Generation of Osteosarcomas From a Combination of Rb Silencing and c-Myc Overexpression in Human Mesenchymal Stem Cells.

: Osteosarcoma (OS) was a malignant tumor occurring with unknown etiology that made prevention and early diagnosis difficult. Mesenchymal stem cells (MSCs), which were found in bone marrow, were claimed to be a possible origin of OS but with little direct evidence. We aimed to characterize OS cells transformed from human MSCs (hMSCs) and identify their association with human primary OS cells and patient survival. Genetic modification with p53 or retinoblastoma (Rb) knockdown and c-Myc or Ras overexpression was applied for hMSC transformation. Transformed cells were assayed for proliferation, differentiation, tumorigenecity, and gene expression profile. Only the combination of Rb knockdown and c-Myc overexpression successfully transformed hMSCs derived from four individual donors, with increasing cell proliferation, decreasing cell senescence rate, and increasing ability to form colonies and spheres in serum-free medium. These transformed cells lost the expression of certain surface markers, increased in osteogenic potential, and decreased in adipogenic potential. After injection in immunodeficient mice, these cells formed OS-like tumors, as evidenced by radiographic analyses and immunohistochemistry of various OS markers. Microarray with cluster analysis revealed that these transformed cells have gene profiles more similar to patient-derived primary OS cells than their normal MSC counterparts. Most importantly, comparison of OS patient tumor samples revealed that a combination of Rb loss and c-Myc overexpression correlated with a decrease in patient survival. This study successfully transformed human MSCs to OS-like cells by Rb knockdown and c-Myc overexpression that may be a useful platform for further investigation of preventive and target therapy for human OS.

SIGNIFICANCE: In the present study, transformation of human mesenchymal stem cells (MSCs) was achieved by the combination of retinoblastoma (Rb) knockdown and c-Myc overexpression. After a robust evaluation with radiographic analyses, micro-positron emission tomography/computed tomography, micro-magnetic resonance imaging, and immunohistochemistry of various osteosarcoma (OS) markers, tumors were found to be formed by intraosseous and subcutaneous injection of these cells that were OS-like tumors. Through the use of microarray with cluster analysis, these transformed cells were shown to have gene profiles more similar to those of patient-derived primary OS cells than their normal MSC counterparts. More important, comparison of OS patient tumor samples revealed that a combination of Rb loss and c-Myc overexpression correlated with a decrease in patient survival. These data strongly suggest that transformed human MSCs by Rb knockdown and c-Myc overexpression are associated with clinical OS tissues and patient survival. Thus, the present study identifies a useful platform for further investigation of prevention and target therapy for human OS.