genome editing and stem cell engineering for disease modeling · 4/13/2017 · drug discovery cell...

Genome editing and stem cell engineering for disease modeling

April13,2017

Adriana S. Beltran Assistant Professor, Department of Pharmacology

Director, Human Pluripotent Stem Cell Core Phone: (919) 537-3996 (office), (919) 537-3995 (lab)

Email: [email protected] Website: http://www.med.unc.edu/humanstemcellcore

The University of North Carolina at Chapel Hill

The human Pluripotent Stem Cell core (HPSCC) is committed to

accelerating collaborative studies in stem cell biology

The HPSCC mission is to serve as the central platform of technical support for investigators seeking to work with human embryonic and

pluripotent stem cells

EmbryonicStemcells

Self-renew Differen5a5on

Pluripotent stem cells

Limitlesspoten5alinregenera5vemedicineanddisease

Rejec5onaBertransplanta5onUsageofhumanembryostocreatethem

CONS

PROS

Somatic cells

ES Cell Differentiation

Self-renewal network-ON Differentiation genes-OFF

Self-renewal network-OFF Differentiation genes-ON

iPS Cell

Oct4

Sox2

Klf4

Reprogramming

c-Myc

Inducing a pluripotent state (iPS)

Pa5ents

pluripotent they can give rise to all three germ layers (Box1). Owing to their pluripotent and self-renewing capacities,ESCs are considered cellular resources for modelinghuman diseases as well as treating damaged humanorgans and tissues [14–17]. iPSCs have been created byreprogramming differentiated cells by the ectopic expres-sion of the four transcription factors Oct4, Sox2, c-Myc, andKlf-4 [18,19]. iPSCs and ESCs share characteristics such asmorphology, expression of surface markers, pluripotentdifferentiation potential, and the capacity for self-renewal.Because iPSCs retain the genetic composition of the par-ental somatic cells, the phenotypes of cells differentiatedfrom iPSCs presumably represent those manifested byparental cells [20–25]. Thus, iPSCs from patients enablethe development of disease-specific cellular models, plat-forms for drug screening, and autologous sources for cellreplacement therapies. Furthermore, iPSCs bypass theethical issues associated with destroying embryos to obtainhuman ESCs and immunological barriers that prevent theuse of heterologous cells [26]. Figure 1 depicts a scheme forstudying ASD using genomics, animal models, and iPSCs.When a person is diagnosed with ASD, genomics methodscan identify the culprit candidate genes and the physiolo-gical functions of the genes can be examined by transgenicmurine models [9]. Meanwhile, iPSCs are readily availableby reprogramming somatic cells from ASD patients. Fol-lowing neuronal differentiation, functional analysis can beperformed to examine in vitro phenotypes manifested byASD iPSCs. Quantifiable measures that are associated

with ASDs, such as neuronal connectivity, synaptic activ-ity, and neuronal migration, can be used as a screeningplatform to test the efficacy of chemicals for amelioratingASD.

When using iPSCs as a cellular model for ASD, theproper selection of standard control determines the inter-pretation of the in vitro phenotypes. Most often, iPSCs fromhealthy persons have been used as controls [21]. Becauseindividual genetic variations have a large influence oncellular physiology, the use of iPSCs derived from closelygenetically related persons, such as siblings or parents, canreduce the compounding genetic effect. Nevertheless,isogenic iPSC lines are the ideal control. One method forgenerating isogenic lines is to take advantage of the factthat when a disease is X-linked and prominent in females,reprogramming produces unique sets of isogenic femaleiPSCs by retaining the active/repressed X chromosomestatus of fibroblasts [27]. Thus, iPSCs can be producedwith either the wild type or mutant allele on the activeX chromosome from female Rett syndrome patients bytaking advantage of this feature (see below and Box 2)[28–31]. However, a recent study showed that the inactiveX chromosome in female iPSCs undergoes erosion ofinactivation over time in culture, raising a concern formodeling X-linked disorders using iPSCs [32]. As geneediting technologies such as zinc finger nuclease (ZFN)or transcription activator-like effector nucleases (TALEN)have improved, manipulating single genes within iPSCshas become possible (Box 3) [22,33]. Gene editing technology

Iden!fica!on of novelgene causing ASD

Advances in gene!csand genomics

ASD pa!ent

Monogenic disorders:Re" syndrome,

Fragile X syndromeTimothy syndrome, etc.

Animalmodel

Gene engineering

Diseasemodeling

Neuronaldifferen!a!on

Drugdiscovery

Celltherapy

iPSC

Soma!c cells

Reprogramming

TRENDS in Molecular Medicine

Figure 1. Overview of adopting induced pluripotent stem cell (iPSC) technology in autism spectrum disorder (ASD) research. ASD is a genetic neurodevelopmentaldisorder. Monogenic syndromic ASDs, including Rett syndrome, Fragile X syndrome, or Timothy syndrome, are caused by well-defined genetic defects. Advancedgenomics tools are used to actively seek novel ASD genes. From skin biopsy or blood samples, patient-derived ASD iPSCs can be generated. Cortical neurons, which aremost relevant neurons for ASD, can be directed from iPSCs using well-established protocols (Table 1). The neuronal cells can be used to elucidate novel diseasepathogenesis and to screen drugs as potential treatments. Recently developed gene editing technologies facilitate correcting the given mutation to provide isogeniccontrols for in vitro disease modeling or cell-based therapy.

Review Trends in Molecular Medicine August 2012, Vol. 18, No. 8

464

iPSCs






ASD pa!ent



Animalmodel

Gene engineering

Diseasemodeling


Drugdiscovery

Celltherapy

iPSC

Soma!c cells

Reprogramming



ReviewTrends in Molecular Medicine August 2012, Vol. 18, No. 8

464

Disease

modelingSpecificcelltype

differen9a9on

Molecular

mechanismof

thediseaseReprograming

Drug

discovery






ASD pa!ent



Animalmodel

Gene engineering

Diseasemodeling


Drugdiscovery

Celltherapy

iPSC

Soma!c cells

Reprogramming




464

Gene9cengineering

Cardiac,neural,

livertoxicity

tests

Specificcelltype

differen9a9on

iPSC as patient-specific pre-clinical disease models

Regenera9ve

Medicine






ASD pa!ent



Animalmodel

Gene engineering

Diseasemodeling


Drugdiscovery

Celltherapy

iPSC

Soma!c cells

Reprogramming



ReviewTrends in Molecular Medicine August 2012, Vol. 18, No. 8

464

Specificcelltype

differen9a9on

Advantage:

Disadvantage:

Longexperienceinusing

Clinicaltrial

Difficulttodesignedand

Construct

Targetsiterestric9ons

Easytodesign

Highspecificity

Laborintensetoconstruct

“T”requirement

Sensi9veto5’methylcytosine

Simplicity

Highefficiency

PAMrequirement

Off-targetconcerns

MashimoT.2014,56,46-52

Tools for genome editing

cussed in relation to genetic analysis and manipulationin animals, especially in rats.

Zinc-finger nucleases

Zinc finger nucleases are chimeric proteins that consistof a specific DNA-binding domain that is made of tan-dem zinc finger-binding motifs fused to a non-specificcleavage domain of the restriction endonuclease FokI(Bibikova et al. 2001; Porteus & Carroll 2005; Wu et al.2007) (Fig. 1). As one zinc finger unit binds with 3-bpof DNA, 9–18 bp sequences can be specifically recog-nized by combining 3–6 different zinc finger units. Bydesigning two zinc finger motifs on either side of 5–6 bp spacer sequences at a target region, the FokInuclease combined with the zinc finger can introducea double-strand break (DSB) within the 5–6 bp spacersequences. Although the DSB is usually repaired vianon-homologous end joining (NHEJ), an arbitrary dele-tion or deletion of base pairs often occurs during therepair process. Consequently, repair by NHEJ is muta-genic and mostly results in a loss-of-function mutation.Moreover, if DNA fragments homologous to the tar-geted sequences are co-injected with the nucleases,homologous recombination (HR) can occur, enablinginsertion of a transgene or replacement of the homolo-gous sequences at the targeted region, which results

in KI mutations. Therefore, artificially designed ZFNscan be used to generate KO or KI alleles at the tar-geted sequences via NHEJ or HR repair, respectively.A summary of how to generate targeted KO rats

using ZFNs is given in Figure. 1. Briefly, two ZFNs aredesigned across the spacer domain to recognize thetargeted DNA sequences. Messenger RNAs are thentranscribed in vitro from the two ZFN plasmids andinjected into the male pronuclei of rat zygotes. Pronu-clear stage embryos are collected from female ratsthat were superovulated by equine chorionic gonado-tropin and human chorionic gonadotropin injection.The ZFN-injected embryos that differentiate into twocells are then transferred to the oviduct of pseudo-pregnant females. This method is based on a similartechnique used to produce conventional transgenicanimals (Palmiter et al. 1982, 1983; Mullins et al.1990), except that mRNA is used. The procedure forthe micromanipulation of embryos is the same for allnucleases, including the below-mentioned TALEN andclustered regularly interspaced short palindromicrepeats (CRISPR) enzymes.The ZFN technology was first reported in the 1990s

(Kim et al. 1996; Chandrasegaran & Smith 1999).From the 2000s, ZFNs have been developed forvarious mammalian cells (Bibikova et al. 2001, 2003;Porteus & Baltimore 2003; Urnov et al. 2005;

Fig. 1. Gene targeting technologies with zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clus-

tered regularly interspaced short palindromic repeats (CRISPR)/Cas in rats. Schematic representation of the genetic engineering methods

used for generating targeted knockout rats.

ª 2013 The AuthorDevelopment, Growth & Differentiation ª 2013 Japanese Society of Developmental Biologists

Genome editing in rats 47

GCCAACCGCACTATGCCTCTATAACTACCGGAGCAGTCGAGGTTGAGCTTCGGTTGGCGTGATACGGAGATATTGATGGCCTCGTCAGCTCCAACTCGAA

FOX1

FOX1

Zinc Finger nucleases (ZFN)FokI nuclease

FokI nuclease

Medical applications of iPS cells

Given the number of drugs that have notoriously been withdrawn from the market because of their tendency to induce arrhythmias, it is highly likely that the current inadequate approaches for assessing cardiotoxic-ity will be complemented by iPS-cell-based assessments of drug effects.

A study from our laboratory explored dyskeratosis congenita, a dis-order of telomere maintenance, and provided an unanticipated insight into the basic biology of telomerase that has therapeutic implications73. In its most severe form, dyskeratosis congenita is caused by a mutation in the dyskerin gene (DKC1), which is X linked, leading to shortened tel-omeres and premature senescence in cells and ultimately manifesting as the degeneration of multiple tissues. Because the reprogramming of cells to an induced pluripotent state is accompanied by the induction of the gene encoding telomerase reverse transcriptase (TERT), we investigated whether the telomerase defect would limit the derivation and mainte-nance of iPS cells from individuals with dyskeratosis congenita. Although the efficiency of iPS-cell derivation was poor, we were able to successfully reprogram patient fibroblasts. Surprisingly, whereas the mean telomere length immediately after reprogramming was shorter than that of the parental fibroblast population, continued passage of some iPS cell lines led to telomere elongation over time. This process was accompanied by upregulation of the expression of TERC, which encodes the RNA subunit of telomerase.

Further analysis established that TERT and TERC, as well as DKC1, were expressed at higher levels in dyskeratosis-congenita-derived iPS cells than in the parental fibroblasts73. We determined that the genes encoding these components of the telomerase pathway — including a cis element in the 3ʹ region of the TERC locus that is essential for a transcriptionally active chromatin structure — were direct binding targets of the pluri-potency-associated transcription factors. Further analysis indicated that transcriptional silencing owing to a 3ʹ deletion in the TERC locus leads to the autosomal dominant form of dyskeratosis congenita by diminishing TERC transcription. Although telomere length is restored in dyskeratosis-congenita-derived iPS cells, differentiation into somatic cells is accompa-nied by a return to pathogenesis with low TERC expression and a decay in telomere length. This finding showed that TERC RNA levels are dynami-cally regulated and that the pluripotent state of the cells is reversible, sug-gesting that drugs that elevate or stabilize TERC expression might rescue defective telomerase activity and provide a therapeutic benefit. Although we set out to understand the pathogenesis of dyskeratosis congenita with

this study, we showed that a high expression level of multiple telomerase components was characteristic of the pluripotent state more generally, illustrating how iPS cells can reveal fundamental aspects of cell biology.

An independent study of the reprogramming of cells from patients with dyskeratosis congenita confirmed the general transcriptional upregula-tion of multiple telomerase components and the maintenance of telomere lengths in clones74; however, in this study, no clones with elongated telom-eres were identified. The different outcomes of these studies highlight the limitations of iPS-cell-based disease models that are imposed by clonal variation as a result of the inherent technical infidelity of reprogram-ming75. This point also introduces an additional important consideration. Before a given iPS-cell disease model can be claimed to be truly represent-ative of the disease, how many patients must be involved, and how many iPS cell lines must be derived from each patient? Although the answers to these questions are unclear, it is crucial to keep these issues in mind when generating disease models and making claims based on these models.

Although iPS cells are an invaluable tool for modelling diseases in vitro, the goal of developing patient-specific stem cells has also been motivated by the prospect of generating a ready supply of immune-compatible cells and tissues for autologous transplantation. At present, the clinical trans-lation of iPS-cell-based cell therapies seems more futuristic than the in vitro use of iPS cells for research and drug development, but two ground-breaking studies have provided the proof of principle in mouse models that the dream might one day be realized. Hanna, Jaenisch and colleagues used homologous recombination to repair the genetic defect in iPS cells derived from a humanized mouse model of sickle-cell anaemia76. Directed differentiation of the repaired iPS cells into haematopoietic progenitors followed by transplantation of these cells into the affected mice led to the rescue of the disease phenotype. The gene-corrected iPS-cell-derived haematopoietic progenitors showed stable engraftment and correction of the disease phenotype.

In another landmark study from Jaenisch’s research group, Wernig and colleagues derived dopaminergic neurons from iPS cells that, when implanted into the brain, became functionally integrated and improved the condition of a rat model of Parkinson’s disease77. The successful implantation and functional recovery in this model is evidence of the therapeutic value of pluripotent stem cells for cell-replacement therapy in the brain — one of the most promising areas for the future of iPS-cell applications.

Figure 2 | Medical applications of iPS cells. Reprogramming technology and iPS cells have the potential to be used to model and treat human disease. In this example, the patient has a neurodegenerative disorder. Patient-specific iPS cells — in this case derived by ectopic co-expression of transcription factors in cells isolated from a skin biopsy — can be used in one of two pathways. In cases in which the disease-causing mutation is known (for example, familial Parkinson’s disease), gene targeting could be used to repair the DNA sequence (right). The gene-corrected patient-specific iPS cells would then undergo directed differentiation into the affected neuronal subtype (for example, midbrain dopaminergic neurons) and be transplanted into the patient’s brain (to engraft the nigrostriatal axis). Alternatively, directed differentiation of the patient-specific iPS cells into the affected neuronal subtype (left) will allow the patient’s disease to be modelled in vitro, and potential drugs can be screened, aiding in the discovery of novel therapeutic compounds.

Patient

cMYC OCT4

KLF4 SOX2

Skin biopsy

Patient-specific iPS cells

In vitrodifferentiation

Affected cell type

Screening fortherapeuticcompounds

Treatmentwith drugs

Disease-specific drugs

Transplantation of geneticallymatched healthy cells

Repaired iPS cells

Healthy cells

Use gene targeting to repairdisease-causing mutation

In vitro differentiation

ening forapeuticpounds

1 9 J A N U A R Y 2 0 1 2 | V O L 4 8 1 | N A T U R E | 3 0 1

REVIEW INSIGHT

© 2012 Macmillan Publishers Limited. All rights reserved

Adaptedfrom:Robinton&Daley,Nature2012

Skinbiopsy,blood,kera5nocytes

Human pluripotent stem cell services

Cell derivation and characterization: •  IPSC generation and characterization •  hES/hiPS directed differentiation into specialized cell

types •  hES/hiPS maintenance, expansion and banking Genome engineering: •  Genome editing of mammalian cells (hESC, iPSC, CSC) •  Engineering effector molecules for gene regulation Training in stem cell culture techniques

Source materials Fibroblast Neural cells Hair follicle Keratinocytes Blood Urine

Avai

labi

lity

Easy to reprogram

Urine cells

Neural stem cells

Keratinocytes

Germline stem cells

Adipose stem cells

T cells Fibroblast

Cord blood stem cells Mesenchymal stem cells

Blood: easy to obtain and easy to reprogram Fibroblast: easy to obtain, frozen stock and expandable Urine: easiest to obtain, easy to reprogram, potential contamination

Method Integra5on Efficiency Cost

Retrovirus Yes 0.001–0.01% ++Len9virus Yes 0.001–0.01% ++Adenoviral Possible 0.0001–0.001% ++++Sendaivirus No 0.01–1% ++++Episomal Possible 0.0001–0.01% +mRNA No >1% ++++

Protein No 0.00001% ++++

Comparison of IPSC derivation technology

Effic

ienc

y

Safety

Lentivirus

Adenovirus

RNA

Protein

Sendai virus

Episomal vector

Patient sample

Primary Cells

Cryopreservation

iPSCs

Reprogramming

Direct differentiation

into specialized cell types

Karyotyping Stem cell markers

Teratoma formation EB differentiation

Gene expression Methylation

MicroRNA profiling Genome engineering

Tissue source: Blood, skin, hair

Methods: Sendai virus Self-replicating RNA vector Protein

1-2 weeks

3-4 weeks

8-12 weeks

Initial Contact the Core

IRB

2-7 weeks

5-15 weeks

1 weeks 2 days

Timeline for iPSC derivation

©20

12 N

atur

e A

mer

ica,

Inc.

All

righ

ts r

eser

ved.

PROTOCOL

NATURE PROTOCOLS | VOL.7 NO.4 | 2012 | 719

the successful reprogramming of mono-nuclear blood cells15–17. In these methods, mononuclear blood cells from donors or frozen samples were infected using retro-virus15 or lentivirus16,17 to express four factors, human OCT3/4, SOX2, KLF4 and c-MYC. In these studies, human T cell reprogramming into iPSCs was achieved, but the efficiency of reprogramming was extremely low (approximately 0.0008–0.01%). Although these methods used less peripheral blood and did not require the pharmacological pretreatment of patients, the problems of transgene genomic insertion and low reprogramming efficiency remained, pre-cluding their wide use in the clinical application of iPSCs. Generating iPSCs with TS-mutated SeV easily erases residual genomic viral RNA from the target cells10, and the method is significantly more efficient (~0.1%) compared with those protocols in which iPSCs were generated from T cells with retrovirus or lentivirus.

Human keratinocytes derived from plucked human hair have also been used as another less invasive method of obtaining iPSCs from patient cells4,18. However, in some cases, these reported methods require several hairs to obtain successful cell outgrowth of keratino-cytes. Dental tissue has also been explored as a potential source of iPSCs19. However, although teeth are routinely removed in many clinics and no further procedures are required with respect to the donor, it is generally difficult to routinely obtain patients’ dental tissues—with specific genetic or nongenetic diseases—for the pur-pose of iPSC studies. In comparison with these outlined methods, our protocol involves harvesting only a small sample of peripheral blood; in addition, T cell proliferation does not need stochastic cell outgrowth. These are clear advantages for clinical application in comparison with the methods reported in the past.

Experimental designBlood sampling. Our protocol is focused on the simple procedure of peripheral venous blood sampling to obtain the donor cells, using a standard process. Patient somatic cells can then be easily and aseptically obtained from the blood sample. In our protocol, 1 ml of whole blood is sufficient to generate TiPSCs (Fig. 2a).

Derivation of activated T cells. Peripheral blood mononuclear cells (PBMCs) can be separated by a Ficoll gradient method from heparinized whole blood samples (Fig. 2b). Although PBMCs contain lymphocytes and monocytes, activation with plate-bound anti-CD3 monoclonal antibody and IL-2 selectively proliferates T cells, and clearly increases the proportion of T cells in the cultured PBMCs11. CD3 protein exists in the complex of TCR proteins on the surface of T cells, and can therefore be used as a T cell–specific marker. Anti-CD3 antibody modulates the TCR-CD3 complex to induce T cell proliferation and activation20, whereas IL-2 also activates general T cell signaling pathways and eventually promotes cytokine transcription, cell survival, cell-cycle entry and growth21. At day 5 of culture with anti-CD3 monoclonal antibody and IL-2, CD3 + cells increased up to ~95% of cultured PBMCs (Fig. 2c–e). With this culture method, users can avoid using a fluorescence-activated cell sorter in which

Blood samplingIsolation of PBMCs

Passagewith cell count

Day 0

IL-2 + anti-CD3 antibody + ESC condition (bFGF +)

T cells beforeactivation

Activation withIL-2 and

anti-CD3 antibody

Oct3/4

Klf4 c-Myc

Sox2

Reprogramming factors

ActivatedT cells

InfectedT cells

Culturingon feeder cells TiPS cells

Day 5 Day 6 Days 20–25

Sendai virusinfection(24 h)

Reseedingonto feeder cells

Picking upTiPS colonies

Figure 1 | Overview of the TiPSC generation protocol. PBMCs are activated for 5 d with IL-2 and anti-CD3 antibody, and then transduced with SeV expressing human OCT3/4, SOX2, KLF4 and c-MYC. TiPSC colonies emerge at 20–25 d after blood sampling.

Beforecentrifuginga b c PBMCs day 0

500 µm 500 µm

PBMCs day 5After

centrifuging d PBMCs day 0CD3-positive

cells 71.2%CD3-positive

cells 95.9%250

200

150

100

50

0

200

150

Cou

nt

Cou

nt

100

50

0102 103

FITC-A104 105 102 103

FITC-A104 105

PBMCs day 5

e 10080

60

40

20

00 3 5

Day

CD

3-po

sitiv

ece

lls (

%)

Figure 2 | Isolation and activation of PBMCs. (a) Whole blood is collected into a 2.5-ml syringe by venipuncture. (b) Before centrifugation, two distinct layers are distinguishable in the 15-ml tube. The upper, dark red layer is the diluted blood and the lower layer is the Ficoll solution. After centrifugation, three distinct layers are apparent. The upper yellow layer contains platelet-rich plasma, whereas the bottom clear layer is the Ficoll solution, and the thin white layer in between contains PBMCs (arrow). (c) Morphology of PBMCs shortly after being seeded in the culture plate and activated using anti-CD3 antibody with IL-2 for 5 d. Proliferated T cells show many clusters in the culture plate at day 5 of activation. (d,e) Flow cytometric analysis of isolated PBMCs and PBMCs activated for 5 d with anti-CD3 antibody and IL-2 gated on the CD45 + cell population. CD3 surface expressions of these populations were examined. The graph represents an average of three independent examinations. Error bars show means ± s.d.

iPSC%medium% hES%medium%

Blood%draw%Isola4on%of%PBMCs%%

Sendai%virus%%infec4on%(24%h)%

Pick%iPS%colonies%

Day%0% Day%9% Day%11% Day%20D23% Day%29D35%

Colonies%emerge%

EM%medium% iPSC%medium%+%Cytokines%

PBMCs%Expansion%

Reseeding%onto%feeder%cells%

Erythroblast+expansion++

CCM#pa&ents#

©20

12 N

atu

re A

mer

ica,

Inc.

All

rig

hts

res

erve

d.

PROTOCOL

NATURE PROTOCOLS | VOL.7 NO.4 | 2012 | 719

the successful reprogramming of mono-nuclear blood cells15–17. In these methods, mononuclear blood cells from donors or frozen samples were infected using retro-virus15 or lentivirus16,17 to express four factors, human OCT3/4, SOX2, KLF4 and c-MYC. In these studies, human T cell reprogramming into iPSCs was achieved, but the efficiency of reprogramming was extremely low (approximately 0.0008–0.01%). Although these methods used less peripheral blood and did not require the pharmacological pretreatment of patients, the problems of transgene genomic insertion and low reprogramming efficiency remained, pre-cluding their wide use in the clinical application of iPSCs. Generating iPSCs with TS-mutated SeV easily erases residual genomic viral RNA from the target cells10, and the method is significantly more efficient (~0.1%) compared with those protocols in which iPSCs were generated from T cells with retrovirus or lentivirus.

Human keratinocytes derived from plucked human hair have also been used as another less invasive method of obtaining iPSCs from patient cells4,18. However, in some cases, these reported methods require several hairs to obtain successful cell outgrowth of keratino-cytes. Dental tissue has also been explored as a potential source of iPSCs19. However, although teeth are routinely removed in many clinics and no further procedures are required with respect to the donor, it is generally difficult to routinely obtain patients’ dental tissues—with specific genetic or nongenetic diseases—for the pur-pose of iPSC studies. In comparison with these outlined methods, our protocol involves harvesting only a small sample of peripheral blood; in addition, T cell proliferation does not need stochastic cell outgrowth. These are clear advantages for clinical application in comparison with the methods reported in the past.

Experimental designBlood sampling. Our protocol is focused on the simple procedure of peripheral venous blood sampling to obtain the donor cells, using a standard process. Patient somatic cells can then be easily and aseptically obtained from the blood sample. In our protocol, 1 ml of whole blood is sufficient to generate TiPSCs (Fig. 2a).

Derivation of activated T cells. Peripheral blood mononuclear cells (PBMCs) can be separated by a Ficoll gradient method from heparinized whole blood samples (Fig. 2b). Although PBMCs contain lymphocytes and monocytes, activation with plate-bound anti-CD3 monoclonal antibody and IL-2 selectively proliferates T cells, and clearly increases the proportion of T cells in the cultured PBMCs11. CD3 protein exists in the complex of TCR proteins on the surface of T cells, and can therefore be used as a T cell–specific marker. Anti-CD3 antibody modulates the TCR-CD3 complex to induce T cell proliferation and activation20, whereas IL-2 also activates general T cell signaling pathways and eventually promotes cytokine transcription, cell survival, cell-cycle entry and growth21. At day 5 of culture with anti-CD3 monoclonal antibody and IL-2, CD3 + cells increased up to ~95% of cultured PBMCs (Fig. 2c–e). With this culture method, users can avoid using a fluorescence-activated cell sorter in which

Blood samplingIsolation of PBMCs

Passagewith cell count

Day 0

IL-2 + anti-CD3 antibody + ESC condition (bFGF +)

T cells beforeactivation

Activation withIL-2 and

anti-CD3 antibody

Oct3/4

Klf4 c-Myc

Sox2

Reprogramming factors

ActivatedT cells

InfectedT cells

Culturingon feeder cells TiPS cells

Day 5 Day 6 Days 20–25

Sendai virusinfection(24 h)

Reseedingonto feeder cells

Picking upTiPS colonies

Figure 1 | Overview of the TiPSC generation protocol. PBMCs are activated for 5 d with IL-2 and anti-CD3 antibody, and then transduced with SeV expressing human OCT3/4, SOX2, KLF4 and c-MYC. TiPSC colonies emerge at 20–25 d after blood sampling.

Beforecentrifuginga b c PBMCs day 0

500 µm 500 µm

PBMCs day 5After

centrifuging d PBMCs day 0CD3-positive

cells 71.2%CD3-positive

cells 95.9%250

200

150

100

50

0

200

150

Cou

nt

Cou

nt

100

50

0102 103

FITC-A104 105 102 103

FITC-A104 105

PBMCs day 5

e 10080

60

40

20

00 3 5

Day

CD

3-po

sitiv

ece

lls (

%)

Figure 2 | Isolation and activation of PBMCs. (a) Whole blood is collected into a 2.5-ml syringe by venipuncture. (b) Before centrifugation, two distinct layers are distinguishable in the 15-ml tube. The upper, dark red layer is the diluted blood and the lower layer is the Ficoll solution. After centrifugation, three distinct layers are apparent. The upper yellow layer contains platelet-rich plasma, whereas the bottom clear layer is the Ficoll solution, and the thin white layer in between contains PBMCs (arrow). (c) Morphology of PBMCs shortly after being seeded in the culture plate and activated using anti-CD3 antibody with IL-2 for 5 d. Proliferated T cells show many clusters in the culture plate at day 5 of activation. (d,e) Flow cytometric analysis of isolated PBMCs and PBMCs activated for 5 d with anti-CD3 antibody and IL-2 gated on the CD45 + cell population. CD3 surface expressions of these populations were examined. The graph represents an average of three independent examinations. Error bars show means ± s.d.

iPSC%medium% hES%medium%

Blood%draw%Isola4on%of%PBMCs%%

Sendai%virus%%infec4on%(24%h)%

Pick%iPS%colonies%

Day%0% Day%9% Day%11% Day%20D23% Day%29D35%

Colonies%emerge%

EM%medium% iPSC%medium%+%Cytokines%

PBMCs%Expansion%

Reseeding%onto%feeder%cells%

Erythroblast+expansion++

CCM#pa&ents#

Passage&3&Passage&0&

ES+like&colonies&

Passage12

iPSCcoloniesinFeeders iPSCsfeeder-free

Pa9ents

RNAvector

Reprogramming blood to iPS cells using a integration-free method

0"0.2"0.4"0.6"0.8"1"

1.2"1.4"1.6"

H7"WT"

24SM"38ED"

40EG1"

26CA3"

14JL2"

25DN5"

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14JL:PBMCs"

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14JL246,XX

09July2014

iPSC1&CCM3+/&+Karyotype

Teratomaforma9on

Quality assurance: characterization of iPSCs

Alkaline phosphatase OCT4

SSEA-4 TRA-1-61

NANOG SOX2

Immunofluorescence of pluripotency markers

qRT-PCR of pluripotency markers

AdrianaBeltran,BryanRichardson

Mesoderm EndodermEctoderm

SMA

CDX2 SOX17

FOXA2NESTIN

PAX6

Differentiation of hES and IPS into three germ layers






ASD pa!ent



Animalmodel

Gene engineering

Diseasemodeling


Drugdiscovery

Celltherapy

iPSC

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Human pluripotent stem cell services

Cell derivation and characterization: •  IPSC Reprogramming and characterization •  hES/hiPS Differentiation •  hES/hiPS Maintenance, expansion and banking Genome engineering: • Genome editing of mammalian cells (hESC, iPSC, CS) •  Engineering effector molecules for gene regulation Training in stem cell culture techniques

/ http://www.sciencemag.org/content/early/recent / 03 January 2013; / Page 4/ 10.1126/science.1232033

Fig. 1. Genome editing in human cells using an engineered type II CRISPR system. (A) RNA-guided gene targeting in human cells involves co-expression of the Cas9 protein bearing a C terminus SV40 nuclear localization signal with one or more guide RNAs (gRNAs) expressed from the human U6 polymerase III promoter. Cas9 unwinds the DNA duplex and cleaves both strands upon recognition of a target sequence by the gRNA, but only if the correct protospacer-adjacent motif (PAM) is

20GG can in principle be targeted. (B) A genomically integrated GFP coding sequence is disrupted by the insertion of a stop codon and a 68bp genomic fragment from the AAVS1 locus. Restoration of the GFP sequence by homologous recombination (HR) with an appropriate donor sequence results in GFP+ cells that can be quantitated by FACS. T1 and T2 gRNAs target sequences within the AAVS1 fragment. Binding sites for the two halves of the TAL effector nuclease heterodimer (TALEN) are underlined. (C) Bar graph depicting HR efficiencies induced by T1, T2, and TALEN-mediated nuclease activity at the target locus, as measured by FACS. Representative FACS plots and

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To evaluate the spreading of DNAme in the SOX2 locus after theinitial induction, we analyzed two additional amplicons (ampliconI and III) flanking the 6ZF-binding site, encompassing ~ 1 kbps up-and downstream of the translation start site (Figures 2c and d).Sequencing analysis of amplicon I (−1069 to − 623 bps upstreamof the translation start site) demonstrated an increase of DNAmeupon ZF598-DNMT3A expression as compared with the MCF7empty vector transduced cell line or ZF598-DNMT3A-E74A-transduced cells. The frequencies of DNAme in amplicon I weresignificantly higher than those immediately adjacent to the ZF-binding site, suggesting that DNAme was possibly spread andeven reinforced in the flanking regions. Most importantly, afterdiscontinuation of the ZF598-DNMT3A expression (Dox removal),the de novo methylation additionally increased up to 97% atspecific CpG sites in Amplicon I (Figure 1c).As last, the MassARRAY analysis of DNAme in amplicon III (+695

to +1055 bps downstream of the translation start site, Figure 2d)revealed no methylation in MCF7 control and ZF598-DNMT3A cellsin the absence of Dox. Upon ZF598-DNMT3A expression (+Dox)CpG methylation increased up to 80%, and no DNAme wasinduced in cells expressing the ZF598-DNMT3A-E74A mutant. Ourtime-course analysis suggests that the de novo DNAme induced bythe artificial methyltransferase results in both, a phase of inductionof gene silencing upon Dox induction, and a phase ofmaintenance and reinforcement of silencing (Dox removal), which

could be associated with propagation or spreading of DNAmefurther away from the 6ZF site during DNA replication. Theseresults further support the increased downregulation of SOX2expression after Dox removal previously observed by quantitativereverse transcription polymerase chain reaction and western blotanalysis and outline the importance of CpG dinucleotides flankingthe core promoter for the regulation of SOX2 expression.

ZF598-DNMT3A expression confers anticancer memory andreduces tumor growth in a breast cancer xenograft in NUDE miceTo analyze whether the ZF598-DNMT3A construct inducedphenotypic memory in vivo we took advantage of our inducibleMCF7 cell lines stably transduced with either the catalyticallyactive ZF598-DNMT3A or the empty vector control. A total of2× 106 MCF7 cells transduced with either ZF598-DNMT3A orcontrol were implanted into the flank of NUDE mice and allowedto grow for 22 days before switching the animals to a Dox-containing diet (Figure 3a). Within each group N= 5 animals weremaintained in a Dox-free diet. At day 19 post induction, half of theZF598-DNMT3A injected animals from the +Dox group wereremoved from the Dox diet (N= 10), to withdraw the expression ofthe ZF methyltransferase (R). In contrast with ZF598-DNMT3A,catalytic dead ZF598-DNMT3A-E74A mutant cells failed to reduce

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Figure 1. Stable downregulation of the SOX2 expression by ZF598-DNMT3A. (a) Schematic illustration of the SOX2 promoter indicating thedomain structure of the ZF598-DNMT3A construct, its binding site and the three amplicons analyzed by sodium bisulfite sequencing orMassARRAYs (amplicon I, blue; II, purple and III, green). (b) Quantification of SOX2 mRNA expression by qRT-PCR in MCF7 cells. Cells werestably transduced with empty vector, ZF598-SKD and ZF598-DNMT3A. The expression of the ZF fusion was controlled by addition orremoval of doxycycline (Dox); R=Dox removal. Cells were induced with Dox every 48 h and either harvested at 72 h after first induction(+Dox) or removed from Dox and subcultured for 8 days and processed for western blot. Error bars represent standard deviation (s.d.)(***Po 0.001, **Po0.01, *Po0.05). (c) Detection of SOX2 by western blot. The C-terminal Hemagglutinin (HA) tag was used forimmunodetection of the ZF proteins. An anti-histone H3 antibody was used as loading control. (d) Cell viability of MCF7 cells assessed by CellTiterGlo assays. Empty vector, ZF598-SKD and ZF598-DNMT3A-transduced cells were induced with Dox for 48 h and removed from Dox after72 h (red arrow). P-values between empty vector and ZF598-DNMT3A and between ZF598-SKD and ZF598-DNMT3A were Po0.001 andPo0.05 respectively, at 144 h.

Programmable oncogenic silencing in breast cancerS Stolzenburg et al

5429

© 2015 Macmillan Publishers Limited Oncogene (2015) 5427 – 5435

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Figure 2. Expression of the ZF598-DNMT3A induces targeted DNA methylation in the SOX2 promoter. (a) Sodium bisulfite-sequencing analysisof DNA derived from MCF7 cells stably transduced with empty vector, ZF598-DNMT3A and ZF598-DNMT3A-E74A mutant and induced withDox. The analyzed amplicon II expands from − 654 to − 279 base pairs (bps) upstream the translation start site and includes the 6ZF-bindingsite (position − 598 relative to the translation start site). Gray circles indicate not analyzed methylation values owing to CpGs with high- or low-mass Dalton peaks falling outside the conservative window of reliable detection for the EpiTYPER software, colored circles indicate variouslymethylated CpGs. (b) MassARRAY analysis of the same amplicon as in (a). Circles indicate the CpG dinucleotides in the amplicon (Color code:yellow=unmethylated CpG to blue= 100% methylated CpG). The percentage of meCpG was determined in MCF7 control-transduced cellsand in MCF7 cells stably transduced with ZF598-DNMT3A or ZF-598-DNMT3A-E74A in absence of Dox (no Dox), presence of Dox (+Dox) orafter Dox removal (R, eight days). (c) MassARRAY analysis of amplicon I (−1069 to − 623 bps upstream of the translation start site) in Dox-freeconditions (no Dox) and after Dox induction (+Dox) Dox removal (R). (d) MassARRAY analysis of amplicon III (+695 to +1055 bps) downstreamof the translation start site, same conditions as above.

Programmable oncogenic silencing in breast cancerS Stolzenburg et al

5430

Oncogene (2015) 5427 – 5435 © 2015 Macmillan Publishers Limited

Targeted silencing of the oncogenic transcriptionfactor SOX2 in breast cancerSabine Stolzenburg1,2, Marianne G. Rots1, Adriana S. Beltran2, Ashley G. Rivenbark2,3,Xinni Yuan2, Haili Qian4, Brian D. Strahl3,5 and Pilar Blancafort2,5,*

1Epigenetic Editing, Department of Pathology and Medical Biology, University Medical Center Groningen,University of Groningen, Hanzeplein 1, 9713 GZ Groningen, The Netherlands, 2Department of Pharmacology,3Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, NC 27599, USA,4State Key Laboratory of Molecular Oncology, Cancer Hospital/Institute, Chinese Academy of MedicalSciences, Pan Jia Yuan Nan Li 17, Chaoyang District, Beijing 100021, P.R. China and 5UNC LinebergerComprehensive Cancer Center, University of North Carolina, Chapel Hill, NC 27599, USA

Received January 17, 2012; Revised March 22, 2012; Accepted April 11, 2012

ABSTRACT

The transcription factor (TF) SOX2 is essential forthe maintenance of pluripotency and self-renewalin embryonic stem cells. In addition to its normalstem cell function, SOX2 over-expression is asso-ciated with cancer development. The ability to se-lectively target this and other oncogenic TFs in cells,however, remains a significant challenge due to the‘undruggable’ characteristics of these molecules.Here, we employ a zinc finger (ZF)-based artificialTF (ATF) approach to selectively suppress SOX2gene expression in cancer cells. We engineeredfour different proteins each composed of 6ZFarrays designed to bind 18bp sites in the SOX2promoter and enhancer region, which controlsSOX2 methylation. The 6ZF domains were linkedto the Kruppel Associated Box (SKD) repressordomain. Three engineered proteins were able tobind their endogenous target sites and effectivelysuppress SOX2 expression (up to 95% repressionefficiencies) in breast cancer cells. Targeteddown-regulation of SOX2 expression resulted indecreased tumor cell proliferation and colony for-mation in these cells. Furthermore, induced expres-sion of an ATF in a mouse model inhibited breastcancer cell growth. Collectively, these findingsdemonstrate the effectiveness and therapeuticpotential of engineered ATFs to mediate potentand long-lasting down-regulation of oncogenic TFexpression in cancer cells.

INTRODUCTION

Transcription factors (TFs) are crucial molecules orches-trating gene programs involved in self-renewal, differenti-ation and organism’s developmental patterning.Maintaining the proper threshold of expression of TFs iscritical for the normal homeostatic function of cells andtissues. Aberrant regulation of TF expression is frequentlyfound in human malignancies and associated with specifictumor subtypes (1). Over-expression of oncogenic TFs iswell documented in the mammary gland, particularly inpoorly differentiated, triple negative breast cancers(TNBCs) (2). TNBCs are characterized by the lack of ex-pression of Estrogen Receptor (ER!), ProgesteroneReceptor (PR!) and Epidermal Growth Factor Receptor2 (Her2!). Recent progress revealed that some TNBCsbelonging to the basal-like and claudin-low intrinsicsubtypes of breast cancers are highly aggressive and resist-ant to treatment (3–5). It has been proposed that thesebreast cancers are enriched in stem cells, which might becritical for tumor initiation, progression and resistance tochemotherapy and radiation (6–11). Albeit their funda-mental role in tumor etiology and progression, TFs arecurrently refractory to target-based drug discoveryapproaches due to their lack of small molecule bindingpockets. Thus, novel strategies are required to efficientlysilence the aberrant expression of oncogenic TFs in cancercells. Ideally these novel approaches should restore andstably maintain the expression pattern of these TFs, likeit is observed in normal epithelial cells.The SOX2 gene encodes a TF belonging to the

high-mobility group (HMG) family (12). SOX2 expressionis critical for the maintenance of self-renewal in embryonicstem cells (ESCs) and neural progenitor cells (13–15).

*To whom correspondence should be addressed. Tel: +1 919 966 1615; Fax: +1 919 966 5640; Email: [email protected]

Published online 4 May 2012 Nucleic Acids Research, 2012, Vol. 40, No. 14 6725–6740doi:10.1093/nar/gks360

! The Author(s) 2012. Published by Oxford University Press.This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

from ATCC (American Type Culture Collection,Manassas, VA, USA) maintained at 37!C and 5% CO2.

ATF construction

The ZF target sites within the SOX2 promoter were se-lected using the website www.zincfingertools.org (42). Theselection of three 18-bp target sites was based on the closeproximity to the transcriptional start site and the highcontent of GNN-triplets in the target sequence. OneATF was designed to target an 18-bp sequence in theSOX2 enhancer region 1, "4-kb upstream of the TSS(Figure 1B). Specific primers were designed coding forthe amino acids in the recognition helix of the ZFs respon-sible for the binding to the target sequence (Figure 1C).The ZF proteins were generated by overlapping PCRas described (43), SfiI-digested fragments were subclonedinto the retroviral vector pMX-IRES-GFP-SKD,generating pMX-ZF552SKD, pMX-ZF598SKD, pMX-ZF619SKD and pMX-ZF4203SKD. Each ATF containsan internal SV40 nuclear localization signal (NLS) and

a terminal hemagglutinin (HA) decapeptide tag. Thecorrect ZF-sequence of the obtained product was con-firmed by plasmid sequencing.

Retrovirus infection of MDA-MB-435s

The pMX retroviral vectors containing the SOX2-ATFswere first co-transfected with the plasmid (pMDG.1) ex-pressing the vesicular stomatitis virus envelope proteininto 293TGagPol cells to produce retroviral particles.Transfection was performed using LipofectamineTM

(Invitrogen, Carlsbad, CA) as recommended by the manu-facturer. For cell proliferation and soft agar assays, cellswere harvested 24 h after the last infection. For flowcytometry analysis, protein, and mRNA extraction,transduced cells were harvested 48 h post-infection.

siRNA transfection

MDA-MB-435s breast cancer cells were transfected witheither a SOX2-specific siRNA pool (siGENOMED-011778-01-04), an irrelevant (non-specific) siRNA

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Figure 1. Design of ATFs to down-regulate SOX2 expression. (A) Schematic representation of a 6 ZF ATF bound to DNA with the orientation ofthe domains depicted. (B) Schematic illustration of the SOX2 promoter outlining the ZF-552SKD, ZF-598SKD, ZF-619SKD and ZF-4203SKDtargeted sequences and their location relative to the transcription start site (TSS). Highlighted are the core promoter (red), regulatory region 1(green), and regulatory region 2 (blue). Arrows show the orientation of the 18-bp binding site in the promoter (from 50 to 30). (C) Alpha-helical ZFamino acid sequences chosen to construct the ATFs. Residues at position –1, +3 and+6 making specific contacts with the recognition triplets areindicated in color (red refers to position –1, blue to position+3 and green to+6 of the ZF recognition helix). (D) Quantification of SOX2 expressionin 12 breast cancer cell lines by western blot.

Nucleic Acids Research, 2012, Vol. 40, No. 14 6727

in tumors derived from empty vector control (data notshown). In contrast, the ZF-598SKD +Dox tumors ex-hibited a more organized tissue with increased amountof intervening stroma separating small islands of tumorcells (Figure 6E, right panel). In addition, immunofluor-escence analyses of the tumor sections demonstrated thatthe ZF proteins were expressed in the nucleus of themajority of tumor cells in ZF-598SKD +Dox animals,but not in un-induced animals (Figure 7A) or controls(data not shown). This induction of ZF expression was

accompanied by a decreased nuclear SOX2 staining(Figure 7A), and by a decreased proliferation of thetumor cells, as indicated by a Ki67 staining of thetumor sections (Figure 7B). In summary, our in vivoanalyses indicated that the tumor suppressive functionsof the engineered silencers were maintained afterlong-term inoculation of the tumor cells, resulting inthe maintenance of the SOX2 down-regulation anddecreased tumor cell proliferation in animal models ofbreast cancer.

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Nucleic Acids Research, 2012, Vol. 40, No. 14 6735

OPEN

ORIGINAL ARTICLE

Stable oncogenic silencing in vivo by programmable andtargeted de novo DNA methylation in breast cancerS Stolzenburg1,2, AS Beltran2, T Swift-Scanlan3, AG Rivenbark4, R Rashwan1 and P Blancafort1,2

With the recent comprehensive mapping of cancer genomes, there is now a need for functional approaches to edit the aberrantepigenetic state of key cancer drivers to reprogram the epi-pathology of the disease. In this study we utilized a programmableDNA-binding methyltransferase to induce targeted incorporation of DNA methylation (DNAme) in the SOX2 oncogene in breastcancer through a six zinc finger (ZF) protein linked to DNA methyltransferase 3A (ZF-DNMT3A). We demonstrated long-lastingoncogenic repression, which was maintained even after suppression of ZF-DNMT3A expression in tumor cells. The de novo DNAmewas faithfully propagated and maintained through cell generations even after the suppression of the expression of the chimericmethyltransferase in the tumor cells. Xenograft studies in NUDE mice demonstrated stable SOX2 repression and long-term breasttumor growth inhibition, which lasted for 4100 days post implantation of the tumor cells in mice. This was accompanied with afaithful maintenance of DNAme in the breast cancer implants. In contrast, downregulation of SOX2 by ZF domains engineered withthe Krueppel-associated box repressor domain resulted in a transient and reversible suppression of oncogenic gene expression. Ourresults indicated that targeted de novo DNAme of the SOX2 oncogenic promoter was sufficient to induce long-lasting epigeneticsilencing, which was not only maintained during cell division but also significantly delayed the tumorigenic phenotype of cancercells in vivo, even in the absence of treatment. Here, we outline a genome-based targeting approach to long-lasting tumor growthinhibition with potential applicability to many other oncogenic drivers that are currently refractory to drug design.

Oncogene (2015) 34, 5427–5435; doi:10.1038/onc.2014.470; published online 16 February 2015

INTRODUCTIONCytosine methylation in mammalian DNA is regarded as a keyepigenetic modification controlling essential processes such asimprinting, silencing of retrotransposons and cell differentiation.1

In addition to DNA methylation (DNAme), repressive histone post-translational modifications reinforce gene inactivation, resulting inthe formation of inactive chromatin, namely heterochromatin.2

Importantly, the epigenetic information must be stably trans-mitted during mitosis for the proper maintenance of cellularidentity, a process referred as epigenetic memory.3 In pathologicalstates such as cancer, the epigenetic landscape of normal cellsbecomes disrupted. Aberrant incorporation or removal of DNAmein cancer cells leads to genome-wide alteration of geneexpression, including inactivation of tumor suppressor genesand reactivation of oncogenes.4 Notably, changes in DNAmepatterns are characteristic hallmarks of the distinct intrinsicsubtypes of breast cancer. Poorly differentiated, highly prolifera-tive basal-like breast cancers associated with stem/progenitor cell-like features are significantly hypomethylated relative to the otherbreast cancer subtypes. Importantly, these tumors are driven byaberrant activation of multiple developmental transcriptionfactors (TFs), which fuel the tumor with sustained proliferation,drug resistance and metastatic capacity.5–7

The High Mobility Group oncogenic TF SOX2 is normallyexpressed in embryonic stem cells and neural progenitor cells,

where it maintains self-renewal.8,9 DNAme in the SOX2 promoterand enhancer regions functions as an epigenetic switch, whichforces cells to activate multiple differentiation pathways.10 SOX2 istherefore not expressed in most normal adult tissues.10–12

Moreover, aberrant reactivation of SOX2 has been detected in~ 43% of basal-like breast cancers and in several other malig-nancies, including glioblastoma, lung, skin, prostate and ovariancarcinomas.13 SOX2 overexpression in tumor specimens has beenassociated with both promoter hypomethylation relative toadjacent normal tissue and copy number amplifications.14,15 Theoverexpression of SOX2 in breast cancer has been shown todirectly activate CYCLIN D1, resulting in an increased mitotic indexand proliferation.16,17 The downregulation of SOX2 by RNAinterference decreased the tumorigenic phenotype in the lung,breast and ovarian cancers.13,16,18 However, a major limitation ofsmall interferin RNA and small hairpin RNA approaches in cancertherapy has been the short half-life of small RNA or themethylation of the virally encoded small hairpin RNA promoter,which results in transient effects in vivo.In light of the essential role of DNAme in the regulation of SOX2

expression we hypothesized that targeted de novo methylation inthe SOX2 promoter would result in an epigenetic 'off' switch,forcing cancer cells to undertake differentiation programs.Furthermore, because DNAme is read and written by endogenousproteins and faithfully transmitted during cell division, we

1Cancer Epigenetics Group, The Harry Perkins Institute of Medical Research, Western Australia & School of Anatomy, Physiology and Human Biology, M309, The University ofWestern Australia, Nedlands, Western Australia, Australia; 2Department of Pharmacology, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA;3Lineberger Comprehensive Cancer Center/School of Nursing, University of North Carolina, Chapel Hill, NC, USA and 4Department of Pathology and Laboratory Medicine,University of North Carolina School of Medicine, Chapel Hill, NC, USA. Correspondence: Professor P Blancafort, Cancer Epigenetics Group, The Harry Perkins Institute of MedicalResearch, Western Australia & School of Anatomy, Physiology and Human Biology, M309, The University of Western Australia, 6 Verdun Street Nedlands, Nedlands, WesternAustralia 6009, Australia.E-mail: [email protected] 14 July 2014; revised 15 November 2014; accepted 9 December 2014; published online 16 February 2015

Oncogene (2015) 34, 5427–5435© 2015 Macmillan Publishers Limited All rights reserved 0950-9232/15

www.nature.com/onc

TheStemCellCoreFacilityoffersaone-to-onetraining

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•  Introduc9ontostemcellbiology•  MorphologyofhESandhiPSCs•  Mediaprepara9on•  Feederprepara9on•  Feeder-freeECMcoa9ng•  PassaginghESandhiPSCsbyenzymeandmanualpicking•  Freezing/thawinghiPSCs

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Going forward

Take home message The Human stem cell core can support your stem cell studies by: 1.  Reprogramming somatic cells into iPS cells. 2.  Differentiate hES and iPS cells into specialized cell types. 3.  Develop protocols for directed differentiation. 4.  Design and engineer gene editing tools for:

•  Making isogenic iPS cells. •  Gene knockouts. •  Deletion of long enhancers and/or short regulatory

elements. •  Making fluorescent reporter lines.

5.  Design and engineer synthetic transcription factors for gene regulation

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