RESUMEN
Blood cell formation is classically thought to occur through a hierarchical differentiation process, although recent studies have shown that lineage commitment may occur earlier in hematopoietic stem and progenitor cells (HSPCs). The relevance to human blood diseases and the underlying regulation of these refined models remain poorly understood. By studying a genetic blood disorder, Diamond-Blackfan anemia (DBA), where the majority of mutations affect ribosomal proteins and the erythroid lineage is selectively perturbed, we are able to gain mechanistic insight into how lineage commitment is programmed normally and disrupted in disease. We show that in DBA, the pool of available ribosomes is limited, while ribosome composition remains constant. Surprisingly, this global reduction in ribosome levels more profoundly alters translation of a select subset of transcripts. We show how the reduced translation of select transcripts in HSPCs can impair erythroid lineage commitment, illuminating a regulatory role for ribosome levels in cellular differentiation.
Asunto(s)
Anemia de Diamond-Blackfan/patología , Ribosomas/metabolismo , Regiones no Traducidas 5' , Anemia de Diamond-Blackfan/genética , Proteínas Reguladoras de la Apoptosis/antagonistas & inhibidores , Proteínas Reguladoras de la Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/metabolismo , Células de la Médula Ósea/metabolismo , Células Cultivadas , Femenino , Factor de Transcripción GATA1/genética , Factor de Transcripción GATA1/metabolismo , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Humanos , Masculino , Mutación Missense , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Proteínas Ribosómicas/antagonistas & inhibidores , Proteínas Ribosómicas/genética , Proteínas Ribosómicas/metabolismo , Ribosomas/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Immunodeficient mouse models have been valuable for studies of human hematopoiesis, but high-fidelity recapitulation of erythropoiesis in most xenograft recipients remains elusive. Recently developed immunodeficient and Kit mutant mice, however, have provided a suitable background to achieve higher-level human erythropoiesis after long-term hematopoietic engraftment. While there has been some characterization of human erythropoiesis in these models, a comprehensive analysis from various human developmental stages has not yet been reported. Here, we have utilized cell surface phenotypes, morphologic analyses, and molecular studies to fully characterize human erythropoiesis from multiple developmental stages in immunodeficient and Kit mutant mouse models following long-term hematopoietic stem and progenitor cell engraftment. We show that human erythropoiesis in such models demonstrates complete maturation and enucleation, as well as developmentally appropriate globin gene expression. These results provide a framework for future studies to utilize this model system for interrogating disorders affecting human erythropoiesis and for developing improved therapeutic approaches.
Asunto(s)
Eritropoyesis , Trasplante de Células Madre Hematopoyéticas , Mutación , Proteínas Proto-Oncogénicas c-kit/metabolismo , Animales , Xenoinjertos , Humanos , Ratones , Ratones Endogámicos NOD , Ratones SCID , Proteínas Proto-Oncogénicas c-kit/genéticaRESUMEN
Genome-wide association studies (GWAS) have identified thousands of variants associated with human diseases and traits. However, the majority of GWAS-implicated variants are in non-coding regions of the genome and require in depth follow-up to identify target genes and decipher biological mechanisms. Here, rather than focusing on causal variants, we have undertaken a pooled loss-of-function screen in primary hematopoietic cells to interrogate 389 candidate genes contained in 75 loci associated with red blood cell traits. Using this approach, we identify 77 genes at 38 GWAS loci, with most loci harboring 1-2 candidate genes. Importantly, the hit set was strongly enriched for genes validated through orthogonal genetic approaches. Genes identified by this approach are enriched in specific and relevant biological pathways, allowing regulators of human erythropoiesis and modifiers of blood diseases to be defined. More generally, this functional screen provides a paradigm for gene-centric follow up of GWAS for a variety of human diseases and traits.
Asunto(s)
Enfermedades Genéticas Congénitas , Predisposición Genética a la Enfermedad , Hematopoyesis/genética , Sitios de Carácter Cuantitativo/genética , Eritrocitos/metabolismo , Eritrocitos/patología , Estudio de Asociación del Genoma Completo , Humanos , Polimorfismo de Nucleótido Simple/genéticaRESUMEN
A 3D cell culture chip was used for high-throughput screening of a human neural progenitor cell line. The differential toxicity of 24 compounds was determined on undifferentiated and differentiating NPCs. Five compounds led to significant differences in IC50 values between undifferentiated and differentiating cultures. This platform has potential use in phenotypic screening to elucidate molecular toxicology on human stem cells.
Asunto(s)
Descubrimiento de Drogas , Ensayos Analíticos de Alto Rendimiento , Células-Madre Neurales/efectos de los fármacos , Células-Madre Neurales/metabolismo , Fenotipo , Técnicas de Cultivo de Célula , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/genética , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Relación Dosis-Respuesta a Droga , Humanos , Células-Madre Neurales/citología , Análisis de Secuencia por Matrices de Oligonucleótidos , Pruebas de Toxicidad AgudaRESUMEN
We have combined molecular beacon (MB) probes with barcoded metal nanowires to enable no-wash, sealed chamber, multiplexed detection of nucleic acids. Probe design and experimental parameters important in nanowire-based MB assays are discussed. Loop regions of 24 bases and 5 base pair stem regions in the beacon probes gave optimal performance. Our results suggest that thermodynamic predictions for secondary structure stability of solution-phase MB can guide probe design for nanowire-based assays. Dengue virus-specific probes with predicted solution-phase DeltaG of folding in 500 mM buffered NaCl of approximately -4 kcal/mol performed better than those with DeltaG > -2 or < -6 kcal/mol. Buffered 300-500 mM NaCl was selected after comparison of several buffers previously reported for similar types of assays, and 200-500 mM NaCl was found to be the optimal ionic strength for the hybridization temperatures (25 and 50 degrees C) and probe designs used here. Target binding to the surface as a function of solution concentration fit a Sips isotherm with Kd = 1.7 +/- 0.3 nM. The detection limit was approximately 100 pM, limited by incomplete quenching. Single base mismatches could be discriminated from fully complementary targets. Oligonucleotide target sequences specific for human immunodeficiency, hepatitis C, and severe acute respiratory viruses were assayed simultaneously in a no-wash, sealed chamber, multiplexed experiment in which each of three probe sequences was attached to a different pattern of encoded nanowires. Finally, we demonstrated that probe-coated nanowires retain their selectivity and sensitivity in a triplexed assay after storage for over 3 months.