RESUMO
Recent studies in non-human model systems have shown therapeutic potential of nucleoside-modified messenger RNA (modRNA) treatments for lysosomal storage diseases. Here, we assessed the efficacy of a modRNA treatment to restore the expression of the galactosidase alpha (GLA), which codes for α-Galactosidase A (α-GAL) enzyme, in a human cardiac model generated from induced pluripotent stem cells (iPSCs) derived from two individuals with Fabry disease. Consistent with the clinical phenotype, cardiomyocytes from iPSCs derived from Fabry-affected individuals showed accumulation of the glycosphingolipid Globotriaosylceramide (GB3), which is an α-galactosidase substrate. Furthermore, the Fabry cardiomyocytes displayed significant upregulation of lysosomal-associated proteins. Upon GLA modRNA treatment, a subset of lysosomal proteins were partially restored to wild-type levels, implying the rescue of the molecular phenotype associated with the Fabry genotype. Importantly, a significant reduction of GB3 levels was observed in GLA modRNA-treated cardiomyocytes, demonstrating that α-GAL enzymatic activity was restored. Together, our results validate the utility of iPSC-derived cardiomyocytes from affected individuals as a model to study disease processes in Fabry disease and the therapeutic potential of GLA modRNA treatment to reduce GB3 accumulation in the heart.
Assuntos
Doença de Fabry , Células-Tronco Pluripotentes Induzidas , Humanos , Miócitos Cardíacos , RNA , Doença de Fabry/genética , Doença de Fabry/terapia , RNA MensageiroRESUMO
Mouse embryonic stem cells (ESCs) grown in serum-supplemented conditions are characterized by an extremely short G1 phase due to the lack of G1-phase control. Concordantly, the G1-phase-specific P53-P21 pathway is compromised in serum ESCs. Here, we provide evidence that P53 is activated upon transition of serum ESCs to their pluripotent ground state using serum-free 2i conditions and that is required for the elongated G1 phase characteristic of ground state ESCs. RNA sequencing and chromatin immunoprecipitation sequencing analyses reveal that P53 directly regulates the expression of the retinoblastoma (RB) protein and that the hypo-phosphorylated, active RB protein plays a key role in G1-phase control. Our findings suggest that the P53-P21 pathway is active in ground state 2i ESCs and that its role in the G1-checkpoint is abolished in serum ESCs. Taken together, the data reveal a mechanism by which inactivation of P53 can lead to loss of RB and uncontrolled cell proliferation.
Assuntos
Ciclo Celular , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Animais , Fase G1 , Regulação da Expressão Gênica , Camundongos , Proteína do Retinoblastoma/metabolismoRESUMO
Background: Injection of adipose-derived mesenchymal stromal cells (ASCs) into murine knee joints after induction of inflammatory collagenase-induced osteoarthritis (CiOA) reduces development of joint pathology. This protection is only achieved when ASCs are applied in early CiOA, which is characterized by synovitis and high S100A8/A9 and IL-1ß levels, suggesting that inflammation is a prerequisite for the protective effect of ASCs. Our objective was to gain more insight into the interplay between synovitis and ASC-mediated amelioration of CiOA pathology. Methods: CiOA was induced by intra-articular collagenase injection. Knee joint sections were stained with hematoxylin/eosin and immunolocalization of polymorphonuclear cells (PMNs) and ASCs was performed using antibodies for NIMP-R14 and CD271, respectively. Chemokine expression induced by IL-1ß or S100A8/A9 was assessed with qPCR and Luminex. ASC-PMN co-cultures were analyzed microscopically and with Luminex for inflammatory mediators. Migration of PMNs through transwell membranes toward conditioned medium of non-stimulated ASCs (ASCNS-CM) or IL-1ß-stimulated ASCs (ASCIL-1ß-CM) was examined using flow cytometry. Phagocytic capacity of PMNs was measured with labeled zymosan particles. Results: Intra-articular saline injection on day 7 of CiOA increased synovitis after 6 h, characterized by PMNs scattered throughout the joint cavity and the synovium. ASC injection resulted in comparable numbers of PMNs which clustered around ASCs in close interaction with the synovial lining. IL-1ß-stimulation of ASCs in vitro strongly increased expression of PMN-attracting chemokines CXCL5, CXCL7, and KC, whereas S100A8/A9-stimulation did not. In agreement, the number of clustered PMNs per ASC was significantly increased after 6 h of co-culturing with IL-1ß-stimulated ASCs. Also migration of PMNs toward ASCIL-1ß-CM was significantly enhanced (287%) when compared to ASCNS-CM. Interestingly, association of PMNs with ASCs significantly diminished KC protein release by ASCs (69% lower after 24 h), accompanied by reduced release of S100A8/A9 protein by the PMNs. Moreover, phagocytic capacity of PMNs was strongly enhanced after priming with ASCIL-1ß-CM. Conclusions: Local application of ASCs in inflamed CiOA knee joints results in clustering of attracted PMNs with ASCs in the synovium, which is likely mediated by IL-1ß-induced up-regulation of chemokine release by ASCs. This results in enhanced phagocytic capacity of PMNs, enabling the clearance of debris to attenuate synovitis.
Assuntos
Interleucina-1beta/fisiologia , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/fisiologia , Neutrófilos/fisiologia , Osteoartrite do Joelho/terapia , Fagocitose , Animais , Artrite Experimental/terapia , Células Cultivadas , Quimiocinas/fisiologia , Feminino , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Neutrófilos/imunologiaRESUMO
Mouse embryonic stem cells (ESCs) cultured in serum are characterized by hyper-phosphorylated RB protein, lack of G1 control, and rapid progression through the cell cycle. Here, we show that ESCs grown in the presence of two small-molecule inhibitors (2i ESCs) have a longer G1-phase with hypo-phosphorylated RB, implying that they have a functional G1 checkpoint. Deletion of RB, P107, and P130 in 2i ESCs results in a G1-phase similar to that of serum ESCs. Inhibition of the ERK signaling pathway in serum ESCs results in the appearance of hypo-phosphorylated RB and the reinstatement of a G1 checkpoint. In addition, induction of a dormant state by the inhibition of MYC, resembling diapause, requires the presence of the RB family proteins. Collectively, our data show that RB-dependent G1 restriction point signaling is active in mouse ESCs grown in 2i but abrogated in serum by ERK-dependent phosphorylation.
Assuntos
Ciclo Celular , Células-Tronco Pluripotentes/citologia , Animais , Fase G1 , Sistema de Sinalização das MAP Quinases , Camundongos , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/enzimologia , Células-Tronco Pluripotentes/enzimologia , Proteína do Retinoblastoma/metabolismoRESUMO
Genome-wide association studies have identified a great number of non-coding risk variants for colorectal cancer (CRC). To date, the majority of these variants have not been functionally studied. Identification of allele-specific transcription factor (TF) binding is of great importance to understand regulatory consequences of such variants. A recently developed proteome-wide analysis of disease-associated SNPs (PWAS) enables identification of TF-DNA interactions in an unbiased manner. Here we perform a large-scale PWAS study to comprehensively characterize TF-binding landscape that is associated with CRC, which identifies 731 allele-specific TF binding at 116 CRC risk loci. This screen identifies the A-allele of rs1800734 within the promoter region of MLH1 as perturbing the binding of TFAP4 and consequently increasing DCLK3 expression through a long-range interaction, which promotes cancer malignancy through enhancing expression of the genes related to epithelial-to-mesenchymal transition.
Assuntos
Neoplasias do Colo/genética , Neoplasias Colorretais/genética , Progressão da Doença , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Alelos , Sistemas CRISPR-Cas , Linhagem Celular Tumoral , Neoplasias Colorretais/metabolismo , Metilação de DNA , Proteínas de Ligação a DNA , Quinases Semelhantes a Duplacortina , Epigênese Genética , Predisposição Genética para Doença , Estudo de Associação Genômica Ampla , Humanos , Proteína 1 Homóloga a MutL/genética , Polimorfismo de Nucleotídeo Único , Regiões Promotoras Genéticas , Proteoma , Proteômica , Fatores de TranscriçãoRESUMO
The t(8;21) acute myeloid leukemia (AML)-associated oncoprotein AML1-ETO disrupts normal hematopoietic differentiation. Here, we have investigated its effects on the transcriptome and epigenome in t(8,21) patient cells. AML1-ETO binding was found at promoter regions of active genes with high levels of histone acetylation but also at distal elements characterized by low acetylation levels and binding of the hematopoietic transcription factors LYL1 and LMO2. In contrast, ERG, FLI1, TAL1, and RUNX1 bind at all AML1-ETO-occupied regulatory regions, including those of the AML1-ETO gene itself, suggesting their involvement in regulating AML1-ETO expression levels. While expression of AML1-ETO in myeloid differentiated induced pluripotent stem cells (iPSCs) induces leukemic characteristics, overexpression increases cell death. We find that expression of wild-type transcription factors RUNX1 and ERG in AML is required to prevent this oncogene overexpression. Together our results show that the interplay of the epigenome and transcription factors prevents apoptosis in t(8;21) AML cells.
Assuntos
Apoptose/genética , Subunidade alfa 2 de Fator de Ligação ao Core/metabolismo , Hematopoese/genética , Leucemia Mieloide Aguda/genética , Proteínas de Fusão Oncogênica/metabolismo , Proteína 1 Parceira de Translocação de RUNX1/metabolismo , Translocação Genética , Acetilação , Sequência de Bases , Linhagem Celular Tumoral , Linhagem da Célula/genética , Sobrevivência Celular/genética , Cromossomos Humanos Par 21/genética , Cromossomos Humanos Par 8/genética , Técnicas de Silenciamento de Genes , Genoma Humano , Histona Desacetilases/metabolismo , Humanos , Leucemia Mieloide Aguda/patologia , Oncogenes , Regiões Promotoras Genéticas , Ligação Proteica/genética , Regulador Transcricional ERG/metabolismoRESUMO
Monocyte differentiation into macrophages represents a cornerstone process for host defense. Concomitantly, immunological imprinting of either tolerance or trained immunity determines the functional fate of macrophages and susceptibility to secondary infections. We characterized the transcriptomes and epigenomes in four primary cell types: monocytes and in vitro-differentiated naïve, tolerized, and trained macrophages. Inflammatory and metabolic pathways were modulated in macrophages, including decreased inflammasome activation, and we identified pathways functionally implicated in trained immunity. ß-glucan training elicits an exclusive epigenetic signature, revealing a complex network of enhancers and promoters. Analysis of transcription factor motifs in deoxyribonuclease I hypersensitive sites at cell-type-specific epigenetic loci unveiled differentiation and treatment-specific repertoires. Altogether, we provide a resource to understand the epigenetic changes that underlie innate immunity in humans.
Assuntos
Diferenciação Celular/genética , Epigênese Genética , Imunidade Inata/genética , Macrófagos/citologia , Monócitos/citologia , Animais , Sítios de Ligação/genética , Desoxirribonuclease I/química , Impressão Genômica , Humanos , Memória Imunológica , Inflamassomos/genética , Inflamassomos/imunologia , Macrófagos/imunologia , Camundongos , Monócitos/imunologia , Fatores de Transcrição/metabolismo , beta-Glucanas/imunologiaRESUMO
OBJECTIVE: In experimental collagenase-induced osteoarthritis (OA) in the mouse, synovial lining macrophages are crucial in mediating joint destruction. It was recently shown that adipose-derived stem cells (ASCs) express immunosuppressive characteristics. This study was undertaken to explore the effect of intraarticular injection of ASCs on synovial lining thickness and its relation to joint pathology in experimental mouse OA. METHODS: ASCs were isolated from fat surrounding the inguinal lymph nodes and cultured for 2 weeks. Experimental OA was induced by injection of collagenase into the knee joints of C57BL/6 mice. OA phenotypes were measured within 8 weeks after induction. Histologic analysis was performed, and synovial thickening, enthesophyte formation, and cartilage destruction were measured in the knee joint. RESULTS: ASCs were injected into the knee joints of mice 7 days after the induction of collagenase-induced OA. On day 1, green fluorescent protein-labeled ASCs were attached to the lining layer in close contact with macrophages. Thickening of the synovial lining, formation of enthesophytes associated with medial collateral ligaments, and formation of enthesophytes associated with cruciate ligaments were significantly inhibited on day 42 after ASC treatment, by 31%, 89%, and 44%, respectively. Destruction of cartilage was inhibited on day 14 (65%) and day 42 (35%). In contrast to early treatment, injection of ASCs on day 14 after OA induction showed no significant effect on synovial activation or joint pathology on day 42. CONCLUSION: These findings indicate that a single injection of ASCs into the knee joints of mice with early-stage collagenase-induced OA inhibits synovial thickening, formation of enthesophytes associated with ligaments, and cartilage destruction.
Assuntos
Condrócitos/imunologia , Articulação do Joelho/imunologia , Osteoartrite do Joelho/imunologia , Osteoartrite do Joelho/terapia , Transplante de Células-Tronco/métodos , Tecido Adiposo/citologia , Animais , Ligamento Cruzado Anterior/imunologia , Ligamento Cruzado Anterior/patologia , Cartilagem Articular/imunologia , Cartilagem Articular/patologia , Movimento Celular/imunologia , Condrócitos/patologia , Condrogênese/imunologia , Colagenases/farmacologia , Ligamentos Colaterais/imunologia , Ligamentos Colaterais/patologia , Modelos Animais de Doenças , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Injeções Intra-Articulares , Articulação do Joelho/patologia , Camundongos , Camundongos Endogâmicos C57BL , Osteoartrite do Joelho/induzido quimicamente , Ligamento Cruzado Posterior/patologia , Membrana Sinovial/imunologia , Membrana Sinovial/patologiaRESUMO
Hematopoietic stem cells (HSCs) accomplish a complex task. On a daily base billions of the 8 different mature cells are delivered in the right proportions. HSCs are located in niches located at several locations in the body. Communication between these spatially separated niches is accomplished by stem cells that leave their niche and migrate to other niches guided by soluble factors. The niche itself comprises all major signaling pathways (Hedgehog, Notch, Wnt, and BMP) and an array of adhesion molecules. The interplay between these components keep HSC in a quiescent state but also speed up production in case of urgent need during infection or excessive blood loss. In this review, we focus on the molecular cues of the niche, functional adhesion molecules and describe recent data obtained with multiphoton microscopy. A vast array of molecules is described that display similar functions as HSC controllers. This points to redundancy in the system that enables HSC to respond to different cues essentially with the same functional response. Apparently, the hematopoietic system is so crucial that it is not dependent on a single cue. When one cue fails to initiate a response, another cue will take over leading to an almost similar response. Another explanation is that every cue adds to an integrated signal that results in reaching the threshold. This integrated signal might be reached from huge signaling by a single cue or the low but additive signals by several cues.