Illuminating the Functional Architecture of iPSC-Derived Neutrophil Granulocytes with Split Fluorescent Proteins
Authors: Petersheim Daniel , Linder Monika I , Kiziltug Mehmet T , Schmid Johanna , Bader Almke , Maier-Begandt Daniela , Walzog Barbara , Renkawitz Jörg , Klein Christoph 
Published: December 2024

The ability to visualize the molecular choreography underlying dynamic biological processes is critical for dissecting the mechanisms guiding cellular differentiation and function, as well as for developing innovative therapeutic strategies. Based on recently developed technological platforms for endogenous gene tagging in cancer cells, we here designed a split fluorescent protein approach aiming to study physiological processes in neutrophil granulocytes at high spatiotemporal resolution.
First, we generated master induced pluripotent stem cell lines with expression cassettes encoding the UbC promoter-driven large fragments of mNeonGreen2 and sfCherry3 (referred to as mNG21-10 and sfCherry31-10) targeted to the AAVS1 and CCR5 safe harbor loci. Upon confirmation of pluripotency of parental iPSCs, complementary split-fluorescent protein fragments (mNG211 or sfCherry311) were introduced by tagging genes expressed in distinct subcellular localizations. Single and multiplex labeling of proteins in iPSCs was highly efficient with editing efficiencies ranging from 20-80%. Successfully tagged genes include LMNB1, ACTB, TUBA1B, H2BC11, SEC61B and TOMM20.
Next, we differentiated iPSC lines expressing mNG211-LaminB1 into neutrophil granulocytes in vitro, aiming to visualize the dynamics of NETosis. Of note, differentiation of mNG211-LaminB1 cells proceeded through the same phenotypically distinct stages of neutrophil maturation (proNeu, preNeu, immatureNeu and matureNeu) as the WT iPSCs, indicating that the differentiation process was not affected by our tagging strategy. The inner nuclear membrane proteins, LAP2beta and LBR, co-localized with mNG211-LaminB1 at the nuclear envelope (NE) of multi-lobed nuclei indicative of correct subcellular insertion of the tagged protein. We then employed WT or mNG211-LaminB1 iPSC-derived neutrophils to characterize the spatiotemporal dynamics of nuclear disassembly during NETosis. Importantly, WT and mNG211-LaminB1 iPSC-derived neutrophils underwent NETosis and disassembled the NE with similar kinetics (NETosis rate 70.8% vs. 74.5% at t = 240min) indicating efficient NE disintegration during NET formation.
To shed light on the dynamic reorganization of the actin cytoskeleton during neutrophil migration we made use of mNG211-ACTB expressing iPSCs. In IL-8/ICAM-1-stimulated iPSC-derived neutrophils, rapid redistribution of mNG211-ACTB to the leading edge and co-localization with phalloidin-stained F-actin was observed. There was no significant effect of tagging on 2D migration of neutrophils with respect to speed (WT 4.0 µm/min vs. tagged 3.4 µm/min), euclidian distance (15.7 µm vs. 12.7 µm) and directionality (0.42 vs. 0.41).
Thus, using marker-free genetic engineering, we have devised a versatile system based on orthogonal split fluorescent proteins for the study of dynamic cellular processes in human iPSC-derived neutrophils. Further studies are ongoing to validate the prospects of the split fluorescent protein platform in the hematopoietic and other organ systems. We anticipate that our system will be useful for a wide range of applications, including the functional assessment of gene variant effects and the implementation of high-content screens.