grow human organs in the bodies of animals

One way to overcome the shortage of organs is to grow organs in the laboratory. Until a few years ago, scientists believed that they could do this using stem cells: take stem cells, which are capable of making different types of tissues, and grow them in molds or artificial scaffolds for the production of new organs. However, researchers have difficulty timing and coordinating the development of the stem cells so that a functional human organ is created. Research in this direction continues, but progress is slow.

A small, but growing, group of researchers, of which I am a member, believes that there may be another way: to let nature do the hard work for us. Evolution has already produced the incredibly complex process in which a handful of cells that are identical to each other turn into tissues and organs with different specializations, which are required to build an entire organism - whether a mouse or a human. This genius show takes place in the weeks and months after a fertilized egg begins to develop into an embryo that grows and develops, without any support from artificial scaffolds, and the embryo becomes a perfect animal that has a heart, lungs, kidneys and other organs. We believe that it is possible to find a way to take organs from animals, such as pigs, and use them in humans.

Of course, a healthy pig's heart will not be of much use to a person in need of a heart transplant. First, our immune system will reject a graft taken from another biological species. (Pig heart valves are a suitable substitute for human tissue only after undergoing chemical treatment that prevents an immune reaction against them, a process that will destroy the functional capacity of complex organs). My colleagues and I believe that it is possible to grow human organs - built from human cells only, or almost only from them - in the body of an animal such as a pig or cow. The animal that will be created is a chimera - a creature whose body is composed of cells of two biological species, such as thegriffon The mythological one, which has the head and wings of an eagle, and the body of a lion. Our dream is to create a chimera by injecting human stem cells into pre-prepared animal embryos so that when they reach their full size, they will contain several organs built from human cells. After the animal is killed, we will remove this single organ from it, a heart, liver or kidney, which is made entirely of human cells, and give it to the person who needs a transplant.

The idea may sound far-fetched, but researchers in the US and Japan have already shown that this is possible in principle. Several different teams injected rat stem cells into mouse embryos, whose genetic makeup was pre-programmed, and transferred the embryos into the wombs of surrogate female mice. After a few weeks of gestation, the surrogates gave birth to animals that looked and behaved like mice, except that they all had rat pancreases. Researchers in my lab and other research groups have gone a step further, injecting human stem cells into pig embryos. Some of these injections were absorbed, and we were able to verify that the human tissue began to develop normally. Then we transferred the chimeric embryos into the uterus of surrogate pigs, where we let them develop for three to four weeks. After conducting a few more experiments in intermediate stages, we will allow the embryos to develop for two to three months, and after this period of time, we will find out how many of their cells are human cells. Assuming that these experiments go well - and that we receive permits from state and local authorities to continue the experiments - we expect to allow the embryos to grow until the end of pregnancy (which lasts four months in pigs).

We are still very far from creating chimeric piglets. We still have a long way to go, and we need to discover the best method for preparing human stem cells and animal embryos in a way that preserves the vitality of the chimeras until the end of pregnancy. Many things can go wrong along the way. But even if we cannot create perfect organs, it is possible that the methods we discover during our work will help us better understand the onset of many serious and deadly diseases, including cancer, their course and their clinical symptoms. If this approach is successful, it could have enormous implications for treatments involving organ transplantation. Transplant waiting lists may become a distant memory when a continuous supply of replacement body parts from the bodies of farm animals is opened to tens of thousands of suffering people all over the world.

learn from nature

In recent years, biologists' knowledge of embryo development has increased so much that we are beginning to very carefully try to adapt the process to our requirements. We also understand how much embryonic growth is subject to the exact location of different cells at different points in time in the developing organism. The cells produce and release special proteins called growth factors, and these in turn activate or silence, depending on changes in their concentrations, a series of genetic instructions that dictate the development processes. Teams in our lab and elsewhere rely on this partial understanding and a lot of trial and error, making changes to pig embryos with the goal of getting them to make tissues that will eventually lead to building a human kidney, pancreas, or other organ.

The raw materials used by us include pig eggs and sperm cells (taken from healthy animals), and human stem cells (grown in tissue cultures). We fertilize a pig's egg with a pig's sperm, and a few hours later the fertilized egg, called zygote, divides into two cells, and then into four cells that appear to be identical to each other. Each of these cells activates the same sets of genes in its DNA, and the activation leads to the production of different proteins whose activity, along with other effects, encourages the cells to continue dividing.

Due to the complex system of reactions between genes and proteins, the cells that were initially identical, begin to move and behave in different ways while dividing and multiplying. Within a few days, a ball of several hundred cells is formed, called Blastocyst. This is the latest point at which we can inject the human stem cells, before different tissues begin to form, which will later produce the organs. Beyond this point in time, the embryo's stem cells will ignore the foreign stem cells, and these will degenerate and die.

During its growth, three layers of cells are formed in the embryo: an outer layer, a middle layer, and an inner layer, and the position of each single cell within the whole complex becomes more important than it had until then. In previous studies it was found, for example, that certain cells within the inner layer activate a gene called Pdx1 in response to signals carried by proteins in their immediate environment. This step in turn activates many other genes that initiate the maturation process of the pancreas. In contrast, some cells in the middle layer activate the Six2 gene in response to external signals, which initiates the formation of the kidneys. Thus, even though all cells in the body contain the same DNA sequences, the specific environment in which any cell is found at a certain stage in embryonic development determines which genes will be activated or silenced, and consequently, which tissue will develop from the cell.

The fact that a single gene such as Pdx1 or Six2 can activate an entire developmental pathway leading to the production of a pancreas or a kidney is of particular importance in our research journey. By missing the one gene essential for pancreas growth (a process my colleagues and I call "emptying the niche" of the stem cells), our lab has been able to create pig embryos that do not develop this vital organ unless we inject them with enough human stem cells that contain the missing gene . If the cells added from the outside develop properly, they will produce an organ because it is all made up of a human body. In the ideal situation, all the other organs of the animal will be built from pig cells only.

As often happens in science, in order to find out how it is possible to empty an embryonic niche and then fill it with stem cells of another biological species, many experiments in rodents were first required. Eventually, in 2010, Hiromitsu Nakauchi, who was then working at the University of Tokyo, reported with his colleagues on success in growing a mouse that had a rat pancreas. Recently, in my lab we were able to engineer the genetic makeup of mouse embryos in a way that caused them to use rat stem cells as a source of cells for their eyes. After three weeks of gestation in the wombs of surrogate mice, these embryos were developed embryos whose eyes contained rat cells.

Challenges

Each step in our journey requires careful consideration of various problems that may arise. Mice are too small to create organs of a size that might be useful for humans in need of transplants, so we are now focusing our efforts on growing pig embryos. Pigs, and their organs, can grow to almost any size needed by surgeons to transplant organs into humans of different body sizes. The gestation period of pigs is also longer than that of mice (pregnancy in mice lasts about 20 days), although it is shorter than that of humans. Since a human embryo needs nine months to reach the end of its development, researchers are trying to develop certain biochemical tricks that should help human stem cells speed up their biological clocks so that they mature, or differentiate, according to the schedule of the surrogate embryo. Presumably it would be easier to adapt the development of human cells to the duration of a pig's gestation than to the much shorter gestation of a mouse.

My colleagues and I are now focusing on growing a pancreas or a kidney from human cells because we know that a single gene initiates the development of these organs in the embryo, so the process is quite simple. In other organs, such as the heart, the onset of development apparently depends on several genes, and this means that emptying the niche would require the silencing of more than one gene, a much more difficult operation. Researchers in the group of George Church Harvard University recently adapted the gene editing tools CRISPR / Cas9 For this purpose and with its help, they can remove several genes located in different locations in the embryo's DNA. In this way, the researchers are preparing for the possibility of performing more complex manipulations to create other organs, as needed.

A bigger problem is ensuring that the human stem cells used in the experiments are sufficiently primary to provide tissues of various types. In the eyes of biologists, this physiological state is "developmentally innocent". Embryonic stem cells, which can be derived from zygotes created in vitro and left in fertility laboratories, are suitable for this purpose. However, their use is controversial.

Over the past decade, researchers have achieved several technical achievements that may, at first glance, solve the dilemma. Researchers have found a way to induce adult cells taken from the skin or intestine of an adult to become like stem cells, called induced pluripotent cells, or iPSC. Experiments with human iPSC cells instead of embryonic stem cells would probably be more ethically acceptable. Their use should have another advantage expressed in the fact that in the future the researchers will be able to create for each patient an organ for transplantation that is genetically adapted to them and therefore will not trigger immune rejection.

However, an in-depth study of iPSC cells, generated in the conventional way until now, suggests that they are not as innocent as they should be to survive in a chimeric embryo. In fact, they are in an advanced state towards becoming specific types of cells, and are no longer able to respond to the biochemical signals coming from the embryonic cells, which tell them to develop into something else. Since iPSC cells do not respond correctly to signals from the rest of the embryo's cells, they are recognized as foreign cells and rejected by the embryo.

recently, John Wu From my laboratory he began to treat human iPSC cells with a special combination of growth factors that allows at least some of them to respond properly to a wider range of signals from embryonic cells. At the time of writing, our group has obtained preliminary results showing that our treated cells can properly integrate into blastocysts. My colleagues and I stopped the growth of the embryos at different time points after fertilization and examined them under a microscope to find out how well the injected cells were able to integrate with the cells of the embryo into which they were inserted. In the future, we plan to allow the embryos to develop for a longer time, until they are six weeks old, an age when the buds of the organs can be seen. At this point in time, the embryos will begin to produce the early structures of the various body tissues and organs.

However, even if we manage to create human iPSC cells that can be fully absorbed into pig embryos, our path is still far from over. From an evolutionary point of view, there is no kinship between humans and pigs similar to that between mice and rats, thanks to which it is possible to create chimeras. Human iPSCs may have lost the ability to recognize all biochemical signals originating from distant biological species, such as pigs. If we fail to find a biochemical workaround, we may have to test our ideas in other biological species, such as cows.

next steps

In 2012 I discussed these and other issues with my co-workers, Joseph Maria Campistol, CEO of the University Hospital in Barcelona, ​​famous throughout the world as an organ transplant center. His advice is etched in my memory: "The only way to know if human iPSC cells can cross barriers between biological species and contribute to the creation of a human organ in a pig's body is to roll up your sleeves and do the experiment," he told me.

Campistol's words spurred me into action. I knew that we would not be able to carry out such a task in our laboratory on our own. Working together with embryologists, veterinarians, stem cell biologists, and bioethicists, we have established an international consortium to test our ideas. In 2015 we started injecting human iPSC cells into pig embryos. I am especially grateful to the Catholic University of San Antonio in Murcia, Spain, and the Moxie Foundation for their support of this early work when no one thought our approach was feasible.

Until now, most of our trials have been conducted in California and Spain, under the supervision of the local and national regulatory institutions. Until now, we have allowed the chimeric pig-human embryos to develop in the wombs of sows up to the age of four weeks of pregnancy - and at this point in time we kill the animals (the guidelines we formulated in cooperation with the authorities require us to kill the surrogates and the embryos).

Overall, the results obtained from these experiments, and others, provided us with basic knowledge about the development of the chimeric embryos. We are beginning to examine what is the best number of human iPSC cells to be injected for the embryo to develop successfully, and what is the most appropriate time to inject them. We have also begun to track the pathways by which human cells migrate to different parts of the developing embryo.

Ethical considerations

At the same time as our scientific work, even when we are immersed in efforts to improve our procedures, we must work with the public and face ethical, social and regulatory challenges raised by this new field of research. Our consortium worked closely with ethics experts and regulators in California and Spain for a year and a half to formulate guidelines for the control of our research.

It goes without saying that we comply with all the accepted rules concerning animal welfare, which should apply to all animal research, rules which require, among other things, to avoid causing unnecessary pain and to provide the animals with space to live and move. However, there are some issues unique to our technology. As mentioned, innocent stem cells really can make tissues of any kind. But we must pay special attention to tissues of three types: nerves, sperm cells and eggs - since the transformation of these tissues into human beings inside the bodies of animals could give rise to creatures that no one would want to create.

Imagine the ethical nightmare that could materialize if, for example, enough human nerve cells were to populate a pig's brain that it would be capable of higher-order thinking. We can prevent such a problem in advance by removing the genetic complex responsible for neural development from all human iPSC cells before injecting them into embryos. Thus, even if the human stem cells manage to migrate to the embryonic niche leading to brain growth, they will not be able to continue developing. The only nerve cells that will be able to grow and develop will be XNUMX% those of a pig.

Another scenario that the researchers are trying to prevent, for reasons that will become clear immediately, is the culture of chimeras among themselves. Even if it is something that has a low probability, there is always a chance that some of the human stem cells we implant will migrate to the embryonic niche that dictates the construction of the reproductive system, instead of remaining in the niche that produces the desired organ (kidney or pancreas). The result could be animals that produce sperm or eggs that are all identical to those of a human. Should these animals be allowed to breed, it would lead to an ethical disaster where a fully human embryo (the result of a human sperm cell from a male pig that fertilized a human egg from a female pig) would begin to grow in the womb of a female pig. The best way to completely prevent such a possibility is to make sure that each chimeric animal used for transplantation starts its life from "zero", in the sense that it is the result of fertilization between reproductive cells of pigs only, and the human stem cells are inserted into the body only after fertilization.

None of this will happen, of course, if it turns out there is no way to overcome the technical challenges. However, even if we fail in our attempt to create functional organs for transplant purposes, I believe that the knowledge we discover and the methods we develop in the course of the work will be of enormous value. One of the fields expected to be hired is cancer research. Studies show that many tumors grow uncontrollably in the bodies of children or adults due to the reactivation of some (though not all) genes that were previously involved in fetal development. Thus, the better the researchers understand the proper signals sent by cells in the embryo, which allow the embryos to grow and tell them when to stop growing, the better it will be to encourage cancer cells to stop their growth.

Scientists are human too, of course. We are passionate about new ideas and new ways of doing things. And we may get carried away in our optimism about what is expected to result from our discoveries - not only in our fields of research but also in what concerns humanity as a whole. However, the preliminary results I described in this article make me cautiously optimistic about the possibility of creating human organs from animal chimeric embryos within the next twenty years.