Stem-cell-derived human microglia transplanted in mouse brain to study human disease

Abstract

Although genetics highlights the role of microglia in Alzheimer's disease, one-third of putative Alzheimer's disease risk genes lack adequate mouse orthologs. Here we successfully engraft human microglia derived from embryonic stem cells in the mouse brain. The cells recapitulate transcriptionally human primary microglia ex vivo and show expression of human-specific Alzheimer's disease risk genes. Oligomeric amyloid-β induces a divergent response in human versus mouse microglia. This model can be used to study the role of microglia in neurological diseases.

Extended Data 1. Gating strategy for the isolation of H9-microglia from the mouse brain and graft efficiency.

(a) Human cells were sorted according to the expression of CD11b, hCD45, and GFP, whereas mouse cells only expressed CD11b but were negative for hCD45 and GFP. (b) H9-microglia graft efficiency. Percentage of CD11b cells in the total sample, and proportion of human cells amongst them. Graph shows mean±SEM, n=6 mice per group.

Extended Data 2. H9-microglia showed a widespread distribution across multiple areas of the brain.

(a) Representative overview of the extent of H9-microglia graft in the mouse brain. Human microglia are stained for P2RY12 across consecutive sections separated by 500μm to capture multiple anatomical areas. Scale bar, 1mm. (b) Higher magnification images of multiple anatomical areas including meninges, cortex, striatum, white matter, choroid plexus and hippocampus. Labelling shows DAPI (in blue), GFP (in green) and P2RY12 (in cyan). Images are representative of a staining performed in n=4 mice. Scale bar, 100 μm.

Extended Data 3. Extended clustering and distribution of in vitro, in vivo (engrafted) H9 and primary microglia.

(a) PCA shows clear separation between in vitro (MNC and MG) and in vivo (engrafted H9 and primary) microglia. The colours correspond to the clustering shown in Figure 2a. (b) t-SNE plots as in Figure 2a, coloured by the combined level of expression of groups of genes that characterise distinct microglial states. The original clusters from Figure 2a are outlined. (c) Selected genes defining the different transcriptomic scores shown in b. The full list of genes is shown in Supplementary Table 3. (d) Distribution of the different samples across the tSNE plot, and (e) percentage of each sample across the different clusters. All the data shown represents 2,246 transplanted H9-microglia (in vivo) (n=3/1, 3 mice in 1 combined sequencing pool), 4496 H9-derived monocytes (n=2/1, 2 differentiations in 1 combined sequencing pool) and 3385 microglia in vitro (n=2/1), and 22,846 human primary microglia obtained from cortical surgical resections (n=7/7 online Methods).

Extended Data 4. Direct comparison of in vitro, in vivo (engrafted) H9 and primary microglia.

(a) Volcano plots showing paired comparisons between average gene expression in vitro MNC, in vitro MG, in vivo (engrafted) MG and primary cells. (b) Individual comparisons of in vivo (engrafted) H9-microglia and each human subject (human cases 1-7). The dashed line corresponds to an arbitrary threshold logFC of 0.2. Blue labels correspond to homeostatic genes whereas red labels correspond to microglial activation genes (Supplementary Table 3). All the data shown represents 2,246 transplanted H9-microglia (in vivo) (n=3/1, 3 mice in 1 combined sequencing pool), 4496 H9-derived monocytes (n=2/1, 2 differentiations in 1 combined sequencing pool) and 3385 microglia in vitro (n=2/1), and 22,846 human primary microglia obtained from cortical surgical resections (n=7/7). In all cases, Wilcoxon Rank Sum test, p-values adjusted with Bonferroni correction based on the total number of genes in the dataset.

Extended Data 5. Characterization of oAβ preparation.

Freshly eluted recombinant Aβ1-42 monomers follow a rapid aggregation course in Tris-EDTA buffer. (a) After 2 hours of incubation, Aβ1-42 monomers oligomerize and run as dimers and trimers (indicated with *) on SDS-PAGE/Coomassie staining, and they are proteinase-K sensitive. (b) Early Aβ1-42 oligomers form A11 and OC-positive aggregates. Two µl of either scrambled or amyloid beta 1-42 from different time points (0 hours, 2 hours and 2 weeks) of incubation was spotted on blots. These dot blots were probed with A11 antibody (Invitrogen; #AHB0052), which recognizes amino acid sequence-independent oligomers of proteins or peptides. A11 epitope is transient and is present only in the early oligomers (2 h), in contrast to the mature fibers (2 w) formed after 2 weeks of incubation. No fibrillary material is detected after 2 hours of incubation. (c) OC antibody (Millipore; #AB2286) recognize epitopes common to monomers, amyloid oligomers, and fibrils. (d) 4G8 antibody (Eurogentec; #SIG-39220) detects N-terminal of the amyloid aggregates (epitope between amino acids 17-24).

Extended Data 6. Preparation of the datasets for the analysis of (a-c) host mouse and (d-f) H9-microglia response to oAβ (see Figure 3).

(a) t-SNE plot of the 13342 cells passing quality control, coloured by clusters. (b) t-SNE plots as in a, coloured by the level of ln normalized expression of selected genes for microglia (Cx3cr1, Tmem119), monocytes (Ccr2) and neutrophils (Ccrl2). (c) Violin plots of selected marker genes for homeostatic microglia (Cx3cr1, Tmem119), CRM (Il1b), ARM (Cd74, H2-Eb1, Ifit3), neutrophils (Ccrl2), monocytes (Ccr2, Ly6c1), astrocytes (Clu), oligodendrocytes (Mbp), neurons (Npy), and cycling cells (Top2a). Analysis shown in Figure 2 was performed after removal of clusters 4 (neutrophils), 7 (monocytes), 10 (astrocytes), 12 (oligodendrocytes) and 9 (neurons). (d) t-SNE plot of the 6444 H9-microglia cells passing quality control, coloured by clusters. (e) t-SNE plot as in a, coloured by treatment (naïve; scrambled peptide, Scr; and oligomeric Aβ, oAβ). (f) t-SNE plot as in a, coloured by the level of ln normalized expression of selected genes for microglia (CX3CR1), cycling cells (MKI67) and brain resident macrophages (MRC1, CD163). Analysis shown in Figure 2 was performed after removal of clusters 1 and 4 (brain resident macrophages), 6 (cycling cells) and 8 (doublets).

Extended Data 7. Expanded analysis, clustering and trajectory inference of the analysis of the response of H9-microglia upon oAβ

(a) PCA of 4880 H9-microglia isolated from the mouse brain (n=3 mice in 1 combined sequencing pool) shows clear separation of the different clusters identified in our analysis in PC1 and PC2. (b) t-SNE plots as in Figure 3a, coloured by the combined level of expression of groups of genes that characterise distinct microglial states. (c) Selected genes defining the different transcriptomic scores shown in Figure 3b: homeostatic score (1), cytokine score (2) activated score (3). The full list of genes is shown in Supplementary Table 3. (d) Volcano plots showing paired comparisons between H9.HM, H9.CRM and H9.PM clusters ((Wilcoxon Rank Sum test, p-values adjusted with Bonferroni correction based on the total number of genes in the dataset). (e) Proportion of the different experimental groups across clusters in Figure 3a (Chi test, *** p < 10^-250). (f, g) Phenotypic trajectory inferred by Monocle 2 as shown in Figure 3a, coloured by (f) treatment and (g) clusters from Figure 3a.

Extended Data 8. Expanded analysis, clustering and trajectory inference of the analysis of the response of mouse host microglia upon oAβ

(a) PCA of 9942 endogenous mouse cells (n=3 mice in 1 combined sequencing pool) shows clear separation of the different clusters identified in our analysis in PC1 and PC2. (b) t-SNE plots as in Figure 3d, coloured by the combined level of expression of groups of genes that characterise distinct microglial states. (c) Selected genes defining the different transcriptomic scores shown in b: homeostatic score (1), cytokine score (2) activated score (3). The full list of genes is shown in Supplementary Table 3. (d) Volcano plots showing paired comparisons between ms.HM, ms.CRM and ms.ARM clusters (Wilcoxon Rank Sum test, p-values adjusted with Bonferroni correction based on the total number of genes in the dataset). (e) Proportion of the different experimental groups across clusters in Figure 3d (Chi test, *** p < 10^-250). (f, g) Phenotypic trajectory inferred by Monocle 2 as shown in Figure 3c, coloured by (f) treatment and (g) clusters from Figure 3d.

Extended Data 9. Cytokine response microglia (CRM) are also present in APP^NL-G-F mice.

(a) Original clustering analysis from Sala Frigerio et al. (2019) consisting of 10,801 microglial cells from 3 to 21 months old APP^NL-G-F mice and aged matched wild type controls. (b) Clusters shown in a, coloured with CRM, HM, ARM and IRM transcriptomic signatures. Note the small population of cells displaying CRM features embeded into the ARM response in APP^NL-G-F microglia. (c) Significant enrichment of either homeostatic (HM) or activated (ARM) microglia gene sets from Sala Frigerio et al. (2019) in our ms.HM and ms.ARM clusters, respectively (ANOVA with Turkey HSD multiple comparisons correction, *** p≈0; box plots represent median, with 25^th and 75^th percentiles and 1.5 times the inter-quartile range as minima and maxima). (d) Subselection of CRM cells from the main clusters shown in a. (e) Microglia cells enriched with a CRM transcriptomic profile are largely located at early stages of the response to amyloid in APP^NL-G-F mice. The left panel shows the trajectory analysis coloured by clusters as represented in panel a, whereas the right panel highlights the cells displaying a CRM profile.

Extended Data 10. Differential responses of human and host mouse microglia to oligomeric Aβ.

(a) Pathway enrichment analysis (GOrilla) shows that the differentially expressed genes in CRM vs. HM clusters are involved in immune and inflammatory processes. (b) Top differentially expressed genes in H9-microglia upon Aβ challenge relative to scrambled peptide, and expression of their mouse orthologs by endogenous mouse cells. Coloured marks indicate the functional category as shown in b. (c) Differentially expressed genes that show opposite behaviour in H9-and mouse host (Rag2^-/- Il2rγ^-/-) microglia. Coloured marks indicate the functional category as shown in b. (d) Volcano plots showing paired comparisons between H9.HM, H9.CRM, but including all genes (even those with no clear orthology to mouse, Wilcoxon Rank Sum test, p-values adjusted with Bonferroni correction based on the total number of genes in the dataset). (e) Further pathway enrichment analysis (GOrilla) performed on the human-specific (with no clear orthology) differentially expressed genes in H9.CRM vs. H9.HM clusters are involved in cytokine/chemokine responses.

Figure 1. Human ESC-derived microglia successfully engraft the mouse brain.

(a) Selection of 44 genes with p>5x10^-8 from 3 landmark studies in the field. See online methods. (b) From these 44 candidate AD risk genes^,,, 15 (marked with a red dot) do not have a clear 1:1 mouse ortholog or display <60% identity between human and mouse at the primary amino acid sequence. Colour scale, green (high similarity) to red (low similarity). (c) Schematic representation of the area of mouse brain covered by transplanted human microglia. Microglia are represented by green dots, and the distance between anatomically consecutive sections is 500μm. (d) H9-microglia successfully engraft the mouse brain and (e) express homeostatic markers TMEM119 and P2RY12 (n=4 mice). Scale bars of 100 and 5μm, respectively. (f) Transplanted cells distribute across the parenchyma forming a mosaic with similar nearest neighbour distance (NND) and density to that of mouse cells from adjacent areas (n=4 mice per group, two-tailed t-test p=0.9, graph shows mean±SEM). H9-microglia are labelled in green (Iba1⁺ GFP⁺), whereas arrowheads highlight few mouse cells (Iba1⁺ GFP^-) co-existing with H9-microglia in the grafted areas of the parenchyma (n=4 mice). Scale bar, 100 μm. (g) Higher magnification microphotographs and 3D reconstruction by Imaris show typical morphology with high complexity branching in H9-microglia (n=4 mice). Scale bar 5 μm.

Figure 2. H9-microglia isolated 8 weeks after transplantation are similar to human primary microglia.

(a) t-SNE plot visualizing 33,144 single cells sorted based on CD11b (primary human), CD11b hCD45 and GFP (engrafted H9-microglia) staining, and in vitro derived monocytes (MNC) and microglia (MG) after quality control, and removal of peripheral cells, cycling cells and doublets. Cells are coloured according to clusters identified with Seurat’s kNN and merging: In vitro-1 MNC, In vitro-2 MG, In vivo-Homeostatic Microglia (HM) and Cytokine Response Microglia (CRM), CNS-Associated Macrophages (CAM), and Neutrophils (Nφ). The assignment of different clusters to distinct cell types/states is based on previous experimental data from our lab and a recent meta-analysis describing multiple modules of microglial transcriptional profiles, as detailed in Extended Data 4a-c and Supplementary Table 3. (b, c) Distribution and percentage of cells from either in vitro, in vivo (engrafted) H9 or primary human microglia across the different clusters identified. (d) Most highly expressed genes in the different samples: in vitro-1 MNC; in vitro-2 MG, in vivo (engrafted) H9 and primary microglia. (e) In situ hybridization for CX3CR1 and P2RY12 (microglia) and MRC1 (perivascular macrophages) confirming the location of the two main distinct identities acquired by H9 engrafted cells (GFP) in the mouse brain (n=4 mice). Scale bar is 25 μm and 10 μm in the left and right panels, respectively. (f, g) Volcano plots showing gene expression differences between average gene expression in (f) 22,846 primary vs. 3385 in vitro MG and (g) 22,846 primary vs. 2,246 engrafted H9-microglial cells (with a logFC threshold of 0.2, Wilcoxon Rank Sum test, p-values adjusted with Bonferroni correction based on the total number of genes in the dataset). Genes associated to homeostatic or activation expression profiles are highlighted in blue and red, respectively (Supplementary Table 3).

Figure 3. Human and host mouse microglial response to oligomeric Aβ.

(a, b) Analysis of the response of H9-microglia upon oAβ exposure. (a) t-SNE plot visualizing the 4880 H9 microglia passing quality control, and after removal of CAM, cycling cells and doublets. Cells are coloured according to clusters identified with Seurat’s kNN (upper panel; H9.HM: Homeostatic Microglia, H9. PM: Primed Microglia, H9.CRM: Cytokine Response Microglia, H9) and treatment (lower panel; Scr: scrambled peptide, oAβ: oligomeric Ab). (b) Plot of the phenotypic trajectory followed by H9-microglia upon oligomeric Aβ exposure, obtained by an unbiased pseudotime ordering with Monocle 2 and coloured by clusters as in d. H9-microglia followed a trajectory from H9.HM and H9.PM, to H9.CRM. The heatmap shows the differential expression of representative genes from each cluster, ordered by pseudotime. (c, d) Analysis of the response of endogenous (Rag2^-/- Il2rγ^-/-) mouse microglia upon oligomeric Aβ challenge. (d) t-SNE plot visualizing the 9942 endogenous mouse microglia passing quality control, and after removal of peripheral cells, CNS-Associated Macrophages (CAM) cycling cells and doublets. Cells are coloured according to clusters identified with Seurat’s kNN (upper panel; ms.HM: (mouse) Homeostatic Microglia, ms.CRM: Cytokine Response Microglia, ms.ARM: Activated Response Microglia) and treatment (lower panel; Scr: scrambled peptide, oAb: oligomeric Ab). (c) Plot of the phenotypic trajectory followed by endogenous mouse microglia upon oligomeric Aβ exposure, obtained by an unbiased pseudotime ordering with Monocle 2 and coloured by clusters as in a. Mouse microglia followed a trajectory from ms.HM to ms.CRM to ms.ARM. The heatmap shows the differential expression of representative genes from each cluster, ordered by pseudotime. (e) Correlation analysis of the log-fold change (logFC) in H9 (y-axis) and host (Rag2^-/- Il2rγ^-/-) mouse (x-axis) microglia upon oligomeric Aβ challenge relative to scrambled peptide (Pearson correlation, R=0.4. Differentially expressed genes are highlighted in green when significant in both species, blue only in H9-microglia or orange only in mouse microglia. Numbers between brackets in the legend represent the amount of up and downregulated genes in each group, respectively. (f) Expression changes induced by Aβ challenge in the selected candidate AD-risk genes (Figure1b). (g) Extension of the table shown in Figure 1a highlighting the important number of putative AD-risk genes in humans that lack good orthologues in mice or show an opposite behaviour upon Aβ challenge (highlighted by red dots). Expression profile of 44 putative AD genes in our datasets (H9-microglia; primary human microglia from 7 patients; and mouse host Rag2^-/- Il2rγ^-/-microglia, mouse RM), and wild type mouse microglia from 2 independent datasets of 12-week-old immunocompetent C57Bl/6 mice (Sala Frigerio et al.,, SF; and Keren-Shaul et al., KS). We identified 15 genes with observed expression in human but not mouse microglia and, that were also observed in H9-microglia.