Identification of Human Hematopoietic Stem Cell-Specific STAT5 Target Genes Involved in Self-Renewal and Transformation.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 568-568
Author(s):  
Szabolcs Fatrai ◽  
Albertus T.J. Wierenga ◽  
Edo Vellenga ◽  
Simon M. G. J. Daenen ◽  
Jan Jacob Schuringa
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Hematopoietic Stem Cells ◽  
Target Gene ◽  
Target Genes ◽  
Differentiation Potential ◽  
Self Renewal ◽  
Hematopoietic Stem

Abstract Abstract 568 The transcription factor STAT5 fulfils an essential role in self-renewal of both mouse and human HSCs and persistent activation of STAT5 contributes to leukemic transformation. In patients with acute myeloid leukemia, increased STAT5 activity has been observed in over 60% of the cases. Yet, little is known about mechanisms that are involved. To gain further insight into these processes we studied whether STAT5-imposed long-term self-renewal is exclusively restricted to HSCs, or whether long-term self-renewal can also be imposed on progenitor cells. Human cord blood (CB) cells were transduced with control and STAT5-ER retroviral vectors allowing the induction of STAT5 activity by treatment of cells with 4-hydroxytamoxifen (4-OHT). Four populations were isolated: hematopoietic stem cells (HSC, CD34+CD38low), common myeloid progenitors (CMP, CD34+CD38+CD123+CD45RA-), granulocyte-macrophage progenitors (GMP, CD34+CD38+CD123+CD45RA+) and megakaryocyte-erythroid progenitors (MEP, CD34+CD38+CD123-CD45RA-). MS5 bone marrow stromal cocultures were initiated and STAT5 activity was induced by 4-OHT. In HSCs, STAT5 overexpression induced a long-term proliferative advantage as well as a significant increase in cobblestone formation. This coincided with elevated levels of Colony Forming Cells (CFCs) that were maintained over 5 weeks. In contrast, STAT5 was unable to induce cobblestone formation in progenitor cocultures and only a transient STAT5-induced increase in cell numbers was observed. CFC numbers dropped significantly after 2 weeks and progenitor initiated cultures could not be maintained longer than 3 weeks regardless of STAT5 activity. Myelopoiesis was blocked and an increase in erythroid differentiation in STAT5-ER-transduced HSC, CMP, and MEP populations was observed, while the differentiation potential of the GMP remained unaffected. Next, we aimed to identify HSC-specific STAT5 target genes by performing microarray analysis on HSC, CMP, GMP and MEP populations transduced with our STAT5-ER vectors. To limit STAT5 mediated effects on erythropoiesis GATA1 was downmodulated in STAT5-transduced CB cells by a lentiviral RNAi approach, which completely abrogated erythropoiesis but maintained enhanced HSC self-renewal. Microarrays were performed on GATA1 downmodulated STAT5-transduced CB cells and controls, and these data sets were compared to the HSC-specific STAT5 target gene lists. This combined approach resulted in the identification of 36 GATA1-independent STAT5 target genes in the HSC population. One of the identified genes was HIF2a. The involvement of HIF2a in STAT5 phenotypes was studied functionally by using a lentiviral HIF2a RNAi approach in STAT5 transduced CB cells. These studies revealed that expansion of STAT5/HIF2a RNAi-transduced cells on MS5 bone marrow stromal cocultures was reduced, coinciding with reduced CFC and LTC-IC frequencies, while differentiation was not affected. In summary, our data show that hematopoietic stem cells, but not progenitors are the exclusive target for STAT5-induced long-term self-renewal. Furthermore, we show that HIF2a is a novel STAT5 target gene which plays an important role in STAT5-induced stem cell phenotypes. Disclosures: No relevant conflicts of interest to declare.

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

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

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

Hematopoietic Stem Cells, but Not Progenitors, Are the Exclusive Target for STAT5-Induced Long-Term Self-Renewal.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1347-1347
Author(s):  
Szabolcs Fatrai ◽  
Albertus T.J. Wierenga ◽  
Edo Vellenga ◽  
Simon M. G. J. Daenen ◽  
Jan J. Schuringa
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Target Genes ◽  
Erythroid Differentiation ◽  
Retroviral Vectors ◽  
Independent Manner ◽  
Differentiation Potential ◽  
Self Renewal ◽  
Hematopoietic Stem

Abstract Constitutive activation of STAT5 has been associated with leukemic transformation. Previously, we demonstrated that overexpression of activated STAT5 in human cord blood (CB) cells results in increased stem cell self-renewal and long-term expansion which coincided with the induction of erythropoiesis. In the present study we investigated whether STAT5-imposed long-term self-renewal is exclusively restricted to HSCs, or whether long-term self-renewal can also be imposed on progenitor cells. We generated retroviral expression systems where STAT5 is fused to the estrogen receptor ligand binding domain (ER) allowing the induction of STAT5 activity by treatment of cells with 4-hydroxytamoxifen (4-OHT). Human CB cells were transduced with control and STAT5-ER retroviral vectors followed by MoFlo sorting into four populations: hematopoietic stem cells (HSC, defined as CD34+CD38low), common myeloid progenitors (CMP, CD34+CD38+CD123+CD45RA−), granulocyte-macrophage progenitors (GMP, CD34+CD38+CD123+CD45RA+) and megakaryocyte-erythroid progenitors (MEP, CD34+CD38+CD123−CD45RA−). Sorted populations were plated on MS5 bone marrow stromal cocultures and STAT5 activity was induced by 4-OHT treatment. HSC cells expressing activated STAT5 displayed a long-term proliferative advantage as well as a significant increase in cobblestone formation. This coincided with elevated levels of Colony Forming Cells (CFCs) that were maintained over 5 weeks. In contrast, STAT5 was unable to induce cobblestone formation in progenitor cocultures and only a transient STAT5-induced increase in cell numbers was observed in cocultures initiated with CMPs and MEPs. Also, CFC numbers dropped significantly after 2 weeks and neither of the cultures could be maintained longer than 3 weeks regardless of STAT5 activity. FACS measurements and cytospins showed a block in myelopoiesis and an increase in erythroid differentiation in STAT5-ER-transduced HSC, CMP, and MEP populations, while the differentiation potential of the GMP remained unaffected. Next, we aimed to identify STAT5 target genes which were upregulated in the STAT5 HSC population that were not responsible for erythroid differentiation and played a role in STAT5-induced self-renewal and long-term expansion. GATA1 was down-modulated in STAT5-transduced CB cells by a lentiviral RNAi approach, which completely abrogated erythropoiesis but maintained enhanced HSC self-renewal. Microarray was performed on both GATA1 downmodulated STAT5-transduced CB cells as well as on STAT5-transduced HSC and progenitor populations. Micoarray data from the two experiments were compared and 39 GATA1- independent STAT5 target genes were identified in the STAT5 HSC population. Many of these genes encoded for membrane proteins or proteins involved in adhesion, migration and signal transduction, and these are currently under investigation. In summary, our data show that hematopoietic stem cells, but not progenitors are the exclusive target for STAT5- induced long-term self-renewal. We identified a set of genes that is upregulated in the STAT5-transduced HSC population in a GATA1-independent manner which is potentially responsible for STAT5-induced self-renewal and long-term expansion.

Superior Impact of p18INK4c over p27kip1 on Long-Term Repopulating Ability of Hematopoietic Stem Cells.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 796-796
Author(s):  
Hui Yu ◽  
Hongmei Shen ◽  
Xianmin Song ◽  
Paulina Huang ◽  
Tao Cheng
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Hematopoietic Stem Cells ◽  
Cell Biology ◽  
Wild Type ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
The Impact

Abstract The G1-phase is a critical window during the cell cycle in which stem cell self-renewal may be balanced with differentiation and apoptosis. Increasing evidence suggests that the cyclin-dependent kinase inhibitors (CKIs) such as p21Cip1/Waf1, p27kip1, p16INK4A, and p18INK4C (p21, p27, p16 and p18 hereafter) are involved in stem cell self-renewal, as largely demonstrated in murine hematopoietic stem cells (HSCs). For example, we have recently demonstrated a significant increase of HSC self-renewal in the absence of p18 (Yuan et al, Nature Cell Biology 2004). But the actual roles of these CKIs in HSCs appear to be distinct as p21 and p18 have opposite effects (Yu H et al, ASH 2004) whereas p16 has a limited effect (Stepanova et al, Blood 2005) on HSC exhaustion after serial bone marrow transfer. Like p18, however, p27 was recently reported to also inhibit HSC self-renewal due to the fact that the competitive repopulating units (CRUs) were increased in p27−/− mouse bone marrow (Walkley et al, Nature Cell Biology 2005) in contrast to the results in a previous report (Cheng T et al, Nature Medicine 2000). To further gauge the impact of p18 versus p27 on the long-term repopulating ability (LTRA) of HSCs, we have generated different congenic strains (CD45.1 and CD45.2) of p18−/− or p27−/− mice in the C57BL/6 background, allowing us to compare them with the competitive repopulation model in the same genetic background. The direct comparison of LTRA between p18−/− and p27−/− HSCs was assessed with the competitive bone marrow transplantation assay in which equal numbers of p18−/− (CD45.2) and p27−/− cells (CD45.1) were co-transplanted. Interestingly, the p18−/− genotype gradually dominated the p27−/− genotype in multiple hematopoietic lineages and p18−/− HSCs showed 4-5 times more LTRA than p27−/− HSCs 12 months after cBMT. Further self-renewal potential of HSCs was examined with secondary transplantation in which primarily transplanted p18−/− or p27−/− cells were equally mixed with wild-type unmanipulated cells. Notably, while the p18−/− cells continued to outcompete the wild-type cells as we previously observed, the p27−/− cells did not behave so in the secondary recipients. Based on the flow cytometric measurement and bone marrow cellularity, we estimated that transplanted p18−/− HSCs (defined with the CD34−LKS immunophenotype) had undergone a 230-fold expansion, while transplanted p27−/− and wild-type HSCs had only achieved a 6.6- and 2.4-fold expansion in the secondary recipients respectively. We further calculated the yield of bone marrow nucleated cells (BMNCs) per HSC. There were approximately 44 x 103, 20.6 x 103, and 15 x 103 BMNCs generated per CD34−LKS cell in p18−/−, p27−/− and wild-type transplanted recipients respectively. Therefore, the dramatic expansion of p18−/− HSCs in the hosts was not accompanied by decreased function per stem cell. Our current study demonstrates that hematopoietic engraftment in the absence of p18 is more advantageous than that in the absence of p27, perhaps due to a more specific role of p18 on self-renewal of the long-term repopulating HSCs.

Protein Arginine Methyltransferase 1 (PRMT1) Dampens Self-Renewal and Promotes Differentiation of Hematopoietic Stem Cells in Mouse Models

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2460-2460 ◽  
Author(s):  
Hairui Su ◽  
Szu-Mam Liu ◽  
Chiao-Wang Sun ◽  
Mark T. Bedford ◽  
Xinyang Zhao
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Bone Marrow Transplantation ◽  
Arginine Methylation ◽  
Poly I:C ◽  
Differentiation Potential ◽  
Self Renewal ◽  
Arginine Methyltransferase ◽  
Hematopoietic Stem

Protein arginine methylation is a common type of post-translational modification. PRMT1, the major type I protein arginine methyltransferase, catalyzes the formation of asymmetric dimethyl-arginine and is implicated in various cellular processes, including hematopoiesis and tumorigenesis. We have shown that PRMT1 expression is naturally low in hematopoietic stem cells (HSCs). However, the functions of PRMT1 in hematopoietic stem cell self-renewal and differentiation are yet to be revealed. We have found a cyanine-based fluorescent probe (E84) that can specifically label PRMT1 protein. E84 staining dynamically captures intracellular PRMT1 level and was used to separate live HSC populations with differential PRMT1 expression by flow cytometry. Subsequent bone marrow transplantation of E84high or E84low Lin−Sca1+cKit+ (LSK) cells showed that E84low LSK cells were much more advantageous in reconstituting each blood cell lineages, compared to the E84high counterparts, meaning that the stem-ness of HSCs is negatively correlated with endogenous PRMT1. Therefore, inhibition of PRMT1 was expected to enhance the number and differentiation potential of functional HSCs. The treatment of a PRMT1-specific inhibitor (MS023) to mice resulted in an enlarged LT-HSC population in bone marrow and decreased frequency of granulocyte progenitor cells. In vitro colony formation assays further demonstrated that PRMT1 is required for GMP differentiation. Then we asked whether copious expression of PRMT1 promotes the differentiation of HSC. In this line, we made a LoxP-STOP-LoxP-PRMT1 transgenic mouse model, which induces PRMT1 overexpression upon the expression of Cre recombinase from tissue-specific promoters. We established Mx1-Cre-PRMT1 (Mx1-Tg) mice. Mx1-Tg mice were injected with poly(I:C) for PRMT1 induction and analyzed at four weeks after the last dose. We found that, as predicted, LT-HSC population was reduced and the Pre-GM population was raised. Accordingly, more CFU-Gs but less GEMMs were grown on CFU assays. We further utilized this animal model to compare the blood reconstitution capabilities of bone marrow cells from Mx1-Tg vs. WT mice in the same repopulating conditions. We performed competitive bone marrow transplantation by injecting Mx1-Tg/WT (CD45.2) bone marrow plus supporting cells (CD45.1) to irradiated mice, followed by 5 doses of poly(I:C) induction. Recipient mice were analyzed during a course of approximately 16 weeks. Mx1-Tg cells were outcompeted by WT cells in reconstituting every blood lineages. Taken together, we conclude that PRMT1 promotes HSC differentiation and accelerates HSC exhaustion during the stress caused by bone marrow irradiation. To understand the mechanism on PRMT1-mediated stress hematopoiesis, we also made Pf4-Cre PRMT1 transgenic mice. When PRMT1 is specifically expressed in MK cells, the number of LT-HSCs was also reduced, implying that PRMT1 affects the self-renewal of LT-HSCs via communication between MK cells and HSCs. Mechanistically, two PRMT1 substrates - RBM15 and DUSP4 - are critical for stem cell self-renewal. We further characterized how PRMT1 activates p38 kinase pathway via directly methylating DUSP4 thus induces ubiquitylation and degradation of DUSP4. The arginine methylation site on DUSP4 has been identified. Moreover, introducing methyl-R mutated DUSP4 back to PRMT1-overexpressing cells partially rescued the loss of HSC differentiation potential. This data adds a new link between arginine methylation and protein phosphorylation mediated by MAP kinases/phosphatases. In addition, we discovered that RBM15 controls alternative RNA splicing and RNA processing in a PRMT1-dosage dependent manner. In this report, we will further address how RBM15 target genes, such as enzymes involved in fatty acid metabolic pathways, affect HSC differentiation. In summary, we report that arginine methylation is a novel regulator for the HSC differentiation via controlling p38-regulated stress pathway and metabolic reprogramming. Disclosures No relevant conflicts of interest to declare.

Regulation of Hematopoietic Stem Cells by Interferons and by DNA Methylation: Implications for Bone Marrow Failure

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. SCI-20-SCI-20
Author(s):  
Margaret A. Goodell
Keyword(s):  
Dna Methylation ◽  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Hematopoietic Stem Cells ◽  
Bone Marrow Failure ◽  
Stem Cell Differentiation ◽  
Primary Myelofibrosis ◽  
Differentiation Potential ◽  
Hematopoietic Stem

Bone marrow failure (BMF), the inability to regenerate the differentiated cells of the blood, has a number of genetic and environmental etiologies, such as mutation of telomere-associated protein genes and immune-related aplastic anemia. Recently, mutations in DNA methyltransferase 3A (DNMT3A) have been found to be associated with approximately 15% of cases of primary myelofibrosis (MF), which can be a cause of BMF. The role of DNMT3A more broadly in hematopoiesis, and specifically in BMF, is currently poorly understood. DNMT3A is one of two de novo DNA methylation enzymes important in developmental fate choice. We showed that Dnmt3a is critical for normal murine hematopoiesis, as hematopoietic stem cells (HSCs) from Dnmt3a knockout (KO) mice displayed greatly diminished differentiation potential while their self-renewal ability was markedly increased1, in effect, leading to failure of blood regeneration or BMF. Combined with loss of Dnmt3b, HSCs exhibited a profound differentiation block, mediated in part by an increase of stabilized b-catenin. While we did not initially observe bone marrow pathology or malignancy development in mice transplanted with Dnmt3a KO HSCs, when we aged a large cohort of mice, all mice succumbed to hematologic disease within about 400 days. Roughly one-third of mice developed frank leukemia (acute lymphocytic leukemia or acute myeloid leukemia), one-third developed MDS, and the remainder developed primary myelofibrosis or chronic myelomonocytic leukemia. The pathological characteristics of the mice broadly mirror those of patients, suggesting the Dnmt3a KO mice can serve as a model for human DNMT3A-mutation associated disease. Strikingly, bone marrow of mice with different disease types exhibit distinct DNA methylation features. These will findings and the implications for disease development will be discussed. We are currently investigating the factors that drive different outcomes in the mice, including stressors such as exposure to interferons. We have hypothesized that HSC proliferation accelerates the Dnnmt3a-associated disease phenotypes. We have previously shown that interferons directly impinge on HSCs in the context of infections. Interferons activate HSCs to divide, generating differentiated progeny and cycling HSCs. Repeated interferon stimulation may permanently impair HSC function and bias stem cell output. When combined with loss of Dnmt3a, interferons may promote BMF. We will discuss broadly how external factors such as aging and infection may collaborate with specific genetic determinants to affect long-term hematopoiesis and malignancy development. Reference: Challen GA, Sun D, Jeong M, et al. Dnmt3a is essential for hematopoietic stem cell differentiation. Nat Genet 2012; 44: 23-31 Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

scholarly journals Age-Associated Characteristics of Murine Hematopoietic Stem Cells

2000 ◽  
Vol 192 (9) ◽  
pp. 1273-1280 ◽  
Author(s):  
Kazuhiro Sudo ◽  
Hideo Ema ◽  
Yohei Morita ◽  
Hiromitsu Nakauchi
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Bone Marrow Cells ◽  
Lymphoid Cells ◽  
Differentiation Potential ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Functional Changes

Little is known of age-associated functional changes in hematopoietic stem cells (HSCs). We studied aging HSCs at the clonal level by isolating CD34−/lowc-Kit+Sca-1+ lineage marker–negative (CD34−KSL) cells from the bone marrow of C57BL/6 mice. A population of CD34−KSL cells gradually expanded as age increased. Regardless of age, these cells formed in vitro colonies with stem cell factor and interleukin (IL)-3 but not with IL-3 alone. They did not form day 12 colony-forming unit (CFU)-S, indicating that they are primitive cells with myeloid differentiation potential. An in vivo limiting dilution assay revealed that numbers of multilineage repopulating cells increased twofold from 2 to 18 mo of age within a population of CD34−KSL cells as well as among unseparated bone marrow cells. In addition, we detected another compartment of repopulating cells, which differed from HSCs, among CD34−KSL cells of 18-mo-old mice. These repopulating cells showed less differentiation potential toward lymphoid cells but retained self-renewal potential, as suggested by secondary transplantation. We propose that HSCs gradually accumulate with age, accompanied by cells with less lymphoid differentiation potential, as a result of repeated self-renewal of HSCs.

scholarly journals Development of adult bone marrow stem cells in H-2-compatible and -incompatible mouse fetuses.

1984 ◽  
Vol 159 (3) ◽  
pp. 731-745 ◽  
Author(s):  
R A Fleischman ◽  
B Mintz
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Fetal Liver ◽  
Donor Strain ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Adult Bone Marrow ◽  
Adult Bone

Bone marrow of normal adult mice was found, after transplacental inoculation, to contain cells still able to seed the livers of early fetuses. The recipients' own hematopoietic stem cells, with a W-mutant defect, were at a selective disadvantage. Progression of donor strain cells to the bone marrow, long-term self-renewal, and differentiation into myeloid and lymphoid derivatives was consistent with the engraftment of totipotent hematopoietic stem cells (THSC) comparable to precursors previously identified (4) in normal fetal liver. More limited stem cells, specific for the myeloid or lymphoid cell lineages, were not detected in adult bone marrow. The bone marrow THSC, however, had a generally lower capacity for self-renewal than did fetal liver THSC. They had also embarked upon irreversible changes in gene expression, including partial histocompatibility restriction. While completely allogeneic fetal liver THSC were readily accepted by fetuses, H-2 incompatibility only occasionally resulted in engraftment of adult bone marrow cells and, in these cases, was often associated with sudden death at 3-5 mo. On the other hand, H-2 compatibility, even with histocompatibility differences at other loci, was sufficient to ensure long-term success as often as with fetal liver THSC.

scholarly journals Microrna 199b Regulates Mouse Hematopoietic Stem Cells Maintenance

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3704-3704
Author(s):  
Aldona A Karaczyn ◽  
Edward Jachimowicz ◽  
Jaspreet S Kohli ◽  
Pradeep Sathyanarayana
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Conflicts Of Interest ◽  
Quiescent State ◽  
Differentiation Potential ◽  
Self Renewal ◽  
Hematopoietic Stem

The preservation of hematopoietic stem cell pool in bone marrow (BM) is crucial for sustained hematopoiesis in adults. Studies assessing adult hematopoietic stem cells functionality had been shown that for example loss of quiescence impairs hematopoietic stem cells maintenance. Although, miR-199b is frequently down-regulated in acute myeloid leukemia, its role in hematopoietic stem cells quiescence, self-renewal and differentiation is poorly understood. Our laboratory investigated the role of miR-199b in hematopoietic stem and progenitor cells (HSPCs) fate using miR-199b-5p global deletion mouse model. Characterization of miR-199b expression pattern among normal HSPC populations revealed that miR-199b is enriched in LT-HSCs and reduced upon myeloablative stress, suggesting its role in HSCs maintenance. Indeed, our results reveal that loss of miR-199b-5p results in imbalance between long-term hematopoietic stem cells (LT-HSCs), short-term hematopoietic stem cells (ST-HSCs) and multipotent progenitors (MMPs) pool. We found that during homeostasis, miR-199b-null HSCs have reduced capacity to maintain quiescent state and exhibit cell-cycle deregulation. Cell cycle analyses showed that attenuation of miR-199b controls HSCs pool, causing defects in G1-S transition of cell cycle, without significant changes in apoptosis. This might be due to increased differentiation of LT-HSCs into MPPs. Indeed, cell differentiation assay in vitro showed that FACS-sorted LT-HSCs (LineagenegSca1posc-Kitpos CD48neg CD150pos) lacking miR-199b have increased differentiation potential into MPP in the presence of early cytokines. In addition, differentiation assays in vitro in FACS-sorted LSK population of 52 weeks old miR-199b KO mice revealed that loss of miR-199b promotes accumulation of GMP-like progenitors but decreases lymphoid differentiation, suggesting that miR199b may regulate age-related pathway. We used non-competitive repopulation studies to show that overall BM donor cellularity was markedly elevated in the absence of miR-199b among HSPCs, committed progenitors and mature myeloid but not lymphoid cell compartments. This may suggest that miR-199b-null LT-HSC render enhanced self-renewal capacity upon regeneration demand yet promoting myeloid reconstitution. Moreover, when we challenged the self-renewal potential of miR-199b-null LT-HSC by a secondary BM transplantation of unfractionated BM cells from primary recipients into secondary hosts, changes in PB reconstitution were dramatic. Gating for HSPCs populations in the BM of secondary recipients in 24 weeks after BMT revealed that levels of LT-HSC were similar between recipients reconstituted with wild-type and miR-199b-KO chimeras, whereas miR-199b-null HSCs contributed relatively more into MPPs. Our data identify that attenuation of miR-199b leads to loss of quiescence and premature differentiation of HSCs. These findings indicate that loss of miR-199b promotes signals that govern differentiation of LT-HSC to MPP leading to accumulation of highly proliferative progenitors during long-term reconstitution. Hematopoietic regeneration via repopulation studies also revealed that miR-199b-deficient HSPCs have a lineage skewing potential toward myeloid lineage or clonal myeloid bias, a hallmark of aging HSCs, implicating a regulatory role for miR-199b in hematopoietic aging. Disclosures No relevant conflicts of interest to declare.

Enhanced Self-Renewal of Hematopoietic Stem Cells Mediated by the Polycomb Gene Product, Bmi-1.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1686-1686
Author(s):  
Hideyuki Oguro ◽  
Atsushi Iwama ◽  
Hiromitsu Nakauchi
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Target Genes ◽  
Ex Vivo ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Ex Vivo Culture ◽  
Deficient Mice

Abstract The Polycomb group (PcG) proteins form multiprotein complexes that play an important role in the maintenance of transcriptional repression of target genes. Loss-of-function analyses show abnormal hematopoiesis in mice deficient for PcG genes including Bmi-1, Mph-1/Rae28, M33, Mel-18, and Eed, suggesting involvement of PcG complexes in the regulation of hematopoiesis. Among them, Bmi-1 has been implicated in the maintenance of hematopoietic and leukemic stem cells. In this study, detailed RT-PCR analysis of mouse hematopoietic cells revealed that all PcG genes encoding components of the Bmi-1-containing complex, such as Bmi-1, Mph1/Rae28, M33, and Mel-18 were highly expressed in CD34−c-Kit+Sca-1+Lin− (CD34−KSL) hematopoietic stem cells (HSCs) and down-regulated during differentiation in the bone marrow. These expression profiles support the idea of positive regulation of HSC self-renewal by the Bmi-1-containing complex. To better understand the role of each component of the PcG complex in HSC and the impact of forced expression of PcG genes on HSC self-renewal, we performed retroviral transduction of Bmi1, Mph1/Rae28, or M33 in HSCs followed by ex vivo culture. After 14-day culture, Bmi-1-transduced but not Mph1/Rae28-transduced cells contained numerous high proliferative potential-colony forming cells (HPP-CFCs), and presented an 80-fold expansion of colony-forming unit-neutrophil/macrophage/Erythroblast/Megakaryocyte (CFU-nmEM) compared to freshly isolated CD34−KSL cells. This effect of Bmi-1 was comparable to that of HoxB4, a well-known HSC activator. In contrast, forced expression of M33 reduced proliferative activity and caused accelerated differentiation into macrophages, leaving no HPP-CFCs after 14 days of ex vivo culture. To determine the mechanism that leads to the drastic expansion of CFU-nmEM, we employed a paired daughter cell assay to see if overexpression of Bmi-1 promotes symmetric HSC division in vitro. Forced expression of Bmi-1 significantly promoted symmetrical cell division of daughter cells, suggesting that Bmi-1 contributes to CFU-nmEM expansion by promoting self-renewal of HSCs. Furthermore, we performed competitive repopulation assays using transduced HSCs cultured ex vivo for 10 days. After 3 months, Bmi-1-transduced HSCs manifested a 35-fold higher repopulation unit (RU) compared with GFP controls and retained full differentiation capacity along myeloid and lymphoid lineages. As expected from in vitro data, HSCs transduced with M33 did not contribute to repopulation at all. In ex vivo culture, expression of both p16INK4a and p19ARF were up-regulated. p16INK4aand p19ARF are known target genes negatively regulated by Bmi-1, and were completely repressed by transducing HSCs with Bmi-1. Therefore, we next examined the involvement of p19ARF in HSC regulation by Bmi-1 using p19ARF-deficient and Bmi-1 and p19ARF-doubly deficient mice. Although bone marrow repopulating activity of p19ARF-deficient HSCs was comparable to that of wild type HSCs, loss of p19ARF expression partially rescued the defective hematopoietic phenotypes of Bmi-1-deficient mice. In addition, transduction of Bmi-1 into p19ARF-deficient HSCs again enhanced repopulating capacity compared with p19ARF-deficient GFP control cells, indicating the existence of additional targets for Bmi-1 in HSCs. Our findings suggest that the level of Bmi-1 is a critical determinant for self-renewal of HSC and demonstrate that Bmi-1 is a novel target for therapeutic manipulation of HSCs.

Zeb2-Defficiency in the Adult Murine Hematopoietic Precursor Cells Leads to Differentiation Defects in Multiple Hematopoietic Lineages and a Myeloproliferative-Like Phenotype

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1199-1199
Author(s):  
Tamara Riedt ◽  
Steven Goossens ◽  
Ines Gütgemann ◽  
Carmen Carrillo-Garcia ◽  
Hichem D Gallala ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Hematopoietic Stem Cells ◽  
Hematopoietic Cells ◽  
Extramedullary Hematopoiesis ◽  
Independent Effect ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Reticular Fibers

Abstract Abstract 1199 The life long replenishment of highly specialized blood cells by a small number of hematopoietic cells (HSC) requires a strict regulation between self-renewal and differentiation in the immature compartment of the bone marrow. Perturbation of this equilibrium can result in stem cell loss or hematologic malignancies. This balance is at least in part controlled by a network of transcription factors. Zeb2 is a transcriptional repressor and plays an important role during the embryonic development as a modulator of the epithelial to mesenchymal transition (EMT) as well as tumor progression and metastasis. We have previously identified the essential role of Zeb2 in murine embryonic hematopoiesis, where selective Zeb2 deficiency in the hematopoietic stem cells resulted in early lethality around day 12.5. The aim of this study was to analyze whether Zeb2 plays a specific role in the regulation of homeostasis in the adult hematopoietic system. Using the Mx1-Cre based inducible Zeb2 conditional knock out mouse model we analyzed the impact of Zeb2 loss on adult hematopoietic stem cell function. Upon the induction of Zeb2 deletion we found a significant decrease in most cell lineages of the peripheral blood, except the neutrophil granulocytes. However, the reduction of mature cells in the blood was not accompanied by reduced bone marrow cellularity, as the cellularity was similar between Zeb2Δ/Δ Mx1-Cre (Zeb2 conditional KO) mice and the control animals (Zeb2+/+Mx1-Cre). However, in the bone marrow of the Zeb2Δ/Δ Mx1-Cre animals the granulocytic lineage was dominating, whereas other lineages e.g. red blood cell precursors and B-lymphoid precursors were drastically reduced. Histological sections of the bone marrow cavity revealed megacaryocytes with abnormal morphology reflecting maturation defects and an increased production of reticular fibers in the BM of Zeb2Δ/Δ Mx1-Cre mice. In addition Zeb2Δ/Δ Mx1-Cre mice displayed a two to three fold increase in spleen size compared to control animals due to an extramedullary hematopoiesis. Analysis of the primitive hematopoietic compartment in the bone marrow and spleens revealed that Zeb2 deletion resulted in a pronounced increase in the most immature hematopoietic cells, defined as Lin-Sca1+cKit+CD48-CD150+ population, and perturbation in different lineage restricted progenitor subpopulations. No difference in cell cycling or apoptotic rate in the stem cell enriched bone marrow population (Lin-Sca1+cKit+CD48-CD150+) was detectable between the genotypes. Upon transplantation into lethally irradiated wild type recipients, Zeb2 deficient stem cells demonstrated significantly reduced ability to differentiate into multiple hematopoietic lineages indicating a niche independent effect of Zeb2 in promoting differentiation of hematopoietic stem cells. On the molecular level, gene expression analysis of hematopoietic stem and progenitor cells using microarray approach revealed increased transcripts of downstream targets of Wnt/ß-Catenin signaling, suggesting increased Wnt signaling activity in absence of Zeb2 in the hematopoietic compartment, which at least in part might be responsible for the observed phenotype. These data indicate that Zeb2 is involved in the regulation of the balance between self-renewal and differentiation at multiple stages of hematopoietic cell maturation. Furthermore the lack of Zeb2 in the hematopoietic compartment leads to a phenotype that resembles the features of human myeloproliferative disorders, especially the early stages of primary myelofibrosis with dominant granulopoiesis, production of reticular fibers in the bone marrow, and morphological abnormalities in megacaryocytes, accompanied by extramedullary hematopoiesis. Disclosures: No relevant conflicts of interest to declare.

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