sca+Lin-CD150+CD48- cells, the expression levels of these two regulators of HSC cell cycle and long-term functions are sensitive to Scl gene dosage. Together, our observations suggest that SCL impedes G0-G1 transition in HSCs and regulates their long-term competence. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Osteoblast ablation reduces normal long-term hematopoietic stem cell self-renewal but accelerates leukemia development

Blood ◽  
2015 ◽  
Vol 125 (17) ◽  
pp. 2678-2688 ◽  
Author(s):  
Marisa Bowers ◽  
Bin Zhang ◽  
Yinwei Ho ◽  
Puneet Agarwal ◽  
Ching-Cheng Chen ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Hematopoietic Stem Cells ◽  
Hematopoietic Stem Cell ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Key Points ◽  
Leukemia Development

Key Points Bone marrow OB ablation leads to reduced quiescence, long-term engraftment, and self-renewal capacity of hematopoietic stem cells. Significantly accelerated leukemia development and reduced survival are seen in transgenic BCR-ABL mice following OB ablation.

Multifunctional Role of Caspase-3 in Regulating Hematopoietic Stem Cells.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 861-861 ◽  
Author(s):  
Viktor Janzen ◽  
Heather E. Fleming ◽  
Michael T. Waring ◽  
Craig D. Milne ◽  
David T. Scadden
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Hematopoietic Stem Cells ◽  
Peripheral Blood ◽  
Caspase 3 ◽  
Brdu Incorporation ◽  
Hematopoietic Stem ◽  
Regulatory Pathways

Abstract The processes of cell cycle control, differentiation and apoptosis are closely intertwined in controlling cell fate during development and in adult homeostasis. Molecular pathways connecting these events in stem cells are poorly defined and we were particularly interested in the cysteine-aspartic acid protease, Caspase-3, an ‘executioner’ caspase also implicated in the regulation of the cyclin dependent kinase inhibitors, p21Cip1 and p27Kip1. These latter proteins are known to participate in primitive hematopoietic cell cycling and self-renewal. We demonstrated high levels of Caspase-3 mRNA and protein in immunophenotypically defined mouse hematopoietic stem cells (HSC). Using mice engineered to be deficient in Caspase-3, we observed a consistent reduction of lymphocytes in peripheral blood counts and a slight reduction in bone marrow cellularity. Notably, knockout animals had an increase in the stem cell enriched Lin−cKit+Sca1+Flk2low (LKSFlk2lo) cell fraction. The apoptotic rates of LKS cells under homeostatic conditions as assayed by the Annexin V assay were not significantly different from controls. However, in-vitro analysis of sorted LKS cells revealed a reduced sensitivity to apoptotic cell death in absence of Caspase-3 under conditions of stress (cytokine withdrawal or gamma irradiation). Primitive hematopoietic cells displayed a higher proliferation rate as demonstrated by BrdU incorporation and a significant reduction in the percentage of cells in the quiescent stage of the cell cycle assessed by the Pyronin-Y/Hoechst staining. Upon transplantation, Caspase-3−/− stem cells demonstrated marked differentiation abnormalities with significantly reduced ability to differentiate into multiple hematopoietic lineages while maintaining an increased number of primitive cells. In a competitive bone marrow transplant using congenic mouse stains Capase-3 deficient HSC out-competed WT cells at the stem cell level, while giving rise to comparable number of peripheral blood cells as the WT controls. Transplant of WT BM cells into Caspase-3 deficient mice revealed no difference in reconstitution ability, suggesting negligible effect of the Caspase-3−/− niche microenvironment to stem cell function. These data indicate that Caspase-3 is involved in the regulation of differentiation and proliferation of HSC as a cell autonomous process. The molecular bases for these effects remain to be determined, but the multi-faceted nature of the changes seen suggest that Caspase-3 is central to multiple regulatory pathways in the stem cell compartment.

Cycling Marrow Stem Cells Are Lost with Purification.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2308-2308
Author(s):  
Laura R Goldberg ◽  
Mark S Dooner ◽  
Mandy Pereira ◽  
Michael DelTatto ◽  
Elaine Papa ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Tritiated Thymidine ◽  
Hematopoietic Stem ◽  
Cell Purification ◽  
Marrow Stem Cell ◽  
Marrow Cells

Abstract Abstract 2308 Hematopoietic stem cell biologists have amassed a tremendous depth of knowledge about the biology of the marrow stem cell over the past few decades, facilitating invaluable basic scientific and translational advances in the field. Most of the studies to date have focused on highly purified populations of marrow cells, with emphasis placed on the need to isolate increasingly restricted subsets of marrow cells within the larger population of resident bone marrow cells in order to get an accurate picture of the true stem cell phenotype. Such studies have led to the dogma that marrow stem cells are quiescent with a stable phenotype and therefore can be purified to homogeneity. However, work from our laboratory, focusing on the stem cell potential in un-separated whole bone marrow (WBM), supports an alternate view of marrow stem cell biology in which a large population of marrow stem cells are actively cycling, continually changing phenotype with cell cycle transit, and therefore, cannot be purified to homogeneity. Our studies separating WBM into cell cycle-specific fractions using Hoechst 33342/Pyronin Y or exposing WBM to tritiated thymidine suicide followed by competitive engraftment into lethally irradiated mice revealed that over 50% of the long-term multi-lineage engraftment potential in un-separated marrow was due to cells in S/G2/M. This is in stark contrast to studies showing that highly purified stem cell populations such as LT-HSC (Lineage–c-kit+sca-1+flk2−) engraft predominantly when in G0. Additionally, by performing standard isolation of a highly purified population of stem cells, SLAM cells (Lineage–c-kit+sca-1+flk2−CD150+CD41−CD48−), and testing the engraftment potential of different cellular fractions created and routinely discarded during this purification process, we found that 90% of the potential engraftment capacity in WBM was lost during conventional SLAM cell purification. Incubation of the Lineage-positive and Lineage-negative fractions with tritiated thymidine, a DNA analogue which selectively kills cells traversing S-phase, led to dramatic reductions in long-term multi-lineage engraftment potential found within both cellular fractions (over 95% and 85% reduction, respectively). This indicates that the discarded population of stem cells during antibody-based stem cell purification is composed largely of cycling cells. In sum, these data strongly support that 1) whole bone marrow contains actively cycling stem cells capable of long-term multi-lineage engraftment, 2) these actively cycling marrow stem cells are lost during the standard stem cell purification strategies, and 3) the protean phenotype of actively cycling cells as they transit through cell cycle will render cycling marrow stem cells difficult to purify to homogeneity. Given the loss of a large pool of actively cycling HSC during standard stem cell isolation techniques, these data underscore the need to re-evaluate the total hematopoietic stem cell pool on a population level in addition to a clonal level in order to provide a more comprehensive study of HSC biology. Disclosures: No relevant conflicts of interest to declare.

Metabolic Regulation of Hematopoietic Stem Cells During Stress

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. SCI-42-SCI-42
Author(s):  
Toshio Suda
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Hematopoietic Stem Cells ◽  
Metabolic Regulation ◽  
Conflicts Of Interest ◽  
Hypoxia Inducible Factor ◽  
Ros Generation ◽  
Hematopoietic Stem

Abstract Abstract SCI-42 Tissue homeostasis over the life of an organism relies on both self-renewal and multipotent differentiation of stem cells. Hematopoietic stem cells (HSCs) are sustained in a specific microenvironment known as the stem cell niche. Adult HSCs are kept quiescent during the cell cycle in the endosteal niche of the bone marrow. Normal HSCs maintain intracellular hypoxia, stabilize the hypoxia-inducible factor-1a (HIF-1a) protein, and generate ATP by anaerobic metabolism. In HIF-1a deficiency, HSCs became metabolically aerobic, lost cell cycle quiescence, and finally became exhausted. An increased dose of HIF-1a protein in VHL-mutated HSCs and their progenitors induced cell cycle quiescence and accumulation of HSCs in the bone marrow (BM), which were not transplantable. This metabolic balance promotes HSC maintenance by limiting the production of reactive oxygen species (ROS), but leaves HSCs susceptible to changes in redox status (1). We have performed the metabolomic analysis in HSCs. Upregulation of pyruvate dehydrogenase kinases enhanced the glycolytic pathway, cell cycle quiescence, and stem cell capacity. Thus, HSCs directly utilize the hypoxic microenvironment to maintain their slow cell cycle by HIF-1a-dependent metabolism. Downregulation of mitochondrial metabolism might be reasonable, since it reduces ROS generation. On the other hand, at the time of BM transplantation, HSCs activate oxidative phosphorylation to acquire more ATP for proliferation. Autophagy also energizes HSCs by providing amino acids during transplantation. ATG (autophagy-related) 7 is essential for transplantation and metabolic homeostasis. The relationship between mitochondrial heat shock protein, mortalin, and metabolism in HSCs will also be discussed. Disclosures: No relevant conflicts of interest to declare.

Scl regulates the quiescence and the long-term competence of hematopoietic stem cells

Blood ◽  
2010 ◽  
Vol 115 (4) ◽  
pp. 792-803 ◽  
Author(s):  
Julie Lacombe ◽  
Sabine Herblot ◽  
Shanti Rojas-Sutterlin ◽  
André Haman ◽  
Stéphane Barakat ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Hematopoietic Stem Cells ◽  
Gene Dosage ◽  
Cell Activity ◽  
Hematopoietic Stem ◽  
Protein Levels ◽  
Competitive Disadvantage ◽  
Term Functions

Abstract The majority of long-term reconstituting hematopoietic stem cells (LT-HSCs) in the adult is in G0, whereas a large proportion of progenitors are more cycling. We show here that the SCL/TAL1 transcription factor is highly expressed in LT-HSCs compared with short-term reconstituting HSCs and progenitors and that SCL negatively regulates the G0-G1 transit of LT-HSCs. Furthermore, when SCL protein levels are decreased by gene targeting or by RNA interference, the reconstitution potential of HSCs is impaired in several transplantation assays. First, the mean stem cell activity of HSCs transplanted at approximately 1 competitive repopulating unit was 2-fold decreased when Scl gene dosage was decreased. Second, Scl+/− HSCs were at a marked competitive disadvantage with Scl+/+ cells when transplanted at 4 competitive repopulating units equivalent. Third, reconstitution of the stem cell pool by adult HSCs expressing Scl-directed shRNAs was decreased compared with controls. At the molecular level, we found that SCL occupies the Cdkn1a and Id1 loci in primary hematopoietic cells and that the expression levels of these 2 regulators of HSC cell cycle and long-term functions are sensitive to Scl gene dosage. Together, our observations suggest that SCL impedes G0-G1 transition in HSCs and regulates their long-term competence.

scholarly journals Adaptation of Marrow Osteoblast Morphology Mediated By a Hematopoietic-Derived Endosteal Cell Is Critical for Donor HSC Engraftment after BMT

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3603-3603 ◽  
Author(s):  
Kathleen Overholt ◽  
Satoru Otsuru ◽  
Victoria Best ◽  
Adam Guess ◽  
Timothy S. Olson ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Hematopoietic Stem Cells ◽  
Diphtheria Toxin ◽  
Fluorescent Protein ◽  
Critical Role ◽  
Hematopoietic Cells ◽  
Hematopoietic Stem ◽  
Cell Engraftment

Abstract Hematopoietic stem cells reside in the bone marrow within specialized microenvironments designated the stem cell niche. The remarkable advances over the past decade have dramatically enhanced our perception of the niche; yet, the operative mechanisms after radioablation in preparation for bone marrow transplantation (BMT) remain poorly understood. We have previously described a profound remodeling of the bone marrow architecture after total body irradiation (TBI). This remodeling, comprised of enlarged, proliferating marrow osteoblasts and megakaryocyte migration from the central marrow space to the endosteal surface, is essential for efficient engraftment of donor cells after BMT; hence, marrow remodeling seems to represent an adaptation of the endosteal niche. To investigate whether hematopoietic cells regulate these changes, we sought to deplete all hematopoietic cells prior to TBI. We generated mice expressing the diphtheria toxin receptor (DTR) in all CD45-derived cells using the Cre/loxP model. To validate this strategy, we first crossed CD45Cre mice, where cre is expressed under the control of the endogenous promoter, with Z/RED mice which will then irreversibly express red fluorescent protein (RFP) in all cells that were derived from CD45-expressing progenitors. Surprisingly, we identified a population of RFP-expressing cells residing among osteoblasts along the endosteal and trabecular bone surfaces (designated red Bone Lining Cell, red BLC). By immunofluorescence staining, these cells lacked expression of CD45, lineage markers (Gr1, CD11b, F 4/80, CD3, B220, Ter119), and cathepsin K indicating it is not a hematopoietic cell, specifically not an osteal macrophage or osteoclast, but was unequivocally derived from CD45-expressing progenitors. We reproduced this fate map by crossing vav1Cre mice with Z/RED mice, confirming the identification and hematopoietic lineage of the red BLC. When crossed with Col2.3GFP transgenic mice, which express green fluorescent protein (GFP) in mature osteoblasts, red BLCs lacked GFP co-expression indicating it is not a generic osteoblast. Interestingly, after TBI, red BLCs markedly proliferate, but do not enlarge, in the metaphysis and epiphysis, but not in the diaphysis, coincident with the osteoblast proliferation suggesting a possible role in marrow remodeling. To pursue our original hypothesis that hematopoietic cells may regulate marrow remodeling, we treated mice expressing DTR in all CD45-derived cells and their non-expressing littermates (controls) with diphtheria toxin (DT) followed by TBI to induce marrow remodeling without the effect of CD45-derived cells. Marrow remodeling ensued; however, the characteristically enlarged endosteal osteoblasts adopted a strikingly flattened morphology (cell thickness, 8.45±0.31 vs. 3.42±0.11 μm, P<0.0001). We then used our competitive secondary transplantation assay to assess engraftment of long-term hematopoietic stem cells (HSCs) in primary recipients. Only 1 of 15 CD45-cell depleted mice engrafted HSCs compared to 10 of 15 control mice (P=0.0017) indicating a critical role of osteoblast morphology, governed by a CD45-derived cell, for donor stem cell engraftment in BMT. Megakaryocytes (Mks) and monocytes/macrophages (MMs) are the two marrow hematopoietic lineages that are recognized to survive short term after TBI and we have shown that the CD45-derived red BLC survives and proliferates after TBI. To determine if these cells regulate osteoblasts, we depleted Mks by treating Mk-specific DTR-expressing mice (generated with PF4Cre mice) with DT (>95%), and in separate cohort, MMs using clondronate (>95%). In each cohort, post-TBI marrow remodeling included the expected enlarged endosteal osteoblasts indistinguishable from controls, suggesting that neither Mks nor MMs direct the acquired osteoblast morphology. Collectively, our data indicate that enlarging of endosteal osteoblasts after marrow ablation is critical for donor cell engraftment, possibly due to altered adhesive properties for primitive hematopoietic cells. During post-TBI marrow remodeling, a CD45-derived cell that survives radioablation governs this osteoblast morphology. Our data implicate the red BLC as this key regulatory element. Understanding the red BLC will likely offer new insight into the niche and may lead to novel strategies to enhance HSC engraftment in BMT. Disclosures No relevant conflicts of interest to declare.

scholarly journals Phenotypic and Functional Changes Induced at the Clonal Level in Hematopoietic Stem Cells After 5-Fluorouracil Treatment

Blood ◽  
1997 ◽  
Vol 89 (10) ◽  
pp. 3596-3606 ◽  
Author(s):  
Troy D. Randall ◽  
Irving L. Weissman
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Cell Population ◽  
Stem Cell Population ◽  
Normal Bone Marrow ◽  
Hematopoietic Stem ◽  
Normal Bone

Abstract A significant fraction of hematopoietic stem cells (HSCs) have been shown to be resistant to the effects of cytotoxic agents such as 5-fluorouracil (5-FU), which is thought to eliminate many of the rapidly dividing, more committed progenitors in the bone marrow and to provide a relatively enriched population of the most primitive hematopoietic progenitor cells. Although differences between 5-FU–enriched progenitor populations and those from normal bone marrow have been described, it remained unclear if these differences reflected characteristics of the most primitive stem cells that were revealed by 5-FU, or if there were changes in the stem-cell population itself. Here, we have examined some of the properties of the stem cells in the bone marrow before and after 5-FU treatment and have defined several activation-related changes in the stem-cell population. We found that long-term reconstituting stem cells decrease their expression of the growth factor receptor c-kit by 10-fold and increase their expression of the integrin Mac-1 (CD11b). These changes begin as early as 24 hours after 5-FU treatment and are most pronounced within 2 to 3 days. This activated phenotype of HSCs isolated from 5-FU–treated mice is similar to the phenotype of stem cells found in the fetal liver and to the phenotype of transiently repopulating progenitors in normal bone marrow. We found that cell cycle is induced concomitantly with these physical changes, and within 2 days as many as 29% of the stem-cell population is in the S/G2/M phases of the cell cycle. Furthermore, when examined at a clonal level, we found that 5-FU did not appear to eliminate many of the transient, multipotent progenitors from the bone marrow that were found to be copurified with long-term repopulating, activated stem cells. These results demonstrate the sensitivity of the hematopoietic system to changes in its homeostasis and correlate the expression of several important surface molecules with the activation state of HSCs.

scholarly journals Searching for hematopoietic stem cells: evidence that Thy-1.1lo Lin- Sca-1+ cells are the only stem cells in C57BL/Ka-Thy-1.1 bone marrow.

1992 ◽  
Vol 175 (1) ◽  
pp. 175-184 ◽  
Author(s):  
N Uchida ◽  
I L Weissman
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Hematopoietic Stem Cells ◽  
Bone Marrow Cells ◽  
Cell Activity ◽  
Hematopoietic Stem ◽  
Stem Cell Activity ◽  
Marrow Cells

Hematopoietic stem cells (HSCs) are defined in mice by three activities: they must rescue lethally irradiated mice (radioprotection), they must self-renew, and they must restore all blood cell lineages permanently. We initially demonstrated that HSCs were contained in a rare (approximately 0.05%) subset of bone marrow cells with the following surface marker profile: Thy-1.1lo Lin- Sca-1+. These cells were capable of long-term, multi-lineage reconstitution and radioprotection of lethally irradiated mice with an enrichment that mirrors their representation in bone marrow, namely, 1,000-2,000-fold. However, the experiments reported did not exclude the possibility that stem cell activity may also reside in populations that are Thy-1.1-, Sca-1-, or Lin+. In this article stem cell activity was determined by measuring: (a) radioprotection provided by sorted cells; (b) long-term, multi-lineage reconstitution of these surviving mice; and (c) long-term, multi-lineage reconstitution by donor cells when radioprotection is provided by coinjection of congenic host bone marrow cells. Here we demonstrate that HSC activity was detected in Thy-1.1+, Sca-1+, and Lin- fractions, but not Thy-1.1-, Sca-1-, or Lin+ bone marrow cells. We conclude that Thy-1.1lo Lin- Sca-1+ cells comprise the only adult C57BL/Ka-Thy-1.1 mouse bone marrow subset that contains pluripotent HSCs.

scholarly journals Neogenin-1 distinguishes between myeloid-biased and balancedHoxb5+mouse long-term hematopoietic stem cells

2019 ◽  
Author(s):  
Gunsagar S. Gulati ◽  
Monika Zukowska ◽  
Joseph Noh ◽  
Allison Zhang ◽  
Rahul Sinha ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Blood Cells ◽  
Surface Marker ◽  
Cycle Phase ◽  
Hematopoietic Stem ◽  
Prospective Isolation

ABSTRACTHematopoietic stem cells (HSCs) self-renew and generate all blood cells. Recent studies with single-cell transplants (1–3) and lineage tracing (4, 5) suggest that adult HSCs are diverse in their reconstitution and lineage potentials. However, prospective isolation of these subpopulations has remained challenging. Here, we identify Neogenin-1 (NEO1) as a unique surface marker on a fraction of mouse HSCs labeled withHoxb5, a specific reporter of long-term HSCs (LT-HSCs) (6). We show that NEO1+Hoxb5+LT-HSCs expand with age and respond to myeloablative stress, while NEO1−Hoxb5+LT-HSCs exhibit no significant change in number. NEO1+Hoxb5+LT-HSCs are more often in the G2/S cell cycle phase compared to NEO1−Hoxb5+LT-HSCs in both young and old bone marrow. Upon serial transplantation, NEO1+Hoxb5+LT-HSCs exhibit myeloid-biased differentiation and reduced reconstitution, while NEO1−Hoxb5+LT-HSCs are lineage-balanced and stably reconstitute recipients. Gene expression comparison reveals increased expression of cell cycle genes and evidence of lineage-priming in the NEO1+fraction. Finally, transplanted NEO1+Hoxb5+LT-HSCs rarely generate NEO1−Hoxb5+LT-HSCs, while NEO1−Hoxb5+LT-HSCs repopulate both LT-HSC fractions. This supports a model in which dormant, balanced, NEO1−Hoxb5+LT-HSCs can hierarchically precede active, myeloid-biased NEO1+Hoxb5+LT-HSCs.SIGNIFICANCE STATEMENTHematopoietic stem cells (HSCs) are rare cells that have the unique ability to regenerate themselves and produce all blood cells throughout life. However, HSCs are functionally heterogeneous and several studies have shown that HSCs can differ in their contribution to major blood lineages. In this study, we discovered that the surface marker, Neogenin-1, can divide mouse HSCs into two subpopulations—one that is more active but biased towards producing myeloid cells and another that is more dormant and capable of equally producing all blood lineages. Neogenin-1 reveals the diversity and hierarchical relationship of HSCs in the mouse bone marrow, enables the prospective isolation of myeloid-biased and balanced HSCs, and opens opportunities to do the same in humans.

scholarly journals Phenotypic and Functional Changes Induced at the Clonal Level in Hematopoietic Stem Cells After 5-Fluorouracil Treatment

Blood ◽  
1997 ◽  
Vol 89 (10) ◽  
pp. 3596-3606 ◽  
Author(s):  
Troy D. Randall ◽  
Irving L. Weissman
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Cell Population ◽  
Stem Cell Population ◽  
Normal Bone Marrow ◽  
Hematopoietic Stem ◽  
Normal Bone

A significant fraction of hematopoietic stem cells (HSCs) have been shown to be resistant to the effects of cytotoxic agents such as 5-fluorouracil (5-FU), which is thought to eliminate many of the rapidly dividing, more committed progenitors in the bone marrow and to provide a relatively enriched population of the most primitive hematopoietic progenitor cells. Although differences between 5-FU–enriched progenitor populations and those from normal bone marrow have been described, it remained unclear if these differences reflected characteristics of the most primitive stem cells that were revealed by 5-FU, or if there were changes in the stem-cell population itself. Here, we have examined some of the properties of the stem cells in the bone marrow before and after 5-FU treatment and have defined several activation-related changes in the stem-cell population. We found that long-term reconstituting stem cells decrease their expression of the growth factor receptor c-kit by 10-fold and increase their expression of the integrin Mac-1 (CD11b). These changes begin as early as 24 hours after 5-FU treatment and are most pronounced within 2 to 3 days. This activated phenotype of HSCs isolated from 5-FU–treated mice is similar to the phenotype of stem cells found in the fetal liver and to the phenotype of transiently repopulating progenitors in normal bone marrow. We found that cell cycle is induced concomitantly with these physical changes, and within 2 days as many as 29% of the stem-cell population is in the S/G2/M phases of the cell cycle. Furthermore, when examined at a clonal level, we found that 5-FU did not appear to eliminate many of the transient, multipotent progenitors from the bone marrow that were found to be copurified with long-term repopulating, activated stem cells. These results demonstrate the sensitivity of the hematopoietic system to changes in its homeostasis and correlate the expression of several important surface molecules with the activation state of HSCs.

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