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. 2017 Jun;44(3):135-142.
doi: 10.1159/000477129. Epub 2017 May 16.

Ex vivo Generation of Genetically Modified Macrophages from Human Induced Pluripotent Stem Cells

Mania Ackermann  1   2 , Alexandra Kuhn  2   3 , Jessica Kunkiel  2   3 , Sylvia Merkert  4   5 , Ulrich Martin  4   5 , Thomas Moritz  2   3 , Nico Lachmann  1   2
Affiliations

Ex vivo Generation of Genetically Modified Macrophages from Human Induced Pluripotent Stem Cells

Mania Ackermann et al. Transfus Med Hemother. 2017 Jun.

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.

Keywords: CBX3-UCOE; Gene therapy; Genome editing; Hematopoiesis; Lentivirus; Macrophages; iPSC.

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Figures

Fig. 1
Fig. 1
CBX3-UCOE-mediated transgene expression in hiPSC-derived macrophages. A Scheme of the lentiviral vector expressing GFP from the EFS (Lv.CBX3.EFS.GFP) (LTR: long terminal repeat). The lentiviral vector was further equipped with a CBX3-UCOE to prevent epigenetic transgene silencing such as methylation and to favor an open chromatin state. B Experimental scheme of lentiviral-mediated transduction of hiPSCs. Lentiviral particles (Lv.CBX3.EFS.GFP) were used to transduce hiPSC monolayer. After transduction, modified iPSCs were subjected to hematopoietic differentiation to study transgene stability. C Flow-cytometric analysis of GFP in transduced CD34iPSC11 over 28 days of culture. D GFP expression in transduced CD34iPSC16 and during hematopoietic differentiation in EBs and MCFCs (scale bars; column 1 and 3: 500 µm, column 2 and 4: 200 µm). E GFP expression in CD34iPSC16-derived macrophages in cell culture (left column) and unstained cytospin (right column) (scale bars: 100 µm). F Flow-cytometric analysis and G stained cytospins of terminally differentiated macrophages.
Fig. 2
Fig. 2
Stable transgene expression from the AAVS1 locus in hiPSC-derived macrophages. A Scheme of ZFN-mediated targeting of the fluorescence reporter genes GFP or RedStarnuc into the AAVS1 safe harbor locus (HAL, homology arm left; HAR, homology arm right). B Schematic outline of nuclease-mediated genome editing of iPSCs. After nucleofection of iPSCs with suitable ZFNs and donor constructs, GFP- or RedStarnuc-positive cells were sorted to obtain transgenic iPS single cell clones. C GFP expression during hematopoietic differentiation (scale bars; column 1 and 2: 200 µm, column 3 and 4: 500 µm) and E in iPSC-derived macrophages (scale bars: 100 µm). D RedStarnuc expression during hematopoietic differentiation (scale bars; column 1 and 2: 200 µm, column 3 and 4: 500 µm) and F in iPSC-derived macrophages (scale bars; column 1: 100 µm and column 2: 50 µm). G Flow-cytometric analysis of terminally differentiated macrophages derived from iPSCs which either express GFP or H RedStarnuc from the AAVS1 locus. I Cytospins of differentiated macrophages derived from iPSCs which either express GFP or J RedStarnuc.
Fig. 3
Fig. 3
iPSC-based gene and cell therapy. Patient-specific iPSCs can be generated from easily accessible blood samples by overexpression of specific transcription factors using viral or non-viral vectors. Genetic modification can be performed in the pluripotent status, thereby allowing sub-cloning and further safety tests. Thereafter, either gene addition using integrating viral vectors or precise genome editing strategies employing designer nucleases (ZFN, TALEN, CRISPR/Cas9) can be applied. Corrected iPSCs (subclones) can subsequently be differentiated into the disease-affected cell type or stem/progenitor cells, which could then be investigated in vivo.
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