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Induction of Pluripotency: 5 s4 ]5 V5 `/ Z7 c) Z$ h
From Mouse to Human7 k. V$ R0 r) `8 u( k$ b) r
Holm Zaehres1) F% @# w" k$ S! @
and Hans R. Schöler1,
" h$ d: A- }$ _0 T: l*. Y0 F8 f5 j0 x/ w
11 \: C% v7 j0 o" m% X' E
Max Planck Institute for Molecular Biomedicine, Department of Cell and Developmental Biology, Münster, NRW 48149, Germany- q$ R; J$ ~: _0 L% u
*Correspondence: schoeler@mpi-muenster.mpg.de3 j# e, A, \7 w) L
DOI 10.1016/j.cell.2007.11.0206 R; s) V5 t" B1 ]3 g7 G9 ~1 u
In this issue of Cell, Takahashi et al. (2007) transfer their seminal work on somatic cell
+ p! l" [* X; Oreprogramming from the mouse to human. By overexpressing the transcription factor
. x9 G7 Z7 Q0 y5 m6 Yquartet of Oct4, Sox2, Klf4, and c-Myc in adult human fbroblasts, they successfully * M% H1 R! d0 t2 p$ @, H9 R% d
isolate human pluripotent stem cells that resemble human embryonic stem cells by all ! J/ s3 o, n6 ?, i1 {: ~
measured criteria. This is a signifcant turning point in nuclear reprogramming research 1 i& g3 c6 S+ H2 U) F
with broad implications for generating patient-specifc pluripotent stem cells for research 9 d3 i) H8 D7 r4 d0 a
and therapeutic applications. v+ C4 ^9 D& L& j! T
This year’s three Physiology or Medi-' a% g+ A( Z) z) p
cine Nobel Laureates—Martin Evans,
. B! k8 h3 y% b0 b+ ~$ a/ d; {) q _Mario Capecchi, and Oliver Smithies—2 ]# ?" k+ g( B" H- D& Y
will be honored in Stockholm in 10 6 j3 b5 T6 o# m+ ?! G
days time for their discovery of DNA
8 g6 l$ H5 L& p7 I1 arecombination and the development
6 z. s3 z; i. W( _! Y% v, a# {of mouse embryonic stem (ES) cell
: Z/ G+ Q0 w9 |5 Wtechnology. It was Martin Evans who ' C% O8 S1 B) W6 j; O
discovered how to make mouse ES 5 P( Q! N) t' f: Q Z' k4 `
cells, enabling any genetic alteration " |4 q% {( v" y
to be transferred to the germline and
4 U9 H. d! w3 `7 X, X7 r* h$ Xhence to the next generation (Evans
- C+ V" a. V+ kand Kaufman, 1981; Martin, 1981).
$ H3 d! }. {& ?; gBefore this breakthrough, researchers : L$ z! w# Y7 A% E$ V$ M {0 M
studied mouse embryonal carcinoma
; D/ g% [3 Y+ g- h7 gcells derived from tumors, which
; c, Q3 ]* [, ^5 l# Jcould form every mouse cell lineage
, E/ y4 m' z- \7 S( [7 S: zexcept the germline. Combining DNA
9 Y& q$ I$ O/ C7 L: k6 }, Y" ]recombination and mouse ES cell $ R) x1 v1 L, Y. m J# g' E
technology revolutionized an entire
3 n, ]4 |, c% h5 {1 Zfeld of research, forming the basis for % K9 e4 V! o# W2 K: b
studying and understanding the roles
6 H! U( T& {0 \" g0 L: b9 A# cof numerous genes in embryonic
2 B* D/ Y; R3 C! ]$ tdevelopment, adult physiology, dis-
5 U: V5 A" o. K6 \4 Pease, and aging. To date, more than
+ @3 n- a& |8 Y( H% \500 mouse models of human disor-
* K q3 m$ V: ?& Z0 t, x8 e! N4 Vders have been generated. Now, with , |) [1 e% g- |* l
the study by Takahashi et al. (2007)
) V$ e' L* E7 kpublished in this issue of Cell, another
, m L" N$ j. L" Himportant revolution is taking place.7 U. R y! a7 }1 `( T+ T/ o/ n. e
Last summer, Takahashi and
5 ]$ M+ p2 ]7 L4 j3 n. ]Yamanaka (2006) stunned the scientifc + x9 H1 C8 g x
community with their study showing / p L) E O4 k. s6 X( l- m3 `
molecular reprogramming of mouse : A6 H" o3 _( N3 c
somatic cells into induced pluripotent + y( i1 {3 v: \: m3 |# d
stem (iPS) cells using just four factors:
1 @/ \! r; E9 a% L' LOct4, Sox2, Klf4, and c-Myc. Their
- R) j" A; @) }elegant but demanding approach of
! v) s* F+ b# o5 Wscreening for a cocktail of factors that ( F! T, H/ L: `; ?1 i- Y
could reprogram mouse fbroblasts
+ f9 b1 `" Y( g/ S ^starting from 24 candidate genes paid ! X- A; q* F. c- T4 R1 A, R0 Y9 [
off with their detailed description of iPS
& T8 c8 _( q( j! Wcells, which are almost indistinguish-
0 @8 A. o, F, p d8 Table from mouse ES cells. As with all
3 |8 P" F G8 D; e( l: {! }scientifc discoveries, these exciting
* a8 E& S- a% N& ^ ~1 y- Z& Xfndings had to be reproduced. Sev-
4 T! Z# A9 v1 Y& u0 ^ Y' Veral studies published this year not 8 k+ ^) I, ?% n: G
only reproduced but also extended
3 X. B7 ?3 }5 X# u6 z4 pthe Takahashi and Yamanaka fndings 8 H: m* q, O0 B) m9 X
by demonstrating the pluripotency and
( O$ S6 s" v4 \$ n/ B* V% }1 a* jdifferentiation potential of mouse iPS
& o; v4 w9 W, v6 rcells in rigorous developmental assays
9 |& L. q# r1 P) \% `(Maherali et al., 2007; Okita et al., 2007;
5 S6 ]' { X1 A& G9 U$ `6 a% \Wernig et al., 2007).) h& ?. B" i2 B6 o4 q: P
In their new study, Takahashi, 4 m! ^$ E# l, t, G4 c1 m
Yamanaka, and their colleagues
& u6 p& E) s( H( u5 e+ e8 o8 r(Takahashi et al., 2007) now translate
( k) `$ D) p! \0 ntheir remarkable fndings from mouse ! V: ]4 E+ R. A- N
to human (see Figure 1). They selected 4 n+ d" n c' W- ~# ?
adult human dermal fbroblasts and 5 j$ q) Q# m1 x# L( |2 _: O
two other human fbroblast popula-
U) f) k2 h* b# X; t T- Htions (from synovial tissue and neo-0 Z& h+ A [3 G4 z9 c& V
natal foreskin) from different human ' m. R" t4 i6 J) P- m
donors as their reprogramming target & }* x* G% d. C3 o6 |0 o
cell populations. They then trans-
G' U" Y- j) Z2 V0 A0 \6 b0 Y& \5 p" L" Kduced the human fbroblast cultures / i* B/ ?8 e( q4 G; s4 f
with retroviral vectors carrying trans-
1 E1 z, A" k7 B! wgenes for the human versions of Oct4,
1 ]! Q. w0 J; j% J) \( S: ]3 iSox2, Klf4, and c-Myc and cultured
% u. ~1 ]1 w; n8 V) k0 N3 ?the cells under human ES cell culture
8 H: m' Y n) g3 ~) q& b0 E ]conditions. Thirty days after transduc-! D( {6 r1 J- ?2 c6 m) }2 S
tion, the culture plates were covered
' x" R E: E4 b8 nwith human ES cell-like iPS colonies
: y+ w2 E, j' H% T6 F3 V% \) T1 R(among other colonies), which could
! n) z- N% {5 o6 |" ]& Ube further propagated and expanded. + m! Y, \( A; j! c: Y+ U/ }; a) ^
The retroviral vectors enabled silenc-5 G' e5 |6 J/ Y. J4 {
ing of all four transgenes after human / x, H% }1 \" A2 S( n M
iPS formation (as found in the mouse
( A J+ T- Z8 S) U- e \system) indicating that the iPS cells
) {8 Q `8 G2 P$ {) }, A4 dare fully reprogrammed and no longer 6 r3 l) }- [% q. r4 `' U3 M& P
depend on transgene expression.# Y$ N x) Y2 L8 g' o3 ~, }5 h
Unlike the mouse study, human & j- v* {3 o" l& Z: j; A
iPS cells were generated without any ; X' y V; H0 X$ }. ?( v
genetic selection procedures. Given
1 y% p+ S' y) q, Hthe lower mitotic index of human ES , w0 ]: M3 `0 A) i
cells, it is not surprising that the gen-% w( Z3 z: p. Y, s6 ]
eration of human iPS cells takes nota-* r' ^8 Z: P, Y, `0 _- [; y j8 f
bly longer than in the mouse system. * B! v2 l& h2 Q, C; ]
The authors subjected their human % Q2 W! o( s6 J5 o; t, }- E2 q3 f+ T
iPS cells to a panel of assays to com-# E3 h9 W# z1 N! E ?* g
pare them with human ES cells. These
+ p) o6 A. @- v8 a4 O: Cassays included morphological stud-
- I k( j1 o+ s$ Bies, surface-marker expression, epi-9 Y: j; n8 \5 [! `* V0 N
genetic status, formation of embryoid
4 t; x' R2 d; g4 fbodies in vitro, directed differentia-
: C( ?. n1 a" Otion into neural cells and beating car-; ~' X+ A* p! e
diomyocytes (according to human
/ w' C6 `; T1 M; iES cell differentiation protocols), and
; G. b5 G6 P2 ^9 z2 k% N$ t0 R+ }# }fnally teratoma formation in vivo. ( C r3 j9 J9 t6 E: k0 K
DNA microarray analysis revealed
' b7 L! S4 L, p0 h' S' ythe remarkable degree of similar-
9 r" ? v8 @( Z( P. N+ ~, Iity between the global gene expres-5 H1 E/ m/ \- O% I0 y g5 S8 I: ^
sion patterns of human iPS cells and
! c% {) \: M" f+ U9 s" phuman ES cells. Notably, genomic
, r+ `/ R+ w' N& H! I. zDNA analysis as well as analysis of
, Q! d+ D+ f: v' R$ _short tandem repeats demonstrated 1 D' e* O. e& \/ ~* S8 q0 v
the genetic origin of independent
0 c, K+ \4 s! \& q0 m3 x. J9 Q+ Uhuman iPS clones from their parental ! |! t( k: G+ X5 }
fbroblast populations.$ R5 Q/ m: e' F2 G1 i8 o
The derivation of mouse and then
7 s& k0 P3 t" b! w! k5 shuman ES cells (Thomson et al., 1998)
- }- R( v% d7 j: g' pas the gold standard of pluripotent 4 {; \ Q |: `, |4 o' `
stem cell populations has necessarily
1 m+ k/ ` g# I" `. bled to emphasis on differences in the 6 V- y( x8 y/ U4 V7 w, i
regulation of self-renewal between ) C+ P; d9 b6 m) c$ B9 L
mouse and human ES cells. For + U# G) ?' [& g7 }/ Z! V
example, human ES cells depend on . N( I: w& T8 m. T1 l
bFGF for self-renewal, whereas their 9 |1 {) n( s: C
mouse counterparts depend on the
0 i& I$ j) P- X3 cLif/Stat3 pathway; BMP is involved in 1 U! D. w/ l2 j6 k
mouse ES cell self-renewal, whereas & ^3 |8 m- p3 [, i4 x$ T9 e! d
in human ES cells it induces differen-1 w* o0 | X# |, N1 g' w% @
tiation. Extrinsic factors and signals
& X' U& A) K, t( |for maintaining pluripotency may dif-
" ?* _6 Y3 x7 n( N* ~9 H# h4 ^fer between mouse and human. How-
- F; ]. Q1 c: f+ t7 F! }. ~ever, the ability to translate somatic
# p% l3 r; Q/ ^cell reprogramming from mouse to
8 q* e5 X- ] Xhuman using the same transcription 0 K9 v! p4 I/ \; e1 N8 v) d6 D, s
factor quartet further emphasizes the 8 a9 Z9 E: q' ]9 P4 ]0 c
conserved nature of the Oct4/Sox2 0 E* n5 `$ o4 z! a" }7 H% N
transcription factor network that
a0 l# e+ b- W3 @9 L4 O! ]controls self-renewal of mouse and 1 N! T; J! \ c+ o
human ES cells (Boyer et al., 2005). 8 y3 s4 {) g/ x; b, n y
Given that Klf4 and c-Myc are chro-
& F9 g( T w3 B4 Q. [* {matin modifers and can immortal-
- F) q& a& S, Rize cells, one might be able to fnd + w p$ l9 w$ l" k% k f
other factors or small molecules that ) ~/ h K* N! \$ b1 `8 I
could replace these two factors in the
8 q$ [" B' c+ @" T6 U# O- Bcocktail (Yamanaka, 2007). In these
$ P; q5 b4 U$ h; k7 B- b* ]studies, the possibility of retroviral 7 k0 _% ~* I* _
insertional mutagenesis, resulting 8 \8 ]9 t0 g3 F0 A
in the activation of other genes con-( H( D! B# S6 j' r$ g1 G
tributing to reprogramming, cannot
* t0 Y% t" a A; D0 ~5 Fbe excluded, providing an opportu-9 Z& |% z% g! r. c' S [" |
nity to potentially identify new repro-& j( h6 ?: \8 ^% N: B
gramming factors beyond the cur-1 W9 l* S* x6 f$ b5 o6 K& O
rent quartet. Also, taking a broader 3 J7 O* F1 ]6 k2 c
screening approach for reprogram-6 c, P5 V8 M# L& ]
ming human fbroblasts (as Takahashi
9 P# }4 l0 O1 A- ?* w. aand Yamanaka did for their mouse
8 r0 b/ T: F V7 \$ Lstudy) might yield other combinations
6 y9 H, R# Z9 w p; _6 Z6 Yof reprogramming factors.
5 A% O) Y+ `: S0 e6 WDirect reprogramming of somatic
6 O6 ]3 A! ^" ^! qcells to a pluripotent state, thus revers-5 c7 O6 I) @' [2 E G8 Q4 }
ing the developmental arrow of time, ' L6 S( A3 h0 c7 z) p0 ~ h- [
is considered by some to be the “holy
. J8 A- F% J. O1 B. fgrail” of stem cell research. Once the
& j+ [7 o+ |; h/ ]results in human cells are confrmed,
6 D6 C- W4 M- N) v+ ]* Pthese advances will enable the cre-
/ ]! R; t1 ?+ jation of patient-specifc stem cell lines
; U; z: ]+ V+ Y6 P' pto study different disease mechanisms
1 D q8 V% S X. N( n% Sin the laboratory. Such cellular models 7 s/ Z' W% J3 \! N7 N3 ^1 L
also have the potential to dramatically ' t/ y {7 `$ o: |( t+ `* d
increase the effciency of drug discov-
9 g# s2 ~2 ?' N6 ]0 R) bery and to provide valuable tools for " X2 i& U) S1 H% E; j
toxicology testing. Furthermore, this & V$ A! N8 j6 i4 _/ O
reprogramming system could make
& Z, l F* F6 S& e6 Zthe idea of customized patient-specifc 3 u9 t" e- g" C* ?3 j" r- f
screening and therapy both possible
7 {8 s. m/ D! {3 X5 w: qand economically feasible. Finally, the
' K; N9 ?) w* k) @( Lwork will have a powerful impact on 7 M- G7 P& d3 ]+ H5 h
the intense debate regarding the moral, ) C/ a% N: j0 g4 ^$ ^* N
religious, and political aspects of ES cell ) s# ~& Q: }! M/ E6 M) D# G+ m8 ]
research. However, a big mistake now
5 l$ p1 _& [$ C6 z8 G( [* gwould be to consider human ES cells
- K3 A# k3 x+ S- j& {" y: ] Lobsolete. There are still many hurdles : g7 l: w# B5 A7 O! b( M8 G" T
to overcome before we ful ly understand
4 v5 I0 C: L0 S' O; w) i4 opluripotency and before we have human $ O4 O; ]* Y) {/ j" W5 y
iPS cells in hand that are suitable for
8 h5 `8 I$ k* C; F: K# Wtherapeutic application. For example,
4 Q0 {$ E6 V! B3 d0 u* Qa signifcant proportion of mice derived
K8 ^) @1 l) M1 r% t& n) jfrom mouse iPS cells develop tumors
' a9 H1 {5 _6 k* e0 g6 s+ Kdue to reactivation of the c-Myc retro-; Z# B9 ^, G! k$ E! F. s, d1 j( [
virus (Okita et al., 2007) compared to ' v' I8 \/ _- W+ @# @
mice derived from ES cells, which are , a. p( T. M% s3 v# S4 x8 r
normal. The search is now on to fnd a
8 C7 |+ o, }7 @2 Zway to reprogram somatic cells without
. `& G+ C# K+ t+ D8 sretroviruses and maybe even using a
4 c' ]2 L ^) _5 |) Lcocktail of small molecules. Given this,
! W6 L. l0 F7 z! D% q/ bit should be emphasized that human # g0 Y i; P- O6 Z
ES cell research is more important than ' ?6 I" K2 C3 M6 V
ever for it will shed light on how iPS
7 b$ [) z4 X- O* {, C5 }) M* H8 Acells can best be maintained in their ) w+ z& R7 W2 R n& ^, p; D
pluripotent state and how they can be - \: x; W5 p/ _ Y( k9 n
induced to differentiate into the cell 3 c4 v* Q4 q3 e- R2 u9 D2 r+ p+ g2 `
lineage of interest. The feld of nuclear . z; T+ w1 L H/ V: Y0 {
reprogramming has come a long way 1 b* Z' Z; ]* i, D
from the initial nuclear transplantation , \7 I E; ^: S1 |( l* T% D4 ?" z
studies in frogs 50 years ago, to the
2 |# S) w/ k# P# G7 Wbirth of Dolly, the frst mammal cloned
1 n, i6 K! P1 Q. S0 O; ufrom adult somatic cells (Wilmut et al., 9 q% Q( I: `4 P& `4 h2 N
1997), to the fallout from the fabricated
5 }/ ^# [- k( Whuman nuclear transfer experiments
& L% F. j2 ~0 hof several years ago, to the landmark / m3 t3 T$ Y0 R8 L
studies of Takahashi, Yamanaka, and 3 \; }7 s- \0 @
their colleagues, frst in mice and now 0 D$ Y7 _* K6 m$ O. [+ J6 `6 S
in humans.. a( ~+ l0 \% m' R0 {
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发表于 2009-4-28 21:35
发表于 2009-5-16 10:41
