RU2567671C1 - Method of pre-differentiation of stromal stem cells of bone marrow in tyrosine hydroxylase-positive cells - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: method of pre-differentiation of stromal stem cells of bone marrow is described. The method involves obtaining a monolayer of culture of human bone marrow stroma, obtaining the cultural supernatant of cerebral peduncles of pig embryo of six week of gestation and the use of this medium for enrichment of population of tyrosine hydroxylase-positive cells of culture of bone marrow stroma by its incubation in the medium containing 50% supernatant of three-day culture of cerebral peduncles of pig embryo.

EFFECT: obtained autologous bone marrow stromal cells can be used for the treatment of neurodegenerative diseases.

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Description

The invention relates to the field of cell biology and biotechnology, specifically to the differentiation of uncommitted bone marrow stromal stem cells into tyrosine hydroxylase - positive cells.

Bone marrow stromal cells are isolated from red bone marrow samples obtained from the iliac crest. Samples are ground, cultured in a medium containing RPMI 1640, F12 culture medium, calf fetal serum in a ratio of 4: 4: 2 (medium 1). After the monolayer of attached cells is formed, they are scattered on culture vessels containing the medium of the following composition: medium 1-50%, culture supernatant of the legs of the brain of a pig embryo of the sixth week of gestation - 50% (medium 2). During cultivation, the bone marrow cell population was enriched with cells bearing tyrosine hydroxylase. As is known, such cells can be precursors of dopamine-producing neurons located in the nigrostrial system of the brain. A decrease in the number or activity of these neurons leads to a decrease in dopamine synthesis, which entails a serious damage to the nervous system, called Parkinson's disease. Thus, the present invention allows to obtain an autologous culture of bone marrow stroma cells enriched with precommitted stem cells carrying a tyrosine hydroxylase marker. These cells may find application in the cellular therapy of Parkinson's disease.

The invention relates to the field of cell biology, biotechnology and transplantation therapy, specifically to the differentiation of uncommitted bone marrow stromal stem cells into tyrosine hydroxylase-positive cells.

In recent years, numerous data have appeared that testify to the high plasticity of the cells of higher animals, the presence of hidden reserves of somatic cell renewal in postnatal ontogenesis due to the maintenance of a stem cell pool - the precursors of various branches of cell differentiation. Today, the concept of both totipotent (zygote) and multipotent stem cell is formulated. A multipotent stem cell is endowed with an invariable ability to self-renewal, maintained throughout the life of the body. These cells are superior to all other cells of the body in proliferative potential. Numerous studies on the properties of mesenchymal and epidermal stem cells have been added to the well-studied ideas about the hematopoietic stem cell. Subsequently, the presence of a neural stem cell in the mammalian brain was shown in both embryos and adult organisms. Actively proliferating neural progenitors persist in specific areas of the central nervous system of adult mammals. They are localized in the subependymal zone, the dentate gyrus of the hippocampus and the olfactory bulb of the brain. These cells give rise to neurons and glia and are capable of supporting local neurogenesis throughout life. Not so long ago, such zones were found in other areas of the central nervous system. Neural precursors include a heterogeneous population of cells, including multipotent stem cells and their oligo- and unipotent descendants.

It is known that the patient’s bone marrow is an affordable source of autologous stem cells for transplantation. It is also known that the pre-differentiation of these cells into nestin-positive cells is possible, according to many data, which are the precursors of neural cells of different stages of differentiation.

We hypothesized that factors secreted by cells at a certain stage in the development of the embryonic brain can differentiate mesinchymally-like cells of the bone marrow stroma into progenital cells, which can later differentiate into dopamine-producing neurons or cells producing tyrosine hydroxylase. This enzyme, which is the key enzyme for dopamine synthesis, is produced in the arcuate nucleus by nedopaminergic neurons. It has been shown that these neurons are involved in the cooperative synthesis of dopamine, which is considered, on the one hand, as a new level of chemical integration of the brain, and on the other hand, as an adaptive reaction in the functional insufficiency of the dopaminergic system. The marker of such cells is known to be the tyrosine hydroxylase enzyme.

State of the art

There is a method of neural differentiation of bone marrow mesenchymal stem cells into neurons in vivo under the influence of a 27-day cultivation in a medium containing epidermal growth factor (EGF) at a concentration of 2 ng / ml (Zemchikhina V., Cytology, T. 47, 2005, No. 7 , pp. 644-648). However, this method does not lead to the formation of tyrosine kinase-positive cells.

The closest method we used as a prototype is a method for the neural differentiation of bone marrow stroma for the treatment of Parkinson's disease with retinoic acid (Schegelskaya E. et al. In vitro differentiation of mouse bone marrow stroma cells into precursor cells of neurons and hepatocytes. Experimental and clinical medicine, No. 1, pp. 16-20, Valoshina et al. Autotherapy with bone marrow stromal cells (BSCM) induced in the nerve in patients with chronic central nervous system diseases (multiple sclerosis, disease Parkinson). 2003 Ukrainsky neyrohirurgichny magazine, number 3, pp. 54-61). Bone marrow stroma was induced into pre-differentiation by the addition of various amounts of a low molecular weight hydrophobic agent, retinoic acid. However, this method does not lead to the formation of tyrosine hydroxylase-positive cells.

Therefore, we have proposed a method for enriching a culture of bone marrow stroma with tyrosine hydroxylase-positive cells. It is known that dopamine in the brain is produced by Purkinje cells located in the nigrostrial system of the legs of the brain. Tyrosine kinase-producing cells are also localized there. Therefore, we obtained the primary culture of the legs of the pig’s brain. The pig was used as one of the animals whose brain is closest to the human in many respects. In addition, due to the fact that a person eats pig meat and offal, including the brain, the human body is adapted to most of the pig viruses. Since a pig embryo obtained under sterile conditions was used to obtain a brain preparation, and the culture was tested for bacterial growth, the possibility of bacterial infection upon receipt of the preparations was excluded.

The preparation of the legs of the brain containing the nigrostrial system, embryos of six-week-old piglets was transferred to the culture by their collagenase processing and further cultivation in a monolayer culture. The supernatant of a monolayer culture at different stages of cultivation was used to attempt to differentiate human bone marrow. Human bone marrow stroma was prepared as follows: 1 ml of red bone marrow from the ilium was crushed and cultured for 3-6 weeks in flat-bottomed vials until a monolayer culture was formed. The culture had the appearance of a fibroblast-like monolayer with slight heterogeneity (Fig. 1). The obtained cells were transferred for experiment into flat-bottomed culture bottles and subjected to treatment with medium containing the supernatant from the culture of the legs of the brain of a pig embryo. After a seven-day incubation, the cells were fixed; immunocytochemistry for tyrosine hydroxylase was performed. For this, the cells of the bone marrow stroma monolayer fixed by methanol were treated with monoclonal antibodies to tyrosine hydroxylase, and then with a conjugate of rabbit antibodies against mouse immunoglobulins labeled with fluorescein. The reaction was taken into account under a luminescent microscope at a magnification of × 400.

As a result of using the supernatant from the weekly cell culture of the legs of the brain of a six-week-old pig embryo, a bone marrow stroma culture was obtained, part of the cells of which gave positive staining for tyrosine hydroxylase (Fig. 2). This indicates the past differentiation towards cells with the potential to stimulate dopamine production by Purkinje cells. The supernatant from a 3-4-week culture of the legs of the brain of a pig embryo did not possess the property of differentiating human bone marrow cells into tyrosine kinase-positive cells.

SUMMARY OF THE INVENTION

Our proposed method for producing a culture of autologous bone marrow stromal cells can be further used to try to treat neurodegenerative diseases, primarily Parkinson's disease, by transplanting them into the brain of patients with an imbalance in the synthesis of tyrosine hydroxylase and dopamine. It was previously shown in animal models with Parkinsonism that the injection of vectors based on adeno-associated viruses carrying the tyrosine hydroxylase gene into the striatum can be used to obtain de novo synthesis of this enzyme and an indirect increase in dopamine synthesis (Haque N., Isacson O. (1997). Exp. Neurol. 144 (1), R. 139-146). At the same time, the problems associated with allogeneic and xenogenic transplantation of similar cells are removed.

Example

A human bone marrow stroma culture was prepared as follows.

Example 1. Bone marrow fragments were obtained by trepanation of the iliac crest of a patient of 65 years of age. On the periosteum, a p-shaped incision was made 1.5 × 1.5 × 1.5 cm in size. The periosteum was bent and 1.5 ml of red bone marrow were removed with a Folksman spoon. Then the periosteum was returned to its original position and sealed with medical glue. The sample was placed in a transport medium of the following composition: RPMI 1640 - 97%, fetal calf serum - 3%, gentamicin - 200 mg per ml. Within no more than 120 minutes, bone marrow fragments were transported into a laminar box for further processing under sterile conditions. Then the fragments were removed and placed in a sterile Petri dish containing medium of the following composition, RPMI 1640 medium 97%, fetal bovine serum 3%, gentamicin 100 mg / ml, as well as fungicone and glutamine. The transport medium with individual cells washed into it was centrifuged for 10 minutes at 1000 rpm. The supernatant was removed and the pellet was resuspended in a fresh portion of the same wash composition. After the third centrifugation, the pellet was resuspended in a culture medium of the following composition: RPMI 1640, DMEM and fetal serum in a ratio of 4: 4: 2, penicillin 100 units / ml, streptomycin 100 mg / ml, amphotericin 0.25 mg / ml, L- glutamine 2 mm at the rate of 250-500 thousand nucleated cells in ml Fragments were crushed and washed intensively, separating from free cells. Then the fragments and pre-centrifuged free cells were placed in separate culture vessels in the culture medium described above. After daily cultivation, non-adherent cells were removed, centrifuged and placed for culture in separate vials with the medium described above. Cells were cultured 6 weeks before the formation of a monolayer. The change of environment was carried out every two days. Monolayer cultures were combined and used for experiments.

The culture of the legs of the brain was obtained as follows. The embryos, without opening the uterus, were delivered to a sterile box within an hour after removal from the mother. Then the uterine horns were opened and the embryos were removed. After removal of the cranial vault and the overlying part of the brain, the brain legs were removed and placed in an environment of the following composition: RPMI 1640 - 97%, fetal calf serum - 3%, gentamicin - 200 mg / ml. Then, the tissue of the brain legs was crushed into fragments with a diameter of 1-2 mm and subjected to collagenase cleavage for 20-120 minutes at a temperature of + 37 ° C in an atmosphere containing 6% CO ₂ . Cells were washed once with medium of the same composition and placed for cultivation in flat-bottomed culture vessels containing medium of the following composition: RPMI 1640, DMEM and fetal calf serum in a ratio of 4: 4: 2, penicillin 100 units / ml, streptomycin 100 mg / ml, amphotericin 0.25 mg / ml, L-glutamine 2 mm at the rate of 250-500 thousand nucleated cells per ml. The environment was changed every two to three days. The supernatant from the culture was frozen at a temperature of -40 ° C and then used for experiments, pre-filtering it through a filter with a pore diameter of 0.22 μm for sterilization and removal of detritus.

For experiments on differentiation, bone marrow stroma cells were scattered at the rate of 200 thousand cells per ml into 12-well polystyrene plates. After the monolayer of attached cells was formed, they were scattered on culture vessels containing a medium of the following composition: medium 1-50%, culture supernatant of the legs of the brain of a pig embryo of the sixth week of gestation - 50% (medium 2). Tyrosine hydroxylase staining was performed using monoclonal antibodies against tyrosine kinase by indirect immunofluorescence.

Claims (1)

A method for the differentiation of bone marrow stromal stem cells, characterized in that the monolayer is incubated in a medium containing 50% of the supernatant of a three-day culture of the legs of the brain of a pig embryo of a sixth week gestation to enrich the population of tyrosine hydroxylase-positive cells.

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