Genetic programming of human iPSC-derived macrophages provides a tool to study the Erythroblastic Island niche in vitro
Lopez Yrigoyen, Martha Beatriz
Macrophages have been attracting much attention as they are present in many tissues and organs, and are involved in homeostatic tissue function and disease. Studies involving human macrophages have been hampered due to the ethical constraints and technical difficulties related to their isolation/derivation for in vitro expansion, and to the difficulty of genetically modifying them to interrogate the role of specific factors in macrophage behaviour. Thus, an off-the-shelf source of macrophages that is amenable to the vast arsenal of genetic manipulation techniques, such as induced pluripotent stem cells (iPSCs), represents a valuable tool in the macrophage field. As a proof of principle, we used a human iPSC-line that was targeted in the safe harbour AAVS1 locus to express the fluorescent protein ZsGreen constitutively (SFCi55-ZsG). We demonstrate efficient production of terminally differentiated macrophages from the SFCi55-ZsG iPSC-line that fluoresce green. Macrophages derived from this targeted cell line are indistinguishable from those generated from their parental line in terms of gene expression, cell surface marker expression and phagocytic ability. Furthermore, genetically modified macrophages could be activated/polarised to an M (LPS+ IFN-ϒ), M (IL10) or M (IL4) phenotype and retained their plasticity-related abilities, as they were able to switch from one activated state to another. These results showed that targeting of iPSCs via the AAVS1 locus allows for the production of fully functional genetically engineered macrophages that could be used for in vivo tracking of macrophages in disease. Furthermore, this strategy provides a platform for the introduction of genes/factors that are predicted to modulate and/or stabilise macrophage phenotype and function in diverse biological settings. We then used the AAVS1 targeting strategy to programme iPSC-derived macrophages into a phenotype comparable to macrophages associated with the erythroid island. Human red blood cell (RBC) precursors proliferate and mature within an erythroblastic island (EI) niche consisting of a central macrophage surrounded by 5-30 erythroblasts. Emulating this EI niche in vitro could provide a tool to study the molecular mechanisms involved in terminal erythropoiesis and might ultimately overcome the limitations associated with the production of RBCs in vitro. The transcription factor KLF1 has been reported to play an important role in murine EI-like macrophages, and we noted the deficient expression of KLF1 in macrophages derived from iPSCs in vitro. We, therefore, hypothesised that enforced expression of KLF1 might programme these macrophages to a more EIlike phenotype. Indeed, activation of KLF1 in iPSC-derived macrophages (iPSC-DM) increased the expression of some EI-associated genes and cell surface markers; and enhanced their phagocytic activity. We established a co-culture system with iPSC-DMs and umbilical cord bloodderived CD34+ haematopoietic progenitor cells, as well as iPSC-derived haematopoietic cells and demonstrated that co-culture with macrophages increased the production of mature and enucleated erythroid cells and this was further enhanced when KLF1 was activated. The effect of KLF1 activation was partially retained when contact with macrophages was inhibited, suggesting that a paracrine effect (secreted factors encoded by KLF1 targets genes) is associated with its mechanisms of action. RNA sequencing of KLF1 activated iPSC-DMs revealed potential cell surface proteins/receptors and secreted factors that might be involved in the enhanced proliferation, enucleation and maturation of erythroid cells. We identified three potential mediators: ANGPTL7, IL33 and SERPINB2 because when added together in erythroid cultures, there was a significant increase in erythroid cell maturation and enucleation in the absence of macrophages. Pilot studies revealed that secreted factors NRG1, IGFBP6, CCL13 and TNFSF10 could also play a role in the later stages of the erythroid differentiation protocol. To our knowledge, this is the first time that macrophage phenotype and function have been manipulated by transcription factor programming. In this particular scenario, our novel co-culture system with programmed macrophages provides a model to study the EI niche and terminal erythropoiesis in vitro, and brings us a step closer to the ultimate goal of replacing blood transfusion with a manufactured product.