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

Blood ◽  
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.

Non-side-population hematopoietic stem cells in mouse bone marrow

Blood ◽  
2006 ◽  
Vol 108 (8) ◽  
pp. 2850-2856 ◽  
Author(s):  
Yohei Morita ◽  
Hideo Ema ◽  
Satoshi Yamazaki ◽  
Hiromitsu Nakauchi
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Cell Function ◽  
Side Population ◽  
Mouse Bone Marrow ◽  
Quiescent State ◽  
Hematopoietic Stem ◽  
Main Population

AbstractMost hematopoietic stem cells (HSCs) are assumed to reside in the so-called side population (SP) in adult mouse bone marrow (BM). We report the coexistence of non-SP HSCs that do not significantly differ from SP HSCs in numbers, capacities, and cell-cycle states. When stained with Hoechst 33342 dye, the CD34-/low c-Kit+Sca-1+lineage marker- (CD34-KSL) cell population, highly enriched in mouse HSCs, was almost equally divided into the SP and the main population (MP) that represents non-SP cells. Competitive repopulation assays with single or 30 SP- or MP-CD34-KSL cells found similar degrees of repopulating activity and frequencies of repopulating cells for these populations. Secondary transplantation detected self-renewal capacity in both populations. SP analysis of BM cells from primary recipient mice suggested that the SP and MP phenotypes are interconvertible. Cell-cycle analyses revealed that CD34-KSL cells were in a quiescent state and showed uniform cell-cycle kinetics, regardless of whether they were in the SP or MP. Bcrp-1 expression was similarly detected in SP- and MP-CD34-KSL cells, suggesting that the SP phenotype is regulated not only by Bcrp-1, but also by other factors. The SP phenotype does not specify all HSCs; its identity with stem cell function thus is unlikely.

scholarly journals Cell Cycle Analysis and Synchronization of Pluripotent Hematopoietic Progenitor Stem Cells

Blood ◽  
1997 ◽  
Vol 90 (6) ◽  
pp. 2293-2299 ◽  
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.

scholarly journals Cell Cycle Analysis and Synchronization of Pluripotent Hematopoietic Progenitor Stem Cells

Blood ◽  
1997 ◽  
Vol 90 (6) ◽  
pp. 2293-2299 ◽  
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.

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

Blood ◽  
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.

scholarly journals Efficient retroviral gene transfer to purified long-term repopulating hematopoietic stem cells

Blood ◽  
1994 ◽  
Vol 84 (1) ◽  
pp. 74-83 ◽  
Author(s):  
SJ Szilvassy ◽  
S Cory
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Cell Biology ◽  
Bone Marrow Cells ◽  
High Titer ◽  
Marker Gene ◽  
Mouse Bone Marrow ◽  
Hematopoietic Stem

Abstract Efficient gene delivery to multipotential hematopoietic stem cells would greatly facilitate the development of effective gene therapy for certain hematopoietic disorders. We have recently described a rapid multiparameter sorting procedure for significantly enriching stem cells with competitive long-term lymphomyeloid repopulating ability (CRU) from 5-fluorouracil (5-FU)-treated mouse bone marrow. The sorted cells have now been tested as targets for retrovirus-mediated delivery of a marker gene, NeoR. They were cocultured for 4 days with fibroblasts producing a high titer of retrovirus in medium containing combinations of the hematopoietic growth factors interleukin-3 (IL-3), IL-6, c-kit ligand (KL), and leukemia inhibitory factor (LIF) and then injected into lethally irradiated recipients, together with sufficient “compromised” bone marrow cells to provide short-term support. Over 80% of the transplanted mice displayed high levels (> or = 20%) of donor- derived leukocytes when analyzed 4 to 6 months later. Proviral DNA was detected in 87% of these animals and, in half of them, the majority of the hematopoietic cells were marked. Thus, infection of the stem cells was most effective. The tissue and cellular distribution of greater than 100 unique clones in 55 mice showed that most sorted stem cells had lymphoid as well as myeloid repopulating potential. Secondary transplantation provided strong evidence for infection of very primitive stem cells because, in several instances, different secondary recipients displayed in their marrow, spleen, thymus and day 14 spleen colony-forming cells the same proviral integration pattern as the primary recipient. Neither primary engraftment nor marking efficiency varied for stem cells cultured in IL-3 + IL-6, IL-3 + IL-6 + KL, IL-3 + IL-6 + LIF, or all four factors, but those cultured in IL-3 + IL-6 + LIF appeared to have lower secondary engraftment potential. Provirus expression was detected in 72% of the strongly marked mice, albeit often at low levels. Highly efficient retroviral marking of purified lymphomyeloid repopulating stem cells should enhance studies of stem cell biology and facilitate analysis of genes controlling hematopoietic differentiation and transformation.

Circadian expression of clock genes in purified hematopoietic stem cells is developmentally regulated in mouse bone marrow

2006 ◽  
Vol 34 (9) ◽  
pp. 1248-1260 ◽  
Author(s):  
Oleg Tsinkalovsky ◽  
Elisabeth Filipski ◽  
Benedikte Rosenlund ◽  
Robert B. Sothern ◽  
Hans Geir Eiken ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Clock Genes ◽  
Mouse Bone Marrow ◽  
Developmentally Regulated ◽  
Hematopoietic Stem ◽  
Circadian Expression

Induction of Adipocyte Differentiation and Reduction of Hematopoesis-Supporting Activity of Mouse Stromal Cells by Suppression of Transcription Factor GATA-2.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1392-1392
Author(s):  
Yoko Okitsu ◽  
Hideo Harigae ◽  
Masanori Seki ◽  
Toru Fujiwara ◽  
Shinichiro Takahashi ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Transcription Factor ◽  
Hematopoietic Stem Cells ◽  
Stromal Cells ◽  
Colony Formation ◽  
Adipocyte Differentiation ◽  
Fetal Liver ◽  
Expression Level ◽  
Hematopoietic Stem

Abstract (Introduction) Aplastic anemia (AA) is characterized by peripheral pancytopenia and fatty bone marrow. An immunological attack to hematopoietic stem cells has been thought to be responsible for the development of the disease. Previously, we reported the expression of transcription factor GATA-2 is significantly decreased in CD34 positive cells in AA. Together with the phenotypes of hematopoietic stem cells in GATA-2 hetero-knockout mice, GATA-2 down-regulation may play a role in the reduction of a stem cell pool observed in AA. On the other hand, GATA-2 has been shown to be essential for the maintenance of immaturity of preadipocytes. If a pathological immune response in AA decreases the level of GATA-2 expression in not only hematopoietic stem cells but also stromal preadipocytes, it may accelerate the maturation of preadipocytes, leading to the formation of fatty bone marrow. To explore this possibility, the phenotypic change of stromal preadipocytes by suppression of GATA-2 was examined in this study. (Method) The GATA-2 expression level was suppressed by using siRNA for GATA-2 in mouse stromal preadipocyte cell lines, TBR9 and TBR343. After the treatment with siRNA, the adipocyte differentiation was induced by the incubation with insulin and dexamethasone for 7days. Then, the maturation level was examined by oil drops formation judged by oil red staining, and by the expression level of adipcin and PPAR-γ mRNA. Supporting activity of hematopoietic colony formation was also evaluated by using mouse fetal liver cells after siRNA treatment. (Results) By using designed siRNA, the GATA-2 expression was suppressed to 30% of control, whereas the expression level of GATA-3, which is co-expressed in preadipocytes, was unchanged. When GATA-2 was suppressed by siRNA, the oil drop formation and adipocyte-specific gene expression was significantly accelerated in both of stromal cells. Furthermore, the number of fetal liver hematopoetic colonies was significantly decreased by suppression of GATA-2, suggesting that GATA-2 down-regulation in stromal preadipocytes results in not only the acceleration of the maturation but also the reduced supporting activity of hematopoietic colony formation (Conclusion) These results suggest that suppression of GATA-2 in hematopoietic tissues induces the characteristic features of AA, i.e., decreased the number of hematopoietic stem cells and increased number of mature adipocytes.

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.

Poly I:C Stimulates Sca-1 Expression in Murine Bone Marrow and Increases the Number of Phenotypic Hematopoietic Stem Cells.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2504-2504
Author(s):  
Russell Garrett ◽  
Gerd Bungartz ◽  
Alevtina Domashenko ◽  
Stephen G. Emerson
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Conflicts Of Interest ◽  
Hematopoietic Cells ◽  
Poly I:C ◽  
Hematopoietic Stem ◽  
Marrow Cells ◽  
Myeloid Progenitors

Abstract Abstract 2504 Poster Board II-481 Polyinosinic:polycytidlyic acid (poly I:C) is a synthetic double-stranded RNA used to mimic viral infections in order to study immune responses and to activate gene deletion in lox-p systems employing a Cre gene responsive to an Mx-1 promoter. Recent observations made by us and others have suggested hematopoietic stem cells, responding to either poly I:C administration or interferon directly, enter cell cycle. Twenty-two hours following a single 100mg intraperitoneal injection of poly I:C into 10-12 week old male C57Bl/6 mice, the mice were injected with a single pulse of BrdU. Two hours later, bone marrow was harvested from legs and stained for Lineage, Sca-1, ckit, CD48, IL7R, and BrdU. In two independent experiments, each with n = 4, 41 and 33% of Lin- Sca-1+ cKit+ (LSK) IL-7R- CD48- cells from poly I:C-treated mice had incorporated BrdU, compared to 7 and 10% in cells from PBS-treated mice. These data support recently published reports. Total bone marrow cellularity was reduced to 45 and 57% in the two experiments, indicating either a rapid death and/or mobilization of marrow cells. Despite this dramatic loss of hematopoietic cells from the bone marrow of poly I:C treated mice, the number of IL-7R- CD48- LSK cells increased 145 and 308% in the two independent experiments. Importantly, the level of Sca-1 expression increased dramatically in the bone marrow of poly I:C-treated mice. Both the percent of Sca-1+ cells and the expression level of Sca-1 on a per cell basis increased after twenty-four hours of poly I:C, with some cells acquiring levels of Sca-1 that are missing from control bone marrow. These data were duplicated in vitro. When total marrow cells were cultured overnight in media containing either PBS or 25mg/mL poly I:C, percent of Sca-1+ cells increased from 23.6 to 43.7%. Within the Sca-1+ fraction of poly I:C-treated cultures, 16.7% had acquired very high levels of Sca-1, compared to only 1.75% in control cultures. Quantitative RT-PCR was employed to measure a greater than 2-fold increase in the amount of Sca-1 mRNA in poly I:C-treated cultures. Whereas the numbers of LSK cells increased in vivo, CD150+/− CD48- IL-7R- Lin- Sca-1- cKit+ myeloid progenitors almost completely disappeared following poly I:C treatment, dropping to 18.59% of control marrow, a reduction that is disproportionately large compared to the overall loss of hematopoietic cells in the marrow. These cells are normally proliferative, with 77.1 and 70.53% accumulating BrdU during the 2-hour pulse in PBS and poly I:C-treated mice, respectively. Interestingly, when Sca-1 is excluded from the analysis, the percent of Lin- IL7R- CD48- cKit+ cells incorporating BrdU decreases following poly I:C treatment, in keeping with interferon's published role as a cell cycle repressor. One possible interpretation of these data is that the increased proliferation of LSK cells noted by us and others is actually the result of Sca-1 acquisition by normally proliferating Sca-1- myeloid progenitors. This new hypothesis is currently being investigated. Disclosures: No relevant conflicts of interest to declare.

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

Blood ◽  
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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