Unknown,Transcriptomics,Genomics,Proteomics

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Cultured human amniotic fluid-derived mesenchymal stromal cells [PIQOR data]

ABSTRACT: Human amniotic fluid-derived mesenchymal stromal cells (hAMSC) have become one of the main cell populations used in regenerative medicine and for the study of various clinical disorders. These cells have a great capacity for proliferation and differentiation, do not form teratomas when transplanted into animal models and their stemness seems to be between embryonic cells and adult mesenchymal cells. Prior to their use in cell therapy they must be cultured and expanded in vitro, but the effect this process has on their fitness, a determining factor for the success or failure of cell therapy, is unknown. We undertook a follow-up of gene and microRNAs (miRNAs) expression using microarray of a hAMSC population kept under in vitro culture conditions for the first 15 passages. Significant changes were noted in the expression of various mRNAs and miRNAs, particularly down-regulation of TP53, increased expression of hsa-miR-125a and up-regulation of CDKN2D. The variations in TP53 and hsa-miR-125a may act as an indicator of the stemness of the hAMSC, whereas CDKN2D may indicate the reduction in the proliferative capacity of these cells in a TP53-independent mechanism. The genes described in this study will help evaluate the fitness of hAMSC, thus guaranteeing their biological quality for use in regenerative medicine. The variations in the expression patterns of the mRNAs were studied using topic-defined PIQOR Stem Cell microarrays. Human amniotic fluid-derived mesenchymal stromal cells pellets of 5 x 10E5 cells were cryopreserved in liquid nitrogen and send to Miltenyi Biotec for their analysis. A pool of non-stimulated lymphocytes (non-mitotic cell cycle) obtained by Ficoll isolation in the Immunology Service of Carlos Haya hospital was used as a control of the expression.

ORGANISM(S): Homo sapiens

SUBMITTER: Jose Maria Miranda-Sayago

PROVIDER: E-GEOD-30064 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:The COP9 signalosome (CSN) is a conserved protein complex occurring in all eukaryotes. The mammalian CSN consists of eight subunits (CSN1 – CSN8) and possesses an intrinsic metalloprotease JAMM motif localised to CSN5. In addition, it is associated with kinases such as protein kinase CK2 and D and the ubiquitin (Ub) specific protease 15. It regulates cullin-RING Ub ligases (CRLs) that label proteins for proteolysis with poly-Ub chains in the Ub proteasome system (UPS). CRLs contain one of the scaffolding cullins 1 – 7, a RING domain protein e. g. Rbx1, and one of hundreds of substrate binding units such as F-box or BTB proteins that specifically target proteins for ubiquitination. The CSN forms functional supercomplexes with CRLs. It removes the Ub-like protein Nedd8 from cullins by its metalloprotease activity and thereby inactivates CRLs. On the other hand, it is essential for CRL function by protecting components and allowing reassembly of the complexes. As a regulator of the UPS the CSN is involved in cell cycle10, DNA repair and development. Overexpression of CSN subunits has been reported in tumour cells. Up to date nothing is known about CSN biogenesis and its regulation. Ten years ago we have observed that overexpression of selected CSN subunits caused a coordinated increase of all CSN subunits and a de novo biosynthesis of the entire complex. Here we show that overexpression of CSN1 mediates an increase of c-Myc which coordinates CSN subunit expression via microRNAs (miRNAs). miRNAs are evolutionarily conserved, endogenous, small, noncoding RNA molecules of about 22 nucleotides that function as posttranscriptional gene regulators. We found that inhibitors of let-7 family miRNAs or upregulation of c-Myc, which represses let-7 miRNAs, resulted in a coordinated increase of CSN subunits and de novo CSN biogenesis. Keywords: Gene expression analysis of human RNA samples using topic-defined PIQOR™ Ubiquitin-PS Microarrays. Two different platforms were used (these are two different microarray batches which are identical in terms of the spotted cDNAs, but differ in the position of some cDNAs) Gene expression analysis of Hela cells overexpressing either CSN2 or CSN1 was performed to study CSN biogenesis and its regulation. Curcumin, Nr. 8 (Curcumin analogon) and Piceatannol were used for the treatment of Hela cells since they inhibit the CSN associated kinases. The aim was to investigate the effect on the signalosome. For each treatment 50 µM Curcumin, Nr. 8 and Piceatannol were used.

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Cultured human amniotic fluid-derived mesenchymal stromal cells [PIQOR data]

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