Dolan MJ, Therrien M, Jereb S, Kamath T, Atkeson T, Marsh SE, Goeva A, Lojek NM, Murphy S, White CM, Joung J, Liu B, Limone F, Eggan K, Hacohen N, Bernstein BE, Glass CK, Leinonen V, Blurton-Jones M, Zhang F, Epstein CB, Macosko EZ, Stevens B. A resource for generating and manipulating human microglial states in vitro. bioRxiv. May 2, 2022. bioRxiv

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  1. Mancuso et al. presented an exciting piece of work, taking advantage of the xenotransplantation model for AD, where human microglia precursors were transplanted into immunodeficient amyloid pathology mouse models. Through single-cell RNA-Seq, they identified cytokine response (CRM), interferon response (IFN), disease-associated (DAM), antigen-presenting response (HLA), and transitory microglia clusters in response to amyloid pathology. All of these clusters have been identified previously in mouse models of amyloid pathology (Ellwanger et al., 2021), except for CRM, which was shown to be APOE- or TREM2-independent. CRM, DAM, and HLA microglia subsets were corroborated in various postmortem human AD snRNA-Seq datasets, confirming the validity of this xenotransplantation model.

    Interestingly, pseudotime analysis suggested the development of CRM fell in a distinct trajectory from the rest of the reactive states. Along the line, it is very intriguing to see that oligomeric Aβ induced the majority of homeostatic microglia to the CRM state, not to others. This is consistent with results from Dolan et al., in that CRM was not observed in response to Aβ fibrils or other CNS substrates. It is nice to see the CRM response was specifically induced by Aβ oligomers, supporting the heterogeneity of microglial responses. The fact that this cluster was not identified previously in mouse models was likely due to lack of resolution, since Ccl3 and Ccl4, which were upregulated in CRM microglia, were also upregulated in the DAM cluster in the 5XFAD model (Zhou et al., 2020). 

    Besides CRM and DAM, it still remains an open question as to how the other reactive states are induced in response to Aβ pathology. Pseudotime analysis suggested that HLA and IFN clusters may derive from DAM. However, lineage-tracing analyses are needed to validate this finding. In addition, the IFN cluster was not observed in any of the postmortem human AD snRNA-Seq datasets reanalyzed, questioning its biological relevance in human pathology.

    Despite the characterization of distinct microglia subsets in amyloid pathology, a more fascinating question, the answer to which remains elusive, is what functions these transcriptionally distinctive cells play. Extensive functional experiments are warranted to determine whether they are beneficial or detrimental before therapeutic interventions can be confidently applied.

    References:

    Ellwanger DC, Wang S, Brioschi S, Shao Z, Green L, Case R, Yoo D, Weishuhn D, Rathanaswami P, Bradley J, Rao S, Cha D, Luan P, Sambashivan S, Gilfillan S, Hasson SA, Foltz IN, van Lookeren Campagne M, Colonna M. Prior activation state shapes the microglia response to antihuman TREM2 in a mouse model of Alzheimer's disease. Proc Natl Acad Sci U S A. 2021 Jan 19;118(3) PubMed.

    Zhou Y, Song WM, Andhey PS, Swain A, Levy T, Miller KR, Poliani PL, Cominelli M, Grover S, Gilfillan S, Cella M, Ulland TK, Zaitsev K, Miyashita A, Ikeuchi T, Sainouchi M, Kakita A, Bennett DA, Schneider JA, Nichols MR, Beausoleil SA, Ulrich JD, Holtzman DM, Artyomov MN, Colonna M. Human and mouse single-nucleus transcriptomics reveal TREM2-dependent and TREM2-independent cellular responses in Alzheimer's disease. Nat Med. 2020 Jan;26(1):131-142. Epub 2020 Jan 13 PubMed. Correction.

    View all comments by Marco Colonna

  2. While the iPSC-derived microglia (iMGL) in vitro model system demonstrates unexpected levels of microglia heterogeneity and inducibility of different phenotypes (as demonstrated by Dolan and colleagues), the coming of age of the xenotransplantation model of iMGLs holds the greatest promise for dissecting the roles of the different microglia phenotypes in a mechanistic way.

    Recently we reported that human microglia exist in several distinct functional states in the aged human brain (Olah et al., 2020), including homeostatic, interferon response, cytokine response, HLA overexpressing with enrichment of the DAM signature, and proliferative. The study by Mancuso and colleagues was able to recapitulate these human microglia states with high fidelity in a xenotransplant mouse model of Alzheimer’s disease (AD). Moreover, speaking to the strength of this approach, Mancuso et al. have shown that the risk alleles APOE4/4 and TREM2R47H both result in the reduction of the HLA phenotype, suggesting that this particular state of microglia is protective in the course of AD pathogenesis—further providing support to the conclusion of our own study of human ex vivo microglia.

    That human iPSC-derived microglia can acquire the exact same phenotypes as human microglia when transplanted to a corresponding mouse brain environment suggests that these transcriptional states are hardwired in the human microglia epigenome and can be evoked by environmental cues. Nonetheless, our own study and now Mancuso and colleagues show that this plasticity/inducibility is probably lost due to aging, and/or genetic predisposition. Accordingly, it will be critical for the field to gain an in-depth understanding of the epigenetic and metabolomic regulation of these different microglia states to be able to restore microglia plasticity, and to fine-tune microglia phenotypes in the context of therapeutic intervention for neurodegenerative diseases.

    In vivo Perturb-Seq experiments will be fundamental in our quest to identify the master regulators of human microglia subset-specific transcriptional programs and to provide us with clues to unlock the full potential of subset-specific differentiation in aged human microglia.

    References:

    Olah M, Menon V, Habib N, Taga MF, Ma Y, Yung CJ, Cimpean M, Khairallah A, Coronas-Samano G, Sankowski R, Grün D, Kroshilina AA, Dionne D, Sarkis RA, Cosgrove GR, Helgager J, Golden JA, Pennell PB, Prinz M, Vonsattel JP, Teich AF, Schneider JA, Bennett DA, Regev A, Elyaman W, Bradshaw EM, De Jager PL. Single cell RNA sequencing of human microglia uncovers a subset associated with Alzheimer's disease. Nat Commun. 2020 Nov 30;11(1):6129. PubMed.

    View all comments by Marta Olah

  3. These two new preprints describe exciting new approaches to utilize iPSC-derived human microglia in vitro and in vivo, which reveal important insights into how cell state relates to functional outcomes, and how the response of human microglia to Alzheimer’s disease pathology differs from what has been observed with mouse microglia.

    Mancuso et al. investigated human microglia states in a xenotransplant amyloid model and observed a remarkable increase in diversity and heterogeneity that more accurately reflects the expanded dynamic range of microglial states in human AD. These findings contrast with mouse microglia whose transcriptional signatures are limited to homeostatic or disease-associated microglial (DAM) states in amyloid models. Importantly, the human microglial states described from the xenotransplant model are similar to those detected in previously published single-nucleus RNA-Seq datasets from AD postmortem tissues, indicating a consensus is forming in the field regarding microglial states present in AD. How microglia states are altered during disease progression remains an open question, and these chimeric models could be useful for parsing states that are specific to certain stages of disease and that may protect against or exacerbate neurodegeneration. Taken together, these results suggest that mouse models markedly underrepresent the biological states in which microglia exist in AD and shine a spotlight on critical species differences. Future translational studies will likely need to address this challenge to accurately assess the therapeutic potential of microglial targets.

    It is our hope that broader implementation of chimeric mouse models like this one will better elucidate the biological effects of AD genetic risk factors such as TREM2 and APOE, and will inform therapeutic strategies for modulating microglia. Our understanding is limited of how transcriptional states impact specific microglial functions and of which microglial functions are beneficial versus deleterious in AD. This study highlights the benefits of assessing AD risk variants in human microglia using xenotransplant models and highlights the risks surrounding mechanistic insights or therapeutic strategies based on deep phenotyping of mouse microglia alone. We are excited about the prospect of future studies using these models to test the impact of candidate therapeutics microglial function and other disease endpoints.

    While leveraging novel in vivo models is extremely valuable to study the complex behavior of microglia in disease, these models are resource- and time-intensive. The ability to conduct some of these experiments using an in vitro system would be highly advantageous. Dolan et al. lay a foundation for leveraging iPSC-derived human microglia for in vitro mechanistic studies to bridge a key gap in our understanding of how microglial state relates to function.

    This work demonstrates, for the first time, that microglia heterogeneity can be recapitulated in a cell culture dish, which was previously thought to be limited by monoculture conditions, yet hinted at in studies that showed only a subset of microglia are phagocytic, for example. Dolan’s work builds confidence in the iMGL system by demonstrating overlap of in vitro and in vivo states of human microglia, and it shows consistency across multiple iPSC lines. Furthermore, Dolan et al. could connect transcriptional cell state to microglial function by demonstrating that different phagocytic substrates contribute to microglial diversity.

    From a technical perspective, the study also shows the ability to genetically manipulate iMGLs using a lentiviral approach that ingeniously leverages an HIV restriction factor inhibitor, Vpx, to enable highly efficient microglial transduction, which is a method well worth pursuing to genetically alter these historically difficult-to-manipulate cells.

    Together, these two papers provide a range of insights into the function of human microglia, their role in AD, and novel insights into how they can be used as an effective research tool in the future.

    View all comments by Kathryn Monroe

  4. In their recent preprint in bioRXiv, Dolan and colleagues report an in vitro platform utilizing human stem-cell-derived microglia (iMGLs) that allows for viral manipulation and high-throughput screens. They show that exposing iMGLs to central nervous system (CNS) substrates can induce their differentiation into in vivo-like microglial subsets. Exposing the iMGLs to apoptotic neurons, or Aβ amyloid fibrils, triggers their differentiation into a state highly analogous to disease-associated microglia (DAMs) seen in vivo. By utilizing virus-like particles containing Vpx, which prevents degradation of deoxynucleoside triphosphates, the authors dramatically augment lentiviral transduction of iMGLs, allowing for manipulation of iMGLs through gene expression. They further validate this platform by utilizing it to identify melanocyte-inducing transcription factor as a critical regulator of DAM gene expression and phagocytosis. Lastly, they demonstrate, as proof of concept, that this platform can be utilized to study samples derived from multiple donors in parallel.

    To date, the study of microglia in vitro mainly has mainly relied on the use of microglial cell lines or on primary microglial cultures. However, these approaches are hindered by their inability to accurately model in vivo microglial states. Additionally, microglia are traditionally not amenable to manipulation with viral vectors, which limits in vitro microglial studies to mostly lower-throughput methods. The model in this study overcomes these technical challenges, as Dolan and colleagues demonstrate from multiple angles. While applications of this technology remain to be seen, it opens possibilities that are certainly very exciting.

    In recent years, microglia have increasingly been shown to play important roles in pathologies of the CNS, particularly in neurodegenerative conditions such as Alzheimer’s disease (AD) (Song and Colonna., 2018). Studies in both mice and humans have demonstrated a microglia subtype in AD, commonly referred to as DAM, marked by expression of a specific set of genes, including APOE, LPL, and TREM2, among others, and the concurrent downregulation of homeostatic microglial signatures, including P2RY12, CX3CR1, etc. (Zhou et al., 2020; Keren-Shaul et al., 2017). Dolan et al., in addition to providing a new method for studying microglia, demonstrated induction of DAM signatures in stem cell-derived microglia exposed to Aβ fibrils or apoptotic neurons, which are elements of the microglial microenvironment in AD. Taken together, these findings suggest an intimate relationship between formation of the DAM subset and hallmarks of AD neuropathology such as Aβ plaques.

    Whether DAMs are the result of pathology, or an active anti-AD cell type remains controversial. DAMs express multiple immune sensing signatures, which suggest an active role in restricting pathology (Deczkowska et al., 2018). On the other hand, a recent study has suggested that DAMs emerge as a result of excessive microglial proliferation, which results in replicative senescence (Hu et al., 2021). Furthermore, it has been suggested that DAMs are not the sole microglia subtype responsible for controlling AD pathology; this is evident by enrichment of AD polygenic risk gene expression across different microglial subsets

    The DAM subset was notable at the time of discovery, in part, because it was associated with disease restriction in AD mouse models. As such, microglia have been proposed to be a therapeutic target in AD. DAM-specific, high-throughput screens using the platform proposed by Dolan et al. will allow us to further understand the role of DAMs in AD, in addition to potentially uncovering druggable molecular targets. In vivo validation of hits found by these screening models will help clarify how, and whether, to target DAMs in AD, as well as determine which, if any, microglial state is desirable for disease restriction.

    References:

    Song WM, Colonna M. The identity and function of microglia in neurodegeneration. Nat Immunol. 2018 Oct;19(10):1048-1058. Epub 2018 Sep 24 PubMed.

    Zhou Y, Song WM, Andhey PS, Swain A, Levy T, Miller KR, Poliani PL, Cominelli M, Grover S, Gilfillan S, Cella M, Ulland TK, Zaitsev K, Miyashita A, Ikeuchi T, Sainouchi M, Kakita A, Bennett DA, Schneider JA, Nichols MR, Beausoleil SA, Ulrich JD, Holtzman DM, Artyomov MN, Colonna M. Human and mouse single-nucleus transcriptomics reveal TREM2-dependent and TREM2-independent cellular responses in Alzheimer's disease. Nat Med. 2020 Jan;26(1):131-142. Epub 2020 Jan 13 PubMed. Correction.

    Keren-Shaul H, Spinrad A, Weiner A, Matcovitch-Natan O, Dvir-Szternfeld R, Ulland TK, David E, Baruch K, Lara-Astaiso D, Toth B, Itzkovitz S, Colonna M, Schwartz M, Amit I. A Unique Microglia Type Associated with Restricting Development of Alzheimer's Disease. Cell. 2017 Jun 15;169(7):1276-1290.e17. Epub 2017 Jun 8 PubMed.

    Deczkowska A, Keren-Shaul H, Weiner A, Colonna M, Schwartz M, Amit I. Disease-Associated Microglia: A Universal Immune Sensor of Neurodegeneration. Cell. 2018 May 17;173(5):1073-1081. PubMed.

    Hu Y, Fryatt GL, Ghorbani M, Obst J, Menassa DA, Martin-Estebane M, Muntslag TA, Olmos-Alonso A, Guerrero-Carrasco M, Thomas D, Cragg MS, Gomez-Nicola D. Replicative senescence dictates the emergence of disease-associated microglia and contributes to Aβ pathology. Cell Rep. 2021 Jun 8;35(10):109228. PubMed.

    View all comments by Marco Colonna

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