IPSK: 10 years later
The human cerebral cortex is cultivated in a Petri dish. Eye diseases are treated with retinal cells derived from the patient's own skin cells. The new drugs are being tested on human cells instead of animal models.
These and other experimental studies and treatment methods originate from the discovery that stirred up the scientific world, made 10 years ago by Dr. Shinya Yamanaka and his graduate student Dr. Kazutoshi Takahashi, who developed a method for reprogramming adult mouse cells and returning them to the state of embryonic cells. The resulting cells from these manipulations are now known as induced pluripotent stem cells (iPSCs).
Induced pluripotent stem cells,
reprogrammed from normal adult cells
and not yet differentiated.
A year later, they managed to replicate their approach on human cells. For this revolutionary research and active work to promote stem cell research, Yamanaka, currently collaborating with Kyoto University and the University of California at San Francisco, in 2012 became one of the laureates of the Nobel Prize in Medicine and Physiology.
Induced pluripotent stem cells According to the definition of the US National Institutes of Health, induced pluripotent stem cells are somatic (adult) cells reprogrammed to enter a state similar to that of embryonic cells by forcibly inducing the expression of factors important for maintaining the "stemness" of embryonic stem cells.
Yamanaka's breakthrough provided researchers with an inexhaustible source of induced pluripotent stem cells that can be differentiated into any specific cell types of an adult organism, ranging from skin cells to heart cells and neurons, for further use in basic research, drug development and disease treatment.
This discovery opened up a practical, and in some critical cases, the only method for directly studying human "diseases in vitro", as well as tracking the early stages of both normal and abnormal development. It also allowed specialists to screen new drugs directly on human cells instead of animal models, which in more cases makes it possible to accurately predict the effect of new drugs on humans.
The emergence of induced pluripotent stem cells has ensured rapid progress in some areas and the emergence of serious difficulties in others. It is already quite obvious that these cells are a godsend for basic research, but the use of new technology in the treatment of diseases remains a very dubious prospect. Some cell types turned out to be difficult to reprogram, and even the protocols of these methods are currently at the stage of constant refinement, since this area as a whole is still very young.
Induced pluripotent stem cells in basic biomedical research
According to Dr. Arnold Kriegstein, director of the Center for Regenerative Medicine and Stem Cell Research of Eli and Edith Broad at the University of California at San Francisco, for many scientists working in the field of biomedical research, the opportunities provided by the technology of creating induced pluripotent stem cells are a dream come true. He explains that induced pluripotent stem cells have provided us with an unprecedented opportunity to look at human development. Kriegstein, who studies the early development of the cerebral cortex, notes that he has never had unlimited access to living cells of the human brain before, whereas now he can take skin cells and grow a model of the cerebral cortex in the laboratory. Thus, the appearance of induced pluripotent stem cells has radically changed the conditions of the game in the field of studying early human development.
Kriegstein is enthusiastic about what researchers can learn from studying "organoids" – grown and induced pluripotent stem cell models of developing pea-sized organs. At the present level, scientists are able to obtain clusters of cells that begin to release molecular signals and differentiate into what should turn into an adult organ in the future.
Such models are very close to what is happening in reality. Recently, Kriegstein and his colleagues demonstrated that even at an early stage, organoids are able to form a complex internal organization, including an anterior-posterior orientation, while their various regions begin to acquire features characteristic of the regions of the embryonic brain.
The data published in a number of scientific papers indicate that organoids allow modeling diseases that develop in adulthood, including those characteristic of late age, such as Alzheimer's disease.
Organoid of the human cerebral cortex, cultured in laboratory conditions.
Kriegstein expresses concern that even though organoids provide an unprecedented opportunity to observe the stages of development, some researchers are getting too ahead of themselves.
He emphasizes that we are talking about the embryonic brain. The longest growth period that we can model corresponds to the full development of the embryo. How likely is it that gene expression, cellular signaling mechanisms and myriad other interactions at this organoid stage will accurately reflect the development of Alzheimer's disease – a disease affecting people aged 60-70 years.
Kriegstein believes that such studies should be approached skeptically. Currently, stem cell technology is changing so rapidly that reproducing the results is a very difficult task. Scientists should develop protocols that allow for a reliable comparison of different methods, and subsequently use such standardized methodologies to promote research and treatment methods. However, he is 100% sure that this will become a reality over time.
Starting from the initial discovery
Yamanaka currently directs the Center for the Study and Application of Induced plurirotent stem cells at Kyoto University with a staff of 500 employees, as well as a research laboratory at the Gladstone Institute of Cardiovascular Diseases in San Francisco. In addition, he holds the position of professor of anatomy at the University of California at San Francisco, while Takahashi is a visiting scientist at the Gladstone Institutes and coordinates the work of the Yamanaki laboratory. Both continue their work with induced pluripotent stem cells, as do other researchers.
In their landmark work, Yamanaka and Takahashi used four genetic factors to induce the return of adult cells to a pluripotent state. Shortly after the breakthrough with induced pluripotent stem cells, Dr. Sheng Ding, who heads the laboratory at the Gladstone Institutes and is a professor in the Department of Pharmaceutical Chemistry at the University of California at San Francisco, began work on improving the reprogramming cocktail.
Eventually, Dean was able to replace genetic transcription factors with drug-like molecules, which eliminates the risks associated with introducing new genetic material into cells. Currently, laboratories around the world are developing and publishing various chemical recipes, in many cases depending on the type of cells to be reprogrammed.
Other recent achievements in the field of pluripotency induction are based on the use of various types of proteins that influence the activity of genes in the cell nucleus. Dr. Robert Blelloch of the Broad Center at the University of California, San Francisco, demonstrated that some small RNA molecules, known as microRNAs, stimulate the "de-differentiation" of adult cells, while others stimulate the opposite – the ability of stem cells to differentiate into adult cells. By influencing the activity of microRNAs, the staff of his laboratory were able to increase the yield of cells a hundredfold during reprogramming.
Blelloch and his colleagues were intrigued by the role of so–called epigenetic factors - naturally occurring or externally introduced molecules that modify proteins inside the nucleus. Manipulations of these molecules can also influence the efficiency of induction of pluripotent cells.
Prospects of therapy
Six years after Yamanaka discovered induced pluripotent stem cells working in a completely different field, researchers have developed a new gene editing technology with unprecedented speed and accuracy and called CRISPR-Cas9. A powerful new tool has radically changed the approaches to gene modification by the "cut and paste" method and has been very quickly adapted by thousands of researchers working in the fields of general biology and drug development.
According to Kriegstein, CRISPR technology has provided specialists with new extraordinary opportunities. It allows you to eliminate the genetic causes or factors contributing to the development of diseases. In addition, researchers can edit mutations in order to determine their criticality in the appearance of early developmental defects.
The speed and accuracy of CRISPR may one day help stem cell scientists achieve their most ambitious goal: reprogramming genetically abnormal cells of patients with hereditary diseases such as sickle cell anemia and Huntington's disease into a pluripotent state, editing their genetic defects in the laboratory and their subsequent differentiation into healthy adult cells. Such cells can be transplanted to patients to restore normal function.
Neurons differentiated from induced stem cells,
obtained from the tissue of patients with Huntington's disease.
While this goal is still very far from being achieved, several early-stage clinical trials are already underway to treat various diseases, ranging from diabetes mellitus and heart disease to Parkinson's disease, using induced pluripotent stem cells.
As part of one of these studies, the first patient has already undergone therapy. In 2014, Japanese researchers isolated induced pluripotent stem cells from the skin cells of a woman with macular degeneration and differentiated them into adult retinal cells. Surgeons transplanted the resulting cells into the patient's eyes in order to rid the patient of the disease. This woman became the first patient to undergo therapy with induced pluripotent stem cells.
The researchers devoted their work to eye disease partly because the differentiation of stem cells into retinal cells is quite simple compared to differentiation into other types of cells. In addition, the procedure of cell transplantation into the eye tissue is relatively simple.
Preparation for the procedure for the second patient had to be stopped, as the researchers found a mutation in one of the genes of induced pluripotent stem cells derived from the patient's own cells. There is no evidence of a relationship between this gene and the development of cancer, but they decided not to use these cells in order to eliminate any potential risks.
The success of treatment partly depends on the high rate of stem cell proliferation. In some cases, hundreds of billions of cells may be needed for transplantation. However, even if several stem cells fail to differentiate into adult target cells, after transplantation they can begin to divide very quickly and eventually form a tumor.
Yamanaka notes that today one of the main tasks of this field is the development of methods that ensure the differentiation of all stem cells before transplantation.
To eliminate the risk of cancer, researchers are conducting "deep sequencing" of the genetic profile of each of the stem cell lines potentially suitable for clinical use. They also decided to use donor cells rather than the patient's own cells. This avoids the large financial costs of quality control, such as deep sequencing, for each patient's pluripotent cell lines.
The use of iPSC in drug screening
Cardiomyocytes obtained by reprogramming
from cells of normal adult skin.
The prospects for the use of stem cells extend to the testing of drugs on induced pluripotent stem cells derived from adult human cells, instead of animal models.
One recent example is the work of Catherine Mummery, a neurologist at the National Clinic of Neurology and Neurosurgery in the UK, who used mature cardiomyocytes derived from induced pluripotent human stem cells – heart cells contracting in a Petri dish – to test two commercially available drugs for the treatment of diseases of the cardiovascular system. She demonstrated that both drugs triggered the same therapeutic effects when used in one dosage and the same toxic reactions in another dosage, as shown in clinical studies.
According to Kriegstein, this impressive work served as proof that the use of animal models of adult human cells derived from induced pluripotent stem cells in drug testing provides reliable results that are more relevant for patients.
Other sources, old and new
Stem cell research is not limited to induced pluripotent stem cells. In some respects, embryonic stem cells (ESCs) remain the gold standard. A specialist in cell biology from the University of California at San Francisco, Dr. Susan Fisher, compares embryonic stem cells at an early stage of development with a blank slate. She explains that they have a "shorter history and less baggage" than induced pluripotent stem cells. However, just like Yamanaka and Kriegstein, she recognizes that the field of stem cell research is still too young to claim the superiority of one strategy over another.
Strong support for the idea of using embryonic stem cells in transplant medicine arose two years ago when a group of researchers from Harvard University demonstrated that cell lines derived from embryonic stem cells can produce an unlimited number of insulin-producing pancreatic islet cells. Currently, early-stage clinical trials are being conducted, the purpose of which is to test the safety and effectiveness of islet cell transplantation in patients for the treatment of type 1 diabetes mellitus.
In 2014, hundreds of clinical trials were conducted in different countries, the main task of which was to test the safety and effectiveness of treatments for various diseases, ranging from heart failure to Parkinson's disease.
To the two well-known strategies for obtaining stem cells, a third has recently been added, called direct reprogramming or transdifferentiation of cells. This method involves the direct transformation of skin cells into target cells - brain, heart, pancreatic cells – excluding the preliminary stage of their return to a fully pluripotent state. This method avoids the risk of developing cancer, which is an integral part of the differentiation of truly pluripotent cells.
This year, Deepak Srivastava, head of research in the field of cardiovascular diseases and stem cells at the Gladstone Institutes and professor of pediatrics, biochemistry and biophysics at the University of California at San Francisco, and a group working under the leadership of Dr. Shen Dean, effectively transformed mouse skin cells into brain cells, as well as contracting heart cells using a combination of chemical compounds. This approach can become an effective method of regeneration of dying or diseased cells and tissues.
Ethical issues and public perception
As scientific progress in many areas has progressed, Yamanaka has come to the conclusion that science is very much ahead of attempts to address ethical issues in some research areas.
When Yamanaka and his colleagues created pluripotent stem cells, their goal was to overcome ethical issues related to embryonic stem cells. Now this work raises new ethical questions. Researchers can create sperm cells or eggs from induced pluripotent stem cells, at least from mice. It is also theoretically possible to grow human organs in pigs or other animals by introducing human induced pluripotent stem cells into animal embryos, creating so-called chimeras.
In addition, he expresses concern about the public perception that the speed of progress may be less than expected. Yamanaka notes that he is "delighted with how fast science is developing. It's just amazing. However, for the most part, the development of new therapies – conducting scientific research, testing the safety and effectiveness of new treatments – requires a lot of financial and time costs. The development of new treatment methods can take 10, 30 and 30 years. This is what we are trying to explain to our patients: we are making great progress, so do not lose hope. However, it takes a lot of time."
Evgeniya Ryabtseva
Portal "Eternal youth" http://vechnayamolodost.ru Based on the materials of the University of California: Despite Major Progress Towards Disease Therapies, Some Technical and Ethical Challenges Still Lie Ahead.
17.10.2016