RU2539750C2 - Method of evaluating immunosuppressive properties of human mesenchymal stromal cells - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to field of medicine, molecular biology and biopharmaceutics. Method of determining immunosuppressive properties of human mesenchymal stromal cells by measuring expression level of molecule HLA-DR on the surface of cell membranes and measuring level expression of molecules IDO, iNOS and COX-2 in cells. As criterion of evaluation serves double increase of level of expression of HLA-DR on the surface of membranes MSC and/or mRNA genes IDO, iNOS and COX-2 in cells.

EFFECT: invention can be applied for evaluation of immunosuppressive properties of cells for diagnostic purposes.

6 dwg, 5 ex

Description

Technical field

The invention relates to medicine, in particular to biopharmacology and medicine, and relates to a method for evaluating the immunosuppressive potential of mesenchymal stromal cells.

State of the art

Recently, it was found that mesenchymal stromal (stem) cells (MSCs) have anti-inflammatory and immunosuppressive properties, which are expressed in the suppression of the proliferation of activated T-lymphocytes, in the suppression of the maturation of dendritic cells and a decrease in the ability of B cells to secrete immunoglobulins.

The immunosuppressive properties of MSCs are not always manifested, but as a result of exposure to the pro-inflammatory microenvironment: (activated lymphocytes) and pro-inflammatory cytokines (IL-1, TNF-alpha, IFN-gamma) [Rubtsov YP, Suzdaltseva YG, Goryunov KV, Kalinina N1, Sysoeva VY, Tkach VA. Acta Naturae. 2012 Jan; 4 (1): 23-31. Regulation of Immunity via Multipotent Mesenchymal Stromal Cells].

Under the influence of these factors, MSCs change their phenotype. In this case, HLA-DR molecules appear on the surface of the cell membrane (HLA class II, class II MHC — the main histocompatibility complex of class 2), which are not characteristic of them in a normal microenvironment [Devine SM, Cobbs C, Jennings M., Bartholomew A., Hoffman R. // Blood. 2003. V.101, P.2999-3001; Krampera M., Glennie S., Dyson J., et al // Blood. 2003. V.101, P.3722-3729; Liu H., Kemeny D.M., Heng B.C., et al // J Immunol. 2006. V.176, P.2864-2871; Romieu-Mourez R., Francois M., Boivin M.N., Stagg J., Galipeau J. // J Immunol. 2007. V.179, P1549-1558].

Under the action of pro-inflammatory cytokines, MSCs secrete molecules that are also not characteristic of them in the normal / normal state. Secreted MSC molecules, in turn, have a pronounced immunosuppressive effect on activated lymphocytes. Alleged molecules involved in the processes of immunosuppression and which are markers of this phenomenon are IDO (indolamine-2,3-dioxigenase, indoleamine-2,3-deoxygenase), iNOS (inducible nitric oxide synthase).

Existing methods for assessing the immunosuppressive properties of MSCs are based on the following principle - control MSCs, as well as MSCs exposed to various factors, are placed in a culture with activated T cells or other types of immune cells (B cells, macrophages, dendritic cells). After joint incubation, the immunosuppressive properties of MSCs characterize their ability to suppress (inhibit) the functions of immune cells, in particular, secrete inflammatory factors (e.g. cytokines), expose characteristic molecules on the surface (so-called activation markers), and slow down the division of activated cells. That is, the ability of MSCs to immunosuppression is determined indirectly by measuring the characteristics inherent in immune cells. The disadvantages of this method include the complexity of the measurement, since the co-cultivation of MSCs and activated immune cells requires a source of these cells, a reliable way to activate them, a method of measuring one or more parameters (fission rate, production level of pro-inflammatory molecules, etc.). An additional problem is that the properties of MSCs are judged by the properties of other cells, which can lead to additional inaccuracies and the need for standardization, since in a complex system it is impossible to exclude the mutual influence of some components on others.

The closest analogue of the claimed invention is a method for determining the phenotype of mesenchymal stem cells, comprising contacting the mesenchymal stem cells with antigen-specific activated immune cells, and measuring the antigen-specific activity of antigen-specific immune cells activated before and after contact with mesenchymal stem cells, in which a decrease in antigen-specific activity as a result of contact with mesenchymal stem cells determines the immuno upressorny potential of mesenchymal stem cells (WO 2009134429). However, a significant disadvantage of this method is its low prognostic value, which leads to its low clinical value.

The present invention provides a method for evaluating the immunosuppressive properties of adipose tissue mesenchymal stromal cells, comprising measuring the level of HLA-DR expression on the surface of MSCs, and measuring the expression level of IDO and iNOS and COX-2 molecules within the cells. The presence of suppressive properties is thus evaluated, in particular, by comparing with the expression levels of the above molecules in MSCs and MSCs activated by pro-inflammatory factors, which were cultured under normal conditions.

The main advantage of the proposed method is that the establishment of a correlation between the level of individual molecules characteristic of MSCs in the immunosuppressive state allows us to further judge the properties of MSCs by comparing these levels in control MSCs and MSCs pre-incubated with various factors or cells. Thus, the proposed method allows you to directly determine the parameter that is inherent in the MSCs themselves, but not the cells with which they are incubated. The proposed method is direct, does not require additional efforts for the preliminary activation of immune cells, is simple from the experimental point of view, and is faster and more reliable in comparison with analogues.

Disclosure of invention

The objective of the present invention is to develop a direct method for determining the immunosuppressive properties of MSCs, which allows you to quickly and reliably determine the presence of immunosuppressive properties of MSCs, in particular in MSCs, which were subjected to various types of stimulation (for example, co-culture in the presence of antigen-activated lymphocytes or incubation in a medium, containing pro-inflammatory protein factors-cytokines).

The inventors of the present invention have found that the immunosuppressive potential of MSCs correlates with the expression level of characteristic marker molecules such as IDO, iNOS, COX-2 within cells, as well as HLA-DR on the surface. The need to measure the level of various markers is due to the fact that under different conditions when an MSC appears in the immunosuppressive phenotype, the level of one or two markers changes. At the same time, the level of the rest may not change. Therefore, in order for this method to be more universal, it is proposed to use a wider set of markers (four at once), which makes it possible to evaluate immunosuppressive properties in a variety of conditions.

The technical result of the present invention is the development of a method by which it is possible to quickly and accurately determine the immunosuppressive potential of MSCs. The proposed method for determining immunosuppressive properties has significant advantages compared with known methods. In particular, the level of MSC molecules themselves is subject to measurement, which directly correlates with their immunosuppressive activity. The level of these molecules determines the ability of MSCs to suppress the functions of immune cells, for example, their division upon activation. The claimed method is direct and faster than existing, since it does not require the presence of pre-activated immune cells in the culture. The proposed method is based on simple experimental approaches that are available in any modern laboratory. In addition, the claimed method is quite easy to quantify and normalize with respect to the control sample of cells, which allows routine measurements.

The stated technical problem is solved in that the method for determining the immunosuppressive properties of human mesenchymal stromal cells involves measuring the level of expression of the HLA-DR molecule on the surface of cell membranes by immunofluorescence staining, followed by measuring the expression level of IDO, iNOS and COX-2 molecules in the cells, while the conclusion about the presence of immunosuppressive properties is made if the expression level of at least one of the above molecules (IDO, iNOS, COX-2) or the level of HLA-DR in the studied MSCs is at least twice as high compared with a control sample of mesenchymal stromal cells.

A particular embodiment of the present invention is the aforementioned method, characterized in that the expression level in the cells is measured by semi-quantitative RT-PCR or RT-PCR in real time.

Short Description of Shapes

The Figure 1 shows a schematic representation of the effect of pro-inflammatory factors secreted by activated T cells on mesenchymal stromal cells (MSCs). Factors released into the culture medium by activated T cells (IFN, TNF, IL-1)? affect MSCs in such a way that MSCs acquire the ability to inhibit T-cell activation (exhibit immunosuppressive properties) due to the synthesis of anti-inflammatory molecules such as iNOS, IDO, COX-2.

The Figure 2 shows the change in the level of surface molecules involved in the presentation of the MSC antigen on the surface of MSCs after incubation with activated human peripheral blood leukocytes (LPC). The figure shows histograms showing the level of factors CD80, CD86 and HLA-DR on the surface of MSCs after the incubation described above. The corresponding proteins were stained using antibodies conjugated with fluorophores, after which the cell mixture was analyzed by flow cytometry, and the data were analyzed using the FlowJo software package. Staining with control isotypic antibodies and specific antibodies is shown. The top row presents the results of the analysis of cultures of naive white blood cells with MSCs, and the bottom row - activated ones.

Figure 3 shows an example of an analysis of agarose gel stained with ethidium bromide, amplification products of IDO cDNA. IDO cDNA was obtained by reverse transcription of total RNA from MSC samples incubated with activated leukocytes in various proportions. As a control, we used samples obtained from MSCs that did not come into contact with white blood cells. The numbers indicate samples that differ in the ratio of MSCs: white blood cells. 1 corresponds to a ratio of 1: 100, 2 - 1:50, 3 - 1:25, and 4 - 1: 12.5, respectively. One can see a change in the amount of cDNA amplification product depending on the ratio of MSCs: leukocytes, which correlates with the manifestation of MSCs immunosuppressive properties, expressed in their ability to slow down the division of leukocytes after activation and increase the level of activation markers on the cell surface. To normalize the amount of template for amplification, a Polymerase Chain Reaction (PCR) was performed with primers specific for actin cDNA.

Figure 4 shows the results of the experiment shown in Fig. 2, presented in the form of histograms. The ratio of MSCs: leukocytes is shown below. The ordinates show the relative units reflecting the amount of amplification product.

Figure 5 shows an example of an analysis on agarose gel stained with ethidium bromide, amplification products of iNOS cDNA. iNOS cDNA was obtained by reverse transcription of total RNA from MSC samples incubated with activated leukocytes in various proportions. As a control, we used samples obtained from MSCs that did not come into contact with white blood cells. The numbers indicate samples that differ in the ratio of MSCs: white blood cells. 1 corresponds to a ratio of 1: 100. 2 - 1:50, 3 - 1:25, and 4 - 1: 12.5, respectively. One can see a change in the amount of the cDNA amplification product depending on the ratio of MSCs: leukocytes, which correlates with the manifestation of MSCs immunosuppressive properties, which is expressed in their ability to slow down the division of leukocytes after activation and increase the level of activation markers on the cell surface. To normalize the amount of template for amplification, a Polymerase Chain Reaction (PCR) was performed with primers specific for actin cDNA.

The Figure 6 shows an example of analysis of agarose gel stained with ethidium bromide, amplification products of COX-2 cDNA. COX-2 cDNA was obtained by reverse transcription of total RNA from MSC samples incubated with activated leukocytes in various proportions. As a control, we used samples obtained from MSCs that did not come into contact with white blood cells. The numbers indicate samples that differ in the ratio of MSCs: white blood cells. 1 corresponds to a ratio of 1: 100, 2 - 1:50, 3 - 1:25, and 4 - 1: 12.5, respectively. One can see a change in the amount of the cDNA amplification product depending on the ratio of MSCs: leukocytes, which correlates with the manifestation of MSCs immunosuppressive properties, which is expressed in their ability to slow down the division of leukocytes after activation and increase the level of activation markers on the cell surface. To normalize the amount of template for amplification, a Polymerase Chain Reaction (PCR) was performed with primers specific for actin cDNA.

The implementation of the invention

In general terms, the proposed invention can be implemented as follows.

The present invention can be used to determine the immunosuppressive properties of any cultures of human mesenchymal stromal cells. Obtaining or isolation of mesenchymal stromal cells can be carried out by standard methods [for example, as described by Luria E.A., Panasyuk A.F., Friedenstein A.Y. // Transfusion. 1971. V.11, P.345-349], well-known specialist in the field of technology to which the present invention relates, and is not limited to the above examples. In particular, bone marrow or adipose tissue MSCs can be used for research.

For the cultivation of human MSCs in vitro, any medium containing the components necessary for the growth of adipose tissue mesenchymal stromal cells can be used, for example, the commercially available DMEM / F12 medium [Resources / media_formulation.55.html].

For a specialist it is obvious that the proposed method can be used to assess the immunosuppressive properties of the various types of exposure. According to the present invention, MSCs previously cultured in the presence of pro-inflammatory protein factors and cytokines can be used as test cells. In this case, intact MSC cells that were cultured in a start-up medium without the addition of the corresponding pro-inflammatory protein factors and cytokines can be used as control cells. In addition, genetically modified MSCs can be used as the studied ones to increase the immunosuppressive properties of MSCs, and parental cells that do not contain genetic modifications can be used as control ones.

According to the present invention, a method for determining the immunosuppressive properties of MSCs involves staining the surface of MSCs with antibodies specific for HLA-DR molecules, and analyzing the proportion and brightness of stained cells by flow cytofluorimetry, as well as isolating a total RNA mixture from MSCs and determining the mRNA level of genes encoding IDO proteins, iNOS and COX-2.

According to the present invention, for the immunofluorescence staining of HLA-DR molecules on the surface of MSCs, any commercially available antibody recognizing HLA-DR, directly conjugated to fluorophores, or unconjugated primary antibodies that are recognized by fluorophore-conjugated secondary antibodies can be used. Immunofluorescence staining (in the case of antibodies directly conjugated with a fluorophore) consists in a short incubation of cells with a solution of the necessary antibodies in phosphate buffer containing 0.1% bovine serum albumin (BSA). The antibody content is 0.5-1 μg per 100 μl of sample. After a 30-minute incubation, the cells are pelleted by centrifugation and washed with a BSA solution in phosphate buffer, after which the cells are pelleted again, resuspended in the above solution and analyzed using a flow cytometer.

Any methods suitable for isolating undegraded total RNA from human cells can be used to isolate total RNA from MSCs. The mRNA level of the analyzed genes (expression level) can be determined by any method well known to those skilled in the art. Particular examples that do not limit the present invention are the methods of semi-quantitative Reverse Transcription - Polymerase Chain Reaction (semi-quantitative RT-PCR) and Reverse Transcription - Polymerase Chain Reaction in real time (the so-called Real-time PCR (RT-PCR in real time time)). These methods are based on the synthesis of a DNA copy (cDNA) on the total mRNA matrix of the studied cells. The resulting cDNA is used as a template for amplification of a specific short DNA fragment using a pair of specific primers. The relative amount of mRNA to be studied is determined by measuring the amount of the product of the polymerase chain reaction either after the reaction (semi-quantitative RT-PCR) or directly during the amplification reaction (RT-PCR real-time). The kinetics of product accumulation depends on the initial number of copies of mRNA in the preparation. To determine the relative change in the level of a specific mRNA, the values obtained in the experimental samples are normalized to the value obtained in the control sample. Thus, it is possible to determine the relative change in the level of the studied mRNA in this sample of cells.

According to the present invention, the conclusion about the presence of immunosuppressive properties in the studied MSCs can be made if the expression level of at least one of the analyzed molecules in the studied mesenchymal stromal cells is at least twice as high as in the control cells.

The composition of the reaction mixture, as well as a list of primers and probes that can be used to analyze the expression level of genes encoding IDO, iNOS, COX-2 proteins by the RT-PCR methods described above, are not limited in any special way. Appropriate nucleotide sequences can be selected using standard programs well known to those skilled in the art, for example, using the Primer 3 package.

The molecules considered by the authors of the present invention can be arbitrarily divided into two groups: surface and intracellular. The surface molecule includes the HLA-DR molecule, which is a molecule of the main histocompatibility complex of the second class. Cells use these molecules to present short antigen peptides on their surface. Such a molecule with a bound peptide is involved in interaction with a T cell receptor on the surface of CD4 T helper cells. This interaction is necessary for the effective activation of the immune response against bacteria and other pathogens. HLA-DR molecules on the surface of cells thus characterize the ability of cells to present an antigen and initiate an immune response. It is known that the appearance of HLA-DR on the surface of MSCs indicates their ability to activate T cells.

Among intracellular molecules, first of all, IDO should be distinguished. This enzyme, indolyl-2,3-deoxygenase, which catalyzes the decomposition of the essential amino acid tryptophan to kinurenine. It is known that tryptophan in large quantities is absorbed by activated T cells, which undergo massive changes in response to stimulation and multiply protein synthesis. On the one hand, depletion of the free tryptophan pool slows down the division of T cells, and on the other hand, the tryptophan cleavage product, kinurinine, is a toxic molecule for T cells and, according to published data, can cause apoptosis in them. Thus, IDO is a factor that negatively affects the function of T cells.

iNOS (inducible NO synthase) is an enzyme whose amount in cells increases dramatically in response to the presence of pro-inflammatory cytokines. According to its name, this protein synthesizes NO released from cells, which is an important factor in the early stages of the immune response, directly participating in the destruction of pathogens. In addition, it was found that NO is an inducer of mitochondrial apoptosis, which depends on the activation of caspase-9 and caspase-3.

COX-2 (cyclooxygenase-2) is an enzyme that is involved in the synthesis of prostaglandin E2 (PGE2), an important small molecule that suppresses the function of immune cells by binding to a specific receptor on the surface of immune cells.

The invention is implemented in a particular case as follows.

Examples

1. Isolation and cultivation of mesenchymal stromal cells of adipose tissue in vitro

Human subcutaneous fat (from 0.5 to 10 ml) was collected during a surgical operation in operating rooms in compliance with aseptic and antiseptic rules. Isolation of cells from the material obtained as a result of the operation was carried out under sterile conditions of a laminar box.

The main steps include:

Grinding tissue in a tissue disintegrator gentleMACSdissiciator (Myltenyi Biotec) to the consistency of a suspension of small (no more than 2 cubic millimeters in size) pieces.

Mixing crushed adipose tissue with solutions of type I collagenase enzymes (200 units / ml, Worthington Biochemical, USA) and dispase (40 units / ml, Sigma, Germany) with a ratio of tissue volume (in ml) to the volume of enzyme solution (in ml) 1 : 2.

Incubation of the sample in a CO ₂ incubator (5% CO ₂ ; 95% air) at 37 ° C for 30-45 min with constant shaking.

Adding at the end of the incubation an equal volume of DMEM / F12 medium containing 100 units / ml penicillin, 100 units / ml streptomycin, 2 mM glutamine, 1 mM sodium pyruvate (HyClone), 10% fetal bovine serum (HyClone).

Centrifugation of the obtained sample at 200g for 5 minutes

Removal using a vacuum pump of a whitish surface layer consisting of mature adipocytes and pieces of enzymatically untreated tissue.

Suspension of a precipitate consisting of stromal cells of adipose tissue and vascular wall and blood cells in sterile deionized water to lyse red blood cells.

Adding to restore the osmotic pressure in the sample 10 times the volume of the medium DMEM / F12.

Filtering the resulting cell suspension through nylon filters with a pore size of 100 μm (BD Falcon Cell Strainer, USA).

Centrifugation of the filtrate at 200 g for 5 minutes

Removal of supernatant.

Determination of the concentration of primary cells extracted from subcutaneous adipose tissue using a Gariaev camera under a microscope or using an automatic cell counter (Countess, Invitrogen) at an air temperature of + 20- + 25 ° C and a relative humidity of 40-70%.

Resuspension of the pellet in DMEM / F12 growth medium containing 100 u / ml penicillin, 100 u / ml streptomycin, 2 mM glutamine, 1 mM sodium pyruvate (HyClone), 10% fetal bovine serum (HyClone) to a concentration of 5 × 10 ⁴ fragments / cm ³ .

Planting the resulting suspension on Petri dishes (Corning Costar) and incubation at 37 ° C, 5% CO ₂ in a CO ₂ incubator for 2 days.

Change media in Petri dishes to remove non-adherent cells. The cell yield should be 4-7x10 ⁴ attached cells per 1 ml of tissue. The growth medium is changed every 2-3 days until a 70-80% monolayer density is reached.

Removal of growth medium from Petri dishes.

Twice washing the monolayer with a solution of DPBS (HyClone).

Monolayer treatment with a mixture of 0.25% trypsin solution and 0.02% EDTA (1: 1) (HyClone).

Incubation for 15 min at 37 ° C, 5% CO ₂ in a CO ₂ incubator.

Suspension of adherent cells by pipetting.

Determination of the suspension concentration of cultured mesenchymal stromal cells of human adipose tissue using a Garyaev camera under a microscope or using an automatic cell counter (Countess, Invitrogen) at an air temperature of + 20- + 25 ° C and a relative humidity of 40-70%.

Adding to the resulting cell suspension 3 times the initial volume of growth medium.

Planting the suspension on Petri dishes in a ratio of 1: 3.

2. Isolation, cultivation and activation of immune cells in vitro

With the consent of healthy donors, in a sterile bottle (Corning Costar) containing a sterile EDTA solution in the amount of 1.2-2 mg / ml of blood, a pH of 7.2 was taken 10-25 ml of peripheral blood from the ulnar vein.

Main steps:

Dilution of native venous blood with 0.01 M sterile PBS solution (pH 7.2) in a ratio of 1: 1.

Add 50 ml Ficoll Paque® (Pharmacia Biotech) with a density of 1.077 to 50 ml conical tubes (Corning Costar).

Layering a diluted suspension of blood cells on Ficoll Paque® in a ratio of 1.5: 1.

Centrifugation of a suspension of blood cells in a density gradient of Ficoll Paque® at 400 g for 30 min.

Transferring interphase cells to a new 50 ml tube, adding 50 ml HBSS solution (HyClone).

Cell deposition by centrifugation at 200 g for 5 min.

Removal of supernatant.

Resuspend the pellet in 50 ml of HBSS solution and centrifuge at 200g for 5 minutes.

Resuspension of sediment in DPBS (HyClone).

Determination of the suspension concentration of cultured mesenchymal stromal cells of human adipose tissue using a Garyaev camera under a microscope or using an automatic cell counter (Countess, Invitrogen) at an air temperature of + 20- + 25 ° C and a relative humidity of 40-70%.

Dilution of a suspension of blood cells to a concentration of 1 × 10 ⁷ cells with DMEM / F12 medium containing 100 u / ml penicillin, 100 u / ml streptomycin, 2 mM glutamine, 1 mM sodium pyruvate (HyClone), 10% fetal bovine serum (HyClone).

Adding phytohemagglutinin cells (PHA, Sigma) to activate the resulting cell suspension to a final concentration of 10 μg / ml.

Incubation of the sample in a CO ₂ incubator (5% CO ₂ ; 95% air) at 37 ° C for 2 hours.

Precipitation of a suspension of activated blood cells by centrifugation at 200 g for 5 minutes

Resuspend the pellet in 50 ml of HBSS solution, centrifuging at 200g for 5 minutes.

Resuspend the pellet in DMEM / F12 growth medium containing 100 units / ml penicillin, 100 units / ml streptomycin, 2 mM glutamine, 1 mM sodium pyruvate (HyClone), 10% fetal bovine serum (HyClone) to a concentration of 1 × 10 ⁷ cells and plating on Petri dishes with pre-cultured MSCs.

Incubation in a CO ₂ incubator (5% CO ₂ ; 95% air) at 37 ° C for 48 hours

3. In vitro activation of MSCs using pro-inflammatory cytokines TNF-alpha, IFN-gamma

Adding to the monolayer of isolated and cocultured MSCs (method 1) TNF-alpha at a concentration of 20 ng / ml and IFN-gamma at a concentration of 30 ng / ml.

Incubation in a CO ₂ incubator (5% CO ₂ ; 95% air) at 37 ° C for 48 hours

4. Assessment of the level of HLA-DR expression on the surface of MSC membranes upon cocultivation with activated lymphocytes

Main steps:

Co-cultivation of mesenchymal stromal cells of adipose tissue with activated peripheral blood lymphocytes or pro-inflammatory cytokines TNF-alpha, IFN-gamma in a CO ₂ incubator (5% CO ₂ ; 95% air) at 37 ° C, 95% air humidity for 48 hours.

Removal of supernatant.

Twice washing the monolayer with a solution of DPBS (HyClone).

Treatment of the monolayer with a mixture of 0.25% trypsin solution and 0.02% EDTA (1: 1) (HyClone), incubation for 15 min at 37 ° C, 5% CO ₂ in a CO ₂ incubator.

Suspension of adherent cells by pipetting.

Centrifugation of the resulting suspension at 200 g 4 ° C for 5 minutes

Resuspend the pellet in 100 μl of phosphate-saline solution containing 1% fetal calf serum.

Preparation of a working solution of antibodies to HLA-DR at the rate of 100 μl per sample.

Adding a working solution of antibodies to the samples, incubation for 20 min in the dark at + 4 ° C.

Cell precipitation by centrifugation 200 g 4 ° C 5 min.

Twice washing of cells with phosphate-saline solution containing 1% fetal calf serum.

Centrifugation Precipitation 200 g 4 ° C 5 min

Resuspend cells in 500 μl of phosphate-saline solution containing 1% fetal calf serum, transfer to FACS tubes.

Taking measurements on a FACS CANTO II instrument (BD biosciences).

After cytometric measurements, it is necessary to analyze the level of the HLA-DR molecule on the surface of normal MSCs and MSCs activated by pro-inflammatory cytokines. For this, appropriate histograms can be constructed using the FlowJo software package. An increase in the proportion of cells carrying an elevated level of HLA-DR on the surface indicates the acquisition of an immunosuppressive phenotype by cells. As a sample for comparison, it is necessary to use intact MSCs each time.

5. Measurement of the level of expression of IDO or iNOS in MSCs uncultivated with activated lymphocytes or pro-inflammatory cytokines.

Co-cultivation of adipose tissue mesenchymal stromal cells with proinflammatory cytokines TNF-alpha, IFN-gamma or with activated peripheral blood lymphocytes in the presence of intercellular contacts to determine IDO or in a contactless system using semi-permeable membranes (transwell) to determine iNOS.

Supernatant Removal

Double washing of cells with a solution of DPBS (HyClone). Isolation from RNA samples:

a. Trisol (Invitrogen) was added to the cell monolayer at a rate of 750 μl per 100 thousand MSC;

b. The resulting lysate was transferred to 1.5 ml Eppendorf tubes, 0.2 ml Chloroform (Himmed) was added, vortexed for 15 s;

c. The phases were separated by centrifugation in a microcentrifuge at 13,000 rpm, T 20 ° C;

d. The aqueous phase was taken into new 1.5 ml tubes, add 0.5 ml of isopropanol (Himmed), vortexed for 15 s, incubated 10 min, T at 20 ° C;

e. Centrifuged on a microcentrifuge for 10 min 13000 rpm, T 20 ° C;

f. The pellets were washed with 500 μl of a 70% ethanol solution, vortexed for 15 s, centrifuged in a microcentrifuge for 5 minutes at 13,000 rpm, T 10 ° C;

g. The supernatant was removed, the washed precipitates were dried on a table at room temperature, but not until completely dry;

h. The precipitates were purified with RNase-purified water (Invitrogen);

i. The concentration was measured on a Nanodrop spectrophotometer, the purity of the obtained total RNA was determined by the ratio of peaks 260/280. The ratio should be more than 1.5, the optimal value of 1.8-1.9;

The implementation of reverse transcription of isolated RNA

a. A mixture of components was prepared: 1 μl Oligo (dT) 20 non-specific primers (Invitrogen) as, 25 mM dNTP dinucleotide triphosphate mixture (Fermentas), 7 μl RNAfree water (Invitrogen), 500 ng of isolated total RNA concentration of 100 ng / μl;

b. Placed in a thermostat, incubated for 5 min at 65 ° C, then cooled on ice at 4 ° C;

c. A mixture of the components was prepared: 4 μl of 5-fold FSbuffer buffer (Invitrogen), 1 μl of 0.1 M dithiotriethol (DTT, Invitrogen), 1 μl of RNAse out ribonuclease inhibitor (Invitrogen), 1 μl of superscript III reverse transcriptase (Invitrogen);

d. Added to the samples, mixed, incubated at 50 ° C for 50 minutes, then inactivated 15 minutes at 70 ° C. Distributed into aliquots;

e. A polymerase chain reaction (PCR) was performed;

f. A mixture containing 5 μl of Fermentas PCR buffer, 5 μl of a 25 mM MgCb solution, 0.5 μl of a 25 mM dNTP dinucleotide triphosphate mixture (Fermentas), 39 μl of water was prepared in 0.2 ml flat-cap PCR tubes RNAse free (Invitrogen), 0.25 μl of a 100 μM IDO primer solution:

IDOp2 Forward 5-AGCCCCTGACTTATGAGAACATGGA-3

IDOp2 Reverse 5-CCAGCCAGACAAATATATGCGAAGAA-3,

or 0.25 μl of a 100 μM iNOS primer solution:

iNOS IDOp1 Forward 5-ATGGCACACGCTATGGAAAACTC-3

iNOS IDOp1 Reverse 5-CTAGACGTGCAAGGCGCTGTGACT-3

or 0.25 μl of a 100 μM primer solution for the SOX-2 gene

COX2p1 Forward 5-TCACGCATCAGTTTTTTCAAGACAGAT-3

COX2pl Reverse 5-TACATCATCAGACCAGGCACCAGA-3

100 ng of obtained cDNA based on 100 ng of the original total RNA. 1 μl Taq polymerase (DNA technology).

g. Tubes were placed in an amplifier;

h. Amplification was performed with the following parameters:

i. 1 cycle - initial denaturation (95 ° C, 5 min);

ii. 35 cycles, including: denaturation (95 ° C, 30 sec), annealing 54 ° C 30 sec, elongation 72 ° C 1 min;

iii. 1 cycle - final elongation (72 ° C, 5 min);

i. Prepared 2% agarose gel prepared on TBE buffer with ethidium bromide;

k. Contributed to one of the wells of the gel, the standard mass of DNA GeneRuler ™ 50 bp DNA Ladder, 50-1000 bp (Fermentas), in the other wells 20 μl of PCR product;

l. 4 μl of a 6-fold dye loading dye buffer (Fermentas) was added to all wells;

m. Electrophoresis was carried out at a voltage of 150 V for 40 min;

n The gel was placed in a Versadoc gel-documenting system (Biorad) and photographed under ultraviolet radiation;

about. After completion of the gel electrophoresis procedure and gel photographing, the product length was estimated.

A comparison of the expression level of IDO, iNOS, COX-2 genes in intact MSCs and MSCs treated with pro-inflammatory cytokines allows us to estimate the level of immunosuppressive activity, which directly correlates with the level of synthesis of these effector molecules. According to the data obtained by the authors of the present invention, an increase in the level of these molecules at least twice indicates the manifestation of an immunosuppressive phenotype by cells.

Claims (2)

1. A method for determining the immunosuppressive properties of human mesenchymal stromal cells, comprising measuring the level of expression of the HLA-DR molecule on the surface of cell membranes by immunofluorescence staining and measuring the expression level of IDO, iNOS and COX-2 molecules in the cells, and the conclusion about the presence of immunosuppressive properties is made, if the expression level of at least one of the above molecules is two times higher compared with a control sample of mesenchymal stromal cells. 2. The method according to claim 1, characterized in that the measurement of the expression level in the cells is carried out by the method of semi-quantitative RT-PCR or RT-PCR in real time.

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