10. Shown is the population doublings (PDs) achieved by Bmi-1, E6, E7 and hTERT
overexpressing cells over 300 days of growth.
H4-1(black line) and UEE16(orange line) were stopped dividing about 200days. ”
”
６
６７
７
６７
16. populationdoublings
days in culture
norm
al
X
M0
X
M1
Regulation of
p16/Rb
telomerase
activation
immortalization
+E6
or TER
T
replicative senescence (mortality stage 1replicative senescence (mortality stage 1:
Mitotic clockMitotic clock
oror
Inappropriate culture condition?Inappropriate culture condition?
+ E7 or Bmi-1+ E7 or Bmi-1
17. A
B
C
D
Neural differentiation
Shown are neural differentiation cells (A-C, and E); no-treatment cells (E and
EE
FF
Human (H4-1)Human (H4-1) Murine (KUSA-A1)Murine (KUSA-A1)
45. 1 week 3 weeks
Recording of action potentials
from spontaneously beating cells
Alexa568
Human marrow stromal cells
with the extended life span
can differentiate into cardiomyocytes
Disorganized
Regular and
stabilized
Recording
microelectrode
50. E-GFP
DAPI E-GFP Trop-I Merge
DAPI E-GFP Trop-I Merge
0
20
40
60
80
100
Co-culture (-)
E-MSC100
non
oxytocin
5-azaC
non
oxytocin
5-azaC
E-MSC214
0
20
40
60
80
100
non
oxytocin
5-azaC
non
oxytocin
5-azaC
(%)Trop-I(+)
E-GFP(+)
(%)
Trop-I(+)
E-GFP(+)
A
B
C D E F
G H I J
Figure3
DAPI Cx43α actinine Merge
DAPI Cx43α actinine Merge
E-MSC100E-MSC214
E-MSC100E-MSC214
K L M N
O P Q R
Co-culture (+)
Co-culture (-) Co-culture (+)
51. 1sec
2sec
Pipette
2 sec 1 sec
APD90 MDP AMP Rate Pacemaker(+)
msec mV mV s n
E-MSC100 326±12 -55±1 56±7 0.9±0.1 3/28
E-MSC214 185 ±16 -48±3 56±3 2.4±0.2 29/41
APD; action potential duration,
MDP; Maximum diastolic potential, AMP; amplitude
Mean ± SEM
Pipette
E-MSC100
A B C
E-MSC214
D E F
G
EPC-100 は同期が強く、活動電位も regular で静止膜電位も深い
EPC-214 は同期は弱く、活動電位は培養経過中 (3W) は、歩調取り電位を有した洞結節型細胞
60. DAPI Cx43α actinine E-GFP Merge
UCBMSC
DAPI E-GFP Trop-I
DAPI E-GFP Trop-I Merge
K
A B C D
F G H I
Figure3
UCBMSCUCBMSC-TERT
L M N O
Merge
DAPI Cx43α actinine Merge E-GFP Merge
UCBMSC-TERT
0
20
40
60
80
100
Co-culture (-)
UCBMSC-TERT
(%)
Trop-I(+)
E-GFP(+)
Co-culture (+)
5-azaC(-)
5-azaC(+)
5-azaC(-)
5-azaC(+)
Merge K+L+M
P Q
R S T U V W
E
J
Merge 60x
Merge 60x
61. 400 ms
Pipette
APD90 MDP AMP Rate Pacemaker(+)
msec mV mV s n
UCBMSC 186±12 -54±2 57±4 0.62±0.11 0/7
UCBMSC-TERT 182±12 -58±2 62±5 0.60±0.08 0/11
APD; action potential duration,
MDP; Maximum diastolic potential, AMP; amplitude
Mean ± SE
Pipette
UCBMSC
A B
UCBMSC-TERT
C D
E
1 s
5%
Site-a
Site-b
Site-c
Site-d
%FS
Time
a
b
c d
F
G
I
Figure5
400 ms
0
2
4
6
8 UCBMSC
%FS
Mean ± SE
n=10 n=10
UCBMSC
TERT
UCBMSC
80. Transplantation of OP-9 pelleted micromasses in the nude
mice
2W 4W 8W
toluidine blue
HE
x200
Editor's Notes
<number>
Expression of p16 level is primarily regulated transcriptionally. Numbers of factors are known to be involved in the regulation Among them MAP cascade is most well characterized. Dr. Serrano’s group found that activated ras does not transform primary fibroblasts but induces premature senescence of the cells. Dr. Hara found that the direct effectors are Ets family proteins especially Ets1 and 2, which are activated by MAP kinases downstream of ras. Furthermore he found Id1 protein directly bind Ets family protein to inhibit the transactivation by Ets proteins. Indeed, transduction of Id1 into primary human keratincytes has been reported to extend their life span.
Bmi-1 is a gene included in polycomb group of genes, and reported to regulate p16 transcription negatively by binding to its promoter region.
So we focused on bmi-1 because if we could inhibit the induction of p16 expression we might be able to immortalize epithelial cells without expecting spontaneous and infrequent methylation of the promoter.
Bmi-1 was cloned as a c-myc cooperating oncogene in murine
lymphomas. It was shown subsequently to be a transcriptional
repressor belonging to the PcG of proteins. Bmi-1-deficient MEFs
overexpressed the INK4a encoded genes p16 and p19ARF
(mouse homologue of human p14ARF)
and underwent premature senescence in culture.
<number>
When HPV E6 and E7 are expressed in human cells they alter the cells proliferative capacity.
Normal human cells will divide in culture for a set number of population doublings which is dependent upon the particular type of cells.
After that, the cells become senescent and stop dividing. This stage has also been referred to as M1 (for mortality stage 1) and marks the end of the proliferative lifespan of normal cells.
If E6 and E7 are expressed, the cells bypass M1 and exhibit an extended lifespan. These cells may or may not exhibit a crisis stage (referred to as M2) before they are immortalized.
E6 or E7 alone immortalize much less efficiently.
When HPV E6 and E7 are expressed in human cells they alter the cells proliferative capacity.
Normal human cells will divide in culture for a set number of population doublings which is dependent upon the particular type of cells.
After that, the cells become senescent and stop dividing. This stage has also been referred to as M1 (for mortality stage 1) and marks the end of the proliferative lifespan of normal cells.
If E6 and E7 are expressed, the cells bypass M1 and exhibit an extended lifespan. These cells may or may not exhibit a crisis stage (referred to as M2) before they are immortalized.
E6 or E7 alone immortalize much less efficiently.
