1 Functional Mature Human Microglia Developed in Human iPSC Microglial Chimeric Mouse Brain

Microglia, the brain-resident macrophages, exhibit highly dynamic functions in neurodevelopment and neurodegeneration. Human microglia possess unique features as compared to mouse microglia, but our understanding of human microglial functions is largely limited by an inability to obtain human microglia under homeostatic states. We developed a human pluripotent stem cell (hPSC)-based microglial chimeric mouse brain model by transplanting hPSC-derived primitive macrophage precursors into neonatal mouse brains. The engrafted human microglia widely disperse in the brain and replace mouse microglia in corpus callosum at 6 months post-transplantation. Single-cell RNA-sequencing of the microglial chimeric mouse brains reveals that xenografted hPSC-derived microglia largely retain human microglial identity, as they exhibit signature gene expression patterns consistent with physiological human microglia and recapitulate heterogeneity of adult human microglia. Importantly, the engrafted hPSC-derived microglia exhibit dynamic response to cuprizone-induced demyelination and species-specific transcriptomic differences in the expression of neurological disease-risk genes in microglia. This model will serve as a novel tool to study the role of human microglia in brain development and degeneration.


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As the resident macrophages of the central nervous system (CNS), microglia play critical roles in 3 maintenance of CNS homeostasis and regulation of a broad range of neuronal responses 1, 2 . Recent 4 studies indicate that dysfunction of microglia contributes to neurodevelopmental and neurodegenerative 5 diseases, including Alzheimer's disease (AD) 3-7 . Moreover, genome-wide association studies have 6 shown that many neurological disease risk genes, particularly neurodegenerative diseases, are highly 7 and sometimes exclusively expressed by microglia [8][9][10] . These observations provide a compelling 8 incentive to investigate the role of microglia in models of abnormal brain development and 9 neurodegeneration. Most studies of microglia largely rely on rodent microglia. However, there is 10 increasing evidence that rodent microglia are not able to faithfully mirror the biology of human microglia 11 11 . In particular, recent transcriptomic studies have clearly demonstrated that a number of immune 12 genes, not identified as part of the mouse microglial signature, were abundantly expressed in human 13 microglia 8,12 . Moreover, a limited overlap was observed in microglial genes regulated during aging and 14 neurodegeneration between mice and humans, indicating that human and mouse microglia age 15 differently under normal and diseased conditions 12, 13 . These findings argue for the development of 16 species-specific research tools to investigate microglial functions in human brain development, aging, 17 and neurodegeneration.

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Functional human brain tissue is scarcely available. In addition, given the considerable 19 sensitivity of microglia to environmental changes 8 , the properties of available human microglia isolated 20 from surgically resected brain tissue may vary significantly, due to different disease states of the 21 patients and the multi-step procedures used for microglial purification. In order to study human 22 microglia in a relatively homeostatic state, many scientists have turned to human pluripotent stem cells 23 (hPSCs). Recent advances in stem cell technology have led to the efficient generation of microglia from 24 hPSCs 14-19 , providing an unlimited source of human microglia to study their function. However, when 25 cultured alone or co-cultured with neurons and astrocytes in 2-dimensional (2D) or 3D 26 organoid/spheroid culture systems, these hPSC-derived microglia best resemble fetal or early postnatal 27 human microglia. This is indicated by much lower expression of key microglial molecules such as 28 TREM2, TMEM119, and P2RY12 in the hPSC-derived microglia, as compared to microglia derived 29 from adult human brain tissue 16,18,20 . Thus, even with these novel in vitro models, it has been 30 challenging to advance understanding of human microglial function in adult ages or in 31 neurodegeneration during aging.

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that are Rag2/IL2rg-deficient and also express the human forms of CSF1, which facilitates the survival 23 of xenografted human myeloid cells and other leukocytes 30,31 . We deposited cells into the white matter 24 overlying the hippocampus and sites within the hippocampal formation ( Figure 1C). In order to visualize 25 the distribution of donor-derived microglia, at 6 months post-transplantation, we stained the mouse 26 brain sections with human-specific antibody recognizing TMEM119 (hTMEM119). TMEM119 is a 27 marker that is only expressed by microglia, but not other macrophages 14, 17, 32 . We found that the 28 donor-derived hTMEM119 + microglia widely disperse in the brain ( Figure 1D). As early as 3 weeks 29 post-transplantation, donor-derived microglia had already migrated along corpus callosum and passed 30 through the rostral migratory stream to the olfactory bulb ( Figure 1E). At 6 months post-transplantation, 31 human microglia widely dispersed in multiple brain regions, including olfactory bulb, hippocampus, and 32 cerebral cortex, and exhibited a highly ramified morphology ( Figure 1F and G). Frequently, we also 33 observed clusters of human microglia in the cerebellum ( Figure 1H), which might be a result from the 34 strong ability of immune cells trafficking along blood vessels and/or the choroid plexus 33 . Similar to our 35 previous studies 24, 25 , we assessed the engraftment efficiency and degree of chimerization by 36 quantifying the percentage of hTMEM119 + cells among total DAPI + cells in the forebrain in sagittal brain 37 sections covering regions from 0.3 to 2.4 mm lateral to midline and found that about 8% of the total 38 cells were human microglia in the 6-month-old mouse brains ( Figure 1D and L). As shown by individual 39 data points overlaid on the bar graphs ( Figure 1L), there were variations in chimerization among 40 animals. In addition to the images showing distribution of donor-derived cells in Figure 1D, we thus also 41 included tile scan images collected from another chimeric mouse brain that represents the lower level 42 of chimerization (supplementary Figure 1A). In the developing brain, microglia are known to use white 43 matter tracts as guiding structures for migration and that they enter different brain regions 34 . In order to 44 examine the migration pattern of our transplanted cells, we deposited PMPs into different sites, the 45 lateral ventricles of P0 mice. As early as three weeks post-transplantation, we found that the majority of 46 donor-derived cells migrated along the anterior corpus callosum, rostral migration stream, and then entered the olfactory bulb. Moreover, some of those cells of migrated posteriorly along the corpus 48 callosum (supplementary Figure 1B), suggesting that engrafted cells likely used corpus callosum to 49 migrate to various brain regions. These results demonstrate that hPSC-derived PMPs survive in mouse 50 brain and that they migrate to a variety of structures.
To examine whether transplanted hPSC-derived PMPs efficiently differentiated to microglia in 1 the mouse brain, we double-stained brain sections for both human nuclei (hN) and hTMEM119. As 2 early as 8 weeks post-transplantation, the vast majority of hN + donor-derived cells (93.2 ± 2.2%, n =7) 3 were positive for hTMEM119 ( Figure 1I and M), indicating the robust and efficient differentiation of 4 hPSC-derived PMPs into microglia. Moreover, the vast majority of the donor-derived cells expressed 5 PU.1, a transcription factor that is necessary for microglial differentiation and maintenance 35-38 , and 6 were positive for human specific CD45 (hCD45), which is expressed by all nucleated hematopoietic 7 cells ( Figure 1J) 39,40 . Similarly, at 6 months post-transplantation, the vast majority of donor-derived 8 cells expressed hTMEM119 (supplementary Figure 2A) and P2RY12 (93.4 ± 3.8%, n = 7; 9 supplementary Figure 2B and C). hTMEM119 and P2RY12 was not expressed in PMP cultures 10 (supplementary Figure 2D). Moreover, we examined the distribution of human donor cells in border 11 regions, including choroid plexus, meninges, and perivascular spaces. We found that most of the 12 hTMEM119 -/hN + donor-derived cells were seen in those border regions. Furthermore, we co-stained 13 hTMEM119 with CD163, an established marker for non-microglial CNS myeloid cells 41, 42 . In choroid 14 plexus, we found that some of the hTMEM119cells co-expressed CD163, suggesting that these 15 transplanted cells differentiated into choroid plexus macrophage (cpMΦ), but not microglia 16 (supplementary Figure 3A). In order to better visualize meninges and perivascular space, we triple-17 stained hN and CD163 with laminin, a marker that has been commonly used to visualize vascular 18 structures in the mammalian brain 43 . There was also a small number of hN + and CD163 + co-expressing 19 cells in these regions, indicating that the transplanted cells differentiated into meningeal macrophage 20 (mMΦ) and perivascular macrophages (pvMΦ) (supplementary Figure 3B and C). There was a 21 possibility that some transplanted cells might remain as progenitors and maintain their hematopoietic 22 progenitor-like cell identity. As shown in supplementary Figure 3D, we found that a small population of 23 hN + cells expressed CD235 in the regions close to the lateral ventricles. Overall, these results 24 demonstrate that the vast majority of engrated hiPSC-derived PMPs differentiate into hTMEM119 + 25 microglia, with a relatively small number giving rise to other of hTMEM119 -CNS myeloid cells in a brain 26 context-dependent manner or remaining as progenitors.

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We next assessed the proliferation of engrafted cells by staining the proliferative marker Ki67.

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As shown in Figure 1K and N, at 3 weeks post-transplantation, about 17% (16.9 ± 5.7%, n = 8) of hN + 29 transplanted cells expressed Ki67, indicating that these cells were capable of proliferating in the mouse 30 brain. At 6 months post-transplantation, the percentage of proliferating cells dramatically decreased and 31 less than 2% (1.7 ± 0.8%, n = 7) of total engrafted cells were Ki67 positive. These Ki67 + proliferating 32 human cells mainly localized in the subventricular zone, the walls along lateral ventricles, corpus 33 callosum, and olfactory bulb ( Figure 1K and supplementary Figure 3E). We also examined the 34 proliferation of mouse host microglia at different brain regions at 3 weeks post-transplantation. We only 35 found a very small number of Ki67 + mouse microglia in the subventricular zone (supplementary Figure   36 3F), which is consistent with a previous report 44 . Taken together, these findings demonstrate that 37 engrafted hPSC-derive PMPs differentiate to microglia, generating a mouse brain with a high degree of 38 human microglial chimerism in the forebrain.

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Human PSC-derived microglia undergo morphological maturation and are functional in the mouse 41 brain 42 Compared with three weeks post-transplantation, hPSCs-derived microglia appeared to exhibit more 43 complex processes at 6 months post-transplantation ( Figure 1E and F). Moreover, even at the same 44 stage, hPSC-derived microglia in the cerebral cortex seemed to exhibit much more complex 45 morphology, compared with the hPSCs-derived microglia in the corpus callosum and cerebellum 46 ( Figure 1G and H and hTMEM119 and Iba1 staining in supplementary Figure 2A and 4, respectively).

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In the corpus callosum, hPSC-derived microglia had fewer branches that aligned with axons; and in the 48 cerebral cortex, the microglia exhibited more complex and ramified processes (supplementary Figure   49 2A and 4), similar to observations from previous studies 34, 45 . This prompted us to further examine the 50 morphological and functional changes of the hPSC-derived microglia along with the development of the 51 mouse brain, particularly in cerebral cortex. Previous studies have shown that there are no changes in 1 microglial number, cytokine levels, and gene expression profiles between wild type and Rag2 -/mice 46 . 2 Building upon that, we also compared the differences between xenografted hPSC-derived microglia vs.
3 host mouse microglia. We double-stained the brain sections with human and mouse specific TMEM119 4 (hTMEM119 and mTMEM119, respectively) antibodies to distinguish hPSC-derived microglia and 5 mouse host microglia (Figure 2 A and B). As shown in Figure 2A, in 6 month old mice, both hPSCs-6 derived microglia and mouse microglia were seen in the cerebral cortex and hippocampus. Notably, we 7 observed that mouse microglia mainly resided in distal regions in the cerebral cortex and hippocampus.

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In particular, in the corpus callosum, mouse microglia were rarely seen, and the vast majority of 9 microglia were hPSC-derived microglia ( Figure 2C), indicating that hPSC-derived microglia replaced 10 the host mouse microglia. In the cerebral cortex, hTMEM119 + hPSC-derived microglia exhibited much 11 more complex processes at 8 weeks and 6 months post-transplantation than those cells at 3 weeks 12 post-transplantation, as indicated by the increased number of endpoints ( Figure 2D). The total length of 13 processes of hPSC-derived microglia also significantly increased from week 3 to week 8 and month 6 14 ( Figure 2E), suggesting the gradual maturation of hPSC-derived microglia in mouse brain. We further 15 examined the morphological differences between hPSC-derived microglia vs. mouse microglia at the

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Microglia have been shown to shape synapse formation by pruning synapses and to maintain 32 oligodendroglial homeostasis by phagocytizing oligodendroglial cells 51-53 . We therefore investigated 33 whether hPSCs-derived microglia are functional in the mouse brain. To examine synaptic pruning 34 function, we employed a super-resolution imaging technique to visualize synapse engulfment by 35 hPSCs-derived microglia. We triple-stained hTMEM119 with both a post-synaptic marker PSD95 and a 36 pre-synaptic marker synapsin I. The 3D reconstruction images show that PSD95 + and synapsin I + 37 puncta are colocalized within hTMEM119 + processes, indicating that these synaptic proteins are 38 phagocytosed by hPSCs-derived microglia at eight weeks post-transplantation in grey matter ( Figure   39 3A). In addition, we also validated the specificity of PDS95 puncta staining by incubating brain sections 40 with the PSD95 antibody together with a PSD95 peptide. We barely detected any PSD95 + puncta 41 signal after the incubation in the presence of PSD95 peptide (supplementary Figure 5A). We also triple-42 staining hTMEM119 and PSD95 with CD68. As shown in Figure 3B Figure 3C). Very few mouse microglia were found to engulf synaptic proteins at 8 weeks posttransplantation (supplementary Figure 6A). To examine the function of phagocytosing oligodendroglia,

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we double-stained hCD45 with PDGFRα, a marker for oligodendroglial progenitor cell. We observed 49 that hCD45 + human microglia were able to engulf PDGFRα + oligodendroglia at 3 weeks post-50 transplantation in the corpus callosum ( Figure 3D). We also double stained hCD45 with the oligodendroglial marker OLIG2 and similarly found that hPSCs-derived microglia in white matter 1 engulfed OLIG2 + oligodendroglia at 3 weeks post-transplantation (supplementary Figure 6B). In 2 addition, we detected that a small population of mouse microglia engulfed OLIG2 + oligodendroglia in 3 the corpus callosum at 3 weeks post-transplantation (supplementary Figure 6C).

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Microglia, together with endothelial cells, pericytes and astrocytes, form the functional blood-5 brain barrier 54-56 . We double-stained brain sections with hCD45 and laminin. We found that hPSC-6 derived microglia clustered around and were closely affiliated with laminin + blood vessels in both grey 7 matter and white matter across different brain regions including the olfactory bulb ( Figure 3E and   8 supplementary Figure 6D). In addition, we also found that mouse microglia similarly had close contact 9 with blood vessels in the cerebral cortex, corpus callosum, and olfactory bulb (supplementary Figure   10 6E). Altogether, these results demonstrate hPSCs-derived are functional in the mouse brain under 11 homeostatic conditions. The human microglia and host mouse microglia exhibited similar microglial 12 functions, including synaptic pruning, phagocytosis of oligodendroglia, and having contact with blood 13 vessels.

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Single-cell RNA-sequencing of hiPSC microglial chimeric mouse brain identifies a gene 16 expression signature consistent with adult human microglia

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Homeostatic human microglia at adult stages are difficult to obtain, because microglia are highly 18 sensitive to environmental changes and microglia derived from adult human brain tissue-derived are 19 usually purified through multi-step procedures that can change their biological properties significantly 8 .

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In addition, microglia derived from hPSCs using all current differentiation protocols largely resemble 21 fetal or early postnatal human microglia 16, 18, 20 . We hypothesize that hPSC microglial chimeric mice 22 may provide a unique opportunity to study biological properties of adult human microglia, because the 23 engrafted hPSC-derived microglia are likely to exhibit an expedited maturation process promoted by the 24 maturing environment in the mouse brain. To test this hypothesis, we examined transcriptomic profiles 25 of hiPSC-derived microglia, developed in the in vivo homeostatic mouse environment, using single-cell 26 RNA-sequencing (scRNA-seq). We collected brain regions where engrafted hiPSCs-derived microglia 27 preferentially dispersed, including the cerebral cortex, hippocampus, corpus callosum, and olfactory 28 bulb, from 6-month-old chimeric mouse brain for scRNA-seq. A previous study has demonstrated that 29 within hours during which microglia are isolated from the brain environment and transferred to culture 30 conditions, microglia undergo significant changes in gene expression 8 . To capture observed 31 expression patterns as close to the "in vivo" patterns as possible, we chose to omit a FACS sorting step 32 since it would have added substantial processing time. Owing to the wide distribution and high 33 abundance of hiPSC-derived microglia in those brain regions, we were able to capture ample numbers 34 of hiPSC-derived microglia for scRNA-seq even without enrichment by FACS sorting. After brain tissue 35 dissociation with papain and centrifugation to remove debris and myelin, single cell suspensions were 36 directly subjected to droplet-based 10X Genomic RNA-seq isolation (Fig. 4A). Using stringent criteria, 37 29,974 cells passed the quality control evaluation (with about 10,000-15,000 reads/cell) from 4 animals 38 for downstream analysis (supplementary Figure 7A).

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We performed dimensionality reduction and clustering using a principal component analysis  Figure 7B). We defined each cluster based on the expression of enriched genes (Table 45 S1) that could be recognized as markers for specific cell types or are reported to be abundantly  Figure 7E). Furthermore, the expression of a set of canonical microglial genes (C1QA, CX3CR, 6 TREM2, CSF1R, and P2RY12) was only detected in Xeno MG and mouse microglia clusters ( Figure   7 4D). Moreover, we performed bulk RNA-seq to analyze the pre-engraftment hiPSC-derived PMPs. We 8 compared transcriptomic profiles between PMPs and Xeno MG from 6 months old chimeric mice.

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Notably, as shown in Figure 4E, compared with PMPs, Xeno MG highly expressed microglial identity 10 markers, such as TMEM119, P2RY12, SALL1, and OLFML3, which were barely detected in PMPs. On 11 the other hand, the expression of markers for hematopoietic progenitor cells, such as CD59, CD44, and 12 CD38, was low in Xeno MG but much higher in PMPs. Furthermore, we compared the transcriptomic 13 profile of Xeno MG with a published dataset generated from human brain tissue-derived human    Figure 8C). Taken together, these results demonstrate that Xeno MG developed in the 1 mouse brain highly resemble mature human microglia and faithfully recapitulate heterogeneity of adult 2 human microglia.

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Transcriptomic profiling analysis reveals differences between co-resident Xeno MG and mouse 5 microglia 6 Previous studies reported differences in transcriptomic profiles between human and mouse microglia 8, 7 12 . In the chimeric mouse brain, as xenografted hiPSC-derived microglia and host mouse microglia 8 developed in the same brain environment, this model may provide a unique opportunity to directly 9 examine the differences between human and mouse microglia. Xeno MG and host mouse microglia 10 clusters obtained from 4 independent samples of 6-month-old chimeric mouse brains were used for the  Figure 8D and Table S2). Importantly, previously-reported signature genes

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Many significantly enriched terms were associated with the innate immune activity of microglia, such as 25 "immune system response," "cellular response to chemical stimulus," and "regulation of cytokine 26 production." This finding of enriched innate immunity-related gene in our Xeno MG could be either 27 reflect the nature of human microglia as reported previously 12, 80 , or the result from differential 28 responses to critical signals from murine molecules, such as fractalkine. These results suggest that, 29 compared to the host mouse microglia, Xeno MG and mouse microglia exhibit overall similar patterns of 30 transcriptomic profile, but numerous species-specific differentially expressed genes were also 31 observed.

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Previous studies have shown that several disease risk genes, such as genes associated with 33 AD, Parkinson's disease (PD), multiple sclerosis (MS), and schizophrenia (SCZ), are preferentially 34 expressed in microglia 8,80,81 . Moreover, relative expression of these genes in human and mouse 35 microglia are also different 8 . Therefore, we examined the expression of disease risk genes in Xeno MG 36 and mouse microglia from our chimeric mouse brain preparation. Expression of disease risk genes, as 37 reported in a recent study 8 , had a highly similar differential expression pattern in co-resident mouse

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we found that out of 32 MS genes, 29 genes, including ZFP36L1, RPL5, and NDFIP1 were more 40 abundantly expressed in Xeno MG than in mouse microglia ( Figure 5D and E). Similarly, out of 14 AD 41 genes, 10 genes including Apoc1, Sorl2, and Mpzl1, were more abundantly expressed in Xeno MG 42 than in mouse microglia (supplementary Figure 8F and I). Out of the 20 PD genes listed in a previous 43 report 8 , 18 genes, such as Vps13c, Snca, Fgf20, Mnnrn1, and Lrrk2, had the same trend of differential 44 expression with greater expression in Xeno MG than in mouse microglia (supplementary Figure 8H and 45 I). We also found that some of the disease genes were preferentially expressed in mouse microglia, 46 such as Syt11and Gba in PD. Altogether, these observations demonstrate that our hPSC microglial chimeric mouse brain can faithfully model disease-relevant transcriptomic differences between human 48 and mouse microglia, and this new model will serve as a new tool for modeling human neurological 49 disorders that involve dysfunction of microglia.

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Human PSC-derived microglia are dynamic in response to cuprizone-induced demyelination 1 To explore whether Xeno MG are functionally dynamic in response to insult, we fed 3 months old 2 chimeric mice with cuprizone-containing diet to induce demyelination. The cuprizone model is one of 3 the most frequently used models to study the pathophysiology of myelin loss in multiple sclerosis 82 . It is 4 appropriate to use our hiPSC microglial chimeric mouse brain to examine the dynamics of human 5 microglia under a demyelination condition, considering our observation that a large number of Xeno MG 6 reside in the corpus callosum at 3 to 4 months post-transplantation and Xeno MG were found nearly 7 exclusively in the corpus callosum at 6 months post-transplantation ( Figure 2C). After 4 weeks of 8 cuprizone treatment, we found that myelin structure, indicated by MBP staining in the corpus callosum, 9 was disrupted and became fragmented in our chimeric mice (Figures 6A), in contrast to the intact and 10 continuous MBP + myelin structure in chimeric mice fed with control diet (supplementary Figure 9). As 11 shown in the super-resolution images in Figure 6B and C, engulfment of MBP + myelin debris by both 12 Xeno MG and mouse microglia were clearly seen in the corpus callosum. Notably, more myelin debris 13 was found inside of mouse microglia, compared with Xeno MG ( Figure 6D). In addition, we also 14 examined the expression of CD74 and SPP1, which is known to be upregulated in multiple sclerosis 83 . Humanized mouse models, in which the immune system is reconstituted with cells of human origin, 3 have been well-established and provide powerful tools for studying cancer, inflammatory and infectious 4 disease, and human hematopoiesis 86 . In this study, by engrafting neonatal mice with hPSC-derived 5 PMPs, we demonstrate the generation of chimeric mouse brains in which hPSC-derived microglia 6 widely disperse. We propose that the following three reasons may account for the generation of human 7 microglial chimeric mouse brain. First, as compared to other types of neural cells, microglial cells are 8 unique in that they turn over remarkably quickly, allowing the vast majority of the population to be 9 renewed several times during a lifetime 87-89 . Previous studies have shown that neonatally transplanted 10 human macroglial or neural progenitor cells can outcompete and largely replace the host mouse brain 11 cells 21, 90, 91 . In this study, we also observe that the hPSC-derived PMPs are highly proliferative prior to 12 transplantation and transplanted cells divide for at least 6 months in the mouse host brain. Therefore, 16, 92 . In our study, we transplanted hPSC-derived PMPs into the mouse brain at the earliest postnatal 27 age, P0, as in general the neonatal brain is more receptive for the transplanted cells and more 28 conducive for their survival and growth 21, 24, 91 . This is also supported by a recent study in which 29 neonatal animals were used for cell transplantation 42 . Moreover, in contrast to those studies that 30 mainly examined donor-derived microglia 2 months after transplantation, we characterized the donor-31 derived microglia up to 6 months post-transplant, which allowed the donor cells to develop for a longer 32 term in the mouse brain. Although there were variations in chimerization among animals, this human 33 microglia chimera model is highly reproducible according to scRNA-seq analysis using four chimeric 34 mouse brains. We caculated the numbers of detected mouse/human microglia in each mouse brain 35 sample and found that human microglia were consistently detected in each sample (legend to 36 supplementary Figure 7). In addition, the high reproducibility of generating such a hiPSC microglial 37 chimeric mouse brain model was also corroborated by two other recent reports 31, 93 .

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Remarkably, we find that xenografted hiPSC-derived microglia developed in the mouse brain retain 39 a human microglial identity. Importantly, xenografted hiPSC-derived microglia showed expression 40 patterns of microglial maturity resembling adult human microglia derived from human brain tissue.

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Therefore, establishment of such a hiPSC microglial chimeric mouse model provides novel 42 opportunities for understanding the biology of human microglia. First, this proof-of-concept study paves 43 the path to interrogating the species differences between human vs. mouse microglia at molecular, 44 functional, and behavioral levels using this hiPSC microglia chimeric mouse brain model. It has been 45 increasingly recognized that as compared to mouse microglia, human microglia possess unique 46 features under conditions of development, aging and disease 8, 11-13 . In our model, human and mouse microglia develop in the same brain, but we have observed that human microglia are morphologically 48 distinct from their mouse counterparts and also exhibit signature gene expression profiles characteristic 49 of human microglia isolated from brain. Microglia are intimately involved in processes of neuronal 50 development, such as neurogenesis, synaptogenesis, and synaptic pruning 94-96 . Building upon the differential expression profiles, our model will be useful to investigate how human and mouse microglia 1 function differently in shaping neuronal development. Similar to a recent report 31 , our engrafted PMPs 2 give to a small population of CNS macrophages. There are also potentially mouse-derived CNS 3 macrophages in the brain tissue that we collected for single-cell RNA-seq, but likely to represent a 4 small fraction of the total cluster. Therefore, within microglial clusters from each species, results may 5 include some similar but distinguishable cell types. However, the primary gene expression differences 6 are likely to be driven by the majority microglia. This was also supported by the observation that 7 previously-reported signature genes expressed in human vs. mouse microglia 8 were differentially 8 expressed in our results. Nevertheless, we take a conservative interpretation and did not claim to 9 identify any novel gene expression differences between human vs. mouse microglia based on the 10 current RNA-seq data. Second, a previous study reports that engrafted human astrocytes modulate 11 other CNS cell types in the mouse brain, particularly enhancing neuronal synaptic plasticity 24 . Thus, in 12 future studies, our hiPSC microglial chimeric mouse model will provide fascinating opportunities to 13 understand how the inclusion of human microglia in the developing brain ultimately impacts neuronal

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Six months old chimeric mice that received transplantation of microglia derived from the hiPSC line 1 were used for single-cell RNA-sequencing experiments. The mice were perfused with oxygenated 2 solution (2.5 mM KCl, 87 mM NaCl, 1.25 mM NaH2PO4, 26 mM NaHCO3, 75 mM sucrose, 20 mM 3 glucose, 2 mM MgSO4, and1 mM CaCl2) as reported 104 and the brain was quickly extracted and kept in 4 the same cold solution for vibratome (VT1200, Leica) sectioning (500µm thickness) and dissection. The 5 brain regions were isolated from where engrafted hiPSCs-derived microglia largely dispersed, including

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To model both human and mouse results in a comparable system, we assigned individual 4 sequencing reads to the optimal species using the alignment score in the bam file, and then separated 5 the original fastq files into individual sets specific by species. These were re-processed with separated 6 mouse or human reference genome indices and then recombined after translating homologous genes.

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In the end, only 147 out of 19,154 barcodes, or 0.76%, included sequences optimally aligning with both 8 species, likely caused by the creation of droplets containing cells from more than one species, so these 9 were eliminated from further consideration. The entire procedure, along with comparisons to alternative 10 strategies, including all required R and Python code, is described elsewhere 110 .

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For analysis of human microglial sub-clusters, extracted human sample/barcode were restricted 12 to human gene symbol results and re-analyzed with Seurat. Gene ontology analysis used the g:Profiler

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Immunostaining and cell counting

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Mouse brains fixed with 4% paraformaldehyde were put in 20% and 30% sucrose for dehydration. After 23 dehydration, brain tissues were blocked with OCT and frozen by solution of dry ice and pure alcohol.

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The tissues were blocked with blocking solution (5% goat or donkey serum in PBS with Triton X-100) in 26 room temperature (RT) for 1 hr. The Triton X-100 concentration was 0.8% for brain tissue. The primary   All data represent mean ± s.e.m. For the data presented as bar-and-data overlap plots. When only 3 two independent groups were compared, significance was determined by two-tailed unpaired t-test with 4 Welch's correction. When three or more groups were compared, one-way ANOVA with Bonferroni post 5 hoc test or two-way ANOVA was used. A P value less than 0.05 was considered significant. The                     orthologs from Xeno MG and mouse microglia clusters, highlighting the differentially expressed genes 4 (DEGs; at least two-fold different) in human Xeno MG (red) or mouse microglia (green) from 6 months 5 old chimeric mouse brain. Significantly different DEGs (less than 5% false discovery rate [FDR] and at 6 least two-fold different) are listed in Table S2.