A human iPSC line capable of differentiating into functional macrophages expressing ZsGreen: a tool for the study and in vivo tracking of therapeutic cells

Abstract

We describe the production of a human induced pluripotent stem cell (iPSC) line, SFCi55-ZsGr, that has been engineered to express the fluorescent reporter gene, ZsGreen, in a constitutive manner. The CAG-driven ZsGreen expression cassette was inserted into the AAVS1 locus and a high level of expression was observed in undifferentiated iPSCs and in cell lineages derived from all three germ layers including haematopoietic cells, hepatocytes and neurons. We demonstrate efficient production of terminally differentiated macrophages from the SFCi55-ZsGreen iPSC line and show that they are indistinguishable from those generated from their parental SFCi55 iPSC line in terms of gene expression, cell surface marker expression and phagocytic activity. The high level of ZsGreen expression had no effect on the ability of macrophages to be activated to an M(LPS + IFNγ), M(IL10) or M(IL4) phenotype nor on their plasticity, assessed by their ability to switch from one phenotype to another. Thus, targeting of the AAVS1 locus in iPSCs allows for the production of fully functional, fluorescently tagged human macrophages that can be used for in vivo tracking in disease models. The strategy also provides a platform for the introduction of factors that are predicted to modulate and/or stabilize macrophage function.This article is part of the theme issue ‘Designer human tissue: coming to a lab near you’.

Human Tumor-Associated Macrophage and Monocyte Transcriptional Landscapes Reveal Cancer-Specific Reprogramming, Biomarkers, and Therapeutic Targets

“…After about 2-3 weeks, the EBs produced macrophage progenitors in the culture supernatant that were harvested and transferred to 10 cm 2 bacteriological dishes in X-VIVOTM 15 medium supplemented with cytokine Mix 3 (100 ng/ml CSF1, 2.0 mM Glutamax, 1% v/v Penicillin/Streptomycin) and allowed to mature for 7 days into iPSC-derived macrophages (iPSC-DM). Macrophage progenitors were harvested every 4 days for approximately 2 months ( Lopez-Yrigoyen et al., 2018 ).…”

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.…”

“…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. This includes patient-specific disease modelling, drug testing, and autologous cell therapy with a reduced risk of immune rejection and infection 6,8,10,11 .…”

“…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.…”

“…To evaluate iPSC-DM phagocytic ability, we used a high content imaging system approach 8,9,11 that makes use of the PerkinElmer Operetta Microscope and pHrodo Zymosan bioparticles (pH-sensitive dye conjugates). Bioparticles were nonfluorescent when added to the cultures ( Figure 4A) but fluoresced bright green in the intracellular acidic pH ( Figure 4B).…”