Ipscs
iPSCs
Carmen Falcone
Postdoctoral scholar position available for highly motivated candidates with a PhD in Neuroscience, Molecular or Cell Biology, Evolutionary or Developmental Biology, Biochemistry or related fields, to join the research group of Carmen Falcone, PhD, at the Department of Neuroscience in SISSA (Trieste, Italy), starting from April 2022. This position will provide the opportunity to be part of a new research team working in an exciting project aimed to study the functions of interlaminar astrocytes in the primate brain, with iPSCs and xenograph mouse models, and molecular, cellular and behavioral techniques. Although the contract for this job is for one year, there is the possibility for it to be renewed for a maximum of 5 years, if the candidate and the lab are a good fit.
Rejuvenating the Alzheimer’s brain: Challenges & Opportunities
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.
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.
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.
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
Analysis of the impact of MnCl2 present in atmospheric particulates on synaptic development using brain models based on hiPSCs derived neurons
FENS Forum 2024
Exploring the function of the synaptic adaptor protein p140Cap in human excitatory neurons derived from iPSCs
FENS Forum 2024
Exploring the impact of chemical and electrical stimulation on human-iPSCs-derived neural networks coupled to high-density arrays
FENS Forum 2024
Integrating network activity with transcriptomic profiling in hiPSCs-derived neuronal networks to understand the molecular drivers of functional heterogeneity in the context of neurodevelopmental disorders
FENS Forum 2024
Interrogating CDKL5 deficiency disorder using human iPSCs-derived cerebral organoids
FENS Forum 2024
Investigating Parkinson's disease using patient-derived iPSCs transplanted in a human-mouse chimera model
FENS Forum 2024