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Generation of pure monocultures of human microglia-like cells from induced pluripotent stem cells.
Microglia are resident tissue macrophages of the central nervous system (CNS) that arise from erythromyeloid progenitors during embryonic development. They play essential roles in CNS development, homeostasis and response to disease. Since microglia are difficult to procure from the human brain, several protocols have been developed to generate microglia-like cells from human induced pluripotent stem cells (hiPSCs). However, some concerns remain over the purity and quality of in vitro generated microglia. Here, we describe a new protocol that does not require co-culture with neural cells and yields cultures of 100% P2Y12+ 95% TMEM119+ ramified human microglia-like cells (hiPSC-MG). In the presence of neural precursor cell-conditioned media, hiPSC-MG expressed high levels of human microglia signature genes, including SALL1, CSF1R, P2RY12, TMEM119, TREM2, HEXB and SIGLEC11, as revealed by whole-transcriptome analysis. Stimulation of hiPSC-MG with lipopolysaccharide resulted in downregulation of P2Y12 expression, induction of IL1B mRNA expression and increase in cell capacitance. HiPSC-MG were phagocytically active and maintained their cell identity after transplantation into murine brain slices and human brain spheroids. Together, our new protocol for the generation of microglia-like cells from human iPSCs will facilitate the study of human microglial function in health and disease.
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Alanine and glutathione targeting of dopamine- or ibuprofen-coupled polypeptide nanocarriers increases both crossing and protective effects on a blood-brain barrier model.
BACKGROUND: Targeting the blood-brain barrier (BBB) is a key step for effective brain delivery of nanocarriers. We have previously discovered that combinations of BBB nutrient transporter ligands alanine and glutathione (A-GSH), increase the permeability of vesicular and polypeptide nanocarriers containing model cargo across the BBB. Our aim was to investigate dopamine- and ibuprofen-coupled 3-armed poly(L-glutamic acid) nanocarriers targeted by A-GSH for transfer across a novel human co-culture model with induced BBB properties. In addition, the protective effect of ibuprofen containing nanoparticles on cytokine-induced barrier damage was also measured. METHOD: Drug-coupled nanocarriers were synthetized and characterized by dynamic light scattering and transmission electron microscopy. Cellular effects, uptake, and permeability of the nanoparticles were investigated on a human stem cell-based co-culture BBB model with improved barrier properties induced by a small molecular cocktail. The model was characterized by immunocytochemistry and permeability for marker molecules. Nanocarrier uptake in human brain endothelial cells and midbrain organoids was quantified by spectrofluorometry and visualized by confocal microscopy. The mechanisms of cellular uptake were explored by addition of free targeting ligands, endocytic and metabolic inhibitors, co-localization of nanocarriers with intracellular organs, and surface charge modification of cells. The protective effect of ibuprofen-coupled nanocarriers was investigated against cytokine-induced barrier damage by impedance and permeability measurements. RESULTS: Targeted nanoformulations of both drugs showed elevated cellular uptake in a time-dependent, active manner via endocytic mechanisms. Addition of free ligands inhibited the cellular internalization of targeted nanocarriers suggesting the crucial role of ligands in the uptake process. A higher permeability across the BBB model was measured for targeted nanocarriers. After crossing the BBB, targeted dopamine nanocarriers subsequently entered midbrain-like organoids derived from healthy and Parkinson's disease patient-specific stem cells. The ibuprofen-coupled targeted nanocarriers showed protective effects against cytokine-induced barrier damage. CONCLUSION: BBB-targeted polypeptide nanoparticles coupled to therapeutic molecules were effectively taken up by brain organoids or showing a BBB protective effect indicating potential applications in nervous system pathologies.
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Understanding the role of steroids in typical and atypical brain development: Advantages of using a "brain in a dish" approach.
Steroids have an important role in growth, development, sexual differentiation and reproduction. All four classes of steroids, androgens, oestrogens, progestogens and glucocorticoids, have varying effects on the brain. Androgens and oestrogens are involved in the sexual differentiation of the brain, and also influence cognition. Progestogens such as progesterone and its metabolites have been shown to be involved in neuroprotection, although their protective effects are timing-dependent. Glucocorticoids are linked with stress and memory performance, also in a dose- and time-dependent manner. Importantly, dysfunction in steroid function has been implicated in the pathogenesis of disease. Moreover, regulating steroid-signalling has been suggested as potential therapeutic avenue for the treatment of a number of neurodevelopmental, psychiatric and neurodegenerative disorders. Therefore, clarifying the role of steroids in typical and atypical brain function is essential for understanding typical brain functions, as well as determining their potential use for pharmacological intervention in the atypical brain. However, the majority of studies have thus far have been conducted using animal models, with limited work using native human tissue or cells. Here, we review the effect of steroids in the typical and atypical brain, focusing on the cellular, molecular functions of these molecules determined from animal models, and the therapeutic potential as highlighted by human studies. We further discuss the promise of human-induced pluripotent stem cells, including advantages of using three-dimensional neuronal cultures (organoids) in high-throughput screens, in accelerating our understanding of the role of steroids in the typical brain, and also with respect to their therapeutic value in the understanding and treatment of the atypical brain.
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Gut mucosal cells transfer α-synuclein to the vagus nerve.
Epidemiological and histopathological findings have raised the possibility that misfolded α-synuclein protein might spread from the gut to the brain and increase the risk of Parkinson's disease. Although past experimental studies in mouse models have relied on gut injections of exogenous recombinant α-synuclein fibrils to study gut-to-brain α-synuclein transfer, the possible origins of misfolded α-synuclein within the gut have remained elusive. We recently demonstrated that sensory cells of intestinal mucosa express α-synuclein. Here, we employed mouse intestinal organoids expressing human α-synuclein to observe the transfer of α-synuclein protein from epithelial cells in organoids to cocultured nodose neurons devoid of α-synuclein. In mice expressing human α-synuclein, but no mouse α-synuclein, α-synuclein fibril-templating activity emerged in α-synuclein-seeded fibril aggregation assays in intestine, vagus nerve, and dorsal motor nucleus. In newly engineered transgenic mice that restrict pathological human α-synuclein expression to intestinal epithelial cells, α-synuclein fibril-templating activity transfered to the vagus nerve and dorsal motor nucleus. Subdiaphragmatic vagotomy prior to induction of α-synuclein expression in intestinal epithelial cells effectively protected the hindbrain from emergence of α-synuclein fibril-templating activity. Overall, these findings highlight a potential non-neuronal source of fibrillar α-synuclein protein that might arise in gut mucosal cells.
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MBNL2 dysfunction in outer radial glial cells is associated with disrupted corticogenesis in congenital myotonic dystrophy.
Myotonic dystrophy type 1 (DM1) arises from toxic CUG-expanded DMPK transcripts that sequester Muscleblind-like (MBNL) proteins, yet how this molecular lesion perturbs brain development in congenital DM1 (CDM) remains unknown. Here, we identify an unanticipated developmental role for MBNL2 in outer radial glial cells, a progenitor population critical for cortical expansion. We demonstrate that MBNL2 is expressed in these cells both in vivo and in forebrain organoids derived from patient-specific human induced pluripotent stem cells (hiPSCs), rendering them particularly sensitive to MBNL2 titration. Using genome editing to excise the CTG repeats in the DMPK gene, we provide evidence that the expanded trinucleotide tract directly contributes to defective neuronal migration and impaired differentiation of late-born cortical neurons in CDM organoids. These findings redefine MBNL2 as a potential regulator of human corticogenesis and uncover a developmental mechanism by which RNA toxicity drives this severe form of DM1. By revealing a prenatal origin for CDM neuropathology linked to MBNL2 dysfunction, this work opens avenues for therapeutic strategies targeting early developmental windows.
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WONOEP appraisal: Modeling early onset epilepsies.
Epilepsy has a peak incidence during the neonatal to early childhood period. These early onset epilepsies may be severe conditions frequently associated with comorbidities such as developmental deficits and intellectual disability and, in a significant percentage of patients, may be medication-resistant. The use of adult rodent models in the exploration of mechanisms and treatments for early life epilepsies is challenging, as it ignores significant age-specific developmental differences. More recently, models developed in immature animals, such as rodent pups, or in three-dimensional organoids may more closely model aspects of the immature brain and could result in more translatable findings. Although models are not perfect, they may offer a more controlled screening platform in studies of mechanisms and treatments, which cannot be done in pediatric patient cohorts. On the other hand, more simplified models with higher throughput capacities are required to deal with the large number of epilepsy candidate genes and the need for new treatment options. Therefore, a combination of different modeling approaches will be beneficial in addressing the unmet needs of pediatric epilepsy patients. In this review, we summarize the discussions on this topic that occurred during the XVI Workshop on Neurobiology of Epilepsy, organized in 2022 by the Neurobiology Commission of the International League Against Epilepsy. We provide an overview of selected models of early onset epilepsies, discussing their advantages and disadvantages. Heterologous expression models provide initial functional insights, and zebrafish, rodent models, and brain organoids present increasingly complex platforms for modeling and validating epilepsy-related phenomena. Together, these models offer valuable insights into early onset epilepsies and accelerate hypothesis generation and therapy discovery.
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The pathophysiology of mixed Alzheimer's disease and vascular dementia.
Mixed dementia is caused most often by the coexistence of Alzheimer's disease (AD) and vascular dementia (VaD) pathologies. This disease presents challenges due to its complex, dual pathology. In this review, we summarize the current understanding of the pathophysiology of AD and VaD, with a particular emphasis on vascular factors that accelerate or exacerbate AD pathology. We then describe animal models, in vitro cell culture systems, and brain organoid models that have been developed or are currently being developed to elucidate the neurodegenerative and vascular components of MD. This review provides an evaluation of the panorama of factors that influence MD pathophysiology and how basic science models can incorporate these factors to advance the knowledge related to the mechanisms of the disease and guide the screening of novel therapeutic approaches.
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Personalized Medicine in Brain Gliomas: Targeted Therapy, Patient-Derived Tumor Models (Review).
Gliomas are the most common type of primary malignant brain tumors. The choice of treatments for these tumors was quite limited for many years, and therapy results generally remain still unsatisfactory. Recently, a significant breakthrough in the treatment of many forms of cancer occurred when personalized targeted therapies were introduced which inhibit tumor growth by affecting a specific molecular target. Another trend gaining popularity in oncology is the creation of patient-derived tumor models which can be used for drug screening to select the optimal therapy regimen. Molecular and genetic mechanisms of brain gliomas growth are considered, consisting of individual components which could potentially be exposed to targeted drugs. The results of the literature review show a higher efficacy of the personalized approach to the treatment of individual patients compared to the use of standard therapies. However, many unresolved issues remain in the area of predicting the effectiveness of a particular drug therapy regimen. The main hopes in solving this issue are set on the use of patient-derived tumor models, which can be used in one-stage testing of a wide range of antitumor drugs.
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Absence of the RING domain in MID1 results in patterning defects in the developing human brain.
The X-linked form of Opitz BBB/G syndrome (OS) is a monogenic disorder in which symptoms are established early during embryonic development. OS is caused by pathogenic variants in the X-linked gene MID1 Disease-associated variants are distributed across the entire gene locus, except for the N-terminal really interesting new gene (RING) domain that encompasses the E3 ubiquitin ligase activity. By using genome-edited human induced pluripotent stem cell lines, we here show that absence of isoforms containing the RING domain of MID1 causes severe patterning defects in human brain organoids. We observed a prominent neurogenic deficit with a reduction in neural tissue and a concomitant increase in choroid plexus-like structures. Transcriptome analyses revealed a deregulation of patterning pathways very early on, even preceding neural induction. Notably, the observed phenotypes starkly contrast with those observed in MID1 full-knockout organoids, indicating the presence of a distinct mechanism that underlies the patterning defects. The severity and early onset of these phenotypes could potentially account for the absence of patients carrying pathogenic variants in exon 1 of the MID1 gene coding for the N-terminal RING domain.
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Functional neurogenomics in autism spectrum disorders: A decade of progress.
Advances in autism spectrum disorder (ASD) genetics have identified many genetic causes, reflecting remarkable progress while at the same time identifying challenges such as heterogeneity and pleiotropy, which complicate attempts to connect genetic risk to mechanisms. High-throughput functional genomic approaches have yielded progress by defining a molecular pathology in the brain of individuals with ASD and in identifying convergent biological pathways through which risk genes are predicted to act. These studies indicate that ASD genetic risk converges in early brain development, primarily during the period of cortical neurogenesis. Over development, genetic perturbations in turn lead to broad neuronal signaling dysregulation, most prominent in glutamatergic cortical-cortical projecting neurons and somatostatin positive interneurons, which is accompanied by glial dyshomeostasis throughout the cerebral cortex. Connecting these developmental perturbations to disrupted neuronal and glial function in the postnatal brain is an important direction in current research. Coupling functional genomic approaches with advances in induced pluripotent stem cell-derived neural organoid development provides a promising avenue for connecting brain pathology to developmental mechanisms.