Hormon 127.07

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

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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p-p53





***

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１１２２３１１２２３

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 ﾚｾﾌﾟﾀｰCD140ｂ

α ﾚｾﾌﾟﾀｰ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 培養ﾌｨｰﾀﾞｰ乳腺上皮細胞： 3T3 培養ﾌｨｰﾀﾞｰ

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

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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


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


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

Hormon 127.07

Science

Transcript of Hormon 127.07