use of genetically modified stem cells

INTRODUCTION

Gene therapy is a novel therapeutic branch ofmodern medicine. Its emergence is a directconsequence of the revolution heralded by the

introduction of recombinant DNA methodology in the1970s. Gene therapy is still highly experimental, buthas the potential to become an important treatmentregimen. In principle, it allows the transfer of geneticinformation into patient tissues and organs. Conse-quently, diseased genes can be eliminated or theirnormal functions rescued. Furthermore, the procedureallows the addition of new functions to cells, such asthe production of immune system mediator proteinsthat help to combat cancer and other diseases.

Originally, monogenic inherited diseases (those causedby inherited single gene defects), such as cystic fibrosis,were considered primary targets for gene therapy.For instance, in pioneering studies on the correctionof adenosine deaminase deficiency, a lymphocyte-associated severe combined immunodeficiency (SCID),was attempted.1 Although no modulation of immunefunction was observed, data from this study, togetherwith other early clinical trials, demonstrated the potentialfeasibility of gene transfer approaches as effective

therapeutic strategies. The first successful clinical trialsusing gene therapy to treat a monogenic disorderinvolved a different type of SCID, caused by mutationof an X chromosome-linked lymphocyte growthfactor receptor.2

While the positive therapeutic outcome was celebratedas a breakthrough for gene therapy, a serious drawbacksubsequently became evident. By February 2005, threechildren out of seventeen who had been successfullytreated for X-linked SCID developed leukemia becausethe vector inserted near an oncogene (a cancer-causinggene), inadvertently causing it to be inappropriatelyexpressed in the genetically-engineered lymphocytetarget cell.3 On a more positive note, a small number ofpatients with adenosine deaminase-deficient SCID havebeen successfully treated by gene therapy without anyadverse side effects.4

A small number of more recent gene therapy clinicaltrials, however, are concerned with monogenicdisorders. Out of the approximately 1000 recordedclinical trials (January 2005), fewer than 10% targetthese diseases (see Figure 4.1). The majority of currentclinical trials (66% of all trials) focus on polygenicdiseases, particularly cancer.

Gene therapy relies on similar principlesas traditional pharmacologic therapy;specifically, regional specificity for thetargeted tissue, specificity of theintroduced gene function in relation todisease, and stability and controllabilityof expression of the introduced gene.To integrate all these aspects into asuccessful therapy is an exceedinglycomplex process that requires expertisefrom many disciplines, includingmolecular and cell biology, geneticsand virology, in addition to bioprocessmanufacturing capability and clinicallaboratory infrastructure.

45

4. USE OF GENETICALLY MODIFIED STEM CELLSIN EXPERIMENTAL GENE THERAPIES

by Thomas P. Zwaka*

Figure 4.1. Indications Addressed by Gene Therapy Clinical Trials.

Phase I 63% (n=678)Phase I/II 20% (n=219)Phase II 14% (n=147)Phase II/III 1.1% (n=12)Phase III 1.9% (n=20)

The Journal of Gene Medicine, ©2005 John Wiley and Sons Ltd www.wiley.co.uk/genmed/clinical

* Center for Cell and Gene Therapy & Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas77030, Email: [email protected]

THE TWO PATHS TO GENE THERAPYGene therapy can be performed either by directtransfer of genes into the patient or by using living cellsas vehicles to transport the genes of interest. Bothmodes have certain advantages and disadvantages.

Direct gene transfer is particularly attractive becauseof its relative simplicity. In this scenario, genes aredelivered directly into a patient’s tissues or bloodstreamby packaging into liposomes (spherical vessels com-posed of the molecules that form the membranes ofcells) or other biological microparticles. Alternately, thegenes are packaged into genetically-engineered viruses,such as retroviruses or adenoviruses. Because of biosafetyconcerns, the viruses are typically altered so that they arenot toxic or infectious (that is, they are replicationincompetent). These basic tools of gene therapists havebeen extensively optimized over the past 10 years.

However, their biggest strength — simplicity — issimultaneously their biggest weakness. In many cases,direct gene transfer does not allow very sophisticatedcontrol over the therapeutic gene. This is because thetransferred gene either randomly integrates into thepatient’s chromosomes or persists unintegrated for arelatively short period of time in the targeted tissue.Additionally, the targeted organ or tissue is notalways easily accessible for direct application of thetherapeutic gene.

On the other hand, therapeutic genes can be deliveredusing living cells. This procedure is relatively complexin comparison to direct gene transfer, and can bedivided into three major steps. In the first step, cellsfrom the patient or other sources are isolated andpropagated in the laboratory. Second, the therapeuticgene is introduced into these cells, applying methodssimilar to those used in direct gene transfer. Finally, thegenetically-modified cells are returned to the patient.The use of cells as gene transfer vehicles has certainadvantages. In the laboratory dish (in vitro), cells canbe manipulated much more precisely than in the body(in vivo). Some of the cell types that continue to divideunder laboratory conditions may be expanded signi-ficantly before reintroduction into the patient.Moreover, some cell types are able to localize toparticular regions of the human body, such as hema-topoietic (blood-forming) stem cells, which return tothe bone marrow. This “homing” phenomenon maybe useful for applying the therapeutic gene withregional specificity.

A major disadvantage, however, is the additionalbiological complexity brought into systems by livingcells. Isolation of a specific cell type requires not onlyextensive knowledge of biological markers, but alsoinsight into the requirements for that cell type to stayalive in vitro and continue to divide. Unfortunately,specific biological markers are not known for many celltypes, and the majority of normal human cells cannotbe maintained for long periods of time in vitro withoutacquiring deleterious mutations.

STEM CELLS AS VEHICLES FORGENE THERAPY

Stem cells can be classified as embryonic or adult,depending on their tissue of origin. The role of adultstem cells is to sustain an established repertoire ofmature cell types in essentially steady-state numbersover the lifetime of the organism. Although adulttissues with a high turnover rate, such as blood, skin,and intestinal epithelium, are maintained by tissue-specific stem cells, the stem cells themselves rarelydivide. However, in certain situations, such as duringtissue repair after injury or following transplantation,stem cell divisions may become more frequent. Theprototypic example of adult stem cells, the hemato-poietic stem cell, has already been demonstrated tobe of utility in gene therapy.4,5 Although they arerelatively rare in the human body, these cells can bereadily isolated from bone marrow or after mobilizationinto peripheral blood. Specific surface markers allowthe identification and enrichment of hematopoieticstem cells from a mixed population of bone marrow orperipheral blood cells.

After in vitro manipulation, these cells may be re-transplanted into patients by injection into thebloodstream, where they travel automatically to theplace in the bone marrow in which they arefunctionally active. Hematopoietic stem cells thathave been explanted, in vitro manipulated, and re-transplanted into the same patient (autologoustransplantation) or a different patient (allogeneictransplantation) retain the ability to contribute to allmature blood cell types of the recipient for anextended period of time (when patients’ cells aretemporarily grown “outside the body” before beingreturned to them, the in vitro process is typicallyreferred to as an “ex vivo” approach).

46

Embryonic Stem Cells

Another adult bone marrow-derived stem cell typewith potential use as a vehicle for gene transfer is themesenchymal stem cell, which has the ability to formcartilage, bone, adipose (fat) tissue, and marrow stroma(the bone marrow microenvironment).6 Recently, arelated stem cell type, the multipotent adult progenitorcell, has been isolated from bone marrow that candifferentiate into multiple lineages, including neurons,hepatocytes (liver cells), endothelial cells (such as thecells that form the lining of blood vessels), and othercell types.7 Other adult stem cells have been identified,such as those in the central nervous system and heart,but these are less well characterized and not as easilyaccessible.8

The traditional method to introduce a therapeutic geneinto hematopoietic stem cells from bone marrow orperipheral blood involves the use of a vector derivedfrom a certain class of virus, called a retrovirus. Onetype of retroviral vector was initially employed to showproof-of-principle that a foreign gene (in that instancethe gene was not therapeutic, but was used as amolecular tag to genetically mark the cells) introducedinto bone marrow cells may be stably maintained forseveral months.9 However, these particular retroviralvectors were only capable of transferring the thera-peutic gene into actively dividing cells. Since mostadult stem cells divide at a relatively slow rate,efficiency was rather low. Vectors derived from othertypes of retroviruses (lentiviruses) and adenoviruseshave the potential to overcome this limitation, sincethey also target non-dividing cells.

The major drawback of these methods is that thetherapeutic gene frequently integrates more or lessrandomly into the chromosomes of the target cell. Inprinciple, this is dangerous, because the gene therapyvector can potentially modify the activity of neighboringgenes (positively or negatively) in close proximity tothe insertion site or even inactivate host genes byintegrating into them. These phenomena are referredto as “insertional mutagenesis.” In extreme cases, suchas in the X-linked SCID gene therapy trials, thesemutations contribute to the malignant transformationof the targeted cells, ultimately resulting in cancer.

Another major limitation of using adult stem cells isthat it is relatively difficult to maintain the stem cellstate during ex vivo manipulations. Under current sub-optimal conditions, adult stem cells tend to lose theirstem cell properties and become more specialized,giving rise to mature cell types through a process

termed “differentiation.” Recent advances in supportiveculture conditions for mouse hematopoietic stem cellsmay ultimately facilitate more effective use of humanhematopoietic stem cells in gene therapy applications.10,11

EMBRYONIC STEM CELL:“THE ULTIMATE STEM CELL”

Embryonic stem cells are capable of unlimited self-renewal while maintaining the potential to differentiateinto derivatives of all three germ layers. Even aftermonths and years of growth in the laboratory, theyretain the ability to form any cell type in the body.These properties reflect their origin from cells of theearly embryo at a stage during which the cellularmachinery is geared toward the rapid expansion anddiversification of cell types.

Murine (mouse) embryonic stem cells were isolatedover 20 years ago,12,13 and paved the way for theisolation of nonhuman primate, and finally humanembryonic stem cells.14 Much of the anticipatedpotential surrounding human embryonic stem cells isan extrapolation from pioneering experiments in themouse system. Experiments performed with humanembryonic stem cells in the last couple of years indicatethat these cells have the potential to make an impor-tant impact on medical science, at least in certainfields. In particular, this impact includes: a) differen-tiation of human embryonic stem cells into various celltypes, such as neurons, cardiac, vascular, hemato-poietic, pancreatic, hepatic, and placental cells, b) thederivation of new cell lines under alternative conditions,c) and the establishment of protocols that allow thegenetic modification of these cells.

THE POTENTIAL OF HUMAN EMBRYONICSTEM CELLS FOR GENE THERAPY

Following derivation, human embryonic stem cells areeasily accessible for controlled and specific geneticmanipulation. When this facility is combined with theirrapid growth, remarkable stability, and ability to maturein vitro into multiple cell types of the body, humanembryonic stem cells are attractive potential tools forgene therapy. Two possible scenarios whereby humanembryonic stem cells may benefit the gene therapyfield are discussed below.

First, human embryonic stem cells could be geneticallymanipulated to introduce the therapeutic gene. This


47

gene may either be active or awaiting later activation,once the modified embryonic stem cell has differ-entiated into the desired cell type. Recently publishedreports establish the feasibility of such an approach.15

Skin cells from an immunodeficient mouse were usedto generate cellular therapy that partially restoredimmune function in the mouse. In these experiments,embryonic stem cells were generated from an immu-nodeficient mouse by nuclear transfer technology. Thenucleus of an egg cell was replaced with that from askin cell of an adult mouse with the genetic immuno-deficiency. The egg was developed to the blastula stageat which embryonic stem cells were derived. Thegenetic defect was corrected by a genetic modificationstrategy designated “gene targeting.” These “cured”embryonic stem cells were differentiated into hema-topoietic “stem” cells and transplanted into immuno-deficient mice. Interestingly, the immune function inthese animals was partially restored. In principle, thisapproach may be employed for treating humanpatients with immunodeficiency or other diseases thatmay be corrected by cell transplantation.

However, significant advances must first be made. Thelevels of immune system reconstitution observed in themice were quite modest (<1% of normal), while themethodology employed to achieve hematopoieticengraftment is not clinically feasible. This methodologyinvolved using a more severely immunodeficient mouseas a recipient (which also had the murine equivalent ofthe human X-linked SCID mutation) and geneticallyengineering the hematopoietic engrafting cells with apotential oncogene prior to transplantation.

Embryonic stem cells may additionally be indirectlybeneficial for cellular gene therapy. Since these cellscan be differentiated in vitro into many cell types,including presumably tissue-specific stem cells, theymay provide a constant in vitro source of cellularmaterial. Such “adult” stem cells derived from embryonicstem cells may thus be utilized to optimize protocolsfor propagation and genetic manipulation techniques.16

To acquire optimal cellular material from clinical samplesin larger quantities for experimental and optimizationpurposes is usually rather difficult since access to thesesamples is limited.

GENETIC MANIPULATION OF STEM CELLSThe therapeutic gene needs to be introduced into thecell type used for therapy. Genes may be introduced

into cells by transfection or transduction. Transfectionutilizes chemical or physical methods to introduce newgenes into cells. Usually, small molecules, such asliposomes, as well as other cationic-lipid based particlesare employed to facilitate the entry of DNA encodingthe gene of interest into the cells. Brief electric shocksare additionally used to facilitate DNA entry into livingcells. All of these techniques have been applied tovarious stem cells, including human embryonic stemcells. However, the destiny of the introduced DNA isrelatively poorly controlled using these procedures. Inmost cells, the DNA disappears after days or weeks, andin rare cases, integrates randomly into host chromo-somal DNA. In vitro drug selection strategies allowthe isolation and expansion of cells that are stablytransfected, as long as they significantly express thenewly introduced gene.

Transduction utilizes viral vectors for DNA transfer.Viruses, by nature, introduce DNA or RNA into cellsvery efficiently. Engineered viruses can be used tointroduce almost any genetic information into cells.However, there are usually limitations in the size of theintroduced gene. Additionally, some viruses (particularlyretroviruses) only infect dividing cells effectively,whereas others (lentiviruses) do not require activelydividing cells. In most cases, the genetic informationcarried by the viral vector is stably integrated into thehost cell genome (the total complement of chromosomesin the cell).

An important parameter that must be carefullymonitored is the random integration into the hostgenome, since this process can induce mutations thatlead to malignant transformation or serious genedysfunction. However, several copies of the therapeuticgene may also be integrated into the genome, helpingto bypass positional effects and gene silencing.Positional effects are caused by certain areas within thegenome and directly influence the activity of theintroduced gene. Gene silencing refers to the pheno-menon whereby over time, most artificially introducedactive genes are turned off by the host cell, amechanism that is not currently well understood. Inthese cases, integration of several copies may help toachieve stable gene expression, since a subset of theintroduced genes may integrate into favorable sites. Inthe past, gene silencing and positional effects were aparticular problem in mouse hematopoietic stemcells.17 These problems led to the optimization of retro-viral and lentiviral vector systems by the addition of

48


genetic control elements (referred to as chromatindomain insulators and scaffold/matrix attachmentregions) into the vectors, resulting in more robustexpression in differentiating cell systems, includinghuman embryonic stem cells.18

In some gene transfer systems, the foreign transgenedoes not integrate at a high rate and remains separatefrom the host genomic DNA, a status denoted“episomal”. Specific proteins stabilizing these episomalDNA molecules have been identified as well as viruses(adenovirus) that persist stably for some time in anepisomal condition. Recently, episomal systems havebeen applied to embryonic stem cells.19

An elegant way to circumvent positional effects andgene silencing is to introduce the gene of interestspecifically into a defined region of the genome by thegene targeting technique referred to previously.20 Thegene targeting technique takes advantage of a cellularDNA repair process known as homologous recom-bination.21 Homologous recombination provides aprecise mechanism for defined modifications ofgenomes in living cells, and has been used extensivelywith mouse embryonic stem cells to investigate genefunction and create mouse models of human diseases.Recombinant DNA is altered in vitro, and the thera-peutic gene is introduced into a copy of the genomicDNA that is targeted during this process. Next,recombinant DNA is introduced by transfection intothe cell, where it recombines with the homologous partof the cell genome. This in turn results in the replace-ment of normal genomic DNA with recombinant DNAcontaining genetic modifications.

Homologous recombination is a very rare event in cells,and thus a powerful selection strategy is necessary toidentify the cells in which it occurs. Usually, theintroduced construct has an additional gene coding forantibiotic resistance (referred to as a selectable marker),allowing cells that have incorporated the recombinantDNA to be positively selected in culture. However,antibiotic resistance only reveals that the cells havetaken up recombinant DNA and incorporated itsomewhere in the genome. To select for cells in whichhomologous recombination has occurred, the end ofthe recombination construct often includes thethymidine kinase gene from the herpes simplex virus.Cells that randomly incorporate recombinant DNAusually retain the entire DNA construct, including theherpes virus thymidine kinase gene. In cells that displayhomologous recombination between the recombinant

construct and cellular DNA, an exchange of homologousDNA sequences is involved, and the non-homologousthymidine kinase gene at the end of the construct iseliminated. Cells expressing the thymidine kinase geneare killed by the antiviral drug ganciclovir in a processknown as negative selection. Therefore, those cellsundergoing homologous recombination are unique inthat they are resistant to both the antibiotic andganciclovir, allowing effective selection with thesedrugs (see Figure 4.2).

Gene targeting by homologous recombination hasrecently been applied to human embryonic stem cells.22

This is important for studying gene functions in vitrofor lineage selection and marking. For therapeuticapplications in transplantation medicine, the controlledmodification of specific genes should be useful forpurifying specific embryonic stem cell-derived, differ-entiated cell types from a mixed population, alteringthe antigenicity of embryonic stem cell derivatives, andadding defined markers that allow the identification oftransplanted cells. Additionally, since the therapeuticgene can now be introduced into defined regions ofthe human genome, better controlled expression ofthe therapeutic gene should be possible. This alsosignificantly reduces the risk of insertional mutagenesis.

FUTURE CHALLENGES FOR STEMCELL-BASED GENE THERAPYDespite promising scientific results with geneticallymodified stem cells, some major problems remain tobe overcome. The more specific and extensive the


49

Figure 4.2. Gene targeting by homologous recombination.

Cell resistant to neomycin and gancyclovir

Cell resistant to neomycin, killed by gancyclovir

=Homologous regions

Random gene insertion

Targeted gene knockout

Knockout cassette

Target gene

necessary, and currently, protocols are being developedto allow the complete depletion of any remainingundifferentiated embryonic stem cells.25 This may beachieved by rigorous purification of embryonic stemcell derivatives or introducing suicide genes that can beexternally controlled.

Another issue is the patient’s immune system response.Transgenic genes, as well as vectors introducing thesegenes (such as those derived from viruses), potentiallytrigger immune system responses. If stem cells are notautologous, they eventually cause immuno-rejection ofthe transplanted cell type. Strategies to circumventthese problems, such as the expression of immunesystem-modulating genes by stem cells, creation ofchimeric, immunotolerable bone marrow or suppression

50


genetic modification, the longer the stem cells have toremain in vitro. Although human embryonic stem cellsin the culture dish remain remarkably stable, the cellsmay accumulate genetic and epigenetic changes thatmight harm the patient (epigenetic changes regulategene activity without altering the genetic blueprint ofthe cell). Indeed, sporadic chromosomal abnormalitiesin human embryonic stem cell culture have beenreported, and these may occur more frequently whenthe cells are passaged as bulk populations. This obser-vation reinforces the necessity to optimize cultureconditions further, to explore new human embryonicstem cell lines, and to monitor the existing celllines.23,24 Additionally undifferentiated embryonic stemcells have the potential to form a type of cancer calleda teratocarcinoma. Safety precautions are therefore

© 2

006

Tere

se W

insl

ow

Figure 4.3. Strategies for Delivering Therapeutic Transgenes into Patients.

The therapeutic geneis packaged into adelivery vehicle suchas a retrovirus andintroduced into thecells.

in vitrodifferentiatedstem cell

ES cellHLA bank

SCNT

OR

OR

Genetically modified ES cells(can block immune rejectionfrom patient)

Therapeuticgene

The genetically modifiedcells are reintroducedinto the patient.

The therapeuticgene is packagedinto a deliveryvehicle such asa retrovirus

Adult stem cells areisolated and propagatedin the laboratory.

Therapeuticgene

...and injectedinto the patient

Target organ(e.g. liver)

Adult stemcells

ES cells

Cell-based DeliveryDirect Delivery

of HLA genes have been suggested.25 In this context,nuclear transfer technology has been recently extendedto human embryonic stem cells.26* Notably, immune-matched human embryonic stem cells have nowbeen established from patients, including an individualwith an immunodeficiency disease, congenitalhypogammaglobulinemia.27* Strategies that combinegene targeting with embryonic stem cell-based therapyare thus potential novel therapeutic options.

The addition of human embryonic stem cells to theexperimental gene therapy arsenal offers great promisein overcoming many of the existing problems of cellularbased gene therapy that have been encountered inclinic trials (see Figure 4.3). Further research is essentialto determine the full potential of both adult andembryonic stem cells in this exciting new field.

REFERENCES1. Mullen CA, Snitzer K, Culver KW, Morgan RA, Anderson WF,

Blaese RM. Molecular analysis of T lymphocyte-directedgene therapy for adenosine deaminase deficiency: long-termexpression in vivo of genes introduced with a retroviralvector. Hum Gene Ther. 1996;7:1123-1129.

2. Hacein-Bey-Abina S, Le Deist F, Carlier F, et al. Sustainedcorrection of X-linked severe combined immunodeficiencyby ex vivo gene therapy. N Engl J Med. Apr 182002;346(16):1185-1193.

3. Hacein-Bey-Abina S, von Kalle C, Schmidt M, et al. Aserious adverse event after successful gene therapy forX-linked severe combined immunodeficiency. N Engl J Med.Jan 16 2003;348(3):255-256.

4. Aiuti A, Slavin S, Aker M, et al. Correction of ADA-SCID bystem cell gene therapy combined with nonmyeloablativeconditioning. Science. 2002;296:2410-2413.

5. Hacein-Bey-Abina S, Le Diest F, Carlier F, et al. Sustainedcorrection of X-linked severe combined immunodeficiencyby ex vivo gene therapy. N Engl J Med. 2002;346:1185-1193.

6. Gregory CA, Prockop DJ, Spees JL. Non-hematopoieticbone marrow stem cells: molecular control of expansionand differentiation. Exp Cell Res. 2005;306:330-335.

7. Jiang Y, Jahagirdar BN, Reinhardt RL, et al. Pluripotency ofmesenchymal stem cells derived from adult marrow.Nature. Jul 4 2002;418(6893):41-49.

8. Stocum DL. Stem cells in CNS and cardiac regeneration.Adv Biochem Eng Biotechnol. 2005;93:135-159.

9. Brenner MK, Rill DR, Holladay MS, et al. Gene marking todetermine whether autologous marrow infusion restoreslong-term haemopoiesis in cancer patients. Lancet.Nov 6 1993;342(8880):1134-1137.

10. Reya T, Duncan AW, Ailles L, et al. A role for Wnt signallingin self-renewal of haematopoietic stem cells. Nature. May22 2003;423(6938):409-414.

11. Willert K, Brown JD, Danenberg E, et al. Wnt proteins arelipid-modified and can act as stem cell growth factors.Nature. May 22 2003;423(6938):448-452.

12. Evans MJ, Kaufman MH. Establishment in culture ofpluripotential cells from mouse embryos. Nature.Jul 9 1981;292(5819):154-156.

13. Martin GR. Isolation of a pluripotent cell line from earlymouse embryos cultured in medium conditioned byteratocarcinoma stem cells. Proc Natl Acad Sci USA.Dec 1981;78(12):7634-7638.

14. Thomson JA, Itskovitz-Eldor J, Shapiro SS, et al. Embryonicstem cell lines derived from human blastocysts. Science.Nov 6 1998;282(5391):1145-1147.

15. Rideout WM, 3rd, Hochedlinger K, Kyba M, Daley GQ,Jaenisch R. Correction of a genetic defect by nucleartransplantation and combined cell and gene therapy.Cell. Apr 5 2002;109(1):17-27.

16. Barberi T, Willis LM, Socci ND, Studer L. Derivation ofmultipotent mesenchymal precursors from humanembryonic stem cells. PLoS Med. 2005;2:e161.

17. Challita PM, Kohn DB. Lack of expression from a retroviralvector after transduction of murine hematopoietic stemcells is associated with methylation in vivo. Proc Natl AcadSci USA. Mar 29 1994;91(7):2567-2571.

18. Ma Y, Ramezani A, Lewis R, Hawley RG, Thomson JA.High-level sustained transgene expression in humanembryonic stem cells using lentiviral vectors. Stem Cells.2003;21(1):111-117.

19. Aubert J, Dunstan H, Chambers I, Smith A. Functionalgene screening in embryonic stem cells implicates Wntantagonism in neural differentiation. Nat Biotechnol.Dec 2002;20(12):1240-1245.

20. Kyba M, Perlingeiro RC, Daley GQ. HoxB4 confers definitivelymphoid-myeloid engraftment potential on embryonicstem cell and yolk sac hematopoietic progenitors. Cell.Apr 5 2002;109(1):29-37.

21. Smithies O. Forty years with homologous recombination.Nat Med. Oct 2001;7(10):1083-1086.

22. Zwaka TP, Thomson JA. Homologous recombination inhuman embryonic stem cells. Nat Biotechnol. Mar2003;21(3):319-321.


51

* Editor’s note: Both papers referenced in 26 and 27 were later retracted.See Science 20 January 2006: Vol. 311. no. 5759, p. 335.

52


23. Draper JS, Smith K, Gokhale P, et al. Recurrent gain ofchromosomes 17q and 12 in cultured human embryonicstem cells. Nat Biotechnol. Jan 2004;22(1):53-54.

24. Cowan CA, Klimanskaya I, McMahon J, et al. Derivationof embryonic stem-cell lines from human blastocysts.N Engl J Med. Mar 25 2004;350(13):1353-1356.

25. Gerecht-Nir S, Itskovitz-Eldor J. Cell therapy usinghuman embryonic stem cells. Transpl Immunol.Apr 2004;12(3-4):203-209.

26. Hwang WS, Ryu YJ, Park JH, et al. Evidence of a pluripotenthuman embryonic stem cell line derived from a clonedblastocyst. Science. Mar 12 2004;303(5664):1669-1674.

27. Hwang WS, Roh SI, Lee BC, et al. Patient-specific embryonicstem cells derived from human SCNT blastocysts. Science.2005;308:1777-1783.

use of genetically modified stem cells

Documents