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1
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Fleischauer J, Bastone AL, Selich A, John-Neek P, Weisskoeppel L, Schaudien D, Schambach A, Rothe M. TGF β Inhibitor A83-01 Enhances Murine HSPC Expansion for Gene Therapy. Cells 2023; 12:1978. [PMID: 37566057 PMCID: PMC10416825 DOI: 10.3390/cells12151978] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/07/2023] [Revised: 07/28/2023] [Accepted: 07/28/2023] [Indexed: 08/12/2023] Open
Abstract
Murine hematopoietic stem and progenitor cells (HSPCs) are commonly used as model systems during gene therapeutic retroviral vector development and preclinical biosafety assessment. Here, we developed cell culture conditions to maintain stemness and prevent differentiation during HSPC culture. We used the small compounds A83-01, pomalidomide, and UM171 (APU). Highly purified LSK SLAM cells expanded in medium containing SCF, IL-3, FLT3-L, and IL-11 but rapidly differentiated to myeloid progenitors and mast cells. The supplementation of APU attenuated the differentiation and preserved the stemness of HSPCs. The TGFβ inhibitor A83-01 was identified as the major effector. It significantly inhibited the mast-cell-associated expression of FcεR1α and the transcription of genes regulating the formation of granules and promoted a 3800-fold expansion of LSK cells. As a functional readout, we used expanded HSPCs in state-of-the-art genotoxicity assays. Like fresh cells, APU-expanded HSPCs transduced with a mutagenic retroviral vector developed a myeloid differentiation block with clonal restriction and dysregulated oncogenic transcriptomic signatures due to vector integration near the high-risk locus Mecom. Thus, expanded HSPCs might serve as a novel cell source for retroviral vector testing and genotoxicity studies.
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Affiliation(s)
- Jenni Fleischauer
- Institute of Experimental Hematology, Hannover Medical School, 30625 Hannover, Germany; (J.F.); (A.L.B.); (A.S.); (P.J.-N.); (L.W.); (A.S.)
- REBIRTH—Research Center for Translational Regenerative Medicine, Hannover Medical School, 30625 Hannover, Germany
| | - Antonella Lucia Bastone
- Institute of Experimental Hematology, Hannover Medical School, 30625 Hannover, Germany; (J.F.); (A.L.B.); (A.S.); (P.J.-N.); (L.W.); (A.S.)
- REBIRTH—Research Center for Translational Regenerative Medicine, Hannover Medical School, 30625 Hannover, Germany
| | - Anton Selich
- Institute of Experimental Hematology, Hannover Medical School, 30625 Hannover, Germany; (J.F.); (A.L.B.); (A.S.); (P.J.-N.); (L.W.); (A.S.)
- REBIRTH—Research Center for Translational Regenerative Medicine, Hannover Medical School, 30625 Hannover, Germany
| | - Philipp John-Neek
- Institute of Experimental Hematology, Hannover Medical School, 30625 Hannover, Germany; (J.F.); (A.L.B.); (A.S.); (P.J.-N.); (L.W.); (A.S.)
- REBIRTH—Research Center for Translational Regenerative Medicine, Hannover Medical School, 30625 Hannover, Germany
| | - Luisa Weisskoeppel
- Institute of Experimental Hematology, Hannover Medical School, 30625 Hannover, Germany; (J.F.); (A.L.B.); (A.S.); (P.J.-N.); (L.W.); (A.S.)
- REBIRTH—Research Center for Translational Regenerative Medicine, Hannover Medical School, 30625 Hannover, Germany
| | - Dirk Schaudien
- Department of Inhalation Toxicology, Fraunhofer Institute for Toxicology and Experimental Medicine ITEM, Nikolai Fuchs Strasse 1, 30625 Hannover, Germany;
| | - Axel Schambach
- Institute of Experimental Hematology, Hannover Medical School, 30625 Hannover, Germany; (J.F.); (A.L.B.); (A.S.); (P.J.-N.); (L.W.); (A.S.)
- REBIRTH—Research Center for Translational Regenerative Medicine, Hannover Medical School, 30625 Hannover, Germany
- Division of Hematology/Oncology, Boston Children’s Hospital, Harvard Medical School, 30625 Hannover, Germany
| | - Michael Rothe
- Institute of Experimental Hematology, Hannover Medical School, 30625 Hannover, Germany; (J.F.); (A.L.B.); (A.S.); (P.J.-N.); (L.W.); (A.S.)
- REBIRTH—Research Center for Translational Regenerative Medicine, Hannover Medical School, 30625 Hannover, Germany
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2
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Transmural myocardial repair with engineered heart muscle in a rat model of heterotopic heart transplantation - A proof-of-concept study. J Mol Cell Cardiol 2022; 168:3-12. [PMID: 35390437 DOI: 10.1016/j.yjmcc.2022.03.013] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/20/2022] [Revised: 03/06/2022] [Accepted: 03/28/2022] [Indexed: 11/23/2022]
Abstract
Engineered heart muscle (EHM) can be implanted epicardially to remuscularize the failing heart. In case of a severely scarred ventricle, excision of scar followed by transmural heart wall replacement may be a more desirable application. Accordingly, we tested the hypothesis that allograft (rat) and xenograft (human) EHM can also be administered as transmural heart wall replacement in a heterotopic, volume-loaded heart transplantation model. We first established a novel rat model model to test surgical transmural left heart wall repair. Subsequently and in continuation of our previous allograft studies, we tested outcome after implantation of contractile engineered heart muscle (EHM) and non-contractile engineered connective tissue (ECT) as well as engineered mesenchymal tissue (EMT) allografts as transmural heart wall replacement. Finally, proof-of-concept for the application of human EHM was obtained in an athymic nude rat model. Only in case of EHM implantation, remuscularization of the surgically created transmural defect was observed with palpable graft vascularization. Taken together, feasibility of transmural heart repair using bioengineered myocardial grafts could be demonstrated in a novel rat model of heterotopic heart transplantation.
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van Heuvel Y, Berg K, Hirch T, Winn K, Modlich U, Stitz J. Establishment of a novel stable human suspension packaging cell line producing ecotropic retroviral MLV(PVC-211) vectors efficiently transducing murine hematopoietic stem and progenitor cells. J Virol Methods 2021; 297:114243. [PMID: 34314749 DOI: 10.1016/j.jviromet.2021.114243] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/16/2020] [Revised: 06/30/2021] [Accepted: 07/15/2021] [Indexed: 12/29/2022]
Abstract
Retroviral vectors derived from murine leukemia virus (MLV) are amongst the most frequently utilized vectors in gene therapy approaches such as the genetic modification of hematopoietic cells. Currently, vector particles are mostly produced employing adherent viral packaging cell lines (VPCs) rendering the scale up of production laborious, and thus cost-intensive. Here, we describe the rapid establishment of a human suspension 293-F cell line derived ecotropic MLV VPC. Using transposon vector technology, a packaging and envelope expression cassette as well as a transfer vector facilitated the establishment of a stable VPC yielding high titers of up to 5.2 × 106 transducing units/mL (TU/mL). Vectors were concentrated using ultrafiltration devices and upon one freeze-thaw-cycle still routinely yielded titers of > 1 × 106 TU/mL. Formation of replication-competent retroviruses was not detected. However and as a first generation transfer vector was used in this proof-of-concept (POC) study, gag gene sequences were transduced into target cells within a range of 1-10 copies per 1000 genomes indicating the homologous recombination of packaging construct elements with the transfer vector. High yield VPC vector productivity was stable over a couple of months and unintended integration of the transposase gene was not observed. Ecotropic MLV vector particles were demonstrated to efficiently transduce primary murine hematopoietic stem and progenitor cells. This novel concept should foster the future establishment of suspension VPCs.
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Affiliation(s)
- Yasemin van Heuvel
- Research Group Pharmaceutical Biotechnology, Faculty of Applied Natural Sciences, TH Köln - University of Applied Sciences, Chempark Leverkusen E28, Kaiser-Wilhelm-Allee, 51368, Leverkusen, Germany; Institute of Technical Chemistry, Leibniz University Hannover, Callinstraße, 530167, Hannover, Germany
| | - Karen Berg
- Research Group Pharmaceutical Biotechnology, Faculty of Applied Natural Sciences, TH Köln - University of Applied Sciences, Chempark Leverkusen E28, Kaiser-Wilhelm-Allee, 51368, Leverkusen, Germany; Research Group Translational Hepatology and Stem Cell Biology, Cluster of Excellence REBIRTH, Department of Gastroenterology, Hepatology, and Endocrinology, Hannover Medical School, Carl-Neuberg-Str. 1, 30625, Hannover, Germany
| | - Tanja Hirch
- Research Group for Gene Modification in Stem Cells, Paul-Ehrlich-Institute, Division of Veterinary Medicine, Paul-Ehrlich-Str. 51-59, 63225, Langen, Germany
| | - Kristina Winn
- Research Group Pharmaceutical Biotechnology, Faculty of Applied Natural Sciences, TH Köln - University of Applied Sciences, Chempark Leverkusen E28, Kaiser-Wilhelm-Allee, 51368, Leverkusen, Germany
| | - Ute Modlich
- Research Group for Gene Modification in Stem Cells, Paul-Ehrlich-Institute, Division of Veterinary Medicine, Paul-Ehrlich-Str. 51-59, 63225, Langen, Germany
| | - Jörn Stitz
- Research Group Pharmaceutical Biotechnology, Faculty of Applied Natural Sciences, TH Köln - University of Applied Sciences, Chempark Leverkusen E28, Kaiser-Wilhelm-Allee, 51368, Leverkusen, Germany.
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4
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Schmiedl A, Bokel K, Huhn V, Ionescu L, Zscheppang K, Dammann CEL. Bone marrow stem cells accelerate lung maturation and prevent the LPS-induced delay of morphological and functional fetal lung development in the presence of ErbB4. Cell Tissue Res 2020; 380:547-564. [PMID: 32055958 DOI: 10.1007/s00441-019-03145-0] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/29/2019] [Accepted: 11/18/2019] [Indexed: 12/28/2022]
Abstract
ErbB4 is a regulator in lung development and disease. Prenatal infection is an important risk factor for the delay of morphologic lung development, while promoting the maturation of the surfactant system. Bone marrow-derived mesenchymal stem cells (BMSCs) have the potential to prevent lung injury. We hypothesized that BMSCs in comparison with hematopoietic control stem cells (HPSCs) minimize the lipopolysaccharide (LPS)-induced lung injury only when functional ErbB4 receptor is present. We injected LPS and/or murine green fluorescent protein-labeled BMSCs or HPSCs into the amniotic cavity of transgenic ErbB4heart mothers at gestational day 17. Fetal lungs were analyzed 24 h later. BMSCs minimized significantly LPS-induced delay in morphological lung maturation consisting of a stereologically measured increase in mesenchyme and septal thickness and a decrease of future airspace and septal surface. This effect was more prominent and significant in the ErbB4heart+/- lungs, suggesting that the presence of functioning ErbB4 signaling is required. BMSC also diminished the LPS induced increase in surfactant protein (Sftp)a mRNA and decrease in Sftpc mRNA is only seen if ErbB4 is present. The reduction of morphological delay of lung development and of levels of immune-modulating Sftp was more pronounced in the presence of the ErbB4 receptor. Thus, ErbB4 may be required for the protective signaling of BMSCs.
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Affiliation(s)
- Andreas Schmiedl
- Institute of Functional and Applied Anatomy, Hannover Medical School, Carl-Neuberg-Straße 1, 30625, Hannover, Germany.
- Biomedical Research in Endstage and Obstructive Lung Disease Hannover (BREATH), Member of the German Center of Lung Research (DZL), Hannover, Germany.
| | - Kyra Bokel
- Institute of Functional and Applied Anatomy, Hannover Medical School, Carl-Neuberg-Straße 1, 30625, Hannover, Germany
| | - Verena Huhn
- Department of Pediatric Pulmonology and Neonatology, Hannover Medical School, Carl-Neuberg-Straße 1, 30625, Hannover, Germany
| | - Lavinia Ionescu
- Department of Pediatrics, University of Alberta, Edmonton, AB, Canada
| | - Katja Zscheppang
- Department of Pediatric Pulmonology and Neonatology, Hannover Medical School, Carl-Neuberg-Straße 1, 30625, Hannover, Germany
| | - Christiane E L Dammann
- Department of Pediatric Pulmonology and Neonatology, Hannover Medical School, Carl-Neuberg-Straße 1, 30625, Hannover, Germany
- Division of Newborn Medicine, Department of Pediatrics, Floating Hospital for Children at Tufts Medical Center, Boston, MA, USA
- Graduate School for Biomedical Sciences, Tufts University, Boston, MA, USA
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5
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Hammerschmidt SI, Werth K, Rothe M, Galla M, Permanyer M, Patzer GE, Bubke A, Frenk DN, Selich A, Lange L, Schambach A, Bošnjak B, Förster R. CRISPR/Cas9 Immunoengineering of Hoxb8-Immortalized Progenitor Cells for Revealing CCR7-Mediated Dendritic Cell Signaling and Migration Mechanisms in vivo. Front Immunol 2018; 9:1949. [PMID: 30210501 PMCID: PMC6120996 DOI: 10.3389/fimmu.2018.01949] [Citation(s) in RCA: 23] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/30/2018] [Accepted: 08/07/2018] [Indexed: 12/30/2022] Open
Abstract
To present antigens to cognate T cells, dendritic cells (DCs) exploit the chemokine receptor CCR7 to travel from peripheral tissue via afferent lymphatic vessels to directly enter draining lymph nodes through the floor of the subcapsular sinus. Here, we combined unlimited proliferative capacity of conditionally Hoxb8-immortalized hematopoietic progenitor cells with CRISPR/Cas9 technology to create a powerful experimental system to investigate DC migration and function. Hematopoietic progenitor cells from the bone marrow of Cas9-transgenic mice were conditionally immortalized by lentiviral transduction introducing a doxycycline-regulated form of the transcription factor Hoxb8 (Cas9-Hoxb8 cells). These cells could be stably cultured for weeks in the presence of doxycycline and puromycin, allowing us to introduce additional genetic modifications applying CRISPR/Cas9 technology. Importantly, modified Cas9-Hoxb8 cells retained their potential to differentiate in vitro into myeloid cells, and GM-CSF-differentiated Cas9-Hoxb8 cells showed the classical phenotype of GM-CSF-differentiated bone marrow-derived dendritic cells. Following intralymphatic delivery Cas9-Hoxb8 DCs entered the lymph node in a CCR7-dependent manner. Finally, we used two-photon microscopy and imaged Cas9-Hoxb8 DCs that expressed the genetic Ca2+ sensor GCaMP6S to visualize in real-time chemokine-induced Ca2+ signaling of lymph-derived DCs entering the LN parenchyma. Altogether, our study not only allows mechanistic insights in DC migration in vivo, but also provides a platform for the immunoengineering of DCs that, in combination with two-photon imaging, can be exploited to further dissect DC dynamics in vivo.
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Affiliation(s)
| | - Kathrin Werth
- Institute of Immunology, Hannover Medical School, Hannover, Germany
| | - Michael Rothe
- Institute of Experimental Hematology, REBIRTH Cluster of Excellence, Hannover Medical School, Hannover, Germany
| | - Melanie Galla
- Institute of Experimental Hematology, REBIRTH Cluster of Excellence, Hannover Medical School, Hannover, Germany
| | - Marc Permanyer
- Institute of Immunology, Hannover Medical School, Hannover, Germany
| | | | - Anja Bubke
- Institute of Immunology, Hannover Medical School, Hannover, Germany
| | - David N. Frenk
- Institute of Immunology, Hannover Medical School, Hannover, Germany
| | - Anton Selich
- Institute of Experimental Hematology, REBIRTH Cluster of Excellence, Hannover Medical School, Hannover, Germany
| | - Lucas Lange
- Institute of Experimental Hematology, REBIRTH Cluster of Excellence, Hannover Medical School, Hannover, Germany
| | - Axel Schambach
- Institute of Experimental Hematology, REBIRTH Cluster of Excellence, Hannover Medical School, Hannover, Germany
| | - Berislav Bošnjak
- Institute of Immunology, Hannover Medical School, Hannover, Germany
| | - Reinhold Förster
- Institute of Immunology, Hannover Medical School, Hannover, Germany
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6
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Pan Z, Yang M, Huang K, Büsche G, Glage S, Ganser A, Li Z. Flow cytometric characterization of acute leukemia reveals a distinctive "blast gate" of murine T-lymphoblastic leukemia/lymphoma. Oncotarget 2018; 9:2320-2328. [PMID: 29416774 PMCID: PMC5788642 DOI: 10.18632/oncotarget.23410] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/05/2017] [Accepted: 12/05/2017] [Indexed: 11/28/2022] Open
Abstract
Immunophenotypic analysis using multiparameter flow cytometry is an indispensable tool for diagnosis and management of acute leukemia. Mouse models have been widely used for medical research for more than 100 years and are indispensable for leukemia research. However, immunophenotypic analysis of murine leukemia was not always performed in published studies, and blast gating for isolation of blasts was shown only in very few studies. No systemic characterization of all types of murine acute leukemia in large cohorts by flow cytometry has been reported. In this study, we used flow cytometry to comprehensively characterize murine acute leukemia in a large cohort of mice. We found that murine T-lymphoblastic leukemia/lymphoma (T-ALL) exhibits a distinctive “blast gate” (CD45bright) with CD45/side scatter gating that differs from the “blast gate” (CD45dim) of human T-ALL. By contrast, murine B-lymphoblastic leukemia and acute myeloid leukemia show the same blast region (CD45dim) as human leukemia. Using blast cell gating, we for first time detected T-ALL development in FLT3-ITD knock-in mice (incidence: 23%). These leukemic cells were selectively killed by the FLT3 inhibitors crenolanib and midostaurin in vitro. These data suggest that FLT3-ITD plays a potential role in the pathogenesis of T-ALL and that FLT3-ITD inhibition is a therapeutic option in the management of patients with T-ALL. Our gating strategy for immunophenotypic analysis can be used for leukemogenesis and preclinical gene therapy studies in mice and may improve the quality of such analyses.
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Affiliation(s)
- Zengkai Pan
- Department of Hematology, Hemostasis, Oncology, and Stem Cell Transplantation, Hannover Medical School, Hannover, Germany
| | - Min Yang
- Department of Hematology, Hemostasis, Oncology, and Stem Cell Transplantation, Hannover Medical School, Hannover, Germany
| | - Kezhi Huang
- Department of Hematology, Hemostasis, Oncology, and Stem Cell Transplantation, Hannover Medical School, Hannover, Germany.,Department of Hematology, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou, China
| | - Guntram Büsche
- Institute of Pathology, Hannover Medical School, Hannover, Germany
| | - Silke Glage
- Institute of Laboratory Animal Science, Hannover Medical School, Hannover, Germany
| | - Arnold Ganser
- Department of Hematology, Hemostasis, Oncology, and Stem Cell Transplantation, Hannover Medical School, Hannover, Germany
| | - Zhixiong Li
- Department of Hematology, Hemostasis, Oncology, and Stem Cell Transplantation, Hannover Medical School, Hannover, Germany
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7
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Yang M, Pan Z, Huang K, Büsche G, Feuerhake F, Chaturvedi A, Nie D, Heuser M, Thol F, von Neuhoff N, Ganser A, Li Z. Activation of TRKA receptor elicits mastocytosis in mice and is involved in the development of resistance to KIT-targeted therapy. Oncotarget 2017; 8:73871-73883. [PMID: 29088753 PMCID: PMC5650308 DOI: 10.18632/oncotarget.18027] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/23/2017] [Accepted: 05/08/2017] [Indexed: 12/03/2022] Open
Abstract
The neurotrophins (NTs) play a key role in neuronal survival and maintenance. The TRK (tropomyosin-related kinase) tyrosine kinase receptors (TRKA, TRKB, TRKC) are high affinity receptors for NTs. There is increasing data demonstrating an important role of the TRK family in cancer initiation and progression. NTs have been known for many years to promote chemotaxis, maturation, and survival of mast cells. However, the role of NT signaling in the pathogenesis of mastocytosis is not well understood. In this study, we demonstrate that activation of TRKA by its ligand nerve growth factor (NGF) is potent to trigger a disease in mice with striking similarities to human systemic mastocytosis (SM). Moreover, activation of TRKA by NGF strongly rescues KIT inhibition-induced cell death of mast cell lines and primary mast cells from patients with SM, and this rescue effect can be efficiently blocked by entrectinib (a new pan TRK specific inhibitor). HMC-1 mast cell leukemia cells that are resistant to KIT inhibition induced by TRKA activation show reactivation of MAPK/ERK (extracellular signal-regulated kinase) and strong upregulation of early growth response 3 (EGR3), suggesting an important role of MAPK-EGR3 axis in the development of resistance to KIT inhibition. Targeting both TRK and KIT significantly prolongs survival of mice xenotransplanted with HMC-1 cells compared with targeting KIT alone. Thus, these data strongly suggest that TRKA signaling can improve neoplastic mast cell fitness. This might explain at least in part why treatment with KIT inhibitors alone so far has been disappointing in most published clinical trials for mastocytosis. Our data suggest that targeting both KIT and TRKs might improve efficacy of molecular therapy in SM with KIT mutations.
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Affiliation(s)
- Min Yang
- Department of Hematology, Hemostasis, Oncology, and Stem Cell Transplantation, Hannover Medical School, Hannover, Germany
| | - Zengkai Pan
- Department of Hematology, Hemostasis, Oncology, and Stem Cell Transplantation, Hannover Medical School, Hannover, Germany
| | - Kezhi Huang
- Department of Hematology, Hemostasis, Oncology, and Stem Cell Transplantation, Hannover Medical School, Hannover, Germany.,Department of Hematology, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou, China
| | - Guntram Büsche
- Institute of Pathology, Hannover Medical School, Hannover, Germany
| | | | - Anuhar Chaturvedi
- Department of Hematology, Hemostasis, Oncology, and Stem Cell Transplantation, Hannover Medical School, Hannover, Germany
| | - Danian Nie
- Department of Hematology, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou, China
| | - Michael Heuser
- Department of Hematology, Hemostasis, Oncology, and Stem Cell Transplantation, Hannover Medical School, Hannover, Germany
| | - Felicitas Thol
- Department of Hematology, Hemostasis, Oncology, and Stem Cell Transplantation, Hannover Medical School, Hannover, Germany
| | - Nils von Neuhoff
- Institute of Pathology, Hannover Medical School, Hannover, Germany
| | - Arnold Ganser
- Department of Hematology, Hemostasis, Oncology, and Stem Cell Transplantation, Hannover Medical School, Hannover, Germany
| | - Zhixiong Li
- Department of Hematology, Hemostasis, Oncology, and Stem Cell Transplantation, Hannover Medical School, Hannover, Germany
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8
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Leukemogenic potency of the novel FLT3-N676K mutant. Ann Hematol 2016; 95:783-91. [DOI: 10.1007/s00277-016-2616-z] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/23/2016] [Accepted: 02/04/2016] [Indexed: 01/22/2023]
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9
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Ripperger T, Manukjan G, Meyer J, Wolter S, Schambach A, Bohne J, Modlich U, Li Z, Skawran B, Schlegelberger B, Steinemann D. The heteromeric transcription factor GABP activates the ITGAM/CD11b promoter and induces myeloid differentiation. BIOCHIMICA ET BIOPHYSICA ACTA-GENE REGULATORY MECHANISMS 2015; 1849:1145-54. [PMID: 26170143 DOI: 10.1016/j.bbagrm.2015.07.005] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 03/30/2015] [Revised: 06/19/2015] [Accepted: 07/09/2015] [Indexed: 11/16/2022]
Abstract
The heteromeric transcription factor GA-binding protein (GABP) consists of two subunits, the alpha subunit (GABPA) carrying the DNA-binding ETS domain, and the beta subunit (GABPB1) harbouring the transcriptional activation domain. GABP is involved in haematopoietic stem cell maintenance and differentiation of myeloid and lymphoid lineages in mice. To elucidate the molecular function of GABP in human haematopoiesis, the present study addressed effects of ectopic overexpression of GABP focussing on the myeloid compartment. Combined overexpression of GABPA and GABPB1 caused a proliferation block in cell lines and drastically reduced the colony-forming capacity of murine lineage-negative cells. Impaired proliferation resulted from perturbed cellular cycling and induction of myeloid differentiation shown by surface markers and myelomonocytic morphology of U937 cells. Depending on the dosage and functional integrity of GABP, ITGAM expression was induced. ITGAM encodes CD11b, the alpha subunit of integrin Mac-1, whose beta subunit, ITGB2/CD18, was already described to be regulated by GABP. Finally, Shield1-dependent proteotuning, luciferase reporter assays and chromatin immunoprecipitation showed that GABP activates the ITGAM/CD11b promoter via three binding sites close to the translational start site. In conclusion, the present study supports the crucial role of GABP in myeloid cell differentiation and identified ITGAM/CD11b as a novel GABP target gene.
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Affiliation(s)
- Tim Ripperger
- Institute of Human Genetics, Hannover Medical School, Carl-Neuberg-Str. 1, 30625 Hannover, Germany.
| | - Georgi Manukjan
- Institute of Human Genetics, Hannover Medical School, Carl-Neuberg-Str. 1, 30625 Hannover, Germany.
| | - Johann Meyer
- Institute of Experimental Haematology, Hannover Medical School, Carl-Neuberg-Str. 1, 30625 Hannover, Germany.
| | - Sabine Wolter
- Institute of Pharmacology, Hannover Medical School, Carl-Neuberg-Str. 1, 30625 Hannover, Germany.
| | - Axel Schambach
- Institute of Experimental Haematology, Hannover Medical School, Carl-Neuberg-Str. 1, 30625 Hannover, Germany; Division of Hematology/Oncology, Boston Children's Hospital, Harvard Medical School, 300 Longwood Avenue, Boston, MA 02115, USA.
| | - Jens Bohne
- Institute of Virology, Hannover Medical School, Carl-Neuberg-Str. 1, 30625 Hannover, Germany.
| | - Ute Modlich
- Institute of Experimental Haematology, Hannover Medical School, Carl-Neuberg-Str. 1, 30625 Hannover, Germany.
| | - Zhixiong Li
- Institute of Experimental Haematology, Hannover Medical School, Carl-Neuberg-Str. 1, 30625 Hannover, Germany.
| | - Britta Skawran
- Institute of Human Genetics, Hannover Medical School, Carl-Neuberg-Str. 1, 30625 Hannover, Germany.
| | - Brigitte Schlegelberger
- Institute of Human Genetics, Hannover Medical School, Carl-Neuberg-Str. 1, 30625 Hannover, Germany.
| | - Doris Steinemann
- Institute of Human Genetics, Hannover Medical School, Carl-Neuberg-Str. 1, 30625 Hannover, Germany.
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10
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Kustikova OS, Stahlhut M, Ha TC, Scherer R, Schambach A, Baum C. Dose response and clonal variability of lentiviral tetracycline-regulated vectors in murine hematopoietic cells. Exp Hematol 2014; 42:505-515.e7. [DOI: 10.1016/j.exphem.2014.03.004] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/24/2013] [Revised: 02/23/2014] [Accepted: 03/06/2014] [Indexed: 12/14/2022]
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11
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Non-integrating gamma-retroviral vectors as a versatile tool for transient zinc-finger nuclease delivery. Sci Rep 2014; 4:4656. [PMID: 24722320 PMCID: PMC3983605 DOI: 10.1038/srep04656] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/30/2013] [Accepted: 03/14/2014] [Indexed: 12/17/2022] Open
Abstract
Designer nucleases, like zinc-finger nucleases (ZFNs), represent valuable tools for targeted genome editing. Here, we took advantage of the gamma-retroviral life cycle and produced vectors to transfer ZFNs in the form of protein, mRNA and episomal DNA. Transfer efficacy and ZFN activity were assessed in quantitative proof-of-concept experiments in a human cell line and in mouse embryonic stem cells. We demonstrate that retrovirus-mediated protein transfer (RPT), retrovirus-mediated mRNA transfer (RMT), and retrovirus-mediated episome transfer (RET) represent powerful methodologies for transient protein delivery or protein expression. Furthermore, we describe complementary strategies to augment ZFN activity after gamma-retroviral transduction, including serial transduction, proteasome inhibition, and hypothermia. Depending on vector dose and target cell type, gene disruption frequencies of up to 15% were achieved with RPT and RMT, and >50% gene knockout after RET. In summary, non-integrating gamma-retroviral vectors represent a versatile tool to transiently deliver ZFNs to human and mouse cells.
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12
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A canonical to non-canonical Wnt signalling switch in haematopoietic stem-cell ageing. Nature 2013; 503:392-6. [PMID: 24141946 DOI: 10.1038/nature12631] [Citation(s) in RCA: 225] [Impact Index Per Article: 18.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/29/2012] [Accepted: 09/03/2013] [Indexed: 02/06/2023]
Abstract
Many organs with a high cell turnover (for example, skin, intestine and blood) are composed of short-lived cells that require continuous replenishment by somatic stem cells. Ageing results in the inability of these tissues to maintain homeostasis and it is believed that somatic stem-cell ageing is one underlying cause of tissue attrition with age or age-related diseases. Ageing of haematopoietic stem cells (HSCs) is associated with impaired haematopoiesis in the elderly. Despite a large amount of data describing the decline of HSC function on ageing, the molecular mechanisms of this process remain largely unknown, which precludes rational approaches to attenuate stem-cell ageing. Here we report an unexpected shift from canonical to non-canonical Wnt signalling in mice due to elevated expression of Wnt5a in aged HSCs, which causes stem-cell ageing. Wnt5a treatment of young HSCs induces ageing-associated stem-cell apolarity, reduction of regenerative capacity and an ageing-like myeloid-lymphoid differentiation skewing via activation of the small Rho GTPase Cdc42. Conversely, Wnt5a haploinsufficiency attenuates HSC ageing, whereas stem-cell-intrinsic reduction of Wnt5a expression results in functionally rejuvenated aged HSCs. Our data demonstrate a critical role for stem-cell-intrinsic non-canonical Wnt5a signalling in HSC ageing.
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13
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Vajen B, Modlich U, Schienke A, Wolf S, Skawran B, Hofmann W, Büsche G, Kreipe H, Baum C, Santos-Barriopedro I, Vaquero A, Schlegelberger B, Rudolph C. Histone methyltransferaseSuv39h1deficiency preventsMyc-induced chromosomal instability in murine myeloid leukemias. Genes Chromosomes Cancer 2013; 52:423-30. [DOI: 10.1002/gcc.22040] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/09/2012] [Accepted: 11/22/2012] [Indexed: 11/09/2022] Open
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14
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Cytological characterization of murine bone marrow and spleen hematopoietic compartments for improved assessment of toxicity in preclinical gene marking models. Ann Hematol 2013; 92:595-604. [PMID: 23307598 DOI: 10.1007/s00277-012-1655-3] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/05/2012] [Accepted: 12/08/2012] [Indexed: 12/30/2022]
Abstract
Gene therapy has proven its potential to cure diseases of the hematopoietic system, but potential adverse reactions related to insertional mutagenesis by integrating gene vectors and chromosomal instability in long-lived repopulating cells have emerged as a major limitation. Preclinical gene therapy in murine models is a powerful model for assessment of gene marking efficiency and adverse reactions. However, changes in the hematologic composition after transplantation with retrovirally modified hematopoietic stem cells have not been well investigated in large cohorts of animals by systematic cytological analyses. In the present study, cytological analyses of bone marrow and spleen were performed in a large cohort (n = 58) of C57BL/6J mice over an extended observation period after gene marking. Interestingly, we observed hematological malignancies in four out of 30 animals transplanted with dLNGFR (truncated form of the human p75 low-affinity nerve growth factor receptor) and tCD34 modified stem/progenitor cells. Our data demonstrate that cytological analysis provides important information for diagnosis of hematological disorders and thus should be included in preclinical studies and performed in each investigated animal. Together with histological analysis, flow cytometric analysis, and other analyses, the quality and predictive value of preclinical gene therapy studies will be improved.
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15
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Cornils K, Bartholomae CC, Thielecke L, Lange C, Arens A, Glauche I, Mock U, Riecken K, Gerdes S, von Kalle C, Schmidt M, Roeder I, Fehse B. Comparative clonal analysis of reconstitution kinetics after transplantation of hematopoietic stem cells gene marked with a lentiviral SIN or a γ-retroviral LTR vector. Exp Hematol 2013; 41:28-38.e3. [DOI: 10.1016/j.exphem.2012.09.003] [Citation(s) in RCA: 22] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/17/2012] [Revised: 08/28/2012] [Accepted: 09/10/2012] [Indexed: 12/13/2022]
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16
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Activation of Evi1 inhibits cell cycle progression and differentiation of hematopoietic progenitor cells. Leukemia 2012; 27:1127-38. [PMID: 23212151 DOI: 10.1038/leu.2012.355] [Citation(s) in RCA: 57] [Impact Index Per Article: 4.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/19/2022]
Abstract
The transcription factor Evi1 has an outstanding role in the formation and transformation of hematopoietic cells. Its activation by chromosomal rearrangement induces a myelodysplastic syndrome with progression to acute myeloid leukemia of poor prognosis. Similarly, retroviral insertion-mediated upregulation confers a competitive advantage to transplanted hematopoietic cells, triggering clonal dominance or even leukemia. To study the molecular and functional response of primary murine hematopoietic progenitor cells to the activation of Evi1, we established an inducible lentiviral expression system. EVI1 had a biphasic effect with initial growth inhibition and retarded myeloid differentiation linked to enhanced survival of myeloblasts in long-term cultures. Gene expression microarray analysis revealed that within 24 h EVI1 upregulated 'stemness' genes characteristic for long-term hematopoietic stem cells (Aldh1a1, Abca1, Cdkn1b, Cdkn1c, Epcam, among others) but downregulated genes involved in DNA replication (Cyclins and their kinases, among others) and DNA repair (including Brca1, Brca2, Rad51). Cell cycle analysis demonstrated EVI1's anti-proliferative effect to be strictly dose-dependent with accumulation of cells in G0/G1, but preservation of a small fraction of long-term proliferating cells. Although confined to cultured cells, our study contributes to new hypotheses addressing the mechanisms and molecular targets involved in preleukemic clonal dominance or leukemic transformation by Evi1.
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17
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Vas V, Senger K, Dörr K, Niebel A, Geiger H. Aging of the microenvironment influences clonality in hematopoiesis. PLoS One 2012; 7:e42080. [PMID: 22879906 PMCID: PMC3412859 DOI: 10.1371/journal.pone.0042080] [Citation(s) in RCA: 66] [Impact Index Per Article: 5.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/01/2012] [Accepted: 07/02/2012] [Indexed: 01/08/2023] Open
Abstract
The mechanisms of the age-associated exponential increase in the incidence of leukemia are not known in detail. Leukemia as well as aging are initiated and regulated in multi-factorial fashion by cell-intrinsic and extrinsic factors. The role of aging of the microenvironment for leukemia initiation/progression has not been investigated in great detail so far. Clonality in hematopoiesis is tightly linked to the initiation of leukemia. Based on a retroviral-insertion mutagenesis approach to generate primitive hematopoietic cells with an intrinsic potential for clonal expansion, we determined clonality of transduced hematopoietic progenitor cells (HPCs) exposed to a young or aged microenvironment in vivo. While HPCs displayed primarily oligo-clonality within a young microenvironment, aged animals transplanted with identical pool of cells displayed reduced clonality within transduced HPCs. Our data show that an aged niche exerts a distinct selection pressure on dominant HPC-clones thus facilitating the transition to mono-clonality, which might be one underlying cause for the increased age-associated incidence of leukemia.
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Affiliation(s)
- Virag Vas
- Department of Dermatology and Allergic Diseases, University of Ulm, Ulm, Germany
| | - Katharina Senger
- Department of Dermatology and Allergic Diseases, University of Ulm, Ulm, Germany
| | - Karin Dörr
- Department of Dermatology and Allergic Diseases, University of Ulm, Ulm, Germany
| | - Anja Niebel
- Department of Dermatology and Allergic Diseases, University of Ulm, Ulm, Germany
| | - Hartmut Geiger
- Department of Dermatology and Allergic Diseases, University of Ulm, Ulm, Germany
- Division of Experimental Hematology and Cancer Biology, Cincinnati Children’s Hospital Medical Center, University of Cincinnati, Cincinnati, Ohio, United States of America
- * E-mail:
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18
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Pfaff N, Lachmann N, Kohlscheen S, Sgodda M, Araúzo-Bravo MJ, Greber B, Kues W, Glage S, Baum C, Niemann H, Schambach A, Cantz T, Moritz T. Efficient hematopoietic redifferentiation of induced pluripotent stem cells derived from primitive murine bone marrow cells. Stem Cells Dev 2011; 21:689-701. [PMID: 21732815 DOI: 10.1089/scd.2011.0010] [Citation(s) in RCA: 26] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/26/2022] Open
Abstract
Heterogeneity among induced pluripotent stem cell (iPSC) lines with regard to their gene expression profile and differentiation potential has been described and at least partly linked to the tissue of origin. Here, we generated iPSCs from primitive [lineage negative (Lin(neg))] and nonadherent differentiated [lineage positive (Lin(pos))] bone marrow cells (BM-iPSC), and compared their differentiation potential to that of fibroblast-derived iPSCs (Fib-iPSC) and embryonic stem cells (ESC). In the undifferentiated state, individual iPSC clones but also ESCs proved remarkably similar when analyzed for alkaline phosphatase and SSEA-1 staining, endogenous expression of the pluripotency genes Nanog, Oct4, and Sox2, or global gene expression profiles. However, substantial differences between iPSC clones were observed after induction of differentiation, which became most obvious upon cytokine-mediated instruction toward the hematopoietic lineage. All 3 BM-iPSC lines derived from undifferentiated Lin(neg) cells yielded high proportions of cells expressing the hematopoietic differentiation marker CD41 and in 2 of these lines high proportions of CD41+/ CD45+ cells were detected. In contrast, little hematopoiesis-specific surface marker expression was detected in 4 Lin(pos) BM-iPSC and 3 Fib-iPSC lines. These results were corroborated by functional studies demonstrating robust colony outgrowth from hematopoietic progenitors in 2 of the Lin(neg) BM-iPSCs only. Thus, in conclusion, our data demonstrate efficient generation of iPSCs from primitive hematopoietic tissue as well as efficient hematopoietic redifferentiation for Lin(neg) BM-iPSC lines, thereby supporting the notion of an epigenetic memory in iPSCs.
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Affiliation(s)
- Nils Pfaff
- REBIRTH Research Group Reprogramming, Hannover Medical School, Hannover, Germany
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19
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Gaussin A, Modlich U, Bauche C, Niederländer NJ, Schambach A, Duros C, Artus A, Baum C, Cohen-Haguenauer O, Mermod N. CTF/NF1 transcription factors act as potent genetic insulators for integrating gene transfer vectors. Gene Ther 2011; 19:15-24. [DOI: 10.1038/gt.2011.70] [Citation(s) in RCA: 26] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022]
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20
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Gutsch R, Kandemir JD, Pietsch D, Cappello C, Meyer J, Simanowski K, Huber R, Brand K. CCAAT/enhancer-binding protein beta inhibits proliferation in monocytic cells by affecting the retinoblastoma protein/E2F/cyclin E pathway but is not directly required for macrophage morphology. J Biol Chem 2011; 286:22716-29. [PMID: 21558273 PMCID: PMC3123039 DOI: 10.1074/jbc.m110.152538] [Citation(s) in RCA: 31] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/15/2022] Open
Abstract
Monocytic differentiation is orchestrated by complex networks that are not fully understood. This study further elucidates the involvement of transcription factor CCAAT/enhancer-binding protein β (C/EBPβ). Initially, we demonstrated a marked increase in nuclear C/EBPβ-liver-enriched activating protein* (LAP*)/liver-enriched activating protein (LAP) levels and LAP/liver-enriched inhibiting protein (LIP) ratios in phorbol 12-myristate 13-acetate (PMA)-treated differentiating THP-1 premonocytic cells accompanied by reduced proliferation. To directly study C/EBPβ effects on monocytic cells, we generated novel THP-1-derived (low endogenous C/EBPβ) cell lines stably overexpressing C/EBPβ isoforms. Most importantly, cells predominantly overexpressing LAP* (C/EBPβ-long), but not those overexpressing LIP (C/EBPβ-short), exhibited a reduced proliferation, with no effect on morphology. PMA-induced inhibition of proliferation was attenuated in C/EBPβ-short cells. In C/EBPβWT macrophage-like cells (high endogenous C/EBPβ), we measured a reduced proliferation/cycling index compared with C/EBPβKO. The typical macrophage morphology was only observed in C/EBPβWT, whereas C/EBPβKO stayed round. C/EBPα did not compensate for C/EBPβ effects on proliferation/morphology. Serum reduction, an independent approach known to inhibit proliferation, induced macrophage morphology in C/EBPβKO macrophage-like cells but not THP-1. In PMA-treated THP-1 and C/EBPβ-long cells, a reduced phosphorylation of cell cycle repressor retinoblastoma was found. In addition, C/EBPβ-long cells showed reduced c-Myc expression accompanied by increased CDK inhibitor p27 and reduced cyclin D1 levels. Finally, C/EBPβ-long and C/EBPβWT cells exhibited low E2F1 and cyclin E levels, and C/EBPβ overexpression was found to inhibit cyclin E1 promoter-dependent transcription. Our results suggest that C/EBPβ reduces monocytic proliferation by affecting the retinoblastoma/E2F/cyclin E pathway and that it may contribute to, but is not directly required for, macrophage morphology. Inhibition of proliferation by C/EBPβ may be important for coordinated monocytic differentiation.
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Affiliation(s)
- Romina Gutsch
- Institute of Clinical Chemistry, Hannover Medical School, Carl-Neuberg-Strasse 1, 30625 Hannover, Germany
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21
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Preuss E, Treschow A, Newrzela S, Brücher D, Weber K, Felldin U, Alici E, Gahrton G, von Laer D, Dilber MS, Fehse B. TK.007: A novel, codon-optimized HSVtk(A168H) mutant for suicide gene therapy. Hum Gene Ther 2011; 21:929-41. [PMID: 20201626 DOI: 10.1089/hum.2009.042] [Citation(s) in RCA: 22] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/12/2022] Open
Abstract
Conditional elimination of infused gene-modified alloreactive T cells, using suicide gene activation, has been shown to be an efficient strategy to abrogate severe graft-versus-host disease (GvHD) in the context of adoptive immunotherapy. To overcome shortcomings of the most widely used suicide gene, wild-type (splice-corrected) herpes simplex virus thymidine kinase (scHSVtk), we generated two new variants: the codon-optimized coHSVtk and, by introducing an additional mutation (A168H), the novel TK.007. We transduced human hematopoietic cell lines and primary T cells with retroviral "sort-suicide vectors" encoding combinations of selection markers (tCD34 and OuaSelect) with one of three HSVtk variants. In vitro we observed higher expression levels and sustained long-term expression of TK.007, indicating lower nonspecific toxicity. Also, we noted significantly improved kinetics of ganciclovir (GCV)-mediated killing for TK.007-transduced cells. In an experimental (murine) allogeneic transplantation model, TK.007-transduced T cells mediated severe GvHD, which was readily abrogated by application of GCV (10 mg/kg). Last, we established a modified allotransplantation model that allowed quantitative comparison of the in vivo activities of TK.007 versus scHSVtk. We found that TK.007 mediates both significantly faster and higher absolute killing at low GCV concentrations (10 and 25 mg/kg). In summary, we demonstrate that the novel TK.007 suicide gene combines better killing performance with reduced nonspecific toxicity (as compared with the frequently used splice-corrected wild-type scHSVtk gene), thus representing a promising alternative for suicide gene therapy.
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Affiliation(s)
- Ellen Preuss
- Clinic for Stem Cell Transplantation, Research Department of Cell and Gene Therapy, University Medical Centre Hamburg-Eppendorf , 20246 Hamburg, Germany
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22
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Lange C, Brunswig-Spickenheier B, Cappallo-Obermann H, Eggert K, Gehling UM, Rudolph C, Schlegelberger B, Cornils K, Zustin J, Spiess AN, Zander AR. Radiation rescue: mesenchymal stromal cells protect from lethal irradiation. PLoS One 2011; 6:e14486. [PMID: 21245929 PMCID: PMC3016319 DOI: 10.1371/journal.pone.0014486] [Citation(s) in RCA: 84] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/26/2010] [Accepted: 12/07/2010] [Indexed: 12/16/2022] Open
Abstract
BACKGROUND Successful treatment of acute radiation syndromes relies on immediate supportive care. In patients with limited hematopoietic recovery potential, hematopoietic stem cell (HSC) transplantation is the only curative treatment option. Because of time consuming donor search and uncertain outcome we propose MSC treatment as an alternative treatment for severely radiation-affected individuals. METHODS AND FINDINGS Mouse mesenchymal stromal cells (mMSCs) were expanded from bone marrow, retrovirally labeled with eGFP (bulk cultures) and cloned. Bulk and five selected clonal mMSCs populations were characterized in vitro for their multilineage differentiation potential and phenotype showing no contamination with hematopoietic cells. Lethally irradiated recipients were i.v. transplanted with bulk or clonal mMSCs. We found a long-term survival of recipients with fast hematopoietic recovery after the transplantation of MSCs exclusively without support by HSCs. Quantitative PCR based chimerism analysis detected eGFP-positive donor cells in peripheral blood immediately after injection and in lungs within 24 hours. However, no donor cells in any investigated tissue remained long-term. Despite the rapidly disappearing donor cells, microarray and quantitative RT-PCR gene expression analysis in the bone marrow of MSC-transplanted animals displayed enhanced regenerative features characterized by (i) decreased proinflammatory, ECM formation and adhesion properties and (ii) boosted anti-inflammation, detoxification, cell cycle and anti-oxidative stress control as compared to HSC-transplanted animals. CONCLUSIONS Our data revealed that systemically administered MSCs provoke a protective mechanism counteracting the inflammatory events and also supporting detoxification and stress management after radiation exposure. Further our results suggest that MSCs, their release of trophic factors and their HSC-niche modulating activity rescue endogenous hematopoiesis thereby serving as fast and effective first-line treatment to combat radiation-induced hematopoietic failure.
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Affiliation(s)
- Claudia Lange
- Clinic for Stem Cell Transplantation, Department of Cell and Gene Therapy, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.
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23
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Ginn SL, Liao SHY, Dane AP, Hu M, Hyman J, Finnie JW, Zheng M, Cavazzana-Calvo M, Alexander SI, Thrasher AJ, Alexander IE. Lymphomagenesis in SCID-X1 mice following lentivirus-mediated phenotype correction independent of insertional mutagenesis and gammac overexpression. Mol Ther 2010; 18:965-76. [PMID: 20354504 PMCID: PMC2890120 DOI: 10.1038/mt.2010.50] [Citation(s) in RCA: 44] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/04/2010] [Accepted: 03/02/2010] [Indexed: 12/13/2022] Open
Abstract
The development of leukemia as a consequence of vector-mediated genotoxicity in gene therapy trials for X-linked severe combined immunodeficiency (SCID-X1) has prompted substantial research effort into the design and safety testing of integrating vectors. An important element of vector design is the selection and evaluation of promoter-enhancer elements with sufficient strength to drive reliable immune reconstitution, but minimal propensity for enhancer-mediated insertional mutagenesis. In this study, we set out to explore the effect of promoter-enhancer selection on the efficacy and safety of human immunodeficiency virus-1-derived lentiviral vectors in gammac-deficient mice. We observed incomplete or absent T- and B-cell development in mice transplanted with progenitors expressing gammac from the phosphoglycerate kinase (PGK) and Wiscott-Aldrich syndrome (WAS) promoters, respectively. In contrast, functional T- and B-cell compartments were restored in mice receiving an equivalent vector containing the elongation factor-1-alpha (EF1alpha) promoter; however, 4 of 14 mice reconstituted with this vector subsequently developed lymphoma. Extensive analyses failed to implicate insertional mutagenesis or gammac overexpression as the underlying mechanism. These findings highlight the need for detailed mechanistic analysis of tumor readouts in preclinical animal models assessing vector safety, and suggest the existence of other ill-defined risk factors for oncogenesis, including replicative stress, in gene therapy protocols targeting the hematopoietic compartment.
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Affiliation(s)
- Samantha L Ginn
- Gene Therapy Research Unit of the Children's Medical Research Institute and The Children's Hospital at Westmead, Westmead, New South Wales, Australia
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24
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Lehmann R, Meyer J, Schuemann M, Krause E, Freund C. A novel S3S-TAP-tag for the isolation of T-cell interaction partners of adhesion and degranulation promoting adaptor protein. Proteomics 2010; 9:5288-95. [PMID: 19798671 DOI: 10.1002/pmic.200900294] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/11/2022]
Abstract
The identification of modular units of cellular function is a major goal for proteomic research. Protein complexes represent important building blocks defining functionality and deciphering their composition remains a major challenge. Here, we have designed a new tandem affinity purification (TAP) tag (termed S3S-tag) for the isolation of protein complexes. Specifically, the immune cell protein ADAP that regulates integrin adhesion was fused either C- or N-terminally to the S3S-tag. After retroviral transduction of a vector containing S3S-tagged ADAP and internal ribosomal entry site encoded enhanced green fluorescent protein (eGFP), Jurkat T cells were sorted according to eGFP expression and further selected for expression of TAP-tagged protein close to endogenous levels. The combination of a cleavable S-tag and a Strep-tag II allowed for the isolation of ADAP and associated proteins. Subsequently, stable isotope labeling with amino acids in cell culture-based mass spectrometric analysis was performed to identify potentially specific interaction partners. Co-purification of the known interaction partner Src kinase-associated phosphoprotein of 55 kDa indicates the validity of our approach, while the identification of the ENA/VASP family member EVL, the guanine nucleotide exchange factor GEF-H1 and the adaptor protein DOCK2 corroborates a link between ADAP-mediated integrin regulation and the cytoskeleton.
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Affiliation(s)
- Roland Lehmann
- Leibniz-Institute for Molecular Pharmacology, Berlin, Germany
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25
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Wicke DC, Meyer J, Buesche G, Heckl D, Kreipe H, Li Z, Welte KH, Ballmaier M, Baum C, Modlich U. Gene therapy of MPL deficiency: challenging balance between leukemia and pancytopenia. Mol Ther 2009; 18:343-52. [PMID: 19844195 DOI: 10.1038/mt.2009.233] [Citation(s) in RCA: 23] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/18/2022] Open
Abstract
Signaling of the thrombopoietin (THPO) receptor MPL is critical for the maintenance of hematopoietic stem cells (HSCs) and megakaryocytic differentiation. Inherited loss-of-function mutations of MPL cause severe thrombocytopenia and aplastic anemia, a syndrome called congenital amegakaryocytic thrombocytopenia (CAMT). With the aim to assess the toxicity of retroviral expression of Mpl as a basis for further development of a gene therapy for this disorder, we expressed Mpl in a murine bone marrow transplantation (BMT) model. Treated mice developed a profound yet transient elevation of multilineage hematopoiesis, which showed morphologic features of a chronic myeloproliferative disorder (CMPD) with progressive pancytopenia. Ten percent of mice (3/27) developed erythroleukemia, associated with insertional activation of Sfpi1 and Fli1. The majority of transplanted mice developed a progressive pancytopenia with histopathological features of a myelodysplastic syndrome (MDS)-like disorder. To avoid these adverse reactions, improved retroviral vectors were designed that mediate reduced and more physiological Mpl expression. Self-inactivating gamma-retroviral vectors were constructed that expressed Mpl from the phosphoglycerate kinase (PGK) or the murine Mpl promoter. Mice that received BM cells expressing Mpl from the Mpl promoter were free of any previously observed adverse reactions.
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Affiliation(s)
- Daniel C Wicke
- Department of Experimental Hematology, Hannover Medical School, Hannover, Germany
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26
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Modlich U, Navarro S, Zychlinski D, Maetzig T, Knoess S, Brugman MH, Schambach A, Charrier S, Galy A, Thrasher AJ, Bueren J, Baum C. Insertional transformation of hematopoietic cells by self-inactivating lentiviral and gammaretroviral vectors. Mol Ther 2009; 17:1919-28. [PMID: 19672245 DOI: 10.1038/mt.2009.179] [Citation(s) in RCA: 304] [Impact Index Per Article: 19.0] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/26/2022] Open
Abstract
Gene transfer vectors may cause clonal imbalance and even malignant cell transformation by insertional upregulation of proto-oncogenes. Lentiviral vectors (LV) with their preferred integration in transcribed genes are considered less genotoxic than gammaretroviral vectors (GV) with their preference for integration next to transcriptional start sites and regulatory gene regions. Using a sensitive cell culture assay and a series of self-inactivating (SIN) vectors, we found that the lentiviral insertion pattern was approximately threefold less likely than the gammaretroviral to trigger transformation of primary hematopoietic cells. However, lentivirally induced mutants also showed robust replating, in line with the selection for common insertion sites (CIS) in the first intron of the Evi1 proto-oncogene. This potent proto-oncogene thus represents a CIS for both GV and LV, despite major differences in their integration mechanisms. Altering the vectors' enhancer-promoter elements had a greater effect on safety than the retroviral insertion pattern. Clinical grade LV expressing the Wiskott-Aldrich syndrome (WAS) protein under control of its own promoter had no transforming potential. Mechanistic studies support the conclusion that enhancer-mediated gene activation is the major cause for insertional transformation of hematopoietic cells, opening rational strategies for risk prevention.
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Affiliation(s)
- Ute Modlich
- Department of Experimental Hematology, Hannover Medical School, Hannover, Germany
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27
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Millington M, Arndt A, Boyd M, Applegate T, Shen S. Towards a clinically relevant lentiviral transduction protocol for primary human CD34 hematopoietic stem/progenitor cells. PLoS One 2009; 4:e6461. [PMID: 19649289 PMCID: PMC2714083 DOI: 10.1371/journal.pone.0006461] [Citation(s) in RCA: 39] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/11/2009] [Accepted: 06/19/2009] [Indexed: 11/21/2022] Open
Abstract
Background Hematopoietic stem cells (HSC), in particular mobilized peripheral blood stem cells, represent an attractive target for cell and gene therapy. Efficient gene delivery into these target cells without compromising self-renewal and multi-potency is crucial for the success of gene therapy. We investigated factors involved in the ex vivo transduction of CD34+ HSCs in order to develop a clinically relevant transduction protocol for gene delivery. Specifically sought was a protocol that allows for efficient transduction with minimal ex vivo manipulation without serum or other reagents of animal origin. Methodology/Principal Findings Using commercially available G-CSF mobilized peripheral blood (PB) CD34+ cells as the most clinically relevant target, we systematically examined factors including the use of serum, cytokine combinations, pre-stimulation time, multiplicity of infection (MOI), transduction duration and the use of spinoculation and/or retronectin. A self-inactivating lentiviral vector (SIN-LV) carrying enhanced green fluorescent protein (GFP) was used as the gene delivery vehicle. HSCs were monitored for transduction efficiency, surface marker expression and cellular function. We were able to demonstrate that efficient gene transduction can be achieved with minimal ex vivo manipulation while maintaining the cellular function of transduced HSCs without serum or other reagents of animal origin. Conclusions/Significance This study helps to better define factors relevant towards developing a standard clinical protocol for the delivery of SIN-LV into CD34+ cells.
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Affiliation(s)
| | - Allison Arndt
- Johnson and Johnson Research Pty Ltd., Eveleigh, New South Wales, Australia
| | - Maureen Boyd
- Johnson and Johnson Research Pty Ltd., Eveleigh, New South Wales, Australia
| | - Tanya Applegate
- Johnson and Johnson Research Pty Ltd., Eveleigh, New South Wales, Australia
| | - Sylvie Shen
- Johnson and Johnson Research Pty Ltd., Eveleigh, New South Wales, Australia
- * E-mail:
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Wyss BK, Donnelly AFW, Zhou D, Sinn AL, Pollok KE, Goebel WS. Enhanced homing and engraftment of fresh but not ex vivo cultured murine marrow cells in submyeloablated hosts following CD26 inhibition by Diprotin A. Exp Hematol 2009; 37:814-23. [PMID: 19540435 PMCID: PMC2700780 DOI: 10.1016/j.exphem.2009.03.005] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/19/2008] [Revised: 03/13/2009] [Accepted: 03/16/2009] [Indexed: 11/17/2022]
Abstract
OBJECTIVE We recently reported that murine marrow cultured ex vivo for gamma-retrovirus transduction engrafts approximately 10-fold less well than fresh marrow upon transplantation into submyeloablated hosts. Here, we evaluated homing efficiency as a potential mechanism for this engraftment disparity, and whether CD26 inhibition with the tripeptide Diprotin A (DipA) would enhance engraftment of ex vivo cultured cells in submyeloablated hosts. MATERIALS AND METHODS Homing and engraftment of fresh and ex vivo cultured lineage-negative (lin(-)) marrow cells in submyeloablated congenic hosts with and without DipA treatment was evaluated. Expression of CXCR4 and CD26 on fresh and cultured lin(-) marrow cells was compared. RESULTS Homing of lin(-) cells cultured for gamma-retrovirus transduction was at least threefold less than that of fresh lin(-) cells 20 hours after transplantation into submyeloablated hosts. DipA treatment of fresh lin(-) cells resulted in at least twofold increased homing and engraftment in submyeloablated hosts. DipA treatment, however, did not significantly improve homing or engraftment of cells undergoing a 3-day culture protocol for gamma-retrovirus transduction in submyeloablated hosts. CXCR4 expression on lin(-) cells was significantly decreased following 3 days of culture; CXCR4 expression was not significantly altered following overnight culture. CONCLUSIONS Ex vivo culture of lin(-) cells for gamma-retroviral transduction downregulates CXCR4 expression and markedly impairs homing and engraftment of murine lin(-) marrow in submyeloablated hosts. While inhibition of CD26 activity with DipA increases homing and engraftment of fresh lin(-) cells, DipA treatment does not improve homing and engraftment of cultured lin(-) marrow cells in submyeloablated congenic hosts.
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Affiliation(s)
- Brandon K. Wyss
- Herman B Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, IN, USA
| | - Abigail F. W. Donnelly
- Herman B Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, IN, USA
| | - Dan Zhou
- Herman B Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, IN, USA
| | - Anthony L. Sinn
- Herman B Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, IN, USA
| | - Karen E. Pollok
- Herman B Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, IN, USA
- Department of Pediatrics, Section of Pediatric Hematology/Oncology, James Whitcomb Riley Hospital for Children, Indiana University School of Medicine, Indianapolis, IN, USA
- Department of Pharmacology and Toxicology, Indiana University School of Medicine, Indianapolis, IN, USA
| | - W. Scott Goebel
- Herman B Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, IN, USA
- Department of Pediatrics, Section of Pediatric Hematology/Oncology, James Whitcomb Riley Hospital for Children, Indiana University School of Medicine, Indianapolis, IN, USA
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Cornils K, Lange C, Schambach A, Brugman MH, Nowak R, Lioznov M, Baum C, Fehse B. Stem cell marking with promotor-deprived self-inactivating retroviral vectors does not lead to induced clonal imbalance. Mol Ther 2009; 17:131-43. [PMID: 19002163 PMCID: PMC2834973 DOI: 10.1038/mt.2008.238] [Citation(s) in RCA: 29] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/09/2008] [Accepted: 09/30/2008] [Indexed: 12/19/2022] Open
Abstract
Stable genetic modification of stem cells holds great promise for gene therapy and marking, but commonly used gamma-retroviral vectors were found to influence growth/survival characteristics of hematopoietic stem cells (HSCs) by insertional mutagenesis. In this article, we show that promoter-deprived gamma-retroviral self-inactivating (pd-SIN) vectors allow stable genetic marking of serially reconstituting murine HSC. In contrast to findings with gamma-retroviral long terminal repeat (LTR) vectors, serial transplantation of pd-SIN-marked HSC in a sensitive mouse model was apparently not associated with induced clonal imbalance of gene-marked HSC. Furthermore, insertions of pd-SIN into protooncogenes, growth-promoting and signaling genes occurred significantly less frequent than in control experiments with LTR vectors. Also, transcriptional dysregulation of neighboring genes potentially caused by the pd-SIN insertion was rarely seen and comparatively weak. The integration pattern of promotor-deprived SIN vectors in reconstituting HSC seems to depend on the transcriptional activity of the respective gene loci reflecting the picture described for LTR vectors. In conclusion, our data strongly support the use of SIN vectors for gene-marking studies and suggest an increased therapeutic index for vectors lacking enhancers active in HSC.
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Affiliation(s)
- Kerstin Cornils
- Experimental Pediatric Oncology and Hematology, Pediatric Clinic III, University Hospital of the Johann Wolfgang Goethe-University, Frankfurt am Main, Germany
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30
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Modlich U, Schambach A, Li Z, Schiedlmeier B. Murine hematopoietic stem cell transduction using retroviral vectors. Methods Mol Biol 2009; 506:23-31. [PMID: 19110617 DOI: 10.1007/978-1-59745-409-4_3] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/18/2022]
Abstract
Hematopoietic stem cells (HSCs) represent an important target cell population in bone marrow transplantation and gene therapy applications. Their progeny cells carry the genetic information of the HSCs and replenish the blood and immune system. Therefore, in the setting of inherited diseases, transduction of HSCs with retroviral vectors (including gammaretro- and lentiviral vectors) offers the possibility to correct the phenotype in all blood lineages as demonstrated in clinical trials for immunodeficiencies (e.g., X-SCID). In the process of developing gene therapy strategies for patient applications, suitable mouse models for the human gene therapy are important to validate the concept. Stem-cell-enriched populations such as lineage negative cells as the functional equivalent of human CD34(+) cells can be isolated from murine bone marrow and efficiently transduced using retroviral vectors. This chapter provides a step-by-step protocol for retroviral transduction of murine lineage negative cells.
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Affiliation(s)
- Ute Modlich
- Department of Experimental Hematology, Hannover Medical School, Hannover, Germany
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31
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Wyss BK, Meyers JL, Sinn AL, Cai S, Pollok KE, Goebel WS. A novel competitive repopulation strategy to quantitate engraftment of ex vivo manipulated murine marrow cells in submyeloablated hosts. Exp Hematol 2008; 36:513-21. [PMID: 18243491 PMCID: PMC2373923 DOI: 10.1016/j.exphem.2007.12.002] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/23/2007] [Revised: 11/26/2007] [Accepted: 12/04/2007] [Indexed: 01/19/2023]
Abstract
OBJECTIVE Standard competitive repopulation assays have proven valuable in evaluating engraftment potential in ablated hosts, permitting comparisons between various test cell populations. However, no similar method exists to compare engraftment of test cells in submyeloablated hosts, which would be helpful given the applications of reduced-intensity conditioning for hematopoietic gene-replacement therapy and other cellular therapies. Here, we developed a novel assay to quantitate engraftment of hematopoietic stem cells in submyeloablated hosts. MATERIALS AND METHODS Engraftment of murine marrow cells transduced with retroviral vectors using two separate protocols was compared to engraftment of fresh untreated competitor cells within low-dose radiation-conditioned hosts using a "three-way" marking system, so that test, competitor, and host cell chimerism could be reliably determined posttransplantation. RESULTS We demonstrate that the repopulating ability of marrow cells transduced using two distinct protocols was reduced approximately 10-fold compared to fresh competitor cells in submyeloablated hosts utilizing the novel "three-way" transplant assay. CONCLUSIONS Murine marrow cells transduced using a clinically applicable protocol acquire an engraftment defect in submyeloablated hosts, similar to cells transduced using a research protocol. We conclude that the submyeloablative competitive repopulation assay described here will be of benefit to comparatively assess the engraftment ability of manipulated hematopoietic stem cells using various culture protocols, such as to test the impact of modifications in transduction protocols needed to attain therapeutic levels of gene-corrected blood cells, or the effect of ex vivo expansion protocols on engraftment potential.
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Affiliation(s)
- Brandon K. Wyss
- Herman B Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, Indiana
| | - Justin L. Meyers
- Herman B Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, Indiana
| | - Anthony L. Sinn
- Herman B Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, Indiana
| | - Shanbao Cai
- Herman B Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, Indiana
| | - Karen E. Pollok
- Herman B Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, Indiana
- Department of Pediatrics, Section of Pediatric Hematology/Oncology, James Whitcomb Riley Hospital for Children, Indiana University School of Medicine, Indianapolis, Indiana
| | - W. Scott Goebel
- Herman B Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, Indiana
- Department of Pediatrics, Section of Pediatric Hematology/Oncology, James Whitcomb Riley Hospital for Children, Indiana University School of Medicine, Indianapolis, Indiana
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32
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Templin C, Kotlarz D, Rathinam C, Rudolph C, Schätzlein S, Ramireddy K, Rudolph KL, Schlegelberger B, Klein C, Drexler H. Establishment of immortalized multipotent hematopoietic progenitor cell lines by retroviral-mediated gene transfer of beta-catenin. Exp Hematol 2008; 36:204-15. [PMID: 18206728 DOI: 10.1016/j.exphem.2007.10.005] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/18/2007] [Revised: 10/22/2007] [Accepted: 10/25/2007] [Indexed: 12/15/2022]
Abstract
OBJECTIVE Hematopoietic stem cells (HSCs) are characterized by their ability to give rise to all mature blood lineages and to renew themselves. In the present study, we show that beta-catenin plays an essential role in the immortalization of hematopoietic progenitor cells (HPCs). MATERIALS AND METHODS Cellular, molecular, and cytogenetic properties of the immortalized HPCs were determined using flow cytometry (immunophenotyping), microscopy (telomere length, spectral karyotyping), telomere repeat amplification protocol assay (telomerase activity), real-time polymerase chain reaction (expression studies), and in vitro and in vivo differentiation assays. RESULTS Retroviral-mediated overexpression of human beta-catenin in lin(-)- and lin(-)Sca-1+c-kit+ hematopoietic cells led to generation of novel, murine interleukin-3-, and stem cell factor-dependent hematopoietic multipotent progenitor cell lines (DK1mix and DK2mix) with stable surface antigen expression of the stem cell markers Sca-1 and c-kit (>92%) in long-term culture. Further, immunophenotypic characterization revealed a CD244+CD150(-)CD48(-) phenotype of DKmix cells, which is consistent with the expression profile of multipotential hematopoietic progenitors. Upon exposure to selective hematopoietic cytokines in vitro, and upon transplantation into irradiated congenic recipients, DKmix cells generated progenies of lymphoid and myeloid lineages. Continuous in vitro proliferation and expansion of DKmix cells were associated with stabilized telomere length and consistent telomerase activity. Interestingly, constitutive expression of beta-catenin was not required for sustained long-term viability and proliferation of immortalized DKmix cells. CONCLUSION In summary, our findings define beta-catenin as an important regulator in HPC self-renewal and function. Further, our results distinguish the importance of beta-catenin in the immortalization process of HPCs, from its dispensable role in their maintenance.
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Affiliation(s)
- Christian Templin
- Department of Cardiology and Angiology, Hannover Medical School, Hannover, Germany.
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33
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Abstract
The possible activation of cellular proto-oncogenes as a result of clonal transformation is a potential limitation in a therapeutic approach involving random integration of gene vectors. Given that enhancer promiscuity represents an important mechanism of insertional transformation, we assessed the enhancer activities of various cellular and retroviral promoters in transient transfection assays, and also in a novel experimental system designed to measure the activation of a minigene cassette contained in stably integrating retroviral vectors. Retroviral enhancer-promoters showed a significantly greater potential to activate neighboring promoters than did cellular promoters derived from human genes, elongation factor-1alpha (EF1alpha) and phosphoglycerate kinase (PGK). Self-inactivating (SIN) vector design reduced but did not abolish enhancer interactions. Using a recently established cell culture assay that detects insertional transformation by serial replating of primary hematopoietic cells, we found that SIN vectors containing the EF1alpha promoter greatly decrease the risk of insertional transformation. Despite integration of multiple copies per cell, activation of the crucial proto-oncogene Evi1 was not detectable when using SIN-EF1alpha vectors. On the basis of several quantitative indicators, the decrease in transforming activity was highly significant (more than tenfold, P < 0.01) when compared with similarly designed vectors containing a retroviral enhancer-promoter with or without a well-characterized genetic insulator core element. In this manner, the insertional biosafety of therapeutic gene vectors can be greatly enhanced and proactively evaluated in sensitive cell-based assays.
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34
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35
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Beaudoin EL, Bais AJ, Junghans RP. Sorting vector producer cells for high transgene expression increases retroviral titer. J Virol Methods 2008; 148:253-9. [PMID: 18249448 DOI: 10.1016/j.jviromet.2007.12.008] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/13/2006] [Revised: 12/11/2007] [Accepted: 12/13/2007] [Indexed: 11/15/2022]
Abstract
Vector producer cells are derived from helper cell lines expressing viral proteins that have been transduced to express a transgene-carrying retroviral genome. Vector producing cells express two relevant forms of RNA in their cytoplasm: vector RNA (vRNA) that is packaged as the actual gene transfer agent, and messenger RNA (mRNA) from which transgene is translated. Two premises underlie this study: (1) vRNA is limiting for virus production and (2) mRNA is proportional to vRNA. Together, these premises predict that transgene expression in the vector producing cells will be predictive of the viral titer from those cells. In this case, sorting the vector producing cells for high transgene expression should select for more virus production in vector producing cell supernatants. This prediction was supported, with a greater than fivefold benefit in viral titer. This demonstrates a rapid and simple method by which to obtain significantly increased viral titers from the same vector producing cell preparation.
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Affiliation(s)
- E L Beaudoin
- Boston University School of Medicine, Department of Surgical Research, North Campus, Roger Williams Medical Center, 825 Chalkstone Avenue, Providence, RI 02908, USA
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36
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Meyer J, Rhein M, Schiedlmeier B, Kustikova O, Rudolph C, Kamino K, Neumann T, Yang M, Wahlers A, Fehse B, Reuther GW, Schlegelberger B, Ganser A, Baum C, Li Z. Remarkable leukemogenic potency and quality of a constitutively active neurotrophin receptor, ΔTrkA. Leukemia 2007; 21:2171-80. [PMID: 17673903 DOI: 10.1038/sj.leu.2404882] [Citation(s) in RCA: 31] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/09/2022]
Abstract
Neurotrophins and their receptors play a key role in neurogenesis and survival. However, we and others have recently obtained evidence for a potential involvement of this receptor system in leukemia. To investigate mechanisms underlying the leukemogenic potential of activated neurotrophin receptor signaling, we analyzed in vivo leukemogenesis mediated by deltaTrkA, a mutant of TRKA (tropomyosin-related kinase A) isolated from a patient with acute myeloid leukemia (AML). Retroviral expression of deltaTrkA in myeloid 32D cells induced AML in syngeneic C3H/Hej mice (n=11/11, latency approximately 4 weeks). C57Bl/6J mice transplanted with deltaTrkA-transduced primary lineage negative (Lin-) bone marrow cells died of a transient polyclonal AML (n=7/15, latency of <12 days). Serial transplantation of AML cells did not re-induce this disease but rather acute lymphoblastic leukemia (ALL, latency >78 days). All primary recipients surviving the early AML developed clonal ALL or myeloid leukemia (latency >72 days) that required additional genetic lesions. PI3K and mTOR-raptor were identified as the crucial mediators of leukemic transformation, whereas STAT and MAP kinase signaling pathways were not activated. Thus, our findings reveal potent and unique transforming properties of altered neurotrophin receptor signaling in leukemogenesis, and encourage further analyses of neurotrophin receptors and downstream signaling events in hematological malignancies.
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Affiliation(s)
- J Meyer
- Department of Experimental Hematology, Hannover Medical School, Hannover, Germany
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37
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Li Z, Kustikova OS, Kamino K, Neumann T, Rhein M, Grassman E, Fehse B, Baum C. Insertional Mutagenesis by Replication-Deficient Retroviral Vectors Encoding the Large T Oncogene. Ann N Y Acad Sci 2007; 1106:95-113. [PMID: 17395733 DOI: 10.1196/annals.1392.003] [Citation(s) in RCA: 15] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/12/2022]
Abstract
Insertion sites of replication-deficient retroviral vectors may trigger clonal dominance of hematopoietic cells in vivo. Here, we tested whether this would also be the case when using vectors that express powerful oncogenes, such as the large tumor antigen (TAg) of simian virus 40. TAg inactivates the tumor-suppressor proteins p53 and Rb by virtue of a chaperone-like activity. Primary hematopoietic stem/progenitor cells transduced with retroviral vectors encoding TAg-induced histiocytic sarcoma (HS) or myeloid leukemia (ML) in transplanted mice (average survival of 21 weeks). Retrovirally introducing TAg into pretransformed 32D cells generated a monocytic leukemia, with faster kinetics ( approximately 8 weeks). Leukemic clones showed retroviral insertions in genes contributing to all known TAg cooperation pathways, acting mitogenic and/or modulating apoptosis (such as BclX, Crk, Pim2, Csfr1/Pdgfrb, Osm/Lif, Axl, Fli, Sema4b, Sox4). 32D-derived monocytic leukemias showed hits in Pim2 and Max proto-oncogenes, or the chaperone Hspa4, plus additional signaling genes. Vector-mediated insertional mutagenesis thus revealed a broad spectrum of potential TAg complementation genes. These findings have important implications for the use of retroviral transgenesis in cancer research, and the expression of signaling genes in somatic gene therapy.
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Affiliation(s)
- Zhixiong Li
- Department of Experimental Hematology, OE6960, Hannover Medical School, Carl-Neuberg-Strasse 1, 30625 Hannover, Germany
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38
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Schambach A, Galla M, Maetzig T, Loew R, Baum C. Improving transcriptional termination of self-inactivating gamma-retroviral and lentiviral vectors. Mol Ther 2007; 15:1167-73. [PMID: 17406345 DOI: 10.1038/sj.mt.6300152] [Citation(s) in RCA: 91] [Impact Index Per Article: 5.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/07/2023] Open
Abstract
Adverse events relating to insertional mutagenesis have reinforced the interest in self-inactivating (SIN) gamma-retroviral and lentiviral vectors without enhancer-promoter sequences in the U3 region of the long terminal repeats. However, SIN vectors suffer from leaky transcriptional termination, increasing the probability of read-through into cellular genes. To improve 3' end processing, we incorporated seven upstream polyadenylation enhancer elements (or upstream sequence elements, USEs) derived from viral or cellular genes into the 3' U3 region of gamma-retroviral and lentiviral SIN vectors. A 100-base-pair sequence representing a recombinant direct repeat of the USE derived from simian virus 40 (2xSV USE) gave the best results, improving both titer and gene expression. In both gamma-retroviral and lentiviral SIN vectors, the 2xSV USE partially substituted for effects provided by the much larger post-transcriptional regulatory element derived from woodchuck hepatitis virus (wPRE). By northern blot and reporter assays, we found that the 2xSV USE greatly improved proper messenger RNA (mRNA) processing at the retroviral termination signal. Importantly, the 2xSV USE was superior to the wPRE in suppressing transcriptional read-through, improving not only vector efficiency but potentially also biosafety.
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Affiliation(s)
- Axel Schambach
- 1Department of Experimental Hematology, Hannover Medical School, Hannover, Germany
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39
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Lange C, Li Z, Fang L, Baum C, Fehse B. CD34 Modulates the Trafficking Behavior of Hematopoietic CellsIn Vivo. Stem Cells Dev 2007; 16:297-304. [PMID: 17521240 DOI: 10.1089/scd.2006.0056] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/14/2022] Open
Abstract
The CD34 surface antigen has been recognized as a marker of hematopoietic stem cells (HSCs) and is widely used for HSC selection as well as for quality control in HSC transplantation. CD34 has been implicated in cytoadhesion signaling, and its expression has been suggested to reflect the activation state of hematopoietic progenitor cells. However, the function of CD34 remains essentially unknown. Here we analyzed the effects of ectopic CD34 expression in vivo in a bone marrow transplantation model. We transduced murine bone marrow stem cells with retroviral vectors encoding either murine full-length or the alternative splice product truncated CD34. Transduced cells were transplanted into syngeneic, marrow ablated hosts. For comparison, "control" animals received either enhanced green fluorescent protein (eGFP)-transduced or mock-transduced cells. Six months post-transplantation, transduced differentiated blood cells ectopically expressing murine CD34 showed decreased migration from peripheral blood to both bone marrow and thymus, an effect that was more pronounced with full-length CD34 than with the truncated variant. In contrast, no influence of transgene expression on trafficking of differentiated blood cells was seen in the eGFP control group. Our data indicate that CD34 expression in mature blood cells has a suppressive effect on cellular trafficking to hematopoietic stroma organs, thereby supporting a modulating role of the CD34 molecule in cytoadhesion.
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Affiliation(s)
- Claudia Lange
- Bone Marrow Transplantation, University Medical Center Hamburg-Eppendorf, 20246 Hamburg, Germany
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40
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Will E, Bailey J, Schuesler T, Modlich U, Balcik B, Burzynski B, Witte D, Layh-Schmitt G, Rudolph C, Schlegelberger B, von Kalle C, Baum C, Sorrentino BP, Wagner LM, Kelly P, Reeves L, Williams DA. Importance of murine study design for testing toxicity of retroviral vectors in support of phase I trials. Mol Ther 2007; 15:782-91. [PMID: 17299409 DOI: 10.1038/sj.mt.6300083] [Citation(s) in RCA: 25] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/17/2023] Open
Abstract
Although retroviral vectors are one of the most widely used vehicles for gene transfer, there is no uniformly accepted pre-clinical model defined to assess their safety, in particular their risk related to insertional mutagenesis. In the murine pre-clinical study presented here, 40 test and 10 control mice were transplanted with ex vivo manipulated bone marrow cells to assess the long-term effects of the transduction of hematopoietic cells with the retroviral vector MSCV-MGMT(P140K)wc. Test mice had significant gene marking 8-12 months post-transplantation with an average of 0.93 vector copies per cell and 41.5% of peripheral blood cells expressing the transgene MGMT(P140K), thus confirming persistent vector expression. Unexpectedly, six test mice developed malignant lymphoma. No vector was detected in the tumor cells of five animals with malignancies, indicating that the malignancies were not caused by insertional mutagenesis or MGMT(P140K) expression. Mice from a concurrent study with a different transgene also revealed additional cases of vector-negative lymphomas of host origin. We conclude that the background tumor formation in this mouse model complicates safety determination of retroviral vectors and propose an improved study design that we predict will increase the relevance and accuracy of interpretation of pre-clinical mouse studies.
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Affiliation(s)
- Elke Will
- Division of Experimental Hematology, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio 45229, USA
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41
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Modlich U, Bohne J, Schmidt M, von Kalle C, Knöss S, Schambach A, Baum C. Cell-culture assays reveal the importance of retroviral vector design for insertional genotoxicity. Blood 2006; 108:2545-53. [PMID: 16825499 PMCID: PMC1895590 DOI: 10.1182/blood-2005-08-024976] [Citation(s) in RCA: 266] [Impact Index Per Article: 14.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/17/2023] Open
Abstract
Retroviral vectors with long terminal repeats (LTRs), which contain strong enhancer/promoter sequences at both ends of their genome, are widely used for stable gene transfer into hematopoietic cells. However, recent clinical data and mouse models point to insertional activation of cellular proto-oncogenes as a dose-limiting side effect of retroviral gene delivery that potentially induces leukemia. Self-inactivating (SIN) retroviral vectors do not contain the terminal repetition of the enhancer/promoter, theoretically attenuating the interaction with neighboring cellular genes. With a new assay based on in vitro expansion of primary murine hematopoietic cells and selection in limiting dilution, we showed that SIN vectors using a strong internal retroviral enhancer/promoter may also transform cells by insertional mutagenesis. Most transformed clones, including those obtained after dose escalation of SIN vectors, showed insertions upstream of the third exon of Evi1 and in reverse orientation to its transcriptional orientation. Normalizing for the vector copy number, we found the transforming capacity of SIN vectors to be significantly reduced when compared with corresponding LTR vectors. Additional modifications of SIN vectors may further increase safety. Improved cell-culture assays will likely play an important role in the evaluation of insertional mutagenesis.
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Affiliation(s)
- Ute Modlich
- Department of Experimental Hematology, Hannover Medical School, Carl-Neuberg-Strasse 1, D-30625 Hannover, Germany
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42
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Schambach A, Galla M, Modlich U, Will E, Chandra S, Reeves L, Colbert M, Williams DA, von Kalle C, Baum C. Lentiviral vectors pseudotyped with murine ecotropic envelope: increased biosafety and convenience in preclinical research. Exp Hematol 2006; 34:588-92. [PMID: 16647564 DOI: 10.1016/j.exphem.2006.02.005] [Citation(s) in RCA: 91] [Impact Index Per Article: 4.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/09/2005] [Revised: 02/06/2006] [Accepted: 02/06/2006] [Indexed: 12/14/2022]
Abstract
OBJECTIVE Lentiviral vectors are increasingly used for preclinical models of gene therapy and other forms of experimental transgenesis. Due to the broad tropism and the ability for concentration by ultracentrifugation, most lentiviral vector preparations are produced using the vesicular stomatitis virus glycoprotein (VSV-g) protein as envelope. Recently, Hanawa and colleagues have demonstrated that the ecotropic envelope protein of murine leukemia viruses allows efficient pseudotyping of HIV-1-derived vector particles. However, this method has found little acceptance, despite potential advantages. MATERIALS AND METHODS We produced lentiviral vectors pseudotyped with murine ecotropic envelope using a four-plasmid transient transfection system and evaluated their performance in murine fibroblasts and hematopoietic cells. RESULTS Titers of lentiviral "ecotropic" supernatants were only slightly lower than those produced with VSV-g, could be concentrated by overnight centrifugation (13,000g), and efficiently transduced murine fibroblasts and hematopoietic cells but not human cells. Our Institutional Biosafety Committee agreed on the production and use of replication-defective lentiviral vectors pseudotyped with murine ecotropic envelope under biosafety level 1 (BL1) conditions with additional BL2 practices. We also obtained useful guidelines for the work with human infectious lentiviral vectors. CONCLUSIONS For the researcher, "ecotropic" lentiviral vectors significantly improve the convenience of daily work, compared to the conditions required for lentiviral pseudotypes that are capable of infecting human cells. High efficiency and superior biosafety in combination with convenient handling will certainly boost the potential applicability of this important vector system.
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Affiliation(s)
- Axel Schambach
- Department of Hematology, Hemostaseology and Oncology, Hannover Medical School, Germany
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Kurre P, Anandakumar P, Kiem HP. Rapid 1-hour transduction of whole bone marrow leads to long-term repopulation of murine recipients with lentivirus-modified hematopoietic stem cells. Gene Ther 2006; 13:369-73. [PMID: 16208421 DOI: 10.1038/sj.gt.3302659] [Citation(s) in RCA: 25] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Abstract
Efficient gene transfer to hematopoietic stem cells by Moloney murine leukemia virus-derived retroviral vectors benefits from ex vivo culture and cytokine support. Both also increase the risks of apoptosis and differentiation among cells targeted for transduction. In an effort to maximize the retention of stem cell properties in target cells, we developed a transduction protocol with a focus on minimizing graft manipulation, cytokine stimulation, and ex vivo exposure duration. Based on their wide host range and ability to transduce quiescent cells, human immunodeficiency virus (HIV)-derived lentivirus vectors are ideally suited for this purpose. Our present studies in a murine model show that whole bone marrow cells are readily transduced after a 1-hour vector exposure in the presence of stem cell factor and CH296 fibronectin fragment. Using this rapid transduction protocol, we achieved long-term, multilineage reconstitution of murine recipients with up to 25% GFP-expressing cells in primary and secondary recipients. Our results demonstrate the unique ability of HIV-derived vectors to transduce hematopoietic stem cells in the absence of enrichment, under minimal cytokine stimulation, and following brief exposures.
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Affiliation(s)
- P Kurre
- Department of Pediatrics, Oregon Health & Science University, Portland, OR, USA.
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Schambach A, Schiedlmeier B, Kühlcke K, Verstegen M, Margison GP, Li Z, Kamino K, Bohne J, Alexandrov A, Hermann FG, von Laer D, Baum C. Towards hematopoietic stem cell-mediated protection against infection with human immunodeficiency virus. Gene Ther 2006; 13:1037-47. [PMID: 16541120 DOI: 10.1038/sj.gt.3302755] [Citation(s) in RCA: 18] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/17/2023]
Abstract
The failure of pharmacological approaches to cure infection with the human immunodeficiency virus (HIV) has renewed the interest in gene-based therapies. Among the various strategies that are currently explored, the blockade of HIV entry into susceptible T cells and macrophages promises to be the most powerful intervention. For long-term protection of both of these lineages, genetic modification of hematopoietic stem cells (HSCs) would be required. Here, we tested whether HSCs and their progeny can be modified to express therapeutic levels of M87o, a gammaretroviral vector encoding an artificial transmembrane molecule that blocks fusion-mediated uptake of HIV. In serial murine bone marrow transplantations, efficient and multilineage expression of M87o was observed for more than 1 year (range 37-75% of mononuclear cells), without signs of toxicity related to the transmembrane molecule. To allow enrichment of M87o-modified HSCs after transplant, we constructed vectors coexpressing the P140K mutant of O(6)-methylguanine-DNA-methyltransferase (MGMT-P140K). This clinically relevant selection marker mediates a survival advantage in HSCs if exposed to combinations of methylguanine-methyltransferase (MGMT) inhibitors and alkylating agents. A bicistronic vector mediated sufficient expression of both M87o and MGMT to confer a selective survival advantage in the presence of HIV and alkylating agents, respectively. These data encourage further investigations in large animal models and clinical trials.
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Affiliation(s)
- A Schambach
- Department of Hematology, Hemostaseology and Oncology, Hannover Medical School, Hannover, Germany
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45
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Fischer M, Schwieger M, Horn S, Niebuhr B, Ford A, Roscher S, Bergholz U, Greaves M, Löhler J, Stocking C. Defining the oncogenic function of the TEL/AML1 (ETV6/RUNX1) fusion protein in a mouse model. Oncogene 2005; 24:7579-91. [PMID: 16044150 DOI: 10.1038/sj.onc.1208931] [Citation(s) in RCA: 73] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/18/2023]
Abstract
The t(12;21) translocation, generating the TEL/AML1 fusion protein, is the most common genetic lesion in childhood cancer. Using a bone marrow transplantation model, we demonstrate that TEL/AML1 expression impinges on normal hematopoietic differentiation, leading to the in vivo accumulation and persistence of an early progenitor compartment with a Sca1(+)/Kit(hi)/CD11b(+) phenotype and an increased self-renewal capacity, as documented by replating assays in vitro. Differentiation of these cells is not blocked, but the frequency of mature blood cells arising from TEL/AML1-transduced progenitors is low. Impaired differentiation is prominently observed in the pro-B-cell compartment, resulting in an proportional increase in early progenitors in vivo, consistent with the t(12;21) ALL phenotype. Despite the accumulation of both multipotent and B-cell progenitors in vivo, no leukemia induction was observed during an observation period of over 1 year. These results are consistent with findings in twins with concordant ALL, showing that TEL/AML1 generates a preleukemic clone in utero that persists for several years in a clinically covert fashion. Furthermore, our studies showed that the pointed domain of TEL/AML1, which recruits transcriptional repressors and directs oligomerization with either TEL/AML1 or wild-type TEL, was essential for the observed differentiation impairment and could not be replaced with another oligomerization domain.
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MESH Headings
- Animals
- B-Lymphocytes
- Bone Marrow Transplantation
- Cell Differentiation
- Cell Transformation, Neoplastic/genetics
- Chromosomes, Human, Pair 12
- Chromosomes, Human, Pair 21
- Core Binding Factor Alpha 2 Subunit/biosynthesis
- Core Binding Factor Alpha 2 Subunit/genetics
- Core Binding Factor Alpha 2 Subunit/physiology
- Hematopoietic Stem Cells
- Humans
- Mice
- Mice, Inbred C57BL
- Oncogene Proteins, Fusion/biosynthesis
- Oncogene Proteins, Fusion/genetics
- Oncogene Proteins, Fusion/physiology
- Phenotype
- Precursor Cell Lymphoblastic Leukemia-Lymphoma/genetics
- Preleukemia/genetics
- Preleukemia/physiopathology
- Translocation, Genetic
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Affiliation(s)
- Meike Fischer
- Molecular Pathology Group, Heinrich-Pette-Institut für Experimentelle Immunologie und Virologie, D-20251 Hamburg, Germany
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46
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Crcareva A, Saito T, Kunisato A, Kumano K, Suzuki T, Sakata-Yanagimoto M, Kawazu M, Stojanovic A, Kurokawa M, Ogawa S, Hirai H, Chiba S. Hematopoietic stem cells expanded by fibroblast growth factor-1 are excellent targets for retrovirus-mediated gene delivery. Exp Hematol 2005; 33:1459-69. [PMID: 16338488 DOI: 10.1016/j.exphem.2005.09.001] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/06/2005] [Revised: 08/31/2005] [Accepted: 09/01/2005] [Indexed: 12/25/2022]
Abstract
OBJECTIVE For the study of the function of genes of interest in hematopoietic stem cells (HSCs) and for successful gene therapy, it is fundamental to develop a method of efficient gene transfer into HSCs. In mice experiments, efforts have been made to raise the transduction efficiency by modifying the vectors, administrating 5-fluorouracil (5-FU) to donor mice, selecting cytokine cocktails to better sustain the long-term repopulating potential of the stem cells, and so on. The objective of this study is to examine whether the use of fibroblast growth factor-1 (FGF-1)-expanded bone marrow cells provide an improved source for retroviral gene delivery to HSCs. MATERIALS AND METHODS Unfractionated bone marrow cells from one mouse were cultured in serum-free medium containing FGF-1. Both floating and attached cells were transferred to retronectin precoated dishes and infected with virus supernatant from MP34 cells stably transduced with pMY/GFP retrovirus. After 3-day infection, the green fluorescence protein-positive fraction was sorted and the cells were transplanted to lethally irradiated mice. RESULTS The experiments illustrated that the number of bone marrow-derived competitive repopulation units (CRUs) was increased from 600 to 9300 per mouse after a 3-week culture period with FGF-1. Following retroviral transduction of the expanded cells, the absolute number of sorted retrovirus-transduced CRUs was 4200. Using these retrovirus-transduced cells in noncompetitive reconstitution assay, we achieved radiation protection and long-term bone marrow reconstitution in 100% of the recipients with average myeloid and lymphoid chimerisms of 70% and 50%, respectively, even if we transplanted 150 recipients with cells derived from a single donor mouse. CONCLUSION In conclusion, FGF-1-expanded bone marrow cells constitute an excellent source of stem cells that could be used in a range of gene delivery protocols.
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47
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Schambach A, Bohne J, Chandra S, Will E, Margison GP, Williams DA, Baum C. Equal potency of gammaretroviral and lentiviral SIN vectors for expression of O6-methylguanine-DNA methyltransferase in hematopoietic cells. Mol Ther 2005; 13:391-400. [PMID: 16226060 DOI: 10.1016/j.ymthe.2005.08.012] [Citation(s) in RCA: 164] [Impact Index Per Article: 8.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/06/2005] [Revised: 08/22/2005] [Accepted: 08/22/2005] [Indexed: 02/07/2023] Open
Abstract
Severe adverse events related to insertional mutagenesis have reinforced interest in self-inactivating (SIN) retroviral vectors lacking enhancer-promoter sequences in the long terminal repeats (LTRs). Here, we have compared the potency of gammaretroviral and lentiviral vectors expressing the P140K mutant of O(6)-methylguanine-DNA methyltransferase (MGMT). MGMT-P140K is a clinically relevant selection marker that mediates a strong survival advantage in hematopoietic cells exposed to alkylating agents. We designed gammaretroviral and lentiviral vectors that contained identical enhancer-promoter sequences located either in the LTR or downstream of the packaging region, for internal initiation of transcription from SIN backbones. Gammaretroviral vectors with intact LTRs containing enhancer-promoter sequences showed both higher titers and higher expression levels than the lentiviral counterparts, likely a result of suboptimal RNA processing of the lentiviral leader region. In the SIN context, gammaretroviral and lentiviral vectors with comparable internal cassettes had similar expression properties. Interestingly, gammaretroviral SIN vectors pseudotyped with RD114/TR had a higher transduction efficiency on proliferating human CD34(+) cells than lentiviral counterparts. These results encourage further investigations into the formation of retroviral hybrid vectors that combine the desired properties of high efficiency and increased biosafety.
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Affiliation(s)
- Axel Schambach
- Department of Hematology, Hemostaseology, and Oncology, Hannover Medical School, Germany
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Lange C, Bassler P, Lioznov MV, Bruns H, Kluth D, Zander AR, Fiegel HC. Liver-specific gene expression in mesenchymal stem cells is induced by liver cells. World J Gastroenterol 2005; 11:4497-504. [PMID: 16052678 PMCID: PMC4398698 DOI: 10.3748/wjg.v11.i29.4497] [Citation(s) in RCA: 72] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
AIM: The origin of putative liver cells from distinct bone marrow stem cells, e.g. hematopoietic stem cells or multipotent adult progenitor cells was found in recent in vitro studies. Cell culture experiments revealed a key role of growth factors for the induction of liver-specific genes in stem cell cultures. We investigated the potential of rat mesenchymal stem cells (MSC) from bone marrow to differentiate into hepatocytic cells in vitro. Furthermore, we assessed the influence of cocultured liver cells on induction of liver-specific gene expression.
METHODS: Mesenchymal stem cells were marked with green fluorescent protein (GFP) by retroviral gene transduction. Clonal marked MSC were either cultured under liver stimulating conditions using fibronectin-coated culture dishes and medium supplemented with SCF, HGF, EGF, and FGF-4 alone, or in presence of freshly isolated rat liver cells. Cells in cocultures were harvested and GFP+ or GFP- cells were separated using fluorescence activated cell sorting. RT-PCR analysis for the stem cell marker Thy1 and the hepatocytic markers CK-18, albumin, CK-19, and AFP was performed in the different cell populations.
RESULTS: Under the specified culture conditions, rat MSC cocultured with liver cells expressed albumin-, CK-18, CK-19, and AFP-RNA over 3 weeks, whereas MSC cultured alone did not show liver specific gene expression.
CONCLUSION: The results indicate that (1) rat MSC from bone marrow can differentiate towards hepatocytic lineage in vitro, and (2) that the microenvironment plays a decisive role for the induction of hepatic differentiation of rMSC.
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Affiliation(s)
- Claudia Lange
- Center of Bone Marrow Transplantation, University Hospital Hamburg-Eppendorf, Martinistrasse 52, D-20246 Hamburg, Germany.
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49
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Lorico A, Bratbak D, Meyer J, Kunke D, Krauss S, Plott WE, Solodushko V, Baum C, Fodstad O, Rappa G. γ-Glutamylcysteine Synthetase and L-Buthionine-(S,R)-Sulfoximine: A New Selection Strategy for Gene-Transduced Neural and Hematopoietic Stem/Progenitor Cells. Hum Gene Ther 2005; 16:711-24. [PMID: 15960602 DOI: 10.1089/hum.2005.16.711] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022] Open
Abstract
In most experimental gene therapy protocols involving stem/progenitor cells, only a small fraction of cells, often therapeutically inadequate, can be transduced and made to express the therapeutic gene. A promising strategy for overcoming this problem is the use of a dominant selection marker, such as a drug resistance gene. In this paper, we explore the potential of the heavy subunit of gamma-glutamylcysteine synthetase (gamma-GCSh) to act as a selection marker. We found that 3T3 fibroblasts transduced with the bicistronic retroviral vector SF91/GCSh-eGFP, encoding gamma-GCSh and the enhanced green fluorescent protein (eGFP), were highly resistant to L-buthionine-(S,R)-sulfoximine (BSO), a gamma-GCS inhibitor with a low clinical toxicity profile. The level of resistance was not proportional to the increase in intracellular glutathione. In fact, cells overexpressing both heavy and light gamma-GCS subunits had higher intracellular GSH levels, and a lower level of resistance to the cytotoxic activity of BSO, compared with cells overexpressing gamma-GCSh alone. 3T3 fibroblasts overexpressing gamma-GCSh could be selected from cultures containing both naive and gene-modified cells by application of exogenous BSO selection pressure for 4 days. Also, primary neural stem/progenitor cells derived from the lateral ventricles of mouse neonatal brains and primary hematopoietic stem/progenitor cells (HSCs/HPCs) from mouse bone marrow, transduced with the gamma-GCSh-eGFP vector, could be selected by BSO treatment in vitro. On ex vivo BSO selection and reimplantation into a syngeneic myeloablated host, donor HSCs/HPCs repopulated the marrow and continued to express the transgene(s). These results provide proof of principle that somatic stem/progenitor cells, transduced simultaneously with a potentially curative gene and gamma-GCSh, can be selected by treatment with BSO before in vivo transplantation.
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Affiliation(s)
- Aurelio Lorico
- Department of Tumor Biology, Norwegian Radium Hospital, Montebello, Oslo 0310, Norway.
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Jaquet K, Krause KT, Denschel J, Faessler P, Nauerz M, Geidel S, Boczor S, Lange C, Stute N, Zander A, Kuck KH. Reduction of Myocardial Scar Size after Implantation of Mesenchymal Stem Cells in Rats: What Is the Mechanism? Stem Cells Dev 2005; 14:299-309. [PMID: 15969625 DOI: 10.1089/scd.2005.14.299] [Citation(s) in RCA: 49] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/14/2022] Open
Abstract
The use of a cellular therapy offers a promising approach for the treatment of heart disease. Besides other precursor cells, bone marrow (BM)-derived stem cells were discovered that migrate into ischemic myocardium and participate in myogenesis as well as angiogenesis. A subpopulation of those are the mesenchymal stem cells (MSC), which may be potential candidates for repairing ischemic heart tissue. MSC are easy to prepare and can be used in an autologous strategy. Here we demonstrate the effect of transplanted MSC in our autologous rat model of myocardial injury. BM was isolated from tibiae and femurs of Wistar rats. After 24 h, the adhering MSC were separated, expanded, retrovirally transduced using green fluorescent protein (GFP), and cloned. A cryo-infarct was generated in the rat hearts, and immediately after this the cells were injected into the border zone of the lesion. After a 10-week follow up, the hearts were excised and the myocardial scar areas were measured using computer-guided morphometry. When comparing transplanted rats (n = 8) with control animals (n = 5) treated rats demonstrated a significant reduction in the width (p < 0.05) of the myocardial scar area. The depth of the scars of the cell therapy rats was less extended (p > 0.05) and the myocardium of these animals was thicker than in the controls (p > 0.05). Immunohistochemical analyses revealed neither evidence of MSC transdifferentiation into cardiomyocytes, nor could an increased neovascularization be found. In conclusion, MSC are responsible for a remarkable reduction of the myocardial scar size in the treated animals. But, whether this strategy is directly transferable to the patient suffering from heart disease has to be determined. In addition, the mechanism by which MSC act in the ischemic heart remains to be determined.
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Affiliation(s)
- Kai Jaquet
- Department of Cardiology/Cell Biology, St. Georg Hospital, Hamburg, Germany.
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