Accepted Manuscript Current status of pig liver xenotransplantation Burcin Ekser, MD, PhD, James F. Markmann, MD, PhD, A.Joseph Tector, MD, PhD PII:
S1743-9191(15)00373-8
DOI:
10.1016/j.ijsu.2015.06.083
Reference:
IJSU 2018
To appear in:
International Journal of Surgery
Received Date: 26 June 2015 Accepted Date: 28 June 2015
Please cite this article as: Ekser B, Markmann JF, Tector AJ, Current status of pig liver xenotransplantation, International Journal of Surgery (2015), doi: 10.1016/j.ijsu.2015.06.083. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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CURRENT STATUS OF PIG LIVER XENOTRANSPLANTATION
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Burcin Ekser, MD, PhD1, James F. Markmann, MD, PhD2, A. Joseph Tector, MD,
(1) Transplant Division, Department of Surgery, Indiana University School of
Medicine, Indianapolis, IN, USA; (2) Division of Transplantation, Department of
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Surgery, Massachusetts General Hospital, Boston, MA, USA
Short Title: Liver xenotransplantation
Address correspondence to: Burcin Ekser, MD, PhD
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Transplant Division, Department of Surgery, Indiana University School of Medicine, 550 University Blvd, Room 4601 Indianapolis, IN, USA
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Telephone: 317-944-4370; Fax 317-948-3268
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Email: [email protected]
KEY WORDS: acute liver failure, liver, pig, nonhuman primate, xenotransplantation
(Word counts: Abstract 161; Text 1,967, Tables 1; Figures 5)
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ABBREVIATIONS β4GalNT2 = beta-1,4-N-acetyl galactosaminyl transferase 2
GTKO = α1,3-galactosyltransferase gene knock-out hDAF = human decay-accelerating factor LSEC = liver sinusoidal endothelial cell
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MELD= model for end-stage liver disease
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CONFLICT OF INTEREST
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Neu5Gc = N-glycolylneuraminic acid WT = wild-type
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GE = genetically-engineered
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The authors declare no conflict of interest.
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ABSTRACT
The shortage of organs from deceased human donors is a major problem limiting the
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number of organs transplanted each year and results in the death of thousands of
patients on the waiting list. Pigs are currently the preferred species for clinical organ xenotransplantation. Progress in genetically-engineered (GE) pig liver
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xenotransplantation increased graft and recipient survival from hours with unmodified pig livers to up to 9 days with normal to near-normal liver function. Deletion of genes
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such as GGTA1 (Gal-knockout pigs) or adding genes such as human complement regulatory proteins (hCD55, hCD46 expressing pigs) enabled hyperacute rejection to be overcome. Although survival up to 9 days was recorded, extended pig graft survival was not achieved due to lethal thrombocytopenia. The current status of GE pig liver
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xenotransplantation with world experience, potential factors causing thrombocytopenia, new targets on pig endothelial cells, and novel GE pigs with more genes deletion to avoid remaining antibody response, such as beta1,4-N-acetyl galactosaminyl
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transferase 2 (β4GalNT2), are discussed.
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INTRODUCTION
Liver transplantation is the only curative therapy for end-stage liver disease. One-year,
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3-year, and 5-year patient survival rates post liver transplantation are 90%, >80%, and >70%, respectively [1]. The current problem with liver transplantation like any other organ transplantation is organ shortage. In the USA, in February 2015, the United
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Network for Organ Sharing waiting list for liver transplantation approached 16,000
patients. This number remains unchanged in the last decade [2]. In 2014, 3,165 patients
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were removed from the waiting list due to death (n= 1,553) or because they became too sick to transplant for the surgical procedure (n=1,612) [2]. In the last 20 years, the total number of patients removed from the liver waiting list without receiving a transplant is
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more than 51,000 [2].
Xenotransplantation, using pig organs, could resolve the shortage of suitable donor organs [3-5]. We have reviewed the current status of pig liver xenotransplantation with
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recent advances in genetically-engineered (GE) pigs and especially in preclinical trials
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with nonhuman primates.
PROGRESS WITH GENETICALLY-ENGINEERED PIGS First studies in experimental preclinical pig-to-nonhuman primate liver xenotransplantation were performed by Calne et al in 1968 [6] using livers from unmodified (wild-type, WT) pigs. The status of WT pig liver xenotransplantation was discussed elsewhere [7-9]. Using GE pigs in xenotransplantation is more limited and not
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as encouraging as other GE pig organ or cell xenotransplantation, though it cleverly raised graft survival from hours with WT pigs to up to 9 days with GE pigs. The world
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experience of GE pig liver xenotransplantation is shown in Table 1.
The first report of GE pig-to-nonhuman primate liver xenotransplantation was by
Ramirez et al [10], where human CD55 (decay-accelerating factor, DAF) expressing
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pigs were used. Two baboon recipients survived 4 and 8 days, respectively. One
baboon recipient died due to the aspiration pneumonia on day 4 and the other baboon
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died due to sepsis and coagulopathy on post-transplant day 8 [10]. By the end of experiments, there were significant increases in direct and total bilirubin (5.4 and 18.9 mg/dl, respectively), indicating cholestatis. In the 8-day survivor baboon, an acute rejection episode on day 2 was treated with three boluses of methylprednisolone [10].
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Thrombocytopenia was evident starting from day 1 in both recipients. On post-operative day 8, the platelet count was 34,000/mm3, associated with a significant reduction in fibrinogen and an elevation of d-dimer, suggesting the development of consumptive
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coagulopathy. Histology of grafts showed no evidence of rejection in the 4-day survivor, but there was evidence of some patchy focal ischemia in the 8-day survivor [10]. The
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same group subsequently reported the use of livers from pigs transgenic for CD55, CD59, and H-transferase [11]. The five recipient baboons survived for only 13 to 24 hours.
The first experience using GTKO (α1,3-galactosyltransferase gene knock-out) pig livers was reported by the Pittsburgh team [12]. Ten orthotopic liver xenotransplants were
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performed in baboons, with two livers being obtained from GTKO pigs and eight from GTKO pigs transgenic for human CD46 (GTKO.hCD46). The Pittsburgh group emphasized the importance of size matching between pig liver and recipient baboon in
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which the pig should be almost 60% smaller in weight than the recipient baboon [12]. Six of 10 baboons survived for 4 to 7 days with normal to near-normal liver function (except for some cholestasis), as evidenced by tests of detoxification, complement
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activity, coagulation parameters, and production of coagulation factors (Figure 1) [13]. Increased bilirubin levels were observed as in the Ramirez et al.’s study [10], but this
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was not associated with mechanical obstruction since viscous bile was present in bile ducts [14]. Kobayashi et al. [15] showed that although human and pig hepatic bile showed similarities, baboon bile is less viscous, containing lower levels of sodium, potassium, calcium, and magnesium, as well as less cholesterol and bilirubin, with
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significantly higher levels of sodium bicarbonate. These observations indicate that bile viscosity seen in pig-to-baboon liver xenotransplants will not be a problem after pig-tohuman liver xenotransplants. All baboons eventually died or euthanized due to severe
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spontaneous bleeding caused by lethal thrombocytopenia (Figure 2) [14, 16].
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More recent GTKO pig-to-baboon liver xenotransplantation studies come from the Boston group. Kim et al were able to achieve 9-day survival using GTKO pig livers in baboons (Table 1) [17]. Three transplanted baboons survived 6, 8, and 9 days. The Boston group preferred to use the same immunosuppressive regimen as in their previous studies, including thymoglobulin and cobra venom factor induction with antiCD154mAb and tacrolimus maintenance (Table 1). The administration of Amicar
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(aminocaproic acid) in 2 cases led to the partial resolution of thrombocytopenia, maintaining platelet counts over 40,000/mm3 throughout the study (Figure 2). Good liver function (normal to near-normal transaminases, coagulation parameters with
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coagulation factors) was documented. However, both recipients died due to bleeding and enterococcal infection. In all three cases, there was no evidence of rejection [17].
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In a more recent study by Yeh et al, the Boston group studied the contribution of
anticoagulant production and clotting pathway deficiencies to fatal bleeding using
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auxiliary GTKO pig liver xenotransplantation in baboons (Table 1) [18]. The donor liver was transplanted in the left upper quadrant by anastomosing the donor portal vein to the confluence of the recipient inferior mesenteric vein and splenic vein (ligating the splenic vein distally post-splenectomy) and the donor infrahepatic inferior vena cava to the
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recipient left renal vein and inferior vena cava confluence. The arterial supply of the auxiliary pig liver was achieved by anastomosing the donor infrahepatic aorta to the recipient infrarenal aorta [18]. There were 3 auxiliary liver xenotransplants with 6, 9, and
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15 days of graft survival. One recipient didn’t receive thymoglobulin induction in order to reduce the infectious complications, however lost the xenograft on day 6 post-transplant
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due to severe acute cellular rejection. This particular baboon recipient survived with his native liver post pig auxiliary liver graftectomy. Interestingly, severe thrombocytopenia recovered immediately after xenograft removal [18]. Pig coagulation factors and proand anti-coagulant levels were measured and compared with orthotopic liver xenotransplantation from the same group [17]. The conclusion was that massive hemorrhage in the setting of orthotopic pig liver xenotransplantation might be avoided
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by supplementation with primate clotting components. However, the remaining native liver continues to be the source of antibodies and coagulation factors which makes the
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model very challenging to make a solid conclusion [18].
The same group recently published a study where they sought to determine the effects of exogenous administration of human coagulation factors infusing human Factor VIIa
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or human prothrombin concentrate complex in GTKO pig-to-baboon liver
xenotransplantation model [19]. In the control baboon which received no human
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coagulation factor, thrombocytopenia was evident throughout the study. Interestingly, the control baboon survived 6 days, however 2 baboons with human prothrombin concentrate complex infusion survived 2 and 4 days. Baboons (n=3) receiving human Factor VIIa infusion survived 6, 6, and 7 days, respectively. Two of them recovered
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platelet counts at the end of the study but eventually died on post-transplant day 6. The longest survived baboon (7 days) with human Factor VIIa infusion had prominent thrombocytopenia at the end of the study [19]. In 3 baboons with human Factor VIIa
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infusion, the histopathology showed minimal to mild inflammation with preserved hepatic architecture on post-operative day 4. Patchy and centrilobular necrosis (10%)
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was also observed with concerns for rejection in one baboon [19]. Unfortunately, there was no data on immunosuppressive regimen and any major bleeding due to thrombocytopenia [19].
FACTORS INFLUENCING THE DEVELOPMENT OF THROMBOCYTOPENIA
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In vivo, ex vivo perfusion, and in vitro studies by the Pittsburgh, Boston, and Indianapolis groups identified several factors influencing the development of survival
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limiting thrombocytopenia and consumptive coagulopathy in GE pig liver xenografts.
The Pittsburgh group found; (i) tissue factor on recipient platelets and PBMCs
(peripheral blood mononuclear cells) (possibly not with activation of donor endothelial
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cells) initiates thrombocytopenia and consumptive coagulopathy independently than immune response starting from 2h post-reperfusion (Figure 3) [16], (ii) no form of
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rejection in 2h biopsies excluding hyperacute rejection, and biopsies at necropsy showed hemorrhagic necrosis, platelet aggregation, platelet-fibrin thrombi, monocyte/macrophage margination mainly in liver sinusoids, and vascular endothelial cell hypertrophy, minimal deposition of IgM, and a near absence of IgG, C3, C4d, C5b-
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9, or cellular infiltrate, suggesting that neither antibody- nor cell-mediated rejection played a major role [12, 14, 16], and (iii) pig von Willebrand factor (vWF) expression on the hepatic vascular endothelium in 2h biopsies and at euthanasia in GTKO pig livers
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and also in WT pig liver when hyperacute rejection occurred [16].
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The Indianapolis group identified; (i) human platelet phagocytosis by pig liver sinusoidal endothelial cells (LSEC) and hepatocytes (Figure 4) [20] via asialoglycoprotein receptor 1 (ASGR1) [21, 22], (ii) differences in human and porcine platelet oligosaccharides influencing platelet phagocytosis by LSECs in vitro [23], and (iii) involvement of CD18 receptor in platelet phagocytosis by porcine Kupffer cells [24]. They also confirmed previous finding of interspecies incompatibilities in CD47/Signal Regulatory Protein
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alpha (SIRPα) self-signaling impacting human platelet uptake by pig LSECs (Figure 5) [25, 26].
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The Boston group, besides confirming the normal to near-normal liver function with good coagulation post GE pig liver xenotransplantation in nonhuman primates [17], showed (i) the partial and complete recovery of thrombocytopenia using aminocapric
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acid [17] and human Factor VIIa infusion (Figure 2) [19]. They also found that massive hemorrhage but not thrombocytopenia post pig liver xenotransplantation could be
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partially ameliorated with auxillary GE pig liver xenotransplantation [18]. (iii) In an in vitro study [27], they confirmed baboon platelet phagocytosis by pig aortic endothelial cells, which was previously shown by the Indianapolis group [20]. However, the pathway
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identified in this work [27] was integrin adhesion pathway involving vWF and MAC-1.
Other significant works published by the Toledo group showing that blocking porcine sialoadhesin improves extracorporeal porcine liver xenoperfusion with human blood
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[28], and by the Maryland group in extracorporeal pig-to-baboon liver xenoperfusion model revealing that glycoprotein Ib receptor blockade through anti-GPIb antibody and
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D-arginine vasopressin improve thrombocytopenia [29].
CONCLUSION AND FUTURE VIEW ON CLINICAL PERSPECTIVE Several groups demonstrated good liver function and the production of porcine proteins and coagulation factors post GE pig liver xenotransplantation in nonhuman primates
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with minimal evidence of rejection [10, 12, 13, 17]. Xenograft survival has been limited by lethal thrombocytopenia and coagulopathy [12, 17, 30] causing hemorrhage in native
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organs as well as the xenografted liver [12, 14, 17].
Recent in vitro and in vivo studies have shed a light on understanding of
thrombocytopenia post pig liver xenotransplantation. Major hemorrhage and/or necrosis
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of pig livers causes limited graft survival despite partial or complete resolution of thrombocytopenia via aminocaproic acid [17] or human Factor VIIa infusion [19]
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indicates that (i) recipient platelets, despite fully present in the peripheral circulation, become non-functional with a shear-stress via porcine LSECs either via ASGR1 receptor or interspecies incompatibility of CD47/SIRPα and/or CD18, MAC-1, vWF/GPIb, or both, and (ii) despite anti-Gal antibodies and complement-related
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immunological injury are mainly avoided via GTKO pigs and human complement regulatory protein expressing pigs, other preformed antibodies (e.g. anti-nonGal antibodies) against proteins, such as β4GalNT2 (beta-1,4-N-acetyl galactosaminyl
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transferase 2) and Neu5Gc (N-glycolylneuraminic acid) may be sufficient to induce an innate immune response, therefore causing an antibody mediated injury in the
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xenograft.
With growing evidence on ASGR1 receptor involvement in xenogeneic platelet phagocytosis in vitro and in a human-to-pig ex vivo perfusion model using porcine forelimbs and livers by Bongoni et al [31], an ASGR1 knockout pig-to-nonhuman primate liver xenotransplantation experiment is of great interest. Recent advancement in
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generating multi-gene knockout pigs, such as GGTA1/CMAH/β4GalNT2 genes [32] using a CRISPR/Cas9 technology [33], or multi-gene expressing pigs such as human CD46, human CD55, human Thrombomodulin, human CD39 [34] will reduce antibody
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binding and may correct consumptive coagulopathy. New preclinical studies in GE pigto-nonhuman primate liver xenotransplantation are warranted using newly available GE pigs, such as ASGR1 knockout, β4GalNT2 knockout, and human SIRPα transgenic
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pigs.
Funding None.
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Ethical approval None.
Research Registration Not required for review article.
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Author contribution
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BE, JFM, AJT – all participated in review of the literature, writing of the manuscript, and final approval of the manuscript
Guarantor
Burcin Ekser, MD, PhD
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[24] R.K. Chihara, L.L. Paris, L.M. Reyes, et al., Primary porcine Kupffer cell phagocytosis of human platelets involves the CD18 receptor, Transplantation. 92 (2011) 739-744.
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Table 1: World experience of preclinical GE pig-to-nonhuman primate liver xenotransplantation Recipient
Type of
N. of
Tx
Tx
Immunosuppression
Survival
Author
Year
Ref
(hours)
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Pig Type
Baboon
Orthotopic
2
CyP+CsA+Cs
hCD55.CD59.
Baboon
Orthotopic
5
CyP+Daclizumab+Rituximab+
HT
CsA+MMF+Cs
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hCD55
#
96, 192
Ramirez
2000
10
13, 18, 20, 21,
Ramirez
2005
11
24
Baboon
Orthotopic
2
ATG+Tacrolimus+MMF+Cs
3, 144
Ekser
2010
12
GTKO.hCD46
Baboon
Orthotopic
8
ATG+Tacrolimus+MMF+Cs
3, 20, 24, 96,
Ekser
2010
12
(n=5),
120, 144, 144,
144, 192, 216
Kim
2012
17
144, 216, 360
Yeh
2014
18
48, 96, 144,
Shah
2015
19
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GTKO
CyP+Tacrolimus+MMF+Cs (n=3) Baboon
Orthotopic
3
ATG+LoCD2b+CVF+anti-
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GTKO
168
CD154mAb+Azathioprine+Cs
Baboon
Auxiliary
3
GTKO
Baboon
Orthotopic
6
ATG+CVF+Tacrolimus+Cs Not available
144, 144, 168
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GTKO
Legend: ATG = anti-thymocyte globulin, CD46 = membrane cofactor protein, CD55 = decay accelerating factor, CD59 = homologous restriction factor (protectin), Cs = corticosteroids, CsA = cyclosporine, CyP = cyclophosphamide, GTKO = α1,3-galactosyltransferase gene-knockout, h = human, HT = H-transferase (α1,2-fucosyltransferase), MMF = mycophenolate mofetil, Tx = transplantation, WT = wild-type.
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Figure Legend: Figure 1: Evidence of genetically-engineered pig liver function in nonhuman primates. A) alanine transaminase pre- and post-transplant in pig-to-baboon liver
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xenotransplantation model; B) International normalized ratio pre- and post-liver
xenotransplantation; C) Coagulation factor Factor V in pre- and post-transplant in pig-tobaboon liver xenotransplantation model with pig (P), baboon (B), and human (H)
average values; D) Western blotting assay to show pig proteins in baboon blood.
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(Obtained with permission from Ekser et al. Transplantation. 90 (2010) 483-493 [13]).
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Figure 2: Platelet counts after in vivo orthotopic GE pig liver xenotransplantation. Platelet counts after pig-to-baboon liver xenotransplantation: Ramirez et al [10]; average of two WT.hCD55 pig liver xenotransplantations. Ekser et al [12]; average of WT (n=1), GTKO (n=2), and GTKO.hCD46 (n=8) pig liver xenotransplantation. Kim et al [17]; average of 3 GTKO pig liver xenotransplantation (estimated from Kim et al. [17]). Shah et al [19]; average of 6 GTKO pig liver xenotransplantation (with available data from the
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abstract)
Figure 3: Tissue factor activity on recipient platelets and PBMCs. Tissue factor activity was measured using the recalcified clotting assay pre-
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transplantation (TX) and 2h after reperfusion of the pig liver (2h) (# = p
S1743-9191(15)00373-8
DOI:
10.1016/j.ijsu.2015.06.083
Reference:
IJSU 2018
To appear in:
International Journal of Surgery
Received Date: 26 June 2015 Accepted Date: 28 June 2015
Please cite this article as: Ekser B, Markmann JF, Tector AJ, Current status of pig liver xenotransplantation, International Journal of Surgery (2015), doi: 10.1016/j.ijsu.2015.06.083. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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CURRENT STATUS OF PIG LIVER XENOTRANSPLANTATION
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Burcin Ekser, MD, PhD1, James F. Markmann, MD, PhD2, A. Joseph Tector, MD,
(1) Transplant Division, Department of Surgery, Indiana University School of
Medicine, Indianapolis, IN, USA; (2) Division of Transplantation, Department of
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Surgery, Massachusetts General Hospital, Boston, MA, USA
Short Title: Liver xenotransplantation
Address correspondence to: Burcin Ekser, MD, PhD
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Transplant Division, Department of Surgery, Indiana University School of Medicine, 550 University Blvd, Room 4601 Indianapolis, IN, USA
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Telephone: 317-944-4370; Fax 317-948-3268
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Email: [email protected]
KEY WORDS: acute liver failure, liver, pig, nonhuman primate, xenotransplantation
(Word counts: Abstract 161; Text 1,967, Tables 1; Figures 5)
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ABBREVIATIONS β4GalNT2 = beta-1,4-N-acetyl galactosaminyl transferase 2
GTKO = α1,3-galactosyltransferase gene knock-out hDAF = human decay-accelerating factor LSEC = liver sinusoidal endothelial cell
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MELD= model for end-stage liver disease
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CONFLICT OF INTEREST
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Neu5Gc = N-glycolylneuraminic acid WT = wild-type
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GE = genetically-engineered
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The authors declare no conflict of interest.
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ABSTRACT
The shortage of organs from deceased human donors is a major problem limiting the
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number of organs transplanted each year and results in the death of thousands of
patients on the waiting list. Pigs are currently the preferred species for clinical organ xenotransplantation. Progress in genetically-engineered (GE) pig liver
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xenotransplantation increased graft and recipient survival from hours with unmodified pig livers to up to 9 days with normal to near-normal liver function. Deletion of genes
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such as GGTA1 (Gal-knockout pigs) or adding genes such as human complement regulatory proteins (hCD55, hCD46 expressing pigs) enabled hyperacute rejection to be overcome. Although survival up to 9 days was recorded, extended pig graft survival was not achieved due to lethal thrombocytopenia. The current status of GE pig liver
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xenotransplantation with world experience, potential factors causing thrombocytopenia, new targets on pig endothelial cells, and novel GE pigs with more genes deletion to avoid remaining antibody response, such as beta1,4-N-acetyl galactosaminyl
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transferase 2 (β4GalNT2), are discussed.
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INTRODUCTION
Liver transplantation is the only curative therapy for end-stage liver disease. One-year,
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3-year, and 5-year patient survival rates post liver transplantation are 90%, >80%, and >70%, respectively [1]. The current problem with liver transplantation like any other organ transplantation is organ shortage. In the USA, in February 2015, the United
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Network for Organ Sharing waiting list for liver transplantation approached 16,000
patients. This number remains unchanged in the last decade [2]. In 2014, 3,165 patients
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were removed from the waiting list due to death (n= 1,553) or because they became too sick to transplant for the surgical procedure (n=1,612) [2]. In the last 20 years, the total number of patients removed from the liver waiting list without receiving a transplant is
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more than 51,000 [2].
Xenotransplantation, using pig organs, could resolve the shortage of suitable donor organs [3-5]. We have reviewed the current status of pig liver xenotransplantation with
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recent advances in genetically-engineered (GE) pigs and especially in preclinical trials
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with nonhuman primates.
PROGRESS WITH GENETICALLY-ENGINEERED PIGS First studies in experimental preclinical pig-to-nonhuman primate liver xenotransplantation were performed by Calne et al in 1968 [6] using livers from unmodified (wild-type, WT) pigs. The status of WT pig liver xenotransplantation was discussed elsewhere [7-9]. Using GE pigs in xenotransplantation is more limited and not
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as encouraging as other GE pig organ or cell xenotransplantation, though it cleverly raised graft survival from hours with WT pigs to up to 9 days with GE pigs. The world
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experience of GE pig liver xenotransplantation is shown in Table 1.
The first report of GE pig-to-nonhuman primate liver xenotransplantation was by
Ramirez et al [10], where human CD55 (decay-accelerating factor, DAF) expressing
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pigs were used. Two baboon recipients survived 4 and 8 days, respectively. One
baboon recipient died due to the aspiration pneumonia on day 4 and the other baboon
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died due to sepsis and coagulopathy on post-transplant day 8 [10]. By the end of experiments, there were significant increases in direct and total bilirubin (5.4 and 18.9 mg/dl, respectively), indicating cholestatis. In the 8-day survivor baboon, an acute rejection episode on day 2 was treated with three boluses of methylprednisolone [10].
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Thrombocytopenia was evident starting from day 1 in both recipients. On post-operative day 8, the platelet count was 34,000/mm3, associated with a significant reduction in fibrinogen and an elevation of d-dimer, suggesting the development of consumptive
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coagulopathy. Histology of grafts showed no evidence of rejection in the 4-day survivor, but there was evidence of some patchy focal ischemia in the 8-day survivor [10]. The
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same group subsequently reported the use of livers from pigs transgenic for CD55, CD59, and H-transferase [11]. The five recipient baboons survived for only 13 to 24 hours.
The first experience using GTKO (α1,3-galactosyltransferase gene knock-out) pig livers was reported by the Pittsburgh team [12]. Ten orthotopic liver xenotransplants were
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performed in baboons, with two livers being obtained from GTKO pigs and eight from GTKO pigs transgenic for human CD46 (GTKO.hCD46). The Pittsburgh group emphasized the importance of size matching between pig liver and recipient baboon in
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which the pig should be almost 60% smaller in weight than the recipient baboon [12]. Six of 10 baboons survived for 4 to 7 days with normal to near-normal liver function (except for some cholestasis), as evidenced by tests of detoxification, complement
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activity, coagulation parameters, and production of coagulation factors (Figure 1) [13]. Increased bilirubin levels were observed as in the Ramirez et al.’s study [10], but this
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was not associated with mechanical obstruction since viscous bile was present in bile ducts [14]. Kobayashi et al. [15] showed that although human and pig hepatic bile showed similarities, baboon bile is less viscous, containing lower levels of sodium, potassium, calcium, and magnesium, as well as less cholesterol and bilirubin, with
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significantly higher levels of sodium bicarbonate. These observations indicate that bile viscosity seen in pig-to-baboon liver xenotransplants will not be a problem after pig-tohuman liver xenotransplants. All baboons eventually died or euthanized due to severe
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spontaneous bleeding caused by lethal thrombocytopenia (Figure 2) [14, 16].
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More recent GTKO pig-to-baboon liver xenotransplantation studies come from the Boston group. Kim et al were able to achieve 9-day survival using GTKO pig livers in baboons (Table 1) [17]. Three transplanted baboons survived 6, 8, and 9 days. The Boston group preferred to use the same immunosuppressive regimen as in their previous studies, including thymoglobulin and cobra venom factor induction with antiCD154mAb and tacrolimus maintenance (Table 1). The administration of Amicar
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(aminocaproic acid) in 2 cases led to the partial resolution of thrombocytopenia, maintaining platelet counts over 40,000/mm3 throughout the study (Figure 2). Good liver function (normal to near-normal transaminases, coagulation parameters with
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coagulation factors) was documented. However, both recipients died due to bleeding and enterococcal infection. In all three cases, there was no evidence of rejection [17].
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In a more recent study by Yeh et al, the Boston group studied the contribution of
anticoagulant production and clotting pathway deficiencies to fatal bleeding using
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auxiliary GTKO pig liver xenotransplantation in baboons (Table 1) [18]. The donor liver was transplanted in the left upper quadrant by anastomosing the donor portal vein to the confluence of the recipient inferior mesenteric vein and splenic vein (ligating the splenic vein distally post-splenectomy) and the donor infrahepatic inferior vena cava to the
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recipient left renal vein and inferior vena cava confluence. The arterial supply of the auxiliary pig liver was achieved by anastomosing the donor infrahepatic aorta to the recipient infrarenal aorta [18]. There were 3 auxiliary liver xenotransplants with 6, 9, and
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15 days of graft survival. One recipient didn’t receive thymoglobulin induction in order to reduce the infectious complications, however lost the xenograft on day 6 post-transplant
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due to severe acute cellular rejection. This particular baboon recipient survived with his native liver post pig auxiliary liver graftectomy. Interestingly, severe thrombocytopenia recovered immediately after xenograft removal [18]. Pig coagulation factors and proand anti-coagulant levels were measured and compared with orthotopic liver xenotransplantation from the same group [17]. The conclusion was that massive hemorrhage in the setting of orthotopic pig liver xenotransplantation might be avoided
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by supplementation with primate clotting components. However, the remaining native liver continues to be the source of antibodies and coagulation factors which makes the
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model very challenging to make a solid conclusion [18].
The same group recently published a study where they sought to determine the effects of exogenous administration of human coagulation factors infusing human Factor VIIa
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or human prothrombin concentrate complex in GTKO pig-to-baboon liver
xenotransplantation model [19]. In the control baboon which received no human
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coagulation factor, thrombocytopenia was evident throughout the study. Interestingly, the control baboon survived 6 days, however 2 baboons with human prothrombin concentrate complex infusion survived 2 and 4 days. Baboons (n=3) receiving human Factor VIIa infusion survived 6, 6, and 7 days, respectively. Two of them recovered
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platelet counts at the end of the study but eventually died on post-transplant day 6. The longest survived baboon (7 days) with human Factor VIIa infusion had prominent thrombocytopenia at the end of the study [19]. In 3 baboons with human Factor VIIa
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infusion, the histopathology showed minimal to mild inflammation with preserved hepatic architecture on post-operative day 4. Patchy and centrilobular necrosis (10%)
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was also observed with concerns for rejection in one baboon [19]. Unfortunately, there was no data on immunosuppressive regimen and any major bleeding due to thrombocytopenia [19].
FACTORS INFLUENCING THE DEVELOPMENT OF THROMBOCYTOPENIA
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In vivo, ex vivo perfusion, and in vitro studies by the Pittsburgh, Boston, and Indianapolis groups identified several factors influencing the development of survival
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limiting thrombocytopenia and consumptive coagulopathy in GE pig liver xenografts.
The Pittsburgh group found; (i) tissue factor on recipient platelets and PBMCs
(peripheral blood mononuclear cells) (possibly not with activation of donor endothelial
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cells) initiates thrombocytopenia and consumptive coagulopathy independently than immune response starting from 2h post-reperfusion (Figure 3) [16], (ii) no form of
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rejection in 2h biopsies excluding hyperacute rejection, and biopsies at necropsy showed hemorrhagic necrosis, platelet aggregation, platelet-fibrin thrombi, monocyte/macrophage margination mainly in liver sinusoids, and vascular endothelial cell hypertrophy, minimal deposition of IgM, and a near absence of IgG, C3, C4d, C5b-
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9, or cellular infiltrate, suggesting that neither antibody- nor cell-mediated rejection played a major role [12, 14, 16], and (iii) pig von Willebrand factor (vWF) expression on the hepatic vascular endothelium in 2h biopsies and at euthanasia in GTKO pig livers
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and also in WT pig liver when hyperacute rejection occurred [16].
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The Indianapolis group identified; (i) human platelet phagocytosis by pig liver sinusoidal endothelial cells (LSEC) and hepatocytes (Figure 4) [20] via asialoglycoprotein receptor 1 (ASGR1) [21, 22], (ii) differences in human and porcine platelet oligosaccharides influencing platelet phagocytosis by LSECs in vitro [23], and (iii) involvement of CD18 receptor in platelet phagocytosis by porcine Kupffer cells [24]. They also confirmed previous finding of interspecies incompatibilities in CD47/Signal Regulatory Protein
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alpha (SIRPα) self-signaling impacting human platelet uptake by pig LSECs (Figure 5) [25, 26].
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The Boston group, besides confirming the normal to near-normal liver function with good coagulation post GE pig liver xenotransplantation in nonhuman primates [17], showed (i) the partial and complete recovery of thrombocytopenia using aminocapric
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acid [17] and human Factor VIIa infusion (Figure 2) [19]. They also found that massive hemorrhage but not thrombocytopenia post pig liver xenotransplantation could be
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partially ameliorated with auxillary GE pig liver xenotransplantation [18]. (iii) In an in vitro study [27], they confirmed baboon platelet phagocytosis by pig aortic endothelial cells, which was previously shown by the Indianapolis group [20]. However, the pathway
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identified in this work [27] was integrin adhesion pathway involving vWF and MAC-1.
Other significant works published by the Toledo group showing that blocking porcine sialoadhesin improves extracorporeal porcine liver xenoperfusion with human blood
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[28], and by the Maryland group in extracorporeal pig-to-baboon liver xenoperfusion model revealing that glycoprotein Ib receptor blockade through anti-GPIb antibody and
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D-arginine vasopressin improve thrombocytopenia [29].
CONCLUSION AND FUTURE VIEW ON CLINICAL PERSPECTIVE Several groups demonstrated good liver function and the production of porcine proteins and coagulation factors post GE pig liver xenotransplantation in nonhuman primates
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with minimal evidence of rejection [10, 12, 13, 17]. Xenograft survival has been limited by lethal thrombocytopenia and coagulopathy [12, 17, 30] causing hemorrhage in native
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organs as well as the xenografted liver [12, 14, 17].
Recent in vitro and in vivo studies have shed a light on understanding of
thrombocytopenia post pig liver xenotransplantation. Major hemorrhage and/or necrosis
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of pig livers causes limited graft survival despite partial or complete resolution of thrombocytopenia via aminocaproic acid [17] or human Factor VIIa infusion [19]
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indicates that (i) recipient platelets, despite fully present in the peripheral circulation, become non-functional with a shear-stress via porcine LSECs either via ASGR1 receptor or interspecies incompatibility of CD47/SIRPα and/or CD18, MAC-1, vWF/GPIb, or both, and (ii) despite anti-Gal antibodies and complement-related
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immunological injury are mainly avoided via GTKO pigs and human complement regulatory protein expressing pigs, other preformed antibodies (e.g. anti-nonGal antibodies) against proteins, such as β4GalNT2 (beta-1,4-N-acetyl galactosaminyl
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transferase 2) and Neu5Gc (N-glycolylneuraminic acid) may be sufficient to induce an innate immune response, therefore causing an antibody mediated injury in the
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xenograft.
With growing evidence on ASGR1 receptor involvement in xenogeneic platelet phagocytosis in vitro and in a human-to-pig ex vivo perfusion model using porcine forelimbs and livers by Bongoni et al [31], an ASGR1 knockout pig-to-nonhuman primate liver xenotransplantation experiment is of great interest. Recent advancement in
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generating multi-gene knockout pigs, such as GGTA1/CMAH/β4GalNT2 genes [32] using a CRISPR/Cas9 technology [33], or multi-gene expressing pigs such as human CD46, human CD55, human Thrombomodulin, human CD39 [34] will reduce antibody
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binding and may correct consumptive coagulopathy. New preclinical studies in GE pigto-nonhuman primate liver xenotransplantation are warranted using newly available GE pigs, such as ASGR1 knockout, β4GalNT2 knockout, and human SIRPα transgenic
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pigs.
Funding None.
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Ethical approval None.
Research Registration Not required for review article.
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Author contribution
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BE, JFM, AJT – all participated in review of the literature, writing of the manuscript, and final approval of the manuscript
Guarantor
Burcin Ekser, MD, PhD
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[24] R.K. Chihara, L.L. Paris, L.M. Reyes, et al., Primary porcine Kupffer cell phagocytosis of human platelets involves the CD18 receptor, Transplantation. 92 (2011) 739-744.
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Table 1: World experience of preclinical GE pig-to-nonhuman primate liver xenotransplantation Recipient
Type of
N. of
Tx
Tx
Immunosuppression
Survival
Author
Year
Ref
(hours)
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Pig Type
Baboon
Orthotopic
2
CyP+CsA+Cs
hCD55.CD59.
Baboon
Orthotopic
5
CyP+Daclizumab+Rituximab+
HT
CsA+MMF+Cs
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hCD55
#
96, 192
Ramirez
2000
10
13, 18, 20, 21,
Ramirez
2005
11
24
Baboon
Orthotopic
2
ATG+Tacrolimus+MMF+Cs
3, 144
Ekser
2010
12
GTKO.hCD46
Baboon
Orthotopic
8
ATG+Tacrolimus+MMF+Cs
3, 20, 24, 96,
Ekser
2010
12
(n=5),
120, 144, 144,
144, 192, 216
Kim
2012
17
144, 216, 360
Yeh
2014
18
48, 96, 144,
Shah
2015
19
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GTKO
CyP+Tacrolimus+MMF+Cs (n=3) Baboon
Orthotopic
3
ATG+LoCD2b+CVF+anti-
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GTKO
168
CD154mAb+Azathioprine+Cs
Baboon
Auxiliary
3
GTKO
Baboon
Orthotopic
6
ATG+CVF+Tacrolimus+Cs Not available
144, 144, 168
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GTKO
Legend: ATG = anti-thymocyte globulin, CD46 = membrane cofactor protein, CD55 = decay accelerating factor, CD59 = homologous restriction factor (protectin), Cs = corticosteroids, CsA = cyclosporine, CyP = cyclophosphamide, GTKO = α1,3-galactosyltransferase gene-knockout, h = human, HT = H-transferase (α1,2-fucosyltransferase), MMF = mycophenolate mofetil, Tx = transplantation, WT = wild-type.
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Figure Legend: Figure 1: Evidence of genetically-engineered pig liver function in nonhuman primates. A) alanine transaminase pre- and post-transplant in pig-to-baboon liver
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xenotransplantation model; B) International normalized ratio pre- and post-liver
xenotransplantation; C) Coagulation factor Factor V in pre- and post-transplant in pig-tobaboon liver xenotransplantation model with pig (P), baboon (B), and human (H)
average values; D) Western blotting assay to show pig proteins in baboon blood.
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(Obtained with permission from Ekser et al. Transplantation. 90 (2010) 483-493 [13]).
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Figure 2: Platelet counts after in vivo orthotopic GE pig liver xenotransplantation. Platelet counts after pig-to-baboon liver xenotransplantation: Ramirez et al [10]; average of two WT.hCD55 pig liver xenotransplantations. Ekser et al [12]; average of WT (n=1), GTKO (n=2), and GTKO.hCD46 (n=8) pig liver xenotransplantation. Kim et al [17]; average of 3 GTKO pig liver xenotransplantation (estimated from Kim et al. [17]). Shah et al [19]; average of 6 GTKO pig liver xenotransplantation (with available data from the
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abstract)
Figure 3: Tissue factor activity on recipient platelets and PBMCs. Tissue factor activity was measured using the recalcified clotting assay pre-
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transplantation (TX) and 2h after reperfusion of the pig liver (2h) (# = p
