RU2458983C1 - Method of producing induced pluripotent stem cells from fibroblasts of patients with huntington's disease - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: described is a method of producing induced pluripotent stem cells (iPS-cells) from skin fibroblasts of a patient with Huntington's chorea, involving production and reproduction of fibroblasts, inoculating said fibroblats on pools with monolayer density 30% in a medium of DMEM+P/S+L-Glu+10% FBS+bFGF 2 ng/mcl, infection of cells when monolayer density reaches 60% with four viruses LeGO-hOct4-MOI 5, LeGO-hSox2-MOI 5, LeGO-hc-Myc-MOI 2.5, LeGO-hKlf4-MOI 5. After four days, the cells are re-inoculated on plastic 1 to 22.5 in a medium of DMEM+P/S+L-Glu+10% FBS; after one day, the medium is replaced with ESmed and cultured for 8-10 days; the medium is replaced once in two days; mechanical re-inoculation is carried out 1 to 1 on matrigel in a medium of mTeSRI; culturing is carried out until growth of columns of morphologically similar to embryonic stem cells; the clones are collected after a week. Disclosed is a cell line obtained using the described method, which is meant for treating Huntington's chorea, which has the following morpho-molecular characteristics: size of the order of 20 mcm, large nucleus, growth in compact colonies with close contact, doubling period as embryonic stem cells, considering error, normal kryotype of 46 chromosomes, expression of Oct4, SOX2, FOXD3, HESX1, Nanog, Sall4.

EFFECT: high treatment efficiency.

2 cl, 5 dwg, 2 tbl, 1 ex

Description

The invention relates to biotechnology, and in particular to a method for producing induced pluripotent stem cells and cell lines obtained by the presented method. The invention is aimed at expanding the arsenal of tools designed to combat Huntington's disease.

Huntington or Huntington (BG) disease (chorea) is one of the most famous extrapyramidal hereditary neurodegenerative diseases. The prevalence of BG varies from 0.1-0.38 (Japan, African countries) to 3-7 (countries of Western and Eastern Europe, USA, Canada) per 100,000 population [Bates et al., 2002], in some populations reaching 15 -17 and above per 100,000 people (Tasmania, Venezuela). For over 100 years, BG has been attracting the attention of researchers around the world. This is primarily due to the particular severity of suffering, characterized by the development and steady progression of choreic hyperkinesis, personality and cognitive impairment, up to the development of severe dementia, and the lack of any effective etiopathogenetic treatment methods to date. It is very dramatic that the clinic of the disease usually develops over 4-5 decades of life (average age of onset 35-44 years [Bates et al., 2002]), and, due to the almost complete penetrance of the mutant BG gene, its carrier inevitably becomes ill. Until the debut of the disease, a person, being a carrier of the BG gene, remains practically healthy, having had time to start a family. Children of the patient are at risk with a probability of transmission of a mutant gene to them of 50%.

This severe neurodegenerative disease with an autosomal dominant inheritance was one of the first in neurology, for which back in 1983 a group of researchers from Harvard University led by J. Gusella mapped a chromosome locus on the short arm of the 4th chromosome (4p16.3 ) when studying the world's largest cohort of patients with HD, the indigenous people of Venezuela (about 3,000 patients) living compactly on the islands of Lake Maracaibo [Wexler et al., 2004]. The cloning of the BG gene in 1993 [HD Collaborative Research Group] was the first major step in the study of the molecular biology of BG. The prevalence of the disease, the regularities of the course of the neurodegenerative process, the high penetrance of the mutant gene and late manifestation make it possible to actually consider BG as a “model” disease in the study of the molecular mechanisms of neurodegeneration, as well as issues of medical genetic counseling and prognostic testing in modern neurogenetics.

The clinical picture of BG consists of a triad of clinical symptoms - a combination of common motor disorders, emotional-volitional and cognitive disorders that are steadily progressing up to the development of dementia due to the selective death of neurons of the subcortical nodes, primarily neostriatum, as well as neurons of mainly IV, V and VI layers cerebral cortex [Van Raamsdonk et al., 2005; Cowan, Raymond, 2006]. Pathological changes are also found in peripheral tissues, including various parts of the digestive tract [Björkqvist et al., 2008; van der Burg et al. 2009]. Conventionally, motor disorders in GB can be divided into two groups - involuntary movements and violation of voluntary movements [Folstein, 1989]. The first group includes characteristic choreic hyperkinesis, athetoid movements, motor anxiety, dystonic manifestations, tremors and, occasionally, myoclonus. The second group usually includes oculomotor disturbances (slow saccades), coordinating disorders, speech, swallowing, and gait disorders. If involuntary movements progress, as a rule, only in the early stages of the disease, then violations of voluntary movements are in a strict linear dependence on the time elapsed since the debut of the BG. Cognitive impairment is expressed in a gradual decrease in memory, mainly short-term, attention, concentration, ability to learn new facts and skills, slow thinking, which ultimately leads to the development of gross subcortical dementia. It was shown that mild cognitive impairment may be present in “asymptomatic” carriers of the BG gene from the risk group long before the first motor manifestations of BG [Klyushnikov, 1998; Bourne et al., 2006; Montoya et al., 2006; Paulsen et al., 2008; Tabrizi et al., 2009; Rupp et al. 2010]. Thus, with this suffering, there is a rather long multi-year stage of “pre-illness” associated with a gradual increase in subclinical neurodegenerative and biochemical changes in the brain substance.

Emotional-volitional and personality disorders initially appear in the form of increased nervousness, irritability, temper, sometimes reaching attacks of aggression in relation to others. Affective disorders in the form of depression, or manic manifestations, and psychopathization of the personality are characteristic. Schizophrenia-like symptoms in the form of hallucinatory-delusional manifestations are frequent [Rosenblatt, 2007]. Choreic dementia develops against the background of disorders in the emotional-volitional sphere, which is very characteristic of subcortical type dementia. In 9% of cases in patients with HD there is an obvious clinic of schizophrenia, while gross personality changes that require the intervention of a psychiatrist are found in more than 70% of cases. Among patients with HD, the frequency of suicides against a background of severe depression is increased - according to Lipe, up to 5-10%.

The clinical manifestations of HD can be combined with each other to varying degrees, which is the basis of a wide phenotypic polymorphism in this disease. At present, hyperkinetic hypertension, in which the presence of common choreic hyperkinesis, and akinetic-rigid, is decisive, is distinguished as the main clinical forms of hypertension. The latter is divided into juvenile and late akinetic-rigid forms. The juvenile form of the disease (Westphalian variant) debuts at the age of 20 years and is manifested by akinetic-rigid syndrome with not pronounced hyperkinesis or their complete absence, often myoclonus and epileptic seizures, gross violations of the intellect and psyche. This atypical form of the disease is most malignant and quickly leads to death. According to Nance, Myers (2001), Gonzalez-Alegre, Afifi (2006), the frequency of the akinetic-rigid form of GB is from 5 to 10% of all cases of the disease. This form of hypertension, as a rule, occurs during the inheritance of hypertension from a sick father. The late form is a consequence of the morphogenesis of a typical hyperkinetic form of the disease, when 15-20 years from its onset, akinetic-rigid syndrome develops with the gradual extinction of hyperkinesis. Some authors also single out the mental form of the disease, in which mental disorders sharply prevail over motor disorders [Wagle et al., 2000].

BG steadily progresses and inevitably ends with a complete disintegration of the personality and immobility of patients requiring constant care. Death usually occurs due to intercurrent infections, general exhaustion, or aspiration of solid or liquid foods. The median survival for HD is 15–18 years (range from 5 to> 25 years), and the average age of death is 54–55 years [Harper et al., 2005].

Cloning of the BG gene in 1993 [HD Collaborative Research Group] made it possible to establish that BG belongs to the group of diseases caused by dynamic mutations: in particular, in BG there is an abnormal increase ("expansion") of trinucleotide cytosine-adenine-guanine (CAG) repeats in the first exon of the HTT gene (IT-15) at the 4p16.3 locus. Elongated mutant alleles are characterized by pronounced genetic instability in the process of DNA replication, especially in male gametogenesis, which often leads to a further change (often an increase) in the number of repeats during gene transfer to the next generation. The NTT gene consists of 67 exons and has a length of more than 200 kb. It is usually expressed as two transcripts (10.3 kb and 13.6 kb), differing in length 3 'UTR. Normal alleles of the HTT gene contain 10–35 repeats (median –18); the most common alleles in a population contain 15–20 CAG repeats [Warby et al., 2009]. Moreover, alleles with the number of CAG repeats 27–35 are called intermediate or “mutable” [Potter et al 2004], since they are a source of expansion of CAG repeats in subsequent generations with a risk of 6–10% [Semaka et al., 2006] however, their carriers have a zero risk of developing the disease. The number of repeats 36–39 is called the “zone of incomplete penetrance,” since in carriers of such alleles, clinically manifest BG develops in rare cases (frequency is not described), often in an atypically mild form and at a very advanced age [Telenius et al., 1993; Britton et al., 1995]. Sometimes such alleles are found in elderly, clinically healthy individuals at risk. In patients with typical clinically manifest forms of GB, the number of CAG triplets is 40 or more, in rare cases, may exceed 100 or more, amounting to more than 60 copies in the juvenile version of BG. Homozygous carriers of the mutant HTT gene do not have the features of clinical manifestations and the course of the disease [Dürr et al., 1999]. Decoding the structure of the HTT gene has become the basis for the development of a reference DNA diagnosis of the disease for cases of expansion of CAG repeats of less than 115 (PCR analysis) and more than 115 (Southern blot analysis, which is also used to confirm cases of homozygous carriage of the mutant gene) [Potter et al., 2004 ; Levin et al., 2006].

Various groups of researchers have identified a variety of significant clinical and genetic correlations in GB. It has been shown that with an increase in the number of copies of CAG repeats, the age of onset of BG decreases [Andrew et al., 1993; Langbehn et al., 2004; Langbehn et al., 2010], the onset age of individual clinical symptoms and the age of death [Rosenblatt et al., 2006; Aziz et al ,, 2009], and the rate of disease progression is also accelerating [Illarioshkin et al., 1994; Illarioshkin et al., 1996]. A study of the molecular biology of the BG gene made it possible to explain these phenomena by the nature of the BG gene encoding the Huntingtin protein, consisting of 3144 amino acid residues, with a molecular weight of 348 kd. Huntingtin is widely expressed in various organs and tissues. In HD, the characteristic intranuclear inclusions, which are high molecular weight amyloid aggregates, are detected in striatum neurons by electron microscopy [Roizin et al., 1979]. The presence of Huntingtin mutant protein in the inclusions is confirmed by immunohistochemical studies [Li et al., 1995; DiFiglia et al., 1997]. Similar huntingtin-positive inclusions are also detected in axons and dendrites. The accumulation of intranuclear aggregates, as a rule, is accompanied by various anomalies of the nuclear membrane (intussusception, increased pore clustering, etc.) [Feigin, 1998]. The function of this protein is still not exactly known; its study seems, therefore, to be one of the urgent problems of modern neurobiology. The homologue of the huntingtin gene is present in a number of lower mammals (for example, the HDh gene in mice), which turned out to be very valuable from an experimental point of view. A number of research groups created lines of mice with the inactivated (“knocked out”) HDh gene [Bates et al., 1997]. In all experiments, the inactivation of both copies of the HDh gene was lethal at an early embryonic stage, which indicates the important role of huntingtin in embryonic development and regulation of the life cycle of cells subject to apoptosis. At the same time, the inactivation of one copy of the HDh gene in most cases was not accompanied by the development of any detectable phenotypic disorders in such animals. Mice in which their own HDh gene was completely inactivated retained normal, early embryonic development provided that a functionally active human huntingtin gene was introduced into their genome with expansion of CAG repeats [White et al., 1997]; this suggests that, in the case of expansion of CAG repeats, the intrinsic normal function of Huntingtin remains intact. Thus, it is assumed that it plays an important role in the early stages of neurogenesis in embryos [Godin et al., 2010], and is also a transcription factor. Huntingtin is associated with microtubules, vesicular membranes and synaptosomes of neurons, which allows us to discuss the participation of this protein in endocytosis and cytoplasmic transport [Albin, Tagle, 1995; Feigin, 1998].

Transgenic BG models in mice were created by embedding in their genome various constructs based on the human NTT gene carrying the expanded (CAG) _n- site with the number of repeats from 48 to 156 [Bates et al., 1997; Reddy et al., 1998; Schilling et al., 1999]. Expression of these genetic constructs led to a progressive decrease in the brain mass of animals, starting from the 12th week of life, death of neurons and astrocytic gliosis in the striatum and other parts of the brain, the appearance of gentingtin-positive inclusions in the nuclei and processes of degenerating neurons, a decrease in total body weight and manifestation in transgenic animals, polymorphic neurological disorders (hyperkinesis, impaired general mobility and gait, epileptic seizures) at the 2nd-5th month of life. With further breeding and the emergence of offspring of transgenic mice homozygous for the expanded (CAG) _n- allele, the age of the manifestation of the disease decreased, and its rate of progression increased. At the same time, transgenic mice that had a CAG region of the gene with the number of repeats below the pathological threshold (6-26 repeats) always remained phenotypically healthy. It was concluded that for the manifestation of the disease in transgenic animals, the necessary condition is not just the expression of a new peptide based on human huntingtin, but the expression in the target tissues of a pathologically elongated protein epitope. These experiments made a key contribution to understanding the pathogenesis of HD and allowed us to formulate the concept of “polyglutamine diseases”. Since the CAG nucleotide triplet encodes the amino acid glutamine, the expansion of CAG repeats leads to a proportional lengthening and change in the conformation of polyglutamine chains in the corresponding proteins, since polyglutamine chains normally play an important role in the implementation of complex intermolecular interactions, including protein-protein interactions [Housman, 1995; Ross, 1995]. Mutant proteins with an extended polyglutamine pathway acquire new, cytotoxic functions. It is assumed that the elongated polyglutamine portion of the molecule promotes the formation of pathological intermolecular bonds with a number of tissue-specific proteins of the central nervous system and transcription factors, aggregation of amyloidogenic protein complexes in the nucleus and cytoplasm, induction of mitochondrial disorders and apoptosis. According to modern concepts, all neurodegenerative diseases caused by the expansion of translated CAG repeats are combined into a single molecular group of polyglutamine diseases, which include BG, 6 forms of autosomal dominant spinocerebellar ataxia (SCA1, 2, 3, 6, 7, 17), dentator Kennedy pallidolysis atrophy and spinal-bulbar amyotrophy [Hardy, Gwinn-Hardy, 1998; Kakizuka, 1998; Paulson, 1999]. The pathophysiology of polyglutamine diseases is characterized by similar mechanisms, allowing them to be distinguished as an independent variety of the so-called conformational brain diseases, combining, in addition to polyglutamine diseases, Parkinson's disease, taupathy, Alzheimer's disease and other forms of cerebral amyloidosis, prion diseases, ALS [Illarioshkin, 2003]. The key links in pathogenesis are [He et al., 2010; Ross, Tabrizi, 2011] a change in the spatial folding (conformation) of proteins with an extended polyglutamine pathway, their precipitation into insoluble amyloid-like formations, the launch of protective intracellular mechanisms aimed at the degradation of abnormal peptides (in particular, expression of chaperone proteins that promote “straightening” of improper folding abnormal proteins and activation of the ubiquitin-proteasome pathway for degradation of abnormal peptides), failure of protective mechanisms, further precipitation of proteins with their accumulation in the nucleus of degene neurons with the formation of intranuclear inclusions, activation of caspase enzymes responsible for triggering the mechanism of programmed cell death by apoptosis type, disruption of intracellular ion channels with activation of glutamate NMDA receptors and excitotoxic effect. Toxic polyglutamines bind a number of intracellular and intranuclear proteins, in particular gene transcription factors, the synthesis of many proteins, energy metabolism are disrupted in the cell, oxidative stress develops, which, ultimately, leads to the death of specific populations of neurons.

Polyglutamine diseases have a number of common clinical and genetic features that are so characteristic for BG due to the nature of the mutation [Illarioshkin et al., 1995; Rosenberg, 1996; Brice, 1998; Stevanin, 2000, Djousse et al., 2003; Aziz et al., 2009; Semaka et al., 2010]: 1) the dominant nature of the implementation of the mutation. All polyglutamine diseases (with the exception of Kennedy's disease) are inherited in an autosomal dominant manner; 2) a high level of expression of mutant genes in various tissues of the body and various parts of the central nervous system, including those that are not involved in the degenerative process and remain intact. Despite the expression of mutant products in all cells of the body, starting from birth, polyglutamine diseases are characterized by a long latent period and the appearance of clinical symptoms only at a relatively late age; 3) close borders of normal and mutant alleles; 4) the severity of the clinical manifestations of polyglutamine diseases is directly proportional to the magnitude of the expansion of CAG repeats; 5) the phenomenon of anticipation due to the instability of the pathological CAG site and the increase in its length during the transmission of the mutant gene to descendants; 6) the effect of "paternal transmission" associated with the predominant extension of the mutant CAG repeat in male gametogenesis and manifested by the manifestation of earlier and more severe cases of the disease in the descendants of the sick father. 7) variable expressivity of polyglutamine diseases - from subclinical and “mild” forms to severe unfolded cases with a fast, fatal course (including even within the same family); 8) somatic mosaicism of the number of CAG repeats — certain differences in the number of trinucleotide repeats in cells of various tissues, due to the instability of the mutant trinucleotide region in the fission of somatic cell nuclei; 9) the origin of de novo mutations from the rare alleles in the population with an “intermediate” number of CAG repeats. Such alleles alone do not lead to disease, but are genetically unstable and are able to go into a “complete mutation” when the gene is transmitted to descendants.

Currently, intensive search work is underway aimed at clarifying the known and searching for new links in the pathogenesis of BG. The role of modifier genes (Glur6, APOE, GRIN2B, HAP1, etc.) that influence the debut and course of the disease is studied [MacDonald et al., 1999; Kehoe et al., 1999; Arning et al., 2007; Metzger et al., 2008]. The mechanisms of aggregation of mutant huntingtin and their cytotoxic effects are analyzed [Arrasate et al., 2004; Poirier et al., 2005; Gidalevitz et al., 2006; Bennett et al., 2007; Jeong et al., 2009], as well as protein-protein interactions [Goehler et al., 2004; Bae et al., 2006; Luo et al., 2008]. Great importance is also given to the study of the role of apoptosis, heat shock proteins, mitochondrial dysfunction, and oxidative stress in the neuronal cytopathology in hypertension [Wyttenbach et al., 2002; Greenamyre, 2007], biosynthesis of cholesterol and its metabolites in the brain [Valenza, Cattaneo 2010; Leoni et al., 2011]. A complete decoding of all the mechanisms of neurodegeneration in hypertension and other similar severe suffering will provide the key to effective etiopathogenetic therapy aimed at preventing and stopping the development of the disease in individuals who have inherited the mutant gene. Currently, the main directions of research of promising pathogenetic methods of treatment of HD are:

1) the search for neuroprotective inhibitors of apoptosis, oxidative stress, excitotoxicity, as well as blocking inflammatory reactions and modulating transglutaminase activity. Within the framework of these studies, a modifying effect on the course of BG (both on animal models and in preliminary clinical trials) was shown for such preparations as coenzyme Q10, idebenone, minocycline, sodium butyrate, a complex of essential fatty acids, racemide, creatine, cystamine, riluzole, memantine [Walker, Raymond, 2004; Bender et al., 2005; Puri et al., 2005; Bonelli, Wenning, 2006; Hersch et al., 2006; Okamoto et al. 2009];

2) the search for substances ("small molecules") that increase the expression of molecular chaperones and the activity of the proteasome complex in order to inhibit the aggregation of polyglutamine proteins and suppress the neurodegenerative process [Zhang et al., 2005; Fecke et al., 2009; Varma, 2010]. Certain achievements have been achieved in vivo;

3) studies of the possibilities of cell therapy with the transplantation of modified stem cells. The results achieved are ambiguous, until recently, the number of observations remains insufficient [Furtado et al., 2005; Bachoud-Levi et al., 2006; Farrington et al. 2006; Dunnett, Rosser, 2007; Dey et al., 2010; Kandasamy et al., 2010; Meyer, 2010; Rossignol et al., 2010; Cicchetti et al., 2011]. This line of research is very promising;

4) gene therapy, first of all, studies of the phenomenon of RNA interference (RNAi) by using "antisense" RNA sequences to block the messenger RNA of target cells in order to suppress the synthesis of mutant huntingtin and reduce the load of cells with products of impaired protein metabolism. This line of research is also very promising and promising [Denovan-Wright et al., 2008; Franich et al., 2008; McBride et al., 2008; Boudreau et al, 2009; Pfister et al., 2009; Maxwell 2009, Hu et al., 2010; Soldati et al., 2011; Sah, Aronin, 2011].

The most promising treatment option for Huntington's disease, in our opinion, is an approach based on cell therapy.

Pluripotent cells are able to differentiate into all types of cells of an adult organism, which makes them a very interesting object for fundamental research of differentiation processes, as well as for medical purposes. For many diseases, there is no medical treatment, such diseases include heart attacks, type 1 diabetes mellitus, various neurodegenerative diseases (Alzheimer's disease, Parkinson's disease, Huntington's disease, Duchenne myodystrophy, etc.). During the development of these diseases, the patient irreversibly loses cells of a certain type, and drug treatment cannot compensate for these losses. Replace the lost cells by the method of cell therapy. Cellular material for transplantation should be homogeneous and consist mainly of cells of the target type. From pluripotent cells, cultures enriched in a particular type of cell can be obtained (Zhang et al., 2009; Paige S.L. et al., 2010; Hu B.-Y. et al., 2010; Ohta S. et al., 2011). Currently, two types of pluripoten cells are known: embryonic stem (ES) cells and induced pluripotent stem (IPS) cells.

Obtaining ES cells and developing methods for manipulating them, of course, should be attributed to one of the most remarkable and important achievements of science at the end of the 20th century. Since mammalian ES cells represent an almost inexhaustible source of undifferentiated and non-transformed cells with a normal diploid karyotype, they will remain an important object for researchers studying the paths of embryonic development, formation and functioning of certain types of tissues in normal conditions and with various pathologies. In addition, human ES cells, in particular genetically modified, in the long term, of course, will certainly find application in the cell therapy of severe human diseases. Nevertheless, the use of ES cells in cell therapy faces a number of difficulties, both medical and ethical in nature.

However, the results of recent studies open up completely new possibilities in the field of cell therapy. We are talking about the reprogramming of human somatic cells into pluripotent stem cells with their further differentiation into cells of various types, as well as, in the future, transplantation of such cells to patients suffering from various serious diseases (Fig. 1).

Previously, it was believed that the development of the body is unidirectional: from totipotent zygotes to highly differentiated cells of various tissues of the adult body. However, it turned out that the process of differentiation can be reversed and pluripotent cells can be obtained from differentiated cells. In 2006, Japanese researchers Takahashi K. and Yamanaka S (Takahashi K. et al, 2006) were able to reprogram adult and embryonic mouse fibroblasts into pluripotent stem cells by introducing four transcription factors Oct3 / 4, Sox2 into these cells using retroviral vectors , c-Myc and Klf4. The authors called these cells induced pluripotent stem (IPA) cells. The IPS cells obtained as a result of this treatment had a morphology similar to ES cells, growth properties and expressed specific markers inherent in ES cells. A year later, Takahashi K. et al. (Takahashi K. et al, 2007) and Nakagawa et al. (Nakagawa, M. et al, 2008) from the same laboratory at Kyoto University reported the successful dedifferentiation of adult fibroblasts and the production of IPA cells using the same factors (Oct4, Sox2, c-Myc and Klf4).

Reprogrammed cells express all major markers of pluripotency inherent in ES cells. The undifferentiated state of pluripotent cells is characterized by various signs. Undifferentiated IPA cells are characterized by high alkaline phosphatase activity (K. Takahashi et al, 2007), expression of TRA-1-60 and TRA-1-81 proteoglycans (Lowry WE et al, 2008) and SSEA-4 glycolipid and, to a lesser extent, SSEA glycolipid -3 (Park I.-H. et al., 2008). SSEA-4 and SSEA-3 are characteristic only of pluripotent cells of humans and primates, iPS mouse cells carry only SSEA-1 glycoprotein on their surface (K. Takahashi et al, 2006).

The intracellular markers of the undifferentiated state are the transcription factors necessary to maintain the pluripotency of IPA, such as Oct4, Nanog, and Sox2 (Maherali N. et al, 2007; Yu J. et al., 2007; Brambrink T. et al., 2008). Also, IPA cells differ from differentiated cells in the epigenetic state of the genome. Thus, in pluripotent cells, unlike differentiated cells, the promoters of the Oct4 and Nanog genes are demethylated (Hanna J. et al., 2008).

In addition to protein markers, there are functional tests for pluripotency. The simplest of these is the spontaneous differentiation of IPA cells in vitro. After cultivation of human IPS in the absence of a feeder from mouse embryonic fibroblasts, the expression of endoderm, mesoderm, and ectoderm markers is detected in differentiated cells. The only possible test for human pluripotent cells for in vivo pluripotency is the formation of teratomas when administered to immunodeficient (SCID) mice. In teratomas, tissues such as the intestinal epithelium (endoderm) are found; cartilage, bone and smooth muscles (mesoderm); neural epithelium (ectoderm) (Takahashi K. et al., 2007; Brambrink T. et al., 2008). IPA cells obtained from differentiated mouse cells can be incorporated not only into the somatic tissues of the chimeric animal, but also form genital cells (Maherali et al., 2007; Zhou et al., 2009).

ES and IPS cells are almost identical in morphological and functional characteristics, however, the attitude of society towards these types of pluripotent cells and their potential areas of application differ significantly. ES cells have a number of disadvantages that limit their use in medicine and laboratory practice (Table 1). Speaking about cell therapy, one of the main disadvantages of ES cells should be noted - the possible development of an immune response to a transplant. Since there are a limited number of ESC lines, it is not possible for all recipients to select a graft suitable for human leukocyte antigens (HLAs). A way out of this situation can be genetically modified human ES cells with the recipient's HLA genes, but this approach is complex and time-consuming, no one knows how many clones will need to be obtained. Another alternative is to obtain a new patient-specific line of ES cells by transferring the somatic cell nucleus to an enucleated oocyte. This technique will avoid the development of an immune response in the recipient, but it involves obtaining a human embryo and its subsequent destruction to isolate ES cells, i.e. this technique combines human cloning and destruction of a human embryo, the two most ethically and politically controversial issues (O ' Mathuna DP et al., 2002). The legal status of the embryo, and therefore the possibility or impossibility of destroying it to obtain ES cells, has been discussed for more than 10 years by politicians, bioethics, human rights activists and journalists.

Another interesting area of application of pluripotent cells is the study of the mechanisms of development of various genetically determined diseases and the screening of drugs on cell cultures carrying a mutation corresponding to a given disease. How to get such "sick" cells? The easiest way is to isolate primary cell cultures from patients suffering from the disease of interest to us. But not all cells of the human body can be in culture for a long time, and some types of cells (for example, neurons) are almost impossible to receive from living donors. Pluripotent cells are able to differentiate into any type of adult cell. When using ES cells from an unclaimed blastocyst in vitro fertilization procedure, the researcher can find out if this embryo carries any mutation, but this is due to the time-consuming procedure of analyzing a single blastomere. When receiving ES cells by transferring the somatic cell nucleus to an enucleated oocyte, the question of the presence of a mutation is resolved, but we return to another stumbling block - human cloning. Thus, obtaining IPA cells with a known mutation from an adult donor is currently the most preferred (table 1). Such cells allow you to study the mechanisms of the development of the disease and test drugs. Also, in combination with the correction of the patient's mutant genes, specific IPS cells can serve as a potentially inexhaustible resource for the cell therapy of human genetic diseases.

Table 1 Comparison of the properties of IPA and ES cells Characteristics ES cells IPS cells Pluripotency + + Source Embryo (blastocyst) Adult body Ethical issues + - The possibility of obtaining "sick" cells - + Patient specificity - +

There is a fairly wide variety of methods for inducing pluripotency. Historically, the first were viral induction systems: retroviral transfection (K. Takahashi et al., 2006), lentiviral transfection (Yu J. et al., 2007). But cells carrying viral inserts in their genome cannot be used for cell therapy because of the danger of activating transgenes in the patient’s body, which can lead to the development of malignant diseases. In this regard, virus-free methods for inducing pluripotency began to be developed: piggyBac transposon carrying transcription factors Oct4, Sox2, c-Myc, Klf4, fused via 2A peptides (Kaji K. et al., 2009), plasmid DNA (Okita K., 2008 ), recombinant transcription factors (Oct4, Sox2, c-Myc, Klf4) (Zhou H. 2009). However, non-viral methods are also not without drawbacks. The transposon-transposase system involves the introduction of two double-strand breaks when embedding the target sequence and when it is removed, the repair of double-strand breaks may fail, which will lead to a potentially dangerous mutation. Reprogramming using non-integrating vectors, as well as using proteins, proceeds with very low efficiency (Table 2). The latest development in this area is the induction of pluripotency by transfection of somatic cells with mRNA of reprogramming factors (Oct4, Sox2, c-Myc, Klf4) (Warren L. et al., 2010). This technique is safe, since mRNA is completely destroyed in the cell, and its in vitro synthesis is not associated with the use of materials of animal origin.

table 2 Reprogramming Efficiency Using Various Techniques Reprogramming method Reprogramming Efficiency (%) Source Lentiviral transfection 0.02-0.2 (Yu J. et al., 2007) (Brambrink T. et al., 2008) Retroviral transfection 0.1 (Takahashi K. et al., 2007) Transposon transposase system 0.1 (4 factors *)
1 (6 factors **) (Yu J .. et al., 2009) Plasmid DNA 0.0001-0.003 (Okita K. et al., 2008) Recombinant Proteins 0.006 (Zhou H. et al., 2009) mRNA 2 (Warren L. et al., 2010) * - Oct4, Sox2, Nanog, LIN28 ** - Oct4, Sox2, Nanog, LIN28, Klf4, c-Myc

As can be seen from Table 2, the reprogramming process, in principle, is low efficient (<2%). However, there are a number of low molecular weight compounds that can increase the efficiency of reprogramming. DNA methyl transferase inhibitors (Huangfu D. et al., 2008a), histone methylases (BIX) (Shi Y. et al., 2008a), histone deacetylases (VPA) (Huangfu D. et al., 2008a) increase the efficiency of reprogramming in several times, since reprogramming is associated with epigenetic rearrangements in somatic cells. Thus, reprogramming with recombinant proteins does not occur in the absence of VPA (Zhou H. et al., 2009). It is also known that the reprogramming efficiency of mouse embryonic fibroblasts can be improved by using the small molecule Bayk8644, which is an antagonist of L-type calcium channels (Shi Y. et al., 2008b). Some small molecules can replace reprogramming factors. Thus, when reprogramming mouse fibroblasts, the kenpaullone compound replaces Klf4 and ensures the induction of Nanog and the production of full-fledged IPA cells in the presence of only three transcription factors (Oct4, Sox2, c-Myc) (Lyssiotis C.A. et al., 2009). The combination of two small molecules, BIX and Bayk8644, allows reprogramming of mouse embryonic fibroblasts using only two transcription factors, Oct4 and Klf4 (Shi Y. et al., 2008b). Human fibroblasts can be returned to the embryonic state using transcription factors Oct4 and Sox2 in the presence of VPA (Huangfu D. et al., 2008b).

The authors developed a method for producing induced pluripotent stem cells from fibroblasts of the skin of patients with Huntington's chorea.

Examples

Isolation of skin fibroblasts

Fibroblasts were isolated from the skin of 2 female patients with a diagnosis of Chorea Huntington. Cause of the disease: expansion of tandem CAG repeats in the 1st exon of the IT-15 gene (chromosome 4p16). The number of copies of CAG repeats in the mutant allele is 42.

Construction of plasmids

Plasmids based on the LeGO vector system LeGO-hOct4, LeGO-hSox2, LeGO-hc-Myc, LeGO-hKlf4 for the assembly of lentiviruses were constructed. In contrast to the original plasmids of the Yamanaka laboratory, in the LeGO plasmids, the transgene is flanked by LoxP sites, which allows it to be potentially removed from the genomic DNA of the cell. Moreover, the new plasmids provide a more efficient assembly of lentiviruses, which leads to a higher titer of the virus in the virus-containing supernatant above the packaging cells. This allows you to abandon the concentration of viruses before use, to simplify and reduce the cost of the procedure for their preparation.

Lentivirus assembly

Lentiviruses LeGO-hOct4, LeGO-hSox2, LeGO-hc-Myc, LeGO-hKlf4 and LeGO-G2 were assembled as a control. The titer of LeGO-hOct4, LeGO-hSox2 viruses was determined, it was shown that the titer of these viruses was the same within the error range, and the expression of transcription factors hOct4, hSox2 was shown.

Getting IPA cells

The skin fibroblasts of a patient of the patient Chorea Huntington were thawed and plated on the wells of a 12-well plate with a density of 30% monolayer in DMEM + P / S + L-Glu + 10% FBS + bFGF 2ng / μl. 2 days after sieving, the cells reached a density of 60% of the monolayer and, at this point, were infected with all four viruses in the following quantities LeGO-hOct4-MOI 5, LeGO-hSox2-MOI 5, LeGO-hc-Myc-MOI 2.5, LeGO- hKlf4-MOI 5. After 4 days, the cells were plated on plastic 1 to 22.5 in DMEM + P / S + L-Glu + 10% FBS. The next day, the medium was replaced by ESmed. Then the cells were cultured in this medium for 8-10 days, the medium was changed every 2 days. At this point, many clones with different morphologies formed on the plates. These clones were mechanically seeded 1 to 1 on matrigel in mTeSR1 medium. After a few days, some of the colonies began to grow and morphologically become more similar to ESCs, while others differentiated and stopped growing.

After a week, clones were selected for a 24-well Matrigel plate; 15 different clones were seeded in mTeSR1 medium, after which they were cultured separately. As a result, 6 clones showed the ability to stably grow on a matrigel substrate in mTeSR1 medium. Two clones were used for further characterization, the remaining 4 were frozen.

Morphological characteristics of iPS cells

The obtained clones of IPA cells, like ESCs, have a size of about 20 μm, a large nucleus, grow in compact colonies with tight contacts (Fig. 2).

IPSCs, within the margin of error, have the same doubling period as ESCs. An analysis of the karyotype of iPSC clones was carried out, both clones have a normal karyotype 46 XX (Fig. 3).

Molecular Characterization of iPSCs

The molecular characterization of iPSC clones was carried out by two different methods: immunocytochemical analysis (ICC) and reverse transcription PCR (RT-PCR). The method of ICC showed the expression of pluripotency markers Oct4 and SSEA4 in both clones (Fig. 4).

RT-PCR showed the expression of genes responsible for maintaining pluripotency: Oct4, SOX2, FOXD3, HESX1, Nanog, Sall4.

Thus, the method proposed by the authors differs from the prior art using new plasmids designed by the authors. Through the use of these plasmids, the efficiency of the method is increased, the procedure for introducing genetic material is simplified (Fig. 5).

Claims (2)

1. A method for producing induced pluripotent stem cells (IPA cells) from the skin fibroblasts of a patient suffering from Huntington’s chorea, including obtaining and propagating fibroblasts, plating said fibroblasts in wells with a density of 30% monolayer in DMEM + P / S + L-Glu + 10% FBS + bFGF 2 ng / ml, cell infection when a density of 60% monolayer is reached with four viruses LeGO-hOct4-MOI 5, LeGO-hSox2-MOI 5, LeGO-hc-Myc-MOI 2.5, LeGO-hKlf4-MOI 5, cell reseeding after 4 days on plastic 1 to 22.5 in DMEM + P / S + L-Glu + 10% FBS, after a day the medium is changed to ESmed and cultured for 8-10 days, while the medium changes they are seeded once every 2 days, 1 to 1 mechanical transfer is performed on matrigel in mTeSR1 medium, cultivation is carried out until colonies morphologically similar to ESCs are grown, clones are selected after a week. 2. The line of IPA cells obtained by the method according to claim 1, intended for the treatment of Huntington’s chorea, having a size of the order of 20 μm, a large nucleus, growth in compact colonies with tight contact, doubling period, as well as ESCs, normal karyotype 46, expression of Oct4, SOX2 , FOXD3, HESX1, Nanog, Sall4.

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