RU2565548C1 - Method of producing of pluripotent stem cells - Google Patents

Abstract

FIELD: biotechnologies.

SUBSTANCE: method of producing of pluripotent stem cells is offered which comprises the process of dedifferentiation of somatic cells, cultivation of induced pluripotent stem cells in the presence of the agent modifying the epigenetic status of the cell, and cultivation of stem cells in the absence of the named agent. The process of dedifferentiation of somatic cells is performed by means of their modification using the reprogramming factors in the medium containing GSK126 methyl transferase inhibitor, and cultivation of the induced pluripotent stem cells in the presence of the agent modifying the epigenetic status of the cell is performed in the medium containing Vakh and Bak proteins.

EFFECT: invention allows to produce the induced pluripotent stem cells capable to full afterripening and capable to be differentiated in cells with low probability of development of their carcinogenesis, increase of rate of producing of the pluripotent stem cells and the differentiated cells produced from them afterwards.

8 cl, 4 ex

Description

The invention relates to the field of biotechnology and can be used in the artificial preparation and cultivation of living tissues and also in the treatment of cancer cells.

Existing ways of reprogramming somatic cells into pluripotent stem cells (Nature, Volume 465, Number 7299, pp. 665-836, 06/10/2010) are accompanied by a number of disadvantages.

The transplantation of nuclei taken from somatic cells into a fertilized egg from which the nucleus has been previously removed is a very complex process and involves the stage of cloned embryos.

The fusion of somatic cells with pluripotent stem cells requires the initially isolated pluripotent stem cells.

Modification of a somatic cell, inducing its transformation into a stem, with the help of: genetic material encoding protein reprogramming factors, recombinant proteins, microRNAs, synthetic self-replicating polycistronic RNA and low molecular weight biologically active substances, in most cases is a very long process. Complete reprogramming of somatic cells into induced pluripotent stem cells can take a whole month.

A common drawback of all known methods of reprogramming is that the differentiated cells formed from the obtained pluripotent stem cells are immature and prone to the formation of a malignant tumor, which is associated with the sudden activation of “stem” genes in undifferentiated cells from which undifferentiated cells are derived, many of which control cell division.

The exception is also not the most effective way of reprogramming cells using 4 Yamanaki factors: Oct4, Klf4, Sox2 and c-Myc (Cell, Volume 126, Issue 4, p. 663-676, 08.25.2006).

There is a differentiation method that converts human embryonic stem cells (hES) into endocrine cells capable of synthesizing pancreatic hormones, insulin, glucagon, somatostatin, pancreatic polypeptide and ghrelin. This process imitates pancreatic organogenesis in vivo, passing cells through stages resembling the formation of a formed endoderm, endoderm of the intestinal tube, pancreatic endoderm and the conversion of endocrine cell precursors into cells expressing endocrine hormones (Nature Biotechnology - 24, 1392-1401, 11.11.2006) .

The main problem of this method is that the isolation of stem cells from the embryo is incompatible with its further development.

A known method of isolating a line of human pluripotent embryonic stem cells, comprising the following stages: isolating human blastocysts, isolating cells from the inner cell mass of the blastocysts, applying the inner cell mass of the blastocysts to embryonic fibroblasts, in which the resulting inner cell mass forms an array of cells, dissociation of the resulting array into dissociated cells, application of dissociated cells to embryonic feeder cells, preparation of a cell colony with dense m rfologicheskoy structure, large nuclei and nucleoli, and culturing colonies of cells prepared to obtain lines of pluripotent human embryonic stem cells (US 6,200,806 B1, C12N 5/073, 13.03.2001).

The multi-stage and complexity of this process is compounded by the fact that human embryonic stem cells are cultivated under very specific conditions, the slightest violation of which leads to a lack of result.

The closest analogue is a method for producing definitive endoderm, mesoderm, ectoderm or endoderm cells, including culturing stem cells in the presence of an agent that changes the epigenetic status of the cell (demethylating agent, histone deacetylase inhibitor, or a combination thereof) and subsequent cultivation of stem cells in the absence of an agent to obtain such image of definitive endoderm, mesoderm, ectoderm or endoderm cells. Stem cells can be obtained by dedifferentiation of somatic cells. The demethylating agent may be 5-azacytidine or 5-aza-2′-deoxycytidine. Histone deacetylase inhibitor (HDAC) can be selected from the group consisting of: trichostatin A, vorinostat (SAHA), proteinostat (PXD101), LAQ824 / LBH589, trapoxin B, entinostat (MS-275), CI994, mocetinostat (MGCD0103) , valproic acid, isovalerate, valerate, valproate, nicotinamide, dihydrocoumarin, naphthopyranone and 2-hydroxynaphthaldehydes, apicidine, FK228 and sodium butyrate (WO 2010065679 A1, C12N 5/071, 06/10/2010).

The main disadvantage of all the described methods is the high likelihood of carcinogenesis of differentiated cells, which is associated with a disturbed process of cell division and apoptosis.

The technical result of the claimed invention is the production of induced pluripotent stem cells capable of full maturation and capable of differentiating into cells with a low probability of developing their carcinogenesis, as well as an increase in the rate of production of pluripotent stem cells and differentiated cells subsequently obtained from them.

The technical result is achieved by the proposed method for producing pluripotent stem cells, including the process of dedifferentiation of somatic cells, cultivation of induced pluripotent stem cells in the presence of an agent that changes the epigenetic status of the cell, and cultivation of stem cells in the absence of the specified agent, while the process of dedifferentiation of somatic cells is carried out by modifying them with using reprogramming factors in a medium containing methyltransferase inhibitor GSK126, and the cultivation of induced pluripotent stem cells in the presence of an agent that changes the epigenetic status of the cell is carried out in a medium containing Bax and Bak proteins.

Preferably, the density of the monolayer of induced cells immediately before the process of dedifferentiation was 30-60%. In this case, it is preferable that the concentration of the GSK126 methyltransferase inhibitor in the medium is 0.8-2 wt.%.

The cultivation of induced pluripotent stem cells is also preferably carried out in the presence of a proliferation inhibitor.

At least one globin peptide chain is preferably used as a proliferation inhibitor.

Stem cell cultivation is preferably carried out in the presence of nitric monoxide.

The cultivation of induced pluripotent stem cells is preferably carried out in the presence of at least one apoptosis inhibitor.

As an apoptosis inhibitor, it is preferable to use a FLIP inhibitor.

The necessity of introducing a methyltransferase inhibitor GSK126 into the environment of dedifferentiation of somatic cells is explained by the following. The presence of the H3K9me3 label impedes the reprogramming of somatic cells into induced stem cells, as it interferes with the landing of protein reprogramming pluripotency factors on target genes. In this regard, cell modification must be carried out with a high content of the H3K27me3 repression marker, which eliminates the work of the H3K9me3 repression marker that impedes the process, and also, among other things, contributes to the preservation of epigenetic memory in the process of cell division, which avoids carcinogenic and destructive phenomena in cells. This can be confirmed by the fact that the installation of the H3K27me3 marker usually occurs during the cell cycle before DNA replication.

In addition, it should be noted that trimethylation of lysine 27 in histone H3 (H3K27me3) is, in principle, a necessary condition for the reprogramming of somatic cells into induced pluripotent stem cells, however, the presence of this marker in small quantities not only inhibits the reprogramming process, but is also fraught with further formation in differentiated cells of inflammatory processes.

In connection with the above, during the dedifferentiation of somatic cells, the need arises for the artificial installation of the H3K27me3 marker. This can be done by introducing N - [(1,2-dihydro-4,6-dimethyl-2-oxo-3-pyridinyl) methyl] -3-methyl-1 - [(1S) -1-methylpropyl] into the dedifferentiation medium -6- [6-1-piperazinyl) -3-pyridinyl] -1H-indole-4-carboxyamide (GSK126 methyltransferase inhibitor).

The GSK126 methyltransferase inhibitor promotes the formation of histone methyltransferase EZH2, which is part of the PRC2 complex of the policomb protein group. The resulting subunit of EZH2, in turn, performs triple methylation of histone H3 with the formation of a repression marker H3K27me3.

According to immunofluorescence analysis, most colonies of induced pluripotent cells obtained by other methods than those proposed give an average signal about the presence of H3K27me3 in the nuclei of 40-100% of the cells in the colony. In this case, at the most stable monolayer density of the induced cells at the beginning of the dedifferentiation process of 30-60% for the formation of the required amount of histone methyltransferase EZH2, it is preferable that the concentration of the GSK126 methyltransferase inhibitor in the medium is 0.8-2 wt.%.

Regarding the improvement of subsequent cultivation and differentiation of stem cells, the following should be noted. It is known that in the body of an average adult, about 50-70 billion cells die every day as a result of apoptosis. For an average child aged 8 to 14 years, the number of cells killed by apoptosis is about 20-30 billion per day. The total mass of cells that undergo destruction during 1 year of life is equivalent to human body mass. In this case, the replenishment of lost cells is ensured by proliferation - an increase in the cell population by division.

The most promising way to obtain pluripotent stem cells is to modify somatic cells, inducing their transformation into stem cells using reprogramming factors - recombinant proteins, microRNAs, synthetic self-replicating polycistronic RNA, or low molecular weight biologically active substances.

One of the known factors mentioned above are Yamanaki factors (Oct4, Klf4, Sox2 and c-Myc).

There are also reprogramming factors, which are low molecular weight compounds that convert adult cells into induced pluripotent stem cells that can maintain their viability at a high level for several months. These compounds include seven factors:

- valproic acid or histone deacetylase inhibitor, which promotes the activation of pluripotency genes and repression of linear differentiation genes;

- GSK3 - glycogen synthase kinase inhibitor 3. Inhibition of GSK3 mimics the activation of the Wnt signaling pathway, as a result of which β-catenin, avoiding degradation, enters the nucleus, where it activates the synthesis of the enzymatic telomerase subunit;

- E-616452 - inhibitor of transforming growth factor beta TGF- [beta], which during reprogramming replaces Sox2, activating Nanog;

- tranylcypromine - a monoamine oxidase inhibitor or demethylase inhibitor that selectively removes methyl groups from lysine at the Lys4 or Lys9 position of histone H3. Acting on chromatin, tranylcypromine causes a global increase in H3K4 methylation and, as a result, gene repression. Demethylase inhibitors have the ability to suppress excessive cell proliferation and thus inhibit the development of cancerous tumors;

- Forskolin - adenylate cyclase activating drug - an enzyme that catalyzes the conversion of ATP to cAMP;

- 3-deazaneplanocin - an inhibitor of S-adenosylhomocysteine hydrolase, which causes the destruction of the PRC2 repressor complex and inhibits histone methylation;

- PD-0325901 - a highly selective and potent synthetic inhibitor of mitogen-activated protein kinase MEK, selectively phosphorylating serine / threonine and tyrosine residues in the activation loop of its substrates.

The variety of existing methods of reprogramming allows the proposed method to obtain stem cells from cells of any maturity - both poorly differentiated and definitive. For example, it is known that, using Yamanaki factors, fibroblasts of any maturity can be differentiated (Journal of Nature Cell Biology, 2006 Aug 25, volume 126 (4), page 663).

A common problem with all known methods of inducing stem cells is that the differentiated cells obtained from them turn out to be immature and correspond to approximately the second month of fetal development.

About 100 mature genes work differently in naturally mature cells and laboratory-derived ones. Half of them are normally active only in stem cells, and over time, these genes cease to function. In differentiated cells, obtained artificially, these genes continue to work. Moreover, the sudden activation of "stem" genes in mature cells, many of which control cell division, is fraught with the appearance of a malignant tumor.

The cause of malignant tumors is a violation of apoptosis and the process of cell division. Somatic mutations of the gene encoding the p53 protein were considered as the main cause of this pathology. About 50% of artificially transformed cells express a mutant form of p53.

The regulation of cell division and apoptosis and bringing them “to condition” before transplantation into the human body, along with an increase in the number of the H3K27me3 repression marker during dedifferentiation, will solve the problem of “overriding” and the occurrence of cancer cells.

Cultivation of the obtained induced pluripotent stem cells in the presence of an agent that changes the epigenetic status of the cell will make the epigenetic status of the cells “mobile,” that is, using mechanisms that do not affect the DNA sequence, will ensure variability in gene expression (cell phenotype).

As an agent that changes the epigenetic status of a cell, for example, as indicated in the prototype, a demethylating agent, a histone deacetylase inhibitor, or a combination thereof can be used. Histone deacetylase inhibitor (HDAC) can be Trichostatin A, Vorinostat (SAHA), Belinostat (PXD101), LAQ824 / LBH589, Trapoxin B, Entinostat (MS-275), CI994, Mocetinostat (MGCD0103), Phenylbutyrate, Valotur, Acid, Valpu valerate, valproate, nicotinamide, dihydrocoumarin, naphthopyranone and 2-hydroxynaphthaldehyde, apicidin, FK228 and sodium butyrate. The HDAC inhibitor may be trichostatin A (TSA). The demethylating agent may be 5-azacytidine or 5-aza-2′-deoxycytidine. The required amount of an agent that changes the epigenetic status of a cell varies depending on its activity, type and concentration of the resulting cells in the culture medium.

In order for the cell destruction rate not to exceed the absorption rate of their destroyed material by neighboring cells, which, in turn, leads to inflammatory processes, it is necessary to regulate the permeability of the outer mitochondrial membrane of cells, which is responsible for the absorption and processing of dead material. A significant role in increasing the permeability of the outer membrane of mitochondria is played by the Bax and Bak proteins. They integrate into the outer mitochondrial membrane and oligomerize. This violates the integrity of the outer membrane of the mitochondria.

The functioning of the Bax and Bak proteins depends on their preliminary activation, for example, Bid and Bim proteins, which belong to the subfamily of BH3 proteins. On the other hand, the activation and functioning of Bax and Bak can be blocked by anti-apoptotic proteins of the Bcl-2 family: Bcl-2, Bcl-xL, Mcl-1, etc. In turn, anti-apoptotic proteins can also be blocked by depressant proteins (for example, Bad) related to the subfamily of BH3 proteins. As a result, combined regulation of the membrane permeability of mitochondria and, accordingly, apoptosis is achieved due to the interaction of apoptotic, antiapoptotic, as well as BH3 activator proteins and depressants. The functions of BH3 proteins are regulated at the level of transcription, the stability of molecules, when interacting with other proteins and with various modifications.

Despite the fact that at this stage it is not possible to establish all the causes of apoptosis disturbance, the addition of bax and bak proteins to the cultivation medium can equalize the rate of destruction and absorption of dead cells by regulating the permeability of the outer mitochondrial membrane. In addition, in the presence of an agent that changes the epigenetic status of the cell, this will make it possible to “cure” the TP53 gene, a disturbed modification of which is found in about 50% of cancerous cells.

This phenomenon can be explained as follows. Environmental factors significantly influence the presence and frequency of TP53 gene mutations, including changes in apoptosis parameters that can be triggered by bax and bak proteins. An increase in the activity of the TP53 gene in the presence of an agent that changes the epigenetic status of the cell can increase the susceptibility of cells to apoptosis entry signals through the indirect effect of the p53 protein on the expression of the bax and bcl2 genes. Such p53-mediated changes in the expression of the bax and bcl2 genes can change the ratio of the amount of Bax and Bcl2 proteins, thereby putting the cell in a state of increased sensitivity to apoptosis. Thus, in addition to chemically equalizing the rate of cell destruction and uptake, bax and bak proteins, through a direct effect on apoptosis, can also exert a reverse effect on the TP53 gene, which consists in preliminary damage to the gene with its subsequent normal structuring.

If stem cell cultivation is carried out in the presence of nitric monoxide, this will allow an easy way to create a stress stimulus for the TP53 gene, thus provoking its additional activation for subsequent treatment.

Moreover, nitrogen monoxide can be added to the atmosphere of cultivation both before the stage of cultivation in the presence of an agent that changes the epigenetic status of cells, and before the stage of cultivation in its absence. The function of creating a stress stimulus for the TP53 gene will be performed in any case, and the preferred time of its action will depend on the type and number of cells received.

It should be noted that due to the strict "post-translational control of p53 protein activation", a high concentration of p53 protein itself does not directly lead to the activation and treatment of the TP53 gene.

To accelerate the "recovery" of induced pluripotent stem cells, their cultivation in the presence of an agent that changes the epigenetic status of the cell, as well as cultivation in the absence of the specified agent, is preferably carried out in the presence of a proliferation inhibitor.

At least one globin peptide chain is preferably used as a proliferation inhibitor. The indicated inhibitor not only allows stem cells to “recover” faster in relation to carcinogenic polymorphisms of genes, but it has also been experimentally established that it helps to regulate the entire development cycle of induced stem cells.

The cultivation of induced pluripotent stem cells is also preferably carried out in the presence of at least one apoptosis inhibitor. Due to the bak and bax proteins, cell death occurs through apoptosis. These proteins also contribute to the regulation of the apoptosis process itself, while an apoptosis inhibitor is desirable to control its speed.

As an apoptosis inhibitor, it is preferable to use a FLIP inhibitor. The specified substance is an intracellular caspase-8 inhibitor that blocks the transmission of apoptosis signal through death receptors. Despite the fact that its role is controversial, since the effect of its overexpression on apoptosis is unknown, when added in small quantities, it is able to adjust the cell division mechanism and increase the processing rate of their dying material.

After cultivation of stem cells, subsequent differentiation of these cells in the presence of at least one growth factor should be carried out, some of which, in addition to the differentiation of cells themselves, can also stimulate their proliferation. Depending on which differentiated cells should ultimately be obtained, various peptide or steroid hormones can be used as growth factors.

Examples of implementation.

Example 1

Chemically induced pluripotent stem cells (HIPSCs) were obtained from fibrocytes of the tail end of the mouse. For this, the fibrocytes were thawed and seeded in a well plate in RPMI-1640 nutrient medium with glutamine. The initial density of the fibrocyte monolayer was 20%. 18.5 hours after sieving, the monolayer density reached 40%.

Upon reaching the desired density, three chemical reprogramming factors were introduced into the nutrient medium, which are low molecular weight compounds, with the following content per unit volume of the cell medium, g / l:

valproic acid - 0.0001,

lithium chloride - 0.00007,

4- [4- (1,3-benzodioxol-5-yl) -5- (2-pyridinyl) -1H-imidazol-2-yl] benzamide (SB-431542 inhibitor, 99% pure) - 0.00009.

A concentrated dimethyl sulfoxide solution of N - [(1,2-dihydro-4,6-dimethyl-2-oxo-3-pyridinyl) methyl] -3-methyl-1 - [(1S) -1-methylpropyl] was also added to the medium -6- [6-1-piperazinyl) -3-pyridinyl] -1H-indole-4-carboxiamide (GSK126 methyltransferase inhibitor, purity 98%) in an amount of 0.8 wt.%.

After 35 hours, the cells were seeded into RPMI-1640 nutrient medium with glutamine, into which Bax protein (grade 6A7), Bak protein (grade (G-23) -G) and an agent that alters the epigenetic status of the cell, in which 5-aza-2′-deoxycytidine was taken, with the following content per unit volume of the cell medium, g / l:

Bax protein (grades 6A7) - 2.5,

Bak protein (grades (G-23) -G) - 2.5,

5-aza-2′-deoxycytidine - 0.00005.

A proliferation inhibitor, which is a composition of a hemoglobin delta globin chain and a pharmaceutically acceptable carrier, was also added to the medium in an amount of 0.00003 g / l of the cell medium.

Under the described conditions, the cells were cultured for 7 hours. After that, the medium was replaced with RPMI-1640 nutrient medium with glutamine. Subsequent cultivation was carried out already in the absence of 5-aza-2′-deoxycytidine for 9 hours in an atmosphere containing nitrogen monoxide.

For the grown induced pluripotent cells, the medium was replaced with Nutrient medium MEM with Earl salts, without glutamine, into which nerve growth factor Mouse Nerve Growth Factor (mNGF) was added, in an amount of 4 g / l of cell medium. Cell differentiation was performed for 32 hours.

The average nuclear cytoplasmic ratio in the cells was 0.04. The nucleolus was poorly expressed. The presence of dispersed chromatin in the nucleus was not detected. These indicators indicate a high degree of cell maturation and the inactive state of the “stem” genes, whose work in mature cells is fraught with the appearance of a malignant tumor.

A 40-day observation of the colony of the obtained cells did not reveal signs of carcinogenesis, as evidenced by the absence of cell atypia and, as a consequence, cell dysplasia. Deviations from the normal structure of the entire tissue complex during the indicated period were not observed.

Example 2

Chemically induced pluripotent stem cells (HIPSCs) were obtained from medium-differentiated myoblasts of the frog hind paw. To do this, these myoblasts were thawed and seeded in a well plate in the nutrient medium Needle MEM with Earle salts, without glutamine. The initial density of the myoblast monolayer was 15%. 15.2 hours after sieving, the monolayer density reached 30%.

Upon reaching the specified density, two chemical reprogramming factors were introduced into the nutrient medium, which are low molecular weight compounds, with the following content per unit volume of the cell medium, g / l:

            tranylcypromine (monoamine oxidase inhibitor) - 0.00008,

PD-0325901 (synthetic inhibitor of mitogen-activated protein kinase MEK) - 0.0001.

A concentrated dimethyl sulfoxide solution of N - [(1,2-dihydro-4,6-dimethyl-2-oxo-3-pyridinyl) methyl] -3-methyl-1 - [(1S) -1-methylpropyl] was also added to the medium -6- [6-1-piperazinyl) -3-pyridinyl] -1H-indole-4-carboxyamide (GSK126 methyltransferase inhibitor, purity 98%) in an amount of 0.6 wt.%.

After 26 hours, the cells were subcultured into Eagle MEM nutrient medium with Earl salts, without glutamine, into which Bax protein (grade B-9), Bak protein (grade Ab-2), and an agent that changes the epigenetic status of the cell were introduced which was a mixture of proteinostat (PXD101) and 5-azacytidine, with the following content per unit volume of the cell medium, g / l:

Bax protein (B-9 grade) - 4,

protein Bak (brand Ab-2) - 2,

Belinostat (PXD101) - 0.00003,

5-azacytidine - 0.00004.

Under the described conditions, the cells were cultured for 6.5 hours. After this, the medium was replaced by the nutrient medium Needle MEM with Earl salts, without glutamine. Subsequent cultivation was carried out already in the absence of an agent that changes the epigenetic status of the cell, for 8 hours (without nitrogen monoxide).

For the grown induced pluripotent cells, the medium was replaced with Eagle MEM nutrient medium with Earl salts, without glutamine, to which Mouse Nerve Growth Factor (mNGF) was added, in an amount of 3.5 g / l of cell medium. The differentiation of the cells was carried out for 30 hours.

The average nuclear cytoplasmic ratio in the cells was 0.035. The nucleolus was poorly expressed. The presence of dispersed chromatin in the nucleus was not detected.

A 40-day observation of the colony of the obtained cells did not reveal signs of carcinogenesis, as evidenced by the absence of cell atypia and, as a consequence, cell dysplasia. Deviations from the normal structure of the entire tissue complex during the indicated period were not observed.

Example 3

Chemically induced pluripotent stem cells (HIPSCs) were obtained from human skin melanocytes. For this, melanocytes were thawed and seeded in a well plate in RPMI-1640 growth medium with alanyl-glutamine. The initial density of the monolayer of melanocytes was 12%. 26 hours after sieving, the monolayer density reached 60%.

Upon reaching the desired density, Yamanaki factors (Oct4, Klf4, Sox2, and c-Myc) were introduced into the nutrient medium in an amount of 0.00015 g / l of the cell medium.

A concentrated dimethyl sulfoxide solution of N - [(1,2-dihydro-4,6-dimethyl-2-oxo-3-pyridinyl) methyl] -3-methyl-1 - [(1S) -1-methylpropyl] was also added to the medium -6- [6-1-piperazinyl) -3-pyridinyl] -1H-indole-4-carboxyamide (GSK126 methyltransferase inhibitor, purity 98%) in an amount of 1.6 wt.%.

After 37 hours, the cells were seeded into RPMI-1640 nutrient medium with alanyl-glutamine, into which Bax protein (grade 6A7), Bak protein (grade (G-23) -G), and apoptosis inhibitor FLIP (FLICE-inhibitory protein) were introduced ) and an agent that changes the epigenetic status of the cell, for which valproate was taken, at their next content per unit volume of the cell medium, g / l:

Bax protein (grades 6A7) - 5,

Bak protein (grades (G-23) -G) - 4,

FLIP (FLICE-inhibitory protein) - 0.00003,

valproate - 0.0002.

A proliferation inhibitor, which is a composition of a hemoglobin delta globin chain and a pharmaceutically acceptable carrier, was also added to the medium in an amount of 0.00003 g / l of the cell medium.

Nitrogen monoxide was injected into the atmosphere of the cell incubator.

Under the described conditions, the cells were cultured for 9.5 hours. After this, the medium was replaced by the nutrient medium Eagle MEM with Earl salts, without glutamine, to which the FLIP-inhibitory protein apoptosis inhibitor was added in an amount of 0.00002 g / l of the cell medium.

Subsequent cultivation was carried out already in the absence of valproate for 10 hours in an atmosphere still containing nitrogen monoxide.

For grown induced pluripotent cells, the medium was replaced with RPMI-1640 nutrient medium with alanyl-glutamine, to which hepatocyte growth factor (HGF) was added in an amount of 6 g / l of cell medium. The differentiation of the cells was carried out for 40 hours.

The average nuclear cytoplasmic ratio in the cells was 0.04. The nucleolus was poorly expressed. The presence of dispersed chromatin in the nucleus was not detected.

A 50-day observation of the colony of the obtained cells did not reveal signs of carcinogenesis, as evidenced by the absence of cell atypia and, as a consequence, cell dysplasia. Deviations from the normal structure of the entire tissue complex during the indicated period were not observed.

Example 4

Chemically induced pluripotent stem cells (HIPSCs) were obtained from rat chondrocytes. For this, chondrocytes were thawed and seeded in a well plate in the nutrient medium Eagle MEM with Earl salts, without glutamine. The initial density of the monolayer of chondrocytes was 25%. 30 hours after sieving, the density of the monolayer reached 67%.

Upon reaching the desired density, three chemical reprogramming factors were introduced into the nutrient medium, which are low molecular weight compounds, with the following content per unit volume of the cell medium, g / l:

GSK3 (glycogen synthase kinase 3 inhibitor) - 0.00002,

tranylcypromine 0.0001,

3-deazaneplanocin 0.00001.

A concentrated dimethyl sulfoxide solution of N - [(1,2-dihydro-4,6-dimethyl-2-oxo-3-pyridinyl) methyl] -3-methyl-1 - [(1S) -1-methylpropyl] was also added to the medium -6- [6-1-piperazinyl) -3-pyridinyl] -1H-indole-4-carboxyamide (GSK126 methyltransferase inhibitor, purity 98%) in an amount of 2 wt.%.

After 33 hours, the cells were transplanted into Eagle MEM nutrient medium with Earl salts, without glutamine, into which Bax protein (grade 2D2), Bak protein (grade Fl-211), apoptosis inhibitor FLIP (FLICE-inhibitory protein), and inhibitor were introduced apoptosis Bcl-w (brand G-21) and an agent that changes the epigenetic status of the cell, which was taken as a mixture of E-616452 (an inhibitor of transforming growth factor beta TGF- [beta]) and valproate, with the following content per unit volume of cell environment, g / l:

Bax protein (brand 2D2) - 2.5,

protein Bak (brands (G-21) - 5,

FLIP (FLICE-inhibitory protein) - 0.00001,

Bcl-w (grades G-21) - 0.00002,

E-616452 (inhibitor of transforming growth factor beta TGF- [beta]) - 0.00008,

valproate - 0.00015.

Nitrogen monoxide was injected into the atmosphere of the cell incubator.

Under the described conditions, the cells were cultured for 7 hours. After this, the medium was replaced with nutrient medium Eagle MEM with Earl salts, without glutamine, to which were added an apoptosis inhibitor FLIP (FLICE-inhibitory protein) in an amount of 0.00001 g / l of cell volume and an apoptosis inhibitor Bcl-w (brand G -21) in an amount of 0.00002 g / l of the volume of the cell medium.

Subsequent cultivation was carried out already in the absence of E-616452 and valproate for 10 hours in an atmosphere still containing nitrogen monoxide.

For grown, induced pluripotent cells, the medium was replaced by Eagle MEM nutrient medium with Earl salts, without glutamine, to which fibroblast growth factor 19 (FGF19) was added in an amount of 10 g / l of cell medium. The differentiation of the cells was carried out for 36 hours.

The average nuclear cytoplasmic ratio in the cells was 0.037. The nucleolus was poorly expressed. The presence of dispersed chromatin in the nucleus was not detected.

A 40-day observation of the colony of the obtained cells did not reveal signs of carcinogenesis, as evidenced by the absence of cell atypia and, as a consequence, cell dysplasia. Deviations from the normal structure of the entire tissue complex during the indicated period were not observed.

Thus, the proposed method allows for a relatively short period of time to obtain mature differentiated cells that are not prone to the formation of a malignant tumor.

The proposed method can find application in bioengineering in the preparation and cultivation of stem cells and their conversion into differentiated cells, in particular in the printing and growing of artificial living tissues and organs.

In conclusion, it can be noted that further study of the reprogramming and differentiation of cells will make it possible to improve the quality of the whole person - starting with his health and ending with his personality. A person consists entirely of cells, the life cycle of each of which is determined by its potency and growth factors, and, therefore, the life cycle and development of the characteristics of each person also depend on the same conditions.

Claims (8)

1. A method of producing pluripotent stem cells, including the process of dedifferentiation of somatic cells, culturing induced pluripotent stem cells in the presence of an agent that changes the epigenetic status of the cell, and culturing stem cells in the absence of the specified agent, characterized in that the process of dedifferentiation of somatic cells is carried out by modifying them with using reprogramming factors in a medium containing a GSK126 methyltransferase inhibitor, and cultivation induced pluripotent stem cells in the presence of an agent that changes the epigenetic status of the cell is carried out in a medium containing Bax and Bak proteins. 2. The method according to p. 1, characterized in that the density of the monolayer of induced cells immediately before the process of dedifferentiation is 30-60%. 3. The method according to p. 2, characterized in that the concentration of the GSK126 methyltransferase inhibitor in the medium is 0.8-2 wt.%. 4. The method according to p. 1, characterized in that the cultivation of induced pluripotent stem cells is also carried out in the presence of a proliferation inhibitor. 5. The method according to p. 4, characterized in that at least one globin peptide chain is used as a proliferation inhibitor. 6. The method according to p. 1, characterized in that the cultivation of stem cells is carried out in the presence of nitric oxide. 7. The method according to p. 1, characterized in that the cultivation of induced pluripotent stem cells is carried out in the presence of at least one apoptosis inhibitor. 8. The method according to p. 7, characterized in that the FLIP inhibitor is used as an apoptosis inhibitor.