RESUMEN
The cleavage of zygotes generates totipotent blastomeres. In human 8-cell blastomeres, zygotic genome activation (ZGA) occurs to initiate the ontogenesis program. However, capturing and maintaining totipotency in human cells pose significant challenges. Here, we realize culturing human totipotent blastomere-like cells (hTBLCs). We find that splicing inhibition can transiently reprogram human pluripotent stem cells into ZGA-like cells (ZLCs), which subsequently transition into stable hTBLCs after long-term passaging. Distinct from reported 8-cell-like cells (8CLCs), both ZLCs and hTBLCs widely silence pluripotent genes. Interestingly, ZLCs activate a particular group of ZGA-specific genes, and hTBLCs are enriched with pre-ZGA-specific genes. During spontaneous differentiation, hTBLCs re-enter the intermediate ZLC stage and further generate epiblast (EPI)-, primitive endoderm (PrE)-, and trophectoderm (TE)-like lineages, effectively recapitulating human pre-implantation development. Possessing both embryonic and extraembryonic developmental potency, hTBLCs can autonomously generate blastocyst-like structures in vitro without external cell signaling. In summary, our study provides key criteria and insights into human cell totipotency.
Asunto(s)
Diferenciación Celular , Empalmosomas , Animales , Humanos , Ratones , Blastocisto/metabolismo , Blastocisto/citología , Blastómeros/metabolismo , Blastómeros/citología , Reprogramación Celular , Desarrollo Embrionario/genética , Estratos Germinativos/metabolismo , Estratos Germinativos/citología , Células Madre Pluripotentes/metabolismo , Células Madre Pluripotentes/citología , Empalme del ARN , Empalmosomas/metabolismo , Células Madre Totipotentes/metabolismo , Células Madre Totipotentes/citología , Cigoto/metabolismo , Células Cultivadas , Modelos Moleculares , Estructura Terciaria de Proteína , Genoma Humano , Análisis de la Célula Individual , Factor 15 de Diferenciación de Crecimiento/química , Factor 15 de Diferenciación de Crecimiento/genética , Factor 15 de Diferenciación de Crecimiento/metabolismo , Epigenómica , Linaje de la CélulaRESUMEN
BACKGROUND: The monitoring of concentration variation of the newly developed growth differentiation factor 15 (GDF15) biomarker in human serum is of great significance for diagnosing cardiovascular diseases. Current methods for the detection of the GDF15 protein mainly are based on antibody-assisted immunoassays, which encounter the limitations in terms of sensitivity, complexity and costs. The development of simple and sensitive biosensors for GDF15 can therefore facilitate the diagnosis of cardiovascular diseases. RESULTS: A new bimetallic quasi-Cu/Co-MOF nanozyme with high catalytic performance for electrochemical reduction of H2O2 is synthesized via simple one-step precipitation and low-temperature calcination method. Such nanozymes are further employed as amplification tags and coupled with cyclic entropy-driven DNA signal enhancement strategies to construct ultrasensitive aptamer-based biosensor for detecting GDF15 in human serums. GDF15 molecules associate with two aptamers and release the ssDNA trigger sequences via target-binding induced displacement reaction. These ssDNAs subsequently initiate cyclic DNA-fueled strand displacement and catalytic hairpin assembly (CHA) reaction cascades for confining many quasi-Cu/Co-MOF nanozymes on sensor electrode, which yield drastically amplified H2O2 reduction current for detecting GDF15 down to 0.12 pg mL-1 with a dynamic range of 0.5 pg mL-1 to 20 ng mL-1. The electrochemical aptasensor also presents good reproducibility and selectivity and exhibits the capability to detect GDF15 in diluent serums. SIGNIFICANCE: Our aptamer-based GDF15 protein electrochemical assay clearly outperforms current existing antibody-based methods and the quasi-Cu/Co-MOF nanozyme/entropy-driven cascaded signal amplification means can be used as a universal strategy for sensitive monitoring of different biomolecular markers for diverse applications.
Asunto(s)
Aptámeros de Nucleótidos , Técnicas Biosensibles , Cobalto , Cobre , Técnicas Electroquímicas , Factor 15 de Diferenciación de Crecimiento , Estructuras Metalorgánicas , Aptámeros de Nucleótidos/química , Factor 15 de Diferenciación de Crecimiento/sangre , Factor 15 de Diferenciación de Crecimiento/química , Cobre/química , Humanos , Estructuras Metalorgánicas/química , Cobalto/química , Técnicas Biosensibles/métodos , Entropía , Peróxido de Hidrógeno/química , Límite de Detección , Técnicas de Amplificación de Ácido Nucleico , ADN/químicaRESUMEN
BACKGROUND: Mitochondrial hepatopathies (MHs) are primary mitochondrial genetic disorders that can present as childhood liver disease. No recognized biomarkers discriminate MH from other childhood liver diseases. The protein biomarkers growth differentiation factor 15 (GDF15) and fibroblast growth factor 21 (FGF21) differentiate mitochondrial myopathies from other myopathies. We evaluated these biomarkers to determine if they discriminate MH from other liver diseases in children. METHODS: Serum biomarkers were measured in 36 children with MH (17 had a genetic diagnosis); 38 each with biliary atresia, α1-antitrypsin deficiency, and Alagille syndrome; 20 with NASH; and 186 controls. RESULTS: GDF15 levels compared to controls were mildly elevated in patients with α1-antitrypsin deficiency, Alagille syndrome, and biliary atresia-young subgroup, but markedly elevated in MH (p<0.001). FGF21 levels were mildly elevated in NASH and markedly elevated in MH (p<0.001). Both biomarkers were higher in patients with MH with a known genetic cause but were similar in acute and chronic presentations. Both markers had a strong performance to identify MH with a molecular diagnosis with the AUC for GDF15 0.93±0.04 and for FGF21 0.90±0.06. Simultaneous elevation of both markers >98th percentile of controls identified genetically confirmed MH with a sensitivity of 88% and specificity of 96%. In MH, independent predictors of survival without requiring liver transplantation were international normalized ratio and either GDF15 or FGF21 levels, with levels <2000 ng/L predicting survival without liver transplantation (p<0.01). CONCLUSIONS: GDF15 and FGF21 are significantly higher in children with MH compared to other childhood liver diseases and controls and, when combined, were predictive of MH and had prognostic implications.