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

1Department of Drthopaedeic Surgery Kyoto University,Kyoto,Japan2Institute for Frontier Medical Sciences Kyoto University,Kyoto,Japan

Yasuko Shima1.2 Takeshi Okamoto1.2 Tatsuya Ishibe1.2

Koichi Nishijo1.2 Tomoki Aoyama2 Tomitaka Nakayama1

Takashi Nakamura1

Junya Toguchida2

transformed cell

normal cell

Transformation of mammalian cells requires multiple steps

SV40 hTERT H-ras12V

E6/E7 hTERT H-ras12V

normal cell transformed cell

Escape from telomere shortening → telomerase expression → hTERTEscape from cell cycle regulation → k/o Rb etc → SV40, E7Escape from programmed cell death → k/o p53 etc → SV40, E6Gain of malignant phenotype → mutation of ras etc → H-ras12V

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

Time (days)

Popula

tion D

oublin

g

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)/ARFdeletion Oncogenic

K-ras mutation

Ink4a(p16)/ARFdeletion

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

Bmi-1

polycomb response elements

polycomb response elements

promoter transcription initiation site

transcription initiation complex

Immortalization of human MSC(hMSC) by the induction of Bmi1

hMSC-Bmi1-hThMSC

Bmi-1hTERT

p16-mRNA

p16-Protein

PD2 PD9

hMSC-E6E7

-hT

hMSC-Bmi1

-hT

hMSCControl(Saos2)

Transformation of immortalized hMSC by the induction of activated H-ras12V

hMSC

Bmi-1hTERT H-ras12V

hMSC-Bmi1-hT

“Parental”

hMSC-Bmi1-hT

-pQCXIP/H-ras12V

“pQCXIP/H-ras12V”

1.Anchorage-independent growth propertycolony formation in soft agar

2.Serum-independent growth propertygrowth curves with 1% serum

3.Acquisition of invasiveness and motilitymatrigel invasion assay

4.Ability to make tumorssubcutaneous tumorigenicity assay

5.Differentiation potentialinduction of adipo and osteo differentiation

endogenous H-

ras(1.1kb)

Parental

Induction of pQCXIP/H-ras12V into hMSC-Bmi1-hT cells

X100 X100 X100

exogenous H-ras12V

(2.5kb)

1 2 3 4 5

1. Parental2. pQCXIP-13. pQCXIP-24. pQCXIP/H-ras12V-15. pQCXIP/H-ras12V-2

Cell Morphology

pQCXIP pQCXIP/H-ras12V

Northern Blot

Growth property with low serum condition

0

2

4

6

8

10

12

14

0 3 4 5 6 8

＊

＊

＊

＊

10% FBS

0

1

2

3

4

5

6

0 3 4 5 6 8

＊

＊

＊

1%FBS

(×105) (×105)＊ : p<0.05

pQCXIP/H-ras12V

pQCXIPParental

pQCXIP/H-ras12V

pQCXIPParental

Cell

Num

ber

Cell

Num

ber

Culture Days Culture Days

＊ : p<0.05

0

10

20

30

40

50

60

70

pQCXIP pQCXIP/H-ras12V

pQCXIP-1

pQCXIP/H-ras12V-1

Colony formation assay in soft agar

x40

colony/cell number

Parental

x40

x200

0

50

100

150

200

250

300

Cell motility (cell counts of control insert)

cell number

ParentalpQCXIP pQCXIP/H-ras12V

pQCXIP-1

pQCXIP-Hras12V 1x200

Parental

Matrigel Invasion Assay

x200

pQCXIP-2

pQCXIP-Hras12V-2

0

2

4

6

8

10

12

14

cell number

pQCXIP pQCXIP/H-ras12V

x200

Tumorigenecity assay

days

volume(mm3)

Right :pQCXIP/H-ras12V

Left :pQCXIP

Inoculation

0

1000

2000

3000

4000

5000

6000

7000

8000

1 3 5 7 9 11 13

pQCXIP/H-ras12V-1pQCXIP/H-ras12V-2

Histopathology of the tumor

Induction (-)

Induction (+)

Parental

Differentiation potential after induction of H-ras12V ー Bone

-ACT

ALP

COLIA1

Induction - - + + 1w

p.c.

OC

ras1 ras2 ras1 ras2

Alizarin Red staining

pQCXIP/H-ras12V

2w 3w

- - + + - - + +

Oil-Red Ostaining

x200

Induction (-)

Induction (+)

Parental pQCXIP/H-ras12V

Differentiation potential after induction of H-ras12V-

Adipo

BACT

p.c.

PPARginduction

Parental1w 2w 3w- + - + - +

BACT

induction p.c.

PPARg

ras1 ras2 ras1 ras2

- - + + 1w 2w 3w

- - + + - - + +

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-rasV12

• 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

hMSC

Bmi-1hTERT H-ras12V

undifferentiatedsarcoma