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

支持能支持能

検体肉眼像および組織像

HE x100 HE x200 HE x200

Type II collagen

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 HEｘ1.25

初代培養細胞　 TBｘ 1.25初代培養細胞　 HEｘ 200

HE X1.5 HE X100

Type II collagen X100 S-100 X200

HE X200

T.Blue X100

P0 軟骨細胞移植部　組織形態像

Human Marrow Stromal Cellsreach senescence after a limited number of cell division

H4-1H4-1

H4-1H4-1

慶應義塾大学医学部慶應義塾大学医学部血液内科血液内科

　木崎 昌弘　木崎 昌弘　福地 由美 　福地 由美

H4-1, senescenceH4-1, senescence

Mori, et al., Mol Cell Biol, 2005

Prolongation of the life span of human marrow stromal cellsby transferring Bmi-1, E6, E7, and TERT

The cells with the extended life span retained the multipotency into osteocytes, chondrocytes, adipocytes, and myocytes in vitro.

ヒト臍帯血間葉系細胞 s（ UCB）

Po

pu

latio

n D

ou

blin

gs

Cultured Days0 80 180

0

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 35

HMEC6/40:LXSN-16E6E7(@p4) p=16 1 0

HMEC6/40:LXSN-16E6E7(@p4) p=16 2 0

HMEC6/8:LXSN-16E6SD(@p4) p=31 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 14

HMEC6/8: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 2

UCB (primary) p=5 2 1 1 2

UCB (primary) p=5 3 0

UCB (primary) p=5 4 0

UCB (primary) p=5 5 1 1 2

UCB (primary) p=5 6 1 1 2

UCB p=9 1 0

UCB p=9 2 0

UCB p=9 3 0

3 4 5 7 8 9 16 HeLa

p16

Actin

Terai, et al. Mol Biol Cell, 2005

0

20

40

60

80

100

120

140

0 50 100 150 200 250

培養日数

E6E7

Mock

Infection

hTERT Mock

hTERT

E6E7

細胞増殖曲線細胞分裂回数

0 0.5 1 2 10

FCS(%)

0

20

40

60

80

100

120

PPL

PLVMSCGM(10%FCS)

UEET-12 　無血清培養

改良培地は無血清下でも血清含有培地と同等の増殖性を有する。

bPAPBhP

bPAbPBbhP

bPABaEV

PaPEPVabbEbVaEaVEV

-

0 20 40 60 80 100 120

mAbs=450nm

MSC äeëùêBàˆéqÇ…ÇÊÇÈëùêBå¯â 

時間

分裂回数血清培地

増殖因子

ＭＳＣＧＭ X

ｐ１６ＩＮＫ４ａ

β tubulin‐

蘇生後の継代 １ １ ２ ２ ３ １ １ ２ ２ ３

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 MSCGM

PDGF+bFGF(ng/ml) 0 1 10 0 1 10 0 1 10

ｂＦＧＦ，ＰＤＧＦが p16発現を誘導する。

MSC bFGF P-AAP-BB

P-AB

+bFGF

P-AA,BB

P-AAP-BB

P-ABPDGF-AA

PDGF-AB

PDGF-BB

A鎖 B鎖

β ﾚｾﾌﾟﾀｰCD140ｂ

α ﾚｾﾌﾟﾀｰCD140ａ

Greb２Nck

Ｒａｓ -GAP

CrkSrc

SHP2PLC-ｒ

PDGF-AA→Competent FactorPDGF-BB→Competent and Prouression Factor

IgG CD140a CD140b

p16

βtubulin

― ＋

MSCの p16発現は PDGF-B鎖による。

(細胞 /H10 8)‐

MSC

aFG

FbF

GF

EGF

VEGF

PDGF

PDGF+aF

GF

PDGF+bFG

FPDG

F+EGF

PDGF+VEG

F

aFG

F+bFGF

aFG

F+EGF

aFG

F+VEGF

bFGF+EG

FbFG

F+VEGF

EGF+VEG

F

HeL

a

p16p16

βtubulinβtubulin

H4-3Normalized Flags 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, Jackson-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, Jackson-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)

1 A 38.3 fgfr4 fibroblast growth factor receptor 41.2821407 A 125.7 fgfr1 fibroblast growth factor receptor 1 (fms-related tyrosine kinase 2, Pfeiffer syndrome)

1 P 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, Jackson-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, Jackson-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, Jackson-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, Jackson-Weiss syndrome)1.0663294 P 2619.3 fgfr1 fibroblast growth factor receptor 1 (fms-related tyrosine kinase 2, Pfeiffer syndrome)

1 A 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, Jackson-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, Jackson-Weiss syndrome)

1 A 67.3 fgfr2 fibroblast growth factor receptor 2 (bacteria-expressed kinase, keratinocyte growth factor receptor, craniofacial dysostosis 1, Crouzon syndrome, Pfeiffer syndrome, Jackson-Weiss syndrome)1 A 8.2 fgfr2 fibroblast growth factor receptor 2 (bacteria-expressed kinase, keratinocyte growth factor receptor, craniofacial dysostosis 1, Crouzon syndrome, Pfeiffer syndrome, Jackson-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 pdgfrb platelet-derived growth factor receptor, beta polypeptide1.0704594 P 4990.3 pdgfra platelet-derived growth factor receptor, alpha polypeptide1.0662563 P 366.9 pdgfrl platelet-derived growth factor receptor-like0.8164289 A 6.2 pdgfra platelet-derived growth factor receptor, alpha polypeptide2.3044996 A 403.1 pdgfra platelet-derived growth factor receptor, alpha polypeptide0.6091959 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。

増殖因子 ストレス

Ras

Raf

MEK1/2

ERK1/2

Ets1/2

MLKｓ、 TAKASK1

MKK3/6

ｐ３８ MAPK

A

B

ｐ１６ INK4a

p16ink4a

β-tubulin

X10%FCS

XA B

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

Mori

National Center for Child Health and Development