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A functional hiPSC-cortical neuron differentiation and maturation model and its application to neurological disorders.

ABSTRACT: The maturation and functional characteristics of human induced pluripotent stem cell (hiPSC)-cortical neurons has not been fully documented. This study developed a phenotypic model of hiPSC-derived cortical neurons, characterized their maturation process, and investigated its application for disease modeling with the integration of multi-electrode array (MEA) technology. Immunocytochemistry analysis indicated early-stage neurons (day 21) were simultaneously positive for both excitatory (vesicular glutamate transporter 1 [VGlut1]) and inhibitory (GABA) markers, while late-stage cultures (day 40) expressed solely VGlut1, indicating a purely excitatory phenotype without containing glial cells. This maturation process was further validated utilizing patch clamp and MEA analysis. Particularly, induced long-term potentiation (LTP) successfully persisted for 1 h in day 40 cultures, but only achieved LTP in the presence of the GABA_A receptor antagonist picrotoxin in day 21 cultures. This system was also applied to epilepsy modeling utilizing bicuculline and its correction utilizing the anti-epileptic drug valproic acid.

SUBMITTER: Autar K

PROVIDER: S-EPMC8758945 | biostudies-literature | 2022 Jan

REPOSITORIES: biostudies-literature

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A functional hiPSC-cortical neuron differentiation and maturation model and its application to neurological disorders.

Autar Kaveena K Guo Xiufang X Rumsey John W JW Long Christopher J CJ Akanda Nesar N Jackson Max M Narasimhan Narasimhan S NS Caneus Julbert J Morgan Dave D Hickman James J JJ

Stem cell reports 20211222 1

The maturation and functional characteristics of human induced pluripotent stem cell (hiPSC)-cortical neurons has not been fully documented. This study developed a phenotypic model of hiPSC-derived cortical neurons, characterized their maturation process, and investigated its application for disease modeling with the integration of multi-electrode array (MEA) technology. Immunocytochemistry analysis indicated early-stage neurons (day 21) were simultaneously positive for both excitatory (vesicula ...[more]

PMID: 34942087

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Project description:IntroductionProlactin (PRL) is a pleiotropic hormone implicated in various physiological processes; however, its contribution to neurodevelopment, particularly early corticogenesis, remains insufficiently characterized. In this study, we investigate PRL's regulatory influence on the initial stages of cortical development, with an emphasis on its effects on neuronal and astrocytic differentiation.MethodsWe employed a standardized in vitro differentiation protocol to generate cortical neurons from mouse embryonic stem cells (mESCs). Prolactin receptor (PRLr) expression was evaluated in pluripotent stem cells, neural stem cells (NSCs), immature neurons, and mature neurons using both PCR and immunofluorescence. These analyses revealed dynamic changes in PRLr expression throughout the differentiation process. Additionally, cells were treated with varying concentrations of PRL during early and late differentiation phases, enabling assessment of its impact on neuronal phenotypic distribution and morphological complexity.ResultsEarly PRL administration significantly enhanced the population of β-tubulin III + immature neurons, promoting neuronal survival without altering NSC proliferation. Furthermore, PRL treatment increased the abundance of Tbr1 + and NeuN + neurons, augmented dendritic complexity, and accelerated neuronal maturation. In contrast, PRL exposure at later stages of neural differentiation did not yield comparable effects. Notably, PRL delayed the maturation of protoplasmic astrocytes, although the total astrocyte population was not affected.DiscussionThese findings highlight PRL's pivotal role as a regulator of early corticogenesis by modulating neuronal survival, dendritic development, and astrocyte maturation. PRL thus emerges as a potential key factor in neurodevelopment, underscoring its importance in the hormonal regulation of neural differentiation and maturation. These insights may have broader implications for understanding the molecular and cellular mechanisms underlying normal and pathological neurodevelopment.

Project description:BackgroundLinks between dissociation and functional neurological disorder (FND)/conversion disorder are well-established, yet the pathophysiology of dissociation remains poorly understood. This MRI study investigated structural alterations associated with somatoform and psychological dissociation in FND. We hypothesized that multimodal, paralimbic cingulo-insular regions would relate to the severity of somatoform dissociation in patients with FND.MethodsFreeSurfer cortical thickness and subcortical volumetric analyses were performed in 26 patients with motor FND and 27 matched healthy controls. Patients with high dissociation as measured by the Somatoform Dissociation Questionnaire-20 (SDQ) or Dissociative Experiences Scale (DES) were compared to controls in stratified analyses. Within-group analyses were also performed with SDQ and DES scores in patients with FND. All cortical thickness analyses were whole-brain corrected at the cluster-wise level.ResultsPatients with FND and high somatoform dissociation (SDQ > 35) showed reduced left caudal anterior cingulate cortex (ACC) cortical thickness compared to controls. In within-group analyses, SDQ scores inversely correlated with left caudal ACC cortical thickness in patients with FND. Depersonalization/derealization scores positively correlated with right lateral occipital cortical thickness. Both within-group findings remained statistically significant controlling for trait anxiety/depression, borderline personality disorder and post-traumatic stress disorder, adverse life events, and motor FND subtypes in post-hoc analyses.ConclusionUsing complementary between-group and within-group analyses, an inverse association between somatoform dissociation and left caudal ACC cortical thickness was observed in patients with FND. A positive relationship was also appreciated between depersonalization/derealization severity and cortical thickness in visual association areas. These findings advance our neuropathobiological understanding of dissociation in FND. Hum Brain Mapp 39:428-439, 2018. © 2017 Wiley Periodicals, Inc.

Project description:Neurodegenerative diseases encompass a group of debilitating conditions resulting from progressive nerve cell death. Of these, Alzheimer's disease (AD) occurs most frequently, but is currently incurable and has limited treatment success. Late onset AD, the most common form, is highly heritable but is caused by a combination of non-genetic risk factors and many low-effect genetic variants whose disease-causing mechanisms remain unclear. By mining the FinnGen study database of phenome-wide association studies, we identified a rare variant, rs148726219, enriched in the Finnish population that is associated with AD risk and dementia, and appears to have arisen on a common haplotype with older AD-associated variants such as rs429358. The rs148726219 variant lies in an overlapping intron of the FosB proto-oncogene (FOSB) and ERCC excision repair 1 (ERCC1) genes. To understand the impact of this SNP on disease phenotypes, we performed CRISPR/Cas9 editing in a human induced pluripotent stem cell (hiPSC) line to generate isogenic clones harboring heterozygous and homozygous alleles of rs148726219. hiPSC clones differentiated into induced excitatory neurons (iNs) did not exhibit detectable molecular or morphological variation in differentiation potential compared to isogenic controls. However, global transcriptome analysis showed differential regulation of nearby genes and upregulation of several biological pathways related to neuronal function, particularly synaptogenesis and calcium signaling, specifically in mature iNs harboring rs148726219 homozygous and heterozygous alleles. Functional differences in iN circuit maturation as measured by calcium imaging were observed across genotypes. Edited mature iNs also displayed downregulation of unfolded protein response and cell death pathways. This study implicates a phenotypic impact of rs148726219 in the context of mature neurons, consistent with its identification in late onset AD, and underscores a hiPSC-based experimental model to functionalize GWAS-identified variants.

Project description:Abstract Background Neuropsychiatric disorders, including schizophrenia (SZ), afflict a significant fraction of the population. Recent genome-wide association studies (GWAS) under the framework of the Psychiatric Genomics Consortium (PGC), along with large-scale sequencing efforts, have identified a plethora of disease risk loci with common and/or rare risk variants. Translating these exciting genomic findings into causation and disease biology offers the promise of developing more tailored therapies in psychiatry. However, understanding the disease biology underlying most GWAS findings remains challenging: (1) The paucity of disease-relevant biological materials for assaying molecular and cellular phenotypes associated with risk loci; (2) Most disease variants lie within poorly-annotated noncoding parts of the genome for which functional interpretation is challenging; and (3) Each locus often contains many genes/variants equivalently associated with the disease due to linkage disequilibrium, and it is difficult to identify which are the likely causal gene/variant. Human neurons derived from induced pluripotent stem cells (iPSCs), both monolayer cultures (2D model) and the emerging brain organoids (3D model), provide a promising alternative to human brains for recapitulating cellular phenotypes relevant to psychiatric disorders. CRISPR/Cas9 editing further strengthens the utility of these models by enabling the generation of isogenic lines with essentially the same genetic background on which allelic effects of a risk variant can be directly compared, thus increasing the sensitivity to detect typically small effects of a GWAS variant. Methods To functionally assess the relevance of noncoding sequences in neuropsychiatric disorders, we hypothesized that disease-relevant noncoding sequences likely overlap with cell-specific open chromatin regions (OCRs). We have carried out a genome-wide OCR profiling of excitatory neuronal differentiation from human iPSCs using an Assay for Transposase-Accessible Chromatin by sequencing (ATAC-seq). Results We found that OCRs in neurons were enriched SZ risk variants in neural OCRs and can help prioritize putatively functional SZ risk variants that may impact OCRs and consequently, cellular development. At a leading SZ-risk locus flanking MIR137, we further examined the functional effects of a prioritized common GWAS SNP rs1198588 in CRISPR/Cas9-edited hiPSCs, and found that SZ-risk allele of rs1198588 altered MIR137 expression, OCR dynamics and dendrite arborization/synapse maturation. To systematically identify such disease risk variants that may affect OCR, we further carried out a proof-of-concept analysis of allele-specific open chromatin (ASoC) of in hIPSC-derived neurons. We found that Heterozygous SNPs showing ASoC are more prevalent in neurons than in hiPSCs. Out of the 12 schizophrenia GWAS-implicated SNPs that we found in neuronal OCRs of this single individual, two SNPs showed ASoC and are thus putatively functional: one lies within the 5’-UTR of CHRNA5 (cholinergic receptor, nicotinic, alpha 5) and the other is in the promoter region of VPS45, a Sec1 family gene involved in synaptic transmission. We are currently in the process of replicating the observed landscape of ASoC in iPSC-derived neurons from a larger sample pool. Discussion Our study suggests that OCR profiling in a human iPSC model of neuron differentiation can predict functional noncoding sequences that regulate neurodevelopment.

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A functional hiPSC-cortical neuron differentiation and maturation model and its application to neurological disorders.

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A functional hiPSC-cortical neuron differentiation and maturation model and its application to neurological disorders.

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