| 1. |
- Subramaniam, Agatheeswaran, et al.
(författare)
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Lysine-specific demethylase 1A (LSD1) restricts ex vivo propagation of human HSCs and is a target of UM171
- 2020
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Ingår i: Blood. - : American Society of Hematology. - 1528-0020 .- 0006-4971. ; 136:19, s. 2151-2161
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Tidskriftsartikel (refereegranskat)abstract
- Culture conditions in which hematopoietic stem cells (HSCs) can be expanded for clinical benefit are highly sought after. Here, we report that inhibition of the epigenetic regulator Lysine-specific histone demethylase 1A (LSD1) induces a rapid expansion of human cord blood derived CD34+ cells and promotes in vitro propagation of long-term repopulating HSCs by preventing differentiation. The phenotype and molecular characteristics of cells treated with LSD1 inhibitors were highly similar to cells treated with UM171, an agent promoting expansion of HSCs through undefined mechanisms, and currently tested in clinical trials. Strikingly, we found that LSD1 as well as other members of the LSD1 containing chromatin remodeling complex CoREST are rapidly poly-ubiquitinated and degraded upon UM171 treatment. CRISPR/Cas9 depletion of the CoREST core member, RCOR1, resulted in expansion of CD34+ cells similar to LSD1 inhibition and UM171. Taken together, LSD1 and CoREST restrict HSC expansion, and are principal targets of UM171, forming a mechanistic basis for the HSC promoting activity of UM171.
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| 2. |
- 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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| 3. |
- 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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| 4. |
- Galeev, Roman, et al.
(författare)
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Forward RNAi screens in human hematopoietic stem cells
- 2017
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Ingår i: Methods in Molecular Biology. - New York, NY : Springer New York. - 1064-3745. ; 1622, s. 29-50
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Bokkapitel (refereegranskat)abstract
- Identifying the genes and pathways that regulate self-renewal and differentiation in somatic stem cells is a central goal in stem cell and cancer biology. Here, we describe a method for RNA interference (RNAi)-based screens combined with next-generation sequencing (NGS) in primary human hematopoietic stem and progenitor cells (HSPCs). These cells are suitable targets for complex, selection-based screens using pooled lentiviral short hairpin RNA (shRNA) libraries. The screening approach presented in this chapter is a promising tool to dissect regulatory mechanisms in hematopoietic stem cells (HSCs) and somatic stem cells in general, and may be particularly useful to identify gene targets and modifiers that can be further exploited in strategies for ex vivo stem cell expansion.
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| 5. |
- Galeev, Roman, et al.
(författare)
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Genome-wide RNAi Screen Identifies Cohesin Genes as Modifiers of Renewal and Differentiation in Human HSCs
- 2016
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Ingår i: Cell Reports. - : Elsevier BV. - 2211-1247. ; 14:12, s. 2988-3000
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Tidskriftsartikel (refereegranskat)abstract
- To gain insights into the regulatory mechanisms of hematopoietic stem cells (HSCs), we employed a genome-wide RNAi screen in human cord-blood derived cells and identified candidate genes whose knockdown maintained the HSC phenotype during culture. A striking finding was the identification of members of the cohesin complex (STAG2, RAD21, STAG1, and SMC3) among the top 20 genes from the screen. Upon individual validation of these cohesin genes, we found that their knockdown led to an immediate expansion of cells with an HSC phenotype in vitro. A similar expansion was observed in vivo following transplantation to immunodeficient mice. Transcriptome analysis of cohesin-deficient CD34(+) cells showed an upregulation of HSC-specific genes, demonstrating an immediate shift toward a more stem-cell-like gene expression signature upon cohesin deficiency. Our findings implicate cohesin as a major regulator of HSCs and illustrate the power of global RNAi screens to identify modifiers of cell fate.
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| 6. |
- Galeev, Roman
(författare)
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Identification of intrinsic regulators in normal and malignant human hematopoiesis. An RNA-interference approach.
- 2018
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Bildandet av blodceller, hematopoesen, utgår från ett litet antal av de så kallade hematopoetiska stamceller som sitter i vår benmärg. Dessa celler har förmågan att ge upphov till alla mogna blodcellstyper av de tre hematopoetiska linjerna, samtidigt som de genom celldelning bibehåller sin egna mängd. Det har uppskattats att vi endast har några tusen hematopoetiska stamceller, men trots deras relativt lilla antal, har de den enastående förmågan att generera runt 11 miljoner mogna blodceller varje sekund, hela livet.Trots att hematopoetiska stamceller sannolikt är de stamceller det forskats mest kring, är vår förståelse kring hur de regleras på molekylär och genetisk fortfarande inkomplett. I syfte att öka vår förståelse om den genetiska regleringen av hematopoetiska stamceller både i normal och malign blodbildning (leukemi), har vi i denna avhandling använt oss av RNA interferens (RNAi) applicerat på blodstamceller från navelsträngsblod i syfte att identifiera gener som styr stamcellernas självförnyelse och celldelning. Genom att använda både ett förvalt och genetiskt komplett RNAi virusbibliotek, har vi funnit att MAPK14, cohesinkomplexet and JARID2 är negativa genetiska regulatorer av blodstamceller. Attenuering av samtliga dessa gener medförde ökad funktionalitet hos stamcellerna.Vi har också använt datan från vår RNAi screen som ett verktyg att preselektera gener i syfte att även identifiera genetiska regulator i malign hematopoes, särskilt akut myeloisk leukemi och myelodysplastiskt syndrom. Genom att sekvensera de preselekterade generna i över 400 patientprover och därefter funktionellt validera våra upptäckter i både humana och murina celler, har vi identifierat NCAPD2 och SDPR som potentiellt AML- och MDS-associerade gener.Sammanfattningsvis har vi i denna avhandling använt oss av en global, helgenomtäckande RNAi screen i mänskliga stamceller isolerade från navelsträngsblod. Vi har visat att detta är en användbar metod att hitta nya genetiska stamcellsregulator och i kombination med helgenomssekvensering att även hitta funktionellt relevanta sjukdomsassocierade gener i hematopoetiska maligniteter.
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| 7. |
- Kinkel, Sarah A, et al.
(författare)
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Jarid2 regulates hematopoietic stem cell function by acting with polycomb repressive complex 2.
- 2015
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Ingår i: Blood. - : American Society of Hematology. - 1528-0020 .- 0006-4971. ; 125:12, s. 1890-1900
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Tidskriftsartikel (refereegranskat)abstract
- Polycomb Repressive Complex 2 (PRC2) plays a key role in hematopoietic stem and progenitor cell (HSPC) function. Analyses of mouse mutants harboring deletions of core components have implicated PRC2 in fine-tuning multiple pathways that instruct HSPC behavior, yet how PRC2 is targeted to specific genomic loci within HSPCs remains unknown. Here we use shRNA-mediated knockdown to survey the function of PRC2 accessory factors that were defined in ES cells, by testing the competitive reconstitution capacity of transduced murine HSPCs. We find that similar to the phenotype observed upon depletion of core subunit Suz12, depleting Jarid2 enhances the competitive transplantation capacity of both fetal and adult mouse HSPCs. Furthermore, we demonstrate that depletion of JARID2 enhances the in vitro expansion and in vivo reconstitution capacity of human HSPCs. Gene expression profiling revealed common Suz12 and Jarid2 target genes that are enriched for the H3K27me3 mark established by PRC2. These data implicate Jarid2 as an important component of PRC2 that has a central role in coordinating HSPC function.
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| 8. |
- Kumar, Praveen, et al.
(författare)
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HMGA2 promotes long-term engraftment and myeloerythroid differentiation of human hematopoietic stem and progenitor cells
- 2019
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Ingår i: Blood Advances. - : American Society of Hematology. - 2473-9529 .- 2473-9537. ; 3:4, s. 681-691
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Tidskriftsartikel (refereegranskat)abstract
- Identification of determinants of fate choices in hematopoietic stem cells (HSCs) is essential to improve the clinical use of HSCs and to enhance our understanding of the biology of normal and malignant hematopoiesis. Here, we show that high-mobility group AT hook 2 (HMGA2), a nonhistone chromosomal-binding protein, is highly and preferentially expressed in HSCs and in the most immature progenitor cell subset of fetal, neonatal, and adult human hematopoiesis. Knockdown of HMGA2 by short hairpin RNA impaired the long-term hematopoietic reconstitution of cord blood (CB)-derived CB CD34+ cells. Conversely, overexpression of HMGA2 in CB CD34+ cells led to overall enhanced reconstitution in serial transplantation assays accompanied by a skewing toward the myeloerythroid lineages. RNA-sequencing analysis showed that enforced HMGA2 expression in CD34+ cells induced gene-expression signatures associated with differentiation toward megakaryocyte-erythroid and myeloid lineages, as well as signatures associated with growth and survival, which at the protein level were coupled with strong activation of AKT. Taken together, our findings demonstrate a key role of HMGA2 in regulation of both proliferation and differentiation of human HSPCs.
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| 9. |
- Moura-Castro, Larissa H., et al.
(författare)
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Sister chromatid cohesion defects are associated with chromosomal copy number heterogeneity in high hyperdiploid childhood acute lymphoblastic leukemia
- 2021
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Ingår i: Genes Chromosomes and Cancer. - : Wiley. - 1045-2257 .- 1098-2264. ; 60:6, s. 410-417
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Tidskriftsartikel (refereegranskat)abstract
- High hyperdiploid acute lymphoblastic leukemia (ALL) is one of the most common malignancies in children. The main driver event of this disease is a nonrandom aneuploidy consisting of gains of whole chromosomes but without overt evidence of chromosomal instability (CIN). Here, we investigated the frequency and severity of defective sister chromatid cohesion—a phenomenon related to CIN—in primary pediatric ALL. We found that a large proportion (86%) of hyperdiploid cases displayed aberrant cohesion, frequently severe, to compare with 49% of ETV6/RUNX1-positive ALL, which mostly displayed mild defects. In hyperdiploid ALL, cohesion defects were associated with increased chromosomal copy number heterogeneity, which could indicate increased CIN. Furthermore, cohesion defects correlated with RAD21 and NCAPG mRNA expression, suggesting a link to reduced cohesin and condensin levels in hyperdiploid ALL. Knockdown of RAD21 in an ALL cell line led to sister chromatid cohesion defects, aberrant mitoses, and increased heterogeneity in chromosomal copy numbers, similar to what was seen in primary hyperdiploid ALL. In summary, our study shows that aberrant sister chromatid cohesion is frequent but heterogeneous in pediatric high hyperdiploid ALL, ranging from mild to very severe defects, and possibly due to low cohesin or condensin levels. Cases with high levels of aberrant chromosome cohesion displayed increased chromosomal copy number heterogeneity, possibly indicative of increased CIN. These abnormalities may play a role in the clonal evolution of hyperdiploid pediatric ALL.
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| 10. |
- Schmiderer, Ludwig, et al.
(författare)
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Efficient and nontoxic biomolecule delivery to primary human hematopoietic stem cells using nanostraws
- 2020
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Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424. ; 117:35, s. 21267-21273
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Tidskriftsartikel (refereegranskat)abstract
- Introduction of exogenous genetic material into primary stem cells is essential for studying biological function and for clinical applications. Traditional delivery methods for nucleic acids, such as electroporation, have advanced the field, but have negative effects on stem cell function and viability. We introduce nanostraw-assisted transfection as an alternative method for RNA delivery to human hematopoietic stem and progenitor cells (HSPCs). Nanostraws are hollow alumina nanotubes that can be used to deliver biomolecules to living cells. We use nanostraws to target human primary HSPCs and show efficient delivery of mRNA, short interfering RNAs (siRNAs), DNA oligonucleotides, and dextrans of sizes ranging from 6 kDa to 2,000 kDa. Nanostraw-treated cells were fully functional and viable, with no impairment in their proliferative or colony-forming capacity, and showed similar long-term engraftment potential in vivo as untreated cells. Additionally, we found that gene expression of the cells was not perturbed by nanostraw treatment, while conventional electroporation changed the expression of more than 2,000 genes. Our results show that nanostraw-mediated transfection is a gentle alternative to established gene delivery methods, and uniquely suited for nonperturbative treatment of sensitive primary stem cells.
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