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Transformation of mesenchymal stem cells by the retrovirus-mediated gene transfer

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

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Transformation of mesenchymal stem cells by the retrovirus-mediated gene transfer

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  1. Transformation of mesenchymal stem cells by the retrovirus-mediated gene transfer 1Department of Drthopaedeic Surgery Kyoto University,Kyoto,Japan 2Institute for Frontier Medical Sciences Kyoto University,Kyoto,Japan Yasuko Shima1.2 Takeshi Okamoto1.2 Tatsuya Ishibe1.2 Koichi Nishijo1.2Tomoki Aoyama2 Tomitaka Nakayama1 Takashi Nakamura1 Junya Toguchida2

  2. H-ras12V hTERT SV40 transformed cell normal cell normal cell H-ras12V hTERT E6/E7 transformed cell Transformation of mammalian cells requires multiple steps 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-ras12V

  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 cells Okamoto T., et al. BBRC., 295: 354-61, 2002 Immortalize hMSC by hTERT+HPVE6/E7

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

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

  6. Immortalization of human MSC(hMSC) by the induction of Bmi1 hTERT Bmi-1 hMSC -Bmi1-hT hMSC hMSC -Bmi1 -hT hMSC -E6E7 -hT hMSC Control (Saos2) PD2 PD9 p16-mRNA p16-Protein

  7. 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 Transformation of immortalized hMSC by the induction of activated H-ras12V Bmi-1 hTERT H-ras12V hMSC -Bmi1-hT -pQCXIP/H-ras12V hMSC -Bmi1-hT hMSC “Parental” “pQCXIP/H-ras12V”

  8. Induction of pQCXIP/H-ras12V intohMSC-Bmi1-hT cells Cell Morphology Parental pQCXIP pQCXIP/H-ras12V X100 X100 X100 1 2 3 4 5 Northern Blot 1. Parental 2. pQCXIP-1 3. pQCXIP-2 4. pQCXIP/H-ras12V-1 5. pQCXIP/H-ras12V-2 exogenous H-ras12V (2.5kb) endogenous H-ras (1.1kb)

  9. pQCXIP/H-ras12V pQCXIP/H-ras12V Growth property with low serum condition (×105) (×105) ＊:p<0.05 ＊:p<0.05 14 6 ＊ pQCXIP pQCXIP Parental Parental 12 ＊ 5 10 4 8 ＊ ＊ 3 Cell Number Cell Number 6 2 ＊ 4 ＊ 1 2 ＊ 0 0 0 3 4 5 6 8 0 3 4 5 6 8 Culture Days Culture Days 10% FBS 1%FBS

  10. Colony formation assay in soft agar colony/cell number 70 pQCXIP-1 60 50 40 x40 pQCXIP/H-ras12V-1 30 20 10 x40 0 pQCXIP Parental pQCXIP /H-ras12V

  11. Cell motility (cell counts of control insert) cell number 300 pQCXIP-1 250 200 150 x200 pQCXIP-Hras12V 1 100 50 0 Parental pQCXIP x200 pQCXIP /H-ras12V

  12. Matrigel Invasion Assay cell number 14 pQCXIP-2 12 10 8 x200 pQCXIP-Hras12V-2 6 4 2 0 x200 Parental pQCXIP pQCXIP /H-ras12V

  13. pQCXIP/H-ras12V-1 pQCXIP/H-ras12V-2 8000 7000 6000 5000 4000 3000 2000 1000 0 1 3 5 7 9 11 13 Tumorigenecity assay volume(mm3) Inoculation Right :pQCXIP/H-ras12V Left :pQCXIP days

  14. Histopathology of the tumor

  15. Differentiation potential after induction of H-ras12VーBone Parental pQCXIP/H-ras12V Induction (-) Alizarin Red staining Induction (+) 1w 2w 3w p.c. - - + + - - + + - - + + Induction OC COLIA1 ALP b-ACT ras1 ras2 ras1 ras2

  16. 1w 2w 3w p.c. induction - - + + - - + + - - + + PPARg BACT ras1 ras2 ras1 ras2 Differentiation potential after induction of H-ras12V-Adipo pQCXIP/H-ras12V Parental Induction (-) Oil-Red O staining Induction (+) x200 Parental 1w 2w 3w p.c. induction - + - + - + PPARg BACT

  17. hMSC was transformed by hTERT+Bmi-1+H-rasV12 Bmi-1 hTERT H-ras12V undifferentiated sarcoma hMSC 1.Serum-independent growth property (+) 2.Anchorage-independent growth property (+) 3.Motility (+) 4.Invasiveness (+) 5.In vivo tumorigenesis (+) 6.Osteogenic differentiation (-) 7.Adipogenic differentiation ? • This is the first time to report of transformation by the combination of hTERT+Bmi1+H-ras12V. Only against hMSC? hMSC is easy to transform?? • What is the cause of sarcoma? Is there the gene equivalent of H-ras12V?hMSC-Bmi1-hTERT is usefull model to screen the sarcoma-related gnes

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