Comparative Global Transcriptome Analysis of Bone Marrow Stromal Cells (BMSC), Human Embryonic Stem (hES) Cells and CD34+ Cells.

Abstract 36

Bone marrow stromal cells (BMSCs, also known as “mesenchymal stem cells) are being used to treat acute graft-versus-host-disease, but their mechanisms of immune modulation are not certain. In vitro studies suggest that the immunosuppressive activity of BMSCs involves multiple factors including transforming growth factor β (TGF-β), hepatocyte growth factor (HGF), TNF-α, IFN-ψ, IL-10, IL-2 and prostaglandin E2 (PGE2). In this study we compared BMSCs with other types of stem cells using global transcriptome and microRNA (miR) expression analysis to identify factors that might contribute to their immunosuppressive effects and to identify biomarkers for assessing the stability, consistency, comparability, and potency of clinical BMSC products. BMSCs (passage 2 or 3) made from marrow aspirates of 4 healthy subjects by culturing in flasks and cell factories with 20% fetal bovine serum (FBS) were compared to 3 human embryonic stem cell lines (hES) and CD34+ cells isolated from G-CSF-mobilized peripheral blood from 3 healthy subjects. The cells were analyzed with an miR expression array with more than 800 probes and an oligonucleotide expression microarray with more than 35,000 probes. Hierarchical clustering analysis of the miR expression data separated the 3 types of cells into 3 distinct groups with unique signatures. MiRNA implicated in cancer and stem cell development that were up-regulated in BMSC compared to CD34+ cells and hES included miR21 and 125b and cancer and stem cell miR down-regulated in BMSCs included miR106a, 106b, 18a, 19b and 20b. When compared to CD34+ cells, several miR in the onco-miR17-92 cluster (miR17, 18a, 20a, 19b-1, and 92-1) and onco-miR106a-363 cluster (106a, 18b, 20b, 19b-2, 92-2, and 363) were down-regulated in BMSCs. Hierarchical analysis of the 4,600 genes that were expressed in greater than 80% of samples and were increased more than 2-fold in at least one sample clustered the 3 cell types into separate groups. Ingenuity pathway analysis revealed that the following pathways contained a significant number of genes that were up-regulated in BMSCs compared to both CD34+ cells and hES: actin based motility by rho, actin cytoskeletal signaling, integrin signaling, androgen signaling, IL-8 signaling VEGF signaling, PTEN signaling, oncostatin M signaling, fMLP signaling in neutrophils, inositol metabolism, cavelolar-mediated endocytosis and NRF2-mediated oxidative stress response pathway. DNA methylation and transcription repression pathway genes were down-regulated in BMSCs. Analysis of specific differentially expressed genes found that the gene most up-regulated in BMSCs was TGF-β1. Its expression was 346-fold fold greater in BMSCs than CD34+ cells and 298-fold greater than in hES. In addition, when compared to CD34+ cells, the expression of IL-6 was up-regulated 13.6-fold in BMSCs, prostaglandin E synthase was up-regulated 13.2-fold, and HGF 8.43-fold. These results support a possible role for TGF-β1, IL-6, HGF and PGE2 in BMSC-mediated immune modulation. TGF-β1, IL-6, HGF and PGE2 synthase are potential BMSC potency biomarkers, but further studies, including the correlation of the expression of these biomarkers in specific BMSC products with the clinical outcomes of patients treated with these products, are needed.