On August 4, 2016, the US National Institutes of Health (NIH) announced that they were going to lift the moratorium on the creation of chimeras. We are talking about ethically controversial experiments in which human stem cells are introduced into animal embryos - as a result, organisms are formed that combine animal and human characteristics. Scientists call them chimeras.
IN Ancient Greece Chimeras were mythological monsters with the head and neck of a lion, the body of a goat and the tail of a snake. The same chimeras are organisms with genetically heterogeneous material. They could serve as convenient biological models for studying various diseases- for example, cancer or neurodegenerative syndromes, could become a source of organs for transplantation. However, when experimental biology approaches science fiction, the public fears that it may lead to unforeseen consequences.
When creating chimeras, stem cells that have the property of pluripotency are used. In other words, they are capable of turning into all the cells of a human embryo. The cells are introduced into the embryonic tissue of model organisms (mice, rats, monkeys, pigs and other animals) at very early stages, after which the embryo is allowed to develop further. In September 2015, the NIH expressed concern that if stem cells were injected into the brains of mice, the result could be rodents with altered cognitive abilities—that is, animals with “superintelligence.” Therefore, the NIH, which awards grants for biomedical research, has decided to withhold funding for experiments with chimeras until its experts examine the ethical issue.
However, some research groups in the United States were already in full swing creating chimeras. MIT Technology Review reports that in 2015, there were about 20 attempts to produce pig-human and sheep-human chimeras. Unfortunately, no scientific work has yet been published, and there have been no reports of successful production of animals with human tissues.
Experiments with chimeric organisms combine both genetic engineering and stem cell biology. It is not enough to simply introduce pluripotent cells into an animal embryo, since in this case the result may be an organism with catastrophic developmental disorders. Scientists typically turn off genes in embryos so that they cannot form specific tissues. In this case, the stem cells take on the task of forming the missing organ, which is no different from a human one, making it suitable for transplantation.
According to cardiologist Daniel Garry, the first tests were carried out in his laboratory this method. The researchers engineered pigs that lacked some skeletal muscle and blood vessels. Such animals would not be viable, but scientists added stem cells from another pig embryo to the embryos. The results so impressed the US military that they awarded Harry a $1.4 million grant to grow human hearts from pigs. The scientist intended to continue his research despite the NIH moratorium, and was one of 11 authors who published a letter criticizing the biomedical center's decision.
Scientists said the NIH moratorium poses a threat to the development of stem cell biology, developmental biology and regenerative medicine, and expressed doubts that using stem cells it is possible to obtain a “humanized” animal with high intelligence. In particular, they pointed out that xenotransplantation experiments in which nerve cells people are implanted into the brains of mice, did not lead to the emergence of overly smart rodents.
Image: Nakauchi et al. / The University of Tokyo
As a precaution, some researchers working on creating chimeras do not allow their creations to be born. Embryologists study embryos to gain information about how much human stem cells contribute to fetal development. However, despite the fact that some laboratories are playing it safe, chimeric animals already exist - for example, mice endowed with the human immune system. Such animals are created through the introduction of liver and thymus cells from aborted human embryos into the body of already born rodents.
Of greatest interest to scientists is the creation of chimeras at the blastocyst stage, when the fetus is a ball consisting of several dozen cells. This method is called embryo complementation. In 2010, researchers from Japan managed to create mice whose pancreas consisted entirely of rat cells. Hiromitsu Nakauchi, the lead author of the paper, later decided to create a “pig-man,” for which he had to move to the United States because scientific committees in Japan do not approve of such experiments. The scientist is now working at Stanford University with a grant from the California Institute of Regenerative Medicine. Most of the pluripotent cells introduced into embryos in his lab are made from his own blood, he said, because bureaucratic barriers prevent him from recruiting outside volunteers.
Most people hear the word "chimera" and think of monsters created by mad scientists. Scientists have to prove that human cells can indeed multiply and form full-fledged and healthy organs in animals. Mice and rats are quite close genetically, so the creation of chimeras in in this case does not pose a problem. In the case of humans and pigs, whose common ancestor lived 90 million years ago, things may be different.
Scientists are already testing the complementation of a pig embryo with human stem cells, but the research began only after the approval of three bioethics commissions. Stanford University, where the research is being conducted, limited the development time of the embryos to 28 days (piglets are born on day 114). However, the fetus will be sufficiently developed so that it can be determined how correctly the rudiments of the organs are formed.
Last week, the NIH proposed replacing the moratorium with additional review by a committee of ethicists and animal welfare experts. They will take into account factors such as the type of human cells, where they are located in the embryo, and possible changes in behavior and appearance animal. The experts' findings will help the NIH decide whether to fund the project under consideration.
An international group of scientists led by the Spaniard Juan Balmonte, known for his work in the field of stem cells, managed to create embryos of human-pig chimeras, which in the future could become a source of donor organs. Another team of researchers cured congenital deafness in mice using viruses. talks about the successes of genetic engineering related to medicine.
The creation of genetically modified organisms is not the only thing that genetic engineering can bring to humanity. Biotechnology makes it possible not only to change genes to improve agricultural plants and animals, but also to treat earlier incurable diseases. Ironically, for this, scientists use the eternal enemies of man - viruses. The latter are used to create vectors that deliver DNA to necessary cells. Another direction that may scare people who are not very knowledgeable in science is the creation of chimera embryos that combine cells from humans and other organisms. However, what at first seems sinister will actually turn out to be a convenient way to create organs.
Kidneys or lungs that are produced by growing chimeric embryos will be suitable for transplantation into people who need them. Those who are afraid of a mutant uprising should think that real benefit from this technology exceeds the vague fears of pessimistic science fiction writers.
Image: Nakauchi et al. / The University of Tokyo
To dispel fears, you need to understand what and how scientists do when they create chimeras. The main material that researchers work with is stem cells, which have pluripotency - the ability to turn into other cells of the body (nerve, fat, muscle, etc.) with the exception of the placenta and yolk sac. They are introduced into the embryos of other organisms, after which the embryo develops further.
Pigmen
This is how an international group of scientists from the USA, Spain and Japan managed to create pig-human, rat-mouse and cow-human chimeras. They reported this in an article published in the journal Cell and became the first document confirming the successful "chimerization" of the Daleks in kinship species.
The main problem is that it is not enough to introduce pluripotent cells into an embryo and expect something good to come out. Instead, the result may be an organism with catastrophic developmental problems, including the formation of teratomas. It is necessary to turn off the genes in the recipient embryos so that they are unable to form specific tissues. In this case, the implanted stem cells take on the task of growing the missing organ.
First, scientists introduced rat stem cells into mouse embryos at the blastocyst stage, when the fetus is a ball of several dozen cells. This method is called embryo complementation. The purpose of the experiment was to find out which factors play a leading role in interspecific chimerism. The embryos were transferred into the body of female mice and then developed into living chimeras, one of which lived to be two years old.
Genes in embryos were turned off using CRISPR/Cas9 technology, which introduces breaks in specific sections of DNA. For example, when testing the approach they used, researchers blocked the activity of a gene that plays an important role in the formation of the pancreas. The mice that were born died as a result, but when pluripotent rat cells were introduced into the embryos, the missing organ developed. The scientists also turned off the Nkx2.5 gene, without which the embryos suffered from serious heart defects and were underdeveloped. Chimerization helped the embryos achieve normal growth, but it was never possible to obtain living chimeras.
Photo: Juan Carlos Izpisua Belmonte / Salk Institute for Biological Studies
Examination of the resulting rat-mice showed that different mouse tissues contained different proportions of rat cells. When scientists tried to introduce rat cells into pig blastocysts and then genetically analyzed four-week-old embryos, they found no rodent DNA. This suggests that not all animals are suitable for chimerization with each other, and the successful grafting of stem cells from some into embryos of others may depend on genetic, morphological or anatomical factors.
The main goal of scientists was to create a human-pig chimera in order to see how human tissue would develop inside the embryo of a non-ruminant artiodactyl animal. They used pig blastocysts and laser beam made microscopic holes for subsequent injection of various groups of pluripotent cells that were grown in different conditions. The embryos were then transplanted into sows, where they developed successfully. Tracking the dynamics of human material was carried out using a fluorescent protein that human stem cells were programmed to produce.
As a result, cells that were precursors were formed in the pig embryo various types tissues, including the heart, liver and nervous system. The pig-human hybrids were allowed to develop for three to four weeks before being destroyed for ethical reasons.
Deaf mice
American scientists from Boston were recently able to restore hearing to mice suffering from a rare genetic disorder. inner ear. For this they used biological system gene delivery (vector) based on neutralized viruses. Researchers have modified an adeno-associated virus that infects people but does not cause disease.
The infectious agent is able to penetrate hair cells - receptors of the auditory system and vestibular apparatus in animals. Biotechnologists used a vector to repair the defective Ush1c gene in the cells of newly born live mice. This mutation causes deafness, blindness and balance problems. As a result, the animals' hearing improved, allowing them to distinguish even quiet sounds.
Genetic engineering, therefore, is not a way to create mutants that threaten humanity. It is an ever-improving set of methods and tools to improve the lives and health of people, especially those who are in great need. Since the creation of chimeras and gene therapy are not so easy to implement and sometimes require ingenious solutions; the development of biotechnology is not happening as quickly as we would like. However, dozens of scientific works, which deepen and enrich our knowledge and skills.
However, something similar to a revolution in medical science really happened. In the end of January Science Magazine Cell published an article by molecular biologist Juan Carlos Izpisua Belmonte, who runs a laboratory at the Salk Institute in California (USA), and 38 of his co-authors. The article tells how scientists managed to create viable embryos consisting of a mixture of pig and human cells.
Who are they
If these creatures were allowed to be born (and biologists did not do this, not least for ethical reasons), they could not be formally assigned to any biological species. Such organisms are called chimeras. Chimeras, which we know from medieval miniatures, have eagle wings attached to the lion's body, and a snake sting to the goat's hooves. Who remembers the mouse with the human auricle on the back - the result of a high-profile experiment 20 years ago, one can easily admit that one can expect something different from biologists. But in this sense, the new creatures from the Belmonte laboratory hardly had a chance to surprise anyone: after birth they would have looked like the most ordinary piglets. It's just that some of the cells in their bodies - about one thousandth of a percent - would contain pure human DNA. And this would make the piglets compare favorably with the long-eared mouse of 1997, which was more of an experiment in plastic surgery and did not have a single human cell.
According to recent estimates, humans have 30-40 trillion cells in total, and pigs have about the same number. Is a thousandth of a percent of such an astronomical figure a lot or a little? It only takes one cell to conceive a child. Therefore, in theory, a chimera pig could become a parent to a human baby.
Donor without a motorcycle
Doctors see pigs not as potential relatives, but as potential donors for transplanting their organs to people. In the United States alone, 27 thousand kidneys, lungs, hearts and intestines are transplanted per year. And in all 27 thousand cases, surgeons deal with organs of living or dead people. But who in their right mind would dare to ask to have one taken from a pig transplanted into their own failing heart, when the procedure with an ordinary, human one has been established and works perfectly? Those who will not get a transplant: 118 thousand people are registered in the United States on the so-called waiting list. According to statistics, approximately 22 of them will die today (and the same number tomorrow, and the same number next Sunday) without waiting for their transplant.
There are too few human donors - and it’s not even that volunteers are very rare. (Unlike the United States, in Russia, by law, a potential donor is considered to be anyone who has not explicitly prohibited the removal of their organs. The law does not require asking consent from relatives.) Only three people out of a thousand, the New Scientist magazine cites British data, die in circumstances that do their organs are suitable for transplantation. The numbers obviously vary from country to country - they depend both on how quickly the ambulance arrives at the scene of an accident or shooting, as a result of which the most promising donors appear, and on how many transplant centers are nearby where the organs can be disposed of correctly. Finally, in a few more hours it is necessary to find and prepare for the operation a patient from the “waiting list” - much stricter compatibility rules apply here than for blood transfusions with its four different groups.
The cells that are least susceptible to rejection are our own. What if we used animals as incubators for kidneys and pancreases grown from human cells (and ideally from the cells of the exact patient who will receive the organ)? The same problem with rejection prevents us from solving the problem head-on: for the ready-made immune system of an adult pig, human cells are no less alien than pig cells are for us.
This means we need to act differently.
Cut and glue
Imagine that before your eyes two people were simultaneously cut in half - say, by a combat laser from a bad science-fiction movie. Then they connected half of one with half of the other, and the glued halves would then live their whole lives as if nothing had happened. The option is even more paradoxical: they took two thin people, pressed them against each other - and got one fat man. If both people are not yet four days from the moment of conception, nothing is impossible. At this stage, the future organism is a ball of identical cells. “You remove the outer protective layer of inanimate matter and physically connect the embryos,” Virginia Papaioannou, a professor at Columbia University (USA), explained in an interview how scientists have been producing chimera mice with the full set of genes of two individuals at the same time since the 1960s. Having touched, two embryos simply form a new, larger ball - almost like soap bubbles meeting in the air. Immune system The ball of cells does not yet have a system that could prevent this - as, indeed, all other systems: they will develop much later.
A more subtle intervention is to add foreign biomaterial to the embryo when its cells have already divided into different varieties. At the blastocyst stage, the embryo - whether in a mouse or a human - is a hollow ball with a small portion of cells locked inside. Only this internal portion will become the future lungs, liver, kidneys, brain, skin and other parts of the adult body, and the entire external portion will turn into a placenta that will not survive childbirth. Biologists prefer to introduce foreign cells at this stage.
It cannot be said that this scenario is pure form opened up exciting opportunities for transplant surgeons. The need for donor organs usually arises later - when a person has already passed the fetal age. How to cross it with another embryo? Take cells of an adult organism that have not acquired a clear mission (like brain or liver cells) and have not lost characteristic of cells The fetus has the ability to transform into anything. They are called stem cells, but they are very rare in the body. In 2012 Nobel Prize in medicine was awarded to the Japanese scientist Shinya Yamanaka for inventing a way to transform ordinary body cells into stem cells - to forget one’s backstory and “fall into childhood.” The full name is induced (because they were forced to change) pluripotent (that is, “capable of anything” - any transformation) stem cells. Chimera researchers use them.
Is it possible to combine embryos like this? different types— for example, rats and mice? This is exactly what the team of Toshihiro Kobayashi from the University of Tokyo first did using stem cells in 2010 - and the American group, which published their results seven years later, brought the method to perfection. How can you be sure that you have actually bred a chimera? Take as a basis embryos doomed to death with specially damaged DNA. Using the newly invented “gene scalpel” CRISPR-Cas9, a method for targeted DNA editing, scientists knocked out genes responsible for the growth of the pancreas or heart. With such a defect there is no chance of survival (or even being born alive). But then rat stem cells were introduced into the embryo. And if a chimera mouse was nevertheless born, scientists could be sure that a rat’s heart was beating inside it.
But the most surprising result concerned the gallbladder. Rats don't have it, but mice do. But chimeras, in which the mouse genes responsible for this organ were disabled, were still born with a working gallbladder- from rat cells. The mouse cells somehow suggested the correct context to the rat cells, and they, succumbing to the influence, formed an organ that was impossible in the rat.
Closer to pigs than to rats
It was not possible to cross a pig and a rat in this way - because these organisms are too different from each other. Different lengths of pregnancy and different sizes organs suggest that cells are programmed to divide at different rates. Finally, will the tiny rat heart of a chimera be able to pump blood through a huge pig liver?
But with people there is no such difficulty: we are much closer to pigs - primarily in the size of our organs. Therefore, pigs (and mini pigs as a separate option) have always been the #1 candidate for xenotransplantation. In parallel with growing human cells in a pig's body, biologists are considering other possibilities - for example, simply taking and hiding from the human immune system those proteins on the surface of pig cells that cause the most acute reaction. Such research has been going on for a long time, so pigs as a candidate for organ transplantation are not new.
A new experiment has shown that there is a possibility, and it is not speculative - or even an incredible coincidence. 2075 embryos were implanted into pigs, and 186 of them reached sufficient maturity, according to scientists. Human cells were tagged with a special tag in their DNA that causes them to produce a fluorescent protein - and 17 mature, healthy embryos glowed confidently in ultraviolet light, proving to scientists that they were definitely chimeras.
From this moment to organs in a living incubator is years, the researchers say. And it’s not just that the proportion of human cells in the chimera’s body is too small. It would be difficult for scientists in any case to see how they grow and what happens to the cells in an adult body.
We are much closer to pigs - primarily in the size of our organs. Therefore, pigs have always been the No. 1 candidate for xenotransplantation
Chimeras of mice and rats, bred earlier, lived a full mouse life at two years. There is no reason to think that human-pig chimeras would have serious problems health problems that prevent you from reaching maturity. It was not biological problems that prevented them from being born, but ethical ones. And so serious that a team from the Salk Institute was forced to conduct research with private money, because the rules of the US National Institutes of Health - an analogue of the Ministry of Health, which funds most of the biomedical research in the country - prohibit spending money on any experiments with the introduction of human stem cells into animal embryos.
What is unethical about giving birth to a pig with a human spleen? Our uncertainty about the results of such an experiment. The proportions of cells in an adult embryo are not the same as those in the embryo. And if pig cells prevail by a million to one ratio, it’s not as scary as if human cells take over. And a creature will be born, more like a person than a pig, with human brain, but with deformities caused by the circumstances of the experiment. For doctors to be able to save people, it seems that they need, among other things, more precise definition human - and a more accurate answer to the question of where people come from.
The embryo is a hybrid of a human and a pig. Biologists from the USA, Japan and Spain introduced human stem cells into a pig egg. Scientists named the embryo grown in the womb of an animal a chimera - in honor of a creature from ancient mythology. In the future, these studies will allow scientists to grow organs for transplantation and study nature genetic diseases. In order for research to move forward, scientists must not only prove the effectiveness of the experiments, but also their ethicality.
What is the essence of the experiment?
A group of American scientists from the Salk Institute for Biological Research in California introduced human stem cells into a pig embryo at early stage development and placed it in the womb of the animal. A month later, the stem cells developed into embryos with the rudiments of human tissue: heart, liver and neurons.
Of the 2,075 embryos transferred, 186 developed to the 28-day stage. The resulting embryos were “extremely unstable,” scientists admit, but so far they are the most successful human hybrid. Scientists write that the resulting chimera is a critical step towards creating animal embryos with functioning human organs.
Source: Cell Press
The ultimate goal is to grow organs that are functional and ready for transplantation; the experiments carried out are the first step towards this, WP writes, citing scientists from California.
The results of a similar study are reported in the first issue of the journal Nature in 2017. As follows from the publication, a group of scientists from Japan and the United States managed to grow a mouse pancreas inside a rat, and then transplant the insulin-producing organ into diabetic mice, which did not cause immune rejection. This was the first confirmation that interspecies organ transplantation is possible, writes Nature.
Why is this necessary?
The main goal of scientists is to grow human organs using embryos of large animals. According to the US Department of Health, 22 people die every day waiting for organs for transplantation. Scientists have long tried to grow artificial tissues outside the human body, but organs developing in a Petri dish (the so-called container for growing microorganisms) are very different from those grown inside a living organism.
The technology for growing artificial organs will most likely be similar to the experiment with mice and rats, writes The Washington Post. The rats that received new cells as part of the studies described in Nature were genetically modified. They couldn't grow their own pancreas, so stem cells "filled the empty space." Some of the glands that appeared in rats were transplanted into sick mice. After surgery, the mice lived with healthy glucose levels for a year—half their life in human terms, writes WP.
The study proved that cross-species transplantation is not only possible, but also effective, senior study author Hiromitsu Nakauchi from Stanford University commented on the results. Scientists managed to “grow” the heart and eyes in the same way.
What are the difficulties?
Scientists from California achieved the first results four years after the start of research. According to them, pigs are ideal animals for the experiment. Their organs are about the same size, but they grow much faster than humans. IN further research The time factor should become the main one, researchers admit.
“So far, the number of human cells in the resulting embryo is very small, and the whole process takes place at the early embryonic stage, so it is too early to talk about creating a full-fledged chimera,” Nakauchi’s colleagues commented on the result. In the resulting embryos, there was only one human cell per 100,000 pig cells (efficiency 0.00001%). "It's enough to achieve an efficiency of 0.1% to 1% of cells," one of the authors of the Californian study explained to the BBC.
After four weeks of development, scientists from the Salk Institute, for ethical reasons, destroyed the resulting embryos to prevent the chimera from fully developing. “We just wanted to answer the question of whether human cells can adapt at all,” explained one of the authors.
Ethical issues
In 2015, the US National Institutes of Health placed a moratorium on funding for research that involves crossing human and animal cells. Since stem cells can develop into any human tissue, an animal with a human brain could eventually be created, some bioethicists believe. Others point to the violation of the “symbolic boundary” between humans and animals, writes WP.
Californian scientists say fears surrounding "chimeras" are more like myths than controlled experiments, but admit the possibility of an animal being born with human cells is a concern.
In August, the US National Institutes of Health allowed funding for chimera research to return. The organization proposes to allow the introduction of human stem cells into embryos at an early stage of development of large animals, with the exception of other primates.
“We have finally been able to prove that this approach to creating organs is possible and safe. I hope people understand this. Many people believe that this is science fiction, but now it is becoming a reality,” Nakauchi commented on the possible lifting of the ban.
Daniil Sotnikov
Preview photo: still from the film “Chimera”
Header photo: WikiCommons
