Characterization of human MSC. (A) Isolated MSC were shown to express MSC markers including SH2, SH4, and Stro-1 as determined by FACs analysis. CD34 (hematopoietic stem cell marker) and CD45 (pan-leukocyte marker), considered to be negative mesenchymal markers, were not expressed in MSC. Control cells are also shown (black). (B) MSC multipotency was confirmed by differentiation (+) to adipocytes, osteocytes, and chondrocytes. Control cells, where differentiation was not induced (−), are also shown.

Transformation of human MSC. (A) Schematic diagram of MSC stepwise transformation. Cell lines were named according to the number of steps or hits introduced sequentially by retroviral transduction (see Table 1). (B) Cell lines were probed by antibodies against introduced oncogenes or their downstream cellular targets. Proliferating cell nuclear antigen (PCNA) reflects cell entry in S phase after pRB inactivation by E7. (C) Anchorage-independent growth of MSC infected with empty retroviruses or combinations of different oncogenes. (D) Tumors formed in mice inoculated with 5 hits MSC (arrows), but not in those inoculated with 4 hits MSC (Left). (E) GEM profiles of five of the MSC tumors generated in mice (open circles) were compared with 96 human sarcomas (23). Multidimensional scale (MDS) shows that the tumors generated in mice clustered with human spindle cell sarcomas.

Functional assays of bioenergetic pathways during stepwise transformation. (A) Growth curve showing the number of cells at 24, 48, and 72 h. Lanes 0–5 indicate cells with different number of genetic hits (see Table 1). (B) Values of glucose uptake and lactate release at 48 h corrected for cell number in MSC and HF during stepwise transformation (∗, P < 0.05; Student's t test). Lanes 0–5 indicate parental cells (0) and each subsequent step in transformation.

Role of PPP during MSC transformation. (A) G6PD activity decreased during MSC transformation (∗, P=0.0132; t test). Western blot confirms the down-regulation of G6PD and the up-regulation of PRPS at the late stages of transformation. (B) MSC transformation also led to a significant decrease in NADPH levels (∗, P=0.0014; Student's t test).

Bioenergetic profiles of tumors originating from MSC in vivo. (A) The heatmaps summarize the average of three samples from cell lines (0–5) and five tumors (T) for glycolytic and TCA cycle genes (representative probes for each gene were chosen). Red indicates up-regulation, and blue down-regulation from the mean. (B) Hypoxia-related genes such as VEGF, hypoxia-inducible protein 2 (HIG2), adrenomedullin (ADM), chemokine CXC motif receptor 4 (CXCR4), and angiopoietin-like 4 (ANGPTL4) were up-regulated in the tumors, compared with in vitro transformed cells. (C) Immunohistochemistry for GLUT1 showed that the central poorly vascularized region of a tumor stained strongly positive (red, 24 × 26 image montage, ×200 magnification). Inset shows higher magnification of GLUT1 staining (red), distinct from the well vascularized region, stained with CD31 (green, 2 × 2 image montage, ×200 magnification). (D) Western blotting confirmed that when transformed MSC were exposed to hypoxia (1% O₂), HIF-1α, GLUT1, hexokinase II (HK II), and CAIX were up-regulated. RAD50 and actin were used as loading controls. (E) Values of glucose uptake at 48 h for parental (0) and 5 hits (5) MSC grown at 21% and 1% O₂. (F) Glucose uptake and lactate release in 5 hits MSC (5) compared with 5 hits MSC explanted from tumors (Exp) and cultured for 3 weeks.