RESUMEN
BACKGROUND: Pooled human embryonic stem cells (hESC) cell lines were profiled to obtain a comprehensive list of genes common to undifferentiated human embryonic stem cells. RESULTS: Pooled hESC lines were profiled to obtain a comprehensive list of genes common to human ES cells. Massively parallel signature sequencing (MPSS) of approximately three million signature tags (signatures) identified close to eleven thousand unique transcripts, of which approximately 25% were uncharacterised or novel genes. Expression of previously identified ES cell markers was confirmed and multiple genes not known to be expressed by ES cells were identified by comparing with public SAGE databases, EST libraries and parallel analysis by microarray and RT-PCR. Chromosomal mapping of expressed genes failed to identify major hotspots and confirmed expression of genes that map to the X and Y chromosome. Comparison with published data sets confirmed the validity of the analysis and the depth and power of MPSS. CONCLUSIONS: Overall, our analysis provides a molecular signature of genes expressed by undifferentiated ES cells that can be used to monitor the state of ES cells isolated by different laboratories using independent methods and maintained under differing culture conditions
Asunto(s)
Embrión de Mamíferos/citología , Perfilación de la Expresión Génica/métodos , Células Madre/química , Células Madre/metabolismo , Diferenciación Celular/genética , Mapeo Cromosómico/métodos , ADN Complementario/genética , Regulación del Desarrollo de la Expresión Génica/genética , HumanosRESUMEN
To identify genes that may be involved in the process of human embryonic stem cell (hESC) differentiation, we profiled gene expression by expressed sequenced tag (EST) enumeration and massively parallel signature sequencing (MPSS) using RNA samples from feeder-free cultures of undifferentiated (passages 40-50) and differentiated (day 14) H1, H7, and H9 lines. MPSS and EST scan analysis showed good concordance and identified a large number of genes that changed rapidly as cultures transition from a pluripotent to a differentiated state. These included known and unknown ES cell-specific genes as well as a large number of known genes that were altered as cells differentiate. A subset of genes that were either up- or down-regulated were selected and their differential expression confirmed by a variety of independent methods, including comparison of expression after further differentiation, publicly available databases, and direct assessments by reverse transcriptase (RT)-PCR and immunocytochemistry. The analysis identified markers unique to the hESC and embryoid bodies (hEBs) stage as well as signaling pathways that likely regulate differentiation. The data generated can be used to monitor the state of hESC isolated by different laboratories using independent methods and maintained under differing culture conditions.
Asunto(s)
Embrión de Mamíferos/citología , Etiquetas de Secuencia Expresada , Perfilación de la Expresión Génica , Células Madre/citología , Técnicas de Cultivo de Célula/métodos , Diferenciación Celular , Línea Celular , Células Cultivadas , Mapeo Cromosómico/métodos , Cromosomas Humanos Par 12 , Bases de Datos como Asunto , Regulación de la Expresión Génica , Genoma Humano , Humanos , Inmunohistoquímica , Familia de Multigenes , ARN/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal , Regulación hacia ArribaRESUMEN
Massively parallel signature sequencing (MPSS) generates millions of short sequence tags corresponding to transcripts from a single RNA preparation. Most MPSS tags can be unambiguously assigned to genes, thereby generating a comprehensive expression profile of the tissue of origin. From the comparison of MPSS data from 32 normal human tissues, we identified 1,056 genes that are predominantly expressed in the testis. Further evaluation by using MPSS tags from cancer cell lines and EST data from a wide variety of tumors identified 202 of these genes as candidates for encoding cancer/testis (CT) antigens. Of these genes, the expression in normal tissues was assessed by RT-PCR in a subset of 166 intron-containing genes, and those with confirmed testis-predominant expression were further evaluated for their expression in 21 cancer cell lines. Thus, 20 CT or CT-like genes were identified, with several exhibiting expression in five or more of the cancer cell lines examined. One of these genes is a member of a CT gene family that we designated as CT45. The CT45 family comprises six highly similar (>98% cDNA identity) genes that are clustered in tandem within a 125-kb region on Xq26.3. CT45 was found to be frequently expressed in both cancer cell lines and lung cancer specimens. Thus, MPSS analysis has resulted in a significant extension of our knowledge of CT antigens, leading to the discovery of a distinctive X-linked CT-antigen gene family.