Section 1 Clinic and Pre-Clinic

**3**

**Chapter 1**

**Abstract**

**1. Introduction**

clinical therapy.

could resolve this issue.

known pathogens and infectious disease.

Biosafety Barrier to

Xenotransplantation

*Wei Wang, Qi Liang, Wei Nie, Juan Zhang and Cheng Chen*

Biosafety barrier is most important for xenotransplantation clinical trial. Source animals used in xenotransplantation should be bred in a closed herd and raised in a well-controlled, pathogen-free environment with high standards of animal welfare. To ensure the source animals' freedom from known pathogens under adequate biosecurity and surveillance, extensive tests must be done. Biosafety of DPF source pig should be proved by animal model before clinical trial. In addition, inclusion criteria for transplant recipients and clinical safe transplantation protocol should be established. Comprehensive anti-immune rejection treatment based on immune tolerance program can significantly prolong the xenograft survival and reduce the adverse impact on the immune system, which is suitable for clinical application. According to the clinical followup plan of the xenograft recipients, the patients should come back to the hospital for a check at regular intervals after the transplantation. The database of clinical trials for xenotransplantation should be established, including specimens, paper documents, and electronic documents. The information and samples of xenotransplantation donors and recipients should be preserved for long time.

**Keywords:** biosafety barrier, donor animal, xenotransplantation, clinical trial

The demand for a new source of organs and cells for clinical transplantation has been exacerbated for decades. And xenotransplantation (e.g., from pigs to human)

In 2008, the WHO and International Xenotransplantation Association (IXA) released a consensus statement on xenotransplantation from pig to human for clinical trials. In this statement, it proposed the criterion for biosafety of source animals in clinical trials. The source animals should be bred in a closed herd for the purpose and kept under a well-controlled and pathogen-free environment with complete animal welfare. Even source animals are housed in appropriate biosecurity and under surveillance, extensive detection must be done to ensure freedom from

Therefore, this chapter will draw attention to the significant biosafety barriers need to be overcome before xenotransplantation from pig to human can become a

#### **Chapter 1**

## Biosafety Barrier to Xenotransplantation

*Wei Wang, Qi Liang, Wei Nie, Juan Zhang and Cheng Chen*

#### **Abstract**

Biosafety barrier is most important for xenotransplantation clinical trial. Source animals used in xenotransplantation should be bred in a closed herd and raised in a well-controlled, pathogen-free environment with high standards of animal welfare. To ensure the source animals' freedom from known pathogens under adequate biosecurity and surveillance, extensive tests must be done. Biosafety of DPF source pig should be proved by animal model before clinical trial. In addition, inclusion criteria for transplant recipients and clinical safe transplantation protocol should be established. Comprehensive anti-immune rejection treatment based on immune tolerance program can significantly prolong the xenograft survival and reduce the adverse impact on the immune system, which is suitable for clinical application. According to the clinical followup plan of the xenograft recipients, the patients should come back to the hospital for a check at regular intervals after the transplantation. The database of clinical trials for xenotransplantation should be established, including specimens, paper documents, and electronic documents. The information and samples of xenotransplantation donors and recipients should be preserved for long time.

**Keywords:** biosafety barrier, donor animal, xenotransplantation, clinical trial

#### **1. Introduction**

The demand for a new source of organs and cells for clinical transplantation has been exacerbated for decades. And xenotransplantation (e.g., from pigs to human) could resolve this issue.

In 2008, the WHO and International Xenotransplantation Association (IXA) released a consensus statement on xenotransplantation from pig to human for clinical trials. In this statement, it proposed the criterion for biosafety of source animals in clinical trials. The source animals should be bred in a closed herd for the purpose and kept under a well-controlled and pathogen-free environment with complete animal welfare. Even source animals are housed in appropriate biosecurity and under surveillance, extensive detection must be done to ensure freedom from known pathogens and infectious disease.

Therefore, this chapter will draw attention to the significant biosafety barriers need to be overcome before xenotransplantation from pig to human can become a clinical therapy.

#### **2. DPF source pig**

The term "DPF" (Designated Pathogen Free) is used to describe animals, animal herds, or animal facilities that have been rigorously documented to be free of specified infectious agents and that are maintained using well-defined routines of testing for designated pathogens and utilizing rigorous SOPs (Standard operating procedures) and practices of herd husbandry and veterinary care to assure the absence of the designated pathogens [1]. So far, there is no normative document specifying the pathogens specified in DPF pig. DPF standards are dynamic and need to be updated over time according to the geographical environment of the animal population and new pathogens emerging. Generally speaking, there are two types of pathogens that need to be excluded from DPF pig: (1) Pathogens that affect animal health; (2) Pathogens that can cause cross-species transmission.

Experts in this field met to agree on the most comprehensive list of bacteria, fungi, parasites and viruses that should not be present in DPF pig [2]. Endogenous viruses are not listed. PERV (Porcine endogenous retrovirus) is the only one endogenous virus we known in pigs [3]. PERV has three subgroups including PERV-A, PERV-B and PERV-C. In general, PERV-A and PERV-B can infect both pig and human cells, but PERV-C can only infect pig cells. It is noteworthy that PERV-A/C recombine were be found in vitro co-culture system using cells from miniature swine, which means PERV-C can also infect human cells in some condition [4]. To monitor the status of DPF pigs, the pigs' samples including blood, serum, tissues and feces must be tested regularly.

The DPF pigs must be raised in biosecure barrier environment. Biosecure barrier facility includes many aspects.

#### **2.1 Facility environment and building**


**5**

*Biosafety Barrier to Xenotransplantation DOI: http://dx.doi.org/10.5772/intechopen.89134*

stainless steel bowls for feed.

deprived, and hand-reared.

wear and wear gloves.

tions will be documented.

**2.3 Health monitoring of DPF pigs**

including animal breeding and genetic records.

should report any such incident in writing.

facility.

**2.2 Facility operation**

7.There will be two rooms of animal pens, a further unit for the sow farrowing, and a quarantine area, all with an air lock entry. The air pressure in all animal areas will be positive to the corridors (monitored by magnehelic gauges). Rooms will have controlled fluorescent lighting, temperature and humidity,

8.Animal pens will have gates of the metal farm type, allowing pigs to see out and receive physical contact from other pigs and staff, with aisles between pens and a drain running in front of each row of pens. There will be windows

9.Each pen will have a valve supplying filtered drinking water and individual

10.Music will be piped into the units by speakers set into the ceiling and serviced from the mezzanine level. Music will be controlled from the main office.

1.The DPF facility will operate as a full sterile barrier facility. Therefore, all goods entering the facility must be sterile and all staff should go through a full shower procedure and gown-up in sterile suits, boots, hats and gloves. All original breeding stock in the facility will be cesarean-derived, colostrum-

2.To enter the facility, staff must shower and don a complete clothing and foot-

3.All activities that take place will be fully documented in the SOP Manual, including inwards receipt of goods through the facility barrier using such

4.There will be SOP-documented regular health screening of pigs and staff.

5.A comprehensive pest control system will be used inside and outside the building and managed by a contracted pest control company. Records of all inspec-

6.Pig care and welfare are a top priority, as described above for the Invercargill

All pigs are uniquely identified and individual records should be maintained,

1.Regular veterinary should attendance at the pig facilities ensures that the staff is trained in disease recognition and that the veterinarian is called immediately in the event of signs and symptoms of disease in any animal. The veterinarian

2.The donor herd should continue to test the porcine pathogens and parasites.

methods as an autoclave, dunk tank, and UV pass-through hatch.

and 15 to 20 air changes per hour of HEPA -filtered air.

in the walls between each pen to allow pigs to see each other.

#### *Biosafety Barrier to Xenotransplantation DOI: http://dx.doi.org/10.5772/intechopen.89134*

*Xenotransplantation - Comprehensive Study*

and feces must be tested regularly.

**2.1 Facility environment and building**

livestock within the area boundary.

supply goods through the barrier.

feed and bedding storerooms.

tected by security alarms 24 hours a day.

and within the barrier (termed "inside the barrier").

facility includes many aspects.

(2) Pathogens that can cause cross-species transmission.

The term "DPF" (Designated Pathogen Free) is used to describe animals, animal herds, or animal facilities that have been rigorously documented to be free of specified infectious agents and that are maintained using well-defined routines of testing for designated pathogens and utilizing rigorous SOPs (Standard operating procedures) and practices of herd husbandry and veterinary care to assure the absence of the designated pathogens [1]. So far, there is no normative document specifying the pathogens specified in DPF pig. DPF standards are dynamic and need to be updated over time according to the geographical environment of the animal population and new pathogens emerging. Generally speaking, there are two types of pathogens that need to be excluded from DPF pig: (1) Pathogens that affect animal health;

Experts in this field met to agree on the most comprehensive list of bacteria, fungi, parasites and viruses that should not be present in DPF pig [2]. Endogenous viruses are not listed. PERV (Porcine endogenous retrovirus) is the only one endogenous virus we known in pigs [3]. PERV has three subgroups including PERV-A, PERV-B and PERV-C. In general, PERV-A and PERV-B can infect both pig and human cells, but PERV-C can only infect pig cells. It is noteworthy that PERV-A/C recombine were be found in vitro co-culture system using cells from miniature swine, which means PERV-C can also infect human cells in some condition [4]. To monitor the status of DPF pigs, the pigs' samples including blood, serum, tissues

The DPF pigs must be raised in biosecure barrier environment. Biosecure barrier

1.The proposed DPF facility will be sited at a property to be confirmed.

2.The building will be on rural land where there are no other pig farms within a radius of 10 km. The grounds of the facility will be protected and planted with trees, and the grass mowed regularly. There are to be no other animals or

3.The building is to be fully protected by a secure fence and electric gate entry. The main entrance door is also to be a security door with key access and pro-

4.The facility is designed with two separate areas, outside the barrier (external)

5.The external area houses a delivery bay, storerooms for feed and bedding, staff lunchroom facilities, office, laundry area, external change rooms, and rooms to

6.Inside the barrier, the building is to have a HEPA (High efficiency particulate air) -filtered air supply and it will only contain goods that are sterile, staff who have showered and are wearing sterile clothes, and the pigs themselves which will be free of all specified diseases. There are to be two rooms holding the pig pens, internal gown-up areas, office, treatment room, reception room, and

**2. DPF source pig**

**4**


#### **2.2 Facility operation**


#### **2.3 Health monitoring of DPF pigs**

All pigs are uniquely identified and individual records should be maintained, including animal breeding and genetic records.


The pigs are conveyed to the DPF breeding center. They must be disinfected in buffer rooms before entering inspection and quarantine where they are isolated for a month. After isolation the pigs give cesarean birth to the first generation of purified pigs. Compared to vaginal births Cesarean section can eliminate or reduce the risk of infecting with pathogens from sow's vajina. These newborns are fed in isolation under aseptic conditions and grow into adulthood. They are then impregnated and naturally deliver the second generation. After being tested for specified pathogens this second generation enters into a DPF area. The first generation of pigs should not be used as source pigs but the pigs in a second or higher generation can be used as DPF source pigs [1, 5].

#### **3. Other biosafety issues for xenotransplantation clinical trials**

Donor pigs are the basis for ensuring the biosafety of xenograft clinical trials. Other biosafety issues are also worthy of attention, including immunosuppression protocols, clinical treatment protocols, sample/data retention programs, and casetracking programs.

#### **3.1 Immunosuppression and tolerance-inducing strategies for xenotransplantation**

The principal challenges that must be faced to make xenotransplantation a clinical reality, which include determining a repeatable strategy for efficient preparation of xenogeneic tissues and organs and tracing the potential transmission of porcine pathogens to human. In addition, it is necessary to overcome the rejection barrier with clinically practicable immunosuppression and tolerance induction strategies. The application of xenotransplantation faces insurmountable immunological barriers, including: (1) hyperacute rejection (complement activation mediated by antibody) which is trigged by natural xenoreactive antibodies against Gal (1,3) and non-Gal antigens, (2) acute rejection of humoral xenograft which is mediated by antibodies that are dependent on T cells, (3) acute cellular xenograft rejection due to T cell mediated cellular responses.

#### *3.1.1 Immunosuppression protocols for xenotransplantation*

Continuous administration of multiple immunosuppressive drugs has been required and attempts to minimize immunosuppression. Immunosuppression in

**7**

*Biosafety Barrier to Xenotransplantation DOI: http://dx.doi.org/10.5772/intechopen.89134*

is still the most commonly utilized option.

pigs), such as CD46, CD55, or CD59.

ity of pig-to-primate xenograft tolerance.

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. One or more rounds of immuno-adsorption or plasmapheresis are essential to remove antibodies from the recipient's circulation. These regimens are often associated with serious side effects such as pancytopenia and sepsis. The xenogeneic T cell response is supposed to be similar to that of typical allogenic responses, even larger. Consider this challenging barrier, most successful immunosuppressive therapy include a T cell depletion method like mono- or polyclonal anti-T cell antibodies, chemotherapeutic agents like cyclophosphamide, or whole body or thymic radiation therapy [6]. And anti-thymocyte globulin (ATG)

The engagement of TCR (T cell receptor) with foreign antigen without costimulatory signal will lead to T cells unresponsive to the antigen (known as T-cell anergy), thereby suppressing antigen induced response. The possible mechanism was that the CTLA4Ig fusion protein blocked CD28/B7 co-stimulatory signaling of the primary pathway, which eventually induced differentiation bias of T helper cells (Th cells [7]). Anti-CD154 antibodies, known to be effective in blocking indirect pathway of allorecognition [6], is also a critical component of effective immunosuppressive strategies in preventing cellular rejection in pig-to-NHPs (Non-human primates) xenotransplantation [8] yet its clinical application is restricted due to high risk of thromboembolic complications [9]. However, in pig-to-NHPs models, immune tolerance achievement approached by utilizing co-stimulatory blocking

The transgenic pigs expressing graft-protecting factors has been shown to require a less toxic immunosuppressive protocol [10] which gives another path to explore. Using advanced gene editing technologies, xenotransplantation from multitransgenic alpha-1,3-galactosyltransferase knockout pigs (GTKO pigs) has demonstrated marked prolongation of xenograft survival. In addition, the incidence of hyperacute rejection was further reduced with organs from the GTKO pigs expressing one or more human complement-regulatory proteins (GTKO/hCRPs

*3.1.2 Tolerance-inducing strategies across xenogeneic immunological barriers*

A better but much more complex approach is to try to achieve immunological tolerance to the xenograft. Three successful tolerance induction approaches have been explored in large animal models: the use of mixed hematopoietic chimerism [11, 12], T regulatory cells [13, 14] and thymic transplantation [15] . It has been demonstrated that tolerance is possible in humans by successful clinical application of the mixed chimerism approach to renal transplantation [16] and by the T regulatory cell approach to liver allografts [17]. Despite the greater immunologic differences between species than within species, both mixed chimerism and thymic transplantation approaches have been shown to be capable of tolerizing human T cells to porcine xenografts in humanized mouse models [18]. Moreover, treatment with in vitro expanded regulatory T cells (Treg) prevents porcine xenograft rejection in humanized NOD-SCID IL-2 receptor gamma null (NSG) mice by the suppression of the T cell-mediated graft destruction, which suggesting the feasibil-

For xenografts, the level of immunosuppressive agents needed to fully suppress immune responses is greater than for allografts, which would likely lead to greater side effects. Thus, adoption of tolerance strategies is inevitable. Even though current immunosuppression seems to be controlling T cell responses in long-term acceptors [19, 20], it appears likely that low levels of T cell-dependent antibodies [21] and

agents and other immunosuppressants in long-term treatments.

#### *Biosafety Barrier to Xenotransplantation DOI: http://dx.doi.org/10.5772/intechopen.89134*

*Xenotransplantation - Comprehensive Study*

separate locations.

separate locations.

locations.

tracking programs.

**xenotransplantation**

to T cell mediated cellular responses.

*3.1.1 Immunosuppression protocols for xenotransplantation*

3.All donor piglets should be necropsied by a veterinarian within 6 hours of cell harvesting. Any pathological changes must be noted and appropriate speci-

4.Donor piglet tissue retention samples collected include brain, heart, kidney, liver, lung, pancreas, and spleen. Duplicate samples are stored at 80°C in two

5.Duplicate donor piglet serum retention samples are also stored in two separate

6.In addition, duplicate final product retention samples are stored at 80°C in two

7.A positive result in any of the infection monitoring tests described in this section, will lead to the donor animal and the batch of isolated islets being discarded.

The pigs are conveyed to the DPF breeding center. They must be disinfected in buffer rooms before entering inspection and quarantine where they are isolated for a month. After isolation the pigs give cesarean birth to the first generation of purified pigs. Compared to vaginal births Cesarean section can eliminate or reduce the risk of infecting with pathogens from sow's vajina. These newborns are fed in isolation under aseptic conditions and grow into adulthood. They are then impregnated and naturally deliver the second generation. After being tested for specified pathogens this second generation enters into a DPF area. The first generation of pigs should not be used as source pigs but

the pigs in a second or higher generation can be used as DPF source pigs [1, 5].

**3. Other biosafety issues for xenotransplantation clinical trials**

**3.1 Immunosuppression and tolerance-inducing strategies for** 

Donor pigs are the basis for ensuring the biosafety of xenograft clinical trials. Other biosafety issues are also worthy of attention, including immunosuppression protocols, clinical treatment protocols, sample/data retention programs, and case-

The principal challenges that must be faced to make xenotransplantation a clinical reality, which include determining a repeatable strategy for efficient preparation of xenogeneic tissues and organs and tracing the potential transmission of porcine pathogens to human. In addition, it is necessary to overcome the rejection barrier with clinically practicable immunosuppression and tolerance induction strategies. The application of xenotransplantation faces insurmountable immunological barriers, including: (1) hyperacute rejection (complement activation mediated by antibody) which is trigged by natural xenoreactive antibodies against Gal (1,3) and non-Gal antigens, (2) acute rejection of humoral xenograft which is mediated by antibodies that are dependent on T cells, (3) acute cellular xenograft rejection due

Continuous administration of multiple immunosuppressive drugs has been required and attempts to minimize immunosuppression. Immunosuppression in

mens taken. The veterinarians' report should be documented.

**6**

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. One or more rounds of immuno-adsorption or plasmapheresis are essential to remove antibodies from the recipient's circulation. These regimens are often associated with serious side effects such as pancytopenia and sepsis.

The xenogeneic T cell response is supposed to be similar to that of typical allogenic responses, even larger. Consider this challenging barrier, most successful immunosuppressive therapy include a T cell depletion method like mono- or polyclonal anti-T cell antibodies, chemotherapeutic agents like cyclophosphamide, or whole body or thymic radiation therapy [6]. And anti-thymocyte globulin (ATG) is still the most commonly utilized option.

The engagement of TCR (T cell receptor) with foreign antigen without costimulatory signal will lead to T cells unresponsive to the antigen (known as T-cell anergy), thereby suppressing antigen induced response. The possible mechanism was that the CTLA4Ig fusion protein blocked CD28/B7 co-stimulatory signaling of the primary pathway, which eventually induced differentiation bias of T helper cells (Th cells [7]). Anti-CD154 antibodies, known to be effective in blocking indirect pathway of allorecognition [6], is also a critical component of effective immunosuppressive strategies in preventing cellular rejection in pig-to-NHPs (Non-human primates) xenotransplantation [8] yet its clinical application is restricted due to high risk of thromboembolic complications [9]. However, in pig-to-NHPs models, immune tolerance achievement approached by utilizing co-stimulatory blocking agents and other immunosuppressants in long-term treatments.

The transgenic pigs expressing graft-protecting factors has been shown to require a less toxic immunosuppressive protocol [10] which gives another path to explore. Using advanced gene editing technologies, xenotransplantation from multitransgenic alpha-1,3-galactosyltransferase knockout pigs (GTKO pigs) has demonstrated marked prolongation of xenograft survival. In addition, the incidence of hyperacute rejection was further reduced with organs from the GTKO pigs expressing one or more human complement-regulatory proteins (GTKO/hCRPs pigs), such as CD46, CD55, or CD59.

#### *3.1.2 Tolerance-inducing strategies across xenogeneic immunological barriers*

A better but much more complex approach is to try to achieve immunological tolerance to the xenograft. Three successful tolerance induction approaches have been explored in large animal models: the use of mixed hematopoietic chimerism [11, 12], T regulatory cells [13, 14] and thymic transplantation [15] . It has been demonstrated that tolerance is possible in humans by successful clinical application of the mixed chimerism approach to renal transplantation [16] and by the T regulatory cell approach to liver allografts [17]. Despite the greater immunologic differences between species than within species, both mixed chimerism and thymic transplantation approaches have been shown to be capable of tolerizing human T cells to porcine xenografts in humanized mouse models [18]. Moreover, treatment with in vitro expanded regulatory T cells (Treg) prevents porcine xenograft rejection in humanized NOD-SCID IL-2 receptor gamma null (NSG) mice by the suppression of the T cell-mediated graft destruction, which suggesting the feasibility of pig-to-primate xenograft tolerance.

For xenografts, the level of immunosuppressive agents needed to fully suppress immune responses is greater than for allografts, which would likely lead to greater side effects. Thus, adoption of tolerance strategies is inevitable. Even though current immunosuppression seems to be controlling T cell responses in long-term acceptors [19, 20], it appears likely that low levels of T cell-dependent antibodies [21] and

activation of innate responses still develop [22], potentially leading to xenograft loss. Tolerance induction has the potential to avoid such persistent immune reactivity and therefore overcome the antibody-mediated response as well. Although tolerance induction in vivo has not yet been achieved in pig-to-baboon models, recent results are encouraging that this goal will be attainable through genetic engineering of porcine donors. It may be that current and future suppressive regimens that fully suppress the immune system will function sufficiently to benefit rejection of xenograft. Regardless of application, the study of tolerance continues to provide an excellent way to explore the functioning and control the immune system.

#### **3.2 Data archive for xenotransplantation clinical trials**

A database of clinical trials for pig islet xenotransplantation should be established, including specimens, paper documents, and electronic documents.

The information of xenotransplantation donors, including the number of animals, test reports, will be preserved for long time. All the samples will be prepared in duplicate and one for long-period preservation in −80° C refrigerator or liquid nitrogen tank. The information of transplant recipients and his/her spouses, such as name, hospital number, clinical data and patient records, will be recorded and maintained for long. When the patient comes to the hospital for review, the sample should be kept, including the following [23]: (1) all serum and plasma of the recipient and his/her spouse will be prepared in duplicate [24]; (2) storage of all samples at −80°C or liquid nitrogen tank for long time; (3) preservation of samples for post-transplant cytokine detection, pathogen detection, etc.; and (4) development of standard operating procedures.

#### **3.3 Postoperative follow-up**

The purpose of follow-up after xenotransplantation is to monitor the occurrence of rejection and adverse events. The goal of patient management is to improve their understanding of the disease, actively participate in and achieve partial self-management, improve compliance and achieve long-term survival and higher quality of life.

Postoperative follow-up of biosafety of clinical trials of recipients and spouses include: time-point, biosafety assays and treatment plan. (1) The patient and their spouses was reviewed 1 month before surgery, 1 month, 3 months, 6 months, 12 months, 2 years, 3 years, 4 years, and 5 years after xenotransplantation, and the sample in duplicate was kept. (2) Biosafety assays include fungal, bacterial, parasitic, viral, nucleic acid, cytokine and lymphocyte population detection. (3) If the biosafety assays are negative, the patient continues the symptomatic treatment, but if positive, then quarantine and treatment, personal protection and report to CDC (Centers for Disease Control and Prevention).

The medical record about postoperative follow-up of a xenograft recipient must contain the following information including the recipient's health status, all xenograft-related information, such as: (1) the contact information system of xenograft recipients. (2) If there is an infection related to xenotransplantation, or the pathogen from xenogeneic origin is identified, the health department of local government and the NHFPC(National health and family planning commission) shall be notified promptly. (3) The institution must have a reliable specimen and data preservation system and a complete information reporting system with the competent department. (4) The protocol must clearly address how patients are monitored for efficacy, biosafety, and period, including the draft clinical follow-up plan of xenotransplantation recipients.

**9**

*Biosafety Barrier to Xenotransplantation DOI: http://dx.doi.org/10.5772/intechopen.89134*

and developed normally in the closed colony.

The authors declare no conflict of interest.

Wei Wang\*, Qi Liang, Wei Nie, Juan Zhang and Cheng Chen

\*Address all correspondence to: cjr.wangwei@vip.163.com

Central-South University, Changsha, China

provided the original work is properly cited.

Institute for Cell Transplantation and Gene Therapy of the Third Xiangya Hospital,

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

Source donor pigs fulfilling the Designated Pathogen-Free (DPF) status have been available from a closed colony by GMP(Good Manufacturing Practice) rigorous routines, operational SOPs and rigorous data retention. Above all are very important for the operation of GMP barrier facility for biosafety of DPF source pig. A list of designated pathogens has been excluded from the DPF donor pig by long-term monitoring program of microbiological surveillance and pathological diagnosis. In addition, the consistently known DPF animals should be bred, grown

The authors would like to thank Pengfei Rong, Xiaoqian Ma, Cejun Yang, Qiong Dong, Shengwang Zhang, Qian Fang, and Chang Xu for their assistance with this

**4. Conclusions**

**Acknowledgements**

**Conflict of interest**

**Author details**

chapter.

### **4. Conclusions**

*Xenotransplantation - Comprehensive Study*

of standard operating procedures.

(Centers for Disease Control and Prevention).

plan of xenotransplantation recipients.

**3.3 Postoperative follow-up**

higher quality of life.

activation of innate responses still develop [22], potentially leading to xenograft loss. Tolerance induction has the potential to avoid such persistent immune reactivity and therefore overcome the antibody-mediated response as well. Although tolerance induction in vivo has not yet been achieved in pig-to-baboon models, recent results are encouraging that this goal will be attainable through genetic engineering of porcine donors. It may be that current and future suppressive regimens that fully suppress the immune system will function sufficiently to benefit rejection of xenograft. Regardless of application, the study of tolerance continues to provide an

excellent way to explore the functioning and control the immune system.

lished, including specimens, paper documents, and electronic documents.

A database of clinical trials for pig islet xenotransplantation should be estab-

The information of xenotransplantation donors, including the number of animals, test reports, will be preserved for long time. All the samples will be prepared in duplicate and one for long-period preservation in −80° C refrigerator or liquid nitrogen tank. The information of transplant recipients and his/her spouses, such as name, hospital number, clinical data and patient records, will be recorded and maintained for long. When the patient comes to the hospital for review, the sample should be kept, including the following [23]: (1) all serum and plasma of the recipient and his/her spouse will be prepared in duplicate [24]; (2) storage of all samples at −80°C or liquid nitrogen tank for long time; (3) preservation of samples for post-transplant cytokine detection, pathogen detection, etc.; and (4) development

The purpose of follow-up after xenotransplantation is to monitor the occurrence of rejection and adverse events. The goal of patient management is to improve their understanding of the disease, actively participate in and achieve partial self-management, improve compliance and achieve long-term survival and

Postoperative follow-up of biosafety of clinical trials of recipients and spouses include: time-point, biosafety assays and treatment plan. (1) The patient and their spouses was reviewed 1 month before surgery, 1 month, 3 months, 6 months, 12 months, 2 years, 3 years, 4 years, and 5 years after xenotransplantation, and the sample in duplicate was kept. (2) Biosafety assays include fungal, bacterial, parasitic, viral, nucleic acid, cytokine and lymphocyte population detection. (3) If the biosafety assays are negative, the patient continues the symptomatic treatment, but if positive, then quarantine and treatment, personal protection and report to CDC

The medical record about postoperative follow-up of a xenograft recipient must contain the following information including the recipient's health status, all xenograft-related information, such as: (1) the contact information system of xenograft recipients. (2) If there is an infection related to xenotransplantation, or the pathogen from xenogeneic origin is identified, the health department of local government and the NHFPC(National health and family planning commission) shall be notified promptly. (3) The institution must have a reliable specimen and data preservation system and a complete information reporting system with the competent department. (4) The protocol must clearly address how patients are monitored for efficacy, biosafety, and period, including the draft clinical follow-up

**3.2 Data archive for xenotransplantation clinical trials**

**8**

Source donor pigs fulfilling the Designated Pathogen-Free (DPF) status have been available from a closed colony by GMP(Good Manufacturing Practice) rigorous routines, operational SOPs and rigorous data retention. Above all are very important for the operation of GMP barrier facility for biosafety of DPF source pig. A list of designated pathogens has been excluded from the DPF donor pig by long-term monitoring program of microbiological surveillance and pathological diagnosis. In addition, the consistently known DPF animals should be bred, grown and developed normally in the closed colony.

### **Acknowledgements**

The authors would like to thank Pengfei Rong, Xiaoqian Ma, Cejun Yang, Qiong Dong, Shengwang Zhang, Qian Fang, and Chang Xu for their assistance with this chapter.

### **Conflict of interest**

The authors declare no conflict of interest.

### **Author details**

Wei Wang\*, Qi Liang, Wei Nie, Juan Zhang and Cheng Chen Institute for Cell Transplantation and Gene Therapy of the Third Xiangya Hospital, Central-South University, Changsha, China

\*Address all correspondence to: cjr.wangwei@vip.163.com

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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[2] Onions D, Cooper DK, Alexander TJ, Brown C, Claassen E, Foweraker JE, et al. An approach to the control of disease transmission in pig-to-human xenotransplantation. Xenotransplantation. 2000;**7**:143-155

[3] Blusch JH, Patience C, Martin U. Pig endogenous retroviruses and xenotransplantation. Xenotransplantation. 2002;**9**:242-251

[4] Wood JC, Quinn G, Suling KM, Oldmixon BA, Van Tine BA, Cina R, et al. Identification of exogenous forms of human-tropic porcine endogenous retrovirus in miniature swine. Journal of Virology. 2004;**78**:2494-2501

[5] Public Health US. Service guideline on infectious disease issues in xenotransplantation. Centers for Disease Control and Prevention. MMWR - Recommendations and Reports. 2001;**50**:1-46

[6] Yamada A, Salama AD, Sayegh MH. The role of novel T cell costimulatory pathways in autoimmunity and transplantation. Journal of the American Society of Nephrology. 2002;**13**:559-575

[7] Tian M, Lv Y, Zhai C, Zhu H, Yu L, Wang B. Alternative immunomodulatory strategies for xenotransplantation: CD80/CD86-CTLA4 pathway-modified immature dendritic cells promote xenograft survival. PLoS One. 2013;**8**:e69640

[8] Cardona K, Korbutt GS, Milas Z, Lyon J, Cano J, Jiang W, et al. Long-term survival of neonatal porcine islets in nonhuman primates by targeting costimulation pathways. Nature Medicine. 2006;**12**:304-306

[9] Schuler W, Bigaud M, Brinkmann V, Di Padova F, Geisse S, Gram H, et al. Efficacy and safety of ABI793, a novel human anti-human CD154 monoclonal antibody, in cynomolgus monkey renal allotransplantation. Transplantation. 2004;**77**:717-726

[10] van der Windt DJ, Bottino R, Casu A, Campanile N, Smetanka C, He J, et al. Long-term controlled normoglycemia in diabetic non-human primates after transplantation with hCD46 transgenic porcine islets. American Journal of Transplantation. 2009;**9**:2716-2726

[11] Fuchimoto Y, Huang CA, Yamada K, Shimizu A, Kitamura H, Colvin RB, et al. Mixed chimerism and tolerance without whole body irradiation in a large animal model. The Journal of Clinical Investigation. 2000;**105**:1779-1789

[12] Yamada Y, Boskovic S, Aoyama A, Murakami T, Putheti P, Smith RN, et al. Overcoming memory T-cell responses for induction of delayed tolerance in nonhuman primates. American Journal of Transplantation. 2012;**12**:330-340

[13] Bashuda H, Kimikawa M, Seino K, Kato Y, Ono F, Shimizu A, et al. Renal allograft rejection is prevented by adoptive transfer of anergic T cells in nonhuman primates. The Journal of Clinical Investigation. 2005;**115**:1896-1902

[14] Yi S, Ji M, Wu J, Ma X, Phillips P, Hawthorne WJ, et al. Adoptive transfer with in vitro expanded human regulatory T cells protects against porcine islet xenograft rejection via interleukin-10 in humanized mice. Diabetes. 2012;**61**:1180-1191

**11**

*Biosafety Barrier to Xenotransplantation DOI: http://dx.doi.org/10.5772/intechopen.89134*

Kumagai N, Yamamoto S, Barth RN, LaMattina JC, et al. Vascularized thymic lobe transplantation in miniature swine: Thymopoiesis and tolerance induction across fully MHC-mismatched barriers. Proceedings of the National Academy of Sciences of the United States of America. 2004;**101**:3827-3832

transplantation correlate with failure of engraftment. Xenotransplantation.

[22] Yang YG. CD47 in xenograft rejection and tolerance induction. Xenotransplantation. 2010;**17**:267-273

[23] Ali KF, San MV, Walsh RM, Bottino R, Stevens T, Trucco M, et al. Change in functional Beta cell capacity

with time following autologous islet transplantation. Pancreas.

[24] Golebiewska JE, Bachul PJ,

Fillman N, Basto L, Kijek MR, Golab K, et al. Assessment of simple indices based on a single fasting blood sample as a tool to estimate beta-cell function after total pancreatectomy with islet autotransplantation—A prospective study. Transplant International.

2013;**20**:458-468

2019;**48**:656-661

2019;**32**:280-290

[15] Kamano C, Vagefi PA,

[16] Strober S, Spitzer TR,

Lowsky R, Sykes M. Translational studies in hematopoietic cell transplantation: Treatment of

[17] Todo S, Yamashita K, Goto R, Zaitsu M, Nagatsu A, Oura T, et al. A pilot study of operational tolerance with a regulatory T-cell-based cell therapy in living donor liver transplantation.

Hepatology. 2016;**64**:632-643

[18] Kalscheuer H, Onoe T,

hematologic malignancies as a stepping stone to tolerance induction. Seminars in Immunology. 2011;**23**:273-281

Dahmani A, Li HW, Holzl M, Yamada K, et al. Xenograft tolerance and immune function of human T cells developing in pig thymus xenografts. Journal of Immunology. 2014;**192**:3442-3450

Larsen CP, et al. Pre-transplant antibody screening and anti-CD154 costimulation blockade promote long-term xenograft survival in a pig-to-primate kidney transplant model. Xenotransplantation.

[20] Iwase H, Hara H, Ezzelarab M, Li T, Zhang Z, Gao B, et al. Immunological and physiological observations in baboons with life-supporting genetically engineered pig kidney grafts. Xenotransplantation.

[21] Liang F, Wamala I, Scalea J, Tena A, Cormack T, Pratts S, et al. Increased levels of anti-non-Gal IgG following

[19] Higginbotham L, Mathews D, Breeden CA, Song M, Farris AR,

2015;**22**:221-230

2017;**24**:12293-12324

pig-to-baboon bone marrow

*Biosafety Barrier to Xenotransplantation DOI: http://dx.doi.org/10.5772/intechopen.89134*

[15] Kamano C, Vagefi PA, Kumagai N, Yamamoto S, Barth RN, LaMattina JC, et al. Vascularized thymic lobe transplantation in miniature swine: Thymopoiesis and tolerance induction across fully MHC-mismatched barriers. Proceedings of the National Academy of Sciences of the United States of America. 2004;**101**:3827-3832

[16] Strober S, Spitzer TR, Lowsky R, Sykes M. Translational studies in hematopoietic cell transplantation: Treatment of hematologic malignancies as a stepping stone to tolerance induction. Seminars in Immunology. 2011;**23**:273-281

[17] Todo S, Yamashita K, Goto R, Zaitsu M, Nagatsu A, Oura T, et al. A pilot study of operational tolerance with a regulatory T-cell-based cell therapy in living donor liver transplantation. Hepatology. 2016;**64**:632-643

[18] Kalscheuer H, Onoe T, Dahmani A, Li HW, Holzl M, Yamada K, et al. Xenograft tolerance and immune function of human T cells developing in pig thymus xenografts. Journal of Immunology. 2014;**192**:3442-3450

[19] Higginbotham L, Mathews D, Breeden CA, Song M, Farris AR, Larsen CP, et al. Pre-transplant antibody screening and anti-CD154 costimulation blockade promote long-term xenograft survival in a pig-to-primate kidney transplant model. Xenotransplantation. 2015;**22**:221-230

[20] Iwase H, Hara H, Ezzelarab M, Li T, Zhang Z, Gao B, et al. Immunological and physiological observations in baboons with life-supporting genetically engineered pig kidney grafts. Xenotransplantation. 2017;**24**:12293-12324

[21] Liang F, Wamala I, Scalea J, Tena A, Cormack T, Pratts S, et al. Increased levels of anti-non-Gal IgG following pig-to-baboon bone marrow

transplantation correlate with failure of engraftment. Xenotransplantation. 2013;**20**:458-468

[22] Yang YG. CD47 in xenograft rejection and tolerance induction. Xenotransplantation. 2010;**17**:267-273

[23] Ali KF, San MV, Walsh RM, Bottino R, Stevens T, Trucco M, et al. Change in functional Beta cell capacity with time following autologous islet transplantation. Pancreas. 2019;**48**:656-661

[24] Golebiewska JE, Bachul PJ, Fillman N, Basto L, Kijek MR, Golab K, et al. Assessment of simple indices based on a single fasting blood sample as a tool to estimate beta-cell function after total pancreatectomy with islet autotransplantation—A prospective study. Transplant International. 2019;**32**:280-290

**10**

*Xenotransplantation - Comprehensive Study*

[1] FDA. Source Animal, Product,

survival of neonatal porcine islets in nonhuman primates by targeting costimulation pathways. Nature Medicine. 2006;**12**:304-306

[9] Schuler W, Bigaud M, Brinkmann V, Di Padova F, Geisse S, Gram H, et al. Efficacy and safety of ABI793, a novel human anti-human CD154 monoclonal antibody, in cynomolgus monkey renal allotransplantation. Transplantation.

[10] van der Windt DJ, Bottino R, Casu A, Campanile N, Smetanka C, He J, et al. Long-term controlled normoglycemia in diabetic non-human primates after transplantation with hCD46 transgenic porcine islets. American Journal of Transplantation. 2009;**9**:2716-2726

[11] Fuchimoto Y, Huang CA, Yamada K, Shimizu A, Kitamura H, Colvin RB, et al. Mixed chimerism and tolerance without whole body irradiation in a large animal model. The Journal of Clinical Investigation.

[12] Yamada Y, Boskovic S, Aoyama A, Murakami T, Putheti P, Smith RN, et al. Overcoming memory T-cell responses for induction of delayed tolerance in nonhuman primates. American Journal of Transplantation. 2012;**12**:330-340

[13] Bashuda H, Kimikawa M, Seino K, Kato Y, Ono F, Shimizu A, et al. Renal allograft rejection is prevented by adoptive transfer of anergic T cells in nonhuman primates. The Journal of Clinical Investigation.

[14] Yi S, Ji M, Wu J, Ma X, Phillips P, Hawthorne WJ, et al. Adoptive transfer

with in vitro expanded human regulatory T cells protects against porcine islet xenograft rejection via interleukin-10 in humanized mice. Diabetes. 2012;**61**:1180-1191

2000;**105**:1779-1789

2005;**115**:1896-1902

2004;**77**:717-726

Preclinical, and Clinical Issues Concerning the Use of Xenotransplantation Products in Humans. U.S. Department of Health and Human Services, Food and Drug Administration, Center for Biologics Evaluation and Research; 2003

**References**

[2] Onions D, Cooper DK,

Alexander TJ, Brown C, Claassen E, Foweraker JE, et al. An approach to the control of disease transmission in pig-to-human xenotransplantation. Xenotransplantation. 2000;**7**:143-155

[3] Blusch JH, Patience C, Martin U. Pig endogenous retroviruses and xenotransplantation.

Xenotransplantation. 2002;**9**:242-251

[4] Wood JC, Quinn G, Suling KM, Oldmixon BA, Van Tine BA, Cina R, et al. Identification of exogenous forms of human-tropic porcine endogenous retrovirus in miniature swine. Journal of Virology. 2004;**78**:2494-2501

[5] Public Health US. Service guideline

[6] Yamada A, Salama AD, Sayegh MH. The role of novel T cell costimulatory pathways in autoimmunity and transplantation. Journal of the American Society of Nephrology.

[7] Tian M, Lv Y, Zhai C, Zhu H, Yu L, Wang B. Alternative immunomodulatory strategies for xenotransplantation: CD80/CD86-CTLA4 pathway-modified immature dendritic cells promote xenograft survival. PLoS One.

[8] Cardona K, Korbutt GS, Milas Z, Lyon J, Cano J, Jiang W, et al. Long-term

on infectious disease issues in xenotransplantation. Centers for Disease Control and Prevention. MMWR - Recommendations and

Reports. 2001;**50**:1-46

2002;**13**:559-575

2013;**8**:e69640

**13**

**Chapter 2**

**Abstract**

*Masayuki Shimoda*

Pig Islet Transplant

transplantation in future clinical trials.

soon as possible after islet isolation.

immunosuppressants.

**1. Introduction**

**Keywords:** xenotransplantation, islet transplantation, porcine islet

Islet transplantation is an effective treatment for insulin-dependent diabetes, but the shortage of donors is a problem. To overcome this, porcine islets have been widely studied as an alternative source. This chapter focuses on recent advances in porcine islet transplantation, placing particular emphasis on new transgenic pig models, islet encapsulation, and biological safety. Genetic modifications aimed at reducing the immunogenicity of islet cells to prolong graft survival or improve insulin secretory function have been reported. Microencapsulation and macroencapsulation of porcine islets may be able to control rejection with little or no immunosuppression. Also, the risk of porcine endogenous retrovirus infection is considered low because several clinical and preclinical studies have found no such evidence. Appropriate pathogen screening, animal selection, and microbiological and quality control measures should improve the safety and efficacy of porcine islet

The islet transplantation protocol used for patients with type 1 diabetes, published by a team of researchers at the University of Alberta in 2000, was called the Edmonton Protocol and became the starting point for clinical islet transplantation [1]. The characteristics of the Edmonton Protocol were that multiple transplants were performed using multiple donors to transplant sufficient amounts of islets, no steroids were used for immunosuppression, and transplants were performed as

Clinical results were reported 5 years after the Edmonton Protocol was announced [2], and several problems were identified. For example, the insulinfree status is not sustained for a long time, the probability of being able to obtain islets of sufficient quality and quantity for transplantation even with islet isolation is about 50%, and there were many side effects, mainly from

Islet transplantation has been found to stabilize blood glucose levels and could prevent severe hypoglycemia, defined as hypoglycemia requiring another person's assistance. Because severe hypoglycemia can be life-threatening for patients with type 1 diabetes, islet transplantation will likely be positioned as a measure for preventing severe hypoglycemia. Indeed, allogeneic islet transplantation is an established treatment for severe hypoglycemia in Canada and other European countries. In addition, in 2016, a phase 3 clinical trial of allogeneic islet transplantation for type 1 diabetes patients with a history of severe hypoglycemia found that islet transplantation has a preventive effect for severe hypoglycemia [3]. Therefore, allogeneic

## **Chapter 2** Pig Islet Transplant

*Masayuki Shimoda*

### **Abstract**

Islet transplantation is an effective treatment for insulin-dependent diabetes, but the shortage of donors is a problem. To overcome this, porcine islets have been widely studied as an alternative source. This chapter focuses on recent advances in porcine islet transplantation, placing particular emphasis on new transgenic pig models, islet encapsulation, and biological safety. Genetic modifications aimed at reducing the immunogenicity of islet cells to prolong graft survival or improve insulin secretory function have been reported. Microencapsulation and macroencapsulation of porcine islets may be able to control rejection with little or no immunosuppression. Also, the risk of porcine endogenous retrovirus infection is considered low because several clinical and preclinical studies have found no such evidence. Appropriate pathogen screening, animal selection, and microbiological and quality control measures should improve the safety and efficacy of porcine islet transplantation in future clinical trials.

**Keywords:** xenotransplantation, islet transplantation, porcine islet

#### **1. Introduction**

The islet transplantation protocol used for patients with type 1 diabetes, published by a team of researchers at the University of Alberta in 2000, was called the Edmonton Protocol and became the starting point for clinical islet transplantation [1]. The characteristics of the Edmonton Protocol were that multiple transplants were performed using multiple donors to transplant sufficient amounts of islets, no steroids were used for immunosuppression, and transplants were performed as soon as possible after islet isolation.

Clinical results were reported 5 years after the Edmonton Protocol was announced [2], and several problems were identified. For example, the insulinfree status is not sustained for a long time, the probability of being able to obtain islets of sufficient quality and quantity for transplantation even with islet isolation is about 50%, and there were many side effects, mainly from immunosuppressants.

Islet transplantation has been found to stabilize blood glucose levels and could prevent severe hypoglycemia, defined as hypoglycemia requiring another person's assistance. Because severe hypoglycemia can be life-threatening for patients with type 1 diabetes, islet transplantation will likely be positioned as a measure for preventing severe hypoglycemia. Indeed, allogeneic islet transplantation is an established treatment for severe hypoglycemia in Canada and other European countries. In addition, in 2016, a phase 3 clinical trial of allogeneic islet transplantation for type 1 diabetes patients with a history of severe hypoglycemia found that islet transplantation has a preventive effect for severe hypoglycemia [3]. Therefore, allogeneic islet transplantation has also come to be recognized as standard treatment for severe hypoglycemia in the United States. Data on allogeneic islet transplantation are registered in the Collaborative Islet Transplant Registry (CITR). According to CITR data, the C-peptide positivity rate after islet transplantation alone was 80% after 1 year and 61% after 3 years, but the severe hypoglycemia prevention rate was 94 and 88%, respectively. This indicates that even if the concentration of C-peptide is below the lower limit of detection for a positive result (0.3 ng/ml), it would be effective in stabilizing blood glucose levels and preventing hypoglycemia. According to the data from the International Pancreas Transplant Registry, the pancreatic graft survival rate in simultaneous kidney and pancreas transplantation was 89% at 1 year and 82% at 3 years after transplantation. In other words, islet transplantation outperforms simultaneous pancreas and kidney transplantation in terms of rates of preventing severe hypoglycemia. The current status and direction of beta cell replacement therapy were discussed at a consensus meeting of the beta cell replacement therapy opinion leaders held at Oxford University in 2014 [4]. According to the consensus report, there are 15–20 million patients with type 1 diabetes worldwide, they are mostly at >20 years after onset of type 1 diabetes, 1 in 6 patients develop hypoglycemia unawareness, and ~10% of deaths in type 1 diabetes patients are due to hypoglycemia. It was announced that β cell replacement therapy was optimal for hypoglycemia in patients with hard-to-control type 1 diabetes. However, only 0.1% of patients with type 1 diabetes could receive beta cell replacement therapy due to a shortage of donors. In Japan, cardiac arrest donor islet transplantation [5] and living donor islet transplantation [6] have been carried out, but in order to fundamentally solve the donor shortage, β cell replacement therapy not relying on human organ donors is considered essential. Under these circumstances, pig organs are attracting attention as an alternative to organs from human donors.

#### **2. Pancreatic islet transplantation using porcine islets**

To realize successful porcine islet transplantation, exploratory clinical research began several decades ago. **Table 1** shows an overview of the history of porcine islet transplantation. In the 1990s in Sweden, Groth et al. transplanted islet cells from fetal pigs into type 1 diabetic patients on immunosuppressants after kidney transplantation [7]. Porcine C-peptide was positive for several months after transplantation, which indicated that porcine islets were successfully engrafted in the human body. Yet, no clinical effect such as a decrease in the amount of insulin injection was observed. In other works, Valdes et al. implanted an angioplasty device with newborn pig islets and Sertoli cells subcutaneously into type 1 diabetes patients [8]. Eleven patients received additional transplantation 6–9 months after the initial transplantation, and four received additional transplantation in the third year. Two patients achieved insulin-free status for several months after transplantation. In New Zealand, Elliott et al. transplanted newborn pig islets encapsulated in hydrogel microcapsules into the peritoneal cavity of type 1 diabetic patients. Because the islets were embedded in the immunoisolation capsule, no immunosuppressant was used. Insulin and glucagon staining of encapsulated pig islets, which were removed after 9.5 years of transplantation, showed that the encapsulated pig islets could be engrafted for a long time [9].

Thus, xenogeneic islet transplantation for type 1 diabetes patients using porcine islets has been performed in several clinical trials overseas. The risk of infection due to xenotransplantation was a concern.

**15**

tives are recommended.

*Pig Islet Transplant*

*DOI: http://dx.doi.org/10.5772/intechopen.88324*

islets and Sertoli cells

South University, China

complementation

**Table 1.**

monkey body for more than 6 months

transplantation of neonatal porcine islets

transplantation of wild-type adult porcine islets

islets were recovered and insulin staining was positive

**3. Designated pathogen-free status and porcine endogenous retrovirus**

tion, the piglet is placed in a biosecure barrier facility.

*Chronological overview of clinical and preclinical islet xenotransplantation.*

Pigs for clinical use must have a designated pathogen-free (DPF) status, which means they are free of pathogens that can infect humans and pigs [10]. DPF status is achieved by delivering a piglet by cesarean section from a sow that has been confirmed to be free of transplacental pathogens, and after cleaning and decontamina-

**Year Events Ref.** 1994 Groth et al. reported that fetal pig islet transplantation to diabetic patients [7] 1997 Patience et al. reported that PERV could infect human cells [11]

[8]

[12]

[13]

[14]

[9]

—

[32]

2005 Valdes-Gonzales et al. reported a 4-year course after transplantation of neonatal pig

2006 Hering et al. achieved long-term insulin-free status in diabetic monkeys by

2006 Cardona et al. achieved long-term insulin independence in diabetic monkeys by

2007 Elliott et al. reported that about 9.5 years after transplantation, encapsulated porcine

2013 Wang et al. commenced neonatal porcine islet transplantation with Tregs at Central

2017 Yamaguchi et al. succeeded in creating a mouse pancreas in a rat using blastocyst

2014 Matsumoto et al. reported porcine islet transplantation under New Zealand regulations [23] 2015 Yang et al. announced that they used CRISPR/Cas9 to inactivate all PERVs [31] 2016 Matsumoto et al. reported clinical efficacy with encapsulated pig islet transplantation [24]

2006 Dufrane et al. showed that encapsulated adult porcine islets survived in the cynomolgus

These facilities are defined at several levels. First, it is necessary that the facility itself be sited away from the pig farming facility. The breeding building must be completely isolated from the outside environment with an air filter, water decontamination system, radiation sterilization, and autoclave for all incoming materials. Piglets are fed with pasteurized milk, not breast milk, and enteric bacteria are provided separately. For waste disposal, especially liquid waste, special consideration is necessary to avoid backflow. Staff must pass through antiseptic showers both when entering and exiting the facility and must change into special sterilized clothes. Routine health checks of personnel are also conducted. In general, all procedures must follow standard operative procedures. It is also important to incorporate current good manufacturing practices in accordance with regulatory guidelines.

Nevertheless, in coculture of PK-15 pig kidney cell line (PK15 cells), and human fetal kidney cells 293 (HEK293 cells), infection of HEK293 cells by porcine endogenous retrovirus (PERV) naturally released from PK15 cells has been reported [11]. The problem of PERV infection via porcine xenotransplantation has emerged, and because PERV-A and PERV-B are integrated into all porcine genes, they are extremely difficult to eliminate. Thus, with regard to PERV, instead of exclusion, denial of infectivity and monitoring of transplanted patients and their close rela-


**Table 1.**

*Xenotransplantation - Comprehensive Study*

islet transplantation has also come to be recognized as standard treatment for severe hypoglycemia in the United States. Data on allogeneic islet transplantation are registered in the Collaborative Islet Transplant Registry (CITR). According to CITR data, the C-peptide positivity rate after islet transplantation alone was 80% after 1 year and 61% after 3 years, but the severe hypoglycemia prevention rate was 94 and 88%, respectively. This indicates that even if the concentration of C-peptide is below the lower limit of detection for a positive result (0.3 ng/ml), it would be effective in stabilizing blood glucose levels and preventing hypoglycemia. According to the data from the International Pancreas Transplant Registry, the pancreatic graft survival rate in simultaneous kidney and pancreas transplantation was 89% at 1 year and 82% at 3 years after transplantation. In other words, islet transplantation outperforms simultaneous pancreas and kidney transplantation in terms of rates of preventing severe hypoglycemia. The current status and direction of beta cell replacement therapy were discussed at a consensus meeting of the beta cell replacement therapy opinion leaders held at Oxford University in 2014 [4]. According to the consensus report, there are 15–20 million patients with type 1 diabetes worldwide, they are mostly at >20 years after onset of type 1 diabetes, 1 in 6 patients develop hypoglycemia unawareness, and ~10% of deaths in type 1 diabetes patients are due to hypoglycemia. It was announced that β cell replacement therapy was optimal for hypoglycemia in patients with hard-to-control type 1 diabetes. However, only 0.1% of patients with type 1 diabetes could receive beta cell replacement therapy due to a shortage of donors. In Japan, cardiac arrest donor islet transplantation [5] and living donor islet transplantation [6] have been carried out, but in order to fundamentally solve the donor shortage, β cell replacement therapy not relying on human organ donors is considered essential. Under these circumstances, pig organs are attracting attention as an alternative to organs from human

**2. Pancreatic islet transplantation using porcine islets**

To realize successful porcine islet transplantation, exploratory clinical research began several decades ago. **Table 1** shows an overview of the history of porcine islet transplantation. In the 1990s in Sweden, Groth et al. transplanted islet cells from fetal pigs into type 1 diabetic patients on immunosuppressants after kidney transplantation [7]. Porcine C-peptide was positive for several months after transplantation, which indicated that porcine islets were successfully engrafted in the human body. Yet, no clinical effect such as a decrease in the amount of insulin injection was observed. In other works, Valdes et al. implanted an angioplasty device with newborn pig islets and Sertoli cells subcutaneously into type 1 diabetes patients [8]. Eleven patients received additional transplantation 6–9 months after the initial transplantation, and four received additional transplantation in the third year. Two patients achieved insulin-free status for several months after transplantation. In New Zealand, Elliott et al. transplanted newborn pig islets encapsulated in hydrogel microcapsules into the peritoneal cavity of type 1 diabetic patients. Because the islets were embedded in the immunoisolation capsule, no immunosuppressant was used. Insulin and glucagon staining of encapsulated pig islets, which were removed after 9.5 years of transplantation, showed that the encapsulated pig islets could be

Thus, xenogeneic islet transplantation for type 1 diabetes patients using porcine islets has been performed in several clinical trials overseas. The risk of infection due

**14**

engrafted for a long time [9].

to xenotransplantation was a concern.

donors.

*Chronological overview of clinical and preclinical islet xenotransplantation.*

#### **3. Designated pathogen-free status and porcine endogenous retrovirus**

Pigs for clinical use must have a designated pathogen-free (DPF) status, which means they are free of pathogens that can infect humans and pigs [10]. DPF status is achieved by delivering a piglet by cesarean section from a sow that has been confirmed to be free of transplacental pathogens, and after cleaning and decontamination, the piglet is placed in a biosecure barrier facility.

These facilities are defined at several levels. First, it is necessary that the facility itself be sited away from the pig farming facility. The breeding building must be completely isolated from the outside environment with an air filter, water decontamination system, radiation sterilization, and autoclave for all incoming materials. Piglets are fed with pasteurized milk, not breast milk, and enteric bacteria are provided separately. For waste disposal, especially liquid waste, special consideration is necessary to avoid backflow. Staff must pass through antiseptic showers both when entering and exiting the facility and must change into special sterilized clothes. Routine health checks of personnel are also conducted. In general, all procedures must follow standard operative procedures. It is also important to incorporate current good manufacturing practices in accordance with regulatory guidelines.

Nevertheless, in coculture of PK-15 pig kidney cell line (PK15 cells), and human fetal kidney cells 293 (HEK293 cells), infection of HEK293 cells by porcine endogenous retrovirus (PERV) naturally released from PK15 cells has been reported [11]. The problem of PERV infection via porcine xenotransplantation has emerged, and because PERV-A and PERV-B are integrated into all porcine genes, they are extremely difficult to eliminate. Thus, with regard to PERV, instead of exclusion, denial of infectivity and monitoring of transplanted patients and their close relatives are recommended.

#### **4. Pig islet transplantation experiment using nonhuman primates**

Dufrane et al. demonstrated that mature pig islets embedded in alginate capsules and transplanted into cynomolgus monkeys without immunosuppressants survived up to 6 months after transplantation [12]. Hering et al. at the University of Minnesota reported that wild-type (unmodified) adult porcine islets transplanted into the portal vein of rhesus monkeys with streptozotocin-induced diabetes mellitus achieved long-term insulin independence [13]. Also, Cardona et al. from the University of Alberta reported that wild-type newborn porcine islets transplanted into the portal vein of monkeys with pancreatectomy-induced diabetes resulted in long-term insulin-free status [14]. Recently, Park et al. reported more advances with modification of immunosuppressants [15]. These reports have brought great hope for islet transplantation using porcine islets. However, the importance of prevention of infections including PERV has been recognized.

#### **5. Guidelines**

While xenotransplantation holds great promise for overcoming donor shortages, the global problem of xenogeneic infection must be considered. Therefore, in 2008 the World Health Organization (WHO) held a conference on xenotransplantation in Changsha, China, and presented the main points as the First WHO Global Consultation on Regulatory Requirements for Xenotransplantation Clinical Trials [16]. This statement, referred to as the Changsha Communique, is the basis for xenotransplantation worldwide. The content summary is shown in **Table 2**.

Based on the Changsha Communique, in 2009 the International Xenotransplantation Association (IXA) announced a consensus statement of conditions for the initiation of clinical trials of porcine islet products for type 1 diabetes [17]. This consensus statement consists of seven chapters and addresses the requirements of the Changsha Communique. Because remarkable progress has been made in research in this field, the statement should be updated.

Since the consensus statement for the initiation of xenogeneic islet transplantation in IXA was announced in 2009, clinical findings of xenotransplantation including clinical xenogeneic islet transplantation in New Zealand have been accumulated, and the consensus statement was updated in 2016 [18]. The contents of the chapters are:

Chapter 1. Key ethical requirements and progress toward the definition of an international regulatory framework: ethical requirements and progress toward establishing an international regulatory framework.

Chapter 2. Source pigs: pig requirements for donor sources.

Chapter 3. Pig islet product manufacturing and release testing: manufacturing, quality control, and release testing.

Chapter 4. Pre-clinical efficacy and complication data required to justify a clinical trial: appropriate pre-clinical trial.

Chapter 5. Strategies to prevent transmission of porcine endogenous retroviruses: concept and prevention strategy for PERV.

Chapter 6. Patient selection for pilot clinical trials of islet xenotransplantation: appropriate patient selection.

Chapter 7. Informed consent and xenotransplantation clinical trials: ideal informed consent procedure.

In particular, because PERV infection and cross-species infection did not occur at all, these infections were regarded as a "theoretical risk" and were considered

**17**

tion" in 2016.

**Table 2.**

*Pig Islet Transplant*

*DOI: http://dx.doi.org/10.5772/intechopen.88324*

diseases by continuous observation

other humans and animals may be infected

and should include both science and ethics

regulatory authorities for a designated period

themselves and to society

suspected infection

recommended to support

*Summary of the contents of the Changsha communique.*

1 Xenotransplantation can be used to treat serious diseases such as diabetes, heart disease, and kidney disease. Also, patients who cannot currently receive transplants may be able to receive transplants 2 Medical animals can provide high-quality cells, tissues, and organs. Genetically modified animals may further improve outcomes. Medical animals are limited to closed colonies. Breeding should be done at a well-controlled pathogen-free facility, with high standards for animal welfare. Medical animals are verified by testing for the absence of known pathogens and, moreover, must be kept free of infectious

3 Xenotransplantation is a complex procedure with risk of rejection, poor graft function, and known or unknown infections. There is a risk of developing serious or new infections, and patients, relatives, or

conducted under strict regulation. Xenotransplantation should not be performed in the absence of national regulations. These regulations should have legal basis and be able to prohibit nonregulatory transplants. Furthermore, this regulatory framework should ensure transparency to the general public

4 Because of the risk to the community at large, clinical trials of xenotransplantation should be

5 Given the risk to the community, the benefit to the patient should be high. In particular, preclinical studies should be conducted using animal experiments with predictable effects to demonstrate the safety and efficacy recommended by the international scientific community. Proposed clinical trials

6 Personnel responsible for clinical trials should explain the inclusion criteria in order to justify the

7 Participation in xenotransplantation usually takes a long time. Samples from donor animals, patients before and after surgery, and all records should be kept. Patients who have had transplants need lifetime follow-up, and close relatives may need similar follow-up. The results of clinical trials should be analyzed rigorously. Patients who have undergone xenotransplantation should be registered in an appropriate database, which should also be able to track donor animals. At the same time, the patient's privacy has to be protected. All records, data, and samples must be prepared for submission to

8 The health-care team must have adequate experience and an understanding of the risks to the patient, the health-care team itself, and the community. Because of the risk of transmission to the community, a system of vigilance and surveillance should be established to ensure that any infection associated

9 There is a need to establish a system for worldwide information exchange, prevention of unregulated xenotransplantation, vigilance and monitoring of xenotransplantation, and response in case of

10 Considering the benefits of successful xenotransplantation, from the early stages, the treatment should be considered widely accepted as the treatment is completed, and the public sector is

clinical trial. Patient selection must be done at the patient's own discretion based on informed consent. Patients and relatives must be effectively educated to ensure compliance and minimize risks to

should be assessed by the relevant regulatory authorities to minimize risk

with the xenotransplant will be identified and addressed immediately

unlikely under the adequate control of suitable donors and recipients. In addition, clinical data have been accumulated, infection diagnostic techniques have progressed, clinical protocols have been improved, the risk of PERV-related infection is better understood, DPF facilities and dietary restriction methods have advanced, and the role of sample archives has been clarified. As a result of these efforts, costeffective generation of donor pigs will be possible, and it is expected that porcine islets will be delivered to many patients who truly need this treatment modality. Some countries have responded to this consensus statement. In Japan, the Ministry of Health, Labour and Welfare also revised the "guidelines on public health infection problems associated with the implementation of xenotransplanta-

#### *Pig Islet Transplant DOI: http://dx.doi.org/10.5772/intechopen.88324*

*Xenotransplantation - Comprehensive Study*

of infections including PERV has been recognized.

in research in this field, the statement should be updated.

establishing an international regulatory framework.

ruses: concept and prevention strategy for PERV.

quality control, and release testing.

appropriate patient selection.

informed consent procedure.

cal trial: appropriate pre-clinical trial.

Chapter 2. Source pigs: pig requirements for donor sources.

**5. Guidelines**

of the chapters are:

**4. Pig islet transplantation experiment using nonhuman primates**

Dufrane et al. demonstrated that mature pig islets embedded in alginate capsules and transplanted into cynomolgus monkeys without immunosuppressants survived up to 6 months after transplantation [12]. Hering et al. at the University of Minnesota reported that wild-type (unmodified) adult porcine islets transplanted into the portal vein of rhesus monkeys with streptozotocin-induced diabetes mellitus achieved long-term insulin independence [13]. Also, Cardona et al. from the University of Alberta reported that wild-type newborn porcine islets transplanted into the portal vein of monkeys with pancreatectomy-induced diabetes resulted in long-term insulin-free status [14]. Recently, Park et al. reported more advances with modification of immunosuppressants [15]. These reports have brought great hope for islet transplantation using porcine islets. However, the importance of prevention

While xenotransplantation holds great promise for overcoming donor shortages, the global problem of xenogeneic infection must be considered. Therefore, in 2008 the World Health Organization (WHO) held a conference on xenotransplantation in Changsha, China, and presented the main points as the First WHO Global Consultation on Regulatory Requirements for Xenotransplantation Clinical Trials [16]. This statement, referred to as the Changsha Communique, is the basis for xenotransplantation worldwide. The content summary is shown in **Table 2**. Based on the Changsha Communique, in 2009 the International

Xenotransplantation Association (IXA) announced a consensus statement of conditions for the initiation of clinical trials of porcine islet products for type 1 diabetes [17]. This consensus statement consists of seven chapters and addresses the requirements of the Changsha Communique. Because remarkable progress has been made

Since the consensus statement for the initiation of xenogeneic islet transplantation in IXA was announced in 2009, clinical findings of xenotransplantation including clinical xenogeneic islet transplantation in New Zealand have been accumulated, and the consensus statement was updated in 2016 [18]. The contents

Chapter 1. Key ethical requirements and progress toward the definition of an international regulatory framework: ethical requirements and progress toward

Chapter 3. Pig islet product manufacturing and release testing: manufacturing,

Chapter 4. Pre-clinical efficacy and complication data required to justify a clini-

Chapter 5. Strategies to prevent transmission of porcine endogenous retrovi-

Chapter 7. Informed consent and xenotransplantation clinical trials: ideal

Chapter 6. Patient selection for pilot clinical trials of islet xenotransplantation:

In particular, because PERV infection and cross-species infection did not occur at all, these infections were regarded as a "theoretical risk" and were considered

**16**


#### **Table 2.**

*Summary of the contents of the Changsha communique.*

unlikely under the adequate control of suitable donors and recipients. In addition, clinical data have been accumulated, infection diagnostic techniques have progressed, clinical protocols have been improved, the risk of PERV-related infection is better understood, DPF facilities and dietary restriction methods have advanced, and the role of sample archives has been clarified. As a result of these efforts, costeffective generation of donor pigs will be possible, and it is expected that porcine islets will be delivered to many patients who truly need this treatment modality.

Some countries have responded to this consensus statement. In Japan, the Ministry of Health, Labour and Welfare also revised the "guidelines on public health infection problems associated with the implementation of xenotransplantation" in 2016.

Recently, in response to the resumption of clinical xenotransplantation, the Third WHO Global Consultation on Regulatory Requirements for Xenotransplantation Clinical Trials was held in 2018, and the contents were announced as the 2018 Changsha Communique [19]. The points of the revision are:


The Communique emphasizes safety while taking into consideration the actual situation of clinical xenotransplantation, social conditions, and technological advances.

#### **6. Encapsulation of islets**

Although islet transplantation has proved to be successful for patients with type 1 diabetes, one of the limitations is the requirement for lifelong immunosuppression. An encapsulation strategy that can prevent rejection of xenogeneic islets can potentially overcome this challenge (**Figure 1**). Such capsules have fine holes that allow the passage of oxygen, glucose, and insulin but not immune cells. Blocking immune cells allows islet transplantation without the need for immunosuppressants. The capsules have been studied in various materials and sizes. There are three main sizes: macro, micro, and nano [20, 21]. The macro-capsule is used to seal islets in centimeter-order devices, which are easy to handle and can be removed and replaced. However, the problem is that substance permeability is low, and foreign body reactions are likely to occur, and the survival rate of internal cells is low. The microcapsule is several hundreds of micrometers to millimeters order in size, is

#### **Figure 1.**

*Schematic representation of an encapsulated porcine islet. Pancreatic islets isolated from DPF pig are encapsulated with an immunoisolation hydrogel. The capsule has fine holes that allow passage of oxygen, glucose, and insulin but not immune cells.*

**19**

of research.

*Pig Islet Transplant*

improved.

*DOI: http://dx.doi.org/10.5772/intechopen.88324*

made mainly of hydrogel, and contains one to several islets. It is compatible in terms of substance permeability and immune isolation ability. However, it is too large for endovascular transplantation and recovery after transplantation is difficult. The nano-capsule has a thin-layered surface coating enclosing pancreatic islets comprising a variety of polymers and therapeutic agents. The permeability is high but stability is an issue. In addition, a surface modification with immune-privileged cells is another concept of encapsulation. Each of these encapsulation techniques

In 1980, Lim and Sun applied microcapsules in diabetes treatment, showing prolonged islet graft survival using alginate-poly-l-lysine-polyethyleneimine microcapsules [22]. Since then, this promising technology has been considerably

In 2014, clinical results were reported in which neonatal porcine islets isolated from DPF pigs encapsulated with alginate and poly-(l)-ornithine were transplanted in 14 patients with type 1 diabetes [23]. The patients were divided into four groups according to transplantation dose, and 5000, 10,000, 15,000, and 20,000 IEQ/kg of encapsulated islets were transplanted intraperitoneally, respectively, according to body weight. No immunosuppressant was used. After transplantation, in the lowdose groups of 5000 and 10,000 IEQ/kg, the frequency of occurrence of hypoglyce-

In 2016, the same group reported results of a clinical trial in which 5000 and 10,000 IEQ/kg of encapsulated neonatal porcine islets were transplanted twice at intervals of 3 months [24]. After transplantation, HbA1c decreased significantly in all patients, and the frequency of occurrence of hypoglycemia unawareness was significantly reduced in the group that received a transplant of 10,000 IEQ/kg twice. Moreover, the group that received a transplant of 10,000 IEQ/kg maintained an average HbA1c of ≤7% over 2 years after transplantation and showed a long-term

**8. Porcine islet transplantation combined with regulatory T cell (Treg)**

in type 1 diabetes patients is underway and is being conducted by Wang et al., Central South University, China (ClinicalTrials.gov Identifier NCT03162237).

immunosuppressants are tacrolimus, mycophenolate mofetil, and belatacept. The primary end point is stable blood glucose level and prevention of ketoacidosis and hypoglycemia and a 30% reduction in required insulin. The authors reported that the condition of these patients improved substantially (http://en.xy3yy.com/document/show\_12/184.html). These results are encouraging and add value to this field

One of the advantages of xenotransplantation is the possibility of genetic modification in the donor. Advances in gene editing, such as the CRISPR/Cas9 system,

A clinical trial of transplantation of neonatal porcine islets and autologous Tregs

/kg of Tregs, and the

has advantages and disadvantages, but the technique is very promising.

**7. Micro-encapsulated neonatal porcine islet transplantation**

mia unawareness was halved compared to that before transplantation.

effect. Clinical effects have been shown in islet xenotransplantation.

The transplanted dose is 10,000 IEQ/kg of islets, 2 × 106

**9. Gene editing and blastocyst complementation**

have facilitated editing of specific genes.

*Xenotransplantation - Comprehensive Study*

fied pigs

**6. Encapsulation of islets**

advances.

Recently, in response to the resumption of clinical xenotransplantation, the Third WHO Global Consultation on Regulatory Requirements for Xenotransplantation Clinical Trials was held in 2018, and the contents were announced as the 2018 Changsha Communique [19]. The points of the revision are:

subsequent prohibition of medical tourism to such countries

2.Emphasis on reproducible preclinical data

4.Deletion of sample retention period requirements

1.Prohibition of clinical trials in countries without national regulations and

3.Development of quality control measures and standards for genetically modi-

The Communique emphasizes safety while taking into consideration the actual

Although islet transplantation has proved to be successful for patients with type 1 diabetes, one of the limitations is the requirement for lifelong immunosuppression. An encapsulation strategy that can prevent rejection of xenogeneic islets can potentially overcome this challenge (**Figure 1**). Such capsules have fine holes that allow the passage of oxygen, glucose, and insulin but not immune cells. Blocking immune cells allows islet transplantation without the need for immunosuppressants. The capsules have been studied in various materials and sizes. There are three main sizes: macro, micro, and nano [20, 21]. The macro-capsule is used to seal islets in centimeter-order devices, which are easy to handle and can be removed and replaced. However, the problem is that substance permeability is low, and foreign body reactions are likely to occur, and the survival rate of internal cells is low. The microcapsule is several hundreds of micrometers to millimeters order in size, is

situation of clinical xenotransplantation, social conditions, and technological

*Schematic representation of an encapsulated porcine islet. Pancreatic islets isolated from DPF pig are encapsulated with an immunoisolation hydrogel. The capsule has fine holes that allow passage of oxygen,* 

**18**

**Figure 1.**

*glucose, and insulin but not immune cells.*

made mainly of hydrogel, and contains one to several islets. It is compatible in terms of substance permeability and immune isolation ability. However, it is too large for endovascular transplantation and recovery after transplantation is difficult. The nano-capsule has a thin-layered surface coating enclosing pancreatic islets comprising a variety of polymers and therapeutic agents. The permeability is high but stability is an issue. In addition, a surface modification with immune-privileged cells is another concept of encapsulation. Each of these encapsulation techniques has advantages and disadvantages, but the technique is very promising.

#### **7. Micro-encapsulated neonatal porcine islet transplantation**

In 1980, Lim and Sun applied microcapsules in diabetes treatment, showing prolonged islet graft survival using alginate-poly-l-lysine-polyethyleneimine microcapsules [22]. Since then, this promising technology has been considerably improved.

In 2014, clinical results were reported in which neonatal porcine islets isolated from DPF pigs encapsulated with alginate and poly-(l)-ornithine were transplanted in 14 patients with type 1 diabetes [23]. The patients were divided into four groups according to transplantation dose, and 5000, 10,000, 15,000, and 20,000 IEQ/kg of encapsulated islets were transplanted intraperitoneally, respectively, according to body weight. No immunosuppressant was used. After transplantation, in the lowdose groups of 5000 and 10,000 IEQ/kg, the frequency of occurrence of hypoglycemia unawareness was halved compared to that before transplantation.

In 2016, the same group reported results of a clinical trial in which 5000 and 10,000 IEQ/kg of encapsulated neonatal porcine islets were transplanted twice at intervals of 3 months [24]. After transplantation, HbA1c decreased significantly in all patients, and the frequency of occurrence of hypoglycemia unawareness was significantly reduced in the group that received a transplant of 10,000 IEQ/kg twice. Moreover, the group that received a transplant of 10,000 IEQ/kg maintained an average HbA1c of ≤7% over 2 years after transplantation and showed a long-term effect. Clinical effects have been shown in islet xenotransplantation.

#### **8. Porcine islet transplantation combined with regulatory T cell (Treg)**

A clinical trial of transplantation of neonatal porcine islets and autologous Tregs in type 1 diabetes patients is underway and is being conducted by Wang et al., Central South University, China (ClinicalTrials.gov Identifier NCT03162237). The transplanted dose is 10,000 IEQ/kg of islets, 2 × 106 /kg of Tregs, and the immunosuppressants are tacrolimus, mycophenolate mofetil, and belatacept. The primary end point is stable blood glucose level and prevention of ketoacidosis and hypoglycemia and a 30% reduction in required insulin. The authors reported that the condition of these patients improved substantially (http://en.xy3yy.com/document/show\_12/184.html). These results are encouraging and add value to this field of research.

#### **9. Gene editing and blastocyst complementation**

One of the advantages of xenotransplantation is the possibility of genetic modification in the donor. Advances in gene editing, such as the CRISPR/Cas9 system, have facilitated editing of specific genes.

Recent advances in genetic engineering and gene editing of donor pigs may overcome the challenge of islet rejection and improve their engraftment and ability to secrete insulin. The required set of genetic modifications will depend on the source of islets (fetal, neonatal, and adult), mode of delivery (encapsulated, free), and the transplantation site. Genetic modification of pigs has been developed mainly via deletion of one or more of the major porcine antigens such as GGTA1, CMAH, and β4GalNT2, and/or insertion of human complement (such as hCD46, hCD55, and hCD59) which suppress the coagulation reaction [25, 26], and/or knockout or insertion of other genes. Simultaneous knockout of two or three major pig antigens has been achieved, and consequently the binding of human antibodies to these cells is significantly reduced. Other genes include the expression of proteins that inhibit co-stimulation of T cells such as hCTLA4Ig [27]. The combinations of multiple gene editing were promising [28, 29]. Currently, the modifications being carried out in pigs span over 24 genes including coagulation regulatory genes, immune cell regulatory genes, and anti-inflammatory genes [30]. Simultaneous modification of more than five genes has been performed in some pigs [30]. These genetically modified pigs will contribute to the improvement of transplantation outcome.

The technology has also been applied to elimination of PERV, and Yang et al. of Harvard University reported inactivation of all PERV genomes using the CRISPR/ Cas9 system [31]. They launched a venture company called eGenesis, aiming to create a human friendly medical pig with the added advantage of PERV inactivation. Thus, it is considered that a medical pig suitable for islet transplantation will be created by gene editing technology.

Yamaguchi et al. of the University of Tokyo complemented mouse-induced pluripotent stem cells (iPS), cells with blastocysts of pancreatic-deficient rats, and succeeded in inducing the rats to develop mouse pancreas [32]. The pancreas derived from mouse iPS, which was produced by this blastocyst complementation method, was the size of the rat pancreas and had a sufficient number of pancreatic islets that could be isolated for transplantation to the mouse. These islets were transplanted with small amounts of an immunosuppressant drug to diabetic mice syngeneic with the iPS cells to normalize blood glucose levels. In addition, this research group also succeeded in inducing apancreatic pigs to produce different pig-derived pancreases by blastocyst complementation [33]. In the future, it may be possible to use a human iPS cell line to generate a medical pig for a human pancreas by blastocyst complementation. If the patient's own iPS-derived pancreas can be obtained from a pig, it is essentially an autologous transplantation, and it thus becomes possible to perform islet transplantation without the need for immunosuppressants.

#### **10. Summary**

Allogeneic islet transplantation is being established as a standard treatment for hypoglycemia unawareness and severe hypoglycemia, but a shortage of human donors has become a problem. Islet xenotransplantation using DPF pigs is considered as a promising fundamental solution to the donor shortage. However, crossspecies infection, especially PERV infection, poses risks to the community, and discussions among key opinion leaders have been implemented by the WHO. As a result, the IXA Consensus Statement was published in 2016, envisioning a future where cost-effective delivery of islet transplants to diabetic patients is facilitated by medical pigs. With the risk of infection always kept in mind, cases of clinical islet xenotransplantation have been accumulated, and steady progress has been made toward a feasible, safe, and effective treatment for diabetic patients. In addition, the development of donor pigs optimal for transplantation using the

**21**

**Author details**

Masayuki Shimoda

Islet Cell Transplantation Project, Diabetes Research Center, Research Institute of

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

National Center for Global Health and Medicine, Tokyo, Japan

\*Address all correspondence to: mshimoda@hosp.ncgm.go.jp

provided the original work is properly cited.

*Pig Islet Transplant*

**Acknowledgements**

*DOI: http://dx.doi.org/10.5772/intechopen.88324*

recently publicized CRISPR/Cas9 technology and blastocyst complementation that could enable the creation of an individual's pancreas in pigs could provide for safer and more effective islet xenotransplantation. Proper pathogen screening, animal selection, microbiological control, and long-term monitoring of recipients will be

I would like to express my deepest appreciation to Dr. Shinichi Matsumoto.

required for clinical application of porcine islet transplantation.

*Pig Islet Transplant DOI: http://dx.doi.org/10.5772/intechopen.88324*

recently publicized CRISPR/Cas9 technology and blastocyst complementation that could enable the creation of an individual's pancreas in pigs could provide for safer and more effective islet xenotransplantation. Proper pathogen screening, animal selection, microbiological control, and long-term monitoring of recipients will be required for clinical application of porcine islet transplantation.

### **Acknowledgements**

*Xenotransplantation - Comprehensive Study*

be created by gene editing technology.

Recent advances in genetic engineering and gene editing of donor pigs may overcome the challenge of islet rejection and improve their engraftment and ability to secrete insulin. The required set of genetic modifications will depend on the source of islets (fetal, neonatal, and adult), mode of delivery (encapsulated, free), and the transplantation site. Genetic modification of pigs has been developed mainly via deletion of one or more of the major porcine antigens such as GGTA1, CMAH, and β4GalNT2, and/or insertion of human complement (such as hCD46, hCD55, and hCD59) which suppress the coagulation reaction [25, 26], and/or knockout or insertion of other genes. Simultaneous knockout of two or three major pig antigens has been achieved, and consequently the binding of human antibodies to these cells is significantly reduced. Other genes include the expression of proteins that inhibit co-stimulation of T cells such as hCTLA4Ig [27]. The combinations of multiple gene editing were promising [28, 29]. Currently, the modifications being carried out in pigs span over 24 genes including coagulation regulatory genes, immune cell regulatory genes, and anti-inflammatory genes [30]. Simultaneous modification of more than five genes has been performed in some pigs [30]. These genetically modified

pigs will contribute to the improvement of transplantation outcome.

perform islet transplantation without the need for immunosuppressants.

Allogeneic islet transplantation is being established as a standard treatment for hypoglycemia unawareness and severe hypoglycemia, but a shortage of human donors has become a problem. Islet xenotransplantation using DPF pigs is considered as a promising fundamental solution to the donor shortage. However, crossspecies infection, especially PERV infection, poses risks to the community, and discussions among key opinion leaders have been implemented by the WHO. As a result, the IXA Consensus Statement was published in 2016, envisioning a future where cost-effective delivery of islet transplants to diabetic patients is facilitated by medical pigs. With the risk of infection always kept in mind, cases of clinical islet xenotransplantation have been accumulated, and steady progress has been made toward a feasible, safe, and effective treatment for diabetic patients. In addition, the development of donor pigs optimal for transplantation using the

The technology has also been applied to elimination of PERV, and Yang et al. of Harvard University reported inactivation of all PERV genomes using the CRISPR/ Cas9 system [31]. They launched a venture company called eGenesis, aiming to create a human friendly medical pig with the added advantage of PERV inactivation. Thus, it is considered that a medical pig suitable for islet transplantation will

Yamaguchi et al. of the University of Tokyo complemented mouse-induced pluripotent stem cells (iPS), cells with blastocysts of pancreatic-deficient rats, and succeeded in inducing the rats to develop mouse pancreas [32]. The pancreas derived from mouse iPS, which was produced by this blastocyst complementation method, was the size of the rat pancreas and had a sufficient number of pancreatic islets that could be isolated for transplantation to the mouse. These islets were transplanted with small amounts of an immunosuppressant drug to diabetic mice syngeneic with the iPS cells to normalize blood glucose levels. In addition, this research group also succeeded in inducing apancreatic pigs to produce different pig-derived pancreases by blastocyst complementation [33]. In the future, it may be possible to use a human iPS cell line to generate a medical pig for a human pancreas by blastocyst complementation. If the patient's own iPS-derived pancreas can be obtained from a pig, it is essentially an autologous transplantation, and it thus becomes possible to

**20**

**10. Summary**

I would like to express my deepest appreciation to Dr. Shinichi Matsumoto.

### **Author details**

Masayuki Shimoda

Islet Cell Transplantation Project, Diabetes Research Center, Research Institute of National Center for Global Health and Medicine, Tokyo, Japan

\*Address all correspondence to: mshimoda@hosp.ncgm.go.jp

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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islets from a type 1 diabetic patient 9.5 yr after xenotransplantation. Xenotransplantation. 2007;**14**:157-161

[10] Spizzo T, Denner J, Gazda L, et al. First update of the international xenotransplantation association consensus statement on conditions for undertaking clinical trials of porcine islet products in type 1 diabetes: Chapter 2a: Source pigs-preventing xenozoonoses. Xenotransplantation. 2016;**23**:25-31

[11] Patience C, Takeuchi Y, Weiss RA. Infection of human cells by an endogenous retrovirus of pigs. Nature Medicine. 1997;**3**:282-286

[12] Dufrane D, Goebbels RM, Saliez A, et al. Six-month survival of microencapsulated pig islets and alginate biocompatibility in primates: Proof of concept. Transplantation. 2006;**81**:1345-1353

[13] Hering BJ, Wijkstrom M, Graham ML, et al. Prolonged diabetes reversal after intraportal xenotransplantation of wild-type porcine islets in immunosuppressed nonhuman primates. Nature Medicine. 2006;**12**:301-303

[14] Cardona K, Korbutt GS, Milas Z, et al. Long-term survival of neonatal porcine islets in nonhuman primates by targeting costimulation pathway. Nature Medicine. 2006;**12**:304-306

[15] Lee JI, Kim J, Choi YJ, et al. The effect of epitope-based ligation of ICAM-1 on survival and retransplantation of pig islets in nonhuman primates. Xenotransplantation. 2018;**25**(1):e12362

[16] The Changsha Communique. First WHO global consultation on regulatory requirements for xenotransplantation clinical trials. Xenotransplantation. 2009;**16**:61-63

**23**

*Pig Islet Transplant*

[17] Hering BJ, Cooper DK, Cozzi E, et al. The international xenotransplantation association consensus statement on conditions for undertaking clinical trials of porcine islet products in type 1 diabetes—Executive summary. Xenotransplantation. 2009;**16**:196-202

[18] Hering BJ, Cozzi E, Spizzo T, et al. First update of the international xenotransplantation association consensus statement on conditions for undertaking clinical trials of porcine islet products in type 1 diabetes—Executive summary. Xenotransplantation. 2016;**23**:3-13

[19] Hawthorne WJ, Cowan PJ, Bühler LH, et al. Third WHO global consultation on regulatory requirements for xenotransplantation clinical trials, Changsha, Hunan, China december 12-14, 2018: "the 2018 Changsha communiqué" the 10-year anniversary of the international consultation on xenotransplantation.

Xenotransplantation. 2019;**26**(2):e12513

Network. 2013;**13**(6):235-239

Science. 1980;**210**:908-910

2014;**46**:1992-1995

[23] Matsumoto S, Tan P, Baker J, et al. Clinical porcine islet xenotransplantation under comprehensive regulation. Transplantation Proceedings.

[24] Matsumoto S, Abalovich A, Wechsler C, et al. Clinical benefit

[21] Vaithilingam V, Bal S, Tuch

[20] Buder B, Alexander M, Krishnan R, et al. Encapsulated islet transplantation: Strategies and clinical trials. Immune

BE. Encapsulated islet transplantation: Where do we stand? The Review of Diabetic Studies. 2017;**14**(1):51-78

[22] Lim F, Sun AM. Microencapsulated islets as bioartificial endocrine pancreas.

*DOI: http://dx.doi.org/10.5772/intechopen.88324*

of islet xenotransplantation for the treatment of type 1 diabetes. eBioMedicine. 2016;**12**:255-262

[25] Estrada JL, Martens G, Li P, et al. Evaluation of human and nonhuman primate antibody binding to pig cells lacking GGTA1/CMAH/ β4GalNT2 genes. Xenotransplantation.

[26] Zhou CY, McInnes E, Copeman L, et al. Transgenic pigs expressing human CD59, in combination with human membrane cofactor protein and human decay-accelerating factor. Xenotransplantation. 2005;**12**:142-148

[27] Bottino R, Wijkstrom M, van der Windt DJ, et al. Pig-to-monkey islet xenotransplantation using multitransgenic pigs. American Journal of Transplantation. 2014;**14**(10):2275-2287

[28] Fischer K, Kraner-Scheiber S, Petersen B, et al. Efficient

gene stacking and gene editing. Scientific Reports. 2016;**6**:29081

[30] Kemter E, Denner J, Wolf E. Will genetic engineering carry xenotransplantation of pig islets to the clinic? Current Diabetes Reports.

[31] Yang L, Guell M, Niu D, et al. Genome-wide inactivation of porcine endogenous retroviruses (PERVs). Science. 2015;**350**:1101-1104

[32] Yamaguchi T, Sato H, Kato-Ito M, et al. Interspecies organogenesis

2018;**18**(11):103

[29] Takahagi Y, Fujimura T, Miyagawa S, et al. Production of alpha 1,3-galactosyltransferase gene knockout pigs expressing both human decay-accelerating factor and N-acetylglucosaminyltransferase III. Molecular Reproduction and Development. 2005;**71**(3):331-338

production of multi-modified pigs for xenotransplantation by 'combineering',

2015;**22**:194-202

#### *Pig Islet Transplant DOI: http://dx.doi.org/10.5772/intechopen.88324*

[17] Hering BJ, Cooper DK, Cozzi E, et al. The international xenotransplantation association consensus statement on conditions for undertaking clinical trials of porcine islet products in type 1 diabetes—Executive summary. Xenotransplantation. 2009;**16**:196-202

[18] Hering BJ, Cozzi E, Spizzo T, et al. First update of the international xenotransplantation association consensus statement on conditions for undertaking clinical trials of porcine islet products in type 1 diabetes—Executive summary. Xenotransplantation. 2016;**23**:3-13

[19] Hawthorne WJ, Cowan PJ, Bühler LH, et al. Third WHO global consultation on regulatory requirements for xenotransplantation clinical trials, Changsha, Hunan, China december 12-14, 2018: "the 2018 Changsha communiqué" the 10-year anniversary of the international consultation on xenotransplantation. Xenotransplantation. 2019;**26**(2):e12513

[20] Buder B, Alexander M, Krishnan R, et al. Encapsulated islet transplantation: Strategies and clinical trials. Immune Network. 2013;**13**(6):235-239

[21] Vaithilingam V, Bal S, Tuch BE. Encapsulated islet transplantation: Where do we stand? The Review of Diabetic Studies. 2017;**14**(1):51-78

[22] Lim F, Sun AM. Microencapsulated islets as bioartificial endocrine pancreas. Science. 1980;**210**:908-910

[23] Matsumoto S, Tan P, Baker J, et al. Clinical porcine islet xenotransplantation under comprehensive regulation. Transplantation Proceedings. 2014;**46**:1992-1995

[24] Matsumoto S, Abalovich A, Wechsler C, et al. Clinical benefit of islet xenotransplantation for the treatment of type 1 diabetes. eBioMedicine. 2016;**12**:255-262

[25] Estrada JL, Martens G, Li P, et al. Evaluation of human and nonhuman primate antibody binding to pig cells lacking GGTA1/CMAH/ β4GalNT2 genes. Xenotransplantation. 2015;**22**:194-202

[26] Zhou CY, McInnes E, Copeman L, et al. Transgenic pigs expressing human CD59, in combination with human membrane cofactor protein and human decay-accelerating factor. Xenotransplantation. 2005;**12**:142-148

[27] Bottino R, Wijkstrom M, van der Windt DJ, et al. Pig-to-monkey islet xenotransplantation using multitransgenic pigs. American Journal of Transplantation. 2014;**14**(10):2275-2287

[28] Fischer K, Kraner-Scheiber S, Petersen B, et al. Efficient production of multi-modified pigs for xenotransplantation by 'combineering', gene stacking and gene editing. Scientific Reports. 2016;**6**:29081

[29] Takahagi Y, Fujimura T, Miyagawa S, et al. Production of alpha 1,3-galactosyltransferase gene knockout pigs expressing both human decay-accelerating factor and N-acetylglucosaminyltransferase III. Molecular Reproduction and Development. 2005;**71**(3):331-338

[30] Kemter E, Denner J, Wolf E. Will genetic engineering carry xenotransplantation of pig islets to the clinic? Current Diabetes Reports. 2018;**18**(11):103

[31] Yang L, Guell M, Niu D, et al. Genome-wide inactivation of porcine endogenous retroviruses (PERVs). Science. 2015;**350**:1101-1104

[32] Yamaguchi T, Sato H, Kato-Ito M, et al. Interspecies organogenesis

**22**

*Xenotransplantation - Comprehensive Study*

[1] Shapiro AM, Lakey JR, Ryan EA, et al. Islet transplantation in seven patients with Type 1 diabetes mellitus using a glucocorticoid-free immunosuppressive regimen. The New England Journal of Medicine.

islets from a type 1 diabetic patient 9.5 yr after xenotransplantation. Xenotransplantation. 2007;**14**:157-161

[10] Spizzo T, Denner J, Gazda L, et al. First update of the international xenotransplantation association consensus statement on conditions for undertaking clinical trials of porcine islet products in type 1 diabetes: Chapter 2a: Source pigs-preventing xenozoonoses. Xenotransplantation.

[11] Patience C, Takeuchi Y, Weiss RA. Infection of human cells by an endogenous retrovirus of pigs. Nature

Medicine. 1997;**3**:282-286

2006;**81**:1345-1353

[12] Dufrane D, Goebbels RM, Saliez A, et al. Six-month survival of microencapsulated pig islets and alginate biocompatibility in primates: Proof of concept. Transplantation.

[13] Hering BJ, Wijkstrom M, Graham ML, et al. Prolonged diabetes reversal after intraportal xenotransplantation

immunosuppressed nonhuman primates. Nature Medicine. 2006;**12**:301-303

[14] Cardona K, Korbutt GS, Milas Z, et al. Long-term survival of neonatal porcine islets in nonhuman primates by targeting costimulation pathway. Nature

of wild-type porcine islets in

Medicine. 2006;**12**:304-306

2009;**16**:61-63

[15] Lee JI, Kim J, Choi YJ, et al. The effect of epitope-based ligation of ICAM-1 on survival and retransplantation of pig islets in nonhuman primates.

Xenotransplantation. 2018;**25**(1):e12362

[16] The Changsha Communique. First WHO global consultation on regulatory requirements for xenotransplantation clinical trials. Xenotransplantation.

2016;**23**:25-31

[2] Ryan EA, Paty BW, Senior PA, et al. Five-year follow-up after clinical islet transplantation. Diabetes.

[3] Hering BJ, Clarke WR, Bridges ND, et al. Phase 3 trial of human islet in type 1 diabetes complicated by severe hypoglycemia. Diabetes Care.

[4] Markmann JF, Bartlett ST, Johnson P, et al. Executive summary of IPITA-TTS opinion leaders report on the future of beta-cell replacement. Transplantation.

[5] Matsumoto S, Okitsu T, Iwanaga Y, et al. Successful islet transplantation from nonheartbeating donor pancreata using modified Ricordi islet isolation method. Transplantation. 2006;**82**:460-465

[6] Matsumoto S, Okitsu T, Iwanaga Y, et al. Insulin independence after living-donor distal pancreatectomy and islet allotransplantation. Lancet.

[7] Groth CG, Korsgren O, Tibell A, et al. Transplantation of porcine fetal pancreatic to diabetic patients. Lancet.

[8] Valdes-Gonzalez RA, Dornates LM, Garibay GN, et al. Xenotransplantation of porcine neonatal islets of langerhans and sertoli cells: A 4-year study. European Journal of Endocrinology.

[9] Elliott RB, Escobar L, Tan PL, et al. Live encapsulated porcine

2000;**343**:230-238

**References**

2005;**54**:2090-2096

2016;**39**:1230-1240

2016;**100**:e25-e31

2005;**365**:1642-1644

1994;**344**:1402-1404

2005;**153**:419-427

generates autologous functional islets. Nature. 2017;**542**:191-196

[33] Matsunari H, Nagashima H, Watanabe M, et al. Blastocyst complementation generates exogenic pancreas in vivo in apancreatic cloned pigs. Proceedings of the National Academy of Sciences of the United States of America. 2013;**110**:4557-4562

**25**

**Chapter 3**

**Abstract**

quantity and function.

**Keypoints**

Porcine Islet Cell

Xenotransplantation

*Rajeswar Chinnuswami, Abid Hussain,* 

*Gopalakrishnan Loganathan, Siddharth Narayanan,* 

This article reviews the rationale, sources and preparation of pig islets for xenotransplantation. Pancreatic islet cell transplantation is an attractive alternative and an effective treatment option for type 1 diabetes, however, donor pancreas shortages prevent islet transplantation from being a widespread solution as the supply cannot possibly equal the demand. Porcine islet xenotransplantation has the potential to address these shortages, and recent preclinical and clinical trials show promising scientific support. Pig islets provide a readily available source for islet transplantation, with the recent trials in non-human primates (NHPs) demonstrating their potential to reverse diabetes. The risk of zoonosis can be reduced by designated pathogen-free breeding of the donor pigs, but porcine endogenous retroviruses (PERVs) which are integrated into the genome of all pigs, are especially difficult to eliminate. However, clinical trials have demonstrated an absence of PERV transmission with a significant reduction in the number of severe hypoglycemic episodes and up to 30% reduction in exogenous insulin doses. A number of methods are currently being tested to overcome the xenograft immune rejection. Some of these methods include the production of various transgenic pigs to better xenotransplantation efficiency and the encapsulation of islets to isolate them from the host immune system. Furthermore, ongoing research is also shedding light on factors such as the age and breed of the donor pig to determine the optimal islet

**Keywords:** type 1 diabetes, xenotransplant, porcine islets, encapsulation, transgenic

• Preclinical studies show improvements in pig islet survival after transplantation.

• Clinical pig islet xenotransplantation studies prove no transmission of PERV.

• Pig islets can be successfully transplanted using encapsulation technology.

*Gene D. Porter and Appakalai N. Balamurugan*

### **Chapter 3**

*Xenotransplantation - Comprehensive Study*

generates autologous functional islets.

complementation generates exogenic pancreas in vivo in apancreatic cloned pigs. Proceedings of the National Academy of Sciences of the United States of America. 2013;**110**:4557-4562

Nature. 2017;**542**:191-196

[33] Matsunari H, Nagashima H, Watanabe M, et al. Blastocyst

**24**

## Porcine Islet Cell Xenotransplantation

*Rajeswar Chinnuswami, Abid Hussain, Gopalakrishnan Loganathan, Siddharth Narayanan, Gene D. Porter and Appakalai N. Balamurugan*

#### **Abstract**

This article reviews the rationale, sources and preparation of pig islets for xenotransplantation. Pancreatic islet cell transplantation is an attractive alternative and an effective treatment option for type 1 diabetes, however, donor pancreas shortages prevent islet transplantation from being a widespread solution as the supply cannot possibly equal the demand. Porcine islet xenotransplantation has the potential to address these shortages, and recent preclinical and clinical trials show promising scientific support. Pig islets provide a readily available source for islet transplantation, with the recent trials in non-human primates (NHPs) demonstrating their potential to reverse diabetes. The risk of zoonosis can be reduced by designated pathogen-free breeding of the donor pigs, but porcine endogenous retroviruses (PERVs) which are integrated into the genome of all pigs, are especially difficult to eliminate. However, clinical trials have demonstrated an absence of PERV transmission with a significant reduction in the number of severe hypoglycemic episodes and up to 30% reduction in exogenous insulin doses. A number of methods are currently being tested to overcome the xenograft immune rejection. Some of these methods include the production of various transgenic pigs to better xenotransplantation efficiency and the encapsulation of islets to isolate them from the host immune system. Furthermore, ongoing research is also shedding light on factors such as the age and breed of the donor pig to determine the optimal islet quantity and function.

**Keywords:** type 1 diabetes, xenotransplant, porcine islets, encapsulation, transgenic

#### **Keypoints**


#### **1. Introduction to islet xeno-transplantation**

Exogenous insulin is the most common treatment option for type I diabetes (insulin-dependent diabetes mellitus), a chronic metabolic disorder caused by the failure of the beta cells of pancreatic islets most often due to T-cell mediated auto immune reaction which result in hyperglycemia [1]. While the standard insulin therapy treats patients with diabetes, however, it does not cure the disease, nor does it prevent the development of the secondary complications leading to end stage organ failures along with its morbidity and mortality [2]. Technical advancements in the production of exogenous insulin, better glucose monitoring system and optimal insulin therapy can reduce HbA1C but still has not addressed the issues of increasing hypoglycemic episodes in patients. Achievement of normoglycemia and exogenous insulin independence is the goal of diabetes treatment. The International Diabetes Federation (IDF) estimated the number of adults suffering from DM in 2017 to be 425 million: this number is expected to increase to 629 million patients in 2040 [3]. Whole pancreas and pancreatic islet transplantation

**Figure 1.**

*Trends in the number of organ donors (blue), organ transplants (green), and patients on the waiting list (Orange) in the US, 2003–2015. In 2003, there were 13,285 donors, 25,473 organ transplants, and 83,731 patients on the waiting list. By 2015, there were 15,068 donors, 30,975 organ transplants and 122,071 patients on the waiting list. Source: http://optn.transplant.hrsa.gov.*

**27**

*Porcine Islet Cell Xenotransplantation*

*DOI: http://dx.doi.org/10.5772/intechopen.90437*

lenges and perspectives for pig islet xenotransplantation.

**2. History of islet xeno-transplantation**

**3. Pig islets as alternative source**

are effective treatment options for diabetes by which insulin independence in T1D patients can be achieved [4]. Unfortunately, both whole organ and cellular transplantation face challenges due to a wide gap between the ever-increasing transplant waiting list and the supply of donor organs [5]. Data from the Organ Procurement and Transplant Network (OPTN) from 2003 to 2015, indicates a 145% increase in the wait list for all organs, while donor availability increased by only 113% (**Figure 1**) [6]. Similarly, the total number of pancreases available is insufficient to match the need for pancreatic islet allo-transplantation [7–9].

Due to this shortage, xenotransplantation using porcine islets has emerged as a potential alternative source for beta cell replacement. Porcine islets have structural and physiological similarities to human islets. Porcine insulin (differs from human insulin by only one amino acid) is used to treat diabetes in clinical practice [10, 11]. Intact functional islets have been successfully isolated from the pig pancreas [12], and these islets have shown the ability to reverse diabetes when transplanted into NHPs [13]. This review article will present the evolution, current practices, chal-

Xenotransplantation has been attempted for the past 300 years or so and blood xenotransfusion was tried as early as the seventeenth century by Jean Baptiste Denis [14]. This was later followed by corneal transplantations from pigs to humans and kidney transplantations in NHP [15, 16]. The first pancreatic xenotransplantation was performed by Watson et al., implanted three ovine fragments into the subcutaneous plane of a diabetic patient. Though clinically significant blood glucose reduction was not demonstrated, the blood sugar level did decrease [17]. This pioneering work was followed by many experimental xenotransplantations, but results were mostly inconclusive [18–21]. Shumakov et al. reported 53 fetal porcine xenotransplants and 18 fetal bovine xenotranaplants in diabetic patients [22]. A century later, Groth et al. performed clinical xenotransplantation trial using fetal porcine islet cell-like clusters (ICCs) and provided preliminary data regarding the function and survival of grafts. After porcine islets were transplanted into 10 insulin-dependent diabetic kidney-transplant patients, detectable levels of porcine C-peptide were identified in the urine for up to 400 days and in one case, renal graft biopsy showed insulin and glucagon positive cells after staining [23]. Several xenotransplantation studies have also been performed in NHPs [20], and have succeeded in reversing diabetes [24–27] and in reducing daily insulin dosage requirement [28]. Transplanted porcine islet grafts were also shown to survive and function in NHPs for longer than 6 months with immunosuppression [25, 27, 29]. The longest survival rate is now over 603 days according to Shin et al.*,* [30]. Studies have also shown that microencapsulation of the transplanted islets and immune-isolation lead to better survival rate without the need for aggressive immunosuppressive therapy [26].

The success of porcine insulin and its role in the treatment of T1D has been well established since its discovery in the 1920s [11, 12, 25]. The structural and physiological similarities between human and pig organs, along with its unlimited supply, have made them an excellent translational research model [25]. Insulin extracted from pig islets has been used for the treatment of diabetes for decades [10, 11, 20, 33]. Because porcine islets produce insulin patterns similar to those found in

#### *Porcine Islet Cell Xenotransplantation DOI: http://dx.doi.org/10.5772/intechopen.90437*

*Xenotransplantation - Comprehensive Study*

**1. Introduction to islet xeno-transplantation**

Exogenous insulin is the most common treatment option for type I diabetes (insulin-dependent diabetes mellitus), a chronic metabolic disorder caused by the failure of the beta cells of pancreatic islets most often due to T-cell mediated auto immune reaction which result in hyperglycemia [1]. While the standard insulin therapy treats patients with diabetes, however, it does not cure the disease, nor does it prevent the development of the secondary complications leading to end stage organ failures along with its morbidity and mortality [2]. Technical advancements in the production of exogenous insulin, better glucose monitoring system and optimal insulin therapy can reduce HbA1C but still has not addressed the issues of increasing hypoglycemic episodes in patients. Achievement of normoglycemia and exogenous insulin independence is the goal of diabetes treatment. The International Diabetes Federation (IDF) estimated the number of adults suffering from DM in 2017 to be 425 million: this number is expected to increase to 629 million patients in 2040 [3]. Whole pancreas and pancreatic islet transplantation

*Trends in the number of organ donors (blue), organ transplants (green), and patients on the waiting list (Orange) in the US, 2003–2015. In 2003, there were 13,285 donors, 25,473 organ transplants, and 83,731 patients on the waiting list. By 2015, there were 15,068 donors, 30,975 organ transplants and 122,071 patients on the* 

**26**

**Figure 1.**

*waiting list. Source: http://optn.transplant.hrsa.gov.*

are effective treatment options for diabetes by which insulin independence in T1D patients can be achieved [4]. Unfortunately, both whole organ and cellular transplantation face challenges due to a wide gap between the ever-increasing transplant waiting list and the supply of donor organs [5]. Data from the Organ Procurement and Transplant Network (OPTN) from 2003 to 2015, indicates a 145% increase in the wait list for all organs, while donor availability increased by only 113% (**Figure 1**) [6]. Similarly, the total number of pancreases available is insufficient to match the need for pancreatic islet allo-transplantation [7–9].

Due to this shortage, xenotransplantation using porcine islets has emerged as a potential alternative source for beta cell replacement. Porcine islets have structural and physiological similarities to human islets. Porcine insulin (differs from human insulin by only one amino acid) is used to treat diabetes in clinical practice [10, 11]. Intact functional islets have been successfully isolated from the pig pancreas [12], and these islets have shown the ability to reverse diabetes when transplanted into NHPs [13]. This review article will present the evolution, current practices, challenges and perspectives for pig islet xenotransplantation.

#### **2. History of islet xeno-transplantation**

Xenotransplantation has been attempted for the past 300 years or so and blood xenotransfusion was tried as early as the seventeenth century by Jean Baptiste Denis [14]. This was later followed by corneal transplantations from pigs to humans and kidney transplantations in NHP [15, 16]. The first pancreatic xenotransplantation was performed by Watson et al., implanted three ovine fragments into the subcutaneous plane of a diabetic patient. Though clinically significant blood glucose reduction was not demonstrated, the blood sugar level did decrease [17]. This pioneering work was followed by many experimental xenotransplantations, but results were mostly inconclusive [18–21]. Shumakov et al. reported 53 fetal porcine xenotransplants and 18 fetal bovine xenotranaplants in diabetic patients [22]. A century later, Groth et al. performed clinical xenotransplantation trial using fetal porcine islet cell-like clusters (ICCs) and provided preliminary data regarding the function and survival of grafts. After porcine islets were transplanted into 10 insulin-dependent diabetic kidney-transplant patients, detectable levels of porcine C-peptide were identified in the urine for up to 400 days and in one case, renal graft biopsy showed insulin and glucagon positive cells after staining [23]. Several xenotransplantation studies have also been performed in NHPs [20], and have succeeded in reversing diabetes [24–27] and in reducing daily insulin dosage requirement [28]. Transplanted porcine islet grafts were also shown to survive and function in NHPs for longer than 6 months with immunosuppression [25, 27, 29]. The longest survival rate is now over 603 days according to Shin et al.*,* [30]. Studies have also shown that microencapsulation of the transplanted islets and immune-isolation lead to better survival rate without the need for aggressive immunosuppressive therapy [26].

#### **3. Pig islets as alternative source**

The success of porcine insulin and its role in the treatment of T1D has been well established since its discovery in the 1920s [11, 12, 25]. The structural and physiological similarities between human and pig organs, along with its unlimited supply, have made them an excellent translational research model [25]. Insulin extracted from pig islets has been used for the treatment of diabetes for decades [10, 11, 20, 33]. Because porcine islets produce insulin patterns similar to those found in

#### *Xenotransplantation - Comprehensive Study*

humans, and because they are readily available [20], studies strongly suggest that islets obtained from pigs could be a promising substitute for human islets in the treatment of T1D. Recent studies on genetically engineered pigs suggest that these pigs are more suitable for xenotransplantation. For example, alpha 1,3-galactosyltransferase gene knockout (GTKO) pigs, have decreased the incidence of immunerejection and improved compatibility between the donor and recipient [31–36].

The major advantages for using pigs as an islet source for xenotransplantation are as follows:


Modified from Ekser et al. [5]; Cooper et al. [37, 38]; Cheng et al. [20].

#### **4. Selection of pig and sources of pig islets**

Islet quantity and quality varies with the breed of pigs. Readily available market pigs have shown to yield lower when compared to the well-studied breeds of pigs like Landrace pigs, Chicago Medical School (CMS) miniature pigs and Chinese Wuzhishan (WZS) miniature pigs [23, 25, 27]. Two major factors which have been studied in relation to the source of pig islets for xenotransplantation are the breed and age of the donors. Some well-studied breeds are the Landrace pigs, Chicago Medical School (CMS) miniature pigs, and the Chinese Wuzhishan (WZS) miniature pigs. Market weight pigs are easily available, but studies have shown lower yields than for other breeds [39]. Landrace pigs have been shown to yield large sized (>250 μm) islets with a high islet volume density [39, 40]. Adult Chicago Medical School (CMS) miniature pigs

**29**

*Porcine Islet Cell Xenotransplantation*

*DOI: http://dx.doi.org/10.5772/intechopen.90437*

Embryonic In the dorsal pancreatic

[43, 57].

Fetal Porcine islets are isolated

(ICCs) [36]. These cellular clusters are composed of <40% endocrine cells (6–8% beta cells) with the majority being the cytokeratinpositive epithelial cells [65]. Their ability to proliferate makes them a potential source of islet cells [27, 36,

66–68].

from fetuses of 60–69 days gestational age [36, 65]. Islets lack a definite shape and capsule and are organized in clusters

primordial, strands of insulin positive cells are seen as early as week 4 [43]. From week 13, cells exhibiting intense immunoreactivity for insulin are distributed throughout the pancreas

are bred under specific pathogen-free (SPF) conditions, and contain large-sized islets. The yield is greater than market or other miniature pigs (9589 ± 2838 IEQ/g), making CMS pigs one of the best sources for obtaining islets [39, 41–45]. Another miniature pig, the Chinese Wuzhishan (WZS) pig has also shown an islet yield greater than that of market pigs [39, 46]**.** Though no consensus has been arrived at the optimal breed for the preclinical/clinical studies, these breeds has been well documented to yield better islets than others. Higher expression of extracellular matrix (ECM) protein in islet capsules makes isolation easier and German Landrace pigs have higher ECM [24].

Additionally, age [43, 44, 47–49] and size of the donor pigs [36, 50–52] are major factors that affect islet isolation outcomes. Some studies have also suggested that gender may play a role in the final islet yield [39, 53, 54]. Pig islets can be obtained at four distinct life-stages: embryonic, fetal, neonatal and adult [55], and **Table 1** summarizes the significance, advantages, and disadvantages of pig islets from

**Islet source Significance Advantages Disadvantages**

cells [43, 58].

complications. Pancreatic primordia obtained on day 28 successfully reversed diabetes in rhesus monkey when compared to that obtained on day 35, which underwent rejection [43, 60, 61].

Isolation process is very simple, involving digestion of the pancreatic tissue to free the islet clusters [65, 69]. No gradient purification

Easily scalable to provide clinical product. Isolation not dependent on the enzyme collagenase, (activity is variable between

necessary.

enzyme lots). The use of alpha 1,3-galactosyltransferase GTKO strains has demonstrated better transplant outcomes than wild-type strains [43, 70].

Embryonic pancreatic tissue exhibit predominantly insulin-positive beta cells without evidence of alpha

Immaturity takes 8–12 weeks (~6 months) for maturation *in vivo*

Poor insulin response post-transplantation due to immaturity [39,

Higher expression of alpha-1,3 galactose (Gal) when compared to adult—more susceptible for humoral rejection. Low yield—only a small number of islets can be isolated, requiring large number of pigs which limits large scale clinical application, with ethical issues.

Cellular culture is required for 5–9 days to form cellular aggregates. Maturation of islets is

delayed Demands higher number of pigs to provide sufficient islets due to lower yield [27, 36, 71]. Because of their clustered appearance, it is difficult to separate islets from the surrounding exocrine and other non-islet cells [36].

[43].

62–64].

Use of embryonic primordial pancreas is better than pluripotent stem cells as they do not need steering toward pancreatic differentiation and have lower risk of teratoma [59].

Following transplantation, the exocrine tissue does not proliferate. Hence, there is decreased immune response and inflammatory

#### *Porcine Islet Cell Xenotransplantation DOI: http://dx.doi.org/10.5772/intechopen.90437*

*Xenotransplantation - Comprehensive Study*

1.Ethically acceptable source.

3.Unlimited availability.

8.Low risk of zoonosis.

donor organ donation.

4.Easy to breed and produce large litters.

6.Significantly low cost of maintenance.

5.Rapid growth into adult organs (6 months).

7.Elective and emergent availability of the organs.

11. Advanced and safe immunosuppression protocols.

13.Islet encapsulation to combat immune challenge.

**4. Selection of pig and sources of pig islets**

9.Facilities available to breed pigs under 'clean' conditions.

10.Obviates 'cultural barriers' to human organ transplant (e.g. Japan); illegal organ trafficking; deleterious effects on organs in brain dead patients; living

12.Cloning and genetic modification of cells to reduce immune destruction.

Modified from Ekser et al. [5]; Cooper et al. [37, 38]; Cheng et al. [20].

Islet quantity and quality varies with the breed of pigs. Readily available market pigs have shown to yield lower when compared to the well-studied breeds of pigs like Landrace pigs, Chicago Medical School (CMS) miniature pigs and Chinese Wuzhishan (WZS) miniature pigs [23, 25, 27]. Two major factors which have been studied in relation to the source of pig islets for xenotransplantation are the breed and age of the donors. Some well-studied breeds are the Landrace pigs, Chicago Medical School (CMS) miniature pigs, and the Chinese Wuzhishan (WZS) miniature pigs. Market weight pigs are easily available, but studies have shown lower yields than for other breeds [39]. Landrace pigs have been shown to yield large sized (>250 μm) islets with a high islet volume density [39, 40]. Adult Chicago Medical School (CMS) miniature pigs

are as follows:

organ.

humans, and because they are readily available [20], studies strongly suggest that islets obtained from pigs could be a promising substitute for human islets in the treatment of T1D. Recent studies on genetically engineered pigs suggest that these pigs are more suitable for xenotransplantation. For example, alpha 1,3-galactosyltransferase gene knockout (GTKO) pigs, have decreased the incidence of immunerejection and improved compatibility between the donor and recipient [31–36]. The major advantages for using pigs as an islet source for xenotransplantation

2.The pig pancreas has structural and physiological similarities to the human

**28**

are bred under specific pathogen-free (SPF) conditions, and contain large-sized islets. The yield is greater than market or other miniature pigs (9589 ± 2838 IEQ/g), making CMS pigs one of the best sources for obtaining islets [39, 41–45]. Another miniature pig, the Chinese Wuzhishan (WZS) pig has also shown an islet yield greater than that of market pigs [39, 46]**.** Though no consensus has been arrived at the optimal breed for the preclinical/clinical studies, these breeds has been well documented to yield better islets than others. Higher expression of extracellular matrix (ECM) protein in islet capsules makes isolation easier and German Landrace pigs have higher ECM [24].

Additionally, age [43, 44, 47–49] and size of the donor pigs [36, 50–52] are major factors that affect islet isolation outcomes. Some studies have also suggested that gender may play a role in the final islet yield [39, 53, 54]. Pig islets can be obtained at four distinct life-stages: embryonic, fetal, neonatal and adult [55], and **Table 1** summarizes the significance, advantages, and disadvantages of pig islets from



#### **Table 1.**

*Different sources of pig islets; significance, advantages, and disadvantages.*

different donor life-stages. Adult pigs have been preferred for their higher yield of mature islet cells that have the potential to secrete insulin soon after transplantation. However, the higher costs, fragility of the islets and the difficulty in isolation are the disadvantages. Neonatal and fetal islets are easy and inexpensive to isolate but the main disadvantage is the significant delay in functioning after transplantation due to their immaturity and their high expression of Galactose-α-1,3-galactose (αGal), the major antigenic target for primate anti-pig antibodies [56].

**31**

follow-up [55, 105].

*Porcine Islet Cell Xenotransplantation*

**5. Pig islet isolation**

79, 95–97].

*DOI: http://dx.doi.org/10.5772/intechopen.90437*

**5.1** *In vitro* **and** *in vivo* **assessment of pig islet function**

Adult pig islet preparation is very similar to human islet isolation methods [55] but the digestion process is a lot more gentler as the porcine islets are extremely fragile. Methods of islet preparation may vary depending on the life-stage of the donor pancreas. Fetal pig ICCs and neonatal pig islets (NPIs) are immature cells and can be easily isolated by enzymatic digestion [55] but must subsequently be cultured prior to transplantation to promote re-aggregation of islet clusters and to help eliminate exocrine cells [55]. The digestion procedure for the adult pig pancreas is significantly different over the fetal or neonatal pancreas. Many factors, such as the type of donor pigs, blood exsanguination, warm ischemia time, cold ischemia time, enzyme lot and activity, perfusate, and the isolation-purification process significantly affect the final islet yield, function and viability [39, 54, 55,

*In vitro* studies investigating the insulin response of islets from donor pigs of different ages have shown that the insulin response from adult pig islets is more pronounced and sustained, and that they have a higher stimulation index over young pigs [36]. Islets from different age of donor pigs have also been compared *in vivo*. Two groups of diabetic nude mice populations were implanted with either young and young adult porcine islets or adult islets. One out of 11 recipients of young and young-adult islets achieved normoglycemia, whereas 32 out of 39 transplanted with adult islets became normal, the blood glucose reaching normal range within 4 weeks post-transplantation. Graft function was confirmed as the cause for normoglycemia, as all 32 mice reverted back to hyperglycemia after islet graft removal [36]. Many studies using NHP models have demonstrated the benefits of the pig islets as xenotransplants, with a potential cure for diabetes [25, 39, 98–101]. These studies have

shown diabetes reversal with prolonged graft survival in diabetic NHPs.

Prevention of the transmission of porcine endogenous retrovirus (PERV) and immunological reactions have been the major hurdles for xenotransplantation in preclinical and clinical trials. Though the risks of zoonosis have been downplayed significantly with the introduction of genetically modified pigs, immunological responses like instant blood mediated inflammatory response (IBMR) dictate the success of the graft survival. One of the most important risk to overcome during xenotransplantation is the prevention of zoonosis [102]. Porcine endogenous retroviruses (PERVs) are of special concern as they are found integrated with porcine genomes and are difficult to eliminate [102]. The degree of risk of PERV being able to infect the human host is unknown, but evidence has shown that PERV can infect human cells when co-cultured with human EK-293 cells [55, 103]. Crossspecies transmission has also been documented in pig to SCID mice xenotransplantation [55, 104]. However, no evidence of transmission has been documented in T1D patients who received porcine islet transplants, even after prolonged

Apart from PERV, other pathogenic organisms including the herpes virus, lymphotropic herpes virus, and cytomegalovirus can also be transmitted. [55]. Methods of combatting these pathogens include careful assessment and screening protocols, designated pathogen-free (DPF) breeding and housing of PERV gene knockout pigs, all of which can help minimize the risk of zoonotic infections [29]. DPF herds

**5.2 Hurdles for xenotransplantation**

### **5. Pig islet isolation**

*Xenotransplantation - Comprehensive Study*

Neonatal The neonatal period is up

74, 75].

Adult Adult pig islets (APIs) are

78–80].

islets [43, 57]. Antigenicity is from N-linked sugars and not from Gal Ag [39, 43, 81–83]. The expression of Gal Ag decreases and becomes negligible as the pig reaches adulthood [43, 81–86]. >2 yrs. is the optimal age [36, 39, 50, 54, 87]. Adult islets are predominantly islet endocrine cells.

The cellular aggregates are composed of <40% endocrine cells (20–25% beta cells) with majority being cytokeratin-positive epithelial cells [65]. About 10–13 days after birth, the ICCs begin to resemble adult islets [43, 57].

the major source of islet cells for xenotransplantation [39,

APIs are well differentiated with distinct and intact capsule and vasculature with very few insulin positive cells outside these

*Different sources of pig islets; significance, advantages, and disadvantages.*

week of life [43]. NPIs comprise ~35% of endocrine cells and ~57% of epithelial cells—islet precursor cells [39, 72, 73]. Correct hyperglycaemia in diabetic animal models as the precursor cells also differentiate and proliferate into beta cells [27, 36, 39,

to 30 days after birth. NPIs are usually obtained from the pancreas within the first

**Islet source Significance Advantages Disadvantages**

[65, 72].

induced injury.

87–90].

[39, 80].

78, 91].

Lower T-cell reactivity than adult pigs [39, 76, 77]. Potential alternative to adult pig islets as xenografts.

Morphologically distinct can be extracted and purified as a single unit [36]. Mature cells—response to hyperglycemia is immediate following transplantation without latency [36, 39, 43,

Insulin independence in diabetic NHPs is achieved when ≥10,000 IEQs are transplanted. (islets pooled from 2 to 4 adult pigs)

Do not require culturing of the isolated islets [65]. Islet yield is greater than for fetal and neonatal pigs [43,

Isolation process is very simple—the process involves digesting pancreatic tissue simply to free islet clusters

Maturation is delayed when compared to adult islets but is faster than for fetal

Cellular culture is required for 5–9 days to form cellular aggregates. Lower yield—limits clinical usage. Only 50,000 aggregates can be obtained from a single pancreas when compared to adult.

Isolation is technically challenging, complex and expensive [36, 43, 65, 79, 92–94]. More fragile islets [65]. Difficult to scale-up [65]. Highly dependent on the enzyme lot and activity [65]. Requires gradient purification [65]. Very high cost of maintenance and breeding in a clean isolated environment [36, 43, 47]. Bigger size of the animal is associated with surgical complications during organ procurement

[36, 50].

ICCs.

No gradient purification. Easily scalable to provide clinical product.

Isolation not dependent on the enzyme collagenase (activity is variable between lots). Isolation process is less expensive than for adult islets. Maintenance of neonates is easy and inexpensive as they are maintained only for few days postpartum. Exhibit strong resistance to inflammatory and hypoxia-

**30**

**Table 1.**

different donor life-stages. Adult pigs have been preferred for their higher yield of mature islet cells that have the potential to secrete insulin soon after transplantation. However, the higher costs, fragility of the islets and the difficulty in isolation are the disadvantages. Neonatal and fetal islets are easy and inexpensive to isolate but the main disadvantage is the significant delay in functioning after transplantation due to their immaturity and their high expression of Galactose-α-1,3-galactose

(αGal), the major antigenic target for primate anti-pig antibodies [56].

Adult pig islet preparation is very similar to human islet isolation methods [55] but the digestion process is a lot more gentler as the porcine islets are extremely fragile. Methods of islet preparation may vary depending on the life-stage of the donor pancreas. Fetal pig ICCs and neonatal pig islets (NPIs) are immature cells and can be easily isolated by enzymatic digestion [55] but must subsequently be cultured prior to transplantation to promote re-aggregation of islet clusters and to help eliminate exocrine cells [55]. The digestion procedure for the adult pig pancreas is significantly different over the fetal or neonatal pancreas. Many factors, such as the type of donor pigs, blood exsanguination, warm ischemia time, cold ischemia time, enzyme lot and activity, perfusate, and the isolation-purification process significantly affect the final islet yield, function and viability [39, 54, 55, 79, 95–97].

#### **5.1** *In vitro* **and** *in vivo* **assessment of pig islet function**

*In vitro* studies investigating the insulin response of islets from donor pigs of different ages have shown that the insulin response from adult pig islets is more pronounced and sustained, and that they have a higher stimulation index over young pigs [36]. Islets from different age of donor pigs have also been compared *in vivo*. Two groups of diabetic nude mice populations were implanted with either young and young adult porcine islets or adult islets. One out of 11 recipients of young and young-adult islets achieved normoglycemia, whereas 32 out of 39 transplanted with adult islets became normal, the blood glucose reaching normal range within 4 weeks post-transplantation. Graft function was confirmed as the cause for normoglycemia, as all 32 mice reverted back to hyperglycemia after islet graft removal [36]. Many studies using NHP models have demonstrated the benefits of the pig islets as xenotransplants, with a potential cure for diabetes [25, 39, 98–101]. These studies have shown diabetes reversal with prolonged graft survival in diabetic NHPs.

#### **5.2 Hurdles for xenotransplantation**

Prevention of the transmission of porcine endogenous retrovirus (PERV) and immunological reactions have been the major hurdles for xenotransplantation in preclinical and clinical trials. Though the risks of zoonosis have been downplayed significantly with the introduction of genetically modified pigs, immunological responses like instant blood mediated inflammatory response (IBMR) dictate the success of the graft survival. One of the most important risk to overcome during xenotransplantation is the prevention of zoonosis [102]. Porcine endogenous retroviruses (PERVs) are of special concern as they are found integrated with porcine genomes and are difficult to eliminate [102]. The degree of risk of PERV being able to infect the human host is unknown, but evidence has shown that PERV can infect human cells when co-cultured with human EK-293 cells [55, 103]. Crossspecies transmission has also been documented in pig to SCID mice xenotransplantation [55, 104]. However, no evidence of transmission has been documented in T1D patients who received porcine islet transplants, even after prolonged follow-up [55, 105].

Apart from PERV, other pathogenic organisms including the herpes virus, lymphotropic herpes virus, and cytomegalovirus can also be transmitted. [55]. Methods of combatting these pathogens include careful assessment and screening protocols, designated pathogen-free (DPF) breeding and housing of PERV gene knockout pigs, all of which can help minimize the risk of zoonotic infections [29]. DPF herds


#### **Table 2.**

*Genetic modifications in pigs to overcome immunological rejection.*

must be free from a comprehensive and list of specified microorganisms [29, 106] and meticulous documentation and standard operating procedures (SOPs) must be implemented to maintain this status [29] including feed restrictions [29].

#### **5.3 Immunological response**

Pig islet cells express different surface proteins that play a major role in the immunological rejection seen following transplantation [102, 107]. Immunological responses are much more complex than seen in allo-transplantation [102]. Immune mediated inflammatory response have been brought down by significantly by genetic modifications as summarized in **Table 2**. Hyper acute rejection (HAR), Instant blood mediated inflammatory response (IBMIR), and cellular rejection are the types of responses seen in graft rejection of which IBMIR is the most crucial. Portal vein site provides good revascularization and drainage for islet transplantation but due to the severe complications like bleeding, thrombosis, and hepatic steatosis, it is no longer an optimal site [108]. Immunological issues observed during xenotransplantation are similar to those seen in allo-transplantation but are much more complex [102]. Pig islets express different types of surface proteins, and these play a critical role in the immunologic rejection seen following transplantation [107]. Multiple genetic modifications in pigs have been proposed to significantly reduce immune mediated inflammatory response, and these are summarized in **Table 2**.

There are four known major routes for islet cell loss following transplantation and these are summarized in the following sections.

#### *5.3.1 Hyper acute rejection (HAR)*

HAR occurs due to the presence of pre-existing host antibodies to surface proteins on the porcine islets. These surface proteins can be broadly categorized into Gal and non-Gal proteins [34, 38, 110]. The Gal epitope is absent in humans, apes and old-world monkeys but many bacteria, NHP and new world monkeys express

**33**

*5.3.3 Cellular rejection*

Cellular rejection, a CD4<sup>+</sup>

tion of macrophages and T-cells (CD4+

*Porcine Islet Cell Xenotransplantation*

*DOI: http://dx.doi.org/10.5772/intechopen.90437*

grow into adults [84, 102, 110, 124, 125].

resulting in substantial islet loss [102, 107, 127].

beneficial even for islets isolated from adult pigs.

sites for transplantation of encapsulated islets [140].

*5.3.2 Instant blood mediated inflammatory reaction (IBMIR)*

the Gal epitope abundantly. In pigs, the expression of Gal antigens decreases as they

As the human body is continuously exposed to micro-organisms (including bacteria), it develops immunity to the Gal antigen and has pre-formed, circulating anti-Gal antibodies [107, 126], which make up around 1% of the circulating antibodies [102, 124]. Once the pigs islets are transplanted, these pre-formed antibodies kill the islet cells rapidly by complement mediated destruction [107, 124]

Antibodies are also produced for other surface epitopes (non-Gal Ag) such as N-glycolylneuraminic acid (NeuGc) also known as Hanganutzu-Deicher and beta 1,4 N-acetylgalactosaminyltransferase (B4GALNT2) [107, 128–130] which are also

There are two known strategies for prevention of HAR. Knockout of genes responsible for adding the Gal epitope and other epitopes such as Neu5Gc to the cell surface can prevent their expression [34, 102]. Secondly, expression of complement regulatory proteins such as hCD46, hCD55 and hCD59 can be induced on the surface of the islet cells [102, 131]. Double knockout pigs (deficient in alpha-gal (GTKO) and Neu5Gc) have been produced, which has significantly reduced the incidence of humoral rejection [102, 132]. The Gal antigen is highly expressed in fetal and neonatal pig pancreas, but its expression decreases as the pigs reach adulthood. The use of GTKO pigs is more validated when using fetal or neonatal pancreas [85, 116], but is not as essential when using adult pigs [116]. However, increasing titres of anti-Gal IgG antibody have been noted when immunosuppression is stopped after adult pig islet transplant [30, 116], so GTKO pigs may prove

Following the intra-portal infusion of the pig islets, the elevated expression of tissue factor by the islets initiates IBMIR [39]. The IBMIR contributes to significant islet loss in the early post-transplant phase through a series of events involving simultaneous complement activation (alternative pathway) [81, 86], activation of intrinsic and extrinsic coagulation pathways, and platelet activation (platelet aggregates around the islets P6) followed by neutrophil and monocyte infiltration [110, 116, 133, 134]. IBMIR can result in 60–80% of islet loss in the immediate post-transplant period [39, 55, 110, 118, 135], but studies in NHPs have shown that if a sufficient number of islet cells survive, they can establish normoglycemia for several months [110]. Genetically modified pigs have been produced [110, 136] to combat IBMIR by decreasing the load of xenoantigens but it failed to provide long-term protection against host response [137]. Experimental studies involving control of complement activation by cobra venom factor, and platelet aggregation and coagulation by anti-platelet agents and low molecular weight heparins are not proven clinically safe, [138, 139]. Peritoneal cavity and omentum offer alternative

T-cell-dependent process [55, 141–143], plays a

cells). Two signaling pathways

major role in islet destruction [39, 118, 144, 145]. Acute cellular rejection occurs within 24 h to 20 days post-transplant, and is characterized by a massive infiltra-

required for the full activation of T cells are the T cell receptor signaling, and the co-stimulatory signaling [55, 146]. Since T cell activation requires double signaling

and CD8+

involved in complement mediated destruction of xenografts [107].

#### *Porcine Islet Cell Xenotransplantation DOI: http://dx.doi.org/10.5772/intechopen.90437*

*Xenotransplantation - Comprehensive Study*

Humoral rejection GTKO

Ischemia/reperfusion injury and inflammatory cytokine related injury

**5.3 Immunological response**

must be free from a comprehensive and list of specified microorganisms [29, 106] and meticulous documentation and standard operating procedures (SOPs) must be

**Immune related islet injury Genetic modifications References**

GTKO pigs/hCRP pigs

ENTPD1 expression Mesenchymal stem cell (MSC)

co-transplantation

GTKO pigs/hCRP pigs

response (immunomodulator)

Expression of human heme oxygenase-1

CD46 (membrane cofactor protein) CD59 (MAC-inhibitory protein) CD55 (decay accelerating factor)

antithrombotic genes (CD39/thrombomodulin)

MSC co-transplantation—downregulate T-cell

[55, 109] [110–112]

[55, 118] [39, 119] [110, 120]

[55, 121] [110–112, 122] [39, 120, 123] [117]

[55, 113–115] [116] [117]

Pig islet cells express different surface proteins that play a major role in the immunological rejection seen following transplantation [102, 107]. Immunological responses are much more complex than seen in allo-transplantation [102]. Immune mediated inflammatory response have been brought down by significantly by genetic modifications as summarized in **Table 2**. Hyper acute rejection (HAR), Instant blood mediated inflammatory response (IBMIR), and cellular rejection are the types of responses seen in graft rejection of which IBMIR is the most crucial. Portal vein site provides good revascularization and drainage for islet transplantation but due to the severe complications like bleeding, thrombosis, and hepatic steatosis, it is no longer an optimal site [108]. Immunological issues observed during xenotransplantation are similar to those seen in allo-transplantation but are much more complex [102]. Pig islets express different types of surface proteins, and these play a critical role in the immunologic rejection seen following transplantation [107]. Multiple genetic modifications in pigs have been proposed to significantly reduce immune mediated inflammatory response, and these are summarized in

There are four known major routes for islet cell loss following transplantation

HAR occurs due to the presence of pre-existing host antibodies to surface proteins on the porcine islets. These surface proteins can be broadly categorized into Gal and non-Gal proteins [34, 38, 110]. The Gal epitope is absent in humans, apes and old-world monkeys but many bacteria, NHP and new world monkeys express

and these are summarized in the following sections.

*5.3.1 Hyper acute rejection (HAR)*

implemented to maintain this status [29] including feed restrictions [29].

IBMIR and coagulation dysfunction TF knockout and overexpression of human

Cellular rejection CTLA4Ig gene expression

*Genetic modifications in pigs to overcome immunological rejection.*

**32**

**Table 2**.

**Table 2.**

the Gal epitope abundantly. In pigs, the expression of Gal antigens decreases as they grow into adults [84, 102, 110, 124, 125].

As the human body is continuously exposed to micro-organisms (including bacteria), it develops immunity to the Gal antigen and has pre-formed, circulating anti-Gal antibodies [107, 126], which make up around 1% of the circulating antibodies [102, 124]. Once the pigs islets are transplanted, these pre-formed antibodies kill the islet cells rapidly by complement mediated destruction [107, 124] resulting in substantial islet loss [102, 107, 127].

Antibodies are also produced for other surface epitopes (non-Gal Ag) such as N-glycolylneuraminic acid (NeuGc) also known as Hanganutzu-Deicher and beta 1,4 N-acetylgalactosaminyltransferase (B4GALNT2) [107, 128–130] which are also involved in complement mediated destruction of xenografts [107].

There are two known strategies for prevention of HAR. Knockout of genes responsible for adding the Gal epitope and other epitopes such as Neu5Gc to the cell surface can prevent their expression [34, 102]. Secondly, expression of complement regulatory proteins such as hCD46, hCD55 and hCD59 can be induced on the surface of the islet cells [102, 131]. Double knockout pigs (deficient in alpha-gal (GTKO) and Neu5Gc) have been produced, which has significantly reduced the incidence of humoral rejection [102, 132]. The Gal antigen is highly expressed in fetal and neonatal pig pancreas, but its expression decreases as the pigs reach adulthood. The use of GTKO pigs is more validated when using fetal or neonatal pancreas [85, 116], but is not as essential when using adult pigs [116]. However, increasing titres of anti-Gal IgG antibody have been noted when immunosuppression is stopped after adult pig islet transplant [30, 116], so GTKO pigs may prove beneficial even for islets isolated from adult pigs.

#### *5.3.2 Instant blood mediated inflammatory reaction (IBMIR)*

Following the intra-portal infusion of the pig islets, the elevated expression of tissue factor by the islets initiates IBMIR [39]. The IBMIR contributes to significant islet loss in the early post-transplant phase through a series of events involving simultaneous complement activation (alternative pathway) [81, 86], activation of intrinsic and extrinsic coagulation pathways, and platelet activation (platelet aggregates around the islets P6) followed by neutrophil and monocyte infiltration [110, 116, 133, 134]. IBMIR can result in 60–80% of islet loss in the immediate post-transplant period [39, 55, 110, 118, 135], but studies in NHPs have shown that if a sufficient number of islet cells survive, they can establish normoglycemia for several months [110]. Genetically modified pigs have been produced [110, 136] to combat IBMIR by decreasing the load of xenoantigens but it failed to provide long-term protection against host response [137]. Experimental studies involving control of complement activation by cobra venom factor, and platelet aggregation and coagulation by anti-platelet agents and low molecular weight heparins are not proven clinically safe, [138, 139]. Peritoneal cavity and omentum offer alternative sites for transplantation of encapsulated islets [140].

#### *5.3.3 Cellular rejection*

Cellular rejection, a CD4<sup>+</sup> T-cell-dependent process [55, 141–143], plays a major role in islet destruction [39, 118, 144, 145]. Acute cellular rejection occurs within 24 h to 20 days post-transplant, and is characterized by a massive infiltration of macrophages and T-cells (CD4+ and CD8+ cells). Two signaling pathways required for the full activation of T cells are the T cell receptor signaling, and the co-stimulatory signaling [55, 146]. Since T cell activation requires double signaling involving TCRs and co-stimulatory molecules [39], blockade of co-stimulatory cell surface molecules such as CD870/86- CD28 and/or CD40L (CD154)- CD40 have significantly improved graft survival, even without immunosuppression [39, 147–149]. The addition of targeted immunosuppression to multi-molecular blockade may further increase effectiveness, and provide an even more promising option to prevent cellular destruction of the transplanted islets [39].

#### *5.3.4 Islet cell revascularization*

Islet revascularization is critical for the survival of transplanted pig islets. Islet grafts are cut off from their native vascular supply and after transplantation, are solely dependent on diffusion for nutrient supply, until functional revascularization is established with the host vasculature. This process takes place within 10–14 days post-transplantation [41, 49, 141].

#### **6. Islet encapsulation approaches**

Islet encapsulation provides the means for islet cell survival in the absence of immunosuppressive drugs. The principle of encapsulation is that transplanted cells are contained within an artificial compartment separated from the immune system by a semipermeable membrane. The capsule should protect the cells from potential damage caused by antibodies, complement proteins, and immune cells. Therefore, the capsule is often referred to as an "immunoisolation device." As well as the protective mechanism provided by the capsules, islet cells within the capsules can also release insulin to control blood glucose levels, since this membrane enables small molecules to diffuse in (glucose, oxygen, and nutrients) and out (metabolic wastes) [39, 150–152]. Thus, the encapsulation system is also regarded as a "bioartificial pancreas." The immunoisolation device or bioartificial pancreas can be commonly separated into two categories, intravascular and extravascular devices. The latter can further be divided into macroencapsulation and microencapsulation devices. Intravascular and extravascular classifications are based on whether or not it is connected directly to the blood circulation.

The macroencapsulation and microencapsulation classifications depend on whether it contains one or more islets in the device [153, 154]. Alginate is the most commonly used capsule material for microencapsulation, but other materials such polyethylene glycol have also been tested [153].

Although the capsule is selectively permeable, islets can be damaged due to hypoxia or inadequate nutrients, and slow glucose and insulin diffusion can delay insulin response to changing glucose levels [155]. Despite the protection offered from direct immune attack, islets can still be damaged by immune responses. Inflammatory cytokines, produced against the capsules can enter the capsule and damage islets. The encapsulated islets themselves may release such cytokines and cause self-damage [156]. Approaches investigated to overcome these problems include testing different sites of implantation, creating biocompatible capsules, and optimizing the capsule size. The use of genetically engineered pig islets within capsules to promote graft survival and function have also been studied [156]. Several clinical trials of encapsulated pig islets to improve long-term survival outcomes of xenografts are currently being conducted around the world [117, 157]. A phase I/IIa clinical study in Moscow has tested the clinical applicability of a commercially available encapsulated pig islet product called Diabecell [39, 158, 159]. Additional phase I/IIa clinical trials are ongoing in New Zealand and Argentina. These trials have demonstrated an absence of PERV transmission, a significant

**35**

**8. Conclusion**

*Porcine Islet Cell Xenotransplantation*

of PERV infection [39, 150].

**7. Regulatory aspects**

*DOI: http://dx.doi.org/10.5772/intechopen.90437*

required before initiating clinical studies [162].

reduction in the number of severe hypoglycaemic episodes and up to 30% reduction in exogenous insulin doses [29, 160]. A 10 year follow up of another study involving xenotransplantation of encapsulated porcine islets into the peritoneum of a T1D patient has shown long-term islet survival and function, with no evidence

Any new therapeutic substance or procedure, safety and efficacy of the drug

Porcine islets represent an excellent alternative source to replace human islets in diabetic patients. Pig islets can be obtained from different life-stages (embryos to adults) and has several other advantages making it an indispensable resource for xenotransplantation. Active research have resulted in standardization of protocols, thereby bettering isolation outcomes. In addition, incorporation of multiple strategies such as generating transgenic pigs together with developing cellular and molecular therapies to sustain long-term xenograft survival have brought porcine islets closer to clinical applications. Despite the risk of zoonosis and other factors which

substance have been inveterate before starting government approved clinical trials. In line with guidance in consensus statements from the International Xenotransplantation Association and the WHO on xenotransplantation, geographical location will impact choice of the microbiological mitigation strategy. Risk management at the source would include the definition of pathogens circulating in the countries of origin [161], establishment of reliable detection, and screening methods and assessment of risk from animal feed. Given the source animals to be utilized will be from specific pathogen-free/designated pathogen-free or high hygienic herds from a single location, the pathogen risk compared with standard slaughter herd animals is significantly reduced. Further testing during the manufacturing process, that is, islet isolation and encapsulation will provide tissue specific data that should further confirm safety of the final product. Moreover, alginate encapsulation allows keeping the islets in culture for longer periods thus giving enough time to perform viral screening on islet products before transplantation. Other release quality controls related to islet morphology, viability, purity, quantity, and potency should also be established in order to guarantee that only well characterized and functional islet preparations are used in patients. The use of genetically modified donor pigs to reduce islet cells immunogenicity and improve their secretory function stipulates that these genetic modifications should be well characterized. Integration of transgene expression cassettes should be in welldefined genomic locations, preferably in the form of a single-targeted integration that would ensure stable expression of the transgene across herds without affecting other cell functions or rendering them tumorigenic. In this context, it should be noted that encapsulation limits the risk of tumor cells spreading since it confines the cells and eliminates the need for immunosuppression meaning that in case the integrity of the encapsulation device would be compromised, xenogeneic pig cells would most probably be rejected by the host immune system. The use of nonhuman primates in research is subjected to very strict ethical and regulatory considerations but the pig-to-primate model is still considered as a gold standard for pig islet xenotransplantation, so that safety and efficacy data obtained using this model are

reduction in the number of severe hypoglycaemic episodes and up to 30% reduction in exogenous insulin doses [29, 160]. A 10 year follow up of another study involving xenotransplantation of encapsulated porcine islets into the peritoneum of a T1D patient has shown long-term islet survival and function, with no evidence of PERV infection [39, 150].

#### **7. Regulatory aspects**

*Xenotransplantation - Comprehensive Study*

*5.3.4 Islet cell revascularization*

post-transplantation [41, 49, 141].

**6. Islet encapsulation approaches**

it is connected directly to the blood circulation.

polyethylene glycol have also been tested [153].

involving TCRs and co-stimulatory molecules [39], blockade of co-stimulatory cell surface molecules such as CD870/86- CD28 and/or CD40L (CD154)- CD40 have significantly improved graft survival, even without immunosuppression [39, 147–149]. The addition of targeted immunosuppression to multi-molecular blockade may further increase effectiveness, and provide an even more promising option

Islet revascularization is critical for the survival of transplanted pig islets. Islet grafts are cut off from their native vascular supply and after transplantation, are solely dependent on diffusion for nutrient supply, until functional revascularization is established with the host vasculature. This process takes place within 10–14 days

Islet encapsulation provides the means for islet cell survival in the absence of immunosuppressive drugs. The principle of encapsulation is that transplanted cells are contained within an artificial compartment separated from the immune system by a semipermeable membrane. The capsule should protect the cells from potential damage caused by antibodies, complement proteins, and immune cells. Therefore, the capsule is often referred to as an "immunoisolation device." As well as the protective mechanism provided by the capsules, islet cells within the capsules can also release insulin to control blood glucose levels, since this membrane enables small molecules to diffuse in (glucose, oxygen, and nutrients) and out (metabolic wastes) [39, 150–152]. Thus, the encapsulation system is also regarded as a "bioartificial pancreas." The immunoisolation device or bioartificial pancreas can be commonly separated into two categories, intravascular and extravascular devices. The latter can further be divided into macroencapsulation and microencapsulation devices. Intravascular and extravascular classifications are based on whether or not

The macroencapsulation and microencapsulation classifications depend on whether it contains one or more islets in the device [153, 154]. Alginate is the most commonly used capsule material for microencapsulation, but other materials such

Although the capsule is selectively permeable, islets can be damaged due to hypoxia or inadequate nutrients, and slow glucose and insulin diffusion can delay insulin response to changing glucose levels [155]. Despite the protection offered from direct immune attack, islets can still be damaged by immune responses. Inflammatory cytokines, produced against the capsules can enter the capsule and damage islets. The encapsulated islets themselves may release such cytokines and cause self-damage [156]. Approaches investigated to overcome these problems include testing different sites of implantation, creating biocompatible capsules, and optimizing the capsule size. The use of genetically engineered pig islets within capsules to promote graft survival and function have also been studied [156]. Several clinical trials of encapsulated pig islets to improve long-term survival outcomes of xenografts are currently being conducted around the world [117, 157]. A phase I/IIa clinical study in Moscow has tested the clinical applicability of a commercially available encapsulated pig islet product called Diabecell [39, 158, 159]. Additional phase I/IIa clinical trials are ongoing in New Zealand and Argentina. These trials have demonstrated an absence of PERV transmission, a significant

to prevent cellular destruction of the transplanted islets [39].

**34**

Any new therapeutic substance or procedure, safety and efficacy of the drug substance have been inveterate before starting government approved clinical trials. In line with guidance in consensus statements from the International Xenotransplantation Association and the WHO on xenotransplantation, geographical location will impact choice of the microbiological mitigation strategy. Risk management at the source would include the definition of pathogens circulating in the countries of origin [161], establishment of reliable detection, and screening methods and assessment of risk from animal feed. Given the source animals to be utilized will be from specific pathogen-free/designated pathogen-free or high hygienic herds from a single location, the pathogen risk compared with standard slaughter herd animals is significantly reduced. Further testing during the manufacturing process, that is, islet isolation and encapsulation will provide tissue specific data that should further confirm safety of the final product. Moreover, alginate encapsulation allows keeping the islets in culture for longer periods thus giving enough time to perform viral screening on islet products before transplantation. Other release quality controls related to islet morphology, viability, purity, quantity, and potency should also be established in order to guarantee that only well characterized and functional islet preparations are used in patients. The use of genetically modified donor pigs to reduce islet cells immunogenicity and improve their secretory function stipulates that these genetic modifications should be well characterized. Integration of transgene expression cassettes should be in welldefined genomic locations, preferably in the form of a single-targeted integration that would ensure stable expression of the transgene across herds without affecting other cell functions or rendering them tumorigenic. In this context, it should be noted that encapsulation limits the risk of tumor cells spreading since it confines the cells and eliminates the need for immunosuppression meaning that in case the integrity of the encapsulation device would be compromised, xenogeneic pig cells would most probably be rejected by the host immune system. The use of nonhuman primates in research is subjected to very strict ethical and regulatory considerations but the pig-to-primate model is still considered as a gold standard for pig islet xenotransplantation, so that safety and efficacy data obtained using this model are required before initiating clinical studies [162].

#### **8. Conclusion**

Porcine islets represent an excellent alternative source to replace human islets in diabetic patients. Pig islets can be obtained from different life-stages (embryos to adults) and has several other advantages making it an indispensable resource for xenotransplantation. Active research have resulted in standardization of protocols, thereby bettering isolation outcomes. In addition, incorporation of multiple strategies such as generating transgenic pigs together with developing cellular and molecular therapies to sustain long-term xenograft survival have brought porcine islets closer to clinical applications. Despite the risk of zoonosis and other factors which

contribute to islet loss post-transplantation, tremendous progress has been made within the field such as developing encapsulated islets to combat host immunity and utilizing host stem cells to aide islet revascularization. Pig islet xenotransplantation currently acts as a bridge between allo-transplantation and stem-cell therapies. With all the tremendous progress made within the field, ongoing research focuses on a better understanding of various factors such as donor characteristics, isolation procedures, microbiological safety, and immunological tolerance to improve pig islet yield, function and transplantation outcomes. Furthering this understanding will require multiple clinical trials directed toward establishing porcine islets as a safe, effective and robust alternative for treating patients with T1D.

### **Acknowledgements**

The authors sincerely thank Kentucky Organ Donor Affiliates (KODA) for the supply of human pancreases for our research programs.

### **Conflicts of interest**

None.

### **Financial support and sponsorship**

The authors thank the Jewish Heritage Fund for Excellence for providing generous support to our program.

#### **Author details**

Rajeswar Chinnuswami, Abid Hussain, Gopalakrishnan Loganathan, Siddharth Narayanan, Gene D. Porter and Appakalai N. Balamurugan\* Clinical Islet Cell Laboratory, Department of Surgery, Cardiovascular Innovation Institute, University of Louisville, Louisville, KY, United States

\*Address all correspondence to: bala.appakalai@louisville.edu

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

**37**

gov

*Porcine Islet Cell Xenotransplantation*

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*Porcine Islet Cell Xenotransplantation DOI: http://dx.doi.org/10.5772/intechopen.90437*

#### **References**

*Xenotransplantation - Comprehensive Study*

**Acknowledgements**

**Conflicts of interest**

ous support to our program.

**Financial support and sponsorship**

None.

contribute to islet loss post-transplantation, tremendous progress has been made within the field such as developing encapsulated islets to combat host immunity and utilizing host stem cells to aide islet revascularization. Pig islet xenotransplantation currently acts as a bridge between allo-transplantation and stem-cell therapies. With all the tremendous progress made within the field, ongoing research focuses on a better understanding of various factors such as donor characteristics, isolation procedures, microbiological safety, and immunological tolerance to improve pig islet yield, function and transplantation outcomes. Furthering this understanding will require multiple clinical trials directed toward establishing porcine islets as a

The authors sincerely thank Kentucky Organ Donor Affiliates (KODA) for the

The authors thank the Jewish Heritage Fund for Excellence for providing gener-

safe, effective and robust alternative for treating patients with T1D.

supply of human pancreases for our research programs.

**36**

**Author details**

Rajeswar Chinnuswami, Abid Hussain, Gopalakrishnan Loganathan, Siddharth Narayanan, Gene D. Porter and Appakalai N. Balamurugan\*

Institute, University of Louisville, Louisville, KY, United States

\*Address all correspondence to: bala.appakalai@louisville.edu

provided the original work is properly cited.

Clinical Islet Cell Laboratory, Department of Surgery, Cardiovascular Innovation

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

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[24] Hering BJ, Walawalkar N. Pig-to-nonhuman primate islet xenotransplantation. Transplant Immunology. 2009;**21**(2):81-86

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[26] Dufrane D, Goebbels R-M, Saliez A, Guiot Y, Gianello P. Six-month survival of microencapsulated pig islets and alginate biocompatibility in primates: Proof of concept. Transplantation. 2006;**81**(9):1345-1353

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[28] Torrie B. More trials of pig cells to help treat type 1 diabetics. The Dominion Post. 2012

[29] Ellis CE, Korbutt GS. Justifying clinical trials for porcine islet xenotransplantation. Xenotransplantation. 2015;**22**(5):336-344

[30] Shin JS, Kim JM, Kim JS, Min BH, Kim YH, Kim HJ, et al. Long-term control of diabetes in immunosuppressed nonhuman primates (NHP) by the transplantation of adult porcine islets. American Journal of Transplantation. 2015;**15**(11):2837-2850

[31] Cooper DK, Koren E, Oriol R. Genetically engineered pigs. Lancet. 1993;**342**(8872):682-683

[32] Koike C, Friday RP, Nakashima I, Luppi P, Fung JJ, Rao AS, et al. Isolation of the regulatory regions and genomic organization of the porcine alpha1,3-galactosyltransferase gene. Transplantation. 2000;**70**(9):1275-1283

[33] Koike C, Fung JJ, Geller DA, Kannagi R, Libert T, Luppi P, et al. Molecular basis of evolutionary loss of the alpha 1,3-galactosyltransferase gene in higher primates. The Journal of Biological Chemistry. 2002;**277**(12):10114-10120

[34] Phelps CJ, Koike C, Vaught TD, Boone J, Wells KD, Chen SH, et al. Production of alpha 1,3-galactosyltransferase-deficient pigs. Science. 2003;**299**(5605):411-414

[35] Kolber-Simonds D, Lai L, Watt SR, Denaro M, Arn S, Augenstein ML, et al. Production of alpha-1,3-galactosyltransferase null pigs by means of nuclear transfer with fibroblasts bearing loss of heterozygosity mutations. Proceedings of the National Academy of Sciences of the United States of America. 2004;**101**(19):7335-7340

**39**

*Porcine Islet Cell Xenotransplantation*

[36] Bottino R, Balamurugan AN, Smetanka C, Bertera S, He J, Rood PP, et al. Isolation outcome and functional characteristics of young and adult pig pancreatic islets for transplantation studies. Xenotransplantation.

[37] de Bock MI, Roy A, Cooper MN, Dart JA, Berthold CL, Retterath AJ, et al. Feasibility of outpatient 24-hour closed-loop insulin delivery. Diabetes

[38] Cooper DK, Ayares D. The immense

[39] Hu Q, Liu Z, Zhu H. Pig islets for islet xenotransplantation: Current status and future perspectives. Chinese Medical Journal. 2014;**127**(2):370-377

[40] Kirchhof N, Hering BJ, Geiss V, Federlin K, Bretzel RG. Evidence for breed-dependent differences in porcine islets of Langerhans. Transplantation Proceedings. 1994;**26**(2):616-617

[41] Kim JH, Kim HI, Lee KW, Yu JE, Kim SH, Park HS, et al. Influence of strain and age differences on the yields of porcine islet isolation: Extremely high islet yields from SPF CMS miniature pigs. Xenotransplantation.

[42] Schuurman HJ. The international xenotransplantation association consensus statement on conditions for undertaking clinical trials of porcine islet products in type 1 diabetes—Chapter 2: Source pigs. Xenotransplantation.

Wijkstrom M, Trucco M, Cooper DK. Islet xenotransplantation: What is the optimal age of the islet-source pig? Xenotransplantation. 2015;**22**(1):7-19

2007;**14**(1):60-66

2009;**16**(4):215-222

[43] Nagaraju S, Bottino R,

potential of xenotransplantation in surgery. International Journal of

Care. 2015;**38**(11):e186-e1e7

Surgery. 2011;**9**(2):122-129

2007;**14**(1):74-82

*DOI: http://dx.doi.org/10.5772/intechopen.90437*

[44] Jay TR, Heald KA, Carless NJ, Topham DE, Downing R. The distribution of porcine pancreatic beta-cells at ages 5, 12 and 24 weeks. Xenotransplantation. 1999;**6**(2):131-140

[45] Shin J-S, Jang J-Y, Park S-K, Choi J-W, Kim S-Y, Min B-H, et al. Extremely high islet yield enables onedonor-one recipient intraportal islet transplantation with enough islet mass in pig-to-non-human primate model. Xenotransplantation. 2013;**20**(5):333

[46] Jiang X, Qian T, Linn T, Cao L, Xiang G, Wang Y, et al. Islet isolation and purification from inbred Wuzhishan miniature pigs. Xenotransplantation.

[47] Jay TR, Heald KA, Downing R. Effect of donor age on porcine insulin secretion. Transplantation Proceedings.

[48] Mueller KR, Balamurugan AN, Cline GW, Pongratz RL, Hooper RL, Weegman BP, et al. Differences in glucose-stimulated insulin secretion in vitro of islets from human,

nonhuman primate, and porcine origin. Xenotransplantation. 2013;**20**(2):75-81

[49] Socci C, Ricordi C, Davalli AM, Staudacher C, Baro P, Vertova A, et al. Selection of donors significantly improves pig islet isolation yield. Hormone and Metabolic Research Supplement. 1990;**25**:32-34

[50] Dufrane D, Goebbels R, Fdilat I, Guiot Y, Gianello P. Impact of porcine islet size on cellular structure and engraftment after transplantation: Adult versus young pigs. Pancreas.

2005;**30**(2):138-147

[51] Hubert T, Jany T, Marcelli-Tourvieille S, Nunes B, Gmyr V,

Kerr-Conte J, et al. Acute insulin response of donors is correlated with pancreatic

2012;**19**(3):159-165

1997;**29**(4):2023

#### *Porcine Islet Cell Xenotransplantation DOI: http://dx.doi.org/10.5772/intechopen.90437*

*Xenotransplantation - Comprehensive Study*

and the risk of contagion: Local

[21] Hitchcock CR, Kiser JC,

[22] Shumakov VI, Bljumkin VN, Ignatenko SN, Skaletsky NN, Slovesnova TA, Babikova RA. The principal results of pancreatic islet cell culture transplantation in diabetes mellitus patients. Transplantation Proceedings. 1987;**19**(1 Pt 3):2372

[23] Groth C, Tibell A, Tollemar J, Bolinder J, Östman J, Möller E, et al. Transplantation of porcine fetal pancreas to diabetic patients. The Lancet. 1994;**344**(8934):1402-1404

[24] Hering BJ, Walawalkar N. Pig-to-nonhuman primate islet xenotransplantation. Transplant Immunology. 2009;**21**(2):81-86

[25] Hering BJ, Wijkstrom M,

of wild-type porcine islets in immunosuppressed nonhuman primates. Nature Medicine.

2006;**12**(3):301-303

2006;**81**(9):1345-1353

Graham ML, Hårdstedt M, Aasheim TC, Jie T, et al. Prolonged diabetes reversal after intraportal xenotransplantation

[26] Dufrane D, Goebbels R-M, Saliez A, Guiot Y, Gianello P. Six-month survival of microencapsulated pig islets and alginate biocompatibility in primates: Proof of concept. Transplantation.

[27] Cardona K, Korbutt GS, Milas Z, Lyon J, Cano J, Jiang W, et al. Longterm survival of neonatal porcine islets in nonhuman primates by targeting costimulation pathways. Nature Medicine. 2006;**12**(3):304-306

[20] Cheng M. Islet xeno/transplantation

[28] Torrie B. More trials of pig cells to help treat type 1 diabetics. The

[29] Ellis CE, Korbutt GS. Justifying clinical trials for porcine islet xenotransplantation. Xenotransplantation. 2015;**22**(5):336-344

immunosuppressed nonhuman primates (NHP) by the transplantation of adult porcine islets. American Journal of Transplantation. 2015;**15**(11):2837-2850

Oriol R. Genetically engineered pigs. Lancet. 1993;**342**(8872):682-683

[32] Koike C, Friday RP, Nakashima I, Luppi P, Fung JJ, Rao AS, et al. Isolation

of the regulatory regions and genomic organization of the porcine alpha1,3-galactosyltransferase gene. Transplantation. 2000;**70**(9):1275-1283

[33] Koike C, Fung JJ, Geller DA, Kannagi R, Libert T, Luppi P, et al. Molecular basis of evolutionary loss of the alpha 1,3-galactosyltransferase

gene in higher primates. The Journal of Biological Chemistry. 2002;**277**(12):10114-10120

[34] Phelps CJ, Koike C, Vaught TD, Boone J, Wells KD, Chen SH, et al. Production of alpha 1,3-galactosyltransferase-deficient pigs. Science. 2003;**299**(5605):411-414

[35] Kolber-Simonds D,

2004;**101**(19):7335-7340

Lai L, Watt SR, Denaro M, Arn S, Augenstein ML, et al. Production of alpha-1,3-galactosyltransferase null pigs by means of nuclear transfer with fibroblasts bearing loss of

heterozygosity mutations. Proceedings of the National Academy of Sciences of the United States of America.

[30] Shin JS, Kim JM, Kim JS, Min BH, Kim YH, Kim HJ, et al. Long-term control of diabetes in

[31] Cooper DK, Koren E,

Dominion Post. 2012

responses from Canada and Australia to an emerging global technoscience. Life Sciences, Society and Policy. 2015;**11**:12

Telander RL, Seljeskog EL. Baboon renal grafts. Journal of the American Medical Association. 1964;**189**(12):934-937

**38**

[36] Bottino R, Balamurugan AN, Smetanka C, Bertera S, He J, Rood PP, et al. Isolation outcome and functional characteristics of young and adult pig pancreatic islets for transplantation studies. Xenotransplantation. 2007;**14**(1):74-82

[37] de Bock MI, Roy A, Cooper MN, Dart JA, Berthold CL, Retterath AJ, et al. Feasibility of outpatient 24-hour closed-loop insulin delivery. Diabetes Care. 2015;**38**(11):e186-e1e7

[38] Cooper DK, Ayares D. The immense potential of xenotransplantation in surgery. International Journal of Surgery. 2011;**9**(2):122-129

[39] Hu Q, Liu Z, Zhu H. Pig islets for islet xenotransplantation: Current status and future perspectives. Chinese Medical Journal. 2014;**127**(2):370-377

[40] Kirchhof N, Hering BJ, Geiss V, Federlin K, Bretzel RG. Evidence for breed-dependent differences in porcine islets of Langerhans. Transplantation Proceedings. 1994;**26**(2):616-617

[41] Kim JH, Kim HI, Lee KW, Yu JE, Kim SH, Park HS, et al. Influence of strain and age differences on the yields of porcine islet isolation: Extremely high islet yields from SPF CMS miniature pigs. Xenotransplantation. 2007;**14**(1):60-66

[42] Schuurman HJ. The international xenotransplantation association consensus statement on conditions for undertaking clinical trials of porcine islet products in type 1 diabetes—Chapter 2: Source pigs. Xenotransplantation. 2009;**16**(4):215-222

[43] Nagaraju S, Bottino R, Wijkstrom M, Trucco M, Cooper DK. Islet xenotransplantation: What is the optimal age of the islet-source pig? Xenotransplantation. 2015;**22**(1):7-19

[44] Jay TR, Heald KA, Carless NJ, Topham DE, Downing R. The distribution of porcine pancreatic beta-cells at ages 5, 12 and 24 weeks. Xenotransplantation. 1999;**6**(2):131-140

[45] Shin J-S, Jang J-Y, Park S-K, Choi J-W, Kim S-Y, Min B-H, et al. Extremely high islet yield enables onedonor-one recipient intraportal islet transplantation with enough islet mass in pig-to-non-human primate model. Xenotransplantation. 2013;**20**(5):333

[46] Jiang X, Qian T, Linn T, Cao L, Xiang G, Wang Y, et al. Islet isolation and purification from inbred Wuzhishan miniature pigs. Xenotransplantation. 2012;**19**(3):159-165

[47] Jay TR, Heald KA, Downing R. Effect of donor age on porcine insulin secretion. Transplantation Proceedings. 1997;**29**(4):2023

[48] Mueller KR, Balamurugan AN, Cline GW, Pongratz RL, Hooper RL, Weegman BP, et al. Differences in glucose-stimulated insulin secretion in vitro of islets from human, nonhuman primate, and porcine origin. Xenotransplantation. 2013;**20**(2):75-81

[49] Socci C, Ricordi C, Davalli AM, Staudacher C, Baro P, Vertova A, et al. Selection of donors significantly improves pig islet isolation yield. Hormone and Metabolic Research Supplement. 1990;**25**:32-34

[50] Dufrane D, Goebbels R, Fdilat I, Guiot Y, Gianello P. Impact of porcine islet size on cellular structure and engraftment after transplantation: Adult versus young pigs. Pancreas. 2005;**30**(2):138-147

[51] Hubert T, Jany T, Marcelli-Tourvieille S, Nunes B, Gmyr V, Kerr-Conte J, et al. Acute insulin response of donors is correlated with pancreatic

islet isolation outcome in the pig. Diabetologia. 2005;**48**(10):2069-2073

[52] Krickhahn M, Buhler C, Meyer T, Thiede A, Ulrichs K. The morphology of islets within the porcine donor pancreas determines the isolation result: Successful isolation of pancreatic islets can now be achieved from young market pigs. Cell Transplantation. 2002;**11**(8):827-838

[53] Jin SM, Shin JS, Kim KS, Gong CH, Park SK, Kim JS, et al. Islet isolation from adult designated pathogenfree pigs: Use of the newer bovine nervous tissue-free enzymes and a revised donor selection strategy would improve the islet graft function. Xenotransplantation. 2011;**18**(6):369-379

[54] Kim HI, Lee SY, Jin SM, Kim KS, Yu JE, Yeom SC, et al. Parameters for successful pig islet isolation as determined using 68 specificpathogen-free miniature pigs. Xenotransplantation. 2009;**16**(1):11-18

[55] Zhu HT, Wang WL, Yu L, Wang B. Pig-islet xenotransplantation: Recent progress and current perspectives. Frontiers in Surgery. 2014;**1**:7

[56] Fang J, Walters A, Hara H, Long C, Yeh P, Ayares D, et al. Antigal antibodies in alpha1,3 galactosyltransferase gene-knockout pigs. Xenotransplantation. 2012;**19**(5):305-310

[57] Alumets J, Hakanson R, Sundler F. Ontogeny of endocrine cells in porcine gut and pancreas. An immunocytochemical study. Gastroenterology. 1983;**85**(6):1359-1372

[58] Eventov-Friedman S, Tchorsh D, Katchman H, Shezen E, Aronovich A, Hecht G, et al. Embryonic pig pancreatic tissue transplantation for the

treatment of diabetes. PLoS Medicine. 2006;**3**(7):e215

[59] Hammerman MR. Development of a novel xenotransplantation strategy for treatment of diabetes mellitus in rat hosts and translation to nonhuman primates. Organogenesis. 2012;**8**(2):41-48

[60] Rogers SA, Chen F, Talcott MR, Faulkner C, Thomas JM, Thevis M, et al. Long-term engraftment following transplantation of pig pancreatic primordia into nonimmunosuppressed diabetic rhesus macaques. Xenotransplantation. 2007;**14**(6):591-602

[61] Rogers SA, Liapis H, Hammerman MR. Normalization of glucose post-transplantation of pig pancreatic anlagen into nonimmunosuppressed diabetic rats depends on obtaining anlagen prior to embryonic day 35. Transplant Immunology. 2005;**14**(2):67-75

[62] Otonkoski T, Ustinov J, Rasilainen S, Kallio E, Korsgren O, Hayry P. Differentiation and maturation of porcine fetal islet cells in vitro and after transplantation. Transplantation. 1999;**68**(11):1674-1683

[63] Tan C, Tuch BE, Tu J, Brown SA. Role of NADH shuttles in glucose-induced insulin secretion from fetal beta-cells. Diabetes. 2002;**51**(10):2989-2996

[64] Bogdani M, Suenens K, Bock T, Pipeleers-Marichal M, In't Veld P, Pipeleers D. Growth and functional maturation of beta-cells in implants of endocrine cells purified from prenatal porcine pancreas. Diabetes. 2005;**54**(12):3387-3394

[65] Korbutt GS. What type of islets should be used? Xenotransplantation. 2008;**15**(2):81-82

**41**

*Porcine Islet Cell Xenotransplantation*

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2001;**71**(11):1671-1677

2005;**14**(5):249-261

2011;**11**(12):2593-2602

1996;**97**(9):2119-2129

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2006;**82**(7):945-952

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isletlike cell clusters. Transplantation.

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[68] Luca G, Nastruzzi C, Calvitti M, Becchetti E, Baroni T, Neri LM, et al. Accelerated functional maturation of isolated neonatal porcine cell clusters: In vitro and in vivo results in NOD mice. Cell Transplantation.

[69] Korsgren O, Jansson L, Eizirik D,

[70] Thompson P, Badell IR, Lowe M, Cano J, Song M, Leopardi F, et al. Islet xenotransplantation using gal-deficient neonatal donors improves engraftment and function. American Journal of Transplantation.

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[73] Dufrane D, Gianello P. Pig islets for clinical islet xenotransplantation.

Andersson A. Functional and morphological differentiation of fetal porcine islet-like cell clusters after transplantation into nude mice. Diabetologia. 1991;**34**(6):379-386

#### *Porcine Islet Cell Xenotransplantation DOI: http://dx.doi.org/10.5772/intechopen.90437*

*Xenotransplantation - Comprehensive Study*

[52] Krickhahn M, Buhler C, Meyer T, Thiede A, Ulrichs K. The morphology of islets within the porcine donor pancreas determines the isolation result: Successful isolation of pancreatic islets can now be achieved from young market pigs. Cell Transplantation.

treatment of diabetes. PLoS Medicine.

[59] Hammerman MR. Development of a novel xenotransplantation strategy for treatment of diabetes mellitus in rat hosts and translation to nonhuman primates. Organogenesis.

Talcott MR, Faulkner C, Thomas JM, Thevis M, et al. Long-term engraftment

Hammerman MR. Normalization of glucose post-transplantation of pig pancreatic anlagen into nonimmunosuppressed diabetic rats depends on obtaining anlagen prior to embryonic day 35. Transplant Immunology. 2005;**14**(2):67-75

following transplantation of pig pancreatic primordia into nonimmunosuppressed diabetic rhesus macaques. Xenotransplantation.

2006;**3**(7):e215

2012;**8**(2):41-48

[60] Rogers SA, Chen F,

2007;**14**(6):591-602

[61] Rogers SA, Liapis H,

[62] Otonkoski T, Ustinov J, Rasilainen S, Kallio E, Korsgren O, Hayry P. Differentiation and maturation of porcine fetal islet cells in vitro and after transplantation. Transplantation.

1999;**68**(11):1674-1683

2002;**51**(10):2989-2996

2008;**15**(2):81-82

[64] Bogdani M, Suenens K, Bock T, Pipeleers-Marichal M, In't Veld P, Pipeleers D. Growth and functional maturation of beta-cells in implants of endocrine cells purified from prenatal porcine pancreas. Diabetes. 2005;**54**(12):3387-3394

[63] Tan C, Tuch BE, Tu J,

Brown SA. Role of NADH shuttles in glucose-induced insulin secretion from fetal beta-cells. Diabetes.

[65] Korbutt GS. What type of islets should be used? Xenotransplantation.

[53] Jin SM, Shin JS, Kim KS, Gong CH, Park SK, Kim JS, et al. Islet isolation from adult designated pathogenfree pigs: Use of the newer bovine nervous tissue-free enzymes and a revised donor selection strategy would improve the islet graft function. Xenotransplantation.

[54] Kim HI, Lee SY, Jin SM, Kim KS, Yu JE, Yeom SC, et al. Parameters for successful pig islet isolation as determined using 68 specificpathogen-free miniature pigs.

Xenotransplantation. 2009;**16**(1):11-18

Wang B. Pig-islet xenotransplantation:

[55] Zhu HT, Wang WL, Yu L,

Recent progress and current perspectives. Frontiers in Surgery.

[56] Fang J, Walters A, Hara H, Long C, Yeh P, Ayares D, et al. Anti-

galactosyltransferase gene-knockout

[58] Eventov-Friedman S, Tchorsh D, Katchman H, Shezen E, Aronovich A, Hecht G, et al. Embryonic pig pancreatic

tissue transplantation for the

gal antibodies in alpha1,3-

pigs. Xenotransplantation.

[57] Alumets J, Hakanson R, Sundler F. Ontogeny of endocrine cells in porcine gut and pancreas. An immunocytochemical study. Gastroenterology. 1983;**85**(6):1359-1372

2012;**19**(5):305-310

2014;**1**:7

islet isolation outcome in the pig. Diabetologia. 2005;**48**(10):2069-2073

2002;**11**(8):827-838

2011;**18**(6):369-379

**40**

[66] Vo L, Tuch BE, Wright DC, Keogh GW, Roberts S, Simpson AM, et al. Lowering of blood glucose to nondiabetic levels in a hyperglycemic pig by allografting of fetal pig isletlike cell clusters. Transplantation. 2001;**71**(11):1671-1677

[67] Korsgren O, Christofferson R, Jansson L. Angiogenesis and angioarchitecture of transplanted fetal porcine islet-like cell clusters. Transplantation. 1999;**68**(11):1761-1766

[68] Luca G, Nastruzzi C, Calvitti M, Becchetti E, Baroni T, Neri LM, et al. Accelerated functional maturation of isolated neonatal porcine cell clusters: In vitro and in vivo results in NOD mice. Cell Transplantation. 2005;**14**(5):249-261

[69] Korsgren O, Jansson L, Eizirik D, Andersson A. Functional and morphological differentiation of fetal porcine islet-like cell clusters after transplantation into nude mice. Diabetologia. 1991;**34**(6):379-386

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[82] Bennet W, Bjorkland A, Sundberg B, Davies H, Liu J, Holgersson J, et al. A comparison of fetal and adult porcine islets with regard to gal alpha (1,3) gal expression and the role of human immunoglobulins and complement in islet cell cytotoxicity. Transplantation. 2000;**69**(8):1711-1717

[83] Rayat GR, Rajotte RV, Hering BJ, Binette TM, Korbutt GS. In vitro and in vivo expression of Galalpha-(1,3)gal on porcine islet cells is age dependent. The Journal of Endocrinology. 2003;**177**(1):127-135

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[85] Diswall M, Angstrom J, Schuurman HJ, Dor FJ, Rydberg L, Breimer ME. Studies on glycolipid antigens in small intestine and pancreas from alpha1,3-galactosyltransferase knockout miniature swine. Transplantation. 2007;**84**(10):1348-1356

[86] Omori T, Nishida T, Komoda H, Fumimoto Y, Ito T, Sawa Y, et al. A study of the xenoantigenicity of neonatal porcine islet-like cell clusters (NPCC) and the efficiency of adenovirus-mediated DAF (CD55) expression. Xenotransplantation. 2006;**13**(5):455-464

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[88] Davalli AM, Bertuzzi F, Socci C, Scaglia L, Gavazzi F, Freschi M, et al. Paradoxical release of insulin by adult pig islets in vitro. Recovery after culture in a defined tissue culture medium. Transplantation. 1993;**56**(1):148-154

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[90] Holmes MA, Clayton HA, Chadwick DR, Bell PR, London NJ, James RF. Functional studies of rat, porcine, and human pancreatic islets cultured in ten commercially available media. Transplantation. 1995;**60**(8):854-860

[91] Ricordi C, Finke EH, Lacy PE. A method for the mass isolation of islets from the adult pig pancreas. Diabetes. 1986;**35**(6):649-653

[92] Dufrane D, Goebbels RM, Guiot Y, Squifflet JP, Henquin JC, Gianello P. A simple method using a polymethylpenten chamber for isolation of human pancreatic islets. Pancreas. 2005;**30**(3):e51-e59

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[97] Anazawa T, Balamurugan AN, Papas KK, Avgoustiniatos ES, Ferrer J, Matsumoto S, et al. Improved method of porcine pancreas procurement with arterial flush and ductal injection enhances islet isolation outcome. Transplantation Proceedings.

[98] Lee JI, Shin JS, Jung WY, Lee G, Kim MS, Kim YS, et al. Porcine islet adaptation to metabolic need of monkeys in pig-to-monkey

intraportal islet xenotransplantation.

Transplantation Proceedings.

[99] Thompson P, Badell IR, Lowe M, Turner A, Cano J, Avila J, et al. Alternative immunomodulatory strategies for xenotransplantation: CD40/154 pathway-sparing regimens

promote xenograft survival.

[100] Thompson P, Cardona K, Russell M, Badell IR, Shaffer V, Korbutt G, et al. CD40-specific costimulation blockade enhances neonatal porcine islet survival in nonhuman primates. American Journal of Transplantation. 2011;**11**(5):947-957

[101] van der Windt DJ, Bottino R, Casu A, Campanile N, Smetanka C, He J, et al. Long-term controlled normoglycemia in diabetic non-human primates after transplantation with hCD46 transgenic porcine islets. American Journal of Transplantation.

2012;**12**(7):1765-1775

2009;**9**(12):2716-2726

American Journal of Transplantation.

2010;**2**(5):265-273

2010;**42**(6):2040-2042

2010;**42**(6):2032-2035

2013;**45**(5):1866-1868

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[102] Denner J. Recent progress in xenotransplantation, with emphasis on virological safety. Annals of Transplantation. 2016;**21**:717-727

2005;**37**(1):496-499

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[105] Valdes-Gonzalez R,

Dorantes LM, Bracho-Blanchet E, Rodríguez-Ventura A, DJG W. No evidence of porcine endogenous retrovirus in patients with type 1 diabetes after long-term porcine islet xenotransplantation. Journal of Medical

Virology. 2010;**82**(2):331-334

[106] Onions D, Cooper DK,

Alexander TJ, Brown C, Claassen E, Foweraker JE, et al. An approach to the control of disease transmission in pig-to-human xenotransplantation. Xenotransplantation. 2000;**7**(2):143-155

[107] Bottino R, Trucco M. Use of genetically-engineered pig donors in islet transplantation. World Journal of

Transplantation. 2015;**5**(4):243

[108] Bhargava R, Senior PA, Ackerman TE, Ryan EA,

Paty BW, Lakey JR, et al. Prevalence of hepatic steatosis after islet transplantation and its relation to graft function. Diabetes. 2004;**53**(5):1311-1317

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*Porcine Islet Cell Xenotransplantation DOI: http://dx.doi.org/10.5772/intechopen.90437*

*Xenotransplantation - Comprehensive Study*

products in type 1 diabetes—Chapter 3: Pig islet product manufacturing and release testing. Xenotransplantation.

Effective islet isolation method with extremely high islet yields from adult pigs. Cell Transplantation.

[88] Davalli AM, Bertuzzi F, Socci C, Scaglia L, Gavazzi F, Freschi M, et al. Paradoxical release of insulin by adult pig islets in vitro. Recovery after culture in a defined tissue culture medium. Transplantation. 1993;**56**(1):148-154

Duvivier V, Darquy S, Larher E, You S, et al. Perifusion analysis of insulin secretion from specific pathogen-free large-white pig islets shows satisfactory functional characteristics for xenografts in humans. Diabetes & Metabolism.

2005;**14**(10):757-762

[89] Gouin E, Rivereau AS,

1998;**24**(3):208-214

1995;**60**(8):854-860

1986;**35**(6):649-653

2005;**30**(3):e51-e59

1995;**103**(Suppl 2):3-14

[90] Holmes MA, Clayton HA, Chadwick DR, Bell PR, London NJ, James RF. Functional studies of rat, porcine, and human pancreatic islets

cultured in ten commercially available media. Transplantation.

[92] Dufrane D, Goebbels RM, Guiot Y, Squifflet JP, Henquin JC, Gianello P. A simple method using a polymethylpenten chamber for isolation of human pancreatic islets. Pancreas.

[93] Brandhorst D, Brandhorst H, Hering BJ, Federlin K, Bretzel RG. Islet isolation from the pancreas of large mammals and humans: 10 years of experience. Experimental and Clinical Endocrinology & Diabetes.

[94] Toso C, Brandhorst D, Oberholzer J, Triponez F, Buhler L, Morel P. Isolation of adult porcine islets of Langerhans. Cell Transplantation. 2000;**9**(3):297-305

[91] Ricordi C, Finke EH, Lacy PE. A method for the mass isolation of islets from the adult pig pancreas. Diabetes.

[81] Komoda H, Miyagawa S, Kubo T, Kitano E, Kitamura H, Omori T, et al. A study of the xenoantigenicity of adult pig islets cells. Xenotransplantation.

[82] Bennet W, Bjorkland A, Sundberg B, Davies H, Liu J, Holgersson J, et al. A comparison of fetal and adult porcine islets with regard to gal alpha (1,3) gal expression and the role of human immunoglobulins and complement in islet cell cytotoxicity. Transplantation.

[83] Rayat GR, Rajotte RV, Hering BJ, Binette TM, Korbutt GS. In vitro and in vivo expression of Galalpha-(1,3)gal on porcine islet cells is age dependent.

The Journal of Endocrinology.

[84] McKenzie IF, Koulmanda M, Mandel TE, Sandrin MS. Pig islet xenografts are susceptible to "anti-pig" but not gal alpha(1,3)gal antibody plus complement in gal o/o mice. Journal of Immunology. 1998;**161**(10):5116-5119

[85] Diswall M, Angstrom J, Schuurman HJ, Dor FJ, Rydberg L, Breimer ME. Studies on glycolipid antigens in small intestine and pancreas from alpha1,3-galactosyltransferase

knockout miniature swine.

2006;**13**(5):455-464

Transplantation. 2007;**84**(10):1348-1356

[87] Yonekawa Y, Matsumoto S, Okitsu T, Arata T, Iwanaga Y, Noguchi H, et al.

[86] Omori T, Nishida T, Komoda H, Fumimoto Y, Ito T, Sawa Y, et al. A study of the xenoantigenicity of neonatal porcine islet-like cell clusters (NPCC) and the efficiency of adenovirus-mediated DAF (CD55) expression. Xenotransplantation.

2009;**16**(4):223-228

2004;**11**(3):237-246

2000;**69**(8):1711-1717

2003;**177**(1):127-135

**42**

[95] Kin T, Shapiro AM. Surgical aspects of human islet isolation. Islets. 2010;**2**(5):265-273

[96] Goto M, Imura T, Inagaki A, Ogawa N, Yamaya H, Fujimori K, et al. The impact of ischemic stress on the quality of isolated pancreatic islets. Transplantation Proceedings. 2010;**42**(6):2040-2042

[97] Anazawa T, Balamurugan AN, Papas KK, Avgoustiniatos ES, Ferrer J, Matsumoto S, et al. Improved method of porcine pancreas procurement with arterial flush and ductal injection enhances islet isolation outcome. Transplantation Proceedings. 2010;**42**(6):2032-2035

[98] Lee JI, Shin JS, Jung WY, Lee G, Kim MS, Kim YS, et al. Porcine islet adaptation to metabolic need of monkeys in pig-to-monkey intraportal islet xenotransplantation. Transplantation Proceedings. 2013;**45**(5):1866-1868

[99] Thompson P, Badell IR, Lowe M, Turner A, Cano J, Avila J, et al. Alternative immunomodulatory strategies for xenotransplantation: CD40/154 pathway-sparing regimens promote xenograft survival. American Journal of Transplantation. 2012;**12**(7):1765-1775

[100] Thompson P, Cardona K, Russell M, Badell IR, Shaffer V, Korbutt G, et al. CD40-specific costimulation blockade enhances neonatal porcine islet survival in nonhuman primates. American Journal of Transplantation. 2011;**11**(5):947-957

[101] van der Windt DJ, Bottino R, Casu A, Campanile N, Smetanka C, He J, et al. Long-term controlled normoglycemia in diabetic non-human primates after transplantation with hCD46 transgenic porcine islets. American Journal of Transplantation. 2009;**9**(12):2716-2726

[102] Denner J. Recent progress in xenotransplantation, with emphasis on virological safety. Annals of Transplantation. 2016;**21**:717-727

[103] Yu P, Zhang L, Li SF, Li YP, Cheng JQ, Lu YR, et al. Long-term effects on HEK-293 cell line after co-culture with porcine endogenous retrovirus. Transplantation Proceedings. 2005;**37**(1):496-499

[104] van der Laan LJ, Lockey C, Griffeth BC, Frasier FS, Wilson CA, Onions DE, et al. Infection by porcine endogenous retrovirus after islet xenotransplantation in SCID mice. Nature. 2000;**407**(6800):90-94

[105] Valdes-Gonzalez R, Dorantes LM, Bracho-Blanchet E, Rodríguez-Ventura A, DJG W. No evidence of porcine endogenous retrovirus in patients with type 1 diabetes after long-term porcine islet xenotransplantation. Journal of Medical Virology. 2010;**82**(2):331-334

[106] Onions D, Cooper DK, Alexander TJ, Brown C, Claassen E, Foweraker JE, et al. An approach to the control of disease transmission in pig-to-human xenotransplantation. Xenotransplantation. 2000;**7**(2):143-155

[107] Bottino R, Trucco M. Use of genetically-engineered pig donors in islet transplantation. World Journal of Transplantation. 2015;**5**(4):243

[108] Bhargava R, Senior PA, Ackerman TE, Ryan EA, Paty BW, Lakey JR, et al. Prevalence of hepatic steatosis after islet transplantation and its relation to graft function. Diabetes. 2004;**53**(5):1311-1317

[109] Yeom HJ, Koo OJ, Yang J, Cho B, Hwang JI, Park SJ, et al. Generation and characterization of human heme oxygenase-1 transgenic pigs. PLoS One. 2012;**7**(10):e46646

[110] Ekser B, Ezzelarab M, Hara H, van der Windt DJ, Wijkstrom M, Bottino R, et al. Clinical xenotransplantation: The next medical revolution? Lancet. 2012;**379**(9816):672-683

[111] Xu XC, Goodman J, Sasaki H, Lowell J, Mohanakumar T. Activation of natural killer cells and macrophages by porcine endothelial cells augments specific T-cell xenoresponse. American Journal of Transplantation. 2002;**2**(4):314-322

[112] Saethre M, Schneider MK, Lambris JD, Magotti P, Haraldsen G, Seebach JD, et al. Cytokine secretion depends on Galalpha(1,3)gal expression in a pig-to-human whole blood model. Journal of Immunology. 2008;**180**(9):6346-6353

[113] Diamond LE, Quinn CM, Martin MJ, Lawson J, Platt JL, Logan JS. A human CD46 transgenic pig model system for the study of discordant xenotransplantation. Transplantation. 2001;**71**(1):132-142

[114] Liu D, Kobayashi T, Onishi A, Furusawa T, Iwamoto M, Suzuki S, et al. Relation between human decayaccelerating factor (hDAF) expression in pig cells and inhibition of human serum anti-pig cytotoxicity: Value of highly expressed hDAF for xenotransplantation. Xenotransplantation. 2007;**14**(1):67-73

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[122] Londrigan SL, Sutherland RM, Brady JL, Carrington EM, Cowan PJ, d'Apice AJ, et al. In situ protection against islet allograft rejection by CTLA4Ig transduction. Transplantation. 2010;**90**(9):951-957

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[110] Ekser B, Ezzelarab M, Hara H, van der Windt DJ, Wijkstrom M, Bottino R, et al. Clinical xenotransplantation: The next medical revolution? Lancet.

[117] Cooper DK, Ekser B,

2016;**238**(2):288-299

2010;**6**(2):219-230

role of genetically engineered pigs in xenotransplantation

Ramsoondar J, Phelps C, Ayares D. The

research. The Journal of Pathology.

[118] Ekser B, Cooper DK. Overcoming the barriers to xenotransplantation: Prospects for the future. Expert Review of Clinical Immunology.

[119] Ma X, Ye B, Gao F, Liang Q, Dong Q, Liu Y, et al. Tissue factor knockdown in porcine islets: An effective approach to suppressing the instant bloodmediated inflammatory reaction. Cell Transplantation. 2012;**21**(1):61-71

Cooper DK. The potential of geneticallymodified pig mesenchymal stromal cells in xenotransplantation. Xenotransplantation. 2010;**17**(1):3-5

[121] Klymiuk N, van Buerck L, Bahr A, Offers M, Kessler B, Wuensch A, et al. Xenografted islet cell clusters from INSLEA29Y transgenic pigs rescue diabetes and prevent immune

rejection in humanized mice. Diabetes.

[122] Londrigan SL, Sutherland RM, Brady JL, Carrington EM, Cowan PJ, d'Apice AJ, et al. In situ protection against islet allograft rejection by CTLA4Ig transduction. Transplantation.

[123] Ezzelarab M, Ezzelarab C, Wilhite T, Kumar G, Hara H,

Ayares D, et al. Genetically-modified pig mesenchymal stromal cells: Xenoantigenicity and effect on human T-cell xenoresponses.

[124] Galili U. The alpha-gal epitope

and the anti-gal antibody in xenotransplantation and in cancer

2012;**61**(6):1527-1532

2010;**90**(9):951-957

Xenotransplantation. 2011;**18**(3):183-195

[120] Ezzelarab M, Ayares D,

[111] Xu XC, Goodman J, Sasaki H, Lowell J, Mohanakumar T. Activation of natural killer cells and macrophages by porcine endothelial cells augments

American Journal of Transplantation.

specific T-cell xenoresponse.

[112] Saethre M, Schneider MK, Lambris JD, Magotti P, Haraldsen G, Seebach JD, et al. Cytokine secretion

depends on Galalpha(1,3)gal expression in a pig-to-human whole blood model. Journal of Immunology.

[113] Diamond LE, Quinn CM,

Martin MJ, Lawson J, Platt JL, Logan JS. A human CD46 transgenic pig model system for the study of discordant xenotransplantation. Transplantation.

[114] Liu D, Kobayashi T, Onishi A, Furusawa T, Iwamoto M, Suzuki S, et al. Relation between human decayaccelerating factor (hDAF) expression

human serum anti-pig cytotoxicity:

Xenotransplantation. 2007;**14**(1):67-73

[115] Le Bas-Bernardet S, Tillou X, Poirier N, Dilek N, Chatelais M, Devalliere J, et al. Xenotransplantation of galactosyl-transferase knockout, CD55, CD59, CD39, and fucosyltransferase transgenic pig kidneys into baboons. Transplantation Proceedings.

in pig cells and inhibition of

Value of highly expressed hDAF for xenotransplantation.

2011;**43**(9):3426-3430

[116] Park CG, Bottino R,

Hawthorne WJ. Current status of islet xenotransplantation. International Journal of Surgery. 2015;**23**(Pt

2008;**180**(9):6346-6353

2001;**71**(1):132-142

2002;**2**(4):314-322

2012;**379**(9816):672-683

**44**

B):261-266

immunotherapy. Immunology and Cell Biology. 2005;**83**(6):674-686

[125] Dai Y, Vaught TD, Boone J, Chen SH, Phelps CJ, Ball S, et al. Targeted disruption of the alpha1,3-galactosyltransferase gene in cloned pigs. Nature Biotechnology. 2002;**20**(3):251-255

[126] Koike C, Uddin M, Wildman DE, Gray EA, Trucco M, Starzl TE, et al. Functionally important glycosyltransferase gain and loss during catarrhine primate emergence. Proceedings of the National Academy of Sciences of the United States of America. 2007;**104**(2):559-564

[127] Kobayashi T, Cooper DK. Anti-Gal, alpha-Gal epitopes, and xenotransplantation. Subcellular Biochemistry. 1999;**32**:229-257

[128] Bouhours D, Pourcel C, Bouhours JE. Simultaneous expression by porcine aorta endothelial cells of glycosphingolipids bearing the major epitope for human xenoreactive antibodies (gal alpha 1-3Gal), blood group H determinant and N-glycolylneuraminic acid. Glycoconjugate Journal. 1996;**13**(6):947-953

[129] Padler-Karavani V, Varki A. Potential impact of the nonhuman sialic acid N-glycolylneuraminic acid on transplant rejection risk. Xenotransplantation. 2011;**18**(1):1-5

[130] Byrne GW, Du Z, Stalboerger P, Kogelberg H, McGregor CG. Cloning and expression of porcine beta1,4 N-acetylgalactosaminyl transferase encoding a new xenoreactive antigen. Xenotransplantation. 2014;**21**(6):543-554

[131] Petersen B, Carnwath JW, Niemann H. The perspectives for porcine-to-human xenografts. Comparative Immunology,

Microbiology and Infectious Diseases. 2009;**32**(2):91-105

[132] Lutz AJ, Li P, Estrada JL, Sidner RA, Chihara RK, Downey SM, et al. Double knockout pigs deficient in N-glycolylneuraminic acid and galactose alpha-1,3-galactose reduce the humoral barrier to xenotransplantation. Xenotransplantation. 2013;**20**(1):27-35

[133] Goto M, Tjernberg J, Dufrane D, Elgue G, Brandhorst D, Ekdahl KN, et al. Dissecting the instant bloodmediated inflammatory reaction in islet xenotransplantation. Xenotransplantation. 2008;**15**(4):225-234

[134] van der Windt DJ, Marigliano M, He J, Votyakova TV, Echeverri GJ, Ekser B, et al. Early islet damage after direct exposure of pig islets to blood: Has humoral immunity been underestimated? Cell Transplantation. 2012;**21**(8):1791-1802

[135] Korsgren O, Lundgren T, Felldin M, Foss A, Isaksson B, Permert J, et al. Optimising islet engraftment is critical for successful clinical islet transplantation. Diabetologia. 2008;**51**(2):227-232

[136] Moberg L, Johansson H, Lukinius A, Berne C, Foss A, Kallen R, et al. Production of tissue factor by pancreatic islet cells as a trigger of detrimental thrombotic reactions in clinical islet transplantation. Lancet. 2002;**360**(9350):2039-2045

[137] Hawthorne WJ, Salvaris EJ, Phillips P, Hawkes J, Liuwantara D, Burns H, et al. Control of IBMIR in neonatal porcine islet xenotransplantation in baboons. American Journal of Transplantation. 2014;**14**(6):1300-1309

[138] Vogel CW, Fritzinger DC, Hew BE, Thorne M, Bammert H. Recombinant cobra venom factor. Molecular Immunology. 2004;**41**(2-3):191-199

[139] Ozmen L, Ekdahl KN, Elgue G, Larsson R, Korsgren O, Nilsson B. Inhibition of thrombin abrogates the instant bloodmediated inflammatory reaction triggered by isolated human islets: Possible application of the thrombin inhibitor melagatran in clinical islet transplantation. Diabetes. 2002;**51**(6):1779-1784

[140] Mourad NI, Gianello PR. Xenoislets: Porcine pancreatic islets for the treatment of type I diabetes. Current Opinion in Organ Transplantation. 2017;**22**(6):529-534

[141] Gill RG, Wolf L, Daniel D, Coulombe M. CD4+ T cells are both necessary and sufficient for islet xenograft rejection. Transplantation Proceedings. 1994;**26**(3):1203

[142] Olack BJ, Jaramillo A, Benshoff ND, Kaleem Z, Swanson CJ, Lowell JA, et al. Rejection of porcine islet xenografts mediated by CD4+ T cells activated through the indirect antigen recognition pathway. Xenotransplantation. 2002;**9**(6):393-401

[143] Koulmanda M, Laufer TM, Auchincloss H Jr, Smith RN. Prolonged survival of fetal pig islet xenografts in mice lacking the capacity for an indirect response. Xenotransplantation. 2004;**11**(6):525-530

[144] Tonomura N, Shimizu A, Wang S, Yamada K, Tchipashvili V, Weir GC, et al. Pig islet xenograft rejection in a mouse model with an established human immune system. Xenotransplantation. 2008;**15**(2):129-135

[145] Scalea J, Hanecamp I, Robson SC, Yamada K. T-cell-mediated immunological barriers to xenotransplantation. Xenotransplantation. 2012;**19**(1):23-30

[146] Trikudanathan S, Sayegh MH. The evolution of the immunobiology of

co-stimulatory pathways: Clinical implications. Clinical and Experimental Rheumatology. 2007;**25**(5 Suppl 46):S12-S21

[147] Tian M, Lv Y, Zhai C, Zhu H, Yu L, Wang B. Alternative immunomodulatory strategies for xenotransplantation: CD80/CD86-CTLA4 pathway-modified immature dendritic cells promote xenograft survival. PLoS One. 2013;**8**(7):e69640

[148] Contreras JL. Extrahepatic transplant sites for islet xenotransplantation. Xenotransplantation. 2008;**15**(2):99-101

[149] Kumagai-Braesch M, Ekberg H, Wang F, Osterholm C, Ehrnfelt C, Sharma A, et al. Anti-LFA-1 improves pig islet xenograft function in diabetic mice when long-term acceptance is induced by CTLA4Ig/ anti-CD40L. Transplantation. 2007;**83**(9):1259-1267

[150] Elliott RB, Escobar L, Tan PL, Muzina M, Zwain S, Buchanan C. Live encapsulated porcine islets from a type 1 diabetic patient 9.5 yr after xenotransplantation. Xenotransplantation. 2007;**14**(2):157-161

[151] Meyer T, Höcht B, Ulrichs K. Xenogeneic islet transplantation of microencapsulated porcine islets for therapy of type I diabetes: Long-term normoglycemia in STZ-diabetic rats without immunosuppression. Pediatric Surgery International. 2008;**24**(12):1375-1378

[152] Zhu HT, Lu L, Liu XY, Yu L, Lyu Y, Wang B. Treatment of diabetes with encapsulated pig islets: An update on current developments. Journal of Zhejiang University Science B. 2015;**16**(5):329-343

[153] Weir GC. Islet encapsulation: Advances and obstacles. Diabetologia: Clinical and Experimental Diabetes and Metabolism. 2013;**56**(7):1458-1461

**47**

*Porcine Islet Cell Xenotransplantation*

[154] Teotia RS, Kadam S, Singh AK, Verma SK, Bahulekar A, Kanetkar S, et al. Islet encapsulated implantable composite hollow fiber membrane based device: A bioartificial pancreas. Materials Science & Engineering C. 2017;**77**:857-866

[155] Korsgren O. Islet encapsulation: Physiological possibilities and

[156] Cooper DK, Matsumoto S, Abalovich A, Itoh T, Mourad NI, Gianello PR, et al. Progress in clinical encapsulated islet xenotransplantation.

Transplantation. 2016;**100**(11):

Garkavenko O, Denner J, Elliott R. Microbiological safety of the first clinical pig islet xenotransplantation trial in New Zealand. Xenotransplantation.

[158] Tan PL. Company profile: Tissue regeneration for diabetes and neurological diseases at Living Cell Technologies. Regenerative Medicine.

[160] Garkavenko O, Durbin K, Tan P, Elliott R. Islets transplantation:

Xenotransplantation. 2011;**18**(1):60

[162] Cooper DK, Bottino R, Gianello P, Graham M, Hawthorne WJ, Kirk AD,

[161] Spizzo T, Denner J, Gazda L, Martin M, Nathu D, Scobie L, et al. First update of the International Xenotransplantation Association consensus statement on conditions for undertaking clinical trials of porcine islet products in type 1 diabetes—Chapter 2a: Source pigs—Preventing xenozoonoses. Xenotransplantation. 2016;**23**(1):25-31

New Zealand experience.

[159] Elliott RB, Living Cell T. Towards xenotransplantation of pig islets in the clinic. Current Opinion in Organ Transplantation. 2011;**16**(2):195-200

[157] Wynyard S, Nathu D,

2014;**21**(4):309-323

2010;**5**(2):181-187

2301-2308

limitations. Diabetes. 2017;**66**:1748-1754

*DOI: http://dx.doi.org/10.5772/intechopen.90437*

et al. First update of the International Xenotransplantation Association consensus statement on conditions for undertaking clinical trials of porcine islet products in type 1 diabetes- chapter 4: Pre-clinical efficacy and complication data required to justify a clinical trial. Xenotransplantation.

2016;**23**(1):46-52

#### *Porcine Islet Cell Xenotransplantation DOI: http://dx.doi.org/10.5772/intechopen.90437*

*Xenotransplantation - Comprehensive Study*

co-stimulatory pathways: Clinical implications. Clinical and Experimental

Rheumatology. 2007;**25**(5 Suppl

[147] Tian M, Lv Y, Zhai C, Zhu H, Yu L, Wang B. Alternative immunomodulatory strategies for xenotransplantation: CD80/CD86-CTLA4 pathway-modified immature dendritic cells promote xenograft survival. PLoS One.

[148] Contreras JL. Extrahepatic transplant sites for islet xenotransplantation. Xenotransplantation. 2008;**15**(2):99-101

[149] Kumagai-Braesch M, Ekberg H, Wang F, Osterholm C, Ehrnfelt C, Sharma A, et al. Anti-LFA-1 improves

Buchanan C. Live encapsulated porcine islets from a type 1 diabetic patient 9.5 yr after xenotransplantation.

Xenotransplantation. 2007;**14**(2):157-161

[152] Zhu HT, Lu L, Liu XY, Yu L, Lyu Y, Wang B. Treatment of diabetes with encapsulated pig islets: An update on current developments. Journal of Zhejiang University Science B.

[153] Weir GC. Islet encapsulation: Advances and obstacles. Diabetologia: Clinical and Experimental Diabetes and Metabolism. 2013;**56**(7):1458-1461

[151] Meyer T, Höcht B, Ulrichs K. Xenogeneic islet transplantation of microencapsulated porcine islets for therapy of type I diabetes: Long-term normoglycemia in STZ-diabetic rats without immunosuppression. Pediatric Surgery International.

2008;**24**(12):1375-1378

2015;**16**(5):329-343

pig islet xenograft function in diabetic mice when long-term acceptance is induced by CTLA4Ig/ anti-CD40L. Transplantation.

2007;**83**(9):1259-1267

[150] Elliott RB, Escobar L, Tan PL, Muzina M, Zwain S,

46):S12-S21

2013;**8**(7):e69640

[139] Ozmen L, Ekdahl KN, Elgue G, Larsson R, Korsgren O, Nilsson B. Inhibition of thrombin abrogates the instant bloodmediated inflammatory reaction triggered by isolated human islets: Possible application of the thrombin inhibitor melagatran in clinical islet transplantation. Diabetes.

2002;**51**(6):1779-1784

2017;**22**(6):529-534

[140] Mourad NI, Gianello PR.

[141] Gill RG, Wolf L, Daniel D, Coulombe M. CD4+ T cells are both necessary and sufficient for islet xenograft rejection. Transplantation Proceedings. 1994;**26**(3):1203

[142] Olack BJ, Jaramillo A,

antigen recognition pathway.

[143] Koulmanda M, Laufer TM, Auchincloss H Jr, Smith RN. Prolonged survival of fetal pig islet xenografts in mice lacking the capacity for an indirect response. Xenotransplantation.

2004;**11**(6):525-530

2008;**15**(2):129-135

[145] Scalea J, Hanecamp I,

immunological barriers to xenotransplantation.

Benshoff ND, Kaleem Z, Swanson CJ, Lowell JA, et al. Rejection of porcine islet xenografts mediated by CD4+ T cells activated through the indirect

Xenotransplantation. 2002;**9**(6):393-401

[144] Tonomura N, Shimizu A, Wang S, Yamada K, Tchipashvili V, Weir GC, et al. Pig islet xenograft rejection in a mouse model with an established human immune system. Xenotransplantation.

Robson SC, Yamada K. T-cell-mediated

Xenotransplantation. 2012;**19**(1):23-30

[146] Trikudanathan S, Sayegh MH. The evolution of the immunobiology of

Xenoislets: Porcine pancreatic islets for the treatment of type I diabetes. Current Opinion in Organ Transplantation.

**46**

[154] Teotia RS, Kadam S, Singh AK, Verma SK, Bahulekar A, Kanetkar S, et al. Islet encapsulated implantable composite hollow fiber membrane based device: A bioartificial pancreas. Materials Science & Engineering C. 2017;**77**:857-866

[155] Korsgren O. Islet encapsulation: Physiological possibilities and limitations. Diabetes. 2017;**66**:1748-1754

[156] Cooper DK, Matsumoto S, Abalovich A, Itoh T, Mourad NI, Gianello PR, et al. Progress in clinical encapsulated islet xenotransplantation. Transplantation. 2016;**100**(11): 2301-2308

[157] Wynyard S, Nathu D, Garkavenko O, Denner J, Elliott R. Microbiological safety of the first clinical pig islet xenotransplantation trial in New Zealand. Xenotransplantation. 2014;**21**(4):309-323

[158] Tan PL. Company profile: Tissue regeneration for diabetes and neurological diseases at Living Cell Technologies. Regenerative Medicine. 2010;**5**(2):181-187

[159] Elliott RB, Living Cell T. Towards xenotransplantation of pig islets in the clinic. Current Opinion in Organ Transplantation. 2011;**16**(2):195-200

[160] Garkavenko O, Durbin K, Tan P, Elliott R. Islets transplantation: New Zealand experience. Xenotransplantation. 2011;**18**(1):60

[161] Spizzo T, Denner J, Gazda L, Martin M, Nathu D, Scobie L, et al. First update of the International Xenotransplantation Association consensus statement on conditions for undertaking clinical trials of porcine islet products in type 1 diabetes—Chapter 2a: Source pigs—Preventing xenozoonoses. Xenotransplantation. 2016;**23**(1):25-31

[162] Cooper DK, Bottino R, Gianello P, Graham M, Hawthorne WJ, Kirk AD,

et al. First update of the International Xenotransplantation Association consensus statement on conditions for undertaking clinical trials of porcine islet products in type 1 diabetes- chapter 4: Pre-clinical efficacy and complication data required to justify a clinical trial. Xenotransplantation. 2016;**23**(1):46-52

Chapter 4

Abstract

goat, lamb

49

1. Introduction

and pig-to-primate combination).

group to develop the procedure in human.

Challenge of Xenotransplantation

in Pediatric Heart Transplantation

Norihide Fukushima, Motohiro Kawauchi, Francois Bouchart,

Although surgical techniques have progressively improved in the field of congenital heart disease (CHD), even such as hypoplastic left heart syndrome, pediatric heart transplantation is the most effective surgical option for complex CHD and cardiomyopathy with severe heart failure. However, even now, donor heart availability has been poor in children. Although technologies for ventricular assist device (VAD) have been progressing even in children, VAD cannot grow as the pediatric recipient grows. Therefore, pediatric cardiac xenotransplantation has a great possibility to save and grow children with end-stage heart failure. In this chapter, I would like to introduce the first pediatric baboon-to-human heart transplantation and its basic animal experiments done by Bailey's group and the following attempts for pediatric cardiac orthotopic xenotransplantation (rhesus monkey-to-baboon

Keywords: concordant and discordant xenogeneic orthotopic heart transplantation, pediatric heart transplantation, clinical trial, antibody absorption, primates, pig,

Clinical heart transplantation (HTx) was the unambiguous goal of the laboratory research at Stanford University in the mid-1960s [1]. They were making tremendous progress in their understanding of the host immune response, and how to control that response with drugs of that era, while at the same time avoiding lethal infection. Then, unexpectedly, on December 3, 1967, Christiaan Barnard et al. performed the first clinical HTx in Cape Town, South Africa [2]. Their recipient survived only 18 days, dying of pneumonia. Nevertheless, it stirred worldwide enthusiasm for HTx, and, more importantly, it opened the door for the Stanford

But with regard to infants, Adrian Kantrowitz at New York attempted HTx in a newborn from an anencephalic baby just 3 days after Barnard's first HTx [3]. The recipient died 6 and a half hour after the procedure, and Kantrowitz never pursued clinical HTx. In the 1970s, there have been great progresses in medical and intensive management and surgical technology for neonates and infants with complex congenital heart. However, almost all neonates and infants with too complexed congenital anomaly, especially hypoplastic left heart syndrome (HLHS), could not survive surgery. Theoretically, these neonates, with naïve immune systems and

Sandra Nehlsen-Cannarella and Leonard L. Bailey

#### Chapter 4

## Challenge of Xenotransplantation in Pediatric Heart Transplantation

Norihide Fukushima, Motohiro Kawauchi, Francois Bouchart, Sandra Nehlsen-Cannarella and Leonard L. Bailey

#### Abstract

Although surgical techniques have progressively improved in the field of congenital heart disease (CHD), even such as hypoplastic left heart syndrome, pediatric heart transplantation is the most effective surgical option for complex CHD and cardiomyopathy with severe heart failure. However, even now, donor heart availability has been poor in children. Although technologies for ventricular assist device (VAD) have been progressing even in children, VAD cannot grow as the pediatric recipient grows. Therefore, pediatric cardiac xenotransplantation has a great possibility to save and grow children with end-stage heart failure. In this chapter, I would like to introduce the first pediatric baboon-to-human heart transplantation and its basic animal experiments done by Bailey's group and the following attempts for pediatric cardiac orthotopic xenotransplantation (rhesus monkey-to-baboon and pig-to-primate combination).

Keywords: concordant and discordant xenogeneic orthotopic heart transplantation, pediatric heart transplantation, clinical trial, antibody absorption, primates, pig, goat, lamb

#### 1. Introduction

Clinical heart transplantation (HTx) was the unambiguous goal of the laboratory research at Stanford University in the mid-1960s [1]. They were making tremendous progress in their understanding of the host immune response, and how to control that response with drugs of that era, while at the same time avoiding lethal infection. Then, unexpectedly, on December 3, 1967, Christiaan Barnard et al. performed the first clinical HTx in Cape Town, South Africa [2]. Their recipient survived only 18 days, dying of pneumonia. Nevertheless, it stirred worldwide enthusiasm for HTx, and, more importantly, it opened the door for the Stanford group to develop the procedure in human.

But with regard to infants, Adrian Kantrowitz at New York attempted HTx in a newborn from an anencephalic baby just 3 days after Barnard's first HTx [3]. The recipient died 6 and a half hour after the procedure, and Kantrowitz never pursued clinical HTx. In the 1970s, there have been great progresses in medical and intensive management and surgical technology for neonates and infants with complex congenital heart. However, almost all neonates and infants with too complexed congenital anomaly, especially hypoplastic left heart syndrome (HLHS), could not survive surgery. Theoretically, these neonates, with naïve immune systems and

uniformly lethal heart disease, should be excellent candidates for HTx that included aortic arch reconstruction. But around for a decade since the first HTx, clinical HTx was limited to only a handful of progressive institutions, and none was spearheading research in neonatal HTx except a little Leonard L Bailey's group at Loma Linda University.

with HLHS named "Baby Fae" was transplanted on October 26, 1984, with the heart of a highly selected infant baboon [8, 9]. She lived for only 20 days, and despite careful observations and analysis, the cause(s) of her death remains somewhat of an enigma. She did heighten awareness, however, and her transplant led directly to the first successful neonatal HTx, again as treatment for HLHS, in November of 1985. That infant is now a 34-year-old man working in Las Vegas. Baby Fae's legacy is found among the hundreds of neonates and small infants who are living today because of primary or secondary HTx in the world. However, donor shortage had been still severe, and continuous experimental efforts to achieve clinical infant

4. The immunological effects of concordant xenograft bridging to

survived 10, 58, 65, 198, and 164 days. Despite a high titer of circulating

(which were ultimately euthanized) was not unlike that expected for

Survival (days)

xenoantibody in each of the host baboons, orthotopic allogeneic engraftment was possible in all five recipients. Each was immunosuppressed with gradations of CSAbased therapy. Survival to 5 and 6 months of the last two consecutive animals

1 A None 11 A None 10 Severe 2 A None 5 A B 58 Moderated to

3 A None 6 A B 65 Moderated to

Survival of xenografts and allografts and host therapy employed in a xenograft bridge to allograft model using

4 A + B B + C 13 A B + C 198<sup>b</sup> None 5 A + B + C None 65 A + B None 164<sup>b</sup> None

Immunosuppression. (A) cyclosporine + azathioprine + solumedrol; (B) goat anti-human T cell IgG; (C)

Therapya Rescue

therapy

Cardiac orthotopic allograft (common olive baboon)

Survival (days)

Allograft rejection

severe

severe

Human neonatal xenoHTx evolved around the idea of xenograft bridging to cardiac allografting. The important question relating to this approach was whether the bridged recipient would develop an antibody response to the initial xenograft that would be cross-reactive with the allograft donor. This question was initially explored by Alonso de Begona [10] using a heterotopic HTx model from African green monkey to juvenile baboons treated with CsA (Table 1). These 5 grafts are rejected over a period of 5–65 days. Lymphocytotoxic xenoantibody was identified in recipient blood samples. The rejected xenografts were removed, and the recipient circulating xenoantibody titers were observed to peak over 24–48 hours. Using cardiopulmonary bypass primed without blood, the immature baboon recipients then underwent orthotopic allogeneic HTx and were treated with varying degrees using a cyclosporine (CSA) protocol. All survived the secondary allogeneic HTx without any evidence of hyperacute, antibody-mediated rejection. The recipients

xenoHTx had been performed in the Bailey's group.

Challenge of Xenotransplantation in Pediatric Heart Transplantation

cardiac allografting in baboon

DOI: http://dx.doi.org/10.5772/intechopen.90321

Cardiac heterotopic xenograft (African green monkey)

therapy

Therapya Rescue

a

b

51

Table 1.

monoclonal antibody.

Electively euthanized.

an immature baboon recipient.

His laboratory was using neonatal goats as recipients, and, initially, goats as donors. In 1981, the Sandoz Laboratory, a pharmaceutical house in Basal, Switzerland, agreed to provide them with an investigational agent called cyclosporine-A (CsA). With CsA immunosuppression alone, they observed remarkable survival, maturation, and reproductive capacity among goats that were orthotopically transplanted as newborns with allografts [4]. Even recipients of cross-species grafts from lamb to goat experienced unprecedented survival [5].

#### 2. Lamb to goat orthotopic concordant xenoHTx

Fourteen newborn (less than 7 days old) goats underwent orthotopic HTx with a size-matched lamb's heart [5]. Ten goats survived longer than 24 hours after HTx. Recipient animals received CsA 48 and 24 hours before HTx and daily after HTx on a gradually reducing daily protocol. Recipients were also given pulse doses of methylprednisolone (100 mg/kg) and azathioprine (3 mg/kg) once a week, the dosage schedule being gradually reduced. Azathioprine was discontinued on postoperative day 60. Survival among the 10 recipients was 24, 32, 44, 47, 60, 60, 78, 90, 120, and 165 days. Average survival was 72 days. Serial left ventricular ejection fractions measured by radionuclide left ventriculography from 1 to 4 months postoperatively in four recipients averaged 50, 58, 45, and 45%. There were no significant infections. Most animals showed mild-to-moderate subacute and chronic graft rejection at autopsy. One host showed no gross or microscopic graft rejection at autopsy on postoperative day 47. Tumor was not observed. These data suggest that long-term survival may be feasible for newborn recipients of cardiac xenografts with CsA therapy and limited supplemental immunosuppression.

#### 3. Attempt of a baboon-to-human orthotopic concordant xenoHTx

Neonatal and small infant heart donors were not available in the early 1980s; hence, the Bailey's group focused on the possibility of using immature baboons as donors for neonates with HLHS. They purchased a panel of infant baboons and studied them extensively for infectious diseases. They performed HLA-typing, twoway mixed lymphocyte cultures, and ex vivo perfusion studies to assess their compatibility with human neonates. They thought it might be possible to actually select a "best" baboon donor for any individual baby with HLHS. They began an arduous 14-month process of obtaining Institutional Review Board (IRB) approval for experimental clinical trials of baboon-to-human baby concordant xenoHTx. Sandra Nehlsen-Cannarella, a transplant immunologist and Medawar protégé, was one of external reviewers, helped their works, and finally joined their team after the IRB was approved in October 1984 [6].

Then, in late July of 1984, Dr. Magdi Yacoub and his team at the National Heart Hospital in London transplanted an 11-day-old newborn with HLHS [7], but the recipient had a complex postoperative course and died of respiratory failure on postoperative day 4. Later that same year, in October, the Bailey's group were confronted with the potential to activate our IRB-approved protocol. A neonate

#### Challenge of Xenotransplantation in Pediatric Heart Transplantation DOI: http://dx.doi.org/10.5772/intechopen.90321

uniformly lethal heart disease, should be excellent candidates for HTx that included aortic arch reconstruction. But around for a decade since the first HTx, clinical HTx

spearheading research in neonatal HTx except a little Leonard L Bailey's group at

His laboratory was using neonatal goats as recipients, and, initially, goats as donors. In 1981, the Sandoz Laboratory, a pharmaceutical house in Basal, Switzerland, agreed to provide them with an investigational agent called cyclosporine-A (CsA). With CsA immunosuppression alone, they observed remarkable survival, maturation, and reproductive capacity among goats that were orthotopically transplanted as newborns with allografts [4]. Even recipients of cross-species grafts

Fourteen newborn (less than 7 days old) goats underwent orthotopic HTx with a size-matched lamb's heart [5]. Ten goats survived longer than 24 hours after HTx. Recipient animals received CsA 48 and 24 hours before HTx and daily after HTx on a gradually reducing daily protocol. Recipients were also given pulse doses of methylprednisolone (100 mg/kg) and azathioprine (3 mg/kg) once a week, the dosage schedule being gradually reduced. Azathioprine was discontinued on postoperative day 60. Survival among the 10 recipients was 24, 32, 44, 47, 60, 60, 78, 90, 120, and 165 days. Average survival was 72 days. Serial left ventricular ejection fractions measured by radionuclide left ventriculography from 1 to 4 months postoperatively in four recipients averaged 50, 58, 45, and 45%. There were no significant infections. Most animals showed mild-to-moderate subacute and chronic graft rejection at autopsy. One host showed no gross or microscopic graft rejection at autopsy on postoperative day 47. Tumor was not observed. These data suggest that long-term survival may be feasible for newborn recipients

was limited to only a handful of progressive institutions, and none was

from lamb to goat experienced unprecedented survival [5].

2. Lamb to goat orthotopic concordant xenoHTx

of cardiac xenografts with CsA therapy and limited supplemental

3. Attempt of a baboon-to-human orthotopic concordant xenoHTx

Neonatal and small infant heart donors were not available in the early 1980s; hence, the Bailey's group focused on the possibility of using immature baboons as donors for neonates with HLHS. They purchased a panel of infant baboons and studied them extensively for infectious diseases. They performed HLA-typing, twoway mixed lymphocyte cultures, and ex vivo perfusion studies to assess their compatibility with human neonates. They thought it might be possible to actually select a "best" baboon donor for any individual baby with HLHS. They began an arduous 14-month process of obtaining Institutional Review Board (IRB) approval for experimental clinical trials of baboon-to-human baby concordant xenoHTx. Sandra Nehlsen-Cannarella, a transplant immunologist and Medawar protégé, was one of external reviewers, helped their works, and finally joined their team after the

Then, in late July of 1984, Dr. Magdi Yacoub and his team at the National Heart Hospital in London transplanted an 11-day-old newborn with HLHS [7], but the recipient had a complex postoperative course and died of respiratory failure on postoperative day 4. Later that same year, in October, the Bailey's group were confronted with the potential to activate our IRB-approved protocol. A neonate

Loma Linda University.

Xenotransplantation - Comprehensive Study

immunosuppression.

50

IRB was approved in October 1984 [6].

with HLHS named "Baby Fae" was transplanted on October 26, 1984, with the heart of a highly selected infant baboon [8, 9]. She lived for only 20 days, and despite careful observations and analysis, the cause(s) of her death remains somewhat of an enigma. She did heighten awareness, however, and her transplant led directly to the first successful neonatal HTx, again as treatment for HLHS, in November of 1985. That infant is now a 34-year-old man working in Las Vegas. Baby Fae's legacy is found among the hundreds of neonates and small infants who are living today because of primary or secondary HTx in the world. However, donor shortage had been still severe, and continuous experimental efforts to achieve clinical infant xenoHTx had been performed in the Bailey's group.

#### 4. The immunological effects of concordant xenograft bridging to cardiac allografting in baboon

Human neonatal xenoHTx evolved around the idea of xenograft bridging to cardiac allografting. The important question relating to this approach was whether the bridged recipient would develop an antibody response to the initial xenograft that would be cross-reactive with the allograft donor. This question was initially explored by Alonso de Begona [10] using a heterotopic HTx model from African green monkey to juvenile baboons treated with CsA (Table 1). These 5 grafts are rejected over a period of 5–65 days. Lymphocytotoxic xenoantibody was identified in recipient blood samples. The rejected xenografts were removed, and the recipient circulating xenoantibody titers were observed to peak over 24–48 hours. Using cardiopulmonary bypass primed without blood, the immature baboon recipients then underwent orthotopic allogeneic HTx and were treated with varying degrees using a cyclosporine (CSA) protocol. All survived the secondary allogeneic HTx without any evidence of hyperacute, antibody-mediated rejection. The recipients survived 10, 58, 65, 198, and 164 days. Despite a high titer of circulating xenoantibody in each of the host baboons, orthotopic allogeneic engraftment was possible in all five recipients. Each was immunosuppressed with gradations of CSAbased therapy. Survival to 5 and 6 months of the last two consecutive animals (which were ultimately euthanized) was not unlike that expected for


a Immunosuppression. (A) cyclosporine + azathioprine + solumedrol; (B) goat anti-human T cell IgG; (C) monoclonal antibody. b Electively euthanized.

#### Table 1.

Survival of xenografts and allografts and host therapy employed in a xenograft bridge to allograft model using an immature baboon recipient.

allotransplanted hosts. Xenoantibody did not appear to alter acute or chronic survival of baboon recipients managed with a clinically applicable regimen of immune regulation. The two chronic survivors had well-functioning allografts that were free of significant rejection injury. These findings have subsequently been confirmed and elaborated on by Michler et al. [11].

methotrexate and splenectomy has produced prolonged host survival in this xenotransplantation model. Results suggest that concordant xenotransplantation

1 6 Moderate cellular rejection None Rejection 2 7 Moderate cellular rejection None Rejection 3 8 Severe cellular rejection None Rejection 4 8 Severe cellular rejection None Rejection 5 9 Severe cellular rejection None Rejection 6 10 Severe cellular rejection None Rejection

1 25 CMV infection, no rejection ALG (21) Systemic CMV

2 32 CMV infection, no rejection None CMV infection

4 53 Mild cellular rejection ALG (68) CMV infection

7 75 Mild cellular rejection ALG + MP (29, 62) During rejection

1 35 Mild cellular rejection None Klebsiella

2 96 No rejection ALG + MP (71) Renal failure

4 234 Toxoplasmosis ALG (94d) Toxoplasmosis

ALG, sheep antilymphocyte globulin; CMV, cytomegalovirus; MP, methyl prednisolone; Tac, tacrolimus; MTX,

Group-C: controls. Group-NP: intravenous sheep antilymphocyte globulin (ALG) induction at 3 and + 5 days perioperatively, daily oral tacrolimus (Tac), and twice weekly intravenous methotrexate (MTX) after

Results of orthotopic cardiac xenotransplantation between immature baboon recipients and rhesus monkey

oral Tac and intravenous MTX) prior to transplantation and the same immunosuppressive therapy after

transplantation. Group-P: two courses of 4-week immunosuppressive therapy (1st course, oral Tac alone; 2nd course,

(38)

Up<sup>b</sup> Tac and MTX (68, 238), ATGAM+MP (392), MP (482)

5 57 Mild cellular rejection ALG + MP (13), Up<sup>b</sup> Tac + ALG + MP

Rescue therapy (onset day after transplant)

Cause of death

infection

(graft)

(lung, kidney)

(lung)

During rejection treatment

embolism

treatment

pneumonia

(lung, kidney)

Liver failure and CMV infection

None CMV infection

Up<sup>b</sup> MTX (25) Pulmonary

None CMV infection

would be a suitable biologic bridge to allotransplantation.

Challenge of Xenotransplantation in Pediatric Heart Transplantation

Histological findings of autopsied xenograft

DOI: http://dx.doi.org/10.5772/intechopen.90321

Survival (days)

Group-C<sup>a</sup>

Group-NP<sup>a</sup>

Group P<sup>a</sup>

a

b

increase dose.

Table 2.

donors.

53

3 43 Cellular infiltration to

6 74 Mild cellular rejection Mild

3 123 Patchy fibrosis in septum and

5 502 Mild cellular rejection Mild

transplantation as for Group-NP.

inferior wall

graft atherosclerosis

Groups NP and P subjects had splenectomy at the time of heart transplantation.

methotrexate; ATGAM, equine anti-thymocyte globulin.

coronary arteries

graft atherosclerosis

#### 5. Rhesus monkey to baboon orthotopic concordant xenoHTx

Orthotopic concordant xenotransplantation in a juvenile primate model was examined [12, 13]. Eighteen donor rhesus monkeys weighing 2.4–3.8 kg (mean 2.9 kg) were matched with juvenile baboons, aged 9–19 months (mean 12.7 months) and weighing 3.2–4.8 kg (mean 3.9 kg), using ABH blood type and mixed lymphocyte culture. In order to examine plasma level of tacrolimus (Tac) in infant baboons and establish immunosuppressive regimen before starting orthotopic xenoHTx experiments [14], seven of these baboons already received two courses of 4-week immunosuppressive therapy prior to HTx. All baboons underwent splenectomy at the time of HTx.

Twelve animals were divided into three groups; five baboons received no immunosuppressive therapy (Group-C). Five baboons were pretreated (Group-P) and the other seven (Group-NP) was not pretreated. Twelve baboons received sheep antilymphocyte globulin (ALG; IV 15 mg/kg) induction for 3 days before the operation and 5 days after xenoHTx and oral tacrolimus (Tac; 18 mg/kg) and intravenous methotrexate (MTX; 0.1–5 mg IV twice weekly) daily after xenoHTx. The baboons in Group-P received two courses of 4-week immunosuppressive therapy prior to xenoHTx; the first course consisted with Tac (18 mg/kg p.o. daily) alone and the second one consisted with Tac (12 mg/kg p.o. daily) and methotrexate (MTX; 25 mg IV weekly). Pretreated baboons had drug-free intervals for 37 days between two courses and for 83–110 days between the second course and xenoHTx. Intravenous methotrexate, methylprednisolone, ALG, and their combination were used as rescue therapy (Table 2).

Baboons in group-C had a mean survival of 8 days; all died as a result of classic severe cellular rejection. Baboons in Group-NP had a mean survival of 51.3 days (25–75 days), and those in Group-P had a mean survival of 198 days (35–502 days). Two in Group-NP died during rescue therapy for rejection, and three in Group-NP and two in Group-P died of cytomegalovirus (CMV) infection. One in Group-NP died of massive micro-pulmonary embolism. The remaining two in Group-P died of Klebsiella pneumoniae and renal failure aggravated by ganciclovir, respectively.

The longest surviving baboon, named Max, had been a healthy, active, growing baboon with normal cardiac function assessed by echocardiography and left ventriculography and coronary arteries normal in size and distribution assessed by coronary arteriograms at 1 year after xenoHTx. After these examinations, we tried to convert him to oral medications, and his level of immunosuppression fluctuated widely, which led to a late, powerful rejection response. This xenograft rejection was reversed successfully using corticosteroids and ALG. The additional bolus immunosuppression, however, permitted the development of generalized CMV disease and eventually bacterial sepsis from which Max (Figure 1) ultimately died. The animal's autopsied xenograft was almost free of cellular rejection but with mild coronary graft atherosclerosis [15].

Management of CMV infection in this splenectomized series of baboon recipients proved to be at least as difficult as controlling the immune response toward their cardiac xenografts. However, Tac coupled with low-dose maintenance

allotransplanted hosts. Xenoantibody did not appear to alter acute or chronic survival of baboon recipients managed with a clinically applicable regimen of immune regulation. The two chronic survivors had well-functioning allografts that were free of significant rejection injury. These findings have subsequently been confirmed

Orthotopic concordant xenotransplantation in a juvenile primate model was examined [12, 13]. Eighteen donor rhesus monkeys weighing 2.4–3.8 kg (mean 2.9 kg) were matched with juvenile baboons, aged 9–19 months (mean 12.7 months) and weighing 3.2–4.8 kg (mean 3.9 kg), using ABH blood type and mixed lymphocyte culture. In order to examine plasma level of tacrolimus (Tac) in infant baboons and establish immunosuppressive regimen before starting orthotopic xenoHTx experiments [14], seven of these baboons already received two courses of 4-week immunosuppressive therapy prior to HTx. All baboons underwent splenectomy at

Twelve animals were divided into three groups; five baboons received no immunosuppressive therapy (Group-C). Five baboons were pretreated (Group-P) and the other seven (Group-NP) was not pretreated. Twelve baboons received sheep antilymphocyte globulin (ALG; IV 15 mg/kg) induction for 3 days before the operation and 5 days after xenoHTx and oral tacrolimus (Tac; 18 mg/kg) and intravenous methotrexate (MTX; 0.1–5 mg IV twice weekly) daily after xenoHTx. The baboons in Group-P received two courses of 4-week immunosuppressive therapy prior to xenoHTx; the first course consisted with Tac (18 mg/kg p.o. daily) alone and the second one consisted with Tac (12 mg/kg p.o. daily) and methotrexate (MTX; 25 mg IV weekly). Pretreated baboons had drug-free intervals for 37 days between two courses and for 83–110 days between the second course and xenoHTx. Intravenous methotrexate, methylprednisolone, ALG, and their combination were

Baboons in group-C had a mean survival of 8 days; all died as a result of classic severe cellular rejection. Baboons in Group-NP had a mean survival of 51.3 days (25–75 days), and those in Group-P had a mean survival of 198 days (35–502 days).

Group-NP and two in Group-P died of cytomegalovirus (CMV) infection. One in Group-NP died of massive micro-pulmonary embolism. The remaining two in Group-P died of Klebsiella pneumoniae and renal failure aggravated by

The longest surviving baboon, named Max, had been a healthy, active, growing

Management of CMV infection in this splenectomized series of baboon recipients proved to be at least as difficult as controlling the immune response toward their cardiac xenografts. However, Tac coupled with low-dose maintenance

Two in Group-NP died during rescue therapy for rejection, and three in

baboon with normal cardiac function assessed by echocardiography and left ventriculography and coronary arteries normal in size and distribution assessed by coronary arteriograms at 1 year after xenoHTx. After these examinations, we tried to convert him to oral medications, and his level of immunosuppression fluctuated widely, which led to a late, powerful rejection response. This xenograft rejection was reversed successfully using corticosteroids and ALG. The additional bolus immunosuppression, however, permitted the development of generalized CMV disease and eventually bacterial sepsis from which Max (Figure 1) ultimately died. The animal's autopsied xenograft was almost free of cellular rejection but with mild

5. Rhesus monkey to baboon orthotopic concordant xenoHTx

and elaborated on by Michler et al. [11].

Xenotransplantation - Comprehensive Study

the time of HTx.

used as rescue therapy (Table 2).

ganciclovir, respectively.

coronary graft atherosclerosis [15].

52

methotrexate and splenectomy has produced prolonged host survival in this xenotransplantation model. Results suggest that concordant xenotransplantation would be a suitable biologic bridge to allotransplantation.


ALG, sheep antilymphocyte globulin; CMV, cytomegalovirus; MP, methyl prednisolone; Tac, tacrolimus; MTX, methotrexate; ATGAM, equine anti-thymocyte globulin.

a Group-C: controls. Group-NP: intravenous sheep antilymphocyte globulin (ALG) induction at 3 and + 5 days perioperatively, daily oral tacrolimus (Tac), and twice weekly intravenous methotrexate (MTX) after transplantation. Group-P: two courses of 4-week immunosuppressive therapy (1st course, oral Tac alone; 2nd course, oral Tac and intravenous MTX) prior to transplantation and the same immunosuppressive therapy after transplantation as for Group-NP.

Groups NP and P subjects had splenectomy at the time of heart transplantation.

b increase dose.

#### Table 2.

Results of orthotopic cardiac xenotransplantation between immature baboon recipients and rhesus monkey donors.

within the first hour. This is due to the binding of the preformed anti-pig antibodies (Ab) to the endothelial cells of the graft. Ab deposits initiate a complement-mediated response with endothelial injury, resulting in thrombosis, interstitial hemorrhage, and edema, with subsequent graft dysfunction [17]. Later, it was determined that Ab bind to the carbohydrate epitope, galactose–a1,3-galactose (Gal), expressed in the pig vascular endothelium. This oligosaccharide is present in other mammals, except humans and primates. These Ab are produced in response to viruses and microorganisms that express Gal and colonize the gastrointestinal tract of primates [18].

The feasibility of transplanting across discordant xenogeneic barriers in an orthotopic newborn pig-to-juvenile baboon model was first explored in the Bailey's

(hours)

1b None None 4.5 HAR Rejection 2 None None 18 HAR Rejection

1c Donor lung None 6.5 HAR Rejection 2 Donor lung None 10 HAR Rejection 3<sup>d</sup> Donor lung None 375 Mild DXR and GCAS CMV

1 Donor lung Blood replacement 117.5 DXR Brain death

Group-C, controls; Group-D, donor lung perfusion; Group-LD, perfusion with another large pig lungs; Group-D + E, donor lung perfusion, exsanguination, and replacement with whole blood pretreated or packed red blood cell

All subjects had pretransplant splenectomy. CMV, cytomegalovirus; RBC, red blood cell; HAR, hyperacute rejection;

Results of orthotopic cardiac xenotransplantation between juvenile baboon recipients and piglet donors.

None 99 Pneumonia Pneumonia

None 111 DXR Rejection

Pathology of autopsied xenograft

100 DXR Rejection

111 DXR Rejection

123 DXR Rejection

174.5 DXR, CR Rejection

Cause of death

infection

7. Pig-to-baboon orthotopic discordant xenoHTx

Challenge of Xenotransplantation in Pediatric Heart Transplantation

DOI: http://dx.doi.org/10.5772/intechopen.90321

Treatment Survival

Exsanguination

Lung perfusion

Group-C<sup>a</sup>

Group-D<sup>a</sup>

Group-LD<sup>a</sup> 1 Large pig lung

2 Large pig lung

Group-D + E<sup>a</sup>

a

b

c

d

55

Table 3.

2c Donor lung RBC/serum

3 Donor lung RBC/serum

4 Donor lung RBC/serum

5 Donor lung RBC/serum

(RBC) and serum pretreated.

No immunosuppression therapy.

replacement

replacement

replacement

replacement

Kidney perfusion in case of suspected antibody-mediated rejection.

Thymic injection with donor myocardium (left atrium).

DXR, delayed xenograft rejection; CR, cellular rejection.

Figure 1.

Max, an immature baboon recipient of an orthotopic cardiac xenotransplant acquired from a donor rhesus monkey.

#### 6. Toward discordant xenoHTx

Although the high degree of evolutionary relatedness between human beings and primates both suggests that xenotransplantation of primate organs and tissue might be successful, particular concerns are raised by the use of primates, such as baboons. The characteristics, for example, of intelligence and complex social interactions of these closely related higher primates appear to be so like those of human beings that use members of those species as sources for xenotransplantation which might well be seen as ethically unacceptable [16]. The potential risk of extinction, even to a species like the baboon that is not currently endangered, must be taken seriously. The possible transmission of disease from higher primates to human beings and the welfare of the animals should be concerned. From these concerns, it is currently agreed that the use of primates would be ethically unacceptable.

Given the ethical concerns raised by the use of primates for xenotransplantation, attention has turned to developing the pig as an alternative source of organs and tissue, because the use of pigs for xenotransplantation raises fewer ethical concerns. Attention has focused in particular on pigs, since their organs are comparable in size to human ones, and they breed rapidly and could thus be used to supply transplant material on a large scale. The use of pigs as a domestic animal that is farmed and eaten is long established, and many would have fewer concerns about their use for xenotransplantation than the use of primates. If pigs are used for xenotransplantation, they are likely to have been genetically modified so the human immune response to the pig organs and tissue is reduced [16].

When a pig organ is transplanted into a human or nonhuman primate, an immediate immune response occurs with hyperacute rejection (HAR). This has been defined as destruction of the graft in less than 24 hours; however, it usually occurs

Challenge of Xenotransplantation in Pediatric Heart Transplantation DOI: http://dx.doi.org/10.5772/intechopen.90321

within the first hour. This is due to the binding of the preformed anti-pig antibodies (Ab) to the endothelial cells of the graft. Ab deposits initiate a complement-mediated response with endothelial injury, resulting in thrombosis, interstitial hemorrhage, and edema, with subsequent graft dysfunction [17]. Later, it was determined that Ab bind to the carbohydrate epitope, galactose–a1,3-galactose (Gal), expressed in the pig vascular endothelium. This oligosaccharide is present in other mammals, except humans and primates. These Ab are produced in response to viruses and microorganisms that express Gal and colonize the gastrointestinal tract of primates [18].

#### 7. Pig-to-baboon orthotopic discordant xenoHTx

The feasibility of transplanting across discordant xenogeneic barriers in an orthotopic newborn pig-to-juvenile baboon model was first explored in the Bailey's


a Group-C, controls; Group-D, donor lung perfusion; Group-LD, perfusion with another large pig lungs; Group-D + E, donor lung perfusion, exsanguination, and replacement with whole blood pretreated or packed red blood cell (RBC) and serum pretreated.

b No immunosuppression therapy.

c Kidney perfusion in case of suspected antibody-mediated rejection.

d Thymic injection with donor myocardium (left atrium).

All subjects had pretransplant splenectomy. CMV, cytomegalovirus; RBC, red blood cell; HAR, hyperacute rejection; DXR, delayed xenograft rejection; CR, cellular rejection.

#### Table 3.

Results of orthotopic cardiac xenotransplantation between juvenile baboon recipients and piglet donors.

6. Toward discordant xenoHTx

Xenotransplantation - Comprehensive Study

Figure 1.

monkey.

54

response to the pig organs and tissue is reduced [16].

Although the high degree of evolutionary relatedness between human beings and primates both suggests that xenotransplantation of primate organs and tissue might be successful, particular concerns are raised by the use of primates, such as baboons. The characteristics, for example, of intelligence and complex social interactions of these closely related higher primates appear to be so like those of human beings that use members of those species as sources for xenotransplantation which might well be seen as ethically unacceptable [16]. The potential risk of extinction, even to a species like the baboon that is not currently endangered, must be taken seriously. The possible transmission of disease from higher primates to human beings and the welfare of the animals should be concerned. From these concerns, it is currently agreed that the use of primates would be ethically unacceptable.

Max, an immature baboon recipient of an orthotopic cardiac xenotransplant acquired from a donor rhesus

Given the ethical concerns raised by the use of primates for xenotransplantation, attention has turned to developing the pig as an alternative source of organs and tissue, because the use of pigs for xenotransplantation raises fewer ethical concerns. Attention has focused in particular on pigs, since their organs are comparable in size to human ones, and they breed rapidly and could thus be used to supply transplant material on a large scale. The use of pigs as a domestic animal that is farmed and eaten is long established, and many would have fewer concerns about their use for xenotransplantation than the use of primates. If pigs are used for xenotransplantation, they are likely to have been genetically modified so the human immune

When a pig organ is transplanted into a human or nonhuman primate, an imme-

diate immune response occurs with hyperacute rejection (HAR). This has been defined as destruction of the graft in less than 24 hours; however, it usually occurs laboratories during the early 1990s. Because HAR was at that time the single most important factor in limiting discordant xenoHTx, early strategies were directed toward eliminating or reducing baboon preformed xeno Ab to pig sugar antigens [19, 20].

The two control animals survived 4.5 and 18 hours, and the pathological changes

of the grafts were compatible with HAR. The other animals survived 125 33 h (10–375 hors). The longest surviving baboon who survived 375 hours was in Group-D, but other two in Group-D died of HAR. All baboons in Group-LD and Group-D + E survived more than 4 days after XenoHTx. One in Group-D died of CMV infection and one in Group-LD died of pneumonia. One in Group-LD and four in Group-D + E died of acute cellular rejection. In summary, examination and echocardiography revealed no evidence of hyperacute rejection in baboons surviving more than 1 day. The longest survivor (375 hours) died of CMV infection with microscopic evidence of mild delayed HAR and graft coronary atherosclerosis. A variable amount of delayed xenograft rejection (DXR) was observed histologically,

Another baboon which underwent large pig lung perfusion and is given Tac + MTX without splenectomy survived 16 days, and the autopsied graft showed

8. The role of anti-pig antibody in pig-to-baboon xenoHTx rejection

Group-L (n = 8) survived more than 24 hr. [19]. Mean survival period was 9.8 3.0 h in Group-S and 151 33 h in Group-L. Baboon anti-pig Ab was

lung absorbed RAb-4 and RAb-37 may play a role in DXR.

57

To investigate the role of anti-pig Ab in discordant xenograft rejection, these 12 baboons were divided into 2 groups: Group-S (n = 4) died within 24 hr. of HTx and

measured before CPB, before circulatory arrest, during AbA, at the end of CPB, and daily after HTx. Anti-RBC Ab was measured by the titration method at temperatures of 4 degrees C and 37 degrees C (RAb-4 and RAb-37). Anti-endothelial cell Ab (EAb) and anti-white blood cell Ab (WAb) titers were measured with enzymelinked immunosorbent assay (ELISA). RAb titration > or = 1/4 and EAb and WAb > or = 1/256 were determined to be seropositive. Seropositive rate of RAb-37 at the end of CPB (endCPB) in Group-L was significantly higher than that in Group-S (8/8 vs. 1/4; P < 0.05). The seronegative rates of RBC-4 and EAb (endCPB) in Group-L were higher than those in Group-S (7/8 vs. 1/4 and 6/8 vs. 1/4, respectively), but not significantly. There was no difference in seronegative rate of WAb (endCPB) between both groups. More than fourfold decrease in RAb-4 and RAb-37 by AbA with a pig lung was observed in 5 and 7 of 8 baboons, while EAb and WAb did not change by AbA. In all of Group-L, RAb-4 reverted to seropositive within 3 days after HTx. In four of Group-L, RAb-37 became S(+), 1 or 2 days before death by rejection. EAb became seropositive in all of Group-S, but five of them survived more than 5 days after seroconversion. It was concluded that a pig

After I came back to Japan, the role of RAb-37 on pig-to-baboon xenoHTx was

investigated using sequential heterotopic HTx [21]. Fifteen pig hearts were obtained from pigs weighing 6.4–91 kg. Eleven hearts from pigs larger than the recipient were used for perfusion, and four hearts from a pig of the same size as the recipient for heterotopic transplant donor heart. Four female baboons weighing 5.9–8.1 kg received Tac (12 mg.kg) and CAM (50 mg/kg) p.o. daily 2 weeks before and after xenoHTx. After perfusion with two or three large pig hearts, a pig heart was heterotopically transplanted in the right neck of recipient baboon. As the second and third recipient baboons died of hypotension during the third pig heart perfusion and could not undergo heterotopic xenoHTx, the last baboon underwent two pig heart perfusion and subsequent heterotopic xenoHTx. All first perfused hearts and two second perfused hearts were hyperacutely rejected within 30 minutes of perfusion, but the other two second and all third

among the other recipient baboons (Table 3 and Figure 2) [20].

Challenge of Xenotransplantation in Pediatric Heart Transplantation

DOI: http://dx.doi.org/10.5772/intechopen.90321

mild DXR and moderate GCAS [20].

All recipient baboon underwent splenectomy 2 weeks before HTx. Donor hearts were obtained from 12 newborn piglets of either sex age 2–7 days and weighing 1.8– 3.1 kg (mean 2.3 0.1 kg) and transplanted orthotopically with deep hypothermia and circulatory arrest in recipient juvenile baboon age 252–459 days (mean 362 19 days) and weighing 2.4–3.5 kg (mean 2.9 0.1 kg). All animals received an infusion of nafamostat mesylate (FUT-175) at a dose of 2 mg/kg/h for 2 h at the time of reperfusion. The recipient baboon received 15 mg/kg CsA orally or 5 mg/kg intravenously and 5 mg/kg 15-deoxyspergualin (DSG) intramuscularly, from the day before HTx until death.

In two baboons, no antibody adsorption (AbA) using pig lungs was performed for control (Group-C). In 10 baboons, the blood in the bypass circuit was perfused into a pig lung to absorb baboon anti-pig antibody during circulatory arrest at the time of HTx. In three baboons (Group-D), the donor lung was perfused, and in two baboons (Group-LD), a lung larger than the donor pig (weighing 5–7 kg) was perfused. In five baboons (Group-D + E), the donor lung was perfused, and exsanguination was also performed at the beginning of cardiopulmonary bypass (CPB), and the baboon blood was replaced with pretreated whole blood (N = 1) or packed red blood cell (RBC) and 50 ml of pretreated plasma (N = 4). The pretreated blood (N = 1) and serum (N = 4) were made by perfusing with other large pig lung (weighing 15 and 20 kg) before xenoHTx. Two baboons underwent pig kidney perfusion using an extracorporeal shunt from the right femoral artery to vein, 5 and 6 days after xenoHTx, because antibody-mediated rejection was suspected.

#### Figure 2.

An immature baboon recipient of an orthotopic cardiac xenotransplant acquired from a donor pig, which survived 6 days after xenotransplant.

Challenge of Xenotransplantation in Pediatric Heart Transplantation DOI: http://dx.doi.org/10.5772/intechopen.90321

laboratories during the early 1990s. Because HAR was at that time the single most important factor in limiting discordant xenoHTx, early strategies were directed toward eliminating or reducing baboon preformed xeno Ab to pig sugar

All recipient baboon underwent splenectomy 2 weeks before HTx. Donor hearts were obtained from 12 newborn piglets of either sex age 2–7 days and weighing 1.8– 3.1 kg (mean 2.3 0.1 kg) and transplanted orthotopically with deep hypothermia and circulatory arrest in recipient juvenile baboon age 252–459 days (mean

362 19 days) and weighing 2.4–3.5 kg (mean 2.9 0.1 kg). All animals received an infusion of nafamostat mesylate (FUT-175) at a dose of 2 mg/kg/h for 2 h at the time of reperfusion. The recipient baboon received 15 mg/kg CsA orally or 5 mg/kg intravenously and 5 mg/kg 15-deoxyspergualin (DSG) intramuscularly, from the

In two baboons, no antibody adsorption (AbA) using pig lungs was performed for control (Group-C). In 10 baboons, the blood in the bypass circuit was perfused into a pig lung to absorb baboon anti-pig antibody during circulatory arrest at the time of HTx. In three baboons (Group-D), the donor lung was perfused, and in two baboons (Group-LD), a lung larger than the donor pig (weighing 5–7 kg) was perfused. In five baboons (Group-D + E), the donor lung was perfused, and exsanguination was also performed at the beginning of cardiopulmonary bypass (CPB), and the baboon blood was replaced with pretreated whole blood (N = 1) or packed red blood cell (RBC) and 50 ml of pretreated plasma (N = 4). The pretreated blood (N = 1) and serum (N = 4) were made by perfusing with other large pig lung (weighing 15 and 20 kg) before xenoHTx. Two baboons underwent pig kidney perfusion using an extracorporeal shunt from the right femoral artery to vein, 5 and

6 days after xenoHTx, because antibody-mediated rejection was suspected.

An immature baboon recipient of an orthotopic cardiac xenotransplant acquired from a donor pig, which

antigens [19, 20].

Figure 2.

56

survived 6 days after xenotransplant.

day before HTx until death.

Xenotransplantation - Comprehensive Study

The two control animals survived 4.5 and 18 hours, and the pathological changes of the grafts were compatible with HAR. The other animals survived 125 33 h (10–375 hors). The longest surviving baboon who survived 375 hours was in Group-D, but other two in Group-D died of HAR. All baboons in Group-LD and Group-D + E survived more than 4 days after XenoHTx. One in Group-D died of CMV infection and one in Group-LD died of pneumonia. One in Group-LD and four in Group-D + E died of acute cellular rejection. In summary, examination and echocardiography revealed no evidence of hyperacute rejection in baboons surviving more than 1 day. The longest survivor (375 hours) died of CMV infection with microscopic evidence of mild delayed HAR and graft coronary atherosclerosis. A variable amount of delayed xenograft rejection (DXR) was observed histologically, among the other recipient baboons (Table 3 and Figure 2) [20].

Another baboon which underwent large pig lung perfusion and is given Tac + MTX without splenectomy survived 16 days, and the autopsied graft showed mild DXR and moderate GCAS [20].

#### 8. The role of anti-pig antibody in pig-to-baboon xenoHTx rejection

To investigate the role of anti-pig Ab in discordant xenograft rejection, these 12 baboons were divided into 2 groups: Group-S (n = 4) died within 24 hr. of HTx and Group-L (n = 8) survived more than 24 hr. [19]. Mean survival period was 9.8 3.0 h in Group-S and 151 33 h in Group-L. Baboon anti-pig Ab was measured before CPB, before circulatory arrest, during AbA, at the end of CPB, and daily after HTx. Anti-RBC Ab was measured by the titration method at temperatures of 4 degrees C and 37 degrees C (RAb-4 and RAb-37). Anti-endothelial cell Ab (EAb) and anti-white blood cell Ab (WAb) titers were measured with enzymelinked immunosorbent assay (ELISA). RAb titration > or = 1/4 and EAb and WAb > or = 1/256 were determined to be seropositive. Seropositive rate of RAb-37 at the end of CPB (endCPB) in Group-L was significantly higher than that in Group-S (8/8 vs. 1/4; P < 0.05). The seronegative rates of RBC-4 and EAb (endCPB) in Group-L were higher than those in Group-S (7/8 vs. 1/4 and 6/8 vs. 1/4, respectively), but not significantly. There was no difference in seronegative rate of WAb (endCPB) between both groups. More than fourfold decrease in RAb-4 and RAb-37 by AbA with a pig lung was observed in 5 and 7 of 8 baboons, while EAb and WAb did not change by AbA. In all of Group-L, RAb-4 reverted to seropositive within 3 days after HTx. In four of Group-L, RAb-37 became S(+), 1 or 2 days before death by rejection. EAb became seropositive in all of Group-S, but five of them survived more than 5 days after seroconversion. It was concluded that a pig lung absorbed RAb-4 and RAb-37 may play a role in DXR.

After I came back to Japan, the role of RAb-37 on pig-to-baboon xenoHTx was investigated using sequential heterotopic HTx [21]. Fifteen pig hearts were obtained from pigs weighing 6.4–91 kg. Eleven hearts from pigs larger than the recipient were used for perfusion, and four hearts from a pig of the same size as the recipient for heterotopic transplant donor heart. Four female baboons weighing 5.9–8.1 kg received Tac (12 mg.kg) and CAM (50 mg/kg) p.o. daily 2 weeks before and after xenoHTx. After perfusion with two or three large pig hearts, a pig heart was heterotopically transplanted in the right neck of recipient baboon. As the second and third recipient baboons died of hypotension during the third pig heart perfusion and could not undergo heterotopic xenoHTx, the last baboon underwent two pig heart perfusion and subsequent heterotopic xenoHTx. All first perfused hearts and two second perfused hearts were hyperacutely rejected within 30 minutes of perfusion, but the other two second and all third


perfused hearts were not rejected within 2 hours after perfusion. The first and last transplanted pig hearts stopped beating 6 days and 18 hours after xenoHTx. Histological examination showed no rejection findings in the myocardium of the graft taken at 1 hour after xenoHTx, but the explanted grafts after cardiac arrest showed massive necrosis with ischemic change which suggested some kinds of DXR. RAb-37 prior to perfusion in all baboons was 1: 256 or 1:512, but that at 1 hour after XenoHTx was less than 1:4 which was considered to be negative. These findings suggested that RAb-37 may play an important role in DXR in pig-

We also investigated the differences between newborn and adult natural heterophile anti-pig red blood cell IgM xenoantibodies as correlates of xenograft survival [22] (Table 4). Newborns and younger infants have significantly lower titers

After coming back to Japan, Kawauchi M also investigated ontogeny of RAb-37 and HAR in 15 macaque monkeys [23]. Ten hearts from newborn Gottingen minia-

macaque monkeys (52, 59, 75, 101, 108, 114, 129, 151, 181, and 192 days old) without immunosuppressive therapy. RAb-37 prior to xenoHTx were gradually increased according to the age of the monkeys. All six donor hearts in the recipients younger than 4 months survived 6 hours, and then the animals were killed while the donor hearts were beating. Donor hearts in four infant recipients ages 129, 151, 181, and 192 days were hyperacutely rejected at 19, 22, 29, and 9 minutes. The pig hearts in

These two findings may suggest that newborn and younger infants may be more

9. Transgenic pig-to-rhesus monkey orthotopic discordant xenoHTx

in the future [24]. Moreover, they showed the possibility that both the

As Miyagawa et al. demonstrated the effect of the human beta-D mannoside beta-1,4-N-acetylglucosaminyltransferase III (GnT-III) gene in downregulating the xenoantigen of pig heart grafts, using a pig to cynomolgus monkey transplantation model suggests that this approach may be useful in clinical xenotransplantation

decay-accelerating factor (DAF) and GnT-III double transgenic pig skin xenografts could be used in place of human skin allografts in the cases of severe burns [25]. Then, after coming back to Japan, the author and Japanese colleagues underwent orthotopic discordant xenoHTx using DAF and GnT-III transgenic pig heart xenografts (unpublished data). Donor hearts were obtained from two F1 pigs, six DAF transgenic pigs (five hetero DAF and one homo DAF), and three GNT-III transgenic pigs and transplanted orthotopically in adult rhesus monkey with deep hypothermia and circulatory arrest. All animals received no immunosuppressive

In two baboons, a F1 pig heart was transplanted for control (Group-C). In three baboons, the blood in the bypass circuit was perfused into a hetero DAF pig heart or lung to absorb baboon anti-pig antibody during circulatory arrest at the time of

In the one control animal, the graft stopped beating 21 minutes after aortic unclamping before weaning from cardiopulmonary bypass (CPB). All other 10 rhesus monkeys could wean from CPB and undergo chest closure, but only one in Group-DAF, one in Group-DAF + P, and two in Group-GNT-III could be removed from a ventilator. Two grafts in Group-C and two perfused pig hearts showed severe HAR. Other grafts showed various degree of HAR. These data suggested that

ture swine (6–12 days old) were heterotopically transplanted into 10 infant

of anti-pig RAb-4 and RAb-37 and anti-pig EAb-IgM than adult.

Challenge of Xenotransplantation in Pediatric Heart Transplantation

DOI: http://dx.doi.org/10.5772/intechopen.90321

the recipients younger than 4 months showed no findings of HAR.

suitable recipient of discordant xenoHTx.

drugs.

59

xenoHTx (Group-DAF + P).

to-baboon combination.

RAb-4 and RAb-37: human anti-pig red blood cell antibody titer at temperature of 4°C and 37°C, respectively. EAb-IgM and EAb-IgG: human anti-pig endothelial cell antibody (immunoglobulin M and G) titers, respectively. \*p < 0.01 vs. adult. \*\*p < 0.05 vs. adult.

\*\*\*p < 0.01 vs. cord blood or infant younger than 38 days old.

#### Table 4.

Human anti-pig antibody against red blood cell and endothelial cell.


DAF, decay-accelerating factor; GnT-III, beta-D mannoside beta-1,4-N-acetylglucosaminyltransferase III; AXC, aortic cross-clamping; CPB, cardiopulmonary bypass; HAR, hyperacute rejection; DXR, delayed xenograft rejection. a Group-C, controls; Group-DAF, transplanted DAF transgenic pig heart; Group-DAF + P, transplanted DAF transgenic pig heart and perfused with another pig heart; Group-GnT-III, transplanted GnT-III transgenic pig heart. b Hetero DAF transgenic pig.

c Homo DAF transgenic pig.

#### Table 5.

Results of orthotopic cardiac xenotransplantation between rhesus monkey recipients and transgenic pig donors.

#### Challenge of Xenotransplantation in Pediatric Heart Transplantation DOI: http://dx.doi.org/10.5772/intechopen.90321

perfused hearts were not rejected within 2 hours after perfusion. The first and last transplanted pig hearts stopped beating 6 days and 18 hours after xenoHTx. Histological examination showed no rejection findings in the myocardium of the graft taken at 1 hour after xenoHTx, but the explanted grafts after cardiac arrest showed massive necrosis with ischemic change which suggested some kinds of DXR. RAb-37 prior to perfusion in all baboons was 1: 256 or 1:512, but that at 1 hour after XenoHTx was less than 1:4 which was considered to be negative. These findings suggested that RAb-37 may play an important role in DXR in pigto-baboon combination.

We also investigated the differences between newborn and adult natural heterophile anti-pig red blood cell IgM xenoantibodies as correlates of xenograft survival [22] (Table 4). Newborns and younger infants have significantly lower titers of anti-pig RAb-4 and RAb-37 and anti-pig EAb-IgM than adult.

After coming back to Japan, Kawauchi M also investigated ontogeny of RAb-37 and HAR in 15 macaque monkeys [23]. Ten hearts from newborn Gottingen miniature swine (6–12 days old) were heterotopically transplanted into 10 infant macaque monkeys (52, 59, 75, 101, 108, 114, 129, 151, 181, and 192 days old) without immunosuppressive therapy. RAb-37 prior to xenoHTx were gradually increased according to the age of the monkeys. All six donor hearts in the recipients younger than 4 months survived 6 hours, and then the animals were killed while the donor hearts were beating. Donor hearts in four infant recipients ages 129, 151, 181, and 192 days were hyperacutely rejected at 19, 22, 29, and 9 minutes. The pig hearts in the recipients younger than 4 months showed no findings of HAR.

These two findings may suggest that newborn and younger infants may be more suitable recipient of discordant xenoHTx.

#### 9. Transgenic pig-to-rhesus monkey orthotopic discordant xenoHTx

As Miyagawa et al. demonstrated the effect of the human beta-D mannoside beta-1,4-N-acetylglucosaminyltransferase III (GnT-III) gene in downregulating the xenoantigen of pig heart grafts, using a pig to cynomolgus monkey transplantation model suggests that this approach may be useful in clinical xenotransplantation in the future [24]. Moreover, they showed the possibility that both the decay-accelerating factor (DAF) and GnT-III double transgenic pig skin xenografts could be used in place of human skin allografts in the cases of severe burns [25].

Then, after coming back to Japan, the author and Japanese colleagues underwent orthotopic discordant xenoHTx using DAF and GnT-III transgenic pig heart xenografts (unpublished data). Donor hearts were obtained from two F1 pigs, six DAF transgenic pigs (five hetero DAF and one homo DAF), and three GNT-III transgenic pigs and transplanted orthotopically in adult rhesus monkey with deep hypothermia and circulatory arrest. All animals received no immunosuppressive drugs.

In two baboons, a F1 pig heart was transplanted for control (Group-C). In three baboons, the blood in the bypass circuit was perfused into a hetero DAF pig heart or lung to absorb baboon anti-pig antibody during circulatory arrest at the time of xenoHTx (Group-DAF + P).

In the one control animal, the graft stopped beating 21 minutes after aortic unclamping before weaning from cardiopulmonary bypass (CPB). All other 10 rhesus monkeys could wean from CPB and undergo chest closure, but only one in Group-DAF, one in Group-DAF + P, and two in Group-GNT-III could be removed from a ventilator. Two grafts in Group-C and two perfused pig hearts showed severe HAR. Other grafts showed various degree of HAR. These data suggested that

RAb-4 RAb-37 EAb-IgM EAb-IgG

Off CPB and chest closure

None 104 Yes No Mild HAR

None 135 Yes Yes Mild HAR

None 126 Yes Yes Mild HAR

Heart 211 Yes No Moderate HAR

Heart 310 Yes No Moderate HAR

Lung 305 Yes Yes Mild HAR

None 73 Yes No Mild to

None 257 Yes Yes Mild to

None 493 Yes Yes Mild to

DAF, decay-accelerating factor; GnT-III, beta-D mannoside beta-1,4-N-acetylglucosaminyltransferase III; AXC, aortic cross-clamping; CPB, cardiopulmonary bypass; HAR, hyperacute rejection; DXR, delayed xenograft rejection.

Group-C, controls; Group-DAF, transplanted DAF transgenic pig heart; Group-DAF + P, transplanted DAF transgenic pig heart and perfused with another pig heart; Group-GnT-III, transplanted GnT-III transgenic pig heart.

Results of orthotopic cardiac xenotransplantation between rhesus monkey recipients and transgenic pig donors.

Extubation Pathology of

autopsied

moderate HAR

moderate HAR

moderate HAR

Adult 582 579 296 291 288 189 853 264 Cord blood <sup>144</sup> 181\* 69/96\* <sup>21</sup> 8.3\*\* <sup>683</sup> <sup>264</sup>

RAb-4 and RAb-37: human anti-pig red blood cell antibody titer at temperature of 4°C and 37°C, respectively. EAb-IgM and EAb-IgG: human anti-pig endothelial cell antibody (immunoglobulin M and G) titers, respectively.

> releasing AXC (minutes)

xenograft Donor pig DAF pig

1b F1 pig None 21 No No Severe HAR 2 F1 pig None 132 Yes No Severe HAR

Infant <sup>&</sup>lt;38 days old <sup>80</sup> <sup>58</sup>\* <sup>30</sup> <sup>19</sup>\* Infant <sup>&</sup>gt; = 38 days old 689 <sup>678</sup>\*\*\* <sup>239</sup> 149\*\*\*

Human anti-pig antibody against red blood cell and endothelial cell.

Treatment Survival after

\*\*\*p < 0.01 vs. cord blood or infant younger than 38 days old.

Xenotransplantation - Comprehensive Study

organ perfusion

\*p < 0.01 vs. adult. \*\*p < 0.05 vs. adult.

Group-C<sup>a</sup>

Group-DAF<sup>a</sup> 1 <sup>c</sup> DAFb transgenic pig

2 DAFb transgenic pig

3 DAF<sup>c</sup> transgenic pig

Group-DAF+P<sup>a</sup> 1 DAFb transgenic pig

2 DAFb transgenic pig

3 <sup>d</sup> DAFb transgenic pig

Group-GnT-III<sup>a</sup> 1 GnT-III transgenic pig

2 <sup>c</sup> GnT-III transgenic pig

3 GnT-III transgenic pig

Hetero DAF transgenic pig.

Homo DAF transgenic pig.

a

b

c

58

Table 5.

Table 4.

transgene of DAF or GNT-III might not be enough to suppress HAR in adult rhesus monkey which had high titers of anti-pig xenoantibodies.

#### 10. Recent concerns about xenotransplantation in children

Xenotransplantation has been proposed as a method of reducing the especially acute shortage of organs for babies and children. Early clinical trials of xenotransplantation will be a form of therapeutic research. Therapeutic research must offer some prospect of genuine benefit for the patient, but it involves greater uncertainties than treatment, and therefore greater caution must be exercised. Many working parties concerning xenotransplantation, such as the British Pediatric Association and the Medical Research Council, have advised that therapeutic research should not involve children if it could equally well be performed with adults. It would be difficult to justify the involvement of children in major and risky xenotransplantation trials before some of the uncertainties have been eliminated in trials involving adults. Therefore, the FDA and WHO also recommend that the first xenotransplantation trials involve adults rather than children.

Then, although the authors tried to continue animal experiment to start clinical pediatric xenoHTx in the mid-2000s, we resigned.

#### 11. Current status of pediatric heart transplantation in the world and Japan

After the Bailey's first xenoHTx, hundreds of neonates and small infants with end-stage heart failure are living today because of primary or secondary HTx in the world. The number of pediatric HTx has been increasing (Figure 3), and their survival has been acceptable in every recipient age (Figure 4).

When the author came back to Japan in 1994, there was no Transplant Act in Japan. In 1988, the Japan Medical Association professed that it would accept brain death as human death. In 1990, the Provisional Commission for the Study on

Brain Death and Organ Transplantation was set up in 1990. The draft of the Organ Transplantation Law was proposed in 1994. Finally, on October 16, 1997, the Organ Transplant Act took effect, which enabled brain dead organ donation only if the person expressed in writing prior to death his/her intent to agree to donate his/her organs. In addition, the Act states that "only persons 15 years and above can express to donate." Then, heart transplants to small children become

Pediatric heart transplants. Kaplan–Meier survival (transplants: January 1982–June 2016).

Challenge of Xenotransplantation in Pediatric Heart Transplantation

DOI: http://dx.doi.org/10.5772/intechopen.90321

So, we started to send children with end-stage heart failure to Dr. Bailey as other pediatricians did (Figure 5) and continued to perform xenoHTx experiments. But as mentioned above, we finished experiments due to the FDA and WHO recommendation against pediatric xenotransplantation. Since 2003, the author and members of Japanese Associations of Transplant patients made many efforts to revise the Act, and finally the Act was revised in 2010. After then, the

impossible.

61

Figure 5.

Figure 4.

Pediatric heart transplant in Japan.

Figure 3. Pediatric heart transplants. Recipient age (in years) distribution by year of transplant.

Challenge of Xenotransplantation in Pediatric Heart Transplantation DOI: http://dx.doi.org/10.5772/intechopen.90321

#### Figure 4.

transgene of DAF or GNT-III might not be enough to suppress HAR in adult rhesus

Xenotransplantation has been proposed as a method of reducing the especially acute shortage of organs for babies and children. Early clinical trials of xenotransplantation will be a form of therapeutic research. Therapeutic research must offer some prospect of genuine benefit for the patient, but it involves greater uncertainties than treatment, and therefore greater caution must be exercised. Many working parties concerning xenotransplantation, such as the British Pediatric Association and the Medical Research Council, have advised that therapeutic research should not involve children if it could equally well be performed with adults. It would be difficult to justify the involvement of children in major and risky xenotransplantation trials before some of the uncertainties have been eliminated in trials involving adults. Therefore, the FDA and WHO also recommend that the first xenotransplantation trials involve adults

Then, although the authors tried to continue animal experiment to start clinical

11. Current status of pediatric heart transplantation in the world and

After the Bailey's first xenoHTx, hundreds of neonates and small infants with end-stage heart failure are living today because of primary or secondary HTx in the world. The number of pediatric HTx has been increasing (Figure 3), and their

When the author came back to Japan in 1994, there was no Transplant Act in Japan. In 1988, the Japan Medical Association professed that it would accept brain death as human death. In 1990, the Provisional Commission for the Study on

monkey which had high titers of anti-pig xenoantibodies.

Xenotransplantation - Comprehensive Study

pediatric xenoHTx in the mid-2000s, we resigned.

survival has been acceptable in every recipient age (Figure 4).

Pediatric heart transplants. Recipient age (in years) distribution by year of transplant.

rather than children.

Japan

Figure 3.

60

10. Recent concerns about xenotransplantation in children

Pediatric heart transplants. Kaplan–Meier survival (transplants: January 1982–June 2016).

Figure 5. Pediatric heart transplant in Japan.

Brain Death and Organ Transplantation was set up in 1990. The draft of the Organ Transplantation Law was proposed in 1994. Finally, on October 16, 1997, the Organ Transplant Act took effect, which enabled brain dead organ donation only if the person expressed in writing prior to death his/her intent to agree to donate his/her organs. In addition, the Act states that "only persons 15 years and above can express to donate." Then, heart transplants to small children become impossible.

So, we started to send children with end-stage heart failure to Dr. Bailey as other pediatricians did (Figure 5) and continued to perform xenoHTx experiments. But as mentioned above, we finished experiments due to the FDA and WHO recommendation against pediatric xenotransplantation. Since 2003, the author and members of Japanese Associations of Transplant patients made many efforts to revise the Act, and finally the Act was revised in 2010. After then, the

number of pediatric HTx has increased and finally exceeded that of HTx abroad (Figures 5 and 6).

Unexpectedly, Dr. Bailey (Figure 7) died of cancer in May 2019.

Figure 6. Pediatric heart transplantation abroad from Japan.

Author details

Norihide Fukushima<sup>1</sup>

Center, Suita, Japan

Charles Nicolle, Rouen, France

Tokyo, Japan

63

\*, Motohiro Kawauchi<sup>2</sup>

Challenge of Xenotransplantation in Pediatric Heart Transplantation

DOI: http://dx.doi.org/10.5772/intechopen.90321

1 Department of Transplant Medicine, National Cerebral and Cardiovascular

2 Department of Cardiovascular Rehabilitation, Itabashi Rehabilitation Hospital,

3 Department of Thoracic and Cardiovascular Surgery, Rouen University Hospital

4 Immunogenetics/HLA Laboratory, Department of Pathology, Detroit Medical Center, Wayne State University Laboratories, Detroit, Michigan, United States

5 Department of Cardiovascular and Thoracic Surgery, Loma Linda University

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

School of Medicine, Loma Linda, California, United States

\*Address all correspondence to: nori@ncvc.go.jp

provided the original work is properly cited.

Sandra Nehlsen-Cannarella<sup>4</sup> and Leonard L. Bailey<sup>5</sup>

, Francois Bouchart<sup>3</sup>

,

Figure 7. The panel of Professor Leonard Bailey's memorial service.

Challenge of Xenotransplantation in Pediatric Heart Transplantation DOI: http://dx.doi.org/10.5772/intechopen.90321

#### Author details

number of pediatric HTx has increased and finally exceeded that of HTx abroad

Unexpectedly, Dr. Bailey (Figure 7) died of cancer in May 2019.

(Figures 5 and 6).

Xenotransplantation - Comprehensive Study

Figure 7.

62

Figure 6.

The panel of Professor Leonard Bailey's memorial service.

Pediatric heart transplantation abroad from Japan.

Norihide Fukushima<sup>1</sup> \*, Motohiro Kawauchi<sup>2</sup> , Francois Bouchart<sup>3</sup> , Sandra Nehlsen-Cannarella<sup>4</sup> and Leonard L. Bailey<sup>5</sup>

1 Department of Transplant Medicine, National Cerebral and Cardiovascular Center, Suita, Japan

2 Department of Cardiovascular Rehabilitation, Itabashi Rehabilitation Hospital, Tokyo, Japan

3 Department of Thoracic and Cardiovascular Surgery, Rouen University Hospital Charles Nicolle, Rouen, France

4 Immunogenetics/HLA Laboratory, Department of Pathology, Detroit Medical Center, Wayne State University Laboratories, Detroit, Michigan, United States

5 Department of Cardiovascular and Thoracic Surgery, Loma Linda University School of Medicine, Loma Linda, California, United States

\*Address all correspondence to: nori@ncvc.go.jp

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

### References

[1] Lower RR, Stoffer RC, Shumway NE. Homovital transplantation of the heart. The Journal of Thoracic and Cardiovascular Surgery. 1961;41: 196-204

[2] Barnard CN. The operation. A human cardiac transplant: An interim report of a successful operation performed at Groote Schuur hospital, Cape Town. South African Medical Journal. 1967;41: 1271-1274

[3] Kantrowitz A, Haller JD, Joos H, et al. Transplantation of the heart in an infant and an adult. The American Journal of Cardiology. 1968;22:782-790

[4] Bailey LL, Li Z, Lacour-Gayet F, et al. Orthotopic cardiac transplantation in the cyclosporine-treated neonate. Transplantation Proceedings. 1983;15 (Suppl 1):2956-2959

[5] Bailey LL, Jang J, Johnson W, et al. Orthotopic cardiac xenografting in the newborn goat. The Journal of Thoracic and Cardiovascular Surgery. 1985;89: 242-247

[6] Bailey LL, Nehlsen-Cannarella S. Observations on cardiac xenotransplantation. Transplantation Proceedings. 1986;18:88-92

[7] Youngest transplant patient dies in Britain after 28 days. New York Times. 1984

[8] Bailey LL, Nehlsen-Cannarella SL, Concepcion W, Jolley WB. Baboon-tohuman cardiac xenotransplantation in a neonate. Journal of the American Medical Association. 1985;254: 3321-3329

[9] Nehlsen-Cannarella SL, Chang L. Immunology and organ transplantation in the neonate and young infant. Critical Care Nursing Clinics of North America. 1992;4:179

[10] Alonso de Begona J, Gundry S, Kawauchi M, Bailey LL, et al. Assessment of baboon lymphocyte subsets and activity in cardiac xenobridging to allotransplantation. Transplantation Proceedings. 1992;24: 453-454

cardiac rejection. Xenotransplantation.

DOI: http://dx.doi.org/10.5772/intechopen.90321

Challenge of Xenotransplantation in Pediatric Heart Transplantation

[25] Fujita T, Miyagawa S, Ezoe K, et al. Skin graft of double transgenic pigs of N-acetylglucosaminyltransferase III (GnT-III) and DAF (CD55) genes survived in cynomolgus monkey for 31 days. Transplant Immunology. 2004;

13(4):259-264

[18] Cooper DKC, Ekser B, Tector AJ.

Immunobiological barriers to xenotransplantation. International Journal of Surgery. 2015;23(Pt B):

[19] Fukushima N, Bouchart F, Gundry SR, Nehlsen-Cannarella S, Gusewitch G, Chang L, et al. The role of anti-pig antibody in pig-to-baboon

rejection. Transplantation. 1994;57:

[20] Fukushima N, Gundry SR, Matsumiya G, Bouchart F, Zuppan C, Bailey LL. Histological findings in heart grafts after orthotopic pig to baboon cardiac transplantation. Transplantation

Proceedings. 1996;28:788-790

Yamaguchi T, Kobayashi Y,

[22] Fukushima N, Fagoaga O, Bouchart F, Grinde S, Gusewitch G, Thorpe R, et al. Comparison of newborn versus adult natural heterophile anti-pig red blood cell IgM xenoantibodies as correlates of xenograft survival. Transplatation Proceedings. 1994;26:

[21] Fukushima N, Shirakura R, Chang J,

Yoshitatsu M, et al. Prolonged survival of pig cardiac xenografts in baboons by sequential cardiac transplantation. Transplantation Proceedings. 1998;30:

[23] Kawauchi M, Nakajima J, Endoh M, Oka T, Takamoto S. Ontogeny of antipig xenoantibody and hyperacute rejection. Transplantation. 2000;70(4):686-688

[24] Miyagawa S, Murakami H, Takahagi Y, et al. Remodeling of the

major pig xenoantigen by Nacetylglucosaminyltransferase III in transgenic pig. The Journal of Biological Chemistry. 2001;276(42):

cardiac xenotransplant

2000;7:31-41

211-216

923-928

3815-3817

1395-1396

39310-39319

65

[11] Michler RE, O'Hair DP, Xu H, Shah AS, Itescu S. Newborn baboon immunity: Lessons in cross-species transplantation. Society of Thoracic Surgeons. 1995;60:S581

[12] Kawauchi M, Gundry SR, Alonso de Begona J, Razzouk AJ, et al. Prolonged survival of orthotopically transplanted heart xenograft in infant baboons. The Journal of Thoracic and Cardiovascular Surgery. 1993;106:779-786

[13] Fukushima N, Fagoaga O, Kawauchi M, et al. Lymphocyte subset markers as predictors of survival after concordant cardiac xenotransplantation. Transplantation Proceedings. 1994;26: 1212-1213

[14] Kawauchi M, Gundry SR, Alonso de Bogona J, Beierle F, Bailey LL. Plasma level of FK 506 in newborn goats and infant baboons. Transplantation Proceedings. 1991;23:2755-2756

[15] Fukushima N, Kawauchi M, Bouchart F, Gundry SR, Zuppan CW, Ruiz CE, et al. Graft atherosclerosis in concordant cardiac transplantation. Transplantation Proceedings. 1994;26: 1059-1060

[16] Toi te Taiao. The cultural, ethical and spiritual aspects of animal-tohuman transplantation. A report on xenotransplantation. Bioethics Council. August 2005. Available at: www.bioeth ics.org.nz

[17] Rose AG, Cooper DK. Venular thrombosis is the key event in the pathogenesis of antibody-mediated Challenge of Xenotransplantation in Pediatric Heart Transplantation DOI: http://dx.doi.org/10.5772/intechopen.90321

cardiac rejection. Xenotransplantation. 2000;7:31-41

References

196-204

1271-1274

[1] Lower RR, Stoffer RC, Shumway NE. Homovital transplantation of the heart.

Xenotransplantation - Comprehensive Study

[10] Alonso de Begona J, Gundry S, Kawauchi M, Bailey LL, et al. Assessment of baboon lymphocyte subsets and activity in cardiac xenobridging to allotransplantation. Transplantation Proceedings. 1992;24:

[11] Michler RE, O'Hair DP, Xu H, Shah AS, Itescu S. Newborn baboon immunity: Lessons in cross-species transplantation. Society of Thoracic

[12] Kawauchi M, Gundry SR, Alonso de Begona J, Razzouk AJ, et al. Prolonged survival of orthotopically transplanted heart xenograft in infant baboons. The Journal of Thoracic and Cardiovascular

Surgeons. 1995;60:S581

Surgery. 1993;106:779-786

[13] Fukushima N, Fagoaga O,

Kawauchi M, et al. Lymphocyte subset markers as predictors of survival after concordant cardiac xenotransplantation. Transplantation Proceedings. 1994;26:

[14] Kawauchi M, Gundry SR, Alonso de Bogona J, Beierle F, Bailey LL. Plasma level of FK 506 in newborn goats and infant baboons. Transplantation Proceedings. 1991;23:2755-2756

[15] Fukushima N, Kawauchi M, Bouchart F, Gundry SR, Zuppan CW, Ruiz CE, et al. Graft atherosclerosis in concordant cardiac transplantation. Transplantation Proceedings. 1994;26:

[16] Toi te Taiao. The cultural, ethical and spiritual aspects of animal-tohuman transplantation. A report on xenotransplantation. Bioethics Council. August 2005. Available at: www.bioeth

[17] Rose AG, Cooper DK. Venular thrombosis is the key event in the pathogenesis of antibody-mediated

453-454

1212-1213

1059-1060

ics.org.nz

[2] Barnard CN. The operation. A human cardiac transplant: An interim report of a successful operation performed at Groote Schuur hospital, Cape Town. South African Medical Journal. 1967;41:

[3] Kantrowitz A, Haller JD, Joos H, et al. Transplantation of the heart in an infant and an adult. The American Journal of

[4] Bailey LL, Li Z, Lacour-Gayet F, et al. Orthotopic cardiac transplantation in the cyclosporine-treated neonate. Transplantation Proceedings. 1983;15

[5] Bailey LL, Jang J, Johnson W, et al. Orthotopic cardiac xenografting in the newborn goat. The Journal of Thoracic and Cardiovascular Surgery. 1985;89:

[6] Bailey LL, Nehlsen-Cannarella S.

xenotransplantation. Transplantation

[7] Youngest transplant patient dies in Britain after 28 days. New York Times.

[8] Bailey LL, Nehlsen-Cannarella SL, Concepcion W, Jolley WB. Baboon-tohuman cardiac xenotransplantation in a neonate. Journal of the American Medical Association. 1985;254:

[9] Nehlsen-Cannarella SL, Chang L. Immunology and organ transplantation in the neonate and young infant. Critical Care Nursing Clinics of North America.

Observations on cardiac

Proceedings. 1986;18:88-92

Cardiology. 1968;22:782-790

(Suppl 1):2956-2959

242-247

1984

3321-3329

1992;4:179

64

The Journal of Thoracic and Cardiovascular Surgery. 1961;41: [18] Cooper DKC, Ekser B, Tector AJ. Immunobiological barriers to xenotransplantation. International Journal of Surgery. 2015;23(Pt B): 211-216

[19] Fukushima N, Bouchart F, Gundry SR, Nehlsen-Cannarella S, Gusewitch G, Chang L, et al. The role of anti-pig antibody in pig-to-baboon cardiac xenotransplant rejection. Transplantation. 1994;57: 923-928

[20] Fukushima N, Gundry SR, Matsumiya G, Bouchart F, Zuppan C, Bailey LL. Histological findings in heart grafts after orthotopic pig to baboon cardiac transplantation. Transplantation Proceedings. 1996;28:788-790

[21] Fukushima N, Shirakura R, Chang J, Yamaguchi T, Kobayashi Y, Yoshitatsu M, et al. Prolonged survival of pig cardiac xenografts in baboons by sequential cardiac transplantation. Transplantation Proceedings. 1998;30: 3815-3817

[22] Fukushima N, Fagoaga O, Bouchart F, Grinde S, Gusewitch G, Thorpe R, et al. Comparison of newborn versus adult natural heterophile anti-pig red blood cell IgM xenoantibodies as correlates of xenograft survival. Transplatation Proceedings. 1994;26: 1395-1396

[23] Kawauchi M, Nakajima J, Endoh M, Oka T, Takamoto S. Ontogeny of antipig xenoantibody and hyperacute rejection. Transplantation. 2000;70(4):686-688

[24] Miyagawa S, Murakami H, Takahagi Y, et al. Remodeling of the major pig xenoantigen by Nacetylglucosaminyltransferase III in transgenic pig. The Journal of Biological Chemistry. 2001;276(42): 39310-39319

[25] Fujita T, Miyagawa S, Ezoe K, et al. Skin graft of double transgenic pigs of N-acetylglucosaminyltransferase III (GnT-III) and DAF (CD55) genes survived in cynomolgus monkey for 31 days. Transplant Immunology. 2004; 13(4):259-264

**67**

Section 2

3D-Bioprinting and

Decellularization

Section 2
