Hoxa9/Meis1 Mediate Leukemic Programming through Microrna-155

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 884-884
Author(s):  
Edith Schneider ◽  
Anna Staffas ◽  
Milijana Mirkovic-Hoesle ◽  
Bernhard Gentner ◽  
Jens Ruschmann ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Colony Formation ◽  
Healthy Donor ◽  
Molecular Mechanisms ◽  
Acute Leukemias ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Donor Bone Marrow

Abstract Synergistic deregulation of HOXA9 and the HOX-gene cofactor MEIS1 is a commonly observed phenomenon in acute myeloid leukemia (AML). The leukemogenic potential of aberrant Hoxa9 and Meis1 expression has been shown in several AML models. However, the molecular mechanisms behind Hoxa9- and Meis1-induced leukemogenesis are still not well understood. In order to identify functionally relevant Meis1-induced microRNAs (miRNA), we profiled the global miRNA expression using a Hoxa9-Meis1 murine AML progression model. This two-step model allowed us to quantify miRNAs at a pre-leukemic stage through the overexpression of the proto-oncogene Hoxa9 (Hoxa9/ctrl), as well as after full leukemic transformation through co-overexpression of Hoxa9 and Meis1 (Hoxa9/Meis1). The pre-leukemic stage is characterized by in vitro immortalization without in vivo engraftment, whereas the transplanted leukemic cells induce full-blown AML in vivo. MiR-155 turned out to be one of the most significant differentially expressed miRNA species and its upregulation was independently validated in Hoxa9/Meis1 cells by qRT-PCR. Subsequent analysis of various AML subtypes (CN-AML, t(11q23), t(8;21), t(15;17), n=38) showed significantly elevated levels of miR-155 in CN-AML with NPM1mut (n=10, p<0.01) and AML with t(11q23) (n=8, p<0.05) compared to healthy donor bone marrow (MNC). These results are in line with overexpression of HOXA9 (CN-AML NPM1mut: p<0.05, t(11q23): p<0.05) and MEIS1 (CN-AML NPM1mut: p<0.01, t(11q23): p<0.05) in these AML samples compared to healthy donor bone marrow cells (MNC). Expression analysis of miR-155 in healthy murine bone marrow (mbm) cells revealed miR-155 enrichment in hematopoietic stem- and progenitor cells compared to mature myeloid cells (p<0.05), mirroring a similar expression pattern as observed for Meis1. Therefore, to dissect the leukemic potential of miR-155 to program mbm, 5-FU-stimulated mbm cells were retrovirally transduced with miR-155, leading to significantly increased proliferation in vitro (p<0.05). This finding suggests enhancement of self-renewal on the stem-/progenitor cell level by miR-155. Furthermore, mbm cells overexpressing Hoxa9 together with miR-155 (Hoxa9/miR-155) significantly increased colony formation (p<0.05) in a methylcellulose assay. In turn, absence of miR-155 (miR-155-/- mbm) significantly reduced colony formation in conjunction with Hoxa9 (p<0.05) and MLL-AF9 (p=0.05), a known positive regulator of Hoxa9 and Meis1. These findings suggest a role for miR-155 in both proliferation and self-renewal indicating that the oncogenic program of Hoxa9/Meis1 relies on the presence of miR-155. The leukemic potency of Hoxa9/miR-155 was further investigated in a murine transplantation model in vivo. Transplantation of mbm co-overexpressing Hoxa9/miR-155 led to significantly increased engraftment levels already after four weeks (wks) (57.8%±31.3, n=16) compared to Hoxa9/ctrl (11.7%±19.3%, p<0.0001, n=17), but less than with Hoxa9/Meis1 (74.5%±20.3%, p<0.01, n=14). In contrast to Hoxa9/ctrl (22±7 wks), mice that received Hoxa9/miR-155 mbm cells had a significantly accelerated onset of a myeloproliferative disease (MPD)-like leukemia within 11 wks (11±6 wks, p<0.0001), but still a less aggressive course of disease compared to mice transplanted with Hoxa9/Meis1 (5±1 wks, p<0.0001). This result is striking considering the aggressive nature of the Hoxa9/Meis1 AML model and given how little is known about its central mechanisms. It also highlights the relevant contribution of miR-155 to the leukemic programming induced by Hoxa9/Meis1 and provides a further rational to target miR-155 in AML. Considering the central role of the Hoxa9/Meis1 in both myeloid and lymphoid acute leukemias, we demonstrate for the first time the leukemogenic relevance of a miRNA within this transcriptional axis. Disclosures No relevant conflicts of interest to declare.

Rapid and Irreversible Alteration of the Ability of Hematopoietic Stem Cells To Execute Both Symmetric and Asymmetric Self-Renewal Divisions by Exposure to Reduced Steel Factor Concentrations with No Effect on Their Survival or Mitogenesis.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 684-684
Author(s):  
David G. Kent ◽  
Brad Dykstra ◽  
Connie J. Eaves
Keyword(s):  
Bone Marrow ◽  
Adult Mouse ◽  
Single Cells ◽  
Mouse Bone Marrow ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Steel Factor ◽  
High Concentrations

Abstract Hematopoietic stem cells (HSCs) are present in the marrow of adult mice at a frequency of 1/104, as measured by limiting dilution transplantation assays for individual cells that produce lymphoid (B and T) as well as myeloid (GM) cells for at least 4 months in irradiated recipients. HSCs thus defined can be reproducibly isolated in the CD45midlin−Rho−SP fraction of adult mouse bone marrow at a purity of &gt;30%. In mice, mutations in c-kit, the receptor for Steel factor (SF) lead to substantial reductions in the adult HSC population. In vitro, SF has been identified as a potent regulator of HSC self-renewal divisions. High concentrations of SF in combination with IL-11 allow adult HSCs to divide with a net 2–4 fold expansion in HSC numbers after 10 days and low concentrations of SF result in loss of HSC activity. To investigate the cellular mechanisms underlying these different outcomes, we cultured 114 CD45midlin−Rho−SP adult mouse bone marrow cells in single cell cultures containing serum-free medium + 20 ng/ml IL-11 and either 300 or 10 ng/ml of SF. Each culture was then examined every 4–6 hr. The kinetics of division of these cells under both conditions was identical with completion of the 1st division occurring between 22–68 hr. During that time none of the input cells died (&lt;1%). After 10 days of culture, during which time all input cells divided at least 5 times (&gt;50 cells), the HSC content of pooled clones (as measured by in vivo transplantation assays) was found to be &gt;10-fold higher in the clones generated under high vs. low SF conditions (p&lt;0.05). To characterize the types of self-renewal divisions undertaken, 9 doublets generated under the high SF condition were harvested between 4 and 8 hr after they underwent their 1st division and then each of the daughters was injected into a separate irradiated mouse. Analysis of the 18 mice showed that for one of the input cells both daughters were HSCs (evidence of a symmetric self-renewal division) and for 3 more, only one of the 2 daughters was an HSC (evidence of an asymmetric self-renewal division). In contrast no daughter HSCs were identified when 6 doublets produced under the low SF condition were assayed. To determine whether the loss of HSC activity under low SF conditions was a pre- or post-mitotic event, additional in vivo HSC assays were performed on cells harvested from individual wells after 8, 16 and 96 hours of incubation. The results revealed no change in the proportion of wells with either low or high concentrations of SF that contained HSCs after 8 hr of incubation (10/36 positive mice injected with starting single cells and 5/17 (low SF) vs. 6/17 (high SF) positive mice injected with 8-hr single cells, respectively). However, a significant difference (p&lt;0.01) was seen after 96 hr (5/35 vs. 2/43 positive mice, respectively) and, after only 16 hr, before a first mitosis was seen under either condition, a decline in HSCs was apparent under the low SF condition (4/15 vs. 1/15 positive mice injected with cells from the high vs. low SF condition). Together, these studies indicate that HSC exposure to different SF concentrations can rapidly and irreversibly alter the ability of HSCs to execute symmetric as well asymmetric self-renewal divisions in vitro.

Cripto Selectively Expands a Distinct Population of Hematopoietic Stem Cells Expressing the Cell Surface Receptor GRP78 and Strongly Induces An Immature Phenotype In Vivo After Ex Vivo Culture

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 405-405
Author(s):  
Kenichi Miharada ◽  
Göran Karlsson ◽  
Jonas Larsson ◽  
Emma Larsson ◽  
Kavitha Siva ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Peripheral Blood ◽  
Distinct Population ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Total Bone Marrow

Abstract Abstract 405 Cripto is a member of the EGF-CFC soluble protein family and has been identified as an important factor for the proliferation/self-renewal of ES and several types of tumor cells. The role for Cripto in the regulation of hematopoietic cells has been unknown. Here we show that Cripto is a potential new candidate factor to increase self-renewal and expand hematopoietic stem cells (HSCs) in vitro. The expression level of Cripto was analyzed by qRT-PCR in several purified murine hematopoietic cell populations. The findings demonstrated that purified CD34-KSL cells, known as highly concentrated HSC population, had higher expression levels than other hematopoietic progenitor populations including CD34+KSL cells. We asked how Cripto regulates HSCs by using recombinant mouse Cripto (rmCripto) for in vitro and in vivo experiments. First we tested the effects of rmCripto on purified hematopoietic stem cells (CD34-LSK) in vitro. After two weeks culture in serum free media supplemented with 100ng/ml of SCF, TPO and 500ng/ml of rmCripto, 30 of CD34-KSL cells formed over 1,300 of colonies, including over 60 of GEMM colonies, while control cultures without rmCripto generated few colonies and no GEMM colonies (p<0.001). Next, 20 of CD34-KSL cells were cultured with or without rmCripto for 2 weeks and transplanted to lethally irradiated mice in a competitive setting. Cripto treated donor cells showed a low level of reconstitution (4–12%) in the peripheral blood, while cells cultured without rmCripto failed to reconstitute. To define the target population and the mechanism of Cripto action, we analyzed two cell surface proteins, GRP78 and Glypican-1, as potential receptor candidates for Cripto regulation of HSC. Surprisingly, CD34-KSL cells were divided into two distinct populations where HSC expressing GRP78 exhibited robust expansion of CFU-GEMM progenitor mediated by rmCripto in CFU-assay whereas GRP78- HSC did not respond (1/3 of CD34-KSL cells were GRP78+). Furthermore, a neutralization antibody for GRP78 completely inhibited the effect of Cripto in both CFU-assay and transplantation assay. In contrast, all lineage negative cells were Glypican-1 positive. These results suggest that GRP78 must be the functional receptor for Cripto on HSC. We therefore sorted these two GRP78+CD34-KSL (GRP78+HSC) and GRP78-CD34-KSL (GRP78-HSC) populations and transplanted to lethally irradiated mice using freshly isolated cells and cells cultured with or without rmCripto for 2 weeks. Interestingly, fresh GRP78-HSCs showed higher reconstitution than GRP78+HSCs (58–82% and 8–40%, p=0.0038) and the reconstitution level in peripheral blood increased rapidly. In contrast, GRP78+HSC reconstituted the peripheral blood slowly, still at a lower level than GRP78-HSC 4 months after transplantation. However, rmCripto selectively expanded (or maintained) GRP78+HSCs but not GRP78-HSCs after culture and generated a similar level of reconstitution as freshly transplanted cells (12–35%). Finally, bone marrow cells of engrafted recipient mice were analyzed at 5 months after transplantation. Surprisingly, GRP78+HSC cultured with rmCripto showed higher reconstitution of the CD34-KSL population in the recipients' bone marrow (45–54%, p=0.0026), while the reconstitution in peripheral blood and in total bone marrow was almost the same. Additionally, most reconstituted CD34-KSL population was GRP78+. Interestingly freshly transplanted sorted GRP78+HSC and GRP78-HSC can produce the GRP78− and GRP78+ populations in the bone marrow and the ratio of GRP78+/− cells that were regenerated have the same proportion as the original donor mice. Compared to cultured cells, the level of reconstitution (peripheral blood, total bone marrow, HSC) in the recipient mice was almost similar. These results indicate that the GRP78 expression on HSC is reversible, but it seems to be “fixed” into an immature stage and differentiate with lower efficiency toward mature cells after long/strong exposure to Cripto signaling. Based on these findings, we propose that Cripto is a novel factor that maintains HSC in an immature state and may be a potent candidate for expansion of a distinct population of GRP78 expressing HSC. Disclosures: No relevant conflicts of interest to declare.

Identification of a Hierarchy of Multipotent Hematopoietic Progenitors in Human Cord Blood.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2237-2237
Author(s):  
Ravindra Majeti ◽  
Christopher Y. Park ◽  
Irving L. Weissman
Keyword(s):  
Bone Marrow ◽  
Cord Blood ◽  
Human Cord Blood ◽  
Self Renewal ◽  
Newborn Mice ◽  
Hematopoietic Stem ◽  
Multipotent Progenitors

Abstract Mouse hematopoiesis is initiated by long-term hematopoietic stem cells (HSC) that differentiate into a series of multipotent progenitors that exhibit progressively diminished self-renewal ability. In human hematopoiesis, populations enriched for HSC have been identified, as have downstream lineage-committed progenitors, but not multipotent progenitors. Previous reports indicate that human HSC are enriched in Lin-CD34+CD38- cord blood and bone marrow, and express CD90. We demonstrate that the Lin-CD34+CD38- fraction of cord blood and bone marrow can be subdivided into three subpopulations: CD90+CD45RA-, CD90-CD45RA-, and CD90-CD45RA+. While, the function of the CD90- subpopulations is unknown, the CD90+CD45RA- subpopulation presumably contains HSC. We report here in vitro and in vivo functional studies of these three subpopulations from normal human cord blood. In vitro, CD90+CD45RA- cells formed all types of myeloid colonies in methylcellulose and were able to replate with 70% efficiency. CD90-CD45RA- cells also formed all types of myeloid colonies, but replated with only 33% efficiency. CD90-CD45RA+ cells failed to form myeloid colonies in methylcellulose. In liquid culture, CD90+CD45RA- cells gave rise to all three subpopulations; CD90-CD45RA- cells gave rise to both CD90- subpopulations, but not CD90+ cells; CD90-CD45RA+ cells gave rise to themselves only. These data establish an in vitro differentiation hierarchy from CD90+CD45RA- to CD90-CD45RA- to CD90-CD45RA+ cells among Lin-CD34+CD38- cord blood. In vivo, xenotransplantation of CD90+CD45RA- cells into NOD/SCID/IL-2R?-null newborn mice resulted in long-term multilineage engraftment with transplantation of as few as 10 purified cells. Secondary transplants from primary engrafted mice also resulted in long-term multilineage engraftment, indicating the presence of self-renewing HSC. Transplantation of CD90-CD45RA- cells also resulted in long-term multilineage engraftment; however, secondary transplants did not reliably result in long-term engraftment, indicating a reduced capacity for self-renewal. Transplantation of CD90-CD45RA+ cells did not result in any detectable human hematopoietic cells, indicating that the function of these cells is undetermined. Finally, transplantation of limiting numbers of CD90-CD45RA- cells (less than 100) resulted in multilineage human engraftment at 4 weeks, that was no longer detectable by 12 weeks. Thus, the CD90-CD45RA- subpopulation is capable of multilineage differentiation while exhibiting limited self-renewal ability. We believe this study represents the first prospective identification of a population of human multipotent progenitors, Lin-CD34+CD38-CD90-CD45RA- cord blood.

Investigating the Biological and Molecular Mechanisms Underpinning the Engraftment/Selection Advantage Conferred to Hematopoietic Stem Cells by HOXB4.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2101-2101
Author(s):  
Michael D. Milsom ◽  
Laura Hollins ◽  
Dorothy Gagen ◽  
Lorna B. Woolford ◽  
Leslie J. Fairbairn
Keyword(s):  
Molecular Mechanisms ◽  
Current Knowledge ◽  
Cellular Transformation ◽  
Model Systems ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Increased Risk ◽  
Vitro Analysis

Abstract We have recently demonstrated that co-expression of HOXB4 enables the enhanced delivery of HSC harbouring a second therapeutic trans-gene. Nonetheless, it is of great importance to elaborate the current knowledge about the mechanism of HOXB4 action in order to both evaluate the safety implications of its use in a clinical strategy, and to gain greater insight into the regulation of HSC self-renewal/expansion. To these ends we have performed an extensive in vitro analysis of the consequences of HOXB4 overexpression in primary murine BMC and in a murine multipotent myeloid progenitor cell line (FDCP-mix). We demonstrate for the first time in murine cells, that ectopic HOXB4 reduces the responsiveness of murine hematopoietic cells to differentiation stimuli. Furthermore, by performing a detailed investigation into the kinetics of FDCP-mix differentiation, we reveal that HOXB4 overexpression results in a specific differentiation delay as opposed to an outright block. We propose that an analogous delay is in operation in repopulating cells in order that the shift to increased assymetrical self-renewal, a requirement for stem cell expansion, is achieved. Notwithstanding this, it is clear that any perturbation in differentiation constitutes an increased risk of cellular transformation if this technology were transferred to a clinical setting. In order to further define the repercussions of ectopic HOXB4 delivery, we have developed a retroviral vector which encodes an activatable version of HOXB4. We have shown that this vector is able to mediate an in vitro differentiation delay in primary murine BMC and FDCP-mix as well as enable enhanced engraftment of BMC in vivo, both dependent upon the addition of the estrogen analogue; tamoxifen. Using this system, we are currently examining the effect of ectopic HOXB4 on the transcriptome of FDCP-mix cells, in addition to performing an in depth study into the biological mechanisms affected by HOXB4 overexpression in BMC in vivo. We envisage that these model systems will be particularly amenable to the manipulation required for target gene identification/validation.

Disease Models and Transformation Mechanisms Mediated by MLL-AF4 Family Oncoproteins in Human Leukemia.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 467-467
Author(s):  
Chi Wai Eric So ◽  
Piu Wong ◽  
Min Lin ◽  
Michael L. Cleary
Keyword(s):  
Fusion Proteins ◽  
Molecular Mechanisms ◽  
Disease Models ◽  
Human Leukemia ◽  
Fusion Partner ◽  
Acute Leukemias ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
In Vitro Transformation

Abstract The Mixed Lineage Leukemia (MLL) gene codes for a histone methyltransferase that is required for hematopoietic development. As a consequence of chromosomal translocations, MLL is fused with over 40 different genes to yield in-frame fusion proteins in acute leukemias. AF4, the most common fusion partner, accounts for 40% of MLL leukemias. The AF4-related proteins, LAF4 and AF5q31, are also fusion partners for MLL in rare cases of leukemia, whereas a fourth family member, FMR2, is a candidate protein for fragile X mental retardation syndrome. MLL fusions with AF4 family proteins manifest as acute biphenotypic or lymphoblastic leukemias that are associated with a poor prognosis, but the lack of appropriate disease models has hampered progress in understanding their underlying molecular mechanisms. Here we report that each of the MLL-AF4 family fusion proteins (the leukemia associated MLL-AF4, MLL-LAF4, and MLL-AF5q31 as well as an artificial MLL-FMR2) is capable of altering the growth and self-renewal properties of primary murine hematopoietic stem/progenitor cells in a serial methylcellulose culture myeloid replating assay. However, significant differences were observed in their respective oncogenic potentials. Cells transduced with MLL-LAF4, MLL-AF5q31 and MLL-FMR2 rapidly expanded during serial replatings, adapted to growth in liquid culture, and induced leukemias in syngeneic recipient mice within 6 months. Conversely, bone marrow cells transduced with MLL-AF4 yielded modest numbers of blast-like colonies in the third round of plating, only transiently expanded in vitro, and quickly underwent terminal differentiation. MLL-LAF4 and MLL-AF5q31 were also capable of enhancing the self-renewal of transduced cells with B lymphoid progenitor phenotypes. A structure/function analysis demonstrated that the previously reported transcriptional transactivation domains of AF4 family proteins were neither necessary nor sufficient for in vitro transformation. Furthermore, the recently reported AF9 interaction motif that is conserved in all AF4 family proteins was also shown to be dispensable for in vitro transformation. These data indicate that transactivation per se, and interaction with AF9 in particular, are not absolutely required for the oncogenic actions of MLL-AF4 family proteins. Conversely, a minimal transformation domain was mapped to the highly conserved carboxy-terminal homology domain shared among all AF4 family proteins, including the Drosophila homolog Lilliputian, and shown to be necessary and sufficient for transformation. Taken together, our studies establish transformation models for MLL-AF4 family fusion proteins and provide critical mechanistic insights into their underlying molecular mechanisms.

Interferon-Gamma Impairs the Self-Renewal of Hematopoietic Stem Cells

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2351-2351
Author(s):  
Alexander M. de Bruin ◽  
Berend Hooibrink ◽  
Martijn A. Nolte
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Interferon Gamma ◽  
Chimeric Mice ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
The Impact

Abstract Abstract 2351 Regulation of hematopoiesis during stress situations, such as bacterial or viral infections, is crucial for the maintenance of sufficient numbers of cells in the blood. It has become clear that activated immune cells provide such feedback signals to the bone marrow. An important mediator in this respect is the pro-inflammatory cytokine Interferon-gamma (IFNγ), which is produced in the bone marrow by activated T cells during the course of an infection. As such, we have previously shown that T cell-derived IFNγ can directly influence the output of myeloid and erythroid cells. To address whether IFNγ can also influence the function of hematopoietic stem cells (HSCs), we cultured highly purified HSCs from murine bone marrow with or without IFNγ and found that IFNγ strongly reduced the absolute number of HSCs in these cultures, both phenotypically and functionally. We confirmed that the functional impact of IFNγ was due to a direct effect on HSCs and not mediated by more differentiated progenitors. In addition, IFNγ does not directly influence the quiescent state of purified HSC, nor their cell cycle entry. By labeling HSCs with CFSE, we found that IFNγ reduces HSC expansion in vitro by decreasing their proliferative capacity, but not their ability to differentiate. To investigate the impact of IFNγ on HSCs in vivo, we infected WT and IFNγ−/− mice with lymphocytic choriomeningitis virus (LCMV) and found that IFNγ severely impaired HSC recovery upon infection. Finally, to exclude indirect effects of IFNγ on other cell types we generated chimeric mice with bone marrow from both WT and IFNγR−/− mice. Infection of these mixed-chimeric mice with LCMV resulted in decreased recovery of WT HSCs, but not of IFNγR−/− HSCs in the same mouse, which formally demonstrates that IFNγ directly impairs the proliferation of HSCs in vivo. Based on these experiments we conclude that IFNγ reduces HSC self renewal both in vitro and in vivo. Importantly, we thereby challenge the current concept in literature that IFNγ would induce the proliferation of HSCs (Baldridge et al, Nature 2010). Our findings thus provide challenging new insight regarding the impact of immune activation on hematopoiesis and will contribute significantly to the scientific discussion concerning this process. Moreover, our data also provide an explanation for the occurrence of anemia and bone marrow failure in several human diseases in which IFNγ is chronically produced. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Distinct Gene Expression Patterns Drive Proliferation of Dominant-Negative RUNX1 Immortalized HSPCs

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1795-1795
Author(s):  
Samantha K Langer ◽  
Alyssa Cull ◽  
Natalie Wossidlo ◽  
Hannes Klump ◽  
Peter A. Horn ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Bone Marrow ◽  
Tumor Suppressor ◽  
Dominant Negative ◽  
Tumor Suppressor Function ◽  
Self Renewal ◽  
Hematopoietic Stem

Abstract The transcription factor RUNX1 is a master regulator of normal hematopoiesis and is involved in cell fate decisions. RUNX1 mutations have been shown to contribute to the development of myeloid neoplasms, and in myelodysplastic syndromes (MDS) it is one of the most frequently mutated genes. Such mutations lead to RUNX1 proteins that lack transactivation activity or DNA-binding ability resulting in a loss of its tumor suppressor function. The dominant-negative short isoform RUNX1a resembles truncated RUNX1 mutants and inhibits the function of the full-length RUNX1 proteins. Additionally, a recently published study identified overexpression of RUNX1a but not full-length RUNX1 in CD34+-cells from patients with myelodysplastic/myeloproliferative disease, which increased with disease progression. This strongly suggests that truncated RUNX1 plays a pivotal role in myelodysplastic disease. However, the precise molecular functions of mutating RUNX1, particularly with respect to the identity of RUNX1 target genes conferring its tumor suppressor function, remain unclear. Previously, our group reported that overexpression of RUNX1a immortalized murine hematopoietic stem and progenitor cells (HSPCs) in vitro. Immunophenotyping of these cells confirmed the expansion of an immature subpopulation defined as Lin- Sca1+ Kit+ (LSK). This phenotype was reversed upon turning RUNX1a-expression off and led to a loss of Sca1 expression (Lin- Kit+, LK). To further understand the molecular consequences of RUNX1a overexpression we sorted the LK cells and LSK cells before and 36h after RUNX1a-expression was turned off. Next, we performed microarray analysis to assess differential gene expression in these different subpopulations. Gene set enrichment analysis (GSEA) identified upregulation of genes highly expressed in hematopoietic stem cells (HSC) and leukemic stem cells (LSCs) in RUNX1a-expressing LSK cells compared to those LSK cells in which RUNX1a expression was turned off. Conversely, a gene signature associated with stemness and self-renewal was lost in LK-cells when RUNX1a expression was turned off. Among the eleven leading edge genes, we found genes implicated in leukemogenesis, stem cell regulation, or both such as Erg, Meis1 and Bcl11a. To further understand the role of RUNX1a in vivo we transplanted C57Bl6 mice (n=29) with HSPCs expressing RUNX1a in a competitive reconstitution setting. Consistent with the immortalization of HSPCs in vitro, RUNX1a-overexpressing HSPCs expanded in the bone marrow of transplanted mice. We observed significantly higher frequencies of LK (2.9-fold) and LSK cells (5-fold) in the RUNX1a-expressing bone marrow cells compared to transplanted control mice. High frequencies of RUNX1a-expressing cells in the bone marrow were associated with lower frequencies of RUNX1a-expressing cells in the peripheral blood indicating a differentiation block. In addition, we found that 85% of the RUNX1a-expressing cells were committed to the myeloid lineage (CD11b+/Ly6G+) at the expense of the lymphoid lineage (B220 and CD3e). Moreover, RUNX1a expression led to an increased percentage (65%) of immature erythroblasts (Ter119-) in the bone marrow compared to control cells (55%). In summary, we have demonstrated that RUNX1a overexpression immortalized HSPCs by upregulation of genes involved in leukemogenesis, stemness and self-renewal. In vivo such HSPCs showed a competitive advantage that was associated with a block of differentiation. Our study, particularly the gene expression analysis, provides novel insights into genetic drivers contributing to the development of myeloid malignancies in patients with RUNX1 mutations. Disclosures No relevant conflicts of interest to declare.

Hematopoietic Stem Cells Overexpressing Smad7 Exhibit Increased Self-Renewal and Regeneration Capacity in Vivo.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 561-561 ◽  
Author(s):  
Ulrika Blank ◽  
Jonas Larsson ◽  
Taiju Utsugisawa ◽  
Mattias Magnusson ◽  
Jenny Klintman ◽  
...  
Keyword(s):  
Stem Cells ◽  
Molecular Mechanisms ◽  
Transforming Growth Factor ◽  
Internal Ribosomal Entry Site ◽  
Entry Site ◽  
Related Factors ◽  
Self Renewal ◽  
Hematopoietic Stem

Abstract The hematopoietic stem cell (HSC) resides in the bone marrow (BM) and can self-renew to generate more stem cells as well as differentiate into all hematopoietic lineages. The precise molecular mechanisms, which govern HSC fate decisions are poorly understood. The Transforming Growth Factor-β (TGF-β) superfamily of ligands, including the TGF-βs, Activins and Bone Morphogenetic Proteins (BMPs), encompasses an important group of growth factors, many of which have been shown to modulate and regulate hematopoiesis. Smad7 is known to block the phosphorylation event of receptor-activated Smads, thus creating a block in the entire signaling cascade downstream of TGF-β and related factors. To assess the effect of blocking the entire Smad signaling pathway downstream of TGF-β/Activin and BMP in HSCs in vivo, we have overexpressed the inhibitory Smad7 by a retroviral gene transfer approach. Both control and Smad7 vectors were MSCV based and expression was driven by the LTR promoter. The Smad7 vector contained the cDNA sequence for murine Smad7 and an internal ribosomal entry site (IRES) followed by GFP, whereas the control vector contained IRES and GFP only. In these experiments BM from wild type C57/B6 mice could efficiently be transduced with Smad7 or control vectors respectively. Upon transduction, cells (Ly5.2) were transplanted in a competitive fashion into lethally irradiated recipients (Ly5.1) and transduced cells were monitored by GFP fluorescence. Smad7 overexpressing cells were able to long-term reconstitute as well as give rise to both lymphoid and myeloid compartments at normal distributions. When self-renewal was assessed by secondary transplantations, Smad7 overexpressing cells showed significantly increased reconstitution ability compared to control transduced cells (blood samples at 12 weeks post transplant: 37.3 ± 5,9 for Smad7 vs. 5.06 ± 1,7 for control. Data represent % GFP positive cells ± SEM). Furthermore, Western blot analysis showed efficient expression of Smad7 protein in BM cells originating from transduced cells of transplanted mice. In addition, Smad2 and Smad1 phosphorylation was blocked upon TGF-β, Activin or BMP stimulation in BM cells, suggesting that Smad7 was functionally active in BM cells in vivo. However, when cultured under serum-free conditions in vitro, Smad7 overexpressing cells exhibited reduced proliferative capacity as compared to control transduced cells (3.5 times fewer cells by day 12 post transduction), suggesting that the in vivo phenotype was dependent on the BM microenvironment. Taken together, our data indicate that blocking of several TGF-β pathways simultaneously increases the self-renewal ability of HSCs in vivo, but not in vitro.

Investigation of Hematopoietic Stem Cell and Progenitor Populations: Implication for Cell Fate Determination and Lineage Commitment.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 801-801 ◽  
Author(s):  
Emmanuelle Passegue ◽  
Camilla Forsberg ◽  
Thomas Serwold ◽  
Scott Kogan ◽  
Irving L. Weissman
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Cell Fate ◽  
Cell Fate Determination ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Hematopoietic Development ◽  
Fate Determination

Abstract A thorough understanding of the lineage potential of each subset of hematopoietic stem cells (HSC) and progenitor populations is critical to establish an accurate map of cell fate determination during hematopoietic development. A controversy exists whether multipotentiality is conserved until a mutually exclusive segregation of myeloid and lymphoid potentials or whether early progenitor populations sequentially lose lineage potential as they differentiate from the long-term self-renewing HSC (LT-HSC), starting with loss of megakaryocyte/erythrocyte (MegE) potential. Hematopoietic cells at different developmental stages can be prospectively isolated based on a combination of cell surface phenotypes and functional assays in vitro and in vivo. However, assessment of lineage potential of cells other than LT-HSC is complicated by the progressive loss of self-renewal activity in progenitor populations and the lack of congenic surface markers on mature cells of the MegE lineage. Using sensitive in vitro and in vivo approaches, we quantitatively and kinetically assessed the MegE potential of Lineage−/c-Kit+/Sca-1+ (KLS) subsets of mouse bone marrow, including LT-HSC (Thy1.1int/Flk-2−), sort-term HSC (ST-HSCF: Thy1.1int/Flk-2+) and multipotent progenitor population (MPPF: Thy1.1−/Flk-2+), and compared it with the MegE potential of downstream myeloid progenitors (CMP, GMP and MEP) and with their ability to give rise to mature myelomonocytic and lymphoid cells. In contrast to previous reports, we demonstrate that Flk2-positive ST-HSCF and MPPF populations have readily detectable but transient MegE potential in vivo that is more robust than committed myeloid progenitors CMP and MEP. We also show that these cells make clonal colonies in vitro and in vivo in the spleen that contained megakaryocytes and erythrocytes. Moreover, we established the kinetics of mature cell production from each stem and progenitor population, hence providing the timing of these early differentiation events in vivo that is of critical importance when investigating lineage potential. Our results demonstrate that multipotentiality is retained in the KLS “stem cell” fraction of the bone marrow and support a model of hematopoietic development with mutually exclusive segregation of myeloid and lymphoid lineage potential. Taken together with previous findings, they indicate that transition from LT-HSC to ST-HSCF and then to MPPF, is accompanied by progressive lose of self-renewal ability, increased proliferation and change in gene expression programs to prepare multipotent cells to leave the stem cell niche and undergo lineage differentiation. This model is by definition a simplification of a complex biological process but accounts for most, if not all, differentiation events, tolerates plasticity in lineage segregation at early steps of commitment and it accommodates intrinsic lineage preferences during ontogeny and aging.

Murine Hematopoietic Stem Cells Require Akt1 and Akt2 for Long-Term Self-Renewal

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 502-502
Author(s):  
Marisa M. Juntilla ◽  
Vineet Patil ◽  
Rohan Joshi ◽  
Gary A. Koretzky
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Fetal Liver ◽  
Akt Signaling ◽  
Self Renewal ◽  
Hematopoietic Stem

Abstract Murine hematopoietic stem cells (HSCs) rely on components of the Akt signaling pathway, such as FOXO family members and PTEN, for efficient self-renewal and continued survival. However, it is unknown whether Akt is also required for murine HSC function. We hypothesized that Akt would be required for HSC self-renewal, and that the absence of Akt would lead to hematopoietic failure resulting in developmental defects in multiple lineages. To address the effect of Akt loss in HSCs we used competitive and noncompetitive murine fetal liver-bone marrow chimeras. In short-term assays, Akt1−/−Akt2−/− fetal liver cells reconstituted the LSK compartment of an irradiated host as well or better than wildtype cells, although failed to generate wildtype levels of more differentiated cells in multiple lineages. When placed in a competitive environment, Akt1−/−Akt2−/− HSCs were outcompeted by wildtype HSCs in serial bone marrow transplant assays, indicating a requirement for Akt1 and Akt2 in the maintainance of long-term hematopoietic stem cells. Akt1−/−Akt2−/− LSKs tend to remain in the G0 phase of the cell cycle compared to wildtype LSKs, suggesting the failure in serial transplant assays may be due to increased quiesence in the absence of Akt1 and Akt2. Additionally, the intracellular content of reactive oxygen species (ROS) in HSCs is dependent on Akt signaling because Akt1−/−Akt2−/− HSCs have decreased ROS levels. Furthermore, pharmacologic augmentation of ROS in the absence of Akt1 and Akt2 results in an exit from quiescence and rescue of differentiation both in vivo and in vitro. Together, these data implicate Akt1 and Akt2 as critical regulators of long-term HSC function and suggest that defective ROS homeostasis may contribute to failed hematopoiesis.

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