Unknown,Transcriptomics,Genomics,Proteomics

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Transcriptome profile in the human synovial MSC-aggregates

ABSTRACT: One of strategies to regenerate cartilage defect is transplantation of mesenchymal stem cells (MSCs). Improvements of therapeutic potential of MSCs are needed to achieve successful cartilage regeneration by transplantation of a limited number of cells. Aggregated culture is a popular method in ES and iPS cells to maintain or enhance their potentials. Here we investigated gene expression profile of aggregated MSCs. 621 genes were up-regulated and 409 genes were down-regulated more than 5-fold in MSC-aggregates compared with the number in MSCs in a monolayer culture. The most up-regulated gene was BMP2, which is one of the genes involved in chondrogenesis. Anti-inflammatory genes were also up-regulated in MSC-aggregates. The microarray data for selected genes were confirmed by real-time PCR. Human synovial MSCs was isolated from synovium of 3 distinct donors. The gene expression profile of MSC-aggregates cultured in hanging drop for 3days was compared with that of MSCs in a monolayer culture.

ORGANISM(S): Homo sapiens

SUBMITTER: Akihiro Umezawa

PROVIDER: E-GEOD-31980 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Fetal cartilage fully regenerates following injury while in adult mammals cartilage injury leads to osteoarthritis (OA). OA is characterized by cartilage breakdown and joint inflammation and associated with significant pain and socioeconomic costs. As no clinically satisfactory treatment is available to date, disease-modifying therapies aimed to achieve cartilage regeneration are urgently required. The inherent regeneration potential of fetal individuals may hold answers to this unmet need. Therefore, to characterize the differences in fetal and adult response to cartilage injury, we carried out histology and comprehensive proteome analyses on fetal (day 80/150-day gestation) and adult cartilage samples one (fetal samples) and three (adult and fetal samples) days after surgical induction of a full-thickness cartilage lesion. In addition, proteins secreted by inflamed fetal MSCs in vitro were compared with the in vivo response to injury to evaluate their therapeutic potential. Histology of synovial samples revealed the presence of neutrophils one day post injury (p.i.) and an influx of macrophages into the subsynovial tissue on day 3 p.i. in fetal samples. In contrast, adult synovial samples showed invasion of neutrophils on day 3 p.i. Activation and migration of Iba1+- macrophages of the synovial lining was observed both in fetal and adult animals. Comparative mass spectrometry revealed 57 proteins significantly up-regulated (> 2FC, FDR<0.05), and 67 proteins significantly down-regulated (<-2 FC) upon injury in adults. Neutrophil-related proteins and acute phase proteins were the two major upregulated protein groups in adult cartilage following injury compared to fetal sheep. In contrast, several immunomodulating proteins and growth factors were significantly higher expressed in the fetus than the adult. Comparison of the in vitro MSCs with the in vivo fetal proteome revealed shared upregulation of 17 proteins, which were considered to be of potential therapeutic interest. The results of this study support our molecular understanding of successful fetal cartilage healing and new therapeutic strategies to induce regeneration in adult articular cartilage by modulating the inflammatory environment. The shared protein upregulation in fetal cartilage in vivo and in fetal MSCS during in vitro inflammation supports the possible therapeutic potential of these factors in specific and fetal MSCs in general.

Project description:We have previously reported that the deficiency of p53 alone or in combination with Rb (Rb-/- p53-/-) in adipose-derived MSCs (ASCs) promotes leiomyosarcoma-like tumors in vivo. Here, we hypothesized that the source of MSCs and/or the cell differentiation stage could determine the phenotype of sarcoma development. To investigate whether there is a link between the source of MSCs and sarcoma phenotype, we generated p53-/- and Rb-/-p53-/- MSCs from bone marrow (BM-MSCs). Both genotypes of BM-MSCs initiated leiomyosarcoma formation similar to p53-/- and Rb-/-p53-/- ASCs. In addition, gene expression profiling revealed a link between p53- or Rb-p53-deficient BM-MSCs and ASCs and muscle-associated sarcomagenesis. These data suggest that the tissue source of MSC does not seem a crucial factor in the development of a particular sarcoma phenotype. To analyze whether the differentiation stage defines the sarcoma phenotype, BM-MSCs and ASCs were induced to differentiate towards the osteogenic lineage, and both p53 and Rb were excised using Cre-expressing adenovectors at different stages along osteogenic differentiation. Regardless of the level of osteogenic commitment, the inactivation of Rb and p53 in BM-MSC-derived, but not in ASC-derived, osteogenic progenitors gave rise to osteosarcoma-like tumors which could be serially transplanted. This indicates that the osteogenic differentiation stage of BM-MSCs imposes the phenotype of in vivo sarcoma development, and that BM-MSC-derived osteogenic progenitors rather than undifferentiated BM-MSCs, undifferentiated ASCs or ASC-derived osteogenic progenitors, represent the cell of origin for osteosarcoma development. To analyse whether the BM-MSC and Fat-MSC (ASC) differentiation stage may define the sarcoma phenotype, RbloxP/loxPp53loxP/loxP BM-MSCs and ASCs were induced to differentiate towards the osteogenic lineage and both Rb and p53 were excised with adenoviral vectors expressing the Cre-recombinase gene (Ad-CMV-Cre) at different stages (day 0 and 10) along osteogenic differentiation. NSG mice were inoculated subcutaneously with 5M-CM-^W10^6 mutant cells. Animals were killed when tumors reached 1 cm3 or 150 days after infusion. Some of the obtained tumors were mechanically disaggregated to establish ex vivo MSC-transformed cell lines. Gene expression analysis was performed using: WT BM-MSCs and ASCs, Rb-/-p53-/- BM-MSCs and ASCs previously differentiated to the osteogenic lineage for 10 days and a tumor cell line derived from p53-/-Rb-/- BM-MSC differentiated to the osteogenic lineage for 10 days.

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Transcriptome profile in the human synovial MSC-aggregates

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