CN117500922A - Extracellular vesicles of umbilical mesenchymal cells for the treatment of osteoarticular diseases and autoimmune diseases - Google Patents
Extracellular vesicles of umbilical mesenchymal cells for the treatment of osteoarticular diseases and autoimmune diseases Download PDFInfo
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Abstract
The present invention relates to a composition of Extracellular Vesicles (EVs) enriched in umbilical mesenchymal cells, the vesicles having a specific marker and miRNA, wherein the composition is for use in the treatment of bone joint diseases and autoimmune diseases. In particular, a composition is sought for protecting Extracellular Vesicles (EVs) enriched in umbilical cord mesenchymal cells, consisting of a group of EVs comprising the following mirnas: hsa-miR-6126, hsa-miR-149-3p and hsa-miR-6780b-5p in 10 most abundant miRNAs, and further comprises at least 5 of the following miRNAs: hsa-miR-574-5p, hsa-miR-6131, hsa-miR-6741-5p, hsa-miR-21-5p, hsa-miR-1290, hsa-miR-4530, hsa-miR-2861, hsa-miR-6088, hsa-miR-1307-5p and/or hsa-miR-6782-5p and has a surface marker CD63, CD81 and CD9; and an osmolality of 250 to 310mOsmol/L and a pH of 6.0 to 8.0. The present invention relates to a process for preparing the composition and its use in the treatment of bone joint disorders and autoimmune diseases.
Description
Technical Field
The present invention relates to the general field of biotechnology, in particular to the field of regenerative medicine based on human mesenchymal cells, in particular extracellular vesicles, and the manufacture of therapeutic products for the treatment of osteoarticular diseases and autoimmune diseases.
Prior Art
Currently, cell therapies form a bridge between research and clinical applications and constitute an advance in tissue engineering to replace, regenerate and/or improve human cells, tissues or organs in order to restore function. Accompanying and encouraging this transformation is the use of stem cells, primarily Mesenchymal Stem Cells (MSCs), in an increasing number of clinical trials. Although the field of cell therapy has grown rapidly in various medical applications, there are several technical problems in its development that require better solutions, which allow on the one hand to reduce the cost of these techniques and on the other hand also to make them more efficient and safer in order to be able to help more people and for new applications.
In this respect, a more recent, safer, less costly approach is to use vesicles that are naturally secreted by cells during cell culture, as they have proven to have great potential in cell-free therapies. Researchers have studied the function of these vesicles in various areas, including their important role in intercellular communication, which affect the formation and/or regulation of disease through their content, which corresponds essentially to proteins and RNAs. It has been found in particular that these vesicles comprise high numbers and species, from 2000 to 3000 types of micrornas or mirnas. These molecules are non-coding RNA strands and play an important role in regulating gene expression. One subset of extracellular vesicles is exosomes (30 to 200 nm), which have been used as carriers for delivering different drugs.
Currently, development of therapies based on extracellular vesicles, including exosomes or small extracellular vesicles (sEV), has advanced in preclinical studies and has tremendous potential for clinical applications, such as those presented in the present invention.
In the prior art, there are different diagnostic applications and treatments using isolated exosomes, for example publication WO 2019038660A1 (Seattle Childrens Hospital DBA Seattle Childrens Res Inst, 2019-02-28) protects a method for diagnosing kawasaki disease by examining exosomes present in a patient's biological fluid.
There are several patent applications in which the exosomes of mesenchymal cells are used for the treatment of some diseases, such as glaucoma, US20190224242A1 (Tomarev et al, 2019-07-25), but the compositions and observed properties used in this document are far different from the compositions of the present invention, for example in this application the exosomes have markers cd11c+ and cd63+, while in the present invention the markers CD63, CD81 and CD9 are required, which is only one difference.
The inventors have not found any literature in the prior art that contemplates the present invention. The present invention is therefore directed to exosome compositions obtained from umbilical cord mesenchymal cells (UC-MSCs) comprising miRNA and/or specific surface markers for the treatment of bone joint diseases and autoimmune diseases.
Drawings
Figure 1 in a murine model of osteoarthritis, treatment with the composition of the invention reduced the severity of the lesions, study 1. A) OA histological scores (or OA scores) show healthy joints (sham treated, or placebo), joints with osteoarthritis, and joints with osteoarthritis treated with the composition (exosomes) of the invention. B) Representative images show tissue sections of "sham treated" control knee (left), OA-induced knee (middle), and knee (right) treated with the compositions of the invention (exosomes). A higher proportion of subchondral bone (P < 0.05) was observed in the knees treated with exosomes than in OA groups.
Figure 2 treatment with the composition of the invention in a murine model of osteoarthritis showed positive results for bone mineral density, study 2. A) Analysis of bone mineral density of the medial condyle of the knee tibia, which corresponds to a histomorphometric analysis of the 3D image. B) Representative images show images of the medial knee tibial condyle observed by μct: "sham treated" control knee (left), OA-induced knee (middle) and treated with exosomes (right), as well as the color scale representing mineralization, leftmost assigned the lowest mineralization and rightmost assigned the highest mineralization. It was observed that treatment with exosomes prevented further bone mineralization, as the observed color with respect to mineralization was similar to that obtained in the "sham-treated" control, i.e. the color on the left side of the scale described above.
Figure 3 treatment with the composition of the invention in a murine model of osteoarthritis showed positive results for bone mineral density, study 3. A) Analysis of bone mineral density of the medial condyle of the knee tibia, which corresponds to a histomorphometric analysis of the 3D image. B) Representative images show images of the medial knee tibial condyle observed by μct: "sham treated" control knee (left), OA-induced knee (middle) and treated with exosomes (right), as well as the color scale representing mineralization, leftmost assigned the lowest mineralization and rightmost assigned the highest mineralization. It was observed that treatment with exosomes prevented further bone mineralization, as the observed color with respect to mineralization was similar to that obtained in the "sham-treated" control, i.e. the color on the left side of the scale described above.
FIG. 4 treatment with the compositions of the invention reduced proliferation of CD4+ Th cells. A) Peripheral blood mononuclear cells were activated with PHA (20. Mu.g/mL) and treated with PBS control and the composition of the present invention, showing proliferation of CD4+ Th cells on the third day of culture. B) Proliferation was carried out on day 5. The compositions of the invention proliferate less than the control at both times.
Fig. 5 treatment with the composition of the invention increases proliferation of Treg cells; which is characterized by having the markers CD4, FOXP3 and CD25. Peripheral blood mononuclear cells were activated with PHA (20. Mu.g/mL) and treated with PBS control and the composition of the invention, showing proliferation of Treg cells on the third day of culture. The compositions of the invention allow for more proliferation than controls.
FIG. 6 treatment with the composition of the present invention positively polarizes macrophage differentiation. Monocytes were isolated from peripheral blood mononuclear cells, cultured and differentiated into macrophages by factors M-CSF and GM-CSF, treated with PBS control and the composition of the invention on day 6, a) 24 hours after stimulation, proliferation of M1 or pro-inflammatory macrophages was reduced by the composition of the invention sEV, B) 24 hours after stimulation, proliferation of M2 or anti-inflammatory macrophages was increased by the composition of the invention sEV.
FIG. 7 shows a graph of the relative concentration of hsa-miRNA in extracellular vesicles present in a composition of the invention for the following reasons.
Specification
The present invention relates to a composition of Extracellular Vesicles (EV) enriched in umbilical mesenchymal cells with specific markers and miRNAs, wherein the composition is useful for the treatment of bone joint diseases and autoimmune diseases, in particular when injected into a lesion to be treated.
In particular, the object of the present invention is a composition of Extracellular Vesicles (EV) enriched in umbilical cord mesenchymal cells, consisting of: a set of EVs comprising the following mirnas: hsa-miR-6126, hsa-miR-149-3p and hsa-miR-6780b-5p in 10 miRNAs with highest abundance comprise at least 5 of the following miRNAs: hsa-miR-574-5p, hsa-miR-6131, hsa-miR-6741-5p, hsa-miR-21-5p, hsa-miR-1290, hsa-miR-4530, hsa-miR-2861, hsa-miR-6088, hsa-miR-1307-5p and/or hsa-miR-6782-5p and has a surface marker CD63, CD81 and CD9;
-and an animal component and protein free medium or carrier having an osmolality of 250-310mOsmol/L and a pH of 6.0-8.0.
In addition, vesicles or exosomes comprise the following mirnas: hsa-miR-8078 in 100 miRNAs with highest abundance; hsa-miR-7150; hsa-miR-6750-5p; hsa-miR-6727-5p; hsa-miR-5585-3p; hsa-miR-4459; hsa-miR-4449; hsa-miR-3687; hsa-miR-3197; hsa-miR-1915-3p; hsa-miR-1285-5p; hsa-miR-1275; hsa-miR-1273h-5p; hsa-miR-1273d; hsa-miR-1237-5p; hsa-miR-874-3p; hsa-miR-671-5p; hsa-miR-424-5p; hsa-miR-376c-3p; hsa-miR-339-3p; hsa-miR-222-3p; hsa-miR-221-3p; hsa-miR-199a-3p; hsa-miR-193a-3p; hsa-miR-181a-5p; hsa-miR-146a-5p; hsa-miR-145-5p; hsa-miR-143-3p; hsa-miR-130a-3p; hsa-miR-125b-5p; hsa-miR-125a-5p; hsa-miR-100-5p; hsa-miR-99a-5p; hsa-miR-92a-3p; hsa-miR-31-5p; hsa-miR-29b-3p; hsa-miR-29a-3p; hsa-miR-27b-3p; hsa-miR-27a-3p; hsa-miR-26a-5p; hsa-miR-24-3p; hsa-miR-23b-3p; hsa-miR-23a-3p; hsa-miR-22-3p; hsa-miR-19b-3p; hsa-miR-16-5p and/or hsa-let-7i-5p.
As mentioned above, EVs are known to contain a large variety of different ratios of mirnas, it is evident to the expert in the art that the subset of 100 most abundant mirnas is small, since concentrations of about 2000 to 3000 mirnas are typically sequenced and calculated. The present inventors have obtained cultures of specific umbilical cord mesenchymal cells (UC-MSCs) from different donors from which they obtained the compositions of the present invention that replicate the specific markers present and the relative concentrations of miRNAs shown. These inventive compounds have proven useful in the treatment of osteoarthropathy and have a positive effect on models associated with the treatment of autoimmune diseases, as will be demonstrated in the examples.
In a second embodiment, the vesicles of the composition of the invention comprise at least 8 of the following mirnas: hsa-miR-574-5p, hsa-miR-6131, hsa-miR-6741-5p, hsa-miR-21-5p, hsa-miR-1290, hsa-miR-4530, hsa-miR-2861, hsa-miR-6088, hsa-miR-1307-5p and/or hsa-miR-6782-5p in the 100 most abundant miRNAs.
In addition, the vesicles may comprise the surface markers N-cadherin and/or CD90 and/or CD44.
In the compositions of the invention, the concentration of vesicles is 5x10 per ml 7 Up to 1x10 13 The total particles have a size of 30 to 300nm.
In a second aspect, the invention relates to a method of obtaining the composition of the invention, wherein the umbilical cord interumbilical cord is amplified from outside the bodyEV is isolated from the supernatant of the mesenchymal cell culture, wherein these cells express at least two of the surface markers N-cadherin, CD44, RANKL, CD105, CD56 and/or CD90 and resuspended in an animal component and protein free medium or carrier having an osmolality of 250-310mOsmol/L and a pH of 6.0-8.0. In the prior art, there are many means of meeting these conditions, both commercial and laboratory. For example, phosphate buffer, PBS, lactate ringer's solution, and the like,Etc. As indicated, the most important are the indicated osmolality and pH.
In addition, the EV is separated from the supernatant by filtration and/or centrifugation, washed at least 2 times with an osmolality of 250 to 310mOsmol/L and a pH of 6.0-8.0 of a culture medium or carrier free of animal components and proteins, wherein and finally resuspended in an osmolality of 250-310mOsmol/L and a pH of 6.0-8.0 of a culture medium or carrier free of animal components and proteins. It is obvious to the expert in the art that different protocols are possible to separate the extracellular vesicles of the supernatant from the culture of mesenchymal cells. The method of the present invention does not depend on the protocol or system used for the separation, as long as it effectively separates vesicles between 30 and 300nm in size, and thus the separation can be performed by any technique or instrument available in the practice of the present invention.
At least 3 of the umbilical cord mesenchymal cells expressing the surface markers N-cadherin, CD44, RANKL, CD105, CD56 and/or CD90 of EV may be obtained therefrom.
The composition of the invention has specific activity, so that the composition can be used for preparing medicines for treating bone joint diseases and autoimmune diseases. For example, the composition has been shown to promote cartilage regeneration; and increasing bone mineral density in clinical cases of osteoarthritis in vivo.
In addition, the composition can be used to reduce proliferation of CD4+ T cells while increasing proliferation of Tregs cells. The composition causes an increase in M2 macrophages or cd206+ HLA-dr+ anti-inflammatory macrophages, and/or a decrease in M1 macrophages or pro-inflammatory macrophages or cd86+ HLA-dr+.
All of these functions, except as exemplified in the examples, indicate that the compositions are useful in the treatment of, for example, osteoarthritis, arthritis, osteoarthritis. In addition, it is useful for treating rheumatoid arthritis, systemic lupus erythematosus, inflammatory bowel disease, multiple sclerosis, type 1 diabetes, guillain-Barre syndrome, chronic inflammatory demyelinating polyneuropathy, psoriasis, graves 'disease, hashimoto thyroiditis, myasthenia gravis, vasculitis, sjogren's syndrome, pernicious anemia, celiac disease, or graft versus host disease.
Examples
Example 1: the composition of the invention is obtained.
Umbilical cord mesenchymal cells were obtained and found to have markers N-cadherin, CD44, RANKL, CD105, CD56 and/or CD90. 3 batches of cells were obtained, for each of which the markers of mesenchymal cells and the selection conditions of the present invention are shown in table 1 below.
TABLE 1 umbilical cord mesenchymal cells used
Cells were cultured with commercially available medium (high glucose DMEM; supplemented with 5% hPL,1% L-Glut,1% pen/Strep) in a total volume of 650mL under normal conditions for any mesenchymal cell culture, until confluence. Subsequently, the cells were washed twice with PBS (phosphate buffer, pH7.2;290 mOsm/L) and then 650mL of induction medium (high sugar DMEM; supplemented with 1% L-Glut only) was added.
After 48 hours of incubation, all supernatant medium was collected from the cells and 650mL induction medium was again added to the culture for a second 48 hour incubation.
The supernatant collected after 1 or 2 times of each incubation was centrifuged at 600g for 10 minutes at 4℃to remove impurities.
The supernatant from the previous centrifugation was treated in an ultracentrifuge at 100,000g for 1 hour and 10 minutes at 4℃and this step was repeated until the total volume of supernatant was treated. The supernatant was removed and the pellet was released with a vortex. Subsequently, the precipitate is washed twice to remove possible contaminants. To this end, the pellet was resuspended in filtered PBS to a final volume of 10ml and centrifuged at 100,000g at 4℃overnight. Subsequently, a final wash was performed to remove the supernatant, and the pellet was resuspended in a final volume of 10ml of ringer's lactate (pH 6.5, 273 mOsm/L) and treated at 100,000g for 1 hour 10 minutes in an ultracentrifuge at 4 ℃. Finally, the supernatant was removed and the pellet was passed through a vortex and stored in aliquots.
The particles of the aliquot are then quantified. Particle quantification was performed by dilution of the pellet (1:100) in filtered PBS using NanoSight NS 300.
From 3 initial cultures, 4 batches of aliquots of the particles or compositions of the invention were obtained. Table 2 below shows the characterization of these particles in terms of the concentration obtained and the presence of the validation marker.
Table 2. Characterization of compositions according to the invention.
Additional analyses were performed on the identity and relative concentrations of the mirnas contained in the 4 batches obtained, with approximately 2500 mirnas in each batch sequenced. Table 3 shows the concentrations of the most relevant mirnas in the present invention. Fig. 7 shows the concentration of Lot a.
| miRNA | Lot A | Lot B | Lot C | Lot D |
| hsa-miR-6126 | 54.33％ | 47.05％ | 51.00％ | 62.31％ |
| hsa-miR-149-3p | 8.79％ | 8.09％ | 7.66％ | 9.59％ |
| hsa-miR-6780b-5p | 5.67％ | 5.53％ | 6.66％ | 4.32％ |
| hsa-miR-21-5p | 1.60％ | 1.55％ | 2.10％ | 1.57％ |
| hsa-miR-6131 | 1.14％ | 0.88％ | 1.14％ | 1.99％ |
| hsa-miR-2861 | 0.95％ | 1.39％ | 0.92％ | 0.70％ |
| hsa-miR-6088 | 0.89％ | 1.15％ | 0.55％ | 0.48％ |
| hsa-miR-574-5p | 0.82％ | 1.36％ | 0.99％ | 0.41％ |
| hsa-miR-1290 | 0.78％ | 0.51％ | 0.96％ | 0.46％ |
| hsa-miR-4530 | 0.71％ | 1.14％ | 0.75％ | 0.25％ |
| hsa-miR-6782-5p | 0.59％ | 0.77％ | 0.90％ | 0.16％ |
| hsa-miR-6741-5p | 0.59％ | 1.10％ | 0.32％ | 0.34％ |
| hsa-miR-1307-5p | 0.43％ | 0.77％ | 0.64％ | 0.02％ |
| Other hsa-miR: | 22.71％ | 28.70％ | 25.41％ | 17.39％ |
table 3. Mirnas present in EVs of the compositions of the invention.
In other sequenced mirs, the following miR concentrations were found to be less than or equal to 0.02% in all batches analyzed:
hsa-miR-8078; hsa-miR-7150; hsa-miR-6750-5p; hsa-miR-6727-5p; hsa-miR-5585-3p; hsa-miR-4459; hsa-miR-4449; hsa-miR-3687; hsa-miR-3197; hsa-miR-1915-3p; hsa-miR-1285-5p; hsa-miR-1275; hsa-miR-1273h-5p; hsa-miR-1273d; hsa-miR-1237-5p; hsa-miR-874-3p; hsa-miR-671-5p; hsa-miR-424-5p; hsa-miR-376c-3p; hsa-miR-339-3p; hsa-miR-222-3p; hsa-miR-221-3p; hsa-miR-199a-3p; hsa-miR-193a-3p; hsa-miR-181a-5p; hsa-miR-146a-5p; hsa-miR-145-5p; hsa-miR-143-3p; hsa-miR-130a-3p; hsa-miR-125b-5p; hsa-miR-125a-5p; hsa-miR-100-5p; hsa-miR-99a-5p; hsa-miR-92a-3p; hsa-miR-31-5p; hsa-miR-29b-3p; hsa-miR-29a-3p; hsa-miR-27b-3p; hsa-miR-27a-3p; hsa-miR-26a-5p; hsa-miR-24-3p; hsa-miR-23b-3p; hsa-miR-23a-3p; hsa-miR-22-3p; hsa-miR-19b-3p; hsa-miR-16-5p and hsa-let-7i-5p.
Example 2: use of the composition of the invention for the treatment of osteoarthritis.
To evaluate the effect of the composition of the present invention on osteoarticular diseases (OA), the composition obtained in example 1 was used alternately in a murine osteoarthritis model.
In this model, disease was induced by injecting collagenase into the joint (in this case the knee) on days 0 and 2, and then corresponding treatments were applied by injection into the joint also on days 7 and 14. Then, on day 40, euthanasia was performed and the joints were analyzed.
In this case, 3 independent studies were performed in which 65 animals were co-treated (study 1:17 animals, study 2:20 animals, study 3:28 animals).
Each group was subdivided into 3 groups, a first control group which received placebo (PBS, 5 μl), a second control group which induced lesions, but no treatment was applied, so they remained in osteoarthritis disease (OA), the last group induced lesions and treatment with the present invention. To induce injury, 5 μl collagenase type VII (1U/5 μl) was injected into the joint at the beginning of the experiment and on day 2 of the experiment for the injury (OA) -only group and those subsequently treated. Subsequently, animals of the third group received treatment as a composition of the invention, which was adjusted to administer 2x10 8 The amount of exosomes, the treatment was administered 2 times in a volume of 5 μl on day 7 and day 14 from the start of the experiment.
After completion of in vivo studies, the treated joints were analyzed, including those with placebo, lesions only, or treated. To analyze the state of the joint, two techniques are used, the first being image studies by a micro CT apparatus, which provides a result of bone mineral density. The second option used was to perform a histological analysis of the treated knee, wherein an OA histological score was established, which corresponds to the clinical signs index of the osteoarthritis mouse model (Cosenza, s., ruiz, m., maumus, m., jorgensen, C.&D.Pathogenic or Therapeutic Extracellular Vesicles in Rheumatic Diseases:Role of Mesenchymal Stem Cell-Derived Vesicles.Int.J.Mol.Sci.18,(2017)。
As noted, the inventors performed 3 in vivo studies, the summary of which is set forth in table 4 below. Notably, in vivo n°1, the results were analyzed by histological analysis, and in vivo 2 and 3, the results were evaluated using microscopic CT analysis.
TABLE 4 summary of in vivo OA studies of the study group
Results
For in vivo study n°1, the results are shown in figure 1. Treatment with the compositions of the invention was observed to have an effect on lesions, i.e. a decrease in OA scores of joints relative to untreated controls (fig. 1A), showing cartilage turnover and/or cartilage regeneration, respectively, which would confirm that the in vivo model is functioning and that the exosomes exhibit prophylactic and/or regenerative activity.
It was understood that the in vivo study corresponds to an isolated experiment, and that the data were confirmed from other studies, the results of which were analyzed by micro-CT.
Regarding n°2 in vivo studies (fig. 2), from the microscopic CT analysis, it can be seen that the compositions or exosomes of the present invention produced by the present inventors successfully prevented lesions produced by osteoarthritis, as they had a lower number of bone mineral density BMDs (fig. 2A) than the control with only lesions, and obtained values similar to those of placebo-treated healthy joints. In particular, imaging analysis of bone mineral density (fig. 2B) showed that the induction model used produced significant damage, but the exosomes would nevertheless be able to produce a regenerative effect.
For the last in vivo study, study 3, as shown in fig. 3, a significant effect on lesions (OA) could be observed with the composition of the invention obtained according to example 1 of UC-MSC cells or sEV. As before, the composition according to the invention has been demonstrated to have a prophylactic and/or regenerative effect on osteoarthritis disease.
These results demonstrate that the use of the compositions of the present invention in osteoarthropathy such as osteoarthritis has a positive effect on the extent of the pathology (as seen in histological studies and OA scores) as well as on the bone mineral density of the joint.
Example 3: immunosuppression assays using the compositions of the invention.
Monocytes were isolated from peripheral blood, activated with PHA (20. Mu.g/mL) and stained with CTV (1:1000) to assess proliferation. Consider two experimental groups: a) 10. Mu.L PBS control and b) 2X10 total particle size with dissolved in 10. Mu.L vehicle 8 Is treated with a single dose of the composition of the invention (sEV's). In both groups, helper T lymphocytes proliferate (abbreviated Th; CD4+) and regulatory T cells (abbreviated Tregs; CD4+Foxp3+CD25+). Fig. 4 shows the percentage proliferation of Th lymphocytes on the third day of culture (fig. 4 a) and the fifth day of culture (fig. 4 b), wherein less proliferation was observed upon treatment with the composition of the invention. Figure 5 shows the percentage of Treg cells, indicating that treatment with the composition of the invention (SeV's) increased the percentage of the population on the third day of culture.
Th cells correspond to a population of cells that play an important role in the adaptive immune response. These cells divide into Th1, th2 and Th17; they all secrete cytokines to stimulate proliferation and differentiation of cells involved in the immune response. There is evidence that there is a greater number of T cells in the organ with OA than in the healthy control group. Tregs, on the other hand, are a subset of cd4+ T cells, maintain homeostasis and tolerance within the immune system, regulate or suppress other cd4+ and cd8+ T cells, possibly also B lymphocytes and dendritic cells. Tregs cells can produce molecules with immunosuppressive functions such as TFG-beta, IL-10 and adenosine.
Example 4: macrophage polarization assay Using the compositions of the invention
Mononuclear cells are isolated from peripheral blood and then obtained using a negative selection kitObtaining the monocytes. These monocytes were cultured using M-CSF and GM-CSF factors and differentiated into macrophages. Six days after incubation, two experimental groups were established: a) Control and b) total particle size 1x10 9 Is treated with a single dose of a sEV composition of the invention.
Flow cytometry was used to evaluate pro-inflammatory macrophages (designated M1; CD86+ HLA-DR+) and anti-inflammatory macrophages (designated M2; CD206+ HLA-DR+), 24 hours after stimulation. FIG. 6 shows the percentage of biscationic cells with M1 (FIG. 6 a) and M2 (FIG. 6 b) markers.
The results indicate that sEV treatment reduced the amount of M1 macrophages and increased the amount of M2 macrophages.
Claims (17)
1.A composition of Extracellular Vesicles (EVs) enriched in umbilical cord mesenchymal cells, characterized in that the composition consists of:
-a set of EVs comprising the following mirnas: hsa-miR-6126, hsa-miR-149-3p and/or hsa-miR-6780b-5p in 10 miRNAs with highest abundance,
and additionally comprising at least 5 of the following mirnas: hsa-miR-574-5p, hsa-miR-6131, hsa-miR-6741-5p, hsa-miR-21-5p, hsa-miR-1290, hsa-miR-4530, hsa-miR-2861, hsa-miR-6088, hsa-miR-1307-5p and/or hsa-miR-6782-5p in the 100 miRNAs with highest abundance,
and has surface markers CD63, CD81 and CD9;
-and an animal component and protein free medium or carrier having an osmolality of 250-310mOsmol/L and a pH of 6.0-8.0.
2. The composition of claim 1, wherein the vesicles comprise at least 8 of the following mirnas: hsa-miR-574-5p, hsa-miR-6131, hsa-miR-6741-5p, hsa-miR-21-5p, hsa-miR-1290, hsa-miR-4530, hsa-miR-2861, hsa-miR-6088, hsa-miR-1307-5p and/or hsa-miR-6782-5p in the 100 most abundant miRNAs.
3. The composition of claim 1, wherein the vesicles additionally have the surface markers N-cadherin and/or CD90 and/or CD44.
4. The composition of claim 1, wherein the vesicles have a concentration of 5x10 per milliliter 7 Up to 1x10 13 Total particles.
5. The composition of claim 4, wherein the vesicles are in the size range of 30 to 300nm.
6. A method of obtaining the composition of claim 1, characterized in that EV at 30 to 300nm is isolated from supernatant of umbilical cord mesenchymal cell culture expanded in vitro and resuspended in an animal and protein component free medium or carrier having an osmolality of 250 to 310mOsmol/L and a pH of 6.0 to 8.0, wherein the cells express at least 2 of the surface markers N-cadherin, CD44, RANKL, CD105, CD56 and/or CD90.
7. The method according to claim 6, characterized in that the EV is separated from the supernatant by ultrafiltration, filtration, ultracentrifugation and/or centrifugation, washed at least 2 times with an animal component and protein free medium or carrier having an osmolality of 250 to 310mOsmol/L and a pH of 6.0 to 8.0, and finally resuspended in an animal component and protein free medium or carrier having an osmolality of 250-310mOsmol/L and a pH of 6.0 to 8.0.
8. The method of claim 7, wherein umbilical cord mesenchymal cells express at least 3 of the surface markers N-cadherin, CD44, RANKL, CD105, CD56, and/or CD90.
9. Use of a composition according to claim 1 for the preparation of a pharmaceutical product for the treatment of bone joint diseases and autoimmune diseases.
10. The use according to claim 9, wherein the composition is for promoting cartilage regeneration.
11. The use according to claim 9, wherein the composition is for preventing cartilage degradation.
12. The use according to claim 9, wherein the composition is for reducing bone mineral density in bone joint diseases.
13. The use according to claim 9, wherein the composition is for reducing proliferation of cd4+ T cells.
14. The use according to claim 9, wherein the composition is for increasing proliferation of T reg cells.
15. The use according to claim 9, wherein the composition is for increasing M2 macrophages or cd206+ HLA-dr+ anti-inflammatory macrophages, and/or decreasing M1 or pro-inflammatory or cd86+ HLA-dr+ macrophages.
16. The use according to claim 9, wherein the composition is for the treatment of osteoarthritis, arthritis, osteoarthritis.
17. The use according to claim 9, wherein the composition is for the treatment of rheumatoid arthritis, systemic lupus erythematosus, inflammatory bowel disease, multiple sclerosis, type 1 diabetes, gillin-barre syndrome, chronic inflammatory demyelinating polyneuropathy, psoriasis, graves 'disease, hashimoto thyroiditis, myasthenia gravis, vasculitis, sjogren's syndrome, pernicious anaemia, celiac disease or graft versus host disease.
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