Multi-level characterization of balanced inhibitory-excitatory cortical neuron network derived from human pluripotent stem cells

Abstract

Generation of neuronal cultures from induced pluripotent stem cells (hiPSCs) serve the studies of human brain disorders. However we lack neuronal networks with balanced excitatory-inhibitory activities, which are suitable for single cell analysis. We generated low-density networks of hPSC-derived GABAergic and glutamatergic cortical neurons. We used two different co-culture models with astrocytes. We show that these cultures have balanced excitatory-inhibitory synaptic identities using confocal microscopy, electrophysiological recordings, calcium imaging and mRNA analysis. These simple and robust protocols offer the opportunity for single-cell to multi-level analysis of patient hiPSC-derived cortical excitatory-inhibitory networks; thereby creating advanced tools to study disease mechanisms underlying neurodevelopmental disorders.

Authors

Aishwarya G Nadadhur, Javier Emperador Melero, Marieke Meijer, Desiree Schut, Gerbren Jacobs, Ka Wan Li, J J Johannes Hjorth, Rhiannon M Meredith, Ruud F Toonen, Ronald E Van Kesteren, August B Smit, Matthijs Verhage, Vivi M Heine

Link

https://doi.org/10.1371/journal.pone.0178533

Affected astrocytes in the spinal cord of the leukodystrophy vanishing white matter

Abstract

Leukodystrophies are often devastating diseases, presented with progressive clinical signs as spasticity, ataxia and cognitive decline, and lack proper treatment options. New therapy strategies for leukodystrophies mostly focus on oligodendrocyte replacement to rescue lack of myelin in the brain, even though disease pathology also often involves other glial cells and the spinal cord. In this study we investigated spinal cord pathology in a mouse model for Vanishing White Matter disease (VWM) and show that astrocytes in the white matter are severely affected. Astrocyte pathology starts postnatally in the sensory tracts, followed by changes in the astrocytic populations in the motor tracts. Studies in post-mortem tissue of two VWM patients, a 13-year-old boy and a 6-year-old girl, confirmed astrocyte abnormalities in the spinal cord. For proper development of new treatment options for VWM and, possibly, other leukodystrophies, future studies should investigate spinal cord involvement.

Authors

Prisca S Leferink, Nicole Breeuwsma, Marianna Bugiani, Marjo S van der Knaap, Vivi M Heine

Link

https://doi.org/10.1002/glia.23289

Patterning factors during neural progenitor induction determine regional identity and differentiation potential in vitro

Abstract

The neural tube consists of neural progenitors (NPs) that acquire different characteristics during gestation due to patterning factors. However, the influence of such patterning factors on human pluripotent stem cells (hPSCs) during in vitro neural differentiation is often unclear. This study compared neural induction protocols involving in vitro patterning with single SMAD inhibition (SSI), retinoic acid (RA) administration and dual SMAD inhibition (DSI). While the derived NP cells expressed known NP markers, they differed in their NP expression profile and differentiation potential. Cortical neuronal cells generated from 1) SSI NPs exhibited less mature neuronal phenotypes, 2) RA NPs exhibited an increased GABAergic phenotype, and 3) DSI NPs exhibited greater expression of glutamatergic lineage markers. Further, although all NPs generated astrocytes, astrocytes derived from the RA-induced NPs had the highest GFAP expression. Differences between NP populations included differential expression of regional identity markers HOXB4, LBX1, OTX1 and GSX2, which persisted into mature neural cell stages. This study suggests that patterning factors regulate how potential NPs may differentiate into specific neuronal and glial cell types in vitro. This challenges the utility of generic neural induction procedures, while highlighting the importance of carefully selecting specific NP protocols.

Authors

Aishwarya G. Nadadhur, Prisca S. Leferink, Dwayne Holmes, Lisa Hinz, Paulien Cornelissen-Steijger, Lisa Gasparotto, Vivi M. Heine

Link

https://doi.org/10.1016/j.scr.2018.08.017

Generation of Isogenic Controls for In Vitro Disease Modelling of X-Chromosomal Disorders

Abstract

Generation of proper controls is crucial in induced pluripotent stem cell (iPSC) studies. X-chromosomal disorders offer the potential to develop isogenic controls due to random X-chromosomal inactivation (XCI). However, the generation of such lines is currently hampered by skewed X-inactivation in fibroblast lines and X-chromosomal reactivation (XCR) after reprogramming. Here we describe a method to generate a pure iPSC population with respect to the specific inactivated X-chromosome (Xi). We used fibroblasts from Rett patients, who all have a causal mutation in the X-linked MeCP2 gene. Pre-sorting these fibroblasts followed by episomal reprogramming, allowed us to overcome skewness in fibroblast lines and to retain the X-chromosomal state, which was unpredictable with lentiviral reprogramming. This means that fibroblast pre-sorting followed by episomal reprogramming can be used to reliably generate iPSC lines with specified X-chromosomal phenotype such as Rett syndrome.

Authors

Lisa Hinz, Stephanie D Hoekstra, Kyoko Watanabe, Danielle Posthuma, Vivi M Heine

Link

https://doi.org/10.1007/s12015-018-9851-8

Neuron-Glia Interactions Increase Neuronal Phenotypes in Tuberous Sclerosis Complex Patient iPSC-Derived Models

Abstract

Tuberous sclerosis complex (TSC) is a rare neurodevelopmental disorder resulting from autosomal dominant mutations in the TSC1 or TSC2 genes, leading to a hyperactivated mammalian target of rapamycin (mTOR) pathway, and gray and white matter defects in the brain. To study the involvement of neuron-glia interactions in TSC phenotypes, we generated TSC patient induced pluripotent stem cell (iPSC)-derived cortical neuronal and oligodendrocyte (OL) cultures. TSC neuron mono-cultures showed increased network activity, as measured by calcium transients and action potential firing, and increased dendritic branching. However, in co-cultures with OLs, neuronal defects became more apparent, showing cellular hypertrophy and increased axonal density. In addition, TSC neuron-OL co-cultures showed increased OL cell proliferation and decreased OL maturation. Pharmacological intervention with the mTOR regulator rapamycin suppressed these defects. Our patient iPSC-based model, therefore, shows a complex cellular TSC phenotype arising from the interaction of neuronal and glial cells and provides a platform for TSC disease modeling and drug development.

Keywords: autism; co-culture; glia; iPSC; in vitro model; myelin; neuron; neuronal activity; oligodendrocyte; tuberous sclerosis complex.

Authors

Aishwarya G Nadadhur, Mouhamed Alsaqati, Lisa Gasparotto, Paulien Cornelissen-Steijger, Eline van Hugte, Stephanie Dooves, Adrian J Harwood, Vivi M Heine

Link

https://doi.org/10.1016/j.stemcr.2018.11.019

Human and mouse iPSC-derived astrocyte subtypes reveal vulnerability in Vanishing White Matter

Abstract

Astrocytes gained attention as important players in neurological disease, including a number of leukodystrophies. Several studies explored the generation of induced pluripotent stem cell-derived astrocytes for drug screening and regenerative studies. Developing robust models of patient induced pluripotent stem cells is challenged by high variability due to diverse genetic backgrounds and long-term culture procedures. While human models are of special interest, mouse-based models have the advantage that for them these issues are less pronounced. Here we present astrocyte differentiation protocols for both human and mouse induced pluripotent stem cells to specifically induce grey and white matter astrocytes. Both subtypes expressed astrocyte-associated markers, had typical astrocyte morphologies, and gave a reactive response to stress. Importantly, the grey and white matter-like astrocytes differed in size, complexity of processes, and expression profile, conform primary grey and white matter astrocytes. The newly presented mouse and human stem cell-based models for the leukodystrophy Vanishing White Matter replicated earlier findings, such as increased proliferation, decreased OPC maturation and modulation by hyaluronidase. We studied intrinsic astrocyte subtype vulnerability in Vanishing White Matter in both human and mouse cells. Oligodendrocyte maturation was specifically inhibited in cultures with Vanishing White Matter white matter-like astrocytes. By performing RNA sequencing, we found more differentially regulated genes in the white than in the grey matter-like astrocytes. Human and mouse astrocytes showed the same affected pathways, although human white matter-like astrocytes presented human-specific disease mechanisms involved in Vanishing White Matter. Using both human and mouse induced pluripotent stem cells, our study presents protocols to generate white and grey matter-like astrocytes, and shows astrocyte subtype-specific defects in Vanishing White Matter. While mouse induced pluripotent stem cell-based cultures may be less suitable to mimic human astrocyte subtype- or patient-specific changes, they might more robustly represent disease mutation-related cellular phenotypes as the cells are derived from inbred mice and the protocols are faster. The presented models give new tools to generate astrocyte subtypes for in vitro disease modeling and in vivo regenerative applications.

Authors

Prisca S. Leferink, Stephanie Dooves, Anne E.J. Hillen, Kyoko Watanabe, Gerbren Jacobs, Lisa Gasparotto, Paulien Cornelissen-Steijger, Marjo S. van der Knaap, Vivi M. Heine

Link

https://doi.org/10.1101/523233

Quantitative proteomic analysis of Rett iPSC-derived neuronal progenitors

Abstract

Background
Rett syndrome (RTT) is a progressive neurodevelopmental disease that is characterized by abnormalities in cognitive, social, and motor skills. RTT is often caused by mutations in the X-linked gene encoding methyl-CpG binding protein 2 (MeCP2). The mechanism by which impaired MeCP2 induces the pathological abnormalities in the brain is not understood. Both patients and mouse models have shown abnormalities at molecular and cellular level before typical RTT-associated symptoms appear. This implies that underlying mechanisms are already affected during neurodevelopmental stages.

Methods
To understand the molecular mechanisms involved in disease onset, we used an RTT patient induced pluripotent stem cell (iPSC)-based model with isogenic controls and performed time-series of proteomic analysis using in-depth high-resolution quantitative mass spectrometry during early stages of neuronal development.

Results
We provide mass spectrometry-based quantitative proteomic data, depth of about 7000 proteins, at neuronal progenitor developmental stages of RTT patient cells and isogenic controls. Our data gives evidence of proteomic alteration at early neurodevelopmental stages, suggesting alterations long before the phase that symptoms of RTT syndrome become apparent. Significant changes are associated with the GO enrichment analysis in biological processes cell-cell adhesion, actin cytoskeleton organization, neuronal stem cell population maintenance, and pituitary gland development, next to protein changes previously associated with RTT, i.e., dendrite morphology and synaptic deficits. Differential expression increased from early to late neural stem cell phases, although proteins involved in immunity, metabolic processes, and calcium signaling were affected throughout all stages analyzed.

Limitations
The limitation of our study is the number of RTT patients analyzed. As the aim of our study was to investigate a large number of proteins, only one patient was considered, of which 3 different RTT iPSC clones and 3 isogenic control iPSC clones were included. Even though this approach allowed the study of mutation-induced alterations due to the usage of isogenic controls, results should be validated on different RTT patients to suggest common disease mechanisms.

Conclusions
During early neuronal differentiation, there are consistent and time-point specific proteomic alterations in RTT patient cells carrying exons 3ÔÇô4 deletion in MECP2. We found changes in proteins involved in pathway associated with RTT phenotypes, including dendrite morphology and synaptogenesis. Our results provide a valuable resource of proteins and pathways for follow-up studies, investigating common mechanisms involved during early disease stages of RTT syndrome.

Authors

Suzy Varderidou-Minasian, Lisa Hinz, Dominique Hagemans, Danielle Posthuma, Maarten Altelaar & Vivi M. Heine

Link

https://doi.org/10.1186/s13229-020-00344-3

Copy number variants (CNVs): a powerful tool for iPSC-based modelling of ASD

Abstract

Patients diagnosed with chromosome microdeletions or duplications, known as copy number variants (CNVs), present a unique opportunity to investigate the relationship between patient genotype and cell phenotype. CNVs have high genetic penetrance and give a good correlation between gene locus and patient clinical phenotype. This is especially effective for the study of patients with neurodevelopmental disorders (NDD), including those falling within the autism spectrum disorders (ASD). A key question is whether this correlation between genetics and clinical presentation at the level of the patient can be translated to the cell phenotypes arising from the neurodevelopment of patient induced pluripotent stem cells (iPSCs).

Here, we examine how iPSCs derived from ASD patients with an associated CNV inform our understanding of the genetic and biological mechanisms underlying the aetiology of ASD. We consider selection of genetically characterised patient iPSCs; use of appropriate control lines; aspects of human neurocellular biology that can capture in vitro the patient clinical phenotype; and current limitations of patient iPSC-based studies. Finally, we consider how future research may be enhanced to maximise the utility of CNV patients for research of pathological mechanisms or therapeutic targets.

Authors

Danijela Drakulic, Srdjan Djurovic, Yasir Ahmed Syed, Sebastiano Trattaro, Nicol├▓ Caporale, Anna Falk, Rivka Ofir, Vivi M. Heine, Samuel J. R. A. Chawner, Antonio Rodriguez-Moreno, Marianne B. M. van den Bree, Giuseppe Testa, Spyros Petrakis & Adrian J. Harwood

Link

https://doi.org/10.1186/s13229-020-00343-4

Neuron-Glia Interactions in Tuberous Sclerosis Complex Affect the Synaptic Balance in 2D and Organoid Cultures

Abstract

Tuberous sclerosis complex (TSC) is a genetic disease affecting the brain. Neurological symptoms like epilepsy and neurodevelopmental issues cause a significant burden on patients. Both neurons and glial cells are affected by TSC mutations. Previous studies have shown changes in the excitation/inhibition balance (E/I balance) in TSC. Astrocytes are known to be important for neuronal development, and astrocytic dysfunction can cause changes in the E/I balance. We hypothesized that astrocytes affect the synaptic balance in TSC. TSC patient-derived stem cells were differentiated into astrocytes, which showed increased proliferation compared to control astrocytes. RNA sequencing revealed changes in gene expression, which were related to epidermal growth factor (EGF) signaling and enriched for genes that coded for secreted or transmembrane proteins. Control neurons were cultured in astrocyte-conditioned medium (ACM) of TSC and control astrocytes. After culture in TSC ACM, neurons showed an altered synaptic balance, with an increase in the percentage of VGAT+ synapses. These findings were confirmed in organoids, presenting a spontaneous 3D organization of neurons and glial cells. To conclude, this study shows that TSC astrocytes are affected and secrete factors that alter the synaptic balance. As an altered E/I balance may underlie many of the neurological TSC symptoms, astrocytes may provide new therapeutic targets.

Keywords: 

astrocytesexcitation/inhibition balancetuberous sclerosis complexEGF signalingastrocyte-conditioned mediumiPSCorganoid

Authors

Stephanie Dooves, Arianne J H van Velthoven, Linda G Suciati, Vivi M Heine

Link

https://doi.org/10.3390/cells10010134

Systematic assessment of variability in the proteome of iPSC derivatives

Abstract

The use of induced pluripotent stem cells (iPSC) to model human complex diseases is gaining popularity as it allows investigation of human cells that are otherwise sparsely available. However, due to its laborious and cost intensive nature, iPSC research is often plagued by limited sample size and putative large variability between clones, decreasing statistical power for detecting experimental effects. Here, we investigate the source and magnitude of variability in the proteome of parallel differentiated astrocytes using mass spectrometry. We compare three possible sources of variability: inter-donor variability, inter- and intra-clonal variability, at different stages of maturation. We show that the interclonal variability is significantly smaller than the inter-donor variability, and that including more donors has a much larger influence on statistical power than adding more clones per donor. Our results provide insight into the sources of variability at protein level between iPSC samples derived in parallel and will aid in optimizing iPSC studies.

Authors

Stephanie D Beekhuis-Hoekstra, Kyoko Watanabe, Josefin Werme, Christiaan A de Leeuw, Iryna Paliukhovich, Ka Wan Li, Frank Koopmans, August B Smit, Danielle Posthuma, Vivi M Heine

Link

https://doi.org/10.1016/j.scr.2021.102512