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Induction of Pluripotency: ; d! G" h2 u1 C. @2 `( {7 H+ R
From Mouse to Human3 {/ \: W5 f7 K+ S' l( h$ g
Holm Zaehres1
% d7 e! d/ X# A+ |! ^% v and Hans R. Schöler1,
4 j9 S+ ^2 T& e/ p) P1 G*# H: j9 o$ T5 a/ o# ]
1) P% Z( `3 R4 @- z1 V7 K
Max Planck Institute for Molecular Biomedicine, Department of Cell and Developmental Biology, Münster, NRW 48149, Germany' ?/ ^% L. I# H8 S) p
*Correspondence: schoeler@mpi-muenster.mpg.de
0 q0 w R& |$ |* {2 S8 W& DDOI 10.1016/j.cell.2007.11.020
2 [2 X, `) z# S, s4 _In this issue of Cell, Takahashi et al. (2007) transfer their seminal work on somatic cell
$ R" j! u3 |8 S4 Lreprogramming from the mouse to human. By overexpressing the transcription factor
; g1 f5 a7 j# n+ vquartet of Oct4, Sox2, Klf4, and c-Myc in adult human fbroblasts, they successfully
! q. j% |" d, ?isolate human pluripotent stem cells that resemble human embryonic stem cells by all * Z! D0 ~2 `- q) k& M. l1 @
measured criteria. This is a signifcant turning point in nuclear reprogramming research 4 |, T# [8 a: z
with broad implications for generating patient-specifc pluripotent stem cells for research
- m2 @/ v+ W0 Q' Mand therapeutic applications.
$ n; x6 O# i# W8 A; z9 wThis year’s three Physiology or Medi-4 w9 z: h4 A* m8 e: }
cine Nobel Laureates—Martin Evans, + f0 c. }: g& j
Mario Capecchi, and Oliver Smithies—
3 w" F& G b6 J3 }& Ewill be honored in Stockholm in 10 ) V4 X3 k7 ~: x [
days time for their discovery of DNA " } N8 ]5 ~% k* g
recombination and the development 9 G; D9 J% M4 Y% U9 `
of mouse embryonic stem (ES) cell " a- s# e2 b* J- j5 c& y7 R+ s- \( j
technology. It was Martin Evans who
, k/ l, C5 g+ }discovered how to make mouse ES
$ N' @6 T S' k! Jcells, enabling any genetic alteration 0 ?* b* b7 ~1 I1 A6 O. I, M( W* T
to be transferred to the germline and ; S2 a" u: h) s% j
hence to the next generation (Evans
& @. P9 m" u! z- M7 n% q) a# @and Kaufman, 1981; Martin, 1981). , m5 o- f6 V; j7 h; a! N
Before this breakthrough, researchers
3 H% x8 R y9 L4 Hstudied mouse embryonal carcinoma
[+ b! d+ w* J# f7 l. qcells derived from tumors, which 9 ~( E# z2 I( i# t5 b2 W, J
could form every mouse cell lineage 1 v' E2 W1 ^2 g; I3 y
except the germline. Combining DNA
, t# I, @0 }9 _; r Rrecombination and mouse ES cell
! I; B( r5 I1 I0 m) Z5 D3 Y* atechnology revolutionized an entire
1 L% @/ Y% x7 v. Ifeld of research, forming the basis for 2 G9 b# T9 N2 G" H+ K# H8 L* I4 y/ [
studying and understanding the roles
# Y9 e$ f/ f7 I; ~8 R& {0 ?of numerous genes in embryonic
* b% v! g$ o( ^* Qdevelopment, adult physiology, dis-4 `( Y( V; K8 X* q9 [& C: ~- _
ease, and aging. To date, more than
: ~6 j8 X5 i( R500 mouse models of human disor-2 l' [$ V' x- `3 t
ders have been generated. Now, with
6 b! z$ c M" ~: L% Z# ?" wthe study by Takahashi et al. (2007)
, V" e7 |2 K& ypublished in this issue of Cell, another , Y: g( B3 n; w8 V/ {% m
important revolution is taking place./ b+ Q2 b4 |, ^2 @9 c
Last summer, Takahashi and " M9 p& @. ?' f4 z
Yamanaka (2006) stunned the scientifc $ ?) d5 s2 b6 x5 \7 x4 O5 X
community with their study showing
6 V' x$ k& B# c& Z7 s9 k+ ~molecular reprogramming of mouse
# j$ F' r# N8 l a* Bsomatic cells into induced pluripotent ' J2 q+ J8 w( v# e* l
stem (iPS) cells using just four factors:
) q V# t. u: J4 pOct4, Sox2, Klf4, and c-Myc. Their
* X5 v3 t- G7 |0 Welegant but demanding approach of # c, U& K# z( J7 |! W
screening for a cocktail of factors that
5 e! Y2 h+ `0 M* |could reprogram mouse fbroblasts ' e: A2 d8 G, M2 x p3 R4 Z3 J
starting from 24 candidate genes paid
5 E' \ U6 x4 r ?off with their detailed description of iPS
5 U7 {* j% L) h) Ucells, which are almost indistinguish-
6 q& c: ~7 |. E: M5 v. U" [able from mouse ES cells. As with all
( L! ~' N/ t( O# ~scientifc discoveries, these exciting / T) N z N' N( B& M
fndings had to be reproduced. Sev- e/ O0 Y9 J+ Y7 M# Y5 T
eral studies published this year not 0 a2 Z& w0 P8 C3 k
only reproduced but also extended
/ S; e6 J `' t( \; I) Xthe Takahashi and Yamanaka fndings / ]1 q2 K1 t7 \* \( b& @7 M: j8 u9 o3 F
by demonstrating the pluripotency and " f4 c8 l; S/ O/ h2 S" |% [8 g
differentiation potential of mouse iPS . |) K$ l9 {1 B) p& ?" H
cells in rigorous developmental assays 2 f6 d. v9 Q: x: e& n/ j
(Maherali et al., 2007; Okita et al., 2007; ; r9 o4 Y* I/ P9 w( Z. X( d
Wernig et al., 2007).
8 N M2 L7 T7 iIn their new study, Takahashi,
+ n F8 B1 F) r! b: ~; eYamanaka, and their colleagues ' T% Q! s, k5 {% v h! f5 b) t# e
(Takahashi et al., 2007) now translate
# n( g. q, M6 ]their remarkable fndings from mouse 9 d% T1 l. n( T* B& N( T
to human (see Figure 1). They selected
1 \& B8 O4 s7 ?! W2 s3 T+ q4 `5 aadult human dermal fbroblasts and * o& G) n2 \* a6 i- U6 W& x% k W# a
two other human fbroblast popula-
! K6 B; A+ A! M+ a {0 m( [tions (from synovial tissue and neo-' {2 t/ o; |9 q8 L9 f
natal foreskin) from different human
$ t" T# V) C" O3 `donors as their reprogramming target ( O' n' y8 F( I. g6 S6 M
cell populations. They then trans-
: y. n0 ?* M, p. A1 e% H5 K Hduced the human fbroblast cultures
, ?- q; q: e8 K' n9 ^6 Wwith retroviral vectors carrying trans-( }* O6 W5 r( g* B' B
genes for the human versions of Oct4, 4 Y; o) g/ |- x: E, I2 c6 g7 `
Sox2, Klf4, and c-Myc and cultured 1 J2 _' ]1 [# L0 e {: h. ~1 S
the cells under human ES cell culture 4 ?: f* p5 A- s c
conditions. Thirty days after transduc-+ ^5 p& ?- P# l7 T S8 @
tion, the culture plates were covered & x- h& L& A, C) E( q+ s
with human ES cell-like iPS colonies
$ K% I9 l# I; h4 }' ~7 v(among other colonies), which could & N6 z* m( v5 T! I: @
be further propagated and expanded. % V8 [* \# H0 h9 k+ g
The retroviral vectors enabled silenc-: f( m0 \) l" g! ]1 Y& d+ Z
ing of all four transgenes after human
2 C6 c% I" v5 G0 Q! p7 L+ FiPS formation (as found in the mouse
2 F3 Z) s @; N! R8 O. \system) indicating that the iPS cells 6 g3 I4 f- Q7 o& Z" r2 y* @: J
are fully reprogrammed and no longer ; K- ~1 }& {1 P I4 X3 M
depend on transgene expression.& Y* L- w& r% j4 U8 F! m
Unlike the mouse study, human
& M3 W1 l+ l- F% \* [* I3 ]iPS cells were generated without any
4 G& R2 C. n+ B6 E$ s6 q0 _genetic selection procedures. Given * p3 v, q5 `7 l7 K0 K8 \4 W5 Q4 H
the lower mitotic index of human ES
$ s( s% r' N ~! }" j6 m6 V( Rcells, it is not surprising that the gen-6 {" r3 L" H! {3 q7 ~- c/ v
eration of human iPS cells takes nota-( I! T" L$ _, e# B1 U! X5 C, `
bly longer than in the mouse system. / v G+ s. S4 p8 ~* r, J7 l3 O
The authors subjected their human + R) Q! X) @- m& q/ G* o/ R! N
iPS cells to a panel of assays to com-6 Q9 }5 v. o/ `
pare them with human ES cells. These 6 w O0 n3 e4 `1 {
assays included morphological stud-
, p, _) h) z" `7 k, K4 P% B' eies, surface-marker expression, epi-
2 \4 f+ o5 l! k9 P2 O$ D7 }4 Cgenetic status, formation of embryoid
" g6 T0 D( H4 `- E/ j) Pbodies in vitro, directed differentia-
# b! f& @/ b3 f3 J) L' k) q2 q. Wtion into neural cells and beating car-/ c2 ^5 j+ m7 U' ]6 Z7 F
diomyocytes (according to human
+ X, C0 x3 N( {, O- `& kES cell differentiation protocols), and
' H# V `0 Y4 G' q' E4 J2 yfnally teratoma formation in vivo.
9 F5 a0 A( H7 S7 hDNA microarray analysis revealed # b ~0 W& C$ c0 s& N; G8 B! N. l
the remarkable degree of similar-5 ^- i) Y' e' t/ K7 m2 }
ity between the global gene expres-
7 I. [9 v6 F. y* bsion patterns of human iPS cells and % Z1 ?& e X" l- K) O* |
human ES cells. Notably, genomic
; i+ q/ h" g. f- b9 I5 O" kDNA analysis as well as analysis of
: y. ~( Q0 b2 A: G, d0 F2 u1 b& j; tshort tandem repeats demonstrated ' y" u8 `) }% Q
the genetic origin of independent
# e5 w) p5 `1 zhuman iPS clones from their parental
8 U6 p0 f; h6 r4 pfbroblast populations.9 B( H7 k! G$ A9 x
The derivation of mouse and then + u6 S) i) b4 @7 o6 p
human ES cells (Thomson et al., 1998)
/ U e' G' i3 p0 m$ p* N+ ^as the gold standard of pluripotent 5 t7 [1 R; c8 U8 F5 Q
stem cell populations has necessarily 6 x6 E4 j2 X/ f
led to emphasis on differences in the - p( s9 }* }# v# a" y
regulation of self-renewal between & o8 u I8 o) M" T: Y% l
mouse and human ES cells. For 9 g' @& x4 {' K q
example, human ES cells depend on
2 I" H) Y$ h+ }2 B$ _bFGF for self-renewal, whereas their 9 O3 k5 T$ e0 M& X
mouse counterparts depend on the 8 w% l$ I: o8 F6 [6 ~( S
Lif/Stat3 pathway; BMP is involved in
0 y8 @ N- K0 v6 d$ |& H/ Dmouse ES cell self-renewal, whereas 9 t8 _. K0 N0 \: m! z. \) ~
in human ES cells it induces differen-3 c7 x0 x% E/ g
tiation. Extrinsic factors and signals - r: N/ |1 U% m4 n$ i2 M2 |
for maintaining pluripotency may dif-
5 x- c( z8 Y, h+ B3 c4 E' ^fer between mouse and human. How-% m: I1 R7 l# G8 Q. e4 D2 W$ h
ever, the ability to translate somatic % n" h2 Y) [; F4 Q: f
cell reprogramming from mouse to 6 C+ D. z, f- n0 |* C7 w7 B
human using the same transcription : k' R. X% E# ^: i i
factor quartet further emphasizes the
$ I; i; M% S* l2 N J* e/ h0 \conserved nature of the Oct4/Sox2
: D2 q0 C; b- f: X" Ztranscription factor network that & b' a' W3 ~& Q8 R& o
controls self-renewal of mouse and 3 g7 ~1 W# d8 ?0 v
human ES cells (Boyer et al., 2005).
# R5 P @6 d& gGiven that Klf4 and c-Myc are chro-: S1 t8 \3 C5 A; Z) X% P
matin modifers and can immortal-
3 |8 e" b* K; rize cells, one might be able to fnd " U' S/ y* w$ l+ {
other factors or small molecules that - x' P4 o9 Y" l
could replace these two factors in the
# s' u: w& C) [ E W7 E4 c$ Ccocktail (Yamanaka, 2007). In these
3 G. S, [. h, T/ O2 T( l1 E4 g8 estudies, the possibility of retroviral
( {* |5 R% x h, u; }insertional mutagenesis, resulting 2 j: H% m; U# h* ^7 T+ @7 \6 M3 t8 w
in the activation of other genes con-& D; Q8 \) c1 s% }* m0 C, O( y
tributing to reprogramming, cannot
/ N# T {9 s6 D. [* d& Lbe excluded, providing an opportu-
& S8 _6 n7 \! y' t" x" I% inity to potentially identify new repro-
! W9 a7 p8 `! c2 c1 qgramming factors beyond the cur-3 d: L) a- q" X8 L- C) R
rent quartet. Also, taking a broader
5 l( N0 {" s7 a' U9 J; q- Pscreening approach for reprogram-2 z/ u, g' G0 u+ J- q
ming human fbroblasts (as Takahashi ' [! D+ I6 @4 J% V: D6 K
and Yamanaka did for their mouse 6 n5 a% `8 H1 U1 p. e
study) might yield other combinations - ?8 \. F$ z1 B+ I! I$ v( y
of reprogramming factors.2 Q9 Z! A f( ?; }) `4 P4 F
Direct reprogramming of somatic 8 @. Y4 z1 n1 q& l; i9 h e, G
cells to a pluripotent state, thus revers-. }/ u; H6 v0 m6 b4 |- A
ing the developmental arrow of time,
' P4 ^9 ~0 O& r7 l2 j2 `/ Nis considered by some to be the “holy
& W/ r1 d0 P; E/ Y; I6 Rgrail” of stem cell research. Once the ) x9 Z9 m3 A: t* E# S g: L
results in human cells are confrmed,
, m2 l- I. Y! [7 [( h: Xthese advances will enable the cre-
3 U: M0 s5 C% q& K1 c; k2 c% N4 gation of patient-specifc stem cell lines
/ U. C5 a& a* J& D9 `/ ato study different disease mechanisms * l- i" z$ Z4 h6 |) I; e& P5 X
in the laboratory. Such cellular models - M0 m. z" [5 x7 v: I0 p% b
also have the potential to dramatically
0 J9 C, u, E4 v% Oincrease the effciency of drug discov-; j; ^& m* k p# Z$ v
ery and to provide valuable tools for 6 a8 n, a1 g* o7 J& s9 [6 V* b
toxicology testing. Furthermore, this 3 m: g8 f! c" j* {2 w
reprogramming system could make
8 z2 a+ }) Z: E% t9 U" A& f0 C5 Pthe idea of customized patient-specifc
% j" n4 _, E5 n; R8 R0 f4 dscreening and therapy both possible
, k6 G5 ]: e: d/ O3 ]8 T$ z4 jand economically feasible. Finally, the & [* A, W9 H- z. k
work will have a powerful impact on 8 Z: t. w6 Y8 G4 z
the intense debate regarding the moral,
( |; U7 P8 c, j) R5 Dreligious, and political aspects of ES cell
3 N6 t" }" R3 }: q5 v2 [research. However, a big mistake now
; t0 E: a+ |6 s; M6 v$ Xwould be to consider human ES cells
2 ^( U, a1 T- |; x+ ?! [5 Y" Q3 j: @obsolete. There are still many hurdles $ m, d+ N* C, a2 s7 z. } k! @
to overcome before we ful ly understand
0 L2 [$ y/ L5 e" w+ o' S) zpluripotency and before we have human ! C7 C6 r# G/ O q4 g Q
iPS cells in hand that are suitable for ; Y' J g' L* C" k2 o0 Q. k- z
therapeutic application. For example,
2 b6 ^( w, X% Fa signifcant proportion of mice derived
" m. c7 g' h3 e* e7 Ofrom mouse iPS cells develop tumors
5 n( o! H* ]5 a) u3 u! h% Ydue to reactivation of the c-Myc retro-+ }9 @9 |+ U/ m; F" Q# `
virus (Okita et al., 2007) compared to 1 B0 G# r7 Z' z/ T; `% \
mice derived from ES cells, which are 1 L* ~5 c; {! O# e9 l
normal. The search is now on to fnd a
( x$ y8 ?6 ~; I- V* sway to reprogram somatic cells without 1 H4 @, Y d8 B& r3 z' r5 x
retroviruses and maybe even using a 9 J; |0 U8 o& ]' {! c! N
cocktail of small molecules. Given this,
; }. t1 f' S, e+ Mit should be emphasized that human
7 q% G, [3 r! K+ ^7 S& gES cell research is more important than ! N! }( z; C7 w& `3 l6 J/ @
ever for it will shed light on how iPS % ^2 Z& ^( l/ Z* z) g5 r, W
cells can best be maintained in their ) `8 P, w2 c& y$ P+ e* _
pluripotent state and how they can be 0 z, Y( k1 K9 k# I2 k
induced to differentiate into the cell 5 ` a/ ? d. x, E/ F; m: d
lineage of interest. The feld of nuclear ! E1 K7 a2 C$ r N- I/ T5 _7 U
reprogramming has come a long way * U" y. _5 H& B0 I
from the initial nuclear transplantation
. Z2 M3 e/ ]! p: `! hstudies in frogs 50 years ago, to the 7 L. `8 N+ M6 T+ o
birth of Dolly, the frst mammal cloned 8 C9 a: g4 K c$ q6 v* `
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发表于 2009-4-28 21:35
发表于 2009-5-16 10:41
