| 1. |
- 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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| 2. |
- Karlsson, Göran, et al.
(författare)
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Gene expression profiling demonstrates that TGF-{beta}1 signals exclusively through receptor complexes involving Alk5 and identifies targets of TGF-{beta} signaling.
- 2005
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Ingår i: Physiological Genomics. - : American Physiological Society. - 1094-8341 .- 1531-2267. ; 21:3, s. 396-403
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Tidskriftsartikel (refereegranskat)abstract
- Transforming growth factor-β 1 (TGF-β) regulates cellular functions like proliferation, differentiation, and apoptosis. On the cell surface, TGF-β binds to receptor complexes consisting of TGF-β receptor type II (Tβ RII) and activin-like kinase receptor-5 (Alk5), and the downstream signaling is transduced by Smad and MAPK proteins. Recent data have shown that alternative receptor combinations aside from the classical pairing of Tβ RII/Alk5 can be relevant for TGF-β signaling. We have screened for alternative receptors for TGF-β and also for gene targets of TGF-β signaling, by performing functional assays and microarray analysis in murine embryonic fibroblast (MEF) cell lines lacking Alk5. Data from TGF-β-stimulated Alk5(-/-) cells show them to be completely unaffected by TGF-β. Additionally, 465 downstream targets of Alk5 signaling were identified when comparing Alk5(-/-) or TGF-β-stimulated Alk5(+/+) MEFs with unstimulated Alk5(+/+) cells. Our results demonstrate that, in MEFs, TGF-β signals exclusively through complexes involving Alk5, and give insight to its downstream effector genes.
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| 3. |
- Lopez de Lapuente Portilla, Aitzkoa, et al.
(författare)
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Genome-wide association study on 13,167 individuals identifies regulators of hematopoietic stem and progenitor cell levels in human blood
- 2021
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Understanding how hematopoietic stem and progenitor cells (HSPCs) are regulated is of central importance for the development of new therapies for blood disorders and stem cell transplantation. To date, HSPC regulation has been extensively studied in vitro and in animal models, but less is known about the mechanisms in vivo in humans. Here, in a genome-wide association study on 13,167 individuals, we identify 9 significant and 2 suggestive DNA sequence variants that influence HSPC (CD34+) levels in human blood. The identified loci associate with blood disorders, harbor known and novel HSPC genes, and affect gene expression in HSPCs. Interestingly, our strongest association maps to the PPM1H gene, encoding an evolutionarily conserved serine/threonine phosphatase never previously implicated in stem cell biology. PPM1H is expressed in HSPCs, and the allele that confers higher blood CD34+ cell levels downregulates PPM1H. By functional fine-mapping, we find that this downregulation is caused by the variant rs772557-A, which abrogates a MYB transcription factor binding site in PPM1H intron 1 that is active in specific HSPC subpopulations, including hematopoietic stem cells, and interacts with the promoter by chromatin looping. Furthermore, rs772557-A selectively increases HSPC subpopulations in which the MYB site is active, and PPM1H shRNA- knockdown increased CD34+ and CD34+90+ cell proportions in umbilical cord blood cultures. Our findings represent the first large-scale association study on a stem cell trait, illuminating HSPC regulation in vivo in humans, and identifying PPM1H as a novel inhibition target that can potentially be utilized clinically to facilitate stem cell harvesting for transplantation.
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| 4. |
- Neddermeyer, Anne H.
(författare)
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Identification and Functional Significance of Aberrant Long Non-coding RNAs in Acute Myeloid Leukemia : Biological and Prognostic Implications
- 2022
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Acute myeloid leukemia (AML) is the most frequently diagnosed type of acute leukemia in adults. It commonly affects people aged 60 or older, as incidence increases with age, and it is characterized by the accumulation of immature hematopoietic progenitor cells in the bone marrow. Despite recent treatment advances and improvements for certain subtypes, as acute promyelocyte leukemia (APL), AML remains difficult to cure. While many patients reach remission after induction treatment, relapses are common and 5-year overall survival remains dismal. Long non-coding RNAs (lncRNAs) are involved in various regulatory cellular functions and, like coding genes, they are frequently dysregulated in cancer. In this thesis, the aim was to elucidate the functional implications of lncRNAs in the biology and treatment response of AML and normal hematopoiesis in order to improve understanding of AML pathology. In Paper I, whole-transcriptome sequencing identified the novel lncRNA MALNC. Clinical correlation analyses and CRISPR-knockout cell models were used to functionally explore its implications in AML. It was identified that enhanced MALNC expression is specifically associated with the AML-subtypes APL and AML with co-mutant NPM1/IDH2R140. Further, it was shown that MALNC is implicated in key factors of leukemogenesis, like differentiation and proliferation, and that MALNC expression associates with better overall survival in AML patients. Moreover, knockout of the MALNC gene sensitized AML cells to arsenic trioxide (ATO), all-trans retinoic acid (ATRA)-ATO combination and venetoclax treatment. In Paper II, three high-throughput functional CRISPR interference screens were performed to identify lncRNAs implicated in proliferation, differentiation or venetoclax response. Several novel lncRNAs were identified to potentially play a positive or negative role in these processes and furthermore were found to implicate AML prognosis. In Paper III, the lncRNA NEAT1 was studied in respect to its role in normal hematopoiesis and AML using CAGE- and RADICL-sequencing. It could be illustrated that NEAT1 expression positively correlates with cell maturity during normal hematopoiesis, in particular monocytes, and associates with core-binding factor AML inv(16) and t(8;21). Further, RADICL-sequencing identified that lncRNA NEAT1 binds to the genomic loci of key hematopoietic transcription factor RUNX2. In contrast to solid cancers, it was demonstrated, that higher NEAT1 expression correlated with better outcome in AML, independent of known risk factors. In summary, these studies have outlined the scope of functional implications of lncRNAs in normal and dysregulated hematopoiesis and have highlighted their potential roles as biomarkers for prognosis and drug sensitivity. These findings support the efforts to understand how lncRNAs could serve as novel biomarkers for personalized treatment.
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| 5. |
- Ali, Nicole, et al.
(författare)
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Forward RNAi screens in primary human hematopoietic stem/progenitor cells
- 2009
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Ingår i: Blood. - : American Society of Hematology. - 1528-0020 .- 0006-4971. ; 113:16, s. 3690-3695
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Tidskriftsartikel (refereegranskat)abstract
- The mechanisms regulating key fate decisions such as self-renewal and differentiation in hematopoietic stem and progenitor cells (HSPC) remain poorly understood. We report here a screening strategy developed to assess modulators of human hematopoiesis using a lentiviral short hairpin RNA (shRNA) library transduced into cord blood-derived stem/progenitor cells. To screen for modifiers of self-renewal/differentiation, we used the limited persistence of HSPCs under ex vivo culture conditions as a baseline for functional selection of shRNAs conferring enhanced maintenance or expansion of the stem/progenitor potential. This approach enables complex, pooled screens in large numbers of cells. Functional selection identified novel specific gene targets (exostoses 1) or shRNA constructs capable of altering human hematopoietic progenitor differentiation or stem cell expansion, respectively, thereby demonstrating the potential of this forward screening approach in primary human stem cell populations. (Blood. 2009; 113: 3690-3695)
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| 6. |
- Baudet, Aurelie, et al.
(författare)
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RNAi screen identifies MAPK14 as a druggable suppressor of human hematopoietic stem cell expansion.
- 2012
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Ingår i: Blood. - : American Society of Hematology. - 1528-0020 .- 0006-4971. ; 119:26, s. 6255-6258
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Tidskriftsartikel (refereegranskat)abstract
- We report on a forward RNAi screen in primary human hematopoietic stem and progenitor cells, using pooled lentiviral shRNA libraries deconvoluted by next generation sequencing. We identify MAPK14/p38α as a modulator of ex vivo stem cell proliferation and show that pharmacological inhibition of p38 dramatically enhances the stem cell activity of cultured umbilical cord blood derived hematopoietic cells. p38 inhibitors should thus be considered in strategies aiming at expanding stem cells for clinical benefit.
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| 7. |
- 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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| 8. |
- Chapellier, Marion, et al.
(författare)
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Arrayed molecular barcoding identifies TNFSF13 as a positive regulator of acute myeloid leukemia-initiating cells
- 2019
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Ingår i: Haematologica. - : Ferrata Storti Foundation (Haematologica). - 1592-8721 .- 0390-6078. ; 104:10, s. 2006-2016
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Tidskriftsartikel (refereegranskat)abstract
- Dysregulation of cytokines in the bone marrow microenvironment promotes acute myeloid leukemia cell growth. Due to the complexity and low throughput of in vivo stem-cell based assays, studying the role of cytokines in the bone marrow niche in a screening setting is challenging. Herein, we developed an ex vivo cytokine screen using 11 arrayed molecular barcodes, allowing for a competitive in vivo readout of leukemia-initiating capacity. With this approach, we assessed the effect of 114 murine cytokines on MLL-AF9 acute myeloid leukemia mouse cells and identified the tumor necrosis factor ligand superfamily member 13 (TNFSF13) as a positive regulator of leukemia-initiating cells. By using Tnfsf13-/- recipient mice, we confirmed that TNFSF13 supports leukemia-initiation also under physiological conditions. TNFSF13 was secreted by normal myeloid cells but not by leukemia mouse cells, suggesting that mature myeloid bone marrow cells support leukemia cells by secreting TNFSF13. TNFSF13 supported leukemia cell proliferation in an NF-κB-dependent manner by binding TNFRSF17 and suppressed apoptosis. Moreover, TNFSF13 supported the growth and survival of several human myeloid leukemia cell lines, demonstrating that our findings translate to human disease. Taken together, using arrayed molecular barcoding, we identified a previously unrecognized role of TNFSF13 as a positive regulator of acute myeloid leukemia-initiating cells. The arrayed barcoded screening methodology is not limited to cytokines and leukemia, but can be extended to other types of ex vivo screens, where a multiplexed in vivo read-out of stem cell functionality is needed.
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| 9. |
- Dudas, Marek, et al.
(författare)
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Epithelial and ectomesenchymal role of the type I TGF-beta receptor ALK5 during facial morphogenesis and palatal fusion
- 2006
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Ingår i: Developmental Biology. - : Elsevier BV. - 1095-564X .- 0012-1606. ; 296:2, s. 298-314
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Tidskriftsartikel (refereegranskat)abstract
- Transforming growth factor beta (TGF-beta) proteins play important roles in morphogenesis of many cramofacial tissues; however, detailed biological mechanisms of TGF-beta action, particularly in vivo, are still poorly understood. Here, we deleted the TGF-beta type I receptor gene Alk5 specifically in the embryonic ectodermal and neural crest cell lineages. Failure in signaling via this receptor, either in the epithelium or in the mesenchyme, caused severe craniofacial defects including cleft palate. Moreover, the facial phenotypes of neural crest-specific Alk5 mutants included devastating facial cleft and appeared significantly more severe than the defects seen in corresponding mutants lacking the TGF-beta type II receptor (TGF beta II), a prototypical binding partner of ALK5. Our data indicate that ALK5 plays unique, non-redundant cell-autonomous roles during facial development. Remarkable divergence between Tgfbr2 and A1k5 phenotypes, together with our biochemical in vitro data, imply that (1) ALK5 mediates signaling of a diverse set of ligands not limited to the three isoforms of TGF-beta, and (2) ALK5 acts also in conjunction with type II receptors other than TGF beta RII. (c) 2006 Elsevier Inc. All rights reserved.
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| 10. |
- Galeev, Roman, et al.
(författare)
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Cohesin in haematopoiesis and leukaemia
- 2018
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Ingår i: Current Opinion in Hematology. - 1065-6251. ; 25:4, s. 259-265
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Forskningsöversikt (refereegranskat)abstract
- Purpose of review Disturbance of the delicate balance between self-renewal and differentiation in haematopoietic stem cells (HSCs) can lead to both leukaemia and bone marrow failure. The regulation of this balance in HSC biology has been intensely investigated in several model systems, and lately the importance of epigenetic modifications as well as the organization and architecture of chromatin has become increasingly recognized. In this review, we will focus on the role of the chromatin organizing protein complex cohesin in regulation of normal and malignant haematopoiesis. Recent findings Several functional studies in both mouse and human systems have implicated cohesin as a critical regulator of self-renewal and differentiation in HSCs. Together with the discovery of recurrent mutations of cohesin genes in myeloid malignancies, this points towards a direct role of perturbed cohesin function in leukemogenesis. Summary The work reviewed here provides new insights about the role of the cohesin complex and chromatin architecture in normal and malignant HSCs, and indicates how cohesin may be specifically targeted for therapeutic benefit in the future.
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