h. niemann, w. a. kues progress in xenotransplantation ... zeitschriften/txmed/2003-1/01... ·...

H. Niemann, W. A. Kues: Progress in Xenotransplantation Research Employing Transgenic Pigs Transplantationsmedizin2003, 15. Jahrg., S. 3

H. Niemann, W. A. Kues

Department of Biotechnology, Institutfür Tierzucht (FAL) Mariensee, Neu-stadt, Germany

Niemann H, Kues WA (2003) Progressin Xenotransplantation Research Em-ploying Transgenic Pigs. Tx Med 15: 3-14

Progress in Xenotransplantation ResearchEmploying Transgenic Pigs

Microinjection of foreign DNA into pronuclei of a fertilized oo-cyte has predominantly been used for the generation of transgeniclivestock. This technology works reliably, but is inefficient and re-sults in random integration and variable expression patterns in thetransgenic offspring. Nevertheless, remarkable achievements havebeen made with this technology with regard to xenotransplanta-tion. Transgenic pigs that express human complement regulatingproteins have been tested in their ability to serve as donors in hu-man organ transplantation (i.e. xenotransplantation). In vitro andin vivo data convincingly show that the hyperacute rejection re-sponse can be overcome in a clinically acceptable manner by suc-cessfully employing this strategy. The recent developments in nu-clear transfer and its merger with the growing genomic data allowtargeted and regulatable transgenesis. Systems for efficient ho-mologous recombination in somatic cells are being developed andthe first knock-out pigs, carrying a deletion in the α-galac-tosyltransferase gene, were recently generated. It is anticipatedthat poly-transgenic pigs will be available as donors for functionalxenografts within a few years. Similarly, pigs may serve as donorsfor a variety of xenogenic cells and tissues. The availability ofthese technologies is essential to maintain “genetic security” andto ensure absence of unwanted side effects.

Key words:transgenic pigs, xenotransplantation, microinjection, nuclear trans-fer

Wissenschaftliche Fortschritte auf dem Weg zur Organspendeaus transgenen Schweinen

Die Mikroinjektion von DNA-Konstrukten in die Vorkerne von Zy-goten war bisher die bevorzugte Methode, um transgene Großtierezu erzeugen. Diese Technologie liefert zwar zuverlässig transgeneNachkommen, ist jedoch mit erheblichen Nachteilen behaftet, wiegroße Ineffizienz (< 5% transgene Nachkommen), zufällige Inte-gration des Fremdgens und variable Expressionsmuster in dentransgenen Nachkommen. Trotzdem sind mit dieser Technologiebeachtliche Fortschritte erzielt worden, insbesondere im Hinblickauf die Xenotransplantation. Humane komplementregulatorischeProteine sind erfolgreich in transgenen Schweinen exprimiert unddiese Tiere im Hinblick auf ihre Eignung in der humanen Organ-transplantation (Xenotransplantation) geprüft worden. Umfang-reiche In vitro- und In vivo-Daten haben gezeigt, dass die hypera-kute Abstoßungsreaktion mit dieser Strategie zuverlässig und inklinisch wirksamer Form überwunden werden kann. Damit ist ein

Transplantationsmedizin H. Niemann, W. A. Kues: Progress in Xenotransplantation Research Employing Transgenic Pigs2003, 15. Jahrg., S. 4

1. Introduction

Microinjection of foreign DNA intopronuclei of a fertilized oocyte has pre-dominantly been used for the genera-tion of transgenic livestock. Microin-jection involves injection of severalthousands of copies of a given geneconstruct into pronuclei of zygotes; zy-gotes are transferred into recipients andresulting offspring are screened for in-tegration of the foreign DNA. Althoughthis procedure works reliable, it is inef-ficient (1-4% transgenic offspring/transferred microinjected zygotes), re-sults in random integration into the hostgenome and variable expression due toposition effects [1,2]. In addition, it istime consuming and requires substantialintellectual, financial and material re-sources [3]. Attributed to the enormousamounts of resources needed for trans-genic livestock production, the costs forone expressing transgenic animal areextraordinary high. It has been esti-mated that one expressing transgenicmouse requires average expenses of121 US$ whereas one expressing trans-genic pig would amount to 25,000 US$,one transgenic sheep 60,000 US$ and

one transgenic cow 546,000 US$ [4].Details of the microinjection technol-ogy and the potential applications oftransgenic livestock have been exten-sively reviewed [1,2,4,5,6,7,8].Despite its inherent limitations, micro-injection has allowed commercial ex-ploitation of transgenic technology pri-marily with animals for biomedicalpurposes. Substantial progress in live-stock transgenesis can be made throughapplication of somatic nuclear transfer.It is anticipated that the merger of nu-clear transfer with molecular tools, suchas targeted genetic modification andconditional gene expression already ex-plored in mice, will provide anotherboost to livestock transgenesis [9,10].Here, we review the present state of re-search aiming at developing transgenicpigs for clinical xenotransplantation andoutline future perspectives.

2. Xenotransplantation

2.1 Transplantation of Solid Organs

Approximately 250,000 people are cur-rently only living because of transplan-

tation of an appropriate human organ(e.g. allotransplantation). In most casesno alternative therapeutic treatment wasavailable and the recipients would havedied without the organ transplantation.However, the enormous progress in or-gan transplantation technology has ledto an acute shortage of appropriate or-gans. Estimations in the USA have re-vealed that approximately 45,000 peo-ple, younger than 65 years need a hearttransplant whereas only 2,000 humanhearts are transplanted annually [11]. Inthe U.S.A., more than 74.000 peopleare awaiting organ transplants and anew patient is added to the waiting listevery 14 minutes. Only 21.000 patientsreceived a transplant in the year 2000[12]. In Germany approximately 2.400kidneys, 730 livers, 540 hearts and 180pancreas are transplanted annually.However, the demand is twice as highas these figures. This has led to the sadand ethically challenging situation thatseveral thousand patients die every yearwho could have survived if appropriateorgans would have been available.To close this growing gap between de-mand and availability of appropriateorgans, xenotransplantation (= thetransplantation of organs between dis-cordant species e.g. from animals tohuman) employing porcine xenograftsis considered as the solution of choice[13,14]. The pig seems to be the opti-mal donor animal because• the organs have a similar size as hu-

man organs,• porcine anatomy and physiology are

not too different from those in hu-mans,

• pigs have short reproduction cyclesand large litters,

• pigs grow rapidly,• maintenance is possible at high hy-

gienic standards at relatively lowcosts,

• pigs are a domesticated species• and transgenic techniques are estab-

lished to modify the immunogenicityof porcine cells and organs.

The process of evaluating transgenicpigs as potential donors for xenotrans-plants involves a variety of complexsteps and is extremely time-, labour-and resource-intensive (Table 1).Essential prerequisites for a successfulxenotransplantation are:1. Prevention of transmission of zoo-

noses from the donor animal to thehuman recipient. This aspect

wichtiger Schritt im Hinblick auf ein längerfristiges Überlebenporciner Xenotransplantate gemacht worden. Die jüngstenEntwicklungen im Bereich des somatischen Kerntransfers in Ver-bindung mit den wachsenden genomischen Daten werden zukün-ftig eine zielgenaue und regulierbare transgene Expression auchin Großtieren erlauben. Systeme für eine effiziente homologe Re-kombination in somatischen Zellen werden z.Zt. entwickelt und dieersten „Knock-out“ Schweine mit einer Deletion des α-Galacto-syltransferase-Gens sind kürzlich generiert worden. Damit sollteeine weitere Verbesserung im Hinblick auf eine Kontrolle der hy-perakuten Abstoßungsreaktion erreicht werden können. Es wirderwartet, dass polytransgene Schweine in einigen Jahren alsSpender für funktionsfähige Xenotransplantate zur Verfügung ste-hen werden. Solche transgenen Schweine werden nicht nur alsSpender für solide Organe, sondern auch für xenogene Zellen undGewebeanteile dienen können. Die Verfügbarkeit dieser neuenTechnologien wird wesentlich für eine Standardisierung dertransgenen Effekte sein und sicherstellen, dass unerwünschte Ne-beneffekte vermieden werden.

Schlüsselwörter:transgene Schweine, Xenotransplantation, Mikroinjektion, Kern-transfer


gained particular significance sincea few years ago it was shown thatporcine endogenous retroviruses(PERV) can be produced by por-cine cell lines and can even infecthuman cell lines [15]. However,until today no infection has beenfound in patients that had receivedvarious forms of living porcine tis-sues (e.g. islet cells, insulin, skin,extracorporal liver) for up to 12years [16]. Recent intensive re-search revealed that PERV do notpresent a noticeable risk for recipi-ents of xenotransplantation pro-vided all necessary precautions aremade [17,18,19, 20]. In addition, astrain of miniature pigs has beenidentified which does not produceinfective PERV [21].

2. Compatibility of the donor organsin anatomy and physiology with

the human organ system, e.g.lifespan differences, growth rate,expected body weight.

3. Overcoming of the immunologicalrejection of the transplanted organ.The immunological hurdles are asfollows [22]:a) Hyperacute rejection response

(HAR) occurs within secondsor minutes. In the case of a dis-cordant organ, e.g. from pig tohuman, naturally occurring an-tibodies react with antigenicstructures on the surface of theporcine organ and induce HARby activating the complementcascade which is achieved viathe antigen-antibody-complex.Ultimately, this results in theformation of the membrane at-tack complex (MAC). How-ever, the complement cascade

can be shut down at variouspoints by expression of regu-latory genes which prevent theformation of MAC. Regulatorsof the complement cascade areCD55 (= Decay AcceleratingFactor DAF), CD46 (= Mem-brane Cofactor Protein, MCP)or CD59. MAC disrupts theendothelial cell layer of theblood vessels which leads to ly-sis, thrombosis, loss of vascularintegrity and ultimately to re-jection of the transplanted or-gan.

b) Acute vascular rejection (AVR)occurs within days. Inducedxenoreactive antibodies arethought to be responsible forAVR. The endothelial cells ofthe graft’s microvasculatureloose their anti-thrombic prop-erties, attract leucocytes,monocytes and platelets leadingto anemia and organ failure.

c) Cellular rejection occurs withinweeks after transplantation. Inthis process the blood vesselsof the transplanted organ aredamaged by T-cells which in-vade the intercellular spacesand destroy the organ. This re-jection is observed after allo-transplantation and normally issuppressed by life-long admi-nistration of immunosuppres-sive drugs.

d) Chronic rejection is a compleximmunological process result-ing in the rejection of the trans-planted organ after severalyears. This process is slow andprogressive and its etiology islargely unknown.

When employing a discordant donorspecies such as the pig, overcoming theHAR and AVR are the preeminentgoals. The most promising strategy toovercome the HAR is the synthesis ofhuman complement regulatory proteinsin transgenic pigs [13,14,23,24]. Fol-lowing transplantation, the porcine or-gan would produce the complementregulatory protein and can thus preventthe complement attack of the recipient.Pigs transgenic for DAF have beengenerated and their hearts have beentransplanted either heterotopically, e.g.in addition to the recipient’s own organor orthotopically (= life supportive) intonon-human primates. Upon heterotopic

Tab. 1: Steps involved in testing transgenic swine as donors for xenotransplantation

1. Generation and propagation of transgenic pigs

2. Determination of the transgenic expression pattern (mRNA, protein, organspecificity, etc.)

3. Selective breeding to expand homozygous transgenic lines

4. In vitro tests to assess the protective function of the transgene against HAR

5. Tests to assess the “safety” of the donor lines (exogenous pathogens, endoge-nous viruses, PERV, etc.)

6. Perfusion studies with isolated porcine organs using human blood

7. In-vivo studies using non-human primates- physiological compatibility- potential transmission of pathogens

8. Registration as a therapeutic treatment

9. Clinical application

Tab. 2: Success rates of RCA-transgenic porcine organs upon transplantation toprimate recipients

RCA Organ/kindof transplant

Recipient Immuno-suppression

Survival(days)

hDAF heart/heterot. Cynomologus +++ ~ 60 dheterotopic “ ++ ~ 90 d

orthopic “ +++ ~ 10 dheterotopic “ + ~ 21 d

kidney/ “ ++ ~ 13 d(max. 35 d)

orthotopichCD59 heart, Baboon ++ ~ 30 h

heterotopichCD46 heart, heterotopic Baboon ++ ~ 23 d

+ = weak immunosuppression; ++ = moderate immunosuppression; +++ = heavy immunosuppres-sion


transplantation, the average survival ofthe recipients reached a maximum of40-90 days whereas the non-transgeniccontrol organs were destroyed within afew minutes. The primates had to betreated with high doses of immunosup-pressive drugs to maintain survival ofthe xenotransplant. Following moderatedoses of immunosuppression survivalrates of 20-25 days could be obtained[25,26]. Employing the genomic cloneof hCD46 (MCP), transgenic pigsshowed a similar expression pattern forthe transgene as found for the endoge-nous gene of the patients. Survival of ahCD46 porcine heart upon transplanta-tion to baboons exceeded 23 days [27].Similarly, transgenic expression ofhCD59 was compatible with an ex-tended survival of porcine hearts fol-lowing transfer into primates [28].Transplantation of hDAF-transgenicporcine kidneys was compatible with anextended survival of the recipients. Thephysiological function of the kidneyswas maintained for up to 3-4 weeks[29] (Table 2). These data show thatHAR can be overcome in a clinicallyacceptable manner by successful em-ploying this strategy [13].Four research groups with strong linksto the pharmaceutical industry have re-ported the generation of transgenic pigswith expression of human complementregulators. In our experiments suchtransgenic pigs were produced withoutrestrictions imposed from the pharma-ceutical industry [30]. Our transgenicpigs show high expression of hCD59predominantly in the heart, kidney andpancreas but also other target organs;several transgenic lines established(Figure 1). Transgenic endothelial cellsand fibroblasts were protected againstcomplement mediated lysis. Perfusionstudies using isolated porcine kidneysemploying human blood revealed a sig-nificant protective effect against HAR.Orthotopic transplantation of a CMV-hCD59 transgenic porcine kidney intocynomolgous monkey was compatiblewith extended survival of >20 days.The use of the CMV promotor provideda more efficient selection of transgenicpigs with an optimized expression pat-tern in 2 out of 5 tested lines [30]. Pre-viously, only one out of 30 linesshowed an expression pattern that wasconsidered to be compatible with a suc-cessful xenotransplantation [26].A novel promising strategy towardssuccessful xenotransplantation is the

Fig. 1: Generation of transgenic pigs for xenotransplantation (data from Niemannet al., 2001).A) Minigene construct for microinjection, PCMV: cytomegalovirus immediate earlypromoter, hCD59: human CD59 (regulator of complement) cDNA, PA: polyade-nylation site, PvuI and AspI: flanking restriction enzyme sites.B) DNA microinjection into one pronucleus of a porcine zygote. Prior to microin-jection the zygote was centrifuged to separate the dark lipids from the cytoplasmaticfraction to allow optic identification of the pronuclei.C) Transgenic founder animal identified by Southern blotting of an ear sample.D) Organ-specific expression of hCD59 protein in an F1-offspring animal deter-mined by Western blotting with a specific monoclonal antibody.E) Cell surface expression of hCD59 in porcine primary cells as determined by flowcytometry. Grey shadowed curves demonstrated presence of hCD59 by usage of amonoclonal antibody, white curves indicate background values by usage of an iso-type matched control antibody.F) Functional expression of human CD59 on transgenic porcine cells as demon-strated by cytotoxicity assay. Porcine cells were incubated with heat treated humanserum and a dilution series of human complement. Specific lysis of porcine cellswere measured by chromium release.G) Ex vivo perfusion of porcine kidneys from F1-animals with human blood. Nearlyall transgenic porcine kidneys could be perfused for 4 hours, whereas non-transgenic control kidneys failed soon after onset of perfusion due to hyperacuterejection. Mean survival times were 207.5 minutes for transgenic and 57.5 minutesfor wildtype kidneys (p<0.005).


knockout of the antigenic structures onthe surface of the porcine organ thatcause HAR (Figure 2). These structuresare known as 1,3-α-gal-epitopes pro-duced by activity of the 1,3-α-galacto-syltransferase. The generation of pigletsin which one allele of the α-galacto-syltransferase locus had been knockedout by homologous recombination inprimary donor cells employed in nu-clear transfer, was recently reported[31,32]. The birth of four healthy pig-lets with disruption of both allelic locifor α-galactosyltransferase has alsobeen published. In this study, the highcytotoxicity of toxin A from Clostrid-ium difficile was used to establish afunctional screen for cells without gal-epitopes. Toxin A binds to gal-epitopesand effectively kills all wildtype cells.Applying toxin A on cells which al-ready carried one deleted α-galactosyl-transferase allele, selected a cell clone,which carried an inactivating pointmutation on the second allele [33].The strength of the cellular response toxenografts can be so great that it is un-likely to be fully controlled by immu-nosuppressive treatment and transgeneexpression. Further improvements ofthe success in xenotransplantationmight arise from the possibility of in-ducing a permanent tolerance acrossxenogenic barriers [34,35]. A promisingstrategy for long-term graft acceptanceseems to induce a permanent chimerismvia intraportal injection of embryonicstem cell like structures [36]. Althoughxenotransplantation poses numerousfurther challenges to research, it is ex-pected that transgenic pigs will beavailable as organ donors within thenext five to ten years [37]. Guidelinesfor the clinical application of porcinexenotransplants are currently being de-veloped in several countries or are al-ready available (USA).

2.2 Use of Xenogenic Cells and Tissue

Another promising area of applicationfor transgenic animals will be the sup-ply of xenogenic cells and tissue. Sev-eral intractable diseases, disorders andinjuries are associated with irreversiblecell death and/or aberrant cellular func-tion. Despite numerous attempts, differ-entiated human cells cannot yet be ex-panded well enough in culture. In thefuture, human embryonic stem -likecells may serve as a source for specific

differentiated therapeutic cell types.Xenogenic cells, in particular from thepig, hold great promise with regard to asuccessful cell therapy for human pa-tients [38]. These cells provide severalsignificant advantages, such as the pos-sibility for manipulation prior to trans-plantation to enhance cell function,banking and cryopreservation, the com-bination with different cell types in thesame graft and the option to introducefail safe mechanisms via suicide genes[38].There are already numerous examplesfor successful application of xenogeniccell therapy. Porcine islet cells havebeen transplanted to diabetic patientsand were shown to be at least partiallyfunctional over a limited period of time[39]. Porcine fetal neural cells weretransplanted into the brain of patientssuffering from Parkinson’s disease andHuntington’s disease [40,41]. In a sin-gle autopsied patient the graft survivedfor more than 7 months and the trans-planted cells formed dopaminergic neu-rons and glial cells. Pig neurons ex-tended axons from the graft site into thehost brain [40]. Further examples forthe potential use of porcine neural cellsare stroke and focal epilepsy [42]. Hu-man, fetal neuronal cells have also beenemployed as transplants into Parkin-

son’s and Huntington’s disease patients.Olfactory ensheathing cells (OECs) orSchwann cells derived from hCD59transgenic pigs promoted axonal regen-eration in rat spinal cord lesion [43].Thus, cells from genetically modifiedpigs may serve as therapeutic measureto restore functional axons across thesite of a spinal cord transsection. Xeno-genic porcine cells may also be usefulas novel therapy for liver diseases.Upon transplantation of porcine hepato-cytes to Watanabe heritable hyperlipi-demic (WHHL) rabbits (a model forfamilial hypercholesterolemia), the xe-nogenic cells migrated out of the ves-sels and integrated into the hepatic pa-renchyma. The integrated porcinehepatocytes provided functional LDLreceptors and thus reduced cholesterollevels by 30-60% for at least 100 days[44].A clone of bovine adrenocortical cellsrestored adrenal function upon trans-plantation to adrenalectomized SCIDmice. This finding shows that func-tional endocrine tissue can be derivedfrom a single somatic cell [45]. Bovineneuronal cells were collected fromtransgenic fetuses, transplanted into thebrain of rats and resulted in significantimprovements of symptoms of Parkin-son’s disease [46]. Furthermore, xeno

Fig. 2: Schematic drawing of gene targeting in livestockGene targeting of somatic primary cells by homologous recombination employing apromoterless targeting vector. Optionally, cell clones with the desired targetingevent could be screened for loss of heterozygosity and subsequently be employed innuclear transfer.


transplantation of retinal pigment epithelial cells holdspromise to treat retinal diseases such as macular degen-eration which is associated with photoreceptor losses.Porcine or bovine fetal cardiomyocytes or myoblastsmay provide a therapeutic approach for the treatment ofischemic heart disease. Similarly, xenogenic porcinecells may be valuable for the repair of skin or cartilagedamage [38].

3. Improvements of Transgenic Technology by Nuclear Transfer

In light of the recent advances, somatic nuclear transferholds the greatest promise for significant improvementsin the generation of transgenic livestock. A major pre-requisite is the availability of suitable primary cells orcell lines compatible with techniques for precise geneticmodifications either for gain or loss of function. Anotherprerequisite is a significantly improved knowledge ofgene sequences and organization of the livestock ge-nome, which currently is lagging much behind that ofmouse and human. In the latter, the putative 3 billion bphave been sequenced in the year 2001. Surprisingly, thehuman genome only contained approximately 30-35.000genes [47]. However, RNA editing and alternativesplicing significantly augments the number of proteinssynthesized from a gene [48,49]. In contrast, in livestockspecies only a minority of genes has been mapped andsequenced until now. However, the technology devel-oped during deciphering the human genome will im-prove and accelerate sequencing of genomes from otherspecies [50]. Even the currently limited genetic informa-tion in livestock species allows the application of cDNAarray technologies and or high density DNA chips toobtain gene expression profiles of nearly any tissue ofinterest. Improvements of RNA isolation and unbiasedamplification of tiny amounts of mRNA (picogram) en-able to analyse even single preimplantation embryos[51].Offspring from nuclear transfer have been born in allmajor livestock species cattle, sheep, goat and swine [52-56]. Our laboratory has cloned cattle already severalyears ago, and recently obtained the first cloned pigletsfrom in vitro matured oocytes (Hoelker et al., in prepa-ration). This forms the basis for future studies on novelapproaches towards genetic modification for xenotrans-plantation. A variety of different cell types of embryonic,fetal and somatic origin has been successfully employedas donors in nuclear transfer. Factors affecting the suc-cess of nuclear transfer are poorly defined and the aver-age percentage of live offspring does not exceed 1-3% ofthe transferred reconstituted embryos [57,58]. A betterunderstanding of the underlying fundamental molecularand cellular processes, such as cell cycle compatibilitiesbetween recipient cytoplasm and donor nucleus [59], cellcycle synchronization of the donor cells [60,61], repro-gramming and the relevance of differentiation versus to-tipotency is urgently needed. Upon serum deprivation ortreatment with chemical cell cycle inhibitors, the majo-rity of porcine donor cells was synchronized at the pre-


sumptive optimal cell cycle stage atGo/G1 without compromising their vi-ability [61,62]. Recently, we have iden-tified expression of Polo-like kinase-1(Plk-1) to be a good marker to indicatethe appropriate cell cycle stage of por-cine donor cells [63]. This contributessubstantially to standardize the nucleartransfer procedures.In cloned offspring predominantly fromruminants approximately 30% are af-flicted by the large offspring syndrome(LOS) which includes an increasedperi- and postnatal mortality and vari-ous other pathologies [52,64,65]. Theseunwanted side effects need to be over-come prior to an eventual commercialexploitation of somatic nuclear transfer.Aberrations of the well orchestratedpattern of gene expression are thoughtto be involved in the high incidence ofLOS. A primary mechanism may bealterations in the methylation of genes,including those that are subject to im-printing [64,66,67]. The first piglets,carrying a knock-out for one allele ofthe α-galactosyltransferase gene did notshow gross abnormalities [31-33].

4. Perspectives and Outlook

The merger of the recent advancementsin the reproductive technologies withthe tools of molecular biology opens thehorizon for a completely new era forgenetic modification of pigs significantfor clinical application of xenotrans-plantation. The recent developments innuclear transfer and the growing geno-mic data will allow the generation ofloss-of function animals, precise tar-geting of transgenes to defined chromo-somal positions, and the establishmentof multi-transgenic pigs for xenotrans-plantation. We are currently testing theusefulness of the tetracycline system forthe conditional expression of trans-plantation related human genes intransgenic pigs [68]. Systems for effi-cient homologous recombination insomatic cells are being developed andthe adaptation of sophisticated molecu-lar tools, already explored in mice, fortransgenic livestock production is un-derway in the international researchcommunity. The availability of thesetechnologies is essential to maintain“genetic security” and to ensure ab-sence of unwanted side effects.

5. Acknowledgements

Research reported in this article hasbeen funded by grants from the BMBFand the DFG. The authors like to ac-knowledge the important contributionfrom many members of the laboratoryand on the experimental farm.

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Prof. Dr. Heiner NiemannAbteilung für Biotechnologie

Institut für TierzuchtMariensee

D-31535 NeustadtE-mail: [email protected]

h. niemann, w. a. kues progress in xenotransplantation ... zeitschriften/txmed/2003-1/01... ·...

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