Silencing Porcine CMAH and GGTA1 Genes Significantly Reduces Xenogeneic Consumption of Human Platelets by Porcine Livers.

BACKGROUND: A profound thrombocytopenia limits hepatic xenotransplantation in the pig-to-primate model. Porcine livers also have shown the ability to phagocytose human platelets in the absence of immune-mediated injury. Recently, inactivation of the porcine ASGR1 gene has been shown to decrease this phenomenon. Inactivating GGTA1 and CMAH genes has reduced the antibody-mediated barrier to xenotransplantation; herein, we describe the effect that these modifications have on xenogeneic consumption of human platelets in the absence of immune-mediated graft injury. METHODS: Wild type (WT), ASGR1, GGTA1, and GGTA1CMAH knockout pigs were compared for their xenogeneic hepatic consumption of human platelets. An in vitro assay was established to measure the association of human platelets with liver sinusoidal endothelial cells (LSECs) by immunohistochemistry. Perfusion models were used to measure human platelet uptake in livers from WT, ASGR1, GGTA1, and GGTA1 CMAH pigs. RESULTS: GGTA1, CMAH LSECs exhibited reduced levels of human platelet binding in vitro when compared with GGTA1 and WT LSECs. In a continuous perfusion model, GGTA1 CMAH livers consumed fewer human platelets than GGTA1 and WT livers. GGTA1 CMAH livers also consumed fewer human platelets than ASGR1 livers in a single-pass model. CONCLUSIONS: Silencing the porcine carbohydrate genes necessary to avoid antibody-mediated rejection in a pig-to-human model also reduces the xenogeneic consumption of human platelets by the porcine liver. The combination of these genetic modifications may be an effective strategy to limit the thrombocytopenia associated with pig-to-human hepatic xenotransplantation.

Reduced human platelet uptake by pig livers deficient in the asialoglycoprotein receptor 1 protein.

BACKGROUND: The lethal thrombocytopenia that accompanies liver xenotransplantation is a barrier to clinical application. Human platelets are bound by the asialoglycoprotein receptor (ASGR) on pig sinusoidal endothelial cells and phagocytosed. Inactivation of the ASGR1 gene in donor pigs may prevent xenotransplantation-induced thrombocytopenia. METHODS: Transcription activator-like effector nucleases (TALENs) were targeted to the ASGR1 gene in pig liver-derived cells. ASGR1 deficient pig cells were used for somatic cell nuclear transfer (SCNT). ASGR1 knock out (ASGR1-/-) fetal fibroblasts were used to produce healthy ASGR1 knock out piglets. Human platelet uptake was measured in ASGR1+/+ and ASGR1-/- livers. RESULTS: Targeted disruption of the ASGR1 gene with TALENs eliminated expression of the receptor. ASGR1-/- livers phagocytosed fewer human platelets than domestic porcine livers during perfusion. CONCLUSIONS: The use of TALENs in liver-derived cells followed by SCNT enabled the production of healthy homozygous ASGR1 knock out pigs. Livers from ASGR1-/- pigs exhibit decreased human platelet uptake. Deletion of the ASGR1 gene is a viable strategy to diminish platelet destruction in pig-to-human xenotransplantation.

Efficient generation of genetically distinct pigs in a single pregnancy using multiplexed single-guide RNA and carbohydrate selection.

BACKGROUND: Manipulating the pig genome to increase compatibility with human biology may facilitate the clinical application of xenotransplantation. Genetic modifications to pig cells have been made by sequential recombination in fetal fibroblasts and liver-derived cells followed by cross-breeding or somatic cell nuclear transfer. The generation of pigs for research or organ donation by these methods is slow, expensive and requires technical expertise. A novel system incorporating the bacterial nuclease Cas9 and single-guide RNA targeting a 20 nucleotide site within a gene can be expressed from a single plasmid leading to a double-strand break and gene disruption. Coexpression of multiple unique single-guide RNA can modify several genetic loci in a single step. We describe a process for increasing the efficiency of selecting cells with multiple genetic modifications. METHODS: We used the CRISPR/Cas system to target the GGTA1, CMAH and putative iGb3S genes in pigs that have been naturally deleted in humans. Cells lacking galactose α-1,3 galactose (α-Gal) were negatively selected by an IB4 lectin/magnetic bead. α-Gal negative multiplexed single-guide RNA-treated cells were used for somatic cell nuclear transfer (SCNT) and transferred to fertile sows. We examined the levels of α-Gal and Neu5Gc expression of 32 day fetuses and piglets and analyzed the targeted genes by DNA sequencing. RESULTS: Liver-derived cells treated with multiple single-guide RNA and selected for an α-Gal null phenotype were significantly more likely to also carry mutations in simultaneously targeted genes. Multiplex single-guide RNA-treated cells used directly for SCNT without further genetic selection produced piglets with deletions in the targeted genes but also created double- and triple-gene KO variations. CRISPR/Cas-treated cells grew normally and yielded normal liters of healthy piglets via somatic cell nuclear transfer. CONCLUSIONS: The CRISPR/Cas system allows targeting of multiple genes in a single reaction with the potential to create pigs of one genetic strain or multiple genetic modifications in a single pregnancy. The application of this phenotypic selection strategy with multiplexed sgRNA and the Cas9 nuclease has accelerated our ability to produce and evaluate pigs important to xenotransplantation.

Creating class I MHC-null pigs using guide RNA and the Cas9 endonuclease.

Pigs are emerging as important large animal models for biomedical research, and they may represent a source of organs for xenotransplantation. The MHC is pivotal to the function of the immune system in health and disease, and it is particularly important in infection and transplant rejection. Pigs deficient in class I MHC could serve as important reagents to study viral immunity as well as allograft and xenograft rejection. In this study, we report the creation and characterization of class I MHC knockout pigs using the Cas9 nuclease and guide RNAs. Pig fetal fibroblasts were genetically engineered using Cas9 and guide RNAs, and class I MHC(-) cells were then used as nuclear donors for somatic cell nuclear transfer. We produced three piglets devoid of all cell surface class I proteins. Although these animals have reduced levels of CD4(-)CD8(+) T cells in peripheral blood, the pigs appear healthy and are developing normally. These pigs are a promising reagent for immunological research.

Efficient selection of Gal-knockout pig cells for somatic cell nuclear transfer.

BACKGROUND: The process of selecting transgenic cells has been one of the bottlenecks in the generation of transgenic animals by somatic cell nuclear transfer (SCNT). In particular, selection for the Gal double-knockout (Gal-DKO) genotype has been time consuming and inefficient. The objective of this work was to generate a highly efficient system to select Gal-DKO cells to be used in SCNT without affecting the efficiency in production of Gal-null pigs. MATERIALS AND METHODS: Fetal liver-derived cells deficient in Gal-expression were initially selected by fluorescence-activated cell sorting (FACS) using IB4 conjugated to a fluorescent dye. Cells recovered by FACS were cultured and expanded, followed by a second round of selection using streptavidin magnetic beads and IB4 lectin biotin. RESULTS: Recovery efficiency of target cells was 0.04% for the first selection using FACS and 0.3% for the second round by magnetic beads. Full reprogramming was obtained on selected Gal-DKO cells after FACS and magnetic beads selection, when used for SCNT to produce the Gal-null piglets. Cells obtained from magnetic beads developed 48 colonies; the Gal-null genotype was found in 44 of them (91.7%). Three of these colonies were used to generate piglets by SCNT. From three recipients receiving embryos, two became pregnant and produced 17 piglets, all of them DKO. CONCLUSIONS: Sequential selection of Gal-DKO cells by FACS/magnetic beads is a highly efficient system to generate null cells. Selected cells were successfully used to generate healthy double-knockout piglets by SCNT.

Fibronectin from alpha 1,3-galactosyltransferase knockout pigs is a xenoantigen.

BACKGROUND: Antibody-mediated rejection continues to be an obstacle for xenotransplantation despite development of α1,3-galactosyltransferase knockout (GTKO) pigs. Fibronectin (Fn) from GTKO pigs was identified as a xenoantigen in baboons. N-glycolylneuraminic acid (Neu5Gc), similar to galactose α1,3-galactose, is an antigenic carbohydrate found in pigs. We evaluated human antibody reactivity and performed initial antigenic epitope characterization of Fn from GTKO pigs. MATERIALS AND METHODS: GTKO pig aortic endothelial cells (AEC) were isolated and assessed for antibody-mediated complement-dependent cytotoxicity (CDC). Human and GTKO pig Fn were purified and analyzed using immunoblots. GTKO pig and human AEC absorbed human sera were assessed for CDC and anti-GTKO pig Fn antibodies. GTKO pig proteins were assessed for Neu5Gc. Immunoaffinity-purified human IgG anti-GTKO pig (hIgG-GTKOp) Fn using a GTKO pig Fn column were evaluated for cross-reactivity with other proteins. RESULTS: GTKO pig AEC had greater human antibody binding, complement deposition and CDC compared with allogeneic human AEC. Human sera absorbed with GTKO pig AEC resulted in diminished anti-GTKO pig Fn antibody. Neu5Gc was identified on GTKO pig Fn and other proteins. The hIgG-GTKOp Fn cross-reacted with multiple GTKO pig proteins and was enriched with anti-Neu5Gc antibody. CONCLUSIONS: Removal of antigenic epitopes from GTKO pig AEC would improve xenograft compatibility. GTKO pig Fn has antigenic epitopes, one identified as Neu5Gc, which may be responsible for pathology and cross-reactivity of hIgG-GTKOp Fn. Genetic knockout of Neu5Gc appears necessary to address significance and identification of non-Neu5Gc GTKO pig Fn antigenic epitopes.

Double knockout pigs deficient in N-glycolylneuraminic acid and galactose α-1,3-galactose reduce the humoral barrier to xenotransplantation.

BACKGROUND: Clinical xenotransplantation is not possible because humans possess antibodies that recognize antigens on the surface of pig cells. Galα-1,3-Gal (Gal) and N-glycolylneuraminic acid (Neu5Gc) are two known xenoantigens. METHODS: We report the homozygous disruption of the α1, 3-galactosyltransferase (GGTA1) and the cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH) genes in liver-derived female pig cells using zinc-finger nucleases (ZFNs). Somatic cell nuclear transfer (SCNT) was used to produce healthy cloned piglets from the genetically modified liver cells. Antibody-binding and antibody-mediated complement-dependent cytotoxicity assays were used to examine the immunoreactivity of pig cells deficient in Neu5Gc and Gal. RESULTS: This approach enabled rapid production of a pig strain deficient in multiple genes without extensive breeding protocols. Immune recognition studies showed that pigs lacking both CMAH and GGTA1 gene activities reduce the humoral barrier to xenotransplantation, further than pigs lacking only GGTA1. CONCLUSIONS: This technology will accelerate the development of pigs for xenotransplantation research.

Immunobiology of liver xenotransplantation.

Pigs are currently the preferred species for future organ xenotransplantation. With advances in the development of genetically modified pigs, clinical xenotransplantation is becoming closer to reality. In preclinical studies (pig-to-nonhuman primate), the xenotransplantation of livers from pigs transgenic for human CD55 or from α1,3-galactosyltransferase gene-knockout pigs+/- transgenic for human CD46, is associated with survival of approximately 7-9 days. Although hepatic function, including coagulation, has proved to be satisfactory, the immediate development of thrombocytopenia is very limiting for pig liver xenotransplantation even as a 'bridge' to allotransplantation. Current studies are directed to understand the immunobiology of platelet activation, aggregation and phagocytosis, in particular the interaction between platelets and liver sinusoidal endothelial cells, hepatocytes and Kupffer cells, toward identifying interventions that may enable clinical application.

Immortalized porcine liver sinusoidal endothelial cells: an in vitro model of xenotransplantation-induced thrombocytopenia.

BACKGROUND: Xenotransplantation has the potential to solve the critical shortage of human organs available for allotransplantation. The major barrier to porcine liver xenotransplantation is sequestration of human platelets causing thrombocytopenia. Porcine liver sinusoidal endothelial cells (LSEC) bind and phagocytose human platelets at least in part through binding of the asialoglycoprotein receptor 1 (ASGR1). Our purpose was to generate an immortalized porcine LSEC (iLSEC) line that mimics primary LSEC in ASGR1 expression and phagocytosis of human platelets. Porcine iLSEC would enable continued study of xenotransplantation-induced thrombocytopenia in vitro with fewer animals sacrificed. METHODS: Primary domestic porcine LSEC were transduced with lentiviral vector expressing the large and small T antigen of SV40 (SV40 TAg). The phenotype and genotype of the immortalized LSEC were compared with primary LSEC. RESULTS: A total of eight clones expressing SV40 TAg were isolated, and one clone was subcultured and analyzed for growth, phenotype, and function during passages 15-40. Expression of the SV40 TAg was confirmed by confocal microscopy and western blot. MTS cell proliferation assay demonstrated that the clone rapidly grew in culture medium with 2-10% fetal bovine serum. iLSEC expressed the endothelial cell marker, CD31, as determined by confocal microscopy and flow cytometry. Activation of iLSEC by treatment with lipopolysaccharide (LPS) resulted in upregulation of the inflammatory cytokine interleukin 6 (IL 6) by qPCR and ELISA. iLSEC phagocytosed human serum albumin and latex beads as measured by flow cytometry. Human platelets were phagocytosed by immortalized porcine LSEC. CONCLUSIONS: Immortalized porcine LSEC retain a phagocytic phenotype, making them a good model for the study of xenotransplantation-induced thrombocytopenia and may provide further insight into the phagocytic role of LSEC.

Differences in human and porcine platelet oligosaccharides may influence phagocytosis by liver sinusoidal cells in vitro.

BACKGROUND: Acute thrombocytopenia was revealed as a limiting factor to porcine liver xenotransplantation from in vitro and in vivo studies using porcine liver in human and baboon transplant models. The asialoglycoprotein receptor 1 (ASGR1) on liver sinusoidal endothelial cells (LSEC) and macrophage antigen complex-1 (Mac-1) on Kupffer cells (KC) mediate platelet phagocytosis and have carbohydrate-binding sites that recognize galactose and N-acetyl glucosamine in the beta conformation. Analysis of these receptor carbohydrate-binding domains and surface carbohydrates on human and porcine platelets may shed light on the mechanism of xenotransplantation-induced thrombocytopenia. METHODS: An amino acid sequence comparison of human and porcine ASGR1 lectin-binding domains was performed. Using fluorescent labeled-lectins, human platelets, domestic and α1,3 galactosyltransferase knockout/human decay accelerating factor, porcine platelets were characterized by flow cytometry and lectin blot analyses. After desialylation, human and porcine platelets were examined by flow cytometry to determine whether sialic acid capping of galactose and N-acetyl glucosamine oligosaccharides in the beta conformation was a factor. Further, desialylated human platelets were studied on primary porcine liver sinusoidal cells with regard to binding and phagocytosis. RESULTS: Human platelets have four times more exposed galactose ß1-4 N-acetyl glucosamine (Galß) and N-acetyl glucosamine ß1-4 N-acetyl glucosamine (ßGlcNAc) than fresh porcine platelets. Galß and ßGlcNAc moieties on porcine platelets were not masked by sialic acid. Removal of sialic acid from human platelets increased binding and phagocytosis by LSEC and KC. CONCLUSIONS: Differences between human and porcine ASGR1 and Mac-1, in combination with a significantly higher number of galactose and N-acetyl glucosamine-containing oligosaccharides on human platelets contribute, in part, to platelet loss seen in porcine liver xenotransplantation.

Primary porcine Kupffer cell phagocytosis of human platelets involves the CD18 receptor.

BACKGROUND: Hepatic failure has been treated successfully with clinical extracorporeal perfusions of porcine livers. However, dog-to-pig and pig-to-baboon liver xenotransplant models have resulted in severe bleeding secondary to liver xenograft-induced thrombocytopenia. Kupffer cells (KC) are abundant phagocytic cells in the liver. KC express the CD11b/CD18 receptor, which has been implicated in chilled platelet binding and phagocytosis through interaction with platelet surface proteins and carbohydrates. We sought to identify the role of KC CD18 in liver xenograft-induced thrombocytopenia. METHODS: Primary pig KC were characterized by flow cytometry, immunoblots, and quantitative polymerase chain reaction. Pig KC were used in inhibition assays with fluorescently labeled human platelets. The CD18 receptor was targeted for siRNA knockdown. RESULTS: Domestic and α1,3-galactosyltransferase double knockout porcine KC cultures were approximately 92% positive for CD18 as detected by quantitative polymerase chain reaction and flow cytometry. Use of CD18 blocking antibodies resulted in reduction of human platelet binding and phagocytosis. Additionally, asialofetuin, not fetuin, inhibited platelet phagocytosis suggesting the involvement of an oligosaccharide-binding site. Furthermore, reduced CD18 expression by siRNA resulted in decreased human platelet binding. CONCLUSIONS: Our data suggest that primary pig KC bind and phagocytose human platelets with involvement of CD18. Further understanding and modification of CD18 expression in pigs may result in a liver xenograft with reduced thrombocytopenic effects, which could be used as a bridge to allogeneic liver transplantation.

ASGR1 expressed by porcine enriched liver sinusoidal endothelial cells mediates human platelet phagocytosis in vitro.

BACKGROUND: Porcine liver xenografts represent a potential solution to the organ shortage, but thrombocytopenia occurs within minutes to hours after xenotransplantation, preventing clinical application. Recently, it was discovered that porcine liver sinusoidal endothelial cells (LSEC) bind and phagocytose human platelets. We examined the role of ASGR1 in binding and removing human platelets by the pig liver endothelium. METHODS: Primary porcine enriched LSEC (eLSEC) were characterized by flow cytometry, immunoblot, quantitative PCR, and immunohistochemistry using confocal microscopy. Phagocytosis inhibition assays using anti-ASGR1 and an ASGR1 substrate were performed. ASGR1 was targeted for siRNA knockdown, and ASGR1-reduced cells were tested for human platelet binding and phagocytosis. RESULTS: ASGR1 is expressed by eLSEC. Human platelet binding and phagocytosis by porcine eLSEC was inhibited by asialofetuin, but not fetuin, suggesting an interaction with galactose ß1-4 N-acetyl glucosamine. Anti-ASGR1 antibodies inhibited human platelet binding in a dose-dependent manner. Knockdown experiments using siRNA reduced ASGR1 expression in asynchronous primary eLSEC by 40%-80%. There was a 20% reduction in translated protein significantly correlated with a 21% decrease in human platelet binding. CONCLUSIONS: ASGR1 on porcine eLSEC mediates phagocytosis of xenogeneic platelets.

Proteomic studies of Syk-interacting proteins using a novel amine-specific isotope tag and GFP nanotrap.

Green fluorescent protein (GFP) and variants have become powerful tools to study protein localization, interactions, and dynamics. We present here a mass spectrometry-based proteomics strategy to examine protein-protein interactions using anti-GFP single-chain antibody V(H)H in a combination with a novel stable isotopic labeling reagent, isotope tag on amino groups (iTAG). We demonstrate that the single-chain V(H)H (GFP nanotrap) allows us to identify interacting partners of the Syk protein-tyrosine kinase bearing a GFP epitope tag with high efficiency and high specificity. Interacting proteins identified include CrkL, BLNK, α- and ß-tubulin, Csk, RanBP5 and DJ-1. The iTAG reagents were prepared with simple procedures and characterized with high accuracy in the determination of peptides in model peptide mixtures and as well as in complex mixture. Applications of the iTAG method and GFP nanotrap to an analysis of the nucleocytoplasmic trafficking of Syk led to the identification of location-specific associations between Syk and multiple proteins. While the results reveal that the new quantitative proteomic strategy is generally applicable to integrate protein interaction data with subcellular localization, extra caution should be taken in evaluating the results obtained by such affinity purification strategies as many interactions appear to occur following cell lysis.

Regulation of Syk by phosphorylation on serine in the linker insert.

The Syk protein-tyrosine kinase is phosphorylated on multiple tyrosines after the aggregation of the B cell antigen receptor. However, metabolic labeling experiments indicate that Syk is inducibly phosphorylated to an even greater extent on serine after receptor ligation. A combination of phosphopeptide mapping and mass spectrometric analyses indicates that serine 291 is a major site of phosphorylation. Serine 291 lies within a 23-amino acid insert located within the linker B region that distinguishes Syk from SykB and Zap-70. The phosphorylation of serine-291 by protein kinase C enhances the ability of Syk to couple the antigen receptor to the activation of the transcription factors NFAT and Elk-1. Protein interaction studies indicate a role for the phosphorylated linker insert in promoting an interaction between Syk and the chaperone protein, prohibitin.

The fate of human platelets perfused through the pig liver: implications for xenotransplantation.

BACKGROUND: Pig liver xenotransplantation could offset the shortage of livers available for orthotopic liver transplantation. Studies in pig and baboon liver xenografts revealed the main obstacle to be a lethal thrombocytopenia that occurred within minutes to hours of transplantation. METHODS: We have created a model of xenotransplantation-induced thrombocytopenia using ex vivo pig liver perfusion with human platelets. Thrombocytopenia was examined using fluorescently labeled platelets during the ex vivo perfusion and coculture with primary liver sinusoidal endothelial cells (LSEC). RESULTS: Ex vivo liver perfusion revealed that 93% of human platelets were removed from circulation after 15 min. Endothelial cells and platelets were not activated based on tissue factor release into the perfusate. Biopsies from the ex vivo perfusion at 15 and 30 min and in vitro analysis indicated that human platelets are phagocytosed by pig LSEC and degraded in phagosomes. Sixty to 120 min after the addition of platelets to the ex vivo perfusion system, we observed platelet fragments and degraded platelets in hepatocytes. Platelet phagocytosis was not mediated by opsonization as Fc blocking had no effect on platelet phagocytosis. In vitro uptake of human platelets by primary LSEC cultures peaked at 15 min followed by a greater than 55% decrease in platelet fluorescence after 3 h. Primary pig LSEC phagosomes containing human platelets were colocalized with lysosomes positive for lysosome-associated membrane protein-1 (LAMP1), indicating the formation of mature phagosomes within pig LSEC. CONCLUSIONS: Our observation of pig LSEC phagocytosis of human platelets describes a novel mechanism of large-particle uptake in the liver. The creation of a model system to study xenotransplantation-induced thrombocytopenia makes possible the investigation into mechanisms that mediate platelet loss.

Total internal reflection fluorescence flow cytometry.

Total internal reflection fluorescence microscopy (TIRFM) has been widely used to explore biological events that are close to the cell membrane by illuminating fluorescent molecules using the evanescent wave. However, TIRFM is typically limited to the examination of a low number of cells, and the results do not reveal potential heterogeneity in the cell population. In this report, we develop an analytical tool referred to as total internal reflection fluorescence flow cytometry (TIRF-FC) to examine the region of the cell membrane with a throughput of approximately 100-150 cells/s and single cell resolution. We use an elastomeric valve that is partially closed to force flowing cells in contact with the glass surface where the evanescent field resides. We demonstrate that TIRF-FC is able to detect the differences in the subcellular location of an intracellular fluorescent protein. Proper data processing and analysis allows TIRF-FC to be quantitative. With the high throughput, TIRF-FC will be a very useful tool for generating information on cell populations with events and dynamics close to the cell surface.

Detection of kinase translocation using microfluidic electroporative flow cytometry.

Directed localization of kinases within cells is important for their activation and involvement in signal transduction. Detection of these events has been largely carried out based on imaging of a low number of cells and subcellular fractionation/Western blotting. These conventional techniques either lack the high throughput desired for probing an entire cell population or provide only the average behaviors of cell populations without information from single cells. Here we demonstrate a new tool, referred to as microfluidic electroporative flow cytometry, to detect the translocation of an EGFP-tagged tyrosine kinase, Syk, to the plasma membrane in B cells at the level of the cell population. We combine electroporation with flow cytometry and observe the release of intracellular kinase out of the cells during electroporation. We found that the release of the kinase was strongly influenced by its subcellular localization. Cells stimulated through the antigen receptor have a fraction of the kinase at the plasma membrane and retain more kinase after electroporation than do cells without stimulation and translocation. We are able to differentiate a cell population with translocation from one without it with the information collected from individual cells of the entire population. This technique potentially allows detection of protein translocation at the single-cell level. Due to the frequent involvement of kinase translocations in disease processes such as oncogenesis, our approach will have utility for kinase-related drug discovery and tumor diagnosis and staging.