RESUMEN
Human induced pluripotent stem cells (hiPSCs) are reprogrammed from somatic cells and are regarded as promising sources for regenerative medicine and disease research. Recently, techniques for analyses of individual cells, such as single-cell RNA-Seq and mass cytometry, have been used to understand the stem cell reprogramming process in the mouse. However, the reprogramming process in hiPSCs remains poorly understood. Here we used mass cytometry to analyze the expression of pluripotency and cell cycle markers in the reprogramming of human stem cells. We confirmed that, during reprogramming, the main cell population was shifted to an intermediate population consisting of neither fibroblasts nor hiPSCs. Detailed population analyses using computational approaches, including dimensional reduction by spanning-tree progression analysis of density-normalized events, PhenoGraph, and diffusion mapping, revealed several distinct cell clusters representing the cells along the reprogramming route. Interestingly, correlation analysis of various markers in hiPSCs revealed that the pluripotency marker TRA-1-60 behaves in a pattern that is different from other pluripotency markers. Furthermore, we found that the expression pattern of another pluripotency marker, octamer-binding protein 4 (OCT4), was distinctive in the pHistone-H3high population (M phase) of the cell cycle. To the best of our knowledge, this is the first mass cytometry-based investigation of human reprogramming and pluripotency. Our analysis elucidates several aspects of hiPSC reprogramming, including several intermediate cell clusters active during the process of reprogramming and distinctive marker expression patterns in hiPSCs.
Asunto(s)
Biomarcadores , Regulación de la Expresión Génica , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Fosfatasa Alcalina/metabolismo , Antígenos de Superficie/metabolismo , Biomarcadores/metabolismo , Ciclo Celular/genética , Ciclo Celular/fisiología , Línea Celular , Reprogramación Celular/genética , Reprogramación Celular/fisiología , Biología Computacional , Técnica del Anticuerpo Fluorescente , Humanos , Citometría de Imagen , Proteína Homeótica Nanog/metabolismo , Factor 3 de Transcripción de Unión a Octámeros/genética , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Fenotipo , Proteoglicanos/metabolismo , Factores de Transcripción SOXB1/metabolismo , Análisis de la Célula IndividualRESUMEN
BACKGROUND: Burkholderia pseudomallei is a facultative intracellular pathogen and the causative agent of melioidosis. A conserved type III secretion system (T3SS3) and type VI secretion system (T6SS1) are critical for intracellular survival and growth. The T3SS3 and T6SS1 genes are coordinately and hierarchically regulated by a TetR-type regulator, BspR. A central transcriptional regulator of the BspR regulatory cascade, BsaN, activates a subset of T3SS3 and T6SS1 loci. RESULTS: To elucidate the scope of the BsaN regulon, we used RNAseq analysis to compare the transcriptomes of wild-type B. pseudomallei KHW and a bsaN deletion mutant. The 60 genes positively-regulated by BsaN include those that we had previously identified in addition to a polyketide biosynthesis locus and genes involved in amino acid biosynthesis. BsaN was also found to repress the transcription of 51 genes including flagellar motility loci and those encoding components of the T3SS3 apparatus. Using a promoter-lacZ fusion assay in E. coli, we show that BsaN together with the chaperone BicA directly control the expression of the T3SS3 translocon, effector and associated regulatory genes that are organized into at least five operons (BPSS1516-BPSS1552). Using a mutagenesis approach, a consensus regulatory motif in the promoter regions of BsaN-regulated genes was shown to be essential for transcriptional activation. CONCLUSIONS: BsaN/BicA functions as a central regulator of key virulence clusters in B. pseudomallei within a more extensive network of genetic regulation. We propose that BsaN/BicA controls a gene expression program that facilitates the adaption and intracellular survival of the pathogen within eukaryotic hosts.
Asunto(s)
Burkholderia pseudomallei/genética , Regulación Bacteriana de la Expresión Génica , Regulón , Factores de Transcripción/metabolismo , Factores de Virulencia/genética , Factores de Virulencia/metabolismo , Eliminación de Gen , Perfilación de la Expresión Génica , Chaperonas Moleculares/metabolismo , Familia de Multigenes , Factores de Transcripción/genéticaRESUMEN
BACKGROUND: Burkholderia pseudomallei is the causative agent of melioidosis, a potentially fatal disease endemic in Southeast Asia and Northern Australia. This Gram-negative pathogen possesses numerous virulence factors including three "injection type" type three secretion systems (T3SSs). B. pseudomallei has been shown to activate NFκB in HEK293T cells in a Toll-like receptor and MyD88 independent manner that requires T3SS gene cluster 3 (T3SS3 or T3SSBsa). However, the mechanism of how T3SS3 contributes to NFκB activation is unknown. RESULTS: Known T3SS3 effectors are not responsible for NFκB activation. Furthermore, T3SS3-null mutants are able to activate NFκB almost to the same extent as wildtype bacteria at late time points of infection, corresponding to delayed escape into the cytosol. NFκB activation also occurs when bacteria are delivered directly into the cytosol by photothermal nanoblade injection. CONCLUSIONS: T3SS3 does not directly activate NFκB but facilitates bacterial escape into the cytosol where the host is able to sense the presence of the pathogen through cytosolic sensors leading to NFκB activation.
