Transformation of mesenchymal stem cells by the retrovirusmediated

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

Transformation of mammalian cells requires multiple steps Escape from telomere shortening Escape from cell cycle regulation Escape from programmed cell death Gain of malignant phenotype SV 40 h. TERT → → telomerase expression k/o Rb etc k/o p 53 etc mutation of ras etc h. TERT SV 40, E 7 SV 40, E 6 H-ras 12 V normal cell transformed cell E 6/E 7 normal cell → → h. TERT H-ras 12 V transformed cell

MSC can be immortalized by the activation of h. TERT? 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-h. TERT MSC-Mock Clonal heterogeneity in differentiation potential of immortalized human mesenchymal stem cells Okamoto T. , et al. BBRC. , 295: 354 -61, 2002 Immortalize h. MSC by h. TERT+HPVE 6/E 7

Inactivation of p 16 may be required to be immortalized Adult human mesenchymal stem cell as a target for neoplastic transformation Population Doubling Serakinci, N. , ---, Kassem, M. Oncogene, 24: 5095 -8, 2004 Ink 4 a(p 16)/ARF deletion Oncogenic K-ras mutation Ink 4 a(p 16)/ARF deletion Time (days)

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 promoter transcription initiation site polycomb response elements Bmi-1 promoter transcription initiation complex transcription initiation site

Immortalization of human MSC(h. MSC) by the induction of Bmi 1 h. TERT Bmi-1 h. MSC -Bmi 1 -h. T h. MSC Control (Saos 2) p 16 -m. RNA p 16 -Protein h. MSC -E 6 E 7 -Bmi 1 -h. T PD 9 h. MSC PD 2

Transformation of immortalized h. MSC by the induction of activated H-ras 12 V h. TERT h. MSC Bmi-1 H-ras 12 V h. MSC -Bmi 1 -h. T -p. QCXIP/H-ras 12 V “Parental” “p. QCXIP/H-ras 12 V” 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

Induction of p. QCXIP/H-ras 12 V into h. MSC-Bmi 1 -h. T cells Cell Morphology p. QCXIP Parental p. QCXIP/H-ras 12 V X 100 Northern Blot exogenous H-ras 12 V (2. 5 kb) endogenous H-ras (1. 1 kb) X 100 1 2 3 4 X 100 5 1. 2. 3. 4. 5. Parental p. QCXIP-1 p. QCXIP-2 p. QCXIP/H-ras 12 V-1 p. QCXIP/H-ras 12 V-2

Growth property with low serum condition (× 105) 14 p. QCXIP/H-ras 12 V ＊: p<0. 05 p. QCXIP Parental 12 (× 105) 6 ＊ p. QCXIP/H-ras 12 V p. QCXIP Parental 5 10 ＊ 4 8 Cell Number ＊: p<0. 05 ＊ 6 4 3 ＊ 2 ＊ ＊ 1 2 0 ＊ 0 3 4 5 Culture Days 10% FBS 6 8 0 0 3 4 5 Culture Days 1%FBS 6 8

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

Cell motility (cell counts of control insert) cell number 300 p. QCXIP-1 250 200 150 p. QCXIP-Hras 12 V 1 x 200 100 50 0 Parental p. QCXIP /H-ras 12 V x 200

Matrigel Invasion Assay cell number 14 p. QCXIP-2 12 10 8 p. QCXIP-Hras 12 V-2 6 x 200 4 2 0 Parental p. QCXIP /H-ras 12 V x 200

Tumorigenecity assay volume(mm 3) 8000 p. QCXIP/H-ras 12 V-1 p. QCXIP/H-ras 12 V-2 7000 6000 5000 4000 3000 2000 1000 0 Inoculation 1 3 5 7 9 11 13 days Right : p. QCXIP/H-ras 12 V Left : p. QCXIP

Histopathology of the tumor

Differentiation potential after induction of H-ras 12 VーBone Parental p. QCXIP/H-ras 12 V Induction (-) Alizarin Red staining Induction (+) 1 w Induction - - 2 w + + OC COLIA 1 ALP b-ACT ras 1 ras 2 - - 3 w + + - - + + p. c.

Differentiation potential after induction of H-ras 12 V-Adipo p. QCXIP/H-ras 12 V Parental Induction (-) Oil-Red O staining Induction (+) x 200 induction PPARg BACT - - 1 w + + - - 2 w + + - - ras 1 ras 2 Parental induction PPARg BACT 1 w - 2 w + - 3 w + - + p. c. 3 w + + p. c.

h. MSC was transformed by h. TERT+Bmi-1+H-ras. V 12 h. TERT Bmi-1 H-ras 12 V undifferentiated sarcoma h. MSC 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 h. TERT+Bmi 1+H-ras 12 V. Only against h. MSC? h. MSC is easy to transform? ? • What is the cause of sarcoma? Is there the gene equivalent of H-ras 12 V? h. MSC-Bmi 1 -h. TERT is usefull model to screen the sarcoma-related gnes