Download Yasuko Shima

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 Nishijo1.2 Tomoki Aoyama2 Tomitaka Nakayama1
Takashi Nakamura1
Junya Toguchida2
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
SV40
hTERT
→
→
→
→
telomerase expression
k/o Rb etc
k/o p53 etc
mutation of ras etc
hTERT
SV40, E7
SV40, E6
H-ras12V
H-ras12V
normal cell
transformed
cell
E6/E7
normal cell
→
→
→
→
hTERT
H-ras12V
transformed
cell
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
Inactivation of p16 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
Ink4a(p16)/ARF
deletion
Oncogenic
K-ras mutation
Ink4a(p16)/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
polycomb response elements
Bmi-1
promoter
transcription
initiation complex
transcription initiation site
Immortalization of human MSC(hMSC) by the induction of Bmi1
hTERT Bmi-1
hMSC
-Bmi1-hT
hMSC
Control
(Saos2)
p16-mRNA
p16-Protein
hMSC hMSC
-E6E7 -Bmi1
-hT
-hT
PD9
hMSC
PD2
Transformation of immortalized hMSC by the induction of activated H-ras12V
hTERT
hMSC
Bmi-1
H-ras12V
hMSC
-Bmi1-hT
hMSC
-Bmi1-hT
-pQCXIP/H-ras12V
“Parental”
“pQCXIP/H-ras12V”
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 pQCXIP/H-ras12V into hMSC-Bmi1-hT cells
Cell Morphology
pQCXIP
Parental
pQCXIP/H-ras12V
X100
Northern Blot
exogenous H-ras12V
(2.5kb)
endogenous H-ras
(1.1kb)
X100
1 2 3
4
X100
5
1.
2.
3.
4.
5.
Parental
pQCXIP-1
pQCXIP-2
pQCXIP/H-ras12V-1
pQCXIP/H-ras12V-2
Growth property with low serum condition
(×105)
14
pQCXIP/H-ras12V
＊: p<0.05
pQCXIP
Parental
12
(×105)
6
＊
＊: p<0.05
pQCXIP/H-ras12V
pQCXIP
Parental
5
＊
10
8
Cell Number
Cell Number
4
＊
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
pQCXIP-1
60
50
40
x40
pQCXIP/H-ras12V-1
30
20
10
x40
0
Parental pQCXIP
pQCXIP
/H-ras12V
Cell motility (cell counts of control insert)
cell number
300
pQCXIP-1
250
200
150
pQCXIP-Hras12V 1
x200
100
50
0
Parental pQCXIP
pQCXIP
/H-ras12V
x200
Matrigel Invasion Assay
cell number
14
pQCXIP-2
12
10
8
pQCXIP-Hras12V-2
6
x200
4
2
0
Parental pQCXIP
pQCXIP
/H-ras12V
x200
Tumorigenecity assay
volume(mm3)
8000
pQCXIP/H-ras12V-1
pQCXIP/H-ras12V-2
7000
6000
5000
4000
3000
2000
1000
0
Inoculation
1
3
5
7
9
11
13 days
Right :pQCXIP/H-ras12V
Left :pQCXIP
Histopathology of the tumor
Differentiation potential after induction of H-ras12VーBone
Parental
pQCXIP/H-ras12V
Induction (-)
Alizarin Red
staining
Induction (+)
1w
Induction
-
-
2w
+
+
OC
COLIA1
ALP
b-ACT
ras1 ras2
ras1 ras2
-
-
3w
+
+
-
-
+
+
p.c.
Differentiation potential after induction of H-ras12V-Adipo
pQCXIP/H-ras12V
Parental
Induction (-)
Oil-Red O
staining
Induction (+)
x200
induction
PPARg
BACT
-
-
1w
+
+
-
-
2w
+
+
-
-
ras1 ras2 ras1 ras2
Parental
induction
PPARg
BACT
1w
-
2w
+
-
3w
+
-
+
p.c.
3w
+
+
p.c.
hMSC was transformed by hTERT+Bmi-1+H-rasV12
hTERT
Bmi-1
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