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Induction of Pluripotency: " V% y$ T5 m5 M1 Q" [) V
From Mouse to Human
) g5 O( l3 O" E$ E* S3 G3 |9 WHolm Zaehres1% v. ~- m4 m, i
and Hans R. Schöler1,1 X, e' O% P: }$ q8 f q
*8 w* [8 N3 f- j
1
& r, B" C- z* [6 W& j( `) `4 M: MMax Planck Institute for Molecular Biomedicine, Department of Cell and Developmental Biology, Münster, NRW 48149, Germany
5 V5 N1 Y: }3 Z3 ~! e( B/ R*Correspondence: schoeler@mpi-muenster.mpg.de0 S7 w( |6 C$ l) l9 j
DOI 10.1016/j.cell.2007.11.020( J1 d: V+ A( _, P) J7 A0 y
In this issue of Cell, Takahashi et al. (2007) transfer their seminal work on somatic cell
5 J% p$ P" X! O6 Preprogramming from the mouse to human. By overexpressing the transcription factor
+ u6 v4 U2 X# \$ Q" hquartet of Oct4, Sox2, Klf4, and c-Myc in adult human fbroblasts, they successfully
2 _ @$ [% T/ J4 r2 F) a) v; ^isolate human pluripotent stem cells that resemble human embryonic stem cells by all
3 R" D3 x, E: L3 L. N- i( tmeasured criteria. This is a signifcant turning point in nuclear reprogramming research - L5 Q8 V7 @9 F% ~% Q0 ]* B
with broad implications for generating patient-specifc pluripotent stem cells for research $ N) [8 X, v& _+ _' o; n7 a
and therapeutic applications.% P# a' z0 j9 L) u
This year’s three Physiology or Medi-6 O5 w' L3 I6 F& \# X5 D
cine Nobel Laureates—Martin Evans,
9 A0 N' U# x; y3 `. A; V2 g( _Mario Capecchi, and Oliver Smithies—- R# z' A$ @/ i1 h6 E1 D7 m1 U
will be honored in Stockholm in 10
K. Z( t4 t7 T% hdays time for their discovery of DNA 5 M6 i) ?5 v+ ]6 A- a
recombination and the development
7 a5 c$ `6 p8 I. |of mouse embryonic stem (ES) cell , Q. K% N* y' B3 z9 @- y
technology. It was Martin Evans who % s! ~0 y2 z& \' |" c: T# K% L
discovered how to make mouse ES " Y5 T; \7 F+ w& p8 _$ ^
cells, enabling any genetic alteration
% B4 T$ a: K! k e0 Nto be transferred to the germline and " k, c6 t8 N$ e6 M" a# @
hence to the next generation (Evans
9 x0 L0 b7 V* U) Zand Kaufman, 1981; Martin, 1981).
! O1 e& Z, ~ `+ E1 QBefore this breakthrough, researchers
- n9 B! X. M9 }studied mouse embryonal carcinoma / N) v6 v$ g; k& c/ K+ N! N/ T
cells derived from tumors, which + J c* V7 @: c% h& z
could form every mouse cell lineage
1 I, |! D' l# @7 g4 q+ hexcept the germline. Combining DNA 9 e8 X" Y1 ?6 U+ O+ g2 Q
recombination and mouse ES cell " x# G. g& t) B/ ~$ O
technology revolutionized an entire 6 g, l5 j$ a- r
feld of research, forming the basis for
$ `& f! \! g$ J$ n/ [studying and understanding the roles 2 d' ~: G M/ O4 C4 X
of numerous genes in embryonic
8 Z: }) R: Q& }4 k' J* Y2 w7 |& ?development, adult physiology, dis-7 R; t. s8 g; M% G+ l
ease, and aging. To date, more than
- `. k- @$ a# [$ X1 T) K1 U500 mouse models of human disor-/ M7 i7 D% ^0 G1 p& u" F
ders have been generated. Now, with
5 C* ?" a0 {& \$ ?1 a* Kthe study by Takahashi et al. (2007) , z+ I9 [9 H. d9 G3 Q
published in this issue of Cell, another
; H" y! h+ G" R0 M" U7 I8 ?important revolution is taking place.9 V4 p1 K# j5 f
Last summer, Takahashi and 9 d( M m% N2 [0 P0 m
Yamanaka (2006) stunned the scientifc 0 A6 I: A- p' M( q" c
community with their study showing 7 T$ P& n( I; d; Y+ E! v
molecular reprogramming of mouse 5 L" n- `3 A. g' I
somatic cells into induced pluripotent
! W) r- W+ j' o0 c' mstem (iPS) cells using just four factors: % u) J8 [7 W9 o7 E
Oct4, Sox2, Klf4, and c-Myc. Their 7 w& d- `7 S: Y) \
elegant but demanding approach of & S- R" J2 K% k4 d. Q
screening for a cocktail of factors that
1 N: z8 g# e; W6 |$ K4 Kcould reprogram mouse fbroblasts / g1 o( E% {1 e0 }( `8 @
starting from 24 candidate genes paid
1 S# A& Y& i9 n) Z/ ?off with their detailed description of iPS 6 a; c5 y8 i3 Q0 e) b# z) z) A
cells, which are almost indistinguish-
& X4 `! t1 i, n+ M8 c6 mable from mouse ES cells. As with all # H1 @- t- ~4 A
scientifc discoveries, these exciting 6 N' M/ a. I2 [1 V. B2 M( c
fndings had to be reproduced. Sev-. ?- Y0 J4 w. w. q9 d
eral studies published this year not
9 y! Y% b# Q8 p' b, f. T* b3 Wonly reproduced but also extended
4 ]1 e6 p; w2 c6 kthe Takahashi and Yamanaka fndings
8 M$ n; ], J5 k. M, L+ g/ xby demonstrating the pluripotency and
, l+ A$ `5 a2 `; t5 \differentiation potential of mouse iPS ( j: K4 f- ]( q4 ~; i8 n
cells in rigorous developmental assays 6 `1 l! b0 L- R) `9 Y' m
(Maherali et al., 2007; Okita et al., 2007;
- p- E, e1 O6 ^% k) @Wernig et al., 2007)." Q. `2 U" d+ h8 k. I6 n
In their new study, Takahashi, 1 p9 I- s* Y: o- w
Yamanaka, and their colleagues $ V0 t. |# B7 S. R/ [
(Takahashi et al., 2007) now translate
# y' ]) V: B0 ?; ~) @0 ztheir remarkable fndings from mouse 5 p: Z0 X3 B& _" A/ X# |
to human (see Figure 1). They selected
3 ]! z) l7 ?$ e' G$ Q& Yadult human dermal fbroblasts and 0 q+ n: c0 ?* L$ s5 y4 J
two other human fbroblast popula-
2 p$ |- ~5 d# a1 `% g6 wtions (from synovial tissue and neo-
: r0 t6 {% b& {. o" X D2 n: { }natal foreskin) from different human
, { x1 ?; i6 Rdonors as their reprogramming target ( U9 j" l3 m' ]
cell populations. They then trans-* L! r2 K5 n& _2 J5 c" B% k$ c
duced the human fbroblast cultures ! o$ B9 u# A* o$ u. h
with retroviral vectors carrying trans-% ?1 F9 }6 \% c8 T3 z0 a
genes for the human versions of Oct4,
9 t+ p1 K" c M2 L* R1 |2 MSox2, Klf4, and c-Myc and cultured
) m( j) f9 f7 Pthe cells under human ES cell culture 6 f( D6 w1 Q: J
conditions. Thirty days after transduc-
1 l) s) G% r( ~( B$ _7 O# Jtion, the culture plates were covered 8 M" J) U5 f, @
with human ES cell-like iPS colonies
0 G: z/ q9 W# a. x! R( @6 A(among other colonies), which could / k% Y1 k9 Z8 O6 m9 o8 R$ O
be further propagated and expanded.
6 {. {) V ^' q! P" o. \- X& ~0 ^- p" jThe retroviral vectors enabled silenc-# N/ S& L% [' f$ X
ing of all four transgenes after human
9 o+ c _+ z5 t! wiPS formation (as found in the mouse 3 b* s& g, c3 \/ F. Y" r- B
system) indicating that the iPS cells
4 n; f9 g! r' [1 }0 z# eare fully reprogrammed and no longer / q- I2 \+ V/ L( M1 J' {
depend on transgene expression.0 u. Z1 Z" k1 ^0 W. C
Unlike the mouse study, human
, T3 p' `% j, }1 KiPS cells were generated without any 3 f) X: b8 @8 {
genetic selection procedures. Given ) y6 t5 P9 v4 a/ f6 {: X
the lower mitotic index of human ES 7 V" d4 T* U. x, [3 J6 D
cells, it is not surprising that the gen-1 `6 Y1 E% h: C* `! G, k! b1 C+ Q% W6 ?
eration of human iPS cells takes nota-; `# y" @! ?5 ?
bly longer than in the mouse system.
; @% N2 t" s+ I! GThe authors subjected their human
/ P0 k9 o7 F. B8 n9 qiPS cells to a panel of assays to com-
- p+ g( B ~( M$ `% L3 t) X: cpare them with human ES cells. These 3 B. l7 l, q/ w( h' d
assays included morphological stud-
6 n# C' m2 t0 a. r6 ]ies, surface-marker expression, epi-
% V7 p/ @, E$ X1 u: W9 ugenetic status, formation of embryoid ; T" E2 Y! K+ ?- ?2 Q
bodies in vitro, directed differentia-
6 [: x% l' ^( r% Ltion into neural cells and beating car-
! X/ w, k6 a/ S6 c9 f. |6 K( Qdiomyocytes (according to human % d- q% ^0 U$ L+ f: r7 U0 O' {
ES cell differentiation protocols), and
; `9 }& g: ?1 [( l2 T# j$ V/ [fnally teratoma formation in vivo.
% ^! H8 x+ z2 q& A9 C6 PDNA microarray analysis revealed
1 @. O" r$ T' o! z( y+ X2 Xthe remarkable degree of similar-3 V1 e; e; l! r) Y4 ~
ity between the global gene expres- }5 V9 e& E, N% f
sion patterns of human iPS cells and # X/ {$ h ?4 F! O* ^# H
human ES cells. Notably, genomic ; [ s: N6 k* Z5 V* W3 i1 U
DNA analysis as well as analysis of ; ~2 ?" ]9 ^8 X, L/ R
short tandem repeats demonstrated ' d5 V) z% u' f. C9 c
the genetic origin of independent
1 ~3 Z/ m" H- m. R$ shuman iPS clones from their parental $ D' A$ ^& D V, N y4 D3 ~
fbroblast populations.8 G' ^2 D+ J+ I, C3 R, ]
The derivation of mouse and then
/ l5 K% [" {% R t' f% m6 |human ES cells (Thomson et al., 1998) ! [" `) O$ ~! _" j9 G7 S( g
as the gold standard of pluripotent ) E2 v' Z' G8 g" U
stem cell populations has necessarily ! s9 _5 n' G# a' u1 ~" [. E
led to emphasis on differences in the
) E" X i" V1 ^' P1 e2 v7 Gregulation of self-renewal between
7 v9 k/ B& c" G0 a# r/ G7 Z! }mouse and human ES cells. For
2 _! c' P D- f' ~) r: f& @3 Rexample, human ES cells depend on
: U$ D) R, Z$ X* C' w4 DbFGF for self-renewal, whereas their
4 S# z& _3 q7 P$ f2 y$ @3 [mouse counterparts depend on the 9 R+ I' a7 V$ \
Lif/Stat3 pathway; BMP is involved in
2 l r, ~3 R3 d# @1 G! vmouse ES cell self-renewal, whereas 9 ~3 E* p; ~& C" F
in human ES cells it induces differen-
( @# n; }2 {7 e* a9 M' o M: E" |tiation. Extrinsic factors and signals
" ~- Z& K5 Q/ z9 i: Z0 [for maintaining pluripotency may dif-9 V |- E1 A' h6 \9 [% J& G
fer between mouse and human. How-
5 [$ u* z! l3 L- W; u, `ever, the ability to translate somatic
2 {: B! |! v5 z3 A1 N. f# jcell reprogramming from mouse to 8 U9 q& K( L. r
human using the same transcription
0 W. X# [% z0 j0 e& R% Gfactor quartet further emphasizes the # U0 S' C3 r0 L
conserved nature of the Oct4/Sox2 # q, X/ E7 n; T6 O' x% s$ Z/ w
transcription factor network that
2 ~, o$ n" u: j) ^, x( Vcontrols self-renewal of mouse and * \! R5 N$ C5 v& I7 w W, g" m
human ES cells (Boyer et al., 2005).
( `2 O7 U6 X, q2 A! sGiven that Klf4 and c-Myc are chro-8 }% _0 C* o3 ?# F4 K5 b# @
matin modifers and can immortal-
' Q1 h0 L# X# y# _: {6 W/ T z/ K* aize cells, one might be able to fnd + Z+ ]0 A* G! T5 c# t. N F
other factors or small molecules that . M; z& i. W& e7 V( t0 `( b
could replace these two factors in the - [7 H, D% I! x# n! b
cocktail (Yamanaka, 2007). In these . Z3 F; g4 o( l
studies, the possibility of retroviral ( ~- F7 P" Q6 |
insertional mutagenesis, resulting
# {- w6 T7 p+ C9 V/ l0 f, din the activation of other genes con-$ a0 l5 L0 k9 L! d- j
tributing to reprogramming, cannot
+ w; T6 ~, k# D! ^* v" w! [8 Tbe excluded, providing an opportu-
# Z. d3 [) C# {8 Y) n `. s+ b# tnity to potentially identify new repro-5 F; X3 v" Z( Q" |: P! ?
gramming factors beyond the cur-
1 ?- f2 m# H2 g# N4 }8 O* srent quartet. Also, taking a broader - W5 Z/ S6 D. }/ I# ^
screening approach for reprogram- {6 z+ M; @3 T' r0 s! a+ z% y& \
ming human fbroblasts (as Takahashi 4 v4 T P- x) u1 ^8 i. W
and Yamanaka did for their mouse
6 G$ I- ]" e( z1 v; o lstudy) might yield other combinations 3 Y4 ?- |! L6 N( E% a/ M2 X
of reprogramming factors.
" r# R; P, W4 ^5 d VDirect reprogramming of somatic
! {/ r9 b5 N( Z; pcells to a pluripotent state, thus revers-
! i! ~# K9 {. O$ g. r- q& F% Ning the developmental arrow of time, + Y. a6 e+ o6 T0 @0 J# ?3 V: U
is considered by some to be the “holy ) l8 h# I1 G n* x
grail” of stem cell research. Once the ' K$ ^9 M% E) h0 K$ m
results in human cells are confrmed, 8 e) |+ ?$ q. B t/ h+ ~, Y! g
these advances will enable the cre-5 H! k$ \" K# p
ation of patient-specifc stem cell lines
0 j6 ?9 X! C6 ?8 t- B# ]2 c# kto study different disease mechanisms
8 |0 i# |$ t6 s2 Y, ~8 j, win the laboratory. Such cellular models / S4 M# |! E. R* O
also have the potential to dramatically ( q9 _6 X( W, o* {; n
increase the effciency of drug discov-# @- t* p% H/ \) \. R, U
ery and to provide valuable tools for
+ l- `- ~8 A2 {1 R4 C9 }: v5 C0 Xtoxicology testing. Furthermore, this * D, ]. b/ R, @, O# t7 r$ R
reprogramming system could make ! n6 y$ S! v3 u$ b
the idea of customized patient-specifc
) P Q; H6 W# \; w uscreening and therapy both possible + U5 I! P. C* m7 u# l; ]9 V
and economically feasible. Finally, the / B! U' p$ _& g, w0 z
work will have a powerful impact on 4 X8 n) f9 Z! p; U" Q. Q: c
the intense debate regarding the moral,
; R0 ~ D" }& N# g/ e: |# o+ {7 ^religious, and political aspects of ES cell
7 {) X9 F& W& q* o0 t4 fresearch. However, a big mistake now 2 m9 {, X8 {6 r* k2 B4 X) D
would be to consider human ES cells
% j. N( s( J3 Tobsolete. There are still many hurdles + d6 v/ p. u/ g4 K7 j
to overcome before we ful ly understand 9 r/ m1 q0 B$ w. ]
pluripotency and before we have human
9 F4 P/ K$ N9 u7 U% z6 [' I8 fiPS cells in hand that are suitable for
' T/ z& \" V8 W4 Y* o' t4 l" Ltherapeutic application. For example,
% M) G' e) j6 |a signifcant proportion of mice derived
/ {; R$ U/ i: z) e' ?3 f4 r. Vfrom mouse iPS cells develop tumors
5 j* w9 n) i1 U, C6 [due to reactivation of the c-Myc retro-
. X/ t( r7 K- s, E' x: [. Vvirus (Okita et al., 2007) compared to
9 {* b1 A- j' N: j( Q: w( Z. p& x1 d2 fmice derived from ES cells, which are 9 M& l! ]* @% n. `/ \6 K! ~
normal. The search is now on to fnd a
! B! E) T6 A$ n; X8 b6 yway to reprogram somatic cells without * w$ H4 D9 V. j' L
retroviruses and maybe even using a $ _; H" I. W" P/ o$ {8 a
cocktail of small molecules. Given this,
- C4 L4 \' l' E4 u! fit should be emphasized that human
) T5 Q9 N: ?- K! X: Q4 VES cell research is more important than 2 q0 T' J! E+ K* A2 k4 H! [9 ]
ever for it will shed light on how iPS ) B5 m: T8 S; W: P( j+ x
cells can best be maintained in their 6 R4 R) x) b) n* S
pluripotent state and how they can be
2 Y$ w# k, B8 B* [induced to differentiate into the cell
- w/ b' a: P# N; M5 x; Glineage of interest. The feld of nuclear 3 u+ |4 A; D4 r4 q6 I
reprogramming has come a long way / j: M" T( h2 B6 ^
from the initial nuclear transplantation
5 T1 |8 A& G8 q1 V& Istudies in frogs 50 years ago, to the 9 l, P2 z \# i: ~
birth of Dolly, the frst mammal cloned
' J' S, p4 i5 \! z% P2 {; Y) ^from adult somatic cells (Wilmut et al., , U/ A7 p! U( V0 o9 L! m$ [. P
1997), to the fallout from the fabricated
1 S' O6 H5 V3 z9 z( Ihuman nuclear transfer experiments 3 _6 Y0 d' l8 ~
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发表于 2009-4-28 21:35
发表于 2009-5-16 10:41
