RUNX1 mutations enhance self-renewal and block granulocytic differentiation in human in vitro models and primary AMLs

Abstract To unravel molecular mechanisms by which Runt-related transcription factor 1 (RUNX1) mutations contribute to leukemic transformation, we introduced the RUNX1-S291fs300X mutation in human CD34+ stem/progenitor cells and in human induced pluripotent stem cells (iPSCs). In both models, RUNX1mut overexpression strongly impaired myeloid commitment. Instead, self-renewal was enhanced, as shown, by increased long-term culture-initiating cell frequencies and enhanced colony-forming cell replating capacity. Long-term suspension cultures with RUNX1mut-transduced cord blood (CB) CD34+ cells continued for more than 100 days, during which the cells displayed an immature granulocyte-macrophage progenitor-like CD34+/CD123+/CD45RA+ phenotype. The CD34+/CD38− hematopoietic stem cell (HSC) population most likely acted as cell of origin, as HSCs provided the best long-term proliferative potential on overexpression of RUNX1mut. CEBPA expression was reduced in RUNX1mut cells, and reexpression of CEBPA partly restored differentiation. RNA-seq analysis on CB/iPSC systems and on primary patient samples confirmed that RUNX1 mutations induce a myeloid differentiation block, and that a common set of RUNX1mut-upregulated target genes was strongly enriched for gene ontology terms associated with nucleosome assembly and chromatin structure. Interestingly, in comparison with AML1-ETO binding in acute myeloid leukemias (AMLs), we found significantly distinct genomic distribution and differential expression for RUNX1mut of genes such as TCF4, MEIS1, and HMGA2 that may potentially contribute to the underlying difference in clinical outcomes between RUNX1mut and AML1-ETO patients. In conclusion, RUNX1mut appears to induce a specific transcriptional program that contributes to leukemic transformation.

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Isolation in a single step of a highly enriched murine hematopoietic stem cell population with competitive long-term repopulating ability

Blood ◽

10.1182/blood.v74.3.930.930 ◽

1989 ◽

Vol 74 (3) ◽

pp. 930-939 ◽

Cited By ~ 49

Author(s):

SJ Szilvassy ◽

PM Lansdorp ◽

RK Humphries ◽

AC Eaves ◽

CJ Eaves

Keyword(s):

Simple Procedure ◽

Hematopoietic Cells ◽

Stem Cell Population ◽

Proliferative Potential ◽

Vitro Assay ◽

Hematopoietic Stem ◽

Marrow Cells

Abstract A simple procedure is described for the quantitation and enrichment of murine hematopoietic cells with the capacity for long-term repopulation of lymphoid and myeloid tissues in lethally irradiated mice. To ensure detection of the most primitive marrow cells with this potential, we used a competitive assay in which female recipients were injected with male “test” cells and 1 to 2 x 10(5) “compromised” female marrow cells with normal short-term repopulating ability, but whose long-term repopulating ability had been reduced by serial transplantation. Primitive hematopoietic cells were purified by flow cytometry and sorting based on their forward and orthogonal light-scattering properties, and Thy-1 and H-2K antigen expression. Enrichment profiles for normal marrow, and marrow of mice injected with 5-fluorouracil (5- FU) four days previously, were established for each of these parameters using an in vitro assay for high proliferative potential, pluripotent colony-forming cells. When all four parameters were gated simultaneously, these clonogenic cells were enriched 100-fold. Both day 9 and day 12 CFU-S were copurified; however, the purity (23%) and enrichment (75-fold) of day 12 CFU-S in the sorted population was greater with 5-FU-treated cells. Five hundred of the sorted 5-FU marrow cells consistently repopulated recipient lymphoid and myeloid tissues (greater than 50% male, 1 to 3 months post-transplant) when co-injected with 1 to 2 x 10(5) compromised female marrow cells, and approximately 100 were sufficient to achieve the same result in 50% of recipients under the same conditions. This relatively simple purification and assay strategy should facilitate further analysis of the heterogeneity and regulation of stem cells that maintain hematopoiesis in vivo.

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Sox2 controls neural stem cell self-renewal through a Fos-centered gene regulatory network

10.1101/2020.03.17.995621 ◽

2020 ◽

Author(s):

Miriam Pagin ◽

Simone Giubbolini ◽

Cristiana Barone ◽

Gaia Sambruni ◽

Yanfen Zhu ◽

...

Keyword(s):

Molecular Mechanisms ◽

Cytokine Signaling ◽

Normal Brain ◽

Suppressor Of Cytokine Signaling ◽

Self Renewal ◽

Upstream Regulator ◽

Socs3 Expression ◽

Pharmacological Manipulations

AbstractThe Sox2 transcription factor is necessary for the long-term self-renewal of neural stem cells (NSC). Its mechanism of action is still poorly defined. To identify molecules regulated by Sox2, and acting in mouse NSC maintenance, we transduced, individually or in combination, into Sox2-deleted NSC, genes whose expression is strongly downregulated following Sox2 loss (Fos, Jun, Egr2). Fos alone rescued long-term proliferation, as shown by in vitro cell growth and clonal analysis. Further, Fos requirement for efficient long-term proliferation was demonstrated by the strong reduction of NSC clones capable of long-term expansion following CRISPR/Cas9-mediated Fos inactivation. Previous work showed that the Suppressor of cytokine signaling 3 (Socs3) gene is strongly downregulated following Sox2 deletion, and its reexpression by lentiviral transduction rescues long-term NSC proliferation. Fos appears to be an upstream regulator of Socs3, possibly together with Jun and Egr2; indeed, Sox2 reexpression in Sox2-deleted NSC progressively activates both Fos and Socs3 expression; in turn, Fos transduction activates Socs3 expression. Based on available SOX2 ChIPseq and ChIA-PET data, as well as results from the literature, we propose a model whereby Sox2 is a direct activator of both Socs3 and Fos, as well as possibly Jun and Egr2; in turn, Fos, Jun and Egr2 may activate Socs3. These results provide the basis for developing a model of a network of interactions, regulating critical effectors of NSC proliferation and long-term maintenance.Significance statementProliferation and maintenance of NSC are essential during normal brain development, and, postnatally, for the maintenance of hippocampal function and memory until advanced age. Little is known about the molecular mechanisms that maintain the critical aspects of NSC biology (quiescence and proliferation) in postnatal age. Our work provides a methodology, transduction of genes deregulated following Sox2 deletion, that allows to test many candidate genes for their ability to sustain NSC proliferation. In principle, this may have interesting implications for identifying targets for pharmacological manipulations.

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Hematopoietic stem/progenitor cells, generation of induced pluripotent stem cells, and isolation of endothelial progenitors from 21- to 23.5-year cryopreserved cord blood

Blood ◽

10.1182/blood-2011-01-330514 ◽

2011 ◽

Vol 117 (18) ◽

pp. 4773-4777 ◽

Cited By ~ 115

Author(s):

Hal E. Broxmeyer ◽

Man-Ryul Lee ◽

Giao Hangoc ◽

Scott Cooper ◽

Nutan Prasain ◽

...

Keyword(s):

Stem Cells ◽

Cord Blood ◽

Progenitor Cells ◽

Proliferative Potential ◽

Hematopoietic Stem ◽

Immunodeficient Mice ◽

Induced Pluripotent

Abstract Cryopreservation of hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs) is crucial for cord blood (CB) banking and transplantation. We evaluated recovery of functional HPC cryopreserved as mononuclear or unseparated cells for up to 23.5 years compared with prefreeze values of the same CB units. Highly efficient recovery (80%-100%) was apparent for granulocyte-macrophage and multipotential hematopoietic progenitors, although some collections had reproducible low recovery. Proliferative potential, response to multiple cytokines, and replating of HPC colonies was extensive. CD34+ cells isolated from CB cryopreserved for up to 21 years had long-term (≥ 6 month) engrafting capability in primary and secondary immunodeficient mice reflecting recovery of long-term repopulating, self-renewing HSCs. We recovered functionally responsive CD4+ and CD8+ T lymphocytes, generated induced pluripotent stem (iPS) cells with differentiation representing all 3 germ cell lineages in vitro and in vivo, and detected high proliferative endothelial colony forming cells, results of relevance to CB biology and banking.

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Interleukin-3 supports expansion of long-term multilineage repopulating activity after multiple stem cell divisions in vitro

Blood ◽

10.1182/blood.v96.5.1748 ◽

2000 ◽

Vol 96 (5) ◽

pp. 1748-1755 ◽

Cited By ~ 90

Author(s):

David Bryder ◽

Sten E. W. Jacobsen

Keyword(s):

Bone Marrow Cells ◽

Ex Vivo ◽

Calf Serum ◽

Self Renewal ◽

Hematopoietic Stem ◽

Cell Divisions ◽

Stem Cell Divisions

Abstract Although long-term repopulating hematopoietic stem cells (HSC) can self-renew and expand extensively in vivo, most efforts at expanding HSC in vitro have proved unsuccessful and have frequently resulted in compromised rather than improved HSC grafts. This has triggered the search for the optimal combination of cytokines for HSC expansion. Through such studies, c-kit ligand (KL), flt3 ligand (FL), thrombopoietin, and IL-11 have emerged as likely positive regulators of HSC self-renewal. In contrast, numerous studies have implicated a unique and potent negative regulatory role of IL-3, suggesting perhaps distinct regulation of HSC fate by different cytokines. However, the interpretations of these findings are complicated by the fact that different cytokines might target distinct subpopulations within the HSC compartment and by the lack of evidence for HSC undergoing self-renewal. Here, in the presence of KL+FL+megakaryocyte growth and development factor (MGDF), which recruits virtually all Lin−Sca-1+kit+ bone marrow cells into proliferation and promotes their self-renewal under serum-free conditions, IL-3 and IL-11 revealed an indistinguishable ability to further enhance proliferation. Surprisingly, and similar to IL-11, IL-3 supported KL+FL+MGDF-induced expansion of multilineage, long-term reconstituting activity in primary and secondary recipients. Furthermore, high-resolution cell division tracking demonstrated that all HSC underwent a minimum of 5 cell divisions, suggesting that long-term repopulating HSC are not compromised by IL-3 stimulation after multiple cell divisions. In striking contrast, the ex vivo expansion of murine HSC in fetal calf serum-containing medium resulted in extensive loss of reconstituting activity, an effect further facilitated by the presence of IL-3.

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Maximal STAT5-Induced Proliferation and Self-Renewal at Intermediate STAT5 Activity Levels

Molecular and Cellular Biology ◽

10.1128/mcb.01025-08 ◽

2008 ◽

Vol 28 (21) ◽

pp. 6668-6680 ◽

Cited By ~ 61

Author(s):

Albertus T. J. Wierenga ◽

Edo Vellenga ◽

Jan Jacob Schuringa

Keyword(s):

Gene Expression ◽

Cell Fate ◽

Target Genes ◽

Expression Patterns ◽

Activity Levels ◽

Dependent Manner ◽

Self Renewal ◽

Hematopoietic Stem ◽

Cell Fate Decisions

ABSTRACT The level of transcription factor activity critically regulates cell fate decisions, such as hematopoietic stem cell (HSC) self-renewal and differentiation. We introduced STAT5A transcriptional activity into human HSCs/progenitor cells in a dose-dependent manner by overexpression of a tamoxifen-inducible STAT5A(1*6)-estrogen receptor fusion protein. Induction of STAT5A activity in CD34+ cells resulted in impaired myelopoiesis and induction of erythropoiesis, which was most pronounced at the highest STAT5A transactivation levels. In contrast, intermediate STAT5A activity levels resulted in the most pronounced proliferative advantage of CD34+ cells. This coincided with increased cobblestone area-forming cell and long-term-culture-initiating cell frequencies, which were predominantly elevated at intermediate STAT5A activity levels but not at high STAT5A levels. Self-renewal of progenitors was addressed by serial replating of CFU, and only progenitors containing intermediate STAT5A activity levels contained self-renewal capacity. By extensive gene expression profiling we could identify gene expression patterns of STAT5 target genes that predominantly associated with a self-renewal and long-term expansion phenotype versus those that identified a predominant differentiation phenotype.

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Induction of Hematopoietic Stem Cell Senescence by Ionizing Radiation.

Blood ◽

10.1182/blood.v106.11.1362.1362 ◽

2005 ◽

Vol 106 (11) ◽

pp. 1362-1362

Author(s):

Yong Wang ◽

Bradley A. Schulte ◽

Amanda C. LaRue ◽

Makio Ogawa ◽

Daohong Zhou

Keyword(s):

Stem Cell ◽

Ionizing Radiation ◽

Hematopoietic Stem Cell ◽

Cellular Senescence ◽

Molecular Mechanisms ◽

Chemotherapeutic Agents ◽

Sublethal Dose ◽

Self Renewal ◽

Hematopoietic Stem

Abstract Exposure to ionizing radiation (IR) and certain chemotherapeutic agents not only causes acute bone marrow (BM) suppression but also leads to long-term residual hematopoietic injury. This later effect has been attributed to the damage to hematopoietic stem cell (HSC) self-renewal. Using a mouse model, we investigated whether IR induces senescence in HSCs, as induction of HSC senescence can lead to the impairment of HSC self-renewal. The results showed that exposure of C57BL/6 mice to a sublethal dose (6.5 Gy) of total body irradiation (TBI) resulted in a long-lasting quantitative and qualitative reduction in HSCs (Lin− c-kit+ Sca-1+ or LKS+ cells). Compared to control HSCs, HSCs from irradiated BM at 4 weeks after TBI exhibited a significant reduction in day-35 CAFC frequency and deficiency in cell proliferation and colony formation in a single cell culture assay stimulated with SCF/TPO and SCF/TPO/IL-3, respectively. In addition, transplantation of irradiated HSCs (500 LKS+ cells/recipient) produced less than 1% long-term (2-month) engraftment in a competitive repopulation assay while transplantation of the same number of control HSCs resulted in 24.8% engraftment. Furthermore, HSCs from irradiated mice expressed increased levels of p16Ink4a and senescence-associated beta-galactosidase (SA-beta-gal), two commonly used biomarkers of cellular senescence. In contrast, hematopoietic progenitor cells (Lin− c-kit+ Sca-1− or LKS− cells) from irradiated mice did not show significant changes in clonogenesity in a CFU assay and expressed minimal levels of p16Ink4a and SA-beta-gal. These results suggest that exposure to IR can induce senescence selectively in HSCs but not in HPCs. Interestingly, this IR- induced HSC senescence was associated with a prolonged elevation of p21Cip1/Waf1, p16Ink4a and p19ARF mRNA expression, whereas the expression of p27Kip1, p18Ink4c and p19 Ink4d mRNA was not increased. This suggests that p21Cip1/Waf1, p16Ink4a and p19ARF may play an important role in IR-induced senescence in HSCs, since their expression has been implicated in the initiation, establishment and maintenance of cellular senescence. Therefore, these findings provide valuable insights into the mechanisms underlying IR-induced long-term BM damage. This could lead to the discovery of novel molecular targets for intervention to circumvent IR-induced BM toxicity. In addition, understanding how normal HSCs senesce after IR and chemotherapy will help us to elucidate the molecular mechanisms whereby leukemia/cancer stem cells evade these cancer treatments and provide better knowledge of organismal aging.

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Inability to Express HOXA Cluster and BCL11A Genes Compromises Self-Renewal and Multipotency of hESC-Derived Hematopoietic Cells

Blood ◽

10.1182/blood.v120.21.1190.1190 ◽

2012 ◽

Vol 120 (21) ◽

pp. 1190-1190 ◽

Cited By ~ 1

Author(s):

Diana R Dou ◽

Arazin Minasian ◽

Maria I Sierra ◽

Pamela Saarikoski ◽

Jian Xu ◽

...

Keyword(s):

Stem Cells ◽

B Cells ◽

Proliferative Potential ◽

Pluripotent Cells ◽

Hoxa Cluster ◽

Self Renewal ◽

Hematopoietic Stem ◽

Cd34 Cells ◽

Hoxa Genes

Abstract Abstract 1190 The inability to derive functional hematopoietic stem cells (HSCs) in vitro from pluripotent cells prevents widespread utilization of HSCs in the clinic; however, the molecular defects compromising the in vitro generated hematopoietic stem/progenitor cells (HSPCs) are unknown. Using a two-step differentiation method in which human embryonic stem cells (hESCs) were first differentiated into embryo bodies (EBs) and then CD34+ cells from hEBs were co-cultured on OP9M2 bone marrow mesenchymal stem cell (MSC) stroma (hEB-OP9), we were able to derive HSPCs expressing the HSC immunophenotype (CD34+CD38−CD90+CD45+) (hereafter termed CD90+HSPCs). Colony forming and stroma co-culture assays demonstrated that the hEB-OP9 CD90+HSPCs were able to differentiate into myelo-erythroid lineages and T-cells. However, when comparing CD90+HSPCs from hEB-OP9 to those from fetal liver (FL)—an in vivo source of HSCs—the former remained severely functionally limited in their proliferative potential and ability to differentiate into B-cells. To identify the basis of the proliferative and differentiation defects, we performed microarray analysis to define gene expression differences between CD90+HSPCs derived from hEB-OP9, FL, early 3–5 week placenta (PL) and an earlier stage of hESC differentiation (hEB). This analysis revealed establishment of the general hematopoietic transcription factor network (e.g. SCL, RUNX1, CMYB, ETV6, HOXB4, MYB), demonstrating the successful differentiation and identification of hematopoietic cells using our two-step culturing techniques and immunophenotype criteria. Moreover, evaluation of Spearman coefficients confirmed CD90+HSPCs isolated from hEB-OP9 culture were brought into closer resemblance of the hFL CD90+HSPCs as compared to to the developmentally immature hEB and hPL CD90+HSPCs. Encouragingly, hEB-OP9 CD90+HSPCs displayed downregulation of expression of genes related to hemogenic endothelium development associated with hEB and hPL while genes critical in HSPC function, including DNA repair and chromatin modification, were upregulated to levels comparable to hFL-HSPCs. However, a subgroup of FL HSPC genes could not be induced in hEB-OP9 HSPCs, including the HOXA cluster genes and BCL11A—implicated in HSC self-renewal and B-cell formation, respectively. Interestingly, absence of HOXA genes and BCL11A and poor proliferative potential were also observed in HSPCs from early placenta, suggesting these defects are not in vitro artifacts but instead reflect an inability of hEB-OP9 HSPCs to complete developmental maturation. To validate the necessity of HOXA genes and BCL11A in proliferation potential and multipotency, we next utilized shRNAs to target MLL—the upstream regulator of the HOXA cluster—, individual HOXA genes, or BCL11A in FL-HSPCs to test whether knockdown was sufficient to recapitulate the defects observed in hESC-derived HSPCs. Knockdown of HOXA7 resulted in the loss of CD34+ cells while HOXA9 shRNA-treated cells displayed a loss of more differentiated CD38hi cells. MLL knockdown depleted both CD38+ and CD34+ populations. BCL11A silencing resulted in the loss of B-cells. These studies identify HOXA genes and BCL11A as developmentally regulated genes essential for generating self-renewing, multipotent HSCs from pluripotent cells. Disclosures: No relevant conflicts of interest to declare.

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MiR-29a Maintains Hematopoietic Stem Cell Self-Renewal and Is Required For Myeloid Leukemogenesis

Blood ◽

10.1182/blood.v122.21.1190.1190 ◽

2013 ◽

Vol 122 (21) ◽

pp. 1190-1190

Author(s):

Wenhuo Hu ◽

James Dooley ◽

Stephen S. Chung ◽

Safak Yalcin ◽

Yu Sup Shin ◽

...

Keyword(s):

Stem Cell ◽

Colony Formation ◽

Target Genes ◽

Conflicts Of Interest ◽

Ectopic Expression ◽

Null Mouse ◽

Cell Cycle Regulators ◽

Self Renewal ◽

Hematopoietic Stem

Abstract microRNAs (miRNAs) are important regulators of both embryonic and adult tissue stem cell self-renewal. We previously showed that ectopic expression of miR-29a, a miRNA highly expressed in HSCs as well as in human acute myeloid leukemia (AML) stem cells, in immature mouse hematopoietic cells is sufficient to induce a myeloproliferative disorder that progresses to AML. During the early phase of this disease, miR-29a induces aberrant self-renewal of committed myeloid progenitors, strongly suggesting a role for miR-29a in regulating HSC self-renewal. In order to determine the role of miR-29a in HSC function, we have evaluated our recently described miR-29a/b1 null mouse. Homozygous deletion of miR-29a/b1 resulted in reduced bone marrow cellularity and reduced colony forming capacity of hematopoietic stem and progenitor cells (HSPCs). The phenotype was mediated specifically by miR-29a since miR-29b expression was not significantly altered in HSCs and reconstitution of miR-29a/b1 null HSPCs with miR-29a, but not miR-29b, rescued in vitro colony formation defects. Self-renewal defects were observed in miR-29a deficient HSCs in both competitive and non-competitive transplantation assays, and these deficits were associated with increased HSC cell cycling and apoptosis. Gene expression studies of miR-29a deficient HSCs demonstrated widespread gene dysregulation including a number of up-regulated miR-29a target genes including DNA methylation enzymes (Dnmt3a, -3b) and cell cycle regulators (e.g. Cdk6, Tcl1, Hbp1, Pten). Knockdown of one of these targets, Dnmt3a, in miR-29a deficient HSCs resulted in partial restoration of colony formation, providing functional validation that Dnmt3a mediates part of miR-29a null HSPCs functional defects. miR-29a loss also abrogated leukemogenesis in the MLL-AF9 retroviral AML model. Together, our results demonstrate that miR-29a positively regulates HSC self-renewal and is required for myeloid leukemogenesis. Disclosures: No relevant conflicts of interest to declare.

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Interleukin-3 supports expansion of long-term multilineage repopulating activity after multiple stem cell divisions in vitro

Blood ◽

10.1182/blood.v96.5.1748.h8001748_1748_1755 ◽

2000 ◽

Vol 96 (5) ◽

pp. 1748-1755 ◽

Cited By ~ 5

Author(s):

David Bryder ◽

Sten E. W. Jacobsen

Keyword(s):

Bone Marrow Cells ◽

Ex Vivo ◽

Calf Serum ◽

Self Renewal ◽

Hematopoietic Stem ◽

Cell Divisions ◽

Stem Cell Divisions

Although long-term repopulating hematopoietic stem cells (HSC) can self-renew and expand extensively in vivo, most efforts at expanding HSC in vitro have proved unsuccessful and have frequently resulted in compromised rather than improved HSC grafts. This has triggered the search for the optimal combination of cytokines for HSC expansion. Through such studies, c-kit ligand (KL), flt3 ligand (FL), thrombopoietin, and IL-11 have emerged as likely positive regulators of HSC self-renewal. In contrast, numerous studies have implicated a unique and potent negative regulatory role of IL-3, suggesting perhaps distinct regulation of HSC fate by different cytokines. However, the interpretations of these findings are complicated by the fact that different cytokines might target distinct subpopulations within the HSC compartment and by the lack of evidence for HSC undergoing self-renewal. Here, in the presence of KL+FL+megakaryocyte growth and development factor (MGDF), which recruits virtually all Lin−Sca-1+kit+ bone marrow cells into proliferation and promotes their self-renewal under serum-free conditions, IL-3 and IL-11 revealed an indistinguishable ability to further enhance proliferation. Surprisingly, and similar to IL-11, IL-3 supported KL+FL+MGDF-induced expansion of multilineage, long-term reconstituting activity in primary and secondary recipients. Furthermore, high-resolution cell division tracking demonstrated that all HSC underwent a minimum of 5 cell divisions, suggesting that long-term repopulating HSC are not compromised by IL-3 stimulation after multiple cell divisions. In striking contrast, the ex vivo expansion of murine HSC in fetal calf serum-containing medium resulted in extensive loss of reconstituting activity, an effect further facilitated by the presence of IL-3.

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Microrna 199b Regulates Mouse Hematopoietic Stem Cells Maintenance

Blood ◽

10.1182/blood-2019-126283 ◽

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.

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