scholarly journals Reprogramming Human Adult Fibroblasts into GABAergic Interneurons

Cells ◽  
2021 ◽  
Vol 10 (12) ◽  
pp. 3450
Author(s):  
Andreas Bruzelius ◽  
Srisaiyini Kidnapillai ◽  
Janelle Drouin-Ouellet ◽  
Tom Stoker ◽  
Roger A. Barker ◽  
...  
Keyword(s):  
Human Fibroblasts ◽  
Autism Spectrum ◽  
Specific Gene ◽  
Gabaergic Interneurons ◽  
Human Adult ◽  
Gene And Protein Expression ◽  
Parkinson’S Diseases ◽  
Fetal Fibroblasts ◽  
Adult Skin ◽  
Adult Fibroblasts

Direct reprogramming is an appealing strategy to generate neurons from a somatic cell by forced expression of transcription factors. The generated neurons can be used for both cell replacement strategies and disease modelling. Using this technique, previous studies have shown that γ-aminobutyric acid (GABA) expressing interneurons can be generated from different cell sources, such as glia cells or fetal fibroblasts. Nevertheless, the generation of neurons from adult human fibroblasts, an easily accessible cell source to obtain patient-derived neurons, has proved to be challenging due to the intrinsic blockade of neuronal commitment. In this paper, we used an optimized protocol for adult skin fibroblast reprogramming based on RE1 Silencing Transcription Factor (REST) inhibition together with a combination of GABAergic fate determinants to convert human adult skin fibroblasts into GABAergic neurons. Our results show a successful conversion in 25 days with upregulation of neuronal gene and protein expression levels. Moreover, we identified specific gene combinations that converted fibroblasts into neurons of a GABAergic interneuronal fate. Despite the well-known difficulty in converting adult fibroblasts into functional neurons in vitro, we could detect functional maturation in the induced neurons. GABAergic interneurons have relevance for cognitive impairments and brain disorders, such as Alzheimer’s and Parkinson’s diseases, epilepsy, schizophrenia and autism spectrum disorders.

Endocannabinoid system in the neurodevelopment of GABAergic interneurons: implications for neurological and psychiatric disorders

2021 ◽  
Vol 0 (0) ◽  
Author(s):  
Chang-geng Song ◽  
Xin Kang ◽  
Fang Yang ◽  
Wan-qing Du ◽  
Jia-jia Zhang ◽  
...  
Keyword(s):  
Psychiatric Disorders ◽  
Endocannabinoid System ◽  
Autism Spectrum ◽  
Network Activity ◽  
Inhibitory Neurons ◽  
Gabaergic Interneurons ◽  
Neural Stem Progenitor Cells ◽  
The Central Nervous System ◽  
Interneuron Development

Abstract In mature mammalian brains, the endocannabinoid system (ECS) plays an important role in the regulation of synaptic plasticity and the functioning of neural networks. Besides, the ECS also contributes to the neurodevelopment of the central nervous system. Due to the increase in the medical and recreational use of cannabis, it is inevitable and essential to elaborate the roles of the ECS on neurodevelopment. GABAergic interneurons represent a group of inhibitory neurons that are vital in controlling neural network activity. However, the role of the ECS in the neurodevelopment of GABAergic interneurons remains to be fully elucidated. In this review, we provide a brief introduction of the ECS and interneuron diversity. We focus on the process of interneuron development and the role of ECS in the modulation of interneuron development, from the expansion of the neural stem/progenitor cells to the migration, specification and maturation of interneurons. We further discuss the potential implications of the ECS and interneurons in the pathogenesis of neurological and psychiatric disorders, including epilepsy, schizophrenia, major depressive disorder and autism spectrum disorder.

scholarly journals An analytical method for the identification of cell type-specific disease gene modules

2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Jinting Guan ◽  
Yiping Lin ◽  
Yang Wang ◽  
Junchao Gao ◽  
Guoli Ji
Keyword(s):  
Disease Gene ◽  
Gene Interaction ◽  
Cell Types ◽  
Autism Spectrum ◽  
Specific Gene ◽  
Cell Type ◽  
Specific Disease ◽  
Cell Type Specific ◽  
Gene Modules ◽  
Disease Associated Genes

Abstract Background Genome-wide association studies have identified genetic variants associated with the risk of brain-related diseases, such as neurological and psychiatric disorders, while the causal variants and the specific vulnerable cell types are often needed to be studied. Many disease-associated genes are expressed in multiple cell types of human brains, while the pathologic variants affect primarily specific cell types. We hypothesize a model in which what determines the manifestation of a disease in a cell type is the presence of disease module comprised of disease-associated genes, instead of individual genes. Therefore, it is essential to identify the presence/absence of disease gene modules in cells. Methods To characterize the cell type-specificity of brain-related diseases, we construct human brain cell type-specific gene interaction networks integrating human brain nucleus gene expression data with a referenced tissue-specific gene interaction network. Then from the cell type-specific gene interaction networks, we identify significant cell type-specific disease gene modules by performing statistical tests. Results Between neurons and glia cells, the constructed cell type-specific gene networks and their gene functions are distinct. Then we identify cell type-specific disease gene modules associated with autism spectrum disorder and find that different gene modules are formed and distinct gene functions may be dysregulated in different cells. We also study the similarity and dissimilarity in cell type-specific disease gene modules among autism spectrum disorder, schizophrenia and bipolar disorder. The functions of neurons-specific disease gene modules are associated with synapse for all three diseases, while those in glia cells are different. To facilitate the use of our method, we develop an R package, CtsDGM, for the identification of cell type-specific disease gene modules. Conclusions The results support our hypothesis that a disease manifests itself in a cell type through forming a statistically significant disease gene module. The identification of cell type-specific disease gene modules can promote the development of more targeted biomarkers and treatments for the disease. Our method can be applied for depicting the cell type heterogeneity of a given disease, and also for studying the similarity and dissimilarity between different disorders, providing new insights into the molecular mechanisms underlying the pathogenesis and progression of diseases.

Generation of Fluorescently Labeled Cell Lines, C3A Hepatoma Cells, and Human Adult Skin Fibroblasts to Study Coculture Models

2013 ◽  
Vol 37 (7) ◽  
pp. E123-E130 ◽  
Author(s):  
Anna Samluk ◽  
Karolina Ewa Zakrzewska ◽  
Krzysztof Dariusz Pluta
Keyword(s):  
Cell Lines ◽  
Hepatoma Cells ◽  
Skin Fibroblasts ◽  
Human Adult ◽  
Adult Skin

scholarly journals Molecular diversity and lineage commitment of human interneuron progenitors

2021 ◽  
Author(s):  
Dmitry Velmeshev ◽  
Manideep Chavali ◽  
Tomasz Jan Nowakowski ◽  
Mohini Bhade ◽  
Simone Mayer ◽  
...  
Keyword(s):  
Gene Expression ◽  
Single Cell ◽  
Autism Spectrum ◽  
Lineage Commitment ◽  
Proper Function ◽  
Specific Gene ◽  
Specific Cell ◽  
Cortical Interneurons ◽  
Human Specific ◽  
Interneuron Development

Cortical interneurons are indispensable for proper function of neocortical circuits. Changes in interneuron development and function are implicated in human disorders, such as autism spectrum disorder and epilepsy. In order to understand human-specific features of cortical development as well as the origins of neurodevelopmental disorders it is crucial to identify the molecular programs underlying human interneuron development and subtype specification. Recent studies have explored gene expression programs underlying mouse interneuron specification and maturation. We applied single-cell RNA sequencing to samples of second trimester human ganglionic eminence and developing cortex to identify molecularly defined subtypes of human interneuron progenitors and immature interneurons. In addition, we integrated this data from the developing human ganglionic eminences and neocortex with single-nucleus RNA-seq of adult cortical interneurons in order to elucidate dynamic molecular changes associated with commitment of progenitors and immature interneurons to mature interneuron subtypes. By comparing our data with published mouse single-cell genomic data, we discover a number of divergent gene expression programs that distinguish human interneuron progenitors from mouse. Moreover, we find that a number of transcription factors expressed during prenatal development become restricted to adult interneuron subtypes in the human but not the mouse, and these adult interneurons express species- and lineage-specific cell adhesion and synaptic genes. Therefore, our study highlights that despite the similarity of main principles of cortical interneuron development and lineage commitment between mouse and human, human interneuron genesis and subtype specification is guided by species-specific gene programs, contributing to human-specific features of cortical inhibitory interneurons.

scholarly journals POGZ controls embryonic stem cell self-renewal and pluripotency by association with esBAF and HP1

2021 ◽  
Author(s):  
Xiaoyun Sun ◽  
Linxi Cheng ◽  
Yuhua Sun
Keyword(s):  
Cell Fate ◽  
Neurodevelopmental Disorders ◽  
Embryonic Stem ◽  
Autism Spectrum ◽  
Specific Gene ◽  
Open Chromatin ◽  
Cell Fate Determination ◽  
Chromatin Remodeler ◽  
Self Renewal ◽  
Fate Determination

AbstractPOGZ, which encodes a multi-domain transcription factor, has been found frequently mutated in neurodevelopmental disorders, particularly autism spectrum disorder (ASD) and intellectual disability (ID). However, little is known about its function in ESC self-renewal and pluripotency, cell fate determination as well as in transcriptional regulation. Here, using embryonic stem cells (ESCs) as model, we show that POGZ plays key roles in the maintenance of ESC and cell fate determination by association with the SWI-SNW chromatin remodeler complex and heterochromatin protein 1 (HP1) proteins. POGZ is essential for the maintenance of ESC undifferentiated state, and loss of POGZ leads to ESC differentiation, likely by up-regulation of primitive endoderm and mesoderm lineage genes and by down-regulation of pluripotency-related genes. Mechanistically, POGZ may control ESC-specific gene expression by association with chromatin remodeler complex esBAF and HP1s, and they can form a PBH triplex. POGZ functions primarily to maintain an open chromatin, as loss of POGZ leads to a reduced chromatin accessibility. Regulation of chromatin under control of POGZ depends on esBAF complex. POGZ is extensively co-localized with OCT4/NANOG genome wide. Taken together, we propose that POGZ is a pluripotency-associated factor, and its absence in ESCs causes failure to maintain a proper ESC-specific chromatin state and transcriptional circuitry of pluripotency, which eventually leads to ESC self-renewal and pluripotency defects. Our work provides important insights into the role of POGZ in ESC self-renewal and pluripotency as well as regulation of transcription, which will be useful for understanding the etiology of neurodevelopmental disorders by POGZ mutation.

Abstract P493: Histone Reader PHF7 Cooperates With The SWI/SNF Complex At Cardiac Super Enhancers To Promote Direct Reprogramming

2021 ◽  
Vol 129 (Suppl_1) ◽  
Author(s):  
Glynnis A Garry ◽  
Svetlana Bezprozvannaya ◽  
Huanyu Zhou ◽  
Hisayuki Hashimoto ◽  
Kenian Chen ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Cardiac Fibroblasts ◽  
Human Fibroblasts ◽  
Cell Types ◽  
Chromatin Accessibility ◽  
Direct Reprogramming ◽  
Ischemic Disease ◽  
Treat Heart Failure ◽  
Cardiac Transcription Factors ◽  
Adult Fibroblasts

Ischemic heart disease is the leading cause of death worldwide. Direct reprogramming of resident cardiac fibroblasts (CFs) to induced cardiomyocytes (iCLMs) has emerged as a potential therapeutic approach to treat heart failure and ischemic disease. Cardiac reprogramming was first achieved through forced expression of the transcription factors Gata4, Mef2c, and Tbx5 (GMT); our laboratory found that Hand2 (GHMT) and Akt1 (AGHMT) markedly enhanced reprogramming efficiency in embryonic and postnatal cell types. However, adult mouse and human fibroblasts are resistant to reprogramming due to staunch epigenetic barriers. We undertook a screen of mammalian gene regulatory factors to discover novel regulators of cardiac reprogramming in adult fibroblasts and identified the epigenetic reader PHF7 as the most potent activating factor. We validated the findings of this screen and found that PHF7 augmented reprogramming of adult fibroblasts ten-fold. Mechanistically, PHF7 localized to cardiac super enhancers in fibroblasts by reading H3K4me2 marks, and through cooperation with the SWI/SNF complex, increased chromatin accessibility and transcription factor binding at these multivalent enhancers. Further, PHF7 recruited cardiac transcription factors to activate a positive transcriptional autoregulatory circuit in reprogramming. Importantly, PHF7 achieved efficient reprogramming through these mechanisms in the absence of Gata4. Collectively, these studies highlight the underexplored necessity of cardiac epigenetic readers, such as PHF7, in harnessing chromatin remodeling and transcriptional complexes to overcome critical barriers to direct cardiac reprogramming.

scholarly journals Conditional Knockdown of Osteopontin Inhibits Breast Cancer Skeletal Metastasis

2019 ◽  
Vol 20 (19) ◽  
pp. 4918 ◽  
Author(s):  
Marineta Kovacheva ◽  
Michael Zepp ◽  
Muriel Schraad ◽  
Stefan Berger ◽  
Martin R. Berger
Keyword(s):  
Breast Cancer ◽  
Bone Metastasis ◽  
Skeletal Metastasis ◽  
Skeletal Metastases ◽  
Specific Gene ◽  
Cell Clones ◽  
Gene And Protein Expression ◽  
Metastasis Model ◽  
Breast Cancer Bone Metastasis

High osteopontin (OPN) expression is linked to breast cancer bone metastasis. In this study we modulated osteopontin levels conditionally and investigated any related antineoplastic effects. Therefore, we established cell clones from human breast cancer MDA-MB-231 cells, in which the expression of OPN is regulated by the Tet-Off tet-off system. These cells, which conditionally express a specific miRNA targeting OPN, were used for in vitro studies as well as for a bone metastasis model in nude rats. Changes in whole-genome expression elicited by conditional OPN knockdown and vesicle formation were also analyzed. The alkylphosphocholine erufosine was used for combination therapy. Conditional OPN knockdown caused mild anti-proliferative, but more intensive anti-migratory and anti clonogenic effects, as well as partial and complete remissions of soft tissue and osteolytic lesions. These effects were associated with specific gene and protein expression modulations following miRNA-mediated OPN knockdown. Furthermore, high levels of OPN were detected in vesicles derived from rats harboring breast cancer skeletal metastases. Finally, the combination of OPN inhibition and erufosine treatment caused an additive reduction of OPN levels in the investigated breast cancer cells. Thus, knockdown of OPN alone or in combination with erufosine is a promising strategy in breast cancer skeletal metastasis treatment.

10q23.31 microduplication encompassing PTEN decreases mTOR signalling activity and is associated with autosomal dominant primary microcephaly

2018 ◽  
Vol 56 (8) ◽  
pp. 543-547 ◽  
Author(s):  
Danyllo Oliveira ◽  
Gabriela Ferraz Leal ◽  
Andréa L Sertié ◽  
Luiz Carlos Caires Jr ◽  
Ernesto Goulart ◽  
...  
Keyword(s):  
Autosomal Dominant ◽  
Autism Spectrum ◽  
Signalling Pathway ◽  
Variable Degree ◽  
Primary Microcephaly ◽  
Dominant Form ◽  
Gene And Protein Expression ◽  
Autosomal Dominant Form ◽  
Mtor Signalling ◽  
Mtor Signalling Pathway

BackgroundHereditary primary microcephaly (MCPH) is mainly characterised by decreased occipitofrontal circumference and variable degree of intellectual disability. MCPH with a dominant pattern of inheritance is a rare condition, so far causally linked to pathogenic variants in the ALFY, DPP6, KIF11 and DYRK1A genes.ObjectiveThis study aimed at identifying the causative variant of the autosomal dominant form of MCPH in a Brazilian family with three affected members.MethodsFollowing clinical evaluation of two sibs and their mother presenting with autosomal dominant MCPH, array comparative genome hybridisation was performed using genomic DNA from peripheral blood of the family members. Gene and protein expression studies were carried out in cultured skin fibroblasts.ResultsA 382 kb microduplication at 10q23.31 was detected, encompassing the entire PTEN, KLLN and ATAD1 genes. PTEN haploinsufficiency has been causally associated with macrocephaly and autism spectrum disorder and, therefore, was considered the most likely candidate gene to be involved in this autosomal dominant form of MCPH. In the patients’ fibroblasts, PTEN mRNA and protein were found to be overexpressed, and the phosphorylation patterns of upstream and downstream components of the mammalian target of rapamycin (mTOR) signalling pathway were dysregulated.ConclusionsTaken together, our results demonstrate that the identified submicroscopic 10q23.31 duplication in a family with MCPH leads to markedly increased expression of PTEN and reduced activity of the mTOR signalling pathway. These results suggest that the most probable pathomechanism underlying the microcephaly phenotype in this family involves downregulation of the mTOR pathway through overexpression of PTEN.

187 Phenotype switch of human fibroblasts into trophoblastic cells

2019 ◽  
Vol 31 (1) ◽  
pp. 218
Author(s):  
S. Arcuri ◽  
E. F. M. Manzoni ◽  
F. Gandolfi ◽  
T. A. L. Brevini
Keyword(s):  
Developmental Disorders ◽  
Human Fibroblasts ◽  
Normal Karyotype ◽  
Small Cells ◽  
Specific Gene ◽  
Trophoblast Cells ◽  
Human Trophoblast ◽  
Trophoblastic Cells

The first differentiation event in mammalian embryos is the formation of the trophectoderm, which is crucial for implantation of the blastocyst and gives rise to specialised populations of trophoblast cells in the definitive placenta. However, our understanding of these early differentiation events is limited, particularly in humans, because of ethical and legal restrictions on the isolation and manipulation of human embryogenesis. Here we describe experiments aimed at converting human fibroblasts into trophoblastic cells, using 5-azacytidine (5-aza-CR) to erase the original phenotype and a cocktail containing bone morphogenic protein 4 (BMP4) and inhibitors of the activin/nodal/ERK signalling pathways, to drive trophoblastic differentiation. The method required 3 main steps: (1) preparation of mouse embryonic fibroblasts (MEF) monolayer, and culture and collection of MEF conditioned medium (CM); (2) culture of human fibroblasts, obtained from a skin biopsy, and epigenetic erasure with 5-aza-CR for 18 h; (3) use of CM, with BMP4 (50 ng mL−1), PD0325901 (1 µM), CHIR99021 (1 µM), and PD173074 (0.1 µM) to differentiate erased human fibroblasts to trophoblast cells. Morphology changes were monitored along the process. In the initial phase of differentiation, a mixture of cell types appeared, including small cells growing in clusters and giant cells possessing very large nuclei. After 2 weeks of culture, cells displayed a distinct trophoblast-like morphology and showed the presence of typical clustering/lacunae monolayer patterning. Cells continued to proliferate and maintained a normal karyotype. BMP4-mediated differentiation was also assessed by quantitative RT-PCR using primers specific for 2 trophoblast markers: keratin 7 (KRT7) and caudal type homeobox 2 (CDX2), that are absent in the original fibroblasts. Their expression appeared by Day 3 of induction and was strong and steady throughout the process, confirming that the acquisition of a trophoblast-like morphology was supported by the activation of trophoblast-specific gene transcription. The results demonstrate the possibility of obtaining trophoblast-like cells through the conversion of human fibroblasts and confirm the involvement of BMP4-together with the inhibition of the activin/nodal/ERK signalling pathway-to activate early trophoblast differentiation in vitro. The challenge for future experiments will be to determine the precise role of BMP signals in human trophoblast definition in vivo, which, to our knowledge, has not been elucidated yet. The method here described is efficient and reproducible and has the advantage of utilising easily accessible cells as a starting population. It can be used for a variety of applications, including drug discovery and stem cell research, as well as to implement studies on the pathogenesis of developmental disorders with trophoblast defects. This research was supported by Carraresi Foundation. Authors are members of the COST Actions CA16119 and CM1406.

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