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. 2010 May 1;24(9):916–932. doi: 10.1101/gad.1899710

Figure 4.

Figure 4.

EWS-FLI-1 expression in hpMSCs grown in KO culture medium generates a cell subpopulation displaying the major in vitro features of ESFT CSCs. (A) Adult and pediatric hMSCDBDMs and hMSCEWS-FLI-1s were compared, by real-time PCR analysis, for expression of OCT4, NANOG, and CD133 that characterize the ESFT CSC molecular phenotype. No induction was observed in hMSCDBDMs. (B) FACS analysis of cell surface CD133 expression in hpMSCEWS-FLI-1s, showing that the 15-fold induction of the CD133 transcript was not accompanied by a concomitant appearance of its glycosylated, stem cell-related epitope, despite marked induction of the NGFR/p75 NCSC marker. (C,D) Experiments described in A and B were repeated using the same hpMSCEWS-FLI-1 population cultured in KO medium. In cells cultured in KO medium, EWS-FLI-1 strongly enhanced OCT4, NANOG, and CD133 transcription (C), and generated a hpMSCEWS-FLI-1 subpopulation expressing the glycosylated cell surface CD133 epitope (D). (E) Real-time PCR analysis of OCT4, NANOG, and EWS-FLI-1 gene expression levels, in CD133-positive and CD133-negative hpMSCEWS-FLI-1-derived RNA, showing that the CD133-positive fraction expresses a higher level of OCT4 and NANOG, but a nearly identical EWS-FLI-1 level. A single representative experiment is shown. All real-time PCR experiments were normalized to cyclophyllin A and done in triplicate. Error bars represent the SD of three independent determinations. (F) hpMSCEWS-FLI-1 CD133-positive fraction expresses a higher level of the ESFT marker LIPI, and of a panel of genes involved in stemness. (G) FACS analysis of cell surface CD133 expression of CD133-positive or CD133-negative hpMSCEWS-FLI-1-derived progeny, cultured in KO medium for 14 d following the initial sorting. (H) Clonogenic assays were performed in triplicate as described in the Materials and Methods. Error bars indicate the difference in the number of spheres formed in the three determinations. (Bottom right) Photomicrograph of a hpMSCEWS-FLI-1-derived sphere. Magnification, 400×.