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Induction of Pluripotency: 0 f- p' p8 e& s, I# d7 W
From Mouse to Human: v1 ]. R; o* c, n; _
Holm Zaehres1/ O0 }" d0 h' G! R. Z+ K# d. \0 F
and Hans R. Schöler1,3 C* x! y: D$ V
* ?, p+ j6 q0 o, C, L2 G+ x* K6 A
1
+ M( i# Q; n1 \7 dMax Planck Institute for Molecular Biomedicine, Department of Cell and Developmental Biology, Münster, NRW 48149, Germany3 V$ Q7 y, m0 v) q! o& Z
*Correspondence: schoeler@mpi-muenster.mpg.de8 L, O1 b8 z% w! ?4 F( E& b
DOI 10.1016/j.cell.2007.11.020
# S1 z, M4 W2 _# H2 \In this issue of Cell, Takahashi et al. (2007) transfer their seminal work on somatic cell
) {# G# U2 i5 k8 ~reprogramming from the mouse to human. By overexpressing the transcription factor
+ E. w& o5 a1 j. L1 n6 pquartet of Oct4, Sox2, Klf4, and c-Myc in adult human fbroblasts, they successfully / }- w9 `7 g) [( w R
isolate human pluripotent stem cells that resemble human embryonic stem cells by all $ F( a" G! x9 h6 b0 d6 E
measured criteria. This is a signifcant turning point in nuclear reprogramming research
0 v8 B% R5 [! O* A1 v6 v0 X7 w- z' pwith broad implications for generating patient-specifc pluripotent stem cells for research
6 J& B& ^6 N2 q1 G9 P' ?, Kand therapeutic applications.- ^* f( Y; v- [' v
This year’s three Physiology or Medi-+ o/ ~7 c" N5 }' w4 A4 A) w4 a
cine Nobel Laureates—Martin Evans,
+ f, x7 `! }4 x) zMario Capecchi, and Oliver Smithies—; h0 h* n8 r/ R* J: _( I. E# Z
will be honored in Stockholm in 10
+ U- G1 L0 ^) k% f& F/ Hdays time for their discovery of DNA
8 B4 V4 m0 l( e. Orecombination and the development
+ ^0 g& N% x$ M7 o: \of mouse embryonic stem (ES) cell # Q9 D7 ?( _ ~, P0 j
technology. It was Martin Evans who , d( B2 S; q& A
discovered how to make mouse ES
" E; S) m/ r+ fcells, enabling any genetic alteration
9 [3 o' p$ H' x2 Y# H/ bto be transferred to the germline and 9 `- A, P8 _6 V6 H* M
hence to the next generation (Evans
9 T' y- b5 a: \7 W8 C3 W2 S- uand Kaufman, 1981; Martin, 1981). - F9 E: S5 r$ U+ y
Before this breakthrough, researchers
r5 q/ ~" y+ E. A/ w9 m& ~8 Rstudied mouse embryonal carcinoma 4 p3 a/ Z9 n7 \7 H
cells derived from tumors, which & a, Y4 o3 v& N' ]( I2 i8 y/ H
could form every mouse cell lineage
9 A$ J( Y$ A% k* t# \& j# P7 C/ Bexcept the germline. Combining DNA 2 M/ k1 D2 j- B' O
recombination and mouse ES cell
/ \* ?0 D4 A. V4 _8 N( n: c) vtechnology revolutionized an entire . u9 o+ F. n1 k2 u
feld of research, forming the basis for
. W4 q9 i! K" ?' Sstudying and understanding the roles . r# O+ d) \9 k, v, a
of numerous genes in embryonic 6 f" C1 F4 S* S3 j+ y
development, adult physiology, dis-
- ^5 a0 o) l9 e, Zease, and aging. To date, more than
. C, d" M! H, G0 A( A5 S+ T2 E500 mouse models of human disor-, Z2 u+ G- X0 ?7 E
ders have been generated. Now, with
$ ?" \" ^7 m9 u/ A u% n( |the study by Takahashi et al. (2007)
, x# q3 j7 o3 p0 ypublished in this issue of Cell, another 4 m: g) F5 P& d9 ]
important revolution is taking place.4 C- x3 ^, m# ^4 z
Last summer, Takahashi and
( F- ]: W8 d+ A9 Q# W% N i8 A0 [; nYamanaka (2006) stunned the scientifc 1 W9 }+ `( ^$ u0 ^' M8 M7 I" H
community with their study showing
* N$ x+ `2 ^; V9 emolecular reprogramming of mouse
( s: W$ N8 ^4 v/ ^) g9 lsomatic cells into induced pluripotent
1 D" _. t7 N7 W% m* Xstem (iPS) cells using just four factors: ( l* U5 i5 ~. F& q. @6 ^. | h
Oct4, Sox2, Klf4, and c-Myc. Their
- i* V* }2 x+ Q Celegant but demanding approach of
, ?0 l% z. B# }- v/ y/ qscreening for a cocktail of factors that . j& I. M* [' f* o* r1 V+ a
could reprogram mouse fbroblasts 9 _) c% s4 M4 d% b! w
starting from 24 candidate genes paid
. x3 y- V# n) { p0 B7 `" woff with their detailed description of iPS $ A7 l- y1 ]$ L6 G! |
cells, which are almost indistinguish-
- |( g& ~5 }& J% \able from mouse ES cells. As with all 0 i2 m; z( G% _" K+ ^5 I9 [# c
scientifc discoveries, these exciting
8 @; V Q/ B. h: M* z4 Wfndings had to be reproduced. Sev-
( ]9 {+ x W: {: s# \eral studies published this year not
( |5 b# n9 \5 Qonly reproduced but also extended - g2 ~' q# _9 U, A% A% W& d
the Takahashi and Yamanaka fndings 5 O3 Q5 \+ ]' E6 W% ?
by demonstrating the pluripotency and
' |! }; v# `, U+ X) {3 Udifferentiation potential of mouse iPS
) i9 u5 V$ K% m, `' k7 x" u) K) v0 { L) B" tcells in rigorous developmental assays
$ m# T% N% b8 G: ]0 h: B2 ]% P0 e(Maherali et al., 2007; Okita et al., 2007; 7 k' ^3 ^' a6 |+ c: h3 {9 r3 V
Wernig et al., 2007). m! l8 a( [1 Z) E7 w$ B
In their new study, Takahashi,
! p( l5 P/ O: i% U, y# Q; KYamanaka, and their colleagues
4 v) l2 ` D3 @9 ~6 y G( ~+ S(Takahashi et al., 2007) now translate - y( d1 l* Q. h* ]4 T: r. C
their remarkable fndings from mouse
# C) z: C5 e/ m* hto human (see Figure 1). They selected 9 B2 L$ Z) c, Y. r
adult human dermal fbroblasts and
9 U# \; D# L! G& k+ qtwo other human fbroblast popula-( g* B U2 ^' Q- s
tions (from synovial tissue and neo-( C5 j' R& \: h8 Y) h. t
natal foreskin) from different human
. n8 I E* R: w' Ldonors as their reprogramming target
3 g o% |# E5 dcell populations. They then trans-/ O: r9 H8 b+ w7 ^8 q9 Y. T
duced the human fbroblast cultures % j7 r5 n3 f% b* v2 g
with retroviral vectors carrying trans-
4 @8 J1 E8 z3 n2 h" h; @- l+ U Mgenes for the human versions of Oct4,
) m- W- z* L' b- z! n. QSox2, Klf4, and c-Myc and cultured
* _5 y6 H+ ]1 h' v7 H' W; V5 pthe cells under human ES cell culture 7 n4 R9 k' X7 S& Y5 u1 p
conditions. Thirty days after transduc-
! a+ B& ?* e9 G T3 d8 W' ition, the culture plates were covered $ j2 ^" u- b0 p3 v. R& S8 J
with human ES cell-like iPS colonies ; K3 b' k) m" y- C$ R2 K
(among other colonies), which could
0 z- y" T3 k) q4 kbe further propagated and expanded. # D1 K; o' Z4 P( K0 h
The retroviral vectors enabled silenc-4 }5 \8 t5 K7 V
ing of all four transgenes after human
3 V4 i8 C& `6 T. siPS formation (as found in the mouse
. ]1 `: n- }7 z' V* Csystem) indicating that the iPS cells
2 J+ r+ k. a4 H8 h+ R) l; ?& i3 pare fully reprogrammed and no longer ' S6 R% O2 T/ M5 J
depend on transgene expression.
- P1 M" y1 r1 ?Unlike the mouse study, human
) q+ e6 ~- Y+ K3 Z7 ^& z) piPS cells were generated without any
1 @( w( e/ ? S4 G2 J: Cgenetic selection procedures. Given ; D% G- C' o. M( X
the lower mitotic index of human ES 1 X n- n# j, D/ E+ c: d0 U
cells, it is not surprising that the gen-
2 J0 a6 |& l8 x5 U! }eration of human iPS cells takes nota-
0 z! C# e! i9 _( F% b6 }$ I3 dbly longer than in the mouse system. ; j3 q# K8 t9 f9 L
The authors subjected their human ) _: q2 x, L7 L9 p! w8 a5 ]6 N
iPS cells to a panel of assays to com-6 N* V8 b7 R9 q' f% k
pare them with human ES cells. These - w; w2 ]# G9 c% |+ c- b3 h" I2 o' ~! I
assays included morphological stud-8 D2 \0 o9 v1 x: S) N
ies, surface-marker expression, epi-% [2 u6 Y! f8 j/ i* z! i; _
genetic status, formation of embryoid ! w, G# @, U1 c' |7 N ]5 n9 J
bodies in vitro, directed differentia-
9 ]4 r% o6 N: X0 x/ @2 ^. ition into neural cells and beating car-9 Z" d) f- M5 }* u" l
diomyocytes (according to human
3 R6 t9 |3 L! l: V& _& C6 yES cell differentiation protocols), and & K+ z9 M# w& Y8 U6 E
fnally teratoma formation in vivo.
; y" w+ t5 g h$ {8 xDNA microarray analysis revealed
* T' D: S6 g4 t$ N8 ?$ tthe remarkable degree of similar-! }1 ` z3 _8 g' L) H
ity between the global gene expres-
" J) E. N1 d ~7 _1 \$ hsion patterns of human iPS cells and $ G; a, A* t$ ^
human ES cells. Notably, genomic # j1 B; A, R" U' G8 h) K
DNA analysis as well as analysis of 2 j$ v T0 E! S |
short tandem repeats demonstrated ) H/ w- z. ~, h% A2 Z$ Z \8 r+ J" n
the genetic origin of independent , _" {- c* C$ r( c2 d
human iPS clones from their parental
* B6 u: e/ x" A. R" Q; ^: Ofbroblast populations./ _ P" f' L# W3 w/ T5 [' K N( p
The derivation of mouse and then
8 G* o. f6 P1 X& yhuman ES cells (Thomson et al., 1998)
A/ P# [ j% p; J7 \* l' Ias the gold standard of pluripotent
8 I# I7 @4 _. G4 l6 z$ Z" ostem cell populations has necessarily $ Y+ @( m! m5 c7 f3 j" ?4 o N6 N
led to emphasis on differences in the 1 w: r4 t1 e& b; C/ z
regulation of self-renewal between 3 n/ g' {4 B) I W1 P* [
mouse and human ES cells. For + J9 a4 t0 |! W* A5 x2 I
example, human ES cells depend on
. t5 J' E; d' p9 N& \/ LbFGF for self-renewal, whereas their 8 M- S# [# `( y' L/ ^" u/ N
mouse counterparts depend on the & r. Z! b# Y9 m8 Z. [, h$ o
Lif/Stat3 pathway; BMP is involved in
# s U- D7 @4 ]4 G9 v' x$ d' O& Qmouse ES cell self-renewal, whereas
9 q9 n5 s2 u& h. Gin human ES cells it induces differen-
_' [4 r# [3 s: ^tiation. Extrinsic factors and signals / f: c9 Y5 |' ?: ?
for maintaining pluripotency may dif-/ `, h4 F- D5 T
fer between mouse and human. How-0 L! ]5 C$ {8 J2 c: g3 _# B
ever, the ability to translate somatic
3 P& X( t6 w% C6 S$ ~cell reprogramming from mouse to
9 \7 z7 K9 T0 z4 w3 ^9 B; qhuman using the same transcription
# n' {8 P; E8 g3 a7 Ffactor quartet further emphasizes the , N2 e; m9 Z* ~$ y
conserved nature of the Oct4/Sox2
0 W; q4 k, H; d- V- M" w6 gtranscription factor network that
7 t% P/ c% U7 J! Scontrols self-renewal of mouse and + {! T8 k4 Y/ \0 k0 j A
human ES cells (Boyer et al., 2005). ; N/ K7 j V% s
Given that Klf4 and c-Myc are chro-; l3 W r# S- ^: o6 @) t/ y
matin modifers and can immortal-
1 n7 t- r1 S4 Xize cells, one might be able to fnd . a+ s! i& B5 w/ b" {' b; K
other factors or small molecules that 2 \ i8 P* U5 ]% e( N6 x; w
could replace these two factors in the
, R+ ?: Y1 R# F! K5 jcocktail (Yamanaka, 2007). In these
* C8 q. q, r/ a8 _; R: qstudies, the possibility of retroviral $ z9 E' L( I t5 [" e
insertional mutagenesis, resulting * m+ c+ X$ M) S3 `; T
in the activation of other genes con-
5 s/ W& @" X" Mtributing to reprogramming, cannot 5 v6 \* @: [. i/ @2 S; @
be excluded, providing an opportu-1 z v$ i# ^' x& y$ |- G
nity to potentially identify new repro-
* u* C# b3 |7 k, Ugramming factors beyond the cur-
# |0 D% Z# c }. `8 [rent quartet. Also, taking a broader 0 j) S' q# B- j' q
screening approach for reprogram-9 E4 j6 C( d1 N
ming human fbroblasts (as Takahashi ( A# s% i3 J" R! i. r
and Yamanaka did for their mouse
' ]8 G- s8 J7 z' t$ ?5 |7 `9 [study) might yield other combinations 7 `" [1 u. N! t
of reprogramming factors.
% P8 s) o& R2 EDirect reprogramming of somatic - Q( G+ a: s8 X4 C$ p4 @9 [
cells to a pluripotent state, thus revers-( i4 r8 x, A0 {
ing the developmental arrow of time,
4 ]8 J1 w: M* A. Tis considered by some to be the “holy
( s% a: B2 u7 C: _9 R/ l7 c& I0 mgrail” of stem cell research. Once the
3 E* S+ `" H% Y$ b) E( C% xresults in human cells are confrmed,
8 v+ Y; g A/ Hthese advances will enable the cre-
. e1 a) d; X( [' ]# j9 yation of patient-specifc stem cell lines
* G8 L% |) d* y" g7 `% nto study different disease mechanisms
7 }3 R0 i6 E8 A( w5 ^9 p+ k) g7 Y I; `7 Vin the laboratory. Such cellular models
5 E) V) J# r- |, V* Ralso have the potential to dramatically # A( N: D( z' S1 u9 t0 t' f
increase the effciency of drug discov-3 `; M& h; s5 s1 H) x
ery and to provide valuable tools for
0 P+ V S; r4 k, ], }toxicology testing. Furthermore, this
- u" U! g6 \, |# r) U) d, Rreprogramming system could make
7 _' ^, d. J; s3 @1 v$ Ethe idea of customized patient-specifc
1 |8 T6 ]% e# {; |& ]7 J' U4 ]screening and therapy both possible ) [/ S1 `/ S6 {6 Y) j# t! p
and economically feasible. Finally, the 2 Q9 Q/ n, c2 a8 p& \4 C
work will have a powerful impact on
* u; i2 @6 p2 M6 n( y: Jthe intense debate regarding the moral, + h3 L4 b. P. L
religious, and political aspects of ES cell
- p! ?, D/ P0 r* g$ Aresearch. However, a big mistake now
/ X7 A5 \5 b! x% g/ Hwould be to consider human ES cells $ u, \+ u s4 d' ~4 y+ E. ^) e f* |1 k/ E
obsolete. There are still many hurdles 0 u2 p; F. ?3 \# y* A- |
to overcome before we ful ly understand
3 S' Y* p: V1 Epluripotency and before we have human 9 Z3 m: @. _8 W/ q" S! a2 ^
iPS cells in hand that are suitable for ) F" O$ h. u1 E$ f" `# {
therapeutic application. For example, ; Q$ X- U& h# E) a& |/ f- k
a signifcant proportion of mice derived
3 K( p) Y7 |0 j0 nfrom mouse iPS cells develop tumors
3 Y, W) x; {$ w- [6 Mdue to reactivation of the c-Myc retro-* n2 ?8 D3 g# m; w. D
virus (Okita et al., 2007) compared to
. [0 A# v/ c1 Q' O$ Z& I! ]( cmice derived from ES cells, which are
. v$ f" e1 ]: E; X( Qnormal. The search is now on to fnd a , E/ P, n3 f! {
way to reprogram somatic cells without
9 s9 j7 F0 M2 A% p/ xretroviruses and maybe even using a 1 b& a. d* Y/ z, M& m
cocktail of small molecules. Given this,
3 |/ D! O( ~% x# V/ Cit should be emphasized that human 2 q( y$ y5 E* k; I) W$ q
ES cell research is more important than
! `6 o! N g% r+ G2 g, [ever for it will shed light on how iPS
5 M; l7 r F+ I. x. L1 j/ T2 O6 b8 kcells can best be maintained in their
* v* z, l5 B) f/ k7 D1 d: Npluripotent state and how they can be
( d& b/ d3 d' i! V4 jinduced to differentiate into the cell : {$ N+ W) N6 y" [( s
lineage of interest. The feld of nuclear Q9 B. ^$ |# T( p2 X5 Q
reprogramming has come a long way
9 X$ @: A/ a3 i cfrom the initial nuclear transplantation
* {$ K! S% i: L) O# J Wstudies in frogs 50 years ago, to the 5 [) b- L( I7 r; [9 Q' N
birth of Dolly, the frst mammal cloned
/ V7 B: u2 z$ ^6 Vfrom adult somatic cells (Wilmut et al.,
& @" a" H# t8 ?3 ^2 f0 r: ?1997), to the fallout from the fabricated # {* w% H: D5 I' @3 \4 N: }$ V
human nuclear transfer experiments ! h5 W2 U# B( F1 B' R- T
of several years ago, to the landmark % H+ e7 z0 {- f/ l* a" d6 k
studies of Takahashi, Yamanaka, and
. r: c) P3 Z ?; S" }2 ztheir colleagues, frst in mice and now . }7 k' U$ a9 w* ~* E" `
in humans." \* p4 V. U1 b/ o: S0 ]
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发表于 2009-4-28 21:35
发表于 2009-5-16 10:41
