In search of cells for IPSC
Step by step to the medicine of the future
Evgenia Alekseeva, "Biomolecule"
The discovery of induced pluripotent stem cells (iPSCs) has become one of the most high-profile and promising achievements in the scientific world in recent years. It would seem that as soon as each person has their own pluripotent stem cells, a huge number of medical problems will be solved. Nevertheless, almost ten years have passed, and the use of IPSC in real practical medicine has not really begun yet. As before, the main problem between the discovery of IPSC and saving the world from all ailments is the methods of induction of pluripotency in cells. The way to bridge this gap may be to search for cell types that are easier to reprogram. One of these types lies literally "on the surface" – dermal papilla cells (it is explained below that this is a connective tissue papilla in the hair bulb – VM).
iPSCs obtained from human dermal papilla cells, stained with the pluripotency marker SSEA4 (red). Drawing from the diploma of the author of the article [16].Stem cells are the panacea of the XXI century
Induced pluripotent stem cells (iPSCs) have made an incredible splash in the scientific community [1]*.
Imagine only – iPSCs of his own genotype can be obtained for each person. This discovery provides a whole range of new opportunities: for example, it solves any issues related to donation at the moment. The resulting iPSCs can be directed in any direction of cellular differentiation and create a donor material that does not need to be searched for. The most important thing is that the tissues and organs obtained in this way will have full immunological compatibility with the patient's tissues, since they will be created from his own cells **.
* – Stem cells can be obtained in other ways: an embryo can share them without any damage to development ("Sparing stem cells" [2]) or... its habitat ("Endometrium as an alternative source of stem cells" [3]). – Ed.** – The article "Organs from the laboratory" tells about the situation in the field of creating artificial organs and technologies for their production [4]. – Ed.
In addition, opportunities for the treatment of genetic diseases are opening up for the first time. Molecular biology also does not stand still: a huge number of methods are being developed that allow you to edit the genome*, correct "errors" in it or add additional elements. But all these methods are ineffective in relation to an adult organism – differentiation of its cells, the formation of organs and tissues occurred under the influence of causal mutations. How can IPSC help? These cells represent the initial form of cellular development. If the genome is corrected at this stage, the subsequent differentiation will take place without deviations.
* – Currently, when developing gene therapy approaches, special hopes are pinned on ZFN, TALEN and CRISPR/Cas9 editing systems based on the site-specific action of nucleases in vivo. These systems usually consist of two modules, one of which recognizes the desired oligonucleotide sequence, and the other cuts the DNA chains: "Should we take a swing at... genome change?" [5], "CRISPR systems: immunization of prokaryotes" [6]. And some of the approaches are already quite "tangible", since the voices of bioethicists sound louder: "Mutagenic chain reaction: genome editing on the verge of fiction" [7]. – Ed.
What problems can there be?Despite the active study of iPSCs, there are a number of problems that make it difficult to use them directly for human treatment.
These problems are related to the method of inducing pluripotency ("reprogramming") of cells. I must say, until quite recently, even the idea of the existence of such methods belonged to the category of heretical, because differentiation was considered an irreversible process – before the "Nobel" works of John Gurdon and Shinya Yamanaka [8]. Yamanaki's group found that only four transcription factors are needed to reprogram terminally differentiated cells: Oct4, Sox2, c-Myc and Klf4 [9]. Transcription factors (TF) are proteins capable of regulating the work of genes. During the operation of these four TF, the cell genome is rearranged into a pluripotent state. At the moment, the only effective way to obtain iPSCs is to introduce TF genes into the cell using lentiviral vector constructs. This method leads to the integration of the vector into the genome of the cell, which can cause unforeseen modifications of its structure and lead to the development of malignant formations. In addition, the viral constructs themselves are antigens for humans, which is also unsafe and limits the possibilities of practical application of IPSC [10].
However...Nevertheless, some iPSCs still got to be applied in practice.
For example, they began to treat sickle cell anemia* with their help. iPSCs were obtained from the patient's cells and the mutation leading to this disease was corrected using specialized genetic methods [11]. Eventually, iPSCs were differentiated into healthy red blood cells and injected into the patient. The fact is that red blood cells are the rare case when the unpredictable integration of TF genes into the cellular genome does not pose any danger, because in the process of differentiation, red blood cells lose the nucleus.
* – Gene and cell engineers in general are not indifferent to blood diseases, and their efforts bring more and more tangible results – both in efficiency and safety: "Summary from the gene therapy fronts. A new strategy for neutralization of hemophilia" [12]. – Ed.
What to do?The above problems can be solved using two independent strategies.
The first strategy is to find alternative safe ways to introduce transcription factors*. To date, a lot of work is being done in this direction. For example, adenoviral constructs that are not embedded in the cellular genome are used as a supplier of TF genes. With another approach, TF is delivered to the cell not in the form of genes, but in the form of RNA matrices or ready-made proteins. Even within the framework of integrative methods of transcription factor delivery, work is underway to reduce the risks of their use by embedding all four genes in one reading frame. There are a lot of options for delivering transcription factors into the cell, but they all lose to the usual lentiviral constructs in two parameters – in terms of efficiency and optimality of material costs and forces. The latter is also important, since in the future reprogramming of cells will be performed not in the laboratory, but on a medical scale.
* – A rare person has not heard about the bad behavior of others and even their own for some reason poorly controlled stem cells: "Stem and branches, stem cells" [1]. The methodology for obtaining iPSCs further increases the degree of oncological alertness: the problem is the incorporation into the genome of not only vector DNA with active promoters, but also proto–oncogenes - yes, the genes of two of the four "magic" TF (c-Myc and Klf4) belong to proto-oncogenes. Therefore, they are trying in every way to find a balance between the effectiveness of reprogramming and safety, changing the spectrum of TF or completely excluding the introduction of genetically engineered structures - there is hope that in the future it will be quite easy to "pohimichit": "A snowball of problems with pluripotence" [10], "There was a simple cell, it became a stem cell" [9]. – Ed.
The second strategy is to find the optimal cell type for reprogramming. It turned out that different cell types can vary significantly in their ability to produce IPSC lines. These differences manifest themselves in unequal speed and efficiency (from the same number of cells of different types, a different number of IPSC clones are obtained) of reprogramming. The ability of a cell type to reprogram depends on various factors. Some cells are multipotent or just actively renewing, which puts them closer than terminally differentiated cells to a pluripotent state.
In addition to the effectiveness of such a transformation, cells differ in the number of transcription factors necessary for their reprogramming. Cells that "respond" to a smaller number of transcription factors not only significantly reduce the risks of even dangerous methods of TF administration, but also increase the effectiveness of safe ones already found. Such cells include, for example, neurons that can be reprogrammed with the introduction of not four, but only one transcription factor [13]. But neurons are not suitable for the ubiquitous creation of iPSCs in medicine due to the complexity of their isolation from the body. In addition to the simplicity of obtaining the optimal cell type should be least susceptible to aggressive environmental influences, which means that skin fibroblasts, most often used for reprogramming, cannot be attributed to such cells [14].
Dermal papilla cells are a possible key to solving problemsFigure 1. The structure of the hair follicle.
The dermal papilla, or hair papilla, is located at the base of the hair bulb.In 2011, it was shown that mouse dermal papilla cells can also be reprogrammed with just one TF – Oct4 [15].
Since then, the idea of reprogramming dermal papilla cells has not been put forward anymore, which seems very strange, since these cells are the best suited for the role of a source of IPSC. The dermal papilla (DP) is a connective tissue papilla in the hair bulb (Fig. 1); it is located in the deep layers of the skin and is less susceptible to aggressive environmental influences. In addition, DP cells are convenient for isolation: a few pulled hairs are enough to obtain a cell culture. But most importantly, these cells are easily reprogrammed and require fewer transcriptome interventions, which is significantly safer compared to classical reprogramming by four transcription factors. Unfortunately, the work was carried out on mouse cells [15], but its results were not verified on human cells.
As a result, in 2014, in the Laboratory of Developmental Genetics of the N.I. Vavilov Institute of General Genetics, the following task was set as part of one of the graduation papers: to determine the minimum set of transcription factors necessary for reprogramming dermal papilla cells. To begin with, a more detailed study of the expression of transcription factor genes in human dermal papilla cells was carried out. It turned out that in this type of cells, the genes of three of the four Yamanaki TF are expressed in the normal state: SOX2, KLF4 and c-MYC (Fig. 2).
Figure 2. The result of RT-PCR analysis of human dermal papilla cells, terminally differentiated fibroblasts and iPSCs for the expression of Yamanaki factors. It can be seen that three of the four transcription factors are present in dermal papilla cells. Drawing from the diploma of the author of the article [16].The fact of expression of KLF4 and c-MYC is not surprising, since their products are involved in the processes of proliferation and regulation of the cell cycle and are present in differentiated cells – for example, in cutaneous fibroblasts.
But Sox2 is a pluripotency factor and is found in already reprogrammed iPSCs, where all TF is present; in this, dermal papilla cells are unique. Thus, if three of the four transcription factors are already synthesized in these cells, there is indeed reason to believe that reprogramming human DP cells using Oct4 factor alone is feasible.
With further research, it turned out that human DP cells are less susceptible to reprogramming than similar mouse cells – it takes much longer and with less efficiency. This is quite logical, since the human hairline has changed its physiological functions in the process of evolution compared to the cover of other mammals. At the moment, reprogramming has been carried out only with the help of two TF – Oct4 and Sox2 (Fig. 3), which is already a good result, since it halves the risks of genome damage described above when using viral constructs [16].
Figure 3. iPSCs obtained from human dermal papilla cells. A – Characteristic morphology of pluripotent stem cells: dense colonies of small cells with a high nuclear-cytoplasmic ratio. B – Staining of iPSCs for pluripotency markers Oct4, Nanog, Tra 1-60 and SSEA4, the presence of which proves the completion of reprogramming. Drawing from the diploma of the author of the article [16].A logical question arises: why did it take more than one transcription factor to reprogram, if three of them are already present in cells?
Apparently, the natural expression of TF genes in dermal papilla cells was not enough to rearrange the genome into a pluripotent state, and for reprogramming by one transcription factor, auxiliary low-molecular agents will be needed to facilitate its work. According to some data, small organic molecules, even without direct influence on the activity of genes, are able to "catalyze" the transfer of cells to a pluripotent state, while replacing part of the TF [9].
Of course, the gap between theory and practice has not been overcome by the author of this article in one year of the thesis, but further research paths are emerging more clearly. The above results showed that dermal papilla cells are indeed closer to pluripotency than ordinary terminally differentiated cells, and studies of reprogramming human DP cells with a single transcription factor need to be continued. Probably, in the near future, in other laboratories that deal with alternative safe ways of introducing transcription factors, there will also be an optimal solution, and by adding two and two, we will still come to the medicine of the future.
Literaturebiomolecule: "Trunk and branches, stem cells";
- biomolecule: "Sparing stem cells";
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- Biomolecule: "And whether we should take a swing at...
- genome change?";Biomolecule: "CRISPR systems: immunization of prokaryotes";
- Biomolecule: "Mutagenic chain reaction: genome editing on the verge of fiction";
- Biomolecule: "Nobel Prize in Physiology or Medicine (2012): induced stem cells";
- biomolecule: "There was a simple cell, it became a stem cell";
- biomolecule: "A snowball of problems with pluripotence";
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- In situ genetic correction of the sickle cell anemia mutation in human induced pluripotent stem cells using engineered zinc finger nucleases. Stem Cells. 29, 1717–1726;biomolecule: "Summary from the gene therapy fronts.
- A new strategy for neutralizing hemophilia";Kim J.B., Zaehres H., Araúzo-Bravo M.J., Schöler H.R. (2009).
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- Single transcription factor reprogramming of hair follicle dermal papilla cells to induced pluripotent stem cells. Stem Cells. 29, 964–971;Alekseeva E.I. Determination of the minimum set of transcription factors sufficient for induction of pluripotency of human dermal papilla cells: thesis.
- – Moscow, 2015 – 56 p.Portal "Eternal youth" http://vechnayamolodost.ru
06.10.2015