PU.1 expression defines distinct functional activities in the phenotypic HSC compartment in a mouse model of inflammatory stress

The transcription factor PU.1 is a critical regulator of lineage fate in blood-forming hematopoietic stem cells (HSC). In response to inflammatory signals, PU.1 expression is increased in HSC, activating myeloid differentiation genes while repressing cell cycle and protein synthesis genes. To address potential functional heterogeneity arising in the phenotypic HSC compartment due to changes in PU.1 expression, here we fractionated phenotypic HSC using the SLAM code in conjunction with PU.1 expression levels using the PU.1-EYFP reporter mouse strain. While PU.1lo SLAM cells contain extensive long-term repopulating activity and a molecular signature corresponding to HSC activity at steady state, under inflammatory conditions the PU.1lo SLAM fraction is comprised almost entirely of HSC-like cells containing extensive short-term megakaryocytic potential. Our data demonstrate that the phenotypic HSC gate is heterogenous, and that similar PU.1 transcription factor levels can be tied to distinct functional activities under steady-state and inflammatory conditions.

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Notch2 Signaling Inhibits Differentiation and Promotes Self Renewal of Hematopoietic Stem and Progenitor Cells During Marrow Regeneration.

Blood ◽

10.1182/blood.v114.22.2544.2544 ◽

2009 ◽

Vol 114 (22) ◽

pp. 2544-2544

Author(s):

Barbara Varnum-Finney ◽

Irwin D. Bernstein

Keyword(s):

Progenitor Cells ◽

Conflicts Of Interest ◽

Ex Vivo ◽

Myeloid Differentiation ◽

Primary Control ◽

Short Term ◽

Self Renewal ◽

Hematopoietic Stem ◽

Post Transplant

Abstract Abstract 2544 Poster Board II-521 Notch regulates numerous lineage choices during vertebrate development, and although ex vivo studies suggest that Notch regulates hematopoietic stem cell (HSC) and multipotential progenitor (MPP) differentiation, a functional role for Notch in HSC/MPP self renewal in vivo remains controversial. We previously reported a Notch2 signaling role during bone marrow (BM) recovery following injection with chemotherapeutic agent 5-fluorouracil (5FU), where Notch2 signaling impedes myeloid differentiation, allowing for generation of sufficient numbers of progenitor cells. Herein, we examine a Notch2 signaling role in HSC as well as progenitor cell self renewal by enumerating generation of HSC and short term repopulating cells in lethally irradiated recipients (Ly5.1+) transplanted with a limiting number (5 × 105) of BM cells from either control mice or from mice bearing Cre-LoxP-inducible Notch2 deletions (Ly5.2+). In recipient mice transplanted with control BM, recovery was evident from Day11 to Day13 post transplant when significantly more than the initial post-irradiation number of 9.0 × 106 BM cells was seen in the recovering marrow. In recovering mice, recipients receiving control cells generated more BM cells than did recipients receiving Notch2-deficient cells. Furthermore, mice receiving control cells generated significantly more donor Sca-1+c-kit+ (SK+) cells than recipients receiving Notch2-deficient BM cells [44.4×103 (s.e.m.+/− 14×103) vs 8.2×103 (s.e.m.+/−1.5×103), respectively, p=0.001]. To quantitate the generation of short term repopulating cells, secondary radioprotection assays were performed. Irradiated secondary recipient mice received 1×106 BM cells from the primary recipients previously transplanted with either control cells or Notch2-deficient cells. Secondary recipients receiving cells from primary control transplants survived significantly longer than those receiving cells from primary Notch2-deficient transplants or than irradiated mice receiving no cells (n=4, p=0.01), indicating Notch2 is required to generate sufficient numbers of cells to provide radioprotection. To quantitate long term HSC generated in the recovering marrow, competitive repopulating units (CRU) were enumerated by performing secondary transplants in which 4-doses of BM cells ranging from 4 × 104 to 5 × 106 cells from primary transplants were injected into secondary recipients along with 1 × 105 Ly5.1+ competing cells. Enumeration of CRU at 2 weeks post transplant confirmed the number of short term repopulating cells was significantly decreased in mice transplanted with Notch2-deficient cells compared to mice transplanted with control cells [(1.3 CRU vs 8.8 CRU / 1×106 BM cells, respectively), p=0.0004)]. Enumeration of CRU at 9 weeks post transplant indicated HSC numbers were also significantly decreased in mice transplanted with Notch2-deficient cells compared to mice transplanted with control cells [(0.1 CRU vs 0.7 CRU / 1×106 BM cells, respectively), p=0.02]. Taken together, our results demonstrate a role for Notch2 in enhancing generation of long term HSC as well as short term repopulating cells and suggests that Notch2 signaling regulates a hierarchy of events to assure the initial repopulation by HSC and MPP, while delaying myeloid differentiation during hematopoietic regeneration. Disclosures: No relevant conflicts of interest to declare.

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E47 regulates hematopoietic stem cell proliferation and energetics but not myeloid lineage restriction

Blood ◽

10.1182/blood-2010-07-297689 ◽

2011 ◽

Vol 117 (13) ◽

pp. 3529-3538 ◽

Cited By ~ 18

Author(s):

Qi Yang ◽

Brandt Esplin ◽

Lisa Borghesi

Keyword(s):

Energy Metabolism ◽

Myeloid Differentiation ◽

Short Term ◽

Hematopoietic Stem ◽

Altered Expression ◽

Helix Loop Helix ◽

Multipotent Progenitors

Abstract The immune system is replenished by self-renewing hematopoietic stem cells (HSCs) that produce multipotent progenitors (MPPs) with little renewal capacity. E-proteins, the widely expressed basic helix-loop-helix transcription factors, contribute to HSC and MPP activity, but their specific functions remain undefined. Using quantitative in vivo and in vitro approaches, we show that E47 is dispensable for the short-term myeloid differentiation of HSCs but regulates their long-term capabilities. E47-deficient progenitors show competent myeloid production in short-term assays in vitro and in vivo. However, long-term myeloid and lymphoid differentiation is compromised because of a progressive loss of HSC self-renewal that is associated with diminished p21 expression and hyperproliferation. The activity of E47 is shown to be cell-intrinsic. Moreover, E47-deficient HSCs and MPPs have altered expression of genes associated with cellular energy metabolism, and the size of the MPP pool but not downstream lymphoid precursors in bone marrow or thymus is rescued in vivo by antioxidant. Together, these observations suggest a role for E47 in the tight control of HSC proliferation and energy metabolism, and demonstrate that E47 is not required for short-term myeloid differentiation.

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ETO2 Controls Hematopoietic Stem Cell Expansion.

Blood ◽

10.1182/blood.v114.22.396.396 ◽

2009 ◽

Vol 114 (22) ◽

pp. 396-396

Author(s):

Stephane Barakat ◽

Julie Lambert ◽

Guy Sauvageau ◽

Trang Hoang

Keyword(s):

Stem Cells ◽

Transcription Factor ◽

Stem Cell ◽

Hematopoietic Stem Cells ◽

Short Term ◽

Self Renewal ◽

Hematopoietic Stem

Abstract Abstract 396 Hematopoietic stem cells that provide short term reconstitution (ST-HSCs) as well as hematopoietic progenitors expand from a small population of long term hematopoietic stem cells (LT-HSCs) that are mostly dormant cells. The mechanisms underlying this expansion remain to be clarified. SCL (stem cell leukemia), is a bHLH transcription factor that controls HSC quiescence and long term competence. Using a proteomics approach to identify components of the SCL complex in erythroid cells, we and others recently showed that the ETO2 co-repressor limits the activity of the SCL complex via direct interaction with the E2A transcription factor. ETO2/CBF2T3 is highly homologous to ETO/CBFA2T1 and both are translocation partners for AML1. We took several approaches to identify ETO2 function in HSCs. We initially found by Q-PCR that ETO2 is highly expressed in populations of cells enriched in short-term HSC (CD34+Flt3-Kit+Sca+Lin-) and lympho-myeloid progenitors (CD34+Flt3+Kit+Sca+Lin-) and at lower levels in LT-HSCs (CD34-Kit+Sca+Lin- or CD150+CD48-Kit+Sca+Lin-). Next, the role of ETO2 was studied by overexpression or downregulation combined with transplantation in mice. Ectopic ETO2 expression induces a 100 fold expansion of LT-HSCs in vivo in transplanted mice associated with differentiation blockade in all lineages, suggesting that ETO2 overexpression overcomes the mechanisms that limit HSC expansion in vivo. We are currently testing the role of the NHR1 domain of ETO2 in this expansion. Conversely, shRNAs directed against ETO2 knock down ET02 levels in Kit+Sca+Lin- cells, causing a ten-fold decrease in this population after transplantation, associated with reduced short-term reconstitution in mice. Finally, proliferation assays using Hoechst and CFSE indicate that ETO2 downregulation affects cell division (CFSE) and leads to an accumulation of Kit+Sca+Lin-cells in G0/G1 state (Hoescht). In conclusion, we show that ETO2 is highly expressed in ST-HSCs and lymphoid progenitors, and controls their expansion by regulating cell cycle entry at the G1-S checkpoint. In addition, ETO2 overexpression converts the self-renewal of maintenance into self-renewal of expansion in LT-HSCs. Disclosures: No relevant conflicts of interest to declare.

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Ing4-deficiency enhances HSC quiescence and confers resistance to inflammatory stress

10.1101/2021.07.14.452366 ◽

2021 ◽

Author(s):

Zanshé Thompson ◽

Georgina A. Anderson ◽

Melanie Rodriguez ◽

Seth Gabriel ◽

Vera Binder ◽

...

Keyword(s):

Gene Expression ◽

Stem Cells ◽

Malignant Cells ◽

Short Term ◽

Regenerative Capacity ◽

Hematopoietic Stem ◽

Inflammatory Stress ◽

Identification And Characterization

Hematopoiesis is tightly regulated by a network of transcription factors and complexes that are required for the development and maintenance of hematopoietic stem cells (HSCs). We recently identified the tumor suppressor, Ing4, as a critical regulator of HSC homeostasis. Though the Ing4 mechanism of action remains poorly characterized, it has been shown to promote stem-like cell characteristics in malignant cells. This activity is, in part, due to Ing4 mediated regulation of several major signaling pathways, including NF-kB and c-Myc. In murine hematopoiesis, Ing4 deficiency induces G0 arrest in HSCs, while simultaneously promoting gene expression signatures associated with differentiation. This results in a poised state for Ing4-deficient HSCs. Long term HSCs are unable to overcome this block, but short-term HSCs convert the poised state into regenerative capacity during hematopoietic challenges, including irradiation and transplantation. Overall, our findings suggest that Ing4 plays a crucial role in the regulation of hematopoiesis. Our model provides key tools for further identification and characterization of pathways that control quiescence and differentiation in HSCs.

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Comparison of the efficacy of hematopoietic stem cell mobilization regimens: a systematic review and network meta-analysis of preclinical studies

Stem Cell Research & Therapy ◽

10.1186/s13287-021-02379-6 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Chengxin Luo ◽

Li Wang ◽

Guixian Wu ◽

Xiangtao Huang ◽

Yali Zhang ◽

...

Keyword(s):

Standard Dose ◽

Meta Analysis ◽

Secondary Outcome ◽

Preclinical Studies ◽

Mice Model ◽

Short Term ◽

Hematopoietic Stem ◽

Support Unit ◽

Meta Analyses

Abstract Background Mobilization failure may occur when the conventional hematopoietic stem cells (HSCs) mobilization agent granulocyte colony-stimulating factor (G-CSF) is used alone, new regimens were developed to improve mobilization efficacy. Multiple studies have been performed to investigate the efficacy of these regimens via animal models, but the results are inconsistent. We aim to compare the efficacy of different HSC mobilization regimens and identify new promising regimens with a network meta-analysis of preclinical studies. Methods We searched Medline and Embase databases for the eligible animal studies that compared the efficacy of different HSC mobilization regimens. Primary outcome is the number of total colony-forming cells (CFCs) in per milliliter of peripheral blood (/ml PB), and the secondary outcome is the number of Lin− Sca1+ Kit+ (LSK) cells/ml PB. Bayesian network meta-analyses were performed following the guidelines of the National Institute for Health and Care Excellence Decision Support Unit (NICE DSU) with WinBUGS version 1.4.3. G-CSF-based regimens were classified into the SD (standard dose, 200–250 μg/kg/day) group and the LD (low dose, 100–150 μg/kg/day) group based on doses, and were classified into the short-term (2–3 days) group and the long-term (4–5 days) group based on administration duration. Long-term SD G-CSF was chosen as the reference treatment. Results are presented as the mean differences (MD) with the associated 95% credibility interval (95% CrI) for each regimen. Results We included 95 eligible studies and reviewed the efficacy of 94 mobilization agents. Then 21 studies using the poor mobilizer mice model (C57BL/6 mice) to investigate the efficacy of different mobilization regimens were included for network meta-analysis. Network meta-analyses indicated that compared with long-term SD G-CSF alone, 14 regimens including long-term SD G-CSF + Me6, long-term SD G-CSF + AMD3100 + EP80031, long-term SD G-CSF + AMD3100 + FG-4497, long-term SD G-CSF + ML141, long-term SD G-CSF + desipramine, AMD3100 + meloxicam, long-term SD G-CSF + reboxetine, AMD3100 + VPC01091, long-term SD G-CSF + FG-4497, Me6, long-term SD G-CSF + EP80031, POL5551, long-term SD G-CSF + AMD3100, AMD1300 + EP80031 and long-term LD G-CSF + meloxicam significantly increased the collections of total CFCs. G-CSF + Me6 ranked first among these regimens in consideration of the number of harvested CFCs/ml PB (MD 2168.0, 95% CrI 2062.0−2272.0). In addition, 7 regimens including long-term SD G-CSF + AMD3100, AMD3100 + EP80031, long-term SD G-CSF + EP80031, short-term SD G-CSF + AMD3100 + IL-33, long-term SD G-CSF + ML141, short-term LD G-CSF + ARL67156, and long-term LD G-CSF + meloxicam significantly increased the collections of LSK cells compared with G-CSF alone. Long-term SD G-CSF + AMD3100 ranked first among these regimens in consideration of the number of harvested LSK cells/ml PB (MD 2577.0, 95% CrI 2422.0–2733.0). Conclusions Considering the number of CFC and LSK cells in PB as outcomes, G-CSF plus AMD3100, Me6, EP80031, ML141, FG-4497, IL-33, ARL67156, meloxicam, desipramine, and reboxetine are all promising mobilizing regimens for future investigation.

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Long-term repopulating hematopoietic stem cells and “side population” in human steady state peripheral blood

Stem Cell Research ◽

10.1016/j.scr.2013.04.003 ◽

2013 ◽

Vol 11 (1) ◽

pp. 625-633 ◽

Cited By ~ 16

Author(s):

Philippe Brunet de la Grange ◽

Marija Vlaski ◽

Pascale Duchez ◽

Jean Chevaleyre ◽

Veronique Lapostolle ◽

...

Keyword(s):

Stem Cells ◽

Steady State ◽

Hematopoietic Stem Cells ◽

Peripheral Blood ◽

Side Population ◽

Hematopoietic Stem

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Functional Resolution of Long-Term-and Short-Term- Hematopoietic Stem Cells

Journal of Stem Cell Research & Therapeutics ◽

10.15406/jsrt.2016.01.00044 ◽

2016 ◽

Vol 1 (6) ◽

Author(s):

Stephen H Bartelmez

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

Short Term ◽

Hematopoietic Stem

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Gata2 is required for HSC generation and survival

Journal of Experimental Medicine ◽

10.1084/jem.20130751 ◽

2013 ◽

Vol 210 (13) ◽

pp. 2843-2850 ◽

Cited By ~ 119

Author(s):

Emma de Pater ◽

Polynikis Kaimakis ◽

Chris S. Vink ◽

Tomomasa Yokomizo ◽

Tomoko Yamada-Inagawa ◽

...

Keyword(s):

Endothelial Cells ◽

Transcription Factor ◽

Developmental Stages ◽

Vascular Endothelial ◽

Vascular Integrity ◽

Hematopoietic Stem ◽

Vascular Endothelial Cadherin ◽

Umbilical Arteries ◽

Specific Deletion

Knowledge of the key transcription factors that drive hematopoietic stem cell (HSC) generation is of particular importance for current hematopoietic regenerative approaches and reprogramming strategies. Whereas GATA2 has long been implicated as a hematopoietic transcription factor and its dysregulated expression is associated with human immunodeficiency syndromes and vascular integrity, it is as yet unknown how GATA2 functions in the generation of HSCs. HSCs are generated from endothelial cells of the major embryonic vasculature (aorta, vitelline, and umbilical arteries) and are found in intra-aortic hematopoietic clusters. In this study, we find that GATA2 function is essential for the generation of HSCs during the stage of endothelial-to-hematopoietic cell transition. Specific deletion of Gata2 in Vec (Vascular Endothelial Cadherin)-expressing endothelial cells results in a deficiency of long-term repopulating HSCs and intra-aortic cluster cells. By specific deletion of Gata2 in Vav-expressing hematopoietic cells (after HSC generation), we further show that GATA2 is essential for HSC survival. This is in contrast to the known activity of the RUNX1 transcription factor, which functions only in the generation of HSCs, and highlights the unique requirement for GATA2 function in HSCs throughout all developmental stages.

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A Transcription Factor Pulse Can Prime Chromatin for Heritable Transcriptional Memory

Molecular and Cellular Biology ◽

10.1128/mcb.00372-16 ◽

2016 ◽

Vol 37 (4) ◽

Cited By ~ 2

Author(s):

Aimee Iberg-Badeaux ◽

Samuel Collombet ◽

Benoit Laurent ◽

Chris van Oevelen ◽

Kuo-Kai Chin ◽

...

Keyword(s):

Transcription Factor ◽

Rna Polymerase Ii ◽

Target Genes ◽

Short Term Memory ◽

Epigenetic Mechanism ◽

Short Term ◽

Term Memory ◽

Pol Ii ◽

Transcriptional Memory

ABSTRACT Short-term and long-term transcriptional memory is the phenomenon whereby the kinetics or magnitude of gene induction is enhanced following a prior induction period. Short-term memory persists within one cell generation or in postmitotic cells, while long-term memory can survive multiple rounds of cell division. We have developed a tissue culture model to study the epigenetic basis for long-term transcriptional memory (LTTM) and subsequently used this model to better understand the epigenetic mechanisms that enable heritable memory of temporary stimuli. We find that a pulse of transcription factor CCAAT/enhancer-binding protein alpha (C/EBPα) induces LTTM on a subset of target genes that survives nine cell divisions. The chromatin landscape at genes that acquire LTTM is more repressed than at those genes that do not exhibit memory, akin to a latent state. We show through chromatin immunoprecipitation (ChIP) and chemical inhibitor studies that RNA polymerase II (Pol II) elongation is important for establishing memory in this model but that Pol II itself is not retained as part of the memory mechanism. More generally, our work reveals that a transcription factor involved in lineage specification can induce LTTM and that failure to rerepress chromatin is one epigenetic mechanism underlying transcriptional memory.

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