Recent advances in the generation of microglia from human induced pluripotent stem cells (iPSCs) have provided exciting new approaches to examine and decipher the biology of microglia. As these ...techniques continue to evolve to encompass more complex in situ and in vivo paradigms, so too have they begun to yield novel scientific insight into the genetics and function of human microglia. As such, researchers now have access to a toolset comprised of three unique “flavors” of iPSC‐derived microglia: in vitro microglia (iMGs), organoid microglia (oMGs), and xenotransplanted microglia (xMGs). The goal of this review is to discuss the variety of research applications that each of these techniques enables and to highlight recent discoveries that these methods have begun to uncover. By presenting the research paradigms in which each model has been successful, as well as the key benefits and limitations of each approach, it is our hope that this review will help interested researchers to incorporate these techniques into their studies, collectively advancing our understanding of human microglia biology.
Stem cell technology is increasingly being used to model human microglia.
In vitro, in situ, and in vivo techniques exist to study human iPSC‐derived microglia.
Application of these techniques has begun to reveal novel aspects of microglia biology.
Research into the function of microglia has dramatically accelerated during the last few years, largely due to recent genetic findings implicating microglia in virtually every neurodegenerative ...disorder. In Alzheimer's disease (AD), a majority of risk loci discovered through genome-wide association studies were found in or near genes expressed most highly in microglia leading to the hypothesis that microglia play a much larger role in disease progression than previously thought. From this body of work produced in the last several years, we find that almost every function of microglia has been proposed to influence the progression of AD from altered phagocytosis and synaptic pruning to cytokine secretion and changes in trophic support. By studying key Alzheimer's risk genes such as TREM2, CD33, ABCA7, and MS4A6A, we will be able to distinguish true disease-modulatory pathways from the full range of microglial-related functions. To successfully carry out these experiments, more advanced microglial models are needed. Microglia are quite sensitive to their local environment, suggesting the need to more fully recapitulate an in vivo environment to study this highly plastic cell type. Likely only by combining the above approaches will the field fully elucidate the molecular pathways that regulate microglia and influence neurodegeneration, in turn uncovering potential new targets for future therapeutic development.
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•Microglia are associated with the progression of AD.•Key microglia functions in AD: cytokine secretion, phagocytosis, and trophic support•Human in vitro models allow for controlled studies of molecular microglia function.•Understanding human microglial function in AD may elucidate new, targeted therapies.
Alzheimer's disease (AD) is a progressive neurodegenerative disease with no cure. Huge efforts have been made to develop anti‐AD drugs in the past decades. However, all drug development programs for ...disease‐modifying therapies have failed. Possible reasons for the high failure rate include incomplete understanding of complex pathophysiology of AD, especially sporadic AD (sAD), and species difference between humans and animal models used in preclinical studies. In this study, sAD is modeled using human induced pluripotent stem cell (hiPSC)‐derived 3D brain organoids. Because the blood–brain barrier (BBB) leakage is a well‐known risk factor for AD, brain organoids are exposed to human serum to mimic the serum exposure consequence of BBB breakdown in AD patient brains. The serum‐exposed brain organoids are able to recapitulate AD‐like pathologies, including increased amyloid beta (Aβ) aggregates and phosphorylated microtubule‐associated tau protein (p‐Tau) level, synaptic loss, and impaired neural network. Serum exposure increases Aβ and p‐Tau levels through inducing beta‐secretase 1 (BACE) and glycogen synthase kinase‐3 alpha / beta (GSK3α/β) levels, respectively. In addition, single‐cell transcriptomic analysis of brain organoids reveals that serum exposure reduced synaptic function in both neurons and astrocytes and induced immune response in astrocytes. The human brain organoid‐based sAD model established in this study can provide a powerful platform for both mechanistic study and therapeutic development in the future.
Brain organoids are exposed to human serum to mimic the serum exposure consequence of the blood–brain barrier (BBB) breakdown in Alzheimer's disease (AD) patient brains, which recapitulates several AD‐like pathologies, including increased amyloid beta (Aβ) and phosphorylated microtubule‐associated tau protein (p‐Tau) level, synaptic loss, and impaired neural network. This human brain organoid‐based sporadic AD (sAD) model provides a powerful platform for both mechanistic study and therapeutic development in the future.
Alzheimer's disease (AD) is the leading cause of age‐related neurodegeneration and is characterized neuropathologically by the accumulation of insoluble beta‐amyloid (Aβ) peptides. In AD brains, ...plaque‐associated myeloid (PAM) cells cluster around Aβ plaques but fail to effectively clear Aβ by phagocytosis. PAM cells were originally thought to be brain‐resident microglia. However, several studies have also suggested that Aβ‐induced inflammation causes peripheral monocytes to enter the otherwise immune‐privileged brain. The relationship between AD progression and inflammation in the brain remains ambiguous because microglia and monocyte‐derived macrophages are extremely difficult to distinguish from one another in an inflamed brain. Whether PAM cells are microglia, peripheral macrophages, or a mixture of both remains unclear. CD11a is a component of the β2 integrin LFA1. We have determined that CD11a is highly expressed on peripheral immune cells, including macrophages, but is not expressed by mouse microglia. These expression patterns remain consistent in LPS‐treated inflamed mice, as well as in two mouse models of AD. Thus, CD11a can be used as a marker to distinguish murine microglia from infiltrating peripheral immune cells. Using CD11a, we show that PAM cells in AD transgenic brains are comprised entirely of microglia. We also demonstrate a novel fluorescence‐assisted quantification technique (FAQT), which reveals a significant increase in T lymphocytes, especially in the brains of female AD mice. Our findings support the notion that microglia are the lead myeloid players in AD and that rejuvenating their phagocytic potential may be an important therapeutic strategy.
Main Points
CD11a expression can distinguish microglia (CD11a−) from peripheral immune cells (CD11a+) in homeostasis, neuroinflammation, and Alzheimer's disease mice.
Plaque‐associated myeloid (PAM) cells do not express CD11a and are microglia.
Fluorescence‐assisted quantification technique (FAQT) reveals a significant increase in microglia numbers and in infiltrating T cells in the brains of AD female mice.
Abstract Microglia, the immune cells of the central nervous system, are dynamic and heterogenous cells. While single cell RNA sequencing has become the conventional methodology for evaluating ...microglial state, transcriptomics do not provide insight into functional changes, identifying a critical gap in the field. Here, we propose a novel organelle phenotyping approach in which we treat live human induced pluripotent stem cell‐derived microglia (iMGL) with organelle dyes staining mitochondria, lipids, lysosomes and acquire data by live‐cell spectral microscopy. Dimensionality reduction techniques and unbiased cluster identification allow for recognition of microglial subpopulations with single‐cell resolution based on organelle function. We validated this methodology using lipopolysaccharide and IL‐10 treatment to polarize iMGL to an “inflammatory” and “anti‐inflammatory” state, respectively, and then applied it to identify a novel regulator of iMGL function, complement protein C1q. While C1q is traditionally known as the initiator of the complement cascade, here we use organelle phenotyping to identify a role for C1q in regulating iMGL polarization via fatty acid storage and mitochondria membrane potential. Follow up evaluation of microglia using traditional read outs of activation state confirm that C1q drives an increase in microglia pro‐inflammatory gene production and migration, while suppressing microglial proliferation. These data together validate the use of a novel organelle phenotyping approach and enable better mechanistic investigation of molecular regulators of microglial state. image
In neurodegenerative diseases, extracellular vesicles (EVs) transfer pathogenic molecules and are consequently involved in disease progression. We have investigated the proteomic profiles of EVs that ...were isolated from four different human‐induced pluripotent stem cell‐derived neural cell types (excitatory neurons, astrocytes, microglia‐like cells, and oligodendrocyte‐like cells). Novel cell type‐specific EV protein markers were then identified for the excitatory neurons (ATP1A3, NCAM1), astrocytes (LRP1, ITGA6), microglia‐like cells (ITGAM, LCP1), and oligodendrocyte‐like cells (LAMP2, FTH1), as well as 16 pan‐EV marker candidates, including integrins and annexins. To further demonstrate how cell‐type‐specific EVs may be involved in Alzheimer's disease (AD), we performed protein co‐expression network analysis and conducted cell type assessments for the proteomes of brain‐derived EVs from the control, mild cognitive impairment, and AD cases. A protein module enriched in astrocyte‐specific EV markers was most significantly associated with the AD pathology and cognitive impairment, suggesting an important role in AD progression. The hub protein from this module, integrin‐β1 (ITGB1), was found to be significantly elevated in astrocyte‐specific EVs enriched from the total brain‐derived AD EVs and associated with the brain β‐amyloid and tau load in independent cohorts. Thus, our study provides a featured framework and rich resource for the future analyses of EV functions in neurodegenerative diseases in a cell type‐specific manner.
Microglia, the principle immune cells of the brain, play important roles in neuronal development, homeostatic function and neurodegenerative disease. Recent genetic studies have further highlighted ...the importance of microglia in neurodegeneration with the identification of disease risk polymorphisms in many microglial genes. To better understand the role of these genes in microglial biology and disease, we, and others, have developed methods to differentiate microglia from human induced pluripotent stem cells (iPSCs). While the development of these methods has begun to enable important new studies of microglial biology, labs with little prior stem cell experience have sometimes found it challenging to adopt these complex protocols. Therefore, we have now developed a greatly simplified approach to generate large numbers of highly pure human microglia.
iPSCs are first differentiated toward a mesodermal, hematopoietic lineage using commercially available media. Highly pure populations of non-adherent CD43
hematopoietic progenitors are then simply transferred to media that includes three key cytokines (M-CSF, IL-34, and TGFβ-1) that promote differentiation of homeostatic microglia. This updated approach avoids the prior requirement for hypoxic incubation, complex media formulation, FACS sorting, or co-culture, thereby significantly simplifying human microglial generation. To confirm that the resulting cells are equivalent to previously developed iPSC-microglia, we performed RNA-sequencing, functional testing, and transplantation studies. Our findings reveal that microglia generated via this simplified method are virtually identical to iPS-microglia produced via our previously published approach. To also determine whether a small molecule activator of TGFβ signaling (IDE1) can be used to replace recombinant TGFβ1, further reducing costs, we examined growth kinetics and the transcriptome of cells differentiated with IDE1. These data demonstrate that a microglial cell can indeed be produced using this alternative approach, although transcriptional differences do occur that should be considered.
We anticipate that this new and greatly simplified protocol will enable many interested labs, including those with little prior stem cell or flow cytometry experience, to generate and study human iPS-microglia. By combining this method with other advances such as CRISPR-gene editing and xenotransplantation, the field will continue to improve our understanding of microglial biology and their important roles in human development, homeostasis, and disease.
Alzheimer's disease (AD) is the leading cause of age‐related dementia, affecting over 5 million people in the U.S. alone. AD patients suffer from progressive neurodegeneration that gradually impairs ...their memory, ability to learn, and carry out daily activities. Unfortunately, current therapies for AD are largely palliative and several promising drug candidates have failed in recent clinical trials. There is therefore an urgent need to improve our understanding of AD pathogenesis, create innovative and predictive models, and develop new and effective therapies. In this review, we will discuss the potential of stem cells to aid in these challenging endeavors. Because of the widespread nature of AD pathology, cell‐replacement strategies have been viewed as an incredibly challenging and unlikely treatment approach. Yet recent work shows that transplantation of neural stem cells (NSCs) can improve cognition, reduce neuronal loss, and enhance synaptic plasticity in animal models of AD. Interestingly, the mechanisms that mediate these effects appear to involve neuroprotection and trophic support rather than neuronal replacement. Stem cells may also offer a powerful new approach to model and study AD. Patient‐derived induced pluripotent stem cells, for example, may help to advance our understanding of disease mechanisms. Likewise, studies of human embryonic and NSCs are helping to decipher the normal functions of AD‐related genes; revealing intriguing roles in neural development. STEM CELLS 2012;30:2612–2618
Microglia play critical roles in brain development, homeostasis, and neurological disorders. Here, we report that human microglial-like cells (iMGLs) can be differentiated from iPSCs to study their ...function in neurological diseases, like Alzheimer’s disease (AD). We find that iMGLs develop in vitro similarly to microglia in vivo, and whole-transcriptome analysis demonstrates that they are highly similar to cultured adult and fetal human microglia. Functional assessment of iMGLs reveals that they secrete cytokines in response to inflammatory stimuli, migrate and undergo calcium transients, and robustly phagocytose CNS substrates. iMGLs were used to examine the effects of Aβ fibrils and brain-derived tau oligomers on AD-related gene expression and to interrogate mechanisms involved in synaptic pruning. Furthermore, iMGLs transplanted into transgenic mice and human brain organoids resemble microglia in vivo. Together, these findings demonstrate that iMGLs can be used to study microglial function, providing important new insight into human neurological disease.
•Fully defined and efficient generation of human microglial-like cells from iPSCs•Whole-transcriptome and functional validation of iPSC-derived microglia (iMGLs)•Novel in vitro and in vivo applications for studying neurological diseases•iMGLs can be used to interrogate AD gene function
Abud et al. describe a fully defined protocol for the generation of human iPSC-derived microglia-like cells (iMGLs). Whole-transcriptome and novel functional analyses were used to validate microglial identity. iMGLs provide a platform for studying microglial function in health and disease.
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