Candidemia-Induced Emergency Granulopoiesis Consists of Successive Dual Waves Triggered by the Shift From C/EBPalpha to C/EBPbeta Dependency.

Abstract Abstract 3778 Introduction: Granulocyte is a major cellular component in the front line of host defense. The number of granulocytes must be tightly tuned to meet the demand, because both the shortage and the excess of granulocytes can be harmful to the host. During emergency situations such as infections, granulocytes are replenished from peripheral pools and bone marrow production. As the half-life of granulocytes is quite short, granulopoiesis, de novo production of granulocytes in bone marrow, plays an important role during emergency. We have previously shown that granulopoiesis at steady state is largely dependent on a transcription factor, C/EBPalpha, whereas emergency granulopoiesis is dependent on C/EBPbeta (Hirai H, et al. Nature Immunol., 2006). However, the precise developmental stage where the shift from C/EBPalpha dependency to C/EBPbeta dependency takes place is almost unknown. The aim of this study is to dissect the process of granulopoiesis by a novel flow cytometric method and to elucidate the molecular mechanisms involved in the regulation of emergency granulopoiesis. Methods: 4 ≂ 106 cfu Candida albicans were intravenously injected to induce emergency granulopoiesis. Mouse bone marrow cells were harvested and stained with a combination of fluorescent-conjugated antibodies including anti-c-kit, anti-CD34, anti-Ly6G antibodies and markers for other lineages. Then the stained cells were analyzed or sorted by flow cytometry. After eliminating the cells which lost potential to give rise to granulocytes, the remaining cells were dissected into five subpopulations (#1≂ #5) according to the expression levels of c-kit and Ly6G. #1 is c-kithigh Ly6Glow cells, @ #2: c-kitint Ly6Glow, #5: c-kitlow Ly6Ghigh, and the cells residing between #2 and #5 are divided into #3 and #4. Cell number, gene expressions and cell cycle status of each population were analyzed before and after inducing emergency granulopoiesis. @ Results and Discussions: Wright-Giemsa staining and qRT-PCR for granule proteins (cathepsin G, myeloperoxidase, neutrophil elastase2, lactoferrin and MMP9) in each population indicated that lower c-kit expression and higher Ly6G expression correlated well with granulocytic differentiation and that the granulopoiesis progresses from # 1 to #5 in this order both at steady state and during emergencies (Figure 1). Then we applied this method to candidemia-induced emergency granulopoiesis. In vivo BrdU incorporation analysis showed immediate acceleration of the cell cycle in the most immature population (#1) and in one of the intermediate populations (#2). Chronological monitoring of each population after inducing candidemia revealed that rapid increase in mature granulocytes (#5) preceded the replenishment from the most immature population (#1). These results suggested that there are two distinct gwavesh in granulopoiesis at the early phase of infection, a rapid supply (first gwaveh) of granulocytes from relatively mature population (#2≂ #4), and a further and sustained supply (second gwaveh) originated from more immature populations (#1) including hematopoietic stem/progenitor cells (Figure 1). Transcripts of C/EBPalpha were significantly downregulated in #1≂ #4 at the early phase of infection, while those of C/EBPbeta were maintained in all the subpopulation (Figure 2), suggesting that shift from C/EBPalpha dependency to C/EBPbeta dependency took place at multiple developmental steps in granulopoiesis. C/EBPbeta has less inhibitory effects on cell cycle than C/EBPalpha while their abilities to induce granulocytic differentiation are similar (Hirai H, et al. Nature Immunol., 2006). The shift toward C/EBPbeta dependency may trigger the dual waves in emergency granulopoiesis, which demands both differentiation and proliferation of granulocytic precursors. Disclosures: No relevant conflicts of interest to declare.

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Impairment of Hematopoietic Stem Cell (HSC) Niche by G-CSF Is Associated with Rapid Mobilization of Serially Reconstituting HSC and Reduced Competitive Repopulation of Mobilized Bone Marrow

Blood ◽

10.1182/blood.v118.21.1889.1889 ◽

2011 ◽

Vol 118 (21) ◽

pp. 1889-1889 ◽

Cited By ~ 1

Author(s):

Jean-Pierre Levesque ◽

Valerie Barbier ◽

Bianca Nowlan ◽

Domenica McCarhty ◽

Ingrid G Winkler

Keyword(s):

Cell Cycle ◽

Bone Marrow ◽

Steady State ◽

Conflicts Of Interest ◽

Brdu Incorporation ◽

Down Regulation ◽

Hematopoietic Stem ◽

Post Transplant ◽

Donor Chimerism ◽

Hsc Niche

Abstract Abstract 1889 We have previously shown that G-CSF administration impairs HSC niches in the mobilized bone marrow (BM). G-CSF causes rapid suppression (within 2 days) of endosteal osteoblasts and bone formation with concomitant down-regulation of Kit ligand, CXCL12 and angiopoietin-1. This effect is mediated by the depletion of specific populations of BM macrophages1. Considering the very rapid impairment of HSC niches in response to G-CSF, we hypothesized that 1) the most primitive HSC should also mobilize at this very early stage within the first 48 hours of G-CSF treatment, and 2) that down-regulation of HSC niche function should also alter the number or function of HSC remaining in the mobilized BM. To test this, 125μg/kg rhuG-CSF was injected twice daily to C57BL/6 mice; blood and BM harvested at days 2 and 5 of G-CSF treatment to be transplanted into congenic recipients in a long-term competitive repopulation assay (LT-CR). Transplantation of 25μL blood showed a gradual increase in the number of LT-CR cells mobilized in response to G-CSF as measured by donor chimerism in myeloid and B lineages at 16 weeks post-transplant. Expectedly repopulating units (RU) per mL blood progressively increased from 0.2 ± 0.0 (n=6) in steady-state to 2.9 ± 1.6 (n=9) and 82.6 ± 40.4 (n=9) at days 2 and 5 of G-CSF treatment respectively. At 16 weeks post-transplant, BM from primary recipients were transplanted into secondary recipients. Surprisingly, secondary recipients of blood samples collected after 2 and 5 days of G-CSF treatment had equivalent levels of donor chimerism (37.2% ± 6.6% for 2 days G-CSF and 47.1% ± 7.8% for 5 days G-CSF, n = 8 per group). Therefore, although the absolute number of RU mobilized at day 2 of G-CSF was 28-fold lower than at day 5 of G-CSF administration, more primitive serially reconstituting HSC were mobilized at equivalent levels at days 2 and 5 of G-CSF treatment. This supports our hypothesis that most potent serially reconstituting HSC are mobilized as early as day 2 of G-CSF treatment consistent with the disappearance of osteoblasts1. To test the potential of HSC remaining in the BM, BM cells from G-CSF mobilized mice were transplanted in competition with BM cells from congenic mice in steady-state. Donor chimerism at 16 weeks post-transplant showed that competitive repopulation of BM cells was severely impaired at day 5 of G-CSF treatment with the number of RU per 200,000 BM cells decreasing from 4.1 ± 1.4 in steady-state and 5.2 ± 1.6 at day 2 of G-CSF treatment, to only 0.14 ± 0.05 at day 5 of G-CSF treatment. To test whether this 29-fold decrease in competitive repopulation was due to increased HSC proliferation, we measured BrdU incorporation for the last 2.5 days prior to BM harvest as well as cell cycle analysis with Ki67 and Hoechst33342. The proportion of quiescent Lin- Sca1+ Kit+ CD48- phenotypic HSC in G0 phase decreased from 62.8 ±4.0% in steady-state to 43.5±8.2% at day 2 of G-CSF, but surged back to 80.5±1.9% and 75.1±3.5% at days 3.5 and 5 of G-CSF treatment. The proportion of HSC in G1 and S/G2/M phases followed the opposite pattern, up at day 2, down at days 3.5 and 5. This was confirmed by BrdU incorporation for 2.5 days with the number of BrdU+ cells among Lin- Sca1+ KIT+ CD48- cells rising from 35.1±4.0% in steady-state, to 51.2±4.5% at day 2 of G-CSF and going down to 18.1±1.9% at day 3.5 and 23.3±5.5% at day 5 of G-CSF. Therefore, G-CSF recruits phenotypic HSC into cell cycle within the first 2 days of administration, but HSC return to quiescence despite continuing G-CSF. Therefore decreased repopulation potential at day5 of G-CSF is not due to increased cycling. Finally, we noted that the number of Lin-Sca1+KIT+CD48-CD150+ HSC and Lin-Sca1+KIT+CD48-CD150- multipotent progenitors were reduced 2.4- and 2.8-fold respectively (p<.05) in G-CSF-mobilized BM. In conclusion, administration of G-CSF rapidly disrupts HSC niches resulting in rapid mobilization of serially-reconstituting LT-CRC as early as day 2 of G-CSF administration. Secondly, the marked reduction of competitive reconstitution potential of mobilized BM was not due to increased HSC cycling but rather to decreased number of HSC remaining in mobilized BM. Disclosures: No relevant conflicts of interest to declare.

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Isolation of a Common Natural Type-I Interferon Producing Cell and Dendritic Cell Progenitor Population in Mouse Bone Marrow.

Blood ◽

10.1182/blood.v108.11.1653.1653 ◽

2006 ◽

Vol 108 (11) ◽

pp. 1653-1653

Author(s):

Nobuyuki Onai ◽

Aya Onai ◽

Markus G. Manz

Keyword(s):

Bone Marrow ◽

Steady State ◽

Type I Interferon ◽

Receptor Expression ◽

Mouse Bone Marrow ◽

Type I ◽

Differentiation Potential ◽

Hematopoietic Stem ◽

Gm Csf

Abstract Most type-I interferon producing cells (IPCs) and dendritic cells (DCs) are non-dividing cells with a short in vivo half-live of several days, and thus need to be continuously replaced. A common differentiation pathway for IPCs and DCs, and accordingly, the existence of common IPC and DC progenitors remains controversial. Flt3-ligand (Flt3L) is a non-redundant cytokine for in vivo IPC and DC development: IPC and DC differentiation potential is confined to Flt3+-hematopoietic progenitors; Flt3L KO mice show massively reduced IPCs and DCs. In contrast to Flt3, the “myeloid” cytokines GM-CSF and M-CSF seem to be less relevant in steady-state IPC and DC differentiation, however, they might be critically important in inflammatory conditions. To identify a candidate common IPC and DC progenitor population, we evaluated Flt3 and “myeloid” cytokine receptor expression in mouse bone marrow. We found that c-kitintlin− cells contained a Flt3+M-CSFR+ fraction that in Flt3L supplemented cultures gave rise to about 95% pure CD11c+MHC class II+ cells, consisting of both CD11c+B220+ IPCs and CD11c+B220− DCs, at a efficiency comparable to that of hematopoietic stem cells. In the presence of GM-CSF, Flt3+M-CSFR+c-kitintlin− cells gave rise to CD11c+CD11b+ DCs but not CD11c−CD11b+ macrophages/monocytes. Furthermore, Flt3+M-CSFR+c-kitintlin− cells possessed very poor, if any activity in myeloid colony forming assays, and lacked pre-B cell colony forming activity. In both, lethally and sub-lethally irradiated mice, transferred Flt3+M-CSFR+c-kitintlin− cells differentiated into CD11c+B220+ IPCs, CD11c+CD8α+, and CD11c+CD8α− conventional DC subsets, while no other hematopoietic cells were detectable. In vivo reconstitution and CFSE-labeling experiments showed that Flt3+M-CSFR+c-kitintlin− cells extensively proliferate in the lethally irradiated mice, reaching peak progeny levels of IPC and DC at day 10 after transplantation, indicating high proliferative, but limited self-renewal capacity of these cells. Quantitative RT-PCR analysis revealed high expression of DC and IPC-development affiliated genes (such as PU.1, STAT3, GM-CSFR, and CX3CR1), but no lymphoid- and erythroid-development affiliated gene transcription. These data suggest the existence of common developmental intermediates for both IPCs and DCs in mouse bone marrow, and thus might provide new insights into the regulation of IPC and DC differentiation in steady-state and inflammation.

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

Blood ◽

10.1182/blood.v108.11.861.861 ◽

2006 ◽

Vol 108 (11) ◽

pp. 861-861 ◽

Cited By ~ 1

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.

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Prostaglandin E2 (PGE2) Treatment Rapidly Increases the Fraction of Non-Dividing Hematopoietic Stem Cells (HSCs)

Blood ◽

10.1182/blood.v112.11.73.73 ◽

2008 ◽

Vol 112 (11) ◽

pp. 73-73

Author(s):

Rebecca L Porter ◽

Benjamin J Frisch ◽

Regis J O’Keefe ◽

Laura M Calvi

Keyword(s):

Cell Cycle ◽

Bone Marrow ◽

Prostaglandin E2 ◽

Flow Cytometric Analysis ◽

Brdu Incorporation ◽

Hematopoietic Stem ◽

Hematopoietic System ◽

Cell Fate Decisions ◽

Flow Cytometric ◽

Incorporated Brdu

Abstract HSCs are pluripotent cells responsible for the establishment and renewal of the entire hematopoietic system. Our group and others have established that osteoblastic cells in the bone marrow microenvironment regulate HSC cell fate decisions. Specifically, Parathyroid hormone (PTH) expands HSCs by activating osteoblasts in the HSC niche. However, the molecular mechanisms for this increase are unknown. PTH increases local production of prostaglandin E2 (PGE2) in osteoblasts by stimulating cyclo-oxygenase 2 (Cox-2). We also recently found that treatment of osteoblastic MC3T3 cells with PTH (10−7 M) rapidly induces PGE2 Synthase expression. Therefore, we hypothesized that PGE2 may act as a mediator of the PTH effect on HSCs. We have shown that in vivo PGE2 treatment caused a 2.75-fold increase in lineage− Sca-1+ c-kit+ (LSK) cells within the bone marrow compared with vehicle treated mice (p=0.0061, n=8/group). Bone marrow mononuclear cells (BMMC) from mice treated with PGE2 also demonstrated superior lymphomyeloid reconstitution in competitive repopulation analyses, suggesting that HSCs are being expanded or modulated to more efficiently reconstitute the hematopoietic system in the recipients. It is known that HSCs that reside in the G0 phase of the cell cycle have increased ability to reconstitute myeloablated recipient mice. Since PGE2 treatment resulted in superior reconstitution, we hypothesized that PGE2 may increase the percentage of HSCs residing in G0. To test this hypothesis, we treated BMMC from male C57b/6 mice with 10−6 M PGE2 or vehicle for 90 minutes. The percentage of cells in G0 vs. G1 was determined by flow-cytometric analysis using the RNA and DNA dyes, Pyronin-Y and Hoechst 33342 respectively. As we predicted, PGE2 treatment increased the percentage of wild-type LSK cells in G0 1.85 fold over vehicle-treated LSK cells (23.63% in vehicle-treated, n=4 vs. 43.7% in PGE2-treated, n=6). Since the PTH-dependent increase in HSCs is Protein Kinase A (PKA) mediated and the PGE2 receptors EP2 and EP4 signal via PKA, we assayed the effect of PGE2 on the percentage of cells in G0 in mice lacking the EP2 receptor (EP2−/− mice). Interestingly, there was no enrichment for HSC in G0 when BMMC from EP2−/− mice were treated with PGE2 (55.25% in vehicle-treated, n=4 vs. 56.06% in PGE2-treated, n=5). These findings suggest that PGE2-dependent regulation of HSC activity may involve increasing the percentage of HSCs that reside in G0 by activation of EP2, thereby augmenting their ability to reconstitute the hematopoietic system of a myeloablated recipient. 5-bromo-2-deoxyuridine (BrdU) incorporation was also used to investigate the effect of PGE2 on cell cycling of HSCs. Male 6–8 week old C57b/6 mice were injected intraperitoneally with 1 mg BrdU and PGE2 (6 mg/kg) or vehicle. After 30, 60, 90 or 120 minutes, mice were sacrificed and BMMC were subjected to flow cytometric analysis for incorporation of BrdU and DNA content in HSCs. As expected for the highly quiescent HSC population, only a small fraction of HSCs incorporated BrdU. After 30 and 60 minutes of treatment, there was no difference in the percentage of cells that incorporated BrdU between vehicle and PGE2-treated mice. However, at the 90 and 120 minute time points, there were significantly less HSCs cycling in the bone marrow from the PGE2 treated mice (12.1% vs. 5.3% at 90 min, n=2 per group; 11.1% vs. 1.8% at 120 min, n=5 per group, p=0.0060), suggesting that fewer PGE2-treated cells were synthesizing DNA. Taken together, the increase in the percentage of HSCs in G0 and the decrease in cycling HSCs after PGE2 treatment indicate that PGE2 could improve engraftment and reconstitution of the hematopoietic system by enriching for HSCs in G0. These results suggest that PGE2 may exert its beneficial effect on bone marrow reconstitution by altering cell cycle dynamics in HSCs. Identification of the molecular events mediating this novel PGE2 action on HSC could provide additional targets for HSC manipulation in clinical situations requiring rapid and efficient bone marrow reconstitution, such as recovery from iatrogenic or pathologic myeloablative injury.

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Gabp Transcription Factor Is Required for Self-Renew and Proliferation of Hematopoietic Stem and Progenitor Cells

Blood ◽

10.1182/blood.v118.21.1284.1284 ◽

2011 ◽

Vol 118 (21) ◽

pp. 1284-1284

Author(s):

Zhongfa Yang ◽

Karen Drumea ◽

James Cormier ◽

Junling Wang ◽

Xuejun Zhu ◽

...

Keyword(s):

Cell Cycle ◽

Bone Marrow ◽

Transcription Factor ◽

Progenitor Cells ◽

Myeloid Cells ◽

Mouse Bone Marrow ◽

Hematopoietic Stem ◽

Significant Difference ◽

Stem And Progenitor Cells ◽

Ets Domain

Abstract Abstract 1284 GABP is an ets transcription factor that regulates genes which are required for normal hematopoietic development. In myeloid cells, GABP is an essential component of a retinoic acid-inducible enhanceosome that mediates granulocytic gene expression and, in lymphoid cells, GABP regulates expression of IL7-R and the essential transcription factor, Pax5. GABP is a tetrameric complex that includes GABPa, which binds DNA via its ets domain, and GABPb, which contains the transcription activation domain. Genetic disruption of mouse Gabpa caused early embryonic lethality. We created mice in which loxP recombination sites flank exons that encode the Gabpa ets domain, and bred them to mice that bear the Mx1Cre recombinase; injection with pIC induced Cre expression and efficiently deleted Gabpa in hematopoietic cells. One half of the Gabpa knock-out (KO) mice died within two weeks of pIC injection in association with widespread visceral hemorrhage. Gabpa KO mice exhibited a rapid loss of mature granulocytes, and residual myeloid cells exhibited myelodysplasia due, in part, to regulation by Gabp of the transcriptional repressor, Gfi-1. We used bone marrow transplantation to demonstrate that the defect in Gabpa null myeloid cells is cell intrinsic. Although hematopoietic progenitor cells in Gabpa KO bone marrow were decreased more than 100-fold compared to pIC treated control mice, there was not a statistically significant difference in the numbers of Lin−c-kit+Sca-1− hematopoietic stem cells (HSCs) between KO and control mice. Genetic disruption of Gfi-1 disruption in HSCs caused increased cell cycle activity – an effect that is diametrically opposite of the effect of Gabpa KO; this suggests that the effect of Gabpa on HSCs is not due to its control of Gfi-1. In contrast, Gabpa KO HSCs exhibited a marked decrease in cell cycle activity, but did not demonstrate increased apoptosis. The defects in S phase entry of Gabpa null HSCs are reminiscent of the cell cycle defects in Gabpa null fibroblasts, in which expression of Skp2 E3 ubiquitin ligase, which controls degradation of the cyclin dependent kinase inhibitors (CDKIs) p21 and p27, was markedly reduced following Gabpa disruption. We showed that Gabpa KO cells express reduced levels of Skp2. We propose that GABP controls self-renewal and proliferation of mouse bone marrow stem and progenitor cells, in part, through its regulation of Skp2. Thus, Gabpa is a key regulator of myeloid differentiation through its control of Gfi-1, but it is required for cell cycle activity of HSCs, by a distinct effect that may be due to its control of Skp2 and CDKIs. Disclosures: No relevant conflicts of interest to declare.

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Cell Cycle Analysis and Synchronization of Pluripotent Hematopoietic Progenitor Stem Cells

Blood ◽

10.1182/blood.v90.6.2293 ◽

1997 ◽

Vol 90 (6) ◽

pp. 2293-2299 ◽

Cited By ~ 71

Author(s):

G. Prem Veer Reddy ◽

Cheryl Y. Tiarks ◽

Lizhen Pang ◽

Joanne Wuu ◽

Chung-Cheng Hsieh ◽

...

Keyword(s):

Cell Cycle ◽

Stem Cells ◽

Bone Marrow ◽

Hematopoietic Stem Cells ◽

Calf Serum ◽

S Phase ◽

G1 Phase ◽

Proliferative Potential ◽

Mouse Bone Marrow ◽

Hematopoietic Stem

Abstract Hematopoietic stem cells purified from mouse bone marrow are quiescent with less than 2% of Lin− Hoechstlow/Rhodaminelow (Lin− Holow/Rholow) and 10% to 15% of Lin−/Sca+ cells in S phase. These cells enter proliferative cycle and progress through G1 and into S phase in the presence of cytokines and 5% heat-inactivated fetal calf serum (HI-FCS). Cytokine-stimulated Lin− Holow/Rholow cells took 36 to 40 hours to complete first division and only 12 hours to complete each of 5 subsequent divisions. These cells require 16 to 18 hours to transit through G0 /G1 period and 28 to 30 hours to enter into mid-S phase during the first cycle. Up to 56% of Lin− Rholow/Holow cells are high-proliferative potential (7 factor-responsive) colony-forming cells (HPP-CFC). At isolation, HPP-CFC are quiescent, but after 28 to 30 hours of culture, greater than 60% are in S phase. Isoleucine-deprivation of Lin−Holow/Rholow cells in S phase of first cycle reversibly blocked them from entering into second cycle. After the release from isoleucine-block, these cells exhibited a G1 period of less than 2 hours and entered into mid-S phase by 12 hours. Thus, the duration of G1 phase of the cells in second cycle is 4 to 5 times shorter than that observed in their first cycle. Similar cell cycle kinetics are observed with Lin−/Sca+ population of bone marrow cells. Stem cell factor (SCF ) alone, in the presence of HI-FCS, is as effective as a cocktail of 2 to 7 cytokines in inducing quiescent Lin−/Sca+ cells to enter into proliferative cycle. Aphidicolin treatment reversibly blocked cytokine-stimulated Lin−/Sca+ cells at G1 /S boundary, allowing their tight synchrony as they progress through first S phase and enter into second G1 . For these cells also, SCF alone is sufficient for their progression through S phase. These studies indicate a very short G1 phase for stem cells induced to proliferate and offer experimental approaches to synchronize murine hematopoietic stem cells.

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Cell Cycle Analysis and Synchronization of Pluripotent Hematopoietic Progenitor Stem Cells

Blood ◽

10.1182/blood.v90.6.2293.2293_2293_2299 ◽

1997 ◽

Vol 90 (6) ◽

pp. 2293-2299 ◽

Cited By ~ 7

Author(s):

G. Prem Veer Reddy ◽

Cheryl Y. Tiarks ◽

Lizhen Pang ◽

Joanne Wuu ◽

Chung-Cheng Hsieh ◽

...

Keyword(s):

Cell Cycle ◽

Stem Cells ◽

Bone Marrow ◽

Hematopoietic Stem Cells ◽

Calf Serum ◽

S Phase ◽

G1 Phase ◽

Proliferative Potential ◽

Mouse Bone Marrow ◽

Hematopoietic Stem

Hematopoietic stem cells purified from mouse bone marrow are quiescent with less than 2% of Lin− Hoechstlow/Rhodaminelow (Lin− Holow/Rholow) and 10% to 15% of Lin−/Sca+ cells in S phase. These cells enter proliferative cycle and progress through G1 and into S phase in the presence of cytokines and 5% heat-inactivated fetal calf serum (HI-FCS). Cytokine-stimulated Lin− Holow/Rholow cells took 36 to 40 hours to complete first division and only 12 hours to complete each of 5 subsequent divisions. These cells require 16 to 18 hours to transit through G0 /G1 period and 28 to 30 hours to enter into mid-S phase during the first cycle. Up to 56% of Lin− Rholow/Holow cells are high-proliferative potential (7 factor-responsive) colony-forming cells (HPP-CFC). At isolation, HPP-CFC are quiescent, but after 28 to 30 hours of culture, greater than 60% are in S phase. Isoleucine-deprivation of Lin−Holow/Rholow cells in S phase of first cycle reversibly blocked them from entering into second cycle. After the release from isoleucine-block, these cells exhibited a G1 period of less than 2 hours and entered into mid-S phase by 12 hours. Thus, the duration of G1 phase of the cells in second cycle is 4 to 5 times shorter than that observed in their first cycle. Similar cell cycle kinetics are observed with Lin−/Sca+ population of bone marrow cells. Stem cell factor (SCF ) alone, in the presence of HI-FCS, is as effective as a cocktail of 2 to 7 cytokines in inducing quiescent Lin−/Sca+ cells to enter into proliferative cycle. Aphidicolin treatment reversibly blocked cytokine-stimulated Lin−/Sca+ cells at G1 /S boundary, allowing their tight synchrony as they progress through first S phase and enter into second G1 . For these cells also, SCF alone is sufficient for their progression through S phase. These studies indicate a very short G1 phase for stem cells induced to proliferate and offer experimental approaches to synchronize murine hematopoietic stem cells.

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A Hypoxic Niche in the Mouse Bone Marrow Diminishes Proliferation and Differentiation of Hematopoietic Stem Cells

Blood ◽

10.1182/blood.v112.11.4777.4777 ◽

2008 ◽

Vol 112 (11) ◽

pp. 4777-4777

Author(s):

Pernilla M Eliasson ◽

Jan-Ingvar Jönsson

Keyword(s):

Cell Cycle ◽

Stem Cells ◽

Bone Marrow ◽

Transcription Factor ◽

Hematopoietic Stem Cells ◽

Active Form ◽

Culture Conditions ◽

Mouse Bone Marrow ◽

Quiescent State ◽

Hematopoietic Stem

Abstract In the bone marrow hematopoietic stem cells (HSCs) reside in specialized niches in close contact with stromal cells and endosteal osteoblasts. It is thought that this environment is hypoxic in nature, where HSCs are maintained in a quiescent state to prevent their depletion. Hypoxia stabilizes the transcription factor HIF-1α which triggers angiogenesis as well as genes slowering the cell cycle, promoting cell survival, and leading to a decrease in cellular metabolism. In this study, hypoxic effects of the maintenance of Lin−Sca1+c-kit+* (LSK) cells derived from mouse bone marrow and the involvement of the transcription factor hypoxia inducible factor 1 α (HIF-1α) were investigated. Hypoxic culture conditions led to an increase in numbers of primitive colony-forming progenitor cells and a preferential expansion of immature blast-like appearing cells. Concurrently, the immature c-kit Sca-1 phenotype was better maintained in hypoxia compared to ambient oxygen levels. Moreover, hypoxia decreased the proliferation of HSCs as measured by CFSE or PKH26 staining. This was confirmed by cell cycle analysis, and hypoxic cultivation decreased the percentage of cells in S-phase whereas cells in G0/G1 phase increased. Cells infected with a constitutively active form of HIF-1α showed the same pattern as cells cultured in hypoxia. To verify that the effect is HIF-1α mediated, we silenced HIF-1α in LSK cells with shRNA. The decrease in proliferation in hypoxic cultivation of cells infected with shRNA against HIF-1α was markedly diminished, indicating that HIF-1α play an important role in controlling proliferation of hematopoietic stem cells. These results suggest that a major function of hypoxia is to counteract proliferation and possibly differentiation, thereby sustaining maintenance. Furthermore, hypoxic culture conditions may have beneficial clinical implications for ex vivo purposes and may improve the yields of stem cells. In our ongoing-studies, we are investigating whether HIF-1α and hypoxia is an absolute prerequisite for the proper maintenance of HSCs in the bone marrow.

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Without GABP Transcription Factor, BCR-ABL Cannot Transform HSCs to Leukemic Stem Cells Nor Induce Chronic Myelogenous Leukemia in Mice

Blood ◽

10.1182/blood.v118.21.965.965 ◽

2011 ◽

Vol 118 (21) ◽

pp. 965-965

Author(s):

Zhongfa Yang ◽

Cong Peng ◽

Yaoyu Chen ◽

Junling Wang ◽

Xuejun Zhu ◽

...

Keyword(s):

Cell Cycle ◽

Stem Cells ◽

Bone Marrow ◽

Transcription Factor ◽

Peripheral Blood ◽

Myeloid Cells ◽

Myelogenous Leukemia ◽

Mouse Bone Marrow ◽

Hematopoietic Stem ◽

Cycle Arrest

Abstract Abstract 965 Chronic Myelogenous Leukemia (CML) is driven by the fusion oncogene, BCR-ABL, which transforms normal hematopoietic stem cells (HSCs) to leukemic stem cells (LSCs). Tyrosine kinase inhibitors, such as imatinib mesylate, control the massive expansion of leukemic cells in most patients with CML, but cannot eradicate CML LSCs. Several genetic pathways have been shown to be critical for the growth and survival of CML LSCs, including signaling molecules, tumor suppressors, and metabolic regulators. However, the role of transcription factors in functional regulation of LSCs in CML has not been widely studied. GA Binding Protein (GABP) is an ets transcription factor that is required for entry of fibroblasts into the cell cycle, and expression of Gabpa (the DNA-binding component of the complex), alone, was sufficient to induce quiescent, serum-starved cells to enter the cell cycle. Thus, Gabp is both necessary and sufficient for cell cycle entry. Conditional deletion of Gabpa in mouse bone marrow decreased hematopoietic progenitor cells more than 100-fold, but hematopoietic stem cells (HSCs) were relatively preserved. Gabpα null HSCs exhibited significant cell cycle arrest. We sought to determine if the cell cycle arrest caused by Gabpa loss could impair development of CML cells in a mouse model. We used retroviral infection of bone marrow from 5-FU-treated mice (to enrich for stem and progenitor cells) to generate a rapidly fatal CML-like syndrome in mice. Bone marrow from mice with loxP-flanked (floxed) Gabpa and wild type control mice was infected with a retrovirus that co-expresses BCR-ABL, Cre recombinase, and green fluorescent protein (GFP). As expected, transplantation into recipient mice of control mouse bone marrow infected with BCR-ABL-Cre-GFP retrovirus caused a rapidly fatal myeloproliferative neoplasm, with a median survival of approximately three weeks; mice died with massive infiltration of GFP+ myeloid cells in peripheral blood cell, spleen, bone marrow, and other organs. In floxed Gabpa bone marrow, the retrovirus deleted floxed Gabpa in cells that express the BCR-ABL fusion oncogene, and these cells were identifiable based on GFP expression. Transplantation of floxed Gabpa bone marrow infected with BCR-ABL-Cre-GFP retrovirus failed to induce CML during six months of observation. Importantly, GFP+ peripheral blood granulocytes were observed for at least 6 months after transplantation; these CD11b+, Gr1+ cells continued to express BCR-ABL and were shown to be Gabpa null. These results indicate that the lack of Gabpa severely impaired the function of LSCs. In addition, secondary transplantation of bone marrow from these mice again demonstrated the presence of BCR-ABL-expressing peripheral blood myeloid cells. We conclude that Gabp transcription factor is required for the transformation of HSCs to LSCs by BCR-ABL. Furthermore, the persistence of BCR-ABL-expressing myeloid cells without the development of leukemia provides a unique model that permits analysis of the biological properties of BCR-ABL in vivo. The continued generation of BCR-ABL-expressing cells without CML development is unprecedented, and represents a unique model of leukemia tumor suppression. Disclosures: No relevant conflicts of interest to declare.

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Induced cell-autonomous neutropenia systemically perturbs hematopoiesis in Cebpa enhancer-null mice

Blood Advances ◽

10.1182/bloodadvances.2021005851 ◽

2021 ◽

Author(s):

Roberto Avelino ◽

Roger Mulet-Lazaro ◽

Marije Havermans ◽

Remco Hoogenboezem ◽

Leonie Smeenk ◽

...

Keyword(s):

Bone Marrow ◽

Stem Cell ◽

Bone Marrow Cells ◽

Flow Cytometric Analysis ◽

Wild Type Mouse ◽

Mouse Bone Marrow ◽

Severe Dysplasia ◽

Wild Type ◽

Hematopoietic Stem ◽

Factor C

The transcription factor C/EBPa initiates the neutrophil gene expression program in the bone marrow. Knockouts of the Cebpa gene or its +37kb enhancer in mice show two major findings: (1) neutropenia in bone marrow and blood; (2) decrease in long-term hematopoietic stem cell (LT-HSC) numbers. Whether the latter finding is cell autonomous (intrinsic) to the LT-HSCs or an extrinsic event exerted on the stem cell compartment remained an open question. Flow cytometric analysis of the Cebpa +37kb enhancer knockout model revealed that the reduction in LT-HSC numbers observed was proportional to the degree of neutropenia. Single cell transcriptomics of wild type mouse bone marrow showed that Cebpa is predominantly expressed in early myeloid-biased progenitors, but not in LT-HSCs. These observations suggest that the negative effect on LT-HSCs is an extrinsic event caused by neutropenia. We transplanted whole bone marrows from +37kb enhancer deleted mice and found that 40% of the recipient mice acquired full blown neutropenia with severe dysplasia and a significant reduction in the total LT-HSC population. The other 60% showed initial signs of myeloid differentiation defects and dysplasia when they were sacrificed, suggesting they were in an early stage of the same pathological process. This phenotype was not seen in mice transplanted with wild type bone marrow cells. Altogether, these results indicate that Cebpa-enhancer deletion causes cell autonomous neutropenia, which reprograms and disturbs the quiescence of HSCs, leading to a systemic impairment of the hematopoietic process.

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