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間葉系細胞間葉系細胞
間葉系細胞の供給源間葉系細胞の供給源
骨髄骨髄多指症多指症
子宮内膜子宮内膜内膜幹細胞内膜幹細胞
月経血月経血臍帯血臍帯血胎盤胎盤
分化能分化能
成育バイオリソース成育バイオリソース
培養した間葉系細胞は、その組織供給源ごとで異なる形質を有するか?
異なる形質を有するとすれば間葉系細胞に共通している分子と組織特異性を規定
する分子は何か?
HE x100 HE x200 HE x200
Type II collagen
軟骨に由来する間葉系細胞は、培養後も軟骨形成能を保持するか?
YUB-B → 骨髄由来間葉系細胞 10%DMEM 4種 MF培地 4種YUB-C → 軟骨細胞 10%DMEM 5種 MF培地 4種YUB-A → 脂肪前駆細胞 10%DMEM 1種 MF培地 1種YUB-S → 繊維芽細胞 10%DMEM 1種YUB-T → すじ 10%DMEM 1種
これまで 5検体。 組織 5種類。
計21種類
小児指組織からの初代培養
軟骨培養細胞の形態像 (Papanicolau 染色、各倍率 200 倍 )
P0 P1
P10 p16免疫染色
P0軟骨細胞皮下移植 2週間後
軟骨培養細胞皮下移植 2週間後
初代培養細胞移植 初代培養細胞移植、浮遊組織を再移植 一回継代細胞移植
初代培養細胞 HE x 1.25 初代培養細胞 TB x 1.25 初代培養細胞 HE x 200
軟骨に由来する間葉系細胞(軟骨芽細胞)は、培養後も軟骨形成能を保持する。
しかし、継代することで脱分化する。
E7, E6, TERT で不死化しても軟骨特異的な遺伝子発現は保持された。
完全長 cDNA sequence における細胞外 matrix(proteoglycan) の発現
H4-1(human stromal cell) rediffrentiation of dedifferentiated chondrocyte
P1 3w
骨に由来する間葉系細胞(骨芽細胞)は、培養後も骨形成能を保持する。
しかし、継代することで脱分化する .
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. ” ”
667
767
p16Ink4a
Ras*
Raf
MEK1/2
ERK1/2
+
Regulation of p16Ink4a expression
MLKs, TAKMLKs, TAKASK1ASK1
MKK3/6MKK3/6
p38MAPKp38MAPK
Ets family?Ets family?Ets2Id-1
Ets1
?
Ets binding sequenceEts binding sequence
De novoCpG methylation
Bmi-1
?
Chromatinremodeling
?
Bmi-1 遺伝子導入による寿命延長
p16INK4ap16INK4a
Bmi-1
ヒト骨髄間質細胞
p1614 15 16 17 19
G1 arrest
p16INK4a
Cdk4/6
RB
E2F 転写抑制
G1 arrest
p16INK4a
Cdk4/6
RB
E2F 転写抑制
G1 arrest
p16INK4a
Cdk4/6
RB
E2F 転写抑制
popu
latio
n do
ublin
gs
days in culture
normal
XM0
XM1
Regulation of p16/Rb
telomerase activation
immortalization
+E6 or T
ERT
replicative senescence (mortality stage 1:M1)replicative senescence (mortality stage 1:M1)
Mitotic clockMitotic clock
oror
Inappropriate culture condition?Inappropriate culture condition?
+ E7 or Bmi-1+ E7 or Bmi-1
A
B
C
D
Neural differentiation
Shown are neural differentiation cells (A-C, and E); no-treatment cells (E and F)
EE
FF
Human (H4-1)Human (H4-1) Murine (KUSA-A1)Murine (KUSA-A1)
IRB 承認 平成 15 年 11 月 27 日 cold blood
1500rpm5min
sub culture
lymphocyte
erythrocyteculture
1500rpm5min
cold blood sub culture
Ficoll-Paque
lymphocyte culture
low culture
erythrocyte
1500rpm40min
臍帯血由来間葉系細胞の培養 301 :臍帯血 32ml → M0 10%FBS-DMEM 302 :臍帯血 57ml → 増殖 10%FBS-DMEM→MSCGM 303 :臍帯血 53ml → M0 MSCGM 304 :臍帯血 22ml → M0 MSCGM 404 :臍帯血 5ml → M0 MSCGM→ 10%FBS-DMEM 405 :臍帯血 5ml → M0 MSCGM→ 10%FBS-DMEM 406 :臍帯血 5ml → M0 MSCGM→ MFⅡcomplete 407 :臍帯血 5ml → M0 MSCGM→ MFⅡcomplete 408 :臍帯血 5ml → 増殖 MSCGM→ MFⅡcomplete
ヒト臍帯血間葉系細胞 s ( UCB )
Popu
latio
n D
oubl
ings
Cultured Days0 80 1800
20
40
Methylation assay of p16 promoterMol.Cell.Biol 1999, 5642-51 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35HMEC6/40:LXSN-16E6E7(@p4) p=16 1 0HMEC6/40:LXSN-16E6E7(@p4) p=16 2 0HMEC6/8:LXSN-16E6SD(@p4) p=31 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 14HMEC6/8:LXSN-16E6SD(@p4) p=31 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 23
UCB (primary) p=5 1 1 1 2UCB (primary) p=5 2 1 1 2UCB (primary) p=5 3 0UCB (primary) p=5 4 0UCB (primary) p=5 5 1 1 2UCB (primary) p=5 6 1 1 2
UCB p=9 1 0UCB p=9 2 0UCB p=9 3 0
3 4 5 7 8 9 16 HeLa
p16
Actin
RB
p16
UC
BM
SCs (
13 P
Ds)
UC
BM
SCs (
21 P
Ds)
UC
BM
SCs (
32 P
Ds)
UC
BTE
RT-
21 (3
2 PD
s)
UC
BTE
RT-
21 (3
8 PD
s)
UC
BTE
RT-
21 (5
3 PD
s)
UC
BTE
RT-
21 (6
9 PD
s)
UC
BE6
E7-2
0 (3
7 PD
s)
UC
BTE
RT-
21 +
H2O
2 (6
9 PD
s)
HeL
a
C33
A
β-actin
p21
p53
p-ATM
1 2 3 4 5 6 7 8 9 10 11
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ǙDZÇÃÉsÉNÉ`ÉÉÇ å©ÇÈÇΩÇflÇ…ÇÕïKóvÇ≈Ç∑ÅB
p-p53
QuickTime˛ Ç∆TIFFÅiLZWÅj êLí£ÉvÉçÉOÉâÉÄ
ǙDZÇÃÉsÉNÉ`ÉÉÇ å©ÇÈÇΩÇflÇ…ÇÕïKóvÇ≈Ç∑ÅB
QuickTime˛ Ç∆TIFFÅiLZWÅj êLí£ÉvÉçÉOÉâÉÄ
ǙDZÇÃÉsÉNÉ`ÉÉÇ å©ÇÈÇΩÇflÇ…ÇÕïKóvÇ≈Ç∑ÅB
***
H4-1
本来の性質(細胞の初期値)を保持したまま増殖させる液性因子は何か?
○寿命延長化ヒト骨髄由来間葉系幹細胞で組成検討
○初代細胞へ適用
・ UEET12 (Mesenchymal stem cells)
1.基礎培地2.増殖因子3.ホルモン、微量元素その他
血清の役割・増殖因子・微量元素・脂質成分・脂質、鉄等のキャリアー・抗酸化作用・細胞接着性の向上・細胞毒性物質の中和
増殖培地=血清(牛胎児)+基礎培地
EGF1003010
31
PDGF1003010
31
bFGF1003010
31
aFGF1003010
31
LIF1003010
31
HGF3010
31
IL610
31
0.30.1
VEGF3010
3
0 50 100 150 200 250 300Cell Growth(Åì,ëŒÇfÇeñ≥ìYâ¡î‰Åj
ëùêBàˆéqÇ…ÇÊÇÈëùêBë£êiå¯â ÅiÇtÇdÇdÇsÅ|ÇPÇQÅj
増殖因子の最適化
◎ PDGF,EGF,b FGF,a FGF/○ LIF/△ VEGF,HGF
( ng/ml )
PDGF
EGFLIF
VEGF
P+EP+LP+V
P+E+LP+E+VP+L+V
0 50 100 150 200 250 300 350Cell Growth(%,ëŒGFñ≥ìYâ¡î‰)
UEET-12Å@ëùêBàˆéqÇÃëgçáÇπ
複数の増殖因子により相乗効果が得られる。
0 10 20 30 40 50 60
106
104
108
1014
1012
1010
#1#2#2#2C
umul
ativ
e ce
ll nu
mbe
r
1016
#2#2
0 10 20 30 40 50 60
106
104
108
1014
1012
1010
Cultuered day
Cum
ulat
ive
cell
num
ber
#1#1#2#2
0 10 20 30 40 50 60
106
104
108
1014
1012
1010
1018
Cum
ulat
ive
cell
num
ber
A
B
C
1016
MSCGM X
p16ink4a
β‐tubulin
培地 X ではp16INK4 a 発現上昇がみとめられる
蘇生後の継代 1 1 2 2 3 1 1 2 2 3
0 5 10 15 20 25day
8
10
12
14
16
18
20
22
PDL MCSGM
X
MSC
培地 X で p16 の発現が高いメカニズムは?
Q
o oo ooo oox oooo ooox ooxo ooxx ox oxo oxx oxxx oxoo
x xx xxx xxo xo xox xoxx xoo xoox xooo xoxo
○ → MSCGM 、 × → 培地 X
hMSC( P-7)
培地 X ×
MSCGM ○培地 X ○ ×
MSCGM ○○
培地 X ××
MSCGM ×○
○○○
○○×○×○
○×××○○
×○×××○
×××
培地により p16 発現は変動する。
p16ink4a
β‐tubulin
p16ink4a
β‐tubulin
X + FCS -IT+FCS
-IT -PDGF-b FGF
-EOP -Sel -P,b+FCS-P,b MSC
p16誘導の主要要因は PDGF,b FGF である。
p16ink4a
β‐tubulin
X-P,b X-P,b/10%FCS MSCGMPDGF+bFGF(ng/ml) 0 1 10 0 1 10 0 1 10
bFGF,PDGFが p16 発現を誘導する。
p16ink4a
β‐tubulin
PDGF +b FGF により p16ink4a 発現が上昇
なぜ培地 X で p16 の発現が高いのか?Q
A
MSC bFGF P-AAP-BB
P-AB
+bFGF
P-AA,BBP-AA
P-BBP-ABPDGF
-AAPDGF
-ABPDGF
-BB
A鎖 B鎖
β レセプターCD140b
α レセプターCD140a
Greb 2Nck
Ras -GAP
Crk SrcSHP2
PLC-r
PDGF-AA→Competent FactorPDGF-BB→Competent and Proguression Factor
IgG CD140a CD140b
p16
βtubulin
― +MSC の p16 発現は PDGF-B鎖による。
( 細胞 /H10‐8)
MSC
aFGF
bFGF
EGFVEGFPDGF
PDGF+aFGF
PDGF+bFGF
PDGF+EGFPDGF+VEGF
aFGF+bFGF
aFGF+EGF
aFGF+VEGF
bFGF+EGF
bFGF+VEGF
EGF+VEGFHeL
a
p16p16
βtubulinβtubulin
H4-3Normal iz e dFlags Raw Common Description
1.0818335 P 2172.2 egfr epidermal growth factor receptor (erythroblastic leukemia viral (v-erb-b) oncogene homolog, avian)1.1522822 P 657.3 egfr epidermal growth factor receptor (erythroblastic leukemia viral (v-erb-b) oncogene homolog, avian)0.6146532 A 337.2 egfr epidermal growth factor receptor (erythroblastic leukemia viral (v-erb-b) oncogene homolog, avian)0.8937527 A 17.8 egfr epidermal growth factor receptor (erythroblastic leukemia viral (v-erb-b) oncogene homolog, avian)1.0583736 A 167.9 egfr epidermal growth factor receptor (erythroblastic leukemia viral (v-erb-b) oncogene homolog, avian)0.8768445 A 358.1 egfr ErbB1-S; Human epidermal growth factor receptor precursor (EGFR) mRNA, complete cds.1.0504522 A 10.4 fgfr2 fibroblast growth factor receptor 2 (bacteria-expressed kinase, keratinocyte growth factor receptor, craniofacial dysostosis 1, Crouzon syndrome, Pfeiffer syndrome, J ackson-Weiss syndrome)0.7824624 A 21.5 fgfr2 fibroblast growth factor receptor 2 (bacteria-expressed kinase, keratinocyte growth factor receptor, craniofacial dysostosis 1, Crouzon syndrome, Pfeiffer syndrome, J ackson-Weiss syndrome)0.417403 A 9.8 fgfr3 fibroblast growth factor receptor 3 (achondroplasia, thanatophoric dwarfism)
0.54518497 A 27.2 fgfr3 fibroblast growth factor receptor 3 (achondroplasia, thanatophoric dwarfism)1A 38.3 fgfr4 fibroblast growth factor receptor 4
1.2821407 A 125.7 fgfr1 fibroblast growth factor receptor 1 (fms- related tyrosine kinase 2, Pfeiffer syndrome)1P 652.2 fgfr1 fibroblast growth factor receptor 1 (fms- related tyrosine kinase 2, Pfeiffer syndrome)
1.4052641 A 14.3 fgfr2 fibroblast growth factor receptor 2 (bacteria-expressed kinase, keratinocyte growth factor receptor, craniofacial dysostosis 1, Crouzon syndrome, Pfeiffer syndrome, J ackson-Weiss syndrome)0.75333506 A 59.2 fgfr2 fibroblast growth factor receptor 2 (bacteria-expressed kinase, keratinocyte growth factor receptor, craniofacial dysostosis 1, Crouzon syndrome, Pfeiffer syndrome, J ackson-Weiss syndrome)1.3886433 P 124.5 fgfr2 fibroblast growth factor receptor 2 (bacteria-expressed kinase, keratinocyte growth factor receptor, craniofacial dysostosis 1, Crouzon syndrome, Pfeiffer syndrome, J ackson-Weiss syndrome)1.1120172 A 13.6 fgfr2 fibroblast growth factor receptor 2 (bacteria-expressed kinase, keratinocyte growth factor receptor, craniofacial dysostosis 1, Crouzon syndrome, Pfeiffer syndrome, J ackson-Weiss syndrome)1.0663294 P 2619.3 fgfr1 fibroblast growth factor receptor 1 (fms- related tyrosine kinase 2, Pfeiffer syndrome)
1A 11.1 fgfr4 fibroblast growth factor receptor 41.1922568 A 46.9 fgfr2 fibroblast growth factor receptor 2 (bacteria-expressed kinase, keratinocyte growth factor receptor, craniofacial dysostosis 1, Crouzon syndrome, Pfeiffer syndrome, J ackson-Weiss syndrome)3.1074321 A 73.5 fgfr2 fibroblast growth factor receptor 2 (bacteria-expressed kinase, keratinocyte growth factor receptor, craniofacial dysostosis 1, Crouzon syndrome, Pfeiffer syndrome, J ackson-Weiss syndrome)
1A 67.3 fgfr2 fibroblast growth factor receptor 2 (bacteria-expressed kinase, keratinocyte growth factor receptor, craniofacial dysostosis 1, Crouzon syndrome, Pfeiffer syndrome, J ackson-Weiss syndrome)1A 8.2 fgfr2 fibroblast growth factor receptor 2 (bacteria-expressed kinase, keratinocyte growth factor receptor, craniofacial dysostosis 1, Crouzon syndrome, Pfeiffer syndrome, J ackson-Weiss syndrome)
1.3065747 P 3125.7 fgfr1 fibroblast growth factor receptor 1 (fms- related tyrosine kinase 2, Pfeiffer syndrome)3.3271298 P 4049.1 pd gf r bplatelet-derived growth factor receptor, beta polypeptide1.0704594 P 4990.3 pd gf r aplatelet-derived growth factor receptor, alpha polypeptide1.0662563 P 366.9 pd gf r lplatelet-derived growth factor receptor- like0.8164289 A 6.2 pd gf r aplatelet-derived growth factor receptor, alpha polypeptide2.3044996 A 403.1 pd gf r aplatelet-derived grow t h f a ct or r ec ep to r, a l p ha p oly pe p t id e0.6091959 A 19.5 flt4 fms- related tyrosine kinase 41.7572612 A 61.6 kdr kinase insert domain receptor (a type III receptor tyrosine kinase)
aFGF-R ○bFGF-R ○EGF-R ◎VEGF-R ×PDGF-R ◎
受容体の有無
p16 発現は増殖因子の種類による。
細胞増殖に都合の良い増殖因子ほど p16 の発現を誘導する?
相関係数:
0.700 50 100 150 200 250cell growth
0
20
40
60
80
100
120
p16
p16/cell growth
増殖因子によるp16発現上昇
→ 他細胞ではどうか?
毛乳頭細胞角化表皮細胞
HUVECHUVEC(ヒト臍帯静脈内皮細胞)(ヒト臍帯静脈内皮細胞)
HBECHBEC(ヒト気管支上皮細胞)(ヒト気管支上皮細胞)HDPCHDPC(( ヒト頭髪毛乳頭細胞ヒト頭髪毛乳頭細胞 ))
44 55 66 77 88 99 1010 111144 55 66 77 88
44 55 66 77 88 99 1010 1111
PassagePassage
Passage Passage
p16p16
22 33 44 55 66
HEECHEEC(( ヒト食道上皮細胞ヒト食道上皮細胞 ))
Passage Passage
33 44 55 77 1010 1313 1717 2020 2525 HFK
p=9HFFHFF
((ヒト新生児皮膚線維芽細胞ヒト新生児皮膚線維芽細胞 ))
p16p16
HFKHFK((ヒト陰茎包皮角化細胞ヒト陰茎包皮角化細胞 ))
44 55 66 88 99 101011 22 33
p16p16
清野先生のデータ
時間
分裂回数
MSC : MSCGM ?毛乳頭細胞: 10%FCS-DMEM表皮角化細胞: 3T3 培養フィーダー乳腺上皮細胞: 3T3 培養フィーダー
MSC :培地 X ?毛乳頭細胞:低血清培地表皮角化細胞:無血清培地乳腺上皮細胞:無血清培地
血清培地
増殖因子
増殖因子 ストレス
Ras
Raf
MEK1/2
ERK1/2
Ets1/2
MLKs、 TAKASK1
MKK3/6
p38MAPK
A
B
p16 INK4a
X10%FCS
X A B
p16ink4a
β‐tubulin
Bone marrow stromal cells
Mori, et al., Mol Cell Biol. 2005, 25(12):5183
UEE-16UET-13UEET-12UBET-7UBT-5
QuickTime˛ Ç∆ êLí£ÉvÉçÉOÉâÉÄ
ǙDZÇÃÉsÉNÉ`ÉÉÇ å©ÇÈÇ…ÇÕïKóvÇ≈Ç∑ÅB
QuickTime˛ Ç∆ êLí£ÉvÉçÉOÉâÉÄ
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1 week 3 weeks
Recording of action potentialsfrom spontaneously beating cells
Alexa568Human marrow stromal cells with the extended life span
can differentiate into cardiomyocytes
Disorganized
Regular and stabilized
Recording microelectrode
Endometrial stem cells
Am J Pathol. 2003, 163(6):2259
Endometrial stem cells
EPC-100, EPC-214
Gene transfer
Primary cultureEPCs
Limiting dilution
hTERTE6 (HPV16)E7 (HPV16)
EPC-100 EPC-214
18S(200bp)
E6(397bp)
E7(178bp)
TERT(144bp)
EPC-100 EPC214
Hum
an H
eart
hEPC
100(
RT-)
hEPC
214(
RT-)
hEPC
100
hEPC
214
DW
A
E-MSC100
E-MSC214
D(+)
18S (200bp)
hANP (406bp)
hBNP (206bp)
Cardiac Actin (400bp)
Csx/Nkx 2.5 (233bp)
GATA4 (475bp)
MLC2a (376bp)
TnT (152bp)
TnI (233bp)
HCN2 (230bp)
Hum
an h
eart
E-M
SC10
0E-
MSC
100(
RT-)
E-M
SC21
4E-
MSC
214(
RT-)
E-M
SC10
0E-
MSC
100(
RT-)
E-M
SC21
4E-
MSC
214(
RT-)
Mou
se h
eart
DW
co-culture (-)
CFigure 2
Gene productPrimer(sense)
Annealing Temperature
(°C)
Productsize(bp)
Primer(anti-sense)
hANP GAACCAGAGGGGAGAGACAGAG CCCTCAGCTTGCTTTTTAGGAG 55 406hBNP CATTTGCAGGGCAAACTGTC CATCTTCCTCCCAAAGCAGC 61 206Cardiac Actin CTTCCGCTGTCCTGAGACAC CCAGACTGGAAGGTAGATGG 61 400hCsx/hNkx2.5 CTTCAAGCCAGAGGCCTACG CCGCCTCTGTCTTCTCCAGC 61 233GATA4 AGACATCGCACTGACTGAGAAC GACGGGTCACTATCTGTGCAAC 61 475MYLC2a GAAGGTGAGTGTCCCAGAGG ACAGAGTTTATTGAGGTGCCCC 61 376cTnT GGCAGCGGAAGAGGATGCTGAA GAGGCACCAAGTTGGGCATGAACGA 61 152cTnI CCCTGCACCAGCCCCAATCAGA CGAAGCCCAGCCCGGTCAACT 61 233hHCN2 CGCCTGATCCGCTACATCCAT AGTGCGAAGGAGTACAGTTCACT61 230E6 GACCCAGAAAGTTACCACAG GCAACAAGACATACATCGAC 60 397E7 ATGACAGCTCAGAGGAGGAGTCCTAGTGTGCCCATTAACAG 60 178TERT CGGAAGAGTGTCTGGAGCAAGGATGAAGCGGAGTCTGGA 60 14418S GTGGAGCGATTTGTCTGGTT CGCTGAGCCAGTCAGTGTAG 60 200
B
E-GFP
DAPI E-GFP Trop-I Merge
DAPI E-GFP Trop-I Merge
020406080
100
Co-culture (-)
E-MSC100
non
oxyt
ocin
5-az
aC
non
oxyt
ocin
5-a z
a C
E-MSC214
020406080
100
non
oxyt
ocin
5-az
aC
non
oxyt
ocin
5-a z
a C
(%)Tr
op-I
(+)
E-G
FP(+
)
(%)
Trop
-I(+
)E-
GFP
(+)
A
B
C D E F
G H I J
Figure3
DAPI Cx43actinine Merge
DAPI Cx43 actinine Merge
E-M
SC10
0E-
MSC
214
E-M
SC10
0E-
MSC
214
K L M N
O P Q R
Co-culture (+)
Co-culture (-) Co-culture (+)
1sec2sec
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
Menstrual BloodMenstrual Blood
EDOM AP 2005/1/26 (8 days)
APD90 MDP AMP BCL Pacemaker(+)
msec mV mV s n
E-Dom 212±28 -46±3 48±3 0.69±0.1 16/16
APD; action potential duration, MDP; Maximum diastolic potential, AMP; amplitude, BCL; Beating cycle lengthMean ± SE
EDOM 細胞の移植による NOGマウスの骨格筋に Vimentin の発現
In vitroによる EDOM細胞の骨格筋の誘導
月経血由来細胞月経血由来細胞
Umbilical Cord Blood
Terai, et al., Mol Biol Cell. 2005, 16(3):1491
Figure 1
Umbilical cord blood
Primary culture
Gene transfer hTERT
Limiting dilution
A
18S(200bp)TERT(144bp)
Hum
an H
eart
UCBM
SCUC
BMSC
(RT-
)UC
BMSC
-TER
TUC
BMSC
-TER
T(RT
-)
DW
UCBMSC
UCBMSC-TERT
UCBMSC
UCBMSC-TERT
B
D
C
A
(+)
18S (200bp)
hANP (406bp)
hBNP (206bp)
Cardiac Actin (400bp)
Csx/Nkx 2.5 (233bp)
GATA4(475bp)
MHC(413)
MLC2 (376bp)
TnT (152bp)
TnI (233bp)
HCN2 (230bp)
Hum
an h
eart
UCBM
SCUC
BMSC
(RT-
)UC
BMSC
-TER
TUC
BMSC
-TER
T(RT
-)M
ouse
hea
rtDW
co-culture (-)
CFigure 2
Gene productPrimer(sense)
Annealing Temperature
(°C)
Productsize(bp)
Primer(anti-sense)
hANP GAACCAGAGGGGAGAGACAGAG CCCTCAGCTTGCTTTTTAGGAG 55 406hBNP CATTTGCAGGGCAAACTGTC CATCTTCCTCCCAAAGCAGC 61 206Cardiac Actin CTTCCGCTGTCCTGAGACAC CCAGACTGGAAGGTAGATGG 61 400hCsx/hNkx2.5 CTTCAAGCCAGAGGCCTACGCCGCCTCTGTCTTCTCCAGC 61 233GATA4 AGACATCGCACTGACTGAGAAC GACGGGTCACTATCTGTGCAAC 61 475MHC GCAAAGTACTGGATGACACGCT GTCATTGCTGAAACCGAGAATG 61 413MYLC2 GAAGGTGAGTGTCCCAGAGG ACAGAGTTTATTGAGGTGCCCC 61 376cTnT GGCAGCGGAAGAGGATGCTGAA GAGGCACCAAGTTGGGCATGAACGA 61 152cTnI CCCTGCACCAGCCCCAATCAGA CGAAGCCCAGCCCGGTCAACT 61 233hHCN2 CGCCTGATCCGCTACATCCAT AGTGCGAAGGAGTACAGTTCACT61 230TERT CGGAAGAGTGTCTGGAGCAAGGATGAAGCGGAGTCTGGA 60 14418S GTGGAGCGATTTGTCTGGTT CGCTGAGCCAGTCAGTGTAG 60 200
B
UCBM
SC-T
ERT
UCBM
SC-T
ERT(
RT-)
CD13 CD14 CD24 CD29 CD31
CD34 CD44 CD45 CD54 CD55
CD59 CDw90 CD105 CD117 CD133
CD164CD140a
SSEA-4SSEA-3SSEA-1
Flk-1CD157 CD166
DAPI Cx43actinine E-GFP Merge
UC
BM
SC
DAPI E-GFP Trop-I
DAPI E-GFP Trop-I Merge
K
A B C D
F G H I
Figure3
UC
BM
SCU
CB
MSC
-TER
T
L M N O
Merge
DAPI Cx43actinine Merge E-GFP Merge
UC
BM
SC-T
ERT
0
20
40
60
80
100
Co-culture (-)
UCBMSC-TERT
(%)
Trop
-I(+
)E-
GFP
(+)
Co-culture (+)
5-az
aC(-
)
5-az
aC(+
)
5-az
aC(-
)
5-az
aC(+
)
Merge K+L+M
P Q
R S T U V W
E
JMerge 60x
Merge 60x
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
E1 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
UCBMSCTERT
UCBMSC
AR (androgen receptor)AR (androgen receptor)
0
50
100
150
200
250
300
350
400
450
500
1687Edom4
1688Edom4HRF
1689Edom5
1690Edom5HRF
1691Edom6
1692Edom6HRF1300EPC1001301EPC214
1840EPCcontrol1841EPCTERT11839EPCTERT2
1112H4-11113UBT51114UBET71115UEET121117UEE161771UET91116UET13
1118UET13NeuralDiff1528MUET13NeuralDiff1
1529UET13NeuralDiff41530UET13NeuralDiff5
1302UCB3021303UCB302D7
1501UCB302TERT
1759UCB408
1760UCB408E6E7-311767UCB408E7-32
1317TdHC1
1319Ligament#10
1617HFDPC1685PL901686PL112
1768H10-2vector1769H10-2Bmi11770H10-2Tert
1772HF7-3
1773BMSC3F0664GSM38627BMSC
GSM38628hES_MSCGSM38629hES_MSC
1902HAdpc28
2009M-1 HAdPC12010M-BM FBS2011M-BM hS2012-Syv FBS2013-Syv hS
1838Yub610_10F0590NHEK-Neo10601RPMI82260734BEAS-2B
0937A5491329mNK921332Raji-1
1502NHBE-11842NCR-G1Ad
1753NCR-G2NAd1754NCR-G2Ad
1299NCR-G3NAd1755NCR-G3Ad1756NCR-G4Ad1757NCR-EW21758NCR-EW3
1623GST6
1805GST6extra1806GST6-5az
1807GST6-5az-extra1898U937control
1899U937HRF1900U937HRFAb
1901U937Ab
GSM31092UCBCD34+GSM31093UCBCD34+GSM31094UCBCD34+GSM31095UCBCD34+GSM31805HEK293GSM31806HEK293
GSM41342hESGSM41343hESGSM41344hES
AR(211110_s_at)AR(211621_at)
ESR1 (estrogen receptor 1)ESR1 (estrogen receptor 1)
0
50
100
150
200
250
300
350
400
450
1687Edom4
1688Edom4HRF
1689Edom5
1690Edom5HRF
1691Edom6
1692Edom6HRF1300EPC1001301EPC214
1840EPCcontrol1841EPCTERT11839EPCTERT2
1112H4-11113UBT51114UBET71115UEET121117UEE161771UET91116UET13
1118UET13NeuralDiff1528MUET13NeuralDiff1
1529UET13NeuralDiff41530UET13NeuralDiff5
1302UCB3021303UCB302D7
1501UCB302TERT
1759UCB408
1760UCB408E6E7-311767UCB408E7-32
1317TdHC1
1319Ligament#10
1617HFDPC1685PL901686PL112
1768H10-2vector1769H10-2Bmi11770H10-2Tert
1772HF7-3
1773BMSC3F0664GSM38627BMSCGSM38628hES_MSCGSM38629hES_MSC
1902HAdpc282009M-1 HAdPC1
2010M-BM FBS2011M-BM hS2012-Syv FBS2013-Syv hS1838Yub610_10F0590NHEK-Neo1
0601RPMI82260734BEAS-2B
0937A5491329mNK921332Raji-1
1502NHBE-11842NCR-G1Ad1753NCR-G2NAd1754NCR-G2Ad1299NCR-G3NAd1755NCR-G3Ad1756NCR-G4Ad1757NCR-EW21758NCR-EW3
1623GST6
1805GST6extra1806GST6-5az
1807GST6-5az-extra
1898U937control1899U937HRF
1900U937HRFAb
1901U937Ab
GSM31092UCBCD34+GSM31093UCBCD34+GSM31094UCBCD34+GSM31095UCBCD34+GSM31805HEK293GSM31806HEK293
GSM41342hESGSM41343hESGSM41344hES
ESR1(205225_at)ESR1(211233_s_at)ESR1(211234_s_at)ESR1(215551_at)ESR1(215552_s_at)
Hierarchical analysis & principal compornent analys
is (PCA)(Cell surface marker associated genes)
MatsumotoTakeda
MoriTsuchiya
NasuNasu
National Research InstituteNational Research Institutefor Child and Health Developmentfor Child and Health Development
TakahashiTakahashi
ShibuyaShibuya
TeraiTerai
Takeda MatsumotoUyama
慶應義塾大学病理学教室森 泰昌
生理学教室岡野栄之島崎琢也
埼玉医科大学五條理志許 俊英
整形外科学教室戸山芳昭今林英明越智健介
微生物学教室瀬川 薫 NIH, USA
洪 実
National Cancer Center, Jpn清野 透
Helix 研究所磯貝 隆夫杉山 友康入江 亮太郎
中外製薬東佐由美大川広行
National Center for Child Health and Development
P1 3w
骨分化誘導前後のALP活性
Kb21
8
5
Telomere length assay
PDs 20 40 70 L H MH4-1UE6E7-16
Telomere length
PDs PDs 5555 8080 80 12080 120 80 120 40 L H 80 120 40 L HUBT-5UBT-5
UBEUBE66T-7T-7UEUE66EE77-12-12UEUE77T-13T-13
Kb21
8
5
Telomere activity analysis (TRAP assay)
Umezawa, A., et al.. Mol. Cell. Biol. 11: 920-927, 1991
Mdx/Scid モデルマウス
ヒト臍帯血間葉系細胞( UCB )Po
pula
tion
Dou
blin
gs
Cultured Days
Infection(P12)
UCB:344 TERT(@P12)
UCB:241 E6E7(@P12)
UCB:108 vec puro(@P12)UCB:Mock infection UCB
0
10
20
30
40
50
60
70
0 20 40 60 80 100 120 140
Serum No serumExcess growth factor
Cell proliferation Senescence
Activated Ras Oxidative stress
Mek-Erk p38
p16
Mek-Erk
Cell proliferation
E
Scheme of the experimetal protocol
Transplantation of OP-9 pelleted micromasses in the nude mice
2W 4W 8W
toluidine blue
HE
x200