IPSK: problems of transition age

A snowball of problems with pluripotencePeter Starokadomsky, "Biomolecule" based on Nature news –

Hayden E.C., Stem cells: The growing pains of pluripotencyIllustrations: California Institute for Regenerative Medicine @ Flickr

Induced pluripotent stem cells are one of the newest areas of biology that is rapidly gaining momentum. Like any other new science, it has now entered a difficult "teenage" period. This is what our story will be about.

Induced pluripotent stem cells (iPSCs) derived from skin cells.
The blue color corresponds to the nuclei of cells; the green "sparks" correspond to the NANOG protein,
present in iPSCs, but absent in "normal" cells;
red dots represent inactivated X chromosomes.

A five–year term in science is a very short period of time. But that's exactly how much time has passed since the first publication of Shinya Yamanaka from Kyoto University of Japan, where he announced the creation of a method for "reprogramming" mouse skin cells into induced pluripotent stem cells (iPSCs). These cells have the ability to transform into many other types of cells, which opens up extraordinary opportunities for regenerative medicine. A year later, the same group of scientists obtained a similar result for human cells.

Cardiac muscle with IPSC. Muscle fibers contain iPSCs that are directly "reprogrammed" into cardiomyocytes.
In the future, the same technique will help mitigate the consequences of a heart attack or other heart lesions.

Like embryonic stem cells, iPSCs can also potentially be used for therapy, creating in vitro models of diseases, or for finding new drugs. However, iPSCs have one indisputable advantage – they can be obtained "here and now" (directly from adult cells of the body), which greatly simplifies the question of the objectivity of the model and the compatibility of future transplants. Another plus is that human embryos are not required to work with IPSC, which removes a number of ethical problems. Now cells can be taken from a specific patient with a certain disease and his disease can be studied in the laboratory, bringing the era of personal genetics and medicine closer.

Scientists predict that iPSCs will change the face of biology and medicine. Over the past five years, hundreds of papers have been published on the study of diseases on various cellular models created with the help of iPSCs – from heart diseases to models of schizophrenia. Everything is going to the fact that soon the treatment with IPSC will become a reality. In California, for example, a group of scientists has been hoping for three years to get permission to treat people with a congenital skin disease – epidermolysis bullosa – using skin grown from the iPSCs of the patients themselves.

However, a huge number of works and results have also revealed a number of problems that arise when using IPSC. So, for example:

• reprogramming may be incomplete or cause mutations;
• iPSCs may not differentiate into all cell types;
• Not all iPSCs are good models for studying diseases.

And although there is no doubt that the prospects for IPSC are huge, we are only at the very beginning of the path, and it is too early to say that the methodology has been worked out.

iPSCs turn into hair sensory cells of the inner ear.
An electronic scanning micrograph (23000×) shows the morphology of iPSCs that have turned into auditory cells.
In the future, they can be transplanted into the ear for the course of some forms of deafness
or use it to test medications against hearing disorders.

In search of a recipeBiologists have been trying from the very beginning to find a safer and more effective way to create iPSCs than the one proposed by Yamanaka.

Recall that he used a retrovirus as a vector for delivering four reprogramming factors into the cell (some of which are potential oncogenes). This type of virus is good because it integrates its genes directly into the genome of the host cell, but this is also the potential danger of the method - violation of the integrity of the genome can unpredictably disrupt the work of other genes, which is a possible prerequisite for malignant degeneration of iPSCs. "A possible prerequisite" does not sound too threatening, but such prerequisites should be excluded in principle before starting any treatment with the use of IPSC.

New reprogramming schemes are published almost every month, but Yamanaki's method still remains unsurpassed in terms of efficiency. This efficiency is 0.01% – that's how many cells become iPSCs after all procedures. For comparison, the adenovirus method (more "gentle", because no integration into the genome occurs) is effective only for 0.0001–0.0018% of cells, and the physico-chemical delivery of reprogramming factors into human cells is only for 0.001%. The lower the efficiency of reprogramming, the higher the cost of the method, and a difference of 10 or 100 times leaves new methods out of business (even if they are safer). In addition, each of them is characterized by its own individual difficulties in obtaining final iPSCs.

One of the actively developed areas is to reduce the potential carcinogenicity of reprogramming factors. First of all, the Icc factor (human transcription factor), which poses the greatest potential threat, falls under suspicion. Published papers where researchers tried not to use Myc, but this further reduced the efficiency of the process. Attempts at post-transformation shutdown of the Myc gene using short RNAs did not give a reliable effect: often the activity of the gene "resurrected" after a short time.

The problems described above have already become one of the hottest topics of induced stem cells. And until now, scientists continue to shuffle various factors in order to get the perfect reprogramming cocktail, and also work out the optimal carrier for its delivery. So, in April 2011, the group of Edward Morris from the University of Pennsylvania announced that they had found a way to reprogram cells with an efficiency two orders of magnitude higher than the usual indicator – about 1% – using a retrovirus to deliver a cluster of microRNAs. Time will tell how much this technology will justify the hopes placed on it.

Old woundsThe study of stem cells has caused an avalanche of new questions that scientists had not even suspected before.

One of them is how critical is the effect of the chromatin epigenetic pattern on the effectiveness of subsequent reprogramming? In June 2010, the groups of George Daley from Boston and Konrad Hochedlinger from Cambridge published papers that iPSCs after induction and reprogramming still retain epigenetic markers of the original cells (although some of them still disappear in the process). Perhaps this is one of the most critical differences between iPSCs and embryonic SC – at the level of epigenetic markers, these two groups of cells differ.

In addition to epigenetic differences, iPSCs contain more "traditional" mutations compared to embryonic SC. The most unpleasant thing here is that a number of mutations and rearrangements of the genome in iPSCs are not inherited from the parent cells, but acquired in the process of reprogramming. It was also unexpectedly discovered that some of these mutations are gradually destroyed during prolonged cultivation of iPSCs (the most likely explanation for this is that cells with the most serious mutations simply die). "However, even with prolonged cultivation, the epigenetic markers of the original cells still do not disappear," writes Joseph Ecker from California. However, others object to him: for example, a group led by Alexander Meissner from Harvard, comparing the epigenetics of 20 embryonic SC with 12 iPSCs, did not find a serious difference between them.

Precursors of insulin-producing cells derived from stem cells.
Transplanted into the pancreas of mice, insulin-producing cells
they will be able to effectively compensate for the loss of insulin in type I diabetes.
In the future, such therapy will be used to treat type 1 diabetes.

Anyway, for the Food and Drug Administration (FDA), all this epigenetics means a lot, and they will not let IPSC into medicine until the issue with epigenetic markers is finally resolved.

Many faces, but not universalAlthough iPSCs are extremely plastic, they are still not universal.

For example, everyone is looking forward to the possibility of obtaining liver cells (hepatocytes) from IPSC. Theoretically, they could replace animals when testing the toxicity of drugs, and also become a panacea for cirrhosis of the liver. But it is still not possible to get hepatocytes – the reason is too complex a pattern of signals that control the differentiation of these cells, and so far it is not possible to find a combination for such reprogramming in vitro. There have already been several publications in the literature about the creation of a reprogramming technique, but subsequent checks revealed certain errors in the evaluation of the results. A new article has recently been published describing a method for producing cells similar to hepatocytes by reprogramming mouse skin cells. However, to what extent this is true and whether they will be able to perform the functions of hepatocytes is not yet clear.

Another hot area of IPSC is substitution therapy for type 1 diabetes. In this disease, as a result of an autoimmune attack, the body's own insulin-producing cells are destroyed. Reprogramming the patient's cells into new insulin-producing cells and implanting them into the pancreas seems to be a very tempting way to treat this ailment, but today no one is able to do this. Scientists simply do not know the pattern of signals that make it possible to "reprogram" SC into Langerhans cells. And although the precursors of these cells have already been obtained – both from embryonic SC and from iPSCs – no one has yet managed to obtain mature insulin-producing cells.

Unified standardTime is money.

In science, this is felt especially clearly. Therefore, it is not surprising that they try to publish a good result as soon as possible – sometimes even to the detriment of the truth. The ease of obtaining iPSCs and the relevance of the topic opened the opportunity to work with them to anyone. This has led to the fact that often groups publishing certain results of work with IPSC characterize them by partial signs, but not by full standards (which would take an order of magnitude longer). As a result, thinking that they are working with iPSCs, they publish the results obtained on cells of an indeterminate breed.

The field of stem cells still requires the introduction of standards that uniquely define the concept of "IPSC", just as the classification of immune cells by combinations of surface markers was introduced at the time. We hope that such standards will be put into practice in the near future.

Natural stem cells. Stem cells in small quantities are constantly present in tissues –
on this slice of the rat brain, they divide to give rise to astrocytes and mature neurons.

How to measure the IQ of neurons in a Petri dish?Researchers can create patient-specific iPSCs to simulate almost any disease.

However, in some cases, the genetic component is not yet the main sign of the disease. We are talking about neurodegenerative diseases. Today, the creation of neuronal cultures for the study of the molecular biology of diseases is one of the most controversial areas. Can the reprogrammed cells of patients with schizophrenia or autism be useful for studying the patterns of the course and treatment of diseases in vitro? In other words, is it possible to measure the IQ of neurons in a Petri dish? Some are trying to prove that this is possible – it is only important to put the questions correctly.

In April of this year, Fred Gage from California showed that neurons obtained as a result of reprogramming from the skin of patients with schizophrenia can be used to select the right treatment. To prove this, with the help of a number of in vitro manipulations, his group managed to eliminate differences in the physiology of normal neurons and neurons of patients. Fred believes that using iPSCs will help to create models and show which genetic factors underlie the development of schizophrenia.

Similar work is carried out in the study of aging. What happens when tissue ages, and to what extent is it programmed into the genome? Indeed, up to this point, the only way to test in vitro the effect of a particular gene that is inactive in a certain disease was to create knockout cell lines in which the gene of interest is inactivated. As a rule, it takes about a year; in addition, it illustrates the effect of the malfunction of only one gene, whereas in many diseases the expression of entire gene families changes. Therefore, knockout cells are a reliable model, but with rather limited capabilities. In comparison with it, iPSCs open up truly unprecedented opportunities. By taking skin cells from any patient we are interested in and reprogramming them into the right type of cells, we can study the entire set of genes that are disrupted in a particular patient. Over time, these data will reliably indicate the difference between pathology and norm. This, in the end, will be able to shed light on the main question of genetics: to what extent are diseases, cancer and aging programmed inside us?

iPSCs bring us huge prospects. It remains to wait until scientists sort out the snowball of problems associated with them and develop a unified theory of working with induced stem cells. However, an excess of problems is a common situation in a new science: the first enthusiasm has passed and has been replaced by a realistic understanding of which issues should be solved first and which should be left for later.

Portal "Eternal youth" http://vechnayamolodost.ru11.07.2011