Unknown,Transcriptomics,Genomics,Proteomics

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RNA-sequencing of human umbilical cord-derived MSC (UCX) in normoxia and hypoxia

ABSTRACT: Human umbilical cord matrix-derived mesenchymal stromal cells (UCM-MSC) are advantageous since can be easily obtained and display special interest as universal and feasible add-on therapy for myocardial infarction (MI). In this study, UCM-MSC from two umbilical cords, UC-A and UC-B, were transplanted in a murine MI model to investigate consistency and durability of the therapeutic benefits. Both cellular products supported sustained and long-term beneficial therapeutic effect. In vitro, the two cell products displayed similar ability to induce the formation of vessel-like structures and comparable transcriptome in normoxia and hypoxia, apart from expression differences in a small subset of genes associated with MHC Class I. These findings support that UCM-MSC are strong candidates to assist the treatment of MI whilst calling for the discussion on methodologies to characterize and select best performing UCM-MSC before clinical application.

INSTRUMENT(S): Ion Torrent S5 XL

ORGANISM(S): Homo sapiens

SUBMITTER: Diana Nascimento

PROVIDER: E-MTAB-9978 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Mesenchymal stem cells (MSC) are multipotent cells which can be obtained from several adult and fetal tissues including human umbilical cord units. We have recently shown that umbilical cord tissue (UC) is richer in MSC than umbilical cord blood (UCB) but their origin and characteristics in blood as compared to the cord remains unknown. Here we compared, for the first time, the exonic protein-coding and intronic noncoding RNA (ncRNA) expression profiles of MSC from match-paired UC and UCB samples, harvested from the same donors, processed simultaneously and under the same culture conditions. The patterns of intronic ncRNA expression in MSC from UC and UCB paired units were highly similar, indicative of their common donor origin. The respective exonic protein-coding transcript expression profiles, however, were significantly different. Hierarchical clustering based on protein-coding expression similarities grouped MSC according to their tissue location rather than original donor. Genes related to systems development, osteogenesis and immune system were expressed at higher levels in UCB, whereas genes related to cell adhesion, morphogenesis, secretion, angiogenesis and neurogenesis were more expressed in UC cells. These molecular differences verified in tissue-specific MSC gene expression may reflect functional activities influenced by distinct niches and should be considered when developing clinical protocols involving MSC from different sources. In addition, these findings reinforce our previous suggestion on the importance of banking the whole umbilical cord unit for research or future therapeutic use. Cy5- and Cy3-labeled cRNAs were obtained using 300 ng total RNA as template for amplification of poly(A) RNA by T7-RNA polymerase with the Agilent Low RNA Input Fluorescent Linear Amplification kit. The T7-polymerase amplified cRNA labeling approach advantageously replaces the reverse-transcriptase cDNA labeling used in early microarray experiments, because T7-RNA polymerase labeling of cRNA preserves the strand orientation of the original mRNA template. Reverse-transcriptase labeling can eventually generate a complementary cDNA second strand and cause artifactual labeling of a target with the opposite sense to that of the original message. Hybridization of 825 ng of Cy3- or Cy5-labeled RNA (dye swap technical replicate) from each UC sample with its paired UCB sample was performed with Agilent in situ Hybridization kit-plus, as recommended by the manufacturer, using a total of 4 intron-exon 44K expression oligoarrays. This array comprises a total of 13,699 exonic probes representing different protein-coding genes, along with custom-designed intronic probes for the antisense or sense strand. Slides were washed and processed according to the Agilent Two-Color Microarray-Based Gene Expression Analysis protocol (Version 5.5) and scanned on a GenePix 4000B scanner (Molecular Devices, Sunnyvale, CA, USA). Fluorescence intensities were extracted using Feature Extraction (FE) software (version 9.0; Agilent). A gene was considered expressed if its probe intensity was significantly higher than the local background intensity, as calculated by the FE software. Then, the software applies local background subtraction and corrects for unequal dye incorporation using the default LOWESS (locally weighted linear regression) method.

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RNA-sequencing of human umbilical cord-derived MSC (UCX) in normoxia and hypoxia

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