RU2774350C2 - Method for obtaining biomedical cell product for treatment of oncological, neurodegenerative and autoimmune diseases - Google Patents

Abstract

FIELD: cell biology.

SUBSTANCE: present invention relates to the field of cell biology and medicine, in particular to a method for obtaining a biomedical cellular product for the treatment of oncological, neurodegenerative, autoimmune and endocrine diseases and a product obtained by this method. To implement the method, first, an autologous mononuclear leukoconcentrate (MNC LC) is obtained from the bone marrow or from peripheral blood. After that, a line of autologous hematopoietic stem cells (HSC) or hematopoietic progenitor cells (HPC) with CD34+, CD45+, HLA DR+ markers is obtained. Next, a line of disease-intact autologous regional mesenchymal stem cells (MSCs) or somatic cells is obtained and their cell mass is increased. At the next stage, reprogramming of said MSCs or somatic cells into XEN-like pluripotent cells is carried out. Then, cell-engineering homotopic replacement of HSC and/or HPC nuclei with nuclei of XEN-like cells or whole XEN-like cells is carried out. At the last stage, the reduced HSC and/or HSC are combined with MNC LC.

EFFECT: invention makes it possible to restore the sanogenetic regulatory and regenerative potential of damaged own HSCs and provide them with the necessary and sufficient amount in the body.

7 cl, 4 dwg, 4 tbl

Description

Technical field

The invention relates to the field of physiology and medicine, and in particular to oncology, immunology, neurology and gerontology. The invention is used for the treatment of cancer and other malignant neoplasms (MNT), neurodegenerative, autoimmune and gerontological diseases, as well as for prolonging the life of mammals and humans by therapy with an autologous biomedical cell product (BMCP).

State of the art

Based on the well-known knowledge and existing biomedical technologies of immunology, oncology, neurology, transplantology, cell biology, molecular biology and cell engineering, the present invention, based on a new theoretical understanding of the insoluble scientific problem of the formation of carcinogenesis, neurodegeneration and autoimmune process, offers a fundamentally new and fairly universal methodological and scientific-practical approach in the treatment of major fatal diseases of civilization (cancer and other malignant diseases, Alzheimer's disease, dementia, Parkinson's disease, type 1 diabetes mellitus, myocardial dystrophy, systemic lupus erythematosus, rheumatism, multiple sclerosis and others), as well as provides a fundamentally new and outstanding result in personalized medicine and life sciences, based on existing and new post-genomic knowledge about the human hematopoietic stem cell.

It is known that the human immune system is normally a “conductor” and “arranger” of all regulatory processes, systemic and local regeneration and sanogenesis of human organs and tissues (Seledtsov V.I., 2019). It has all the necessary range of humoral factors and cellular tools to regulate these processes. This conceptual position can be fully attributed to the processes of regeneration and restoration of the nervous tissue of the human brain and spinal cord in case of damaged brain disease, neurodegenerative and autoimmune processes, and oncological diseases (Bryukhovetsky A.S., 2016). The role of the immune system in the biology of cancer and the mechanisms of carcinogenesis in other malignant tumors, as well as in neurodegenerative processes, is central and decisive (Bryukhovetsky A.S., Khotimchenko Yu.S., 2019).

Intrinsic immunocompetent cells (ICCs) of the human immune system are damaged in their genetic and postgenomic epigenetic structure in absolutely different ways, and also functionally suffer in different ways in various diseases, and this manifests itself in different ways in violations of their effector functions and diverse clinical manifestations. In all cases of autoimmune, neurodegenerative and oncological diseases, various genetic breakdowns, disorders of transcriptomic and protein metabolism, and severe metabolomic disorders are always detected. But they are different each time and depend in each case on the characteristics of the pathogenesis of the disease, the topical localization of the disease, the patient's condition at the time of the action of etiopathogenetic factors, and on a number of other reasons that we do not know or do not take into account. For example, in cases of oncological process, killer IKCs of innate immunity, such as NK cells, NKT cells, ϒδT cells, cytotoxic blood lymphocytes stop destroying tumor cells (TC), become absolutely tolerant to tumor antigens and transformed TC, and instead of performing genetically and epigenetically determined functions of elimination of OK, cease to recognize and destroy them. Leading oncologists and oncoimmunologists of the country believe that TCs easily evade the destruction of innate immunity by cellular systems (Kadagidze Z.G., 2017; Grivtsova L.Yu., Tupitsyn N.N., 2017), but in fact it is the ICCs that stop responding on TC at the molecular biological level, as they received transcriptomic and proteomic modification of the genome and epigenome and all their intracellular signaling pathways under the influence of horizontal and vertical exchange of tumor-specific proteins (RSP) from TC, which led to informational reprogramming of their genetically determined transcriptomic algorithms of killer functions (Bryukhovetskiy A.S., 2014; Bryukhovetskiy A.S., 2019). Similar transformations as a result of carcinogenesis occur with IKC of acquired immunity. Thus, macrophages and dendritic cells, which should destroy TCs, present tumor antigens on their surface, and instruct cytotoxic T-lymphocytes how to destroy TCs, also cease to perform their effector functions, which leads to their immunotolerance to TCs and unhindered tumor growth. It is the insufficiency of the immune system, manifested by hyporeactivity or unreactivity of the ICC of innate and acquired immunity, that becomes the main reason for the unimpeded growth of the tumor.

In autoimmune and neurodegenerative processes, absolutely opposite molecular biological processes take place, as a result of which own lymphocytes, on the contrary, become immunologically hyperactive, autoaggressive and hyperreactive to certain specialized highly differentiated cells and tissues of the human body and begin to destroy their own cells and tissues of the body, which manifests itself in the outcome degenerative, dystrophic or atrophic process. In systemic lupus erythematosus and polymyositis, the connective tissue becomes the target of autoreactive ICC, in multiple sclerosis and multiple encephalomyelitis, the myelin sheath of axons of neurons of the nervous tissue becomes the target of these ICC. In type 1 diabetes mellitus, pancreatic β-cells become the target of autoaggressive own T-lymphoid cells. In Alzheimer's disease and hydrocyanic cortical atrophy, cortical neurons become the target of autoaggressive own ICCs. In Parkinson's disease, these aggressive ICCs damage the neurons of the striopallidar zone, and in amyotrophic lateral sclerosis (ALS) they damage motor neurons, so in modern classifications of ALS they even began to be called motor neuron disease.

The main thing that unites all these diseases is that they arise as a result of the aggressive expansion of their own lymphocytes into specialized cells, tissues and organs of the body, into which they penetrate as a result of a violation of the hematotissue barriers or the blood-brain barrier due to the impact of various etiopathogenetic factors of diseases and age. This leads to systemic and / or local aseptic inflammation in certain organs, tissues and systems of the body, a powerful release of pro-inflammatory cytokines by leukocytes (cytokine storm) and the occurrence of genetic mutations and postgenomic (transcriptomic, proteomic, metabolomic, secretomic) disorders in the core of specialized differentiated or tissue-specific stem cells (SC), that is, these specialized and highly differentiated cells of different tissues become target cells for lymphocytes, in which pathological proliferative or degenerative interstitial processes are triggered. Systemic or local aseptic inflammation in a specialized (nervous, endocrine, connective, etc.) tissue in both cases causes excitotoxicity, metabolic disorders in cells and the development of oxidative stress in all elements of the tissue continuum involved in the pathological process of the organ, which increases the spectrum genetic and postgenomic disorders in specialized cells. This leads to the accumulation of various pathological proteins in the target cells, from which, as a rule, the target cell tries unsuccessfully to free itself. In a number of cells, tumor-specific proteins accumulate and TC and tumor stem cells (TCCs) are formed, while in other cells, tau proteins (Alzheimer's disease), FUS and SOD1 proteins (amyotrophic lateral sclerosis, hydrocyanic cortical atrophy, ALS dementia) accumulate, Lewy bodies (Parkinson's disease) and global atrophic and neurodegenerative changes are formed in these cells. These pathological specific proteins are cell gravediggers resulting from mutations in genes for which there are no regulatory tools in the body. These are proteins not of our evolutionary species (Homo sapiense), also called tumor-specific proteins (TSPs), intracellular accumulation proteins, taupathies, etc.). They accumulate in the cell, cannot be brought out by it due to their uniform diffusion and global distribution over all compartments of the cell, and become the main cause of cell death or the death of the whole organism. Thus, RSD in glioblastoma, breast cancer, and lung cancer is involved in all signaling pathways of intracellular transduction, and only 10 pathways are only partially damaged by them and are capable of real functioning (Bryukhovetsky A.S., 2014).

In other words, one of the fundamental mechanisms of most diseases of civilization (cancer and other malignancies, accumulation diseases and autoimmune diseases), aging and death of a person as a result of extreme or chronic exposure to adverse environmental factors and endogenous factors is the appearance in a specialized cell of the human body of genome mutations and transcriptome disorders, which leads to the accumulation of pathospecific proteomic bodies in the cell and to the impossibility of independent release from these extraspecies pathological protein bodies. The cell is always trying to get rid of them in a fairly standard way in the form of removing them in the form of microvesicles or division and thereby save itself. A primitive, but usually effective solution to the problem that has arisen through microvesicular transport, mitosis and reproductive cellular behavior, chosen by a specialized and highly differentiated cell of the body when pathological protein bodies accumulate in it, is genetically determined and evolutionarily worked out. Cell division, on the one hand, allows you to get rid of pathological proteins by removing them outside or into a daughter cell. However, if this instrument of cell sanogenesis through repeated and unsuccessful division is ineffective, it begins and carries out plastic restructuring of the entire intracellular signaling apparatus of the cell, which leads to the formation of a neoplastic process in one of the cases and the formation of tumors. On the other hand, when the division of the affected cell is immediately impossible due to its diverse intercellular connections and contacts, as well as the systemic integration of cells in the organ and tissue, then in order to bring out these pathological proteins, the specialized cell begins to prepare for division and rebuild its entire genome. protemic structure for the process of purification from pathological proteins, destroys intercellular connections, intercellular interactions and membrane receptors, which leads to its degeneration and atrophy, information blockade and death. This is the universal fundamental cellular mechanism for the development of the primary stages of both carcinogenesis and neurodegeneration and degenerative-atrophic processes.

These diseases do not occur in one day or one or two months, they “flow” for years against the background of chronic functional pathospecific insufficiency of the immune system, and manifest only when the resource of regulation and sanogenesis of the immune system is completely exhausted or there is a failure in the vital life support and regulation systems. immune system caused by exogenous (trauma, radiation, etc.) or endogenous (impaired blood rheology, lymph formation, etc.) factors. For example, ALS and Alzheimer's disease appear and can be diagnosed only when the patient has already lost 70-80% of specialized neurons in the brain and spinal cord, which occurs during the several previous years during which the disease forms.

However, the mechanism by itself for the appearance and accumulation of pathological proteins in a specialized cell of the body as a result of mutations is natural, obvious and has the right to occur due to possible errors in the processes of cell mitosis, the number of which in the human body is several billion a day. The process becomes pathological in violation of the mechanisms of regulation and control over it by the human immune system. The first line of supervision and control is the peripheral blood (PC) ICC, whose function is the destruction of pathological cells. The second line of regulatory and sanogenetic protection of the body is carried out by regional SCs, and the third line of cellular and humoral immune protection is the involvement of hematopoietic stem cells (HSCs) in this process.

The central systemic mechanism of generalization and chronicization of the carcinogenic, autoimmune and neurodegenerative process in the human body is the involvement of regional and systemic SCs in the pathological process. Both tissue-specific SCs and the main regulatory cells of the body, HSCs, are involved in these processes. It is believed that HSC is the slowest cell in the human and mammalian body, its cell cycle in humans is 360 days and therefore all cells of the body are checked against it and regulated by it in accordance with the systemic law of nature - the slowest phase is the control in any systemic process ( N. Wiener, 1960).

Despite the fact that HSC is the main hematopoietic cell and the ancestor of all hematopoietic and immune cells of the body, it is almost always involved in the processes of tissue-specific regulation of all organs and systems of the body through the exchange of regulatory proteins. It has been shown (Milkina E.V. et al., 2016; Bryukhovetsky I.S., 2017) that HSCs during intercellular interaction with TC and CSC of various types of cancer (lung cancer, breast cancer, glioblastoma multiforme) secrete a huge amount of regulatory proteins, which, through exosomal transport, enter damaged cells and radically change their fate, but at the same time they themselves often receive a large amount of RSP inside the cytoplasm, therefore, autologous HSCs themselves can become CSCs and be the main cause of hemablastosis. The amount of RSP in HSC with different types of intercellular interaction with OC can vary from 15 to 67% of the proteomic structure of the cell (Bryukhovetskiy A.S., Shevchenko V.E., 2016; Bryukhovetskiy A.S., 2019).

This universal mechanism of intercellular protein exchange between HSC and pathological cells leads to its genomic-epigenome and global proteomic changes and reprogramming of its functions. This scientific fact has been confirmed by proteomic mapping and profiling of normal cells, healthy SCs and CSCs and is described in a number of scientific articles and monographs in Russia and the USA (Bryukhovetskiy A.S., 2014; Bryukhovetskiy A.S., 2019). The descendants of the proteome-modified OSB of their own HSC in the form of a whole pool of ICC with pathological properties inherit these changes in their genome and epigenome and have pathological effector functions inherited from them.

Thus, the fundamental scientific fact and the main cause of systemic and disease-forming disturbances in the processes of regulation and sanogenesis of the human immune system is the genomic-epigenomic transformation of the protein, DNA and RNA structure of HSC by pathospecific protein bodies obtained by it during horizontal and vertical information exchange from damaged TC or degenerative cells. . Obviously, pathospecific protein bodies (OSB, Lewy bodies, tau proteins, etc.) appear in the cytoplasm, mitochondria, genome, and epigenome of highly specialized cells as a result of the accumulation of mutations in them during various oncological, autoimmune, endocrine, and neurodegenerative processes in tissues organism. This phenomenon of HSC proteomic degeneration as a result of the pathogenesis of these diseases is characteristic not only for cancer and other malignant oncological diseases, but also for the formation of a number of autoimmune, neurodegenerative, degenerative-atrophic and endocrine diseases. Therefore, the use of any molecularly targeted and targeted oncoimmune drugs for the treatment of these diseases will only be temporarily effective, but will still lead to a recurrence of the disease and its progression, since the main cause of the disease, genomic-epigenome transformed HSC, producing or hyporeactive (tolerant) HSCs will not be eliminated. , or hyperreactive (aggressive cytotoxic) ICC.

If, as a result of these proteomic intercellular interactions between the pathological cell and SC or HSC, a minimal transformation of the epigenome occurred, and the HSC genome was practically not affected, then the main changes in the epigenome occurred mainly in peripheral mononuclear cells, and after 100-120 days the disease itself will stop without treatment or for against the background of ongoing therapy, since this is the maximum life time of affected autoreactive or immunotolerant PC lymphocytes. If, in case of severe epigenetic damage to HSCs, their genome is not damaged or partially damaged and only in a small part of these cells, then the disease can be stopped by transplantation of autologous HSCs after high-dose chemotherapy. This has been shown with great success today in the transplantation of autologous HSCs in some patients with multiple sclerosis, polymyositis and systemic lupus erythematosus. High-dose chemotherapy before transplantation makes it possible to "empty" the patient's bone marrow from pathological HSCs and kill all HSCs incapable of migration and mobilization, restore normal hematopoiesis and "restart" the entire immune system from healthy HSCs.

If, however, during intercellular interaction with a pathological cell, HSCs have received irreversible changes in their genomic and postgenomic status, and all HSCs of the patient are affected by global proteomic disorders, then transplantation of autologous HSCs will be ineffective, and any treatment will be effective only temporarily. Studies have shown that if, as a result of intercellular interaction with pathological cells, the HSC proteomic structure was damaged by more than 30-35%, then the process is practically irreversible (Bryukhovetskiy A.S., 2019).

Thus, a fundamentally important theoretical and practical scientific fact has been established and proven, which consists in the fact that the majority of oncological, neurodegenerative, autoimmune, gerontological and endocrine human diseases are not unique diseases of certain highly differentiated specialized cells and tissues (motor, cortical or cerebellar neurons, neurons striopallidar zone, fibroblasts, neuroglia and microglia, pancreatic β-cells, cardiomyocytes or endotheliocytes, fibroblasts, etc.), as it was generally recognized in the world, but are a genome-proteomic disease of the patient's own hematopoietic SC, provoked and actively supported by its descendants in the form of autologous ICC with the corresponding genomic and proteomic modifications of the protein structure (Bryukhovetsky A.S., Khotimchenko Yu.S., 2018). Carcinogenic, autoimmune and neurodegenerative degeneration of specialized cells in the human body as a result of the pathogenesis of these diseases is a natural outcome of the disease, and not its cause and a tool for the progression and recurrence of the disease. The cause of system formation and the factor of chronicity of the pathological process are genomic and epigenomic disorders of HSCs and their descendants, as well as similar changes in regional SCs involved in the pathological process.

From these new theoretical and methodological positions, both involutional degenerative-atrophic and senile changes in organs and tissues should be considered not as a natural age-related process of decrepitude and deterioration of cells and tissues of the body, but as a to degenerative-dystrophic changes or proliferative processes in specialized cells of organs and tissues in human ontogenesis. Aging is not an age norm, but a real transcriptomic-proteomic pathology of HSCs and regional human SCs, due to a decrease in their quality control, supervisory and regulatory characteristics and properties, a deficiency in their quantity with age and disease, as well as a violation of the regulatory, sanogenetic and regenerative abilities of these hematopoietic cellular systems and all immunity. Decreased immune surveillance and control of HSC and its descendants over cells of specialized tissues leads to their untimely replacement or restoration. A universal system of control and supervision over the effective work of all specialized cells of the body and their regulation and replacement are genetically incorporated into the program of HSCs and tissue-specific SCs, and that is why in the process of evolution an additional antitumor or neurodegenerative defense mechanism was not laid down to fight cancer and other diseases of civilization. It exists in human hematopoiesis and immunopoiesis in the very structure of HSCs and is brilliantly realized in healthy HSCs, especially at a young age. Damage to the genomic and postgenomic structure of HSCs in humans over time is primarily due to adverse environmental conditions due to human intervention in nature, and only in the last place are individual damage to HSCs by etiological factors of the disease (trauma, infection, etc.). This alternative scientific view on the main diseases of civilization, on the age-related changes in cells and tissues with age, and on the process of human aging itself opens up new ways to early prevention of a number of diseases of civilization and the formation of longevity paths for all mankind.

The manifestation of clinical and paraclinical manifestations of most fatal diseases of human civilization reveals not the cause of the disease in the form of damage to specialized cells of organs and systems, but the natural outcome of the disease , due to violations of immunological surveillance and control of target cells in body tissues, a decrease in the sanogenetic potential of ICC, which are descendants of the gene - and epigenome-transformed HSCs. Clinical manifestations of an oncological, autoimmune, or neurodegenerative disease are a manifestation of a deep tissue defect due to damage to specialized cells and disruption of their function. That is why the authors of the present invention are convinced that ALS, today an absolutely incurable disease, is not a disease of the motor neuron, as is generally recognized in the world classifications (ICD 11 and ICD 12) and modern textbooks on neurology, namely, the genomic epigenomic disease of the patient's HSC, and in this view, this disease in the early stages can be completely cured using the BMCP of the present invention. In particular, it was shown that the neurospecific profile of HSC in familial ALS makes it possible to detect the presence of a compensated disease in healthy family members who do not yet have a clinic of motor damage (Bryukhovetsky A.S., Grivtsova L.Yu., 2018).

Thus, it is possible to state the most important biological scientific fact, which consists in the fact that the real main conductor and the main leader in the human immune system itself is autologous human HSCs . And if in the course of a long life of a person and/or as a result of informational regulatory exchange of pathological proteins with damaged somatic specialized cells of the body, his own HSCs accumulate a significant amount of pathospecific protein bodies, then these pathospecific proteins begin to negatively affect the genome and epigenome of HSCs, damage them, due to which forms a pathospecific (oncospecific, tau-protein-specific, FUS-protein specific, SOD-1 protein-specific, etc.) proteomic-transcriptomic transformation of the entire atomic-molecular structure of its own HSC.

In various cases of powerful one-time or repeated insignificant combined impact on a person of etiological environmental factors, pressure of macro- and microeconomic factors of society and the negative impact of the socio-psychological microenvironment, these multi-vector effects become etiopathogenetic factors and causes of the main diseases of civilization, and in some cases cancer develops and other malignant tumors, and in other cases, these same factors lead to autoimmune and degenerative processes in the cells of the body, the outcome of which is involution, degenerative or generally degenerative-atrophic and fibrous damage in specialized cells of the body. It is obvious that the multidirectionality of damage to highly differentiated cells due to local or systemic aseptic inflammation depends on the functional state of the specialized cell, the phase of the cell cycle, which is affected by the pathological factor, and on the degree of immune deficiency due to the level of genomepigenomic damage to HSCs and the state of the body's ICC, which leads to not only to various genomic and postgenomic changes in specialized cells in the target tissues of the affected organs, but also to information perturbations in the genomic and postgenomic structure of ICC and HSC.

Therefore, transcriptomic and proteomic rearrangement in ICC and HSC can form both a proliferative and degenerative vector of changes in the molecular structure of ICC and HSC, which in the clinic can manifest as malignant oncological processes and be accompanied by “plus-tissue” phenomena (the appearance of an uncontrolled amount of CSC, pathological division of CSCs, excessive reproduction of TCs, migration of TCs, insufficient immune antitumor functionality of ICCs), and go in the opposite direction, with the formation of degeneration and atrophy and the appearance of the tissue phenomenon "minus-tissue" (degeneration, atrophy, fibrosis and sclerosis of the tissue, disruption and loss of function and excess immune hyperreactive functionality).

As noted above, the life span of ICC PC, even pathological ones, is limited to 100-120 days of life of lymphocytes and granulocytes in the human body, therefore, the chronization of the process, the systemic nature and severity of chronic inflammation in tissues, the presence of oxidative stress and excitotoxicity of most fatal diseases of civilization can only be supported by fundamental causes of the pathogenesis of the disease, leading to specific immune deficiency, and this is possible only at the level of formation of the pathology of the HSC genome and epigenome. Therefore, the main fundamental cause of these diseases of civilization and the formation of age-related involution of organs and tissues is not a local pathology of specialized cells of organs and tissues, but genomic-epigenomic degeneration and transformation of the molecular proteomic structure of one's own HSC. This scientific fact has been proven by proteomic mapping and profiling of HSCs and other SCs in cancer and a number of neurodegenerative diseases (application for invention of the Russian Federation No. 2018145635, filed on 12/21/2018).

Summarizing all of the above, we can make the following logical conclusion from all the modern proven facts and scientific knowledge at our disposal about the genomic-epigenomic changes in HSCs. The solution to the problem of curing most diseases of human civilization can be, on the one hand, only the restoration of genetically and epigenetically determined functions of HSCs, due to its atomic and molecular structure, and the restoration of their functional hematopoietic and immunopoietic properties and characteristics. On the other hand, it is necessary to restore a sufficient number of healthy HSCs. Qualitative and quantitative restoration of HSCs of an elderly or sick person and the restoration of their regulatory potential will completely block any processes of carcinogenesis, degenerative-dystrophic involution and fibrosis in body tissues, stop oncological, autoimmune and neurodegenerative diseases and actually prolong the active life of human organs and systems by 30- 40 years. It is the HSC, or rather the restoration or reconstruction of the damaged HSC in any person, that is the “golden key” to his long active life without old age and the main diseases of civilization, which he “collected” in the genome and epigenome of the HSC during his life up to 40 years or more.

As analogues (prior art) of the present invention, one can point to the currently existing standard technologies for transplantation of cell nuclei used in in vitro fertilization (IVF), which implement the principle of replacing the nucleus (genome) of a donor egg cell with the nucleus (genome) of a recipient somatic cell ( patent application RU 2004117092 A, 11/20/2005; WO 0052145, 09/08/2000; patent RU 2280462 C2, 07/27/2006; patent RU 2252252 C1, 05/20/2005, Stojkovic M., et.al., 2005) and domestic technologies) hybrid human SCs (patent RU 2352637 C2 /WO/2009/002223) and their use in humans (Teplyashin A.S. et al., 2009). Also an analogue of the present invention is the patent RU 2535972, 20/12/2014 (Antineoplastic individual proteome-based targeted cell drug, a method for its production and the use of this drug for the treatment of cancer and other malignant neoplasms), as well as the work of a group of Chinese professor Hongkui Deng (Zhao Y., Zhao T., Guan J., Zhang X., et al., 2015, Xiang Li et al., Zhen Chai, Hongkui Deng, 2017). All of the listed known technical solutions do not provide the restoration of their own damaged HSCs, which would lead to the cure of a mammal, including humans, from cancer and other malignant neoplasms, neurodegenerative and autoimmune diseases.

Accordingly, the purpose of the present invention is to obtain a radical solution to the problem of curing a person from cancer and other cancers, neurodegenerative and autoimmune diseases by providing a technical result, which consists in restoring the sanogenetic regulatory and regenerative potential of damaged own HSCs, the ancestors of all cellular systems of hematopoiesis and immunopoiesis, and providing them necessary and sufficient amount in the body. It is the biorestoration of the molecular biological structure of HSC, which has suffered as a result of the negative influence of environmental factors and etiological factors of HSC disease, as the main system-forming cause of all major diseases of modern civilization and involutionary age-related processes in the body, the present invention is directed.

The essence of the invention

The above purpose of the present invention with obtaining the specified technical result is achieved by the fact that a method is proposed for obtaining a biomedical cell product (BMCP) for the treatment of oncological, neurodegenerative, autoimmune and endocrine diseases and prolonging the life of a mammal and a person, including:

obtaining an autologous mononuclear leukoconcentrate (MNC) from bone marrow exfusate or from a leukopheresis product of mobilized mononuclear cells (LC MNC) of peripheral blood;

obtaining a line of autologous hematopoietic stem cells (HSC) with CD34+, CD45+, HLA DR+ markers and/or hematopoietic progenitor cells (HPC) with CD34+, CD45-, HLA DR- markers differentiation;

obtaining a line of disease-intact autologous regional multipotent stem cells or somatic cells and increasing their cell mass;

reprogramming said autologous regional multipotent stem cells or somatic cells into XEN-like pluripotent cells by chemical induction;

carrying out cell-engineering homotopic replacement of HSC and/or HCP nuclei with the nuclei of XEN-like cells or with whole XEN-like cells by obtaining cytoplasts from native HSCs and/or HCPs by treating these cells with cytocholasin B and transplanting karyoplasts obtained from XEN-like cells by their treatment with cytocholasin B, or whole XEN-like cells into HSC and/or HCP cytoplasts, thereby obtaining reduced HSC and/or HCP containing multipotent HSC cytoplasts and pluripotent karyoplasts of XEN-like cells;

combination of restored HSCs and/or GCPs with LC PBMCs from which the original HSCs and/or GCPs were isolated to obtain a target biomedical cell product containing a line of postgenomically restored multipotent genetically homogeneous autologous HSCs with markers
CD34+Lin-, CD38", HLA-DR+, CD45+.

In addition, according to the present invention, reduced HSCs and/or HCPs coupled to LK MNCs are preferably cultured in a medium that prevents differentiation to increase their cell mass, form and mature their properties of multipotency in the hematopoietic direction, and control these properties by flow cytometry.

At the same time, in the case of oncology or neurodegenerative diseases of the brain and spinal cord, as well as in the case of autoimmune diseases, mesenchymal stromal stem cells (MSSCs) with markers CD10+, CD13+, CD44+, CD90+ (Thy-1), CD105+, CD34 are used as cells undamaged by the disease. -, CD45- and CD117- and/or mesenchymal stromal progenitor cells (MSPCs) with markers CD10+, CD13+, CD44-, CD90+ (Thy-1), CD105+, CD34+, CD45+ and CD117+, and in the case of oncology of solid organs as intact cell disease, neural stem cells (NSCs) with the CD 133+ marker and/or neural progenitor cells (NPCs) with the CD 133+ marker are used.

According to the present invention, reprogramming by chemical induction is preferably carried out with a cocktail containing small molecular weight substances selected from the group consisting of VPA, TD114-2, CHIR, 616452, tranylcypromine, formskolin, AM580, EPZ004777, CH55 and vitamin C (VC), and transplantation of the resulting from XEN-like cells of karyoplasts or whole XEN-like cells into cytoplasts, HSCs and/or HPCs are carried out in vitro by electroporation or by laser-optical method.

In addition, according to the present invention, a biomedical cell product (BMCP) obtained by the method of the present invention and containing autologous LA MNCs depleted in their own HSCs and/or HCPs as a result of their immunoseparation, with a line of genetically homogeneous autologous hematopoietic cells with markers CD34+Lin- , CD38", HLA-DR+CD45+.

List of graphics

In FIG. 1 shows the dependence of the survival of animals on their age, obtained as a result of comparative experiments on bone marrow transplantation and BMCP according to the present invention (see Example 11).

In FIG. 2A-2B show the results of GFP fluorescence of HSCs in donor, control and experimental groups of animals described in Example 11.

Detailed disclosure of the invention

When creating the present invention, the question arose - what can be proposed to restore the molecular-structural and functional capabilities of HSCs with genomic and epigenomic disorders? Any attempt at extremely complex genomic editing of pathological HSCs using modern genetic engineering tools (zinc fingers, CRISP / Cas9 technologies, etc.) seems to be ineffective, since in each specific case there will be a personalized type of molecular damage to the DNA structure of the HSC genome and specific epigenetic changes in the core of HSCs in each patient, and elucidation of the location of these structural abnormalities in these cells, even using the most cutting-edge diagnostic technologies of genome sequencing, full-transcriptome analysis with microarraying of the expression of 34,000 genes, or extensive protemic mapping of 8,000 proteins, will only allow to outline and designate the main violations genomic-transcriptome-proteomic intracellular construct, but this approach will not provide real treatment and will be very expensive. Therefore, it is obvious that the search for specific genes, methylation disorders and damaged histones, as well as the detection of transcriptomic, proteomic and metabolomic disorders in HSCs for the occurrence of a particular disease is not constructive and will not give the desired result in practice. In each case, DNA damage in the HSC nucleus will be individual and specific and will depend on the nature and strength of the etiological effect of pathospecific proteins that have entered the HSC and affect its genome and epigenetic structures, as well as on the characteristics of the transformation of the proteomic profile of the pathological specialized highly differentiated target cell. in human organs and tissues.

Therefore, the author of the present invention abandoned the most modern genome editing technologies and decided to approach the process quite radically, using the more “old” and well-studied and proven cell engineering technologies used for transplantation of cell nuclei, since this approach makes it possible to technologically remove the pathological genome and make impossible to reverse aberrant epigenetic modifications from HSC and replace it with a healthy genome of another somatic cell of the body (application for invention RU 2004117092 A, IPC C12N 5/00, 11/20/2005; WO 00/52145, IPC C12N 5/06, 09/08/2000; patent RU 2280462 C2, IPC A61K 35/55, 07/27/2006; patent RU 2252252 C1, IPC C12N 5/08, 05/20/2005). This assumption is due to the fact that it is HSC and HCP, as well as tissue-specific cells of the tissue line where the pathological process unfolds, that are the main instrument of regulation in sanogenesis. Other cellular systems that are not involved in the pathological process are practically not exposed to the same number of adverse effects, and their genome and epigenome may practically not have such damage. Reprogramming of somatic and SCs not involved in the pathological process can be implemented using modern technologies of chemically induced reprogramming of somatic cells to obtain multipotent HSCs.

The use of cell engineering as a scientific biotechnological solution has already proven itself very well in the clinical technologies of in vitro fertilization (IVF) and the creation of heterogeneous SCs for cell therapy. Techniques and techniques for replacing the genome of one cell with the genome of another cell are solved in modern medical biotechnologies by replacing their nuclei from somatic cells, described and patented (Teplyashin A.S. et al., 2009). In modern IVF, this is a fairly routine procedure, when a genome is extracted from one donor egg, which is packaged in the nucleus of this cell, and in its place is put the nucleus (genome) extracted from the somatic cell of the recipient (future mother), and an embryo is grown, which is then implanted into the uterus of the woman herself for engraftment. However, as soon as we start obtaining SCs from artificially created cell-engineered blastocysts or human embryos of different stages of gestation (blastocysts, zygotes, embryos, etc.), we are forced to destroy an already living human body, which is prohibited by law in the Russian Federation. All such procedures for creating genetically homogeneous SCs refer to reproductive and therapeutic cloning, since these technologies are based on the creation of a living hybrid patient of a compatible embryonic organism, whose cells, when it is destroyed, are used to create SC cell lines. Therefore, it was necessary to propose a biotechnology that would not use fertilized or cell-engineered human and animal oocytes and would not use embryonic material of various stages of gestation.

One of the modern approaches to realizing the goal of replacing HSCs is to obtain chemically induced pluripotent stem cells (iPSCs or iPSCs) by reprogramming them from any somatic cell (fibroblast, neuron, adipose tissue SCs, blood cells) using modern known genome transcription factors (Oct , Sox2 , Klf4 and c-Myc). These iPSCs can be reprogrammed into specialized multipotent SCs using other transcription factors. Thus, as a result of the use of four transcription factors (Gata2, Gfi1b, cFos, and Etv6), it has already been possible to obtain viable HSCs (CD34+, CD45+, CD133+) from mouse skin fibroblast, which have the entire spectrum of multipotency and are capable of producing all types of hematopoietic and immunopoetic cells (CF Pereira , B. Chang, J. Qiu et al., 2013). In another study, after transferring to iPSCs using six transcription factors (Run1t1, Hlf, Lmo2, Prdm5, Pbx1, and Zfp37), human HSCs with full multipotency and capable of restoring all types of blood cells were obtained in another study (J. Riddell, R Gazit, BS Garrison et al., 2014). Today, pluripotent SCs can differentiate into almost all cell types in the body. This unique potential provides promising avenues for cell therapy to repair tissues or organs destroyed by injury, degenerative disease, aging, or cancer. The discovery of iPSC production technology suggests a possible strategy for the creation of tissue-specific multipotent SCs for the patient. However, due to concerns about the specificity, efficacy, kinetics, and safety of iPSC reprogramming, improvements or fundamental changes in this process are required before their effective clinical use, since the introduction of transcription factors into the genome is carried out using adenoviruses, lentiviruses, or plasmids, which is genetically unsafe. both for the patient himself and for his offspring (T.Ma, M.Xie, T.Laurent, S.Ding, 2013).

There is another serious drawback associated with this method of obtaining iPSCs. Any type of cells in the body is characterized by its own individual epigenome - a certain spectrum and pattern of post-translational covalent modifications of histones, DNA methylation, and the presence of a set of small non-coding RNAs. The combination of these factors forms a unique chromatin structure inherent in a particular type of cell. The chromatin of pluripotent cells is characterized by an "open" architecture and a decompacted state. Chromatin in this configuration during differentiation is able to quickly undergo various post-translational modifications of histones, DNA methylation and demethylation. Pluripotent cells also contain "bivalent domains", that is, regions enriched simultaneously with marks of both active and inactive chromatin. Most of the bivalent domains are associated with transcription start points of genes involved in development. In pluripotent cells, these genes are characterized by a low level of transcription, while during differentiation, bivalent domains turn into monovalent domains containing marks of either active or inactive chromatin, and, accordingly, activation or repression of genes is observed, which determines the specialization of cells of a particular type (cited by Vaskova E. A., Stekleneva A.E., Medvedev S.P., Zakian S.M., 2013). Therefore, when choosing a source for reprogramming, one should take into account the fact that iPSCs retain epigenetic memory of the tissues from which they originated, which affects their ability to directed differentiation (J.G. Crompton, M. Rao, N.P. Restifo, 2013; J.M. Polo, S. Liu, M. E. Figueroa, et al. & K. Hochedlinger, 2010; K. Kim, A. Doi, B. Wen et al., 2010; K. Kim, R. Zhao, A. Doi et al., 2011 ; O. Bar-Nur, H.A. Russ, S. Efrat, N. Benvenisty, 2011; Denker H.-W., 2012; Jong-Hee Lee, Jung Bok Lee, Zoya Shapovalova et al., 2014; Vaskova, E. A., Stekleneva, A.E., Medvedev, S.P., & Zakian, S.M., 2013; Kyttälä, R. Moraghebi, C. Valensisi et al., 2015). Residual epigenetic memory does not necessarily appear at the stage of pluripotency. iPSCs obtained from different tissues have the proper morphology, genes characteristic of pluripotency are active in them, and they are able to differentiate into tissues of three embryonic layers both in vitro and in vivo (Vaskova, E.A., Stekleneva, A.E. ., Medvedev, S. P., & Zakian, S. M., 2013; A. Kyttälä, R. Moraghebi, C. Valensisi et al., 2015). However, this epigenetic memory may appear later, during re-differentiation into specific cell types, which requires the activation of loci that retain elements of residual epigenetic memory (J.G. Crompton, M. Rao, N.P. Restifo, 2013; J.M. Polo, S. Liu, M.E. Figueroa , et al. & K. Hochedlinger, 2010; K. Kim, A. Doi, B. Wen et al., 2010; K. Kim, R. Zhao, A. Doi et al., 2011; O. Bar-Nur , H.A. Russ, S. Efrat, N. Benvenisty, 2011; Denker H.-W., 2012). It is necessary to find a solution how to bypass the mechanism of reversion (return) of epigenetic memory in these cells for their wide use in the clinic.

In the field of biomedicine, iPSCs are the most strategically advantageous because they can be obtained from somatic cells and are autologous with respect to the somatic cell donor. To date, it is known that iPSCs are very similar in their properties to embryonic SCs, that is, they express a similar range of genes and form teratomas containing derivatives of all three germ layers. At the same time, many works show that, as a result of reprogramming, iPCC lines acquire a number of genetic and epigenetic aberrations, including disturbances in the functioning of imprinted genes, changes in the number of gene copies, point mutations, an aberrant pattern of DNA methylation, and a number of others. At the same time, differences in the epigenomes and transcriptomes of embryonic SCs and ISPCs are due not only to aberrations acquired in the process of reprogramming, but also to the preservation of a number of epigenetic marks of the original somatic cells. Such inheritance of the properties of iPSC epigenomes and transcriptomes is called “epigenetic memory” (Vaskova E.A., Stekleneva A.E., Medvedev, S.P., & Zakian, S.M., 2013).

Thus, despite the great success of modern biotechnologies in the creation and potential use of iPSCs in the creation of HSCs from somatic cells by reprogramming them with transcription factors, the disadvantages of these technologies outweigh all their advantages, and a fundamentally new approach to this problem is needed to exclude viral intervention in human genome, prevent the formation of teratomas, and find a mechanism to overcome the reversal of residual epigenetic memory after transdifferentiation of one cell into another.

An alternative approach to the transdifferentiation of somatic cells without the use of transcription factors and the rejection of their viral carriers was proposed in 2013 by the Chinese professor Hongkui Deng and his scientific group of scientists from Peking University (Zhao Y., Zhao T., Guan J., Zhang X. et al., 2015). They solved the problem of reprogramming somatic cells into the desired phenotypes of other cell lines by creating chemically induced iPSCs, more precisely by creating an innovative biotechnological platform based on the XEN-like state (a state similar to the embryonic endoderm) of reprogrammable cell systems. The XEN-like state of cells they obtained, induced by certain cocktails of low molecular weight chemicals, became a bridge that connects somatic cells with pluripotency during chemical reprogramming and, as it were, bypasses the pluripotent embryonic state of the cell. The extraembryonic XEN-like state mediates the process of iPSC generation. XEN-like cells are transcriptionally and functionally similar to the in vivo counterpart. XEN cells are ready for Oct4 expression and pluripotency establishment. The induction of iPSCs is greatly enhanced by highly precise manipulation of small molecules. The chemical reprogramming process requires earlier formation of embryonic endoderm-like (XEN) cells and a late transition from XEN-like cells to chemically induced iPSCs in a unique way that is fundamentally different from the reprogramming pathway using transcription factors. Moreover, precisely manipulating the transition of cell fate stepwise through the XEN-like state allows the identification of small molecular weight accelerators and the creation of a reliable chemical reprogramming system with a yield up to 1000 times greater than in a previously reported protocol (J. Riddell, R. Gazit, B.S. Garrison et al. , 2014) using six transcription factors (Run1t1, Hlf, Lmo2, Prdm5, Pbx1, and Zfp37) and the authors’ own protocol for chemical induction in obtaining iPSCs from 2013 (Xiang Li et al., Zhen Chai, Hongkui Deng, 2017). These results indicate that chemical reprogramming is a promising approach to manipulate cell fate. In other words, in addition to the possibility of obtaining patient-specific autologous tissues and organs from iPSCs, another attractive strategy for regenerative medicine by Chinese scientists is the strategy of chemical transdifferentiation - direct transformation of one type of somatic cells into another through the so-called cell bridge in the form of XEN cell systems. . This transdifferentiation not only detects and exploits the developing plastic intermediates generated during iPSC reprogramming, but also produces a very wide range of cells, including expandable tissue-specific progenitor cells. Cells in the induced XEN-like state readily expanded for at least 20 passages and retained genome stability and lineage specification potential. Therefore, a groundbreaking study by Beijing scientists establishes a multifunctional pathway for chemical reprogramming of the lineage and may provide a platform for generating diverse cell types through the application of this extensible XEN-like state (Xiang Li et al., Zhen Chai, Hongkui Deng, 2017).

The method of the present invention uses non-reproductive and therapeutic cloning and non-transdifferentiation of somatic cells using transcription factors to obtain genetically homogeneous HSCs. In the proposed method for chemical induction in order to transdifferentiate somatic and stem cells into genetically homogeneous HSCs, exclusively autologous cell lines of adult somatic cells (fibroblasts, karyocytes, fat cells) and lines of multipotent non-hematopoietic SCs of the patient (neutral SCs (NSCs), adipose tissue SCs, mesenchymal stromal SCs (MSSCs), etc.) isolated from humans in vivo. The method of the present invention, in principle, does not involve obtaining a zygote, blastocyst or embryo, as is customary in all types of cloning and its subsequent destruction to obtain multipotent stem cells, and also does not involve the deprivation of life of an unborn embryonic hybrid creature or the introduction of one's own cells into the genome structure viral and vector carriers and plasmids. The invention proposes to work with one of two or three lines of adult somatic cells or non-hematopoietic stem and progenitor cells already secondarily differentiated by the patient's body towards a certain multipotency, extracted from one person and located exclusively in the postnatal period of ontogenesis. All manipulations with transdifferentiation of cells into a XEN-like state should be performed by gene-oriented chemical induction with small molecules using the technology of the group of Professor H. Deng (Zhao Y., Zhao T., Guan J., Zhang X., et al., 2015; Xiang Li et al., Zhen Chai, Hongkui Deng, 2017). This will make it possible to obtain full-fledged iPSCs from somatic or SCs, bypassing the stage of embryonic development, using a stepwise algorithmic processing of cells in an induction medium with low molecular weight chemicals (perturbogens) at a given concentration and exposure. The resulting iPSCs are cultivated in a special medium that prevents the differentiation of these cells and induces the activation of the expression of cell nucleus genes towards hematopoiesis and immunopoiesis by using a cascade of cytokines that activate the hematopoietic orientation of the resulting cells, also by using cultivation in a medium with a complex of specific perturbogens - specially selected low molecular weight chemical compounds for bioinformatics , that is, from the pluripotent XEN state of the cell, it must be made into a multipotent HSC. To do this, the next step is to transplant the nuclei (karyoplasts) of XEN-like cells into HSC cytoplasts obtained from the patient. Thus, HSC-like autologous cell-engineered systems with a genetically homogeneous XEN-like cell nucleus are obtained, with the goal being to obtain viable multipotent HSCs from this cell-engineered mix of the patient's own cells. At this stage, the authors of the present invention took advantage of the methodological approach proposed earlier in the patent for invention RU 2535972, IPC A61K 48/00, 12/20/2014 on "Antineoplastic individual proteome-based targeted cell drug, a method for its preparation and use of this drug for cancer therapy and other malignant neoplasms. This patent discloses a targeted cell preparation molecularly targeting the integrin pathway and focal adhesion in CSCs by selecting perturbogens (low molecular weight chemical compounds) from the Massachusetts Institute of Technology (MIT) cell transcriptome database. This database of transcriptomic gene expression profiles of cells contains the results of the work of various researchers on mapping and profiling gene expression before and after manipulations with cells on the Affymetrix platform devices. Therefore, in connection with the present invention, it was proposed to remove the full transcriptome gene expression profile of HSC-like cells with a nucleus in the XEN-like state, and to find perturbogens by bioinformatics, which would lead the cell-engineering system to cell systems with a transcriptome profile by chemical induction. expression of healthy HSC genes. The problem of reversal (return) of epigenetic residual memory in the resulting cell-engineered cells is proposed to be solved by adding to the cultured medium a set of standard chemical molecules widely used for the epigenetic balance of pregnant woman cells at the early stages of human embryo formation (folic acid, vitamin B12, vitamin C and etc.). As a result of cell engineering in the proposed method, an equivalent replacement of the damaged genome and epigenome (nucleus) of a multipotent HSC is carried out with the genome and epigenome (nucleus) of another healthy multipotent HSC-oriented cell in an XEN-like state, not involved in pathological processes in the patient's body. The process of fusion of cytoplasts of the HSC of the patient and the karyoplast of hematopoietically oriented cell-engineered (GOKI) HSC can be realized by their electroporation. The "ripening" of the obtained GOKI HSCs is carried out in an environment that prevents the differentiation of HSCs, with ionomycin and folic acid, vitamin C and vitamin B12. Thus, the present invention proposes a new solution to combat the residual epigenetic memory of HSCs created from a somatic cell through a chemically induced XEN-like state by performing cell engineering of autologous HSCs and removing the nucleus of this cell from the patient's pathological HSC, in which its entire damaged genome, and its replacement with the genome of a healthy transdifferentiated somatic or SC of a non-hematopoietic series of the same organism, but which, by chemical induction, became GOKI HSC.

When creating the present invention, the author proceeded from his own scientific hypothesis about the altered proteomic and transcriptomic structure of HSC in patients with oncological, autoimmune and neurodegenerative diseases and, of course, the pathospecific proteomic profile of the patient's multipotent HSC (Bryukhovetsky A.S., 2014; Bryukhovetskiy A.S., 2019 ). Therefore, a possible solution to the problem of combating these diseases is the functional and molecular restoration of damaged HSCs due to its cell-engineering reconstruction and the restoration of their necessary quantitative balance for homeostasis in the human body. The material for HSC reconstruction was the genome and epigen (nucleus) of any other healthy somatic or multipotent SC of a non-hematopoietic line in the patient's body. Obviously, it will not be difficult to find a healthy somatic or tissue-specific multipotent SC of another tissue-specific lineage in the human body. There are no moral, ethical, social, legal and legal restrictions on the use of somatic cells as a source of nuclei for their transfer to other somatic cells. This fully applies to all types of SC.

Obviously, the effectiveness of the method increases significantly when using the nuclei of their own SC or progenitor cells. Moreover, the tissue from which cells can be isolated as a source must be accessible and versatile. The most accessible and versatile are adipose tissue, bone marrow and the olfactory lining of the patient's nose. But autologous multipotent liver SCs and SCs obtained from the menstrual blood of a sick woman or testes of a man can also be used as regional multipotent SCs or somatic cells to achieve this goal. In this regard, MSSCs isolated from bone marrow or adipose tissue are most preferred. These cells have unique characteristics in culture: the ability to proliferate for a long time and, upon induction to differentiation, to form tissue cells of mesenchymal origin, they can be quite easily obtained from each person, including the patient. When such cells are transplanted into the enucleated HSC of a patient, it is possible to obtain cellular objects that are genetically identical to the patient, i.e. having genetically homogeneous characteristics (Teplyashin A.S. et al., 2009).

At the same time, in cases of oncological (for example, glioma multiforme), neurodegenerative (for example, ALS or ALS dementia), autoimmune (for example, multiple sclerosis) diseases of the brain or spinal cord, not only HSCs, but also NSCs suffer, therefore, the best source for identifying a healthy genome (nucleus) of cells in the body in this case can become cells of the mesenchymal series. They are isolated from the patient's bone marrow, adipose tissue, or mobilized peripheral blood (PB) mononuclear concentrate (MNC). In the case of oncological diseases of solid organs (for example, breast cancer, lung cancer, kidney cancer, etc.), as a rule, HSCs and MSSCs are affected, but NSCs, as well as tissue-specific multipotent SCs obtained from trans-barrier organs (eyes, testicles, etc.), SCs of the liver and adipose tissue are almost never damaged. Thus, if a patient has a process in the nervous tissue, then for the reconstruction of HSCs it is advisable to use MSSCs of the bone marrow and adipose tissue, which are not involved in the pathological process, and if the process is localized in solid organs, the best source of the autologous genome of healthy cells will be NSCs obtained from olfactory lining of the nose.

The very process of replacing the HSC core with the core of another tissue-specific multipotent SC is currently technically implemented on somatic cells in IVF and therapeutic cloning and does not present any technical difficulties. It is well established today by most molecular biologists, and this technology has now become routine. The biotechnological process of replacing the pathological HSC genome with the genome of another healthy multipotent SC is equivalent to replacing the core of one autologous HSC with the core of another autologous tissue-specific multipotent SC. Therefore, this biotechnological process in the present description is not called nuclear transplantation (as in therapeutic and reproductive cloning), but the replacement or substitution of the genome or nucleus of HSCs. substitution it's well known in biology term (late lat.substitutio, from lat.substitute - I put instead, I appoint in return), meaning substitution in the process of evolution of one organ by another in the same organism, which occupies a similar position in the organism and performs a biologically equivalent function. In the case of genome replacement, it is reasonable to speak of homotopic substitution or substitution (homotopic substitution) or equivalent replacement , which means the replacement in the process of evolution of one organ by another, occupying a similar position in the body and performing a biologically equivalent function (the replaced organ is reduced in this case). For example, in a number of cases there is a reduction of the replaced organ and a progressive development of the replacement. So, in chordates, the axial skeleton of the notochord is replaced first by a cartilaginous, then a bony spine; in cactus-like plants, leaves (photosynthetic organs) are replaced by stems. The term "substitution" was introduced by N. Kleinenberg (1886) (Gilyarov M.S. et al., 1986) and this term very accurately reflects the essence of the proposed phenomenon of artificial replacement of the genome (nucleus) located in one autologous pathological cell with the genome (nucleus ) another healthy autologous cell to perform the same function.

In order not to confuse the method proposed in the present invention with various forms of reproductive and therapeutic cloning, it can be called the method of equivalent substitution of the cellular genome (nucleus) or the method of homotopic substitution (replacement) of the cellular genome (nucleus). The purpose of the therapeutic cloning procedure is to obtain hybrid SCs that are genetically compatible with the donor organism, and the purpose of the method of homotopic substitution of the cellular genome in the present invention is the reconstruction (restoration) of damaged own HSCs with genetic biomaterial obtained exclusively from the patient himself. It would also be incorrect to use the terminology of therapeutic cloning in the present invention, calling the result of cell engineering a "hybrid". A hybrid (from Latin hibrida, hybrida - crossbreed) is an organism or cell obtained as a result of crossing genetically different forms, and in the method of the present invention, only homogeneous, autologous genetic material is processed.

Regarding manipulations to block epigenetic memory. We propose to designate the fact of the return of epigenetic memory in cell-engineered HSCs can be called the reversion of epigenetic memory. Reversion (from Latin reversio - return, return) is an ambiguous term and can mean: Reversion (biology) - a tendency to return parts with mixed characteristics to their original parental forms. Therefore, genetically homogeneous HSCs with homotopic genome substitution and a blocked mechanism of epigenetic memory reversal obtained as a result of cell engineering manipulations can be designated as HSCs without epigenetic memory reversal .

Thus, only healthy somatic or multipotent patient SCs, such as NSCs, MSSCs, hepatic SCs, even eye SCs, should be used as a source for obtaining the genome (nucleus) in the present invention. However, the use of SCs and progenitor cells in the restoration of HSCs is most preferable for the synchronization of cell cycles of cells connected by cell engineering. HSCs are usually in 70-80% of cases in the G ₀ phase of the cell cycle, and other SCs are also found in cell culture predominantly in the same phase of the cell cycle. Therefore, their membrane, cytoplasmic and nuclear proteins and metabolic factors are practically balanced in the phase of the cell cycle. Transcription of HSC _stemness factors is better ensured in the G0 phase of the cell cycle, as well as the maintenance of these cells with genome substitution by cultivation in an induction medium that prevents differentiation and favors an increase in their number, followed by the creation of a therapeutic line of healthy HSCs in the patient.

The present invention is based on the equivalent cell-engineering replacement of the damaged genome of autologous HSCs of a patient with a fatal disease with a healthy genome of a somatic or SC of another cell line, but not the hematopoietic line of the same patient's organism, transferred to an XEN-like state, by chemical induction with small molecules and genetically homogeneous HSCs with homotopic substitution of the genome, and the subsequent formation of a cell line of autologous cell-engineered HSCs for the manufacture of BMCPs. The obtained BMCPs can be used to treat a number of diseases in order to completely stop the progression and prevent recurrence of the disease in patients with oncological, neurodegenerative, autoimmune or endocrinological diseases or provide an anti-aging (anti-aging) clinical effect and significantly prolong the active life of a person. Technologically, substitution of the HSC genome (nucleus) is realized by immunoseparation of HSCs on magnetic beads using an immunosorter from the bone marrow or from LC mobilized MNCs of PCs, subsequent substitution of HSC nuclei for the nuclei of somatically healthy cells (stem or progenitor) and further cultivation and growth of the HSC cell mass in environment that prevents differentiation.

The main step of the method according to the present invention is to obtain two autologous cell lines of multipotent SCs, one of which will always be the line of the patient's own HSCs. The source of obtaining the HSC line will be either the patient's bone marrow or the leukoconcentrate of mobilized PB MNCs. Another source of SC can be adipose tissue, the olfactory lining of the nasal mucosa, bone marrow or leukoconcentrate of MNC SC, and so on. Cell lines isolated from the patient are cultured in standard ways.

The next and central step of the proposed method is the chemical induction of a somatic or multipotent regional stem cell according to the protocols of Professor Hongkui Deng (Zhao Y., Zhao T., Guan J., Zhang X., et al., 2015, Xiang Li et al., Zhen Chai, Hongkui Deng, 2017). Strict implementation of these protocols allows a somatic cell (for example, a fibroblast) or a regional SC (neural SC or MSSC) to be reprogrammed into an XEN-like state by gene-oriented chemical induction with cocktails containing various small molecules. This will make it possible to obtain full-fledged XEN-like cells from somatic or stem cells, bypassing the stage of embryonic development, using a step-by-step chemical treatment of cells in an induction medium with small molecules (perturbogens) at a given concentration and exposure. The obtained XEN-like cells will be cultivated in a special medium that prevents the differentiation of these cells and induces the activation of cell nucleus gene expression towards hematopoiesis and immunopoiesis by using a cascade of cytokines that activate the hematopoietic orientation of the resulting cells.

The next step of the method according to the present invention is the transplantation of the nuclei (karyoplasts) of XEN-like cells into HSC cytoplasts obtained from the patient. Thus, we obtain HSC-like autologous cell-engineering systems with a genetically homogeneous XEN-like cell nucleus, that is, from the available two patient cell lines (HSC and multipotent SC or somatic cells), several options are offered for obtaining genetically homogeneous HSC with homotopic substitution of the cell genome (kernels). The first option for obtaining such a therapeutic line of HSCs is carried out by isolating a suspension of enucleated cells (HSC cytoplasts) from the recipient HSC cell line of the patient by incubating them with cytochalasin B, and from the donor line of XEN-like SCs, the nucleus (karyoplast) is also isolated by 15 minutes of treatment with cytochalasin B XEN-like MSSCs, XEN-like NSCs, etc.) and their subsequent fusion by pulsed electrical stimulation (electroporation). Another option also suggests the possibility of isolating a culture of enucleated cells (HSC cytoplasts) from the recipient HSC cell line of the patient, as in the first option, and whole XEN-like multipotent cells are used as a karyoplast (genome) without isolating the nucleus. The subsequent connection of the HSC cytoplast with a donor multipotent cell occurs by fusion of their membranes and cytoplasms using electrical impulse stimulation (electroporation) to obtain reduced HSCs.

The final step of the method according to the present invention is the combination of reduced HSCs with the patient's PC MNC leucoconcentrate and co-cultivation in a medium that prevents differentiation, containing ionomycin, factors stimulating the hematopoietic properties of these cells and containing folic acid, vitamin C and vitamin B12. The presence of a microenvironment in the form of the entire spectrum of MNCs of SCs of cell-engineered HSCs with homotopopic substitution of the genome (nucleus) with the presence of factors blocking epigenetic memory (folic acid, vitamin C, and vitamin B12) at the earliest stages of their cultivation leads to the formation of a hematopoietic transcriptome profile of gene expression already on the 7th-9th day of cultivation in SBU and the appearance of all major HSC markers: CD34+CD45+CD133+HLADR+. Further, the obtained BMCP is cryopreserved with DMSO in liquid nitrogen at a temperature of -196°C.

The main inventive finding and the fundamental difference between the method for obtaining BMCP according to the present invention based on a line of genetically homogeneous HSCs with genome substitution from reproductive and therapeutic cloning technologies is that in any type of cloning, oocytes are always used as a donor cell, and any somatic cell can be a recipient and as a result of their fusion, a zygote is formed from it. Subsequently, the zygote is destroyed and stem cells or so-called patient-specific cells are obtained from it. As a result of the implementation of the present invention, the recipient and donor of cells will be exclusively the patient himself, which has almost never been used either in the experiment, or in the clinic, or in obtaining genetically homogeneous SCs. This removes from the agenda all the moral and ethical issues of cell nuclear transplantation associated with therapeutic and reproductive cloning and fears of creating cell and tissue hybrids and mutants, but it is precisely this that urgently raises questions about assessing the hematopoietic and immune functionality of the obtained autologous HSCs and the need for pre-transplantation confirmation of the fact of ability them to a full-fledged hematopoietic function and the possibility of participation of the patient's cell-engineered HSCs in full-fledged hematopoiesis and immunopoiesis.

The production of autologous healthy (restored) HSCs proposed in the present invention is explained by the unique biology of SCs in one organism, which for some reason was not taken into account earlier, and by the fact that 256 types of different human cells are formed during human embryogenesis and the return of cells to the past stages of embryogenesis that has already occurred. (recapitulation of embryogenesis - repetition of already passed stages of embryogenesis of ancestors) does not exist, that is, bypassing the mechanism of recapitulation of embryogenesis for somatic cells is not normally programmed in human biology in principle. Once, having received a certain differentiation vector during embryogenesis, cell systems will produce only similar cell systems according to the “patterns” of this phenotype and its molecular structure and will never move to another cell lineage, that is, they will not transform into other types of cells. Therefore, in embryogenesis, immediately after the formation of totipotent SCs and their transformation into pluripotent SCs, multipotent SCs appear from them, forming tissue-specific SCs (HSCs, MSSCs, NSCs, etc.), which for the rest of a person’s life will be absolutely autonomous in their linearity, since the paths of formation and ontogenesis of these cells practically do not intersect. So after the birth of a person, each of them is solely responsible for the formation of cells of a certain tissue line. Transdifferentiation or transition of one SC to another SC bypassing the recapitulation of embryogenesis in nature is also not possible.

Under artificial laboratory conditions, it was demonstrated that, under certain special basic conditions (SBU), SC transdifferentiation is possible and real. It has been shown that, under specific culturing conditions, HSCs can be obtained from XEN-like NSCs and XEN-like MSSCs. On experimental animals, which, after high-dose chemotherapy and bone marrow devastation, were transplanted into the blood of cell-engineered HSCs, it was shown that donor cell-engineered HSCs obtained from XEN-like cell systems took root in the bone marrow, became recipient HSCs and formed the entire hematopoiesis and immunopoiesis.

Giving genetically homogeneous HSCs with homotopic substitution of the genome of patients with hematopoietic characteristics is carried out in SBU cultivation, as a result of which the resulting HSCs acquire distinct markers of the HSC cell surface. However, the cultivation of cell-engineered HSCs in SBU cannot yet guarantee their proven hematopoietic stem properties. Therefore, the proposed cell-engineering manipulation, which is a well-known method of nuclear transplantation (transplantation) by removing the nucleus from the patient's pathological HSC and implanting the nucleus (genome) of autologous XEN-like MSSCs or XEN-like NSCs or other SCs into it, and creating healthy cell-engineering HSC does not solve the problem of guaranteeing these cellular systems distinct hematopoietic properties, which after transplantation will need to restore all the oppressed hematopoiesis and immunopoiesis in the human body. Therefore, in order to confer additional stem and hematopoietic cell functions to the resulting cell-modified autologous HSCs. it is necessary to activate and cultivate them in a medium that prevents the differentiation of HSCs and increases (induces) the hematopoietic properties of the obtained autologous HSCs. Such media are known and the technique for increasing the hematopoietic stemness of the HSC culture has already been implemented in many cell laboratories (Bykovskaya S.N., 2015).

At the same time, understanding all the existing risks of transplantation of cell-engineered genetically homogeneous HSCs with homotopic substitution of the genome (nucleus), it is necessary to reduce the risks of obtaining a negative result of using the obtained HSCs in bone marrow transplantation (BMT), and also to be sure that these newly obtained healthy autologous HSCs will be able to restore the entire hematopoiesis and immunopoiesis of the human body after high-dose myeloablative chemotherapy during autologous transplantation of HSCs and BMT. It is necessary not only to evaluate immunophenotypically their stemness by markers of their cell surface and their compliance with existing HSC standards, but also to make biological samples with this biomaterial on experimental animals (immune-deficient mice, rats), on models of hematopoiesis disorders by lethal bone marrow irradiation in these animals and evaluate the in vivo hematopoietic functions of these HSC cell systems. However, even with positive results of biological samples, it is necessary to make a duplicate transplant biomaterial from non-manipulated HSCs and mobilized leuconcentrate of PBM or bone marrow by performing a second collection of HSCs on the 6th day of stimulation with granulocyte colony-stimulating factor (G-CSF) in order to be able to re-transplant the patient if hematopoiesis will not recover 1-2 months after transplantation of cell-engineered autologous HSCs.

The most important scientific and practical issue of the present invention was the question of the molecularly targeted effect of the donor HSC cytoplasm on the nucleus of XEN-like pluripotent SCs and its ability to activate genes responsible for hematopoiesis and immunopoiesis in the patient's body in the transplanted nucleus. The positive results of the experiments showed the possibility of inducing the recipient cytoplasm on the nucleus of a donor cell in an XEN-like state, when it was suggested that there are substances in mammalian cells that can directly or indirectly affect the cell nucleus, change its functional state with the help of a positive or negative regulation of expression of a certain part of genes. However, a complex of objective reasons (genome heterogeneity, often with a significant deficiency of chromosomes, as well as chromosomal rearrangements that occur in the environment of a heterogeneous cytoplasm), inherent in the method of hybridization of somatic cells, did not allow us to identify the substances involved in the expression of the genome, as well as to establish their chemical structure. and mechanism of action. In the case of the present invention, these unfavorable factors are excluded, since the nucleus will be transferred to the autologous cytoplasm in the appropriate phase of the cell cell cycle and will be surrounded by native, i.e., autologous compartments of the cell.

It turned out that in the nuclei previously removed from the differentiated cell and placed in the cytoplasm of the egg, the processes of cell differentiation began, and in many cases a normal organism developed, that is, the nuclei of specialized cells contained the full amount of information necessary for the development of a full-fledged organism. These experiments showed that the cytoplasm regulates nuclear activity in specialized cells and many nuclear functions are inhibited by components of the cytoplasm . HSC cytoplasmic factors will have an activating effect on the transcription of stemness and hematopoietic differentiation factors. It was fully confirmed that the inhibitory effect coming to the nucleus from the cytoplasm of a differentiated cell is eliminated in a suitable chemical composition of the cytoplasmic environment, and the processes that determine cell differentiation can again take place in the nuclei (Gurdan JB, 1977). For a long time, the question was whether the inhibitory effect of the transplanted nucleus of non-hematopoietic multipotent SC would be overcome by HSC cytoplasmic factors. Today we have received an answer and we know that the placement of the nucleus of a healthy (without genomic and postgenomic changes) multipotent SC, transferred to the XEN-like state, into the HSC cytoplasm will have the effect of turning on the HSC genes in the genome of the transplanted nucleus, which was shown by full-transcriptom studies of the expression of the genes of these cells . The most important point of these studies will be the synchronization of the cell cycle between the donor nucleus and the recipient HSC cytoplasm.

At present, the use of cytochalasins, substances synthesized by fungi, has played a significant role in the development of modern cell-engineering methods for transplanting mammalian cell nuclei. The main effect of cytochalasin B is that it sharply increases the resistance of the cell to mechanical damage during enucleation, causing decondensation of the cytoskeleton microfilaments and dramatically increasing the plasticity and adaptability of the surface membrane to various mechanical influences. At the same time, after treatment of the cell with cytocholasin B, a cytoplasmic cord is formed, the final thinning of which leads to the formation, as it were, of two "cells" or, more precisely, two isolated compartments - a cytoplast (devoid of a HSC nucleus) and a karyoplast (the nucleus of a multipotent HSC), i.e. essentially an artificial division takes place. In other words, cytochalasin B, destroying the structure of microfilaments, contributes to the unique arrangement of the nucleus, and it remains connected to the cell as a thin "stalk" in the form of a "mini-umbilical cord" cytoplasmic cord. During centrifugation, this cytoplasmic cord is torn in most of these cells, and enucleated (non-nuclear) cells (cytoplasts) and nuclei separated during centrifugation (karyoplasts or mini-cells) are formed, surrounded by a thin layer of cytoplasm and a plasma membrane. Enucleation efficiency can be monitored by the Giemsa method by staining a cell monolayer located on the surface of one of the cups or discs.

Methods for isolating karyoplasts are somewhat more complex and include a number of operations for centrifugation, density gradient separation, etc. In some cases, tantalum particles with a diameter of 1-3 microns are added to the mixture of cells and karyoplasts. They penetrate the cells and never enter the karyoplast, so heavier cells settle faster than karyoplasts. Cytoplasts contain all types of organelles inherent in a normal cell, retain the ability to attach to the substrate, form a folded membrane, move, and carry out pinocytosis. Karyoplasts are surrounded by a thin layer of cytoplasm (about 10% of the entire cellular cytoplasm), contain a compact endoplasmic reticulum, several mitochondria and ribosomes. In some cell lines, 1/10 karyoplasts are able to restore all the lost volume of the cytoplasm and recover into viable cells.

For cell reconstruction, a suspension of karyoplasts in saline buffer is added to a cytoplast culture monolayer at a ratio of 100 karyoplasts per 1 cytoplast. Previously, the fusion of cytoplasts with karyoplasts was carried out by coating them with inactivated viral particles and incubating them for 45 minutes at 4°C, and then for another 45 minutes at 37°C and washing with Earl's solution to remove unfused karyoplasts. Today, there are more sparing procedures for the fusion of karyoplasts and cytoplasts of different cells by electrical stimulation with electric current in a culture solution.

Thus, using the well-known technology of transplantation of somatic cell nuclei, modified for the purpose of obtaining healthy autologous HSCs of the patient himself by using the method of destroying the structure of microfilaments in HSCs with cytochalasin B, it is possible to achieve enucleation of the nuclei of pathological HSCs isolated from LC PB MNCs by immunosorting with CD34+ monoclonal antibodies, CD45+. Using the technology of cell induction of somatic and multipotent SCs of a non-hematopoietic line, HSCs with a nucleus in a XEN-like state are obtained in the claimed method. As a result of pretreatment of the patient's own HSCs with cytocholasin B, a series of laboratory steps, cell isolation on fecol, and centrifugation, cytoplasts (enucleated cells) are obtained, and karyoplasts (isolated nuclei of these cells) are removed from HSCs and sent for proteomic mapping (if necessary). Autologous HSC cytoplasts will be used further in the current biotechnological cell engineering process. Similar actions are carried out with a donor culture of healthy XEN-like SCs (NSCs, MSSCs, etc.) and they are separated into cytoplasts and karyoplasts, but autologous karyoplasts of healthy cells are isolated from them in the technological process and left. With further joining of HSC cytoplasts and XEN-like stem karyoplasts of tissue-specific SCs, their fusion into new nucleated autologous HSCs is achieved by culturing in SBU. SBU can be created by their joint incubation, as well as using the known technology of electrical stimulation with electric current or by using non-invasive optical-laser techniques for nuclear transplantation in mammals (Sviridova-Chailokhyan T.A., Kantor G.M., 2010). Upon completion of the process of fusion of HSC karyoplasts with SC cytoplasts, a preparation of healthy cell-engineered HSCs is obtained and purified by removing unconnected karyoplasts, cytoplasts and fragments from destroyed cells using staged centrifugation. The resulting HSCs are tested for viability with trypan blue and transferred to a culture laboratory for further cultivation and obtaining a cell line in a medium that prevents HSC differentiation, and increasing their cell mass to the required amount (1-2% of the total number of MNC SC cells). Their qualitative and quantitative characteristics are assessed by the cytofluorimetric method on a flow cytometer after 4-5 days of cultivation and, if they comply with the standard, they are frozen in liquid nitrogen with DMSO.

When carrying out TCM after high-dose standard chemotherapy in the myeloablative mode, we perform defrosting of the patient's PC MNC concentrate and the culture of healthy genetically homogeneous HSCs with homotopic substitution of the genome, wash them from the cryoprotector and DMSO by double centrifugation with 0.9% sodium chloride solution, mix them in a bag for transfusion and carry out intravenous administration in TCM in the standard mode or use it for intrathecal administration. In other cases, when the maturation of cell-engineered HSCs occurred in the MNC SC concentrate, then for use we defrost the finished BMCP and use it for bone marrow transplantation or for intrathecal injections.

Technically, the manufacture of BMCP according to the present invention in the form of a cell-modified HSC with homotopic substitution of the genome consists of creating and obtaining its main cellular components for BMCP, their joint cultivation and cryopreservation, and subsequent thawing and mixing (if necessary) directlyex tempore before clinical use or use for HSC transplantation. Obtaining and using BMCP for autologous BMT in the treatment of a number of fatal and so far incurable human diseases begins with diagnosing the presence of HSC proteomic pathology in a patient. For the earliest diagnosis of HSC pathology, the method of early molecular biological diagnosis of malignant neoplasms and amyotrophic lateral sclerosis, disclosed in the RF application for invention No.

Depending on the localization of the pathological process, it is necessary to determine which cells will be used for the manufacture of cell-modified HSCs with homotopic genome substitution. In the case of pathology of the central nervous system, the patient's autologous line of MSSCs from adipose tissue is used. In the case of diseases of solid organs of the connective tissue or other diseases, when the leading role in the restoration of damage or tissue proliferation belongs to MSSCs, then NSCs obtained from the olfactory lining of the nose or the nucleus of hepatic SCs, SCs of the eye, or other multipotent SCs will act as a nuclear donor.

The production method of the BMCP of the present invention is described in more detail below.

Stage 1. Obtaining MNC leucoconcentrate from non-manipulated bone marrow, mobilized peripheral blood

Obtaining MNC leucoconcentrate of non-manipulated bone marrow

Requirements for bone marrow donors. Donors should be pre-screened for HIV 1 and HIV 2 infections, immunodeficiency syndrome; syphilis, hepatitis B and C; acute infectious diseases.

Getting bone marrow. The sampling of material for subsequent cell isolation is carried out by a surgeon who has practical experience in this procedure. Bone marrow exfusion is performed in the operating room by puncture of the distal iliac crest under general or local anesthesia. The puncture is carried out with a sternal needle; bone marrow is taken with a Luer type syringe, the bone marrow suspension is transferred into a bag with isotonic anticoagulant.

Mobilization of GSK in PC

In order to increase the amount of SC in the PC, the patient receives 8 injections of G-CSF subcutaneously with an interval of 10-12 hours for 4 days. In the first three days, the dose of the drug is 2.5 mcg / kg, on the last day the dose is doubled. A general blood test is performed daily and on the 4th-5th day an ultrasound of the abdominal cavity and fibrogastroscopy are performed.

Collection of GSK and GKP

Collection No. 1 is carried out on the 5th day from the start of G-CSF stimulation, and collection No. 2 on the 6th day from the start of stimulation on the COBE-Spectra blood separator using a disposable separation system and standard solutions. The duration of the procedure is 3-4 hours depending on the speed of the procedure, the weight of the donor and the parameters of the blood test. The procedure is carried out by taking blood from one vein, processing it inside the separator, taking a certain amount of SC and returning the rest of the blood components to the donor through another vein. Venous access is carried out by puncture of two peripheral veins or through a double-lumen central catheter installed in the subclavian vein for the duration of the session. The average volume of collected material is 300-400 ml. The collected material is evaluated by two parameters: by the total number of nuclear cells in the compartment and by the number of CD34+ cells per kilogram of the patient's weight. Nuclear cells in the separate are determined by counting in the Goryaev chamber before any manipulations. The percentage of CD34+ cells in the cell suspension obtained during cytopheresis was determined by flow cytometry. All the material obtained is collected in cryobag No. 1. All material collected on the 6th day of stimulation of the patient is collected in a cryobag No. 2 in a similar way. The further procedure for manipulating non-biomaterial in bags does not differ from each other.

Definition of peripheral SCs and GPCs

The determination of the subpopulation composition of CD34+ cells is carried out cytofluorimetrically using the triple label method (simultaneous staining of cells with antibodies to three different antigens loaded with different fluorochrome dyes).

Preparation standardization

The number of progenitor cells is determined cytofluorimetrically in a direct immunofluorescence reaction (RIF).

A double labeling method is carried out with simultaneous staining of the cell substrate with monoclonal antibodies to the CD34+ antigen as the main marker of the cells of the pool of autologous multipotent hematopoietic SCs and to the CD45- leukocyte antigen molecule that determines HSC. This technique allows you to immediately calculate the ratio of the number of CD34+ cells and all HSCs (CD45+) in the material. To assess the levels of non-specific binding, a portion of the cells are stained with isotype controls. As isotype controls, murine isotype IgG1 immunoglobulins labeled with dyes similar to those used for monoclonal antibodies (PE, FITC, PerCP) are used as standard.

Preparation of cell samples. Before setting up the reaction, PC cells and the cytopheresis product are freed from erythrocyte impurities by a standard lysis procedure and subsequent washing in PBS-BSA by centrifugation at 1000g for 5 minutes. PBS-BSA can be replaced with Hank's medium or 199.

With isolated cells, direct RIF is carried out in a 96-well plate, while a panel of 3 wells is used to determine the number of cells in any material: unstained cells; cells stained with isotype controls with a label corresponding to the label of the used monoclonal antibodies (MCA); cells stained simultaneously with MCA for CD34 and CD45 antigens.

It should be noted that mAbs to CD34+ are preferably used with phycoerythrin (PE) or peridinine chlorophyll (PerCP) tags. These fluorochromes have a higher level of specific signal compared to fluorescein isothiocyanate (FITC).

To determine the number of CD34+ cells, antibodies to the CD34 antigen of the HPCA-2 clone (8G12), IgG1 isotype, are optimal.

Thus, the standard panel looks like

1. IgG1 PE control + IgG1FITC control ;

2. control IgG1 PE + MCA to CD45 FITC ;

3. MCA to CD34 PE ( HPCA-2) + MCA to CD45 FITC .

RIF staging

1. Cells are added to the wells in an amount of at least 500,000 cells per well.

2. Next, an antibody cocktail is added to each well in accordance with the panel and gently resuspended with a pipette. Each MCA is taken in the amount of 10 μl per well, the total volume of MCA in the well is 20 μl.

3. Cells are incubated with antibodies for 30 minutes at 4°C.

4. At the end of the incubation time, the cells are washed twice from unbound antibodies by centrifugation for 5-7 minutes at 1000g.

5. Cells are transferred to special plastic tubes for counting on a flow cytometer.

6. The volume of the cell suspension in each tube is adjusted to 200-500 µl by adding PBS-BSA to the cells.

The account should be carried out immediately after setting the reaction.

Counting and recording on a flow cytometer

Evaluation of the reaction on a 5-parameter flow cytometer. This scheme is applicable to the flow cytometer of any configuration. CD34+ cells in PC represent a small cell population. Even under conditions of preliminary stimulation of hematopoiesis, the maximum percentage of these cells is 1.0-3.0%. Therefore, when counting these cells in each analyzed sample, at least 20,000 cell events should be accumulated.

Cell collection and analysis is performed in a CD45+ cell gate that includes all HSCs. In this context, a gate implies an event accumulation area limited by certain parameters. In this case SSC/ FL-1 (CD45 FITC). That is, along the abscissa (FL-1) on the dot cytogram, all CD45+ events will be visible, and along the ordinate (SSC parameter - side light scattering), cell events will be located in accordance with their granularity (cytometric, not morphological term). The absolute number of CD34+ cells in 1 µl of blood and cytoconcentrate was calculated based on the number of leukocytes in the hemogram on the day of the study.

The principle of determining CD34+ cells in hematopoietic tissue. Sample recording.

1. Selecting the area of analysis - the gate.

The Acquisition cell sample recording menu is open in the instrument. At the first stage, in the Setup mode (cell sample viewing mode without recording), samples No. 1 (IgG1PE+ IgG1 FITC) and No. 2 (IgG1 PE +CD45 FITC) are viewed and CD45+ events are detected. These events are located to the right of the values of 10 ¹ (the average threshold value of the specific signal level for fluorescein FITC) along the abscissa - FL-1 (CD45+cells) and are quite clearly visualized compared to control sample No. 1.

For sample No. 1, the entire area located to the right of the main cell density is limited - the gate, which includes only CD45+ events in sample No. 2 and contains the minimum number of cellular events in sample No. 1.

2. The instrument is switched to sample recording mode (normal), and sample #1 is collected and recorded for 20,000 cell events without a gate (whole cell population). Recording this sample in the gate does not make sense, since it does not contain mAbs to the CD45 antigen. As shown above, this probe is necessary to correctly select a gate containing only CD45+ events.

3. Samples #2 and #3 are collected and recorded in a CD45+ gate. This gate is identical (that is, the gate parameters do not change during the collection process) for both the 2nd and 3rd samples. The minimum number of cell events is 20,000 for each sample.

Analysis of recorded samples

1. Go to the Analysis menu of recorded samples. We open the cytogram (dot-plot) of sample No. 2 in the SSC/FL-2 parameters, in the gate R1 - CD45+ (the gate was selected by us earlier at the time of recording the collection and was automatically transferred to the analysis mode). In this case, the ordinate axis will reflect the SSC parameter - the granularity of events that fell into the analyzed gate, and the abscissa axis FL-2 - a detector that reflects the levels of nonspecific binding for the phycoerythrin dye, detected by antibodies to mouse IgG1PE immunoglobulins.

2. According to this cytogram, a control marker is set in such a way that there are practically no cellular events in the lower right quadrant. On average, for the vertical bar of the marker, the values will be about 130 (to the right of 10 ² ), and for the horizontal bar of the marker, the average values will be 60, which corresponds to the concept of SSC-low. That is, cells with a minimum number of cellular inclusions. Thus, the levels of non-specific binding will be equal to or close to zero.

3. Automatically go to sample number 3. All instrument readings taken for the second sample are saved. That is, the cytogram is opened in the SSC/FL-2 parameters, the gate selected earlier when collecting for CD45+ cell events is saved, and the values of the control marker set for sample No. 2 are saved. as in sample 2, but specific CD34+ events. The percentage of CD34+ cells is displayed automatically by the device.

Isolation of hematopoietic CD34+ CD133+ Lin- cells from LC MNCs of SC

This stage of cultivating HSCs in the proposed method is performed in case of detecting a small (less than 0.5-1%) amount of HSCs in LC MNCs of PC and the need to increase their number and activate HSCs. If the number of HSCs in LC MNCs of PC is more than 1.0-1.5% of the total number of lymphocytes, then you can immediately proceed to the next step of the proposed method - immunoseparation (see paragraph B of this section).

A. Cultivation under conditions of limited differentiation

as a way to increase the amount of HSC from LC MNC PC in a cryobag.

The purpose of this biotechnological stage is to increase the number of the patient's own HSCs for further cell engineering manipulations. The main problem of cultivating highly plastic undifferentiated HSCs with CD34+, CD133+Lin- markers is their rapid release into differentiation, since in order to solve the problems described above, it is necessary to increase the number of these cells without developing colonies of differentiated cells. At the same time, the ability for subsequent differentiation in such cultured cells should be preserved, if possible, in its original form. The objective of this stage is both to increase the number of undifferentiated HSCs of the patient ex vivo, and to completely free from erythrocytes and granulocytes and slightly increase them by culturing them in a growth medium containing a mixture of cytokines, consisting of SC growth factor (SCF), a ligand of tyrosine kinase 3 from fetal liver (Flt3L) and thrombopoietin (TPO), the difference of which is that the above mixture of cytokines additionally contains ionomycin in the following ratio of components per 1 ml of growth medium: SCF - (50-150) 10 ³ ng, TPO - (50 -150)⋅10 ³ ng, Flt3L - (10-50)⋅10 ³ ng, ionomycin - (10-100)⋅10 ³ ng.

The entire LC is divided into test tubes of 50 ml and each tube is treated individually. Ionomycin has been used as a growth stimulator for undifferentiated SC cells ex vivo. SC growth factor (SCF) stimulates SC proliferation by interacting with a specific receptor (protooncogenic c-kit) with tyrosine kinase activity (Witte ON, 1990), inducing kinase activation and transphosphorylation of receptor chains. Phosphotyrosine residues of the receptor function as docking sites for proteins that bind c-kit to signaling pathways, which induces proliferation activation (Aronica SM et al., 1996). Flt3 ligand (Flt3L) stimulates HSC proliferation to no lesser extent than SCF. Flt3 is a class III tyrosine kinase receptor expressed in early HPCs. The interaction of Flt3 with the ligand (Flt3L) activates signaling pathways and subsequent SC proliferation (Matthews Wea, 1991). Thrombopoietin (TRO) is a glycoprotein hormone that regulates the production of platelets by bone marrow stem cells. TPO was first characterized as a ligand that binds to the surface receptor c-MP1. TPO belongs to class I hematopoietic cytokines. In suspension culture, TPO in combination with SCF stimulates the proliferation of multipotent SCs and increases their number (Ku et al., 1996). Ionomycin increases the concentration of intracellular Ca ²⁺ , starting with the mobilization of intracellular stores, which are then maintained by the influx of extracellular Ca ²⁺ , induces the hydrolysis of phosphoinositides and stimulates the activation of protein kinase C, stimulating the entry of the cation by regulated calcium stores, but not by direct action on the plasma membrane. (A. J. Morgan and R. Jacob, 1994, Biochem. J. 1994, 300 (665-672)). Thus, ionomycin is an agent of primary cell activation (spherilization of cells), see, for example, patent RU 2108579. Ionomycin has been used as a growth agent for differentiated cells, such as hybridomas and dendritic cells. The required level of HSC stemness is ensured by the fact that from an equivalent list of known cytokines, such a mixture of them was identified that, in certain selected concentrations, was able, together with a new proliferation stimulating factor for SC - ionomycin, to induce only cell proliferation and thus did not require the use of additional agents for inhibition of cell differentiation. In addition, an unexpected result for this technical solution was the achievement of very high cell proliferation rates, which made it possible to reduce the cell culture time to 7-9 days and thus avoid the problems of long-term cell culture. Moreover, cultivation in this medium made it possible not to change the basic percentages of fundamental cellular elements in LC MNCs and, at the same time, to increase the amount of HSCs to 1.5-2%.

Thus, summarizing all of the above, in this method, HSCs are not isolated from autologous bone marrow or autologous concentrate of mobilized PK, as was customary in a number of studies, but are cultivated directly in the LC MNC PK. The resulting suspension is cultured in Dulbecco M medium (IMDM) supplemented with insulin and transferrin, as well as growth factor CK (SCF), tyrosine kinase 3 ligand from fetal liver (Flt3L), thrombopoietin (TPO), and ionomycin (Ca ²⁺ ionophore). After 3-7 days of cultivation, the grown cell suspension contains about 1.5-2% of CD34+ cells. The technique allows for a short period of time to clear LC from blood impurities (erythrocytes) and granulocytes, slightly increase the increased number of poorly differentiated HSCs, which can be used for therapeutic purposes as a component of BMCP according to the present invention.

Carry out the following procedures:

1. Transportation of bags with bone marrow cells, mobilized blood or LC MNC PC is carried out in ThermoCont thermal containers at room temperature (18-25°C).

2. The collection of cells from the mobilized blood bag is carried out in a class II laminar cabinet located in the sterile block;

2.1. The cell suspension is taken with a syringe, piercing the membrane of the connecting tube on the bag and divided into 50 ml test tubes.

3. Obtaining the MNC fraction: cell suspension taken with a syringe is diluted in a ratio of 1:4 with phosphate buffer solution (PBS) without calcium and magnesium ions (PBS Ca ²⁺ Mg ²⁺ free) and then layered on a gradient solution (d=1.077 g /ml, MP Biomedicals) in 50 ml tubes.

3.1. The tubes are centrifuged at 400g for 30 min at 20°C;

3.2. As a result of gradient centrifugation, erythrocytes and granulocytes sink to the bottom of the tube, and the mononuclear fraction in the form of a dense ring is concentrated between two layers on the surface of the gradient;

3.3. the MNC fraction is taken with a pipette, diluted in fresh PBS and washed three times by centrifugation for 10 min at 300 g and a temperature of 10°C.

4. Cell count and phenotypic analysis of the mononuclear fraction:

4.1. To determine the number of live MNCs, 20 µl of trypan blue (Gibco) was added to 20 µl of the cell suspension. Cells are counted in a hemocytometer, the proportion of living cells should not be lower than 95%.

4.2. To assess the number of CD34+ and CD133+ cells of the mononuclear fraction, 0.1 ml of the cell suspension is stained with the corresponding antibodies and the proportion of HSCs in the initial cell suspension is determined on a FACSCalibur instrument (Beckton Dickinson).

Example 1 Phenotypic analysis data of MNCs in one indicative experiment conducted on LC cells of donor H. Proportions of cells in LC MNCs of SC: HSC (CD34+,CD45+) of the donor was 0.5%, the number of neural cells was 5%, T-lymphocytes 84%, B -cells -1.2%.

Cultivation . The liquid culture system consists of Iscove's modified Dulbecco's Medium IMDM (Gibco, UK) supplemented with 0.5 g/l bovine serum albumin (Sigma Aldrich), 0.39 µg/ml human insulin and 60 µg/ml transferrin (Gibco). SC growth factor (SCF), another name c-Kit-ligand (c-KitL, R&D Systems), is added to the culture medium at a concentration of 100 ng/ml, as well as Flt3-ligand (Flt3L) at a concentration of 20 ng/ml, ( R&D Systems), and ionomycin (Sigma Aldrich) at a concentration of 0.1 μM/ml.

Cultivation method. Cells are cultured at 37°C in a humid atmosphere with an influx of 5% CO₂. Fresh medium and cytokines are added every third day. The cultivation time is 3-7 days. The growth factors used promote SC proliferation without the need to add agents that suppress differentiation. The obtained suspensions of SC cells of any individual can be stored frozen or can be introduced into BMCP immediately after cultivation and applied to patients.

Table 1

Evaluation of the Purity of the Cell Suspension after Cultivation

Marker Percentage of antigen-positive lymphocytes Hematopoietic SCs CD34+CD45+ 2.2 CD34+CD45- 1.1 T cell antigens CD3+CD56-D16- 81.2 CD3+CD56+CD16+ 2.06 CD3+CD4+ 50.2 CD3+CD8+ 31.4 Cd3+ γδ+ 1.2 NK cell antigens CD94+CD16+CD56-CD3- 10.63 CD16+CD56+ 0.12 CD16+CD56low 0.0 CD16-CD56+CD94-CD3- 3.3 Subpopulations of NK cells (within CD56 ^- CD16 ⁺ ) CD94- 100.0%

Conclusion. The material is preserved. Based on flow cytometry data, lymphocytes that can be analyzed are 84.7%. Among them, mature T cells account for 81.2% with a predominance of CD4+ subpopulations. γδ-T cells amounted to 1.2%. Heterogeneous NK cells are represented by populations of CD94+, CD16+, CD56- and CD94-, CD56+, CD16- and CD16+, CD56+, totaling 14.0% of lymphocytes. No CD16+, CD56low populations were found.

The efficiency of obtaining the proposed BMCP component, which makes it possible to cultivate SCs with cytokines that promote proliferation, but not differentiation of SCs, opens up prospects for the use of a patient's own SCs for the treatment of such diseases as coronary heart disease, burns, long-term non-healing wounds and injuries of the extremities, spinal cord and brain. Cell-engineered autologous healthy HSCs can be isolated and stored frozen for immediate use. At the same time, the ability for subsequent differentiation in such cultured cells is retained at the level of initially isolated SCs.

LC MNCs of the patient's PC, enriched with HSC, are collected in a bag from test tubes under sterile conditions in a laminar flow hood and transferred to the stage of immunoseparation.

B. Immunoseparation as a method for isolating HSCs from LC PB MNCs

This stage is carried out either in a sterile licensed operating room or in a culture laboratory using a certified and approved for clinical use in Russia CliniMACS® plus immunoseparator (Germany) using CliniMACS TCR 34+/133+ Kit antibodies and specialized consumables for this device in accordance with the manufacturer's instructions. To do this, the resulting LC is placed in a 400 ml disposable package for cryopreservation, standard antibodies to CD34+ CD133+ markers on magnetic beads are added to it, connected to the apparatus, and immunoseparation is carried out in the usual mode. HSCs are collected in an immunoseparator and transferred to a culture laboratory for further manipulations, and CD34+ CD133+ leukoconcentrate depleted from HSCs is transferred to a bone marrow bank for cryopreservation in a 400 ml cryo-resistant bag with DMSO.

C. Cryopreservation of cellular material

A sterile DMSO solution at a final concentration of 10% is added to the cooled cell suspension in autologous plasma. The suspension is packaged in cryopackages or the required number of cryotubes, labeled, packaged and frozen. After freezing, the package or cryovials with PBMNCs are transferred to a Dewar vessel for storage in nitrogen vapor.

D. Cryopreservation of LA MNC without HSC

The traditional method of cryopreservation is to add DMSO to the cell suspension at a final concentration of 10% and standard freezing at 1 degree per minute to -80°C or -120°C using standard electronic software freezing programs and storage in liquid nitrogen or liquid nitrogen vapor.

E. Isolation of the HSC cell fraction

The separated HSCs are concentrated by centrifugation at 2000 rpm for 10 min at +18°C. With the help of a manual plasma extractor, plasma is removed from the container as much as possible, the cells remain in a volume of 40-60 ml.

E. Addition of cryoprotectant

Highly purified dimethyl sulfoxide (DMSO) is used as a cryoprotectant for hematopoietic cells. An equal volume of polyglucin with DMSO is added to the obtained cells with constant stirring. Mixing of DMSO with polyglucin occurs as an exothermic reaction with the release of a moderate amount of heat. The concentration of DMSO in polyglucin is 10-12%. Thus, its final concentration in the frozen material will be 5-6%.

The use of polyglucin made it possible to halve the amount of DMSO, to 5-6% of the final concentration, since polyglucin has the following properties: firstly, the ability to disaggregate cells and thereby improve the penetration of cryophylactic into cells, and secondly, polyglucin (6% dextran) itself is a cryophylactic.

J . Counting the number of cryopreserved cells

At the next stage, it is necessary to count the nucleated cells, as well as the CD 34+ cells remaining in the LK MNC to be frozen.

Selection of the optimal volume of material to be frozen

In cryopreservation, to create the best conditions, the ratio of the volume of the plastic container and the volume of the material frozen in it, as well as the concentration of frozen cells, are important. As it was found, the optimal concentration of cells to be frozen is (40-100)⋅10 ⁶ cells/ml.

Freezing LC of mobilized MNCs

Depending on the final volume of the frozen material, the number of bags or polymer ampoules (20-25) for deep program freezing is selected. Next, the cell suspension is transferred into these heat-resistant plastic tubes.

To freeze ampoules with cell suspension, they are placed in an electronic software freezer. The rate of cooling of the biomaterial at a temperature from 0°C to -40°C is 1.1 degrees per minute, and then 1 degree per minute to -80°C or -120°C. After the completion of the program electronic freezing cycle, the tubes with frozen material are transferred to the storage for long-term storage. The test tubes with the material to be frozen are then placed in liquid nitrogen vapor at -165° to -170° C. where they will remain in liquid nitrogen or its vapor until use.

The proposed method takes into account and implements the basic requirements for a biomaterial subjected to cryopreservation, namely, the maximum preservation of SC viability; the minimum volume (100-120 ml) of the frozen material with the maximum number of cells contained (up to 100⋅10 ⁶ /ml), for PC the minimum volume is 50-60 ml; minimum volume of autograft. The advantages of cryopreservation in liquid nitrogen vapor are the good safety of storage of bags and tubes with cellular biomaterial, as well as a significantly lower nitrogen consumption.

Stage 2. Obtaining a line (lines) of autologous regional SCs

for cell engineering

The MSSC line is made from 50 ml of bone marrow exfusate or LC MNC PC, their cryobag No. 1, obtained during the first collection of HSC.

A. Method for isolating and cultivating the MSSC line

MSSCs are isolated and isolated from various sources according to well-known international protocols: from bone marrow (P.Penfornis, R. Pochampally et al., 2011), from cord blood (Can A. and Balci D., 2011) or from adipose tissue (Zachar V., Rasmussen J. G., and Fink T., 2011) of the patient himself. For the isolation of MSSCs from adipose tissue, a domestic modification was used (Teplyashin A.A., 2012), and for the isolation and isolation of MSSCs from the bone marrow, a modification by Professor A.G. Konoplyannikov was used. (2014).

Bone marrow sampling material was used to obtain MSSCs. 50 ml of bone marrow was exfused from the iliac crest with special biopsy needles.

Example 2 Isolation of MSSCs from bone marrow.

After settling the bone marrow sample for 1-2 hours at room temperature, the isolated upper fraction of cells is taken with a Pasteur pipette and resuspended in the growth medium. The growth medium is RPMI-1640 containing penicillin-streptomycin (100 U/ml), amphotericin (100 ng/ml), L-glutamine 2 mM, 20% fetal calf serum. Cultivation is carried out in plastic bottles with a bottom area of 25 cm², into which (2-10)⋅10⁵ cells/cm² in 8 ml of growth medium. Vials are placed in CO₂-incubator (5% CO₂). After 48 hours, the medium with non-attached cells is removed and replaced with fresh. Upon reaching a confluent monolayer (90-100%), the cells are subcultured using a 0.25% trypsin-EDTA solution into new vials, initially with a bottom area of 75 cm², and subsequently, with an increase in cell mass, into large culture flasks with a bottom area of 175 cm². This method allows, by the end of 5-6 weeks, to obtain a population of MSSCs in the amount of (1-2) ⋅ 10^eight enough cells for transplantation into recipients. The study of specific markers of the obtained MSSC cultures by flow cytometry shows that they belong to the CD10 population.^lowCD34^- CD45^- CD44⁺ CD90⁺ CD105⁺ cells, which is typical for cells derived from multipotent mesenchymal SCs of an adult organism.

The resulting cellular biomaterial is washed from the culture medium with 0.9% physiological NaCl solution by centrifuging the cells twice at 2000g, and the material can be used immediately for cell engineering. It is washed from the culture medium with saline and transferred to 200 ml of saline with 10 units/ml of heparin. If the material is planned for intrathecal administration, then it should be packaged into aliquots of 1⋅10 ⁶ MSSCs in 2 ml cryotubes and frozen in a program freezer. If the material is to be used for intravenous or intra-arterial administration, then the entire volume (1-2)⋅10 ⁸ MSSCs is cryopreserved in 5-10 ml cryotubes or special bags for bone marrow storage.

Cryopreservation of cell material

A sterile DMSO solution at a final concentration of 10% is added to the cooled cell suspension in autologous plasma. The suspension is packaged in the required number of cryotubes, marked, packaged and frozen. After freezing, the cryovials are transferred to a Dewar vessel for storage in nitrogen vapor.

Use of cellular material for transplantation

1. The material intended for transplantation is washed from the culture medium with saline and transferred to 200 ml of saline with 10 units/ml of heparin.

2. Transportation of cell material to the cell engineering laboratory is carried out on ice in a thermally insulated container.

3. The cell engineering material is transferred to the culture laboratory and the cell engineering laboratory.

B. Isolation of MSSCs from adipose tissue

Example 3 Adipose tissue was taken by biopsy in a volume of 10 g. The obtained 10 g were washed three times with PBS (Gibco) at pH 7.2, without Ca ions.²⁺ and Mg²⁺containing a single solution of antibiotics and antimycotics (Gibco), the final concentration of penicillin is 100 units / ml, streptomycin 100 μg / ml, amphotericin B 0.25 μg / ml, and impurities of dense connective tissue are removed. Next, mechanical fragmentation of the tissue is carried out with medical scissors in culture dishes with a diameter of 10 cm (Costar) until a finely dispersed mass is obtained, which is transferred into two 50 ml tubes with a conical bottom (Costar) and each sample is suspended in 25 ml of DMEM medium containing antibiotics and an antimycotic. Next, an enzyme treatment step is carried out: 1 ml of a 2% solution of type I collagenase (Gibco) in a PBS buffer solution without Ca ions is injected into the resulting suspensions.²⁺ and Mg²⁺ to a final enzyme concentration of 0.075%, incubated at 37°C for 30 minutes with slow shaking. The resulting mixture is thoroughly mixed, then an equivalent volume of DMEM containing 10% FBS and antibiotics and antimycotic is added and centrifuged for 10 minutes at 1000g. The supernatant and fat droplets are removed, the precipitates are combined and suspended in 10 ml of cold lysis buffer (+4°C) containing 155 mM NH4Cl, 10 mM KHCO3, 0.1 mM Na2EDTA. The mixture is thoroughly mixed and incubated for 3-5 minutes at room temperature. Next, 10 ml of DMEM containing antibiotics and an antimycotic are added to the cell suspension. The cells are pelleted by centrifugation for 10 minutes at 1000g. The supernatant is removed and a washing step is carried out. The cell pellet is resuspended in 30 ml of DMEM medium containing antibiotics and antimycotics, and centrifuged at 1000g for 10 minutes. The pellet obtained is resuspended in 25 ml of DMEM medium containing antibiotics and an antimycotic. The resulting cell suspension is passed through a filter with a pore size of 100 μm and centrifuged for 10 minutes at 300g. The pellet is resuspended in DMEM supplemented with antibiotics and antimycotics, 10% FBS, and a 25 ml volume of non-essential amino acids (Non-Essential Amino Acids, Gibco) once. The suspension is passed through a filter with a pore size of 10 μm. As a result, a homogeneous fraction of cells is obtained, free from cellular debris and blood cells.

The resulting cell suspension is evaluated by counting in a Goryaev chamber and seeded into vials with an area of 75 cm ² at the rate of 106 cells/cm ² . The proportion of attached cells is approximately 1-1.5%. Thus, the number of MSSCs isolated from 1 g of adipose tissue is about (1.5-3)⋅10 ⁴ cells. After 24 hours, the medium is changed to DMEM containing antibiotics (100 U/ml penicillin, 100 μg/ml streptomycin, Gibco), 10% FBS and a single solution of non-essential amino acids (Non - Essential Amino Acids, Gibco). Upon reaching the monolayer, the cells are subcultivated, a visual assessment of the cell morphology is carried out by means of phase-contrast microscopy, the mitotic index and the time of cytogeneration are assessed. According to the results of morphological analysis, two subpopulations were identified in the cell fraction obtained after isolation. The first type of cells is a subpopulation of small fusiform cells with a diameter of 10-15 μm with a clearly defined nucleus and homogeneous cytoplasm. The second type is represented by cells of a round shape with a flat outgrowth of the cytoplasm elongated on one side, the size of the cells reaches 40 microns; there is a dark nucleus, shifted to one edge, heterogeneous cytoplasm and increased granularity in the nuclear region. In the phase of logarithmic cell growth, the mitotic index and cytogeneration time are calculated. The ratio of the number of mitotic cells to the total number of cells is 34%. Doubling time 54-62 hours. The resulting cells are immunophenotyped by indirect immunofluorescence. Using a flow cytometer (Beckman Coulter), a high level of expression of the following antigens CD10, (CALLA), CD13 (APN), CD44 (hyaluronic acid receptor), CD90 (Thy-1), CD105 (endoglin) (Becton Dickinson) was detected. The absence of expression of HSC markers CD34, CD45 and CD117 was also shown (Becton Dickinson). The results of immunophenotyping show that the population of obtained cells corresponds to MSSCs in terms of the expression of surface antigens.

B. Method for isolating and culturing NSC CD133+

from the olfactory lining of the human nose

Requirements for donors of the olfactory lining of the nose. Donors, that is, the patients themselves, must be pre-screened for HIV 1 and HIV 2 infections, immunodeficiency syndrome; syphilis, hepatitis B and C; acute infectious diseases. The technique and protocol for obtaining NSC is described in the works of Zhang X. et al., (2004-2006). Olfactory nasal lining tissue is obtained from patients and processed according to the standard culture protocol of CT Marshal et al., (2005-2006) . In domestic practice, a modification of the method proposed by prof. I.V. Viktorov (2012).

Example 4 To obtain healthy NSCs, the tissue of the olfactory lining of the nose is taken by endoscopic resection of a piece of mucosa (10 mm x 10 mm) of the upper choanas of the donor's nose. The resulting tissue is delivered to the laboratory in a chilled Ca-free Hank's solution.²⁺ and Mg²⁺ (HBSS) containing antibiotics and antimycotics (1:100; Gibco). The delivery time does not exceed 2 hours. After repeated washing in the same solution, blood vessels are removed from the tumor tissue, after which the tissue is crushed and incubated for 40 minutes at 36.5°C in a trypsin/EDTA 025% solution prepared at 0.01 M phosphate buffer (PBS, pH 7.4). The action of the enzymes is blocked with DMEM (Gibco) containing 3% serum, the tissue is washed in three changes of Hanks' balanced salt solution (HBSS, Hanks' balanced salt solution, Sigma) and dissociated by repeated pipetting in the nutrient medium. Medium composition: Minimum Eagle medium (MEM, Sigma) 90%, fetal calf serum (Fetal bovine serum, FBS, Gibco, Ivitrogen), glucose 0.8%, glutamine 2mM (Gibco), B27 supplements (Sigma), HEPES 20mM , growth factors (only for primary cultures) - fibroblast growth factor (FGF2, 1ng/ml, Sigma), neurogrowth factor (NGF, 2 ng/ml, Sigma).

The resulting cell suspension is centrifuged (3 min at 1200 rpm), the pellet is resuspended in a nutrient medium of the same composition. The number and viability of dissociated cells are determined in a Goryaev chamber after staining with 0.1% trypan blue solution. For subsequent cultivation, cell suspensions with only 85-95% of viable cells are used. Dissociated cells (5×10 ⁵ cells/ml) were cultured in 12-well plates on polylysine-laminin substrate for 14 days (36.5°C, 5% CO ₂ ). A partial change of 1/3 of the nutrient medium is performed twice a week. The primary culture after the formation of a confluent monolayer is removed using a solution of trypsin/EDTA. After washing in HBSS and centrifugation, the cells are resuspended in nutrient medium. The cell suspension (10,000-12,000 cells/cm ² ) is transferred into 12-well plates or flasks (area 25 cm ² ). Cultures are passaged in this manner 4 times until a confluent monolayer is formed. Formed in the cell monolayer attached to the substrate and free-floating neurospheres are selected using Pasteur pipettes and dissociated by the enzyme treatment method described above. Isolation of the neurospheres makes it possible to separate them from the parietal glial cells, fibroblasts, and stromal (supporting) cells attached to the substrate. The cell suspension of neurosphere cells after washing and centrifugation is resuspended in a nutrient medium and cultured in 12-well plates (10000-12000 cells/cm ² ) and on cover slips (18x18 mm) in Petri dishes until a confluent monolayer is formed. The resulting cultures are used for cytological and immunocytochemical studies. Some of the last passage cells are frozen in cryopreservation medium (90% serum, 10% dimethyl sulfoxide) and stored in liquid nitrogen.

The cell monolayer is fixed in a 4% paraformaldehyde solution prepared in 0.01 M phosphate buffer (pH 7.4) for 30 min. After washing in PBS (30 min), the cells are incubated for 24 h at 4°C with primary antibodies to β-tubulin (1:300; Chemicon), nestin (1:100, Chemicon) and neuronal specific enolase (1:100). After washing in PBS, cells are sequentially treated with biotinylated antibodies with avidin-biotin complex (ABC, Vector Laboratories, Inc), diaminobenzidine solution prepared in phosphate buffer (DBA 0.5 mg/ml, hydrogen peroxide 0.03%,). The preparations are dehydrated and placed under coverslips in synthetic resin (Entellan, Merk).

The material is washed from the culture medium with physiological saline and transferred to a test tube with saline. Since the material is planned for cell engineering, it should be packaged into aliquots of (0.5-0.6⋅10 ⁶ NSC) in 2 ml cryotubes and frozen in a program freezer.

Stage 3. Chemically induced transdifferentiation (reprogramming) of autologous somatic and multipotent MSSCs and NSCs into XEN-like cell systems

A detailed protocol for chemical induction of somatic or regional SCs is given in Xiang Li et al., Zhen Chai, Hongkui Deng (2017) Direct Reprogramming of Fibroblasts via a Chemically Induced XEN-like State. Cell Stem Cell DOI:10.1016/j.stem.2017.05.019

(https://www.cell.com/cell-stem-cell/fulltext/S1934-5909(17)30185-6?_returnURL=https%3A%2F%2Flinkinghub.elsevier.com%2Fretrieve%2Fpii%2FS1934590917301856%3Fshowall %3Dtrue).

In the method of chemical induction used to obtain cells in the XEN state from somatic and SC, there are three essential steps in the process of chemical reprogramming. In these stages, a cocktail of five small molecules "VC6TF" (VPA, CHIR99021, 616452, tranylcypromine and forskolin) was used in stage 1 for 16-20 days; another small molecule, DZNep, was added at the start of step 2 over the next 20-24 days; and 2i medium (double inhibition of ERK and GSK3 with PD0325901 and CHIR99021 respectively) was used in step 3 for the last 12-16 days. In general, the chemical reprogramming process can take up to 48-60 days. To analyze this process, it is necessary to carefully monitor the change in cell morphology during chemical reprogramming at each stage. Notably, several epithelial colonies formed at the end of stage 1, and these epithelial cells proliferated rapidly during stage 2. More importantly, dynamic changes in cell fate during chemical reprogramming should be monitored, as iPSCs predominantly originate from within these epithelial cell colonies. In some experiments, 100% iPSC was obtained from these colonies even when the cells were replanted at a lower density; epithelial cell colonies grew to <20% confluence. These data indicate that iPSCs can be generated from these epithelial cells.

The XEN-like state is the main technological platform for the industrial production of chemically induced HSCs. This is preceded by fine tuning of the chemical recipe to produce XEN-like cells in a reliable manner. To induce iPSCs from fibroblasts or regional SCs, XEN-like primary colonies can be induced at an early stage of reprogramming, and for this purpose, a modification of the cell transdifferentiation method using a seven-component cocktail (VPA, CHIR99021, 616452, tranylcypromine, forskolin, AM580 and EPZ004777; VC6TFAE ) (Zhao Y., Zhao T., Guan J., Zhang X., Fu Y., Ye J., Zhu J., Meng G., Ge J., Yang S., Cheng L., Du Y., Zhao C., Wang T., Su L., Yang W., Deng H. A XEN-like State Bridges Somatic Cells to Pluripotency during Chemical Reprogramming (Eng.) // Cell. - 2015. - Vol. 163, no 7. - P. 1678-1691. - DOI:10.1016/j.cell.2015.11.017. - PMID 26686652) (see Table 2 below). Modification of the XEN-like induction made it possible to optimize the chemical recipe by replacing each of the seven small molecules with functional analogues. TD114-2 has been identified as a preferred GSK3-beta inhibitor over CHIR99021 (Table 2). By replacing CHIR99021 with TD114-2, primary colony yield was increased 3-fold without changing the establishment of the XEN-like state. Thus, the low molecular weight cocktail was identified to induce XEN-like cells from fibroblasts in a significantly improved manner. Small molecular weight cocktails that are used in chemical reprogramming have been identified by phenotypic screening and do not involve direct activation of classical reprogramming factors such as Oct4, Sox2, Klf4 and c-Myc. In addition, a major advantage of the chemical approach is that small molecules can be fine-tuned in terms of their concentrations, durations, structures, and combinations, allowing more precise control of the iPSC generation process at each reprogramming step. Therefore, the identification of key molecular events and intermediate cell states will greatly improve the efficiency of reprogramming, allowing fine-tuning of small molecules and culture conditions based on the markers of each reprogramming step.

The duration of treatment of low molecular weight cells and the density of cells to be recharged were extremely important in the later stages of reprogramming. Although the old protocol supports a re-seeding density of 300,000 cells per well, the new protocol supports a density of 50,000-100,000 cells per well of a 6-well plate. In addition, the modified chemical induction protocol had an optimal duration of stage 2 of 12 days. Thus, the transition of cells from XEN to pluripotent SCs was significantly improved with the addition of new small molecules, changing the structure of small molecules and optimizing the density of charge exchange and processing time of small molecules.

table 2

Small molecules used to carry out transdifferentiation

somatic and regional SCs into XEN-like cellular states

Full name Abbreviation Dose Source Molecular weight VPA V 500 (μM) Sigma, cat. no.P4543 166.19 CHIR99021 FROM 10-20 Synthesized by WUXI APPTEC 465.34 616452 6 ten Synthesized by WUXI APPTEC 287.12 Tranylcypromine T 5-10 Enzo, cat. no.BML-EI217-0005 182.23 Forskolin F 10-50 Enzo, cat. no.BML-CN100-0100 410.50 AM580 A 0.05 Tocris, cat. no.0760 351.44 EPZ004777 E 5 Selleckchem, cat. no.S7353 539.67 TD114-2 TD114-2 2-4 Synthesized by WUXI APPTEC 529 Dorsomorpin D 1-2 Tocris, cat. no. 3093 472.41 LDN193189 L 0.1 Selleckchem, cat. no.S2618 472.41 SB431542 S 2-10 Tocris, cat. no. 1614 384.39 CH55 5 one Tocris, cat. no. 2020 364.47 L-Ascorbic acid 2-phosphate VC 50 (µg/ml) Sigma, cat. no.A8960 289.54

A robust iPSC induction protocol was created by modulating cellular transitions through a XEN-like state, as optimized reprogramming conditions for steps #1-3 were combined. Using this new protocol, a well of 50,000 original fibroblasts was induced and the cells were expanded to >1,000,000 or more replanted cells (transplanted in 10-15 wells). At the end of the reprogramming period, a total of 1,000-9,000 iPSC colonies were obtained with a total induction time of 40 days (16, 12, and 12 days for stages #1, #2, and #3, respectively). In addition, this new protocol has been independently replicated over 30 times, and iPSCs could also be generated from adult dermal and lung fibroblasts with significantly increased efficiency. Using culture conditions based on TD114-2, XEN-like colonies can be expanded over a long period of time (at least 20 passages tested) even when single colony and single cells are selected at an average ratio of 1:20-30 every 3-4 day. Despite their highly expandable properties, tumorigenesis was not detected after injection of XEN-like cells into severe combined immunodeficient mice (XEN-like cells in passage 6 and passage 19 testing). In addition, as shown by the results of karyotype analysis and comparative genomic hybridization, long-term extended XEN-like intermediates retained the genetic integrity and stability of the genome. Most importantly, in addition to maintaining XEN-like expression patterns, these expanded XEN-like cells retained their functional specific potential without reducing their HSC inducing efficiency even after 20 passages.

Protocol for chemically inducing XEN-like cells

from fibroblasts and regional SCs

Preparation of small molecules.

The small molecules used in this protocol, including: VPA, TD114-2, 616452, Tranylcypromine, Forskolin, AM580, EPZ004777, and others, are listed above in Table 2. Small molecules were prepared according to the manufacturer's instructions. TD114-2 was synthesized according to the chemical structure (Kuo et al., 2003, Storm et al., 2007).

Preparation of nutrient medium.

Stage No. 1 of the preparation of a nutrient medium for all types of somatic and regional stem cells.

KnockOut DMEM (GIBCO) supplemented with 10% KSR (GIBCO), 10% FBS (Pan-Biotech), 1% GlutaMAX™-I (GIBCO), 1% NEAA (GIBCO), 0.055 mM β-ME (Sigma), 1 % penicillin-streptomycin (GIBCO), 100 ng/ml bFGF (Origene) containing a small molecular weight chemical induction cocktail: 0.5 mM VPA, 2-4 µM TD114-2 or 20 µM CHIR, 10 µM 616452, 10 µM Tranylcypromine, 10-50 µM forskolin, 0.05 µM AM580 and 5 µM EPZ004777. To significantly increase the efficiency of fibroblast induction, 1 μM CH55 and 50 μg ml of vitamin C (VC) are recommended.

Stage number 2 of the preparation of the nutrient medium.

A. for somatic cells and cells of mesenchymal origin.

Dulbecco M medium (IMDM) supplemented with insulin and transferrin should contain a mixture of cytokines consisting of stem cell growth factor (SCF), fetal liver tyrosine kinase ligand 3 (Flt3L) and thrombopoietin (TPO), ionomycin in the following ratio: SCF( 50-150)⋅10 ³ ng , TPO(50-150)⋅10 ³ ng , Flt3L (10-50)⋅10 ³ ng, ionomycin (10-100)⋅10 ³ ng per ml growth medium, 1% penicillin -streptomycin (GIBCO), 25 ng/ml bFGF, and also contain a low molecular weight cocktail for chemical induction: 10-50 μM forskolin, 3 μM CHIR, 1 μM dorsomorphine.

B. For neural SCs.

Neurobasal (GIBCO) with 0.5% N-2 (GIBCO), 1% B-27 (GIBCO), 1% GlutaMAX™-I (GIBCO), 1% penicillin-streptomycin (GIBCO), 25 ng/mL bFGF, 20 ng/ml BDNF, 20 ng/ml GDNF containing a small molecule cocktail: 10-50 uM forskolin, 3 uM CHIR, 1 uM dorsomorphine.

Chemical induction.

1. Somatic cells (fibroblasts) or regional SCs were seeded at a density of 30,000-50,000 cells per well of a 6-well plate or 300,000 cells per 100 mm dish (day 1). Culture dishes or dishes were pre-coated with 0.1% gelatin.

2. Changed medium to stage 1 culture medium (day 0). The culture medium was updated every 4 days. XEN-like colonies will form on days 6-8.

3. On the 12th day, the induced cells should be transferred to the culture medium of the hematopoietic induction stage No. 2. The medium was updated every 4 days.

Optional: XEN-like colonies at the end of stage 1 were compact. Reseeding operation on day 12 can expand XEN-like colonies and increase cell yield. Primary XEN-like cells were trypsinized and reseeded at a ratio of 1:10-20 in the stage 1 culture medium. high concentration of 50 µM, was toxic to passaged XEN-like cells at stage 2, a concentration of 10-20 µM in hematopoietic induction culture medium on passaged XEN-like cells and 50 µM at stage 2 for non-passing induction was recommended.

4. After 8-12 days in stage #2 medium, XEN-like colonies become hematopoietic-positive. Increasing the induction period can lead to more hematopoietic-positive colonies and increased maturity, which is manifested in the appearance of markers of hematopoiesis. These hematopoietic-positive cells can be characterized or transplanted for co-cultivation with primary PK MNCs under low differentiation conditions for further maturation.

XEN-like cells: Passaging and expansion.

On day 12, induced XEN-like colonies were trypsinized from the whole well and replated on gelatin-coated culture plates at a ratio of 1:10-20 in stage 1 medium for the first passage. After the XEN-like cells in passage 1 become 100% confluent, the cells were passivated at a ratio of 1:20-30 every 3-4 days in stage 1 medium. XEN-like cells can be expanded continuously in stage 1 medium for more than 20 passages with clone-determining potentials.

One primary XEN-like colony passage requires feeder layers of mitomycin C-treated MEFs. One primary XEN-like colony is taken with a 1 ml syringe, trypsinized for 5 minutes and neutralized with stage 1 culture medium, then pipetted up and down to obtain suspension of individual cells. The cell suspension was seeded onto feed beds with stage 1 growth medium for further expansion. Subsequent passaging of XEN-like cells can be without a feeder.

For hematopoietic induction from passaged XEN-like cells, cells were seeded at 50,000-100,000 cells per well of a 6-well plate (gelatin coated) in stage 1 medium (day 0). Until passaged cells converge 70-80% on day 2 or 3, switch XEN-like cells to stage 2 hematopoietic induction medium. After 12-20 days, hematopoietic-positive colonies will appear.

Stage 4. Cell engineering and obtaining genetically homogeneous hematopoietic stem cells with homotopic genome substitution

A. Obtaining a cell-engineered HSC preparation with homotopic genome substitution for transplantation

Option 1. Cell-engineering method for the reconstruction of damaged HSC by fusion of the HSC cytoplast with the karyoplast of chemically induced cells in the XEN-like state (Ci XEN-like SC).

For enucleation of the nuclei, the technology of treatment of cell culture with cytochalasin B was used, when the concentration of cytochalasin B was calculated in such a way that the amount of cytochalasin B reached 10 μg per 1 ml of the usual nutrient medium in which the cells were cultivated. During centrifugation, in most of these cells pretreated with cytocholasin B, the cytoplasmic cord ruptures and enucleated (non-nuclear) cells (cytoplasts) and nuclei separated during centrifugation (karyoplasts) are formed, surrounded by a thin layer of cytoplasm and a plasma membrane, that is, the next cell-engineering operation should have been be aimed at obtaining a population of cytoplasts or obtaining non-nuclear HSC cells with cell surface markers CD34+, CD45+, CD133+, and it was produced using the separation of caryplasts from whole cells. In some cases, the efficiency of enucleation does not exceed 50%. In order to increase the percentage of enucleation, enucleated (cytoplasts) and whole cells contained on the surface of plastic dishes and glass discs were removed and treated with trypsin, resulting in 10 ⁶ whole cells and cytoplasts in 1 ml of saline with phosphate buffer. The cell-cytoplastic mixture was placed on 14 ml of a linear gradient of renographin-76, which was placed in centrifuge tubes, the volume concentration of which varied from 15 to 30%, and centrifuged at 1000g and a temperature of 25°C for 5 min. Clearly demarcated layers of whole cells and cytoplasts were removed from centrifuge tubes and used as directed. The cytoplasts obtained in the renographin-76 density gradient were not stained with trypan blue, which served as a control for separation efficiency. They retained the ability to reattach to the surface of the culture dish and could be used later for the reconstruction of viable cells by fusion with autologous karyoplasts.

B. Methods for obtaining nuclear biomaterial from CiXEN-like SC (karyoplasts) varied.

Option 1. Methods for enucleation from CiXEN-like SC were similar to those used for HSC and at the same dosage of cytocholasin B (10 μg/ml) to obtain karyoplasts and cytoplasts. Cytochalasin B solution is prepared by adding a DMSO-based stock solution to TCM 199 medium supplemented with 4% BSA and antibiotics at a final concentration of 5-7.5 µg/ml. CiXEN-like SC cells intended for the isolation of karyoplasts were seeded on plastic plates cut from culture flasks 2 days before enucleation, before being placed in 50-ml centrifuge tubes filled with normal nutrient medium, they were arranged so that the surfaces of these plates with attached cells were outside. The patient's CiXEN-like SC lines were centrifuged at 9500g and 35°C for 15 min. The purpose of this procedure was to reduce the number of whole cells in the sediment of karyoplasts to be obtained. After removing the cells of the plastic plate with monolayer cells weakly attached to the substrate, the remaining cells were transferred into test tubes, where the concentration of cytochalasin B per 1 ml of the usual nutrient medium reaches 10 μg. The contents of the tubes were incubated at 37°C for 15 min, the duration of centrifugation at 35°C and 7000 rpm was 45 min. The resulting supernatant was discarded, and the sediment, represented mainly by CiXEN-like SC karyoplasts, was resuspended in a normal nutrient medium.

Fragments of the cytoplasm, dead karyoplasts, and whole cells were found in caryplast preparations prepared according to the described method. Separation of cytoplasmic fragments is carried out by deposition in a Ficoll gradient of 1-6% concentration at 1g and 37°C for 90 min in a humidified atmosphere containing 5% CO ₂ . Fragments of the cytoplasm located in the upper layer were aspirated and removed, and the sediment of СiXEN-like SC karyoplasts formed after removal and dilution with nutrient medium was again precipitated by centrifugation and resuspended in fresh nutrient medium. Whole cells (their 0.4-4%) were removed by double 90-minute incubation of the suspension of karyoplasts in culture dishes. Incubation for 3 hours dramatically reduces whole-cell contamination of karyoplasts and is mandatory for all nuclear transplantation experiments.

The next operation is the separation of viable caryplasts from those that died. It included resuspending the pellet in nutrient medium to a concentration of 10 ⁷ mini-cells per 1 ml, layering the suspension on Ficoll-paque (solution containing 5% ficoll and 9% sodium diatrizoate) in centrifuge tubes, and carefully adding more than 1 ml of the medium so so that the added medium and the suspension of karyoplasts form two independent layers. The next centrifugation at 800 rpm (130 g) for 75 min at room temperature allows you to get a pellet, which consisted of 99% of dead karyoplasts. At the border of the nutrient medium and Ficoll-paque, there is a layer consisting of 98% viable karyoplasts, which were used further in the work.

Other methods can be used to clean karyoplasts, in particular, tantalum particles with a size of 1-3 microns. By the time of enucleation, almost all cells contain more than 12 tantalum particles. Due to the fact that the density of tantalum is more than 15 times higher than the density of the cell, components containing tantalum particles are deposited much faster than karyoplasts in which tantalum is absent.

Option 2. As a second option for obtaining a biomaterial for cell engineering, native tissue-specific CiXEN-like SC of the patient, obtained by cultivation according to the technology described above, can be used.

AT . Cell-engineered bioassembly of autologous reconstructed HSCs.

The next operation was bioassembly and reconstruction of cell-engineered HSCs. In the first variant, for the reconstruction (recovery) of HSCs, a suspension of CiXEN-like SC karyoplasts in saline buffer was added to a monolayer of HSC cytoplast culture in the proportion of 100 CiXEN-like SC karyoplasts per 1 HSC cytoplast. HSC cytoplasts should already be coated with inactivated Sindai virus particles (according to McGras and Solter, 2005). Incubate for 45 minutes at 4°C and then another 45 minutes at 37°C. Washed with Earle's solution to remove unfused karyoplasts, since 10% of karyoplasts of some cell lines are able to restore the entire volume of cytoplasm lost during enucleation and again turn into viable cells. The ability of karyoplasts to regenerate the cytoplasm lost during enucleation and form viable cell colonies depends on the amount of cytoplasm surrounding the nucleus. In the fraction of CiXEN-like SC karyoplasts, whose nuclei contain 2-4% of the amount of cytoplasm contained in an intact cell (small karyoplasts), only one of 10 ⁶ purified elements can form a viable cell colony. For the fusion of cytoplasts and karyoplasts of CiXEN-like SC , electroporation (electrofusion) was used using a multiporator - a device capable of acting with pulses of a given voltage. During the impact of the pulse, due to the formation of pores in the cytoplasmic membranes of the combined cells, their cytoplasms are combined. The patient's reshaped cell-engineered XEN-like HSCs are placed in an electrofusion chamber, which is pre-filled with a weak hypotonic buffer (200-240 mOsmol/kg) containing sorbitol. The fusion is carried out after a preliminary eligment of 0.3 kV/cm lasting 10 seconds by means of two successive pulses lasting 30 seconds with a voltage of 1-1.4 kV/cm. After that, the resulting autologous HSCs with homotopic substitution of the genome are thoroughly washed from the buffer in the washing medium and transferred to the post-activation solution. The use of a weak hypotonic buffer in combination with low voltage, on the one hand, makes it possible to increase the percentage of fusion of CiXEN-like SC karyotypes and HSC cytoplasts of the patient, and, on the other hand, to ensure the best survival of the obtained reconstructed cells. It is possible to increase the efficiency of fusion of HSC cytoplasts and CiXEN-like SC karyoplasts using the technology of optical-mechanical laser fusion, proposed by Russian scientists L.M. Chailokhyan, T.A. Sviridova-Chailokhyan (2010).

Option 2. Cell-engineering method for HSC reconstruction by fusion of the HSC cytoplast with whole-cell CiXEN-like SCs.

This variant of cell engineering involves the removal of the nucleus from the patient's HSC and the transplantation of somatic cell nuclei not by isolating the karyoplast from CiXEN-like SC , but by fusion of the cytoplast with the whole CiXEN-like SC . The procedure for obtaining cytoplasts from HSCs does not differ from the first variant. The HSC cytoplasts obtained as a result of enucleation are transferred from the medium preventing differentiation and maturation into the TCM 199 medium containing 0.1% hyaluronidase and incubated at 37°C for 1 minute. HSC cytoplasts are removed by gentle pipetting in washing medium and transferred to cytochalasin B solution for 5 minutes. Cytochalasin B solution is also prepared by adding a DMSO-based stock solution to TCM 199 medium supplemented with 4% BSA and antibiotics at a final concentration of 5-7.5 µg/ml. The next stage of the proposed variant of cell engineering consists in the fusion of whole-cell CiXEN-like SC with the cytoplast of enucleated HSC to obtain cell-engineered genetically homogeneous HSC of the patient.

To create cell-engineered HSCs of the patient capable of development, the cytoplasm of the transferred CiXEN-like SC should be combined with the cytoplasm of the enucleated HSC, followed by activation of the resulting cell-engineered HSCs of the patient. This is usually done using electrofusion, as described above. Activation is carried out simultaneously with electrofusion. The fusion and activation procedure is carried out at a temperature of 25-28°C. Compliance with this temperature regime provides optimal conditions for the effect of the buffer and, as a result, increases the permeability of cytoplasmic membranes, providing a better effect of the generated voltage without harming the viability of oocytes and cells.

G. Post-activation of HSCs with homotopic substitution of the genome.

It is known that, to prevent methylation processes, the resulting reconstructed HSCs are post-activated and only then cultivated to the desired stage. Activated cells are cultured first for 30 minutes in a solution of cytochalasin B of the above composition, and then in a culture medium containing DMAP (the medium is prepared by adding 100 × stock solution of DMAP based on DMSO at a final concentration in medium of 2 mM). After 2-4 hours of post-activation, the reconstructed HSCs are analyzed and those that have combined cytoplasts and karyoplasts are selected, after which the reconstructed HSCs, in which fusion is visualized, are transferred to a medium of a suitable composition for further cultivation. The composition of the growth medium culture medium containing the mixture of cytokines consisted of stem cell growth factor (SCF), tyrosine kinase ligand 3 from fetal liver (Flt3L) and thrombopoietin (TPO), the difference of which is that the above mixture of cytokines, also as in the cultivation of LC MNC PC, additionally contains ionomycin in the following ratio of components per 1 ml of growth medium: SCF (50-150)⋅10 ³ ng , TPO (50-150)⋅10 ³ ng, Flt3L (10-50)⋅ 10 ³ ng, ionomycin (10-100)⋅10 ³ ng. Post-activation and cultivation is carried out in media that do not contain phenol red. This substance is added as a pH indicator, but it has toxic properties, which reduces cell viability.

The efficiency of obtaining the proposed BMCP component, which makes it possible to cultivate SCs with cytokines that promote proliferation, but not differentiation of SCs, opens up prospects for the use of a patient's own SCs for the treatment of such diseases as coronary heart disease, burns, long-term non-healing wounds and injuries of the extremities, spinal cord and brain. Autologous SCs can be isolated and stored frozen for immediate use. At the same time, the ability for subsequent differentiation in such cultured genetically engineered cells is retained at the level of initially isolated SCs.

Stage 5 Obtaining a line of genetically homogeneous HSCs with homotopic substitution of the genome (nucleus) without epigenetic memory reversal

The purpose of this biotechnological stage is the "ripening" of the resulting cell systems to complete hematopoietic differentiation and multipotency, allowing these cells to differentiate into all types of blood cells, increasing the number of received cell-engineered HSCs of the patient and completely blocking the reversion of epigenetic memory of somatic and non-hematopoietic SCs in these cells. from which they were derived. The tasks of this stage are to carry out the "ripening" of the obtained cell-engineered cells to a multipotent hematopoietic state and the appearance of highly plastic undifferentiated HSCs with CD34+, CD133+Lin- markers, but without the development of differentiated cell colonies. At the same time, the ability for subsequent differentiation in such cultured cells should be preserved, if possible, in its original form. This task can be ensured by the influence of HSC own cytoplasm on the transplanted cell nucleus in the XEN-like state, the appropriate hematopoietic microenvironment of the cell-engineered cell with blood cells during cultivation, and the presence of factors under culture conditions that stimulate cell maturation in the hematopoietic direction. It is also very important at this early stage of formation of the hematopoietic orientation of the cell to apply factors blocking in it the epigenetic memory of DNA modifications and epigenetic changes in histones associated with the previous stages of cell differentiation in the "past" life, before cell engineering.

The most important component of this stage is the combination of cell-engineered HSCs with LAs of own MNCs of SCs for their joint cultivation. All cryopreserved LA is thawed, washed from cryoprevention and 10% DMSO by centrifugation with physiological saline, divided into 50 ml tubes, and each tube is treated individually. A cell suspension consisting of 98.5-99.5% of LC MNCs of the patient's PC and 0.5-1.0% of cell-engineered HSCs is cultured in Dulbecco M medium (IMDM) with the addition of insulin and transferrin, as well as growth factor stem cells (SCF), tyrosine kinase 3 ligand from fetal liver (Flt3L), thrombopoietin (TPO), and ionomycin (Ca ²⁺ ionophore). After 3-7 days of cultivation, the grown cell suspension contains about 1.5-2% of CD34+ cells. The difference of this medium is that the above mixture of cytokines additionally contains ionomycin in the following ratio of components per 1 ml of growth medium: SCF (50-150)⋅10 ³ ng, TPO (50-150)⋅10 ³ ng, Flt3L (10- 50)⋅10 ³ ng, ionomycin (10-100)⋅10 ³ ng. As in step 1 of the method of the present invention (when increasing the number of HSCs), ionomycin is added to the culture medium as a stimulating agent for the growth of undifferentiated SCs in ex vivo conditions. The properties of the components of the culture medium used are described above in the description of step 1 of the method of the present invention.

iPSCs (iPSCs) derived from neural progenitors and fibroblasts are characterized by residual methylation of the loci responsible for the formation of the hematopoietic lineage, which causes a decrease in the level of differentiation of these iPSCs in the corresponding hematopoietic direction. Treatment with small molecules of valproic acid (an inhibitor of histone deacetylase 1 (HDAC1)) of clones of cell-engineered HSCs in which the imprinted Dlk1-Dio3 locus is repressed leads to reactivation of the genes of this locus in a number of lines. Such iPSCs were able to contribute to the development of the organism with tetraploid complementation (Stadtfeld M., Apostolou E., Akutsu H., Fukuda A., Follett P., Natesan S., Kono T., Shioda T., Hochedlinger K. // Nature 2010. V. 465. No. 7295. P. 175-181).

To block epigenetic memory, cells that have received cell-engineering reconstruction are pretreated with valproic acid, vitamin B12 and ascorbic acid from the very first days of their cultivation. It is known that aberrant silencing of the imprinted Dlk1-Dio3 locus was observed in certain iPSC clones, including those derived from GPC, which are also characterized by a low level of transcription of this locus. This effect is thought to be caused by "epigenetic memory". Due to transcriptional disturbances in Dlk1-Dio3, iPSC loci become unable to efficiently form a chimera and cannot form a new chimeric organism through tetraploid complementation. Treatment with valproic acid, a histone deacetylase inhibitor, leads to transcriptional activation at the Dlk1-Dio3 locus and restores the ability of iPSCs to tetraploid complementation and efficient formation of chimeric animals. These low molecular weight substances have proven themselves as inducers of proper cell differentiation of cells in fetal embryogenesis when these substances are taken by pregnant women in the very early period of pregnancy. Today, ascorbic acid (vitamin C) has been shown to influence the DNA methylation pattern. Thus, in iPSCs cultivated with the addition of ascorbic acid to the medium, depending on its dose, reactivation of the imprinted Meg3 gene of the Dlk1-Dio3 locus was observed. However, ascorbic acid does not lead to large-scale demethylation of the entire genome, it is only able to prevent aberrant methylation of the Dlk1-Dio3 locus during reprogramming, but is not able to lead to DNA demethylation in already stable iPSC clones (Stadtfeld M., Apostolou E., Ferrari F. , Choi J., Walsh R.M., Chen T., Ooi S.S., Kim S.Y., Bestor T.H., Shioda T., et al., Nat Genet 2012 V 44 No 4 pp 398-405). At the same time, the ability of cells to differentiate in a certain direction and the methylation profile can be restored byrepeated rounds of reprogramming. It has been shown that, for example, iPSCs derived from neural progenitor cells had a very low ability to form HSC colonies. But when such colonies were reprogrammed, the formation of hematopoietic colonies by "secondary" iPSCs increased significantly (Kim K., Doi A., Wen B., Ng K., Zhao R., Cahan P., Kim J., Aryee M.J., Ji H., Ehrlich L.I., et al., Nature 2010 V 467 No 7313 P 285-290. If iPSCs derived from neuronal progenitors differentiate into HSC lines, and secondary iPSCs are subsequently derived from these derivatives, these secondary iPSCs will have a higher potential to differentiate into blood cells.

Thus, an additional inventive finding in the present invention was the cultivation of cell-engineered HSCs together with LC PBMs under conditions of limited differentiation and in the presence of factors that stimulate the hematopoietic characteristics of these cells and ensure the formation of genetically homogeneous multipotent HSCs with homotopic substitution of the genome. When counting cells and phenotypic analysis of the mononuclear fraction to determine the number of live MNCs, 20 μl of trypan blue (Gibco) is added to 20 μl of the cell suspension. Cells are counted in a hemocytometer, the proportion of living cells should not be lower than 95%. To assess the number of CD34+ and CD133+ cells of the mononuclear fraction, 0.1 ml of the cell suspension is stained with the corresponding antibodies and the proportion of HSCs in the initial cell suspension is determined on a FACSCalibur instrument (Beckton Dickinson).

Example 5 Phenotypic analysis of MNCs in one representative experiment conducted on LC cells of patient C. Cell proportions in LC MNCs of SC: HSC CD34+CD45+ of the donor was 0.7%, the number of neural cells was 15%, T-lymphocytes 74%, B cells -10.2%.

Cultivation . The liquid culture system consists of Iscove's modified Dulbecco's Medium IMDM (Gibco, UK) supplemented with 0.5 g/l bovine serum albumin (Sigma Aldrich), 0.39 µg/ml human insulin and 60 µg/ml transferrin (Gibco). SC growth factor (SCF), another name c-Kit-ligand (c-KitL, R&D Systems), is added to the culture medium at a concentration of 100 ng/ml, as well as Flt3-ligand (Flt3L) at a concentration of 20 ng/ml, ( R&D Systems), and ionomycin (Sigma Aldrich) at a concentration of 0.1 μM/ml. To block epigenetic memory, a cocktail of valproic acid (Merck.) (VPA) at a concentration of 1 μM (Sigma), Vitamin B1 (Vb1) (Sigma) at a concentration of 9 mg/l, and ascorbic acid (VC) (Sigma) at a concentration of 25 or 50 mcg/ml. VC was added from the second day until the end of the cultivation at this stage. VPA was added from day 3 to day 7 of human HSC generation, respectively.

Cultivation method. Cells are cultured at 37°C in a humid atmosphere with an influx of 5% CO₂. Fresh medium and cytokines are added every third day. The cultivation time is 3-7 days. The growth factors used promote SC proliferation without the need to add agents that suppress differentiation. The resulting SC suspensions of any individual can be stored frozen or can be introduced into BMCP immediately after cultivation and applied to patients.

Table 3

Evaluation of the Purity of the Cell Suspension after Cultivation

Marker Percentage of antigen-positive lymphocytes Hematopoietic SCs CD34+CD45+ 1.8 CD34+CD45- 1.6 T cell antigens CD3+CD56-CD16- 79.5 CD3+CD56+CD16+ 3.1 CD3+CD4+ 51.2 CD3+CD8+ 33.1 Cd3+ γδ+ 5, 2 NK cell antigens CD94+CD16+CD56-CD3- 9.3 CD16+CD56+ 1.1 CD16+CD56low 1.0 CD16-CD56+CD94-CD3- 3.3 Subpopulations of NK cells (within CD56 ^- CD16 ⁺ ) CD94- 100.0%

Conclusion: The material is intact. Based on flow cytometric data, HSCs are 1.8%, and SMCs are 1.6% without disturbing the percentage of subpopulations of all lymphocytes.

Stage 6. Cryopreservation of cell material

Cryopreservation at this stage is essentially carried out as in stage 1 of the method of the present invention.

Stage 7. Use of cellular material for transplantation

The material intended for transplantation, in the form of a concentrate of PK MNCs with a line of cell-engineered HSCs of the patient, is thawed in a water bath, washed off by double centrifugation from DMSO and culture medium with physiological saline, and placed in a sterile bag with 200 ml of saline and 10 units/ml of heparin. The number of cell-modified HSCs should be at least 1.5-2% of the total volume of transplanted cells mobilized by PB MNCs.

Transportation of cellular material is carried out on ice in a thermally insulated container.

Cellular material is administered intravenously using a transfusion system for 1 hour. 10 minutes before the start of the procedure, the patient receives 4 mg of metipred.

The present invention is further illustrated by the following examples, which should not be construed as limiting the scope of the present invention.

Example 6 .Patient S-howl L.V., 42 years old, diagnosed with amyotrophic lateral sclerosis,high form with severe muscle atrophy of the limbs, pseudobulbar syndrome and flaccid tetraparesis. With informed consent, stimulation of hematopoiesis with tevagramtin at a dose of 300 mg intramuscularly for 4 days was started, and on the 5th day a double dose of G-CSF was introduced. On the 6th day after catheterization of the central vein, leukocytopheresis was performed on the Spectra Coba apparatus. Received 480 ml of LA PBMC, which were transferred to the Bone Marrow Bank, where after the sedimentation of cellular LA on the ficoll gradient, LA was purified from erythrocytes and granulocytes. The purified LA was transferred to the operating room of the clinic, where, using monoclonal antibodies CD133+CD34+CD45+kit on magnetic beads on a CliniMACS cell sorter, the patient's HSC was immunosorbed. HSCs were obtained in a volume of 5 ml in physiological saline with a cell concentration of 5⋅10⁹ in 10 ml. The concentration of HSCs in LC MNCs of SC was 1.5%, therefore, HSCs were sent for research to the laboratory of hematopoiesis immunology for flow cytometry.

Example 7 Enucleation of HSCs from LC MNCs of SC in S-voi L.V., 42 years old.

The HSCs obtained in Example 6 were treated for 1 minute with a 0.1% hyaluronidase solution (Sigma) prepared by adding 90 μl of 10× stock solution to an anti-differentiation wash with ionomycin. Washed many times in this medium and transferred to a fresh washing medium containing cytochalasin B. The solution was prepared by adding 100 × stock (final concentration 5-7.5 μg/ml) solution of cytochalasin B. In this medium, autologous HSCs were cultivated in an incubator at 38 5°C and 5% CO ₂ for 30 minutes. Autologous HSCs cultured in a solution of cytochalasin B were placed in a test tube and centrifuged twice at 2000 rpm in the medium in which the enucleation procedure was performed. As a result of centrifugation, the HSC cytoplasts floated in the supernatant liquid, and the karyoplasts settled at the bottom of the tube. The cytoplasts were pipetted into the culture medium. After that, the cytoplasts were stained with Hoechst 33342 (5 kg/ml). Next, the cytoplasts were thoroughly washed in the manipulation medium and transferred to a drop of fresh medium under a layer of mineral oil. The quality of DNA removal was assessed using an Olympus microscope in ultraviolet light. Only HSCs that do not contain their own DNA were used for fusion. After confirming the fact of enucleation, the cytoplasts were frozen with DMSO medium in a program freezer to a temperature of -196°C and placed in a dewar with liquid nitrogen.

Example 8 .Obtaining multipotent MSSCs in patient S-voi L.V., 42 years old.

MMSCs were obtained from the adipose tissue of the patient, for which the adipose tissue was taken openly during a surgical operation from the subcutaneous fat of the anterior abdominal wall of the patient through a 3 cm incision made under local anesthesia with 1% novocaine solution, and thoroughly washed in phosphate-buffered saline , minced with small sharp scissors and subjected to enzymatic treatment with 0.075% collagenase type I solution in DMEM medium at 37°C for 30 minutes. Collagenase was washed out with an equivalent volume of nutrient medium (DMEM supplemented with 10% fetal calf serum) and centrifuged at 1000g for 5 minutes. The resulting pellet was resuspended and passed through filters with a pore diameter of 100 μm to remove cell debris. The cell concentration for seeding was 5×10 ⁶ cells per 75 cm ³ culture flask. MSSCs were cultured in DMEM medium (Gibco) with low glucose content, supplemented with fetal calf serum, a single solution of non-essential amino acids and antibiotics at 37°C and 5% CO ₂ in air. After two days of cultivation, colonies of attached cells were observed. The medium was changed every 4 days, and upon reaching 90% strength of the monolayer, they were subcultivated and frozen. At the right time, an aliquot of frozen cells was thawed and washed from the cryoprotectant by double centrifugation at 1500 rpm for 5 minutes in DMEM nutrient medium containing 10% fetal calf serum. After that, the cells were seeded in culture Petri dishes and cultured under the conditions used before freezing until a dense monolayer was obtained. For the last two days, the cells were cultured in a serum-free medium. Immediately before the nuclear transfer procedure, the cells were removed from the substrate with a trypsin-EDTA solution, washed, and diluted in a small amount of TCM 199 manipulation medium to obtain a cell suspension.

Example 9 .Electrofusion and activation of the obtained reconstructed (restored) autologous HSCs.

Autologous cytoplasts (enucleated HSCs) and autologous karyoplasts (MSSCs in XEN-like state obtained from adipose tissue S-voi L.V., 42 years old) were combined by electrofusion in a microchamber using an Eppendorf multiporator. The reconstructed HSCs were transferred to the electrofusion chamber with 50 µl of sorbitol solution (200-240 mOsmol/kg) Fusion and activation was carried out after a preliminary elugment at 0.3 kV/cm for 10 minutes with two successive pulses at 1-1.4 kV/cm for 30 seconds. sorbitol solution in this case was 25-28 ° C. After exposure to pulses, the restored HSCs were thoroughly washed from the sorbitol solution in the medium for manipulations.

Example 10 .Cultivation of the obtained recovered HSCs.

The electrofusion-treated reduced HSCs were first cultured in NCSU-23 medium containing 5 µg/mL cytochalasin B for 30 minutes and then in NCSU-23 medium supplemented with DMAP for 3 hours (DMAP stock solution diluted in DMSO was added to medium at a final concentration of 2 mM). At the end of this period, HSCs were selected, in which the cytoplast and MSSC contents were completely combined. Selected cell complexes were cultured for 5-6 days in an incubator at 38.5°C, 5% CO ₂ in air and maximum humidity. In the process of cultivation, the amount of obtained HSCs was estimated. Within 5 days, due to the optimization of enucleation techniques and transplantation of MSSCs from adipose tissue, 54.7% of HSCs were successfully reconstructed. Electrofusion modes and compositions of buffer solutions ensure the fusion of cytoplasmic membranes of HSCs and transplanted MMSCs in 75% of cases. Activated cell complexes have the ability to complete hematopoiesis, as evidenced by HSC markers in the CD34+ gate, which is presented in Table 4 - the results of an immunophenotypic study of the HSC culture of the patient S-voi L.V., 42 years old, date of receipt of the material: 10.01.2019 .).

Table 4

The result of an immunophenotypic study of restored HSCs

STEM CELL CONTENT (%) CD34+ (in Syto-16+ gate) 1.61 SUBPOPULATIONS OF CD34+ CELLS (%) CD45 92 HLA-DR CD34 ⁺
CD38 98.6 96.2 CD33CD34 ⁺
CD13 88.4 99.6 CD71CD34 ⁺
CD117 73.2 92.9 CD90CD34 ⁺
CD56 95.6 62.6 CD19CD34 ⁺
CD61 3.4 6.85 CD7CD34 ⁺
CD10 3.02 91.4 CD2CD34 ⁺
CD62L 0 0.3 CD8CD34 ⁺
CD44 98.7 97.3 CD133CD34 ⁺
CD28 95.5 92.0

Lines of genetically homogeneous HSCs with homotopic substitution of the genome obtained as a result of cell engineering or transdifferentiation should always be transplanted only in combination with LC of mobilized HSC MNCs, since the isolated use of cells of this line will be clinically ineffective.

So, when transplanting genetically homogeneous HSCs with homotopic genome substitution, it is fundamentally important that the cells of the resulting line during transplantation be in the microenvironment of mobilized PC lymphocytes, since this balanced combination of lymphocytes works as a single bone marrow cluster, the mechanism of action of which was described by the Japanese researcher K. Ono (2002).

At the same time, during transplantation, it is necessary to observe the quantitative parameters of the content of cells of the obtained HSC line and not to reduce their number to less than 1% of the total cellular content of PB MNCs.

Example 11 . Checking the effectiveness of BMCP.

The animal study was carried out in accordance with the recommendations of the Moscow Private Medical University "Reaviz" and the protocol of preclinical studies, which was approved by the local ethics committee of this university (protocol 02-2018). Three groups of recipients were used for the experiments. All recipients were GFP-negative B10-GFP mice, which are heterozygous for the GFP transgene. The control group initially consisted of 20 animals. In the first experimental group there were 56 animals, five of which were used to control the level of chimerism and were excluded from mortality statistics, and in the second experimental group there were 50 animals. Starting at the age of 15 months, when about 50% of mice remained alive in all groups, the experimental animals of the first group received a series of intravenous injections of bone marrow from young (from 3 to 15 weeks) donor mice that were GFP-positive individuals of the same B10 strain. -GFP, and the animals of the second experimental group received intravenous injections of BMCP obtained on the basis of their (autologous) HSC according to the present invention. Mice aged 3-15 weeks and heterozygous for the green fluorescent protein transgene (ie expressing GFP) of the same B10-GFP strain were used as bone marrow donors. All mice were purchased from the same nursery where this line of mice was kept as a small herd, thereby increasing their inbredness and reducing their life expectancy.

Isolation and transplantation of bone marrow were performed according to the procedure described by Colvin et al. (2004) with minor modifications. Animals were euthanized by neck dislocation and sterilized for 3-5 minutes in 70% ethanol. The bones of the entire skeleton (skull, spine and hips) were demuscled and ground in a sterile mortar and pestle (treated with ethanol and UV light) in 5 ml of sterile Hank's Balanced Salt Solution (HBSS) (Life Technologies Gibco BRL) with 10 U/ml heparin . The resulting suspension was filtered through a 70 μm filter (BD Biosciences), then the filter was washed with 2 ml of buffer, and the two filtrates were combined and centrifuged for 5 minutes at 340g. The pellet was resuspended in 8 ml HBSS/heparin and centrifuged for 5 minutes at 340g. The pellet was resuspended in the same buffer solution (total volume 2-4 ml) and cells were counted in a Goryaev chamber or using an automatic cell counter. Typically, the yield was (2-4)⋅10 ⁸ nucleated cells ((5-6)⋅10 ⁷ nucleated cells per ml) depending on the age of the donor. Immediately prior to injection (within 20 minutes), the cells were further filtered through a 40 μm filter (BD Biosciences). A total of (15-20)⋅10 ⁶ cells per recipient animal was slowly injected over about 1 min in a volume of 200-300 µl per animal with an insulin syringe (0.33GX 12.7 mm needle) through the tail vein; the tail was preheated in a water bath for 2-3 minutes at 44°C. In 15 animals, bone marrow cells were transplanted during each day of transplantation. Transplantations were repeated 6 times within 3 months with 10-20-day intervals in experimental animals of the first and second groups . To control the level of chimerism (degree of engraftment of donor cells) the proportion of GFP+ cells in recipient cells was analyzed. For this, 200 μl of cell suspension containing 10-20 million cells per 1 ml was placed on a glass slide, 10 μl of NucBlue solution (Thermo Scientific), and after incubation for 5 minutes, the cells were covered with a coverslip. Images were acquired with a NICON 2000 fluorescent microscope, which took 10-20 wavelength excitation photographs for NucBlue (Hoechst 33342) and GFP using Cellinsight CX7 (Thermo Scientific), which are evenly distributed throughout the slide. The average percentage of fluorescent cells was determined automatically using the software. For the analysis of fluorescent structures in hard tissue, a piece of tissue with a volume of 1×3 mm was crushed between a glass slide and a cover slip, and photographs were taken using a NICON 2000 fluorescent microscope. % of most long-lived mice. The MF for control mice (MFK) was determined in two ways. The first value of MLS K1 was a direct estimate of the average life expectancy of the 10% longest-lived animals from the first group of mice (ie 2 out of 20) and secondly, MLS K2 was calculated using a survival approximation. For the survival curve in the experimental group of mice that died of natural causes prior to bone marrow transplantation, see FIG. 1. The approximation was made using the non-linear approximation function from Nonlinear Model Fit of the MATHEMATICA software package. Confidence interval for P = 0.05 was calculated in a standard way using Student's t-test. The two values (MFC1 and MFC2) thus obtained were compared and the resulting value of MFK was calculated. This method increased the accuracy of the calculation of MFK since the number of animals was limited.

Results. In FIG. 1 shows the effect of syngeneic bone marrow transplantation of young donors (first experimental group, open circles) and syngeneic BMCD transplantation according to the present invention (second experimental group, black circles) on the life expectancy of recipients. The control group (black squares) was used to directly determine the maximum lifespan of untreated mice (MTL K1). The age of death of the last 10% of this group (two mice) was 17.6 and 19.3 months, which gives a direct estimate for the control group of mice: MRV C1 = 18.45 ± 2.6 months. This value of MRV K1 has a high standard deviation due to the rather small size of the control group. In order to improve the accuracy of NRV C, we used data on mortality in the experimental group before transplantation (for example, before the age of 15 months), when mortality was due to natural causes. Taking the standard shape of the survival curve according to the Gompertz-Makham law (Makeham, 1860):

f (t) = N0 ⋅ Exp - Exp (μ0 + μ1t) μ1, where

N0 = 100% ⋅ Experience - Exp (μ0),

μ1, μ1 and μ2 are the parameters that allow, on their basis, to minimize the quadratic deviation of the model curve from the natural death of animals.

The numerical values of the parameters were determined as follows:

μ0 = -11.4 and μ1 = 0.7.

This allows us to calculate the maximum lifespan, i.e. the average lifespan of animals that lived from t10 to 1, for mice in the first experimental group before treatment (transplantation), thus serving as an additional control (MTV K2):

MTW K2 = ∞. R t10 t ⋅ f (t) d t∞ R t 10f (t) dt = 17.2 ± 0.6 months,

where t 10 is the point in time when 10% of the animals remained in the population.

The MRV K2 value is in good agreement with the MRV K1 obtained using the first method for estimating the MRV in the control group. There is little difference between the two methods. Estimation of MLV is needed because of the lower reliability of the first method under conditions when random deviations become significant, that is, if the number of mice is small.

Therefore, in order to compensate for the low number at the end of the control study (two mice), this fact can be corrected by averaging the values obtained from the two methods for determining MMT:

MVA C = 12 ⋅ MVA K1 + 11 ⋅ MVA K2( 11 + 12) = 17.4 ± 0.5 months,

where 11 and 12 are the standard deviations of MF K1 and MF K2, respectively.

At the age of 15 months (beginning of donor bone marrow injections), 31 mice of the first experimental group and 26 mice of the second experimental group survived. In the first experimental group, 9 mice died during the injection on the operating table (embolism) during transplantation of BM of young mice. An additional 5 mice were sacrificed for tissue sampling and the rest (42 mice) died naturally. In the second experimental group, 5 mice died from embolism (bone marrow mixed with BMCP of the present invention) during transplantation, 2 mice were sacrificed for sampling, and the remaining mice (19 mice) died naturally. As can be seen, the lethality curves of the animals of the first and second groups practically do not differ. Immediate death was caused in 9 embolic mice of the first experimental group due to the inaccuracy of the researcher in the elimination of aggregates from the injected material, as well as by induced activation of platelets in the bloodstream. An additional control was carried out on 15 old animals of the same breed, which received a total of 75 injections of 300 μl of HBSS-heparin, and showed no lethality. Thus, embolic risk requires further study. It is possible that the oldest individuals died after injections of bone marrow. Taking this consideration into account, it was decided to include embolic mice in the analysis statistics for calculating MRV. In the statistical analysis, embolic mice were counted as if they had died of natural causes. The stepped curves in Fig. 1 reflect the dynamics of mortality adjusted for embolic animals. Mice killed for sampling were not counted in the statistics and plotted in FIG. one.

To calculate the life expectancy after bone marrow transplantation, the maximum age of 10% of the longest-lived mice from the experimental groups was used, which corresponds to the age of the last 5 mice. The maximum life expectancy after bone marrow transplantation is 21.0, 22.5, 22.7, 23.8, and 24.2 months, which gives MRV exp = 22.8 ± 0.8 months.

Thus, non-ablative syngeneic bone marrow transplantation increased the maximum lifespan of mice in the first experimental group by 100% × (22.8–17.4)/17.4 = 31 ± 5%, and the survival time from the start of transplantation increased by (22 .8-15) / (17.4-15) = 3.25 ± 0.3 times.

The time of maximum life expectancy in the second experimental group increased by 100% x (50 -26) (-11.4 + 0.7) = 25.6 ± 8%, and the survival time from the start of transplantation increased by (22.8-15 )/ (25.6-19) = 1.2 ± 0.5 times.

At the age of 19.3 months, when the last mouse of the control group died sedentary, almost immobile (hunched appearance, bad hair), and the mice of the experimental groups were active, had a straight spine and a shiny even coat.

GFP fluorescence (green) of HSCs in the bone marrow of young donor mice is shown in FIG. 2A. GFP fluorescence of HSCs in aged control mice is shown in FIG. 2B. GFP fluorescence of recipient bone marrow cells after transplantation of both native (first experimental group) and cell-engineered HSCs in the BMCP according to the present invention is practically indistinguishable (Fig. 2C). In both experimental groups, six months after transplantation, GFP fluorescence was detected in 7% of recipient bone marrow cells. Considering that only a quarter (25%) of progenitor cells fluoresce in the bone marrow of a heterozygous donor (Fig. 2A), the chimeric rate of the transplanted recipient was (7/25) × 100% = 28%. Thus, more than a quarter of recipient bone marrow cells 6 months after non-ablative transplantation were of donor origin. Therefore, life extension should be due not only to the paracrine effect of HSCs, but also to cell replacement. Fluorescent patterns were also visualized in other tissues of the recipients such as liver, spleen, kidney and muscle 6 months after the last injection.

List of abbreviations

ALS - amyotrophic lateral sclerosis

BMCP - biomedical cell product according to the present invention

HSC - hematopoietic stem cells

G-CSF - granulocyte colony stimulating factor

HPC - hepomoetic progenitor cells

GOKI - hematopoietically oriented cell-engineering (cell)

MN - malignant neoplasms

ICC - immunocompetent cells

iPSCs - induced pluripotent stem cells

LC - leucoconcentrate

MCA - monoclonal antibodies

MNCs - mononuclear cells

MSSC - mesenchymal stromal stem cells

NSC - neural stem cells

OSB - tumor-specific proteins

OK - tumor cells

TSC - tumor stem cells

PC - peripheral blood

RIF - immunofluorescence reaction

SBU - special basic conditions

SC - stem cells

TCM - bone marrow transplant

IVF - in vitro fertilization

Claims (13)

1. A method for obtaining a biomedical cell product for the treatment of oncological, neurodegenerative, autoimmune and endocrine diseases, including obtaining an autologous mononuclear leukoconcentrate (MNC) from bone marrow exfusate or from a leukopheresis product of mobilized mononuclear cells (LC MNC) of peripheral blood; obtaining a line of autologous hematopoietic stem cells (HSC) with CD34+, CD45+, HLA DR+ markers and/or hematopoietic progenitor cells (HPC) with CD34+, CD45-, HLA DR- markers differentiation; obtaining a line of disease-intact autologous regional multipotent stem cells or somatic cells and increasing their cell mass; reprogramming said autologous regional multipotent stem cells or somatic cells into XEN-like pluripotent cells by chemical induction; carrying out cell-engineering homotopic replacement of HSC and/or HCP nuclei with the nuclei of XEN-like cells or with whole XEN-like cells by obtaining cytoplasts from native HSCs and/or HCPs by treating these cells with cytocholasin B and transplanting karyoplasts obtained from XEN-like cells by their treatment with cytocholasin B, or whole XEN-like cells into HSC and/or HCP cytoplasts, thereby obtaining reduced HSC and/or HCP containing multipotent HSC cytoplasts and pluripotent karyoplasts of XEN-like cells; combination of restored HSCs and/or GPCs with LC PBMCs from which the original HSCs and/or GPCs were isolated to obtain a target biomedical cell product containing a line of postgenomically restored multipotent genetically homogeneous autologous HSCs with markers CD34+Lin-, CD38", HLA- DR+, CD45+. 2. The method according to claim 1, characterized in that the reduced HSCs and/or HCPs, connected to LA MNCs, are cultivated in a medium that prevents differentiation to increase their cell mass, form and mature their properties of multipotency in the hematopoietic direction and control these properties flow cytometry. 3. The method according to claim 1, characterized in that in the case of oncology or neurodegenerative diseases of the brain and spinal cord, as well as in the case of autoimmune diseases, mesenchymal stromal stem cells (MSSCs) with markers CD10+, CD13+, CD44+ are used as cells undamaged by the disease, CD90+ (Thy-1), CD105+, CD34-, CD45- and CD117- and/or mesenchymal stromal progenitor cells (MSPCs) with CD10+, CD13+, CD44-, CD90+ (Thy-1), CD105+, CD34+, CD45+ and CD117+. 4. The method according to claim 1, characterized in that in the case of oncology of solid organs, neural stem cells (NSCs) with the CD 133+ marker and/or neural progenitor cells (NPCs) with the CD 133+ marker are used as cells undamaged by the disease. 5. The method according to claim 1, characterized in that the reprogramming by chemical induction is carried out with a cocktail containing low molecular weight substances selected from the group consisting of VPA, TD114-2, CHIR, 616452, tranylcypromine, formskolin, AM580, EPZ004777, CH55 and vitamin C(VC). 6. The method according to claim 1, characterized in that the transplantation of karyoplasts obtained from XEN-like cells or whole XEN-like cells into HSC and/or HCP cytoplasts is carried out in vitro by electroporation or by a laser-optical method. 7. Biomedical cell product for the treatment of cancer, neurodegenerative, autoimmune and endocrine diseases, obtained by the method according to one of paragraphs. 1-6 and containing an autologous leukoconcentrate of mobilized mononuclear cells, depleted in its own HSCs and/or GPCs as a result of their immunoseparation, with a line of genetically homogeneous autologous hematopoietic cells with markers CD34+Lin–, CD38", HLA-DR+CD45+.

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