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Induction of Pluripotency: ) v4 J8 [2 R7 C1 G& o
From Mouse to Human+ a- a% P4 q% \( B7 @% S1 `3 C
Holm Zaehres1
. Q* g# a% ^7 ]* w6 z+ m, ? and Hans R. Schöler1,
0 m% p3 Z) h/ T3 Z. ]% D4 ~*- K" a1 L# Q" q& p% ~) U
1 `7 y) l3 ~& S
Max Planck Institute for Molecular Biomedicine, Department of Cell and Developmental Biology, Münster, NRW 48149, Germany
' i- Y) M) Q5 E7 ^6 @8 ~*Correspondence: schoeler@mpi-muenster.mpg.de/ [# F! o* B7 F, R K- l( a8 {
DOI 10.1016/j.cell.2007.11.020
$ w6 H( @4 A) E" }! }In this issue of Cell, Takahashi et al. (2007) transfer their seminal work on somatic cell & z: f8 \( W# Y# b! X. _, B1 U8 Y
reprogramming from the mouse to human. By overexpressing the transcription factor
9 |/ I. c- d2 n& tquartet of Oct4, Sox2, Klf4, and c-Myc in adult human fbroblasts, they successfully $ C; D# i' q" g6 y( I" D+ }: p( k
isolate human pluripotent stem cells that resemble human embryonic stem cells by all 4 U3 F% T. R- A( w
measured criteria. This is a signifcant turning point in nuclear reprogramming research - U" T; [. g- u) K
with broad implications for generating patient-specifc pluripotent stem cells for research
% A! f+ m" \/ s3 _+ Mand therapeutic applications.$ I2 Y t" t" w8 n1 a
This year’s three Physiology or Medi-/ ^- M% Z/ _! n) H
cine Nobel Laureates—Martin Evans, 0 }0 H3 Q; L6 }5 j# _- j, A# z5 |
Mario Capecchi, and Oliver Smithies—
s B; |" k* S: i r$ `3 ~will be honored in Stockholm in 10 + z- N8 c. e' L
days time for their discovery of DNA
% y0 u' [, ?% `: D4 e+ \recombination and the development
7 E3 V6 B7 l4 x; p! Tof mouse embryonic stem (ES) cell % h9 y% s" f8 e. q5 H- c
technology. It was Martin Evans who
9 i c) z$ D& o: h5 U l8 Q0 k( gdiscovered how to make mouse ES
# i- M1 R. G3 L ]: l( S) ~9 u+ }6 lcells, enabling any genetic alteration ( Y: t5 F3 B6 F! u' p
to be transferred to the germline and 7 [# i$ ?# A( u( Y9 ?, k0 B
hence to the next generation (Evans
7 p1 ~# \6 O7 j2 k8 K4 {9 oand Kaufman, 1981; Martin, 1981). " E( z" s1 p5 e5 \: \6 W1 k2 c& B
Before this breakthrough, researchers
y, x% x# v, @& z4 O$ L& v, _0 M9 b% _studied mouse embryonal carcinoma
. s" A: X2 I; w3 Q5 Q8 d% Mcells derived from tumors, which ' s' y' {- V: G! W' r7 \
could form every mouse cell lineage 4 V' I8 m& h3 _7 }: y; T6 w
except the germline. Combining DNA 1 Z) t& g+ J& N& l4 T! z
recombination and mouse ES cell
' E7 |2 ?% X+ J/ A K! Wtechnology revolutionized an entire # i' w1 ^' w' \6 \
feld of research, forming the basis for $ n% }0 r4 n" u: U
studying and understanding the roles
, c" M+ W# F8 E; h1 V/ d9 Zof numerous genes in embryonic
8 d/ r1 g, O3 z$ Adevelopment, adult physiology, dis-
9 E* ]( f G+ Zease, and aging. To date, more than * m, s, `; i5 A9 _& W& N- Y9 `2 {
500 mouse models of human disor-! u: F- Q+ m& {# ]
ders have been generated. Now, with 0 `; I1 d& m3 F/ z
the study by Takahashi et al. (2007) # ?! S6 Q: y$ ^7 Z
published in this issue of Cell, another 0 {( Q' [7 E" r0 |; x: @ ^
important revolution is taking place.
( u8 T2 y; g1 I6 R) VLast summer, Takahashi and
. M* R _6 \& }1 n) [. `' l0 vYamanaka (2006) stunned the scientifc
( \2 F8 w6 h; u' ucommunity with their study showing & m, O7 N5 c7 Y. `. `1 V1 O
molecular reprogramming of mouse
; l! d' u9 [1 \! r" X# O5 @2 R0 Hsomatic cells into induced pluripotent
}+ O$ P Y( L& Estem (iPS) cells using just four factors: $ P, e" r8 M- Y1 ^6 ]
Oct4, Sox2, Klf4, and c-Myc. Their
. I% o, K9 S+ b |elegant but demanding approach of
2 D% k8 y( ], Iscreening for a cocktail of factors that
( V% W- n T- C& Hcould reprogram mouse fbroblasts
, A8 x _. ~: t2 G5 tstarting from 24 candidate genes paid
6 J# I3 N' X1 Y' J. @' Goff with their detailed description of iPS 5 T1 s. {. u( V, V
cells, which are almost indistinguish-6 _% g* P E+ i# n+ J3 K
able from mouse ES cells. As with all ; ~ z" I" }0 ?, T$ m0 g& h
scientifc discoveries, these exciting
8 q9 ^# L8 @; Z7 \1 u1 kfndings had to be reproduced. Sev-' v$ O0 v& P3 a! a
eral studies published this year not
! z9 |1 @6 N# Lonly reproduced but also extended
|. v. y: p. j) ?5 y# ~0 ^the Takahashi and Yamanaka fndings
" u1 `4 m$ b# F! @: Zby demonstrating the pluripotency and
! J0 i r& `& ~9 A5 o$ @differentiation potential of mouse iPS
0 z9 }4 R; y7 H) y8 T- @% Z" X# Ecells in rigorous developmental assays
% H! }8 x! C8 |0 X9 q(Maherali et al., 2007; Okita et al., 2007;
# u- W7 b. I# J5 {# IWernig et al., 2007).
& Z4 S% c! Y6 T8 t5 t& Q. qIn their new study, Takahashi, ; l- J; v& X% E% r
Yamanaka, and their colleagues - s4 }. l, Q$ ?
(Takahashi et al., 2007) now translate
1 K2 a- e+ q9 Ntheir remarkable fndings from mouse 7 q7 l1 g% N: Y) L3 z
to human (see Figure 1). They selected
6 x* @, K# }: n* ~0 H- tadult human dermal fbroblasts and
" b3 N) ]8 X4 Q# e) q3 X. \two other human fbroblast popula-6 A8 ^: g6 [! L# O1 N4 B* \
tions (from synovial tissue and neo-
) x% x, \4 f, _7 n X( wnatal foreskin) from different human
3 O1 X1 q1 g! i6 d) cdonors as their reprogramming target
/ g# n( v4 g- e. Y! C9 g0 Tcell populations. They then trans-8 |( t! H, F/ h( v
duced the human fbroblast cultures . }8 S$ M& G8 C0 u! `2 Y
with retroviral vectors carrying trans-
- j( v w# u& s. f' j+ |genes for the human versions of Oct4,
1 q; I" _" D+ {; `+ DSox2, Klf4, and c-Myc and cultured
1 n, A! @ b" L5 cthe cells under human ES cell culture
5 v% V6 B- v* w! A" G+ qconditions. Thirty days after transduc-
* V! u- M. M# f6 [$ |- C' Ution, the culture plates were covered 3 L# g) U2 W* u- K7 h
with human ES cell-like iPS colonies ' U2 R7 k8 p) ?# l) S# i( ^7 c
(among other colonies), which could # B2 L7 A& U- Y: W
be further propagated and expanded.
7 F' G* a" h) t# `) SThe retroviral vectors enabled silenc-" Y& W+ W9 F! B7 R
ing of all four transgenes after human
% h/ y& n$ T# u% ]' B4 riPS formation (as found in the mouse
% y5 f. K7 Q3 u! k4 {system) indicating that the iPS cells
" b# i6 y, [; uare fully reprogrammed and no longer ! Z& u4 ?) \: n
depend on transgene expression.
$ U! c; a4 A; `9 u$ O9 c: q% tUnlike the mouse study, human 4 X* W# g& A% `- k8 P
iPS cells were generated without any 6 T2 e$ r. z% |6 _% \& l. ^2 c% {# P
genetic selection procedures. Given 1 ]0 K" a7 P& q$ N- C
the lower mitotic index of human ES
/ a6 y& y7 k9 P0 P( T/ F. \cells, it is not surprising that the gen-
6 q. d% U. `9 o- W! m& k6 Leration of human iPS cells takes nota-
! S- U. d& c1 Q8 b# _: @bly longer than in the mouse system. 1 b: r& u x* A0 Z
The authors subjected their human / L! O! B( k' O1 D0 b# W& W) v/ H
iPS cells to a panel of assays to com-
9 z. V7 s" M- H- L# \pare them with human ES cells. These
* v# {* R. ^& A0 m2 Uassays included morphological stud-
' h4 ^: P$ F; o- Sies, surface-marker expression, epi-: T0 k6 q; J' F
genetic status, formation of embryoid 5 [' m! z M5 p" A' ?7 D4 J6 L, b! f7 k
bodies in vitro, directed differentia-9 f! g8 a+ i T
tion into neural cells and beating car-' z" \* o: o0 E6 g
diomyocytes (according to human ) S$ K2 G# y! ~, P/ {! x+ I
ES cell differentiation protocols), and , ~( C8 b) ^% h$ H6 h
fnally teratoma formation in vivo.
- B# n0 d6 _1 S5 |* S6 B, b3 hDNA microarray analysis revealed
! ?8 p3 ~2 Q. u# N4 ?2 x% ethe remarkable degree of similar-4 Z( d0 J5 Y1 ^. G) h( \0 c6 M
ity between the global gene expres-$ t+ c3 d! C' k: |5 T! n# Z
sion patterns of human iPS cells and
$ ?& V: I( ^* P, e) b. c) B" uhuman ES cells. Notably, genomic
8 I) U8 l) r C0 Z; {: rDNA analysis as well as analysis of ' u* d, r! H$ P5 M
short tandem repeats demonstrated 3 b) F' L i4 Z! ?$ \
the genetic origin of independent . e- c& M+ b+ n4 m9 K
human iPS clones from their parental 9 n* b1 y/ |, I
fbroblast populations.
$ h6 ]: u) }& w5 ?" v3 hThe derivation of mouse and then
" Q( s) Z5 l' w5 M* ?2 r# nhuman ES cells (Thomson et al., 1998)
# }% f6 j% t! ` e8 Tas the gold standard of pluripotent ! p7 F+ F" O& Y# g% P$ z
stem cell populations has necessarily
( A! n& O2 J# s$ wled to emphasis on differences in the & V+ R) D- q4 z; E
regulation of self-renewal between % [+ m- P: h( s! T$ Y) p
mouse and human ES cells. For 7 d# c# J, [. j* l
example, human ES cells depend on " _" h& z% u6 b! I
bFGF for self-renewal, whereas their
! h$ t7 k9 i6 }2 Q% m5 ]mouse counterparts depend on the
# M* j$ j4 K) d$ s, Y4 }$ nLif/Stat3 pathway; BMP is involved in
6 Y' _# t: [% ^' b7 I. c* h4 omouse ES cell self-renewal, whereas 1 z2 M* x* r A; s x3 s) F
in human ES cells it induces differen-( F; I* X- V0 s2 |( D+ R0 N3 m! ]8 i; ^; I
tiation. Extrinsic factors and signals
5 R, d4 E5 _' h* ?for maintaining pluripotency may dif-. u% g9 K6 Z: z6 F
fer between mouse and human. How-
* t% T0 h" @* `/ qever, the ability to translate somatic
+ J+ @2 M) P: ]8 z' Wcell reprogramming from mouse to
7 D W9 e4 W0 O6 O/ N. R( d4 t* Whuman using the same transcription
; z4 S8 K( u1 q0 qfactor quartet further emphasizes the
' }7 f* E; ?+ tconserved nature of the Oct4/Sox2
?: ~8 K; {* P# ^% U! @& x- }5 {transcription factor network that n! [1 s J+ V6 C, v
controls self-renewal of mouse and
7 O" j$ g4 i5 hhuman ES cells (Boyer et al., 2005). : m8 ]! ~( _/ B# s" p9 y* }
Given that Klf4 and c-Myc are chro-+ N) K6 C4 N- U3 h) g% }4 T
matin modifers and can immortal-3 h; A4 X$ v; [, f, e- O
ize cells, one might be able to fnd 0 Q% E- t) X6 B7 b$ @
other factors or small molecules that ; x* ~- `: w$ v8 g A) w2 M) m
could replace these two factors in the
3 }( Y8 W+ n2 R9 f! ?cocktail (Yamanaka, 2007). In these
- G* e( J" C4 u8 j: {studies, the possibility of retroviral
" I) K( A/ H2 G6 oinsertional mutagenesis, resulting
. w; W+ [9 |5 b0 F a6 j2 Din the activation of other genes con-8 V/ S# Y" k7 }( V3 C2 l' _& K
tributing to reprogramming, cannot
w) z! V2 E, ^% i& R$ ybe excluded, providing an opportu-
* a* \5 P5 g0 Q+ Qnity to potentially identify new repro-
7 K& U! d+ l6 `& s( |gramming factors beyond the cur-
0 |2 W* l2 b# `" r: H( ~4 U8 ^7 f8 jrent quartet. Also, taking a broader 1 h. A, r. \3 t6 G
screening approach for reprogram-
0 s {9 r# ?+ ^/ c4 t& cming human fbroblasts (as Takahashi % L" \) G/ v* Y( A+ I c# z, u
and Yamanaka did for their mouse
+ V5 i4 P1 s2 {4 l+ ystudy) might yield other combinations 6 k' @6 Z, M- ?
of reprogramming factors.
" B% m- {, } eDirect reprogramming of somatic 2 w; P% o$ P# `7 X3 F& B0 t( I; x! h
cells to a pluripotent state, thus revers-
, A( B( _. S7 e8 w% F9 h& Ving the developmental arrow of time, 3 Z7 {" Z' ?7 D2 X& ]
is considered by some to be the “holy
7 m7 d. o, p' Xgrail” of stem cell research. Once the
1 r+ z3 V2 Q6 W$ m/ D. Uresults in human cells are confrmed, 9 B! J ~) x f" {6 w z: a$ j
these advances will enable the cre-
( D6 \( G9 Z9 ~' M! Vation of patient-specifc stem cell lines
# K& x2 B& |; @: uto study different disease mechanisms 6 U0 |7 Z5 a8 P# G
in the laboratory. Such cellular models % \( P4 u6 L2 v3 r8 i
also have the potential to dramatically
8 ]: f$ \, y4 @6 T$ ^$ E/ ?increase the effciency of drug discov-$ R5 A6 ?/ P% H9 `
ery and to provide valuable tools for + z8 ?7 B4 N& D
toxicology testing. Furthermore, this
3 o/ I: K/ @: p, f4 o, n$ Y1 Greprogramming system could make + m1 @) g( c" t4 g7 F( Q
the idea of customized patient-specifc
# W5 j& ]6 N" _' k7 {" Nscreening and therapy both possible , T/ O& i4 E8 K( b5 M% G9 y
and economically feasible. Finally, the ! C" G: O6 A+ ?# x4 ?! q0 j C
work will have a powerful impact on
; P) L: u/ I* \7 _+ nthe intense debate regarding the moral,
! Q' C% w. T5 ]& A3 jreligious, and political aspects of ES cell ' O: o; {; @ r
research. However, a big mistake now $ l7 B3 J: S3 l
would be to consider human ES cells 7 a. {* F* m: C% T& P
obsolete. There are still many hurdles ( O, W6 K! [2 Q# _
to overcome before we ful ly understand - X6 h1 L: j |1 s- u% b; m
pluripotency and before we have human & n" {& P$ q+ l+ m- c& S- N
iPS cells in hand that are suitable for
, R4 Z. r7 g% f' C" f/ Rtherapeutic application. For example, 6 i+ c1 I0 P# ?7 e' m5 h
a signifcant proportion of mice derived - ~' W: r$ Y" Q
from mouse iPS cells develop tumors
, Z$ f% v( w; z, i, Rdue to reactivation of the c-Myc retro-& U7 r2 l' `! a/ w. l
virus (Okita et al., 2007) compared to
5 E0 a- [% T# y" n Q: D* x. Bmice derived from ES cells, which are
# w6 _: A A( `# e( R( \normal. The search is now on to fnd a * R" s! g* ^8 ?- E/ f
way to reprogram somatic cells without / ^7 ?7 v4 c, ?& r. ]
retroviruses and maybe even using a
. D: H& T- D/ f4 W0 B0 P# Scocktail of small molecules. Given this, 9 h+ L2 `% G; h" _
it should be emphasized that human 2 @0 b/ l! |1 g a( |( {
ES cell research is more important than ; G& N9 Y/ p' O) _, M6 _
ever for it will shed light on how iPS 2 ^* Y) {. P" ?* }
cells can best be maintained in their , r1 x( v0 |4 b" d, }% ?- n8 z
pluripotent state and how they can be
; n; v) l/ q( H& o _# y) `induced to differentiate into the cell + {: e0 p# o6 U6 u$ a
lineage of interest. The feld of nuclear % }# o) ~# }/ Q9 Q0 n7 A
reprogramming has come a long way " m) s& D& I& j; G; T. `& e4 k7 y3 R
from the initial nuclear transplantation * D: l' P0 U. c- T/ `+ I4 g
studies in frogs 50 years ago, to the * ^6 M6 Q4 ]3 n3 I
birth of Dolly, the frst mammal cloned
4 c- c. c. B# m+ j3 Rfrom adult somatic cells (Wilmut et al.,
I* h+ l2 ]. J, X0 v/ {. i5 W1997), to the fallout from the fabricated
' ^1 H9 x' T# [ Rhuman nuclear transfer experiments
6 h& _$ {2 |( a. E' zof several years ago, to the landmark
5 i/ F# f. H2 c9 F' `studies of Takahashi, Yamanaka, and ( j5 \# z" L- n$ @
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发表于 2009-4-28 21:35
发表于 2009-5-16 10:41
