Camilleri, Emily T.

AbstractImproving the effectiveness of adipose‐tissue derived human mesenchymal stromal/stem cells (AMSCs) for skeletal therapies requires a detailed characterization of mechanisms supporting cell proliferation and multi‐potency. We investigated the molecular phenotype of AMSCs that were either actively proliferating in platelet lysate or in a basal non‐proliferative state. Flow cytometry combined with high‐throughput RNA sequencing (RNASeq) and RT‐qPCR analyses validate that AMSCs express classic mesenchymal cell surface markers (e.g., CD44, CD73/NT5E, CD90/THY1, and CD105/ENG). Expression of CD90 is selectively elevated at confluence. Self‐renewing AMSCs express a standard cell cycle program that successively mediates DNA replication, chromatin packaging, cyto‐architectural enlargement, and mitotic division. Confluent AMSCs preferentially express genes involved in extracellular matrix (ECM) formation and cellular communication. For example, cell cycle‐related biomarkers (e.g., cyclins E2 and B2, transcription factor E2F1) and histone‐related genes (e.g., H4, HINFP, NPAT) are elevated in proliferating AMSCs, while ECM genes are strongly upregulated (>10‐fold) in quiescent AMSCs. AMSCs also express pluripotency genes (e.g., POU5F1, NANOG, KLF4) and early mesenchymal markers (e.g., NES, ACTA2) consistent with their multipotent phenotype. Strikingly, AMSCs modulate expression of WNT signaling components and switch production of WNT ligands (from WNT5A/WNT5B/WNT7B to WNT2/WNT2B), while upregulating WNT‐related genes (WISP2, SFRP2, and SFRP4). Furthermore, post‐proliferative AMSCs spontaneously express fibroblastic, osteogenic, chondrogenic, and adipogenic biomarkers when maintained in confluent cultures. Our findings validate the biological properties of self‐renewing and multi‐potent AMSCs by providing high‐resolution quality control data that support their clinical versatility. J. Cell. Biochem. 115: 1816–1828, 2014. © 2014 Wiley Periodicals, Inc.

Ewing sarcoma is an aggressive pediatric small round cell tumor that predominantly occurs in bone. Approximately 85% of Ewing sarcomas harbor the EWS/FLI fusion protein, which arises from a chromosomal translocation, t(11:22)(q24:q12). EWS/FLI interacts with numerous lineage‐essential transcription factors to maintain mesenchymal progenitors in an undifferentiated state. We previously showed that EWS/FLI binds the osteogenic transcription factor RUNX2 and prevents osteoblast differentiation. In this study, we investigated the role of another Runt‐domain protein, RUNX3, in Ewing sarcoma. RUNX3 participates in mesenchymal‐derived bone formation and is a context dependent tumor suppressor and oncogene. RUNX3 was detected in all Ewing sarcoma cells examined, whereas RUNX2 was detected in only 73% of specimens. Like RUNX2, RUNX3 binds to EWS/FLI via its Runt domain. EWS/FLI prevented RUNX3 from activating the transcription of a RUNX‐responsive reporter, p6OSE2. Stable suppression of RUNX3 expression in the Ewing sarcoma cell line A673 delayed colony growth in anchorage independent soft agar assays and reversed expression of EWS/FLI‐responsive genes. These results demonstrate an important role for RUNX3 in Ewing sarcoma. J. Cell. Physiol. 229: 2049–2056, 2014. © 2014 Wiley Periodicals, Inc.