Unknown,Transcriptomics,Genomics,Proteomics

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Characterization of microRNA-offset RNAs in human embryonic stem cells (hESCs)

ABSTRACT: Small RNA molecules, including microRNAs (miRNAs), play critical roles in regulating pluripotency, proliferation and differentiation of embryonic stem cells. miRNA-offset RNAs (moRNAs) are similar in length to miRNAs, align to miRNA precursor (pre-miRNA) loci and are therefore believed to derive from processing of the pre-miRNA hairpin sequence. Recent next generation sequencing (NGS) studies have reported the presence of moRNAs in human neurons and cancer cells and in several tissues in mouse, including pluripotent stem cells. In order to gain additional knowledge about human moRNAs and their putative development-related expression, we applied NGS of small RNAs in human embryonic stem cells (hESCs) and fibroblasts. We found that certain moRNA isoforms are notably expressed in hESCs from loci coding for stem cell-selective or cancer-related miRNA clusters. In contrast, we observed only sparse moRNAs in fibroblasts. Consistent with earlier findings, most of the observed moRNAs derived from conserved loci and their expression did not appear to correlate with the expression of the adjacent miRNAs. We provide here the first report of moRNAs in hESCs, and their expression profile in in comparison to fibroblasts. Moreover, we expand the repertoire of hESC miRNAs. These findings provide an expansion on the known repertoire of small non-coding RNA contents in hESCs. We performed small RNA-seq analysis from three samples: one from hESC line HS401, one from hESC line HS181 and one from human foreskin fibroblast line HFF-1

ORGANISM(S): Homo sapiens

SUBMITTER: Suvi Asikainen

PROVIDER: E-GEOD-62501 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Recent advances in the study of human embryonic stem cells (hESCs) and induced-pluripotent stem cells (iPS) highlight their importance as both model systems for basic research and avenues for therapeutic applications. To gain better insight into these cells, a clearer understanding of their molecular properties is crucial. In this study, we analyze changes in the expression profile of microRNAs (miRNAs) and mRNAs in nine different, National Institutes of Health (NIH)-approved hESC lines. By examining both undifferentiated hESCs and cells exposed to an undirected differentiation scheme at early stages, we found those miRNAs and mRNAs enriched in the hESC lines, and those miRNAs and mRNAs initially regulated upon commitment. In comparing these profiles with those of an embryonal carcinoma (EC) cell line, NTera-2, we observed distinct patterns of miRNA expression in hESCs. Furthermore, we identify several new hESC-enriched miRNAs that respond rapidly to differentiation cues, in addition to miRNAs from a large cluster of hESC-enriched miRNAs located on chromosome 19, and miRNAs from the miR-302 cluster and the miR-17~92 family of clusters. We show that key miRNAs in the chromosome 19 cluster are highly enriched in hESCs in comparison to NTera-2, and might therefore serve as future diagnostic markers for stem cell capacity. Examination of changes in mRNA expression during early commitment further reveals that many differentiation-regulated genes are possible candidates for regulation by these hESC-enriched miRNAs. A set of 9 different NIH-approved hESC lines were treated with conditions to promote either maintenance of an undifferentiated state (mouse embryonic fibroblast- conditioned media) or undirected differentiation of the hESCs into various cell lineages (DMEM + 20% FBS). RNA was harvested from the cells and subjected to miRNA microarray analysis. An embryonal carcinma cell line, NTera-2, which shows similar growth characteristics to hESCs was used an additional comparative sample. Additionally, human placental RNA samples were used as internal controls for miRNA microarray slide (Agilent Technologies). The miRNA expression data was used to analyze differential miRNA expression in each specific cell line, as well as large-scale comparisons of the undifferentiated and differentiated hESC lines.

Project description:The MLL-AF4 fusion gene is a hallmark genomic aberration in high-risk acute lymphoblastic leukemia in infants. Although it is well-established that MLL-AF4 arises pre-natally during human development, its effects on hematopoietic development in utero remains unexplored. We have created a human-specific in vitro system to study early hemato-endothelial development in MLL-AF4-expressing human embryonic stem cells (hESCs). Differentiation and functional studies as well as clonal analyses and gene expression profiling reveal that expression of MLL-AF4 in hESCs has a phenotypic, functional and gene expression impact. It enhances the specification of hemogenic precursors from hESCs and impairs further hematopoietic commitment of these precursors in favour of the endothelial cell fate. Similar to that reported in cord blood CD34+ hematopoietic stem/progenitor cells (HSPCs), MLL-AF4 expression is not sufficient to transform hESC-derived hematopoietic cells in vitro or in vivo, indicating that additional events may be required to initiate leukemogenesis or that embryonic hematopoiesis is not the appropriate human cellular target for MLL-AF4-mediated leukemogenesis. This work illustrates how hESCs can provide unique insights into human development and further our understanding of how leukemic fusion genes known to arise pre-natally regulate human embryonic hematopoietic specification. MLL is involved in transcriptional regulation and most MLL translocations appear to result in increased expression of Hox genes and hematopoietic genes. We therefore assessed the impact of MLL-AF4 expression on the transcriptome of hESCs. Gene expression profiling performed in MLL-AF4 hESCs revealed that MLL-AF4 preferentially activates transcription. 1826 out of the 3001 genes (61%) expressed were up-regulated in MLL-AF4 hESCs. Human ESC samples were collected during the exponential cell growth phase and stabilized in RNA later. 500 ng of each total RNA sample was labelled with Cy3 using the Quick-Amp Labelling kit and hybridized with the Gene Expression Hybridization kit to a Whole Human Genome Oligo Microarray (Agilent Technologies) following the Manufacturer’s instructions. Each cell line was analyzed as independent duplicates. NEO-expressing (empty lentivector) hESC line was used as the baseline.

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Characterization of microRNA-offset RNAs in human embryonic stem cells (hESCs)

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