LETTERS
Pluripotentstemcellsinducedfromadultneuralstemcells by reprogramming with two factors
Jeong Beom Kim
1
*
, Holm Zaehres
1
*
, Guangming Wu
1
, Luca Gentile
1
, Kinarm Ko
1
, Vittorio Sebastiano
1
,Marcos J. Arau´zo-Bravo
1
, David Ruau
2
, Dong Wook Han
1
, Martin Zenke
2
& Hans R. Scho¨ler
1
Reprogramming of somatic cells is a valuable tool to understandthe mechanisms of regaining pluripotency and further opens upthe possibility of generating patient-specific pluripotent stemcells. Reprogramming of mouse and human somatic cells intopluripotent stem cells, designated as induced pluripotent stem(iPS) cells, has been possible with the expression of the transcrip-tion factor quartet Oct4 (also known as Pou5f1), Sox2, c-Myc andKlf4 (refs 1–11). Considering that ectopic expression of c-Myccauses tumorigenicity in offspring
2
and that retroviruses them-selves can cause insertional mutagenesis, the generation of iPScells with a minimal number of factors may hasten the clinicalapplication of this approach. Here we show that adult mouseneural stem cells express higher endogenous levels of Sox2 andc-Myc than embryonic stem cells, and that exogenous Oct4togetherwitheitherKlf4orc-MycissufficienttogenerateiPScellsfrom neural stem cells. These two-factor iPS cells are similar toembryonicstemcellsatthemolecularlevel,contributetodevelop-ment of the germ line, and form chimaeras. We propose that, ininducing pluripotency, the number of reprogramming factors canbe reduced when using somatic cells that endogenously expressappropriate levels of complementing factors.
Mouse and human somatic cells can be reprogrammed into iPScells by the expression of a defined set of factors (
Oct4
,
Sox2
,
c-Myc
and
Klf4
, as well as
Nanog
and human
LIN28
)
1–11
. It is possible togenerate iPS cells from mouse and human fibroblasts in the absenceof
c-Myc
retrovirus
6,7
,andthereforeitwassuggestedthatendogenousexpression ofc-Myccouldhavearoleinthereprogrammingprocess.Neural stem cells (NSCs) endogenously express Sox2, which may function in maintaining the stemness and multipotency of NSCs
12,13
, and Sox2 was suggested in maintaining cellular pluripo-tencybyregulatingtheexpressionofOct4(ref.14).NSCswereestab-lished from adult Oct4–GFP (OG2)/ROSA26 heterozygoustransgenic mouse brains
15–17
, expressing green fluorescent protein(GFP) under the control of the
Oct4
promoter (Oct4–GFP) andthe
Escherichia coli lacZ
transgene from the constitutive
ROSA26
locus (
ROSA26 lacZ
). The established NSCs had the capacity forself-renewal and multipotency (Supplementary Fig. 1).Compared to embryonic stem cells (ESCs), expression of
Sox2
wasapproximately twofold higher in NSCs.
c-Myc
and
Klf4
were alsoexpressed at levels about tenfold higher and eightfold lower inNSCs than in ESCs, respectively (Fig. 1a). Western blot analysesshowed that the relationship between protein and RNA levels inNSCs corresponded to that in ESCs for Oct4, Sox2 and Klf4; thec-Myc protein level was comparable in NSCs and ESCs (Fig. 1b).In this study, we attempted to reprogram NSCs into iPS cells by introducing different combinations of the four factors
Oct4
,
Sox2
,
c-Myc
and
Klf4
(Supplementary Table 1) using the retroviral MX vector system. We assessed the ability of 15 different transcriptionfactorcombinationstoinduceOct4–GFP-positivecolonyformation.Sixcombinationswereabletoinducethegeneration ofiPScellsfromNSCs, as judged by the formation of GFP
1
colonies and the estab-lishment of iPS cell lines. We observed GFP
1
cells 4days after trans-ductionwiththecombinationcontainingallfourfactors—thatis,thecontrolcombination—andnotedagradualincreaseinthenumberof GFP
1
colonies during the first 2 weeks post-infection(Supplementary Fig. 2a). We established four-factor iPS cells fromGFP
1
ESC-likecoloniesonday14.Thesefour-factoriPScellsstainedpositive for stage-specific embryonic antigen-1 (SSEA-1) and alkal-ine phosphatase (Supplementary Fig. 2b), showed messenger RNAexpression patterns similar to those in ESCs (Fig. 2a, Supplementary Fig. 2c), and led to teratoma formation on injection into nude mice(Supplementary Fig. 2d). Our results demonstrate that NSCs can bereprogrammed into iPS cells by the four transcription factors: Oct4,Sox2, c-Myc and Klf4.Three different combinations of three factors were also capable of generating iPS cells from NSCs:
Oct4
,
Klf4
and
c-Myc
(OKM);
Oct4
,
Klf4
and
Sox2
(OKS); and
Oct4
,
c-Myc
and
Sox2
(OMS;Supplementary Table 1). We did not observe GFP
1
colonies forthe three-factor combinations that did not include Oct4. GFP
1
col-onies were observed 1week after transduction with the OKM com-bination (without
Sox2
). However, GFP
1
colony formation wasobserved only after 14–21days with the OKS combination (without
c-Myc
), and was even more delayed with the OMS combination(without
Klf4
; Supplementary Table 1). Nonetheless, these OKM,OKS and OMS three-factor iPS cells had similar gene expressionprofilestoESCs(SupplementaryFig.3a),andalltypesofthree-factoriPS cells differentiated into all three germ layers (Supplementary Fig.3b). Taken together, these results indicate that three-factor iPS cellscould be generated in the absence of
Sox2
,
Klf4
or
c-Myc
retrovirusesin NSCs, which endogenously express these three factors.We next assessed the ability of two-factor combinations to inducethe generation of iPS cells from NSCs. Only two combinations weresuccessful in reprogramming NSCs. We first observed GFP
1
coloniesinNSCculturesinfectedwith
Oct4
and
Klf4
(OK)and1–2weekslaterin those infected with
Oct4
and
c-Myc
(OM; Supplementary Table 1).The two-factor OM iPS cells showed an ESC-like gene expressionpattern and contributed to the three germ layers in teratomas(Supplementary Fig. 3a, b). Klf4 and c-Myc may exert similar func-tions.ItisknownthatKlf4hasaroleintheinactivationofthe
p53
(alsoknownas
Trp53
)tumour-suppressorgene,whichleadstocellimmor-talization; Klf4 also works in conjunction with the RAS
V12
oncogenicsignal transduction protein to stimulate cellular proliferation
18,19
.
*
These authors contributed equally to this work.
1
Department of Cell and Developmental Biology, Max Planck Institute for Molecular Biomedicine, Ro¨ntgenstrasse 20, 48149 Mu¨nster, NRW, Germany.
2
Institute for BiomedicalEngineering, Department of Cell Biology, RWTH Aachen University Medical School, Pauwelsstrasse 30, 52074 Aachen, NRW, Germany.
1
©2008
Macmillan Publishers Limited. All rights reserved
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