RESUMEN
Precise control of gene expression levels is essential for normal cell functions, yet how they are defined and tightly maintained, particularly at intermediate levels, remains elusive. Here, using a series of newly developed sequencing, imaging, and functional assays, we uncover a class of transcription factors with dual roles as activators and repressors, referred to as condensate-forming level-regulating dual-action transcription factors (TFs). They reduce high expression but increase low expression to achieve stable intermediate levels. Dual-action TFs directly exert activating and repressing functions via condensate-forming domains that compartmentalize core transcriptional unit selectively. Clinically relevant mutations in these domains, which are linked to a range of developmental disorders, impair condensate selectivity and dual-action TF activity. These results collectively address a fundamental question in expression regulation and demonstrate the potential of level-regulating dual-action TFs as powerful effectors for engineering controlled expression levels.
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Factores de Transcripción , Animales , Humanos , Ratones , Regulación de la Expresión Génica , Mutación , Proteínas Represoras/metabolismo , Proteínas Represoras/genética , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , Línea CelularRESUMEN
PURPOSE: In order to make the postoperative effect of open double eyelid more close to the physiological and anatomical structure of double eyelid, we improved the traditional open double eyelid operation according to the anatomical characteristics of the upper eyelid. We fixed part of the orbicularis oculi muscle above the incisal margin with the orbital septum flap to make the double eyelid formed after surgery more natural and beautiful. MATERIALS AND METHODS: A total of 76 patients who received open double blepharoplasty in department of plastic surgery from February 2019 to May 2022 were selected as this study objects, all of whom were female. Their ages ranged from 18 to 32 years, with a mean of (23.6 ± 5.2) years. The surgical method is open double blepharoplasty by fixing part of the orbicularis oculi muscle above the incisal margin with the flap of the orbital septum. RESULTS: In this study, all 76 patients underwent successful surgery, with an average operation time of (1.5 ± 0.2) h. The postoperative double eyelid curvature was smooth and the double eyelid width was basically symmetrical. In terms of the doctors' satisfaction evaluation of the postoperative effect, 64 cases were very satisfied and 12 cases were satisfied. In terms of patients' satisfaction evaluation of the postoperative effect, 60 patients were very satisfied, 15 patients were satisfied and 1 patient was dissatisfied. CONCLUSIONS: Through this study, we found that this surgical method invented by us has short operation time, good operation effect, few postoperative complications and high patient satisfaction, which is worthy of promotion and application in plastic surgery clinical practice. LEVEL OF EVIDENCE IV: This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to www.springer.com/00266 .
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Blefaroplastia , Técnicas de Sutura , Humanos , Femenino , Adolescente , Adulto Joven , Adulto , Masculino , Párpados/cirugía , Blefaroplastia/métodos , Músculos Faciales/cirugía , Satisfacción del Paciente , Estudios RetrospectivosRESUMEN
BACKGROUND: Sebaceous glands (SGs) synthesize and secret sebum to protect and moisturize the dermal system via the complicated endocrine modulation. Dysfunction of SG are usually implicated in a number of dermal and inflammatory diseases. However, the molecular mechanism behind the differentiation, development and proliferation of SGs is far away to fully understand. METHODS: Herein, the rat volar and mammary tissues with abundant SGs from female SD rats with (post-natal day (PND)-35) and without puberty onset (PND-25) were arrested, and conducted RNA sequencing. The protein complex of Neuropeptide Y receptor Y2 (NPY2R)/NPY5R/Nuclear factor of activated T cells 1 (NFATc1) was performed by immunoprecipitation, mass spectrum and gel filtration. Genome-wide occupancy of NFATc1 was measured by chromatin immunoprecipitation sequencing. Target proteins' expression and localization was detected by western blot and immunofluorescence. RESULTS: NPY2R gene was significantly up-regulated in volar and mammary SGs of PND-25. A special protein complex of NPY2R/NPY5R/NFATc1 in PND-25. NFATc1 was dephosphorylated and activated, then localized into nucleus to exert as a transcription factor in volar SGs of PND-35. NFATc1 was especially binding at enhancer regions to facilitate the distal SG and sebum related genes' transcription. Dual specificity tyrosine phosphorylation regulated kinase 1A (DYRK1A) contributed to NFATc1 phosphorylation in PND-25, and inactivated of DYRK1A resulted in NFATc1 dephosphorylation and nuclear localization in PND-35. CONCLUSIONS: Our findings unmask the new role of NPY2R/NFATc1/DYRK1A in pubertal SG, and are of benefit to advanced understanding the molecular mechanism of SGs' function after puberty, and provide some theoretical basis for the treatment of acne vulgaris from the perspective of hormone regulation.
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Acné Vulgar , Glándulas Sebáceas , Animales , Femenino , Ratas , Acné Vulgar/metabolismo , Factores de Transcripción NFI/metabolismo , Ratas Sprague-Dawley , Glándulas Sebáceas/metabolismo , Sebo/metabolismo , Quinasas DyrKRESUMEN
In mice, only the zygotes and blastomeres from 2-cell embryos are authentic totipotent stem cells (TotiSCs) capable of producing all the differentiated cells in both embryonic and extraembryonic tissues and forming an entire organism1. However, it remains unknown whether and how totipotent stem cells can be established in vitro in the absence of germline cells. Here we demonstrate the induction and long-term maintenance of TotiSCs from mouse pluripotent stem cells using a combination of three small molecules: the retinoic acid analogue TTNPB, 1-azakenpaullone and the kinase blocker WS6. The resulting chemically induced totipotent stem cells (ciTotiSCs), resembled mouse totipotent 2-cell embryo cells at the transcriptome, epigenome and metabolome levels. In addition, ciTotiSCs exhibited bidirectional developmental potentials and were able to produce both embryonic and extraembryonic cells in vitro and in teratoma. Furthermore, following injection into 8-cell embryos, ciTotiSCs contributed to both embryonic and extraembryonic lineages with high efficiency. Our chemical approach to totipotent stem cell induction and maintenance provides a defined in vitro system for manipulating and developing understanding of the totipotent state and the development of multicellular organisms from non-germline cells.
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Células Madre Totipotentes , Animales , Ratones , Blastómeros , Diferenciación Celular/efectos de los fármacos , Células Madre Embrionarias/citología , Células Madre Embrionarias/efectos de los fármacos , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/efectos de los fármacos , Células Madre Totipotentes/citología , Células Madre Totipotentes/efectos de los fármacos , Teratoma/patología , Linaje de la Célula/efectos de los fármacosRESUMEN
Cryopreservation of human T lymphocytes has become a key strategy for supporting cell-based immunotherapy. However, the effects of ice seeding on the cryopreservation of cells under relatively slow cooling have not been well researched. The cryopreservation strategy with a nontoxic, single-ingredient, and injectable cryoprotective solution remains to be developed. We conducted ice seeding for the cells in a solution of normal saline with 1% (v/v) dimethyl sulfoxide (Me2SO), 0.1 M trehalose, and 4% (w/v) human serum albumin (HSA) under different slow cooling rates. With the positive results, we further applied seeding in the solution of 0.2 M trehalose and 4% (w/v) HSA under the same cooling rates. The optimal concentration of trehalose in the Me2SO-free solutions was then investigated under the optimized cooling rate with seeding, with control groups without seeding, and in a freezing container. In vitro toxicity of the cryoprotective solutions to the cells was also tested. We found that the relative viability of cells (1% [v/v] Me2SO, 0.1 M trehalose and 4% [w/v] HSA) was improved significantly from 88.6% to 94.1% with ice seeding, compared with that without seeding (p < 0.05). The relative viability of cells (0.2 M trehalose and 4% [w/v] HSA) with seeding was significantly higher than that without seeding, 96.3% and 92.0%, respectively (p < 0.05). With no significant difference in relative viability between the solutions of 0.2 M trehalose or 0.3 M trehalose with 4% (w/v) HSA (92.4% and 94.6%, respectively, p > 0.05), the solution of 0.2 M trehalose and 4% (w/v) HSA was selected as the optimized Me2SO-free solution. This strategy could cryopreserve human T lymphocytes without any toxic cryoprotectant and boost the application of cell products in humans by intravenous injection, with the osmolality of the low-concentration cryoprotective solution close to that of human plasma.
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Hielo , Trehalosa , Humanos , Trehalosa/farmacología , Linfocitos T , Crioprotectores/farmacología , Criopreservación/métodos , Dimetilsulfóxido/farmacología , Supervivencia CelularRESUMEN
Rationale: Traditional treatments for leukemia fail to address stem cell drug resistance characterized by epigenetic mediators such as histone lysine-specific demethylase 4 (KDM4). The KDM4 family, which acts as epigenetic regulators inducing histone demethylation during the development and progression of leukemia, lacks specific molecular inhibitors. Methods: The KDM4 inhibitor, SD49-7, was synthesized and purified based on acyl hydrazone Schiff base. The interaction between SD49-7 and KDM4s was monitored in vitro by surface plasma resonance (SPR). In vitro and in vivo biological function experiments were performed to analyze apoptosis, colony-formation, proliferation, differentiation, and cell cycle in cell sub-lines and mice. Molecular mechanisms were demonstrated by RNA-seq, ChIP-seq, RT-qPCR and Western blotting. Results: We found significantly high KDM4A expression levels in several human leukemia subtypes. The knockdown of KDM4s inhibited leukemogenesis in the MLL-AF9 leukemia mouse model but did not affect the survival of normal human hematopoietic cells. We identified SD49-7 as a selective KDM4 inhibitor that impaired the progression of leukemia stem cells (LSCs) in vitro. SD49-7 suppressed leukemia development in the mouse model and patient-derived xenograft model of leukemia. Depletion of KDM4s activated the apoptosis signaling pathway by suppressing MDM2 expression via modulating H3K9me3 levels on the MDM2 promoter region. Conclusion: Our study demonstrates a unique KDM4 inhibitor for LSCs to overcome the resistance to traditional treatment and offers KDM4 inhibition as a promising strategy for resistant leukemia therapy.
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Histonas , Leucemia Mieloide Aguda , Animales , Ciclo Celular , Histona Demetilasas/metabolismo , Histonas/metabolismo , Humanos , Histona Demetilasas con Dominio de Jumonji/metabolismo , Leucemia Mieloide Aguda/metabolismo , Ratones , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Células Madre/metabolismoRESUMEN
Phycocyanin (PC) is a natural pigment-protein complex in food dye applications. In this study, a phycocyanin-epigallocatechin gallate (EGCG) complex (PE) was prepared and the effects of EGCG on the structure and color stability of PC were evaluated. The fluorescence results showed that the binding number n was 62.1 ± 3.41 (EGCG/PC) and the binding constant K was 4.39 (±0.2) × 105 M-1, indicating a weak-binding interaction. Fourier transform-infrared analysis showed that EGCG caused structural changes in PC by partially uncoiling α-helix and increasing ß-sheet content. The EGCG induced a PC association at a reaction molar ratio above 40:1 (EGCG/PC). Moreover, EGCG protected phycocyanobilin against color fading, making PE more stable relative to PC under 8-days storage in light. This study provides a novel scheme to stabilize PC by forming a complex with polyphenols, which will facilitate the PC application as a natural blue pigment in food.
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Catequina , Ficocianina , Catequina/análogos & derivados , PolifenolesRESUMEN
Heart disease is the leading cause of human deaths worldwide. Due to lacking cardiomyocytes with replicative capacity and cardiac progenitor cells with differentiation potential in adult hearts, massive loss of cardiomyocytes after ischemic events produces permanent damage, ultimately leading to heart failure. Cellular reprogramming is a promising strategy to regenerate heart by induction of cardiomyocytes from other cell types, such as cardiac fibroblasts. In contrast to conventional virus-based cardiac reprogramming, non-viral approaches greatly reduce the potential risk that includes disruption of genome integrity by integration of foreign DNAs, expression of exogenous genes with oncogenic potential, and appearance of partially reprogrammed cells harmful for the physiological functions of tissues/organs, which impedes their in-vivo applications. Here, we review the recent progress in development of non-viral approaches to directly reprogram somatic cells towards cardiomyocytes and their therapeutic application for heart regeneration.
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Reprogramación Celular/fisiología , Miocitos Cardíacos/metabolismo , Medicina Regenerativa/métodos , Animales , Humanos , RatonesRESUMEN
The aim of the present work is to isolate a series of triterpene derivatives with rhamnosyl linking acetyl groups from Glechoma longituba according to the structural characteristics of previously described triterpene saponins. The extract ion chromatography spectrum of the crude extract of G.â longituba was detected and analyzed by HPLC-HR-ESI-MS to determine possible components, and these metabolites were traced and separated by combining high-resolution mass spectrometry and predicted liquid chromatography retention time. Three 11α, 12α-epoxypentacyclic oleanolic acid triterpene saponins (glechomanosidesâ H-J) and one ursane triterpene aldehyde saponin with a C-28 aldehyde group were isolated from G.â longituba. The structure of these compounds was confirmed by NMR and compared with those of previously characterized compounds. The strategy described in this report enables a rapid, reliable, and complete analysis of glycoside compounds containing different numbers of acetyl groups at different positions on the sugar.
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Lamiaceae/química , Extractos Vegetales/análisis , Ramnosa/análisis , Saponinas/análisis , Triterpenos/análisis , Acetilación , Cromatografía Líquida de Alta Presión , Conformación Molecular , Extractos Vegetales/metabolismo , Ramnosa/metabolismo , Saponinas/metabolismo , Espectrometría de Masas en Tándem , Triterpenos/metabolismoRESUMEN
Ferritin, a protein with an 8-nm cage structure, can encapsulate and deliver bioactive molecules. In this study, succinylation was adopted to modify plant ferritin to fabricate succinylated red been ferritin (SRBF) at pH 8.0. The SRBF was retained as a cage-like shape (12 nm diameter), while its secondary structure was altered, rendering higher negative charge accompanies by decreased surface hydrophobicity. The SRBF also demonstrated favorable property of reversible assembly regulated by pH-transitions (pH 2.0/7.0), thus enabled successful encapsulation of epigallocatechin gallate (EGCG) for fabrication of EGCG-loaded SRBF complexes with a diameter of ~12 nm. Succinylation enhanced the thermal stabilities of ferritin and the embedded EGCG. Moreover, SRBF markedly improved the transport efficiency of EGCG in Caco-2 monolayers relative to EGCG and that encapsulated in unmodified ferritin. These findings have extended the succinylation reaction for the cage-like protein modification, and facilitated the usage of ferritin variant in delivery of bioactive molecules.
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Portadores de Fármacos/química , Ferritinas/química , Nanoestructuras/química , Polifenoles/farmacocinética , Células CACO-2 , Catequina/análogos & derivados , Catequina/química , Estabilidad de Medicamentos , Ferritinas/farmacocinética , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Polifenoles/químicaRESUMEN
BACKGROUND: The functionality of pea proteins is relatively weak relative to that of soybean proteins, which limits the application of pea proteins in food and nutritional applications. Glycosylation is a promising approach to influence the protein structure and in turn change the functional properties of pea proteins. RESULTS: In this study, the effect of transglutaminase-induced oligochitosan glycosylation on the structural and functional properties of pea seed legumin was studied. Different oligochitosan-modified legumin complexes (OLCs) were prepared by applying different molar ratios of legumin to oligochitosan (1:1 to 1:4) induced by transglutaminase (10 U g-1 protein). Results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), glucosamine, and free amino analysis showed that the legumin could be covalently bonded with the oligochitosan and were influenced by the applying dose of the oligochitosan. Infrared spectroscopy, fluorescence, and scanning electron microscopy analysis indicated that the structure of the different OLC samples could be changed to different extents. Moreover, although the emulsifying activity decreased, the emulsification stability, thermal stability, and in vitro digestive stability of the OLCs were remarkably improved relative to that of the untreated legumin. CONCLUSION: Oligochitosan glycosylation could change the structure of the legumin and consequently improve its emulsification stability, thermal stability, and in vitro digestive stability. This study will facilitate the legumin functionalization by the glycosylation approach to fabricate protein-oligochitosan complex for potential food and nutritional applications. © 2020 Society of Chemical Industry.
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Quitina/análogos & derivados , Proteínas de Guisantes/química , Pisum sativum/química , Secuencia de Aminoácidos , Quitina/química , Quitosano , Electroforesis en Gel de Poliacrilamida , Glicosilación , Calor , Oligosacáridos , Estabilidad Proteica , Semillas/químicaRESUMEN
A new series of millepachine derivative containing aminophosphonate ester moieties were designed and synthesized, and evaluated for their anticancer activities using MTT assay. Among all the compounds, compound 9m exhibited the most potent cytotoxic activity against all tested human cancer cell lines including multidrug resistant phenotype, which inhibited cancer cell growth with IC50 values ranging from 0.85 to 3.09 µM, respectively. In addition, its low cytotoxicity toward human normal liver cells HL-7702 and sensitivity toward to doxorubicin or cisplatin-resistant cells indicated the possibility for cancer therapy. Furthermore, 9m significantly induced cell apoptosis and cell cycle arrest in G2/M phase and dramatically disrupted the microtubule organization. Moreover, a decrease in MMP, an increase in reactive oxygen species (ROS) generation and Bax/Bcl-2 ratio, accompanied by activated caspase-3 and -9, were observed in HepG-2 cells after incubation with 9m, indicating that the mitochondrial pathway was involved in the 9m-mediated apoptosis.
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Antineoplásicos/farmacología , Chalconas/farmacología , Ésteres/farmacología , Organofosfonatos/farmacología , Tubulina (Proteína)/metabolismo , Antineoplásicos/síntesis química , Antineoplásicos/química , Apoptosis/efectos de los fármacos , Ciclo Celular/efectos de los fármacos , Línea Celular , Proliferación Celular/efectos de los fármacos , Chalconas/síntesis química , Chalconas/química , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Ésteres/química , Humanos , Estructura Molecular , Organofosfonatos/química , Relación Estructura-ActividadRESUMEN
Ectopic expression of Oct4, Sox2, Klf4, and c-Myc can reprogram differentiated somatic cells into induced pluripotent stem cells (iPSCs). For years, Oct4 has been considered the key reprogramming factor core of the four factors. Here, we challenge this view by reporting a core function of Sox2 and Klf4 in reprogramming. We found that polycistronic expression of Sox2 and Klf4 was sufficient to induce pluripotency in the absence of exogenous Oct4, and the stoichiometry of Sox2 and Klf4 was essential. Sox2 and Klf4 cooperatively bound across the genome, leading to epigenetic remodeling of their targets, including pluripotency genes and gradual activation of the pluripotency network. Interestingly, cells of different germ layer origins, fibroblasts (mesoderm) and neural progenitor cells (ectoderm), showed convergent reprogramming trajectories and similar efficiency. This work demonstrates a core function of Sox2 and Klf4 in pluripotency induction and shows that this mechanism is independent of germ layer origin.
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Técnicas de Reprogramación Celular , Reprogramación Celular , Células Madre Embrionarias de Ratones/metabolismo , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Factores de Transcripción SOXB1/metabolismo , Animales , Células HEK293 , Humanos , Factor 4 Similar a Kruppel , Ratones , Células Madre Embrionarias de Ratones/citología , Factor 3 de Transcripción de Unión a Octámeros/genética , Factores de Transcripción SOXB1/genéticaRESUMEN
Metabolism has been shown to integrate with epigenetics and transcription to modulate cell fate and function. Beyond meeting the bioenergetic and biosynthetic demands of T-cell differentiation, whether metabolism might control T-cell fate by an epigenetic mechanism is unclear. Here, through the discovery and mechanistic characterization of a small molecule, (aminooxy)acetic acid, that reprograms the differentiation of T helper 17 (TH17) cells towards induced regulatory T (iTreg) cells, we show that increased transamination, mainly catalysed by GOT1, leads to increased levels of 2-hydroxyglutarate in differentiating TH17 cells. The accumulation of 2-hydroxyglutarate resulted in hypermethylation of the Foxp3 gene locus and inhibited Foxp3 transcription, which is essential for fate determination towards TH17 cells. Inhibition of the conversion of glutamate to α-ketoglutaric acid prevented the production of 2-hydroxyglutarate, reduced methylation of the Foxp3 gene locus, and increased Foxp3 expression. This consequently blocked the differentiation of TH17 cells by antagonizing the function of transcription factor RORγt and promoted polarization into iTreg cells. Selective inhibition of GOT1 with (aminooxy)acetic acid ameliorated experimental autoimmune encephalomyelitis in a therapeutic mouse model by regulating the balance between TH17 and iTreg cells. Targeting a glutamate-dependent metabolic pathway thus represents a new strategy for developing therapeutic agents against TH17-mediated autoimmune diseases.
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Diferenciación Celular , Epigénesis Genética , Linfocitos T Reguladores/citología , Linfocitos T Reguladores/metabolismo , Células Th17/citología , Células Th17/metabolismo , Ácido Aminooxiacético/farmacología , Ácido Aminooxiacético/uso terapéutico , Animales , Aspartato Aminotransferasa Citoplasmática , Diferenciación Celular/efectos de los fármacos , Encefalomielitis Autoinmune Experimental/tratamiento farmacológico , Encefalomielitis Autoinmune Experimental/inmunología , Epigénesis Genética/efectos de los fármacos , Femenino , Factores de Transcripción Forkhead/genética , Glutaratos/metabolismo , Ácidos Cetoglutáricos/metabolismo , Masculino , Ratones , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/metabolismo , Linfocitos T Reguladores/efectos de los fármacos , Linfocitos T Reguladores/inmunología , Células Th17/efectos de los fármacos , Células Th17/inmunología , Transaminasas/antagonistas & inhibidoresRESUMEN
Reprogramming somatic fibroblasts into alternative lineages would provide a promising source of cells for regenerative therapy. However, transdifferentiating human cells into specific homogeneous, functional cell types is challenging. Here we show that cardiomyocyte-like cells can be generated by treating human fibroblasts with a combination of nine compounds that we term 9C. The chemically induced cardiomyocyte-like cells uniformly contracted and resembled human cardiomyocytes in their transcriptome, epigenetic, and electrophysiological properties. 9C treatment of human fibroblasts resulted in a more open-chromatin conformation at key heart developmental genes, enabling their promoters and enhancers to bind effectors of major cardiogenic signals. When transplanted into infarcted mouse hearts, 9C-treated fibroblasts were efficiently converted to chemically induced cardiomyocyte-like cells. This pharmacological approach to lineage-specific reprogramming may have many important therapeutic implications after further optimization to generate mature cardiac cells.
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Técnicas de Reprogramación Celular , Reprogramación Celular/efectos de los fármacos , Fibroblastos/efectos de los fármacos , Miocitos Cardíacos/citología , Bibliotecas de Moléculas Pequeñas/farmacología , Animales , Linaje de la Célula/efectos de los fármacos , Linaje de la Célula/genética , Transdiferenciación Celular , Cromatina/química , Cromatina/metabolismo , Modelos Animales de Enfermedad , Fibroblastos/citología , Corazón/embriología , Humanos , Masculino , Ratones , Ratones Endogámicos NOD , Infarto del Miocardio/cirugía , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/trasplante , Cadenas Pesadas de Miosina/genética , Organogénesis/genética , Conformación Proteica , Piel/citología , Bibliotecas de Moléculas Pequeñas/química , Transcriptoma , Transducción GenéticaRESUMEN
Pancreatic beta cells are of great interest for biomedical research and regenerative medicine. Here we show the conversion of human fibroblasts towards an endodermal cell fate by employing non-integrative episomal reprogramming factors in combination with specific growth factors and chemical compounds. On initial culture, converted definitive endodermal progenitor cells (cDE cells) are specified into posterior foregut-like progenitor cells (cPF cells). The cPF cells and their derivatives, pancreatic endodermal progenitor cells (cPE cells), can be greatly expanded. A screening approach identified chemical compounds that promote the differentiation and maturation of cPE cells into functional pancreatic beta-like cells (cPB cells) in vitro. Transplanted cPB cells exhibit glucose-stimulated insulin secretion in vivo and protect mice from chemically induced diabetes. In summary, our study has important implications for future strategies aimed at generating high numbers of functional beta cells, which may help restoring normoglycemia in patients suffering from diabetes.
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Diferenciación Celular/fisiología , Fibroblastos/fisiología , Células Secretoras de Insulina/fisiología , Células Madre/fisiología , Animales , Técnicas de Cultivo de Célula , Diabetes Mellitus Experimental , Fibroblastos/citología , Glucosa/metabolismo , Humanos , Insulina/metabolismo , Secreción de Insulina , Células Secretoras de Insulina/citología , Células Secretoras de Insulina/metabolismo , Ratones , Células Madre/citologíaRESUMEN
Successful generation of induced pluripotent stem cells entails a major metabolic switch from mitochondrial oxidative phosphorylation to glycolysis during the reprogramming process. The mechanism of this metabolic reprogramming, however, remains elusive. Here, our results suggest that an Atg5-independent autophagic process mediates mitochondrial clearance, a characteristic event involved in the metabolic switch. We found that blocking such autophagy, but not canonical autophagy, inhibits mitochondrial clearance, in turn, preventing iPSC induction. Furthermore, AMPK seems to be upstream of this autophagic pathway and can be targeted by small molecules to modulate mitochondrial clearance during metabolic reprogramming. Our work not only reveals that the Atg5-independent autophagy is crucial for establishing pluripotency, but it also suggests that iPSC generation and tumorigenesis share a similar metabolic switch.
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Autofagia , Reprogramación Celular , Células Madre Pluripotentes Inducidas/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Mitocondrias/metabolismo , Proteínas Quinasas Activadas por AMP/metabolismo , Aminoimidazol Carboxamida/análogos & derivados , Aminoimidazol Carboxamida/farmacología , Animales , Proteína 5 Relacionada con la Autofagia , Western Blotting , Células Cultivadas , Embrión de Mamíferos/citología , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Fibroblastos/ultraestructura , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Ratones Endogámicos C57BL , Ratones Noqueados , Microscopía Electrónica , Microscopía Fluorescente , Proteínas Asociadas a Microtúbulos/genética , Factor 3 de Transcripción de Unión a Octámeros/genética , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Interferencia de ARN , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Ribonucleótidos/farmacología , Sirolimus/farmacologíaRESUMEN
The bacterial CRISPR-Cas9 system has emerged as an effective tool for sequence-specific gene knockout through non-homologous end joining (NHEJ), but it remains inefficient for precise editing of genome sequences. Here we develop a reporter-based screening approach for high-throughput identification of chemical compounds that can modulate precise genome editing through homology-directed repair (HDR). Using our screening method, we have identified small molecules that can enhance CRISPR-mediated HDR efficiency, 3-fold for large fragment insertions and 9-fold for point mutations. Interestingly, we have also observed that a small molecule that inhibits HDR can enhance frame shift insertion and deletion (indel) mutations mediated by NHEJ. The identified small molecules function robustly in diverse cell types with minimal toxicity. The use of small molecules provides a simple and effective strategy to enhance precise genome engineering applications and facilitates the study of DNA repair mechanisms in mammalian cells.
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Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Reparación del ADN/efectos de los fármacos , Ingeniería Genética/métodos , Genoma Humano/genética , Células Madre Pluripotentes/efectos de los fármacos , Células Madre Pluripotentes/metabolismo , Bibliotecas de Moléculas Pequeñas/farmacología , Relación Dosis-Respuesta a Droga , Células HeLa , Ensayos Analíticos de Alto Rendimiento , Humanos , Estructura Molecular , Bibliotecas de Moléculas Pequeñas/química , Relación Estructura-ActividadRESUMEN
Pancreatic ß cells are of great interest for the treatment of type 1 diabetes. A number of strategies already exist for the generation of ß cells, but a general approach for reprogramming nonendodermal cells into ß cells could provide an attractive alternative in a variety of contexts. Here, we describe a stepwise method in which pluripotency reprogramming factors were transiently expressed in fibroblasts in conjunction with a unique combination of soluble molecules to generate definitive endoderm-like cells that did not pass through a pluripotent state. These endoderm-like cells were then directed toward pancreatic lineages using further combinations of small molecules in vitro. The resulting pancreatic progenitor-like cells could mature into cells of all three pancreatic lineages in vivo, including functional, insulin-secreting ß-like cells that help to ameliorate hyperglycemia. Our findings may therefore provide a useful approach for generating large numbers of functional ß cells for disease modeling and, ultimately, cell-based therapy.