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Current progress in xenotransplantation and organ bioengineering
International Journal of Surgery 13 (2015) 239e244
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International Journal of Surgery journal homepage: www.journal-surgery.net
Review
Current progress in xenotransplantation and organ bioengineering Sebastian G. Michel a, b, *, Maria Lucia L. Madariaga a, c, Vincenzo Villani a, Kumaran Shanmugarajah a, d a
Transplantation Biology Research Center, Massachusetts General Hospital, Building 149, 13th Street, Charlestown, Boston, MA 02114, USA €t München, Munich D-81377, Germany Department of Cardiac Surgery, Ludwig-Maximilians-Universita c Department of Surgery, Massachusetts General Hospital, 55 Fruit Street, Boston, MA 02129, USA d Division of Surgery, Imperial College London, Exhibition Road, London SW7 2AZ, United Kingdom b
h i g h l i g h t s Xenotransplantation and bioengineering could solve the donor organ shortage crisis. Bioengineered organs and xenografts are challenged by immunobiological barriers. Further work is needed to generate transplantable complex organs or xenografts.
a r t i c l e i n f o
a b s t r a c t
Article history: Received 27 July 2014 Received in revised form 30 November 2014 Accepted 7 December 2014 Available online 11 December 2014
Organ transplantation represents a unique method of treatment to cure people with end-stage organ failure. Since the first successful organ transplant in 1954, the field of transplantation has made great strides forward. However, despite the ability to transform and save lives, transplant surgery is still faced with a fundamental problem the number of people requiring organ transplants is simply higher than the number of organs available. To put this in stark perspective, because of this critical organ shortage 18 people every day in the United States alone die on a transplant waiting list (U.S. Department of Health & Human Services, http://organdonor.gov/about/data.html). To address this problem, attempts have been made to increase the organ supply through xenotransplantation and more recently, bioengineering. Here we trace the development of both fields, discuss their current status and highlight limitations going forward. Ultimately, lessons learned in each field may prove widely applicable and lead to the successful development of xenografts, bioengineered constructs, and bioengineered xeno-organs, thereby increasing the supply of organs for transplantation. © 2014 Surgical Associates Ltd. Published by Elsevier Ltd. All rights reserved.
Keywords: Organ shortage Xenotransplantation Bioengineering
1. Introduction
2. History of clinical xenotransplantation
Regenerative medicine and xenotransplantation share the same goal e that of replacing diseased organs with newly functioning ones. Both of these fields have the potential to mitigate the evergrowing demand for transplantable organs and reduce waiting list mortality by creating a new, inexhaustible supply of organs.
Clinical animal-to-human solid organ transplants have been performed on a number of occasions in critical situations (Table 1). In 1964, James D. Hardy attempted a chimpanzee to human heart transplant [1]. Subsequently Christiaan Barnard's team performed heart xenotransplants using baboon and chimpanzee donors [2]. In these early cases patients survived hours to days, with death attributed to the small size of primate hearts being unable to support the circulation of adult humans. Other cases highlighted the immunological barriers that had to be overcome for xenotransplants to work. Notably, a team lead by Leonard L. Bailey in California performed a heart transplant on Baby Fae, in which size was not an issue but the graft failed at 20 days due to humoral rejection [3]. Despite the poor outcomes in initial xenotransplants,
* Corresponding author. Transplantation Biology Research Center, Massachusetts General Hospital and Harvard Medical School, Building 149, 13th Street, Charlestown, Boston MA 02114, USA. E-mail addresses: [email protected] (S.G. Michel), [email protected] (K. Shanmugarajah). http://dx.doi.org/10.1016/j.ijsu.2014.12.011 1743-9191/© 2014 Surgical Associates Ltd. Published by Elsevier Ltd. All rights reserved.
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Table 1 Milestones in clinical xenotransplantation. Year
Donor
Patient survival
1963/64 (13) 1963/64 (6) 1964 1977 (2)
Chimpanzee kidneys Baboon kidneys
Up to 9 Keith Reemtsma [4] months 19 to 98 days Thomas Starzl [5]
Chimpanzee heart Baboon and chimpanzee hearts Baboon heart Baboon liver
2h 6 h and 4 days 20 days 70 days
1984 1992
Surgeon
James Hardy Christiaan Barnard Leonard Bailey Thomas Starzl
Refs.
[1] [2] [3] [6,7]
a few cases provided optimism that this form of organ replacement could work. In a series of chimpanzee-to-human kidney transplants performed by Keith Reemtsma, a patient was reported to have survived for nine months [4]. Thomas E. Starzl's group reported the survival 19e98 days in 6 cases of baboon-to-human transplantation [5]. In 1992, Starzl and colleagues reported 70 day survival in baboon-to-human liver xenotransplants. [6,7] 3. Optimal donor species for xenotransplantation While non-human primates are phylogenetically the most similar to humans, the use of these animals as xenotransplant donors has several drawbacks. The most pertinent of these include their small sizes, infection risks, long gestation and growth periods and ethical concerns. For these reasons, swine are currently considered the most likely xenotransplant donors. Swine are easy to breed, can be produced in germ-free conditions, their organs reach a size that can provide life-supporting functions to human recipients and the ethical barriers precluding nonhuman primate research are present to a lesser degree. Furthermore, swine can be genetically manipulated to permit immune challenges to be overcome. However, there are still major obstacles before swine can be successfully used as a source of organs. First, the immunological responses of the recipient against the graft need to be controlled. These include hyperacute rejection (characterized by binding of naturally-occuring xenoreactive antibodies that trigger the complement cascade), acute antibody-mediated rejection (acute humoral xenograft rejection, AHXR), acute cellular rejection, and chronic rejection. Second, xenografts may have physiological limitations, such as molecular incompatibilities in the coagulation system, which preclude their use in providing functional replacement of a failing organ. Third, risk of transmissible infections such as porcine endogenous retroviruses (PERVs) affecting humans. To date there has been no case where PERVs have caused an infection in humans that were exposed to porcine tissues [8]. However, pig cells can transmit PERV to human cells in vitro [9], which makes it mandatory that all potential recipients of porcine organs and cells, as well as their families be monitored closely. The risk of contracting West Nile virus, rabies and HIV from swine organs is considered vanishingly low [10]. 4. Overcoming the immune response to pig xenotransplants by genetic engineering Unlike the rejection of allografts, which is mainly governed by T cells, the immune response to porcine xenografts primarily occurs hyperacutely, mediated by pre-formed, natural antibodies to the Galactosyl-alpha(1,3)galactose (Gal) epiotope that is highly expressed on porcine endothelium. Knocking out the gene for a1,3galactosyltransferase prevents the expression of the Gal epitope on porcine tissues. This helped overcome hyperacute rejection in pig-
to-primate studies [11]. Despite this, non-Gal antibodies may still activate the porcine endothelium, leading to microvascular thrombosis and graft loss. Byrne and colleagues identified many non-Gal antigens on porcine endothelium which are members of the heat shock protein family [12]. If the next generation of genetically modified pigs can address these non-Gal antigens, another significant step towards controlling the humoral response to xenotransplants will be made. Aside from the humoral response, efforts have also been made to control the innate immune response to xenotransplantation by engineering pigs that: 1) express human CD47, a marker for “self”, to prevent organ damage caused by macrophages [13,14]; 2) express the inhibitory receptor HLA-E to control the NK cell response [15]; or 3) express complement regulators, such as human decay accelerating factor (hDAF) [16] or membrane cofactor protein (hCD46). However, more important for graft survival was the supraphysiologic expression of these complement regulators rather than the fact that they were human [17]. Thrombotic microangiopathy caused by immunologic mechanisms is compounded in the xenotransplant setting due the molecular incompatibilities in the coagulation cascade between pig and primate. To overcome this, efforts are underway to allow for the expression of human anticoagulants on pig endothelium. These include expression of human CD39 to inhibit platelets, human thrombomodulin to allow activation of the human anticoagulant protein C and tissue factor pathway inhibitor to prevent the initiation of the extrinsic pathway of coagulation [18]. 5. Immunosuppression protocols for xenotransplantation Immunosuppression in preclinical models of xenotransplantation usually consists of B-cell and plasma cell therapeutics like Rituximab and Bortezomib in addition to the standard triple drug immunosuppression [19]. For example, peritransplant B-cell depletion using 4 weekly doses of anti-CD20 antibody, along with ATG, anti-CD154 and MMF-based immunosuppressive regimen resulted in prolonged survival of Gal-knock out (Gal-KO)/ human CD46 transgenic pig cardiac xenografts (up to 236 days) [20]. One or more rounds of immuno-adsorption or plasmapheresis are necessary to remove antibodies from the recipient's circulation. These regimens are often associated with severe side effects like pancytopenia and sepsis. Considering that systemic immunosuppression needs to be higher in xenotransplantation than in allotransplantation, another strategy to counteract this effect is to express immunosupppressive molecules like CTLA-4Ig on the pig endothelium [21]. However, the step-wise approach of gene knock-in or gene knock-out is limited in scope; kidneys from pigs who were Gal-KO and transgenic for human CD55 (hCD55), hCD59, hCD39, and fucosyl-transferase (hHT) showed limited improved survival in baboons [22]. An alternative but much more complex approach is to try to achieve immunological tolerance to the xenograft. In clinical allotransplantation, the tolerance approach has already proven to be successful by achieving transient mixed hematopoietic chimerism by donor bone marrow co-transplantation with kidney allografts. A number of patients at Massachusetts General Hospital have been living immunosuppression-free for several years now after receiving their kidney along with bone marrow of the donor [23,24]. Apart from generating bone marrow-based chimerism, another tolerance approach would be to transplant xenogeneic thymus to promote recipient thymopoiesis, which could induce T cell tolerance to solid organ xenografts. Indeed, based on this approach, Yamada and colleagues have reported over 80 day survival of a life-supporting kidney transplant in a pig-to-primate model [25,26].
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Other experimental protocols are based on studies by Billingham and colleagues showing that the environment during early development can induce immune tolerance [27]. Chimeric pig hearts were harvested from pigs rendered chimeric by intrauterine injection of sheep mononuclear cells [28]. These chimeric pig hearts, when transplanted into sheep and maintained on single-agent immunosuppression, showed superior freedom from acute rejection and graft survival compared to non-chimeric donor hearts [28]. 6. Current status of specific xenografts
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recipients [43]. 6.4. Lung Reports of orthotopic pig-to-primate pulmonary transplantation has achieved only very limited survival rates 72 h [44] and 4.5 days [45] compared to other solid organs. Lungs release large amounts of von Willebrand Factor that binds GPIb on human/ primate platelets. This leads to pronounced platelet activation and clotting. A possible future strategy to control this includes using transgenic pigs that express tissue factor pathway inhibitor or CD39.
6.1. Heart 6.5. Islets of langerhans In a preclinical life-supporting orthotopic pig-to-baboon model, Ekser and colleagues have achieved median survival of 40 days (maximum of 57 days) of heart xenografts using Gal-KO/CD46 transgenic pigs and immunosuppression with ATG induction, tacrolimus, sirolimus and tapering steroids [29]. Despite these results, a limiting factor of orthotopic xenoheart transplant has been early graft failure not attributable to hyperacute rejection [30]. Possible causes for this phenomenon, termed “perioperative cardiac xenograft dysfunction” are sensitivity of the pig heart to the extracorporeal circulation and ischemia reperfusion injury or the inflammatory effect of preformed non-Gal antibodies [30]. Importantly, these problems are not insurmountable due to advances in critical care.
Pig islet transplantation has already been performed clinically in countries with limited national regulations like Mexico, Russia and China [46], but most researchers agree that more preclinical data is necessary before this treatment should be applied to patients. In the pig-to-nonhuman primate model, encapsulation has been shown to be effective to prevent damage to the islets caused by T cells and antibodies. The islets functioned for 6 months without immunosuppression [47]. Valdez-Gonzalez grafted neonatal porcine islets into the subcutaneous tissue of 12 children with IDDM without immunosuppression which lead to reduction in insulin requirement [48]. 7. Applying bioengineering to organ transplantation
6.2. Kidney Using a tolerance approach, over 80 days of kidney graft survival was achieved in two baboons who received grafts from Gal-KO pigs [11]. The baboons underwent thymectomy and splenectomy prior to combined kidney and vascularized thymus transplantation. Immunosuppression consisted of T-cell depletion with ATG and anti-CD2, cobra venom factor, anti-CD154, MMF and tapering steroids. 6.3. Liver Extracorporeal liver perfusion devices containing pig hepatocytes to treat acute liver failure have been used in over 100 clinical patients [31e35]. However, a significant survival benefit has not been shown. In addition, whole organ extracorporeal pig liver perfusions in acute liver failure have been performed in 11 patients in the United States as a bridge-to-transplant with a mortality of 50% [36,37]. In a preclinical pig-to-primate liver transplant model, 8-day survival was reported using hDAF-expressing pigs. In this study, newly synthesized proteins by the porcine liver were demonstrated [38]. Baboon recipients of liver grafts from alpha1,3-galactosyl transferase knockout pigs survived 6, 8 and 9 days before succumbing to infection and coagulopathy [39]. The functionality of liver factors and proteins when transplanted into non-condordant species remains undetermined. Recent studies demonstrate successful generation of humanized rat and mouse livers. In these small animal models, human hepatocytes are successfully engrafted into host livers after treating host animals with injury-, adenovirus- or toxin-based techniques [40e42]. Apart from being useful for pharmacologic and infectious studies, if this technology can be scaled up to a human size, e.g. humanized pig livers, then this may prove to be an important method to enhance hepatocyte acceptance across species. As proofof-principle, chimeric mouse livers seeded with rat hepatocytes were able to sustain liver function for up to 6 months in rat
Similar to xenotransplantation, regenerative medicine has the potential to replace diseased organs with functional ones. Tremendous growth in the field of regenerative medicine occurred in the 1990s with the advent of developments in nuclear transfer technology, stem cell isolation, and tissue engineering, in which the principles of biology and engineering are applied to develop functional substitutes for damaged tissue and organs [49e51]. In addition, regenerative medicine may also bolster existing organ transplant practices by improving the quality of organs deemed ineligible for transplant [52]. Furthermore, bioengineered organs could also be transplanted without need for life-long immunosuppression, erasing the heavy burden associated with its side effects and cost. These advantages may allow patients to be transplanted earlier, when they are healthier and better able to withstand surgery. 8. Principles of organ bioengineering In current approaches to developing bioartificial tissue, a target organ is first decellularized, then reseeded with appropriate cell types, and then matured in a bioreactor [53]. Target organs can be harvested up to 4e6 h postmortem, up to the point at which proteolysis begins. Decellularization refers to the process in which an organ is stripped of its viable tissue usually by a mixture of detergents until all that remains are the basic protein and polysaccharide components. This biocompatible backbone is a natural, ideal platform for organ bioengineering. It is already geometrically and spatially organized and provides an essential source of signaling cues for cells to grow, expand, and exert their functiondwitness the improved outcomes of whole pancreas transplantation over islet cell transplantation [54]. After generating a scaffold, there are different options for reseeding. The appropriate cell to repopulate a construct needs to take into account expansion, functionality, and replicative senescence. Currently, stem cells, progenitor cells, and human embryonic
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stem cells are difficult to obtain, may have limited expansion capabilities and are fraught with ethical concerns [49]. The ectopic expression of transcription factors can generate autologous cells (induced pluripotent stem cells) for organ replacement; however, these cells have tumorigenic traits since reprogramming is often accompanied by genetic and epigenetic alterations [55]. Another option is to rely on repopulation by host cells in vivo, a process which has been shown in implanted dermal matrices and trachea [49]. To date the most successful bioengineered organs to have been used in the clinical setting are relatively simple, hollow, avascular structures without reconnection to a vascular supply (Table 2). In 2004, a 30 year old female with end-stage bronchomalacia underwent implantation of a totally bioengineered human trachea constructed from a deceased donor scaffold seeded with autologous cells derived from mesenchymal stem cells and epithelial cells [56]. In 2006, human bladder matrices were seeded with autologous bladder cells grown from culture and implanted into 10 patients who did well with a mean follow-up of nearly 4 years [57]. In 2007, blood vessels engineered from autologous skin and superficial vein cells were also successfully implanted into 10 patients with end-stage renal disease on hemodialysis [58]. Currently over 160 patients have received successful constructs of tracheas, bladders, and blood vessels without the need for immunosuppressive medication [59,60]. However, these constructs rely on diffusion for nutrients and oxygen exchange and are therefore limited in size by risk of ischemia and subsequent graft failure.
9. Current status of specific bioengineered organs 9.1. Heart Initial forays into cardiac bioengineering focused on repairing damaged cardiac muscle with targeted delivery of myoblasts [61e63]. Recently, the perfusion decellularization technique was applied to rat hearts to generate whole organ scaffolds. These structures were repopulated with neonatal rat cardiomyocytes and the resulting organ was able to demonstrate contractility of up to 2% of normal contractile function [64].
9.3. Liver In contrast to patients with kidney failure on dialysis, patients in liver failure have no recourse for liver replacement therapy. Attempts to create an extracorporeal bioartifical liver system containing functional, viable hepatocytes have fallen short of expectations. Worldwide, 78 patients have been recipients of hepatocyte infusions: genetically modified hepatocytes were infused into 21 patients with metabolic disease and genetically unmodified hepatocytes were infused into 20 patients with chronic liver disease and 37 patients with acute fulminant liver failure [68] with mixed results. Current technology is limited by the inability to generate sufficient liver mass to sustain physiological function. Recently, new protocols have induced human fibroblasts to a multipotent progenitor cell state from which hepatocytes could successfully differentiate, proliferate, and repopulate mouse livers [69]. Other groups have successfully seeded liver scaffolds with adult hepatocytes and implanted the recellularized scaffolds into rats for up to 8 h [70]. This bioengineered organ was able to support hepatocyte survival and liver-specific function, such as production of albumin, urea synthesis, and cytochrome P450 expression. 9.4. Kidney Decellularized kidneys have been made with rat, pig, and rhesus monkey kidneys [71,72]. Renal extracellular matrices produced from porcine kidneys were implanted into pigs in vivo as proof-ofconcept and demonstrated the ability to withstand physiologic blood pressure without extravasation; however, despite preservation of renal architecture at 2 weeks, the naked vasculature was thrombosed by 24 h after implantation [73]. An important contribution was made in 2013 when Song and colleagues reported that rat kidneys seeded with epithelial and endothelial cells produced rudimentary urine in vivo. [74] Future advances depend on further understanding the developmental biology of the kidney and identification of potential renal progenitor cells [51,75,76]. 9.5. Pancreas Pancreatic scaffolds have been used to help produce large numbers of functional pancreatic endocrine cells [77] and a bioengineered rat pancreas seeded with rat islets was able to modulate glucose metabolism and reduce the need for exogenous insulin in diabetic rats [78].
9.2. Lung 9.6. Intestine Lung bioengineering is challenging because of the complexity of the tissue and multiple cell types involved. Two landmark studies in 2010 established the feasibility of bioengineered lungs in small animal models. These organs were generated by decellularizing rat lungs, repopulating them with epithelial and endothelial cells, and implanting them into rats in vivo for up to 6 h to demonstrate successful gas exchange [65,66]. In an effort to scale these findings up to a clinically relevant model, porcine lungs were recently successfully decellularized to produce lung scaffolds with in vitro properties resembling those of human lungs [67].
Table 2 Milestones in clinical implantation of bioengineered organs. Date
Organ
Clinical outcome
Refs.
2004 2006 2007
Trachea Bladder Blood vessels
Proximal graft necrosis Successful implantation up to 4 years Successful implantation up to 13 months
[56] [57] [58]
In dogs, composite esophaguses were developed into welldifferentiated lumen of stratified squamous cells surrounded by a thick smooth muscle-like tissue that could be surgically manipulated in mock esophagectomies [79]. 10. Xenobioengineered organs An intriguing prospect is to combine the strengths of both xenotransplantation and bioengineering to generate “semi-xenografts” where the scaffold would be animal-derived and the repopulated cells would be human-derived [51]. This would make it easier to control the quality of the scaffold, as pathogen-free herds and post-harvest processing would eliminate most known pathogens [49]. For example, bioartificial human tissue with an innate vascularized network was created using a porcine small bowel platform [59,80]. Though the majority of proteins in the extracellular matrix are highly conserved across species [81], the significant
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barriers facing xenotransplantation still pertain, such as the antigenicity of the Gal epitope on swine [82]. 11. Limitations of current technology and future challenges The scientific barriers to a successfully engineered organ can be grouped into three main levels: scaffolds, cells, and construct [51]. Scaffolds must be biocompatible, promote cell growth, allow for angiogenesis and remodeling, and avoid immunogenicity. Cells need to be functional and sufficient in number without becoming tumorigenic or contaminated. The entire construct, to be clinically viable, needs to withstand physiologic blood pressure, avoid clotting, and be accepted by the host immune system. Further study is needed to understand mechanisms of organ development/regeneration, cell to cell or extracellular matrix interactions, and vascularization. Practically, bioengineered organs must meet quality standards. Imaging, in vitro assays, and bioinformatics can aid in assessing the quality of the construct and the state of the tissue and cells within it. Finally, cost. If organ bioengineering remains a highly individualistic process, the high cost of generating stem cells for each individual alone is prohibitive [83]. Once the technology is more mature, a comparative analysis of cost is necessary to determine whether this method represents a cost-effective manner in which to increase the organ donor pool.
[3]
[4] [5]
[6] [7] [8]
[9] [10] [11]
[12]
[13]
[14]
[15]
12. Conclusion [16]
Xenotransplantation and bioengineering of organs are two promising new technologies to treat terminal organ failure. Both provide potential solutions to the worsening organ shortage facing allo-transplantation today. Time will determine which of the two approaches, if at all, will live up to expectations. In the meantime, efforts should be invested in both technologies to eventually tailor therapeutic strategies to each individual patient.
[17]
[18] [19]
[20]
Ethical approval None. Financial support None. Author contribution KS e conception and design, analysis, writing of manuscript and critical review. VV e conception and design, analysis, writing of manuscript and critical review. LM e conception and design, analysis,writing of manuscript and critical review. SM e conception and design, analysis, writing of manuscript and critical review. Conflicts of interest
[21]
[22]
[23]
[24]
[25]
[26] [27]
[28]
[29] [30]
None. [31]
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International Journal of Surgery journal homepage: www.journal-surgery.net
Review
Current progress in xenotransplantation and organ bioengineering Sebastian G. Michel a, b, *, Maria Lucia L. Madariaga a, c, Vincenzo Villani a, Kumaran Shanmugarajah a, d a
Transplantation Biology Research Center, Massachusetts General Hospital, Building 149, 13th Street, Charlestown, Boston, MA 02114, USA €t München, Munich D-81377, Germany Department of Cardiac Surgery, Ludwig-Maximilians-Universita c Department of Surgery, Massachusetts General Hospital, 55 Fruit Street, Boston, MA 02129, USA d Division of Surgery, Imperial College London, Exhibition Road, London SW7 2AZ, United Kingdom b
h i g h l i g h t s Xenotransplantation and bioengineering could solve the donor organ shortage crisis. Bioengineered organs and xenografts are challenged by immunobiological barriers. Further work is needed to generate transplantable complex organs or xenografts.
a r t i c l e i n f o
a b s t r a c t
Article history: Received 27 July 2014 Received in revised form 30 November 2014 Accepted 7 December 2014 Available online 11 December 2014
Organ transplantation represents a unique method of treatment to cure people with end-stage organ failure. Since the first successful organ transplant in 1954, the field of transplantation has made great strides forward. However, despite the ability to transform and save lives, transplant surgery is still faced with a fundamental problem the number of people requiring organ transplants is simply higher than the number of organs available. To put this in stark perspective, because of this critical organ shortage 18 people every day in the United States alone die on a transplant waiting list (U.S. Department of Health & Human Services, http://organdonor.gov/about/data.html). To address this problem, attempts have been made to increase the organ supply through xenotransplantation and more recently, bioengineering. Here we trace the development of both fields, discuss their current status and highlight limitations going forward. Ultimately, lessons learned in each field may prove widely applicable and lead to the successful development of xenografts, bioengineered constructs, and bioengineered xeno-organs, thereby increasing the supply of organs for transplantation. © 2014 Surgical Associates Ltd. Published by Elsevier Ltd. All rights reserved.
Keywords: Organ shortage Xenotransplantation Bioengineering
1. Introduction
2. History of clinical xenotransplantation
Regenerative medicine and xenotransplantation share the same goal e that of replacing diseased organs with newly functioning ones. Both of these fields have the potential to mitigate the evergrowing demand for transplantable organs and reduce waiting list mortality by creating a new, inexhaustible supply of organs.
Clinical animal-to-human solid organ transplants have been performed on a number of occasions in critical situations (Table 1). In 1964, James D. Hardy attempted a chimpanzee to human heart transplant [1]. Subsequently Christiaan Barnard's team performed heart xenotransplants using baboon and chimpanzee donors [2]. In these early cases patients survived hours to days, with death attributed to the small size of primate hearts being unable to support the circulation of adult humans. Other cases highlighted the immunological barriers that had to be overcome for xenotransplants to work. Notably, a team lead by Leonard L. Bailey in California performed a heart transplant on Baby Fae, in which size was not an issue but the graft failed at 20 days due to humoral rejection [3]. Despite the poor outcomes in initial xenotransplants,
* Corresponding author. Transplantation Biology Research Center, Massachusetts General Hospital and Harvard Medical School, Building 149, 13th Street, Charlestown, Boston MA 02114, USA. E-mail addresses: [email protected] (S.G. Michel), [email protected] (K. Shanmugarajah). http://dx.doi.org/10.1016/j.ijsu.2014.12.011 1743-9191/© 2014 Surgical Associates Ltd. Published by Elsevier Ltd. All rights reserved.
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Table 1 Milestones in clinical xenotransplantation. Year
Donor
Patient survival
1963/64 (13) 1963/64 (6) 1964 1977 (2)
Chimpanzee kidneys Baboon kidneys
Up to 9 Keith Reemtsma [4] months 19 to 98 days Thomas Starzl [5]
Chimpanzee heart Baboon and chimpanzee hearts Baboon heart Baboon liver
2h 6 h and 4 days 20 days 70 days
1984 1992
Surgeon
James Hardy Christiaan Barnard Leonard Bailey Thomas Starzl
Refs.
[1] [2] [3] [6,7]
a few cases provided optimism that this form of organ replacement could work. In a series of chimpanzee-to-human kidney transplants performed by Keith Reemtsma, a patient was reported to have survived for nine months [4]. Thomas E. Starzl's group reported the survival 19e98 days in 6 cases of baboon-to-human transplantation [5]. In 1992, Starzl and colleagues reported 70 day survival in baboon-to-human liver xenotransplants. [6,7] 3. Optimal donor species for xenotransplantation While non-human primates are phylogenetically the most similar to humans, the use of these animals as xenotransplant donors has several drawbacks. The most pertinent of these include their small sizes, infection risks, long gestation and growth periods and ethical concerns. For these reasons, swine are currently considered the most likely xenotransplant donors. Swine are easy to breed, can be produced in germ-free conditions, their organs reach a size that can provide life-supporting functions to human recipients and the ethical barriers precluding nonhuman primate research are present to a lesser degree. Furthermore, swine can be genetically manipulated to permit immune challenges to be overcome. However, there are still major obstacles before swine can be successfully used as a source of organs. First, the immunological responses of the recipient against the graft need to be controlled. These include hyperacute rejection (characterized by binding of naturally-occuring xenoreactive antibodies that trigger the complement cascade), acute antibody-mediated rejection (acute humoral xenograft rejection, AHXR), acute cellular rejection, and chronic rejection. Second, xenografts may have physiological limitations, such as molecular incompatibilities in the coagulation system, which preclude their use in providing functional replacement of a failing organ. Third, risk of transmissible infections such as porcine endogenous retroviruses (PERVs) affecting humans. To date there has been no case where PERVs have caused an infection in humans that were exposed to porcine tissues [8]. However, pig cells can transmit PERV to human cells in vitro [9], which makes it mandatory that all potential recipients of porcine organs and cells, as well as their families be monitored closely. The risk of contracting West Nile virus, rabies and HIV from swine organs is considered vanishingly low [10]. 4. Overcoming the immune response to pig xenotransplants by genetic engineering Unlike the rejection of allografts, which is mainly governed by T cells, the immune response to porcine xenografts primarily occurs hyperacutely, mediated by pre-formed, natural antibodies to the Galactosyl-alpha(1,3)galactose (Gal) epiotope that is highly expressed on porcine endothelium. Knocking out the gene for a1,3galactosyltransferase prevents the expression of the Gal epitope on porcine tissues. This helped overcome hyperacute rejection in pig-
to-primate studies [11]. Despite this, non-Gal antibodies may still activate the porcine endothelium, leading to microvascular thrombosis and graft loss. Byrne and colleagues identified many non-Gal antigens on porcine endothelium which are members of the heat shock protein family [12]. If the next generation of genetically modified pigs can address these non-Gal antigens, another significant step towards controlling the humoral response to xenotransplants will be made. Aside from the humoral response, efforts have also been made to control the innate immune response to xenotransplantation by engineering pigs that: 1) express human CD47, a marker for “self”, to prevent organ damage caused by macrophages [13,14]; 2) express the inhibitory receptor HLA-E to control the NK cell response [15]; or 3) express complement regulators, such as human decay accelerating factor (hDAF) [16] or membrane cofactor protein (hCD46). However, more important for graft survival was the supraphysiologic expression of these complement regulators rather than the fact that they were human [17]. Thrombotic microangiopathy caused by immunologic mechanisms is compounded in the xenotransplant setting due the molecular incompatibilities in the coagulation cascade between pig and primate. To overcome this, efforts are underway to allow for the expression of human anticoagulants on pig endothelium. These include expression of human CD39 to inhibit platelets, human thrombomodulin to allow activation of the human anticoagulant protein C and tissue factor pathway inhibitor to prevent the initiation of the extrinsic pathway of coagulation [18]. 5. Immunosuppression protocols for xenotransplantation Immunosuppression in preclinical models of xenotransplantation usually consists of B-cell and plasma cell therapeutics like Rituximab and Bortezomib in addition to the standard triple drug immunosuppression [19]. For example, peritransplant B-cell depletion using 4 weekly doses of anti-CD20 antibody, along with ATG, anti-CD154 and MMF-based immunosuppressive regimen resulted in prolonged survival of Gal-knock out (Gal-KO)/ human CD46 transgenic pig cardiac xenografts (up to 236 days) [20]. One or more rounds of immuno-adsorption or plasmapheresis are necessary to remove antibodies from the recipient's circulation. These regimens are often associated with severe side effects like pancytopenia and sepsis. Considering that systemic immunosuppression needs to be higher in xenotransplantation than in allotransplantation, another strategy to counteract this effect is to express immunosupppressive molecules like CTLA-4Ig on the pig endothelium [21]. However, the step-wise approach of gene knock-in or gene knock-out is limited in scope; kidneys from pigs who were Gal-KO and transgenic for human CD55 (hCD55), hCD59, hCD39, and fucosyl-transferase (hHT) showed limited improved survival in baboons [22]. An alternative but much more complex approach is to try to achieve immunological tolerance to the xenograft. In clinical allotransplantation, the tolerance approach has already proven to be successful by achieving transient mixed hematopoietic chimerism by donor bone marrow co-transplantation with kidney allografts. A number of patients at Massachusetts General Hospital have been living immunosuppression-free for several years now after receiving their kidney along with bone marrow of the donor [23,24]. Apart from generating bone marrow-based chimerism, another tolerance approach would be to transplant xenogeneic thymus to promote recipient thymopoiesis, which could induce T cell tolerance to solid organ xenografts. Indeed, based on this approach, Yamada and colleagues have reported over 80 day survival of a life-supporting kidney transplant in a pig-to-primate model [25,26].
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Other experimental protocols are based on studies by Billingham and colleagues showing that the environment during early development can induce immune tolerance [27]. Chimeric pig hearts were harvested from pigs rendered chimeric by intrauterine injection of sheep mononuclear cells [28]. These chimeric pig hearts, when transplanted into sheep and maintained on single-agent immunosuppression, showed superior freedom from acute rejection and graft survival compared to non-chimeric donor hearts [28]. 6. Current status of specific xenografts
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recipients [43]. 6.4. Lung Reports of orthotopic pig-to-primate pulmonary transplantation has achieved only very limited survival rates 72 h [44] and 4.5 days [45] compared to other solid organs. Lungs release large amounts of von Willebrand Factor that binds GPIb on human/ primate platelets. This leads to pronounced platelet activation and clotting. A possible future strategy to control this includes using transgenic pigs that express tissue factor pathway inhibitor or CD39.
6.1. Heart 6.5. Islets of langerhans In a preclinical life-supporting orthotopic pig-to-baboon model, Ekser and colleagues have achieved median survival of 40 days (maximum of 57 days) of heart xenografts using Gal-KO/CD46 transgenic pigs and immunosuppression with ATG induction, tacrolimus, sirolimus and tapering steroids [29]. Despite these results, a limiting factor of orthotopic xenoheart transplant has been early graft failure not attributable to hyperacute rejection [30]. Possible causes for this phenomenon, termed “perioperative cardiac xenograft dysfunction” are sensitivity of the pig heart to the extracorporeal circulation and ischemia reperfusion injury or the inflammatory effect of preformed non-Gal antibodies [30]. Importantly, these problems are not insurmountable due to advances in critical care.
Pig islet transplantation has already been performed clinically in countries with limited national regulations like Mexico, Russia and China [46], but most researchers agree that more preclinical data is necessary before this treatment should be applied to patients. In the pig-to-nonhuman primate model, encapsulation has been shown to be effective to prevent damage to the islets caused by T cells and antibodies. The islets functioned for 6 months without immunosuppression [47]. Valdez-Gonzalez grafted neonatal porcine islets into the subcutaneous tissue of 12 children with IDDM without immunosuppression which lead to reduction in insulin requirement [48]. 7. Applying bioengineering to organ transplantation
6.2. Kidney Using a tolerance approach, over 80 days of kidney graft survival was achieved in two baboons who received grafts from Gal-KO pigs [11]. The baboons underwent thymectomy and splenectomy prior to combined kidney and vascularized thymus transplantation. Immunosuppression consisted of T-cell depletion with ATG and anti-CD2, cobra venom factor, anti-CD154, MMF and tapering steroids. 6.3. Liver Extracorporeal liver perfusion devices containing pig hepatocytes to treat acute liver failure have been used in over 100 clinical patients [31e35]. However, a significant survival benefit has not been shown. In addition, whole organ extracorporeal pig liver perfusions in acute liver failure have been performed in 11 patients in the United States as a bridge-to-transplant with a mortality of 50% [36,37]. In a preclinical pig-to-primate liver transplant model, 8-day survival was reported using hDAF-expressing pigs. In this study, newly synthesized proteins by the porcine liver were demonstrated [38]. Baboon recipients of liver grafts from alpha1,3-galactosyl transferase knockout pigs survived 6, 8 and 9 days before succumbing to infection and coagulopathy [39]. The functionality of liver factors and proteins when transplanted into non-condordant species remains undetermined. Recent studies demonstrate successful generation of humanized rat and mouse livers. In these small animal models, human hepatocytes are successfully engrafted into host livers after treating host animals with injury-, adenovirus- or toxin-based techniques [40e42]. Apart from being useful for pharmacologic and infectious studies, if this technology can be scaled up to a human size, e.g. humanized pig livers, then this may prove to be an important method to enhance hepatocyte acceptance across species. As proofof-principle, chimeric mouse livers seeded with rat hepatocytes were able to sustain liver function for up to 6 months in rat
Similar to xenotransplantation, regenerative medicine has the potential to replace diseased organs with functional ones. Tremendous growth in the field of regenerative medicine occurred in the 1990s with the advent of developments in nuclear transfer technology, stem cell isolation, and tissue engineering, in which the principles of biology and engineering are applied to develop functional substitutes for damaged tissue and organs [49e51]. In addition, regenerative medicine may also bolster existing organ transplant practices by improving the quality of organs deemed ineligible for transplant [52]. Furthermore, bioengineered organs could also be transplanted without need for life-long immunosuppression, erasing the heavy burden associated with its side effects and cost. These advantages may allow patients to be transplanted earlier, when they are healthier and better able to withstand surgery. 8. Principles of organ bioengineering In current approaches to developing bioartificial tissue, a target organ is first decellularized, then reseeded with appropriate cell types, and then matured in a bioreactor [53]. Target organs can be harvested up to 4e6 h postmortem, up to the point at which proteolysis begins. Decellularization refers to the process in which an organ is stripped of its viable tissue usually by a mixture of detergents until all that remains are the basic protein and polysaccharide components. This biocompatible backbone is a natural, ideal platform for organ bioengineering. It is already geometrically and spatially organized and provides an essential source of signaling cues for cells to grow, expand, and exert their functiondwitness the improved outcomes of whole pancreas transplantation over islet cell transplantation [54]. After generating a scaffold, there are different options for reseeding. The appropriate cell to repopulate a construct needs to take into account expansion, functionality, and replicative senescence. Currently, stem cells, progenitor cells, and human embryonic
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stem cells are difficult to obtain, may have limited expansion capabilities and are fraught with ethical concerns [49]. The ectopic expression of transcription factors can generate autologous cells (induced pluripotent stem cells) for organ replacement; however, these cells have tumorigenic traits since reprogramming is often accompanied by genetic and epigenetic alterations [55]. Another option is to rely on repopulation by host cells in vivo, a process which has been shown in implanted dermal matrices and trachea [49]. To date the most successful bioengineered organs to have been used in the clinical setting are relatively simple, hollow, avascular structures without reconnection to a vascular supply (Table 2). In 2004, a 30 year old female with end-stage bronchomalacia underwent implantation of a totally bioengineered human trachea constructed from a deceased donor scaffold seeded with autologous cells derived from mesenchymal stem cells and epithelial cells [56]. In 2006, human bladder matrices were seeded with autologous bladder cells grown from culture and implanted into 10 patients who did well with a mean follow-up of nearly 4 years [57]. In 2007, blood vessels engineered from autologous skin and superficial vein cells were also successfully implanted into 10 patients with end-stage renal disease on hemodialysis [58]. Currently over 160 patients have received successful constructs of tracheas, bladders, and blood vessels without the need for immunosuppressive medication [59,60]. However, these constructs rely on diffusion for nutrients and oxygen exchange and are therefore limited in size by risk of ischemia and subsequent graft failure.
9. Current status of specific bioengineered organs 9.1. Heart Initial forays into cardiac bioengineering focused on repairing damaged cardiac muscle with targeted delivery of myoblasts [61e63]. Recently, the perfusion decellularization technique was applied to rat hearts to generate whole organ scaffolds. These structures were repopulated with neonatal rat cardiomyocytes and the resulting organ was able to demonstrate contractility of up to 2% of normal contractile function [64].
9.3. Liver In contrast to patients with kidney failure on dialysis, patients in liver failure have no recourse for liver replacement therapy. Attempts to create an extracorporeal bioartifical liver system containing functional, viable hepatocytes have fallen short of expectations. Worldwide, 78 patients have been recipients of hepatocyte infusions: genetically modified hepatocytes were infused into 21 patients with metabolic disease and genetically unmodified hepatocytes were infused into 20 patients with chronic liver disease and 37 patients with acute fulminant liver failure [68] with mixed results. Current technology is limited by the inability to generate sufficient liver mass to sustain physiological function. Recently, new protocols have induced human fibroblasts to a multipotent progenitor cell state from which hepatocytes could successfully differentiate, proliferate, and repopulate mouse livers [69]. Other groups have successfully seeded liver scaffolds with adult hepatocytes and implanted the recellularized scaffolds into rats for up to 8 h [70]. This bioengineered organ was able to support hepatocyte survival and liver-specific function, such as production of albumin, urea synthesis, and cytochrome P450 expression. 9.4. Kidney Decellularized kidneys have been made with rat, pig, and rhesus monkey kidneys [71,72]. Renal extracellular matrices produced from porcine kidneys were implanted into pigs in vivo as proof-ofconcept and demonstrated the ability to withstand physiologic blood pressure without extravasation; however, despite preservation of renal architecture at 2 weeks, the naked vasculature was thrombosed by 24 h after implantation [73]. An important contribution was made in 2013 when Song and colleagues reported that rat kidneys seeded with epithelial and endothelial cells produced rudimentary urine in vivo. [74] Future advances depend on further understanding the developmental biology of the kidney and identification of potential renal progenitor cells [51,75,76]. 9.5. Pancreas Pancreatic scaffolds have been used to help produce large numbers of functional pancreatic endocrine cells [77] and a bioengineered rat pancreas seeded with rat islets was able to modulate glucose metabolism and reduce the need for exogenous insulin in diabetic rats [78].
9.2. Lung 9.6. Intestine Lung bioengineering is challenging because of the complexity of the tissue and multiple cell types involved. Two landmark studies in 2010 established the feasibility of bioengineered lungs in small animal models. These organs were generated by decellularizing rat lungs, repopulating them with epithelial and endothelial cells, and implanting them into rats in vivo for up to 6 h to demonstrate successful gas exchange [65,66]. In an effort to scale these findings up to a clinically relevant model, porcine lungs were recently successfully decellularized to produce lung scaffolds with in vitro properties resembling those of human lungs [67].
Table 2 Milestones in clinical implantation of bioengineered organs. Date
Organ
Clinical outcome
Refs.
2004 2006 2007
Trachea Bladder Blood vessels
Proximal graft necrosis Successful implantation up to 4 years Successful implantation up to 13 months
[56] [57] [58]
In dogs, composite esophaguses were developed into welldifferentiated lumen of stratified squamous cells surrounded by a thick smooth muscle-like tissue that could be surgically manipulated in mock esophagectomies [79]. 10. Xenobioengineered organs An intriguing prospect is to combine the strengths of both xenotransplantation and bioengineering to generate “semi-xenografts” where the scaffold would be animal-derived and the repopulated cells would be human-derived [51]. This would make it easier to control the quality of the scaffold, as pathogen-free herds and post-harvest processing would eliminate most known pathogens [49]. For example, bioartificial human tissue with an innate vascularized network was created using a porcine small bowel platform [59,80]. Though the majority of proteins in the extracellular matrix are highly conserved across species [81], the significant
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barriers facing xenotransplantation still pertain, such as the antigenicity of the Gal epitope on swine [82]. 11. Limitations of current technology and future challenges The scientific barriers to a successfully engineered organ can be grouped into three main levels: scaffolds, cells, and construct [51]. Scaffolds must be biocompatible, promote cell growth, allow for angiogenesis and remodeling, and avoid immunogenicity. Cells need to be functional and sufficient in number without becoming tumorigenic or contaminated. The entire construct, to be clinically viable, needs to withstand physiologic blood pressure, avoid clotting, and be accepted by the host immune system. Further study is needed to understand mechanisms of organ development/regeneration, cell to cell or extracellular matrix interactions, and vascularization. Practically, bioengineered organs must meet quality standards. Imaging, in vitro assays, and bioinformatics can aid in assessing the quality of the construct and the state of the tissue and cells within it. Finally, cost. If organ bioengineering remains a highly individualistic process, the high cost of generating stem cells for each individual alone is prohibitive [83]. Once the technology is more mature, a comparative analysis of cost is necessary to determine whether this method represents a cost-effective manner in which to increase the organ donor pool.
[3]
[4] [5]
[6] [7] [8]
[9] [10] [11]
[12]
[13]
[14]
[15]
12. Conclusion [16]
Xenotransplantation and bioengineering of organs are two promising new technologies to treat terminal organ failure. Both provide potential solutions to the worsening organ shortage facing allo-transplantation today. Time will determine which of the two approaches, if at all, will live up to expectations. In the meantime, efforts should be invested in both technologies to eventually tailor therapeutic strategies to each individual patient.
[17]
[18] [19]
[20]
Ethical approval None. Financial support None. Author contribution KS e conception and design, analysis, writing of manuscript and critical review. VV e conception and design, analysis, writing of manuscript and critical review. LM e conception and design, analysis,writing of manuscript and critical review. SM e conception and design, analysis, writing of manuscript and critical review. Conflicts of interest
[21]
[22]
[23]
[24]
[25]
[26] [27]
[28]
[29] [30]
None. [31]
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