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

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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.

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Fig. 1: Human ESC-derived microglia successfully engraft mouse brain.
Fig. 2: H9-microglia isolated 8 weeks after transplantation are similar to human primary microglia.
Fig. 3: Human and host mouse microglial response to oligomeric amyloid-β.

Data availability

The data generated in this study are available from Gene Expression Omnibus (GEO; GSE137444). The data and code are also available at https://scope.bdslab.org. Data from Keren-Shaul et al.16 are available from GEO (GSE98969). Data from Sala Frigerio et al.12 are available from GEO (GSE127893).

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Acknowledgements

Work in the laboratory of B.D.S. was supported by the European Union (grant no. ERC-834682 CELLPHASE_AD), the Fonds voor Wetenschappelijk Onderzoek, KU Leuven, Flanders Institute for Biotechnology, UK Dementia Research Institute (Medical Research Council, Alzheimer’s Research UK and Alzheimer’s Society), a Methusalem grant from KU Leuven and the Flemish Government, Vlaams Initiatief voor Netwerken voor Dementie Onderzoek (Strategic Basic Research Grant no. 135043), the ‘Geneeskundige Stichting Koningin Elisabeth’, Opening the Future campaign of the Leuven Universitair Fonds, the Belgian Alzheimer Research Foundation and the Alzheimer’s Association USA. B.D.S. is holder of the Bax-Vanluffelen Chair for Alzheimer’s Disease. B.D.S. receives funding from the Medical Research Council, the Alzheimer’s Society and Alzheimer’s Research UK via the Dementia Research Institute. Cell sorting was performed at the KU Leuven FACS core facility, and sequencing was carried out by the Flanders Institute for Biotechnology Nucleomics Core. R.M. recieves funding from Fonds voor Wetenschappelijk Onderzoek (grant no.G0C9219N) and is a recipient of a postdoctoral fellowship from the Alzheimer’s Association USA.

Author information

R.M. conceived and designed the study, performed all of the experiments and wrote the manuscript. J.V.D.D. conceived and designed the study, performed all of the experiments and wrote the manuscript. N.F. conceived and designed the study, performed all of the experiments and wrote the manuscript. L.W. performed all of the experiments. S.B. contributed to the preparation of oligomeric amyloid-β and intracerebral injections. O.B. assisted with the flow cytometry experiments. A.L. contributed to the interpretation of the data. A.S. assisted on human genetics and human-to-mouse orthology. Y.F. assisted with the analysis of single-cell RNA sequencing data. S.P. assisted with the single-cell RNA sequencing experiments. A.A.-M. optimized the xenograft experiments. C.S.F. optimized the single-cell sequencing experiments and library preparations. C.C. assisted with the differentiation of microglia from ESCs. L.S. established and maintained the mouse colonies. T.T. recruited the human subjects, performed the neurosurgeries and provided the human tissue specimens. V.H.P. contributed to the design of the study and interpretation of the data. C.V. contributed to the design of the study and interpretation of the data. M.F. contributed to the design of the study, and analysis and interpretation of the data. B.D.S. conceived and designed the study and wrote the manuscript. All authors discussed the results and commented on the manuscript.

Correspondence to Renzo Mancuso or Bart De Strooper.

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Competing interests

The authors declare no competing interests. B.D.S. receives grants from different companies that support his research and is a consultant for several companies, but nothing is directly related to the current publication.

Additional information

Peer review information Nature Neuroscience thanks Frederick Bennett and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 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 ± s.e.m., n = 6 mice per group.

Extended Data Fig. 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 500 μm 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 Fig. 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 Fig. 2a. (b) t-SNE plots as in Fig. 2a, coloured by the combined level of expression of groups of genes that characterize distinct microglial states. The original clusters from Fig. 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 Methods).

Extended Data Fig. 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 Fig. 5 Characterization of oligomeric amyloid-β preparation.

Freshly eluted recombinant amyloid β1–42 monomers follow a rapid aggregation course in Tris-EDTA buffer. (a) After 2 hours of incubation, amyloid β1–42 monomers oligomerize and run as dimers and trimers (indicated with *) on SDS-PAGE/Coomassie staining, and they are proteinase-K sensitive. (b) Early amyloid β1–42 oligomers form A11 and OC-positive aggregates. Two µl of either scrambled or amyloid-β1–42 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-β oligomers, and fibrils. (d) 4G8 antibody (Eurogentec; #SIG-39220) detects N-terminal of the amyloid-β aggregates (epitope between amino acids 17–24).

Extended Data Fig. 6 Preparation of the datasets for the analysis of (a-c) host mouse and (d-f) H9-microglia response to oligomeric amyloid-β.

(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 Fig. 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 amyloid-β, 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 Fig. 2 was performed after removal of clusters 1 and 4 (brain resident macrophages), 6 (cycling cells) and 8 (doublets).

Extended Data Fig. 7 Expanded analysis, clustering and trajectory inference of the analysis of the response of H9-microglia upon oligomeric amyloid-β.

(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 Fig. 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 Fig. 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 Fig. 3a (Chi2 test, *** P < 10−250). (f, g) Phenotypic trajectory inferred by Monocle 2 as shown in Fig. 3a, coloured by (f) treatment and (g) clusters from Fig. 3a.

Extended Data Fig. 8 Expanded analysis, clustering and trajectory inference of the analysis of the response of mouse host microglia upon oligomeric amyloid-β.

(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 Fig. 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 Fig. 3d (Chi2 test, *** P < 10−250). (f, g) Phenotypic trajectory inferred by Monocle 2 as shown in Fig. 3c, coloured by (f) treatment and (g) clusters from Fig. 3d.

Extended Data Fig. 9 Cytokine response microglia (CRM) are also present in APPNL-G-F mice.

(a) Original clustering analysis from Sala Frigerio et al. (2019)11 consisting of 10,801 microglial cells from 3 to 21 months old APPNL-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 APPNL-G-F microglia. (c) Significant enrichment of either homeostatic (HM) or activated (ARM) microglia gene sets from Sala Frigerio et al. (2019)11 in our ms.HM and ms.ARM clusters, respectively (ANOVA with Turkey HSD multiple comparisons correction, *** P ≈ 0; box plots represent median, with 25th and 75th 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-β in APPNL-G-F mice11. The top panel shows the trajectory analysis coloured by clusters as represented in panel a, whereas the bottom panel highlights the cells displaying a CRM profile.

Extended Data Fig. 10 Differential responses of human and host mouse microglia to oligomeric amyloid-β.

(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 oligomeric amyloid-β 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.

Supplementary information

Supplementary Information

Supplementary Figs. 1 and 2.

Reporting Summary

Supplementary tables

Supplementary Tables 1–4.

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Mancuso, R., Van Den Daele, J., Fattorelli, N. et al. Stem-cell-derived human microglia transplanted in mouse brain to study human disease. Nat Neurosci (2019) doi:10.1038/s41593-019-0525-x

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