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

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.

TALEN-mediated functional correction of human iPSC-derived macrophages in context of hereditary pulmonary alveolar proteinosis

“…With the emergence of RNA-guided nucleases, CRISPR/Cas9-mediated correction of congenital lympho-hematopoietic diseases were reported 9 , 45 – 47 . Several of these studies targeted the gene of interest into the well-characterized AAVS1 safe harbor site 3 , 7 , 16 , 48 . This included approaches to functionally correct granulocytes derived from X-CGD iPSCs employing the same TALEN pair as utilized here to direct site specificity of the integration 7 , 31 .…”

“…In our hands, this design already allowed for high and sustained expression of the CSF2RA coop transgene from a mono-allelic copy of the expression cassette in pluripotent and myeloid cells without signs of silencing during the differentiation process. Should variegation of transgene expression during the differentiation process occur in the future, epigenetic regulatory elements including ubiquitous chromatin opening elements (UCOEs) may be incorporated into the expression constructs to promote an even stronger anti-silencing environment 37 , 43 , 48 , 50 . Likewise, if the constitutive expression of CSF2RA from the CAG promoter turns out problematic e.g.…”

Concise Review: Towards the Clinical Translation of Induced Pluripotent Stem Cell-Derived Blood Cells—Ready for Take-Off

“…The resulting cells are of high purity, transcriptionally closely related to their in vivo counterparts, and seem to be fully functional. Because of its simplicity and efficiency, this protocol has since become a standard for differentiating macrophages and meanwhile been further advanced to generate macrophages and granulocytes from human iPSCs . In addition, similar techniques have been developed to also generate microglia‐like cells from human iPSCs, further demonstrating the potential to produce various macrophage subsets .…”

Production and Characterization of Human Macrophages from Pluripotent Stem Cells

“…Technologies that allow for the genetic manipulation of human pluripotent stem cells (PSCs), such as Zinc Finger Nuclease, TALENS, and CRISPR-Cas9, have revolutionised medical research 1,2,3,4 . Genetic manipulation of human PSCs is a particularly attractive strategy when the primary cell of interest is difficult to expand and/or to maintain in vitro, or is difficult to genetically manipulate, such as is the case for macrophages 5,6,7,8,9 . As human iPSCs can be derived from any somatic cell, they circumvent the ethical limitations associated with ESCs, and provide a strategy for delivering personalized medicine.…”

“…The formation of three-dimensional aggregates, known as embryoid bodies (EBs) initiates differentiation of iPSCs. Bone morphogenetic protein (BMP4), stem cell factor (SCF), and vascular endothelial growth factor (VEGF) are added to drive mesoderm specification and support emerging hematopoietic cells 7,8,9,11,12 . The differentiating cells within the EBs also initiate the activation of endogenous signalling pathways such as Wnt and Activin.…”