RESUMO
We have used single cell transcriptome analysis to re-examine the substates of early passage, karyotypically Normal, and late passage, karyotypically Abnormal ('Culture Adapted') human embryonic stem cells characterized by differential expression of the cell surface marker antigen, SSEA3. The results confirmed that culture adaptation is associated with alterations to the dynamics of the SSEA3(+) and SSEA3(-) substates of these cells, with SSEA3(-) Adapted cells remaining within the stem cell compartment whereas the SSEA3(-) Normal cells appear to have differentiated. However, the single cell data reveal that these substates are characterized by further heterogeneity that changes on culture adaptation. Notably the Adapted population includes cells with a transcriptome substate suggestive of a shift to a more naïve-like phenotype in contrast to the cells of the Normal population. Further, a subset of the Normal SSEA3(+) cells expresses genes typical of endoderm differentiation, despite also expressing the undifferentiated stem cell genes, POU5F1 (OCT4) and NANOG, whereas such apparently lineage-primed cells are absent from the Adapted population. These results suggest that the selective growth advantage gained by genetically variant, culture adapted human embryonic stem cells may derive in part from a changed substate structure that influences their propensity for differentiation.
Assuntos
Antígenos Glicosídicos Associados a Tumores/genética , Diferenciação Celular , Antígenos Embrionários Estágio-Específicos/genética , Antígenos Glicosídicos Associados a Tumores/metabolismo , Células Cultivadas , Análise por Conglomerados , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Células-Tronco Embrionárias Humanas , Humanos , Proteína Homeobox Nanog , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Antígenos Embrionários Estágio-Específicos/metabolismo , TranscriptomaRESUMO
TaqMan Array Cards are high-throughput, accurate, sensitive, and simple-to-use tools for quantitative analysis of mRNA or miRNA transcripts using a real-time PCR protocol. They utilize a microfluidic card with 384 reaction chambers and eight sample loading ports. For studies of coding transcripts, the reaction chambers are preloaded with user selected or predefined panels of Applied Biosystems TaqMan Gene Expression Assays. These assays enable real-time monitoring of a PCR reaction via hydrolysis of an oligonucleotide probe which has been dual labeled with fluorescent dye and quencher. Applications of TaqMan Array Cards include verification and follow on testing of microarray results, as well as hypothesis driven testing of panels of genes selected for their biological functions and relationships. This chapter describes a protocol for assaying transcription in cultured cells using methods optimized to minimize hands-on time and pipetting steps by skipping RNA isolation and generating cDNA directly in Ambion Cells-to-C(T) lysis solution.
Assuntos
Indústria Farmacêutica/métodos , Análise de Sequência com Séries de Oligonucleotídeos/instrumentação , Taq Polimerase/metabolismo , Animais , Extratos Celulares/química , Células Cultivadas , Indústria Farmacêutica/instrumentação , Humanos , Camundongos , Técnicas Analíticas Microfluídicas , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Reação em Cadeia da Polimerase , Ratos , Transcrição Reversa , Fatores de TempoRESUMO
The unique properties of embryonic stem cells (ESCs) to self-renew indefinitely or to differentiate to any cell type have great potential for clinical applications in regenerative medicine. MicroRNAs (miRNAs) are emerging as important regulators of post-transcriptional gene expression and have been implicated as crucial elements in regulating ESCs. Here, we review recent progresses in characterizing the role of miRNAs in the maintenance and development of ESCs.
