Animai Science Papers and Reports vol. 22 (2004) Supplement l, 119-126Institute of Genetics and Animai Breeding, Jastrzębiec, Poland

Presented at the Conjerence"Somatie cloning as a tool in animai biotechnology"held at the "ANIMBIOGEN" Cen/re oj Excellencein Genomics and Biotechnology lmproving FunctionalTraits in Farm Animals and Quality ojtheir Products,18-19 January 2004, WIerzba, Poland

Genetic manipulation of human embryonic stem cells

Jim McWhir*, Alex Didomenico, Zoe Hewitt,Helen Priddle1, Alison Thomson

Department ofGene Expression and DevelopmentRosIin Institute, Rosiin, Midlothian EH 2S 9PS, UK

Human ES cells have the potential to develop into all cell types, providing both a model of humandevelopment and a source of therapeutically useful cells.Although the methods for their transfectionare less robust than with their murine equivalents, genes can now be added and existing genesmodified by gene targeting, Genetic modification of hES cells will allow us to identify, purify andamplify progenitor cells for many lineages and may be brought to bear upon problems of immunerejection and enhanced vasculartsation of grafted rissue,

KEY WORDS: embryonic stem cells I genetic manipulation I human I progenitor cells Iregenerative medicine

Embryonic stem (ES) cells are permanent cell lines isolated from explantedblastocysts [Evans and Kauffman 1981, Martin 1981] and share an unusual set ofproperties with the stem cells of germ cell tumours (EC cells). They can be cultured

"e-mail: jim.mcwhir@bbsrc.ac.uk'Present address: School ofHuman Development, Obstetrics & Gynaecology, D Floor East Block,Queens Medical Centre, Nottingham NG7 2UH, UK

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under conditions in which they cycle indefinitely, or under altered conditions in whichthey terminally differentiate into a variety of specialized ceIls. When undifferentiatedmurine ES (mES) or EC cells are returned to the preimpIantation embryo they resumeanormai programme of development and give rise to chimaeric animaIs. However,only mES cells give rise to germ-line contributions. In mice the importance of mEScells has been to effect precise genetic changes in transgenie animals by introducingmodified ES cells to the blastocyst. However, in vitro differentiation of mES cells alsosuggests therapeutic applications for ES cells in the treatment of degenerative and meta-bolic disease.

The potential role for ES cells in regenerative medicine has become a matter ofintensive study since the isolation of the first human ES (hES) cell lines in 1998[Thomson et al. 1998, Reubinoff et al. 2000]. Evidence is rapidly accumulating that,like mES cells, differentiation ofhuman ES cells gives rise to a wide range of differen-tiated ceIl types [reviewed by McWhir et al. 2003]. The premise ofthis presentation isthat the development of procedures for long-term functional engraftment of hES-de-rived cells in clinical applications can be greatly enhanced by genetic modification.Reliable gene addition and gene targeting can now be achieved with hES cells and thisis illustrated beIow by examples both from the literature and from work in progress inour own laboratory.

Why modify hES cells?

Protection against tumorigenesis

Ensuring the destruction of potentially tumorigenie undifferentiated hES cells from aheterogenous population of differentiated cells can be achieved using the expression ofnegatively selectable markers from an undifferentiated hES cell-specific promoter. 'Sui-cide' gene therapy has already been described for treating cancer [Freytag et al. 2002].Adapting this approach for hES cells does not require targeted delivery in vivo and hEScells have been modified to constitutively express the thymidine kinase gene such thatapplication of the prodrug gancyclovir causes ablation of hES cells in vitro and theirderivatives in vivo [Schuldiner et al. 2003]. In a slightly different approach we have modi-fied hES cells to express the xenotransplantation antigen galactosyl transferase under thecontrol ofthe ES-specific hTERT promoter (Priddle, Hewitt and McWhir, unpublished).The success of this altemative approach is presently under investigation.

Prevention of rejection

Rejection of stem cell-derived grafts by an allogenic host could be circumventedin a number of ways [reviewed by Bradley et al. 2002]. The most practical solutionsmay be either to render the hES cell and its derivatives 'invisible' to the immunesystem or to render the immune system tolerant to the graft. The former may be possi-ble by genetic modification of those loei of the hES ceIl genome responsible forimmunogenicity. Strategies of this type however, may entail heightened risk of viral

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infection and tumorigenesis. Tolerization approaches that leave the nomlal immunercsponse unchanged are intuitively much more attractive.

Enhancing differcntiarion Ol' cngrnfrmcnt

Understanding the pathways involved in commitment to a specific lineage can beaided by modification of genes in mousc ES cells folIowed by in vitro differentiationto determine their role in development [e.g. Hidaka et al. 1999]. A similar approachhas been employed to study thc undifferentiated statc in 1ll0USC ES celi s [e.g. Niwa et al.2000]. Similar studies with human ES cclls will both cnlighten human dcvelopmentalprocesses and enable celi biologists to manipulate gene pathways to improve differentia-tion to required celi types. Transgenes can be used to uprcgulate pathways that promotecommitment to the desired Iineage [e.g. Kyba et al. 2002], to provide a lineage-specificmarker to assist in the purification of a defined celi population [e.g. Ying et al. 2003] or toprovide factors that enhance vasculogenesis such as VEGF [e.g. Dei Rio et al. 99].

Models of human discase

Many disease conditions result in the deterioration oftissues due to specific, knowngenetic lesions. For ex ample, Lesch Nyhan syndrome is caused by dysfunction at thehypoxanthine phosphoribosyltransferase (HPRT) loeus; Cystic Fibrosis is caused bylesions in the Cystic Fibrosis transconductance regulator gene (CFTR). Others such asmotoneuron disease (ALS) have associations with mutations (SODl for ALS) that,though not directly causal, place carriers at heightened risk. Human ES celi s carryingthese mutations could, in principle, be obtained from embryos carrying these lesions,but the logistics ofidentifying such embryos and then isolating ES Iines is daunting. Insome cases it would be far easier to generate those lesions in existing hES celi s bygene targeting, although for the more common lesions for which pre-implantation ge-netic screening is perfonned routinely, it may be possible to isolate hES celi s directlyfrom screened/donated embryos.

Procedures for transfection of hES celis

Additive rransgemcs

Human ES celi s can be transfected by lipofection and electroporation [reviewedby Priddle 2004], adenovirus and adeno-associated virus [Smith Arica et al. 2003],retroviruses and lentivirus [Ma et al. 2003]. In general transfection efficiencies are notas high as with mES celi s and this probably reflects the still less than optimal defini-tion of conditions for the culture and transfection of hES cells. Many hES celi Iinescannot be enzymatically disaggregated to a single celi suspension and exhibit lowcloning efficiencies that in tum are reflected in low transfection rates. The extent towhich this is problematic varies from laboratory to laboratory and from line to line andit appears that some hES lines can in at least some hands, be adapted to enzymaticdisagregation.

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Although random integration of transgenes is the simplest method of gene intro-duction it is frequently associated with variation in transgene expression due to ele-ments at the site ofintegration, and with position effect variegation in which stochasticevents at the integration site lead to gene silencing in a proportion of transgenie cells.The most elegant way to introduce transgenes is by directing their integration to aspecific site in the genorne with predictable effect. Figure l shows examples of GFPtransgene expression in two random sites in the hES genome. Clones with high andlow levels of position effect variegation (pev) are contrasted by FACS and immuno-histological analyses in Figure l.

2

Fig. 1. FACS analysis (left) ofhES clones expressing galactosyl transferase transgene with, respectively,low position effect variegation (top) and high position effect variegation (bottom). Right hand pancis arestained with galT-specific fluorescent lectin (FITC-BS-IB)4

Gcnc targcting

Gene targeting in hES cells has been demonstrated at the hypoxanthinephosphoribosyl transferase (HPRT) locus and at the Oct4 locus [Zwaka and Thomson2003]. The HPRT locus is particularly useful as a model target for the development oftargeting systems, as it is possible in male celi s (HPRT is X-linked), to select directlyfor loss of function arising as a consequence of homologous recombination. For tar-geting HPRT, Zwaka and Thomson used a targeting construct containing 12 kb ofhomology, achieving a transfection efficiency of 2.3Xl OE-5and a targeting frequency(measured as a proportion of transfectants) of 2%. In our own laboratory we haveobtained 6tg-resistant colonies in hES cells using a smaller HPRT targeting vectorcontaining only 6 Kb ofhomology (Fig. 2) and, perhaps predictably, observed a lower

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l,

A LoxM LoxP

Hlndlll Hindlll

6.9 Kb L....- _

HPRT 2 3

LoxM LoxP

B

IIl0m (lG141) BuoHI (lUlG)BuoHI(U'II)

•.Fig. 2. Structures ofthe HPRT targeting construct (A) and targeled locus (8). Lox M and Lox P sites aredesigned to facilitate subsequent introduction by Cre-mediated recombination of any transgene tlanked bythe same incompatible lox sites.

L2Kb 3.9Kb UKb•... .... ..Sm:U Sm:U

Purified 6.7 kb peR pro duet

BamHI DamHI+---------------.. .-----.SKIt -1.7 KIt

Lane l: BamHI 1'2Lane 2: BamHI M2

Lane 3: Uncut 1'2PCR pruduetLane 4: Smal 1'2Lane 5: Smal M2Lane 6:UDCutM2 PCRproduct

Fig. 3. Restriction digest of an RT-PCR band generated with one primer within the selectable marker and asecond outwith the region of homology showing the predicted band sizes.

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6TGR clones 1-18kb ;;::::;:==::::=::::;::;:;;::-::::==-:==:::;:;:::;;;::::;;;;;:;23- fi.•

9.4-6.6-4.4 -

2.3-

wt HPRT IOCUS: exo=sn2 exo~ I I Nco I: >20 kb

targeted locus r----- : __N_---,I Nco I: 2.9 kbIN prObe N

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Male human ES cells transduced with AAV-HPe3PN

Fig. 4. Southern ana1ysis of 6TG-resistant hES cells following transduction with AAV-based targetingvector, showing correct targeting in 14 of 18 clones (2.5 Kb diagnostic band).

proportion of 6TG-resistant colonies of 0.2% [DiDomenico and Me Whir, unpublished].Preliminary RT-PCR data are presented in Figure 3 and are still to be formally eon-firmed by Southern.

In collaboration with David Russell ofthe University ofWashington we have alsodemonstrated the use of AAV-based vectors for gene targeting in hES cells (Thomson,Priddle, Russell and McWhir, unpublished) - Figure 4. For reasons not yet well under-stood the use of AAV vectors allows targeting at much higher frequency than withplasmid-based vectors, though we do not yet achieve the very high frequencies previ-ously reported for human somatic cells [Hirata et al. 2002].

Conclusions

Although there are concerns about the therapeutic use of genetically modifiedcells there is much that we can learn about the processes of differentiation and aboutthe characteristics of progenitor cells by applying genetic modification to hES cells. Itmay be that the use of these cells as a tool for basie science is ultimately as, or evenmore important, than their direct clinical application. Several of the major challenge sto the development of regenerative medicine can all be addressed using transgenie

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approaches: purification of progenitors (by engineering lineage-specific markers), am-plification of progenitors (by lineage-restricted telomerization), achieving long termengraftment (inducible expression of angiogenic factors), and generating hES cellscarrying specific alle1es (gene targeting). The technology has now developed to thepoint where all of these possibilities can be and are being explored.

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15. SMITH-ARICA 1., THOMSON A, ANSELL R., CHIORINI 1., DAVIOSON B., MCWHIR J.,2003 - Infection efficiency of human and mouse embryonic stem cells using adenoviral and adeno-associated virai vectors. Cloning and Steni Cells 5 (I), 51-62.

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Jim McWhir, Alex Didomenico, Zoe Hewitt,Helen Priddle, Alison Thomson

Manipulacje genetyczne na pierwotnychkomórkach zarodkowych człowieka

Streszczenie

Ponieważ ludzkie pierwotne komórki zarodkowe (hES cells)mają zdolność do różnicowania się wewszystkie rodzaje komórek są one zarówno modelem do badań rozwoju czlowieka, jak i źródlem komórekterapeutycznych. Chociaż metody ich transfekcji nie sa tak dobrze opracowane jak w przypadku mysichpierwotnych komórek zarodkowych, tym niemniej można już do nich wprowadzać obce geny, jak równieżpreprowadzać modyfikacje ich własnych genów metodą gen e targeling. Genetyczne modyfikacjepierwotnych komórek zarodkowych czlowieka pozwolą nam zarówno na identyfikację, izolację oraznamnażanie komórek progenitorowych wielu rodzajów komórek jak również pomogą, być może, wrozwiązaniu wielu problemów dotyczących odrzucania przeszczepów oraz możliwości zwiększenia ichukrwienia.

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