supplementary materials supplementary methods cell...

Supplementary Materials Banito et al. p1

SUPPLEMENTARY MATERIALS

SUPPLEMENTARY METHODS

Cell culture. EBV-transformed hB clones were maintained in RPMI supplemented with 10%

FCS, 2 mM L-glutamine and antibiotics (10 µg/ml Penicillin and Streptomycin). E14Tg2a

mouse ES cells were grown and maintained undifferentiated on irradiated SNL feeder layers.

ES cells were grown in KO-DMEM medium plus 10% FCS, non-essential amino acids, L-

glutamine, 2-mercaptoethanol, antibiotics and 1000 U/ml of leukaemia inhibitory factor

(ESGRO-LIF). Human ES cell lines H1, H7 and H9 cells were cultured as previously

described (Pereira et al. 2008).

Experimental Heterokaryons. Heterokaryons were generated by fusing ES cells and human

B-lymphocytes using 50% polyethylene glycol, pH 7.4 (PEG 1500; Roche Diagnostics,

Mannheim, Germany). The procedure has been described in detail on (Pereira et al. 2008).

For the experimental heterokaryons experiments the qRT-PCR was performed as described

in (Pereira et al. 2008). All primers used for the experimental heterokaryons experiments were

previously tested to assuage that were human-specific for genes and did not crossreact with

the murine counterpart. Primer sets used are listed in the Sup. Table II.

High Content Analysis. For the High Content Analysis (HCA), cells where plated in 96-well

plates (Nunc) and stained by immunofluorescence. Two fluorescence images corresponding

to DAPI and gene-primary antibody/AlexaFluor488-secondary antibody were acquired for

each condition with the InCell 1000 automated epifluorescence microscope at 20x

magnification (GE). HCA was performed using In Cell investigator Software (GE). For the

analysis, cells and nuclei of the cell were defined using the DAPI staining. The nuclei were

segmented using top-hat segmentation defining a minimum nucleus area of 100 µm2. To

define the cell segmentation a collar segmentation routine was used, specifying a ratio of

1µm. To define the expression of the analyzed proteins by cell, the average intensity of pixels

in the reference channel (Alexa488) within the defined nuclear region (Object Nuclear

Intensity) was measured. Once each cell has associated a nuclear intensity for the specific

expression, a threshold filter defining positive and negative expressing cells was set.

Threshold filter uses a histogram for data visualization (Sup. Fig 4A-C). To specify the filter

settings the nuclear intensity measure was selected. The threshold filter defining cells with

nuclear intensities above or below a given value as positive or negative respectively for the

expression of the protein was set. The cut-off of the filter was set as follows: first expression

in the control was measured (i.e. p16INK4a expression in wells with empty vector infected cells,

Sup. Fig 4A) to define the negative population (red population on the histogram). Secondly,

the analysis of the positive control (i.e p16INK4a expression in wells with RasV12 infected cells,

Sup. Fig 4B) was performed to define the high expressing population (green population of the


histogram). Once the cut-off was set up, the analysis of the whole experiment was carried out

(Sup. Fig 4C). As a result, the program assigns to each cell the definition of positive or

negative for the expression of the protein analyzed (i.e. p16INK4a positive (p16) and negative

(neg) expressing cells in empty vector versus RasV12 infected cells, Sup. Fig 4D), and

generates a percentage of both cell populations (positive and negative) per well. The mean of

the nuclear intensity was also routinely analyzed and equivalent results were obtained. The

antibodies used for the analysis were tested with robust controls (shRNAs) to test their

specificity. Where possible, alternative antibodies were tested in control experiments to

confirm the results obtained.

Immunoblot. Protein extracts were resolved by SDS–PAGE and transferred to Protran

nitrocellulose membranes (Whatman, Dassel, Germany). Membranes were blocked in 5%

milk powder, 0.05% Tween-20 in PBS for 1 h and incubated with the primary antibody at 4°C

overnight. Membranes were washed on 0.05% Tween-20 in PBS for 4 x 15 min at room

temperature. Depending on the experiment, sheep anti-mouse HRP or donkey anti-rabbit

HRP (GE Healthcare) were used as secondary antibodies. Antibody binding was visualized

using ECL reagents (GE Healthcare).

Reprogramming of human fibroblasts. Highly purified retroviral particles encoding each of

the human genes OCT4, SOX2, C-MYC and KLF4 were obtained from Vectalys. On day 1,

105 IMR90 cells for each condition were seeded in 6 well plates in medium containing Fetal

Bovine Serum (FBS). The following day the cells were transduced with the four viruses at a

MOI of 10 each for 24 hours. On day 3, the cells were washed three times in PBS and cells

grown in medium containing FBS for three additional days. On day 7, the cells were passaged

to plates containing irradiated MEFs and grown for two more days in medium containing FBS.

After day 9, cells were grown in standard hESC culture conditions (KSR medium: Knock out

DMEM + 20% Serum Replacer + FGF2 4ng/ml). The first hIPSC colonies appeared 15 days

later and were picked 5 days later. Individual colonies were picked and transferred to 12 well

plates containing MEFs feeders in KSR + FGF. Colonies were expanded by enzymatic

dissociation. The resulting hIPSCs lines were characterised for the expression of pluripotency

markers (Oct-4 (Santa Cruz), Sox2 (R&D system), Nanog (R&D system) and Tra-1-60 (Santa

Cruz) by immunostaining.

In vitro differentiation of human IPS. It was performed as described in (Vallier et al. 2009).

Briefly, human ESCs grown in feeder free and serum free conditions were harvested using 1

mg/ml Dispase then split into plates pre-coated with fibronectin. hIPSCs were grown for the

first two days in CDM supplemented with recombinant Activin (Kindly provided by Marko

Hyvonen) and FGF2 (12 ng/ml, R&D systems). To obtain extra-embryonic tissue, hIPSCs

were grown for 7 days in CDM in the presence of BMP4 (10 ng/ml) and SB431542 (10

microM, Tocris). To obtain neuroectoderm progenitors, hESCs were grown in CDM in the

presence of SB431542 (10 µM Tocris) and FGF2 (12 ng/ml, R&D systems) and Noggin (200

ng/ml) for 10 additional days. The resulting cells were grown in non-adherent conditions for 10


days and then plated back for 5 additional days in CDM to obtain fully differentiated neuronal

cells. To obtain mesendoderm precursors, hIPSCs were grown for the 3 following days in

CDM in the presence of BMP4 (10ng/ml, R&D systems), FGF2 (20 ng/ml, R&D systems) and

Activin (100 ng/ml, R&D systems) and LY29002 (10 µM, Promega).


SUPPLEMENTARY FIGURE LEGENDS

Supplementary Figure S1. The expression of the four reprogramming factors from a

polycistronic vector or from 4 independent plasmids induces growth arrest in IMR90

human fibroblasts. A. Expression of the 4 reprogramming factors (Sox2, Oct4, Klf4 and c-Myc) using the

polycistronic cassette (Carey et al. 2009) cloned in pBABE was confirmed by

immunofluorescence in IMR90 cells.

B. Quantification of BrdU incorporation in IMR90 transduced with OSKM construct (OSKM),

with the 4 factors in independent plasmids (4F) and controls.

C. Quantification of SA-βGal positive cells in IMR90 infected with OSKM, 4F and controls

(Left). Representative microphotographs showing that cells infected with OSKM or 4F display

SA-βGal activity (Right). This figure is an extension of the experiment presented in Fig. 1E.

D. Quantification of the percentage of cells in different stages of the cell cycle as analyzed by

PI staining followed by flow cytometry.

E. Expression of the 4 reprogramming factors in IMR90 cells do not induce a significant

amount of apoptosis as estimated by IF using an antibody recognizing cleaved caspase 3.

The positive control is IMR90 cells treated with cyclohexamide (100 µg/ml) for 1h and TNF α

(50 ng/ml) for 18 hours.

Supplementary Figure S2. Expression of the 4 reprogramming factors induces

senescence in BJs and MEFs.

A. Crystal Violet stained plates (top panels) and bright field images (middle) showing the

arrest induced in BJs (human foreskin fibroblasts) as compared with the empty vector and

cells expressing a hairpin against p21CIP1 (105 cells were seeded after selection and fixed 14

days later). Analysis of mRNA levels for p53, p21CIP1 (CDKN1a) and p16INK4a (INK4a) in the

indicated cells (bottom).

B. Crystal Violet stained plates of MEFs infected with the 4 factors (pMXs-Oct4, pMXs-Sox2,

pMXs-Klf4 and pMXs-Myc T58A) and with a hairpin targeting p16Ink4a/p19Arf (105 cells were

seeded and fixed 15 days later). Protein levels of p21CIP1 and p16INK4a were determined by

Western blot.

Supplementary Figure S3. Dissecting the effect of the individual reprogramming

factors on senescence. A. Expression of reprogramming factors (Sox2, Oct4, Klf4 and c-Myc) affects the cell growth

of IMR90 human diploid fibroblasts. Cells were transduced using pBABE retroviral plasmids

encoding the 4 factors and selected for 6 days. 105 cells were seeded per 10 cm dish, fixed

and stained with crystal violet 15 days later.

B. Growth curve of IMR90 infected with the each reprogramming factors, Nanog and controls.

Cells were seeded in triplicate in 24-well plates; one plate was fixed every two days and


stained with crystal violet. The relative cell number corresponds to the absorbance at each

time point relative to absorbance at day 0 (day after seeding, Left panel). Quantification of

BrdU incorporation in IMR90 cells infected individually with the reprogramming factors (12

days post infection, right panel).

C. Quantification of the SA β-gal positive cells and representative microphotographs showing

an increase in SA β-gal positive cells after expression of the individual reprogramming factors.

Oncogenic RAS induces SA β-gal, while infection with an shRNA targeting p53 or with Nanog

does not.

D. Control immunofluorescence showing Nanog expression.

Supplementary Figure S4. High content analysis of protein expression by immunofluorescence.

IMR90 cells infected with empty vector and RasV12 were seeded in 96-well plates at a

density of 4,000 cells per well. The following day, cells were stained by immunofluorescence

for p16INK4a and DAPI (nucleus) and subjected to High Content Analysis (HCA). To create a

Threshold Filter to distinguish cells expressing high or low levels of p16INK4a we generated a

cut-off based on the histograms of p16INK4a nuclear intensity (Nuc Intensity Reference 2) for

cells infected with empty vector (A), RasV12 (B) or both (C). In this example the cut-off was

set above a value of nuclear intensity of 235.

Supplementary Figure S5. Upregulation of DNA damage, oxidative stress and

senescence markers in response to expression of the 4 reprogramming factors.

A. 8-oxo-Guanine levels increase in response to expression of the 4 reprogramming factors.

Detection of 8 oxo-guanine (a common DNA lesion resulting from oxidative stress) in IMR90s

infected with OSKM and controls.

B-E. Expression of the 4 reprogramming factors results in an increase in the cells positive for

DNA damage (as measured by pST/Q IF), p53, p21CIP1 and p16INK4a. Representative images

are shown of the experiment quantified in Fig. 2A-D.

Supplementary Figure S6. Induction of p16Ink4a and p21Cip1 upon expression of the 4

reprogramming factors in MEFs.

The graphs presented were generated from data published by (Mikkelsen et al. 2008). The

latter refers to gene expression profiles obtained during doxycycline treatment (days 4, 8, 12,

and 16) of MEFs carrying integrated doxycycline-inducible lentiviral vectors expressing the

four reprogramming factors. Expression levels of p16INK4a and p21CIP1 upon Dox treatment

are relative to the levels of expression in day 0.

Supplementary Figure S7. Time course for the expression of p53, p21CIP and p16Ink4a in

IMR90 cells expressing the 4 reprogramming factors.


The levels of p53, p21CIP and p16Ink4a were determined by immunofluorescence over the

course of 10 days. The time line corresponds to the one presented in Figure 1.

Supplementary Figure S8. Upregulation of senescence effectors during heterokaryon-based reprogramming.

A. Cartoon summarizing the system.

B. Expression of human transcripts linked to pluripotency is upregulated after cell fusion.

C. Upregulation of human INK4a (encoding for p16INK4a) and human CDKN1a (encoding for

p21CIP1) transcripts during reprogramming induced by heterokaryon formation between

human B cells (hB) and mouse ES cells. Human ES cells (hES).

D. Levels of human HPRT as a control.

Supplementary Figure S9. Levels of p16Ink4a and p21Cip1 are elevated in partially

reprogrammed iPS cells when compared with fully reprogrammed iPS cells.

A. RNA samples from incompletely and fully reprogrammed mouse embryonic fibroblasts

(MEFs) were analyzed by qRT-PCR for the expression of the cyclin dependent kinase

inhibitors p16Ink4a (Ink4a) and p21Cip1 (Cdkn1a). Pre-IPS samples exhibit ES cell morphology,

but do not express all pluripotency associated genes, maintain the expression of viral

transgenes, retained the epigenetic silencing of the X chromosome, are irresponsive to LIF

and are unable to colonise chimeras.

B. Values were obtained from gene expression profiling data presented in Supplemental

Material of (Mikkelsen et al. 2008). Values of expression for p16Ink4a (Ink4a) and p21Cip1

(Cdkn1a) in partially reprogrammed cell lines (MCV6 and MCV8), IPS cell line (MCV8.1) and

ES cells are relative to expression levels in MEFs. Partially reprogrammed cells

characteristics are defined in detail in (Mikkelsen et al. 2008).

C. Values presented for p16Ink4a and p21Cip1 correspond to the logarithm (base 2) of the

expression in partially reprogrammed (Pre-iPS) and ES and were obtained from

Supplemental Data (Table S1) of (Sridharan et al. 2009). The values are normalized to the

expression in ES cells.

Supplementary Figure S10. ChIP analysis of the INK4a/ARF locus in IMR90s expressing

the 4 reprogramming factors from independent plasmids (4F).

A, B. Expression of the 4 reprogramming factors from independent plasmids (4F), results in a

loss of H3K27me3 marks and an increase in H3K4me3 marks in the INK4b/ARF/INK4a locus,

as seen with OSKM cells (Figure 3).

C. Expression of the 4 reprogramming factors results in increased recruitment of RNA pol II to

the INK4a promoter.

Supplementary Figure S11. Upregulation of RBL2 (p130) during reprogramming and

effect of expressing miR-302a-d.


A. RBL2 (p130) is upregulated during reprogramming-induced senescence. IMR90s infected

with OSKM vector were subjected to immunofluorescence using antibodies recognizing p130

(RBL2, retinoblastoma-like protein 2).

B-C. Infection with a vector expressing miRNAs miR-302a-d prevents the upregulation of

p21CIP1a and p130 in response to the 4 reprogramming factors. Representative images of the

data shown in Fig. 4D, E.

Supplementary Figure S12. Comparison of the expression levels of ES cells specific

microRNAs during RIS and in iPS cells.

miR-302b, is detected at lower levels in OSKM arrested cells when compared to iPS or

human ES cells, exhibiting the same expression pattern as the related miRNAs, miR-302a,

miR-302c and miR-302d (Figure 4). Values are relative to IMR90 cells infected with a control

vector. miR-372 and miR-373, which also target CDKN1a (encoding for p21CIP1) and RBL2

(encoding for p130), are undetected in human fibroblast as well as in OSKM arrested cells.

Values are normalized to hES cells; u.d.l., under the detection limit.

Supplementary Figure S13. Inhibition of senescence effectors improves

reprogramming efficiency of BJ and IMR90 cells.

A. Colonies of the reprogramming experiment performed with BJ human fibroblast and

described in Fig.5, were analyzed by immunofluorescence for NANOG and TRA-1-60

expression. Representative pictures of double positive colonies are shown. The quantification

is presented in Fig. 5B.

B. Bright filed images showing morphology of fully and partially reprogrammed colonies

derived from BJ cells.

C. Count of partially reprogrammed colonies in BJs (human foreskin fibroblasts) empty vector

and with knockdown for p53, p21Cip1and p16Ink4a.

D. IMR90 human fibroblasts were infected with the indicated viruses and cells selected. Q-

RT-PCR showing the levels of the transcripts for p53, CDKN1A (encoding for p21CIP1) and

INK4a (encoding for p16INK4a) in the cells used for the reprogramming experiments.

E, F. Count of partially (E) and fully (F) reprogrammed colonies. IMR90 fibroblasts expressing

shRNAs targeting p16INK4a, p21CIP1 or p53 were transduced with lentiviruses expressing Oct-4,

Sox2, Klf4 and c-Myc and grown in culture conditions compatible with pluripotent stem cells

growth. Partially or fully reprogrammed colonies (as defined by morphological characteristics)

were counted 3 weeks after transduction. Number of colonies is shown as the mean ± SD

from three informative experiments. Microphotographs of representative colonies are shown

at the right side.

G. Fully reprogrammed colonies generated from IMR90 fibroblasts expressing shRNA

targeting p16INK4a were picked and expanded. After amplification, immunostaining analysis

was performed to detect the expression of the pluripotency markers Oct4, Sox2, Nanog and

Tra-1-60.


Supplementary Figure S14. Enhanced efficiency of reprogramming in MEFs knockout

or knockdown for senescence effectors.

A. MEFs were infected with the indicated vectors and selected. Q-RT-PCR showing the levels

of the transcripts for Tp53, Cdkn1a (encoding for p21Cip1) and Ink4a (encoding for p16Ink4a) in

the cells used for the reprogramming experiments showed in (B).

B. MEFS expressing shRNAs targeting p16INK4a/p19Arf, p21CIP1 or p53 were transduced with

lentiviruses expressing Oct-4, Sox2, Klf4 and c-Myc and grown in culture conditions

compatible with pluripotent stem cells growth. The number of alkaline phosphatase (AP)

positive colonies obtained after seeding 35,000 cells was quantified.

C. Wildtype (WT) MEFs or MEFs knockout for p53, p21 (p53-/-, p21-/-) were transduced with

lentiviruses expressing Oct-4, Sox2, Klf4 and c-Myc, and grown in culture conditions

compatible with pluripotent stem cells growth The number of alkaline phosphatase (AP)

positive colonies obtained after seeding 35,000 cells was quantified.

Supplementary Figure S15. iPS-2 and iPS-3 clones derived from BJs shp16 and shp53

express pluripotency related genes.

qRT-PCR for hDNMT3b, hRex1, hCripto and hTert in BJs and IPS-2 (derived from BJs

shp16) and iPS-3. Values are relative to iPS2. These are representative q-RT-PCR of the

data summarized in the table presented in Fig. 5D.

Supplementary Figure S16. Senescence as an early barrier for reprogramming.

Reprogramming induced senescence (RIS) is an early barrier to the process. Partially

reprogrammed (Pre-iPS) cells are trapped in a late state in the reprogramming process due to

incomplete epigenetic resetting. Strategies including inhibition of DNA methylation,

knockdown of lineage specific genes (Mikkelsen et al. 2008) or treatment with 2 inhibitors

(Silva et al. 2008) can convert Pre-iPS cells to fully reprogrammed iPS cells or increase the

proportion of fully vs. partially reprogrammed cells (middle panel). Here, we show how

knocking down critical senescence effectors results in increased numbers of both partially and

fully reprogrammed cells, presumably by reducing the proportion of cells that succumb to the

first barrier (right panel).


SUPPLEMENTARY REFERENCES

Carey, B.W., Markoulaki, S., Hanna, J., Saha, K., Gao, Q., Mitalipova, M., and Jaenisch, R. 2009. Reprogramming of murine and human somatic cells using a single polycistronic vector. Proc Natl Acad Sci U S A 106(1): 157-162.

Mikkelsen, T.S., Hanna, J., Zhang, X., Ku, M., Wernig, M., Schorderet, P., Bernstein, B.E., Jaenisch, R., Lander, E.S., and Meissner, A. 2008. Dissecting direct reprogramming through integrative genomic analysis. Nature 454(7200): 49-55.

Pereira, C.F., Terranova, R., Ryan, N.K., Santos, J., Morris, K.J., Cui, W., Merkenschlager, M., and Fisher, A.G. 2008. Heterokaryon-based reprogramming of human B lymphocytes for pluripotency requires Oct4 but not Sox2. PLoS Genet 4(9): e1000170.

Silva, J., Barrandon, O., Nichols, J., Kawaguchi, J., Theunissen, T.W., and Smith, A. 2008. Promotion of reprogramming to ground state pluripotency by signal inhibition. PLoS Biol 6(10): e253.

Sridharan, R., Tchieu, J., Mason, M.J., Yachechko, R., Kuoy, E., Horvath, S., Zhou, Q., and Plath, K. 2009. Role of the murine reprogramming factors in the induction of pluripotency. Cell 136(2): 364-377.

Vallier, L., Touboul, T., Chng, Z., Brimpari, M., Hannan, N., Millan, E., Smithers, L.E., Trotter, M., Rugg-Gunn, P., Weber, A., and Pedersen, R.A. 2009. Early cell fate decisions of human embryonic stem cells and mouse epiblast stem cells are controlled by the same signalling pathways. PLoS One 4(6): e6082.


Supplementary Table I. Details of plasmids generated for this study.

Vector Insert cloned from Addgene number Reference

pBABEpuro-OSKM TetO-FUW-OSKM Addgene 20321 Carey et al., 2009

pBABEpuro-SO FUW-SO Addgene 20329 Carey et al., 2009

pBABEpuro-Oct4 pMXs-Oct3/4 Addgene 13366 Takahasi and Yamanaka, 2006

pBABEpuro-Sox2 pMXs-Sox2 Addgene 13367 Takahasi and Yamanaka, 2006

pBABEpuro-Klf4 pMXs- Klf4 Addgene 13370 Takahasi and Yamanaka, 2006

pBABEpuro-Nanog pMXs- Nanog Addgene 13354 Takahasi and Yamanaka, 2006


Supplementary Table II. Details of oligonucleotide primers used in this study.

Primers used for qRT-PCR Target Forward primer Reverse primer Human p16: CGGTCGGAGGCCGATCCAG GCGCCGTGGAGCAGCAGCAGCT (Run at 66°C) Human p53: CCGCAGTCAGATCCTAGCG AATCATCCATTGCTTGGGACG Human p21: CCTGTCACTGTCTTGTACCCT GCGTTTGGAGTGGTAGAAATCT Human Oct4: TCGAGAACCGAGTGAGAGGC CACACTCGGACCACATCCTTC Human Nanog: CCAACATCCTGAACCTCAGCTAC GCCTTCTGCGTCACACCATT Human Cripto: AGAAGTGTTCCCTGTGTAAATGCTG CACGAGGTGCTCATCCATCA Human HPRT: TCCTTGGTCAGGCAGTATAATCC GTCAAGGGCATATCCTACAACAAA Human GAPDH: TGATGACATCAAGAAGGTGGTG TCCTTGGAGGCCATGTGGGCCA Human RPS14: TCACCGCCCTACACATCAAACT CTGCGAGTGCTGTCAGAGG Mouse p21: CCTGGTGATGTCCGACCTG CCATGAGCGCATCGCAATC Mouse p53: CACGTACTCTCCTCCCCTCAAT AACTGCACAGGGCACGTCTT Mouse p16: GTGTGCATGACGTGCGGG GCAGTTCGAATCTGCACCGTAG Mouse RPS14: GACCAAGACCCCTGGACCT CCCCTTTTCTTCGAGTGCTA TaqMan probes Target Applied Biosystems Reference Human mir-302a 529 Human mir-302b 531 Human mir-302c 533 Human mir-302d 535 Human mir-372 560 Human mir-373 561 Human RNU 48 1006 Human JMJD3 Hs 00389749_m1 Human GAPDH 4333764-0803023

Primers used for generating shRNA constructs:

Human p21, forward oligo:

TCCCACAATGCTGAATATACAGATCCCCTCCCACAATGCTGAATATACATTCAAGAGATGTATATTCAG

CATTGTGGGATTTTTA

Human p21, reverse oligo:

AGCTTAAAAATCCCACAATGCTGAATATACATCTCTTGAATGTATATTCAGCATTGTGGGAGGG


Supplementary Table III. Details of antibodies used in this study. Target Source BrdU A21300, Invitrogen

Human p16INK4a JC-8, CRUK,

Human p53 DO-1, sc-126, Santa Cruz Biotechnology

Human γH2AX 05-636, Upstate

Human pST/Q 2851, Cell Signaling

Human and mouse β- Actin sc-47778, Santa Cruz Biotechnology

Human p21 CIP1 18209, Abcam

Human and mouse p21CIP1 p1484, Sigma

Human JMJD3 ARP40102, Aviva

Human and mouse Oct4 sc-5279, Santa Cruz Biotechnology

Mouse Sox2 15830, Abcam

Human and mouse Klf4 34814, Abcam

Human and mousec-Myc ssc-764, Santa Cruz Biotechnology

8 oxo-guanine Mab3560, Millipore

Human Cleaved Caspase-3 9664, Cell Signaling

Human Tra-81 56000017, BD Pharmingen

Human Tra-60 sc-21705, Santa Cruz Biotechnology

Mouse Nanog REC-RCAB0002PF, CosmoBio

Human Pax6 PRB-278P-100, Covance

Human CDX2 MU392A-UC, BioGenex

Human Gata6 ab22600, Abcam

Human Sox2 AF2018, R&D


Human Brachyury AF2085, R&D


GENESDEV/2009/121525/Banito_Fig S1

4 Factors

% P

ositi

ve c

ells

Vector RASshp53OSKM 4-F0

10203040506070

BrdU

SA β-Gal

01020304050607080

Vector RASshp53OSKM 4-F

% P

ositi

ve c

ells

D E

0

10

20

30

40

50

60

G1 S G2

Vector

OSKMRAS

shp53

% C

ells

0102030405060708090

TNF+CHX

Vector RASOSKM shp53

% P

ositi

ve c

ells

Cleaved Caspase 3

C

A

B

Vector OSKM

RAS

Vector OSKM shp21

Vector OSKM shp21

0

1

2

3

4

Vector OSKM0

1

2

3

4

Vector OSKM0

1

2

3

4

Vector OSKM

TP53 CDKN1a INK4a

Rel

ativ

e m

RN

A le

vels

A

BVector 4 factors shInk4a/arf

Vector

OSKM4F

p21

p16

β-Actin


A

B

Vector RASSox2Oct4 c-MycKlf4

Sox2Oct4 c-MycKlf4

Vector RAS shp53 NanogC

% B

rdU

pos

itive

cel

ls

Vector RASSox2Oct4 Klf4 c-Myc0

5

10

15

20

25

30

35

40

45

D

Vector

Nanog

DAPI Nanog Merge


A B C

D

Vector

RAS


05

1015202530354045

VectorOSKMRAS shp53

8oxoG

% P

ositi

ve c

ells

DAPI OxoG Merge

Vector

OSKM


p21CIP1DAPI Merge

Vector

OSKM

DAPI p16INK4a

Vector

OSKM

DAPI p53 Merge

Vector

OSKM

DAPI pST/Q Merge

Vector

OSKM

Merge

A

B C

D E

0123456789

0

5

10

15

20

25

30

Ctrl 4 8 12 16

Ink4a

Time after Dox. Induction (d)

Ctrl 4 8 12 16Rel

ativ

e m

RN

A le

vels

Cdkn1a

Time after Dox. Induction (d)

Rel

ativ

e m

RN

A le

vels


p53

p21

p16


C

D

B

AR

elat

ive

mR

NA

leve

lsR

elat

ive

mR

NA

leve

ls

Rel

ativ

e m

RN

A le

vels

Rel

ativ

e m

RN

A le

vels

Rel

ativ

e m

RN

A le

vels

Rel

ativ

e m

RN

A le

vels


0

10

20

30

40

50

60

Rel

ativ

e m

RN

A le

vels

Ink4a

0

0.5

1

1.5

2

2.5

Rel

ativ

e m

RN

A le

vels

Cdkn1a

MEFs Pre-iPS ES MEFs Pre-iPS ES

A

B

C

00.5

11.5

22.5

33.5

ESPre-IPS

02468

1012141618

Rel

ativ

e m

RN

A le

vels

0

1

2

3

4

5

6R

elat

ive

mR

NA

leve

ls

00.5

11.5

22.5

33.5

ESPre-IPS

Rel

ativ

e m

RN

A le

vels

MEFs MCV6 MCV8 iPS ESPre-iPS

Ink4a Cdkn1a

MEFs MCV6 MCV8 iPS ESPre-iPS

Cdkn1aInk4a

Rel

ativ

e m

RN

A le

vels


0

0.05

0.1

0.15

0.2

0.25

1 3 5 7 12

Vector4F

0

0.05

0.1

0.15

0.2

0.25

Vector4F

H3K4me3

H3K27me3H

3K27

me3

/Tot

al H

3H

3K4m

e3/T

otal

H3

2 4 6 8

1 3 5 7 122 4 6 8

Primer sets:

Primer sets:


A

B

C

00.05

0.10.15

0.20.25

0.30.35

RNA pol II

Sign

al/T

otal

H3

hES

VectorOSKM

INK4a pr. 3’ regionp.s. 6 p.s. 12

DAPI p130 Merge

Vector

OSKM

p130


OSKM

Ctrl. 302Vector

DAPI

p21CIP1

DAPI

p130

OSKMCtrl. 302Vector

A

B

C

u.d.l.u.d.l.

u.d.l.


G

0

20

40

60

80

100

120

pRS shp16

Rel

ativ

e m

RN

A le

vels

0

20

40

60

80

100

120

pRS shp53

Rel

ativ

e m

RN

A le

vels

0

20

40

60

80

100

120

pRS shp21 shp53

Rel

ativ

e m

RN

A le

vels

A

Oct-4 Sox2 Nanog Tra-1-60

p53 CDKN1a INK4a

E

F

Col

onie

s

0

2

4

6

8

10

12

Vector shp16 shp21 shp53

Fully Reprogrammed (IMR90)

Col

onie

s

Partially Reprogrammed (IMR90)

050

100150200250300350400450500


hiPSCs derived from IMR90-shp16

0

40

80

120

160

200


Nan

og a

nd T

RA

-1-6

0 n

egat

ive

colo

nies

Partial Reprogrammed (BJs)

D

IMR90

FullyReprogrammed

PartiallyReprogrammed



NA

NO

GTR

A-1

-60

DA

PI

B

C

BJBJ

A

B

0200400600800

10001200140016001800

Vector shp53 shp21 shInk4a/Arf

0

0.2

0.4

0.6

0.8

1

1.2

Vector shp53

Tp53

0

0.2

0.4

0.6

0.8

1

1.2

Vector shp21

Cdkn1a

0

0.2

0.4

0.6

0.8

1

1.2

Vector shInk4a/Arf

Ink4a

Rel

ativ

e m

RN

A le

vels

MEFs

AP p

ositi

ve c

lolo

nies

mouse iPS

0

200

400

600

800

1000

1200

1400

WT p53-/- p21-/-

AP

Posi

tive

Col

onie

s

Cmouse iPS


‘Standard’ Reprogramming

Low efficiency

SOMATIC CELLS

Senescence

Intermediate State

iPS CELLSPre-iPS

Inhibition of senescence

Increased numbersof both Fully and

Partialreprogrammed iPS

SOMATIC CELLS

Senescence

Intermediate State

iPS CELLSPre-iPS

Inhibition of DNA methylation

Increased proportionof Fully vs. Partialreprogrammed iPS

SOMATIC CELLS

Senescence

Intermediate State

iPS CELLS

Pre-iPS


supplementary materials supplementary methods cell...

Documents