MODELING TISSUE-RESIDENT MACROPHAGE DEVELOPMENT FROM MOUSE PLURIPOTENT STEM CELLS

Abstract

Tissue-resident macrophages (TRMs) are innate immune cells that participate in tissue development, homeostasis, and immune surveillance. Extensive efforts have been made to recapitulate TRM development from pluripotent stem cells (PSCs) in vitro to study molecular and cellular mechanisms of TRM development and to create cellular models of disease. However, available PSC models of mouse TRM development exhibit low overall efficiencies of TRM generation, produce heterogeneous off-target populations, and rely upon undefined media components, thus limiting their reproducibility, scalability, and widespread application as an experimental platform for TRM biology. To address these important limitations, we developed an efficient and reproducible protocol to faithfully recapitulate the stepwise differentiation of mouse PSCs (epiblast stem cells) into unspecialized, proliferative TRMs through the pro-definitive hematopoietic program under defined conditions. These immature TRMs can stably integrate into developing mouse neural organoids in vitro and acquire features of microglia. In addition, PSC-derived immature TRMs can stably engraft into the lung niche in vivo and adopt alveolar macrophage characteristics. This new platform for modeling mouse TRM development represents a powerful experimental model system for studying TRM function and dysfunction in development and disease.

Figure 1:. Identification of conditions for generating yolk sac pre-hematopoietic mesoderm (PHM) from mouse EpiSCs

A. Overview of developmental stages from pluripotency through yolk sac hematopoiesis and TRM differentiation, including relevant stages of embryonic development in mouse (bottom row). B. Overview of approach for differentiation of EpiSCs into immature TRMs, including stage-specific marker genes. C. Summary of the first two stages of the TRM differentiation protocol. The optimal levels of Wnt and TGF-β signaling to promote KDR(+) PDGFRa(−) yolk sac mesoderm/PHM differentiation during Stage 2 were not known. D. Representative flow cytometry plots (left) of D2 PHM yield observed at different levels of Wnt pathway activation. CHIR99021 was used to activate the canonical Wnt pathway, and LGK479 was used to inhibit Wnt signaling. Activin A (AA) was kept constant at 10 ng/mL during stage 2 for each condition. Summary of flow cytometry data across 3 independent differentiation experiments (right). Significance was determined using ordinary one-way ANOVA, corrected for multiple comparisons with Tukey test (***P < 0.001). E. Representative flow cytometry plots (left) showing D2 PHM yield observed at different levels of TGF-β pathway activation. Activin A was used to stimulate TGF-β signaling and SB431542 was used to block TGF-β signaling. Only endogenous Wnt signaling (same as the No Wnt condition in [D] was used in Stage 2). Summary of flow cytometry data across 3 independent differentiation experiments (right). Significance was determined using ordinary one-way ANOVA, corrected for multiple comparisons with Tukey test (*P < 0.05; **P < 0.01; ***P < 0.001). F. Representative flow cytometry plots (left) measuring PHM yield at the end of Stage 2 in optimized PHM conditions (No Wnt, 10 ng/mL Activin A). Percentage of CXCR4 within the KDR(+) PDGFRa(−) population is also shown. Summary of flow cytometry data from 2 distinct cell lines (right). B6/129 EpiSCs (n = 13 independent differentiations [technical replicates]). DO-202 EpiSCs (n = 4 independent differentiations [technical replicates])

Figure 2:. Efficient generation of erythro-myeloid progenitors from pre-hematopoietic yolk sac mesoderm

A. Detailed overview of signaling conditions for hemogenic endothelium differentiation and hematopoiesis. These conditions are adapted (with modifications) from Guttikonda et al., 2021. B. Evaluation of EMP emergence during in vitro hematopoiesis (left). Generation of Kit(+) CD41(+) CD16/32(+) EMPs and myeloid marker expression (CD45) was evaluated using flow cytometry at the indicated timepoints. Flow cytometry plots are gated for Kit(+) cells (top row). Under these conditions, essentially all EMPs also express CD45 (bottom row). Quantification of EMP generation (D8) across 4 independent differentiation experiments from the indicated cell lines (right). C. Immunofluorescence staining of differentiating cultures at D8 for RUNX1, a transcription factor expressed in yolk sac hemogenic endothelia and EMPs. EHT = endothelial-to-hematopoietic transition. Scale bars = 25 μM.

Figure 3:. Differentiation of EMPs into immature TRMs and characterization of immature TRMs

A. Overview of conditions used for differentiation of EMPs into immature TRMs and TRM expansion. B. Brightfield images of immature TRMs at D10 and D16 (top row) and immunofluoresence staining immature TRMs (D16) for pan-macrophage markers IBA1 and PU.1 (bottom row). C. Assessment of TRM surface markers CX₃CR1, CD11b, CD45, and P2RY12 by flow cytometry to evaluate immature TRM differentiation efficiency (left). Summary of differentiation efficiency (% CX₃CR1(+) cells) obtained from the indicated EpiSC lines (right). Each dot represents an independent differentiation experiment. D. Heatmap of gene expression (RNA-seq) of a panel of cell type-specific marker genes in CX₃CR1(+) immature TRMs purified by FACS at D16. Log2 normalized counts (transcripts per million [TPM]) are plotted for each gene (average expression level from n = 2 independent differentiations [technical replicates] from each of the indicated cell lines). E. Violin plots of expression levels (log2 normalized counts, TPM) of individual genes from signatures of yolk sac-derived pMacs and yolk sac-derived macrophages obtained from E10.25 and E10.5 embryos (Mass et. al., 2016). Each dot represents a single gene, and the value for the expression level of the gene is the average expression across TRMs from all of the 3 EpiSC lines. F. Confocal microscopy images (brightfield overlaid with fluorescence) of D20 immature TRMs containing pH-sensitive CypHer 5E-labeled phagocytosed synaptosomes (left) and apoptotic neurons (right). Arrows indicate engulfed cellular material. Scale bars = 25 μm.

Figure 4:. Immature TRMs integrate into developing cortical organoids and adult mouse lung niche

A. Overview of strategy for co-culture of immature TRMs and mouse cerebral cortex organoids generated from EpiSCs. B. Quantification of CD11b(+) TRMs in cortical organoids using flow cytometry at D24 and D32. C. Confocal imaging of tissue-cleared cortical organoids with integrated TRMs. Immunofluorescence staining for TRM markers PU.1 and IBA1. High magnification image (right) showing ramified morphology of TRMs. D. Overview of experimental design for depletion of endogenous alveolar macrophages and subsequent intratracheal delivery of immature TRMs into mouse lungs. CD45.1 mice were used so that transplanted CD45.2-expressing TRMs can be distinguished from CD45.1(+) host macrophages. E. Representative flow cytometry plots for lung tissue from mice transplanted with TRMs at the indicated timepoints. Alveolar macrophages are CD11b(−) SiglecF(+) CD11c(+). F. Summary of results from lung transplantation experiments at D3 and D7 post-transplant. Individual dots represent results from a single mouse (biological replicates).