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Induction of Pluripotency: - P3 c: ^4 N$ Q' p8 y8 d0 W
From Mouse to Human: F0 t! Q* q2 a
Holm Zaehres1
) | P3 A, e8 b and Hans R. Schöler1,# Z, p- `: s+ s) C5 L7 F
*/ p, ]. d) L6 a5 X n. V9 B+ `' R7 b
1- v9 B! K2 d1 L# W, x
Max Planck Institute for Molecular Biomedicine, Department of Cell and Developmental Biology, Münster, NRW 48149, Germany
4 X3 N8 j' V( a, [. e9 G2 t6 G3 o*Correspondence: schoeler@mpi-muenster.mpg.de4 A2 m2 @. U" O& ?! p
DOI 10.1016/j.cell.2007.11.0204 |9 T0 ?3 b: O/ m3 D. L
In this issue of Cell, Takahashi et al. (2007) transfer their seminal work on somatic cell - v. m9 U) N, O. H5 H
reprogramming from the mouse to human. By overexpressing the transcription factor $ x. t8 ~4 P* \ s9 C) y
quartet of Oct4, Sox2, Klf4, and c-Myc in adult human fbroblasts, they successfully ! \) u* }0 ]* x# \$ s& z7 y2 D
isolate human pluripotent stem cells that resemble human embryonic stem cells by all 3 L' @/ D; |' F8 w% O1 d2 ?3 B1 p
measured criteria. This is a signifcant turning point in nuclear reprogramming research 8 O% s* x U9 D! A/ o# M' o) V
with broad implications for generating patient-specifc pluripotent stem cells for research
. m9 r( }3 {0 T Aand therapeutic applications.6 u0 ]9 ?) c8 j& |
This year’s three Physiology or Medi-$ k8 H& m0 o# ~5 t9 E
cine Nobel Laureates—Martin Evans,
% r+ o0 L. N/ _5 }3 c" DMario Capecchi, and Oliver Smithies—
" d& r @. l! iwill be honored in Stockholm in 10
) N. x5 z% g3 P4 Q0 Gdays time for their discovery of DNA 8 @$ m) d3 j9 E" p
recombination and the development
2 J* O7 H$ _5 x; X9 g" [6 Z7 N. Fof mouse embryonic stem (ES) cell 2 s! q* {; ?3 i' s
technology. It was Martin Evans who
p/ g$ T2 P& Gdiscovered how to make mouse ES
2 l6 @& v+ V0 D, t3 X: ~2 d) rcells, enabling any genetic alteration - l) }: s7 a. C4 j& F5 L
to be transferred to the germline and ( p5 M5 p# ~' Z. @
hence to the next generation (Evans
2 G+ g* s/ `5 O& }3 O7 t. `and Kaufman, 1981; Martin, 1981).
! i' R! M ~% }$ D0 JBefore this breakthrough, researchers 0 R0 @( n! A6 N
studied mouse embryonal carcinoma
5 }6 Y4 U; n3 m* b, ~8 ]( gcells derived from tumors, which
, N4 T3 X2 r! L( m/ D0 qcould form every mouse cell lineage
9 x6 W2 R' G9 _4 wexcept the germline. Combining DNA
# `6 v' G* I) F3 i3 drecombination and mouse ES cell ( Z& ~2 N( G$ q5 W
technology revolutionized an entire
( k6 w. B4 E" r7 ?' Z8 L a4 Ffeld of research, forming the basis for 0 y% v4 A6 E9 Y5 C+ O8 R8 M4 V
studying and understanding the roles ; N0 K0 Y- ] w) n! H' q
of numerous genes in embryonic
- l. M5 B. B% ?/ i. ]development, adult physiology, dis-6 N2 \6 v" }/ Y
ease, and aging. To date, more than , \) U) I: I! v; k$ c
500 mouse models of human disor-0 D4 ]" n+ Q1 \
ders have been generated. Now, with
, B" l/ H1 X* e- s6 d) ~0 Z$ Gthe study by Takahashi et al. (2007)
* D& | y7 R3 ^" c F8 s3 T, T+ I& xpublished in this issue of Cell, another R( @7 }: b% H/ m* ~
important revolution is taking place.; M( @' w& A2 ?) `/ Q# \1 w
Last summer, Takahashi and : M5 v/ S9 F' n. z5 ~
Yamanaka (2006) stunned the scientifc
" h0 ^4 V' f- D" y& |. H% ~community with their study showing
0 L) @3 S- H3 U4 hmolecular reprogramming of mouse $ w# x3 p- y/ d* v3 o
somatic cells into induced pluripotent " [, T# B) I0 p0 [+ A4 [
stem (iPS) cells using just four factors:
4 _ h' M# d% pOct4, Sox2, Klf4, and c-Myc. Their
( Y, A& a# z% M0 W) Xelegant but demanding approach of 7 G8 F# D$ L: B8 p2 N& ^
screening for a cocktail of factors that . J3 _2 E5 T* M% F; _6 p
could reprogram mouse fbroblasts ' l7 B! M! ~6 f5 s! p! j
starting from 24 candidate genes paid 8 n8 d& o7 j, P2 U2 _7 @" o
off with their detailed description of iPS
& g: ~" `& d9 V$ A! ocells, which are almost indistinguish-9 ?/ W ^2 x9 U# ~+ u% u1 z
able from mouse ES cells. As with all
+ y4 C. a- }$ E2 i4 sscientifc discoveries, these exciting , x! g- z& u; m( ?- J0 I
fndings had to be reproduced. Sev-7 L8 s1 r0 k1 H$ I/ ?% F
eral studies published this year not 5 e. @5 e6 B5 P
only reproduced but also extended
7 P+ C1 V) K: ~the Takahashi and Yamanaka fndings 6 h) W2 h S z9 {' l
by demonstrating the pluripotency and * D) \/ \3 }1 ^, f3 e% B8 B
differentiation potential of mouse iPS ( @3 W( G$ y$ z* a; s1 i
cells in rigorous developmental assays
4 `: s$ r6 B: V% `$ s- N7 ?; W(Maherali et al., 2007; Okita et al., 2007; # ?3 h" c. Y% |
Wernig et al., 2007).
, u3 _3 n, c# O3 Q7 |In their new study, Takahashi, % C7 v8 i. X; l x- V! i/ r% h2 r8 X
Yamanaka, and their colleagues ( g; P* o3 F r
(Takahashi et al., 2007) now translate
! w# P& t+ E4 j, K; e/ Qtheir remarkable fndings from mouse
7 ^/ w/ |* b, W7 q+ [& x0 w9 [- ?to human (see Figure 1). They selected
1 u m$ p! `5 _: I' ?( Z6 T+ c/ Padult human dermal fbroblasts and
; |3 R3 n+ |& z( z0 v/ c+ ^: mtwo other human fbroblast popula-
. x' G2 ]6 w! r' |4 ]: @tions (from synovial tissue and neo-4 K8 u1 D+ ?5 k. ?" ]7 A
natal foreskin) from different human # R/ y+ m( t, i
donors as their reprogramming target
3 A) n4 k, ^, Qcell populations. They then trans-
( w7 M ?& C+ L: t3 v+ {' h. Pduced the human fbroblast cultures : B% B- |0 V; }7 g' K2 j' Y( |
with retroviral vectors carrying trans-5 a2 G. t. h; ~7 M+ E
genes for the human versions of Oct4, ( ?5 O& N. f# j9 u; Q4 N% m
Sox2, Klf4, and c-Myc and cultured
. m8 `( G& w* N1 |the cells under human ES cell culture 9 a1 h3 g5 J5 d/ z5 ]5 ^
conditions. Thirty days after transduc-0 D ~6 ]9 }; |2 I7 @% I
tion, the culture plates were covered
# x" z4 K |: y* A+ `2 u: P4 x" Wwith human ES cell-like iPS colonies
0 n' D& n% i% r& v(among other colonies), which could 1 k5 R$ o2 ~2 m2 _- N5 u
be further propagated and expanded.
+ z; L! E' ^' G' j' dThe retroviral vectors enabled silenc-
% L3 |6 Y4 \# Z/ U9 ming of all four transgenes after human ( ]4 @6 S9 c" d6 H5 t! [6 C( e
iPS formation (as found in the mouse
4 v7 o: C% `5 R/ \/ W8 zsystem) indicating that the iPS cells
; i R! }* o; s7 j8 Kare fully reprogrammed and no longer
7 h- N# c ^) E/ s! _: Ndepend on transgene expression.8 ~! r% W* [( N% ~! k: @2 I" |
Unlike the mouse study, human
: K/ q1 s) Q) xiPS cells were generated without any 6 w1 e$ i( u7 i0 z" j
genetic selection procedures. Given
$ X3 n; [3 `0 S! p( }8 l* `, Lthe lower mitotic index of human ES ; b7 `, r" y* h' F+ i0 J
cells, it is not surprising that the gen-0 z# Y- j; N) L
eration of human iPS cells takes nota-
, h/ p5 x+ R9 dbly longer than in the mouse system.
5 b7 s! e' C: [' s" D! K% BThe authors subjected their human
1 [$ ~4 |) I, P0 E' GiPS cells to a panel of assays to com-
4 V. b, ]& b" n9 y; V% [pare them with human ES cells. These
* |( F* R" }7 Hassays included morphological stud-
9 J8 [$ F" V5 [, ]ies, surface-marker expression, epi-
M# ], _% T7 U9 B) w8 d( I. R* Zgenetic status, formation of embryoid
7 A% W7 @ O6 j# C, r* I4 abodies in vitro, directed differentia-" G. B& x; A' j% n1 ~$ \
tion into neural cells and beating car-! f8 x! H) A3 L4 p
diomyocytes (according to human " h. M* _2 m8 k4 G
ES cell differentiation protocols), and
8 n) Z* d" Z3 L. c5 G& @. Ffnally teratoma formation in vivo. @9 E0 _' W9 [4 q1 [2 K. s; q
DNA microarray analysis revealed 8 Y& p4 ~) C9 }
the remarkable degree of similar-
; y" E; U! @$ {$ uity between the global gene expres-
6 M" F% T% X8 E0 W1 R$ Usion patterns of human iPS cells and
! U4 b. ]5 s6 @5 f3 f; H6 ~human ES cells. Notably, genomic
8 i4 L' z h6 ^DNA analysis as well as analysis of
, J0 [, `9 ^. ~4 Z5 L& c f+ e \short tandem repeats demonstrated
$ N7 J/ D' j! o/ cthe genetic origin of independent ( d1 o: R3 Z" }* y9 g2 M" W( k
human iPS clones from their parental 0 G5 q3 \7 p. B8 }' d" o
fbroblast populations.
- p7 P$ ]$ n4 d2 e" mThe derivation of mouse and then 4 y5 b! |- w" `* z
human ES cells (Thomson et al., 1998) ; u% }6 S! Z: y+ X
as the gold standard of pluripotent
& i" h' u$ ]- d; Cstem cell populations has necessarily * c0 k& u2 h: S! S! R2 a4 `# d
led to emphasis on differences in the 2 L7 |6 ?5 Q0 \ v9 z5 Y7 t
regulation of self-renewal between
, e* u6 G* |: D7 m$ ?, [ wmouse and human ES cells. For
I& g: [! x2 p6 d2 y1 W5 kexample, human ES cells depend on 5 r0 f6 ^& e" W2 r* [
bFGF for self-renewal, whereas their
% a; N+ i6 c8 s( m1 Wmouse counterparts depend on the 0 Z9 z5 r' s( n; i: O6 D
Lif/Stat3 pathway; BMP is involved in 9 K" D% N5 n( T/ Z. T
mouse ES cell self-renewal, whereas % j0 |1 l) P" m7 O2 s$ h' _; c
in human ES cells it induces differen-
1 S1 A0 e h* _tiation. Extrinsic factors and signals - d. T% I" y' j9 _3 Q" e
for maintaining pluripotency may dif-3 K1 h3 P0 E' }# q3 p5 x- l
fer between mouse and human. How-8 c5 z- h0 s3 y6 Y0 B9 D7 ?
ever, the ability to translate somatic
8 }$ r7 ~# S) E @! d" f- ]cell reprogramming from mouse to 8 P/ A+ c2 s6 q/ A# V
human using the same transcription 2 H" C7 b) I3 E
factor quartet further emphasizes the ' L' w3 v/ {! {/ J: X$ h+ }
conserved nature of the Oct4/Sox2 2 c5 U1 Z$ z/ N8 d
transcription factor network that
. f1 K+ N& E7 X( xcontrols self-renewal of mouse and % o+ Y. P4 I$ d
human ES cells (Boyer et al., 2005).
; g) u) T- h( I% mGiven that Klf4 and c-Myc are chro-
+ U9 I) c2 T& @0 v; amatin modifers and can immortal-
3 V" m+ X$ y b% P7 Zize cells, one might be able to fnd 2 @' o8 w) I$ T* s1 N9 j$ c
other factors or small molecules that
' J' ], H; M( K9 Tcould replace these two factors in the % a3 w, ?. W! M. j- O9 f
cocktail (Yamanaka, 2007). In these
& w! B' {; x( \* cstudies, the possibility of retroviral
$ S7 u5 ^; m# Yinsertional mutagenesis, resulting 0 m0 h7 p/ ?4 f3 }4 u' o2 c
in the activation of other genes con-
# j$ P2 ?7 ]( k1 e( Mtributing to reprogramming, cannot " v/ c2 m$ b% K- a7 C5 u+ D
be excluded, providing an opportu-" ^" M5 M# z4 w/ @/ Z
nity to potentially identify new repro-- ~. d" A0 M$ T4 b& t* z/ D' J
gramming factors beyond the cur-
- w" E- E* G+ f" Jrent quartet. Also, taking a broader ; Z2 V5 U: `" s
screening approach for reprogram-
. E; E5 ?6 v- g6 T5 G$ b/ D Dming human fbroblasts (as Takahashi 2 t" o+ @' i4 y; y5 G7 ~7 j
and Yamanaka did for their mouse ' S& U* t" j9 ~) V
study) might yield other combinations
& _6 @, i' K1 b9 Iof reprogramming factors." v1 O' h$ t8 y- W
Direct reprogramming of somatic
' s* O- z0 Y( K! [cells to a pluripotent state, thus revers-# [4 W V/ w: b# K
ing the developmental arrow of time, ; N6 C- d5 @6 m3 |" D
is considered by some to be the “holy ( z+ C% B( e/ d0 V
grail” of stem cell research. Once the
3 C; `. p9 ?! l- G$ R# @+ }: P1 u' E& @+ wresults in human cells are confrmed, 6 l, Q0 c- P& {
these advances will enable the cre-
+ s- h x s/ c7 d; Y) _- d0 sation of patient-specifc stem cell lines * D7 g2 \* e, W6 A( M
to study different disease mechanisms 0 Z2 G1 C8 ?: y# i2 \
in the laboratory. Such cellular models # l0 {. T* A3 s: |+ j) @6 p' t
also have the potential to dramatically
# o3 G1 b! X @0 j3 R" k: [3 S) X wincrease the effciency of drug discov-
9 o3 Q4 _1 j! I/ e3 P1 ? X8 |# Wery and to provide valuable tools for & s4 _+ d' C0 _
toxicology testing. Furthermore, this 5 [; {* r, H" D4 G+ i+ O
reprogramming system could make
1 z" I; V% L) ?, kthe idea of customized patient-specifc & }. T& s9 s* I) Q% F& B
screening and therapy both possible 3 l$ d; b% I" o1 b A; T
and economically feasible. Finally, the
6 s" m! v! n2 |) v6 Fwork will have a powerful impact on 1 D3 [: S# w t" N
the intense debate regarding the moral, , W. Q/ b7 l$ b5 ^( N* X
religious, and political aspects of ES cell 0 Y' C4 E5 A/ ]
research. However, a big mistake now
q% |0 p- `: g' N6 `9 }would be to consider human ES cells 1 i3 m M% k4 G# D8 W0 e
obsolete. There are still many hurdles
1 o, y& U# N2 L" gto overcome before we ful ly understand ; _" N5 V/ ]2 m7 H# @- B9 [& k
pluripotency and before we have human
8 t5 O& b% O: E( {9 M, V diPS cells in hand that are suitable for
2 n: k. T# w. s: Ftherapeutic application. For example, ' I5 K- j. U- a# W! ?8 t4 H8 q$ a3 A
a signifcant proportion of mice derived G7 A5 \" }/ O+ g4 V* Z& @3 d
from mouse iPS cells develop tumors # r5 a) F x1 x
due to reactivation of the c-Myc retro-
7 r7 M. c7 C6 ^virus (Okita et al., 2007) compared to
+ t* M7 [: Y# D W6 Y( umice derived from ES cells, which are # d2 a* i0 ^# d) a8 E, z% }% {2 E
normal. The search is now on to fnd a # M& I ?, i5 k& ?5 a
way to reprogram somatic cells without
1 a3 e8 L' D4 tretroviruses and maybe even using a
; X! f( |" a5 s$ ]7 i! [* Ucocktail of small molecules. Given this, ( D' l- ~, U6 g7 B) k- l" w
it should be emphasized that human $ I/ d# Q* I* k0 x, t7 \% x/ E
ES cell research is more important than & E2 b- o0 A, |7 h) W6 }
ever for it will shed light on how iPS ; |6 T- \$ J+ F J: n; @# _
cells can best be maintained in their 7 C9 B! J: ?1 i3 E- x
pluripotent state and how they can be 9 v" _2 H* T/ \; R% i
induced to differentiate into the cell
( r& L/ C. N5 L! `7 Flineage of interest. The feld of nuclear 9 w- C4 K( Q5 g) V/ F
reprogramming has come a long way
) K4 v( s4 k. a% l7 S: Xfrom the initial nuclear transplantation
/ k2 x- _2 e! e3 Q6 Pstudies in frogs 50 years ago, to the
3 r% d; j: j1 j$ I4 gbirth of Dolly, the frst mammal cloned
! |9 ?9 l/ L) o. ifrom adult somatic cells (Wilmut et al., & q& W1 f+ r3 _* s
1997), to the fallout from the fabricated / Y% G7 i7 o- ]. J G$ S, d
human nuclear transfer experiments ; a9 N2 \6 N+ k Z* e0 W
of several years ago, to the landmark
/ v& E$ G& ^8 o# a; _8 rstudies of Takahashi, Yamanaka, and
9 W7 q/ s# R( a/ A7 j* L6 O. Ctheir colleagues, frst in mice and now
* s; h# T7 Q l- Cin humans.7 @6 G/ k$ J1 l3 N, w1 n
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发表于 2009-4-28 21:35
发表于 2009-5-16 10:41
