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Induction of Pluripotency: / F5 C2 d" n) P7 o t6 p
From Mouse to Human- T, d. u; D+ R: j Y. o9 X
Holm Zaehres1
! e3 P; B0 \ ^9 _& x$ O and Hans R. Schöler1,
4 P2 {$ k ]. x*
3 O! y6 \) X$ C, W0 ?, p! U, R. k13 B- T$ U( q5 P, e5 E# F( z
Max Planck Institute for Molecular Biomedicine, Department of Cell and Developmental Biology, Münster, NRW 48149, Germany
1 q5 U7 @9 I' ?6 h0 V*Correspondence: schoeler@mpi-muenster.mpg.de
' z0 n6 P$ ?; l7 y) {DOI 10.1016/j.cell.2007.11.0203 [& w3 L. U- Z
In this issue of Cell, Takahashi et al. (2007) transfer their seminal work on somatic cell 3 ?# m* J- Z$ K2 k k
reprogramming from the mouse to human. By overexpressing the transcription factor
v; M8 y- }# equartet of Oct4, Sox2, Klf4, and c-Myc in adult human fbroblasts, they successfully
6 y- J1 u1 M5 [, u1 Lisolate human pluripotent stem cells that resemble human embryonic stem cells by all
1 w+ q! T. l2 n# ?: ?- B* mmeasured criteria. This is a signifcant turning point in nuclear reprogramming research
6 T+ h% e; A R- G, Z8 Wwith broad implications for generating patient-specifc pluripotent stem cells for research
# ~0 s- ?. u+ u" H+ d( C2 Jand therapeutic applications.3 s, @; N0 e% T% P' A, B
This year’s three Physiology or Medi-
4 [! ?9 c: m; X" \% `cine Nobel Laureates—Martin Evans, ! v4 G: _/ ~+ _+ W1 ]" E2 Z
Mario Capecchi, and Oliver Smithies—
, f9 G1 z/ Q' w( O. q7 zwill be honored in Stockholm in 10 , l5 {8 Y6 P4 h9 X2 w
days time for their discovery of DNA
) u( O" y7 q, r' ~) hrecombination and the development
! Z: D$ R/ }/ b( C3 }# k8 bof mouse embryonic stem (ES) cell
. V6 |# w f+ R/ ^3 x! x$ ]% [/ n, Etechnology. It was Martin Evans who
7 v; ?% ^, A( B" R( b7 ^; Q: Wdiscovered how to make mouse ES
5 k8 @, M6 K& n( |- n dcells, enabling any genetic alteration . D; j4 K1 o/ A- p
to be transferred to the germline and / w) V# G* r$ N6 ~4 n+ W
hence to the next generation (Evans 6 y/ J9 B3 c6 M) x
and Kaufman, 1981; Martin, 1981).
* X0 r/ ?# z% ?. c- y/ HBefore this breakthrough, researchers
+ ~5 u3 Z" V X' t" }studied mouse embryonal carcinoma
1 h. b8 m* o6 M9 `1 vcells derived from tumors, which
# R, W1 A4 j; V9 l8 ecould form every mouse cell lineage
+ H7 K5 r9 L$ U j) m( w0 R+ H; bexcept the germline. Combining DNA
8 b: r3 [& K) u, ~8 i+ T h/ k: \. t; N! [recombination and mouse ES cell
3 h+ ^9 ] m _) Q0 _) mtechnology revolutionized an entire % [: v9 H0 J. o0 U1 w. D; b4 ^
feld of research, forming the basis for
: J5 ~7 y' f/ K- Qstudying and understanding the roles 7 M! Y8 s. D) {2 G( s# F
of numerous genes in embryonic
' h5 w q% a8 s- h8 u/ Ldevelopment, adult physiology, dis-; o' t. w4 o3 g4 j- D
ease, and aging. To date, more than ! s* ^% R$ @; {' s: C9 t
500 mouse models of human disor-
' h( F! l2 ? M U- ?9 w. @ders have been generated. Now, with
- ~" {8 E, q9 B! q1 H4 Qthe study by Takahashi et al. (2007) 7 |7 z Y8 V H, _: d, t- V
published in this issue of Cell, another
. C# \8 B% F d& y& [important revolution is taking place.
% c. ]! p6 ?+ j) {Last summer, Takahashi and + D. [6 g9 S/ B; Z
Yamanaka (2006) stunned the scientifc - L, a( f. F: D$ Z. U
community with their study showing
! P6 T( A! M# | q% @3 V# kmolecular reprogramming of mouse
6 b3 N0 S( J1 M2 ]7 Vsomatic cells into induced pluripotent
" S- _5 [# j/ a, g% |stem (iPS) cells using just four factors:
$ O' {' q2 Y7 y" ^6 TOct4, Sox2, Klf4, and c-Myc. Their $ B! `; _; W$ I: e7 T
elegant but demanding approach of
3 J6 E( G) Y9 {* o8 R2 Tscreening for a cocktail of factors that 5 A2 X$ p: |5 _% K1 _
could reprogram mouse fbroblasts
. S6 s4 J( s( U1 `2 c: `starting from 24 candidate genes paid
8 {- u, w( x6 _5 i# s+ i+ D3 coff with their detailed description of iPS
4 }1 X0 V$ z5 o1 ^# K! pcells, which are almost indistinguish-
) ?& |# c/ I2 u* uable from mouse ES cells. As with all " H n" H/ m* b- y9 ~6 s
scientifc discoveries, these exciting
6 q I T. m: F+ G3 I! Kfndings had to be reproduced. Sev-# Q" y/ m( G$ L" Q3 G, K V: u
eral studies published this year not
A" h9 P: [& V) x& ^, \( Lonly reproduced but also extended * X9 h2 p4 Q3 _) P3 S
the Takahashi and Yamanaka fndings
$ L h2 G5 Z0 }3 P; A* H) `by demonstrating the pluripotency and
* r( c2 \0 \% P* Q/ p( S) jdifferentiation potential of mouse iPS
: H. L- k( B1 \" j# E& B' a' rcells in rigorous developmental assays ( m. N! m5 y& g* b* N
(Maherali et al., 2007; Okita et al., 2007;
+ F! a7 {' m9 J5 x' {$ `* iWernig et al., 2007).
6 X: g" N) P3 x( gIn their new study, Takahashi, $ M% }5 x- L) z; c1 ~
Yamanaka, and their colleagues
# ?8 V' f" B% C: V3 s7 N/ b4 [* g' C(Takahashi et al., 2007) now translate - J9 D- @5 R8 T: T5 X
their remarkable fndings from mouse / ^5 @/ _$ K" w& R
to human (see Figure 1). They selected ! o" A$ f R8 q
adult human dermal fbroblasts and 3 c6 m9 y+ A" x& K. }- V
two other human fbroblast popula-) Z6 M* E' K! g* X% K9 L
tions (from synovial tissue and neo-& M" [8 w$ {8 t$ X: v
natal foreskin) from different human , R: a# Z6 l1 f& Z% T: c/ J
donors as their reprogramming target - a$ Z* T! a' Q0 s, |4 x
cell populations. They then trans-
! _2 A M0 I, _; iduced the human fbroblast cultures 9 f3 L3 j! A( ^ |; X: n1 A, T
with retroviral vectors carrying trans-
$ V0 m7 a; c* f8 Egenes for the human versions of Oct4,
: v+ o, j) K6 f XSox2, Klf4, and c-Myc and cultured " g! L2 w) c; w/ n
the cells under human ES cell culture 2 r; S J0 W2 [5 x I5 V+ _& ^
conditions. Thirty days after transduc-# b1 T9 N. c9 x+ Z/ H8 ^3 N. E
tion, the culture plates were covered
3 {" |- k2 A/ {3 d0 s; Vwith human ES cell-like iPS colonies
& I- ?' x& P! q: J9 A7 H" U(among other colonies), which could
$ }( z. S, ]! A7 }& M* Rbe further propagated and expanded. # z2 _: O4 Z; m& Y8 u2 J, |
The retroviral vectors enabled silenc-
& A( } n4 ~! G Ming of all four transgenes after human
& t5 C B# J3 o$ I. S3 P: b5 ?; BiPS formation (as found in the mouse
6 E' z( k7 E) O+ I% P# gsystem) indicating that the iPS cells 2 m- z& i/ h# y0 W- \& R6 z/ I2 S
are fully reprogrammed and no longer
* O' m% s6 B( ` i- g3 cdepend on transgene expression.1 L1 E/ h% N( l9 @5 Q- \
Unlike the mouse study, human - q1 B7 z E* E+ Q
iPS cells were generated without any
! i" P/ O. u) P# S+ H) Egenetic selection procedures. Given ' W$ Q* K: ^* T( Q( N8 f: b
the lower mitotic index of human ES 2 ]0 x6 q0 D+ b5 A9 }
cells, it is not surprising that the gen-
% b' f7 K: ^: t$ ~' f) Jeration of human iPS cells takes nota-
6 }0 m, L+ u. x/ E- S, }1 c+ Mbly longer than in the mouse system.
, i6 V4 h( @1 i& sThe authors subjected their human . e9 P' d9 Q0 {- @; P7 p/ F" \* \% V& C
iPS cells to a panel of assays to com-
2 D4 G; A6 P4 G2 f) q$ opare them with human ES cells. These * O9 n4 k- e- k' x" l9 j
assays included morphological stud-: N$ z* T! p6 U6 m! J5 |4 I
ies, surface-marker expression, epi-
- X; M' n/ ?% z& M% d, o0 _5 \' P; Ngenetic status, formation of embryoid
1 p. E& O4 q2 M. p' C# N$ vbodies in vitro, directed differentia-
, p# w4 `; a! `% w( u" Rtion into neural cells and beating car-" v- ~0 b/ `6 s' x% I) {- j
diomyocytes (according to human & i3 }2 n b2 n0 \+ |
ES cell differentiation protocols), and & a D- V. a% z% k, q" W. B
fnally teratoma formation in vivo. . \& D2 p2 L" i- V: K9 l4 }
DNA microarray analysis revealed # l8 Z! |+ x: J$ [/ T& W
the remarkable degree of similar-
0 k- T l0 ^. [ity between the global gene expres-$ E, a+ ~* R4 `$ ?5 e
sion patterns of human iPS cells and + }3 D8 t' T+ ~1 N/ d
human ES cells. Notably, genomic
" Y, z( k9 L6 C2 _* D8 \DNA analysis as well as analysis of 8 s" \7 {& E' S: ?( h: J4 l. X
short tandem repeats demonstrated ) S8 O$ f( d+ s7 w6 n4 o
the genetic origin of independent
- e9 E' o( Q& n+ _7 G( e- z5 nhuman iPS clones from their parental ; j! I0 |9 r! A' m
fbroblast populations.# G/ k$ J& E+ h* u8 z2 l2 D1 s
The derivation of mouse and then
4 |( n' W- E0 Mhuman ES cells (Thomson et al., 1998) ) |& `4 ^' y% ^; B9 E* z
as the gold standard of pluripotent ) v \: s2 h$ C8 O" _
stem cell populations has necessarily ( {3 A- f# u ?
led to emphasis on differences in the
: Y( x$ {, ^7 H l7 k# lregulation of self-renewal between ( u* S& x! V1 B8 i
mouse and human ES cells. For 3 P0 b# p1 W# {' v& U5 X
example, human ES cells depend on
" K; X5 C5 e- O* FbFGF for self-renewal, whereas their 0 c) C/ |9 v; {. n) c; }
mouse counterparts depend on the
' g/ @. I$ ~' W. |+ V# f! fLif/Stat3 pathway; BMP is involved in
* Y8 N% Y) F0 ` f1 ~mouse ES cell self-renewal, whereas " k J3 F# r: v C' N/ H
in human ES cells it induces differen-
& A3 v) ?' p! g1 R. f* Atiation. Extrinsic factors and signals 7 a! \; B- |8 a+ b, [) `7 X' S
for maintaining pluripotency may dif-9 C9 N' U3 A. l: f
fer between mouse and human. How-3 B9 U, W# J6 I% P
ever, the ability to translate somatic # E* h8 S+ Y1 ]) s& ~! D/ H
cell reprogramming from mouse to
- R% `# r0 R, C$ E! K* ^, f/ ~human using the same transcription
5 L+ e# t, {9 D O3 k) afactor quartet further emphasizes the ) P& ~; k5 V* n7 Q' W+ Q% s
conserved nature of the Oct4/Sox2
$ E4 \1 g7 U9 d' v9 \4 e3 X# _& Ktranscription factor network that 2 u$ ~' J; P' ]2 B, f' O
controls self-renewal of mouse and
1 f, m3 I. W4 v( ihuman ES cells (Boyer et al., 2005).
! G% x8 Z9 h+ `# _$ PGiven that Klf4 and c-Myc are chro-8 [; E) L6 ]- F
matin modifers and can immortal-1 t3 S! Q9 n0 E+ L
ize cells, one might be able to fnd % L( _7 Q9 z( g. o
other factors or small molecules that
( E* S C( r. I0 o1 G! tcould replace these two factors in the
( V+ w: y4 b3 _cocktail (Yamanaka, 2007). In these ! }# j8 L! X0 F. k' m
studies, the possibility of retroviral * B3 L6 a$ c: W/ \& U$ S
insertional mutagenesis, resulting
2 A6 s! R& z7 E4 uin the activation of other genes con-
2 h9 `8 {9 `" Z9 o6 dtributing to reprogramming, cannot
# S* n" z; Q; ]9 G* q1 p. Pbe excluded, providing an opportu-+ b" g/ m- g" _" o; e
nity to potentially identify new repro-! b5 c' ?& @! F3 \0 R# ]8 v
gramming factors beyond the cur-3 J( b% [( u1 {! P
rent quartet. Also, taking a broader + e- t0 }. S% h3 G* G+ O
screening approach for reprogram-
+ T6 x( H5 `, q' v$ w! l/ t1 ^ming human fbroblasts (as Takahashi
1 F+ [; _$ V, e" L7 Rand Yamanaka did for their mouse
/ C4 ~/ T4 V7 `& Vstudy) might yield other combinations 4 [; a! q. [/ @* h* p/ z
of reprogramming factors.% _+ y1 a2 n2 d1 z
Direct reprogramming of somatic
: j. V5 \& c) L( O! Ycells to a pluripotent state, thus revers-
8 Z6 l& m: \ B) Ming the developmental arrow of time, # r: j" |* R% C9 B) @( @& z+ p
is considered by some to be the “holy - r9 P% B/ N% B' {( S
grail” of stem cell research. Once the 1 x$ R) x6 P) a, ]2 Q
results in human cells are confrmed,
! G! Q D7 `$ l6 `4 Q# x7 t2 rthese advances will enable the cre-
0 q" \2 D9 [8 s( \# |4 a& Sation of patient-specifc stem cell lines
3 w8 p. t5 q, a; {& _to study different disease mechanisms + @# E1 o, G1 A1 S! o* A+ W9 Y
in the laboratory. Such cellular models
( w; @# I% H3 ], Z+ xalso have the potential to dramatically
* H- L1 f* B; tincrease the effciency of drug discov-$ r/ l! B& V/ [, S+ P3 ^) U
ery and to provide valuable tools for ; y# _2 _) K, a& z3 c( q L
toxicology testing. Furthermore, this 6 C7 \/ @9 p# D. d3 Q$ X
reprogramming system could make
0 J3 c- q. r" ethe idea of customized patient-specifc 5 k7 }1 B" i; _7 D7 M) l
screening and therapy both possible
+ w- N% C- ~" x5 |- r6 r/ q4 Eand economically feasible. Finally, the * o7 Z4 {3 T/ F2 l
work will have a powerful impact on 4 v8 S2 H' \; [+ ]; |8 B6 |# ?) O3 h
the intense debate regarding the moral, 0 h P. m7 W' v. u. K7 D2 n- H
religious, and political aspects of ES cell ; k4 O. g$ W, T
research. However, a big mistake now ^9 V$ E5 L0 y- x
would be to consider human ES cells + L3 J' c+ J& B9 D' t
obsolete. There are still many hurdles 6 P: |6 Y, @7 I' H% ?$ [" E+ u! x
to overcome before we ful ly understand $ ?& U1 ]# a% j+ u3 ^3 z% @
pluripotency and before we have human
A# i. s5 S# ~iPS cells in hand that are suitable for
) D/ T) u. D5 ?" c0 e3 H; d6 qtherapeutic application. For example,
. H. E* V) g- a$ k. G0 fa signifcant proportion of mice derived
# w: b) [$ s$ J4 q1 Z8 Ofrom mouse iPS cells develop tumors ( ~* O+ ~9 }% t) |( J
due to reactivation of the c-Myc retro-
- P, u2 R9 g+ T! {" L: W @virus (Okita et al., 2007) compared to
( C9 w( a. |$ H+ m& P8 Y+ a& rmice derived from ES cells, which are
2 z+ @$ h2 F# rnormal. The search is now on to fnd a
* o6 g |: G# Z0 nway to reprogram somatic cells without # d1 K0 F* v n
retroviruses and maybe even using a
) o" P7 C5 f( p/ N1 b1 }8 Z. Ecocktail of small molecules. Given this,
' V- d) _- `8 x0 P1 X9 H% Qit should be emphasized that human
6 C, q, E4 P! e' |ES cell research is more important than
' @' y% ^( M- i3 X" I# |ever for it will shed light on how iPS
4 m6 U- @: U% M, N7 S4 @cells can best be maintained in their
! s0 f+ k9 \, Z0 d0 F1 y" f' apluripotent state and how they can be
. B1 E/ W: W. l! K2 Dinduced to differentiate into the cell
6 t9 a# b, _4 p! }, T3 Alineage of interest. The feld of nuclear
' g+ o) |, ~' n4 A0 z& k4 c8 _reprogramming has come a long way $ {" [, ]9 U4 a
from the initial nuclear transplantation $ _# w N8 {% @1 C) @. C! H- G# F
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/ e( J* b0 k# k2 s2 _1 q/ x, Lbirth of Dolly, the frst mammal cloned
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1997), to the fallout from the fabricated
5 |$ A, u8 p! D1 @9 h& J( L( Hhuman nuclear transfer experiments & f+ n& i. e6 }/ R6 h# S9 I
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* E' R- f/ f0 \( Estudies of Takahashi, Yamanaka, and 6 M7 y- z4 ?. I. i o( t
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发表于 2009-4-28 21:35
发表于 2009-5-16 10:41
