iPSC

Rejuvenating the Alzheimer’s brain: Challenges & Opportunities

Unlocking the Secrets of Microglia in Neurodegenerative diseases: Mechanisms of resilience to AD pathologies

Epigenetic rewiring in Schinzel-Giedion syndrome

During life, a variety of specialized cells arise to grant the right and timely corrected functions of tissues and organs. Regulation of chromatin in defining specialized genomic regions (e.g. enhancers) plays a key role in developmental transitions from progenitors into cell lineages. These enhancers, properly topologically positioned in 3D space, ultimately guide the transcriptional programs. It is becoming clear that several pathologies converge in differential enhancer usage with respect to physiological situations. However, why some regulatory regions are physiologically preferred, while some others can emerge in certain conditions, including other fate decisions or diseases, remains obscure. Schinzel-Giedion syndrome (SGS) is a rare disease with symptoms such as severe developmental delay, congenital malformations, progressive brain atrophy, intractable seizures, and infantile death. SGS is caused by mutations in the SETBP1 gene that results in its accumulation further leading to the downstream accumulation of SET. The oncoprotein SET has been found as part of the histone chaperone complex INHAT that blocks the activity of histone acetyltransferases suggesting that SGS may (i) represent a natural model of alternative chromatin regulation and (ii) offer chances to study downstream (mal)adaptive mechanisms. I will present our work on the characterization of SGS in appropriate experimental models including iPSC-derived cultures and mouse.

Integration of 3D human stem cell models derived from post-mortem tissue and statistical genomics to guide schizophrenia therapeutic development

Schizophrenia is a neuropsychiatric disorder characterized by positive symptoms (such as hallucinations and delusions), negative symptoms (such as avolition and withdrawal) and cognitive dysfunction1. Schizophrenia is highly heritable, and genetic studies are playing a pivotal role in identifying potential biomarkers and causal disease mechanisms with the hope of informing new treatments. Genome-wide association studies (GWAS) identified nearly 270 loci with a high statistical association with schizophrenia risk; however each locus confers only a small increase in risk therefore it is difficult to translate these findings into understanding disease biology that can lead to treatments. Induced pluripotent stem cell (iPSC) models are a tractable system to translate genetic findings and interrogate mechanisms of pathogenesis. Mounting research with patient-derived iPSCs has proposed several neurodevelopmental pathways altered in SCZ, such as neural progenitor cell (NPC) proliferation, imbalanced differentiation of excitatory and inhibitory cortical neurons. However, it is unclear what exactly these iPS models recapitulate, how potential perturbations of early brain development translates into illness in adults and how iPS models that represent fetal stages can be utilized to further drug development efforts to treat adult illness. I will present the largest transcriptome analysis of post-mortem caudate nucleus in schizophrenia where we discovered that decreased presynaptic DRD2 autoregulation is the causal dopamine risk factor for schizophrenia (Benjamin et al, Nature Neuroscience 2022 https://doi.org/10.1038/s41593-022-01182-7). We developed stem cell models from a subset of the postmortem cohort to better understand the molecular underpinnings of human psychiatric disorders (Sawada et al, Stem Cell Research 2020). We established a method for the differentiation of iPS cells into ventral forebrain organoids and performed single cell RNAseq and cellular phenotyping. To our knowledge, this is the first study to evaluate iPSC models of SZ from the same individuals with postmortem tissue. Our study establishes that striatal neurons in the patients with SCZ carry abnormalities that originated during early brain development. Differentiation of inhibitory neurons is accelerated whereas excitatory neuronal development is delayed, implicating an excitation and inhibition (E-I) imbalance during early brain development in SCZ. We found a significant overlap of genes upregulated in the inhibitory neurons in SCZ organoids with upregulated genes in postmortem caudate tissues from patients with SCZ compared with control individuals, including the donors of our iPS cell cohort. Altogether, we demonstrate that ventral forebrain organoids derived from postmortem tissue of individuals with schizophrenia recapitulate perturbed striatal gene expression dynamics of the donors’ brains (Sawada et al, biorxiv 2022 https://doi.org/10.1101/2022.05.26.493589).

Cell-type specific alterations underpinning convergent ASD phenotypes in PACS1 neurodevelopmental disorder

Bridging the gap between artificial models and cortical circuits

Artificial neural networks simplify complex biological circuits into tractable models for computational exploration and experimentation. However, the simplification of artificial models also undermines their applicability to real brain dynamics. Typical efforts to address this mismatch add complexity to increasingly unwieldy models. Here, we take a different approach; by reducing the complexity of a biological cortical culture, we aim to distil the essential factors of neuronal dynamics and plasticity. We leverage recent advances in growing neurons from human induced pluripotent stem cells (hiPSCs) to analyse ex vivo cortical cultures with only two distinct excitatory and inhibitory neuron populations. Over 6 weeks of development, we record from thousands of neurons using high-density microelectrode arrays (HD-MEAs) that allow access to individual neurons and the broader population dynamics. We compare these dynamics to two-population artificial networks of single-compartment neurons with random sparse connections and show that they produce similar dynamics. Specifically, our model captures the firing and bursting statistics of the cultures. Moreover, tightly integrating models and cultures allows us to evaluate the impact of changing architectures over weeks of development, with and without external stimuli. Broadly, the use of simplified cortical cultures enables us to use the repertoire of theoretical neuroscience techniques established over the past decades on artificial network models. Our approach of deriving neural networks from human cells also allows us, for the first time, to directly compare neural dynamics of disease and control. We found that cultures e.g. from epilepsy patients tended to have increasingly more avalanches of synchronous activity over weeks of development, in contrast to the control cultures. Next, we will test possible interventions, in silico and in vitro, in a drive for personalised approaches to medical care. This work starts bridging an important theoretical-experimental neuroscience gap for advancing our understanding of mammalian neuron dynamics.

Investigating activity-dependent processes in cerebral cortex development and disease

The cerebral cortex contains an extraordinary diversity of excitatory projection neuron (PN) and inhibitory interneurons (IN), wired together to form complex circuits. Spatiotemporally coordinated execution of intrinsic molecular programs by PNs and INs and activity-dependent processes, contribute to cortical development and cortical microcircuits formation. Alterations of these delicate processes have often been associated to neurological/neurodevelopmental disorders. However, despite the groundbreaking discovery that spontaneous activity in the embryonic brain can shape regional identities of distinct cortical territories, it is still unclear whether this early activity contributes to define subtype-specific neuronal fate as well as circuit assembly. In this study, we combined in utero genetic perturbations via CRISPR/Cas9 system and pharmacological inhibition of selected ion channels with RNA-sequencing and live imaging technologies to identify the activity-regulated processes controlling the development of different cortical PN classes, their wiring and the acquisition of subtype specific features. Moreover, we generated human induced pluripotent stem cells (iPSCs) form patients affected by a severe, rare and untreatable form of developmental epileptic encephalopathy. By differentiating cortical organoids form patient-derived iPSCs we create human models of early electrical alterations for studying molecular, structural and functional consequences of the genetic mutations during cortical development. Our ultimate goal is to define the activity-conditioned processes that physiologically occur during the development of cortical circuits, to identify novel therapeutical paths to address the pathological consequences of neonatal epilepsies.

Human stem cell models of Alzheimer’s disease and frontotemporal dementia

The development of human induced pluripotent stem cells (iPSC) and their subsequent differentiation into neurons has provided new opportunities for the generation of physiologically-relevant, in vitro disease models. I will present our work using iPSC to modal familial Alzheimer's Disease (fAD) and Frontotemporal Dementia (FTD). We have investigated the mutation-specific effects of APP and PSEN1 mutations on Abeta generation in neurons generated from individuals with fAD, revealing distinct mechanisms that may contribute to clinical heterogeneity in disease. I will also discuss our work to understand the developmental and pathological changes to tau that occur in iPSC-neurons, particularly the challenges of understanding tau pathology in a developmental system, tau proteostasis and how iPSC-neurons may help us identify early signatures of tau pathology in disease.

2nd In-Vitro 2D & 3D Neuronal Networks Summit

The event is open to everyone interested in Neuroscience, Cell Biology, Drug Discovery, Disease Modeling, and Bio/Neuroengineering! This meeting is a platform bringing scientists from all over the world together and fostering scientific exchange and collaboration.

2nd In-Vitro 2D & 3D Neuronal Networks Summit

The event is open to everyone interested in Neuroscience, Cell Biology, Drug Discovery, Disease Modeling, and Bio/Neuroengineering! This meeting is a platform bringing scientists from all over the world together and fostering scientific exchange and collaboration.

Using Human Stem Cells to Uncover Genetic Epilepsy Mechanisms

Reprogramming somatic cells to a pluripotent state via the induced pluripotent stem cell (iPSC) method offers an increasingly utilized approach for neurological disease modeling with patient-derived cells. Several groups, including ours, have applied the iPSC approach to model severe genetic developmental and epileptic encephalopathies (DEEs) with patient-derived cells. Although most studies to date involve 2-D cultures of patient-derived neurons, brain organoids are increasingly being employed to explore genetic DEE mechanisms. We are applying this approach to understand PMSE (Polyhydramnios, Megalencephaly and Symptomatic Epilepsy) syndrome, Rett Syndrome (in collaboration with Ben Novitch at UCLA) and Protocadherin-19 Clustering Epilepsy (PCE). I will describe our findings of robust structural phenotypes in PMSE and PCE patient-derived brain organoid models, as well as functional abnormalities identified in fusion organoid models of Rett syndrome. In addition to showing epilepsy-relevant phenotypes, both 2D and brain organoid cultures offer platforms to identify novel therapies. We will also discuss challenges and recent advances in the brain organoid field, including a new single rosette brain organoid model that we have developed. The field is advancing rapidly and our findings suggest that brain organoid approaches offers great promise for modeling genetic neurodevelopmental epilepsies and identifying precision therapies.

Application of Airy beam light sheet microscopy to examine early neurodevelopmental structures in 3D hiPSC-derived human cortical spheroids

The inability to observe relevant biological processes in vivo significantly restricts human neurodevelopmental research. Advances in appropriate in vitro model systems, including patient-specific human brain organoids and human cortical spheroids (hCSs), offer a pragmatic solution to this issue. In particular, hCSs are an accessible method for generating homogenous organoids of dorsal telencephalic fate, which recapitulate key aspects of human corticogenesis, including the formation of neural rosettes—in vitro correlates of the neural tube. These neurogenic niches give rise to neural progenitors that subsequently differentiate into neurons. Studies differentiating induced pluripotent stem cells (hiPSCs) in 2D have linked atypical formation of neural rosettes with neurodevelopmental disorders such as autism spectrum conditions. Thus far, however, conventional methods of tissue preparation in this field limit the ability to image these structures in three-dimensions within intact hCS or other 3D preparations. To overcome this limitation, we have sought to optimise a methodological approach to process hCSs to maximise the utility of a novel Airy-beam light sheet microscope (ALSM) to acquire high resolution volumetric images of internal structures within hCS representative of early developmental time points.

Translational upregulation of STXBP1 by non-coding RNAs as an innovative treatment for STXBP1 encephalopathy

Developmental and epileptic encephalopathies (DEEs) are a broad spectrum of genetic epilepsies associated with impaired neurological development as a direct consequence of a genetic mutation, in addition to the effect of the frequent epileptic activity on brain. Compelling genetic studies indicate that heterozygous de novo mutations represent the most common underlying genetic mechanism, in accordance with the sporadic presentation of DEE. De novo mutations may exert a loss-of-function (LOF) on the protein by decrementing expression level and/or activity, leading to functional haploinsufficiency. These diseases share several features: severe and frequent refractory seizures, diffusely abnormal background activity on EEG, intellectual disability often profound, and severe consequences on global development. One of major causes of early onset DEE are de novo heterozygous mutations in syntaxin-binding-protein-1 gene STXBP1, which encodes a membrane trafficking protein playing critical role in vesicular docking and fusion. LOF STXBP1 mutations lead to a failure of neurotransmitter secretion from synaptic vesicles. Core clinical features of STXBP1 encephalopathy include early-onset epilepsy with hypsarrhythmic EEG, or burst-suppression pattern, or multifocal epileptiform activity. Seizures are often resistant to standard treatments and patients typically show intellectual disability, mostly severe to profound. Additional neurologic features may include autistic traits, movement disorders (dyskinesia, dystonia, tremor), axial hypotonia, and ataxia, indicating a broader neurologic impairment. Patients with severe neuro-cognitive features but without epilepsy have been reported. Recently, a new class of natural and synthetic non-coding RNAs have been identified, enabling upregulation of protein translation in a gene-specific way (SINEUPs), without any increase in mRNA of the target gene. SINEUPs are translational activators composed by a Binding Domain (BD) that overlaps, in antisense orientation, to the sense protein-coding mRNA, and determines target selection; and an Effector Domain (ED), that is essential for protein synthesis up regulation. SINEUPs have been shown to restore the physiological expression of a protein in case of haploinsufficiency, without driving excessive overexpression out of the physiological range. This technology brings many advantages, as it mainly acts on endogenous target mRNAs produced in situ by the wild-type allele; this action is limited to mRNA under physiological regulation, therefore no off-site effects can be expected in cells and tissues that do not express the target transcript; by acting only on a posttranscriptional level, SINEUPs do not trigger hereditable genome editing. After bioinformatic analysis of the promoter region of interest, we designed SINEUPs with 3 different BD for STXBP1. Human neurons from iPSCs were treated and STXBP1 levels showed a 1.5-fold increase compared to the Negative control. RNA levels of STXBP1 after the administration of SINEUPs remained stable as expected. These preliminary results proved the SINEUPs potential to specifically increase the protein levels without impacting on the genome. This is an extremely flexible approach to target many developmental and epileptic encephalopathies caused by haploinsufficiency, and therefore to address these diseases in a more tailored and radical way.

CRISPR-based functional genomics in iPSC-based models of brain disease

Human genes associated with brain-related diseases are being discovered at an accelerating pace. A major challenge is an identification of the mechanisms through which these genes act, and of potential therapeutic strategies. To elucidate such mechanisms in human cells, we established a CRISPR-based platform for genetic screening in human iPSC-derived neurons, astrocytes and microglia. Our approach relies on CRISPR interference (CRISPRi) and CRISPR activation (CRISPRa), in which a catalytically dead version of the bacterial Cas9 protein recruits transcriptional repressors or activators, respectively, to endogenous genes to control their expression, as directed by a small guide RNA (sgRNA). Complex libraries of sgRNAs enable us to conduct genome-wide or focused loss-of-function and gain-of-function screens. Such screens uncover molecular players for phenotypes based on survival, stress resistance, fluorescent phenotypes, high-content imaging and single-cell RNA-Seq. To uncover disease mechanisms and therapeutic targets, we are conducting genetic modifier screens for disease-relevant cellular phenotypes in patient-derived neurons and glia with familial mutations and isogenic controls. In a genome-wide screen, we have uncovered genes that modulate the formation of disease-associated aggregates of tau in neurons with a tauopathy-linked mutation (MAPT V337M). CRISPRi/a can also be used to model and functionally evaluate disease-associated changes in gene expression, such as those caused by eQTLs, haploinsufficiency, or disease states of brain cells. We will discuss an application to Alzheimer’s Disease-associated genes in microglia.

Functional characterization of human iPSC-derived neurons at single-cell resolution

Recent developments in induced pluripotent stem cell (iPSC) technology have enabled easier access to human cells in vitro. With increasing availability of human iPSC-derived neurons, both healthy and disease cell lines, screening compounds for neurodegenerative diseases on human cells can potentially be performed in the earlier stages of drug discovery. To accelerate the functional characterization of iPSC-derived neurons and the effect of compounds, reproducible and relevant results are necessary. In this webinar, the speakers will: Introduce high-resolution functional imaging of human iPSC-derived neurons Showcase how to extract functional features of hundreds of cells in a cell culture sample label-free Discuss electrophysiological parameters for characterizing the differences among several human neuronal cell lines

ARID1B-haploinsufficiency leads to delayed neuronal network development of iPSC-derived excitatory neurons

Assessing functionality of iPSC- based neurons models of Familial Dysautonomia

Assessment of vascularization and neurogenesis in an iPSC-derived 16p11.2 deletion organoid model

Characterization of hiPSC-derived endothelial cells role in the formation of cerebral amyloid angiopathy related to Alzheimer’s disease

Development and characterization of an in vitro model of SSADH deficiency using patient IPSC-derived neurons to support unbiased screening of novel therapeutic approaches to treatment

Development of functional in vitro model in Dravet syndrome patient hiPSC-derived cortical neurons

Elucidating the role of FUS and FMRP in regulating SYNGAP1 expression in human iPSC-derived neurons

Exploring the impact of APOE polymorphism on the molecular, morphological and functional profile of iPSC-derived astrocytes from Alzheimer's patients

Extracellular vesicles from human iPSC-derived neural stem cells alleviate microglial response and cognitive impairments in a chronic neuroinflammation model

Functional integration of grafted hiPSC derived dopamine neurons in a mouse model of Parkinson's disease

Generating sleep oscillations using primary and hiPSC-derived thalamo-cortical cultures

Generation of a Blood-Brain Barrier Model using Cryopreserved Human iPSC-derived Brain Microvascular Endothelial Cells, Pericytes, and Astrocytes

Generation of a patient specific hIPSC-derived neuronal model for Congenital Central Hypoventilation Syndrome (CCHS)

Human iPSC derived neural progenitors and cortical neurons as a model to study SARS-CoV-2 infection

Human iPSC-based cellular systems to model Autosomal dominant leukodystrophy

Human iPSC-based millifluidic model of the BBB/brain as part of the Microbiota-Gut-Brain axis MINERVA platform

Human iPSC-derived tridimensional-full-networks model to study microglia heterogeneity in Alzheimer’s disease

Human IPSCs-derived oligodendrocytes and astrocytes as the first Autosomal Dominant Leukodystrophy-relevant cellular models

Identification of pro-angiogenic factors for in vitro vascularization of hiPSC-derived brain organoids

Impaired neuronal maturation in a human iPSC derived cortical organoid model of Tauopathy

Insight into the role of the primary cilium in hIPSC-derived neuronal networks

Interferon gamma exposure of human iPSC-derived neurons alters major histocompatibility complex I and synapsin I protein expression

iPSC-derived cortical neurons and patterned cortical organoids to dissect the neurodevelopmental roots of Fragile X Syndrome

An all iPSC-derived cortico-striato-nigral minicircuit modelling Parkinson’s Disease revealed electrophysiological changes in medium spiny neurons cocultured with dopaminergic neurons carrying GBA N370S mutation

Long-term evaluation of intranigral transplantation of human iPSC-derived dopamine neurons in a Parkinson’s disease mice model

Modelling Dravet syndrome using human iPSC-derived neural circuits

Neurons, astrocytes, and oligodendrocytes are present in spinal organoids derived from human induced pluripotent stem cells (hIPSC)

N-glycosylation of induced pluripotent stem cells (iPSCs) and neural stem cells (NSCs) derived from a person with Down Syndrome (DS) caused by Trisomy 21 (T21)

Patient-derived iPSCs and cortical differentiation: A novel model for cerebral methylmalonic aciduria

Sialic acid biosynthesis is essential for network formation of iPSC-derived excitatory neurons

Standardization criteria of hiPSC derived neurons for Brain-on-Chip applications

Studying sporadic and familial Alzheimer´s disease on iPSC-derived hippocampal and cortical neurons: effect of APOE and Presenilin1

Unravelling the role of the STX1B gene in genetic epilepsy syndromes using an iPSC-derived autaptic culture system

Validation of iPSC-derived blood-brain barrier model on microfluidic chip

Altered autophagy in KANSL1 haploinsufficient iPSC-derived astrocytes

FENS Forum 2024

Analysis of the impact of MnCl2 present in atmospheric particulates on synaptic development using brain models based on hiPSCs derived neurons

FENS Forum 2024

Characterisation of Magi-family synaptic scaffolding proteins in human iPSC-derived neurons

FENS Forum 2024

Characterization of the autophagic-lysosomal pathway in Parkinson’s disease using patient iPSC-derived dopaminergic neurons containing a LRRK2 G2019S mutation

FENS Forum 2024

Decoding retinitis pigmentosa: Unveiling PRPF31 mutation effects on human iPSC-derived retinal organoids in vitro models

FENS Forum 2024

Development of iPSC-derived neural progenitor cells with enhanced migration to stroke tissue and inducible ablation systems

FENS Forum 2024