Bone Abstracts

Improving the effectiveness of adipose-derived human mesenchymal stromal/stem cells (AMSCs) for skeletal therapies requires a detailed molecular characterization of mechanisms supporting cell proliferation and multi-potency of AMSCs. We compared gene expression data obtained by high-throughput RNA sequencing and RT-qPCR of actively proliferating and uninduced post-proliferative AMSCs with flow cytometry data for mesenchymal stem cell markers (e.g., CD44, CD73/NT5E, CD90/THY1 and CD105/ENG). Immuno-detection of CD markers corresponds well with mRNA expression results. AMSCs also express mesenchymal markers (NES and ACTA2) and pluripotency genes (e.g., POU5F1, NANOG, KLF4). Proliferating AMSCs express mRNAs for cell cycle-related biomarkers (e.g., cyclins CCND1 and CCNB2). These genes are down-regulated in confluent AMSC cultures. Thus, cessation of AMSC proliferation occurs by selective suppression of the cell cycle machinery. Quiescent AMSCs exhibit >tenfold upregulation of proteins involved in tissue matrix formation, including several leucine-rich repeat proteins (ASPN, ECM2, FMOD, OGN and PODN). Quiescent AMSCs also modulate expression of WNT signaling components, including the WNT-inducible gene WISP2 and the decoy receptors SFRP2 and SFRP2, while switching production of WNT5A, WNT5B and WNT7B to WNT2 and WNT2A. Furthermore, we show that post-proliferative AMSCs spontaneously transition into fibroblastic, osteogenic, chondrogenic and adipogenic cell fates. Our findings provide fundamental quality control data to support the clinical versatility of AMSCs.