RU2722669C1 - Method for producing associates of hematopoietic and stromal progenitor cells capable of suppressing activation and proliferation of allogenic lymphocytes - Google Patents

Abstract

FIELD: biotechnologies.SUBSTANCE: invention relates to the biotechnology. Described is a method for producing associates of hematopoietic and stromal progenitor cells. Preparation of stromal sublayer of MSC from stromal-vascular fraction of adipose tissue with stop of cell divisions with mitomycin C, mononuclear fraction of umbilical blood is added and are co-cultivated for 144 hours to obtain associates of HSPC with atMSC, capable also effectively, as atMSC in monoculture, to suppress activation and proliferation of allogenic lymphocytes. Method enables producing associates of hematopoietic stem and progenitor cells of umbilical cord blood (HSPC) and atMSC, in which the population of HSPC is more than by 80 % presented by precursor cells with complex phenotype CD34/CD133associated with stromal sublayer atMSC.EFFECT: invention can be used for producing associates, in which multipotent mesenchymal stromal cells from adipose tissue (atMSC) retain immunosuppressive activity.4 cl, 4 dwg, 2 tbl, 1 ex

Description

The invention relates to medicine, namely to biotechnology, and can be used to obtain associates of hematopoietic and stromal progenitor cells in which multipotent mesenchymal stromal cells (MSCs) retain immunosuppressive activity.

Currently, MSCs from various tissue sources are considered as promising material for cell therapy and regenerative medicine (Bianco et al., 2013; Caplan, 2015). This is due to their high functional activity. MSCs are involved in physiological and reparative remodeling, as well as in the formation of various tissue niches, primarily hematopoietic (Kumar et al., 2017). The hematopoiesis-supporting activity of MSCs was found to be in demand in ex vivo protocols of expansion of hematopoietic stem and progenitor cells (HSPCs) from umbilical cord blood to overcome the problem of HSPA deficiency in one sample. In contrast to suspension monocultures of HSPC, where in the overwhelming majority of cases hematopoietic cells are used for expansion after selection, most often by the CD34 antigen, in the co-culture, both the initial nucleated cells of the PC and individual fractions are expanded. However, some protocols consist of sequential stages, including different options for co-cultivation. For example, in Robinson et al., (2006), at first, cord-containing cord blood cells were co-cultivated with MSCs for 7 days, then the suspension fraction was collected and cultured in suspension with the addition of hepatopoietic cytokines, and the attached HSPCs were amplified for another 7 days. Then, the newly grown floating HSPCs were added to the previously selected suspension. Such a protocol made it possible to obtain a GSPK population significantly enriched in both undifferentiated (CD34 ⁺ ) and committed (CFU) hematopoietic precursors. Cellular products prepared as described have already passed limited clinical trials (De Lima et al., 2012).

However, the possibilities of co-culture of hematopoietic and stromal precursors as a source of various cellular products have not yet been fully exploited. As the achieved result, the number of suspension hematopoietic precursors or their derivatives, forming hematopoietic colonies in semi-liquid media, or capable of supporting hematopoiesis for a long time in vivo (Flores-Guzman et al., 2009), is usually taken into account. Moreover, HSPCs associated with the stromal sublayer are usually not analyzed. However, as is well known, the interaction of HSPC with stromal cells, in particular with MSCs, is accompanied by the distribution of hematopoietic precursors into various compartments relative to the stromal layer. Hematopoietic precursors may be present in the suspension in an unattached state, attached to the surface of the MSC or located in the space under the layer of the MSC, as was already demonstrated by Dexter et al. (1977). HSPCs under the MSC monolayer are less mature and have less proliferative activity, whereas hematopoietic precursors adhered to the surface of MSCs actively divide. Hematopoietic cells are able to move from one compartment to another. Hematopoietic precursors with the CD34 ⁺ CD38 ^- phenotype predominantly migrate through the MSC layer (Jing et al., 2010). It is possible that conditions are created in the space under the MSC for maintaining hematopoietic precursors in an immature state, or immature precursors are selectively attracted there (Wagner et al., 2007). Thus, in the GSPK / MSC co-culture there are always two fractions of GSPK: the suspension fraction, which is characterized in sufficient detail, and the much less studied population - stroma-associated GSPK, according to some data representing the least committed part of hematopoietic precursors (Wagner et al., 2007; Jing et al., 2010).

Due to the fact that MSCs are immunoconductive, they are used for allogeneic administration in various clinical protocols (Miguel et al., 2012), including to improve the engraftment of HSPC during transplantation. From this point of view, cell products consisting of MSCs and related HSPCs are of significant interest. Firstly, there is no need to separate stromal and hematopoietic cells before transplantation. Secondly, other MSC activity can be used, such as the production of biologically active mediators.

Therefore, the objective of the invention was to develop a technology that allows the production of associates consisting of hematopoietic predecessors of cord blood and MSCs (MSC-HSPC), in which MSCs retain the ability to suppress the activity of allogeneic immune cells.

To obtain MSC-GSPK associates, the following steps were taken:

- MSCs were isolated from the stromal-vascular fraction of adipose tissue (gMSC) and were constantly cultured at a concentration of O ₂ in the medium of 5%,

- preparation of the stromal sublayer was carried out, treating the zhMSC with mitomycin C to stop cell divisions,

- the mononuclear fraction of umbilical cord blood (MNC PK) was added to the monolayer of zhtMSC treated with mitomycin C, and they were cultured for 72 hours together,

- selection of hematopoietic precursors from MNC PK was performed due to adhesion to zhMSC,

- cultured zhMSCs with PC MNCs attached to them for 72 hours with the formation of a stroma-associated population of HSPC (zhtMSK-HSPC).

The following steps were performed to analyze the immunosuppressive activity of zMTSC in associates:

- co-cultivation of the associations of zhtMSK-HSPK with allogeneic mitogen-stimulated peripheral blood T cells (T cells) for 72 hours,

- determination of the proportion of T cells expressing a marker of late activation of histocompatibility class II antigen (HLA-DR),

- determination of the proportion of proliferating T cells.

The technical result of the proposed method is to obtain associates GSPK and zhTMSK, in which:

- the HSPC population is more than 80% represented by progenitor cells with the CD34 ⁺ / CD133 ⁺ phenotype associated with the stromal sublayer from zhMSC and forming cobblestone regions (COOB).

- zhTMSK as a part of associates are capable of inhibiting, as well as zhMSK in a monoculture, the activation and proliferation of allogeneic lymphocytes, which can be claimed when using the obtained associates as hematopoietic grafts.

Brief Description of the Drawings

FIG. 1 - Scheme for the production of HSPC after co-cultivation of MNC PK with mitomycin C-treated zhtMSK.

FIG. 2 - Associations zhtMSK-GSPK after 144 hours of ex vivo expansion. gtMSC-associated HSPCs on the surface of mitomycin C-treated gtMSCs and under the stromal sublayer — COOB.

FIG. 3 - GSPK and zhTMSK constituting associates, when analyzed on a flow cytometer, the distribution of size and granularity.

FIG. 4 - GSPK and zhTMSK constituting associates, when analyzed on a flow cytometer, immunophenotyping by expression of CD45 and CD90 antigens.

Tab. 1 - The proportion of phytohemagglutin-stimulated allogeneic lymphocytes expressing the antigen of the major histocompatibility complex class II (HLA-DR), and the proliferative index in the presence of zhMSC and zhMSC-HSPK associates.

The results are presented as the multiplicity of differences in gene expression in gTMSC from associates against gTMSC in monoculture. Data from three independent experiments.

Tab. 2 - Differential expression of genes encoding immunosuppressive molecules in gTMSC (gTMSK from associates against gTMSC).

The results are presented as the multiplicity of differences in gene expression in gTMSC from associates against gTMSC in monoculture. Data from three independent experiments.

IL8 - interleukin 8; LIF - a factor inhibiting leukemia; IDO-indolamine-2,3-dioxinase; CCL2 (MCP-1) - monocytic chemoattractant protein-1; IL6 - interleukin 6; VEGF - vascular endothelial growth factor; PTGS2 - prostaglandin-endoperoxide-synthase-2.

DETAILED DESCRIPTION OF THE INVENTION

Isolation of the mononuclear fraction of cord blood (MNC PC).

The procurement of PCs was carried out with written informed consent of the examined healthy women in labor at the departments of the Scientific Center for Obstetrics, Gynecology and Perinatology named after Acad. IN AND. Kulakova. Blood was collected in bags of the donor system with the anticoagulant TSFDA-1 and processed for 24 hours. The preparation of the “nucleated cell concentrate” was carried out by the double centrifugation method in accordance with the registered medical technology (FS2009 / 387 of 11/23/2009). After erythrocyte sedimentation and removal of excess plasma, the fraction of nucleated PC cells was resuspended in autologous plasma with the addition of 10% dimethyl sulfoxide (Sigma, USA) and 1% Dextran-40, packaged in cryovials, and subjected to program freezing to a final temperature of -90 ° C in accordance with Standard stem cell bank operating procedures. On the day of the experiment, the cells were thawed in a water bath at + 37 ° C and washed from the cryoprotectant in excess of the culture medium (see below). After assessing viability in the trypan blue test, the cell concentration was adjusted to 1.5-2.5 × 10 ⁶ cells / ml and used for 30 minutes.

Isolation and cultivation

To obtain MSCs, the stromal-vascular fraction of human adipose tissue was used. Cells were isolated according to the standard method and cultured with zMTSCs continuously at 5% O ₂ in a multigas incubator (Sanyo, Japan) in α-MEM medium (Gibco, USA) with the addition of 10% inactivated fetal bovine serum (Hyclone, USA), 100 units / ml of penicillin and 100 μg / ml of streptomycin (Biolot, Russia). Upon reaching 70-80% confluency, the cells were reseeded. For experiments, MSCs of 2-4 passages were used (Buravkova et al., 2009).

Preparation of the stromal sublayer to obtain zhtMSK-GSPK associates

Mitomycin C (1.5 μg / ml) was added to gtMSCs, which reached 70-80% of the confluent monolayer, in growth medium for 18 hours. After this, the cells were thoroughly washed with isotonic phosphate buffer, detached from the substrate using trypsin-EDTA solution and scattered into 60 mm diameter Petri dishes at the rate of 8 × 10 ³ cells / cm ² and placed in a CO ₂ incubator at 20% O ₂ (Sanyo, Japan). The density of the monolayer zhtMSK after attachment and spreading was 70-80%. After 48 hours, zhTMSK was used for co-cultivation with PCS MNCs. Obtaining zhtMSK-associated GSPK

The scheme for HSPC preparation upon co-cultivation of PC MNCs with zhMSC is shown in Figure 1. Co-cultivation was carried out for 144 hours. First, a suspension of PC MNCs (10 × 10 ⁶ ) in 5 ml of medium was added to the premonolayer treated with mitomycin C ztMSC in Petri dishes 60 mm in diameter and cultivated for 72 hours. Then, non-adherent cells were selected and the culture medium was replaced with fresh. Co-cultivation led to adhesion of a portion of the MNCs of PK. These cells, upon further cultivation for another 72 hours, gave rise to a new population of suspension HSPCs. At the same time, part of the GSPCs remained associated with zhTMSCs, forming two compartments - on the surface and under the monolayer of zhTMSCs. After co-cultivation, the newly formed suspension of HSPC was removed, and the remaining htMSC-HSPC complex was trypsinized and samples were prepared to characterize the immunophenotype, determine viability and immunosuppressive activity using flow cytometry.

Immunophenotypic analysis

Phenotyping was carried out by flow cytometry using the appropriate antibodies conjugated with FITC, PE, or APC dyes: for zhMSCs, CD73, CD90, CD 105 (IO Test, Beckman Coulter, USA); for GSPK - CD45, CD34 (BD Biosciences, USA); for T cells - CD3, HLA-DR (IO Test, Beckman Coulter). To prepare samples, zhTMSK or zhMSK-HSPK associates were detached from the substrate using a trypsin-EDTA solution. Suspensions of T cells, trypsinized gtMSCs or gtMSC-HSPC associates were stained with antibodies according to the manufacturer's instructions. Cells were analyzed on an Accuri C6 flow cytometer (Becton Dickinson, USA). To evaluate the level of non-specific staining, an isotopic control (anti-IgG1 and anti-IgG2a, BD Biosciences) was used. The data obtained were analyzed in a program for collecting, auto-compensation, data processing and image acquisition BD Accuri C6 software.

Gene expression

To assess the mRNA level of genes responsible for immunosuppressive activity, the zMTSCs obtained after magnetic immunoseparation were lysed using Trizol reagent (Qiagen, United States) and mRNA was isolated, cDNA was synthesized on its matrix using the Quantitech Reverse Transcription Kit (Qiagen, United States). Using RT-PCR using appropriate primers (Qiagen) assessed the level of gene expression.

Immunomodulatory activity of T cells

Allogenic mitogen-stimulated peripheral blood T cells in RPMI-1640 medium (5% FCS, 10 μg / ml PHA) in a ratio of 1:10 were added to a suspension of trypsinized gtMSC or gtMSC-HSPC associates (cultured for 72 hours). All non-adherent cells were then collected. T cells in which the level of activated (HLA-DR-positive) and proliferated cells was assessed by cytofluorimetric analysis.

T cell proliferation

Before the experiment, T cells were pretreated with carboxyfluorescein succinimidyl ether (CFSE, Invitrogen, USA) according to the standard protocol. The proliferation level was estimated by the cytofluorimetric method according to the proportional decrease in the CFSE fluorescence intensity during T cell division.

Statistics. Statistical analysis was performed using the Microsoft Excel 2000 and Statistica 10.0 software package and the Mann-Whitney test. Differences were considered significant at p <0.05.

Example 1

A suspension of MNC PK in the amount of 2 × 10 ⁶ cells / ml was added to the premonolayer of mitomycin C-treated zhtMSCs (70-80% confluency), cultured at 5% O ₂ .

The co-cultivation of MNC PK and gtMSC was carried out at a concentration of O ₂ in the medium of 20%. PC MNCs that were not adhered for 72 hours were removed and the remaining cells were further cultured for another 72 hours at an O ₂ concentration of 20% in the medium with the formation of a stroma-associated population of HSPCs.

In the course of the work, it was shown that as a result of co-cultivation for 144 hours, a population of hematopoietic cells associated with the stromal sublayer is formed. The figure 2 shows a representative image of the associates zhtMSK-GSPK. Thin white arrows indicate gtMSC-associated HSPCs on the surface of gtMSCs, thin black arrows indicate gtMSC-associated HSPCs under the stromal sublayer - “cells forming cobblestone pavement areas - (COOB)”, stars indicate gtMSCs (Giemsa differential staining, bright field, scale segment - 50 μm).

zhMSCs in cell associates had a typical fibroblast-like phenotype, had high viability (more than 99%) and expressed markers characteristic of stromal differential cell: CD90, CD73, CD 105.

The viability of HSPC and zhMSC in associates according to the trypan blue test was more than 95%.

After trypsinization of the zhMMSK-HSPK associates, samples were obtained for cytofluorimetric analysis containing both types of cells.

A representative distribution diagram of zhMMSCs and HSPCs from associates in size and granularity is shown in Figure 3. Analysis by size / structure did not reveal clearly distinguishable subpopulations. The ratio of gtMSC and HSPC in associates according to flow cytofluorimetry averaged 3: 1.

Next, the cells were immunophenotyped in suspension. Hematopoietic cells were identified by expression of the panleukocyte antigen CD45, and gtMSK by expression of the CD90 antigen, as shown in figure 4. Next, the analysis was carried out among CD45-positive cells, among which cells expressing only CD45 (upper left quadrant) were identified, as well as cells carrying both markers (CD45 ⁺ CD90 ⁺ ) (upper right quadrant). As you know, CD90 is expressed not only by stromal cells, but also by early HSPCs (Pineault and Abu-Khader, 2015). It can be assumed that cells with the immunophenotype CD45 ⁺ CD90 ⁺ are earlier precursors than CD45 ⁺ CD90 ⁺ cells. Primitive HSPCs with the phenotype CD45 ⁺ / CD90 ⁺ / CD34 ⁺ were identified. Among cells associated with zhMSCs, the proportion of uncommitted HSPCs (CD34 ⁺ ) averaged 63%.

To assess the ability of gTMSCs to immunomodulate, their effect on the activation and proliferation of mitogen-stimulated T cells was determined. In a suspension of trypsinized gTMSCs in monoculture and in co-culture with HSPC, PHA-activated allogeneic peripheral blood mononuclear cells (Lymphocytes) were added. Table 1 shows the results of determining the proportion of T cells expressing a marker of late activation of HLA-DR, as well as the proportion of proliferating T cells. After 72 hours, the suppressive effect on T cells was similar for zhMSC in monocultures and associates: a twofold decrease in the proportion of HLA-DR-positive T cells and a twofold decrease in the proliferative index of T cells were revealed.

Differential analysis of the expression of gTMSC genes encoding the main immunosuppressive molecules showed that gTMSCs in associations with HSPCs were characterized by increased expression of the IDO, PTGS2, LIF genes encoding the corresponding molecules, as shown in Table 2.

Currently, MSCs are used in various protocols of cell therapy and regenerative medicine, due to their potential to create a regenerative microenvironment through the production of various biologically active mediators (Murphy et al., 2013; Syed and Evans, 2013). zhMSCs are considered as one of the most attractive sources of MSCs (Bruun et al., 2018). This is primarily due to the immunomodulatory activity of the obtained gTMSCs (Bartholomew et al., 2002).

In the work of Bartholomew et al. (2002), which became fundamental in studies of the immunosuppressive activity of MSCs, showed an increase in the time of rejection of allogeneic skin flaps in monkeys that were injected with allogeneic MSCs. A similar effect was described in experiments on corneal transplantation with presensitized rats in which the administration of allogeneic MSCs prevented rejection of transplanted tissues (Lohan et al., 2018). Clinical studies have shown a decrease in the severity of the graft versus host (GVHD) reaction in patients who are resistant to steroids with the introduction of MSCs (Sanchez-Guijo et al., 2014; Amorin et al., 2014). In addition, co-transplantation of MSCs improved engraftment and reduced the risk of rejection during hematopoietic cell transplantation (Jitschin et al., 2013; Amorin et al., 2014). The ability to suppress the activity of the immune cells of the recipient allows the use of stromal precursors from adipose tissue - zhMSC as a “third tissue” to reduce the severity of GVHD (Jurado et al., 2017).

zhMSCs, as well as bone marrow-derived MSCs, have the ability to support hematopoiesis, which can be used for ex vivo expansion of hematopoietic precursors (Wagner et al., 2007; Nakao et al., 2010; Nishiwaki et al., 2012; Andreeva et al ., 2015, 2018). This is especially true for cord blood HSPCs when the HSPC content in one sample is insufficient for transplantation (Broxmeyer and Farag, 2013).

We have shown that, as a result of co-cultivation of PS MNCs and zhtMSKs, it is possible to obtain a flotation and associated with zhtMSK HSPK populations. Part of the GSPK in the associates showed a primitive phenotype, confirmed by their ability to form “cobblestone pavement areas”. It is believed that such HSPCs are capable of long-term maintenance of hematopoiesis in vivo (de Haan and Van Zant, 1997). It is possible that zhMSCs used for co-cultivation may be of interest not only as a feeder layer for HSPCs, but also demonstrate activity characteristic of MSCs used as “third tissue”.

In the present work, as a result of ex vivo expansion of umbilical cord blood mononuclear cells into the sublayer of stromal cells from adipose tissue, heterocellular associates consisting of undifferentiated hematopoietic precursors - HSPC and stromal cells - zhMSCs were obtained. HSPC-associated zhMSCs showed an increase in the expression of genes responsible for immunosuppressive activity (IDO, LIF, PTGS2) and inhibited the activation and proliferation of allogeneic stimulated T cells.

Thus, we have proposed an effective method for producing cell preparations representing associates of uncommitted HSPCs and zhMSCs that are able to maintain ex vivo hematopoiesis and retain the ability to immunosuppress, which may be in demand when using zhMMSK-HSPK cell associates to restore blood formation.

Bibliography

Buravkova L.B., Grinakovskaya O.S., Andreeva E.R., Zhambalova A.P., Kozionova M.P. Characterization of mesenchymal stromal cells from human lipoaspirate cultured at a low oxygen content. Cytology. 2009.51 (1): 5.

Amorin B., Alegretti A.R., Valim V., Pezzi A., Laureano A.M., da Silva M.A., Wieck A., Silla L. Mesenchymal stem cell therapy and acute graft-versus-host disease: a review. Hum Cell. 2014.27 (4): 137-150.

Andreeva E.R., Andrianova I.V., Sotnezova E.V., Buravkov S.V., Bobyleva P.I., Romanov Y.A., Buravkova LB. Human adipose-tissue derived stromal cells in combination with hypoxia effectively support ex vivo expansion of cord blood haematopoietic progenitors. Plos one. 2015.10 (4): e0124939.

Andreeva E.R., Andrianova I.V., Gornostaeva A.N., Gogiya B.S., Buravkova L.V., Evaluation of committed and primitive cord blood progenitors after expansion on adipose stromal cells. Cell Tissue Res. 2018.372 (3): 523-533.

Bartholomew A., Sturgeon C., Siatskas M., Ferrer K., Mcintosh K., Patil S., Hardy W., Devine S., Ucker D., Deans R., Moseley A., Hoffman R. Mesenchymal stem cells suppress lymphocyte proliferation in vitro and prolong skin graft survival in vivo. Exp Hematol. 2002.30 (1): 42-8.

Bianco P., Cao X., Frenette P.S., Mao J.J., Robey P.G., Simmons P.J., Wang C.Y. The meaning, the sense and the significance: translating the science of mesenchymal stem cells into medicine. Nat Med. 2013.19 (1): 35-42.

Broxmeyer H.E. and Farag S. Background and future considerations for human cord blood hematopoietic cell transplantation, including economic concerns. Stem Cells Dev. 2013.22 (Suppl 1): 103-110.

Bruun K., Schermer E., Sivendra A., Valaik E., Wise R. B., Said R., Bracht J. R. Therapeutic applications of adipose-derived stem cells in cardiovascular disease. Am J Stem Cells. 2018.7 (4): 94-103.

Caplan A.I. Adult mesenchymal stem cells: when, where, and how. Stem cells international. 2015.2015: 628767.

De Lima M., McNiece I., Robinson SN, Munsell M, Eapen M., Horowitz M., Alousi A., Saliba R., McMannis JD, Kaur L, Kebriaei P., Parmar S., Popat U., Hosing C, Champlin R., Bollard C, Molldrem JJ, Jones RB, Nieto Y., Andersson BS, Shah N., Oran B., Cooper LJ, Worth L., Qazilbash MH, Korbling M., Rondon G., Ciurea S ., Bosque D., Maewal I., Simmons PJ, Shpall EJ. Cord-blood engraftment with ex vivo mesenchymal-cell coculture. N Engl J Med. 2012.367 (24): 2305-2315.

Dexter T.M., Allen T. D., Lajtha L. G., Conditions controlling the proliferation of haemopoietic stem cells in vitro. J Cell Physiol. 1977.91 (3): 335-344.

de Haan G., and Van Zant G. Intrinsic and extrinsic control of hemopoietic stem cell numbers: mapping of a stem cell gene. J. Exp. Med. 1997.186 (4): 529-536.

G.,

V., Valencia-Plata I.,

G., Mayani H. Individual and combined effects of mesenchymal stromal cells and recombinant stimulatory cytokines on the in vitro growth of primitive hematopoietic cells from human umbilical cord blood, Cytotherapy. 2009. 11(7): 886-896.Flores-Guzman P., Flores-Figueroa E., Montesinos JJ,

G.,

V., Valencia-Plata I.,

G., Mayani H. Individual and combined effects of mesenchymal stromal cells and recombinant stimulatory cytokines on the in vitro growth of primitive hematopoietic cells from human umbilical cord blood, Cytotherapy. 2009.11 (7): 886-896.

Jing D., Fonseca AV, Alakel N., Fierro FA, Muller K., Bornhauser M., Ehninger G., Corbeil D., Ordemann R., Hematopoietic stem cells in co-culture with mesenchymal stromal cells-modeling the niche compartments in vitro. Haematologica, 2010, 95 (4): 542-550.

S., Moll G., Ringden O., Kiessling R., Linder S., Le Blanc K. Alterations in the cellular immune compartment of patients treated with third-party mesenchymal stromal cells following allogeneic hematopoietic stem cell transplantation. Stem Cells. 2013. 31(8): 1715-1725.Jitschin R., Mougiakakos D., Von Bahr L.,

S., Moll G., Ringden O., Kiessling R., Linder S., Le Blanc K. Alterations in the cellular immune compartment of patients treated with third-party mesenchymal stromal cells following allogeneic hematopoietic stem cell transplantation. Stem Cells. 2013.31 (8): 1715-1725.

A.,

E., Espinosa О., Remigia M.J., Moratalla L., Goterris R.,

R., Ruiz-Cabello F.,

S., Pascual M.J.., Espigado I., Solano C. Adipose tissue-derived mesenchymal stromal cells as part of therapy for chronic graft-versus-host disease: A phase I/II study. Cytotherapy. 2017. .19(8): 927-936..Jurado M., De La Mata C.,

A.,

E., Espinosa O., Remigia MJ, Moratalla L., Goterris R.,

R., Ruiz-Cabello F.,

S., Pascual MJ., Espigado I., Solano C. Adipose tissue-derived mesenchymal stromal cells as part of therapy for chronic graft-versus-host disease: A phase I / II study. Cytotherapy. 2017. .19 (8): 927-936 ..

Kumar S., Geiger H. HSC niche biology and HSC expansion ex vivo. Trends Mol Med. 2017.23 (9): 799-819

Lohan P., Murphy N., Treacy O., Lynch K., Morcos M., Chen B., Ryan AE, Griffin MD, Ritter T. Third-party allogeneic mesenchymal stromal cells prevent rejection in a pre-sensitized high-risk model of corneal transplantation. Front Immunol. 2018.; 9: 2666 ..

Miguel M. P. De, Fuentes-Julian S., Blazquez-Martinez A., Pascual C. Y., Aller M. A., Arias J., Arnalich-Montiel F. Immunosuppressive properties of mesenchymal stem cells: advances and applications. Curr Mol Med. 2012.12 (5): 574-591.

Nakao N., Nakayama T., Yahata T., Muguruma Y, Saito S., Miyata Y, Yamamoto K., Naoe T. Adipose tissue-derived mesenchymal stem cells facilitate hematopoiesis in vitro and in vivo: advantages over bone marrow-derived mesenchymal stem cells. Am J Pathol. 2010.177 (2): 547-554.

Nishiwaki S., Nakayama T., Saito S., Mizuno H., Ozaki T., Takahashi Y., Maruyama S., Nishida T., Murata M., Kojima S., Naoe T. Efficacy and safety of human adipose tissue -derived mesenchymal stem cells for supporting hematopoiesis. Int J Hematol. 2012.96 (3): 295-300.

Murphy M.V., Moncivais K., Caplan A.I. Mesenchymal stem cells: environmentally responsive therapeutics for regenerative medicine. Exp Mol Med. 2013.45: e54.

Robinson SN, Ng J., Niu T, Yang H., McMannis JD, Karandish S., Kaur I., Fu P., Del Angel M., Messinger R., Flagge F., de Lima M., Decker W. , Xing D., Champlin R., Shpall EJ Superior ex vivo cord blood expansion following co-culture with bone marrow-derived mesenchymal stem cells, Bone Marrow Transplant. 2006.37 (4): 359-366.

F.,

Т.,

O., Redondo A., Parody R.,

C.,

E., Andreu E.,

F.,

M., Regidor C., Villaron E.,

L., Caballero D.,

M.C.,

J.A.. Sequential third-party mesenchymal stromal cell therapy for refractory acute graft-versus-host disease. Biol Blood Marrow Transplant. 2014. 20(10): 1580-1585.

F.,

T.,

O., Redondo A., Parody R.,

C.,

E., Andreu E.,

F.,

M., Regidor C., Villaron E.,

L., Caballero D.,

MC

JA. Sequential third-party mesenchymal stromal cell therapy for refractory acute graft-versus-host disease. Biol Blood Marrow Transplant. 2014.20 (10): 1580-1585.

Syed, B.A., Evans, J.B. Stem cell therapy market. (2013) Nat Rev Drug Discov 12 (3): 185-186.

Wagner W., Wein F., Roderburg C., Saffrich R., Faber A., Krause U., Schubert M., Benes V., Eckstein V., Maul H., Ho A.D. Adhesion of hematopoietic progenitor cells to human mesenchymal stem cells as a model for cell-cell interaction, Exp. Hematol. 2007.35 (2): 314-325.

Claims (4)

1. A method of obtaining associates of hematopoietic and stromal progenitor cells capable of inhibiting the activation and proliferation of allogeneic lymphocytes, comprising preparing a stromal rod from multipotent stromal cells isolated from the stromal-vascular fraction of adult adipose tissue (gMSC), during which gMSCs are constantly cultivated by 5% O ₂ until the required number of cells is obtained, then the cell divisions of zhMTSK are stopped by mitomycin C, washing from mitomycin C is carried out, and zhMSK is reseeded to obtain a support layer for HSPC; after that, a mononuclear fraction of umbilical cord blood in the amount of 2 × 10 ⁶ cells / ml is added to the formed layer from gMSC and a joint cultivation is carried out for 144 hours. 2. The method according to p. 1, characterized in that after the first 72 hours of cultivation, non-adherent cells are selected and the culture medium is replaced with fresh. 3. The method according to p. 1, characterized in that the hematopoietic progenitor cells are hematopoietic stem and progenitor cells (HSPC) from umbilical cord blood. 4. The method according to p. 1, characterized in that the stromal progenitor cells are multipotent adipose tissue cells (zhMSK).

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