RNA EDITOME ANALYSIS OF HEMATOPOIETIC CELLS REVEALS ANTIZYME INHIBITOR 1 AS A FUNCTIONAL REGULATOR IN HEMATOPOIETIC STEM AND PROGENITOR CELLS

Abstract Abstract 2581 The bone marrow (BM) microenvironment consists of a heterogeneous population including mesenchymal stem cells and as well as more differentiated cells like osteoblast and adipocytes. These cells are believed to be crucial regulators of hematopoetic cell development, however, so far, their identity and specific functions has not been well defined. We have by using Ebf2 reporter transgenic Tg(Ebf2-Gfp) mice found that CD45−TER119−EBF2+ cells are selectively expressed in non-hematopoietic cells in mouse BM and highly enriched with MSCs whereas the EBF2− stromal cells are very heterogenous (Qian, et al., manuscript, 2010). In the present study, we have subfractionated the EBF2− stromal cells by fluorescent activated cell sorter (FACS) using CD44. On contrary to previous findings on cultured MSCs, we found that the freshly isolated CD45−TER119−EBF2+ MSCs were absent for CD44 whereas around 40% of the CD45−TER119−EBF2− cells express CD44. Colony forming unit-fibroblast (CFU-F) assay revealed that among the CD45−LIN−EBF2− cells, CD44− cells contained generated 20-fold more CFU-Fs (1/140) than the CD44+ cells. The EBF2−CD44− cells could be grown sustainably in vitro while the CD44+ cells could not, suggesting that Cd44− cells represents a more primitive cell population. In agreement with this, global gene expression analysis revealed that the CD44− cells, but not in the CD44+ cells expressed a set of genes including connective tissue growth factor (Ctgf), collagen type I (Col1a1), NOV and Runx2 and Necdin(Ndn) known to mark MSCs (Djouad et al., 2007) (Tanabe et al., 2008). Furthermore, microarray data and Q-PCR analysis from two independent experiments revealed a dramatic downregulation of cell cycle genes including Cdc6, Cdca7,-8 and Ki67, Cdk4-6) and up-regulation of Cdkis such as p57 and p21 in the EBF2−CD44− cells, compared to the CD44+ cells indicating a relatively quiescent state of the CD44− cells ex vivo. This was confirmed by FACS analysis of KI67 staining. Furthermore, our microarray analysis suggested high expression of a set of hematopoietic growth factors and cytokines genes including Angiopoietin like 1, Kit ligand, Cxcl12 and Jag-1 in the EBF2−CD44− stromal cells in comparison with that in the EBF2+ or EBF2−CD44+ cell fractions, indicating a potential role of the EBF2− cells in hematopoiesis. The hematopoiesis supporting activity of the different stromal cell fractions were tested by in vitro hematopoietic stem and progenitor assays- cobblestone area forming cells (CAFC) and colony forming unit in culture (CFU-C). We found an increased numbers of CAFCs and CFU-Cs from hematopoietic stem and progenitor cells (Lineage−SCA1+KIT+) in culture with feeder layer of the EBF2−CD44− cells, compared to that in culture with previously defined EBF2+ MSCs (Qian, et al., manuscript, 2010), confirming a high capacity of the EBF2−CD44− cells to support hematopoietic stem and progenitor cell activities. Since the EBF2+ cells display a much higher CFU-F cloning frequency (1/6) than the CD44−EBF2− cells, this would also indicate that MSCs might not be the most critical regulators of HSC activity. Taken together, we have identified three functionally and molecularly distinct cell populations by using CD44 and transgenic EBF2 expression and provided clear evidence of that primary mesenchymal stem and progenitor cells reside in the CD44− cell fraction in mouse BM. The findings provide new evidence for biological and molecular features of primary stromal cell subsets and important basis for future identification of stage-specific cellular and molecular interaction pathways between hematopoietic cells and their cellular niche components. Disclosures: No relevant conflicts of interest to declare.

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Investigating The Role Of ARAP3 In The Regulation Of Hematopoietic Stem and Progenitor Cells

Blood ◽

10.1182/blood.v122.21.2409.2409 ◽

2013 ◽

Vol 122 (21) ◽

pp. 2409-2409

Author(s):

Yiwen Song ◽

Sonja Vermeren ◽

Wei Tong

Keyword(s):

Endothelial Cells ◽

Plasma Membrane ◽

Progenitor Cells ◽

Hematopoietic Cells ◽

Conditional Knockout ◽

Ph Domain ◽

Hematopoietic Stem ◽

Normal Peripheral Blood ◽

Stem And Progenitor Cells

Abstract ARAP3 is a member of the dual Arf-and-Rho GTPase-activating proteins (GAP) family, functioning specifically to inactivate its substrates Arf6 and RhoA GTPases. ARAP3 is translocated to the plasma membrane after PIP3 binding to the first two of its five PH domains, facilitating its GAP activity in a PI3K-mediated manner. Rho family GTPases are found to play critical roles in many aspects of hematopoietic stem and progenitor cells (HSPCs), such as engraftment and migration, while a role for Arf family GTPases in hematopoiesis is less defined. Previous studies found that either exogenous ARAP3 expression in epithelial cells or RNAi-mediated ARAP3 depletion in endothelial cells disrupts F-actin or lamellipodia formation, respectively, resulting in a cell rounding phenotype and failure to spread. This implies that ARAP3 control of Arf6 and RhoA is tightly regulated, and maintaining precise regulation of ARAP3 levels is crucial to actin organization in the cell. Although ARAP3 was first identified in porcine leukocytes, its function in the hematopoietic system is incompletely understood. Germline deletion of Arap3 results in embryonic lethality due to angiogenic defects. Since endothelial cells are important for the emergence of HSCs during embryonic development, early lethality precludes further studying the role of ARAP3 in definitive hematopoiesis. Therefore, we generated several transgenic mouse models to manipulate ARAP3 in the hematopoietic compartment: (1) Arap3fl/fl;Vav-Cretg conditional knockout mice (CKO) deletes ARAP3 specifically in hematopoietic cells, (2) Arap3fl/fl;VE-Cadherin -Cretg CKO mice selectively deletes ARAP3 in embryonic endothelial cells and thereby hematopoietic cells, and (3) Arap3R302,3A/R302,3A germline knock-in mice (KI/KI) mutates the first PH domain to ablate PI3K-mediated ARAP3 activity in all tissues. We found an almost 100% and 90% excision efficiency in the Vav-Cretg- and VEC-Cretg- mediated deletion of ARAP3 in the bone marrow (BM), respectively. However, the CKO mice appear normal in steady-state hematopoiesis, showing normal peripheral blood (PB) counts and normal distributions of all lineages in the BM. Interestingly, we observed an expansion of the Lin-Scal+cKit+ (LSK) stem and progenitor compartment in the CKO mice. This is due to an increase in the multi-potent progenitor (MPP) fraction, but not the long-term or short-term HSC (LT- or ST-HSC) fractions. Although loss of ARAP3 does not alter the frequency of phenotypically-characterized HSCs, we performed competitive BM transplantation (BMT) studies to investigate the functional impact of ARAP3 deficiency. 500 LSK cells from Arap3 CKO (Arap3fl/fl;Vav-Cretg and Arap3fl/fl;VEC-Cretg) or Arap3fl/fl control littermate donors were transplanted with competitor BM cells into irradiated recipients. We observed similar donor-derived reconstitution and lineage repopulation in the mice transplanted with Arap3fl/fl and Arap3 CKO HSCs. Moreover, Arap3 CKO HSCs show normal reconstitution in secondary transplants. Arap3 KI/KI mice are also grossly normal and exhibit an expanded MPP compartment. Importantly, Arap3KI/KI LSKs show impaired reconstitution compared to controls in the competitive BMT assays. Upon secondary and tertiary transplantation, reconstitution in both PB and BM diminished in the Arap3KI/KI groups, in contrast to sustained reconstitution in the control group. Additionally, we observed a marked skewing towards the myeloid lineage in Arap3KI/KI transplanted secondary and tertiary recipients. These data suggest a defect in HSC function in Arap3KI/KI mice. Myeloid-skewed reconstitution also points to the possibility of selection for “myeloid-primed” HSCs and against “balanced” HSCs, as HSCs exhaust during aging or upon serial transplantation. Taken together, our data suggest that ARAP3 plays a non-cell-autonomous role in HSCs by regulating HSC niche cells. Alternatively, the ARAP3 PH domain mutant that is incapable of locating to the plasma membrane in response to PI3K may exert a novel dominant negative function in HSCs. We are investigating mechanistically how ARAP3 controls HSC engraftment and self-renewal to elucidate the potential cell-autonomous and non-cell-autonomous roles of ARAP3 in HSCs. In summary, our studies identify a previously unappreciated role of ARAP3 as a regulator of hematopoiesis and hematopoietic stem and progenitor cell function. Disclosures: No relevant conflicts of interest to declare.

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Deletion of proapoptotic Puma selectively protects hematopoietic stem and progenitor cells against high-dose radiation

Blood ◽

10.1182/blood-2009-10-248872 ◽

2010 ◽

Vol 115 (23) ◽

pp. 4707-4714 ◽

Cited By ~ 61

Author(s):

Lijian Shao ◽

Yan Sun ◽

Zhonghui Zhang ◽

Wei Feng ◽

Yongxing Gao ◽

...

Keyword(s):

Bone Marrow ◽

Progenitor Cells ◽

Hematopoietic Cells ◽

High Dose ◽

Adverse Side Effect ◽

Hematopoietic Stem ◽

Stem And Progenitor Cells ◽

Novel Strategy ◽

Induced Apoptosis ◽

Dose Radiation

Abstract Bone marrow injury is a major adverse side effect of radiation and chemotherapy. Attempts to limit such damage are warranted, but their success requires a better understanding of how radiation and anticancer drugs harm the bone marrow. Here, we report one pivotal role of the BH3-only protein Puma in the radiosensitivity of hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs). Puma deficiency in mice confers resistance to high-dose radiation in a hematopoietic cell–autonomous manner. Unexpectedly, loss of one Puma allele is sufficient to confer mice radioresistance. Interestingly, null mutation in Puma protects both primitive and differentiated hematopoietic cells from damage caused by low-dose radiation but selectively protects HSCs and HPCs against high-dose radiation, thereby accelerating hematopoietic regeneration. Consistent with these findings, Puma is required for radiation-induced apoptosis in HSCs and HPCs, and Puma is selectively induced by irradiation in primitive hematopoietic cells, and this induction is impaired in Puma-heterozygous cells. Together, our data indicate that selective targeting of p53 downstream apoptotic targets may represent a novel strategy to protecting HSCs and HPCs in patients undergoing intensive cancer radiotherapy and chemotherapy.

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Hematopoietic stem and progenitor cells regulate the regeneration of their niche by secreting Angiopoietin-1

eLife ◽

10.7554/elife.05521 ◽

2015 ◽

Vol 4 ◽

Cited By ~ 84

Author(s):

Bo O Zhou ◽

Lei Ding ◽

Sean J Morrison

Keyword(s):

Bone Marrow ◽

Progenitor Cells ◽

Stromal Cells ◽

Leptin Receptor ◽

Hematopoietic Cells ◽

Perivascular Niche ◽

Hematopoietic Stem ◽

Stem And Progenitor Cells ◽

And Function ◽

Angiopoietin 1

Hematopoietic stem cells (HSCs) are maintained by a perivascular niche in bone marrow but it is unclear whether the niche is reciprocally regulated by HSCs. Here, we systematically assessed the expression and function of Angiopoietin-1 (Angpt1) in bone marrow. Angpt1 was not expressed by osteoblasts. Angpt1 was most highly expressed by HSCs, and at lower levels by c-kit+ hematopoietic progenitors, megakaryocytes, and Leptin Receptor+ (LepR+) stromal cells. Global conditional deletion of Angpt1, or deletion from osteoblasts, LepR+ cells, Nes-cre-expressing cells, megakaryocytes, endothelial cells or hematopoietic cells in normal mice did not affect hematopoiesis, HSC maintenance, or HSC quiescence. Deletion of Angpt1 from hematopoietic cells and LepR+ cells had little effect on vasculature or HSC frequency under steady-state conditions but accelerated vascular and hematopoietic recovery after irradiation while increasing vascular leakiness. Hematopoietic stem/progenitor cells and LepR+ stromal cells regulate niche regeneration by secreting Angpt1, reducing vascular leakiness but slowing niche recovery.

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In Vitro Hematopoietic Lineage Interconversion from Human Bone Marrow Stem and Progenitor Cells.

Blood ◽

10.1182/blood.v104.11.4160.4160 ◽

2004 ◽

Vol 104 (11) ◽

pp. 4160-4160

Author(s):

Ling Chen ◽

Stephanie Jean-Noel ◽

Kevin Hall ◽

Ying Shi ◽

Griffin P. Rodgers

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

Cell Surface ◽

Progenitor Cells ◽

Hematopoietic Cells ◽

Human Bone ◽

Human Bone Marrow ◽

Hematopoietic Stem ◽

Stem And Progenitor Cells ◽

Two Populations

Abstract The cell surface antigen, CD133, marks a fraction of hematopoietic stem and progenitor cells and has been successfully used to study their differential biology. To evaluate the differentiating capacity of stem/progenitor cells, we cultivated purified normal human bone marrow CD133 selected cells for 2 weeks with erythropoietin (EPO) or granulocyte colony-stimulating factor (G-CSF) to induce erythroid or myeloid differentiation, respectively. After the second week of cultivation, we reversed the seeding environment of the two populations by placing EPO treated cells into a G-CSF environment and G-CSF treated cells into an EPO environment for an additional 2-week culture. The cells produced in the culture were phenotypically defined by morphology and flow cytometry, and genotypically by RNA and proteomic analyses. Three-color flow cytometry was used for identifying CD133+ progenitors, CD36+ erythroid and CD13+ myeloid cells, as shown in Table 1. The morphology of the cultured cells, assessed by Wright-Giemsa staining, is consistent with the conversion of cellular specific markers. Rapid analysis of gene expression demonstrated co-expression of 76% of 266 genes analyzed among the erythroid and myeloid lineages. Furthermore, proteomic analysis exhibited the sharing of 33% of 9518 expressed protein spots assayed in the two populations after the first 2-week culture, and 32% after 2 weeks of the switch culture. Our data clearly demonstrate that the committed erythroid and myeloid precursors are able to change their fate and can switch into the opposite cell type by a conversion pathway under a specifically defined condition. We termed this switch as interconversion, considering conversion of hematopoietic cells to non-hematopoietic cells. Furthermore, the observations presented in this study show that cytokines used can improve the conversion. We are developing a mathematical model describing the kinetics of hematopoietic stem/progenitor cell transitions into specific lineages, along with the conversion of committed cells based on multiple potential energy wells corresponding to different cell states and cytokines. Table 1. Expression of cell surface markers after 4-week culture D0 1 week 2 weeks 4 weeks CD expression (%) E G E G E2w →G2w → G2w E2w Data are presented as a mean of at least 2 experiments. E: EPO; G: G-CSF; E2w or G2w: EPO or G-CSF treatment for two weeks. CD133+ 96.19 15.74 13.6 0.24 0.36 0.01 0.63 CD36+ 0 60.37 27.39 96.37 25.87 45.41 68.54 CD13+ 0.43 35.41 57.29 24.41 92.1 85.87 37.76 CD133+ / CD36+ 0.44 22.24 15.97 0.12 0.18 1.55 7.65 CD133+ / CD13+ 1.24 19.43 13.36 0.36 1.09 13.31 14.92 CD36+ / CD13+ 0.09 41.25 17.80 23.69 54.1 46.60 79.41

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A comparative gene-expression analysis of CD34+ hematopoietic stem and progenitor cells grown in static and stirred culture systems

Cellular & Molecular Biology Letters ◽

10.2478/s11658-006-0039-x ◽

2006 ◽

Vol 11 (4) ◽

Cited By ~ 14

Author(s):

Qunliang Li ◽

Qiwei Liu ◽

Haibo Cai ◽

Wen-Song Tan

Keyword(s):

Gene Expression ◽

Progenitor Cells ◽

Hematopoietic Cells ◽

Genetic Changes ◽

Hematopoietic Stem ◽

Culture Systems ◽

Cd34 Cells ◽

Stem And Progenitor Cells ◽

Culture Strategies ◽

Differential Transcription

AbstractStatic and stirred culture systems are widely used to expand hematopoietic cells, but differential culture performances are observed between these systems. We hypothesize that these differential culture outcomes are caused by the physiological responses of CD34+ hematopoietic stem and progenitor cells (HSPCs) to the different physical microenvironments created in these culture devices. To understand the genetic changes provoked by culture microenvironments, the gene expression profiling of CD34+ HSPCs grown in static and stirred culture systems was compared using SMART-PCR and cDNA arrays. The results revealed that 103 and 99 genes were significantly expressed in CD34+ cells from static and stirred systems, respectively. Of those, 91 have similar levels of expression, while 12 show differential transcription levels. These differentially expressed genes are mainly involved in anti-oxidation, DNA repair, apoptosis, and chemotactic activity. A quantitative molecular understanding of the influences of growth microenvironments on transcriptional events in CD34+ HSPCs should give new insights into optimizing culture strategies to produce hematopoietic cells.

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Developmental Stage and Sex-Specific Role of IGF/IGF1R Signaling in Hematopoietic Stem and Progenitor Cells

Blood ◽

10.1182/blood.v126.23.1157.1157 ◽

2015 ◽

Vol 126 (23) ◽

pp. 1157-1157

Author(s):

Ying Xie ◽

Xin Hu ◽

Zhe Li

Keyword(s):

Developmental Stage ◽

Progenitor Cells ◽

Hematopoietic Cells ◽

Conditional Knockout ◽

Specific Role ◽

Hematopoietic Stem ◽

Male And Female ◽

Stem And Progenitor Cells ◽

Igf1r Signaling

Abstract Insulin-like growth factors (IGFs) are critical regulators of cell growth, proliferation and survival. Their activities are mainly mediated through insulin-like growth factor 1 receptor (IGF1R). Both IGFs and IGF1R are commonly involved in human cancers, including leukemia. Thus, a better understanding of the role of IGF/IGF1R signaling in normal hematopoiesis will enhance our understanding of leukemogenesis. We showed previously a developmental stage-specific role of this pathway in regulating fetal (but not adult) megakaryocytic progenitors via interplay with GATA1 (Genes Dev. 24:1659-72). To determine whether IGF/IGF1R signaling also plays differential roles in fetal and adult hematopoiesis in general, we analysed Igf1r conditional knockout mice using Mx1-Cre and Vav-Cre for the adult stage, and Tie2-Cre for the fetal stage. Although both fetal and adult stages of hematopoiesis were not significantly perturbed, loss of Igf1r in fetal (but not in adult) hematopoietic cells led to a significant reduction in the number of CFUs in clonogenic assays. In adult Igf1r -null Lin-Sca-1+c-Kit+ (LSK) hematopoietic stem and progenitor cells (HSPCs), analysis of the long-term hematopoietic stem cell (LT-HSC), short-term HSC (ST-HSC) and multipotent progenitor (MPP) subsets of LSK cells revealed an increase in the percentage of LT-HSCs when compared to that of wild-type (WT) mice, consistent with a recent study (Nature. 500:345-9). These data suggest that IGF/IGF1R signaling may have a developmental stage-restricted role in fetal progenitors; in adults, this pathway may play a role in the transition from LT-HSCs to MPPs. Recently it was shown that estrogen signaling plays an important role in regulating proliferation of HSCs (Nature. 505:555-8). Since during development, both male and female fetuses are exposed to the same high level of maternal estrogen, whereas in adults, male and female hematopoietic cells are exposed to different levels of circulating estrogen, we hypothesized that adult (but not fetal) HSPCs from male and female mice might exhibit different phenotypes in response to Igf1r -loss. To test this, we analysed the adult LSK population by separating males and females. Interestingly, we found that in females, but not in males, the LSK population is significantly reduced upon Igf1r -loss. Conversely, a preliminary study in fetal liver CFU assay revealed that both Igf1r -null female and male fetuses exhibited a similar reduction in their CFUs compared to matched WT controls. To understand the female-specific role of IGF/IGF1R signaling in adult HSPCs, we analysed the cell cycle status of HSCs. Similar to the reported observation (Nature. 505:555-8), we found that in WT female adults, there were significantly more Ki67+ cycling LT-HSCs than those in males. Intriguingly, Igf1r -loss significantly reduced the percentage of cycling LT-HSCs in females to a level comparable to that of WT males, but had a neglectable effect on cycling LT-HSCs in males. Administration of estradiol (E2) revealed that in females, E2 injection led to an increase in the percentage of Ki67+ LT-HSCs and this increase was partially abolished when under the Igf1r- null background; in males, E2 injection also increased the percentage of Ki67+ LT-HSCs, although we did not observe a notable reduction in this population when under the Igf1r -null background. Overall, our data suggest that although IGF/IGF1R signaling is not essential for normal hematopoiesis, it may play a more important role under conditions (e.g., fetal development, pregnancy) when there is a higher demand for the output from the hematopoietic system; in particular, this pathway may play an important role in mediating the effect of estrogen on self-renewal and proliferation of HSPCs. Disclosures No relevant conflicts of interest to declare.

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JAK/STAT Signal Transduction Pathway Activation During Hematopoietic Differentiation From Human Embryonic Stem Cells (hESC)

Blood ◽

10.1182/blood.v118.21.2346.2346 ◽

2011 ◽

Vol 118 (21) ◽

pp. 2346-2346

Author(s):

Chun Fan ◽

Richard Yunkang Liu ◽

Kristine Li ◽

Kenneth S. Zuckerman

Keyword(s):

Signal Transduction ◽

Stem Cells ◽

Stem Cell ◽

Progenitor Cells ◽

Hematopoietic Cells ◽

Embryonic Stem ◽

Hematopoietic Differentiation ◽

Hematopoietic Stem ◽

Stem And Progenitor Cells ◽

Undifferentiated Hesc

Abstract Abstract 2346 The ability to produce hematopoietic cells from human embryonic stem cells (hESC) has been demonstrated, using different multistage culture systems with multiple growth factor combinations. However, very little is understood about the molecular mechanisms that regulate the differentiation from hESC to hematopoietic stem and progenitor cells and further to specific lineages of differentiated hematopoietic cells. Among many signaling pathways involved in stem and progenitor cell differentiation, the JAK/STAT pathways are known to play critical roles in hematopoietic stem cell maintenance and hematopoietic differentiation. STAT3 activation is known to be essential for maintenance of murine ESC, but not human ESC, but it appears not to play a major role in myeloid cell differentiation. Although different levels of JAK2 and STAT5 signaling are important for erythroid and megakaryocytic differentiation, JAK/STAT signaling is not thought to play a role in hESC maintenance or differentiation and is not known to be essential for early stages of differentiation to hematopoietic stem and progenitor cells (HSC/HPC). We have established a serum-free, feeder cell-free system for maintaining hESC (H1 and H9 cells) and for differentiating the hESC to embryoid bodies (EB), from which end-stage hematopoietic cells, notably megakaryocytes and platelets, are produced. We used a multi-stage culture system to produce megakaryocytes and platelets from EBs, including 2 days with vascular endothelial growth factor (VEGF) and bone morphogenic protein (BMP4), 2 more days with VEGF, BMP4, stem cell factor (SCF), Flt3 ligand (FL), and thrombopoietin (TPO), 10 days with VEGF, BMP4, TPO, SCF, FL, IL3, IL6, and IL11, and 2–6 weeks with TPO, SCF, FL, IL3, IL6, and IL11. We used serial western blots, immunofluorescence with confocal microscopy and systematically observed changes of JAK/STAT signal transduction molecule activation. We found a consistent, dynamic change of STAT5 protein phosphorylation during the hematopoietic differentiation process. Interestingly, although JAK2, STAT3 and STAT5 protein were present, and JAK2 and STAT3 were phosphorylated in hESC, there was no evidence of STAT5 phosphorylation/activation in the undifferentiated hESC. During the early phases of differentiation of hESC-derived EBs toward hematopoietic progenitors in the presence of hematopoiesis-related cytokines, STAT5 was phosphorylated and activated in CD34+ HSCs and in CD61+/CD235a (glycophorin A)+ or CD41+/CD235a+ early megakaryocytic/erythroid progenitor cells (MEP). Although there was no detectable change in total STAT5 protein expression levels through hematopoietic differentiation, there was a slowly progressive decrease in phosphorylated/activated STAT5 with further maturation to megakaryocytes that express CD42b+, platelet factor 4, and von Willebrand factor and form proplatelets and platelets. Thus, in spite of hESC containing abundant phosphorylated JAK2, which is a known activator of STAT5, there was no phosphorylation/activation of STAT5 in undifferentiated hESC or early EBs. However, STAT5 became phosphorylated/activated early in hematopoiesis and declined over the course of progressive differentiation along the megakaryocytic lineage. These findings imply that activated JAK2 does not drive the activation of STAT5 that is an early event in differentiation from EBs and mesoderm to HSC and HPC in vitro. Disclosures: No relevant conflicts of interest to declare.

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Fli-1 Transcriptionally Integrates Microenvironmental Cues Sensing By Self-Renewing Hematopoietic Stem and Progenitor Cells

Blood ◽

10.1182/blood-2019-126190 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 725-725

Author(s):

Tomer Itkin ◽

Chaitanya R Badwe ◽

Sean Houghton ◽

Yang Lin ◽

Ying Liu ◽

...

Keyword(s):

Progenitor Cells ◽

Hematopoietic Cells ◽

Premature Mortality ◽

Self Renewal ◽

Hematopoietic Stem ◽

Transgenic Model ◽

Vascular Niche ◽

Isolated Lung ◽

Stem And Progenitor Cells

During adulthood and embryogenesis, fate decisions of hematopoietic stem and progenitor cells (HSPCs), such as specification, self-renewal, and differentiation are tightly regulated by their neighboring niche cells. Moreover, distinct types of niches supply differential cues to direct alternative cell fates for HSPCs. Yet, currently the intrinsic mechanisms balancing HSPC response obliqueness to microenvironmental signals are unknown. Friend Leukemia integration-1 (Fli-1), is an ETS transcription factor expressed by vascular beds and hematopoietic lineages. Fli-1 belongs to the "heptad factors" which are hypothesized to specify and sustain a hematopoietic cell fate. While Fli-1 overexpression is linked to leukemia, the functional role Fli-1 plays in HSPC specification and maintenance remains undefined. We show that inducible deletion of Fli-1 using a Rosa-CreERT2 transgenic adult mice (Fli-1ROSAΔ), results in a rapid thrombocytopenia-associated mortality. Transplantation of Fli-1ROSAΔ bone marrow (BM) cells into WT recipients, to exclude vascular-mediated defects, followed by induction of Fli-1 deletion, resulted with the same phenotype. In a set of modulated competitive transplantation experiments (differential induction time points pre- or post-transplant), we observed defective ability of Fli-1ROSAΔ HSPCs to lodge, engraft, and to sustain hematopoiesis post repopulation. Fli-1 deficient HSPCs exhibited reduced quiescent cell cycling status, a hallmark of stemness, and displayed enhanced apoptosis. Thus, Fli-1 is essential for previously unrecognized cell-autonomous HSPC functions. To determine whether Fli-1 modulates HSPC specification, Fli-1 was conditionally deleted using a developmental VE-cadherin (CDH5)-Cre transgenic model (Fli-1CDH5Δ). This resulted with premature mortality of Fli-1CDH5Δ embryos, accompanied with a hemorrhagic phenotype. Reduced numbers of hematopoietic cells were still detected in the AGM of e10.5 Fli-1CDH5Δ embryos. Conditional Fli-1 deletion using a developmental hematopoietic Vav-1 Cre transgenic model (Fli-1Vav-1Δ) resulted again with premature mortality. Reduced presence of embryonic Fli-1Vav-1Δ liver HSPCs was observed at e12.5. We also applied two in vitro co-culture systems, to study Fli-1 in endothelial to hematopoietic transition (EHT). First, isolated hemogenic endothelial cells (HEC) from WT and Fli-1ROSAΔ embryos were co-cultured with AGM-derived vascular niche. HECs isolated from Fli-1ROSAΔ AGM were still able to convert to CD45+ cells, however these cells did not expand on a vascular niche. Secondly, we have applied an endothelial to hematopoietic reprogramming system in which isolated lung ECs are virally introduced with DOX inducible FosB, Gfi1, Runx1, and Spi1 (FGRS) factors and co-cultured with vascular niche cells. Both WT and Fli-1ROSAΔ ECs were able to acquire a hemogenic like state resulting with a final capacity to convert into hematopoietic cells. Again, Fli-1ROSAΔ cells displayed lesser numbers of CD45+ cells at the end point, presumably due to impaired interaction with the vascular niche. Indeed, reduced expansion capacity was observed both for mature CD45+ and for HSPC derived from Fli-1CDH5Δ AGM region. Adult Fli-1ROSAΔ HSPCs exhibited the same niche-dependent expansion defect. Induction of Fli-1 deletion in vitro in adult HSPCs revealed loss of dependency on vascular niche inductive signals, as no additive expansion effect was observed for Fli-1ROSAΔ HSPCs in the presence of a vascular niche. Hence, Fli-1 is essential for HSPC expansion rather than hematopoietic specification. Differential RNA-seq analysis combined with epigenetic studies of expanding WT and Fli-1ROSAΔ HSPCs, revealed dysregulation of Fli-1-controlled pathways involved in transduction of microenvironmental signals for self-renewal. Unexpectedly, H3K27Ac analysis, a marker for transcriptional priming, revealed increased global acetylation of Fli-1ROSAΔ HSPCs' chromatin. Therefor, Fli-1 may not only perform as transcription activator, but foremostly as a genomic suppressor via modulation of histone acetylation status. Decrypting the mechanism(s) by which Fli-1 orchestrates HSPC self-renewal, may promote an improved expansion protocol of human HSPC pre-transplantation, and provide additional insights for microenvironmental sensing by Fli-1-dependent leukemic cells. Disclosures No relevant conflicts of interest to declare.

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Increased hematopoietic stem cell mobilization in aged mice

Blood ◽

10.1182/blood-2005-12-010272 ◽

2006 ◽

Vol 108 (7) ◽

pp. 2190-2197 ◽

Cited By ~ 113

Author(s):

Zhenlan Xing ◽

Marnie A. Ryan ◽

Deidre Daria ◽

Kalpana J. Nattamai ◽

Gary Van Zant ◽

...

Keyword(s):

Bone Marrow ◽

Stem Cell ◽

Progenitor Cells ◽

Close Association ◽

Hematopoietic Cells ◽

Dimensional Structure ◽

Hematopoietic Stem ◽

Aged Mice ◽

Stem And Progenitor Cells ◽

Cell Mobilization

Abstract Hematopoietic stem and progenitor cells (HSPCs) are located in the bone marrow in close association with a highly organized 3-dimensional structure formed by stroma cells, referred to as the niche. Mobilization of HSPCs from bone marrow to peripheral blood in response to granulocyte colony-stimulating factor (G-CSF) requires de-adhesion of HSPCs from the niche. The influence of aging of HSPCs on cell-stroma interactions has not been determined in detail. Using a mouse model of G-CSF–induced mobilization, we demonstrated that the ability to mobilize hematopoietic stem cells is approximately 5-fold greater in aged mice. Competitive mobilization experiments confirmed that enhanced mobilization ability was intrinsic to the stem cell. Enhanced mobilization efficiency of primitive hematopoietic cells from aged mice correlated with reduced adhesion of hematopoietic progenitor cells to stroma and with elevated levels of GTP-bound Cdc42. These results might indicate that stroma–stem cell interactions are dynamic over a lifetime and result in physiologically relevant changes in the biology of primitive hematopoietic cells with age.

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