Unknown,Transcriptomics,Genomics,Proteomics

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Transcriptome analysis of Stem Cells from Human Umbilical Cord Blood

ABSTRACT: Uncovering the molecular mechanism of stem cell self-renewal is critical to broaden current therapeutic applications and to understand how its de-regulation may lead to pathological conditions. In this work, we report changes in gene expression and related cell processes during conditions of high in vitro expansion of CD133+/CD34+ primitive stem cells from human umbilical cord blood (UCB). Based on DNA microarray assays, we demonstrate that primary metabolism and cell cycle genes are specifically up-regulated, as well as members of the Wnt and Notch signaling pathways. Estradiol (E2) treatment resulted in enhanced cell expansion, which was correlated with activation of MEK/ERK signaling genes and inhibition of GLI2, a member of the Hedgehog pathway. Most notably, E2-induced CD133+/CD34+ cell expansion was highly associated to regulation of genes disrupted in cancer, such as the recently described DOCK4 and SPARCL1 tumor suppressor genes. Quantitative real-time PCR analysis confirmed down-regulation of DOCK4 and SPARCL1 in E2-treated CD133+/CD34+ cells and parallel results were verified when comparing their expression in mononuclear blood cells of chronic myeloid leukemia patients and healthy individuals. The striking differential expression of cancer-associated genes found reveals potential molecular targets for oncogenic transformation of CD133+/CD34+ cells and strengthens the importance of pre-clinical studies assessing safety of stem cell expansion protocols for therapeutic application. Keywords: dose response Gene expression intensities were measured using CodeLink Human Whole Genome Bioarrays.

ORGANISM(S): Homo sapiens

SUBMITTER: Ricardo VÃªncio

PROVIDER: E-GEOD-4609 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Common molecular pathways involved in human CD133+/CD34+ progenitor cell expansion and cancer.

Okamoto Oswaldo Keith OK Carvalho Ana Carolina S R AC Marti Luciana C LC Vêncio Ricardo Z RZ Moreira-Filho Carlos A CA

Cancer cell international 20070608

<h4>Background</h4>Uncovering the molecular mechanism underlying expansion of hematopoietic stem and progenitor cells is critical to extend current therapeutic applications and to understand how its deregulation relates to leukemia. The characterization of genes commonly relevant to stem/progenitor cell expansion and tumor development should facilitate the identification of novel therapeutic targets in cancer.<h4>Methods</h4>CD34+/CD133+ progenitor cells were purified from human umbilical cord b ...[more]

PMID: 17559657

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Project description:(This section will be revisited in good time prior to publication, for final approval) Research within the field of functional genomics provides a more profound understanding of living organisms, by elucidating the interaction between genes and gene products. This is of increasing importance in many fields, and especially within cancer research. With the use of microarray technology, the gene expression of a vast number of genes can be measured simultaneously. This allows us insight into tumor biology, and an opportunity to obtain knowledge of diagnostic and prognostic value, as well as hints towards the causes of cancer. In this study the gene expression in a selection of human neuroendocrine (NE) and non-neuroendocrine tumor cell lines were compared. Both qualitative and quantitative methods were applied to get an insight into the NE tumor biology. NE cells are found in practically every organ and tissue in the body and are assigned to synthesize peptide hormones and take up amino acids and transform these into biogenic amines by carboxylation. NE tumors arise after malignant transformation of neuroendocrine cells and are usually highly differentiated and have a low growth rate, which makes this a high prevalence tumor type. These rather rare tumors characteristically express NE markers, such as chromogranin A, and contains characteristic dense-secretory core granules. By using cDNA microarray analysis, the gene expression profiles of various NE tumor cell lines (e. g. carcinoid, neuroblastoma, medullary thyroid carcinoma and endocrine pancreatic tumor) were compared with those of non-NE tumor cells. As a reference the commercially available Universal human RNA reference (Stratagene) was used. Verification of the results were done by real time RT-PCR analysis after the NE nature of the cell lines was stated by immunohistochemical, electron microscopic and light microscopic examinations. A large number of genes were differentially expressed in NE cell lines vs. the non-NE cell lines, and were found to be of interest in NE tumor biology. Some of these genes were previously known in NE tumor biology, whereas the vast majority of the specified genes are novel in the context of NE tumor biology. Genes with little previous information were also identified and thought to be an asset in the search of new NE markers. A feasible prospective of the study would be expansion with additional samples cell lines and a more thorough verification with real time RT-PCR analysis. This would enhance the credibility and thus the utility of the findings. Future studies on the biological functions of the candidate genes and the mechanisms of their regulation, will hopefully enable suggestions of new NE markers of prognostic or diagnostic value, and eventually new drug targets. Keywords: neuroendocrine, neuroendocrine markers, gene expression profiling, cDNA microarray The hybridisation was performed by an experienced laboratory technician Eli Helge, (Dept. of Laboratory Medicine, Children`s and Women`s health, NTNU) at the microarray core facility. Three separate runs with 10, 11 and 3 slides was needed to obtain the 20 hybridisations (dyeswap x 10 cell lines) planned for this study. Of the 10 cell lines 6 were neuroendocrine and 4 were non-neuroendocrine. For comparison between these two groups the universal human reference RNA (Stratagene) was used as the "control" on each slide. An adapted version of the 3DNA Array 350 protocol (Genisphere) was used. The slides were scanned with a ScanArrayTMExpressHT (Packard BioScience) twice immediately after the hybridisation was completed to avoid photo bleaching of the fluorescent dyes.

Project description:It is now well established that mature mammalian spermatozoa carry a population of mRNA molecules, at least some of which are transferred to the oocyte at fertilisation. However, the function of the sperm transcriptome remains largely unclear. To shed light on the evolutionary conservation of this feature of sperm biology, we analysed highly purified populations of mature sperm from the fruitfly, Drosophila melanogaster. As with mammalian sperm, we found a consistently enriched population of mRNA molecules that are not likely to be derived from contaminating somatic cells or immature sperm. Using tagged transcripts for three of the spermatozoal mRNAs, we demonstrate that they are transferred to the oocyte at fertilisation and can be detected at least until the onset of zygotic gene expression. We find a remarkable conservation in the functional annotations associated with fly and human spermatozoal mRNAs, in particular a highly significant enrichment for transcripts encoding Ribosomal Proteins. The identification of a conserved set of spermatozoal transcripts opens the possibility of using the power of Drosophila genetics to address the function of this enigmatic class of molecules. RNA extracted from three biological replicates of purified sperm (Sperm rep1, Sperm rep2 and Sperm rep3) was used as a template for oligo-dT-primed reverse transcription, amplification, labelling of dye swapped technical replicates and hybridisation to long oligonucleotides microarrays. As a control, RNA from two biological replicates of dissected adult testis plus accessory glands (Testis_rep1, Testis_rep2) was amplified, labelled (dye-swap technical replicate) and hybridised to similar arrays. To help with the spot-finding of the arrays genomic DNA was co-hybridised in some cases (this genomic DNA data was excluded from further analysis). Genes with an intensity level below 200 in at least one channel across the Sperm or Testis set were removed (5579 transcripts present in all three sperm replicates, 5358 transcripts from the testis/accessory gland samples and 4295 transcripts common to both data sets). Then the quantile normalisation was independently applied to the Sperm replicate samples and Testis replicates.

Dataset Information

Transcriptome analysis of Stem Cells from Human Umbilical Cord Blood

Publications

Common molecular pathways involved in human CD133+/CD34+ progenitor cell expansion and cancer.

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