1. Transformation of mesenchymal stem cells by the retrovirus-mediated gene transfer 1 Department of Drthopaedeic Surgery Kyoto University,Kyoto,Japan 2 Institute for Frontier Medical Sciences Kyoto University,Kyoto,Japan Yasuko Shima 1.2 Takeshi Okamoto 1.2 Tatsuya Ishibe 1.2 Koichi Nishijo 1.2 Tomoki Aoyama 2 Tomitaka Nakayama 1 Takashi Nakamura 1 Junya Toguchida 2

2. transformed cell normal cell Transformation of mammalian cells requires multiple steps SV40 hTERT H-ras 12V E6/E7 hTERT H-ras 12V normal cell transformed cell Escape from telomere shortening → telomerase expression → hTERT Escape from cell cycle regulation → k/o Rb etc → SV40, E7 Escape from programmed cell death → k/o p53 etc → SV40, E6 Gain of malignant phenotype → mutation of ras etc → H-ras 12V

3. MSC can be immortalized by the activation of hTERT? Telomerase expression extends the proliferative life-span and maintains the osteogenic potential of human bone marrow stromal cells Simonsen, J.L., ---, Kassem, M. Nature Biotech., 20: 592-6, 2002 MSC-hTERT MSC-Mock Clonal heterogeneity in differentiation potential of immortalized human mesenchymal stem cellsOkamoto T., et al. BBRC., 295: 354-61, 2002 Immortalize hMSC by hTERT+HPVE6/E7

4. Time (days) Population Doubling Adult human mesenchymal stem cell as a target for neoplastic transformation Serakinci, N., ---, Kassem, M. Oncogene, 24: 5095-8, 2004 Inactivation of p16 may be required to be immortalized Ink4a(p16)/ARF deletion Oncogenic K-ras mutation Ink4a(p16)/ARF deletion

5. Bmi-1 (B cell-specific Mo-MLV integration site 1) First isolated as an oncogene that cooperates with c-myc in the generation of mouse lymphomas. Required to maintain stable repression of target genes promotertranscription initiation site Bmi-1 polycomb response elements polycomb response elements promotertranscription initiation site transcription initiation complex

6. Immortalization of human MSC(hMSC) by the induction of Bmi1 hMSC -Bmi1-hT hMSC Bmi-1 hTERT p16-mRNA p16-Protein PD2PD9 hMSC -E6E7 -hT hMSC -Bmi1 -hT hMSC Control (Saos2)

7. Transformation of immortalized hMSC by the induction of activated H-ras 12V hMSC Bmi-1 hTERT H-ras 12V hMSC -Bmi1-hT “Parental” hMSC -Bmi1-hT -pQCXIP/H-ras 12V “pQCXIP/H-ras 12V ” 1.Anchorage-independent growth property colony formation in soft agar 2.Serum-independent growth property growth curves with 1% serum 3.Acquisition of invasiveness and motility matrigel invasion assay 4.Ability to make tumors subcutaneous tumorigenicity assay 5.Differentiation potential induction of adipo and osteo differentiation

8. endogenous H-ras (1.1kb) Parental Induction of pQCXIP/H-ras 12V into hMSC-Bmi1-hT cells X100 exogenous H-ras 12V (2.5kb) 1234512345 1. Parental 2. pQCXIP-1 3. pQCXIP-2 4. pQCXIP/H-ras 12V -1 5. pQCXIP/H-ras 12V -2 Cell Morphology pQCXIP pQCXIP/H-ras 12V Northern Blot

9. Growth property with low serum condition 0 2 4 6 8 10 12 14 034568 ＊ ＊ ＊ ＊ 10% FBS 0 1 2 3 4 5 6 034568 ＊ ＊ ＊ 1%FBS (×10 5 ) ＊ : p<0.05 pQCXIP/H-ras 12V pQCXIP Parental pQCXIP/H-ras 12V pQCXIP Parental Cell Number Culture Days ＊ : p<0.05

10. 0 10 20 30 40 50 60 70 pQCXIP /H-ras 12V pQCXIP-1 pQCXIP/H-ras 12V -1 Colony formation assay in soft agar x40 colony/cell number Parental x40

11. x200 0 50 100 150 200 250 300 Cell motility (cell counts of control insert) cell number ParentalpQCXIP /H-ras 12V pQCXIP-1 pQCXIP-Hras 12V 1 x200

12. Parental Matrigel Invasion Assay x200 pQCXIP-2 pQCXIP-Hras 12V -2 0 2 4 6 8 10 12 14 cell number pQCXIP /H-ras 12V x200

13. Tumorigenecity assay days volume(mm3) Right :pQCXIP/H-ras 12V Left :pQCXIP Inoculation 0 1000 2000 3000 4000 5000 6000 7000 8000 135791113 pQCXIP/H-ras 12V -1 pQCXIP/H-ras 12V -2

14. Histopathology of the tumor

15. Induction (-) Induction (+) Parental Differentiation potential after induction of H-ras 12V ー Bone  -ACT ALP COLIA1 Induction - - + + 1w p.c. OC ras1ras2ras1ras2 Alizarin Red staining pQCXIP/H-ras 12V 2w3w - - + +

16. Oil-Red O staining x200 Induction (-) Induction (+) Parental pQCXIP/H-ras 12V Differentiation potential after induction of H-ras 12V- Adipo BACT p.c. PPARg induction Parental 1w2w3w - + BACT induction p.c. PPARg ras1ras2ras1ras2 - - + + 1w2w3w - - + +

17. 1.Serum-independent growth property(+) 2.Anchorage-independent growth property(+) 3.Motility(+) 4.Invasiveness(+) 5.In vivo tumorigenesis(+) 6.Osteogenic differentiation(-) 7.Adipogenic differentiation? hMSC was transformed by hTERT+Bmi-1+H-ras V12 This is the first time to report of transformation by the combination of hTERT+Bmi1+H-ras 12V. Only against hMSC? hMSC is easy to transform?? What is the cause of sarcoma? Is there the gene equivalent of H-ras 12V ? hMSC-Bmi1-hTERT is usefull model to screen the sarcoma-related gnes hMSC Bmi-1 hTERT H-ras 12V undifferentiated sarcoma