| 1. |
- Björnsson, Jon Mar, et al.
(författare)
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Proliferation of primitive myeloid progenitors can be reversibly induced by HOXA10
- 2001
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Ingår i: Blood. - 1528-0020 .- 0006-4971. ; 98:12, s. 3301-3308
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Tidskriftsartikel (refereegranskat)abstract
- Recent studies show that several Hox transcription factors are important for regulation of proliferation and differentiation in hematopoiesis. Among these is HOXA10, which is selectively expressed at high levels in the most primitive subpopulation of human CD34(+) bone marrow cells. When overexpressed, HOXA10 increases the proliferation of early progenitor cells and can lead to the development of myeloid leukemia. To study the effects of HOXA10 on primitive hematopoietic progenitors in more detail, transgenic mice were generated with regulatable HOXA10 expression. The transgenic mouse model, referred to as tetO-HOXA10, contains the HOXA10 gene controlled by a tetracycline-responsive element and a minimal promoter. Thus, the expression of HOXA10 is inducible and reversible depending on the absence or presence of tetracycline or its analog, doxycycline. A retroviral vector containing the tetracycline transactivator gene (tTA) was used to induce expression of the HOXA10 gene In bone marrow cells from the transgenic mice. Reverse transcription-polymerase chain reaction analysis confirmed regulatable HOXA10 expression in several transgenic lines. HOXA10 induction led to the formation of hematopoietic colonies containing blastlike cells and megakaryocytes. Moreover, the induction of HOXA10 resulted in significant proliferative advantage of primitive hematopoietic progenitors (spleen colony-forming units [CFU-S-12]), which was reversible on withdrawal of induction. Activation of HOXA10 expression in tet0-HOXA10 mice will therefore govern proliferation of primitive myeloid progenitors in a regulated fashion. This novel animal model can be used to identify the target genes of HOXA10 and better clarify, the specific role of HOXA10 in normal and malignant hematopoiesis.
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| 2. |
- Björnsson, Jon Mar, et al.
(författare)
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Reduced proliferative capacity of hematopoietic stem cells deficient in hoxb3 and hoxb4
- 2003
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Ingår i: Blood. - 0006-4971 .- 1528-0020. ; 23:11, s. 3872-3883
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Tidskriftsartikel (refereegranskat)abstract
- Several homeobox transcription factors, such as HOXB3 and HOXB4, have been implicated in regulation of hematopoiesis. In support of this, studies show that overexpression of HOXB4 strongly enhances hematopoietic stem cell regeneration. Here we find that mice deficient in both Hoxb3 and Hoxb4 have defects in endogenous hematopoiesis with reduced cellularity in hematopoietic organs and diminished number of hematopoietic progenitors without perturbing lineage commitment. Analysis of embryonic day 14.5 fetal livers revealed a significant reduction in the hematopoietic stem cell pool, suggesting that the reduction in cellularity observed postnatally is due to insufficient expansion during fetal development. Primitive Lin(-) Scal(+) c-kit(+) hematopoietic progenitors lacking Hoxb3 and Hoxb4 displayed impaired proliferative capacity in vitro. Similarly, in vivo repopulating studies of Hoxb3/Hoxb4-deficient hematopoietic cells resulted in lower repopulating capability compared to normal littermates. Since no defects in homing were observed, these results suggest a slower regeneration of mutant HSC. Furthermore, treatment with cytostatic drugs demonstrated slower cell cycle kinetics of hematopoietic stem cells deficient in Hoxb3 and Hoxb4, resulting in increased tolerance to antimitotic drugs. Collectively, these data suggest a direct physiological role of Hoxb4 and Hoxb3 in regulating stem cell regeneration and that these genes are required for maximal proliferative response.
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| 3. |
- Blank Savukinas, Ulrika, et al.
(författare)
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Signaling pathways governing stem cell fate.
- 2008
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Ingår i: Blood. - : American Society of Hematology. - 1528-0020 .- 0006-4971. ; 111:2, s. 492-503
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Forskningsöversikt (refereegranskat)abstract
- Hematopoietic stem cells (HSCs) are historically the most thoroughly characterized type of adult stem cell, and the hematopoietic system has served as a principal model structure of stem-cell biology for several decades. However, paradoxically, although HSCs can be defined by function and even purified to near-homogeneity, the intricate molecular machinery and the signaling mechanisms regulating fate events, such as self-renewal and differentiation, have remained elusive. Recently, several developmentally conserved signaling pathways have emerged as important control devices of HSC fate, including Notch, Wingless-type (Wnt), Sonic hedgehog (Shh), and Smad pathways. HSCs reside in a complex environment in the bone marrow, providing a niche that optimally balances signals that control self-renewal and differentiation. These signaling circuits provide a valuable structure for our understanding of how HSC regulation occurs, concomitantly with providing information of how the bone marrow microenvironment couples and integrates extrinsic with intrinsic HSC fate determinants. It is the focus of this review to highlight some of the most recent developments concerning signaling pathways governing HSC fate.
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| 4. |
- Blank Savukinas, Ulrika, et al.
(författare)
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Smad7 promotes self-renewal of hematopoietic stem cells in vivo.
- 2006
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Ingår i: Blood. - : American Society of Hematology. - 1528-0020 .- 0006-4971. ; 108:13, s. 4246-4254
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Tidskriftsartikel (refereegranskat)abstract
- The Smad-signaling pathway downstream of the transforming growth factor–beta superfamily of ligands is an evolutionarily conserved signaling circuitry with critical functions in a wide variety of biologic processes. To investigate the role of this pathway in the regulation of hematopoietic stem cells (HSCs), we have blocked Smad signaling by retroviral gene transfer of the inhibitory Smad7 to murine HSCs. We report here that the self-renewal capacity of HSCs is promoted in vivo upon blocking of the entire Smad pathway, as shown by both primary and secondary bone marrow (BM) transplantations. Importantly, HSCs overexpressing Smad7 have an unperturbed differentiation capacity as evidenced by normal contribution to both lymphoid and myeloid cell lineages, suggesting that the Smad pathway regulates self-renewal independently of differentiation. Moreover, phosphorylation of Smads was inhibited in response to ligand stimulation in BM cells, thus verifying impairment of the Smad-signaling cascade in Smad7-overexpressing cells. Taken together, these data reveal an important and previously unappreciated role for the Smad-signaling pathway in the regulation of self-renewal of HSCs in vivo.
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| 5. |
- Blank Savukinas, Ulrika, et al.
(författare)
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TGF-β signaling in the control of hematopoietic stem cells.
- 2015
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Ingår i: Blood. - : American Society of Hematology. - 1528-0020 .- 0006-4971. ; 125:23, s. 3542-3550
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Tidskriftsartikel (refereegranskat)abstract
- Blood is a tissue with high cellular turnover, and its production is a tightly orchestrated process that requires constant replenishment. All mature blood cells are generated from hematopoietic stem cells (HSCs), which are the self-renewing units that sustain life-long hematopoiesis. HSC behavior, such as self-renewal and quiescence, are regulated by a wide array of factors, including external signaling cues present in the bone marrow. The Transforming Growth Factor-β (TGF-β) family of cytokines constitutes a multifunctional signaling circuitry, which regulates pivotal functions related to cell fate and behavior in virtually all tissues of the body. In the hematopoietic system, TGF-β signaling controls a wide spectrum of biological processes, from homeostasis of the immune system to quiescence and self-renewal of HSCs. Here, we review key features and emerging concepts pertaining to TGF-β and downstream signaling pathways in normal HSC biology, featuring aspects of aging, hematological disease, and how this circuitry may be exploited for clinical purposes in the future.
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| 6. |
- Brun, Ann, et al.
(författare)
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Hoxb4-deficient mice undergo normal hematopoietic development but exhibit a mild proliferation defect in hematopoietic stem cells
- 2004
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Ingår i: Blood. - : American Society of Hematology. - 1528-0020 .- 0006-4971. ; 103:11, s. 4126-4133
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Tidskriftsartikel (refereegranskat)abstract
- Enforced expression of Hoxb4 dramatically increases the regeneration of murine hematopoietic stem cells (HSCs) after transplantation and enhances the repopulation ability of human severe combined immunodeficiency (SCID) repopulating cells. Therefore, we asked what physiologic role Hoxb4 has in hematopoiesis. A novel mouse model lacking the entire Hoxb4 gene exhibits significantly reduced cellularity in spleen and bone marrow (BM) and a subtle reduction in red blood cell counts and hemoglobin values. A mild reduction was observed in the numbers of primitive progenitors and stem cells in adult BM and fetal liver, whereas lineage distribution was normal. Although the cell cycle kinetics of primitive progenitors was normal during endogenous hematopoiesis, defects in proliferative responses of BM Lin(-) Sca1(+) c-kit(+) stem and progenitor cells were observed in culture and in vivo after the transplantation of BM and fetal liver HSCs. Quantitative analysis of mRNA from fetal liver revealed that a deficiency of Hoxb4 alone changed the expression levels of several other Hox genes and of genes involved in cell cycle regulation. In summary, the deficiency of Hoxb4 leads to hypocellularity in hematopoietic organs and impaired proliferative capacity. However, Hoxb4 is not required for the generation of HSCs or the maintenance of steady state hematopoiesis.
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| 7. |
- Flygare, Johan, et al.
(författare)
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Deficiency of ribosomal protein S19 in CD34+ cells generated by siRNA blocks erythroid development and mimics defects seen in Diamond-Blackfan anemia
- 2005
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Ingår i: Blood. - : American Society of Hematology. - 0006-4971 .- 1528-0020. ; 105:12, s. 4627-4634
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Tidskriftsartikel (refereegranskat)abstract
- Diamond-Blackfan anemia (DBA) is a congenital red cell aplasia in which 25% of the patients have a mutation in the ribosomal protein S19 (RPS19) gene. To study effects of RPS19 deficiency in hematopoiesis we transduced CD34+ umbilical cord blood (CB) and bone marrow (BM) cells with 3 lentiviral vectors expressing small interfering RNA (siRNA) against RPS19 and 1 scrambled control vector. All vectors also express green fluorescent protein (GFP). Transduction with the siRNA vectors reduced RPS19 mRNA levels to various degrees, which resulted in erythroid defects, correlating to the degree of RPS19 down-regulation, and was rescued by expression of an siRNA-resistant RPS19 transcript. Erythroid colony formation capacity conjointly decreased with RPS19 levels in CD34+ CB and BM cells. In liquid culture supporting erythroid differentiation, RPS19-silenced as well as DBA patient CD34+ cells exhibited reduced proliferative capacity and impaired erythroid differentiation resulting in fewer erythroid colony-forming units (CFU-Es). When assaying myeloid development, a less pronounced influence on proliferation was seen. This study shows for the first time that RPS19 silencing decreases the proliferative capacity of hematopoietic progenitors and leads to a defect in erythroid development.
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| 8. |
- Flygare, Johan, et al.
(författare)
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Diamond-Blackfan Anemia: Erythropoiesis Lost in Translation
- 2007
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Ingår i: Blood. - : American Society of Hematology. - 1528-0020 .- 0006-4971. ; 109:8, s. 3152-3160
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Diamond-Blackfan anemia (DBA) is a congenital erythroid aplasia that usually presents as macrocytic anemia during infancy. Linkage analysis suggests that at least four genes are associated with DBA of which two have been identified so far. The known DBA genes encode the ribosomal proteins S19 and S24 accounting for 25% and 2% of the patients respectively. This study shows that RPS19 gene transfer improves the proliferation defect and erythroid development in RPS19 deficient DBA patient CD34+ cells in vitro. These results indicate that RPS19-deficient DBA patients are potential candidates for gene therapy. We next created two disease models for RPS19-deficient DBA. We were able to induce a DBA phenotype in normal cells by reducing RPS19 expression using RNA interference (RNAi) to silence RPS19 expression in human CD34+ BM cells. Analogous in vitro DBA models were created using erythroid leukemia TF-1 and UT7 cell lines that harbor Doxycycline-dependent RNAi-mediated RPS19 silencing. When induced to silence RPS19 expression, TF-1 cell proliferation decreased together with a marked reduction in the number of erythroid cells. The DBA disease model cell lines were next used in a study showing that RPS19-deficient TF-1 cells and DBA patient cells share a defect in 18S rRNA processing which ultimately hampers ribosomal 40S subunit maturation. We predict that these RPS19-deficient cell lines can be used for further mechanistic studies on RPS19 deficiency in erythropoiesis. The study is concluded by a discussion where links between ribosomal proteins and erythropoiesis are reviewed together with considerations regarding future directions of DBA research.
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| 9. |
- Flygare, Johan, et al.
(författare)
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Human RPS19, the gene mutated in Diamond Blackfan anemia, encodes a ribosomal protein required for the maturation of 40S ribosomal subunits.
- 2007
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Ingår i: Blood. - : American Society of Hematology. - 1528-0020 .- 0006-4971. ; 109:3, s. 980-986
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Tidskriftsartikel (refereegranskat)abstract
- Diamond-Blackfan anemia (DBA) typically presents with red blood cell aplasia that usually manifests in the first year of life. The only gene currently known to be mutated in DBA encodes ribosomal protein S19 (RPS19). Previous studies have shown that the yeast RPS19 protein is required for a specific step in the maturation of 40S ribosomal subunits. Our objective here was to determine whether the human RPS19 protein functions at a similar step in 40S subunit maturation. Studies where RPS19 expression is reduced by siRNA in the hematopoietic cell line, TF-1, show that human RPS19 is also required for a specific step in the maturation of 40S ribosomal subunits. This maturation defect can be monitored by studying rRNA-processing intermediates along the ribosome synthesis pathway. Analysis of these intermediates in CD34(-) cells from the bone marrow of patients with DBA harboring mutations in RPS19 revealed a pre-rRNA-processing defect similar to that observed in TF-1 cells where RPS19 expression was reduced. This defect was observed to a lesser extent in CD34(+) cells from patients with DBA who have mutations in RPS19.
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| 10. |
- Hamaguchi, Isao, et al.
(författare)
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Gene transfer improves erythroid development in ribosomal protein S19-deficient Diamond-Blackfan anemia.
- 2002
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Ingår i: Blood. - 1528-0020. ; 100:8, s. 2724-2731
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Tidskriftsartikel (refereegranskat)abstract
- Diamond-Blackfan anemia (DBA) is a congenital bone marrow failure syndrome characterized by a specific deficiency in erythroid progenitors. Forty percent of the patients are blood transfusion-dependent. Recent reports show that the ribosomal protein S19 (RPS19) gene is mutated in 25% of all patients with DBA. We constructed oncoretroviral vectors containing the RPS19 gene to develop gene therapy for RPS19-deficient DBA. These vectors were used to introduce the RPS19 gene into CD34(+) bone marrow (BM) cells from 4 patients with DBA with RPS19 gene mutations. Overexpression of the RPS19 transgene increased the number of erythroid colonies by almost 3-fold. High expression levels of the RPS19 transgene improved erythroid colony-forming ability substantially whereas low expression levels had no effect. Overexpression of RPS19 had no detrimental effect on granulocyte-macrophage colony formation. Therefore, these findings suggest that gene therapy for RPS19-deficient patients with DBA using viral vectors that express the RPS19 gene is feasible. (Blood. 2002;100:2724-2731)
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