Porcine Xenotransplantation Research Articles

NECESSITY IN XENOTRANSPLANTATION: ISLAMIC PERSPECTIVES REVISITED

Organ failures cause significant problems and sufferings which affect the livelihood, quality of life and decrease in life expectancy to the person. The caregivers, family and community also carry the disease burden and costs of the organ failures. Allotransplantation (AT) offers probable cures to organ failure but is limited by the supply of human organs. Organ shortages have resulted in numerous biomedical ethical problems and illegal activities. Xenotransplantation (XT) and specifically Porcine Xenotransplantation (PXT) has high expectations from researchers and clinicians to be the alternative to AT. From the Islamic perspectives, multiple deliberations and fatwas are issued that vary from total prohibition to conditional permissibility and full permissibility. Often, ḍarūrah or dire necessity is invoked to make XT or PXT permissible. The relevant biomedical issues and whether pre-conditions for ḍarūrah have been satisfied are discussed.

Procedure of Adult Porcine Islet Isolation.

Islet transplantation is a useful therapeutic choice for severe diabetes mellitus; however, limited donor supplies have interfered with the use of this treatment. Therefore, the establishment of alternative donor sources and engineered tissue, which enables to produce appropriate insulin for controlling blood glucose, is an important challenge. The adult pig is a promising and feasible donor source and materials for the engineered tissue for the clinical setting among various candidates. The recent progress of gene-editing technology contributes to possible clinical porcine xenotransplantation, including porcine islet xenotransplantation. For the success of future clinical porcine islet xenotransplantation, establishing an islet isolation technique for acquiring adequate, good-quality porcine islets is equally important to use a gene-edited pig. However, the characteristics of porcine islets are different from other species; therefore, establishing a suitable technique for porcine islets is challenging. Impact statement Recent technological progress promotes the feasibility of xenotransplantation, including islet xenotransplantation, for clinical setting. Adult pig is a promising and feasible donor source for islet xenotransplantation and engineered tissue, which enables to control blood glucose in recipients. It is important to acquire porcine islets in good qualities for the promotion, however, establishing a technique for adult porcine islet isolation is important but challenging because of the vulnerability of adult porcine islets. Deciding the proper timing of stopping pancreatic digestion is one of the important factors for obtaining adult porcine islets in good quality.

Reevaluating the Ethical Issues in Porcine-to-Human Heart Xenotransplantation.

A major limiting factor with heart allotransplantation remains the availability of organs from deceased donors. Porcine heart xenotransplantation could serve as an alternative source of organs for patients with terminal heart failure. A first-in-human porcine xenotransplantation that occurred in January 2022 at the University of Maryland Medical Center provided an opportunity to examine several ethical issues to guide selection criteria for future xenotransplantation clinical trials. In this article, the authors, who are clinicians at UMMC, discuss the appropriate balancing of risks and benefits and the significance, if any, of clinical equipoise. The authors also review the alleged role of the psychosocial evaluation in identifying patients at an elevated risk of posttransplant noncompliance, and they consider how the evaluation's implementation might enhance inequities among diverse populations. The authors argue that, based on the principle of reciprocity, psychosocial criteria should be used, not to exclude patients, but instead to identify patients who need additional support. Finally, the authors discuss the requirements for and the proper assessment of informed and voluntary consent from patients being considered for xenotransplantation.

Extensive germline genome engineering in pigs.

The clinical applicability of porcine xenotransplantation-a long-investigated alternative to the scarce availability of human organs for patients with organ failure-is limited by molecular incompatibilities between the immune systems of pigs and humans as well as by the risk of transmitting porcine endogenous retroviruses (PERVs). We recently showed the production of pigs with genomically inactivated PERVs. Here, using a combination of CRISPR-Cas9 and transposon technologies, we show that pigs with all PERVs inactivated can also be genetically engineered to eliminate three xenoantigens and to express nine human transgenes that enhance the pigs' immunological compatibility and blood-coagulation compatibility with humans. The engineered pigs exhibit normal physiology, fertility and germline transmission of the 13 genes and 42 alleles edited. Using in vitro assays, we show that cells from the engineered pigs are resistant to human humoral rejection, cell-mediated damage and pathogenesis associated with dysregulated coagulation. The extensive genome engineering of pigs for greater compatibility with the human immune system may eventually enable safe and effective porcine xenotransplantation.

Regulatory changes in China on xenotransplantation and related products.

Given the persistence and the worldwide shortage of organs from both the deceased and living donors for clinical transplantation, pig organs or tissues hold immense promises for the patients on the waiting list, and xenotransplantation is deemed as one of the solutions to the organ shortage crisis. Indeed, the emerging gene editing technologies, such as CRISPR/Cas9, have led to tremendous progress in the generation of genetically modified pigs to overcome many barriers associated. We presented a description of the xenotransplantation regulations in China and the related products. Several groups in China have successfully generated transgenic pigs with the elimination of immune rejection or coagulation-related genes, and both pre-clinical and clinical studies have been reported. However, the pre-clinical evaluation and clinical application of porcine xenotransplantation raises ethical and regulatory considerations. Herein, in this review, we will summarize and discuss the progress in xenotransplantation in China and xenotransplantation-related products from the regulatory perspective. There has been remarkable progress in both the genetically modified pigs and pre-clinical studies in China, and China will be the first country to successfully fulfill the xenotransplantation from pig organ to human in the near future.

Comparison of Islet Characterization from Use of Standard Crude Collagenase to GMP-Grade Collagenase Enzyme Blends in Preweaned Porcine Islet Isolations

For the advancement of porcine xenotransplantation for clinical use in type 1 diabetes mellitus, the concerns of a sustainable and safe digestion enzyme blend must be overcome. Incorporating good manufacturing practices (GMP) can facilitate this through utilizing GMP-grade enzymes. In conjunction, still taking into account the cost-effectiveness, a wide concern. We evaluated how GMP-grade enzyme blends impact our piglet islets and their long-term effects. Preweaned porcine islets (PPIs) were isolated from 8- to 10-day-old pigs. Digestion enzyme blends, collagenase type V (Type V), collagenase AF-1 GMP-grade with collagenase NB 6 GMP-grade (AF-1 and NB 6), and collagenase AF-1 GMP-grade with collagenase neutral protease AF GMP-grade (AF-1 and NP AF) were compared. Islet quality control assessments, islet yield, viability, and function, were performed on days 3 and 7, and cell content was performed on day 7. GMP-grade AF-1 and NB 6 (17,209 ± 2,730 islet equivalent per gram of pancreatic tissue [IE/g] on day 3, 9,001 ± 1,034 IE/g on day 7) and AF-1 and NP AF (17,214 ± 3,901 IE/g on day 3, 8,833 ± 2,398 IE/g on day 7) showed a significant increase in islet yield compared to Type V (4,618 ± 1,240 IE/g on day 3, 1,923 ± 704 IE/g on day 7). Islet size, viability, and function showed comparable results in all enzyme blends. There was no significant difference in islet cellular content between enzyme blends. This study demonstrated a comparison of GMP-grade collagenase enzyme blends and a standard crude collagenase enzyme in preweaned-aged porcine, a novel topic in this age. GMP-grade enzyme blends of AF-1 and NB 6 and AF-1 and NP AF resulted in substantially higher yields and as effective PPIs compared to Type V. In the long run, considering costs, integrity, and sustainability, GMP-grade enzyme blends are more favorable for clinical application due to high reproducibility in comparison to undefined manufacturing processes of standard enzymes.

Class I Major Histocompatibility Complex Antigen Identification and Engineering in Porcine Xenotransplantation

Class I Major Histocompatibility Complex Antigen Identification and Engineering in Porcine Xenotransplantation

Genetic strategies to bring islet xenotransplantation to the clinic.

Despite the benefits of islet and pancreas allotransplantation, their widespread use in type 1 diabetes is limited because of the paucity of suitable donors. Porcine xenotransplantation offers an alternative, and advances in genetic modification of pigs have opened up new potential for its clinical use. This review outlines the barriers to successful islet xenotransplantation, and genetic modifications that have been tested to overcome these. Islets from pigs lacking α1,3-galactosyltransferase, to prevent hyperacute rejection, are now used as a background strain for further genetic modifications. The instant blood-mediated inflammatory reaction is overcome by expressing complement regulators including CD46, CD55 and CD59. Prevention of immune-mediated rejection mediated by T cells, macrophages and natural killer cells remains a challenge. The use of immunosuppressive antibodies, such as anti-CD154 or anti-CD2, can be protective, and may be useful if they are produced by the islets themselves. The field of xenotransplantation has benefited enormously from the development of new genetic modification strategies. With the possibility of multiple genetic modifications in the same animal, and a detailed knowledge of the mechanism of xenograft rejection, the challenge now is to develop islets that provide long-term graft survival without systemic immunosuppression.

Efficient production of multi-modified pigs for xenotransplantation by 'combineering', gene stacking and gene editing.

Xenotransplantation from pigs could alleviate the shortage of human tissues and organs for transplantation. Means have been identified to overcome hyperacute rejection and acute vascular rejection mechanisms mounted by the recipient. The challenge is to combine multiple genetic modifications to enable normal animal breeding and meet the demand for transplants. We used two methods to colocate xenoprotective transgenes at one locus, sequential targeted transgene placement - ‘gene stacking’, and cointegration of multiple engineered large vectors - ‘combineering’, to generate pigs carrying modifications considered necessary to inhibit short to mid-term xenograft rejection. Pigs were generated by serial nuclear transfer and analysed at intermediate stages. Human complement inhibitors CD46, CD55 and CD59 were abundantly expressed in all tissues examined, human HO1 and human A20 were widely expressed. ZFN or CRISPR/Cas9 mediated homozygous GGTA1 and CMAH knockout abolished α-Gal and Neu5Gc epitopes. Cells from multi-transgenic piglets showed complete protection against human complement-mediated lysis, even before GGTA1 knockout. Blockade of endothelial activation reduced TNFα-induced E-selectin expression, IFNγ-induced MHC class-II upregulation and TNFα/cycloheximide caspase induction. Microbial analysis found no PERV-C, PCMV or 13 other infectious agents. These animals are a major advance towards clinical porcine xenotransplantation and demonstrate that livestock engineering has come of age.

Genetic Modification of Porcine Endogenous Retrovirus (PERV) Sequences in Cultured Pig Cells as a Model for Decreasing Infectious Risk in Xenotransplantation

Xenotransplantation of porcine (pig) organs into human patients has been proposed as a solution to the shortage of transplantable organs in human medicine. A potential safety barrier to clinical implementation of xenotransplantation is the infectious risk associated with expression of endogenous retroviral sequences in the porcine genome. Notably, Porcine Endogenous Retroviruses (PERVs) have been shown capable of infecting human cells in culture. The ability to specifically modify genomic loci in a wide range of in vitro and in vivo models has recently become widely accessible due to development of synthetic engineered nuclease technologies. Among them, Tal Effector Nucleases (TALENs) can be designed to recognize and catalyze double stranded DNA breaks (DSBs) in investigator‐specified genomic loci. These DSBs, when resolved by endogenous DNA repair mechanisms, commonly leave mutagenic insertions or deletions (indels) in target loci. This pilot study addresses the possibility of genetically modifying the porcine genome to specifically ablate PERV sequences via Tal Effector Nuclease (TALEN) genome editing techniques. TALEN sequences were designed and constructed against PERV pol and gag genes and were transfected into porcine PK(15) cells. Targeted pol and gag loci in PK(15) cells displayed TALEN‐induced genetic modification. These results suggest the potential utility of genome engineering strategies in ablating PERV gene expression in porcine xenotransplantation.

Detailed Mapping of Determinants within the Porcine Endogenous Retrovirus Envelope Surface Unit Identifies Critical Residues for Human Cell Infection within the Proline-Rich Region

Replication-competent porcine endogenous retroviruses (PERVs) are either human cell tropic (PERV-A and PERV-B) or non-human cell tropic (PERV-C). We previously demonstrated that PERV in vitro cell tropism is modulated by 2 residues within the C terminus of SU and that the PERV receptor binding domain (RBD) extends beyond the variable regions A and B (VRA and VRB, respectively), to include the proline rich-region (PRR) of SU (M. Gemeniano et al., Virology 346:108-117, 2000; T. Argaw et al., J. Virol. 82:7483-7489, 2008). The present study aimed to identify the specific elements within the PERV RBD that interact with the C-terminal elements of SU to facilitate human cell infection. We constructed a series of chimeric and mutated envelopes between PERV-A and PERV-C and using pseudotyped retroviral vectors to map the human cell tropism-determining sequences within the PERV RBD. We show that the PRR from PERV-A is both necessary and sufficient to allow human cell infection when substituted into the homologous region of the PERV-C envelope carrying two C-terminal amino acid substitutions shown to influence human cell tropism, Q374R and I412V (PERV-Crv). Furthermore, substitution of a single amino acid residue in the PRR of the non-human-tropic PERV-Crv envelope allows vectors carrying this envelope to infect human cells. Receptor interference assays showed that these modified PERV-C envelopes do not bind either of the human PERV-A receptors, suggesting the presence of a distinct human PERV-C receptor. Finally, vectors carrying these modified PERV-C envelopes infect primary human endothelial cells, a cell type likely to be exposed to PERV in clinical use of certain porcine xenotransplantation products.

Innate cellular immunity and xenotransplantation.

This review assesses the recent progress in xenograft rejection by innate immune responses, with a focus on innate cellular xenoreactivity. Current literature was reviewed for new insights into the role of innate cellular immunity in xenograft rejection. Increasing evidence confirms that vigorous innate immune cell activation is accounted for by a combination of xenoantigen recognition by activating receptors, and incompatibility in inhibitory receptor-ligand interactions. Although both innate humoral and cellular xenoimmune responses are predominantly elicited by preformed and induced xenoreactive antibodies in nonhuman primates following porcine xenotransplantation, innate immune cells can also be activated by xenografts in the absence of antibodies. The latter antibody-independent response will likely persist in recipients even when adaptive xenoimmune responses are suppressed. In addition to xenograft rejection by recipient innate immune cells, phagocytic cells within liver xenografts are also deleterious to recipients by causing thrombocytopenia. Strategies of overcoming innate immune responses are required for successful clinical xenotransplantation. In addition to developing better immunosuppressive and tolerance induction protocols, endeavors towards further genetic modifications of porcine source animals are ultimately important for successful clinical xenotransplantation.

Multiplex high resolution melting assay for estimation of Porcine Endogenous Retrovirus (PERV) relative gene dosage in pigs and detection of PERV infection in xenograft recipients

Porcine Endogenous Retrovirus (PERV) poses an infectious risk in the field of xenotransplantation. This risk may be mitigated by breeding selectively animals bearing favorable PERV genetic characteristics including pigs with low levels of PERV integrated in the genome. A real-time quantitative polymerase chain reaction (PCR) assay employing the Roche High Resolution Melting (HRM) Master was used to estimate the relative gene dosage of PERV pol integrated within the pig genome. When assessed across 99 pigs of the Auckland Island breed numerous animals bearing low gene dosage were identified. The assay was adapted further to perform multiplex PCR for the detection of PERV infection within xenograft recipients. Besides PERV, amplification targets for the multiplex PCR include a pig cell marker for the determination of microchimerism and an internal amplification control (IAC) to assess the efficiency of nucleic acid isolation and effects of PCR inhibition. When 12 patients who had received porcine islet transplants were tested no evidence of PERV infection was found. The assay was shown to be specific, highly reproducible with superior performance over conventional nested PCR. This assay can be used as both a screening tool for PERV proviral levels within donor pigs and as a diagnostic tool to examine PERV transmission in human patients treated with porcine xenotransplantation material.

Porcine Xenotransplantation to Primates

Xenotransplantation is a hot topic currently, since the demand for diverse organs is increasing in patients. Among many species, pigs are suitable animals for xenotranplantation as they share many anatomical and physiological characteristics with humans. This review article provides an overview of porcine xenotransplantation and the rejection of pig xenotransplants in primates, and use of genetically modified and cloned pigs in xenotransplantation. It also highlights major target organs in porcine xenotransplantation and virus infection in xenotransplantation.

Real-Time Quantitative Polymerase Chain Reaction With SYBR Green I Detection for Estimating Copy Numbers of Porcine Endogenous Retrovirus From Chinese Miniature Pigs

Porcine endogenous retrovirus (PERV) in the pig genome represents a potential infectious risk in xenotransplantation. Chinese miniature pigs have been considered to be potential organ donors in China. However, an adequate level of information on PERV from Chinese miniature pigs has not been available. We established an SYBR Green I-based real-time quantitative polymerase chain reaction (PCR) assay for estimating copy numbers of PERV integrated in the host genome. The assay was 100-fold more sensitive compared with conventional PCR. We also evaluated the specificity and reproducibility of the assay. We statistically analyzed the difference in PERV copy numbers integrated into the genomes of Wuzhishan pigs versus Bama minipigs. This approach will be useful to screen donor pigs as well as to examine clinical samples from human subjects treated with porcine xenotransplantation products for evidence of PERV transmission.

Aims and status of the German DFG ‐ Transregio Research Group Xenotransplantation

Allotransplantation is the therapy of choice for endstage diseases. Due to the organ shortage, three times respectively twice as many patients are currently waiting for kidneys or hearts as are actually transplanted annually in Germany. Consequently, the mortality rate on the heart transplant list is around 16%; if patients have to wait more than 24 months for a kidney, the graft survival after 10 years will be less than half when compared to those who are able to receive an organ within 6 months. Therefore, porcine xenotransplantation of kidneys and hearts will be a very much needed alternative clinical technique – including (porcine) xenogenic islet cell transplantation for diabetic patients with hypoglycaemic attacks – a large number of persons, if one considers that the number of people suffering from diabetes worldwide increased by nearly 50% over the last decade.Of course, much more work is needed before clinical xenotransplantation will become a safe and long lasting alternative. The α(1,3)‐galactosyltransferase KO, PERV C free pigs – held in specific pathogen free units – must express several human transgenes, like a complement regulator molecule (in order to suppress non Gal‐AB's) and HLA‐E (protection from NK‐cell cytotoxicity). Some kind of accommodation will be necessary (e.g. via Hemoxygenase‐1 or PDL‐1 – a negative costimulatory receptor which also increases the CD4+ CD25high Fox p3+ subset). An overexpression of thrombomodulin (TM) would finally be necessary in order to overcome the disparity between porcine (TM) and human (Thrombin) proteins and possibly avoid microvascular thrombotic disease.And to top all these desires of a clinical transplant specialist, all the genetic engineering should be achieved in close connection within one chromosome, preferably an artificial one, in order to make all these changes hereditary.The just mentioned strategies are the main topics of the DFG‐Transregio Research Group. The following abstracts will highlight the respective progress and will also inevitably evaluate arising problems. International leaders in our field will give their results and opinion – an interchange which is necessary within the small but very active xenotransplantation research field.It is my sincere belief that a clinical scenario of xenotransplantation will be possible in the near future.

Absence of Replication of Porcine Endogenous Retrovirus and Porcine Lymphotropic Herpesvirus Type 1 with Prolonged Pig Cell Microchimerism after Pig-to-Baboon Xenotransplantation

Porcine endogenous retrovirus (PERV), porcine cytomegalovirus (PCMV), and porcine lymphotropic herpesvirus (PLHV) are common porcine viruses that may be activated with immunosuppression for xenotransplantation. Studies of viral replication or transmission are possible due to prolonged survival of xenografts in baboon recipients from human decay-accelerating factor transgenic or alpha-1,3-galactosyltransferase gene knockout miniature swine. Ten baboons underwent xenotransplantation with transgenic pig organs. Graft survival was 32 to 179 days. Recipient serial samples of peripheral blood mononuclear cells (PBMC) and plasma were analyzed for PCMV, PERV, and PLHV-1 nucleic acids and viral replication using quantitative PCR assays. The PBMC contained PERV proviral DNA in 10 animals, PLHV-1 DNA in 6, and PCMV in 2. PERV RNA was not detected in any PBMC or serum samples. Plasma PLHV-1 DNA was detected in one animal. Pig cell microchimerism (pig major histocompatibility complex class I and pig mitochondrial cytochrome c oxidase subunit II sequences) was present in all recipients with detectable PERV or PLHV-1 (85.5%). Productive infection of PERV or PLHV-1 could not be demonstrated. The PLHV-1 viral load did not increase in serum over time, despite prolonged graft survival and pig cell microchimerism. There was no association of viral loads with the nature of exogenous immune suppression. In conclusion, PERV provirus and PLHV-1 DNA were detected in baboons following porcine xenotransplantation. Viral detection appeared to be due to persistent pig cell microchimerism. There was no evidence of productive infection in recipient baboons for up to 6 months of xenograft function.

Identification of Residues outside of the Receptor Binding Domain That Influence the Infectivity and Tropism of Porcine Endogenous Retrovirus

Identification of determinants of human tropism of porcine endogenous retrovirus (PERV) is critical to understanding the risk of transmission of PERV to recipients of porcine xenotransplantation products. Previously, we showed that a chimeric envelope cDNA encoding the 360 N-terminal residues of the human-tropic PERV envelope class A (PERV-A) SU and the 130 C-terminal residues of the pig-tropic PERV-C SU and all of TM (PERV-A/C) showed a 100-fold decrease in infectivity titer on human cells (M. Gemeniano, O. Mpanju, D. R. Salomon, M. V. Eiden, and C. A. Wilson, Virology 346:108-117, 2006). To identify residues important for human cell infection, we performed site-directed mutagenesis on each of the nine residues, singly or in combination, that distinguish the C-terminal region of PERV-C from PERV-A. Of the nine amino acids, two single-amino-acid substitutions, Q374R and I412V, restored the infectivity of human cells to the chimeric PERV-A/C to a titer equivalent to that of PERV-A. In contrast, PERV-A/C mutant envelope Q439P resulted in undetectable infection of human cells and an approximately 1,000-fold decrease in control pig cells. Mutation of K441R rescued mutants that carried Q439P, suggesting an incompatibility between the proline residue at this position and the presence of KK in the proteolytic cleavage signal. We confirmed this incompatibility with vectors carrying PERV-A envelope mutant R462K that were also rendered noninfectious. Finally, tropism of vectors carrying PERV-C envelope mutants with only four amino acid changes in the C terminus of PERV-C envelope, NHRQ436YNRP plus K441R, was shifted to one similar to that of PERV-A. Our results show an important and previously unrecognized role for infectivity and tropism for residues at the C terminus of SU.

193 HUMAN CYTOMEGALOVIRUS PROTEIN US11 DOWN-REGULATES THE EXPRESSION OF SWINE LEUKOCYTE ANTIGEN-I IN MINIPIG CELLS

The CD8+ cytotoxic T lymphocyte (CTL)-mediated immune response is important in porcine xenotransplantation, and pigs could be used as a good model for organ donor if these immunological barriers are overcome. Human cytomegalovirus (HCMV) encodes unique short (US) 11 gene, which interferes with cellular immune responses by inducing rapid degradation of newly synthesized heavy chains of major histocompatibility (MHC) class I. The destruction of heavy chains by US11 helps the virus to hide from recognition by cytotoxic T lymphocytes at early stage. In this study we demonstrated the inhibitory effect of US11 on the cytotoxicity of CTL cells by down-regulation of swine leukocyte antigen (SLA)-I expression. We established five US11 clonal cell lines by transfection into minipig fetal fibroblasts and confirmed the integration of US11 gene by PCR and Southern blot assay. The reduction of SLA-I, which was expressed on the cell surface, was also detected by flow cytometry assay. The level (14.6% to 21.2%) of SLA-1 expression in US11 clonal cell lines was decreased relative to that in the control. In the CTL assay, the rate of CD8+ T cell-mediated cytotoxicity was significantly reduced to 31.9 � 11.3%, compared to that of the control (81.4 � 5.3%; P < 0.01; n = 4). These results indicate that HCMV viral protein US11 can effectively suppress the presentation of SLA-I in pig fetal fibroblast cells. This work was supported by a grant (Code # 20070101034005) from the BioGreen 21 Program, Rural Development Administration, Republic of Korea.

Lack of evidence for PERV expression after apoptosis‐mediated horizontal gene transfer between porcine and human cells

Evidence for porcine endogenous retrovirus (PERV) infection of human cells has provoked a public health debate over the proposed use of porcine xenografts to alleviate the worldwide shortage of human allografts. Nevertheless, the potential relevance of PERV transmission by apoptosis-mediated horizontal DNA transfer, a documented means of infection-independent retrovirus delivery, appears to have been overlooked in this discussion. To examine the hypothesis that apoptotic cell death during porcine xenograft rejection is capable of fostering horizontal DNA transfer, we have now assessed in vitro cocultures, consisting of phagocytic human fibroblasts and apoptotic or necrotic porcine B-lymphoblastoid cells, for evidence of cross-species PERV exchange and eventual replication. Using real-time polymerase chain reaction (PCR) assays, designed to differentiate nuclear and cytoplasmic DNA derived from either porcine or human cells, we now report evidence for the presence of porcine DNA, including PERV, in the nucleus of human fibroblasts exposed to apoptotic porcine cells. This novel demonstration of apoptosis-mediated horizontal PERV transfer is characterized by a low efficiency of transfer and a transient nature, being present in only 0.22% of the cocultured human cells and disappearing to undetectable levels within 4 weeks of exposure to apoptotic porcine cells. In contrast, using PERV-specific real-time reverse-transcriptase PCR (RT-PCR) and ultra-sensitive product-enhanced reverse transcriptase (PERT) assays, we find no evidence for human fibroblast-derived cellular PERV RNA or coculture supernatant-based RT-activity, indicating a lack of subsequent PERV replication. Together, these results suggest that apoptosis-mediated horizontal PERV transfer does not present an overt hazard within the framework of porcine xenotransplantation. However, we also present arguments against extrapolation of these in vitro observations directly to clinical circumstances.