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1.
Int J Mol Sci ; 21(23)2020 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-33266139

RESUMO

Induced pluripotent stem cell (iPSC)-derived neural progenitor cells (NPCs) are promising tools to model complex neurological or psychiatric diseases, including schizophrenia. Multiple studies have compared patient-derived and healthy control NPCs derived from iPSCs in order to investigate cellular phenotypes of this disease, although the establishment, stabilization, and directed differentiation of iPSC lines are rather expensive and time-demanding. However, interrupted reprogramming by omitting the stabilization of iPSCs may allow for the generation of a plastic stage of the cells and thus provide a shortcut to derive NPSCs directly from tissue samples. Here, we demonstrate a method to generate shortcut NPCs (sNPCs) from blood mononuclear cells and present a detailed comparison of these sNPCs with NPCs obtained from the same blood samples through stable iPSC clones and a subsequent neural differentiation (classical NPCs-cNPCs). Peripheral blood cells were obtained from a schizophrenia patient and his two healthy parents (a case-parent trio), while a further umbilical cord blood sample was obtained from the cord of a healthy new-born. The expression of stage-specific markers in sNPCs and cNPCs were compared both at the protein and RNA levels. We also performed functional tests to investigate Wnt and glutamate signaling and the oxidative stress, as these pathways have been suggested to play important roles in the pathophysiology of schizophrenia. We found similar responses in the two types of NPCs, suggesting that the shortcut procedure provides sNPCs, allowing an efficient screening of disease-related phenotypes.


Assuntos
Diferenciação Celular , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Biomarcadores , Diferenciação Celular/genética , Células Cultivadas , Regulação da Expressão Gênica no Desenvolvimento , Glutamina/metabolismo , Humanos , Imuno-Histoquímica , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Transdução de Sinais
2.
Mol Cell Neurosci ; 88: 222-230, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29425968

RESUMO

Pluripotent stem cell derived human neuronal progenitor cells (hPSC-NPCs) and their mature neuronal cell culture derivatives may efficiently be used for central nervous system (CNS) drug screening, including the investigation of ligand-induced calcium signalization. We have established hippocampal NPC cultures derived from human induced PSCs, which were previously generated by non-integrating Sendai virus reprogramming. Using established protocols these NPCs were differentiated into hippocampal dentate gyrus neurons. In order to study calcium signaling without the need of dye loading, we have stably expressed an advanced calcium indicator protein (GCaMP6fast) in the NPCs using the Sleeping Beauty transposon system. We observed no significant effects of the long-term GCaMP6 expression on NPC morphology, gene expression pattern or neural differentiation capacity. In order to compare the functional properties of GCaMP6-expressing neural cells and the corresponding parental cells loaded with calcium indicator dye Fluo-4, a detailed characterization of calcium signals was performed. We found that the calcium signals induced by ATP, glutamate, LPA, or proteases - were similar in these two systems. Moreover, the presence of the calcium indicator protein allowed for a sensitive, repeatable detection of changes in calcium signaling during the process of neurogenesis and neuronal maturation.


Assuntos
Cálcio/metabolismo , Giro Denteado/citologia , Células-Tronco Pluripotentes Induzidas/citologia , Neurônios/citologia , Células-Tronco Pluripotentes/citologia , Diferenciação Celular/fisiologia , Processos de Crescimento Celular/fisiologia , Hipocampo/citologia , Humanos , Células-Tronco Neurais/metabolismo , Neurogênese/fisiologia
3.
Neuropsychopharmacol Hung ; 18(4): 188-198, 2016 12.
Artigo em Húngaro | MEDLINE | ID: mdl-28259862

RESUMO

Over the past decade we witnessed the birth of a new scientific area that lies at the borders of developmental biology, stem cell biology, basic and clinical neuroscience. In vitro disease modeling refers to the approach that exploits the capacity of stem cells for self-renewal and pluripotency by generating specific cell types that are relevant for a given disorder. Based on this method, neurological and psychiatric disorders can be investigated by differentiating stem cells into neurons in a dish, and studying the relevant neuronal populations affected in the pathophysiology of the disorder in terms of specific cellular phenotypes. The advent of induced pluripotent stem cells (IPSCs) has made it possible to reprogram IPSCs from somatic cells of patients carrying specific genetic risk variants, and to analyze the in vitro cellular findings in the context of the clinical picture. Pluripotent stem cell based disease modeling offers an alternative solution for invasive and mostly not performable central nervous system biopsies in neuropsychiatric disorders, and is an appealing laboratory method for studying biomarkers of these disorders and for future drug development. This review summarizes the pluripotent stem cell based disease modeling literature in two important neuropsychiatric disorders, Alzheimer's disease and schizophrenia.


Assuntos
Doença de Alzheimer/terapia , Células-Tronco Pluripotentes Induzidas , Esquizofrenia/terapia , Biomarcadores , Humanos , Neurônios
4.
Biomolecules ; 14(6)2024 Jun 13.
Artigo em Inglês | MEDLINE | ID: mdl-38927091

RESUMO

BACKGROUND: Induced pluripotent stem cell (iPSC) based neuronal differentiation is valuable for studying neuropsychiatric disorders and pharmacological mechanisms at the cellular level. We aimed to examine the effects of typical and atypical antipsychotics on human iPSC-derived neural progenitor cells (NPCs). METHODS: Proliferation and neurite outgrowth were measured by live cell imaging, and gene expression levels related to neuronal identity were analyzed by RT-QPCR and immunocytochemistry during differentiation into hippocampal dentate gyrus granule cells following treatment of low- and high-dose antipsychotics (haloperidol, olanzapine, and risperidone). RESULTS: Antipsychotics did not modify the growth properties of NPCs after 3 days of treatment. However, the characteristics of neurite outgrowth changed significantly in response to haloperidol and olanzapine. After three weeks of differentiation, mRNA expression levels of the selected neuronal markers increased (except for MAP2), while antipsychotics caused only subtle changes. Additionally, we found no changes in MAP2 or GFAP protein expression levels as a result of antipsychotic treatment. CONCLUSIONS: Altogether, antipsychotic medications promoted neurogenesis in vitro by influencing neurite outgrowth rather than changing cell survival or gene expression. This study provides insights into the effects of antipsychotics on neuronal differentiation and highlights the importance of considering neurite outgrowth as a potential target of action.


Assuntos
Antipsicóticos , Diferenciação Celular , Haloperidol , Hipocampo , Células-Tronco Pluripotentes Induzidas , Células-Tronco Neurais , Neurogênese , Olanzapina , Risperidona , Humanos , Olanzapina/farmacologia , Risperidona/farmacologia , Neurogênese/efeitos dos fármacos , Hipocampo/citologia , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Haloperidol/farmacologia , Antipsicóticos/farmacologia , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Neurais/efeitos dos fármacos , Células-Tronco Neurais/metabolismo , Células-Tronco Neurais/citologia , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Crescimento Neuronal/efeitos dos fármacos
5.
Schizophr Res ; 2024 Jan 29.
Artigo em Inglês | MEDLINE | ID: mdl-38290943

RESUMO

BACKGROUND: Schizophrenia (SCZ) is a severe neuropsychiatric disorder of complex, poorly understood etiology, associated with both genetic and environmental factors. De novo mutations (DNMs) represent a new source of genetic variation in SCZ, however, in most cases their biological significance remains unclear. We sought to investigate molecular disease pathways connected to DNMs in SCZ by combining human induced pluripotent stem cell (hiPSC) based disease modeling and CRISPR-based genome editing. METHODS: We selected a SCZ case-parent trio with the case individual carrying a potentially disease causing 1495C > T nonsense DNM in the zinc finger MYND domain-containing protein 11 (ZMYND11), a gene implicated in biological processes relevant for SCZ. In the patient-derived hiPSC line the mutation was corrected using CRISPR, while monoallelic or biallelic frameshift mutations were introduced into a control hiPSC line. Isogenic cell lines were differentiated into hippocampal neuronal progenitor cells (NPCs) and functionally active dentate gyrus granule cells (DGGCs). Immunofluorescence microscopy and RNA sequencing were used to test for morphological and transcriptomic differences at NPC and DGCC stages. Functionality of neurons was investigated using calcium-imaging and multi-electrode array measurements. RESULTS: Morphology in the mutant hippocampal NPCs and neurons was preserved, however, we detected significant transcriptomic and functional alterations. RNA sequencing showed massive upregulation of neuronal differentiation genes, and downregulation of cell adhesion genes. Decreased reactivity to glutamate was demonstrated by calcium-imaging. CONCLUSIONS: Our findings lend support to the involvement of glutamatergic dysregulation in the pathogenesis of SCZ. This approach represents a powerful model system for precision psychiatry and pharmacological research.

6.
Front Cell Dev Biol ; 9: 719636, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34604221

RESUMO

Studies on neural development and neuronal regeneration after injury are mainly based on animal models. The establishment of pluripotent stem cell (PSC) technology, however, opened new perspectives for better understanding these processes in human models by providing unlimited cell source for hard-to-obtain human tissues. Here, we aimed at identifying the molecular factors that confine and modulate an early step of neural regeneration, the formation of neurites in human neural progenitor cells (NPCs). Enhanced green fluorescent protein (eGFP) was stably expressed in NPCs differentiated from human embryonic and induced PSC lines, and the neurite outgrowth was investigated under normal and injury-related conditions using a high-content screening system. We found that inhibitors of the non-muscle myosin II (NMII), blebbistatin and its novel, non-toxic derivatives, initiated extensive neurite outgrowth in human NPCs. The extracellular matrix components strongly influenced the rate of neurite formation but NMII inhibitors were able to override the inhibitory effect of a restrictive environment. Non-additive stimulatory effect on neurite generation was also detected by the inhibition of Rho-associated, coiled-coil-containing protein kinase 1 (ROCK1), the upstream regulator of NMII. In contrast, inhibition of c-Jun N-terminal kinases (JNKs) had only a negligible effect, suggesting that the ROCK1 signal is dominantly manifested by actomyosin activity. In addition to providing a reliable cell-based in vitro model for identifying intrinsic mechanisms and environmental factors responsible for impeded axonal regeneration in humans, our results demonstrate that NMII and ROCK1 are important pharmacological targets for the augmentation of neural regeneration at the progenitor level. These studies may open novel perspectives for development of more effective pharmacological treatments and cell therapies for various neurodegenerative disorders.

7.
Stem Cell Res ; 51: 102140, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33503521

RESUMO

Here we describe the generation of induced pluripotent stem cell lines from each member - male proband, mother, father - of a schizophrenia case-parent trio that participated in an exome sequencing study, and 3 de novo mutations were identified in the proband. Peripheral blood mononuclear cells were obtained from all three individuals and reprogrammed using Sendai virus particles carrying the Yamanaka transgenes. These 3 iPSC lines (iPSC-SZ-HU-MO 1, iPSC-SZ-HU-FA 1, and iPSC-SZ-HU-PROB 1) represent a resource for examining the functional significance of the identified de novo mutations in the molecular pathophysiology of schizophrenia.


Assuntos
Células-Tronco Pluripotentes Induzidas , Esquizofrenia , Células Clonais , Humanos , Leucócitos Mononucleares , Masculino , Mutação/genética , Proteínas de Ligação a RNA , Receptores KIR2DL1 , Esquizofrenia/genética , Sialoglicoproteínas , Transativadores
8.
Stem Cell Res Ther ; 11(1): 504, 2020 11 27.
Artigo em Inglês | MEDLINE | ID: mdl-33246498

RESUMO

BACKGROUND: De novo mutations (DNMs) have been implicated in the etiology of schizophrenia (SZ), a chronic debilitating psychiatric disorder characterized by hallucinations, delusions, cognitive dysfunction, and decreased community functioning. Several DNMs have been identified by examining SZ cases and their unaffected parents; however, in most cases, the biological significance of these mutations remains elusive. To overcome this limitation, we have developed an approach of using induced pluripotent stem cell (iPSC) lines from each member of a SZ case-parent trio, in order to investigate the effects of DNMs in cellular progenies of interest, particularly in dentate gyrus neuronal progenitors. METHODS: We identified a male SZ patient characterized by early disease onset and negative symptoms, who is a carrier of 3 non-synonymous DNMs in genes LRRC7, KHSRP, and KIR2DL1. iPSC lines were generated from his and his parents' peripheral blood mononuclear cells using Sendai virus-based reprogramming and differentiated into neuronal progenitor cells (NPCs) and hippocampal dentate gyrus granule cells. We used RNASeq to explore transcriptomic differences and calcium (Ca2+) imaging, cell proliferation, migration, oxidative stress, and mitochondrial assays to characterize the investigated NPC lines. RESULTS: NPCs derived from the SZ patient exhibited transcriptomic differences related to Wnt signaling, neuronal differentiation, axonal guidance and synaptic function, and decreased Ca2+ reactivity to glutamate. Moreover, we could observe increased cellular proliferation and alterations in mitochondrial quantity and morphology. CONCLUSIONS: The approach of reprograming case-parent trios represents an opportunity for investigating the molecular effects of disease-causing mutations and comparing these in cell lines with reduced variation in genetic background. Our results are indicative of a partial overlap between schizophrenia and autism-related phenotypes in the investigated family. LIMITATIONS: Our study investigated only one family; therefore, the generalizability of findings is limited. We could not derive iPSCs from two other siblings to test for possible genetic effects in the family that are not driven by DNMs. The transcriptomic and functional assays were limited to the NPC stage, although these variables should also be investigated at the mature neuronal stage.


Assuntos
Transtorno Autístico , Células-Tronco Pluripotentes Induzidas , Esquizofrenia , Humanos , Leucócitos Mononucleares , Masculino , Mutação , Fenótipo , Proteínas de Ligação a RNA , Esquizofrenia/genética , Sialoglicoproteínas , Transativadores
9.
FEBS Open Bio ; 8(4): 584-592, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29632811

RESUMO

5-Hydroxymethylcytosine (5hmC) is produced from 5-methylcytosine (5mC) by Ten-eleven translocation (TET) dioxygenases. The epigenetic modification 5hmC has crucial roles in both cellular development and differentiation. The 5hmC level is particularly high in the brain. While 5mC is generally associated with gene silencing/reduced expression, 5hmC is a more permissive epigenetic mark. To understand its physiological function, an easy and accurate quantification method is required. Here, we have developed a novel LC-MS/MS-based approach to quantify both genomic 5mC and 5hmC contents. The method is based on the liberation of nucleobases by formic acid. Applying this method, we characterized the levels of DNA methylation and hydroxymethylation in mouse brain and liver, primary hepatocytes, and various cell lines. Using this approach, we confirm that the treatment of different cell lines with the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine leads to a decrease in 5mC content. This decrease was accompanied by an increase in 5hmC levels in cell lines of hematopoietic origin. Finally, we showed that ascorbate elevates the levels of 5hmC and augments the effect of 5-aza-2'-deoxycytidine without significantly influencing 5mC levels.

10.
Stem Cells Int ; 2016: 7909176, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26839567

RESUMO

Neuronal differentiation of induced pluripotent stem cells and direct reprogramming represent powerful methods for modeling the development of neurons in vitro. Moreover, this approach is also a means for comparing various cellular phenotypes between cell lines originating from healthy and diseased individuals or isogenic cell lines engineered to differ at only one or a few genomic loci. Despite methodological constraints and initial skepticism regarding this approach, the field is expanding at a fast pace. The improvements include the development of new differentiation protocols resulting in selected neuronal populations (e.g., dopaminergic, GABAergic, hippocampal, and cortical), the widespread use of genome editing methods, and single-cell techniques. A major challenge awaiting in vitro disease modeling is the integration of clinical data in the models, by selection of well characterized clinical populations. Ideally, these models will also demonstrate how different diagnostic categories share overlapping molecular disease mechanisms, but also have unique characteristics. In this review we evaluate studies with regard to the described developments, to demonstrate how differentiation of induced pluripotent stem cells and direct reprogramming can contribute to psychiatry.

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