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1.
Nat Commun ; 12(1): 5255, 2021 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-34489438

RESUMO

Monocytes are part of the mononuclear phagocytic system. Monocytes play a central role during inflammatory conditions and a better understanding of their dynamics might open therapeutic opportunities. In the present study, we focused on the characterization and impact of monocytes on brown adipose tissue (BAT) functions during tissue remodeling. Single-cell RNA sequencing analysis of BAT immune cells uncovered a large diversity in monocyte and macrophage populations. Fate-mapping experiments demonstrated that the BAT macrophage pool requires constant replenishment from monocytes. Using a genetic model of BAT expansion, we found that brown fat monocyte numbers were selectively increased in this scenario. This observation was confirmed using a CCR2-binding radiotracer and positron emission tomography. Importantly, in line with their tissue recruitment, blood monocyte counts were decreased while bone marrow hematopoiesis was not affected. Monocyte depletion prevented brown adipose tissue expansion and altered its architecture. Podoplanin engagement is strictly required for BAT expansion. Together, these data redefine the diversity of immune cells in the BAT and emphasize the role of monocyte recruitment for tissue remodeling.


Assuntos
Tecido Adiposo Marrom/citologia , Monócitos/fisiologia , Adiponectina/genética , Tecido Adiposo Marrom/fisiologia , Animais , Diferenciação Celular/genética , Contagem de Leucócitos , Macrófagos/citologia , Macrófagos/fisiologia , Glicoproteínas de Membrana/metabolismo , Camundongos Transgênicos , Monócitos/citologia , Tomografia por Emissão de Pósitrons , Receptores CCR2/genética , Receptores CCR2/metabolismo
2.
Nat Commun ; 12(1): 4813, 2021 08 10.
Artigo em Inglês | MEDLINE | ID: mdl-34376664

RESUMO

Differences in immune responses to viruses and autoimmune diseases such as systemic lupus erythematosus (SLE) can show sexual dimorphism. Age-associated B cells (ABC) are a population of CD11c+T-bet+ B cells critical for antiviral responses and autoimmune disorders. Absence of DEF6 and SWAP-70, two homologous guanine exchange factors, in double-knock-out (DKO) mice leads to a lupus-like syndrome in females marked by accumulation of ABCs. Here we demonstrate that DKO ABCs show sex-specific differences in cell number, upregulation of an ISG signature, and further differentiation. DKO ABCs undergo oligoclonal expansion and differentiate into both CD11c+ and CD11c- effector B cell populations with pathogenic and pro-inflammatory function as demonstrated by BCR sequencing and fate-mapping experiments. Tlr7 duplication in DKO males overrides the sex-bias and further augments the dissemination and pathogenicity of ABCs, resulting in severe pulmonary inflammation and early mortality. Thus, sexual dimorphism shapes the expansion, function and differentiation of ABCs that accompanies TLR7-driven immunopathogenesis.


Assuntos
Envelhecimento/imunologia , Linfócitos B/imunologia , Diferenciação Celular/imunologia , Lúpus Eritematoso Sistêmico/imunologia , Fatores Etários , Envelhecimento/genética , Animais , Linfócitos B/citologia , Linfócitos B/metabolismo , Antígeno CD11c/imunologia , Antígeno CD11c/metabolismo , Diferenciação Celular/genética , Células Cultivadas , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/imunologia , Proteínas de Ligação a DNA/metabolismo , Feminino , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/imunologia , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Estimativa de Kaplan-Meier , Lúpus Eritematoso Sistêmico/genética , Lúpus Eritematoso Sistêmico/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Antígenos de Histocompatibilidade Menor/genética , Antígenos de Histocompatibilidade Menor/imunologia , Antígenos de Histocompatibilidade Menor/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/imunologia , Proteínas Nucleares/metabolismo , Fatores Sexuais , Proteínas com Domínio T/imunologia , Proteínas com Domínio T/metabolismo
3.
Nat Commun ; 12(1): 4923, 2021 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-34389713

RESUMO

With increasing age of the population, countries across the globe are facing a substantial increase in osteoporotic fractures. Genetic association signals for fractures have been reported at the RSPO3 locus, but the causal gene and the underlying mechanism are unknown. Here we show that the fracture reducing allele at the RSPO3 locus associate with increased RSPO3 expression both at the mRNA and protein levels, increased trabecular bone mineral density and reduced risk mainly of distal forearm fractures in humans. We also demonstrate that RSPO3 is expressed in osteoprogenitor cells and osteoblasts and that osteoblast-derived RSPO3 is the principal source of RSPO3 in bone and an important regulator of vertebral trabecular bone mass and bone strength in adult mice. Mechanistic studies revealed that RSPO3 in a cell-autonomous manner increases osteoblast proliferation and differentiation. In conclusion, RSPO3 regulates vertebral trabecular bone mass and bone strength in mice and fracture risk in humans.


Assuntos
Osso Esponjoso/metabolismo , Fraturas Ósseas/genética , Predisposição Genética para Doença/genética , Polimorfismo de Nucleotídeo Único , Trombospondinas/genética , Animais , Densidade Óssea , Osso Esponjoso/lesões , Diferenciação Celular/genética , Proliferação de Células/genética , Células Cultivadas , Humanos , Análise da Randomização Mendeliana/métodos , Camundongos Knockout , Camundongos Transgênicos , Osteoblastos/citologia , Osteoblastos/metabolismo , Fatores de Risco , Trombospondinas/deficiência
4.
Nat Commun ; 12(1): 4897, 2021 08 12.
Artigo em Inglês | MEDLINE | ID: mdl-34385432

RESUMO

Precise control of mammalian gene expression is facilitated through epigenetic mechanisms and nuclear organization. In particular, insulated chromosome structures are important for regulatory control, but the phenotypic consequences of their boundary disruption on developmental processes are complex and remain insufficiently understood. Here, we generated deeply sequenced Hi-C data for human pluripotent stem cells (hPSCs) that allowed us to identify CTCF loop domains that have highly conserved boundary CTCF sites and show a notable enrichment of individual developmental regulators. Importantly, perturbation of such a boundary in hPSCs interfered with proper differentiation through deregulated distal enhancer-promoter activity. Finally, we found that germline variations affecting such boundaries are subject to purifying selection and are underrepresented in the human population. Taken together, our findings highlight the importance of developmental gene isolation through chromosomal folding structures as a mechanism to ensure their proper expression.


Assuntos
Diferenciação Celular/genética , Perfilação da Expressão Gênica/métodos , Genoma Humano/genética , Células-Tronco Embrionárias Humanas/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Elementos Reguladores de Transcrição/genética , Sítios de Ligação/genética , Western Blotting , Fator de Ligação a CCCTC/genética , Fator de Ligação a CCCTC/metabolismo , Linhagem Celular , Elementos Facilitadores Genéticos/genética , Células-Tronco Embrionárias Humanas/citologia , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Regiões Promotoras Genéticas/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de DNA/métodos
5.
Int J Mol Sci ; 22(16)2021 Aug 10.
Artigo em Inglês | MEDLINE | ID: mdl-34445302

RESUMO

With the intent to achieve the best modalities for myocardial cell therapy, different cell types are being evaluated as potent sources for differentiation into cardiomyocytes. Embryonic stem cells and induced pluripotent stem cells have great potential for future progress in the treatment of myocardial diseases. We reviewed aspects of epigenetic mechanisms that play a role in the differentiation of these cells into cardiomyocytes. Cardiomyocytes proliferate during fetal life, and after birth, they undergo permanent terminal differentiation. Upregulation of cardiac-specific genes in adults induces hypertrophy due to terminal differentiation. The repression or expression of these genes is controlled by chromatin structural and epigenetic changes. However, few studies have reviewed and analyzed the epigenetic aspects of the differentiation of embryonic stem cells and induced pluripotent stem cells into cardiac lineage cells. In this review, we focus on the current knowledge of epigenetic regulation of cardiomyocyte proliferation and differentiation from embryonic and induced pluripotent stem cells through histone modification and microRNAs, the maintenance of pluripotency, and its alteration during cardiac lineage differentiation.


Assuntos
Diferenciação Celular/genética , Epigênese Genética/fisiologia , Miócitos Cardíacos/fisiologia , Animais , Terapia Baseada em Transplante de Células e Tecidos/métodos , Células Cultivadas , Células-Tronco Embrionárias/fisiologia , Humanos , Células-Tronco Pluripotentes Induzidas/fisiologia , Medicina Regenerativa/métodos , Engenharia Tecidual/métodos
6.
Int J Mol Sci ; 22(16)2021 Aug 11.
Artigo em Inglês | MEDLINE | ID: mdl-34445348

RESUMO

The periodontal ligament is a soft connective tissue embedded between the alveolar bone and cementum, the surface hard tissue of teeth. Periodontal ligament fibroblasts (PDLF) actively express osteo/cementogenic genes, which contribute to periodontal tissue homeostasis. However, the key factors maintaining the osteo/cementogenic abilities of PDLF remain unclear. We herein demonstrated that PPARγ was expressed by in vivo periodontal ligament tissue and its distribution pattern correlated with alkaline phosphate enzyme activity. The knockdown of PPARγ markedly reduced the osteo/cementogenic abilities of PDLF in vitro, whereas PPARγ agonists exerted the opposite effects. PPARγ was required to maintain the acetylation status of H3K9 and H3K27, active chromatin markers, and the supplementation of acetyl-CoA, a donor of histone acetylation, restored PPARγ knockdown-induced decreases in the osteo/cementogenic abilities of PDLF. An RNA-seq/ChIP-seq combined analysis identified four osteogenic transcripts, RUNX2, SULF2, RCAN2, and RGMA, in the PPARγ-dependent active chromatin region marked by H3K27ac. Furthermore, RUNX2-binding sites were selectively enriched in the PPARγ-dependent active chromatin region. Collectively, these results identified PPARγ as the key transcriptional factor maintaining the osteo/cementogenic abilities of PDLF and revealed that global H3K27ac modifications play a role in the comprehensive osteo/cementogenic transcriptional alterations mediated by PPARγ.


Assuntos
Fibroblastos/fisiologia , Histonas/metabolismo , PPAR gama/fisiologia , Ligamento Periodontal/fisiologia , Acetilação , Diferenciação Celular/genética , Células Cultivadas , Cementogênese/genética , Cementogênese/fisiologia , Regulação da Expressão Gênica , Histona Acetiltransferases/metabolismo , Histonas/química , Humanos , Osteogênese/genética , Osteogênese/fisiologia , Ligamento Periodontal/citologia , Processamento de Proteína Pós-Traducional/genética
7.
Int J Mol Sci ; 22(15)2021 Jul 29.
Artigo em Inglês | MEDLINE | ID: mdl-34360910

RESUMO

Modified mRNA (modRNA)-based somatic reprogramming is an effective and safe approach that overcomes the genomic mutation risk caused by viral integrative methods. It has improved the disadvantages of conventional mRNA and has better stability and immunogenicity. The modRNA molecules encoding multiple pluripotent factors have been applied successfully in reprogramming somatic cells such as fibroblasts, mesenchymal stem cells, and amniotic fluid stem cells to generate pluripotent stem cells (iPSCs). Moreover, it also can be directly used in the terminal differentiation of stem cells and fibroblasts into functional therapeutic cells, which exhibit great promise in disease modeling, drug screening, cell transplantation therapy, and regenerative medicine. In this review, we summarized the reprogramming applications of modified mRNA in iPSC generation and therapeutic applications of functionally differentiated cells.


Assuntos
Diferenciação Celular/genética , Reprogramação Celular/genética , Células-Tronco Pluripotentes Induzidas/citologia , RNA Mensageiro/genética , Transfecção/métodos , Animais , Transdiferenciação Celular/genética , Transplante de Células/métodos , Humanos , Camundongos , Medicina Regenerativa/métodos
8.
Nat Commun ; 12(1): 4208, 2021 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-34244516

RESUMO

The transcriptional regulators underlying induction and differentiation of dense connective tissues such as tendon and related fibrocartilaginous tissues (meniscus and annulus fibrosus) remain largely unknown. Using an iterative approach informed by developmental cues and single cell RNA sequencing (scRNA-seq), we establish directed differentiation models to generate tendon and fibrocartilage cells from mouse embryonic stem cells (mESCs) by activation of TGFß and hedgehog pathways, achieving 90% induction efficiency. Transcriptional signatures of the mESC-derived cells recapitulate embryonic tendon and fibrocartilage signatures from the mouse tail. scRNA-seq further identify retinoic acid signaling as a critical regulator of cell fate switch between TGFß-induced tendon and fibrocartilage lineages. Trajectory analysis by RNA sequencing define transcriptional modules underlying tendon and fibrocartilage fate induction and identify molecules associated with lineage-specific differentiation. Finally, we successfully generate 3-dimensional engineered tissues using these differentiation protocols and show activation of mechanotransduction markers with dynamic tensile loading. These findings provide a serum-free approach to generate tendon and fibrocartilage cells and tissues at high efficiency for modeling development and disease.


Assuntos
Fibrocartilagem/crescimento & desenvolvimento , Células-Tronco Embrionárias Murinas/fisiologia , Tendões/crescimento & desenvolvimento , Engenharia Tecidual/métodos , Ativação Transcricional , Animais , Diferenciação Celular/genética , Embrião de Mamíferos , Fibrocartilagem/citologia , Regulação da Expressão Gênica no Desenvolvimento , Proteínas Hedgehog/metabolismo , Mecanotransdução Celular/genética , Camundongos , RNA-Seq , Transdução de Sinais/genética , Análise de Célula Única , Tendões/citologia , Fator de Crescimento Transformador beta/metabolismo , Tretinoína/metabolismo
9.
Int J Mol Sci ; 22(12)2021 Jun 17.
Artigo em Inglês | MEDLINE | ID: mdl-34204414

RESUMO

Background: Applying mesenchymal stem cells (MSCs), together with the distraction osteogenesis (DO) process, displayed enhanced bone quality and shorter treatment periods. The DO guides the differentiation of MSCs by providing mechanical clues. However, the underlying key genes and pathways are largely unknown. The aim of this study was to screen and identify hub genes involved in distraction-induced osteogenesis of MSCs and potential molecular mechanisms. Material and Methods: The datasets were downloaded from the ArrayExpress database. Three samples of negative control and two samples subjected to 5% cyclic sinusoidal distraction at 0.25 Hz for 6 h were selected for screening differentially expressed genes (DEGs) and then analysed via bioinformatics methods. The Gene Ontology (GO) terms and Kyoto Encyclopaedia of Genes and Genomes (KEGG) pathway enrichment were investigated. The protein-protein interaction (PPI) network was visualised through the Cytoscape software. Gene set enrichment analysis (GSEA) was conducted to verify the enrichment of a self-defined osteogenic gene sets collection and identify osteogenic hub genes. Results: Three hub genes (IL6, MMP2, and EP300) that were highly associated with distraction-induced osteogenesis of MSCs were identified via the Venn diagram. These hub genes could provide a new understanding of distraction-induced osteogenic differentiation of MSCs and serve as potential gene targets for optimising DO via targeted therapies.


Assuntos
Biologia Computacional/métodos , Perfilação da Expressão Gênica , Osteogênese/genética , Células-Tronco/citologia , Células-Tronco/metabolismo , Transcriptoma , Biomarcadores , Diferenciação Celular/genética , Perfilação da Expressão Gênica/métodos , Ontologia Genética , Redes Reguladoras de Genes , Humanos , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Mapeamento de Interação de Proteínas , Mapas de Interação de Proteínas
10.
J Immunol ; 207(3): 837-848, 2021 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-34282004

RESUMO

Dendritic cells (DCs) are critical for pathogen recognition and Ag processing/presentation. Human monocyte-derived DCs (moDCs) have been extensively used in experimental studies and DC-based immunotherapy approaches. However, the extent of human moDC and peripheral DCs heterogeneity and their interrelationship remain elusive. In this study, we performed single-cell RNA sequencing of human moDCs and blood DCs. We identified seven subtypes within moDCs: five corresponded to type 2 conventional DCs (cDC2s), and the other two were CLEC10A+CD127+ cells with no resemblance to any peripheral DC subpopulations characterized to date. Moreover, we defined five similar subtypes in human cDC2s, revealed the potential differentiation trajectory among them, and unveiled the transcriptomic differences between moDCs and cDC2s. We further studied the transcriptomic changes of each moDC subtype during maturation, demonstrating SLAMF7 and IL15RA as maturation markers and CLEC10A and SIGLEC10 as markers for immature DCs. These findings will enable more accurate functional/developmental analyses of human cDC2s and moDCs.


Assuntos
Células Dendríticas/fisiologia , Monócitos/fisiologia , Análise de Célula Única/métodos , Adulto , Diferenciação Celular/genética , Células Cultivadas , Citocinas/metabolismo , Feminino , Perfilação da Expressão Gênica , Humanos , Lectinas/genética , Lectinas Tipo C/genética , Masculino , Receptores de Superfície Celular/genética , Receptores de Interleucina-15/genética , Análise de Sequência de RNA , Família de Moléculas de Sinalização da Ativação Linfocitária/genética , Células Th2/imunologia , Adulto Jovem
11.
Nucleic Acids Res ; 49(13): 7437-7456, 2021 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-34197623

RESUMO

Despite its prominence, the mechanisms through which the tumor suppressor p53 regulates most genes remain unclear. Recently, the regulatory factor X 7 (RFX7) emerged as a suppressor of lymphoid neoplasms, but its regulation and target genes mediating tumor suppression remain unknown. Here, we identify a novel p53-RFX7 signaling axis. Integrative analysis of the RFX7 DNA binding landscape and the RFX7-regulated transcriptome in three distinct cell systems reveals that RFX7 directly controls multiple established tumor suppressors, including PDCD4, PIK3IP1, MXD4, and PNRC1, across cell types and is the missing link for their activation in response to p53 and stress. RFX7 target gene expression correlates with cell differentiation and better prognosis in numerous cancer types. Interestingly, we find that RFX7 sensitizes cells to Doxorubicin by promoting apoptosis. Together, our work establishes RFX7's role as a ubiquitous regulator of cell growth and fate determination and a key node in the p53 transcriptional program.


Assuntos
Regulação da Expressão Gênica , Redes Reguladoras de Genes , Genes Supressores de Tumor , Fatores de Transcrição de Fator Regulador X/metabolismo , Estresse Fisiológico/genética , Proteína Supressora de Tumor p53/metabolismo , Animais , Antibióticos Antineoplásicos/farmacologia , Apoptose , Diferenciação Celular/genética , Linhagem Celular Tumoral , DNA/metabolismo , Doxorrubicina/farmacologia , Humanos , Camundongos , Neoplasias/genética , Neoplasias/mortalidade , Prognóstico , Regiões Promotoras Genéticas , Fatores de Transcrição de Fator Regulador X/fisiologia , Transdução de Sinais , Transativadores/metabolismo , Transcriptoma
12.
Int J Mol Sci ; 22(13)2021 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-34203569

RESUMO

Propolis is a honeybee product with various biological activities, including antidiabetic effects. We previously reported that artepillin C, a prenylated cinnamic acid derivative isolated from Brazilian green propolis, acts as a peroxisome proliferator-activated receptor γ (PPARγ) ligand and promotes adipocyte differentiation. In this study, we examined the effect of baccharin, another major component of Brazilian green propolis, on adipocyte differentiation. The treatment of mouse 3T3-L1 preadipocytes with baccharin resulted in increased lipid accumulation, cellular triglyceride levels, glycerol-3-phosphate dehydrogenase activity, and glucose uptake. The mRNA expression levels of PPARγ and its target genes were also increased by baccharin treatment. Furthermore, baccharin enhanced PPARγ-dependent luciferase activity, suggesting that baccharin promotes adipocyte differentiation via PPARγ activation. In diabetic ob/ob mice, intraperitoneal administration of 50 mg/kg baccharin significantly improved blood glucose levels. Our results suggest that baccharin has a hypoglycemic effect on glucose metabolic disorders, such as type 2 diabetes mellitus.


Assuntos
Adipócitos/metabolismo , Hiperglicemia/metabolismo , Própole/química , Animais , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Glicerolfosfato Desidrogenase/genética , Glicerolfosfato Desidrogenase/metabolismo , Hiperglicemia/genética , Camundongos
13.
Nat Commun ; 12(1): 4640, 2021 07 30.
Artigo em Inglês | MEDLINE | ID: mdl-34330896

RESUMO

Cranial sutures are major growth centers for the calvarial vault, and their premature fusion leads to a pathologic condition called craniosynostosis. This study investigates whether skeletal stem/progenitor cells are resident in the cranial sutures. Prospective isolation by FACS identifies this population with a significant difference in spatio-temporal representation between fusing versus patent sutures. Transcriptomic analysis highlights a distinct signature in cells derived from the physiological closing PF suture, and scRNA sequencing identifies transcriptional heterogeneity among sutures. Wnt-signaling activation increases skeletal stem/progenitor cells in sutures, whereas its inhibition decreases. Crossing Axin2LacZ/+ mouse, endowing enhanced Wnt activation, to a Twist1+/- mouse model of coronal craniosynostosis enriches skeletal stem/progenitor cells in sutures restoring patency. Co-transplantation of these cells with Wnt3a prevents resynostosis following suturectomy in Twist1+/- mice. Our study reveals that decrease and/or imbalance of skeletal stem/progenitor cells representation within sutures may underlie craniosynostosis. These findings have translational implications toward therapeutic approaches for craniosynostosis.


Assuntos
Suturas Cranianas/metabolismo , Craniossinostoses/genética , Modelos Animais de Doenças , Perfilação da Expressão Gênica/métodos , Células-Tronco/metabolismo , Animais , Proteína Axina/genética , Proteína Axina/metabolismo , Diferenciação Celular/genética , Proliferação de Células/genética , Células Cultivadas , Suturas Cranianas/citologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Sistema Musculoesquelético/citologia , Sistema Musculoesquelético/metabolismo , Células-Tronco/citologia , Proteína 1 Relacionada a Twist/genética , Proteína 1 Relacionada a Twist/metabolismo , Via de Sinalização Wnt/genética , Proteína Wnt3A/genética , Proteína Wnt3A/metabolismo
14.
Nat Genet ; 53(8): 1221-1232, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34294917

RESUMO

Driver mutations in genes encoding histone H3 proteins resulting in p.Lys27Met substitutions (H3-K27M) are frequent in pediatric midline brain tumors. However, the precise mechanisms by which H3-K27M causes tumor initiation remain unclear. Here, we use human hindbrain neural stem cells to model the consequences of H3.3-K27M on the epigenomic landscape in a relevant developmental context. Genome-wide mapping of epitope-tagged histone H3.3 revealed that both the wild type and the K27M mutant incorporate abundantly at pre-existing active enhancers and promoters, and to a lesser extent at Polycomb repressive complex 2 (PRC2)-bound regions. At active enhancers, H3.3-K27M leads to focal H3K27ac loss, decreased chromatin accessibility and reduced transcriptional expression of nearby neurodevelopmental genes. In addition, H3.3-K27M deposition at a subset of PRC2 target genes leads to increased PRC2 and PRC1 binding and augmented transcriptional repression that can be partially reversed by PRC2 inhibitors. Our work suggests that, rather than imposing de novo transcriptional circuits, H3.3-K27M drives tumorigenesis by locking initiating cells in their pre-existing, immature epigenomic state, via disruption of PRC2 and enhancer functions.


Assuntos
Elementos Facilitadores Genéticos , Histonas/metabolismo , Células-Tronco Neurais/fisiologia , Complexo Repressor Polycomb 2/genética , Rombencéfalo/citologia , Animais , Neoplasias Encefálicas/genética , Diferenciação Celular/genética , Linhagem Celular , Proteína Potenciadora do Homólogo 2 de Zeste/genética , Epigenoma , Regulação da Expressão Gênica no Desenvolvimento , Glioma/genética , Histonas/genética , Humanos , Lisina/metabolismo , Masculino , Camundongos Endogâmicos , Mutação , Células-Tronco Neurais/transplante , Oncogenes , Complexo Repressor Polycomb 2/antagonistas & inibidores , Complexo Repressor Polycomb 2/metabolismo , Regiões Promotoras Genéticas , Rombencéfalo/fisiologia
15.
Methods Mol Biol ; 2352: 1-12, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34324176

RESUMO

Forced expression of specific neuronal transcription factors in mouse embryonic fibroblasts (MEFs) can lead to their direct conversion into functional neurons. Direct neuronal reprogramming has become a powerful tool to characterize individual factors and molecular mechanisms involved in forced and normal neurogenesis and to generate neuronal cell types for in vitro studies. Here we provide a detailed protocol for the isolation of MEFs devoid of neural tissue and their direct reprogramming into functional neurons by overexpression of neuronal reprogramming factors (Ascl1, Brn2, and Myt1l) using lentiviral vectors. This method enables quick and efficient generation of mouse neurons in vitro for versatile functional and mechanistic characterization.


Assuntos
Separação Celular/métodos , Técnicas de Reprogramação Celular , Reprogramação Celular , Fibroblastos/citologia , Fibroblastos/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Animais , Técnicas de Cultura de Células , Diferenciação Celular/genética , Reprogramação Celular/genética , Criopreservação , Vetores Genéticos/biossíntese , Vetores Genéticos/genética , Humanos , Camundongos , Neurogênese/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transdução Genética
16.
Methods Mol Biol ; 2352: 13-29, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34324177

RESUMO

Spontaneous neuronal replacement is almost absent in the postnatal mammalian nervous system. However, several studies have shown that both early postnatal and adult astroglia can be reprogrammed in vitro or in vivo by forced expression of proneural transcription factors, such as Neurogenin-2 or Achaete-scute homolog 1 (Ascl1), to acquire a neuronal fate. The reprogramming process stably induces properties such as distinctly neuronal morphology, expression of neuron-specific proteins, and the gain of mature neuronal functional features. Direct conversion of astroglia into neurons thus possesses potential as a basis for cell-based strategies against neurological diseases. In this chapter, we describe a well-established protocol used for direct reprogramming of postnatal cortical astrocytes into functional neurons in vitro and discuss available tools and approaches to dissect molecular and cell biological mechanisms underlying the reprogramming process.


Assuntos
Astrócitos/citologia , Astrócitos/metabolismo , Reprogramação Celular , Neurônios/citologia , Neurônios/metabolismo , Animais , Diferenciação Celular/genética , Separação Celular/métodos , Células Cultivadas , Reprogramação Celular/genética , Camundongos , Neocórtex/citologia , Neuroglia/citologia , Neuroglia/metabolismo , Cultura Primária de Células , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
17.
Methods Mol Biol ; 2352: 45-55, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34324179

RESUMO

Astrocytes play important roles in neurodevelopment and diseases. Previous studies described ways to derive astrocytes from somatic cells by going through iPSC or iNSC/iNPC intermediates. Here we describe a method to directly convert mouse fibroblasts into functional astrocytes using small molecules without transgenes or viral transduction. The direct chemical reprogramming method described in this study provides a more rapid way to derive astrocytes from fibroblasts.


Assuntos
Astrócitos/citologia , Astrócitos/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Técnicas de Reprogramação Celular , Reprogramação Celular/efeitos dos fármacos , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Animais , Astrócitos/metabolismo , Biomarcadores , Diferenciação Celular/genética , Separação Celular , Células Cultivadas , Reprogramação Celular/genética , Fibroblastos/metabolismo , Expressão Gênica , Humanos , Imuno-Histoquímica , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/metabolismo , Camundongos , Fatores de Transcrição/genética , Transgenes
18.
Methods Mol Biol ; 2352: 97-115, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34324182

RESUMO

Since the first demonstration of direct dopaminergic neuronal reprogramming, over a dozen methods have been developed to generate induced dopaminergic neurons from various sources of cells. Here, we first present an overview of the different methods to generate induced neurons of a generic type and of different subtypes, with a particular focus on induced dopaminergic neurons generated from human fibroblasts. We then describe a protocol to generate induced dopaminergic neurons from commercially available human fetal lung fibroblasts. These cells could serve for various biomedical application, including regenerative medicine for conditions such as Parkinson's disease.


Assuntos
Transdiferenciação Celular , Técnicas de Reprogramação Celular , Reprogramação Celular , Neurônios Dopaminérgicos/citologia , Neurônios Dopaminérgicos/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Diferenciação Celular/genética , Linhagem Celular , Células Cultivadas , Expressão Gênica , Vetores Genéticos/administração & dosagem , Vetores Genéticos/genética , Humanos , Lentivirus/genética , Fatores de Transcrição/genética
19.
Methods Mol Biol ; 2352: 127-132, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34324184

RESUMO

Human motor neurons are important materials for the research of the pathogenesis and drug discovery of motor neuron diseases. Various methods to generate motor neurons (MNs) from embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs) by the addition of signaling molecules have been reported. However, they require multiple steps and complicated processes. Here we describe an approach for generating human MNs from ESCs/iPSCs using a single Sendai virus vector encoding three transcription factors-Lhx3, Ngn2, and Isl1. This approach enabled us to generate MNs in one step, adding Sendai virus vector in culture medium. This simple method significantly reduces the efforts to generate MNs, and it provides a useful tool for motor neuron disease research.


Assuntos
Diferenciação Celular , Células-Tronco Embrionárias/citologia , Vetores Genéticos , Células-Tronco Pluripotentes Induzidas/citologia , Neurônios Motores/citologia , Vírus Sendai , Diferenciação Celular/genética , Linhagem Celular , Células-Tronco Embrionárias/metabolismo , Expressão Gênica , Vetores Genéticos/genética , Humanos , Imuno-Histoquímica , Células-Tronco Pluripotentes Induzidas/metabolismo , Neurônios Motores/metabolismo , Vírus Sendai/genética , Fatores de Transcrição/genética , Transgenes
20.
Methods Mol Biol ; 2352: 149-170, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34324186

RESUMO

Oligodendrocytes are the main glial cell type in the central nervous system supporting the axonal part of neurons via myelin and lactate delivery. Both the conductive myelin formation and the energy support via lactate can be affected in diseases, such as multiple sclerosis and amyotrophic lateral sclerosis, respectively. Therefore, human disease modeling is needed to gain more mechanistic insights to drive drug discovery research. Here, patient-derived induced pluripotent stem cells (iPSCs) serve as a necessary tool providing an infinite cell source for patient-specific disease modeling, which allows investigation of oligodendrocyte involvement in human disease.Small molecule-based differentiation protocols to generate oligodendrocytes from pluripotent stem cells can last more than 90 days. Here, we provide a transcription factor-based, fast and efficient protocol for generating O4+ oligodendrocytes in just 20-24 days. After a neural induction phase of 8-12 days, SOX10 is overexpressed either with the use of lentiviral vectors or via engineered iPSCs, which inducibly overexpress SOX10 after doxycycline addition. Using this last method, a pure O4+ cell population is achieved after keeping the SOX10-overexpressing neural stem cells in culture for an additional 10 days. Furthermore, these O4+ cells can be co-cultured with iPSC-derived cortical neurons in 384-well format, allowing pro-myelinating drug screens. In conclusion, we provide a fast and efficient oligodendrocyte differentiation protocol allowing both in vitro human disease modeling and a high-throughput co-culture system for drug discovery.


Assuntos
Diferenciação Celular/genética , Expressão Gênica , Oligodendroglia/citologia , Oligodendroglia/metabolismo , Fatores de Transcrição SOXE/genética , Fatores de Transcrição/genética , Técnicas de Cultura de Células , Células Cultivadas , Clonagem Molecular , Ordem dos Genes , Vetores Genéticos/administração & dosagem , Vetores Genéticos/biossíntese , Vetores Genéticos/genética , Humanos , Separação Imunomagnética , Lentivirus/genética , Neurogênese
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