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Induction of Pluripotency:
# }) {: I G/ p" n% PFrom Mouse to Human
3 Z% ?# O" b. t# [4 n% C; i6 jHolm Zaehres1
; V# @* J4 u8 L9 y9 T and Hans R. Schöler1,3 M9 w6 p& v4 U, k
*
4 Q- C" N# }$ T" S1* ]% _* \+ k5 f9 e. g5 x" k
Max Planck Institute for Molecular Biomedicine, Department of Cell and Developmental Biology, Münster, NRW 48149, Germany7 M0 J" W V8 B. _
*Correspondence: schoeler@mpi-muenster.mpg.de
# C6 J9 X5 ]& v9 DDOI 10.1016/j.cell.2007.11.020
% |9 m6 D9 w% j! B( HIn this issue of Cell, Takahashi et al. (2007) transfer their seminal work on somatic cell , d; C/ o7 y+ ^: ?1 {
reprogramming from the mouse to human. By overexpressing the transcription factor 8 Q' E4 k/ m6 k( t+ J, G4 s2 `
quartet of Oct4, Sox2, Klf4, and c-Myc in adult human fbroblasts, they successfully 9 @# ]0 T# f& q: e2 V) P3 M' i
isolate human pluripotent stem cells that resemble human embryonic stem cells by all
" |* a3 \/ H m( L2 {measured criteria. This is a signifcant turning point in nuclear reprogramming research 6 ~- E) Y9 I5 _/ j5 ]
with broad implications for generating patient-specifc pluripotent stem cells for research . r& F( b; `3 M: _0 u/ r
and therapeutic applications.
& ]# S8 ~+ P! _9 ?- I+ jThis year’s three Physiology or Medi-0 K7 _4 v, X% B' W- J4 e( ?
cine Nobel Laureates—Martin Evans,
2 z, z9 \* L2 W4 H: q5 ?/ O$ F5 nMario Capecchi, and Oliver Smithies—' O! Y2 \$ B3 {: I, B: j8 t
will be honored in Stockholm in 10 . l* x ^6 N1 C- x; N6 i
days time for their discovery of DNA
5 {* @, [/ B crecombination and the development 3 H, N: y# q1 x, a1 U! A$ T
of mouse embryonic stem (ES) cell 4 o' Q1 M% j" a. m. T- k4 W4 s
technology. It was Martin Evans who * N: P4 X& [3 p+ E
discovered how to make mouse ES 7 O+ n1 x; ?' h9 [8 D9 G* i) N" c
cells, enabling any genetic alteration
# ^0 R* O* R/ {' M; ?to be transferred to the germline and
3 Y; \, U% q a; [5 I8 U$ rhence to the next generation (Evans 4 F* R2 M( E+ m) n3 J: a
and Kaufman, 1981; Martin, 1981).
( ^4 p; v3 z m9 N) A z3 OBefore this breakthrough, researchers
& {" n, q; [% ^+ j; O# Gstudied mouse embryonal carcinoma ' h' o- _& o% R7 r" Y2 c
cells derived from tumors, which
3 h+ f1 }! Q) u A. ~could form every mouse cell lineage
, l% j9 ~. n! `" f- oexcept the germline. Combining DNA
4 B7 R7 o# o/ T/ w2 Nrecombination and mouse ES cell - a& B0 \/ t9 c1 e+ n
technology revolutionized an entire
3 b# g' k+ k( cfeld of research, forming the basis for , I5 }, U5 ?7 p5 a
studying and understanding the roles
: f- W& Y/ R% J# G, T9 M& o( ?' zof numerous genes in embryonic
! g# I7 r9 F- g( ^( s7 \& O5 e2 J. @1 Fdevelopment, adult physiology, dis-
3 e( K! l6 X+ Oease, and aging. To date, more than
) C) |8 n6 d; E+ I6 j500 mouse models of human disor-
! A7 c, ?5 n+ V0 @) kders have been generated. Now, with + j* @1 c. K* m6 U* R. z( }/ Y
the study by Takahashi et al. (2007) ! O5 W, u$ l1 D+ d$ {) E
published in this issue of Cell, another : W) k$ L; J/ h: ^% |
important revolution is taking place.
! f' [7 u* r# w$ M9 \( w4 mLast summer, Takahashi and @: S3 P" U; c
Yamanaka (2006) stunned the scientifc
5 l# w3 k* ]& Y) H5 H. }# X& Ycommunity with their study showing
6 q7 j _4 [( _6 t! R( A% [molecular reprogramming of mouse ! z- e4 \5 e( z7 v
somatic cells into induced pluripotent 8 W0 y0 J6 X; ]+ T, m
stem (iPS) cells using just four factors: ; B/ T% L! @& S: u- R+ b$ S5 n4 t
Oct4, Sox2, Klf4, and c-Myc. Their R; \# I; m3 Y! Y: z6 R
elegant but demanding approach of
7 Q: h( N! K, l( @1 Pscreening for a cocktail of factors that
0 z# q ]9 H. ^- }7 Q& Vcould reprogram mouse fbroblasts
3 e( i, Z' k0 P A+ zstarting from 24 candidate genes paid
0 r8 _, J- `: q8 Soff with their detailed description of iPS $ v H. S( w6 L5 g( \
cells, which are almost indistinguish-
2 l9 c" K7 b% \able from mouse ES cells. As with all
/ E2 o X/ h6 M+ C& S5 O/ Q9 a5 vscientifc discoveries, these exciting
9 O& E% |1 ?8 j, Q, H5 H: k9 Q ~9 X) tfndings had to be reproduced. Sev-
2 D; }% t& ^9 [) @7 K$ U9 aeral studies published this year not
4 Z! P' h4 @8 T* J2 xonly reproduced but also extended
7 d0 w! r' w1 j; t/ Xthe Takahashi and Yamanaka fndings
/ m* Q& w# d vby demonstrating the pluripotency and
% ~- ]; }$ W& l, C; tdifferentiation potential of mouse iPS " [. l9 C: ~# G7 j" E' W
cells in rigorous developmental assays ! v$ V4 Y1 E" ], {% M: ^
(Maherali et al., 2007; Okita et al., 2007; " |/ N+ r6 t9 z
Wernig et al., 2007).
+ @" s$ l4 I# }In their new study, Takahashi,
# j& l& \; P5 |1 K% w( {Yamanaka, and their colleagues
4 M4 D% D$ q7 o7 e# r# }; J+ t(Takahashi et al., 2007) now translate 2 J& ?5 _) ^ J3 |: Y9 P" B( Z
their remarkable fndings from mouse
; l- x2 @& T$ L2 l" m4 Eto human (see Figure 1). They selected # z: l4 d8 H: j6 y+ ]& J
adult human dermal fbroblasts and & O2 g5 j- k$ O# g* S. L
two other human fbroblast popula-
& ~: l9 f! ~- l2 Q. ]tions (from synovial tissue and neo-
3 T& M' S. F4 Xnatal foreskin) from different human - q6 D; [9 g7 z& `
donors as their reprogramming target
/ S( I* ^- {. I3 Hcell populations. They then trans-5 N1 W8 d7 P- Y% G# \
duced the human fbroblast cultures
1 n) M' w) P- Z/ _with retroviral vectors carrying trans-9 ?9 L2 F3 a$ A/ r) f9 [1 a
genes for the human versions of Oct4,
2 X& K! ^9 f* F1 T4 L% @; YSox2, Klf4, and c-Myc and cultured , r! \% T; o5 I9 X
the cells under human ES cell culture
1 J5 U H) q: ~, r gconditions. Thirty days after transduc-
0 ~& C5 _* Y* G" \ Etion, the culture plates were covered
' r& }# w/ x/ x# p2 T: nwith human ES cell-like iPS colonies
+ W% ^ `1 V5 |% f5 f" T! J% a7 ^6 J(among other colonies), which could $ |2 R, D* E, ?) `9 o! C
be further propagated and expanded.
; ^' C( N K( Y7 A2 n$ B6 {( {* T' ~! OThe retroviral vectors enabled silenc-! {- X& M( G5 g: U7 e# {- q
ing of all four transgenes after human 6 B' _4 K3 [0 s- F
iPS formation (as found in the mouse
) T( d Z e/ u: k* g0 ~$ Osystem) indicating that the iPS cells ; Z7 F6 H( ~" k' k
are fully reprogrammed and no longer
$ f% h9 [" F8 H# j- Z E6 \. {depend on transgene expression.& l6 h" V K) t# e9 Q) ~3 u
Unlike the mouse study, human 0 _& s- G' e6 N' X( Q7 C1 u
iPS cells were generated without any
+ J* n* ^7 T( a" Egenetic selection procedures. Given
& }4 I5 f7 X% S0 Hthe lower mitotic index of human ES
) c7 |. f+ w( t1 Qcells, it is not surprising that the gen-- k3 C& {5 S* u0 |
eration of human iPS cells takes nota-
: L3 O! ~! x8 U3 _/ e: I$ `bly longer than in the mouse system. ( F# ] S/ l3 Y
The authors subjected their human
( k- T/ F! h; ]) i0 u" ~4 s' C, qiPS cells to a panel of assays to com-; G9 x+ l8 v+ H2 B; @ _
pare them with human ES cells. These
$ D! o+ L9 Y- c6 K( tassays included morphological stud-
1 X9 U% Q$ l, b# O3 e/ ~% y2 Wies, surface-marker expression, epi-
" Y, B8 U' Q* wgenetic status, formation of embryoid 0 L8 v1 |0 a# h7 v W! q8 D: e
bodies in vitro, directed differentia-' [8 E( A) m. p
tion into neural cells and beating car-* x5 A8 i5 p7 @9 W* n# v; d
diomyocytes (according to human
7 _% }" }* d+ f, J: e4 ]( R0 aES cell differentiation protocols), and
2 n5 h0 j, {% ?) s2 Q/ y! @) Ofnally teratoma formation in vivo. - L% x' Q }+ Q. b }8 z
DNA microarray analysis revealed
8 g3 h# @5 _9 ?' r& \, ?the remarkable degree of similar-
^/ T$ w% ~. ]( e4 X" L9 fity between the global gene expres-
8 J, y( k- K c2 J6 f6 Ksion patterns of human iPS cells and % h9 h. H( r, J1 t; \9 a
human ES cells. Notably, genomic - B2 k& G4 T* | d( T. Z# F) Y
DNA analysis as well as analysis of
% s. o9 q+ M; d$ T j$ p+ {short tandem repeats demonstrated - x; L0 y/ G: P, [- H
the genetic origin of independent 9 \: W0 K+ D5 E- n* A; V
human iPS clones from their parental
' L. p, n: D0 z/ ?, r- A5 w1 kfbroblast populations.. U8 h8 F* M" H( ~: w! R+ U" t4 f; J
The derivation of mouse and then ( q) L/ d# f+ i8 v. u
human ES cells (Thomson et al., 1998)
9 p+ \ |/ R0 R U# J+ Eas the gold standard of pluripotent " ?9 S; ^' Y# D( s4 V8 O
stem cell populations has necessarily
; D" i& E) @6 J0 Oled to emphasis on differences in the
0 n) G. a2 l9 J% s2 p% y; a4 I# @regulation of self-renewal between 2 Q3 Y9 {9 [+ `- g
mouse and human ES cells. For 3 F1 Y! |( ~% r3 [( s( n
example, human ES cells depend on
& T, V, j- `5 a4 [bFGF for self-renewal, whereas their 5 G4 s1 k1 }9 N
mouse counterparts depend on the # z$ E& f$ V A8 k3 L
Lif/Stat3 pathway; BMP is involved in 2 i) K7 ~( D" C& W
mouse ES cell self-renewal, whereas 0 N- t6 o& K; v, {
in human ES cells it induces differen- [1 N4 N+ J; F" Q9 Q
tiation. Extrinsic factors and signals
& Q1 G/ v% h8 l' M0 k6 {6 z0 _for maintaining pluripotency may dif-% e' E$ r( a* |$ Y4 z
fer between mouse and human. How-
1 w( r3 s& S" t: b4 never, the ability to translate somatic
2 t0 B0 r2 ] e' i, O3 `7 dcell reprogramming from mouse to * l8 v/ t/ B u) h: \, J) o$ R: `
human using the same transcription
' y/ P; \, N L1 h' g* v* Jfactor quartet further emphasizes the
) q5 g9 k5 V7 _, Yconserved nature of the Oct4/Sox2
( ? O) n4 n4 g* k! stranscription factor network that
* K1 x6 g! ^' M0 ucontrols self-renewal of mouse and
3 q. P- s6 m" I) _" O' t+ v" K khuman ES cells (Boyer et al., 2005).
( P2 z3 E/ U) y* y$ Y. j! y4 NGiven that Klf4 and c-Myc are chro-2 I2 Q( w6 [+ Z+ `- F5 \" h
matin modifers and can immortal-6 J% w" a# w1 K5 U% S" T1 [& L5 V
ize cells, one might be able to fnd
! S- n1 A- D- B: m! M& n. sother factors or small molecules that 4 N9 u( k! \$ m% i% r
could replace these two factors in the
9 Y# B1 N! m& Z3 @" Tcocktail (Yamanaka, 2007). In these 4 g+ u L, g; |( d
studies, the possibility of retroviral ' c" |! O: k/ i) y- I4 J9 ]
insertional mutagenesis, resulting
/ a: e2 q+ T6 P% Q/ Kin the activation of other genes con-
( U( C* [ E; Htributing to reprogramming, cannot % v$ c, A3 D$ M, A
be excluded, providing an opportu-8 T- u2 ~8 A( y3 h: A3 D" g: S
nity to potentially identify new repro-' P" \' @9 i: R" z$ K
gramming factors beyond the cur-+ h2 [5 _$ E. `: F
rent quartet. Also, taking a broader
" i. n# v4 j% E( `screening approach for reprogram-
- m4 a- W! d, P" ~, o; Yming human fbroblasts (as Takahashi
' z; H( ?) I* Y1 E. [and Yamanaka did for their mouse
) A. B5 U; z& d* w' O8 \* i5 D* Mstudy) might yield other combinations ) R; K" r; t* I
of reprogramming factors." P8 ^' r) c" x/ {3 B# T4 y
Direct reprogramming of somatic
* @+ \1 }6 f) Z/ B9 b7 m8 Tcells to a pluripotent state, thus revers-
4 ]3 r9 y* n1 |' q5 r: ]+ K* Jing the developmental arrow of time, ' k: J, K Y1 G0 ]/ v9 B
is considered by some to be the “holy 2 ~2 A8 E% D$ @: N6 r( P% L
grail” of stem cell research. Once the - s% G) x9 i8 a# E+ X5 I
results in human cells are confrmed, 9 r5 D* _6 O2 A* o4 V+ o" m
these advances will enable the cre-0 c6 ?# P, V' e& X
ation of patient-specifc stem cell lines * C' x0 z3 I% U0 B3 Q% D" v- G
to study different disease mechanisms 5 p3 U/ i) {% _% A7 I% h' X0 L9 R
in the laboratory. Such cellular models 1 H: i- O6 {. Z" ?6 p1 `! s8 `& R1 O
also have the potential to dramatically
" c4 x3 R# n! g N/ _2 oincrease the effciency of drug discov-
) h4 i; P5 b# W6 L9 Iery and to provide valuable tools for # \1 P9 x/ ~) z5 `- H ~# y
toxicology testing. Furthermore, this * ^# M6 _. s) l
reprogramming system could make
: }4 k( a7 d% O& A6 t1 v: C$ E. Jthe idea of customized patient-specifc
/ u& |- z9 P$ U( }0 B6 cscreening and therapy both possible 3 n: ^ H& }) i" @: g
and economically feasible. Finally, the
' c( B( h1 ~. O- h; Z+ {3 Owork will have a powerful impact on
- K) `5 f" l( z; E& \; z2 ythe intense debate regarding the moral, ) O8 ` w7 x$ B7 b
religious, and political aspects of ES cell
, [' k0 f% C4 p3 ^research. However, a big mistake now 9 `$ `/ ~9 J) H% z1 n4 c
would be to consider human ES cells , v* F% n/ D# U. [* ?4 S
obsolete. There are still many hurdles ! p; p. g) [! k$ K2 t) Q9 e/ f4 T0 n
to overcome before we ful ly understand % L: X5 O. Y8 `' [
pluripotency and before we have human # V$ {' h. ]: a# j8 K$ ?* q
iPS cells in hand that are suitable for # Z% }. \ G" f! T1 r
therapeutic application. For example,
; ]/ \# i: U9 s% p) la signifcant proportion of mice derived * D& o6 ?/ `; _( z' @' {
from mouse iPS cells develop tumors
5 _% p4 p9 E# _due to reactivation of the c-Myc retro-
7 }9 Z3 Y8 g E9 lvirus (Okita et al., 2007) compared to
) K, y. u' l7 ^* }$ F; g- Mmice derived from ES cells, which are
3 v4 H, {9 J* ^/ Qnormal. The search is now on to fnd a
/ Y3 _9 B, w! B& D, t3 x1 q3 N2 oway to reprogram somatic cells without : q% U2 O H" ]2 J3 b6 G
retroviruses and maybe even using a 8 Y% k5 y8 \& E* m
cocktail of small molecules. Given this,
# n6 f5 ?- z6 z" {9 Tit should be emphasized that human
# r5 W1 ~& E" BES cell research is more important than
# M0 C2 J" b K" uever for it will shed light on how iPS " @* J$ B- r4 O5 T
cells can best be maintained in their
" K9 ?% ~% ? _pluripotent state and how they can be
9 U0 ^1 G, |/ Q9 w2 c6 Vinduced to differentiate into the cell
2 ?/ h) c# h# flineage of interest. The feld of nuclear
* X {; t4 ?% |3 k- F1 P+ |reprogramming has come a long way
! H& ~; c) o/ X# B6 K; Wfrom the initial nuclear transplantation
+ N9 R0 u6 ?7 h2 kstudies in frogs 50 years ago, to the $ \' t3 B* `/ N7 I: C1 d4 A* R
birth of Dolly, the frst mammal cloned
; l- n- K/ B$ l4 Z" L, v# Sfrom adult somatic cells (Wilmut et al.,
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发表于 2009-4-28 21:35
发表于 2009-5-16 10:41
