RU2805066C2 - Extracellular vesicles originating from mesenchymal stem cells - Google Patents

Abstract

FIELD: pharmaceuticals.

SUBSTANCE: invention relates to therapeutic, diagnostic, veterinary or cosmetic compositions containing extracellular vesicles derived from CD106^highCD151⁺nestin⁺ mesenchymal stem cells (MSC) of placental tissue, for use as a drug for the treatment of subjects suffering from ischemic disease, system disorders circulatory, immunopathological, organ damage or organ dysfunction.

EFFECT: invention also relates to a specific method of obtaining these extracellular vesicles.

41 cl, 5 dwg, 7 ex

Description

Field of invention

The present invention discloses a composition containing extracellular vesicles (EVs) derived from CD106 ^high CD151 ^{+ nestin +} mesenchymal stem cells (MSC) of placental tissue.

According to a first aspect, the invention relates to a specific method for producing these EVs.

According to a second aspect, the invention relates to a therapeutic, diagnostic, veterinary or cosmetic composition containing extracellular vesicles obtained by said specific method.

According to a third aspect, the invention relates to a composition containing these EVs for use as a medicament for the treatment of subjects suffering from ischemic disease, circulatory disease, immunopathology, organ damage or organ dysfunction.

Prior Art

Comprehensive studies of the therapeutic potential of mesenchymal stem cells (MSCs) for the repair and regeneration of damaged tissues have been carried out at both preclinical and clinical stages. MSCs mediate immunomodulation as well as pro-regenerative effects. For example, MSCs have been shown to improve wound healing in diabetic mice by promoting epithelialization, angiogenesis, and granulation tissue formation. Other studies have shown that bone marrow-derived MSCs are effective in treating wounds, including chronic wounds, by promoting angiogenesis and scar removal. Direct application of MSCs derived from bone marrow or umbilical cord to patients with chronic non-healing wounds leads to wound closure and skin restoration.

In particular, PCT/EP 2017/082316 relates to a method for producing CD106 ^high CD151 ⁺ nestin ⁺ MSC for various therapeutic applications, such as, for example, in the case of vascular diseases and wounds. These MSCs exhibit increased proangiogenic activities among other activities.

The mechanisms underlying the healing properties of MSCs have been studied. Interestingly, Shabbir et al.'s previous results from wound healing assays did not conclude that MSCs themselves differentiate to replace damaged tissue (fibroblasts). Instead, MSCs have been shown to promote fibroblast migration, which is integral to the healing process, even without direct contact (Shabbir et al., 2015). This highlights the importance of paracrine signaling between MSCs and diseased cells. This paracrine effect is thought to result from the secretion of various factors and extracellular vesicles (EVs) by these MSCs. These vesicles contain a lipid bilayer membrane similar to that of parental MSCs and carry various proteins, messenger RNAs, and microRNAs (cargos) derived from these MSCs, factors that enhance endogenous repair and regeneration mechanisms.

Extracellular vesicles (EVs), such as exosomes and microvesicles, have indeed previously been identified as major mediators of these paracrine cellular effects, acting as signal transduction mediators in immune biological processes (Raposo et al., 1996). Since then, EVs have been found to mediate interactions between different types of immune cells and also between tumor and immune cells. Depending on the cell source, EVs can stimulate or suppress proinflammatory responses.

Cells and especially MSCs can release many different types of vesicles into their surrounding extracellular environment. Collectively referred to as EVs, they include exosomes (30–150 nm), microvesicles that form as appendages of the plasma membrane (100–1000 nm), and apoptotic bodies (>500 nm). They contain lipids, proteins and RNA. They mediate targeted intercellular signal transmission in the processes of physiological and pathophysiological communication.

MSC-derived EVs are gaining popularity as a new strategy to achieve the therapeutic effects of stem cells without the risks and hassles of administering cells to patients. MSC-derived EVs do have several key advantages over cell-based products that justify efforts to bring EVs into the clinic:

- administration of MSC-derived EVs may be safe.

MSC-derived EVs have been used in an increasing number of different animal models and have been tested in a patient suffering from steroid-resistant acute graft-versus-host disease (GVHD). - graft-versus-host disease), as well as in a group of patients with chronic kidney disease (Giebel et al., 2017). To date, administration of MSC-derived EVs appears to be safe in humans and in all animal models tested.

Unlike cellular products, EVs are not capable of self-replication and therefore lack any potential for endogenous tumor formation.

It is known that the biological properties and functions of cells can be influenced and reprogrammed by environmental factors. Because EVs lack well-described metabolic activities, it is unlikely that their function can be reprogrammed by the environment. Thus, the biological activity and functional properties of EVs can be determined and controlled with greater precision than in the case of cells.

With average sizes less than 200 nm, EVs can be sterilized by filtration. This significantly reduces the risk of biological contamination of the respective therapeutic agents.

- EVs are much easier to work with than cells.

Freezing, thawing, and storage conditions appear to be less critical for EVs than for cells. To prepare cell grafts at the bedside, it is necessary to have specially trained personnel, which can be dispensed with when preparing EV-based therapeutics at the bedside.

Therapeutic EVs can be obtained from supernatants of cell lines whose cells are not intended to be used for therapies using the cells themselves. Thus, scaling up the resulting EVs can be done much more easily than with cell-based therapeutics.

The use of MSC-derived EVs for cell-free therapy is gaining popularity (Phinney & Pittenger, Stem Cells, 2017), and several studies have demonstrated a role for MSC-secreted EVs in angiogenesis:

- exosomes secreted by MSCs stimulate angiogenesis of endothelial cells through the transfer of microRNA-125a (Liang et al., J. Cell Sci., 2016),

- exosomes released from MSCs derived from human induced pluripotent stem cells promote skin wound healing by stimulating collagen synthesis and angiogenesis (Zhang J. and DR., 2015),

- MSC-derived exosomes induce fibroblast proliferation and migration in common and chronic wounds and enhance angiogenesis in vitro (Shabbir et al., 2015),

- exosomes derived from human umbilical cord MSCs enhance angiogenesis through the WNT4/catenin pathway (Zhang B. et al., 2015).

A 2017 review by Than UTT et al describes other studies that do show that EVs are involved in the regulation of a number of cellular processes required for wound healing. In particular, EVs could influence the coagulation process, cell proliferation, migration, angiogenesis, collagen formation, and extracellular matrix remodeling. In addition to transmitting information from parent secreting cells, messenger RNA (mRNA) and microRNA from EVs can also stimulate biological processes, including proliferation, angiogenesis, and apoptosis.

EV-mediated regulation of cell proliferation, an important process for wound healing, has been demonstrated using EVs derived from multiple cell types, such as MSCs, fibroblasts, mouse embryonic stem cells, and human endothelial progenitor cells. In particular, internalization of MSC-derived EVs by fibroblasts results in a dose-dependent increase in the proliferation and migration of these fibroblasts, regardless of whether they are derived from normal donors or from patients with chronic wounds.

EVs can stimulate cell proliferation by activating not only signaling pathways directly involved in cell cycle stimulation, but also signaling pathways involved in the regulation of growth factor expression. In addition, such overexpression of growth factors may in turn act as paracrine or autocrine signals to promote cell proliferation.

EVs released from cells such as human keratinocytes or MSCs have also been shown to influence endothelial cell migration, which is critical for vascular repair and regeneration.

All types of EVs (microvesicles and exosomes) contribute to varying degrees to the regulation of vascular formation by enhancing the expression of proangiogenic factors. For example, exosomes released by human fetal MSCs and human endothelial cells enhance angiogenesis by promoting proliferation and migration of endothelial cells to the wound site. More blood vessels were observed in exosome-treated areas compared to control treatment.

Based on all these analyses, it can be assumed that MSC-derived EVs can be safely used in wound treatment and regenerative medicine for vascular pathologies.

Dongdong Ti et al (Journal of translational medicine, 2015) described extracellular vesicles purified from umbilical cord-derived MSCs that were pretreated with the proinflammatory factor lipopolysaccharide (LPS).

Yunbing Wu et al (BioMed Research International, 2017) described the anti-inflammatory properties of extracellular vesicles derived from unstimulated mesenchymal stem cells obtained from human umbilical cord.

Neither of these two publications suggests adding interleukin(IL)1β and/or IL4 to stimulate MSCs to generate EVs enriched for the angiogenic marker CD106.

More broadly, none of the prior art documents even suggest treating MSCs with IL4, let alone improving their pro-angiogenic properties. In this context, the present inventors showed in WO 2018/108859 that culturing MSCs together with IL4 and IL1β leads to a significant and unexpected increase in the surface level of pro-angiogenic surface markers such as CD106. In particular, as shown in Example 4 of WO 2018/108859, the increase in CD106 expression observed with the combination of IL1β and IL4 is 3 times greater than that observed with IL1β alone, and the increase in CD106 expression observed with the combination of IL1β and IL4, 5 times the increase observed with IL4 alone. This effect has never been observed before.

The work presented here demonstrates that the proangiogenic and anti-inflammatory activities of CD106 ^high CD151 ⁺ nestin ⁺ MSCs obtained according to the method described in PCT/EP 2017/082316 (WO 2018/108859) are essentially mediated by EVs derived from these MSCs. Therefore, the inventors propose to use a biological product containing these EVs since, due to their production by MSCs, they exhibit increased levels of pro-angiogenic/pro-inflammatory surface proteins. Accordingly, this biological product exhibits the same enhanced pro-angiogenic activity as the parent MSCs, without restrictions on their use in clinical practice and industry.

In addition to their therapeutic potential, EVs can be used as biomarkers, particularly in the diagnosis of cancer. Among the 35 ongoing clinical trials related to the use of exosomes in cancer, approximately two to three are diagnostic and the rest are therapeutic (Roya et al., 2018).

Detailed Description of the Invention

Thus, according to a first aspect, the present invention relates to a composition containing extracellular vesicles (EVs) of CD106 ^high CD151 ⁺ nestin ⁺ MSC, which were obtained according to the method described in the application PCT/EP 2017/082316 (published as WO 2018/108859) .

This production method involves the following two general steps:

(1) culturing mesenchymal stem cells derived from biological tissue or fluid in a first culture medium free of growth factors so as to create a population of cultured undifferentiated mesenchymal stem cells,

And

(2) contacting said population of cultured undifferentiated mesenchymal stem cells with a second culture medium containing pro-inflammatory growth factors or inflammatory mediators, resulting in the formation of CD106 ^high CD151 ⁺ nestin ⁺ mesenchymal stem cells of interest, which will be used to obtain EVs by invention.

Said “undifferentiated MSC population” can be obtained by collecting mononuclear cells present in biological tissue or fluid and growing them in a first culture medium. These mononuclear cells can be obtained by any conventional method, such as enzymatic digestion or explant culture of perinatal tissue pieces (Otte et al., 2013), or isolation from biological fluids (Van Pham et al., 2016).

Explant culture is a particularly preferred method for obtaining such MSCs from umbilical cord, as shown below in Example 3.

Typically, this method requires removing the sample from the transport solution, cutting it into sections (approximately 2-3 cm long), disinfecting them with antibiotics and antifungal agents, which are then washed to restore the tissue and get rid of pieces of said tissue in the flasks to which Attachment will occur (preferably without medium, at room temperature), after which complete medium is carefully added to the attached explants and incubated at 37°C for several days. Ultimately, the migrating cells are collected using appropriate apparatus and maintained in culture in the appropriate first medium (see below) until they reach the intended confluency.

Said "first culture medium" may be any classical medium commonly used to promote the growth of living primary cells. Preferably, it does not contain any growth factors or any differentiation factors.

The person skilled in the art is well aware of what type of culture media can be used as the "first culture medium". Examples include DMEM, DMEM/F12, MEM (minimal maintenance medium), MEM alpha (α-MEM), IMDM (Iscove's modified Dulbecco's medium), or RPMI (Roswell Park Memorial Institute media). Preferably, said first culture medium is DMEM (Dulbecco's modified Eagle's medium) or DMEM/F12 (Dulbecco's modified Eagle's medium with Ham's F12 nutrient mixture).

More preferably, said first culture medium contains 2-20% or 2-10% fetal calf serum. Alternatively, said first culture medium may contain 1-5% platelet lysate. The most preferred medium contains 2-20% or 2-10% fetal bovine serum and 1-5% platelet lysate.

In addition, it is possible to use a medium that does not contain blood serum or platelet lysate as the first culture medium, provided that it contains other appropriate agents that promote the growth of primary living cells.

In a preferred embodiment, said "biological tissue" is any part of placental tissue or umbilical cord. In particular, it may include or be composed of placental cotyledons, amniotic membrane or placental chorionic membrane. In addition, Wharton's jelly, found in the umbilical cord, may be present. It may or may not include veins and/or arteries.

In another embodiment, said "body fluid" is a sample of umbilical cord blood, placental blood or amniotic fluid that has been collected from a woman or mammal generally without causing harm. For example, these tissues and fluids can be obtained after the birth of a child or procreation without any invasive procedures.

Said population of undifferentiated MSCs is preferably a plastic-plated population of mesenchymal stem cells that has been cultured in said first culture medium lacking any growth factor until the cells reach 85-90% confluency.

Systematically, cells are characterized phenotypically by fluorescence-activated cell sorting (FACS) or any conventional method to determine the level of surface markers CD73, CD90, CD105, CD166, CD45, CD34 and HLA-DR (human leukocyte antigens, DR sublocus).

If 95% of the cells express positive surface markers CD73, CD90, CD105 and CD166, and less than 2% express negative surface markers CD45, CD34 and HLA-DR, then the cells are trypsinized and reseeded at a low density, for example, at a density of 1000-5000 MSC per cm ² into the second culture medium.

Preferably, said "second culture medium" is any classical medium commonly used to promote the growth of living primary cells. This may be the same medium as the "first culture medium" or it may be another medium selected from, for example, DMEM, DMEM/F12, MEM, alpha-MEM (α-MEM), IMDM or RPMI. More preferably, said second culture medium is DMEM (Dulbecco's modified Eagle's medium) or DMEM/F12 (Dulbecco's modified Eagle's medium with Ham's F12 nutrient mixture).

Even more preferably, said second culture medium contains blood serum or platelet lysate, for example in the range of 2-20% fetal bovine serum and/or 1-5% platelet lysate. The most preferred second medium is DMEM containing 2-20% fetal bovine serum and 1-5% platelet lysate. In addition, it is possible to use a medium that does not contain blood serum or platelet lysate as the second culture medium, provided that it contains other appropriate agents that promote the growth of primary living cells.

When the cells reach 40-50% confluency, pro-inflammatory growth factors or inflammatory mediators are added to the second culture medium and the cells are cultured in this medium until they reach 90-95% confluency.

Said “pro-inflammatory growth factors” are typically interleukins or chemokines, which are known to have a pro-inflammatory effect. Examples of interleukins that may be added to the second culture medium include tumor necrosis factor alpha (TNFα), IL1, IL4, IL12, IL18, and interferon gamma (IFNγ). Examples of chemokines that can be added to the second culture medium include CXC-type chemokines CXCL8, CXCL10, CXCL1, CXCL2, CXCL3, CC-type chemokines CCL2 and CCL5. Other mediators involved in inflammation (such as anti-inflammatory agents) can be used.

In a preferred embodiment, at least two pro-inflammatory growth factors are added to the second culture medium defined above. These at least two pro-inflammatory growth factors are selected from the group consisting of: TNFα, IL1, IL4, IL12, IL18 and IFNγ. In a more preferred embodiment, said pro-inflammatory growth factors are selected from IL1, IL4, IL12, IL18. Even more preferably they are IL1 and IL4.

Typical concentrations of growth factor(s) that may be added to MSCs are in the range of 1-200 ng/ml, preferably in the range of 1-100 ng/ml, more preferably in the range of 10- 80 ng/ml. Preferably, the duration of the MSC culture step along with the growth factor(s) is at least one day, more preferably two days.

The term "IL1" as used herein means any isoform of interleukin 1, in particular IL1α and IL1β. IL1 isoforms can come from different sources, depending on the intended application. For example, IL1 of animal origin can be used for veterinary applications. Preferably, only IL1β is added to the second culture medium of the invention. In this particular embodiment, the concentration of interleukin 1β added may be in the range of 1-100 ng/ml, preferably in the range of 1-50 ng/ml, more preferably in the range of 10-40 ng/ml.

Human IL1-beta (IL1β or IL1b) is referenced under accession number NP_000567.1. GMP (Good Manufacturing Practice) compliant recombinant protein is commercially available (RnD Systems, Thermofisher, Cellgenix, Peprotech).

The term "IL4" as used herein means any isoform of interleukin 4. IL4 can be derived from a variety of sources, depending on the intended use. For example, IL4 of animal origin can be used for veterinary applications.

Human IL4 is referenced under accession number AAA59149. Recombinant protein that meets the conditions is commercially available (RnD Systems, Thermofisher, Cellgenix, Peprotech).

Any mixture of different pro-inflammatory growth factors can be used in said second medium. In particular, a preferred embodiment is the use of a mixture of IL1 and IL4, more particularly IL1β and IL4, as described below in the experimental section.

In this particular embodiment, the added interleukin 1β has a concentration in the range of 1-100 ng/ml, preferably in the range of 1-50 ng/ml, more preferably in the range of 10-40 ng/ml, and the added IL4 has a concentration in the range 1-100 ng/ml, preferably in the range of 1-50 ng/ml, more preferably in the range of 10-40 ng/ml in the second culture medium. Preferably, the duration of the culture step together with interleukin 1β and IL4 is at least one day, more preferably two days. Typically, the concentration of added interleukin 1β is 10 ng/ml, and the concentration of added interleukin IL4 is 10 ng/ml. Therefore, the cells are preferably cultured in a culture medium containing both interleukin 1β and IL4 at a concentration of 10 ng/ml, for example two days.

To determine the level of surface markers CD73, CD90, CD105, CD166, CD45, CD34 and HLA-DR during their production, the phenotypic characteristics of cells can be assessed by any conventional means. These markers are well known in the art. All antibodies useful for determining the expression levels of these markers are commercially available.

First of all, the expression of these cell surface markers can be assessed using well-known technologies, such as staining of cell membranes using biotinylation or other equivalent methods, followed by immunoprecipitation with specific antibodies, flow cytometry, Western blotting, enzyme-linked immunosorbent assay (ELISA). ELISA) or by enzyme-linked immunospot testing (ELISPOT), using antibody microarrays or tissue microarrays in combination with immunohistochemistry. Other suitable methods include fluorescence resonance energy transfer (FRET) or bioluminescence resonance energy transfer (BRET), microscopy or histochemistry techniques on single cells using one or more excitation wavelengths and using any of the adapted optical techniques, and electrochemical techniques (techniques voltametry and amperometry), atomic force microscopy and radiofrequency methods, e.g. multipole resonance spectroscopy, confocal and non-confocal, fluorescence, luminescence, chemiluminescence, absorption, reflection, transmission and birefringence or refractive index signal determination (e.g. surface plasmon resonance, ellipsometry, resonance mirror method, grating coupler waveguide method or interferometry), magnetic resonance imaging, analysis by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE); fractionation using high performance liquid chromatography (HPLC); time-of-flight mass spectroscopy with ionization by laser desorption of ions from the matrix (MALDI-TOF); liquid chromatography/mass spectrometry/mass spectrometry (LC-MS/MS). Preferably, cell surface marker levels are assessed by FACS.

Specifically, to obtain MSCs of interest it is usually necessary to:

a) collect mononuclear cells contained in biological tissue or fluid of perinatal origin,

b) provide conditions for the growth of said mononuclear cells in the first culture medium until they reach a confluence of 85-90%, preferably on a plastic surface,

c) after 95% of cells express positive markers CD73, CD90, CD105 and CD166, and less than 2% express negative markers CD45, CD34 and HLA-DR, seed cells at a density of 1000-5000 MSCs per cm ² in second culture medium,

d) add inflammatory mediators or pro-inflammatory growth factors in the concentration range of 1-100 ng/ml after the cells reach 40-50% confluency,

e) collect cells when they reach 90-95% confluency.

The collected cells can then be characterized phenotypically by FACS or any conventional method to determine the level of surface markers CD73, CD90, CD105, CD166, CD45, CD34 and HLA-DR. Said first and second culture media are described above.

In step d) of this method, the typical concentration of the added growth factor(s) is in the range of 1-200 ng/ml, preferably in the range of 1-100 ng/ml, more preferably in the range of 10-80 ng/ml. Preferably, the duration of the culture step together with the growth factor(s) is at least one day, more preferably two days.

In step d) of this method, the concentration of added interleukin 1β or IL4 may be in the range of 1-100 ng/ml, preferably in the range of 1-50 ng/ml, more preferably in the range of 10-40 ng/ml. Preferably, the duration of the culture step together with interleukin 1β and IL4 is at least one day, more preferably two days. Typically, the concentration of added interleukin 1β is 10 ng/ml, and the concentration of added interleukin IL4 is 10 ng/ml. Therefore, the cells are preferably cultured in a culture medium containing both interleukin 1β and IL4 at a concentration of 10 ng/ml, and the duration of this culture stage is, for example, two days.

The final collected cells will be "MSCs of the invention", "cell culture of interest", or "CD106 ^high CD151 ⁺ nestin ⁺ MSCs of interest", or "EV-producing cells". This cell culture typically contains greater than 60%, preferably greater than 60 to 70%, preferably greater than 70%, preferably greater than 80%, more preferably greater than 90%, and even more preferably greater than 95% of cells expressing CD106. In addition, it contains over 98%, preferably over 99% of cells expressing CD151. In addition, it contains over 98%, preferably over 99% of cells expressing nestin. Finally, it contains over 95%, preferably over 96%, preferably over 97%, preferably over 98% of cells expressing positive markers CD73, CD90, CD105 and CD166, and contains less than 2% of cells expressing negative markers CD45, CD34 and HLA-DR.

CD106 (also known as VCAM-1, which stands for vascular cell adhesion molecule 1) is known to have three isoforms. The sequences of these isoforms a, b and c are NP_001069.1, NP_542413.1 and NP_001186763.1, respectively. Antibodies for detecting the expression level of this particular biomarker are commercially available (eg, manufactured by Thermofisher, Abcam, OriGen, etc.). The expression of this marker on the surface of MSCs is a very important property since it triggers pro-angiogenic activities, which are essential for their therapeutic applications.

The biomarker nestin in humans is registered under the number NP_006608.1. Antibodies for detecting the expression level of this particular biomarker are commercially available (eg, manufactured by Thermofisher, Abcam, etc.).

The biomarker CD151 in humans is registered under the number NP_620599. Antibodies to detect the expression level of this particular biomarker are commercially available (eg, manufactured by Invitrogen, Sigma-Aldrich, Abeam, etc.).

More specifically, to obtain MSCs of interest it is usually necessary to:

a) collect, possibly separately, placental tissues from several donors;

b) it is possible to wash placental tissue cut into 1 mm ³ cubes three times using 1x PBS (phosphate buffered saline) and wash the tissue cubes again to remove most of the blood from the tissue;

c) it is possible to carry out the process of digestion of placental tissue from each donor separately with collagenase, centrifugation of the digested tissue and collection of mononuclear cells;

d) seed the collected mononuclear cells into the culture medium;

e) carry out trypsinization and subculture of cells after they reach 85-90% confluency;

f) characterize the cells based on the percentage of cells that express positive markers CD73, CD90, CD105 and CD166 and negative markers CD45, CD34 and HLA-DR;

g) inoculate cells in a culture medium containing 90% Dulbecco's modified Eagle's medium/F12-KnockOut (DMEM/F12-KO) and 10% FBS (fetal bovine serum) and growth factors, at a seeding density of 1000-5000 MSC per cm ² , when among them there are at least 95% expressing positive markers, and no more than 2% expressing negative markers;

h) add interleukin 1β in the range of 1-100 ng/ml and possibly IL4 in the range of 1-100 ng/ml, once the cells reach 40-50% confluency,

i) trypsinize and harvest the cells once they reach 90-95% confluency; And

j) it is possible to characterize cells based on the percentage of cells that express positive markers CD73, CD90, CD105 and CD166 and negative markers CD45, CD34 and HLA-DR.

Cells obtained through these methods are further used to produce EVs according to the invention.

The term "extracellular vesicles" (EV) is a general term for cellular vesicles whose size ranges from approximately 30 nm to several microns. Among them, exosomes constitute the most fully characterized class of EVs. Exosomes have a diameter of less than approximately 150 nm and are derived from the endosomal compartment. EVs contain cytosolic and membrane proteins derived from parental cells. The protein content of EVs depends on their cellular origin, and EVs are enriched in certain molecules, especially endosome-associated proteins (eg, CD63) and proteins involved in multivesicular body formation, and also contain targeting/adhesion molecules. Notably, EVs contain not only proteins, but also functionally active mRNA, long noncoding RNAs, and microRNAs, and have been shown in some cases to deliver this genetic material to recipient cells.

By “extracellular vesicles” or “EVs” we mean membrane vesicles released by cells into their microenvironment from their plasma membrane, or intracellular vesicles recovered after disruption of the cell membrane. In the context of the present invention, EVs typically have a diameter of less than or equal to about 500 nm, in particular from about 30 to about 500 nm, or from about 40 to about 500 nm, or from about 50 to about 250 nm. EVs are surrounded by a phospholipid membrane, which preferentially contains cholesterol, sphingomyelin and ceramide at relatively high levels and also preferably contains detergent-resistant membrane domains (lipid rafts).

The protein composition of EV membranes is similar to that of cell membranes. EVs are typically characterized by the presence of actin β, proteins involved in membrane transport and membrane fusion (such as Rab, GTPases (guanosine triphosphatases), annexins and flotillin), components of the endosomal sorting complexes required for transport (ESCRT, from the English endosomal sorting complexes required for transport) (such as Alix), tumor susceptibility gene 101 (TSG101), heat shock proteins (HSPs, such as HSPA8, HSP90AA1, HSC70 and HSC90), integrins (such as CD62L, CD62E or CD62P) and tetraspanins (especially CD63, CD81, CD82, CD53, CD9 and/or CD37). In the examples below, their presence is determined by the combination of CD9 and CD81 markers and the absence of calnexin.

In addition, within the scope of the invention is the use of the secretome of CD106 ^high CD151 ⁺ nestin ⁺ MSC cells, which were obtained according to the method described in PCT/EP 2017/082316. As used herein, the term “secretome” refers to all factors that are secreted by a cell, including EVs, proteins (growth factors, chemokines, cytokines, adhesion molecules, proteases, etc.), lipids, microRNAs, mRNA. The secretome of CD106 ^high CD151 ⁺ nestin ⁺ MSC cells, which were obtained according to the method described in PCT/EP 2017/082316, is believed to have the same pro-angiogenic biological effects as CD106 ^high CD151 ⁺ nestin ⁺ MSC described in PCT/EP 2017/082316.

EVs can be purified from MSC cells of interest by various methods, such as those described in Konoshencko et al., 2018 or Lai et al., 2010.

Differential centrifugation

The method includes several stages, including at least the following three stages from 1) to 3):

1) centrifugation at low speed to remove cells and cellular debris (less than 10,000 × g),

2) centrifugation at higher speeds to eliminate larger vesicles and, finally

3) high-speed centrifugation to sediment EVs (above 100,000 × g).

The resulting EV preparation is further purified and the isolated vesicles are selected according to their size using suspension microfiltration. Density Gradient Centrifugation

This approach combines ultracentrifugation and the application of a sucrose density gradient. More specifically, density gradient centrifugation is used to separate EVs from non-vesicle particles such as proteins and protein/RNA aggregates. Thus, this method allows the separation of vesicles from particles with a different density. Proper centrifugation time is very important, otherwise contaminants may still be present in the EV fractions if they are close in density. Recent studies recommend using EVs as pellets from ultracentrifugation for sucrose density gradient centrifugation.

Gel Permeation Chromatography

Gelprone chromatography is used to separate macromolecules based on their size and shape rather than molecular weight. This method uses a column filled with porous polymer beads containing numerous pores and channels. The passage of molecules through these balls depends on the diameter of the molecules. Molecules with a small radius take longer to migrate through the pores in the column beads, while macromolecules elute from the column sooner. Gel permeation chromatography allows for the precise separation of large and small molecules. In addition, different elution solutions can be used in this method.

Ultrafiltration

Ultrafiltration membranes can also be used to isolate EVs. Depending on the size of the microvesicles, this method allows the separation of EVs from proteins and other large molecular weight macromolecules. In addition, EVs can be released by trapping them as they pass through a porous structure. The most common filtration membranes have pore sizes of 0.8 µm, 0.45 µm or 0.22 µm and can be used to collect EVs above 800 nm, 400 nm or 200 nm. In particular, to isolate EVs with a size of 40–100 nm, a ciliated structure based on micropillar porous silicon ciliated structure was developed. At the initial stage, larger vesicles are removed. In the next step, the aggregate of EVs is concentrated on a filtration membrane. The isolation step is relatively short, but the method requires pre-incubation of the silica structure with a PBS-based buffer. In the next step, the aggregate of EVs is concentrated on a filtration membrane.

Deposition using polymers

The polymer-based sedimentation method typically involves mixing a biological fluid with a polymer-containing precipitation solution, incubating at 4°C, and ultracentrifuging. One of the most common polymers used for polymer-based deposition is polyethylene glycol (PEG), preferably PEG 6000 or PEG 8000. Deposition using this polymer has a number of advantages, including moderate impact on EV release and use of neutral pH. Several commercial kits have been developed that use PEG to isolate EVs. The most commonly used kit is ExoQuick™ (System Biosciences, Mountain View, CA, USA). Recent studies have demonstrated that the highest EV yields were obtained using the ultracentrifugation method in conjunction with the ExoQuick™ kit.

Immunological isolation

Several methods have been developed for the immunological isolation of EVs, based on the use of proteins external or internal to the EV membrane surface or intracellular EV proteins. However, these methods are commonly used to detect, analyze, and quantify EV proteins.

The examples below use ultrafiltration.

EVs obtained by purification from MSC cells obtained according to the above-mentioned production method are hereinafter referred to as "EVs according to the invention". They exhibit increased proangiogenic activity (as do the MSCs that produce them) compared to prior art EVs.

As shown in Example 8 below, these EVs are characterized in particular by the fact that they demonstrate the presence of:

(1) CD106 at a detectable level, and

(2) CD200 at detectable levels.

Importantly, they demonstrate the presence of the proangiogenic markers CD106/VCAM and CD200 at higher levels than prior art EVs (see Figure 3). They also demonstrate the presence of a number of other angiogenic markers such as fibroblast growth factor 7 (FGF7), CCL2 and angiopoietin 1 (see Fig. 4).

These markers are well known in the art, and antibodies that detect them are commercially available. Their presence can be assessed by any traditional method such as Western blotting.

According to the present invention, an EV "demonstrates the presence of a marker at a detectable level" if the level of presence of said marker is significant, i.e. if the signal associated with staining of a specified marker (usually obtained using an antibody recognizing the specified marker, the antibody, for example, associated with a fluorescent dye) that is measured for the specified EV is greater than the signal corresponding to the staining of EVs that are known not to demonstrate the presence of the specified marker. One skilled in the art will be well versed in how to identify these cells/markers, so there is no need to describe these protocols in detail herein.

The composition according to the invention mainly contains extracellular vesicles (EV) according to the invention, formed by MSC cells described in PCT/EP 2017/082316.

In the context of the present invention, the amount of EV present in the composition is preferably determined using a NanoSight device (manufactured by Malvern), in which case the amount of EV is designated as "pp", which corresponds to the number of particles detected by the NanoSight device.

Typically, the composition of the invention contains from 1×10 ⁸ to 1×10 ¹⁴ ppEV per ml, more preferably from 1×10 ¹¹ to 1×10 ¹² ppEV per ml.

According to a second aspect, the present invention also relates to a method for preparing a composition containing EVs from MSCs obtained by the above-mentioned method, comprising:

a) culturing said MSCs in an EV-free culture medium under conditions allowing their proliferation; And

b) purification of EVs from these cells.

The term "EV-free culture medium" which may be used herein refers, for example, to a classical basal medium without additives (such as alpha-MEM, DMEM, DMEM/F12...) or to a classical basal medium supplemented with no additives. vesicles with platelet lysate in an amount of from 1 to 10%, preferably 5%, more preferably 8%. Before being brought into contact with the MSC of the invention, it does not contain any exogenous EV (after contact with the MSC of the invention, EVs that are produced by the MSC of the invention will begin to be present in this environment). Thus, it contains neither serum nor platelet lysate, which may contain exogenous vesicles.

Said EV-free culture medium is preferably supplemented with added growth factor(s), the concentration of which is in the range of 1-200 ng/ml, preferably in the range of 1-100 ng/ml, more preferably in the range of 10-80 ng/ml. More preferably, said culture medium is supplemented with interleukin 1β and/or IL4, the concentration of which is in the range of 1-100 ng/ml, preferably in the range of 1-50 ng/ml, more preferably in the range of 10-40 ng/ml. Typically, the concentration of added interleukin 1β in said medium is 10 ng/ml, and the concentration of added interleukin IL4 in said medium is 10 ng/ml. Therefore, the cells are preferably cultured in an EV-free culture medium that contains both interleukin 1β and IL4 at a concentration of 10 ng/ml, for example two days.

Conditions allowing the proliferation of MSC cells have been described above. An important point is that the cells must be in good condition, since due to cell death and the presence of apoptotic bodies, the EV preparation may become contaminated. Thus, conditions that allow MSC cell amplification and maintenance of exponential growth should be used, and EV purification should be carried out before the end of the exponential growth phase, i.e. until reaching a plateau, when cell death becomes significant. For media containing components of animal origin (e.g. blood serum), EV depletion of the media should be carried out. This can be done by centrifuging the culture medium at 100,000 x g for 8-16 hours (eg overnight) at approximately 4°C.

Cultivation of MSCs under these conditions usually lasts from 1 to 7 days, preferably from 1 to 5 days, more preferably from 2 to 3 days, even more preferably for 72 hours.

For therapeutic purposes, this entire method should preferably be carried out under sterile conditions.

Purification of EVs can be done by any of the above methods (preferably through ultrafiltration, as shown in the experimental section below).

The results described below also show that the use of the methods of the invention enables the generation of EVs exhibiting high levels of the membrane protein CD106/VCAM1. Importantly, this protein is associated with the expression of proangiogenic cytokines and proinflammatory proteins (Han Z.C., et al., Bio-medical Materials and Engineering, 2017, and Du W. et al., Stem Cell research & therapy, 2016). Thus, EVs of the invention that exhibit high levels of CD106 protein can be used due to their proangiogenic/proinflammatory efficacy.

Katoh & Katoh [Stem Cell Investig., 2019, 6: 10] mention that CD200 is involved in a variety of physiological and pathological processes at the interface of vascular remodeling and immune regulation. CD200 is a transmembrane protein that is expressed in a number of cells, such as B and T lymphocytes, endothelial cells, neurons and pancreatic islet cells, and whose expression is upregulated by IL4. CD200 signals through the CD200 receptor (CD200R), a transmembrane protein. CD200-CD200R-mediated signaling plays a critical role in cancer and non-cancer diseases through the regulation of immunity and angiogenesis. For example, compared with CD200− B16 melanoma cells, CD200+ B16 melanoma cells demonstrate enhanced tumorigenesis due to proliferation of cells of this myeloid lineage and increased tumor angiogenesis, as shown in Cd200r knockout mice.

In this regard, according to a third aspect, the present invention relates to a composition containing extracellular vesicles derived from CD106 ^high CD151 ⁺ nestin ⁺ MSC, obtained according to the method described in PCT/EP 2017/082316, for use in the treatment of subjects suffering from ischemic diseases, circulatory system disorders, immunopathologies, organ damage or organ dysfunction. In other words, the invention relates to the use of said EVs for the preparation of a medicament intended for use in the treatment of subjects suffering from coronary artery disease or a circulatory system disorder. In addition, the medicinal product according to the invention can be applied to the capillary network of blood vessels of the skin and can be included in dermatological and cosmetic compositions applied to the skin.

Preferably, the composition according to the invention does not contain any cells; in particular, it does not contain any MSC cells. Typically it contains only the EV according to the invention as the only active principle. In addition, it may contain a pharmaceutical carrier or adjuvant as specified below.

The EV-containing composition of the invention is administered in therapeutically effective amounts.

As used herein, the term “therapeutically effective amount” refers to an amount sufficient for the intended use. In the case of proangiogenic compositions according to the invention, it refers to an amount sufficient to induce migration and/or proliferation of endothelial cells.

The dose administered may vary depending on the age of the subject, body surface area or body weight, or the route of administration and associated bioavailability. Such dose adaptation is well known to those skilled in the art.

Any mammal can be treated with the compositions/EVs of the invention. Said mammal may be a domestic animal (dog, cat, horse, etc.) or a livestock animal (sheep, goat, cow, etc.). It will be apparent to one skilled in the art that if an animal is treated according to the method of the invention, the initial undifferentiated MSC will be obtained from a biological sample from the same animal species (allogeneic graft) or from a similar species (heterologous graft), and the growth factors that are used in the second culture environment will correspond to the growth factors of an animal of the same species. For example, if a cat is being treated, the initial MSCs will be derived from perinatal tissue or body fluid of the cat, and feline IL1β (recombinant or not) will be added to the second culture medium, possibly along with feline IL4.

In a preferred embodiment, said mammal is a human. In this case, the initial MSCs would be derived from perinatal tissue or body fluid obtained from the woman, and human IL1β (recombinant or not) would be added to the second culture medium, possibly along with human IL4.

For this purpose, the composition of the invention can be administered or applied topically to said subject by any conventional method. In this case, the present invention relates to a method of treating a subject suffering from coronary artery disease, circulatory system disorder, immunopathology, organ damage or organ dysfunction, said method comprising the step of administering the above-described composition to said subject. Such administration may be accomplished through the use of an implantable reservoir, or by in situ injection of the EV into the muscle, or by intravenous injection, or using any suitable delivery system. In addition, administration can be accomplished locally by directly bringing the EV into contact with the skin or mucosa, or by applying the EV to the skin or any mucosa using a device, or by delivering the EV using any suitable delivery system to the skin or any mucosa .

Preferably, said disease or disorder is selected from the group consisting of: diabetes mellitus type 1, diabetes type II, GVHD, aplastic anemia, multiple sclerosis, Duchenne muscular dystrophy, rheumatoid arthritis, cerebral stroke, idiopathic pulmonary fibrosis, dilated cardiomyopathy, osteoarthritis, cirrhosis , liver failure, renal failure, peripheral arterial occlusive disease, critical limb ischemia, peripheral vascular disease, heart failure, diabetic ulcer or any degenerative disease, synechia, endometrial pathology or fibrotic lesion of the gastrointestinal tract such as rectal fistula. More preferably, said disease or disorder is peripheral arterial occlusive disease, critical limb ischemia, peripheral vascular disease or diabetic ulcer. In a specific embodiment, said disease or disorder is a skin or mucosal disease, including (but not limited to) a diabetic ulcer, ulcer, injury, burn, steam or liquid burn, wound or wound healing problem, bedsore, wart, and so on.

EV according to the invention can be more specifically used in a dermatological preparation, the purpose of which is to treat skin pathologies such as burns, wounds, ulcers, scars, warts or other diseases such as synechia or fibrous lesions of the gastrointestinal tract (for example, fistula rectum intestines).

In another specific embodiment, said disease or disorder is a rectal fistula or endometrial injury.

Other applications are covered by this application. In particular, it is possible to use EVs and compositions according to the invention to solve diagnostic, dermatological and cosmetic problems, for example, to regenerate cells of the skin or mucous membrane, improve the appearance of the skin or mucous membrane, correct a defect in the skin or mucous membrane, or heal a burned area of the skin or mucous membrane.

To increase the effectiveness and facilitate the administration of the drug of the invention, the EV of the invention can be mixed with any agent, composition of agents, or combined with other biocompatible material or device. In addition, the EVs of the invention can be encapsulated or included in any suitable delivery system or any suitable biocompatible material. EVs or compositions containing them can be used in conjunction with a medical device such as an endoscope, stent or syringe, for example. It can also be applied topically by bringing the EV into contact with the skin or mucous membrane.

In the case of intravenous, intratumoral or intranasal administration, aqueous suspensions, isotonic saline solutions or sterile injection solutions that contain pharmacologically compatible dispersing agents and/or wetting agents can be used. As an excipient you can use water, alcohols, polyols, glycerin, vegetable oils and so on.

In the case of local administration, the compositions may be presented in the form of a gel, paste, ointment, cream, lotion, aqueous or aqueous-alcoholic liquid suspension, oily solution, lotion or serum-type dispersion, anhydrous or lipophilic gel, liquid or semi-solid milk-type emulsion, prepared by dispersing a fatty phase in an aqueous phase or vice versa, suspensions or emulsions of a soft or semi-solid consistency such as a cream or gel or, alternatively, microemulsions, microcapsules, microparticles or vesicular dispersions of the ionic and/or non-ionic type. These compositions are prepared according to standard methods. In addition, a surfactant can be included in the composition to provide deeper penetration of the EV. The penetration enhancing agent may be selected from, for example, mineral oil, ethanol, triacetin, glycerol and propylene glycol; the cohesive agents are selected, for example, from the group consisting of polyisobutylene, polyvinyl acetate, polyvinyl alcohol and thickeners.

Suitable oral formulations for unit dosage administration primarily include tablets, film-coated tablets, pills, capsules and soft gelatin capsules, oral powders, granules, solutions and suspensions.

If a solid composition is prepared in tablet form, the main active ingredient may be mixed with a pharmaceutical diluent such as gelatin, starch, lactose, stearic acid or magnesium stearate, talc, gum acacia or analogues. The tablets may be coated with sucrose or other suitable materials, or even processed so that they have a sustained or delayed action and continuously release a predetermined amount of the active ingredient.

The capsule formulation can be prepared by mixing the active ingredient with a diluent and filling the resulting mixture into soft or hard capsules along with excipients such as vegetable oils, waxes, fats, semi-solid or liquid polyols and so on.

The preparation in the form of a syrup or elixir may contain the active ingredient together with a sweetener, an antiseptic, a flavoring agent and a suitable coloring agent. Excipients such as water, polyols, sucrose, invert sugar, glucose and so on can be used.

Powders or water-dispersible granules may contain the active ingredient mixed with dispersing agents, wetting agents and suspending agents together with flavor correctors and sweeteners.

For subcutaneous administration, any suitable pharmaceutically acceptable diluent may be used. In particular, a pharmaceutically acceptable oily diluent such as sesame oil may be used.

The present invention is also directed to the development of a medical device containing the EV of the invention. By “medical device” as used herein is meant any instrument, device, fixture, machine, equipment, implant, reagent for administering a therapeutic composition. In the context of the present invention, said medical device is, for example, a patch, a stent, an endoscope or a syringe.

The present invention is also directed to the development of a delivery system containing the EVs of the invention. By "delivery system" as used herein is meant any system (medium or carrier) for administering a pharmaceutical product to a patient. This may be an oral delivery system or a controlled release system. In the context of the invention, said delivery system is, for example, liposomes, proliposomes, microspheres, micro- or nanovesicles made of biopolymers, lipids or nanoparticles.

In a preferred embodiment of the invention, the EVs of the invention are included in a hydrogel or other biocompatible material or excipient. Said hydrogel may primarily include a sodium alginate hydrogel, a hyaluronic acid hydrogel, a chitosan hydrogel, a collagen hydrogel, a hydroxypropyl methylcellulose hydrogel. (HPMC), poly-L-lysine hydrogel, poly-L-glutamic acid hydrogel, polyvinyl alcohol (PVA) hydrogel, polyacrylic acid hydrogel, polymethylacrylic acid hydrogel, polyacrylamide (PAM) hydrogel ) and hydrogel based on poly-N-acrylamide (PNAM).

The present invention also relates to a hydrogel containing the EV of the invention and possibly other biocompatible material or excipient. In the present invention, an alginate-based hydrogel such as the alginate-based hydrogel described in CN 106538515 is preferred.

In the context of the present invention, "biocompatible materials" are materials traditionally used in biomedical applications. These are, for example, metals (such as stainless steel, cobalt alloys, titanium alloys), ceramics (aluminum oxide, zirconium dioxide, calcium phosphates), polymers (silicones, polyethylene, polyvinyl chloride, polyurethanes, polylactides) or natural polymers (alginate, collagen , gelatin, elastin, etc.). These materials can be synthetic or natural. Biocompatible excipients are well known in the art and therefore need not be described in detail.

This hydrogel can be used for cosmetic or therapeutic purposes.

The present invention also relates to a pharmaceutical or veterinary composition containing the EVs of the invention, as well as its use for the treatment of diseases and disorders mentioned above. It also relates to a dermatological or cosmetic composition containing an EV according to the invention.

Such pharmaceutical, veterinary or cosmetic composition may further contain other biocompatible agents (eg, hydrogel) as described above.

Said composition preferably contains from 1×10 ⁸ to 1×10 ¹⁴ ppEV per ml; more preferably 1×10 ¹¹ to 1×10 ¹² ppEV per ml.

Description of the figures

In FIG. Figure 1 shows the results of Western blotting demonstrating the presence/absence of CD9, CD81 and calnexin markers in extracellular vesicles, the purification of which is described in the Examples section. Lane A - cell lysate of MDA cells; Lane B - MSC cell lysate according to the invention in alpha-MEM; Lane C - EV according to the invention in alpha-MEM.

In FIG. Figure 2 presents the results of a Boyden chamber chemotactic migration assay demonstrating the paracrine effect of the cells used to obtain the EVs of the invention on ECFC (endothelial colony forming cells) in the presence or absence of vascular endothelial growth factor (VEGF) . Column T is the level of migration in the control medium in the absence (-)/presence (+) of VEGF (50 ng/ml). Column 1 - level of cell migration from batch 1 in the absence (-)/presence (+) of VEGF (50 ng/ml). Column 2 - level of cell migration from batch 2 in the absence (-)/presence (+) of VEGF (50 ng/ml). Column 3 - level of cell migration from batch 3 in the absence (-)/presence (+) of VEGF (50 ng/ml).

In FIG. 3 shows Western blot results showing the content of EVs derived from MSCs pretreated under three different conditions: Lane A - EVs derived from unstimulated MSCs; Lane B - EVs derived from MSCs stimulated with IL1β and IL4 (EVs of the invention) , and track C - EVs originating from MSCs stimulated with LPS, as described in example 8. Six markers were tested (CD9, CD81, Alix (ALG-2-interacting protein X) , calnexin, VCAM (English vascular cell adhesion molecule - vascular cell adhesion molecule) and CD200). 20 µg EV was added to the well.

In FIG. Figure 4 demonstrates the differential abundance of different angiogenesis-related proteins determined using a panel of proteins for MSC-derived EVs pretreated under three different conditions (unstimulated, IL-stimulated, and LPS-stimulated). The average pixel intensity is given for each protein.

In FIG. 5 shows Western blot results showing the contents of MSCs pretreated under three different conditions: Lane A - unstimulated MSCs, Lane B - MSCs stimulated with IL1β and IL4 (MSCs of the invention), and Lane C - MSCs stimulated with LPS as described in example 8. Six markers were tested (CD9, CD81, Alix, calnexin, VCAM and CD200). Lysate was added to the well in an amount corresponding to 20 μg of protein.

Examples

For simplicity and illustrative purposes, the present invention is described with reference to its typical embodiments. However, it will be apparent to one of ordinary skill in the art that the present invention can be practiced without limitation to these specific details. In other cases, well-known methods are not described in detail so as not to unduly obscure the understanding of the present invention.

All steps 1-4 below are carried out as described in the Examples section of application PCT/EP 2017/082316, which is hereby incorporated by reference.

1. Isolation of pro-angiogenic umbilical cord-derived mesenchymal stem cells (UCMSC)

The umbilical cord was removed from the transport solution and cut into 2-3 cm sections. Each segment containing a blood clot that could not be removed was discarded to avoid contamination by attached blood cells. These sections were then disinfected by washing in an antibiotic and antifungal bath containing αMEM plus vancomycin (1 g/L) plus amoxicillin (1 g/L) plus amikacin (500 mg/L) plus amphotericin B (50 mg/L), for 30 min at room temperature (RT). Antibiotics were dissolved in sterile water for injection extemporaneously.

Umbilical cord sections were removed from the bath and quickly washed in 1× PBS at room temperature (RT). Light incisions were made on the surface layer of the epithelium without affecting the vessels. The section was then divided into smaller 0.5 cm thick sections and placed in the bottom of a 150 cm ² plastic flask with a lid. Slices of 6 to 10 per flask were placed so that there was a free space of at least 1 cm radius around each slice and allowed to attach for 15 min at RT without adding medium.

Once attached, complete medium (aMEM plus 5% clinical grade platelet lysate plus heparin (2 U/ml)) was carefully added to keep the explants attached to the bottom of the flask. Then the contents of the flasks were incubated at 37°C, 90% humidity and 5% CO ₂ .

The culture medium was changed after 5-7 days.

On the 10th day after isolation, cell migration from the explants was monitored using an inverted microscope. If a halo of attached cells was noted around most of the explants, they were carefully removed by lifting them out of the flask through the lid using sterile, disposable tweezers.

Starting from this stage, the confluency of the cells was visually checked every other day and, if necessary, the medium was replaced on the 17th day.

When the cells reached 70-90% confluency or D20 value, the medium was removed and the cells were washed using 30 ml of 1× PBS per flask. Cells were then detached using trypsin, collected along with the old medium, and centrifuged for 10 min at 300 × g. The supernatant was discarded, and the cells were then suspended in a cryopreservation solution containing αMEM plus human serum albumin (HSA; 100 mg/ml) plus 10% dimethyl sulfoxide (DMSO) and cryopreserved.

2. Thawing and culturing MSC cells

Cells were thawed following the classical protocol. Briefly, cryovials were removed from liquid nitrogen and quickly immersed in a 37°C water bath. Once the tube was free of ice, cells were diluted in prewarmed (37°C) complete medium (αMEM plus 0.5% (v/v) ciprofloxacin plus heparin (2 U/mL) plus 5% (v/v) ciprofloxacin). /vol.) platelet lysate (PL)) and quickly centrifuged (300×g RT, 5 min).

After centrifugation, cells were suspended in prewarmed complete medium and assessed for number and viability (using trypan blue/Malasses hemocytometer).

Cells were seeded at a density of 4000 cells/cm ² in a plastic culture flask containing complete medium and incubated (90% humidity, 5% CO ₂ , 37°C).

3. Stimulation of MSC cells

After several days of propagation, cells were checked for confluency. When confluence reached 30–50%, the old medium was discarded and replaced with either fresh complete medium for unstimulated growth conditions or fresh medium supplemented with IL-1β (10 ng/ml) and IL-4 (10 ng/ml).

The cells were then incubated for at least 2 days, after which the experiment was performed using flow cytometry.

This stimulation step was important in conferring a pro-angiogenic phenotype on MSCs, as described in PCT/EP 2017/082316.

The angiogenic activity of cells from several batches was tested in a Boyden chamber assay (see Figure 2).

Briefly, ECFC migration in response to a gradient of proangiogenic substances was assessed in a 24-well modified Boyden chamber on an 8-μm polycarbonate membrane filter (BD Biosciences) coated with fibronectin (20 μg/ml) from bovine plasma (Sigma- Aldrich - F1141). Before the experiment, the bottom of the wells of a 24-well plate was seeded with MSC cells at a density of 6000 cells/cm ² in six replicates and the cells were cultured for 4 days in αMEM plus 1% SVF (stromal vascular fraction). A series of control media wells filled with αMEM plus 1% FBS were prepared.

After overnight fasting in EBM-2 basal medium (Endothelial Cell Growth basal medium-2) (Lonza - CC-3156) supplemented with 0.2% FBS, 200,000 ECFCs in starvation medium were loaded into the top of the microchamber cells/well.

As a positive control for each condition, VEGF (Miltenyi - 130-109-383) was added to three of the six wells at a concentration of 50 ng/ml.

After 5 hours of incubation, cells were removed from the upper surface of the membrane filter by wiping with a cotton swab. All membranes were then stained using the May-Grünwald-Gemsa (MGG) method, mounted, and photographed.

In FIG. 2, all columns with a (-) sign represent the number of migrated ECFCs in the absence of VEGF, whereas all columns with a (+) sign represent the number of migrated ECFCs in the presence of VEGF. For control conditions (T- and T+), it was shown that ECFCs are able to enhance their migration in the presence of VEGF. Test conditions (1, 2, 3) show that while batch 1 appears to have no effect on ECFC behavior, both batches 2 and 3 enhance the effect of VEGF on ECFC, and furthermore, batch 3 even enhances ECFC migration in the absence of VEGF.

Since cell-to-cell contact is prevented in Boyden's chambers, this effect is certainly mediated by soluble extracellular mediators, either soluble proteins or extracellular vesicles.

4. Collection and cryopreservation of MSC cells

After 2-3 days of expansion/stimulation, cells were checked for confluency. If confluence was up to 80% inclusive, then cells were collected. Briefly, old media was discarded and cells were washed with 1× DPBS (Dulbecco's phosphate buffered saline). Trypsin-EDTA (ethylenediaminetetraacetic acid) was added and the cells were incubated for 5 min at 37°C. The effect of trypsin was neutralized by adding at least two times the volume of medium, and the cell suspension was collected and assessed for quantity and viability.

Cells were centrifuged for 10 min at 300×g. The supernatant was discarded, and the cells were then suspended in a cryopreservation solution containing αMEM plus HSA (100 mg/ml) plus 10% DMSO and cryopreserved.

5. Thawing MSC cells and preparing conditioned medium

Cells were thawed following the classical protocol. Briefly, cryovials or cryopouches were removed from liquid nitrogen and quickly immersed in a 37°C water bath. Once the tube was free of ice, cells were diluted in prewarmed (37°C) αMEM and quickly centrifuged (300 × g, RT, 5 min).

After centrifugation, cells were suspended in prewarmed complete medium and assessed for number and viability (using trypan blue/Malasses hemocytometer).

Cells were seeded at a density of 2000 cells/cm ² in the required number of plastic culture flasks with an area of 300 cm ² to obtain the required amount of conditioned medium (1 (one flask) T300 corresponds to 30 ml of conditioned medium). Cells were incubated at 90% humidity, 5% CO ₂ , 37°C in αMEM supplemented with 0.5% (v/v) cilofloxacin, heparin (2 units/ml) and 8% (v/v) centrifuged PL. Centrifuged PL was obtained by centrifuging the PL for 1 h at 6000 × g and 10 °C and recovering the supernatant.

After 5 days, the medium was replaced. When the cells reached 80% confluency (on day 6), the medium was removed and the cells were washed 3 times with PBS. Unsupplemented αMEM was added at a rate of 50 ml/T300 and maintained for 24 hours. After 24 hours, the medium was replaced with 30 ml of either unsupplemented αMEM or αMEM supplemented with 8% vesicles without PL (platelet lysate).

The cells were left to secrete for 36 hours, the medium was removed, centrifuged for 5 minutes at 400×g and RT in Heraeus Multifuge 3 S-R. The supernatant was removed and frozen at -80°C.

Cells were trypsinized and analyzed for cell number and viability.

6. Isolation and characterization of extracellular vesicles

Extracellular vesicles derived from such MSCs can be isolated by any methods known in the art, such as, but not limited to, ultracentrifugation, ultrafiltration, density gradient centrifugation, gel permeation chromatography, use of a precipitation kit, immunoaffinity capture, application of microfluidic devices.

In this experiment, the fraction enriched in extracellular vesicles was separated by ultrafiltration of the conditioned medium followed by gel permeation liquid chromatography (SEC).

Analysis of the number and size distribution of extracellular vesicles was carried out using the nanoparticle trajectory analysis method (Nanosight).

In addition, the content of the EVs of the invention can be assessed by Western blotting using the following conditions.

Materials and reagents

Lysis buffer (RIPA) (from the English radioimmunoprecipitation assay buffer - radioimmunoprecipitation assay):

- sodium deoxycholate 1%;

- SDS 0.1%;

- Tris⋅Cl, pH 7.4 20 mM;

- EDTA 1 mM;

- NaCl 150 mM;

- Triton x-100 1%;

- protease inhibitor cocktail (CIP, 100x), Sigma catalog number P8340.

Cell lysis

100 μl of cold RIPA buffer containing CIP at 1× final concentration was added to MSC cells in an amount of 1×10 ⁶ .

The resulting mixture was incubated for 10 min on ice, centrifuged for 15 min at 4°C for 20 min, and then the supernatant was recovered.

Protein concentration determinations were performed using the Bicinchoninic Acid (BCA) Trace Protein Assay Kit, Pierce part number 23235.

Electrophoresis was carried out in 4-12% bis-Tris gels with a layer thickness of 1.5 mm, having 10 wells, Novex Nosex (Life Technologies, NP0335PBOX) or NuPAGE® (working buffer based on 3-(N -morpholino)propanesulfonic acid (MOPS) with SDS (20x)) (Life Technologies, NP0001). Samples were denatured for 10 min at 70°C using ¼ volume lithium dodecyl sulfate (LDS) buffer (Invitrogen, NP0007, 4x) with or without 500 mM dithiothreitol (DTT). Transfer was carried out electrophoretically using a Mini Trans-Blot membrane system, part number: 170-3930, to a polyvinylidene fluoride (PVDF) membrane (Amersham, part number: RPN303LFP) activated with absolute ethanol and subjected to washing.

The following substances were used to identify proteins:

Tris-buffered saline (TBS), 10x, BioRad part number 1706435;

blocking buffer (TBS milk): 1x TBS_0.1% Tween_5% skim milk;

Wash buffer (TBS): 1x TBS_0.1% Tween;

AC buffer (TBS_AC): 1x TBS_0.1% Tween_0.3% milk.

Analysis of the presence of membrane proteins CD9 (Biolegend - 312102) and CD81 (Biolegend - 349501) was carried out using Western blotting (Fig. 1). Both markers, CD9 and CD81, were present in the purified EV fraction (lane C), as well as in the MDA cell lysate (lane A - positive control) and in the original cell lysate (column B). The reticulum marker calnexin (Elabscience - E-AB-30723) was absent, indicating that this fraction was purified correctly.

Fluorimetric analysis was performed using an antibody labeled with Alexa Fluor 680, GAM (goat anti-mouse - goat anti-mouse IgG; catalog number: A21058) or GAR (goat anti-rabbit - goat anti-rabbit IgG) ; catalog number: A21076) from Thermofisher, at a dilution of 1/10000.

Chemiluminescence assays were performed using horseradish peroxidase (HRP)-conjugated antibody, GAM (cat. no.: 170-6516) or GAR (cat. no.: 170-6515) from BioRad, at a dilution of 1/5000.

7. Comparison of UCMSC-derived EVs stimulated with LPS and UCMSC-derived EVs stimulated with a combination of IL1β and IL4 (EVs of the invention)

Dongdong Ti et al (Journal of translational medicine, 2015) describe extracellular vesicles purified from umbilical cord-derived MSCs that were pretreated with the proinflammatory factor LPS.

Yunbing Wu et al (BioMed Research International, 2017) describe the anti-inflammatory properties of extracellular vesicles derived from unstimulated human umbilical cord-derived mesenchymal stem cells.

None of these publications suggest adding IL1β and/or IL4 to stimulate MSCs from which the EVs of the invention are derived.

The purpose of the presented example is to demonstrate that EVs derived from CD106 ^high CD151 ⁺ nestin ⁺ MSCs of the invention differ from those described in prior art documents in that they are secreted by cells that have been cultured in a specific conditioning medium containing pro-inflammatory growth factors such as IL1β and IL4.

Comparison of the EVs of the invention with EVs derived from unstimulated MSCs or from MSCs stimulated with LPS demonstrated that the EVs of the invention exhibit molecular properties that truly differentiate them from prior art EVs, for example in terms of protein content or surface markers.

7.1. Preparation of conditioned media (culture supernatants) of umbilical cord-derived MSCs cultured in 3 different conditioning media

7.1.1. Preparation of 3 conditioning media

Three conditioning media were prepared as follows.

NS indicates control medium (no pro-inflammatory factor treatment).

For a 675 ml packet of αMEM (final concentration: 10 ng/ml).

1. Place the insertion device into one of the exit ports.

2. Using a needle, inject 290 µl of heparin solution (5000 units/ml; final concentration 2 units/ml). Verify by suction/draining that there is no heparin in the administration device.

3. Place the injection device into the outlet port of the clinical platelet lysate (CPL) bag and withdraw 56.8 mL of centrifuged CPL using a 50 mL syringe with a Luer-Lock (LL) needle.

S1 denotes conditioning medium containing IL1β and IL4 ("second medium" in the method described in WO 2018/108859).

For a 675 ml packet of αMEM (final total interleukins concentration: 10 ng/ml).

4. Place the insertion device into one of the exit ports.

5. Using a needle, inject 290 µl of heparin solution (5000 units/ml; final concentration 2 units/ml). Verify by suction/draining that there is no heparin in the administration device.

6. Place the insertion device into the outlet port of the CPL bag and withdraw 56.8 ml of centrifuged CPL using a 50 ml LL syringe.

7. Inject CPL into the aMEM bag and homogenize using a syringe.

8. Add cytokines:

1) defrost an aliquot (73.2 μl) of each interleukins (IL) (C = 100 μg/ml);

2) to select 400 μl of complete medium and mix it with IL, use a 1 ml syringe equipped with a needle;

3) take the environment containing the IL and add it to the complete environment package.

Conditioning medium containing LPS

For a 675 ml packet of αMEM (final LPS concentration: 100 ng/ml).

1. Place the insertion device into one of the exit ports.

2. Using a needle, inject 290 µl of heparin solution (5000 units/ml; final concentration 2 units/ml). Verify by suction/draining that there is no heparin in the administration device.

3. Place the insertion device into the exit port of the CPL bag and withdraw 56.8 ml of centrifuged CPL using a 50 ml LL syringe.

4. Inject CPL into the aMEM bag and homogenize using a syringe.

5. Add LPS:

1) defrost an aliquot (73.2 μl) of LPS (C = 1 mg/ml);

2) to select 400 μl of complete medium and mix it with LPS, use a 1 ml syringe equipped with a needle;

3) take the environment containing LPS and add it to the complete environment package.

Preparation of Centrifuged Platelet Lysate for Clinical Use (CPL)

To begin “clearing” cells of exogenous CPL-derived EVs, centrifuged CPL was used in the stimulation step.

1. Place the insertion device into the exit port.

2. Transfer CPL into 50 ml plastic tubes.

3. Perform centrifugation at 6000×g and 4°C for 1 hour.

4. Transfer the supernatant to a new container. 7.1.2. Thawing and amplification

Umbilical cord-derived mesenchymal stem cells, stored in nitrogen gas after isolation and first subculture, were thawed following the classical protocol. Briefly, the bag was removed from storage and quickly immersed in a 37°C water bath. Once no ice remained in the bag, cells were diluted in prewarmed (37°C) complete medium (αMEM plus 0.5% (v/v) ciprofloxacin plus heparin (2 units/ml) plus 5% (v/v) ciprofloxacin. /vol.) PL) and quickly centrifuged (300×g, RT, 5 min).

After centrifugation, cells were suspended in prewarmed complete medium and assessed for cell number and viability (using trypan blue/Malasses hemocytometer).

Cells were seeded at a density of 2000 cells/cm ² in two 1-layer CellSTACK-1 (CS1) cell culture chambers in complete medium and incubated (90% humidity, 5% CO ₂ , 37°C) for seven days.

After seven days, CS1 was washed using 100 ml of PBS, and Trypzean was added to collect cells at a rate of 25 ml per CS1. After 10 min in the incubator, Trypzean was neutralized by adding a total of 100 ml of complete medium (at a rate of 50 ml per CS1). Cells were pooled and assessed for number and viability.

7.1.3. Stimulation

Seeding in T300 was carried out at a density of 6000 cells/ ^cm2 .

After one day, the medium was replaced with the appropriate conditioned medium. Cell stimulation was carried out for 2 days without changing the medium.

7.1.4. Fasting and EV secretion

After stimulation, the medium was discarded and the cells were washed three times with PBS. Cells were then starved for 24 h under αMEM plus/minus pro-inflammatory factor (combination of IL1β (10 ng/ml) and IL4 (10 ng/ml) or LPS (100 ng/ml)).

After the starvation period, the cells were washed three more times with PBS, then each flask was loaded with 30 ml of αMEM plus/minus pro-inflammatory factor (IL or LPS) for 72 hours.

The supernatant was collected in 50 ml plastic tubes and centrifuged for 5 min at 400 × g at room temperature. The supernatant for each condition was combined into 500 ml bottles and, together with the bottles, a small 1 ml aliquot was separately frozen at -80°C.

To estimate the number of cells for each condition, trypsinization was performed for 3 T300 flasks, and the cells were cryopreserved in cryovials.

Cell culture and processing reagents

7.2. Phenotypic characteristics of cells contained in 3 conditioned media

To assess the effectiveness of stimulation using cytometric analysis, phenotypic characterization of cells cultured using 3 conditioned media was performed.

As expected, cells pretreated in S1 and LPS medium exhibited different phenotypes, with the proangiogenic marker CD106 expressed at higher levels in S1 conditioned medium (due to cell stimulation with IL, as described in WO 2018/108859 ).

Cytometry reagents used for cytometric analysis:

7.3. EV Cleaning

In this experiment, fractions enriched in extracellular vesicles were isolated by ultrafiltration of 3 conditioned media.

The number and size distribution of extracellular vesicles were analyzed using the nanoparticle trajectory analysis method (Nanosight 300 from Malvern-Panalytical).

EV purification results are presented below.

Exit

Size

Output/Cages

7.3. Characterization of MSC-derived EVs pretreated with NS/S1/LPS

7.3.1. Analysis of protein content in EVs and MSCs using Western blotting

The contents of EVs and MSCs obtained under 3 conditions (NS/S1/LPS) were assessed by Western blotting using the following conditions.

Materials and reagents

Lysis buffer (for RIPA):

- sodium deoxycholate 1%;

- SDS 0.1%;

- Tris⋅Cl, pH 7.4 20 mM;

- EDTA 1 mM;

- NaCl 150 mM;

NP40 (from the English nonyl phenoxypolyethoxyethanol - nonylphenoxypolyethoxyethanol) 1%;

- protease inhibitor cocktail (CIP, 100x), Sigma catalog number P8340.

Cell lysis (in case of MSC)

To MSC cells in an amount of 1x10 ⁶ add 100 µl of cold RIPA buffer containing CIP in 1x final concentration.

Incubate for 10 min on ice, centrifuge at 4°C for 20 min at 10,000 x g and then remove the supernatant.

The EV-containing fraction was solubilized after adding an equal volume of ice-cold 2× RIPA lysis buffer.

Protein concentration determinations were performed using the Micro BCA Protein Assay Kit, Pierce part number 23235.

Electrophoresis was carried out in 4-12% bis-Tris gels with a layer thickness of 1.5 mm, having 10 wells, Novex Nosex (Life Technologies, NP0335PBOX) or NuPAGE® (MOPS-based working buffer with SDS) used for protein separation. 20x)) (Life Technologies, NP0001). Samples were denatured for 10 min at 70°C using ¼ volume of sample buffer containing LDS (Invitrogen, NP0007, 4×) with or without DTT (500 mM). Transfer was carried out electrophoretically using a Mini Trans-Bio membrane system, part number: 170-3930, to a PVDF membrane (Amersham, part number: RPN303LFP) activated with absolute ethanol and subjected to washing.

The following substances were used to identify proteins: TBS, 10x, BioRad catalog number 1706435;

blocking buffer (TBS milk): 1x TBS_0.1% Tween_5% skim milk;

Wash buffer (TBS): 1x TBS_0.1% Tween;

AC buffer (TBS_AC): 1x TBS_0.1% Tween_0.3% milk.

Fluorimetric analysis was performed using Alexa Fluor 680-labeled antibody, GAM (cat. no.: A21058) or GAR (cat. no.: A21076) from Thermofisher, at a dilution of 1/10,000.

Chemiluminescence assays were performed using an HRP-conjugated antibody, GAM (cat. no.: 170-6516) or GAR (cat. no.: 170-6515) from BioRad, at a dilution of 1/5000.

Antibodies to CD9: Biolegend - 312102.

Antibodies to CD81: Biolegend - 349501.

Antibodies to calnexin: Elabscience - E-AB-30723.

Antibodies to VCAM: Bio-Rad - VMA00461.

Antibodies to CD200: Bio-Techne - 2AF2724.

results

As expected, the proangiogenic marker CD106/VCAM was detected in lane B (MSC of the invention) and was completely absent in lane C (MSC in LPS medium) and lane A (negative control). The membrane glycoprotein CD200, a second proangiogenic marker, was present in MSCs from the S1 fraction, and was not present in either MSCs from the LPS fraction or in lane A (negative control) (see Fig. 5).

In purified EVs (Fig. 3), both membrane proteins CD9 and CD81 were present in all fractions (lanes A, B, and C) as well as in the MDA cell lysate (positive control) (Fig. 3).

The reticulum marker calnexin was not detected, indicating that the EV fraction was purified correctly.

The proangiogenic marker CD106/VCAM was detected in lane B (EV of the invention) and was completely absent from lane C (EV in LPS medium) and lane A (negative control), indicating that the MSC membrane markers of the invention transferred to EV (Figure 3).

The membrane glycoprotein CD200, a second proangiogenic marker, was present in EVs from the S1 fraction, and was not present in either EVs from the LPS fraction or in lane A (negative control) (see Fig. 3).

7.3.2. Set of proteins involved in angiogenesis

EVs derived from MSCs cultured in NS, S1 and LPS conditioned media were compared with the Angiogenesis Proteomics Kit (R&D Systems - ARY007).

Lysis buffer was prepared as follows.

1. Based on 50 ml: dissolve 157.6 mg Tris-HCl in 10 ml of water; 400.3 mg NaCl in 10 ml water and 37.2 mg EDTA in 10 ml water.

2. Adjust pH value to 8.

3. Add 0.5 ml Triton X-100, 5 ml glycerol, 50 µl aprotinin solution (10 mg/ml), 50 µl leupeptin solution (10 mg/ml) and 500 µl pepstatin solution (1 mg/ml).

4. Add sufficient sterile water (QSP) to 50 ml.

EVs derived from MSCs obtained in NS, S1 and LPS media were solubilized in lysis buffer:

- NS: 150 μl EVs were suspended in 1 ml lysis buffer;

- S1: 130.4 μl EVs were suspended in 1 ml lysis buffer;

- LPS: 125 μl EVs were suspended in 1 ml lysis buffer.

Using the BCA assay, the total protein content (300 μg) in the lysates was determined, thereby confirming the efficiency of lysis.

EV lysates were further resuspended by pipetting up and down and gently vortexed at 2-8°C for 30 minutes, then centrifuged using a microcentrifuge at 14,000×g for 5 minutes. The supernatant was transferred to a clean tube and analyzed following the manufacturer's instructions.

Angiogenesis Proteomics Kit Reagents

In the graph presented in Fig. Figure 4 shows results for proteins that were significantly different in expression levels across the 3 samples. For 28 proangiogenic proteins out of 59 analyzed in this set, differences in expression are presented in the S1 and LPS samples, and this confirms that the EVs of the invention exhibit characteristics different from those of prior art EVs, in particular with regard to the content of proangiogenic proteins.

Bibliographical references

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Claims (110)

1. A composition for the treatment of liver cirrhosis, containing extracellular vesicles (EVs) derived from CD106 ^high CD151 ⁺ nestin ⁺ mesenchymal stem cells (MSCs), and a biocompatible material, wherein said extracellular vesicles are obtained by: (1) culturing mesenchymal stem cells derived from perinatal tissue in a first growth factor-free culture medium so as to create a population of cultured undifferentiated mesenchymal stem cells, (2) contacting said population of cultured undifferentiated mesenchymal stem cells with a second culture medium containing a mixture of interleukin (IL)-4 and IL1β, resulting in the formation of CD106 ^high CD151 ⁺ nestin ⁺ mesenchymal stem cells, (3) culturing said MSCs in a culture medium free of extracellular vesicles under conditions allowing their proliferation; And (4) purifying extracellular vesicles from cells obtained in step (3). 2. A composition for the treatment of diabetes containing extracellular vesicles (EVs) derived from CD106 ^high CD151 ⁺ nestin ⁺ mesenchymal stem cells (MSCs), and a biocompatible material, wherein said extracellular vesicles are obtained by: (1) culturing mesenchymal stem cells derived from perinatal tissue in a first growth factor-free culture medium so as to create a population of cultured undifferentiated mesenchymal stem cells, (2) contacting said population of cultured undifferentiated mesenchymal stem cells with a second culture medium containing a mixture of interleukin (IL)-4 and IL1β, resulting in the formation of CD106 ^high CD151 ⁺ nestin ⁺ mesenchymal stem cells, (3) culturing said MSCs in a culture medium free of extracellular vesicles under conditions allowing their proliferation; And (4) purifying extracellular vesicles from cells obtained in step (3). 3. A composition for the treatment of aplastic anemia, containing extracellular vesicles (EVs) derived from CD106 ^high CD151 ⁺ nestin ⁺ mesenchymal stem cells (MSCs), and a biocompatible material, wherein said extracellular vesicles are obtained by: (1) culturing mesenchymal stem cells derived from perinatal tissue in a first growth factor-free culture medium so as to create a population of cultured undifferentiated mesenchymal stem cells, (2) contacting said population of cultured undifferentiated mesenchymal stem cells with a second culture medium containing a mixture of interleukin (IL)-4 and IL1β, resulting in the formation of CD106 ^high CD151 ⁺ nestin ⁺ mesenchymal stem cells, (3) culturing said MSCs in a culture medium free of extracellular vesicles under conditions allowing their proliferation; And (4) purifying extracellular vesicles from cells obtained in step (3). 4. A composition for inducing angiogenesis containing extracellular vesicles (EVs) derived from CD106 ^high CD151 ⁺ nestin ⁺ mesenchymal stem cells (MSCs), and a biocompatible material, wherein said extracellular vesicles are obtained by: (1) culturing mesenchymal stem cells derived from perinatal tissue in a first growth factor-free culture medium so as to create a population of cultured undifferentiated mesenchymal stem cells, (2) contacting said population of cultured undifferentiated mesenchymal stem cells with a second culture medium containing a mixture of interleukin (IL)-4 and IL1β, resulting in the formation of CD106 ^high CD151 ⁺ nestin ⁺ mesenchymal stem cells, (3) culturing said MSCs in a culture medium free of extracellular vesicles under conditions allowing their proliferation; And (4) purifying extracellular vesicles from cells obtained in step (3). 5. Composition according to any one of paragraphs. 1-4, wherein said extracellular vesicles are derived from undifferentiated MSCs derived from perinatal tissue selected from the placenta, umbilical cord or placental membranes or components thereof, or from perinatal fluid. 6. Composition according to any one of paragraphs. 1-5, wherein said extracellular vesicles are derived from undifferentiated MSCs derived from umbilical cord blood, placental blood or amniotic fluid. 7. Composition according to any one of paragraphs. 1-6, wherein said extracellular vesicles are obtained from undifferentiated umbilical cord-derived MSCs, preferably by isolating cells from an umbilical cord fragment explant. 8. Composition according to any one of paragraphs. 1-7, wherein said extracellular vesicles are obtained from undifferentiated placenta-derived MSCs, preferably by isolating cells from a piece of placental tissue. 9. A pharmaceutical composition for the treatment of liver cirrhosis, containing extracellular vesicles (EVs) derived from CD106 ^high CD151 ⁺ nestin ⁺ mesenchymal stem cells (MSCs), and a biocompatible material, wherein said extracellular vesicles are obtained by: (1) culturing mesenchymal stem cells derived from perinatal tissue in a first growth factor-free culture medium so as to create a population of cultured undifferentiated mesenchymal stem cells, (2) contacting said population of cultured undifferentiated mesenchymal stem cells with a second culture medium containing a mixture of IL-4 and IL1β, resulting in the formation of CD106 ^high CD151 ⁺ nestin ⁺ mesenchymal stem cells, (3) culturing said MSCs in a culture medium free of extracellular vesicles under conditions allowing their proliferation; And (4) purifying extracellular vesicles from cells obtained in step (3). 10. A pharmaceutical composition for the treatment of diabetes containing extracellular vesicles (EVs) derived from CD106 ^high CD151 ⁺ nestin ⁺ mesenchymal stem cells (MSCs), and a biocompatible material, wherein said extracellular vesicles are obtained by: (1) culturing mesenchymal stem cells derived from perinatal tissue in a first growth factor-free culture medium so as to create a population of cultured undifferentiated mesenchymal stem cells, (2) contacting said population of cultured undifferentiated mesenchymal stem cells with a second culture medium containing a mixture of IL-4 and IL1β, resulting in the formation of CD106 ^high CD151 ⁺ nestin ⁺ mesenchymal stem cells, (3) culturing said MSCs in a culture medium free of extracellular vesicles under conditions allowing their proliferation; And (4) purifying extracellular vesicles from cells obtained in step (3). 11. A pharmaceutical composition for the treatment of aplastic anemia, containing extracellular vesicles (EVs) derived from CD106 ^high CD151 ⁺ nestin ⁺ mesenchymal stem cells (MSC), and a biocompatible material, wherein said extracellular vesicles are obtained by: (1) culturing mesenchymal stem cells derived from perinatal tissue in a first growth factor-free culture medium so as to create a population of cultured undifferentiated mesenchymal stem cells, (2) contacting said population of cultured undifferentiated mesenchymal stem cells with a second culture medium containing a mixture of IL-4 and IL1β, resulting in the formation of CD106 ^high CD151 ⁺ nestin ⁺ mesenchymal stem cells, (3) culturing said MSCs in a culture medium free of extracellular vesicles under conditions allowing their proliferation; And (4) purifying extracellular vesicles from cells obtained in step (3). 12. A pharmaceutical composition for inducing angiogenesis containing extracellular vesicles (EVs) derived from CD106 ^high CD151 ⁺ nestin ⁺ mesenchymal stem cells (MSCs), and a biocompatible material, wherein said extracellular vesicles are obtained by: (1) culturing mesenchymal stem cells derived from perinatal tissue in a first growth factor-free culture medium so as to create a population of cultured undifferentiated mesenchymal stem cells, (2) contacting said population of cultured undifferentiated mesenchymal stem cells with a second culture medium containing a mixture of IL-4 and IL1β, resulting in the formation of CD106 ^high CD151 ⁺ nestin ⁺ mesenchymal stem cells, (3) culturing said MSCs in a culture medium free of extracellular vesicles under conditions allowing their proliferation; And (4) purifying extracellular vesicles from cells obtained in step (3). 13. Pharmaceutical composition according to any one of paragraphs. 9-12, where the specified biocompatible material is a hydrogel. 14. Cosmetic composition for skin cell regeneration containing extracellular vesicles (EVs) derived from CD106^highCD151⁺nestin⁺ mesenchymal stem cells (MSC), and a biocompatible material, wherein said extracellular vesicles are obtained by: (1) culturing mesenchymal stem cells derived from perinatal tissue in a first growth factor-free culture medium so as to create a population of cultured undifferentiated mesenchymal stem cells, (2) contacting said population of cultured undifferentiated mesenchymal stem cells with a second culture medium containing a mixture of IL-4 and IL1β, resulting in the formation of CD106 ^high CD151 ⁺ nestin ⁺ mesenchymal stem cells, (3) culturing said MSCs in a culture medium free of extracellular vesicles under conditions allowing their proliferation; And (4) purifying extracellular vesicles from cells obtained in step (3). 15. A cosmetic composition for the regeneration of mucosal cells containing extracellular vesicles (EVs) derived from CD106 ^high CD151 ⁺ nestin ⁺ mesenchymal stem cells (MSC), and a biocompatible material, wherein said extracellular vesicles are obtained by: (1) culturing mesenchymal stem cells derived from perinatal tissue in a first growth factor-free culture medium so as to create a population of cultured undifferentiated mesenchymal stem cells, (2) contacting said population of cultured undifferentiated mesenchymal stem cells with a second culture medium containing a mixture of IL-4 and IL1β, resulting in the formation of CD106 ^high CD151 ⁺ nestin ⁺ mesenchymal stem cells, (3) culturing said MSCs in a culture medium free of extracellular vesicles under conditions allowing their proliferation; And (4) purifying extracellular vesicles from cells obtained in step (3). 16. A cosmetic composition for improving the appearance of the skin, containing extracellular vesicles (EVs) derived from CD106 ^high CD151 ⁺ nestin ⁺ mesenchymal stem cells (MSC), and a biocompatible material, wherein said extracellular vesicles are obtained by: (1) culturing mesenchymal stem cells derived from perinatal tissue in a first growth factor-free culture medium so as to create a population of cultured undifferentiated mesenchymal stem cells, (2) contacting said population of cultured undifferentiated mesenchymal stem cells with a second culture medium containing a mixture of IL-4 and IL1β, resulting in the formation of CD106 ^high CD151 ⁺ nestin ⁺ mesenchymal stem cells, (3) culturing said MSCs in a culture medium free of extracellular vesicles under conditions allowing their proliferation; And (4) purifying extracellular vesicles from cells obtained in step (3). 17. A cosmetic composition for improving the appearance of the mucous membrane, containing extracellular vesicles (EVs) derived from CD106 ^high CD151 ⁺ nestin ⁺ mesenchymal stem cells (MSCs), and a biocompatible material, wherein said extracellular vesicles are obtained by: (1) culturing mesenchymal stem cells derived from perinatal tissue in a first growth factor-free culture medium so as to create a population of cultured undifferentiated mesenchymal stem cells, (2) contacting said population of cultured undifferentiated mesenchymal stem cells with a second culture medium containing a mixture of IL-4 and IL1β, resulting in the formation of CD106 ^high CD151 ⁺ nestin ⁺ mesenchymal stem cells, (3) culturing said MSCs in a culture medium free of extracellular vesicles under conditions allowing their proliferation; And (4) purifying extracellular vesicles from cells obtained in step (3). 18. A cosmetic composition for correcting a skin defect, containing extracellular vesicles (EVs) derived from CD106 ^high CD151 ⁺ nestin ⁺ mesenchymal stem cells (MSCs), and a biocompatible material, wherein said extracellular vesicles are obtained by: (1) culturing mesenchymal stem cells derived from perinatal tissue in a first growth factor-free culture medium so as to create a population of cultured undifferentiated mesenchymal stem cells, (2) contacting said population of cultured undifferentiated mesenchymal stem cells with a second culture medium containing a mixture of IL-4 and IL1β, resulting in the formation of CD106 ^high CD151 ⁺ nestin ⁺ mesenchymal stem cells, (3) culturing said MSCs in a culture medium free of extracellular vesicles under conditions allowing their proliferation; And (4) purifying extracellular vesicles from cells obtained in step (3). 19. A cosmetic composition for correcting a mucosal defect, containing extracellular vesicles (EVs) derived from CD106 ^high CD151 ⁺ nestin ⁺ mesenchymal stem cells (MSCs), and a biocompatible material, wherein said extracellular vesicles are obtained by: (1) culturing mesenchymal stem cells derived from perinatal tissue in a first growth factor-free culture medium so as to create a population of cultured undifferentiated mesenchymal stem cells, (2) contacting said population of cultured undifferentiated mesenchymal stem cells with a second culture medium containing a mixture of IL-4 and IL1β, resulting in the formation of CD106 ^high CD151 ⁺ nestin ⁺ mesenchymal stem cells, (3) culturing said MSCs in a culture medium free of extracellular vesicles under conditions allowing their proliferation; And (4) purifying extracellular vesicles from cells obtained in step (3). 20. A cosmetic composition for healing a skin lesion or disorder, comprising extracellular vesicles (EVs) derived from CD106 ^high CD151 ⁺ nestin ⁺ mesenchymal stem cells (MSCs), and a biocompatible material, wherein said extracellular vesicles are obtained by: (1) culturing mesenchymal stem cells derived from perinatal tissue in a first growth factor-free culture medium so as to create a population of cultured undifferentiated mesenchymal stem cells, (2) contacting said population of cultured undifferentiated mesenchymal stem cells with a second culture medium containing a mixture of IL-4 and IL1β, resulting in the formation of CD106 ^high CD151 ⁺ nestin ⁺ mesenchymal stem cells, (3) culturing said MSCs in a culture medium free of extracellular vesicles under conditions allowing their proliferation; And (4) purifying extracellular vesicles from cells obtained in step (3). 21. Use of a cosmetic composition according to any one of paragraphs. 14-20 for correction of age spots. 22. Use of a cosmetic composition according to any one of paragraphs. 14-20 for spot correction. 23. Use of a cosmetic composition according to any one of paragraphs. 14-20 for acne correction. 24. Use of a cosmetic composition according to any one of paragraphs. 14-20 for wrinkle correction. 25. Use of a cosmetic composition according to any one of paragraphs. 14-20 to correct dryness. 26. Use of a cosmetic composition according to any one of paragraphs. 14-20 for burn healing. 27. Use of a cosmetic composition according to any one of paragraphs. 14-20 for scar healing. 28. Use of a cosmetic composition according to any one of paragraphs. 14-20 for angioma healing. 29. Use of a cosmetic composition according to any one of paragraphs. 14-20 for nevus healing. 30. Use of a cosmetic composition according to any one of paragraphs. 14-20 for wart healing. 31. A system for administering extracellular vesicles (EVs) derived from CD106 ^high CD151 ⁺ nestin ⁺ mesenchymal stem cells (MSCs) to a patient, wherein said extracellular vesicles are obtained by: (1) culturing mesenchymal stem cells derived from perinatal tissue in a first growth factor-free culture medium so as to create a population of cultured undifferentiated mesenchymal stem cells, (2) contacting said population of cultured undifferentiated mesenchymal stem cells with a second culture medium containing a mixture of IL-4 and IL1β, resulting in the formation of CD106 ^high CD151 ⁺ nestin ⁺ mesenchymal stem cells, (3) culturing said MSCs in a culture medium free of extracellular vesicles under conditions allowing their proliferation; And (4) purification of extracellular vesicles from cells obtained in step (3), and wherein said system is liposomes, proliposomes, microspheres, micro- or nanovesicles of biopolymers, lipids or nanoparticles comprising said EVs. 32. The system according to claim 31, which is micro- or nanovesicles made of biopolymers, lipids or nanoparticles. 33. A method for producing extracellular vesicles, wherein said method includes the stages of: (1) culturing mesenchymal stem cells obtained from perinatal tissue in a first growth factor-free culture medium so as to create a population of cultured undifferentiated mesenchymal stem cells, (2) contacting said population of cultured undifferentiated mesenchymal stem cells with a second culture medium containing a mixture of IL-4 and IL1β, resulting in the formation of CD106 ^high CD151 ⁺ nestin ⁺ mesenchymal stem cells, (3) culturing said MSCs in a culture medium free of extracellular vesicles under conditions that allow them to multiply; And (4) purify extracellular vesicles from cells obtained in step (3). 34. The method of claim 33, wherein said undifferentiated MSCs are derived from perinatal tissue selected from the placenta, umbilical cord or placental membranes or components thereof, or from perinatal fluid. 35. The method of claim 33, wherein said undifferentiated MSCs are obtained by isolating cells from a perinatal tissue explant. 36. The method of claim 33, wherein said undifferentiated MSCs are derived from the umbilical cord, preferably by isolating cells from an umbilical cord fragment explant. 37. The method of claim 33, wherein said undifferentiated MSCs are derived from the placenta, preferably by isolating cells from a fragment of placental tissue. 38. Method according to any one of paragraphs. 33-37, wherein said second culture medium contains interleukin 1β in the range of 1 to 100 ng/ml. 39. Method according to any one of paragraphs. 33-38, wherein said second culture medium contains interleukin 4 in the range of 1 to 100 ng/ml. 40. Method according to any one of paragraphs. 33-39, wherein said extracellular vesicle-free culture medium is supplemented with a mixture of IL1 and IL4, preferably IL1β and IL4. 41. Method according to any one of paragraphs. 33-40, where extracellular vesicles are purified by ultrafiltration.