nature cell biology - mir-34 mirnas provide a barrier for somatic cell reprogramming

8/12/2019 Nature Cell Biology - MiR-34 MiRNAs Provide a Barrier for Somatic Cell Reprogramming

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miR-34 miRNAs provide a barrier for somatic cell

reprogrammingYong Jin Choi1,7, Chao-Po Lin1,7, Jaclyn J. Ho1, Xingyue He2, Nobuhiro Okada1, Pengcheng Bu1, Yingchao Zhong1,Sang Yong Kim3, Margaux J. Bennett1, Caifu Chen4, Arzu Ozturk5, Geoffrey G. Hicks5, Greg J. Hannon6

and Lin He1,8

Somatic reprogramming induced by defined transcription

factors is a low-efficiency process that is enhanced by p53

deficiency15. So far, p21 is the only p53 target shown to

contribute to p53 repression of iPSC (induced pluripotent stem

cell) generation1,3, indicating that additional p53 targets may

regulate this process. Here, we demonstrate that miR-34

microRNAs (miRNAs), particularly miR-34a, exhibit

p53-dependent induction during reprogramming. Mir34a

deficiency in mice significantly increased reprogramming

efficiency and kinetics, with miR-34a and p21 cooperatively

regulating somatic reprogramming downstream of p53. Unlike

p53 deficiency, which enhances reprogramming at the expense

of iPSC pluripotency, genetic ablation of Mir34apromoted

iPSC generation without compromising self-renewal or

differentiation. Suppression of reprogramming by miR-34a wasdue, at least in part, to repression of pluripotency genes,

includingNanog, Sox2 andMycn(also known as N-Myc). This

post-transcriptional gene repression by miR-34a also regulated

iPSC differentiation kinetics. miR-34b and c similarly

repressed reprogramming; and all three miR-34 miRNAs acted

cooperatively in this process. Taken together, our findings

identified miR-34 miRNAs as p53 targets that play an

essential role in restraining somatic reprogramming.

Differentiated somatic cells can be induced to generate pluripotent

stem cells that functionally resemble embryonic stem cells (ESCs;

ref. 6). This reprogramming process is rooted in the remarkable cellular

plasticity retained during differentiation. The process can be triggered

by exogenous expression of a set of defined ESC-specific transcription

factors, Pou5f1 (also known as Oct4), Sox2, Klf4 and c-Myc (refs69),

which constitute the core regulatory circuits controlling pluripotency

and self-renewal. Enforced expression of these reprogramming factors

1Division of Cellular and Developmental Biology, Molecular and Cell Biology Department, University of California at Berkeley, Berkeley, California 94705, USA. 2 40

Landsdowne Street, Cambridge, Massachusetts 02139, USA. 3 Transgenetic facility, 500 Sunnyside Blvd., Woodbury, New York 11797-2924, USA. 4 Genomic Assays

R&D, Life Technologies Corporation, 850 Lincoln Centre Dr., Foster City, California 94404, USA. 5 Manitoba Institute of Cell Biology, 675 McDermot Ave. Rm.

ON5029, Winnipeg, MB R3E 0V9, Canada. 6 Cold Spring Harbor Laboratory, Watson School of Biological Sciences, 1 Bungtown Road, Cold Spring Harbor, New York

11724, USA. 7 These authors contributed equally to this work.8Correspondence should be addressed to L.H. (e-mail:[email protected])

Received 10 May 2011; accepted 22 September 2011; published online 23 October 2011; DOI: 10.1038/ncb2366

generates iPSCs with low efficiency and slow kinetics, suggesting the

existence of cellular and molecularbarriersto theprocess10.

Recent studies have revealed considerable mechanistic overlap

between somatic cell reprogramming and malignant transformation11.

Cellular mechanisms that enhance reprogramming, including cell

proliferation and survival, evasion of DNA-damage response, and cell

immortalization, have long been known to promote tumorigenesis35,12.

Several oncogenes and tumour suppressors also serve as essential

regulators for reprogramming6,10,11. Notably, the inactivation of p53,

one of the most important tumour suppressors, significantly enhances

iPSC generation15,13. As a tumour suppressor, p53s transcriptional

regulation converges onto multiple target genes that collectively

mediate its downstream effects, including cell-cycle arrest, cellular

senescence, apoptosis, DNA-damage response and genomic stability14.

Similarly, p53s role in repressing reprogramming is probably alsomediated through multiple targets. To date, the cell-cycle regulator

p21 is the only p53 target with a demonstrated role in repressing

reprogramming. However,p21deficiency only partially phenocopies

that ofp53(refs1,3,15), suggesting that p53 represses iPSC generation

through as-yetunidentified mechanism(s) and target(s).

Previous studies have identified the miR-34 microRNAs (miRNAs)

as bona fide p53 transcriptional targets, whose overexpression

triggers cell-cycle arrest or apoptosis in a cell-type- and context-

dependent manner1618. miRNAs, a large family of small non-coding

RNAs, primarily repress gene expression post-transcriptionally, by

pairing with partially complementary messenger RNA targets19,20.

In response to p53 activation, inducedMir34miRNAs can mediate

p53 downstream effects by repressing specific target genes, including

cyclin D1, cyclin E2, Cdk4, Cdk6, Bcl2 and c-Met (ref. 21).

Although p53 is primarily characterized for its role in transcriptional

activation, it acts as a global gene regulator that both activates

and represses gene expression14. Direct transcriptional repression,

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together with indirect post-transcriptional repression through miRNAs

such as miR-34, constitute two major mechanisms for p53-

mediated gene repression.

The miR-34 miRNAs belong to an evolutionarily conserved family,

with three mammalian homologues, miR-34a, b and c, localized to two

distinct genomic loci,Mir34aandMir34b/c(Fig. 1a; ref.16). All three

Mir34miRNAs were significantly induced when mouse embryonic

fibroblast (MEF) reprogramming was triggered with Sox2, Oct4 and

Klf4 in the presence or absence of c-Myc (Fig. 2a, data not shown). In

both cases,Mir34induction, similarly to that ofp21, was dependent on

the activation of intact p53 (Fig. 2a). We therefore investigated whether

miR-34 miRNAs are components of the reprogramming regulatory

circuit downstream of p53. Among all miR-34 miRNAs, miR-34a

exhibited the highest induction level during reprogramming, whereas

miR-34b was the lowest. We therefore focused initially on the role of

Mir34ain iPSC induction.

We generatedMir34aknockout mice using C57BL/6 ESCs(Fig. 1a),

confirmed the germline transmission of the targeted allele (Fig. 1b)

and verified the genetic ablation ofMir34a by expression studies

(Fig. 1c). Mir34a/

mice were born at the expected Mendelianratio, without obvious developmental or pathological abnormalities

up to 12 months. However, when we induced the Mir34a/

MEFs for reprogramming, we observed a significant increase in the

reprogramming efficiency(Fig. 2b,d and Supplementary Fig. S1a).

Three-factor-infectedMir34a/ MEFs exhibited a 4.5-fold increase

in alkaline phosphatase-positive colonies with typical iPSC morphology

(Fig. 2b), whereas four-factor-infected Mir34a/ MEFs yielded a

4-fold increase (Supplementary Fig. S1a). Furthermore, when we

plated infected MEFs into 96-well plates at a density of one cell

per well, Mir34a deficiency caused a similar increase in alkaline

phosphatase-positive colonies with typical iPSC morphology(Fig. 2c).

Using MEFs carrying an Oct4Gfp knockin reporter allele22, we

confirmed the effect of miR-34a in promoting reprogramming, scoring

fully reprogrammed iPSCs on the basis of endogenousOct4expression

indicated by green fluorescent protein (GFP). Consistently, a greater

than fourfold increase in reprogramming efficiency was observed in

Mir34a/; Oct4Gfp/+MEFs after three- or four-factor transduction

(Fig. 2d). A significant increase in reprogramed Oct4Gfp-positive

colonies could also be achieved using a locked nucleic acid (LNA)

inhibitor against miR-34a (Supplementary Fig. S1b). Notably, Mir34a

deficiency both enhanced the overall efficiency of iPSC generation and

led to more rapid reprogramming kinetics. Small iPSC-like colonies

first appeared 7 days post-infection in four-factor-transduced wild-type

MEFs, but as early as post-infection day 5 inMir34a/ MEFs.

As miR-34b and c share the miR-34a seed sequence and are similarlyinduced during reprogramming(Fig. 2a), they probably also regulate

iPSC generation. We generatedMir34b/cknockout mice (Fig. 1ac). As

with miR-34a deficiency,Mir34b/cknockout alone promoted somatic

reprogramming, although to a lesser degree (Fig. 2e). Interestingly,

MEFs deficient for all three miR-34 miRNAs exhibited an even

greater increase in iPSC generation(Fig. 2e), suggesting a cooperative

effect among these genes, although the exact molecular and cellular

mechanisms underlying this cooperation still remained unclear. As the

Mir34a/;Mir34b/c/ MEFs did not completely phenocopyp53/

MEFs, extra mechanisms may act downstream of p53 to mediate the

suppression of reprogramming.

Previous studies have identified p21 as an important mediator

of p53 suppression of reprogramming1,12. Mir34 and p21 both

exhibitedp53-dependent induction during reprogramming (Fig. 2a).

p21 inductionalso wasobservedinMir34a/ MEFs(Fig. 3a). Whereas

MEFs deficient for Mir34aalone or p21 alone showed comparable

increases in iPSC generation, MEFs deficient for both Mir34aandp21

exhibited a cooperative increase that recapitulated a significant fraction

of the p53 effect (Fig. 3c). Given the functional similarities among

the three miR-34 miRNAs, the cooperative effects among p21 and all

miR-34 miRNAs could be even greater. Thus, the miR-34 miRNAs,

together with p21, constitute important downstream effectors of p53

to mediate the repression of reprogramming.

p21 represses reprogramming efficiency primarily through its

repression of cell proliferation12. Although miR-34a and p21 repress

reprogramming efficiency similarly (Fig. 3c), their effects on cell

proliferation are quite different. The increased cell proliferation in

p21/ MEFs was highly significant (Fig. 3b), yet within the time

frame of our reprogramming experimentsMir34a/ MEFs exhibited

little increase up to passage 6. We cannot completely exclude the

possibility that a mild increase in Mir34a/

MEF proliferationcontributed to the increased reprogramming. Yet, unlike p21, whose

effects on reprogramming are largely attributed to its inhibition

of cell proliferation12, miR-34a is likely to act mostly through a

separate mechanism independent of cell proliferation. These two

distinct mechanisms may underlie the cooperative regulation of

reprogramming by miR-34a and p21.

Mir34a/ iPSCs resemble wild-type iPSCs and ESCs, exhibiting

ESC-like morphology (Fig. 4a and Supplementary Fig. S1c) and

expressing key molecular markers for pluripotency. High levels of Oct4,

Nanog and SSEA1 were detected (Fig. 4b and Supplementary Fig. S1d).

Mir34a/ iPSCs injected into immunocompromised nude mice

yielded differentiated teratomas, containing terminally differentiated

cell types from all three germ layers (Fig. 4c,d and Supplementary

Fig. S1e). Furthermore, three independent lines of four-factor-induced

Mir34a/ iPSCs all yielded healthy adult chimaeric mice with a high

percentage of iPSC contribution (Fig. 4e and Supplementary Fig. S1g,

Table S1). Taken together,Mir34adeficiency enhances the efficiency of

iPSC generation without compromising self-renewal or pluripotency.

Although p53deficiency induced reprogramming more efficiently

than Mir34a deficiency, the p53/ iPSCs exhibited compromised

self-renewal and differentiation capacity1,4. As also reported in

ref. 1, we have observed that four-factor-induced p53/ iPSCs

lost ESC-like morphology after five to six passages in culture,

and failed to generate highly differentiated teratomas. In contrast,

four-factor-induced Mir34a/ iPSCs remained stable beyondpassage 26, with no significant differences in self-renewal when

compared with wild-type iPSCs(Supplementary Fig. S1f).Furthermore,

generation of healthy adult chimaeras from p53/ iPSCs was

difficult. The percentage of p53/ iPSC contribution was low,

and the majority of such chimaeras succumbed to tumorigenesis

before 7 weeks1. In contrast,Mir34a/ iPSCs exhibited functional

pluripotency: all three four-factor-induced Mir34a/ iPSC lines

tested gave rise to healthy adult chimaeras with a high percentage

of iPSC contribution (Fig. 4e and Supplementary S1g, Table S1),

which remained tumour free for 6 months (to the time of

manuscript preparation).

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: loxP

E P

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robe

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robe

3p

robe

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robe

5probe 3probe

3probeMir34a E

ELacZ

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E P

Kozaksequence : FRT E : EcoRI P : PsiI X : XhoI

X E

M : MfeI S : SpeI

: loxP

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b

c

FRT

Figure 1Generation ofMir34aand Mir34b/cknockout MEFs. (a) Diagrams

of endogenousMir34aand Mir34b/cgene structure (WT) and the knockout

construct (). Using recombineering, we engineered the Mir34atargeting

vector with a 6-kilobase (kb) homologous arm on both 5 and 3

ends, flanking a Kozak sequence, a lacZ complementary DNA and an

FRTNeoFRT cassette. The Mir34b/c targeting vector (Neo) contains

a 6-kb homologous arm at each end, with the Mir34b/c gene and a

Neo selection cassette flanked by loxP sites. (b) Validating the germline

transmission of the Mir34a and Mir34b/c targeted allele using Southern

analysis. Putative wild-type, Mir 34a+/ and Mir 34a/ animals derived

from three independently targeted ESC lines were analysed by Southern

blotting using probes either 5 or 3 to the homologous arms (left). Similar

validation was carried out for wild-type, Mir 34b/c/+ and Mir 34b/c+/

animals (right). (c) Confirming loss of miR-34 expression in Mir34a and

Mir34b/cknockout MEFs. Littermate-controlled wild-type,Mir 34a+/ and

Mir 34a/ MEFs were analysed by real-timePCR to quantify theexpression

of Mir34a. Whereas wild-type MEFs showed robust miR-34a induction

on culture stress, no miR-34a expression was detected in Mir 34a/

MEFs. The miR-34a level in Mir 34a+/ MEFs was approximately half

that of wild-type MEFs. Similar validation was carried out for Mir 34b/c/

MEFs. Error bar, s.d., n= 3. Uncropped images of blots are shown in

Supplementary Fig. S6.

Consistent with these differences, we observed that four-factor-

induced p53/ iPSCs, but not Mir34a/ or wild-type iPSCs,

failed to silence retroviral transgenes, thus exhibiting lower levels

of the corresponding endogenous genes (Supplementary Fig. S3ac).

The exogenous expression of reprogramming factors, particularly

c-Myc (Supplementary Fig. S3e), may contribute to the impaired

self-renewal and differentiation (Supplementary Fig. S3d), and

promote tumorigenesis1. In contrast, pluripotency was established

easily in Mir34a/ iPSCs by the endogenous transcription factor

circuitry, leaving the exogenous transgenes dispensable for the

maintenance of pluripotency.

Similar toMir34a/ iPSCs,Mir34a/;Mir34b/c/ iPSCs also

exhibited robust self-renewal and differentiation capacity, with typical

ESC morphology, key pluripotency markers and the ability to generate

differentiated teratomas (Supplementary Fig. S2a

c). Their ability togenerate chimaeras remains to be determined.

The increased reprogramming efficiency and compromised pluripo-

tency ofp53/ MEFs may result from aberrant apoptosis and DNA

damage response4. However,Mir34adeficiency failed to protect cells

from apoptosis or DNA-damage response during reprogramming

(Supplementary Fig. S4ac). Thus, the enhanced reprogramming effi-

ciency observed inMir34a/ MEFs may reflect a mechanism largely

independent of apoptosis and DNA damage response. These findings

contrast with the ability ofMir34aoverexpression to trigger apoptosis

in specific tumour cells17,18. These Mir34a effects on apoptosis are

probably cell-type dependent, differing between primary fibroblasts16

and specific cancer cell lines17,18. Cells deficient forMir34amay exhibit

apoptotic defects under conditions other than reprogramming. It is

also possible that the lack of apoptotic protection in reprogramming

Mir34a/ MEFs reflectsthe functional redundancy ofmiR-34b andc.

Enhanced reprogramming in p53-deficient cells has also been at-

tributed to cell immortalization and increased cell proliferation15,12,15.

Whereas cell immortalization greatly promotes iPSC generation from

p53/ MEFs (ref.5), increased cell proliferation is a key mechanism

driving stochastic reprogramming ofp53KD B cells12. AlthoughMir34

overexpression induced growth arrest and cellular senescence in pri-

mary fibroblasts16, no defects in senescence response were observed in

Mir34a/ MEFs during reprogramming (Supplementary Fig. S4d and

data not shown). As described above, we did not observe a significant

MEF proliferation difference caused byMir34adeletion within the

time frame of our reprogramming experiment. Presumably, miR-34aregulates reprogramming efficiency largely through a mechanism

independent of cell proliferation and cell senescence.

To better define the molecular mechanism of miR-34a in reprogram-

ming, we initiated a search for its targets, specifically by examining

genes that promotes the iPSC generation for possible miR-34a-binding

sites. RNA22 identified a number of such genes with predicted miR-34a

sites23 (Supplementary Fig. S5d). Of all the genes tested,Nanog,Sox2

andMycn(N-Myc) emerged as top candidates(Fig. 5a and Supplemen-

tary Fig. S5a). All three exhibitedMir34a-dependent repression (Fig. 5b

and Supplementary Fig. S5c), and each had potent effects in promoting

reprogramming24,25. ESCs over-expressing miR-34a, miR-34b or

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WT

WT

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p53/

2,500 infected

cells per well2,500infectedcells perwell

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P< 0.01 P< 0.01

NumberofAP-positivecolonies


WT 34a/ p53/


P< 0.05

P< 0.005

P< 0.0001

n= 3

n= 3

n= 3

pri-miR-34b/c

Foldupregulation

Mature miR-34b

WT WT34

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pri-miR-34a

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miR-34a p21

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ositivecolonies

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c/

0

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Phase Oct4Gfp

Mir34a/

Uninfected Infected Uninfected Infected Uninfected Infected

P< 0.01 P< 0.02

Figure 2Deficiency of miR-34 miRNAs increases reprogramming efficiency.

(a) Four reprogramming factors triggered p53-dependent induction of

miR-34 miRNAs. Three days after transduction, pri-miR-34a (the precursor

transcript from the Mir34alocus), mature miR-34a and p21 were measuredin uninfected and four-factor-induced wild-type (WT) and p 53

/MEFs.

Induction of pri-miR-34a was dependent on the intact p53 response, and was

comparable to that of p21. Induction of pri-miR-34b/c and mature miR-34b

and miR-34c was determined in wild-type, Mir 34a/ andMir 34b/c/

MEFs. Error bar, s.d., n=3. (b) Mir34adeficiency significantly enhanced

three-factor-induced MEF reprogramming. 2,500 three-factor-infected

wild-type,Mir 34a/ orp 53/ MEFs were plated to score reprogramming

by alkaline phosphatase (AP)-positive colonies with characteristic ESC

morphology. A representative image and quantitative analysis is shown

out of five independent experiments, comparing littermate-controlled

wild-type and Mir 34a/ MEFs (left, P< 0.01), as well as wild-type and

p53/

MEFs (right, P< 0.01). Error bar, s.d., n=4. (c) Single-sorted,

four-factor-infected MEFs were cultured at a density of one cell per well.

Four weeks post-plating, alkaline phosphatase-positive colonies with typical

iPSC morphology were scored for wild-type, Mir 34a/ and p53/

iPSCs. Four independent experiments confirmed this finding. P< 0.05

for comparison between wild-type and Mir34a/ MEFs. Error bar, s.e.m.,

n= experiments with independent MEF lines. (d) Mir34adeficiencysignificantly enhanced MEF reprogramming as measured by Oct4Gfp

reporter expression. Three- or four-factor-infected wild-type and Mir 34a/

MEFs that carry an Oct4Gfpallele were sorted at the density of 2,500 cells

per well and 1,000 cells per well, respectively. Reprogramming efficiency

was quantified by GFP-positive clones. Images of Oct4Gfp-positive iPSCs

are shown on the left. A quantitative analysis for reprogramming efficiency

triggered by four factors (left, P< 0.01) or three factors (right, P< 0.02)

is shown. Scale bar, 100 m. Error bar, s.d., n=3. OSKM, Oct4, Sox2, Klf4

and Myc; OSK, Oct4, Sox2 and Klf4. (e) miR-34a, b and c cooperatively

regulate somatic reprogramming. Deficiency in Mir34a or Mir34b/calone

significantly promoted somatic reprogramming, yet deficiency in all Mir34

miRNAs exhibited further increase. Two independent experiments confirmed

this finding. n, experiments with independent MEFs. All P-values were

calculated on the basis of a two-tailed Students t-test.



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Tuj1/DAPI SMA/DAPI Foxa2/DAPI

Oct4 DAPI SSEA1 DAPI

Wild type

Wildtype

Wildtype

M

ir34a

/

Mir34a

/

Mir34a/

Mir34a/

Oct4/DAPI SSEA1/DAPI

Epidermis Cart ilage Cil liated epithelium

Neural rosette Bone-like Gut-like epithelium

Ectoderm Mesoderm Endoderm Ectoderm Mesoderm Endoderm

Epidermis Cartilage Gut-l ike epithel ium

Hair foll icle Muscle Cil liated epithelium

Tuj1/DAPI SMA/DAPI Foxa2/DAPI

Wild type

a e

b

c

d

Figure 4 Mir34a/ iPSCs functionally resemble wild-type iPSCs. (a) iPSCs

derived from both wild-type and Mir 34a/ MEFs exhibited ESC-like

morphology in culture, with robust alkaline phosphatase expression.

Scale bar, 20m for the left panel, 100 m for the middle and right

panels. (b) Both wild-type and Mir 34a/ iPSCs expressed pluripotency

markers, including nucleus-localized Oct4 and membrane-localized SSEA1.

Scale bar, 20 m. (c,d) Wild-type and Mir 34a/ iPSCs both generated

differentiated teratomas. Teratomas derived from four-factor-inducedwild-type (left) and Mir 34a/ (right) iPSCs were collected from nude

mice 46 weeks after subcutaneous injection. Haematoxylin and eosin

staining (c), as well as immunofluorescence staining (d), revealed

terminally differentiated cell types derived from all three germ layers.

Scale bar in c, 25 m; in d, 50 m. (e) Four-factor-induced Mir 34a/

iPSCs efficiently contribute to adult chimaeric mice. We injected three

independent lines of passage 7 Oct4Gfp/+, Mir 34a/ iPSCs into

albino-C57BL/6/cBrd/cBrd/cr blastocysts. The iPSC contribution to adult

chimaeric mice was determined by coat colour pigmentation. DAPI,4,6-diamidino-2-phenylindole.

global gene expression31. Along with transcription factors, miRNAs

have emerged as essential gene regulators in self-renewal and

differentiation of pluripotent stem cells32,33. ESCs with deficient

global miRNA biogenesis exhibit proliferation and differentiation

defects34. In addition, the RNA-binding protein LIN28, which

induces reprogramming by suppressing let-7 biogenesis8,3537, has been

identified as a major reprogramming factor in humans. Beside let-7

miRNAs, a number of miRNAs are specifically enriched or depleted in

pluripotent stem cells and are demonstrated to regulate self-renewal

and differentiation30,32,34,3840. Here, we identify the miR-34 miRNAs as

regulators that suppress reprogramming downstream of p53, providing

direct evidence that modulation of miRNA abundance could constitute

a critical step in establishing pluripotency. As miRNA functions

can be manipulated by oligonucleotide-based inhibitors or mimics,

our findings suggest a paradigm for promoting reprogramming by

manipulating specific miRNA functions.

Although p53 directly regulates hundreds of target genes14, miR-34

miRNAs and p21 are the main downstream targets that cooperatively



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a

c

d

e

b

No. 1 No. 2 No. 3 No. 1 No. 2 No. 3

OSKM OSK

No. 1 No. 2 No. 3 No. 1 No. 2 No. 3 No. 1 No. 2 No. 3

0.3

Wild type Mir34a/;

Mir34b/c/

OSK

0.16

43

43

34

72

43

34

55

43

34

72

43

34

55

43

34

34

55

Mr(K)

Mr(K) Mr(K)

Wildtype

Wildtype

Mir34a

/

LIF feeder(day 2)Untreated

Wild type

Wild type

Mir34a/

Mir34a/

(Days) (Days)

Nanog Oct4 Sox2

Mir34a

/

Rela

tivemRNAlevels

(Days) (Days)

RelativemRNAlevels

Rela

tivemRNAlevels

Relat

ivemRNAlevels

RelativemRNAlevels

RelativemRNAlevels

Nanog

UGUUGGUCGAU-----UCUGUGA

AGACACU--CCAUC

UUC

GGUC

Sox2

UG UG UCGAUUCU GACGGU

UU AU AGCUGAGA UUGCCA

GUU

U

G

A AU

miR-34a

N-Myc

UC GUGACGGU

CCA CGG AG CACUGCCA

UGU GUCUG

AUUG

CACUGUAGCUG

miR-34a

miR-34a

AC UCA

Oct4

Tub

Nanog

Sox2

N-Myc

0.04 0.05 0.02 0.06

1 0.93

1.12 0.84

0.95

0.99 0.95 1.05 1.10 1.06 1.14

1.08 0.98 1.85 1.57 1.78

1.09 1.07 3.54 2.98 3.17 0.87

0.96 1.03 1.01 1.52 1.40

1.16

0.96 1.05 0 .981 .20 1.18 1.16

1.07 0.76 1.74 1.70 1.72

1.46

0 .85 1.32 2 .97 2.95 3.06

1.16 1.11 0.73 1.79 2.04 1.93

0.93

1.03 0.99 0.98 1.06 1.11 1.03

0.97 0.920.961.10 1.04

1.07 0.80 2.01 2.45 1.28

0.53 0.59 0.49

1 0.97 0.70 0.53 0.61

1 0.87 0.95 0.90 0.83

1 1.11 1.06 1.08 1.21

Nanog

Oct4

Tub

Sox2

Wild type Mir34a/ Mir34a/Wild type

No. 1 No. 2 No. 3

Nanog

Oct4

Tub

Sox2

N-Myc

0.03

0.02 0.03

0.12 0.01

0.010.03

Mock

GFPsiR

NA

miR

-34a

miR

-34b

miR

-34c

0.29 0.14 0.04 0.01

0.05 0.07 0.00 0.06

0.04 0.11 0.03 0.04

0.07

0.040.05

0.14 0.04

0.03 0.02

0.09

0.09 0.20.09

0 1 2 3

(Days)

40

0.2

0.4

0.6

0.8

1.0

0 1 2 3 0

0.4

0.8

1.2

1.6

0 1 2 3 40

0.5

1.0

1.5

0

0.2

0.4

0.6

0.8

1.0

0 1 2 3

(Days)

0 1 2 30

0.3

0.6

0.9

1.2

1.5

0

0.2

0.4

0.6

0.8

1.0

0 1 2 3

LIF feeder + RA

(day 2)Untreated

Nanog Oct4 Sox2

1 1.05 0.62 0.45 0.53

0.94 0.99 1.07 1.16 1.11 1.13

1.00 1.03 0.97 1.13 1.08 1.15

Figure 5 Mir34a represses Nanog, Sox2 and N-Myc expression

post-transcriptionally. (a) Schematic representation of the Nanog, Sox2and N-Myc 3UTR, and the predicted miR-34 binding sites. The mouse

Nanog, Sox2 and N-Myc each contains one putative miR-34a-binding

site within its 3UTR. (b) Enforced expression of Mir34a, Mir34b

and Mir34c in ESCs reduced the protein levels of Nanog, Sox2 and

N-Myc, but not Oct4. Feeder-free ESCs were transfected with miRNA

mimics for miR-34a, miR-34b and miR-34c, and a negative control,

Gfp short interfering RNA (siRNA). At 48 h post transfection, western

analysis indicated a significant reduction in the protein levels of Nanog,

Sox2 and N-Myc, but not Oct4. The value of each band indicates the

relative expression level normalized by the internal control, -tubulin,

averaged between two independent experiments, and presented as mean

s.e.m. (c) Derepression of Nanog, Sox2 and N-Myc was observed in

Mir34-deficient iPSCs. A significant increase of Nanog and Sox2, but

not Oct4, was observed in four-factor-induced Mir 34a/ iPSCs, when

compared with passage-matched, littermate-controlled wild-type iPSCs. A

similar comparison was made for passage-matched, three-factor-inducedwild-type and Mir 34a/; Mir 34b/c/ double-knockout iPSCs, where

derepression of Nanog, Sox2 and N-Myc was observed. For this western

analysis, the quantitation of each band was carried out by Quantity One

software, and was normalized against its own internal tubulin control. The

s.e.m. of three independent iPSC lines are shown for each genotype. ( d,e)

Mir34a-deficient iPSCs exhibited slower kinetics during differentiation.

Wild-type and Mir 34a/ iPSCs were both triggered to differentiate

by withdrawal of LIF in the absence (d) or presence (e) of retinoic

acid treatment. Images of a typical iPSC culture two days after each

differentiation condition are shown on the left, and quantitative analyses

of the decline of Nanog, Sox2 and Oct4 transcripts in response to these

differentiating conditions are shown on the right. Error bar, s.e.m., n=3.P


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L E T T E R S

repress iPSC generation. Previous studies indicate that p53 effects on

reprogramming reflect changes in cell proliferation, immortalization,

apoptosis and DNA-damage response4,5,12. p21, a key downstream

target of p53, represses cell proliferation. Interestingly, Mir34a

deficiency alters MEF reprogramming not through cell proliferation

but, at least in part, by posttranscriptional derepression of pluripotency

genes. Thus, miR-34 miRNAs, together with other p53 targets,

including p21, collectively mediate the suppression of somatic

reprogramming through multiple critical cellular pathways.

METHODS

Methods and any associated references are available in the online

version of the paper athttp://www.nature.com/naturecellbiology

Note: Supplementary Information is available on the Nature Cell Biology website

ACKNOWLEDGEMENTS

We thank B. Zaghi, A. Basila, A. Perez, G. Lai, M. Foth, A. Kemp, A. Fresnoza,

A. Valeros, A. Fritz, D. Schichnes and H. Noller for technical assistance and

A. Economides for discussions and input. We also thank J. M. Halbleib, D. Schaffer

andR. M.Harland forreading themanuscripts, andI. Lemischka, Q. Li,M. Hemann

and B. Vogelstein for sharing reagents. In particular, we are grateful for the

pathological expertise R. Van Andel offered us. L.H. is a Searle Scholar, and is

supported by a new faculty award by California Institute for Regenerative Medicine(RN2-00923-1) and an R01 (R01 CA139067) and an R00 grant (R00 CA126186)

from the National Cancer Institute (NCI). G.J.H. is a Howard Hughes Medical

Institute investigator, and is supported by a program project grant from the NCI.

C-P.L. is supported by a Siebel postdoctoral fellowship.

AUTHOR CONTRIBUTIONS

Y.J.C., C-P.L. and L.H. designed all experiments and carried out the majority of

the experiments shown in all figures and supplementary figures. J.J.H. and Y.Z.

carried out immunofluorescence analyses and teratoma analyses to characterize

the pluripotency of the iPSCs. L.H., X.H., P.B. and G.J.H. generated knockout

constructs for Mir34a and Mir34b/c, and identified the correctly targeted ESC

clones forMir34a. N.O. and P.B. identified the correctly targeted ESC clone for

Mir34b/c, validated Mir34a and Mir34b/ctargeting in mice by Southern blotting

and generated Mir34a/, Mir34b/c/ andMir34 triple knockout MEFs. S.Y.K.

and G.J.H. carried out the blastocyst injection for Mir34a

+/

and Mir34b/c

+/

ESC clones. A.O., S.Y.K. and G.G.H. characterized the pluripotency of iPSCs using

chimaera assays. M.J.B. and C.C. contributed to the identification of miR-34a

targets.

COMPETING FINANCIAL INTERESTS

The authors declare no competing financial interests.

Published online at http://www.nature.com/naturecellbiology

Reprints and permissions information is available online at http://www.nature.com/

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DOI: 10.1038/ncb2366 M E T H O D S

METHODSAnimals.Using recombineering, we engineered the Mir34a knockout constructwith a 6-kb homologous arm on both the 5 and 3 ends, which flanked a Kozak

sequence, a LacZ cDNA and an FRTNeoFRT cassette. The Mir34b/c targeting

vector contains a 6-kb homologous arm at each end, with the Mir34b/c gene

and a Neo selection cassette flanked byloxPsites. The deleted region forMir34a

knockoutcontainedtheMir34a pre-miRNAand 20-base pair(bp) flankingsequenceat each end. The deleted region for Mir34b/c knockout contained Mir34b and c

pre-miRNA with a 200-bp flanking sequence at each end. To facilitate homologous

recombination, theMir34aandMir34b/cknockout constructs were electroporatedinto Bruce4 ESCs and v6.5 ESCs, respectively. 300 Neo-resistant ESC colonies for

each knockout construct were screened for homologous recombination. Correctly

targeted ESCs were validated with Southern analysis using both 5 and 3 probes.

Three independently targeted ESC clones for Mir34a and two for Mir34b/c were

injected into blastocysts, all of which gave rise to chimaeric animals able to produce

germline transmission.

Oct4Gfpmice were acquired from Jackson Laboratory (catalogue no. 008214),

and maintained on a mixed C57BL/6 and 129S4Sv/Jae genetic background. The

ACTFLP (catalogue no. 005703) and the p21/ mice (catalogue no.003263)

were purchased from the Jackson Laboratory. Mir34a, p53 and p21 knockout

mice were bred and maintained on the isogenic C57BL/6J background to generate

littermate-controlled MEFs.NCr-nu/nufemale athymic mice were purchased fromTaconic (catalogue no. NCRNU).

Cell culture.Primary MEFs were isolated from littermate-controlled E13.5

embryos. MEFs and ecotropic Phoenix cells were cultured in DMEM (Invitrogen,catalogue no. 11995-073) with 10% fetal bovine serum and 1% 100 penicillin

and streptomycin (Invitrogen, catalogue no. 15140-163). iPSCs were cultured onto

irradiated MEF feeder layers in the ESC medium, which contained Knockout

DMEM (Invitrogen, catalogue no. 10829-018), 15% ES-grade fetal bovine serum

(Invitrogen, catalogue no. 16141079), 2 mM l-glutamine (Invitrogen, catalogue no.

25030-164), 1104 M MEM non-essential amino acids (Invitrogen, catalogue no.

11140-076), 1104 M 2-mercaptoethanol (Sigma, catalogue no. M3148) and 1%

100 penicillin and streptomycin. Feeder-free ESCs, provided by I. Lemischka,were

cultured in the same ESC medium on gelatin-coated plates 41. Dicer/ HCT116

cells, provided by B. Vogelstein, were cultured in McCoy 5A medium (Invitrogen,catalogue no. 16600082) with 10% fetal bovine serum and 1% 100 penicillin and

streptomycin42.

iPSC induction and reprogramming efficiency. Ecotropic phoenix cells weretransfected with the pMX retroviral vectors encoding mouse Oct4, Sox2, c-Myc

and Klf4 (Addgene, catalogue nos 13366, 13367, 13375 and 13370) by the calcium

phosphate transfection method43. One day before infection, MEFs were seeded at1.3105 cells per well in a six-well plate. MEFs were infected twice, and cultured for

48 h before we sorted 1,000four-factor-infectedMEFs or 2,500three-factor-infected

MEFs into each well ofa 12-wellplate.Oncethe iPSC-likecoloniesappeared,alkaline

phosphatase staining or Oct4 staining was carried out to evaluate reprogramming

efficiency. To establish stable iPSC lines, single iPSC-like colonies were each picked

into one well of a 96-well plate, before expanding on irradiated MEF feeders. To

determine reprogramming efficiency usingOct4Gfp/+ MEFs, we collected 1,000

four-factor-infectedMEFs or 2,500 three-factor-infectedMEFs. The reprogrammediPSC colonies were scored by GFP expression.

To determine if serial transfection of miR-34a LNA inhibitor promotes somatic

reprogramming, we transfected miR-34a LNA oligo (Exiqon, catalogue no. 41840-

00) and a control LNA oligo (Exiqon, catalogue no. 199004-00) into Oct4Gfp/+MEFsevery 6 daysduring the course of somatic reprogramming.The reprogrammed

colonies were scored by GFP-positive clones with characteristic ESC morphology.

Alkaline phosphatase and immunofluorescent staining.Alkaline phosphatasestaining was carried out using an Alkaline Phosphatase Detection Kit (Millipore,

catalogue no. SCR004) according to the manufacturers protocol. For immunocyto-

chemical staining, iPSCs were fixed with 4% paraformaldehyde and incubated with

blocking solution (0.1%Triton X-100 and5% normalgoat serumin PBS) for30 min

at room temperature. To detect the expression of pluripotency markers, cells were

stained with antibody against Oct3/4 (1:100, Santa Cruz Biotechnology, catalogue

no. sc-5279), SSEA1 (1:200, Santa Cruz Biotechnology, catalogue no. sc-21702) andNanog (1:100, Abcam, ab80892). To measure DNA-damage response, wild-type,

Mir34a/ andp53/ MEFs infected with four reprogramming factors were col-

lectedon days 3,6 and9 post-infection.After beingfixedwith4% paraformaldehyde,

cells were stained with antibody against -H2AX (1:200, Millipore, catalogue no.

05-636).

Teratoma formation and chimaera generation. 1 106 wild-type, Mir34a/

orMir34a/ Mir34b/c/ iPSCs were injected into dorsal flanks of 67-week-old

immune-deficient nu/nu mice. Resulting teratomas were fixed in 10% formalin,

embedded in paraffin, sectioned to 6 m and stained with haematoxylin and eosin.

To detect differentiated cell types, we stained the cryosection slides with antibody

against Tuj1 (1:100, Abcam,cat.no. ab7751), SMA(1:100, Abcam,cat.no. ab18147)

and Foxa2 (1:500, Abcam, cat. no. ab40874) to detect cell lineages derived from

ectoderm, mesoderm and endoderm, respectively.

To determine the ability ofMir34a/ iPSCs to contribute to adult chimaeras,

we generated Oct4Gfp/+, Mir34a/, Aw/a iPSCs on a mixed C57BL/6and 129S4Sv/Jae genetic background. Three stable Mir34a/ iPSC lines were

established and injected into albino-C57BL/6/cBrd/cBrd/cr blastocysts at passage

7. Chimeric blastocysts were subsequently transferred to day 2.5 pseudopregnantrecipient CD-1 females, and the percentage of chimaera contribution was estimated

by scoring the level of coat colour pigmentation.

iPSC differentiation assay.We triggered differentiation of wild-type andMir34a/ iPSCs with LIF withdrawal in the presence or absence of retinoic acid4,34.

To remove the feeder cells, iPSCs cultured in the complete media with LIF were

trypsinized and plated on 0.1% gelatin-coated plates for 1 h before the collection.

Subsequently, feederless iPSCs were plated on gelatin-coated plates in LIF-free

media. 1M retinoic acid was added to iPSC cultures at day 1. The differentiating

iPSCs were collecteddailyin thecourse of 4 days, andthe expression of pluripotency

genes was analysed by real-time PCR.

Real-time PCR analyses.TaqMan miRNA assays (Applied Biosystems) were usedto measure the level of mature miR-34 miRNAs. The mRNA and pri-miRNA levels

were determined by real-time PCR with SYBR (Kapa Biosystems, catalogue no.

KK4604). The real-time PCR primers used for endogenous and exogenous Oct4,Sox2andKlf4have been described in ref. 44. The primers for total Sox2,Oct4,Klf4

and c-Mychave been describedin ref. 45.The primers forNanoghavebeen described

in ref. 43. The primers for pri-miR-34a, pri-miR-34b/c, p21 and Actin have been

described in ref. 16.

Western analyses. Annealed miR-34a, miR-34b and miR-34c oligos and con-trol siRNA oligos were each transfected into ESCs at a final concentration

of 50 nM using Lipofectamine 2000 (Invitrogen, catalogue no. 11668027). 48 h

post-transfection, ESCs transfected with miR-34a, miR-34b, miR-34a and con-

trol Gfp siRNA, as well as the mock transfected control, were collected intoradioimmunoprecipitation assay buffer, 20 mM Tris at pH 7.5, 150 mM NaCl,

1% Nonidet P-40, 0.5% sodium deoxycholate, 1 mM EDTA and 0.1% SDS with

protease inhibitor cocktail (Roche, catalogue no. 11836153001). Cell lysates were

subjected to western analyses. For iPSC collection, trypsinized iPSCs and feeder

MEFs were both plated on a gelatin-coated plate. After one hour of culture,

feeder MEFs were largely attached to the plates, whereas most iPSCs remained

in the supernatant or lightly attached to the MEF feeders. iPSCs were thenseparated from the feeders, and subjected to western analyses. Antibodies against

mouse Nanog (Abcam, ab80892), Sox2 (Millipore, ab5603), Oct3/4 (Santa Cruz,

sc-5279), N-Myc (Abcam, ab18698), Klf4 (Abcam, ab26648), p21 (Santa Cruz,

sc-6246) and p53 (Vector Laboratories, CM5) were used at 1:1,000 dilution.

-tubulin (Sigma, clone B-5-1-2) was used at a 1:4,000 dilution as a loading

control. The quantitation of all western analysis was carried out with Quantity

One (Bio-Rad).

Proliferation, apoptosis and senescence assay. Cell proliferation was measuredby the cumulative population doublings. For each passage, cells were plated at a

concentration of 6104 cells per well of a 12-well plate. After every two days of

culture, cells were trypsinized, counted and seeded at the same concentration.

To determinethe percentageof apoptoticcells in infectedMEFs, cellswere stained

with APC-annexin V (BD Pharmingen, catalogue no. 559925) in 10 mM HEPES at

pH7.4,140mMNaCl, 2.5 mMCaCl2, then incubatedwith7-AAD(BD Pharmingen,

catalogue no. 550475). The percentages of early-apoptotic cells, late-apoptotic cells

and necrotic cells were determined by FACS analysis.

The senescent cells were determined by senescence-associated -galactosidase

(SA--Gal) staining. Infected MEFs were fixed in 2% (v/v) formaldehyde and

0.2% (w/v) glutaraldehyde, then stained with freshly prepared staining solution

(5mM K3Fe(CN)6; 5mM K4Fe(CN)6; 30 mM citric acid/phosphate buffer, 150 mM

NaCl, 2 mM MgCl2; 1 mgml1 X-Gal at pH 6.0) at 37 C for 1620 h (ref. 46).

The percentages of SA--Gal-positive cells were calculated in three independentrepresentative fields, and the total number of cells counted for each measurement

exceeded 400.

Luciferase assays. The mouse Sox2 3UTR containing one miR-34a-bindingsite was cloned into the PENTR/TOPO-D vector (Invitrogen, catalogue no.

K2400-20, forward primer, 5-CACCGGAGAAGGGGAGAGATTTTCAAAG-3;

reverse primer, 5-TACATGGATTCTCGGCAGCCTGAT-3). Similarly, a fragment

containing the mouseNanog3 UTR and a small portion of its open reading frame

was cloned (forward primer, 5-CACCAACCAAAGGATGAAGTGCAAGCGG-3;

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M E T H O D S DOI: 10.1038/ncb2366

reverse primer, 5-TCAGGAGGCAAAGATAAGTGGGCA-3). Mutagenesis of the

miR-34a-binding sites within these 3UTR fragments was carried out using the

following primers:Nanogforward, 5-ACTGTAGCTGTCTTCGAAGAGGGCGTC-

AGATCTTGTTACG-3;Nanogreverse, 5-TCTGACGCCCTCTTCGAAGACAGC-

TACAGTGTACTTACAT-3;Sox2forward, 5-ATGTCCATTGTTTATGGCGCGC-

CAATATATTTTTCGAGGAAAGGGTTCTTG-3; Sox2reverse, 5-TTCCTCGAA-

AAATATATTGGCGCGCCATAAACAATGGACATTTGATTGCCA-3. Wild-typeand mutated 3UTR constructs were each cloned downstream of a firefly luciferase

(Luc) reporter. Each Luc construct (100 ng) was transfected intoDicer-deficient

HCT116 cells together with aRenillaluciferase construct (10ng) as a normalizationcontrol, and either 50 nM control Gfp siRNA or miR-34a mimics, respectively.

The same experiment was carried out in feeder-free ESCs. The firefly and Renilla

luciferase activity of each transfection was determined by dual luciferase assay

(Promega, catalogue no. E1960) 48 or 72 h post-transfection.

41. Schaniel, C. et al. Smarcc1/Baf155 couples self-renewal gene repression with

changes in chromatin structure in mouse embryonic stem cells. Stem Cells 27,

29792991 (2009).

42. Cummins, J. M.et al. The colorectal microRNAome. Proc. Natl Acad. Sci. USA103,

36873692 (2006).

43. Takahashi, K. & Yamanaka, S. Induction of pluripotent stem cells from

mouse embryonic and adult fibroblast cultures by defined factors. Cell 126,

663676 (2006).

44. Judson, R. L., Babiarz, J. E., Venere, M. & Blelloch, R. Embryonic stem cell-specific

microRNAs promote induced pluripotency.Nat. Biotechnol. 27, 459461 (2009).

45. Brambrink, T. et al. Sequential expression of pluripotency markers during direct

reprogramming of mouse somatic cells. Cell Stem Cell 2, 151159 (2008).

46. Debacq-Chainiaux, F., Erusalimsky, J. D., Campisi, J. & Toussaint, O. Protocols to

detect senescence-associated-galactosidase (SA-betagal) activity, a biomarker of

senescent cells in culture and in vivo. Nat. Protoc. 4, 17981806 (2009).

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S U P P LE M E N T A RY I N F O R M AT I O N

WWW.NATURE.COM/NATURECELLBIOLOGY 1

DOI: 10.1038/ncb2366

Figure S1 mir-34deficiency significantly enhanced reprogramming without

compromising iPSC pluripotency. a.1000 four-factor infected WT and mir-

34a-/-MEFs (left), or WT and p53-/-MEFs (right) were collected by FACS

sorting and plated in each well of a 12 well plate. Alkaline phosphatase-

positive colonies with ESC-like morphology were counted 2 weeks post-

infection. A representative image and quantitative analysis are shown out of

five independent experiments comparing littermate-controlled WT and mir-

34a-/-(left, **P< 0.01), as well as WT andp53-/-MEFs (right, **P< 0.01)

for reprogramming efficiency. Error bar, standard deviation, n=4. b. Inhibition

of miR-34aby LNA promoted somatic reprogramming.Oct4Gfp/+MEFs were

transfected with 100nM miR-34aLNA oligos or a control LNA oligo every 6

days during reprogramming. A representative quantitative analysis was shown

out of two independent experiments. Error bar, standard deviation, n=3.

Scale bar, 20mm. c.Representative images of three- (left) or four- (right)

factor induced iPSCs derived from both WT and mir-34a-/-MEFs. Three

independently derived iPSC lines were shown for each genotype, all of which

exhibited ES-like morphology. OSKM, Oct4, Sox2, Klf4 and c-Myc; OSK, Oct4,

Sox2 and Klf4. Scale bar, 100mm. d.Both WT and mir-34a-/-iPSCs induced

by three reprogramming factors expressed pluripotency markers, exhibiting

nuclear expression of Oct4 and Nanog, as well as membrane expression of

SSEA1. Scale bar, 20mm. e.Three-factor induced WT (left) and mir-34a-/-

iPSCs (right) gave rise to terminally differentiated teratomas. H&E staining

revealed terminally differentiated tissue types from all three germ layers,

including epidermis and neural rosette from ectoderm, cartilage and muscle

from mesoderm, and gut-like epithelia and ciliated epithelia from endoderm.

Scale bar, 25mm. f.Four-factor-inducedOct4-Gfp/+, mir-34a-/-iPSCs can

be maintained in culture for 20 passages without losing the characteristic

morphological features (phase-contrast image, left) and Oct4expression (Oct4-

Gfpexpression, right). Scale bar, 100mm. g.Representative image of chimeric

animals derived from three independent Oct4-Gfp/+, mir-34a-/-iPSC lines.

a

0

40

80

120

160

200

40

60

80

100

WT

mir-34a-/-

1000 infected

cells/well

WT 34a-/-

#1

#2

#1

#2

#ofAPpositivecolonies

#ofAPpositivecolonies

p53-/-

WT p53-/-

#1

#2

#1

#2

WT

1000 infected

cells/well

** P


12/17

S U P P LE M E N T A RY I N F O R M A T I O N

2 WWW.NATURE.COM/NATURECELLBIOLOGY

Figure S2 34a-/-; mir-34b/c-/- iPSCs functionally resemble WT iPSCs.

a.Representative phase-contrast images of three- factor induced

iPSCs derived from mir-34a-/-; mir-34b/c-/- MEFs. Two independently

derived iPSC lines were shown to exhibit ES-like morphology. Scale

bar, 100mm. b. mir-34a-/-; mir-34b/c-/- iPSCs induced by three

reprogramming factors expressed pluripotency markers, including

Oct4 and SSEA1. Scale bar, 20mm. c. Three-factor induced mir-34a-/-;

mir-34b/c-/- iPSCs gave rise to differentiated teratomas. H&E staining

revealed terminally differentiated tissue types from all three germ

layers. Scale bar, 25mm.

mir-34a ; mir-34b/c

a

Line

#1

Line#

2

-/- -/-

b

c

SSEA-1 / DAPISSEA-1DAPI

Oct4 / DAPIOct4DAPI

Cartilage Cilliated EpitheliumNeural Rosette

Ectoderm Mesoderm Endoderm

mir-34a ; mir-34b/c iPSCs-/- -/-

mir-34a;

mir-34b/c-/- -/-


13/17


WWW.NATURE.COM/NATURECELLBIOLOGY 3

Figure S3 Endogenous, exogenous and total levels of reprogramming factors

in four-factor induced iPSCs.RNAs were prepared from WT, mir-34a-/-and

p53-/-MEFs, four-factor induced iPSCs, as well as V6.5 ES cells. The levels of

endogenous (middle), exogenous (right) and total (left) Sox2(a), Klf4 (b) and

Oct4(c) were each determined by real-time PCR analyses. The levels of total

Nanog (d) and c-Myc (e) were also determined. Error bar, standard deviation, n=3.

a

b

WT

34a-/-

p53-/-

#1

#2

#3 #

1#2

#3 #

1#2

#3

ES

MEF WT iPS 34a iPS-/-

p53 iPS-/-

c

WT

34a-/-

p53-/-

#1

#2

#3 #

1#2

#3 #

1#2

#3

ES


p53 iPS-/-

WT

34a

p53

#1

#2

#3 #

1#2

#3 #

1#2

#3

ES


p53 iPS-/-

Foldu

pregulation

Fold

upregulation

Foldupregulation

Fo

ldupregulation

WT

34a-/-

p53-/-

#1

#2

#3 #

1#2

#3 #

1#2

#3

ES


p53 iPS-/-

Foldu

pregulation

0

0.2

0.4

0.6

0.8

1

1.2

WT

34a-/-

p53-/-

#1

#2

#3 #

1#2

#3 #

1#2

#3

ES


p53 iPS-/-

Nanog

d e

Fold

upregulation

WT

34a-/-

p53

#1 #

2#3

#1

#2

#3 #

1#2

#3

ES


p53 iPS-/-

Total c-Myc

Foldupregulation

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6 Exogenous Sox2

WT

34a-/-

p53-/-

#1

#2

#3 #

1#2

#3 #

1#2

#3

ES


p53 iPS-/-

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6Exogenous Klf4

0

0.2

0.4

0.6

0.8

1

1.2

1.4

WT

34a-/-

p53-/-

#1

#2

#3 #

1#2

#3 #

1#2

#3

ES


p53 iPS-/-

EndogenousOct4

0

0.2

0.4

0.6

0.8

1

1.2 Total Oct4

0

1

2

3

4

5

6

7 Total Sox2

0

0.2

0.4

0.6

0.8

1

1.2

-/-

0

0.2

0.4

0.6

0.8

1

1.2

1.4

WT

34a

p53

#1

#2

#3 #

1#2

#3 #

1#2

#3

ES


p53 iPS-/-

EndogenousSox2

Foldupregulation

0

1

2

3

4

5

6

7

8

WT

34a-/-

p53

#1

#2

#3 #

1#2

#3 #

1#2

#3

ES


p53 iPS-/-

-/-

Total Klf4

0

0.2

0.4

0.6

0.8

1

1.2

1.4

WT

34a-/-

p53-/-

#1

#2

#3 #

1#2

#3 #

1#2

#3

ES


p53 iPS-/-

EndogenousKlf4

Fo

ldupregulation

Fo

ldupregulation

Fo

ldu

pregulation

ExogenousOct4

0

0.5

1

1.5

2

2.5

WT

34a-/-

p53-/-

#1

#2

#3 #

1#2

#3 #

1#2

#3

ES


p53 iPS-/-


14/17



Figure S4Deficiency of mir-34adoes not significantly impair apoptoticresponse, DNA damage response, and senescence during reprogramming.

a. Three-factor transduced WT, mir-34a-/-and p53-/-MEFs were collected

before infection and 9 days post-infection. The apoptotic response of

each cell line was measured using Annexin-V and 7-amino-actinomycin

D (7-AAD) staining. Representative FACS profiles of MEFs with three

different genotypes were shown before and after the transduction of three

reprogramming factors. b.A quantitative analysis was carried out to compare

the percentage of early and late apoptotic populations of each genotype

before infection and 9 days after infection. A representative bar graph

was shown out of four independent experiments. Although loss of p53

significantly impaired the apoptotic response in three-factor infected MEFs,

mir-34a-/-MEFs exhibited no evasion of apoptosis during reprogramming.

c.Four-factor transduced WT, mir-34a-/-and p53-/-MEFs were collected

six days post-infection, and the DNA damage was measured by the extent

of DNA double strand breaks usingg-H2AX (red) staining. The nuclei were

stained by DAPI (blue). Although loss of p53significantly impaired the DNA

damage response in the four-factor infected MEFs, both WT and mir-34a-/-

MEFs exhibited very little g-H2AX staining. Scale bar, 20mm. d.Wildtype,

mir-34a-/-, and p53-/-MEFs were each infected with three reprogramming

factors. SA-b-Gal staining was performed 3 days post infection to determine

the percentage of senescent population for each genotype. The quantitative

analyses were shown for SA-b-Gal staining in MEFs with three different

genotypes.

ba

WT mir-34a-/- p53-/-

Annexin V

7-AAD

Uninfected

OSK,

D9p

ostinfection

0

2

4

6

8

10

12

Uninfected D9 post infection

WT #1

WT #2

34a #1

34a #2

p53

Percentageofearlyand

lateapoptoticcells(%) -/-

-/-

-/-

c d

Percen

tageofSA--gal

pos

itivecells(%)

WT#1

WT#2

34a#1-/-

34a#2-/-

p53

0

10

20

30

40

50

60

WT mir-34a p53-/- -/-

H

2AX/DAPI

-/-


15/17


16/17



Figure S6 The uncropped western blots shown in the main figures.a. The

uncropped western blots shown in Fig. 3a. with the locations of molecular

weight markers. b. The uncropped western blots shown in Fig. 5b with the

locations of molecular weight markers. c, d, e. The uncropped western blots

shown in the left (c), middle (d), and right (e) panel of Fig. 5c with the

locations of molecular weight markers. MW, molecular weight.

a b

c

d e


17/17


Table S1 Contributions of mir-34adeficientiPSCs to chimeric animals.

9

mir-34a-/- iPSC line #3 92

mir-34a-/-

iPSC line #1

10

Supplementary Table S1. Contributions of mir-34a-/-

iPSCs to chimeric animals

Percentage of

Chimerism

50% (2/3)

60% (1/3)

40% (1/2)

35% (1/2)

Genetic background for

blastocysts

C57BL/6J

C57BL/6-cBrd/cBrd/Cr and

C57BL/6J

No. of live

Chimeras

3 (33%)

2 (20%)

10% (2/17)

20% (2/17)

30% (2/17)

40% (1/17)50% (6/17)

60% (1/17)

70% (3/17)

iPS cell line

mir-34a-/-

iPSC line #2

# of injected

blastocysts

# of live

pups

45 17 (38%) C57BL/6-cBrd/cBrd/Cr

20

34

nature cell biology - mir-34 mirnas provide a barrier for somatic cell reprogramming

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