Physiological basis for xenotransplantation from genetically modified pigs to humans.

The collective efforts of scientists over multiple decades have led to advancements in molecular and cellular biology-based technologies including genetic engineering and animal cloning that are now being harnessed to enhance the suitability of pig organs for xenotransplantation into humans. Using organs sourced from pigs with multiple gene deletions and human transgene insertions, investigators have overcome formidable immunological and physiological barriers in pig-to-nonhuman primate (NHP) xenotransplantation and achieved prolonged pig xenograft survival. These studies informed the design of Revivicor's (Revivicor Inc, Blacksburg, VA) genetically engineered pigs with 10 genetic modifications (10 GE) (including the inactivation of 4 endogenous porcine genes and insertion of 6 human transgenes), whose hearts and kidneys have now been studied in preclinical human xenotransplantation models with brain-dead recipients. Additionally, the first two clinical cases of pig-to-human heart xenotransplantation were recently performed with hearts from this 10 GE pig at the University of Maryland. Although this review focuses on xenotransplantation of hearts and kidneys, multiple organs, tissues, and cell types from genetically engineered pigs will provide much-needed therapeutic interventions in the future.

Humanized von Willebrand factor reduces platelet sequestration in ex vivo and in vivo xenotransplant models.

The transplantation of organs across species offers the potential to solve the shortage of human organs. While activation of human platelets by human von Willebrand factor (vWF) requires vWF activation by shear stress, contact between human platelets and porcine vWF (pvWF) leads to spontaneous platelet adhesion and activation. This non-physiologic interaction may contribute to the thrombocytopenia and coagulation pathway dysregulation often associated with xenotransplantation of pig organs in nonhuman primates. Pigs genetically modified to decrease antibody and complement-dependent rejection (GTKO.hCD46) were engineered to express humanized pvWF (h*pvWF) by replacing a pvWF gene region that encodes the glycoprotein Ib-binding site with human cDNA orthologs. This modification corrected for non-physiologic human platelet aggregation on exposure to pig plasma, while preserving in vitro platelet activation by collagen. Organs from pigs with h*pvWF demonstrated reduced platelet sequestration during lung (p ≤ .01) and liver (p ≤ .038 within 4 h) perfusion ex vivo with human blood and after pig-to-baboon lung transplantation (p ≤ .007). Residual platelet sequestration and activation were not prevented by the blockade of canonical platelet adhesion pathways. The h*pvWF modification prevents physiologically inappropriate activation of human or baboon platelets by porcine vWF, addressing one cause of the thrombocytopenia and platelet activation observed with xenotransplantation.

Cutting Edge: IFN-γ Produced by Brain-Resident Cells Is Crucial To Control Cerebral Infection with Toxoplasma gondii.

In vitro studies demonstrated that microglia and astrocytes produce IFN-γ in response to various stimulations, including LPS. However, the physiological role of IFN-γ production by brain-resident cells, including glial cells, in resistance against cerebral infections remains unknown. We analyzed the role of IFN-γ production by brain-resident cells in resistance to reactivation of cerebral infection with Toxoplasma gondii using a murine model. Our study using bone marrow chimeric mice revealed that IFN-γ production by brain-resident cells is essential for upregulating IFN-γ-mediated protective innate immune responses to restrict cerebral T. gondii growth. Studies using a transgenic strain that expresses IFN-γ only in CD11b(+) cells suggested that IFN-γ production by microglia, which is the only CD11b(+) cell population among brain-resident cells, is able to suppress the parasite growth. Furthermore, IFN-γ produced by brain-resident cells is pivotal for recruiting T cells into the brain to control the infection. These results indicate that IFN-γ produced by brain-resident cells is crucial for facilitating both the protective innate and T cell-mediated immune responses to control cerebral infection with T. gondii.

Pigs in Transplantation Research and Their Potential as Sources of Organs in Clinical Xenotransplantation.

The pig has long been used as a research animal and has now gained importance as a potential source of organs for clinical xenotransplantation. When an organ from a wild-type (i. e., genetically unmodified) pig is transplanted into an immunosuppressed nonhuman primate, a vigorous host immune response causes hyperacute rejection (within minutes or hours). This response has been largely overcome by 1) extensive gene editing of the organ-source pig and 2) the administration to the recipient of novel immunosuppressive therapy based on blockade of the CD40/CD154 T cell costimulation pathway. Gene editing has consisted of 1) deletion of expression of the 3 known carbohydrate xenoantigens against which humans have natural (preformed) antibodies and 2) the introduction of human 'protective' genes. The combination of gene editing and novel immunosuppressive therapy has extended life-supporting pig kidney graft survival to greater than 1 y and of pig heart survival to up to 9 mo. This review briefly describes the techniques of gene editing, the potential risks of transfer of porcine endogenous retroviruses with the organ, and the need for breeding and housing of donor pigs under biosecure conditions.

Developmental expression of the cellular prion protein (PrP(C) ) in bovine embryos.

The mammalian cellular prion protein (PrP(C) ) is a highly conserved glycoprotein that may undergo conversion into a conformationally altered isoform (scrapie prion protein or PrP(Sc) ), widely believed to be the pathogenic agent of transmissible spongiform encephalopathies (TSEs). Although much is known about PrP(Sc) conversion and its role in TSEs, the normal function of PrP(C) has not been elucidated. In adult mammals, PrP(C) is most abundant in the central nervous tissue, with intermediate levels in the intestine and heart, and lower levels in the pancreas and liver. PrP(C) is expressed during neurogenesis throughout development, and it has recently been proposed that PrP(C) participates in neural cell differentiation during embryogenesis. In order to establish the developmental timing and to address the cell-specific expression of PrP(C) during mammalian development, we examined PrP(C) expression in bovine gametes and embryos through gestation Day 39. Our data revealed differential levels of Prnp mRNA at Days 4 and 18 in pre-attachment embryos. PrP(C) was detected in the developing central and peripheral nervous systems in Day-27, 32-, and -39 embryos. PrP(C) was particularly expressed in differentiated neural cells located in the marginal regions of the central nervous system, but was absent from mitotically active, periventricular areas. Moreover, a PrP(C) cell-specific pattern of expression was detected in non-nervous tissues, including liver and mesonephros, during these stages. The potential participation of PrP(C) in neural cell differentiation is supported by its specific expression in differentiated states of neurogenesis.

Expression and knockdown of cellular prion protein (PrPC) in differentiating mouse embryonic stem cells.

The mammalian cellular prion protein (PrP(C)) is a highly conserved glycoprotein that may undergo conversion into a conformationally altered isoform (scrapie prion protein or PrP(Sc)), widely believed to be the pathogenic agent of transmissible spongiform encephalopathies (TSEs). Although much is known about pathogenic PrP conversion and its role in TSEs, the normal function of PrP(C) is poorly understood. Given the abundant expression of PrP(C) in the developing mammalian CNS and the spatial association with differentiated stages of neurogenesis, recently it has been proposed that PrP(C) participates in neural cell differentiation. In the present study, we investigated the role of PrP(C) in neural development during early embryogenesis. In bovine fetuses, PrP(C) was differentially expressed in the neuroepithelium, showing higher levels at the intermediate and marginal layers where more differentiated states of neurogenesis were located. We utilized differentiating mouse embryonic stem (ES) cells to test whether PrP(C) contributed to the process of neural differentiation during early embryogenesis. PrP(C) showed increasing levels of expression starting on Day 9 until Day 18 of ES cell differentiation. PrP(C) expression was negatively correlated with pluripotency marker Oct-4 confirming that ES cells had indeed differentiated. Induction of ES cells differentiation by retinoic acid (RA) resulted in up-regulation of PrP(C) at Day 20 and nestin at Day 12. PrP(C) expression was knocked down in PrP-targeted siRNA ES cells between Days 12 and 20. PrP(C) knockdown in ES cells resulted in nestin reduction at Days 16 and 20. Analysis of bovine fetuses suggests the participation of PrP(C) in neural cell differentiation during early embryogenesis. The positive association between PrP(C) and nestin expression provide evidence for the contribution of PrP(C) to ES cell differentiation into neural progenitor cells.

Production of interspecies somatic/pluripotent heterokaryons using polyethylene glycol (PEG) and selection by imaging flow cytometry for the study of nuclear reprogramming.

Fusion of somatic cells to embryonic stem cells induces reprogramming of the somatic nucleus and can be used to study the effect of trans-acting factors from the pluripotent cell over the differentiated nucleus. However, fusion only occurs in a small fraction of the cells exposed to fusogenic conditions, hence the need for a protocol that produces high fusion rate with minimal cell damage, coupled with a method capable of identifying and selecting these rare events. Here, we describe a protocol to induce formation of bi-species mouse pluripotent/bovine somatic heterokaryons, as well as same-species homokaryons, using polyethylene glycol (PEG). To identify bi-species fusion products, heterokaryons were labeled using cell type-specific fluorescent antibodies and selected using imaging (Amnis ImageStream Mark II) and traditional (BD FACSAria I) flow cytometry. Heterokaryons selected with this method produced ES cell-like colonies in vitro. This procedure can be combined with downstream applications such as nucleic acid isolation for RT-PCR and RNA-Seq, and used as a tool to study somatic cell nuclear reprogramming.

Sperm morphology and preparation method affect bovine embryonic development.

This study was conducted to evaluate the effect of sperm separation methods of semen samples collected from bulls subjected to scrotal insulation on embryonic development after in vitro fertilization (IVF) and to determine whether IVF results would be affected by various heparin concentrations. Morphologically abnormal semen samples were obtained and cryopreserved from Holstein bulls following scrotal insulation for 48 hours. Standard protocols using the Percoll gradient (90%/45%) method and the swim-up method were used to separate spermatozoa fractions in experiment I. The pellet (A(p)) and the 45% layer (B(p)) were isolated from the Percoll separation, while for the swim-up separation, the supernatant (A(s)) and the interphase (B(s)) were isolated. The overall blastocyst rate for our laboratory control semen was 23.1 +/- 2.1% for Percoll separations (A(p) and B(p)) and 18.2 +/- 2.0% for swim-up (A(s) and B(s)) separations. This rate was higher (P <.01) than the rate observed for the semen from the bull that had the greatest response to scrotal insult 5 days prior to the insult, when it was 9.2 +/- 2.1% for the Percoll separation and 20.7 +/- 2.3% for the swim-up separation, while semen from 27 days after scrotal insulation (D +27) resulted in no blastocyst formation for the Percoll separation and a 4.2 +/- 2.1% rate for the swim-up separation. In experiment II, semen was sampled from the bulls that responded in the greatest and least degrees to scrotal insult 5 days before scrotal insulation (D -5) and on days 23 (D +23) and 34 (D +34) after scrotal insulation. These samples were exposed to IVF mediums with 3 different heparin concentrations (0.1, 1.0, and 10 microg/mL). There was a significant difference (P <.05) in developmental scores between the D -5 (1.08 +/- 0.08), D +23 (0.9 +/- 0.08), and D +34 (0.8 +/- 0.08) samples, but no differences were observed in blastocyst formation based on the number of cleaved embryos. Increasing the heparin concentration resulted in higher (P <.01) embryonic developmental scores. In conclusion, when semen samples with high percentages of abnormal spermatozoa are used for IVF, semen separation preparation methods affect results. Our results show that the separation methods used under these conditions were inadequate in their ability to provide potentially competent sperm for IVF. However, selecting appropriate sperm separation procedures could improve in the IVF embryonic development of semen from bulls used in artificial insemination. Also, an increase in the heparin concentration was able to partially overcome deficiencies, which suggests that morphologically abnormal spermatozoa undergo capacitation despite possible structural changes to the plasma membrane.

Quantitative and qualitative analysis of cellular prion protein (PrP(C)) expression in bovine somatic tissues.

The host encoded cellular prion protein (PrP(C)) is an N-linked glycoprotein tethered to the cell membrane by a glycophosphatidylinositol (GPI) anchor. Under certain conditions, PrP(C) can undergo conversion into a conformationally-altered isoform (PrP(Sc)) widely believed to be the pathogenic agent of transmissible spongiform encephalopathies (TSEs). Understanding the tissue-specific expression of PrP(C) is crucial considering that cells expressing high levels of PrP(C) bear a risk for conversion and accumulation of PrP(Sc). In the present study, fifteen bovine somatic tissues were analyzed for PrP(C) expression by quantitative western blot and immunohistochemistry. Quantitative western blot analysis revealed highest expression of PrP(C) in cerebellum, obex and spinal cord. Intermediate levels were detected in thymus, intestine, nerve, heart and spleen, and lower levels in lung, muscle, kidney, lymph node, skin, pancreas and liver. Immunohistochemical analysis detected intense cellular-specific PrP(C) staining in neurons, thymocytes and lymphocytes. PrP(C) was also detected in the enteric wall, pancreatic islets of langerhans, myocardium, pulmonary alveolar sacs, renal glomeruli and dermal epithelial cells. This study demonstrated the quantitatively varied, wide-spread, tissue- and cell-specific expression pattern of PrP(C) in bovine somatic tissues. The importance of this study is to lay the foundation for understanding the tissue-specific expression of PrP(C) and to consider the potential participation of more bovine tissues in the transmission of BSE infection.