| 1. |
- Bäckström, Alexandra, et al.
(författare)
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Combinatorial gene targeting in primary human hematopoietic stem and progenitor cells
- 2022
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Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 12:1
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Tidskriftsartikel (refereegranskat)abstract
- The CRISPR/Cas9 system offers enormous versatility for functional genomics but many applications have proven to be challenging in primary human cells compared to cell lines or mouse cells. Here, to establish a paradigm for multiplexed gene editing in primary human cord blood-derived hematopoietic stem and progenitor cells (HSPCs), we used co-delivery of lentiviral sgRNA vectors expressing either Enhanced Green Fluorescent Protein (EGFP) or Kusabira Orange (KuO), together with Cas9 mRNA, to simultaneously edit two genetic loci. The fluorescent markers allow for tracking of either single- or double-edited cells, and we could achieve robust double knockout of the cell surface molecules CD45 and CD44 with an efficiency of ~ 70%. As a functional proof of concept, we demonstrate that this system can be used to model gene dependencies for cell survival, by simultaneously targeting the cohesin genes STAG1 and STAG2. Moreover, we show combinatorial effects with potential synergy for HSPC expansion by targeting the Aryl Hydrocarbon Receptor (AHR) in conjunction with members of the CoREST complex. Taken together, our traceable multiplexed CRISPR/Cas9 system enables studies of genetic dependencies and cooperation in primary HSPCs, and has important implications for modelling polygenic diseases, as well as investigation of the underlying mechanisms of gene interactions.
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| 2. |
- Lopez de Lapuente Portilla, Aitzkoa, et al.
(författare)
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Genome-wide association study on 13 167 individuals identifies regulators of blood CD34+cell levels
- 2022
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Ingår i: Blood. - : American Society of Hematology. - 0006-4971 .- 1528-0020. ; 139:11, s. 1659-1669
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Tidskriftsartikel (refereegranskat)abstract
- Stem cell transplantation is a cornerstone in the treatment of blood malignancies. The most common method to harvest stem cells for transplantation is by leukapheresis, requiring mobilization of CD34+ hematopoietic stem and progenitor cells (HSPCs) from the bone marrow into the blood. Identifying the genetic factors that control blood CD34+ cell levels could reveal new drug targets for HSPC mobilization. Here we report the first large-scale, genome-wide association study on blood CD34+ cell levels. Across 13 167 individuals, we identify 9 significant and 2 suggestive associations, accounted for by 8 loci (PPM1H, CXCR4, ENO1-RERE, ITGA9, ARHGAP45, CEBPA, TERT, and MYC). Notably, 4 of the identified associations map to CXCR4, showing that bona fide regulators of blood CD34+ cell levels can be identified through genetic variation. Further, the most significant association maps to PPM1H, encoding a serine/threonine phosphatase never previously implicated in HSPC biology. PPM1H is expressed in HSPCs, and the allele that confers higher blood CD34+ cell levels downregulates PPM1H. Through functional fine-mapping, we find that this downregulation is caused by the variant rs772557-A, which abrogates an 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, PPM1H knockdown increases the proportion of CD34+ and CD34+90+ cells in cord blood assays. Our results provide the first large-scale analysis of the genetic architecture of blood CD34+ cell levels and warrant further investigation of PPM1H as a potential inhibition target for stem cell mobilization.
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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. |
- Lopez de Lapuente Portilla, Aitzkoa, et al.
(författare)
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Identification of regulators of hematopoietic stem and progenitor cells in vivo in humans using population genetics
- 2020
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Konferensbidrag (refereegranskat)abstract
- Introduction: Understanding how hematopoietic stem- and progenitor cells (HSPCs) are regulated is of central importance for the development of new therapies for blood disorders and for regenerative medicine. Traditionally, however, HSPC regulation has been studied in model systems, and little is known about the situation in vivo in humans. Methods: To learn how HSPCs are regulated under native conditions in humans, we carried out a first large-scale genome-wide association study on CD34+ cells, representing HSPCs in blood. We used circulating CD34+ levels as a proxy trait to expose regulators of key phenomena like HSPC pool size, migration, and early differentiation. We created a unique phenotyping platform based on high-throughput, high-resolution flow-cytometry and machine learning-based algorithms for automated flow data analysis, and quantified CD34+ cells in 9,936 adults.Results: We identified 8 genome-wide (P<5x10-8) and 20 suggestive loci (P<5x10-6) associated with CD34+ levels. The two strongest were the HSPC migration receptor CXCR4 and a novel protein phosphatase never previously implicated in stem cell biology. Using eQTL, ATAC-seq, and promoter capture Hi-C analysis in isolated HSPCs, we pinpoint likely causal variants, including variants in distant regulatory elements selectively active in specific HSPC subpopulations. Furthermore, shRNA knockdown in primary CD34+ cells supports that some of the identified genes affect CD34+ proliferation and differentiation.Conclusions: We report the first large-scale analysis of the genetic architecture of HSPC regulation, with potential implications for stem cell transplantation and the treatment of hematologic malignancies.Grant information: European Research Council, Swedish Research Council, Swedish Cancer Society.
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| 5. |
- Safaee Talkhoncheh, Mehrnaz, et al.
(författare)
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Ciclopirox Ethanolamine Preserves the Immature State of Human HSCs by Mediating Intracellular Iron Content
- 2023
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Ingår i: Blood Advances. - 2473-9529. ; 7:24, s. 7407-7417
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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. To elucidate regulatory mechanisms governing the maintenance and propagation of human HSCs ex vivo, we screened libraries of annotated small molecules in human cord blood (CB) cells using an optimized assay for detection of functional HSCs during culture. We found that the antifungal agent ciclopirox ethanolamine (CPX) selectively supported immature CD34+CD90+ cells during culture and enhanced their long-term in vivo repopulation capacity. Purified HSCs treated with CPX showed a reduced cell division rate and an enrichment of HSC- specific gene expression patterns. Mechanistically, we found that the HSC stimulating effect of CPX was directly mediated by chelation of the intracellular iron pool, which in turn affected iron-dependent proteins and enzymes mediating cellular metabolism and respiration. Our findings unveil a significant impact of iron homeostasis in regulation of human HSCs, with important implications for both basic HSC biology and clinical hematology.
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| 6. |
- 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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| 7. |
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| 8. |
- 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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| 9. |
- Yudovich, David, et al.
(författare)
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Combined lentiviral- and RNA-mediated CRISPR/Cas9 delivery for efficient and traceable gene editing in human hematopoietic stem and progenitor cells
- 2020
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Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 10:1
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Tidskriftsartikel (refereegranskat)abstract
- The CRISPR/Cas9 system is a versatile tool for functional genomics and forward genetic screens in mammalian cells. However, it has been challenging to deliver the CRISPR components to sensitive cell types, such as primary human hematopoietic stem and progenitor cells (HSPCs), partly due to lentiviral transduction of Cas9 being extremely inefficient in these cells. Here, to overcome these hurdles, we developed a combinatorial system using stable lentiviral delivery of single guide RNA (sgRNA) followed by transient transfection of Cas9 mRNA by electroporation in human cord blood-derived CD34+ HSPCs. We further applied an optimized sgRNA structure, that significantly improved editing efficiency in this context, and we obtained knockout levels reaching 90% for the cell surface proteins CD45 and CD44 in sgRNA transduced HSPCs. Our combinatorial CRISPR/Cas9 delivery approach had no negative influence on CD34 expression or colony forming capacity in vitro compared to non-treated HSPCs. Furthermore, gene edited HSPCs showed intact in vivo reconstitution capacity following transplantation to immunodeficient mice. Taken together, we developed a paradigm for combinatorial CRISPR/Cas9 delivery that enables efficient and traceable gene editing in primary human HSPCs, and is compatible with high functionality both in vitro and in vivo.
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| 10. |
- Žemaitis, Kristijonas, et al.
(författare)
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RNAi Screen Identifies MTA1 as an Epigenetic Modifier of Differentiation Commitment in Human HSPCs
- 2022
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Ingår i: Experimental Hematology. - : Elsevier BV. - 1873-2399 .- 0301-472X. ; 115, s. 20-29
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Tidskriftsartikel (refereegranskat)abstract
- The molecular mechanisms regulating key fate decisions of hematopoietic stem cells (HSCs) remain incompletely understood. Here, we targeted global shRNA libraries to primary human hematopoietic stem and progenitor cells (HSPCs) to screen for modifiers of self-renewal and differentiation, and identified metastasis-associated 1 (MTA1) as a negative regulator of human HSPC propagation in vitro. Knockdown of MTA1 by independent shRNAs in primary human cord blood (CB) HSPCs led to a cell expansion during culture and a relative accumulation of immature CD34 +CD90 + cells with perturbed in vitro differentiation potential. Transplantation experiments in immunodeficient mice revealed a significant reduction in human chimerism in both blood and bone marrow from HSPCs with knockdown of MTA1, possibly caused by reduced maturation of blood cells. We further found that MTA1 associates with the nucleosome remodeling deacetylase (NuRD) complex in human HSPCs, and on knockdown of MTA1, we observed an increase in H3K27Ac marks coupled with a downregulation of genes linked to differentiation toward the erythroid lineage. Together, our findings identify MTA1 as a novel regulator of human HSPCs in vitro and in vivo with critical functions for differentiation commitment.
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