Xenotransplantation: The Obsolete Waiting List

Imagine yourself.

Living life, living happy. You have plans to travel over summer, start a new hobby, and go out with friends on the weekend. But one day, you’re told you can’t. Well, you can but you have to wait until the proper arrangements are made.

So you wait.

But slowly your wait goes from a few hours to a few days to a few weeks and it seems like there’s no end in sight. Your life is put on pause. This may sound absurd — maybe not right now with quarantining — but for thousands of people this is a reality.

They want exactly what you do:

• the freedom to travel
• meet friends
• and have fun

But they can achieve this only by undergoing an organ transplant.

Currently, the 112,880 patients are on the national organ waitlist in America. Yet, in 2019, only 39,718 transplants were performed. This large annual difference results in 20 people dying every day waiting for an organ transplant — and this is just in America alone.

There are over 1 million people on the global organ waiting list. Yet, only 10% get the crucial transplant they need to survive. This results in hundreds of thousands of deaths from the waiting patients; on top of, people being murdered to obtain these organs through the black market in many Asian communities.

All due to the huge shortage of organs.

• only 3 in 1,000 people die in a way that doesn’t damage organs
• 90% of U.S. adults support organ donation but only 60% are registered donors
• The average wait time for an organ transplant is 3–5 years

That last one shocked me too.

However, all of this doesn’t have to be a reality in the near future. With advances in the field of xenotransplantation, an organ waitlist might soon become obsolete. But first, where did transplantation even begin?

Mythical Beginnings

The earliest reports of organ transplantation date back to ancient civilization. Greek, Roman and Chinese myths featured powerful gods and healers performing transplants with cadavers and animals. Early accounts of skin grafting on patients also date back to Indian doctors in 800 B.C.

However, with the arrival of recorded history, comes a less questionable origin: Italian surgeon, Gasparo Tagliacozzi.

Tagliacozzi with his illustrated De Curtorum Chirurgia per Insitionem

In the sixteenth century, Tagliacozzi was the first to use skin transplants for plastic reconstruction. He used grafted skin from patients’ arms to treat sword fight wounds — often deconstructed noses.

He compiled all his work into a text called De Curtorum Chirurgia per Insitionem (roughly translating to The Surgical Restoration of Defects by Grafting). Along with insights on plastic surgery, this book was also the first to describe the immunologic rejection of skin taken from different donors, which Tagliacozzi coined the “the force and power of individuality”. (basically hinting on the power of our immune system on organ rejection, more on that later😉)

For the next few centuries, no real progress was made on advancing organ transplantation. That was until two technological breakthroughs set the stage:

1. Anaesthetics

“a drug or agent used to abolish the sensation of pain, to achieve adequate muscle relaxation during surgery, to calm fear and allay anxiety, and to produce amnesia for the event.” The American Heritage® Medical Dictionary

• tl;dr: blocks neurotransmitter receptors in the brain for pain perception
• Wide-scale medical anesthesia used in the early 1800s
• Used for recreational use for centuries before
• Early anesthesia was inhaled chemicals such as nitrous oxide (laughing gas) or diethyl ether

2. Antiseptics

“antimicrobial substances that are applied to living tissue/skin to reduce the possibility of infection, sepsis, or putrefaction” — Clinical Microbiology Reviews

• tl;dr: reduced chances of infection during surgery
• Formally documented by the British surgeon Joseph Lister in 1867

His beard game was also terrific

• Inspired by Louis Pasteur’s work in microbiology
• Involved hand-washing, sterilizing wounds, instruments, and operating rooms

First Successful Transplant

Both of these innovations greatly advanced the field of medicine and organ transplantation. In 1905, Eduard Zirm, an Austrian ophthalmologist, performed the world’s first corneal transplant. The groundbreaking procedure restored vision to the previously blinded 45-year-old Alois Gloga.

Both corneas — the eyes’ transparent, protective outer layer — were replaced in a procedure called penetrating keratoplasty and was the first successful allograft: transplantation carried between genetically different same species. Gloga survived for 3 years before dying to leukemia.

Fast forward a decade…

The same year, he also received the Nobel Prize in Physiology or Medicine for his pioneering work.

French surgeon, Alexis Carrel, develops a new method for vascular anastomoses — connection of two blood vessels. This aided him in 1912, when Dr. Carrel connected the blood vessel of one dog’s kidneys to another dog receiving it and *technically* performed the first successful kidney transplant.

Fast forward another few decades…

At this point, enough research and trials had been done for doctors to realize the immunological barriers of transplantation. Many surgeons had utilized immunosuppression techniques. But no one had achieved a successful long term transplant. Enter the father of modern transplantation: Dr. Joseph Murray.

Murray(left) accepting his Nobel Prize for Medicine in 1990

Murray was the first to successfully transfer a human kidney into another human in 1954. The patient, 23-year-old Ronald Herrick, received the donation from his twin brother Richard. It was the first successful human isograft: a graft taken from a genetically identical person, such as two genetically identical twins.

Although Ukrainian doctor Yu Yu Voronoy also transplanted a kidney from a deceased donor in 1936, the recipient died two days after because of rejection and the kidneys were not functional either.

Murray and a team of doctors at Boston’s Peter Bent Brigham Hospital carrying out the surgey

This triumph boosted morale and sparked motivation for clinicians and researchers in the field of organ transplantation. In the following years, the world marveled at similar accomplishments such as simultaneous liver and pancreas transplants in the early 60s and the first heart transplant in 1967 by South African surgeon Christiaan Barnard.

But the shortage of organs still remained a big problem. Scientists had found a way to do transplantation but didn’t have a consistent supply to help patients.

This lead to the possibility of using non human organs for transplants instead.

Although many scientists attempted to conduct organ transplants between baboons, chimpanzees, and pigs in the early twentieth century, all of them proved to be unsuccessful. It wasn’t until 50 years later when real progress was made.

The best organ candidate became the kidney mainly due to the high amounts of incurable renal (kidney-related) issues and disorders at the time. The chimpanzee was also selected as the non-human organ donor for primarily three reasons:

1. Their close taxonomic relationship to homo sapiens
2. Their renal function and size corresponding closely to homo sapiens
3. Their similar blood types of A and O and thereby ease of blood transfusion because of universal donor

Once all the stones were set in place, the first trials started. Transplant surgeon, Keith Reemtsma of Tulane University, conducted the first chimpanzee to human transplants from 1963 to 1964.

Keith Reemtsma of Tulane University

All the trial patients were terminal uremics (had failed kidneys) and were maintained on painful and expensive dialysis. They were given the option of:

• supportive treatment only
• an allograft from a relative
• a cadaveric allograft if available
• or a heterograft (graft from non-human species).

If the first three options were unable to be met, the patients agreed to a revolutionary yet potentially life-threatening transplant. With the help of pretransplantation treatment with immunosuppressant aids like azathioprine, actinomycin C, and steroids, six patients in total received the renal heterotransplants from chimpanzees.

The donor chimpanzees also received pretransplantation treatment with endotracheal anesthesia(inhaled down the trachea/throat with a tube), monitoring of blood pressure, electrocardiogram, and body temperature.

Once the graft was ready to attach, the external iliac artery (kidney artery) and vein were anastomosed — shoutout Dr. Carrel — to the graft. It took from 36 to 43 minutes until blood flow was established in the graft and the patient. But after that, it was complete.

Although the six patients only lived between 19 to 91 days, the procedure marked the start of xenotransplantation.

What is Xenotransplantation?

As defined by the Food and Drug Administration, xenotransplantation is any procedure that involves the transplantation, implantation or infusion into a human recipient of either

1. live cells, tissues, or organs from a nonhuman animal source or
2. human body fluids, cells, tissues or organs that have had ex vivo contact with live nonhuman animal cells, tissues or organs.

It can be more simply summed as:

“the process of grafting or transplanting organs or tissues between members of different species.”

With thousands dying every year from waiting on a life-saving transplant, xenotransplantation provides a hopeful solution to the growing need of organs. With kidney dialysis treatments also averaging around $1.45 million per patient for life, xenotransplants could save billions of dollars. The American Journal of Transplantation, in 2016, estimated around $46 billion dollars and $12 billion dollars saved per year in healthcare costs taxpayer money, respectively.

Initially, chimpanzees were chosen as potential donors by researchers such as Reemtsma. However, non-human primates were soon ruled out as donors for practical and ethical reasons including:

• Highest risk of transmitting viruses capable of infecting humans
• HIV originated from chimpanzees
• Public reluctance to exploit due to humanistic features
• Difficulty maintaining sufficient supply with breeding

Due to this, the next thing became:

wait no, not like this, like this:

Pigs are the perfect animal donor that fits all the boxes necessary for xenotransplantation:

• They can be raised in a clean environment and have a reduced risk of infection
• There is no supply issue since they are already widely bred for the food industry
• They have similar anatomy (structure) and physiology (function) to human organs
• Materials from pigs have also been regularly used for medical purposes for heart valve replacement and protective skin grafts
• If treated humanely, they also have a less ethical issue

However, since it is a non-human donor, far greater issues arise beyond normal graft rejection, including many critical metabolic and immune incompatibilities. More drugs are often required to allow the graft to be accepted and remain functioning an, oftentimes, immunosuppressing is useless because of the human immune system.

Problems with (Xeno)Transplantation

Before we can get into xenotransplantation issues between pig donors and humans hosts, its necessary to understand the problems with allograft transplantation between a human donor and another human host.

There are essentially three different types of organ transplant rejections: hyperacute, acute, and chronic. If you know your immunology, here’s a graph explaining each one (courtesy of Dr. Brian McDaniel). If not, lets quickly go over it together!

Since grafted organs express different antigens(tiny ‘Iabel’ proteins outside of molecules) than its host, the immune system attacks the graft thinking it is a harmful foreign invasion. During transplants, most damage is present in the blood vessels since they are the interface between donated tissues and patient.

Due to this, graft antigens lining the endothelium (outer tissue) come into immediate contact with the immune system in the host’s blood. This damage often can result in mild fibrosis(tissue scarring causing thickening) to a hemorrhage(ruptured blood vessel). This leads us to the first type of rejection:

Hyperacute Rejection

Quick Facts:

• Immediate onset
• Mediated by preformed antibodies against blood type incompatibility
• Hypersensitivity reaction type I

This is the first and most apparent type of rejection. So much so, in fact, that it’s often present during organ transparent surgery.

It is caused by ABO blood type mismatch and results in pre-formed antibodies(proteins that bind and marks antigens for attacking) going to the graft and triggering attack. For example, a person with type B blood would already have preformed antibodies against type A blood.

This mismatch would result in complexities such as thrombosis(blood clot inside a blood vessel) and occlusion(the blockage or closing of a blood vessel) of the graft vessel.

It is luckily very visible with noticeable pale areas of axemia(blood removed from circulation but remaining in certain areas)and bleeding. This reaction is also classified as hypersensitivity type I reaction, which is just a metric use to measure extreme physical sensitivity to particular substances or conditions (such as the donor tissue)

Acute Rejection

Quick Facts:

• Onset in weeks to months
• Mediated by T-cell response against foreign MHC
• Hypersensitivity reaction type IV

The human immune system is a complex machine inside of all of us. It is a large army with millions of molecular soldiers, guards, weapons factories, and more, that are constantly fighting off bacteria and infections in our bodies.

The immune system has nearly a dozen responsibilities, from producing markers for harmful invaders to coordinating an attack with over 21 different types of cells. One of the most important cells is a T cell or a lymphocyte. These cells differentiate into different functions but primarily act as guards. They also play a big role in transplant rejection.

If the organ has been in the body for quite some time, acute rejection can kick in. This is the most common type of rejection and is a T cell-mediated response against foreign major histocompatibility complex(MHC).

MHC (also called HLA or human leukocyte antigen) is a unqiue section of a vertebrate’s DNA that codes for cell surface proteins essential for the adaptive immune system (immune system responsible for new pathogens). Each human expresses a unqiue set of 12 to 14 different MHC alleles(one of multiple alternative forms of a gene on a single spot on a chromosome) with very high variation.

Only twins — because of their similar genetic makeup — have same MHC molecules, making transplantation easier. MHC peptide antigens specifically provoke a immune response from cytotoxic(killer) T cells and contribute to rejection.

MHC on its own is very complicated and demands its own seperate article.

Chronic Rejection

Quick Facts:

• Onset in months to years
• Mediated by T-cell response against foreign MHC that looks like local MHC
• Hypersensitivity reaction type III and IV

Out of the three, chronic rejection is the least understood. Its exact mechanisms are still not understood by scientists. What we do know, is that it also involves the major histocompatibility complex; when the recipient’s anti-HLA antibodies attack HLA molecules on transplanted endothelial tissue.

This results in intimal tissue thickening, fibrosis of graft vessels, and organ atrophy (organ shrinkage). Compared to acute rejection, this is a slow progressive decline in organ dysfunction. In a way, it can be thought of as accelerated aging, while acute is rapid.

Similarly, while acute is curable with treatment and immunosuppressants, patients that reach chronic rejection need to receive a completely new organ transplant.

Graft vs. Host

On rare occasions, immune cells of the organ draft may also attack the recipient. This is mostly seen in bone marrow transplants and leads to hypersensitivity reaction IV in various organ systems in the body. Similar to acute rejection, this can also be treated by immunosuppressants.

Xenotransplantation

Now that we got through traditional graft problems, let’s get into the nitty-gritty with xenotransplantation.

To simplify things a bit, let’s divide the problems into two types:

1. Immunological
2. Infectious

Here’s a quick timeline chronologically paving the progress in xenograft rejection:

Now, lets get into some of the key immunological problems.

One of the primary problems in pig xenotransplantation to humans is caused by an antigen called Galactose-alpha-1,3-galactose. Alpha-gal, for short, is a carbohydrate found in most mammalian cell membranes — excluding primates and humans, who have lost the GGTA1 gene that codes for alpha-gal.

Chemical composition of Alpha gal

Many bacteria and microbes have alpha-gal on their surfaces so anti-alpha gal immunoglobulin G antibodies are very common in our bodies. Due to this, the antibodies would immediately bind to the endothelial cells of a pig graft and cause immediate hyperacute rejection or delayed xenograft rejection.

However, in the early 2000s, this was luckily overcome by Dr. Mohammed Mohiuddin, director of the Cardiac Xenotransplantation Program at the University of Maryland, through genetic engineering pigs with the Alpha-gal gene knocked out were created.

Another innate immune barrier to xenografts is natural killer cells. These cells are specialized large granular lymphocytes that don’t need antigens to bind to or the presence of a foreign MHC — known as swine leukocyte antigen(SLA) in pigs. Because these cells don’t need initial activation from the immune system they are often times unaffected by traditional immunosuppression used against hyperacute rejections.

Along with natural killer cells, macrophages also pose a huge immune barrier. They are a type of white blood cell that engulfs and digests dangerous foreign objects. A transmembrane protein, known as CD47, is expressed in all of our cells and signals macrophages not to attack. A regulatory membrane glycoprotein on macrophages, known as the signal-regulatory protein alpha or SIRPα, acts as an inhibitory receptor and lets macrophages know not to engulf human cells in a process called phagocytosis.

Example of a few immune cells

However, porcine CD47 cells do not transmit that inhibitory signal to human SIRPα which results in rapid destruction of tissue on the graft. As a solution, it was hypothesized that putting human CD47 cells into PBSC (peripheral blood stem cells) pigs might help create tolerance for this issue. This was proven successful in 2014.

Now, lets touch onto the infectious side.

After a decade of technological advancements and discoveries, in 1997, governments made further xenotransplantation research illegal globally. This might seem like another example of government regulation coming in between the private sector, but the choice was an extremely important one — especially now.

This choice was made due to fears of a pig virus pandemic. Porcine endogenous retrovirus is a type of retrovirus that can be derived from the pig’s genome. Mutations in the genome can lead to the virus becoming harmful to humans and harming many as swine flu has over history.

With pig grafts, the genome still contains the PERV gene which if transplanted and mutated can lead to damage to the person with the transplant and potential to spread to others and take many people’s lives.

This, however, was overcome by a leading figure in the field of xenotransplantation research: George Church. His team used gene editing with CRISPR-Cas to inactive all the places with PERVs in pig embryonic fibroblasts. They then used somatic cell nuclear transfer (SCNT) to produce embryos from the fibroblasts and transferred them into surrogate pigs. Using such an approach, they successfully generated PERV-inactivated pigs.

The same approach is what Church’s company Egenisis specializes in: gene editing pigs. They are currently working on groundbreaking research in the immunology and virology problems of xenotransplantation.

Overview of Church’s process

I know what you’re thinking.

There still isn’t a solution to some of the immunological problems so what’s the point?

Well, let’s get into it.

Solutions for Xenotransplantation

Ironically, many people believe that the solution for xenotransplantation is not xenotransplantation. Tissue engineering is believed to be a much better and ideal alternative to the difficulties of xenotransplantation. Why wouldn’t it be? You can just engineer tissues to grow into fully functional organs using those new things called stem cells, right? Wrong.

Stem cell tissue engineering where we can print out full organs is far from being a reality in the near future. Although scientists have found a way to engineer bone marrow to provide and extract stem cells, they can’t reliably and easily convert those stem cells to any desired type of cell.

Even so, organs are insanely complex. Let’s take one of the (relatively) simplest ones: the kidney.

Looks pretty simple, blood travels down from the thoracic aorta into the renal artery and goes inside the kidney. The blood vessels diffuse waste out and pump the clean blood out of the renal vein and into the vena cava. Basically like a smaller version of those sink filters, right? Nope, wrong again.

The working and functioning are extremely precise to a fine granularity that is very difficult to recreate with the current level of scientific advancements. Even with that being, were missing tiny components such as the specialized nephrons on the endothelial tissue, combining the slender ureter with the waste from the neurons, and the complex functioning and communication of the tiny external tissues that help initiate steps infiltration, such as the Bowman’s capsule.

Heres a more detailed diagram to help:

The reality is that the scientific timeline of being able to use tissue engineering instead of organ transplants is long and far one. On the other hand, innovations in the field of xenotransplantation are only 2–5 years away from being a reality.

With that out of the way, there are two very promising paths being taken currently towards clinical trials with xenotransplantation:

1. Mixed chimerism

2. Thymic trnasplantaion

Mixed Chimerism

Stem cells are these special types of cells that are undifferentiated and can transform into any type of cell in the body from brain cells (neurons), blood cells (hematopoietic cells) and even bone cells(osteocytes).

Hemopoietic stem cell transplantation is the replacement of damaged or harmful hematopoietic stem cells with healthy hematopoietic stem cells. It is most commonly done through a bone marrow transplant and due to its nature, can result in the coexistence of immune cells from both the donor and patient, all in immunological tolerance harmony.

That is known as mixed chimerism which is also the inspiration behind one of the most promising solutions to xenotransplantation. This approach relies on mixed chimerism leading to T cell and B cell (produces antibodies for T cells) immune tolerization.

Coexistence of donor and host cells

This theory was tested with non-myleoblative lab mice(which basically means their immune systems were tweaked) with a similar tolerance model of a human immune system. This involved radiation and monoclonal antibodies against T cells and NKC (anti-CD4, anti-CD8, etc) from a bone marrow donor.

These mice successfully developed mixed tolerism and accepted the pig skin graft without immunosuppression (for up to a 100+ days). It is important to note, that this is only specific tolerance with the bone marrow donor and its antibodies. NOT general tolerance to all pig grafts as a third party skin graft was also tested and rejected. By introducing the antibodies, the mice were able to create conditions for the pig cells and antigens and were able to create tolerance of anti-Gal secreting B cell and T cell tolerance.

This discovery also proved that pig cells can be coexisting with humans and induced curiosity for the second possible treatment.

Thymic Transplantation

The thymus is a lymphoid gland part of our endocrine system which is the system that manages our chemical messages. It is also where T cells are matured and adapted to different foreign invaders. This also makes it an ideal candidate for manipulation to allow for T cell toleration.

Thymus interaction with T cells

Back in the 1990’s, the first experiments with thymus transplantation took place. Immunocompetent (normal immune system) mice had their thymus removed and T cells deprived before being given a pig thymus. The pig thymus would then generate a new collection of pig T cells that would help create antibodies that are resistant to porcine cells.

This same practice was then applied in larger primates like a baboon to create T cell tolerance and potentially allow for xenograft acceptance. It was proven success and a baboon carried a life-supporting pig kidney got 6 months with no issues. An in vitro (studied in a test tube) study showed specific pig resistance in the baboon which allowed for the transplantation.

This can also be worked in the human immune system with human immune mice. Like the mixed chimerism trials, these results proved the principle that human central T cell tolerance to porcine xenografts can be induced.

Although these results may apply to only human immune mice or primates, they indicate that porcine xenotransplantation can actually be possible and pushes that one important step closer to clinical trials.

It’s imperative that we get there since only then will we be truly able to understand pure human immune system response and once we do, we might see a future where thousands of deaths are preventable and the organ waitlist is obsolete.

Key Takeaways:

• Innovations over the years, such as immunosuppression, have made long term organ transplantation a reality

• Organ shortage is a problem that affects over a million people and leads to the death of thousands of people

• Xenotransplantation from pigs is physiologically possible to humans provides a potential solution to the problem

• Organ rejection has huge immunologicalhurdles but genetic engineering has a solution

• Creating antibody, T cell, B cell, and natural killer cell tolerance might be the key graft acceptance

• Lab trials with mice proved that creating tolerance is possible

Resources

Here are some of the resources I used in my rabbit hole of curiosity and understanding. Like most things, there are minor inconsistencies between some data points, statistics, but nothing provoking. Huge shoutout to all of the amazing educators, writers, and annotators listed below.

Informative Videos

Research Papers / Journals

Inactivation of porcine endogenous retrovirus in pigs using CRISPR-Cas9

A brief history of cross-species organ transplantation

Historical Overview of Transplantation

Transgenic Expression of Human CD47 Markedly Increases Engraftment in a Murine Model of Pig-to-Human Hematopoietic Cell Transplantation

Genetically Engineered Pigs And Target Specific Immunomodulation Provide Significant Graft Survival And Hope For Clinical Cardiac Xenotransplantation

Xenotransplantation: Current Status in Preclinical Research

Websites / Articles

History of medicine: Organ transplantation

TRANSPLANT REJECTION: HYPERACUTE, ACUTE, CHRONIC & GRAFT VERSUS HOST

Meet the pigs that could solve the human organ transplant crisis

Xenotransplantation: using pigs as organ and tissue donors for humans

Xenotransplantation: Can pigs save human lives?

Thanks you for reading and getting to the end of the article. If you only skimmed through it but still got something out of it, thank you as well. If you wanna share your opinions or thoughts, leave them in the comments or tweet me.

Wait! Before you go:

Hi! I’m Sabeeh and I’m a curious 17-year-old who is super passionate about emerging technologies such as artificial intelligence, brain-computer interfaces, gene-editing, and more. I would love to connect and learn more about you as well — Shoot me a DM!

This article is also part of an ongoing project with my friends Aaron, Adam, and Veda. For more information on us and the future of xenotransplantation, click here.

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