Functional Mature Human Microglia Developed in Human Ipsc Microglial Chimeric Mouse Brain 2 3 Ranjie Xu 1, Andrew J
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bioRxiv preprint doi: https://doi.org/10.1101/594721; this version posted December 30, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 1 Functional Mature Human Microglia Developed in Human iPSC Microglial Chimeric Mouse Brain 2 3 Ranjie Xu 1, Andrew J. Boreland 1, Xiaoxi Li 1, Anthony Posyton1, Kelvin Kwan1, Ronald P. Hart1, Peng 4 Jiang1 ,* 5 6 1Department of Cell Biology and Neuroscience, Rutgers University, Piscataway, NJ 08854, USA. 7 8 9 10 11 12 13 14 15 16 17 18 19 *Address correspondence to: 20 Peng Jiang, Ph.D. 21 Assistant Professor 22 Department of Cell Biology and Neuroscience 23 Rutgers University 24 604 Allison Road, Piscataway, NJ 08854 25 Email: [email protected] 26 Phone: 848-445-2805 1 bioRxiv preprint doi: https://doi.org/10.1101/594721; this version posted December 30, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 1 Abstract 2 3 Microglia, the brain-resident macrophages, exhibit highly dynamic functions in neurodevelopment and 4 neurodegeneration. Human microglia possess unique features as compared to mouse microglia, but 5 our understanding of human microglial functions is largely limited by an inability to obtain human 6 microglia under homeostatic states. We developed a human pluripotent stem cell (hPSC)-based 7 microglial chimeric mouse brain model by transplanting hPSC-derived primitive macrophage precursors 8 into neonatal mouse brains. The engrafted human microglia widely disperse in the brain and replace 9 mouse microglia in corpus callosum at 6 months post-transplantation. Single-cell RNA-sequencing of 10 the microglial chimeric mouse brains reveals that xenografted hPSC-derived microglia largely retain 11 human microglial identity, as they exhibit signature gene expression patterns consistent with 12 physiological human microglia and recapitulate heterogeneity of adult human microglia. Importantly, the 13 engrafted hPSC-derived microglia exhibit dynamic response to cuprizone-induced demyelination and 14 species-specific transcriptomic differences in the expression of neurological disease-risk genes in 15 microglia. This model will serve as a novel tool to study the role of human microglia in brain 16 development and degeneration. 17 18 2 bioRxiv preprint doi: https://doi.org/10.1101/594721; this version posted December 30, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 1 Introduction 2 3 As the resident macrophages of the central nervous system (CNS), microglia play critical roles in 4 maintenance of CNS homeostasis and regulation of a broad range of neuronal responses 1, 2. Recent 5 studies indicate that dysfunction of microglia contributes to neurodevelopmental and neurodegenerative 6 diseases, including Alzheimer’s disease (AD) 3-7. Moreover, genome-wide association studies have 7 shown that many neurological disease risk genes, particularly neurodegenerative diseases, are highly 8 and sometimes exclusively expressed by microglia 8-10. These observations provide a compelling 9 incentive to investigate the role of microglia in models of abnormal brain development and 10 neurodegeneration. Most studies of microglia largely rely on rodent microglia. However, there is 11 increasing evidence that rodent microglia are not able to faithfully mirror the biology of human microglia 12 11. In particular, recent transcriptomic studies have clearly demonstrated that a number of immune 13 genes, not identified as part of the mouse microglial signature, were abundantly expressed in human 14 microglia 8, 12. Moreover, a limited overlap was observed in microglial genes regulated during aging and 15 neurodegeneration between mice and humans, indicating that human and mouse microglia age 16 differently under normal and diseased conditions 12, 13. These findings argue for the development of 17 species-specific research tools to investigate microglial functions in human brain development, aging, 18 and neurodegeneration. 19 Functional human brain tissue is scarcely available. In addition, given the considerable 20 sensitivity of microglia to environmental changes 8, the properties of available human microglia isolated 21 from surgically resected brain tissue may vary significantly, due to different disease states of the 22 patients and the multi-step procedures used for microglial purification. In order to study human 23 microglia in a relatively homeostatic state, many scientists have turned to human pluripotent stem cells 24 (hPSCs). Recent advances in stem cell technology have led to the efficient generation of microglia from 25 hPSCs 14-19, providing an unlimited source of human microglia to study their function. However, when 26 cultured alone or co-cultured with neurons and astrocytes in 2-dimensional (2D) or 3D 27 organoid/spheroid culture systems, these hPSC-derived microglia best resemble fetal or early postnatal 28 human microglia. This is indicated by much lower expression of key microglial molecules such as 29 TREM2, TMEM119, and P2RY12 in the hPSC-derived microglia, as compared to microglia derived 30 from adult human brain tissue 16, 18, 20. Thus, even with these novel in vitro models, it has been 31 challenging to advance understanding of human microglial function in adult ages or in 32 neurodegeneration during aging. 33 Recent studies from us 21, 22 and others 23-25 have demonstrated that neonatally engrafted 34 human neural or macroglial (oligodendroglial and astroglial) progenitor cells can largely repopulate and 35 functionally integrate into the adult host rodent brain or spinal cord, generating widespread chimerism. 36 This human-mouse chimeric approach provides unique opportunities for studying the pathophysiology 37 of the human cells within an intact brain. In this study, we developed a hPSC microglial chimeric mouse 38 brain model, by transplanting hPSC-derived microglia into neonatal mouse brains. The engrafted 39 hPSC-derived microglia can proliferate, migrate, and widely disperse in the brain. We hypothesize that 40 the limited functional maturation of hPSC-derived microglia in in vitro models is primarily caused by the 41 fact that those microglia are maintained in an environment that lacks the complex cell-cell/cell-matrix 42 interactions existing in an in vivo brain environment 8. To test this hypothesis, we employed single-cell 43 RNA-sequencing and super-resolution confocal imaging to examine the identity and function of hPSC- 44 derived microglia developed for six months in the mouse brain under both homeostatic and toxin- 45 induced demyelination conditions. 3 bioRxiv preprint doi: https://doi.org/10.1101/594721; this version posted December 30, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 1 Results 2 3 Generation of hPSC microglial chimeric mouse brains 4 Microglia originate from yolk sac erythromyeloid progenitors (EMPs) during primitive hematopoiesis. 5 EMPs further develop to primitive macrophage precursors (PMPs) that migrate into the developing neural 6 tube and become microglia with ramified processes within the CNS environment 1. We first derived PMPs 7 from hPSCs, including one human induced pluripotent stem cell (hiPSC) line and one human embryonic 8 stem cell (hESC) line, using a published protocol 18. Briefly, the yolk sac embryoid bodies (YS-EBs) were 9 generated by treating the EBs with bone morphogenetic protein 4 (BMP4), vascular endothelial growth 10 factor (VEGF), and stem cell factor (SCF). Next, the YS-EBs were plated into dishes with interleukin-3 11 (IL-3) and macrophage colony-stimulating factor (M-CSF) to promote myeloid differentiation. At 2–3 12 weeks after plating, hPSC-derived PMPs emerged into the supernatant and were continuously produced 13 for more than 3 months. The cumulative yield of PMPs was around 40-fold higher than the number of 14 input hPSCs (Figure 1A), similar to the results from previous studies 16, 18, 26. PMPs are produced in a 15 Myb-independent manner that closely recapitulated primitive hematopoiesis 1, 18, 27. We confirmed the 16 identity of these hPSC-derived PMPs by staining with CD235, a marker for YS primitive hematopoietic 17 progenitors 28, 29, and CD43, a marker for hematopoietic progenitor-like cells28, 29. As shown in Figure 1B, 18 over 95% of the hPSC-derived PMPs expressed both markers. Moreover, the human PMPs are highly 19 proliferative as indicated by Ki67 staining (95.4 ± 2.2%, n = 4) (Figure 1B). Using this method, we routinely 20 obtain ample numbers of hPSC-derived PMPs with high purity as required for cell transplantation 21 experiments. 22 We engrafted hPSC-derived PMPs into the brains of postnatal day 0 (P0) immunodeficient mice 23 that are Rag2/IL2rg-deficient and also express the human forms of CSF1, which facilitates the survival 24 of xenografted human myeloid cells and other leukocytes30, 31. We deposited cells into the white matter 25 overlying the hippocampus and sites within the hippocampal formation (Figure 1C). In order to visualize 26 the distribution of donor-derived microglia, at 6 months post-transplantation, we stained the mouse 27 brain sections with human-specific antibody recognizing TMEM119 (hTMEM119). TMEM119 is a 28 marker that is only expressed by microglia, but not other macrophages 14, 17, 32. We found that the 29 donor-derived hTMEM119+ microglia widely disperse in the brain (Figure 1D). As early as 3 weeks 30 post-transplantation, donor-derived microglia had already migrated along corpus callosum and passed 31 through the rostral migratory stream to the olfactory bulb (Figure 1E).