RESUMO
Extracellular matrix (ECM) stiffness is a major driver of stem cell fate. However, the involvement of the three-dimensional (3D) genomic reorganization in response to ECM stiffness remains unclear. Here, we generated comprehensive 3D chromatin landscapes of mesenchymal stem cells (MSCs) exposed to various ECM stiffness. We found that there were more long-range chromatin interactions, but less compartment A in MSCs cultured on stiff ECM than those cultured on soft ECM. However, the switch from compartment B in MSCs cultured on soft ECM to compartment A in MSCs cultured on stiff ECM included genes encoding proteins primarily enriched in cytoskeleton organization. At the topologically associating domains (TADs) level, stiff ECM tends to have merged TADs on soft ECM. These merged TADs on stiff ECM include upregulated genes encoding proteins enriched in osteogenesis, such as SP1, ETS1, and DCHS1, which were validated by quantitative real-time polymerase chain reaction and found to be consistent with the increase of alkaline phosphatase staining. Knockdown of SP1 or ETS1 led to the downregulation of osteogenic marker genes, including COL1A1, RUNX2, ALP, and OCN in MSCs cultured on stiff ECM. Our study provides an important insight into the stiff ECM-mediated promotion of MSC differentiation towards osteogenesis, emphasizing the influence of mechanical cues on the reorganization of 3D genome architecture and stem cell fate.
Assuntos
Diferenciação Celular , Matriz Extracelular , Células-Tronco Mesenquimais , Osteogênese , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Osteogênese/genética , Matriz Extracelular/metabolismo , Diferenciação Celular/genética , Humanos , Células Cultivadas , AnimaisRESUMO
The primed epiblast acts as a transitional stage between the relatively homogeneous naïve epiblast and the gastrulating embryo. Its formation entails coordinated changes in regulatory circuits driven by transcription factors and epigenetic modifications. Using a multi-omic approach in human embryonic stem cell models across the spectrum of peri-implantation development, we demonstrate that the transcription factors ZIC2 and ZIC3 have overlapping but essential roles in opening primed-specific enhancers. Together, they are essential to facilitate progression to and maintain primed pluripotency. ZIC2/3 accomplish this by recruiting SWI/SNF to chromatin and loss of ZIC2/3 or degradation of SWI/SNF both prevent enhancer activation. Loss of ZIC2/3 also results in transcriptome changes consistent with perturbed Polycomb activity and a shift towards the expression of genes linked to differentiation towards the mesendoderm. Additionally, we find an intriguing dependency on the transcriptional machinery for sustained recruitment of ZIC2/3 over a subset of primed-hESC specific enhancers. Taken together, ZIC2 and ZIC3 regulate highly dynamic lineage-specific enhancers and collectively act as key regulators of human primed pluripotency.
Assuntos
Diferenciação Celular , Proteínas de Homeodomínio , Células-Tronco Embrionárias Humanas , Células-Tronco Pluripotentes , Fatores de Transcrição , Humanos , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Proteínas de Homeodomínio/metabolismo , Proteínas de Homeodomínio/genética , Diferenciação Celular/genética , Células-Tronco Pluripotentes/metabolismo , Células-Tronco Pluripotentes/citologia , Células-Tronco Embrionárias Humanas/metabolismo , Células-Tronco Embrionárias Humanas/citologia , Camadas Germinativas/metabolismo , Camadas Germinativas/citologia , Elementos Facilitadores Genéticos , Proteínas Cromossômicas não Histona/metabolismo , Proteínas Cromossômicas não Histona/genética , Regulação da Expressão Gênica no Desenvolvimento , Cromatina/metabolismo , Proteínas NuclearesRESUMO
Human induced pluripotent stem cells (iPSCs) have great potential in research, but pluripotency testing faces challenges due to non-standardized methods and ambiguous markers. Here, we use long-read nanopore transcriptome sequencing to discover 172 genes linked to cell states not covered by current guidelines. We validate 12 genes by qPCR as unique markers for specific cell fates: pluripotency (CNMD, NANOG, SPP1), endoderm (CER1, EOMES, GATA6), mesoderm (APLNR, HAND1, HOXB7), and ectoderm (HES5, PAMR1, PAX6). Using these genes, we develop a machine learning-based scoring system, "hiPSCore", trained on 15 iPSC lines and validated on 10 more. hiPSCore accurately classifies pluripotent and differentiated cells and predicts their potential to become specialized 2D cells and 3D organoids. Our re-evaluation of cell fate marker genes identifies key targets for future studies on cell fate assessment. hiPSCore improves iPSC testing by reducing time, subjectivity, and resource use, thus enhancing iPSC quality for scientific and medical applications.
Assuntos
Diferenciação Celular , Células-Tronco Pluripotentes Induzidas , Controle de Qualidade , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/citologia , Humanos , Diferenciação Celular/genética , Biomarcadores/metabolismo , Aprendizado de Máquina , Endoderma/citologia , Endoderma/metabolismo , Transcriptoma , Mesoderma/metabolismo , Mesoderma/citologia , Linhagem Celular , Ectoderma/metabolismo , Ectoderma/citologia , Organoides/metabolismo , Perfilação da Expressão Gênica/métodos , Proteína Homeobox Nanog/genética , Proteína Homeobox Nanog/metabolismo , Proteínas com Domínio TRESUMO
White adipose tissue (WAT) is essential for lipid storage and systemic energy homeostasis. Understanding adipocyte formation and stability is key to developing therapies for obesity and metabolic disorders. Through a high-throughput cDNA screen, we identified PATZ1, a POZ/BTB and AT-Hook Containing Zinc Finger 1 protein, as an important adipogenic transcription factor. PATZ1 is expressed in human and mouse adipocyte precursor cells (APCs) and adipocytes. In cellular models, PATZ1 promotes adipogenesis via protein-protein interactions and DNA binding. PATZ1 ablation in mouse adipocytes and APCs leads to a reduced APC pool, decreased fat mass, and hypertrophied adipocytes. ChIP-Seq and RNA-seq analyses show that PATZ1 supports adipogenesis by interacting with transcriptional machinery at the promoter regions of key early adipogenic factors. Mass-spec results show that PATZ1 associates with GTF2I, with GTF2I modulating PATZ1's function during differentiation. These findings underscore PATZ1's regulatory role in adipocyte differentiation and adiposity, offering insights into adipose tissue development.
Assuntos
Adipócitos , Adipogenia , Regiões Promotoras Genéticas , Fatores de Transcrição , Adipogenia/genética , Animais , Camundongos , Humanos , Adipócitos/metabolismo , Adipócitos/citologia , Regiões Promotoras Genéticas/genética , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Diferenciação Celular/genética , Tecido Adiposo Branco/metabolismo , Tecido Adiposo Branco/citologia , Masculino , Células 3T3-L1 , Camundongos Endogâmicos C57BL , Regulação da Expressão GênicaRESUMO
Primary cultures of proximal tubule cells are widely used to model the behavior of kidney epithelial cells in vitro. However, de-differentiation of primary cells upon culture has been observed and appreciated for decades, yet the mechanisms driving this phenomenon remain poorly understood. This confounds the interpretation of experiments using primary kidney epithelial cells and prevents their use to engineer functional kidney tissue ex vivo. In this report, we measure the dynamics of cell-state transformations in early primary culture of mouse proximal tubules to identify key pathways and processes that correlate with and may drive de-differentiation. Our data show that the loss of proximal-tubule-specific genes is rapid, uniform, and sustained even after confluent, polarized epithelial monolayers develop. This de-differentiation occurs uniformly across many common culture condition variations. Changes in early culture were strongly associated with the loss of HNF4A. Exogenous re-expression of HNF4A can promote expression of a subset of proximal tubule genes in a de-differentiated proximal tubule cell line. Using genetically labeled proximal tubule cells, we show that selective pressures very early in culture influence which cells grow to confluence. Together, these data indicate that the loss of in vivo function in proximal tubule cultures occurs very early and suggest that the sustained loss of HNF4A is a key regulatory event mediating this change.
Assuntos
Fator 4 Nuclear de Hepatócito , Túbulos Renais Proximais , Animais , Túbulos Renais Proximais/metabolismo , Túbulos Renais Proximais/citologia , Fator 4 Nuclear de Hepatócito/genética , Fator 4 Nuclear de Hepatócito/metabolismo , Camundongos , Células Epiteliais/metabolismo , Células Cultivadas , Diferenciação Celular/genética , Cultura Primária de Células , Regulação da Expressão Gênica , Linhagem CelularRESUMO
This study aimed to investigate the effects of Shuanglongjiegu pill (SLJGP) on the osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) and explore its mechanism based on miR-217/RUNX2 axis. Results found that drug-containing serum of SLJGP promoted BMSCs viability with a dose-dependent effect. Under osteogenic differentiation conditions, SLJGP promoted the expression of ALP, OPN, BMP2, RUNX2, and the osteogenic differentiation ability of BMSCs. In addition, SLJGP significantly reduced miR-217 expression, and miR-217 directly targeted RUNX2. After treatment with miR-217 mimic, the promoting effects of SLJGP on proliferation and osteogenic differentiation of BMSCs were significantly inhibited. MiR-217 mimic co-treated with pcDNA-RUNX2 further confirmed that the miR-217/RUNX2 axis was involved in SLJGP to promote osteogenic differentiation of BMSCs. In addition, analysis of Wnt/ß-catenin pathway protein expression showed that SLJGP activated the Wnt/ß-catenin pathway through miR-217/RUNX2. In conclusion, SLJGP promoted osteogenic differentiation of BMSCs by regulating miR-217/RUNX2 axis and activating Wnt/ß-catenin pathway.
Assuntos
Diferenciação Celular , Subunidade alfa 1 de Fator de Ligação ao Core , Medicamentos de Ervas Chinesas , Células-Tronco Mesenquimais , MicroRNAs , Osteogênese , Via de Sinalização Wnt , MicroRNAs/genética , MicroRNAs/metabolismo , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Osteogênese/efeitos dos fármacos , Osteogênese/genética , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Via de Sinalização Wnt/efeitos dos fármacos , Via de Sinalização Wnt/genética , Via de Sinalização Wnt/fisiologia , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Medicamentos de Ervas Chinesas/farmacologia , Células Cultivadas , Humanos , Animais , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/metabolismo , Proliferação de Células/efeitos dos fármacosRESUMO
BACKGROUND: Adipose tissue affects not only the meat quality of domestic animals, but also human health. Adipocyte differentiation is regulated by a series of regulatory genes and cyclins. Four and half-LIM protein (FHL2) is positively correlated with the hypertrophy of adipocytes and can cause symptoms such as obesity and diabetes. RESULT: In the transcriptome sequencing analysis of intramuscular adipocytes after three days of differentiation, the differentially expressed gene FHL2 was found. To further explore the biological significance of the differentially expressed gene FHL2, which was downregulated in the mature adipocytes. We revealed the function of FHL2 in adipogenesis through the acquisition and loss of function of FHL2. The results showed that the overexpression of FHL2 significantly increased the expression of adipogenic genes (PPARγ, C/EBPß) and the differentiation of intramuscular and subcutaneous adipocytes. However, silencing FHL2 significantly inhibited adipocyte differentiation. The overexpression of FHL2 increased the number of adipocytes stained with crystal violet and increased the mRNA expression of proliferation marker genes such as CCNE, PCNA, CCND and CDK2. In addition, it significantly increased the rate of EdU positive cells. In terms of apoptosis, overexpression of FHL2 significantly inhibited the expression of P53 and BAX in both intramuscular and subcutaneous adipocytes, which are involved in cell apoptosis. However, overexpression of FHL2 promoted the expression of BCL, but was rescued by the silencing of FHL2. CONCLUSIONS: In summary, FHL2 may be a positive regulator of intramuscular and subcutaneous adipocyte differentiation and proliferation, and acts as a negative regulator of intramuscular and subcutaneous adipocyte apoptosis. These findings provide a theoretical basis for the subsequent elucidation of FHL2 in adipocytes.
Assuntos
Adipócitos , Adipogenia , Cabras , Proteínas com Homeodomínio LIM , Proteínas Musculares , Animais , Cabras/genética , Adipócitos/metabolismo , Adipócitos/citologia , Adipogenia/genética , Proteínas com Homeodomínio LIM/genética , Proteínas com Homeodomínio LIM/metabolismo , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Apoptose/genética , Diferenciação Celular/genética , Proliferação de Células , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Gordura Subcutânea/metabolismo , Gordura Subcutânea/citologia , Perfilação da Expressão GênicaRESUMO
Naïve pluripotency is sustained by a self-reinforcing gene regulatory network (GRN) comprising core and naïve pluripotency-specific transcription factors (TFs). Upon exiting naïve pluripotency, embryonic stem cells (ESCs) transition through a formative post-implantation-like pluripotent state, where they acquire competence for lineage choice. However, the mechanisms underlying disengagement from the naïve GRN and initiation of the formative GRN are unclear. Here, we demonstrate that phosphorylated AKT acts as a gatekeeper that prevents nuclear localisation of FoxO TFs in naïve ESCs. PTEN-mediated reduction of AKT activity upon exit from naïve pluripotency allows nuclear entry of FoxO TFs, enforcing a cell fate transition by binding and activating formative pluripotency-specific enhancers. Indeed, FoxO TFs are necessary and sufficient for the activation of the formative pluripotency-specific GRN. Our work uncovers a pivotal role for FoxO TFs in establishing formative post-implantation pluripotency, a critical early embryonic cell fate transition.
Assuntos
Redes Reguladoras de Genes , Células-Tronco Pluripotentes , Animais , Camundongos , Células-Tronco Pluripotentes/metabolismo , Diferenciação Celular/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , PTEN Fosfo-Hidrolase/metabolismo , PTEN Fosfo-Hidrolase/genética , Fatores de Transcrição Forkhead/metabolismo , Fatores de Transcrição Forkhead/genética , Proteína Forkhead Box O1/metabolismo , Proteína Forkhead Box O1/genética , Fosforilação , Células-Tronco Embrionárias Murinas/metabolismo , Células-Tronco Embrionárias/metabolismo , Regulação da Expressão Gênica no DesenvolvimentoRESUMO
Tissue-resident memory CD8+ T cells serve as a first-line defense against many pathogens. In this issue of Immunity, Buquicchio et al. unveil the epigenomic landscapes of virus-specific CD8+ T cell subsets, highlighting common and organ-specific regulators driving their differentiation.
Assuntos
Linfócitos T CD8-Positivos , Epigenômica , Memória Imunológica , Memória Imunológica/imunologia , Memória Imunológica/genética , Humanos , Linfócitos T CD8-Positivos/imunologia , Animais , Epigênese Genética/imunologia , Diferenciação Celular/imunologia , Diferenciação Celular/genética , Especificidade de Órgãos/imunologia , Especificidade de Órgãos/genéticaRESUMO
Glioblastoma remains one of the deadliest brain malignancies. First-line therapy consists of maximal surgical tumor resection, accompanied by chemotherapy and radiotherapy. Malignant cells escape surgical resection by migrating into the surrounding healthy brain tissue, where they give rise to the recurrent tumor. Based on gene expression, tumor cores can be subtyped into mesenchymal, proneural, and classical tumors, each being associated with differences in genetic alterations and cellular composition. In contrast, the adjacent brain parenchyma where infiltrating malignant cells escape surgical resection is less characterized in patients. Using spatial transcriptomics (n = 11), we show that malignant cells within proneural or mesenchymal tumor cores display spatially organized differences in gene expression, although such differences decrease within the infiltrated brain tissue. Malignant cells residing in infiltrated brain tissue have increased expression of genes related to neurodevelopmental pathways and glial cell differentiation. Our findings provide an updated view of the spatial landscape of glioblastomas and further our understanding of the malignant cells that infiltrate the healthy brain, providing new avenues for the targeted therapy of these cells after surgical resection.
Assuntos
Neoplasias Encefálicas , Encéfalo , Regulação Neoplásica da Expressão Gênica , Glioblastoma , Receptores Notch , Transdução de Sinais , Humanos , Glioblastoma/genética , Glioblastoma/patologia , Glioblastoma/metabolismo , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Neoplasias Encefálicas/metabolismo , Receptores Notch/metabolismo , Receptores Notch/genética , Encéfalo/metabolismo , Encéfalo/patologia , Transcriptoma , Sinapses/metabolismo , Masculino , Feminino , Linhagem Celular Tumoral , Neuroglia/metabolismo , Neuroglia/patologia , Diferenciação Celular/genéticaRESUMO
Cardiac biological pacing (BP) is one of the future directions for bradyarrhythmias intervention. Currently, cardiac pacemaker cells (PCs) used for cardiac BP are mainly derived from pluripotent stem cells (PSCs). However, the production of high-quality cardiac PCs from PSCs remains a challenge. Here, we developed a cardiac PC differentiation strategy by adopting dual PC markers and simulating the developmental route of PCs. First, two PC markers, Shox2 and Hcn4, were selected to establish Shox2:EGFP; Hcn4:mCherry mouse PSC reporter line. Then, by stepwise guiding naïve PSCs to cardiac PCs following naïve to formative pluripotency transition and manipulating signaling pathways during cardiac PCs differentiation, we designed the FSK method that increased the yield of SHOX2+; HCN4+ cells with typical PC characteristics, which was 12 and 42 folds higher than that of the embryoid body (EB) and the monolayer M10 methods respectively. In addition, the in vitro cardiac PCs differentiation trajectory was mapped by single-cell RNA sequencing (scRNA-seq), which resembled in vivo PCs development, and ZFP503 was verified as a key regulator of cardiac PCs differentiation. These PSC-derived cardiac PCs have the potential to drive advances in cardiac BP technology, help with the understanding of PCs (patho)physiology, and benefit drug discovery for PC-related diseases as well.
Assuntos
Diferenciação Celular , Miócitos Cardíacos , Células-Tronco Pluripotentes , Animais , Camundongos , Diferenciação Celular/genética , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/citologia , Células-Tronco Pluripotentes/metabolismo , Células-Tronco Pluripotentes/citologia , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/genética , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/metabolismo , Corpos Embrioides/citologia , Corpos Embrioides/metabolismoRESUMO
Rationale: Regulatory processes of transcription factors (TFs) shape heart development and influence the adult heart's response to stress, contributing to cardiac disorders. Despite their significance, the precise mechanisms underpinning TF-mediated regulation remain elusive. Here, we identify that EBF1, as a TF, is highly expressed in human heart tissues. EBF1 is reported to be associated with human cardiovascular disease, but its roles are unclear in heart. In this study, we investigated EBF1 function in cardiac system. Methods: RNA-seq was utilized to profile EBF1 expression patterns. CRISPR/Cas9 was utilized to knock out EBF1 to investigate its effects. Human pluripotent stem cells (hPSCs) differentiated into cardiac lineages were used to mimic cardiac development. Cardiac function was evaluated on mouse model with Ebf1 knockout by using techniques such as echocardiography. RNA-seq was conducted to analyze transcriptional perturbations. ChIP-seq was employed to elucidate EBF1-bound genes and the underlying regulatory mechanisms. Results: EBF1 was expressed in some human and mouse cardiomyocyte. Knockout of EBF1 inhibited cardiac development. ChIP-seq indicated EBF1's binding on promoters of cardiogenic TFs pivotal to cardiac development, facilitating their transcriptional expression and promoting cardiac development. In mouse, Ebf1 depletion triggered transcriptional perturbations of genes, resulting in cardiac remodeling. Mechanistically, we found that EBF1 directly bound to upstream chromatin regions of cardiac hypertrophy-inducing genes, contributing to cardiac hypertrophy. Conclusions: We uncover the mechanisms underlying EBF1-mediated regulatory processes, shedding light on cardiac development, and the pathogenesis of cardiac remodeling. These findings emphasize EBF1's critical role in orchestrating diverse aspects of cardiac processes and provide a promising therapeutic intervention for cardiomyopathy.
Assuntos
Perfilação da Expressão Gênica , Miócitos Cardíacos , Transativadores , Animais , Humanos , Camundongos , Transativadores/genética , Transativadores/metabolismo , Miócitos Cardíacos/metabolismo , Diferenciação Celular/genética , Coração/fisiopatologia , Camundongos Knockout , Células-Tronco Pluripotentes/metabolismo , Transcriptoma/genética , Sistemas CRISPR-Cas/genéticaRESUMO
Dedifferentiated liposarcoma (DDLPS) is the most frequent high-grade soft tissue sarcoma subtype. It is characterized by a component of undifferentiated tumor cells coexisting with a component of well-differentiated adipocytic tumor cells. Both dedifferentiated (DD) and well-differentiated (WD) components exhibit MDM2 amplification, however their cellular origin remains elusive. Using single-cell RNA sequencing, DNA sequencing, in situ multiplex immunofluorescence and functional assays in paired WD and DD components from primary DDLPS tumors, we characterize the cellular heterogeneity of DDLPS tumor and micro-environment. We identify a population of tumor adipocyte stem cells (ASC) showing striking similarities with adipocyte stromal progenitors found in white adipose tissue. We show that tumor ASC harbor the ancestral genomic alterations of WD and DD components, suggesting that both derive from these progenitors following clonal evolution. Last, we show that DD tumor cells keep important biological properties of ASC including pluripotency and that their adipogenic properties are inhibited by a TGF-ß-high immunosuppressive tumor micro-environment.
Assuntos
Adipócitos , Evolução Clonal , Lipossarcoma , Proteínas Proto-Oncogênicas c-mdm2 , Microambiente Tumoral , Humanos , Lipossarcoma/genética , Lipossarcoma/patologia , Lipossarcoma/metabolismo , Adipócitos/patologia , Adipócitos/metabolismo , Microambiente Tumoral/genética , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Proteínas Proto-Oncogênicas c-mdm2/genética , Células-Tronco Neoplásicas/patologia , Células-Tronco Neoplásicas/metabolismo , Análise de Célula Única , Feminino , Desdiferenciação Celular/genética , Masculino , Diferenciação Celular/genética , Fator de Crescimento Transformador beta/metabolismo , Pessoa de Meia-Idade , IdosoRESUMO
Long known as the site of ribosome biogenesis, the nucleolus is increasingly recognized for its role in shaping three-dimensional (3D) genome organization. Still, the mechanisms governing the targeting of selected regions of the genome to nucleolus-associated domains (NADs) remain enigmatic. Here, we reveal the essential role of ZNF274, a SCAN-bearing member of the Krüppel-associated box (KRAB)-containing zinc finger protein (KZFP) family, in sequestering lineage-specific gene clusters within NADs. Ablation of ZNF274 triggers transcriptional activation across entire genomic neighborhoods-encompassing, among others, protocadherin and KZFP-encoding genes-with loss of repressive chromatin marks, altered the 3D genome architecture and de novo CTCF binding. Mechanistically, ZNF274 anchors target DNA sequences at the nucleolus and facilitates their compartmentalization via a previously uncharted function of the SCAN domain. Our findings illuminate the mechanisms underlying NAD organization and suggest that perinucleolar entrapment into repressive hubs constrains the activation of tandemly arrayed genes to enable selective expression and modulate cell differentiation programs during development.
Assuntos
Nucléolo Celular , Família Multigênica , Nucléolo Celular/metabolismo , Nucléolo Celular/genética , Animais , Humanos , Camundongos , Fator de Ligação a CCCTC/metabolismo , Fator de Ligação a CCCTC/genética , Cromatina/metabolismo , Cromatina/genética , Linhagem da Célula/genética , Dedos de Zinco/genética , Diferenciação Celular/genética , Ligação ProteicaRESUMO
Age-related osteoporosis is a prevalent bone metabolic disorder distinguished by an aberration in the equilibrium between bone formation and resorption. The reduction in the stemness of Bone Marrow Mesenchymal Stem Cells (BMSCs) plays a pivotal role in the onset of this ailment. Comprehending the molecular pathways that govern BMSCs stemness is imperative for delineating the etiology of age-related osteoporosis and devising efficacious treatment modalities. The study utilized single-cell RNA sequencing and miRNA sequencing to investigate the cellular heterogeneity and stemness of BMSCs. Through dual-luciferase reporter assays and functional experiments, the regulatory effect of miR-183 on CTNNB1 (ß-catenin) was confirmed. Overexpression and knockdown studies were conducted to explore the impact of miR-183 and ß-catenin on stemness-related transcription factors Oct4, Nanog, and Sox2. Cell proliferation assays and osteogenic differentiation experiments were carried out to validate the influence of miR-183 and ß-catenin on the stemness properties of BMSCs. Single-cell analysis revealed that ß-catenin is highly expressed in both high stemness clusters and terminal differentiation clusters of BMSCs. Overexpression of ß-catenin upregulated stemness transcription factors, while its suppression had the opposite effect, indicating a dual regulatory role of ß-catenin in maintaining BMSCs stemness and promoting bone differentiation. Furthermore, the confluence of miRNA sequencing analyses and predictions from online databases revealed miR-183 as a potential modulator of BMSCs stemness and a novel upstream regulator of ß-catenin. The overexpression of miR-183 effectively diminished the stemness characteristics of BMSCs by suppressing ß-catenin, whereas the inhibition of miR-183 augmented stemness. These outcomes align with the observed alterations in the expression levels and functional assessments of transcription factors associated with stemness. This study provides evidence for the essential involvement of ß-catenin in preserving the stemness of BMSCs, as well as elucidating the molecular mechanism through which miR-183 selectively targets ß-catenin to modulate stemness. These results underscore the potential of miR-183 and ß-catenin as molecular targets for augmenting the stemness of BMSCs. This strategy is anticipated to facilitate the restoration of bone microarchitecture and facilitate bone tissue regeneration by addressing potential cellular dysfunctions, thereby presenting novel targets and perspectives for the management of age-related osteoporosis.
Assuntos
Diferenciação Celular , Células-Tronco Mesenquimais , MicroRNAs , Osteogênese , Osteoporose , beta Catenina , MicroRNAs/genética , MicroRNAs/metabolismo , beta Catenina/metabolismo , beta Catenina/genética , Osteoporose/genética , Osteoporose/metabolismo , Osteoporose/patologia , Células-Tronco Mesenquimais/metabolismo , Osteogênese/genética , Animais , Diferenciação Celular/genética , Humanos , Proliferação de Células/genética , Análise de Célula Única , Regulação da Expressão Gênica , CamundongosRESUMO
Unlike conventional CD4+ T cells, which are phenotypically and functionally plastic, invariant NKT (iNKT) cells generally exist in a terminally differentiated state. Naïve CD4+ T cells can acquire alternative epigenetic states in response to different cues, but it remains unclear whether peripheral iNKT cells are epigenetically stable or malleable. Repetitive encounters of liver-resident iNKT cells (LiNKTs) with alpha-galactosylceramide (αGalCer)/CD1d-coated nanoparticles (NPs) can trigger their differentiation into a LiNKT cell subset expressing a T regulatory type 1 (TR1)-like (LiNKTR1) transcriptional signature. Here we dissect the epigenetic underpinnings of the LiNKT-LiNKTR1 conversion as compared to those underlying the peptide-major histocompatibility complex (pMHC)-NP-induced T-follicular helper (TFH)-to-TR1 transdifferentiation process. We show that gene upregulation during the LINKT-to-LiNKTR1 cell conversion is associated with demethylation of gene bodies, inter-genic regions, promoters and distal gene regulatory elements, in the absence of major changes in chromatin exposure or deposition of expression-promoting histone marks. In contrast, the naïve CD4+ T cell-to-TFH differentiation process involves extensive remodeling of the chromatin and the acquisition of a broad repertoire of epigenetic modifications that are then largely inherited by TFH cell-derived TR1 cell progeny. These observations indicate that LiNKT cells are epigenetically malleable and particularly susceptible to gene de-methylation.
Assuntos
Metilação de DNA , Epigênese Genética , Fígado , Células T Matadoras Naturais , Células T Matadoras Naturais/imunologia , Células T Matadoras Naturais/metabolismo , Animais , Fígado/imunologia , Fígado/citologia , Fígado/metabolismo , Camundongos , Diferenciação Celular/genética , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo , Camundongos Endogâmicos C57BL , Galactosilceramidas , Transcrição Gênica , Antígenos CD1d/genética , Antígenos CD1d/metabolismoRESUMO
Rare genetic diseases with neurodevelopmental disorders (NDDs) encompass several heterogeneous conditions (autism spectrum disorder (ASD), intellectual disability (ID), attention deficit hyperactivity disorder (ADHD), specific learning disorder (SLD), among others). Currently, few treatments are available for these patients. The difficulty in accessing human brain samples and the discrepancies between human and animal models highlight the need for new research approaches. One promising approach is the use of the cerebral organoids. These 3D, self-organized structures, generated from induced pluripotent stem cells (iPSCs), enable the reproduction of the stages of human brain development, from the proliferation of neural stem cells to their differentiation into neurons, oligodentrocytes, and astrocytes. Cerebral organoids hold great promise in understanding brain development and in the search for treatments.
Title: Des organoïdes cérébraux pour la compréhension et la thérapie des maladies génétiques rares avec troubles neurodéveloppementaux. Abstract: Les maladies génétiques associées à des troubles neurodéveloppementaux (TND) regroupent plusieurs maladies pour lesquelles peu de traitements sont proposés. L'impossibilité d'accéder à des échantillons de cerveaux humains pour des études ex vivo, et les divergences entre l'homme et les modèles animaux rendent nécessaires de nouvelles approches de recherche. L'organoïde cérébral, une structure en trois dimensions, auto-organisée, et générée à partir de cellules souches pluripotentes induites, permet de reproduire les étapes de développement du cerveau humain, de la prolifération des cellules souches neurales à leur différenciation en neurones, en oligodendrocytes, ou en astrocytes. L'intérêt de ce modèle est désormais prouvé pour la compréhension du développement cérébral et pour la recherche de traitements. Après une présentation des cellules souches pluripotentes induites et des organoïdes, nous exposerons comment cette technique est actuellement déployée, en particulier pour étudier les mécanismes physiopathologiques résultant de variations génétiques pathogènes de gènes candidats de TND.
Assuntos
Transtornos do Neurodesenvolvimento , Organoides , Doenças Raras , Humanos , Transtornos do Neurodesenvolvimento/genética , Transtornos do Neurodesenvolvimento/terapia , Transtornos do Neurodesenvolvimento/patologia , Doenças Raras/genética , Doenças Raras/terapia , Animais , Células-Tronco Pluripotentes Induzidas/transplante , Encéfalo/patologia , Doenças Genéticas Inatas/terapia , Doenças Genéticas Inatas/genética , Doenças Genéticas Inatas/patologia , Diferenciação Celular/genéticaRESUMO
OBJECTIVE: This study was designed to evaluate TFAP2A-AS1 expression in the dental pulp of teeth with or without pulpitis and to determine the function of TFAP2A-AS1 in pulp cells. METHODS: GSE92681 was analyzed to filter out differentially expressed lncRNAs. Pulp samples from teeth with pulpitis and healthy teeth (control) were examined using real-time (RT) quantitative polymerase chain reaction (qPCR). Human dental pulp stem cells (hDPSCs) were cultured in a specific medium for osteogenic induction, or treated with lipopolysaccharide (LPS) to simulate inflammation. The viability and apoptosis of human DPSCs (hDPSCs) were determined by XTT assay and apoptosis detection kit. Inflammation was induced by LPS and assessed by measuring the expression and release of inflammatory cytokines after TFAP2A-AS1 knockdown. Osteogenic differentiation of hDPSCs was investigated by determining expression levels of osteogenic markers and alkaline phosphatase (ALP) activity after TFAP2A-AS1 overexpression. The downstream microRNA (miRNA) was predicted. Dual-luciferase reporter was used to confirm the binding between miR-32-5p and TFAP2A-AS1. RESULTS: The expression of TFAP2A-AS1 was evaluated in inflamed pulp using RT-qPCR. TFAP2A-AS1 had a discriminatory ability for healthy individuals and patients with pulpitis. The expression of TFAP2A-AS1 decreased upon the osteogenic differentiation of hDPSCs, and increased upon the LPS induction. TFAP2A-AS1 can reverse the osteogenic differentiation of hDPSCs, as evidenced by decreased levels of dentine sialophosphoprotein, dentin matrix protein-1, and ALP activity. TFAP2A-AS1 knockdown can promote cell proliferation of hDPSCs and relieve LPS-induced inflammation, as evidenced by decreased levels of TNF-α, IL-1ß, and IL-6. miR-32-5p was identified as a downstream miRNA of TFAP2A-AS1. CONCLUSION: This study demonstrated the expression and potential function of TFAP2A-AS1 in the human dental pulp. TFAP2A-AS1 can inhibit odontogenic differentiation but promote inflammation in pulp cells.
Assuntos
Polpa Dentária , MicroRNAs , Pulpite , RNA Longo não Codificante , Fator de Transcrição AP-2 , Humanos , MicroRNAs/genética , RNA Longo não Codificante/genética , Polpa Dentária/metabolismo , Polpa Dentária/patologia , Pulpite/metabolismo , Pulpite/genética , Pulpite/patologia , Fator de Transcrição AP-2/metabolismo , Fator de Transcrição AP-2/genética , Diferenciação Celular/genética , Osteogênese/genética , Apoptose/genética , Regulação da Expressão Gênica , Células Cultivadas , Lipopolissacarídeos , Células-Tronco/metabolismoRESUMO
BACKGROUND: The molecular mechanisms and signaling pathways involved in tooth morphogenesis have been the research focus in the fields of tooth and bone development. However, the cell population in molars at the late bell stage and the mechanisms of hard tissue formation and mineralization remain limited knowledge. RESULTS: Here, we used the rat mandibular first and second molars as models to perform single-cell RNA sequencing (scRNA-seq) analysis to investigate cell identity and driver genes related to dental mesenchymal cell differentiation during the late bell hard tissue formation stage. We identified seven main cell types and investigated the heterogeneity of mesenchymal cells. Subsequently, we identified novel cell marker genes, including Pclo in dental follicle cells, Wnt10a in pre-odontoblasts, Fst and Igfbp2 in periodontal ligament cells, and validated the expression of Igfbp3 in the apical pulp. The dynamic model revealed three differentiation trajectories within mesenchymal cells, originating from two types of dental follicle cells and apical pulp cells. Apical pulp cell differentiation is associated with the genes Ptn and Satb2, while dental follicle cell differentiation is associated with the genes Tnc, Vim, Slc26a7, and Fgfr1. Cluster-specific regulons were analyzed by pySCENIC. In addition, the odontogenic function of driver gene TNC was verified in the odontoblastic differentiation of human dental pulp stem cells. The expression of osteoclast differentiation factors was found to be increased in macrophages of the mandibular first molar. CONCLUSIONS: Our results revealed the cell heterogeneity of molars in the late bell stage and identified driver genes associated with dental mesenchymal cell differentiation. These findings provide potential targets for diagnosing dental hard tissue diseases and tooth regeneration.
Assuntos
Diferenciação Celular , Células-Tronco Mesenquimais , Dente Molar , RNA-Seq , Análise de Célula Única , Animais , Diferenciação Celular/genética , Ratos , Análise de Célula Única/métodos , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/citologia , RNA-Seq/métodos , Odontogênese/genética , Análise da Expressão Gênica de Célula ÚnicaRESUMO
LMNA-related dilated cardiomyopathy (DCM) is an autosomal-dominant genetic condition with cardiomyocyte and conduction system dysfunction often resulting in heart failure or sudden death. The condition is caused by mutation in the Lamin A/C (LMNA) gene encoding Type-A nuclear lamin proteins involved in nuclear integrity, epigenetic regulation of gene expression, and differentiation. The molecular mechanisms of the disease are not completely understood, and there are no definitive treatments to reverse progression or prevent mortality. We investigated possible mechanisms of LMNA-related DCM using induced pluripotent stem cells derived from a family with a heterozygous LMNA c.357-2A>G splice-site mutation. We differentiated one LMNA-mutant iPSC line derived from an affected female (Patient) and two non-mutant iPSC lines derived from her unaffected sister (Control) and conducted single-cell RNA sequencing for 12 samples (four from Patients and eight from Controls) across seven time points: Day 0, 2, 4, 9, 16, 19, and 30. Our bioinformatics workflow identified 125,554 cells in raw data and 110,521 (88%) high-quality cells in sequentially processed data. Unsupervised clustering, cell annotation, and trajectory inference found complex heterogeneity: ten main cell types; many possible subtypes; and lineage bifurcation for cardiac progenitors to cardiomyocytes (CMs) and epicardium-derived cells (EPDCs). Data integration and comparative analyses of Patient and Control cells found cell type and lineage-specific differentially expressed genes (DEGs) with enrichment, supporting pathway dysregulation. Top DEGs and enriched pathways included 10 ZNF genes and RNA polymerase II transcription in pluripotent cells (PP); BMP4 and TGF Beta/BMP signaling, sarcomere gene subsets and cardiogenesis, CDH2 and EMT in CMs; LMNA and epigenetic regulation, as well as DDIT4 and mTORC1 signaling in EPDCs. Top DEGs also included XIST and other X-linked genes, six imprinted genes (SNRPN, PWAR6, NDN, PEG10, MEG3, MEG8), and enriched gene sets related to metabolism, proliferation, and homeostasis. We confirmed Lamin A/C haploinsufficiency by allelic expression and Western blot. Our complex Patient-derived iPSC model for Lamin A/C haploinsufficiency in PP, CM, and EPDC provided support for dysregulation of genes and pathways, many previously associated with Lamin A/C defects, such as epigenetic gene expression, signaling, and differentiation. Our findings support disruption of epigenomic developmental programs, as proposed in other LMNA disease models. We recognized other factors influencing epigenetics and differentiation; thus, our approach needs improvement to further investigate this mechanism in an iPSC-derived model.