Exercises with Nuclei

From a scientific point of view, the name is, of course, wrong and the researchers do not use it seriously. But in order for such children to be born, besides mom and dad, another woman is really needed from whom the child will inherit 37 genes in addition to many thousands from their real parents. Although these 37 are very important for our body, they will not affect eye color, character and ability to play chess directly.

The story of such children in Ukraine can be traced to 2014. Back at the clinic, where Pavlo Mazur works, it was decided to develop a new technology to solve the problem of age-related infertility. Biological laws work in such a way that at approximately 37 the woman’s ability to conceive, even with assisted reproductive technologies, starts dropping. After 40 it is very difficult to do, though, is still possible and a lot of positive examples can be found. But usually this requires huge efforts and even they guarantee nothing.

Our researchers suggested a hypothesis which in lay terms can be formulated as follows. The problem of older women is that their oocytes (female sex cells, which are often erroneously referred to as ova) have “old” cytoplasm and it is the exact reason for their infertility.

Here it is worth recalling the school, to be more precise, the lessons where the structure of cells is studied. It is enough for us to understand that the cell (including oocyte) has a nucleus with DNA (the genes containing the whole information about our body) and cytoplasm. The cytoplasm is the “inner ocean” of the cell – everything that surrounds the nucleus. In this ocean there are many different organelles that every second make extraordinary operations without which no life in the truest sense of the word is possible. The only organelle that we need to know for this story is the mitochondrium, but we will return to it later.

In order to solve the problem, our researchers suggested replacing “old” cytoplasm with “young” one. This requires a donor – a young woman with good reproductive health, who will donate her oocytes which have not yet matured. The researchers will remove the nucleus with genes from it and replace it with the same nucleus (i.e., genes) of the older woman who wants to be a mom. Such techniques are referred to as “nuclear transfers”. As a result of all these complex manipulations we get the oocyte with the “old” genes of the mother and “young” donor cytoplasm. And then – just following the procedure, familiar to any experienced embryologist.

But the hypothesis was not confirmed. Experiments have shown that rejuvenation of the cytoplasm does not help older women to have children. In addition, if to inject young donor’s nuclei into the oocytes of older patients, everything works well with a resulting embryo that would normally develop. So, the problem of age-related infertility is not in the “advanced age” of the cytoplasm.

In science, as we know, a negative result is also a result. But can the technique that Pavlo Mazur has mastered, be applied to generate a positive result?

There is a category of patients who are unable to conceive children through the problems associated with the cytoplasm. As already mentioned, there are approximately 3% of the young women, who were treated in clinics of reproductive medicine. From a biological point of view, these problems are different in nature: in some situations, science understands the mechanism, in others – it does not, but it is clear that somehow the mechanism is linked with the cytoplasm.

“In our clinic we have a proven method, supplies, needles, reagents, pipettes, media – everything needed for almost all nuclear transfers. Why not try?” says Pavlo Mazur. “We tried, and we did it!”

At the beginning of 2017, a healthy child was born after the procedure of nuclear transfer – the first in Ukraine and the second in the world. A year earlier, in Mexico, the first child was born after the procedure of nuclear transfer, which was carried out along a slightly different methodology than the one used by the Ukrainian researchers.

Together with donor cytoplasm, these children have the same mitochondria, which we have already mentioned. Unlike any other organelles, they have their own genes (those 37), this time not their mom’s ones, as any of us, but those inherited from the female donor or, if you prefer, the “third parent”.

Pride and Surprise

Science in Ukraine (including medical) still survives and gives the results “in spite of, not thanks to”. With regard to the implementation of scientific results in practice, the situation is even more deplorable. But this story shows that the Ukrainian researcher together with his colleagues was ahead of the entire (almost entire) world and has got real results that crool, toddle, then get up on their feet, pronounce different words and almost for sure (if they want) one day they will have their own healthy children.

As Pavlo Mazur says, the embryologist should be “pretty good with their hands” for such a work. No doubt that it is owing to his hands (and now to the hands of his other colleagues) that “children from three parents” are born in Ukraine. But it is only part of the story. Assisted reproductive medicine, like any other, is governed by the laws, which may differ in different countries: they can be more conservative in some and less conservative in others. In some country you can choose the gender of your baby, while in the other you cannot. Somewhere the HIV-positive man cannot become a sperm donor, and somewhere – it is not a problem (no, it presents no danger if everything is done correctly).

In the US, for example, in the beginning of 2000s, there were the nuclear transfer experiments conducted. But after they failed, the FDA (Food and Drug Administration) has forbidden to carry out such studies.

At the same time, the UK, for instance, has permitted nuclear transfer since 2015, but so far, as known, no child after this procedure has been born.

In Ukraine the use of such techniques is not prohibited, so we have absolutely legitimately born 12 children, who, besides the mom’s and dad’s genes, have the genes of a third person. Most of them were born owing to Pavlo Mazur’ hands.

Besides Ukraine and Mexico, last year “the child from three parents” came into the world in Greece.

And What About Two “Fathers”?

The most important thing about this story is, of course, not the fact that a boy or girl has the “third father”, but the fact that a man and a woman who previously could not have biological children even for all the money in the world, now have got the opportunity. Yes, so far, not in all countries, and the total number of such children is very small as compared with the need. But this is only the beginning and subsequently nuclear transfer will be increasingly used in reproductive medicine. Perhaps, same-sex couples can have children with the help of this technique.

Pavlo Mazur says that today lesbian couples come with this question. What they want is that one of the women gave her “nuclear material” (actually, becoming the mother) and the other – the cytoplasm with mitochondrial genes. They still need a man to be able to have a child who will inherit the genes from both women. As we know, technically it is possible. But not legally, as strict medical indications are required for such procedures. If none of the women suffer from absolute infertility, then there are no indications.

If not to take into account ethical issues, is it possible to inject two female or two male nuclei into a single oocyte?

“It is possible!” insists Pavlo Mazur. “But it will not conceive a child.

The fact is that maternal and paternal DNA differ in being specially “tagged”, which means that the embryo even will not start to develop.

But today, the researchers conduct laboratory experiments in mice to obtain offspring from same-sex couples. There is some progress, and it is quite possible that in a few years it will be technically possible for humans. It is obvious that the ethical side of the issue is still present, but that is a different story.”

It Is a Fungus

Pneumonias can be very different. There are common ones, caused by the usual bacteria called pneumococci. In this case, they are treated at home with ordinary antibiotics and after three weeks the patient is practically healthy.

And there are atypical pneumonias which are caused by atypical pathogens, such as, for example, some coronaviruses. However, not only by them but by that very influenza virus, measles virus, Mycoplasma, Klebsiella, and many others. Such pneumonias, as we now know, can be very treacherous and even deadly. Fortunately, not always.

Among the atypical pathogens, there is one about which most people, unless they are doctors, have not heard and with which have not faced.

Its scientific name is Pneumocystis jirovecii and it causes the so-called Pneumocystis pneumonia. This is the problem primarily faced by patients with AIDS and oncohematological diseases, and patients after organ transplantation. If you do not belong to a risk group, you can be sure 100% that you will not get infected.

Researchers have long argued regarding the reference of the causative agent of Pneumocystis pneumonia to some systematic rank until they came to the conclusion that it belongs to fungi. This is an important point because what we consider an ordinary “microbe”, in fact may be a virus, a bacterium (which are also different), a protozoan (many of which are animals), or a fungus. This means that the diseases caused by such pathogens, are treated quite differently (everybody knows that antibiotics against flu are not only useless, but can cause even greater problems).

The same situation is with the pathogen of Pneumocystis pneumonia. Ordinary antibiotics that treat regular pneumonia do not effect it. Although there are drugs that do cope with this fungus. In general, it is possible to cure this disease, although there are difficulties. But primarily it should be diagnosed. And here is where the difficulty arises.

The Difficulties of Diagnosis

The clinical performance of this pneumonia has its own characteristics, but it can be easily confused with some other varieties of pneumonia. X-ray or CT scan results can help, but they also do not guarantee accurate diagnosis. And in order to treat Pneumocystis pneumonia, it should be diagnosed with complete accuracy to have clear understanding that it is exactly this type and not some other.

If it were a bacterium, it could be cultivated in a special medium, what is usually done with different pathogenic bacteria, to determine their species rank and antibiotic sensitivity. But this fungus does not grow in a Petri dish.

Our medicine is armed with microscopic method to determine pneumocyst. This means that the sputum sample of a patient is placed under a microscope to see what kind of microorganisms can be seen. Sometimes pneumocyst can be seen, but very often it cannot be seen, despite its being the cause of the disease. In such situations the method is reported to have low sensitivity.

Therefore, it often happens in practice that patients are treated for pneumocyst when the pneumonia treatment showed no improvement. Often this treatment starts too late. According to some estimates (no official statistics), approximately 10% of AIDS patients in Ukraine die from Pneumocystis pneumonia.

But what about PCR?

Polymerase chain reaction, which is so much spoken about lately, has become the “gold standard” in identifying a variety of pathogens. Using a fragment of genetic material, it allows with great accuracy to state if there is, for example, hepatitis C virus, the causative agent of herpes, HPV, or, finally, SARS-CoV-2 in the human body. Test systems based on PCR are widely used in a large number of laboratories.

Why not diagnose Pneumocystis pneumonia using this method? Many countries do it. But not we.

About 4 years ago microbiologists-clinicians from one medical center where they treat AIDS patients addressed Olena Moshynets, Candidate of Biological Sciences and senior research officer of the Institute of Molecular Biology and Genetics (IMBG). They asked her to develop a test system in order to diagnose and treat patients with Pneumocystis pneumonia.

They did not consider the option to purchase an imported test system. The problem was not even in the cost, but in the fact that they were absent at the market (at least, at the moment). In developed countries, medical institutions that often diagnose Pneumocystis pneumonia, produce such test system in-house for themselves or order in other scientific institutions that are ready to execute such an order. A commercial product did not exist, most probably because it represented no special benefit for the business. In other words, even abroad you cannot go to a pharmacy, online store or pharmacy depot and buy such a test system.

PCR is familiar to molecular biologists and geneticists who constantly use it for the variety of their research tasks. Therefore, the group of Olena Moshynets decided to start working on this task. They managed to find funding for reagents, study supplies and salary for the staff of six people for one of the programs of the National Academy of Sciences of Ukraine – UAH 200 thousand.

It took them a little less than a year to develop a test system. In the fall of 2016 it was checked on patients in the Kyiv City Center for Prevention and Fight of AIDS on the basis of Kyiv City Clinical Hospital No. 5. It turned out to be quite sensitive: it “saw” pneumocyst in those samples where microscopic methods failed to find it.

Non-Profit Diagnosis

In order to legally apply such a test system, it must be certified in accordance with applicable law. Olena Moshynets turned to one of the law firms that deemed that, taking into account all discounts (for non-profit entity), the legal support for this procedure will cost approximately the same as the development of test systems, i.e., about UAH 200 thousand.

It is not worth mentioning that this expenditure for NASU was unaffordable (while it is worth recalling that such developments of applied nature should not be among the priorities of NASU, but it is a different story).

The developers of the test system tried to find sponsors or investors willing to fund its certification, but they failed. The reason was that people found nothing of interest in it: pneumocyst does not threaten a relatively healthy person, and those who predominantly suffer from it are the representatives of socially vulnerable layers of the society (one can hardly earn on them).

The situation has not changed for subsequent years. In our country today accurate and quick determination of pneumocyst is possible neither at public medical institutions nor at a qualified commercial laboratory.

“There is the test, there is the problem, there is no decision yet,” concludes Olena Moshynets.

From time to time the doctors from commercial clinics address her on an individual basis with a request to determine pneumocyst in a biological sample of a patient. It is not difficult technically. But it is impossible legally. Olena Moshynets claims that she has no personal commercial benefit in this development. But, of course, as the author, she still hopes that there is someone who will help implement this socially significant project.

A Change of Function

Their idea is to get the fibroblasts to change their function and to “transform” into heart muscle cells. In other words, to “rewind” changes after a heart attack.

From the point of view of fundamental science, the mission is quite feasible. The cells of our body can be very different both in the appearance, and in the tasks they perform. For example, a muscle cell in our biceps is designated to contract, while a nerve cell – to transmit nerve impulses. But despite this, each of them contains exactly the same set of genes.

Cells differ because different genes are activated in different types (while all other genes do not disappear, but they are “asleep” because they are not involved in the specific work the cell performs). Researchers already know how to “activate” and “deactivate” the necessary genes in order to transform the cells of one type into the cells of another, or even into non-specific cells – the non-stem cells. Further, they can be “grown” as the cells of any “function”. The Japanese researcher Shinya Yamanaka received the Nobel prize in Physiology or Medicine in 2012 for the research in this area.

But if to speak about a specific task, when it is required to “transform” the cells of the connective tissues into cardiomyocytes, at this, to do it not “in vitro” but “in vivo”, then, according to Oksana Piven, no one has ever done it worldwide. There is no blueprint of such tasks: it should be still created, and this is usually done by “cut and try” method.

Five Elements

The researchers from IMBG are currently working with rat cell culture. To put it simply, they have rat embryonic fibroblasts in the Petri dish that are to be “transformed” into cardiomyocytes. It can be achieved by “waking up” five exact specific genes known as GATA4, MyoD, Tbx5, Mef2c and HAND2. Each of them can accommodate a cascade of other genes, and then the cell becomes a cardiomyocyte and performs all its inherent functions.

There are several different ways to “activate” the required genes. Each has its advantages and disadvantages, for example, some may cause cancer (if we are talking about working with an animal or human body). Our researchers decided to use a system called CRISPR/Cas9, well known as “gene scissors” outside of the professional community.

It is really frequently used in a wide variety of experiments in order to cut the DNA molecule in the place where the researcher needs. But the other modification is used in our situation that does not cut anything (nor it should) but finds a specific gene, attached to it, and “activates it”.

Our researchers have managed to configure this molecular system for it to activate the very five genes in the cells of rat fibroblasts. Consequently, various genes typical for cardiomyocytes actually function in the cells. This is good news showing that the process of “transformation” has begun. But the problem is that the cells are still unable to contract, as cardiomyocytes do.

Now the researchers are trying to understand why this does not happen. In the near future they want to add some substances to the “chemical cocktail” that can make cells shift away from their fibroblast function to a greater extent. The results of the experiment will show whether it is a success.

From Rat to Man

A parallel team under the supervision of Oksana Piven has started to work on the second phase of a large job. The already covered path should be repeated on the culture of human cells. This means that molecular system CRISPR/Cas9 should be re-set, as “rat” variant is different from the human one for four genes of five.

First it was decided to work with the stem cells of the funiculus (it is not abortive material; the material is legally and ethically obtained with the consent of parturient women). After the success is achieved at this stage, the researchers will get to experiment on more complex “material” – the fibroblasts of adult human body.

There is no doubt that this work will result in a valuable scientific research data that the authors will publish in one of several international journals. A more challenging issue is whether these studies will help treat people from the consequences of myocardial infarction. To be perfectly accurate and honest, nobody knows the answer. The reason is the same: it is a new branch of research and it is impossible to predict all difficulties on this path. It may well be that some of them are impossible to solve. This is an absolutely typical situation for the research work.

The optimistic scenario can be as follows: after a successful transformation of rat fibroblasts to cardiomyocytes in a Petri dish the same should be done in the organism of a laboratory rat, which has been “forced” to survive a heart attack before this.

If all goes well here, the same experiment should be repeated in animals whose body is more similar to human. Most likely, in pigs. This is non-clinical trial. And only then clinical trial will take its turn, that is, the experiments in people. First, it is necessary to make sure that this “treatment” does not harm, and then – that it heals.

“As far as I can predict, the worst thing that can happen to a patient as a result of this therapy is nothing,” says Oksana Piven. “Everything will remain as it is, but it cannot harm a person.”

All these stages of the research can take place in Ukraine, although it is evident, they need funding, which is a stumbling point for our researchers. But that is another story. Even in case of the most optimistic scenario, the clinical application of new medical technologies will be possible not earlier than in a few years.