Mania Ackermann1,2, Alexandra Kuhn2,3, Jessica Kunkiel2,3, Sylvia Merkert4,5, Ulrich Martin4,5, Thomas Moritz2,3, Nico Lachmann1,2. 1. JRG Translational Hematology, REBIRTH Cluster of Excellence, Hanover Medical School, Hanover, Germany. 2. Institute of Experimental Hematology, Hanover Medical School, Hanover, Germany. 3. RG Reprogramming and Gene Therapy, REBIRTH Cluster of Excellence, Hanover Medical School, Hanover, Germany. 4. Leibniz Research Laboratories for Biotechnology and Artificial Organs (LEBAO), Department of Cardiothoracic, Transplantation and Vascular Surgery (HTTG), REBIRTH-Cluster of Excellence, Hanover Medical School, Hanover, Germany. 5. Biomedical Research in Endstage and Obstructive Lung Disease (BREATH), Member of the German Center for Lung Research (DZL), Hanover, Germany.
Abstract
BACKGROUND: Pluripotent stem cells, including induced pluripotent stem cells (iPSCs), have the capacity to differentiate towards all three germ layers and have been highlighted as an attractive cell source for the field of regenerative medicine. Thus, stable expression of therapeutic transgenes in iPSCs, as well as thereof derived progeny of hematopoietic lineage, may lay the foundation for innovative cell replacement therapies. METHODS: We have utilized human iPSC lines genetically modified by lentiviral vector technology or targeted integration of reporter genes to evaluate transgene expression during hematopoietic specification and differentiation towards macrophages. RESULTS: Use of lentiviral vectors equipped with an ubiquitous chromatin opening element (CBX3-UCOE) as well as zinc finger nuclease-mediated targeting of an expression cassette into the human adeno-associated virus integration site 1 (AAVS1) safe harbor resulted in stable transgene expression in iPSCs. When iPSCs were differentiated along the myeloid pathway into macrophages, both strategies yielded sustained transgene expression during the hematopoietic specification process including mature CD14+ and CD11b+ macrophages. CONCLUSION: Combination of human iPSC technology with either lentiviral vector technology or designer nuclease-based genome editing allows for the generation of transgenic iPSC-derived macrophages with stable transgene expression which may be useful for novel cell and gene replacement therapies.
BACKGROUND: Pluripotent stem cells, including induced pluripotent stem cells (iPSCs), have the capacity to differentiate towards all three germ layers and have been highlighted as an attractive cell source for the field of regenerative medicine. Thus, stable expression of therapeutic transgenes in iPSCs, as well as thereof derived progeny of hematopoietic lineage, may lay the foundation for innovative cell replacement therapies. METHODS: We have utilized human iPSC lines genetically modified by lentiviral vector technology or targeted integration of reporter genes to evaluate transgene expression during hematopoietic specification and differentiation towards macrophages. RESULTS: Use of lentiviral vectors equipped with an ubiquitous chromatin opening element (CBX3-UCOE) as well as zinc finger nuclease-mediated targeting of an expression cassette into the humanadeno-associated virus integration site 1 (AAVS1) safe harbor resulted in stable transgene expression in iPSCs. When iPSCs were differentiated along the myeloid pathway into macrophages, both strategies yielded sustained transgene expression during the hematopoietic specification process including mature CD14+ and CD11b+ macrophages. CONCLUSION: Combination of human iPSC technology with either lentiviral vector technology or designer nuclease-based genome editing allows for the generation of transgenic iPSC-derived macrophages with stable transgene expression which may be useful for novel cell and gene replacement therapies.
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