WO2013186418A1

WO2013186418A1 - Marker for determining the therapeutic potential of mesenchymal stem cells, an inhibitor of the marker for therapeutic use and stem cells with low expression of the marker for therapeutic use

Info

Publication number: WO2013186418A1
Application number: PCT/ES2013/070381
Authority: WO
Inventors: Manuel Angel GONZÁLEZ DE LA PEÑA; Antonio Bernad Miana; María TOMÉ PIZARRO
Original assignee: Fundación Centro Nacional De Investigaciones Cardiovasculares Carlos Iii
Priority date: 2012-06-12
Filing date: 2013-06-12
Publication date: 2013-12-19
Also published as: WO2013186418A9; ES2434853B1; ES2434853A1

Abstract

The invention relates to methods for determining the therapeutic potential and immunoregulatory capacity of stems cells, based on expression levels of microRNA miR-335. This determination can be used as a criterion for the release of groups of stem cells for therapeutic use. In addition, the invention also relates to in vitro methods for increasing the immunoregulatory capacity of a stem cell, in which said methods comprise treating the stem cell with an miR-335 inhibitor. Finally, the invention relates to therapeutic methods and uses for treating a patient with an autoimmune disease, an inflammatory disease, graft-versus-host disease or a disease requiring transplant tolerance induction or regeneration and/or repair of tissue, said therapeutic methods and uses comprising the administration of a stem cell having reduced miR-335 levels of expression and/or activity.

Description

MOLECULAR MARKER OF THERAPEUTIC POWER OF MOTHER CELLS SENQUIMALE S HUMANAS AND ITS USES

FIELD OF THE INVENTION

The present invention falls within the field of molecular biology and biomedicine. Specifically, the present invention relates to the use of miR-335 as a molecular marker to quantify the therapeutic potency of human mesenchymal stem cells employed in regenerative medicine.

BACKGROUND OF THE INVENTION

Mesenchymal stem cells (MSC) are a type of adult stem cells especially promising for their therapeutic applications. Human mesenchymal stem cells (hMSC) were isolated and characterized for the first time in the bone marrow stroma, constituting a totally different population of hematopoietic stem cells, and their role is to contribute to the regeneration of non-hematopoietic connective tissues (bone , cartilage, muscle, ligament, tendon, adipose tissue and stroma). At present, if we exclude hematopoietic stem cells, traditionally used in bone marrow transplants, they are the main type of cells used in clinical trials for the treatment of very varied pathologies. MSCs have a great therapeutic potential in tissue regeneration, since isolated and expanded MSCs in culture are able to differentiate into osteoblasts, chondrocytes, myocytes and adipocytes, among other cell types, and can be subsequently reintroduced into the human body to Repair lost or damaged tissues. Another characteristic that is even more valuable in clinical practice is its immunomodulation capacity, which makes them extremely useful for the treatment of immune-based diseases. However, very little is still known about the mechanisms that regulate its biological and therapeutic properties.

For the vast majority of therapeutic applications it is necessary to have a much higher number of hMSC than is possible directly from a biopsy. Consequently, it is necessary to perform ex vivo expansion procedures that allow to achieve the minimum therapeutic doses (10-100 million). However, over the last few years, evidence has been accumulating that said expansion process is not carried out under optimal conditions and that it is advisable to minimize the expansion time and reduce the oxygen tension used. Extensive culture of murine hMSC and MSC (mMSC) promotes genetic instability associated with the progress of culture senescence. In addition, the behavior of a biopsy during cell expansion is practically unpredictable, although there seems to be a significant relationship with the donor's age and / or medical history.

Recently it has been published that hMSCs obtained from donors / older patients result in preparations with reduced therapeutic properties. The authors propose that in mMSC the overexpression of PEDF (pigment epithelium-derived factor) that occurs with age is a critical factor that affects the therapeutic properties of cells. Other evidences mark alterations in various routes. Regarding its osteogenic capacity, it has been confirmed that the main difference between MSCs derived from young and old donors is their reduced proliferation capacity and their greater tendency to senescence.

Likewise, it has been possible to confirm that there are epigenetic modifications in hMSC in relevant genes associated with both the age of the donor and the ex vivo culture. It has been striking to confirm that these deficiencies may be related to the poor quality of the extracellular matrix.

The MSCs used for cell therapy are considered medications, so their use must comply with the Drug Law, be handled according to GMP, etc. Usually a therapeutic entity that is going to be used as a medicine must go through sterility controls (eg, mycoplasma free), identity (markers), purity (contaminants), and potency. The release of lots of MSC for clinical use should be in less than 24 hours because if the cells do not die or deteriorate, so no power control is currently performed because all known methods take weeks for each batch.

A problem of cell banks for clinical use is that MSCs have great variability in their therapeutic potential. At present, this potential cannot be known a priori (expensive and time-delayed studies are necessary), so many samples that are really suboptimal can be stored, with the consequent expense and possible harm to patients. There is therefore a need to develop a method that allows to quickly and efficiently determine the therapeutic potential of the MSC batches in order to determine their suitability for administration to a patient.

SUMMARY OF THE INVENTION

The authors of the present invention have discovered that, surprisingly, there is a correlation between the expression levels of the miR-335 microRNA and the therapeutic potential of human mesenchymal stem cells (hMSC). Specifically, a correlation between the expression levels of miR-335 and the immunoregulatory capacity or power of said cells has been observed, so that a cell expressing high levels of miR-335 has a low capacity or immunoregulatory power, and therefore a low therapeutic potential. Thus, as seen in Figure 5, hMSCs modified so that they overexpress miR-335 have a capacity to inhibit the proliferation of human lymphocytes significantly less than control cells. Additionally, as seen in Figure 6, the administration of MSCs that overexpress miR-335 to animals results in an increased mortality in response to the administration of LPS with respect to mice that did not receive cells, indicating that the MSC have lost part of the ability to regulate the response to LPS. Therefore, in a first aspect the present invention relates to a method for determining the immunoregulatory capacity or potency of a stem cell, which comprises determining in said stem cell the levels of expression and / or activity of miR-335, where levels high with respect to a reference value are indicative that said stem cell has a low immunoregulatory capacity, or where decreased levels with respect to a reference value are indicative that said stem cell has a high immunoregulatory capacity.

In another aspect, the invention relates to a method for selecting a batch of stem cells for therapeutic applications related to the immunoregulatory activity of said stem cells which comprises determining in the stem cells of said batch the miR expression levels and / or activity -335, where if the expression and / or activity levels of miR-335 are lower than a reference value, the batch of stem cells is selected for therapeutic applications related to the immunoregulatory activity of said stem cells.

In another aspect, the invention relates to a method for selecting a batch of stem cells for therapeutic applications related to the immunoregulatory activity of said stem cells comprising determining the levels of expression and / or activity of miR335 in stem cells of said batch and in the stem cells of a control lot previously selected as suitable for therapeutic applications related to immunoregulatory activity, where if the levels of expression and / or activity of miR335 in the stem cells are similar to the levels of expression and / or activity of miR335 in the cells of the control lot, then the batch of stem cells is selected for therapeutic applications related to the immunoregulatory activity of said stem cells.

In another aspect, the invention relates to a kit for determining the immunoregulatory capacity of a stem cell comprising

(i) suitable reagents to determine the expression level of miR-335 and

(ii) suitable reagents to determine the level of expression of a reference RNA.

In another aspect, the invention relates to an in vitro method for increasing the immunoregulatory capacity of a stem cell, which comprises inhibiting the expression and / or activity of miR-335 in said stem cell.

In another aspect, the invention relates to a use of a stem cell characterized in that it has reduced levels of expression and / or activity of miR-335 to prepare a medicament for the treatment of a disease selected from the group formed by an autoimmune disease, a inflammatory disease, graft versus host disease, type 1 diabetes, type 2 diabetes and cardiovascular disease.

In another aspect, the invention relates to a use of a stem cell characterized in that it has reduced levels of expression and / or activity of miR-335 to prepare a medicament for inducing tolerance to a transplant. In another aspect, the invention relates to a use of a stem cell characterized in that it has reduced levels of expression and / or activity of miR-335 to prepare a medicament for tissue repair and regeneration.

In another aspect, the invention relates to an in vitro method for generating regulatory T cells, which comprises contacting a population of cells containing T cells with a population of stem cells characterized in that they have reduced levels of expression and / or activity of miR-335 under conditions suitable for the generation of regulatory T cells.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1. Expression levels of miR-335 in hMSC are directly related to the age of the donor. Relative expression of miR-335 (using the expression of RNU6B as an endogenous control) measured by quantitative real-time RT-PCR in different types of hMSC from different donors. A) Expression level of miR-335 measured in hMSC from bone marrow. B) Level of expression of miR-335 measured in hMSC from adipose tissue.

Figure 2. Cellular senescence levels in hMSC cultures are directly related to miR-335 expression levels. A) Overexpression of miR-335 was induced in two different isolates of hMSC (19, white bars, and 33, gray bars) by transduction with a lentiviral vector encoding miR-335, and β expression levels were measured -galactosidase by specific staining. The same hMSC transduced with the same lentiviral vector but without coding said miRNA was used as a negative control. B) Cellular senescence was induced in the same two hMSC isolates by treatment with gamma radiation, and the endogenous expression levels of miR-335 were measured by RT-PCR in the treated (irradiated) or untreated (wild) cells. Error bars correspond to the standard error (N = 3).

Figure 3. The expression level of miR-335 is inversely related to telomeric length in hMSC. A) Telomeric length was measured in hMSC (white bars) and hMSC transfected with a lentiviral construct that allows overexpression of telomerase (gray bars). The measurements were taken at 21 and 89 days of culture. B) Relative expression of miR-335 measured by quantitative RT-PCR a real time (using the RNU6B measurement as an endogenous control) of hMSC (soft bars) and hMSC-hTERT (gray bars) at 5, 21 and 89 days of remaining in culture.

Figure 4. Relationship between the endogenous levels of miR-335 and the partial pressure of O2 during culture. HMSC of adipose tissue from three different donors (Ad 36, Ad 40 and Ad 43) were grown at 3% (white bars) or 20% (black bars) of 0 ₂ for one week. Endogenous levels of miR-335 were measured by quantitative real-time PCR.

Figure 5. Overexpression of miR-335 decreases the in vitro immunoregulatory capacity of hMSCs. Human peripheral blood mononuclear cells (PBMC) were stimulated with SEB (Ing / mL), in the absence or presence of an increasing number of hMSC, which were transduced with the lentiviral construct pLV-EmGFP-MIR335 or pLV-EmGFP- mock The antiproliferative response was determined at 96 hours of culture by measuring the incorporation of BrdU into the PBMC.

Figure 6. Overexpression of miR-335 decreases the in vivo immunoregulatory capacity of hMSCs. Death by endotoxemia was induced in a murine model (BALBc mice) by intraperitoneal (ip) injection of 400 μg of LPS. Half an hour after the administration of LPS, bone marrow hMSC was administered in order to avoid such death by endotoxemia. In this experiment, 10 ⁵ cells were administered to each animal (except for the negative controls) and 10 mice were used for each of the following experimental groups: negative control (LPS 400 μg + PBS), unmodified hMSC (LPS 400 μg + bm-hMSC 19 wt), hMSC transduced with a lentiviral control vector, which encodes a miRNA without targets in the human genome (LPS 400 μg + bm-hMSC 19 mock), and hMSC transduced with a lentiviral vector encoding miR-335 ( LPS 400 μg + bm-hMSC 19 335). The figure shows the survival curve of the animals of each experimental group during the 96 h after administration of the LPS.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based on the discovery that miR-335 expression levels are inversely related to the immunomodulatory activity of hMSC, that is, that elevated miR-335 levels result in cells that have a lower immunomodulatory capacity and that , so levels Reduced miR-335 results in cells that have a greater immunomodulatory capacity. Since the therapeutic activity of hMSCs depends mainly on their immunomodulatory / anti-inflammatory activity, the present inventors propose that the expression levels of said miR-335 are predictive of the therapeutic potency of said cells, in particular their immunoregulatory capacity. This finding has a high utility in medicine since it allows the use of miR-335 levels to predict the in vivo therapeutic activity of a batch of hMSC for applications in regenerative medicine as well as potency testing within the regulatoryly accepted batch release criteria. of stem cells for therapeutic applications.

Indeed, the inventors have observed that the overexpression of miR-335 produces a significant reduction in the immunoregulatory capacity of said hMSC both in vitro and in vivo (Example 3).

Based on this discovery, the inventive aspects that are explained in detail below have been developed.

Method to determine the immunoregulatory capacity of stem cells

In a first aspect, the present invention relates to a method, hereinafter the first method of the invention, for determining the immunoregulatory capacity or potency of a stem cell, which comprises determining in said stem cell the levels of expression and / or activity of miR-335, where high levels with respect to a reference value are indicative that said stem cell has a low potency or immunoregulatory capacity, or where decreased levels with respect to a reference value are indicative that said stem cell has a high power or immunoregulatory capacity.

The term "stem cell", as used herein, refers to a totipotent, pluripotent or multipotent cell, capable of generating one or more differentiated cell types, and which also has the ability to regenerate itself, ie , to produce more stem cells. Totipotent stem cells can give rise to both the embryonic components (such as the three embryonic layers, the germ lineage and the tissues that will give rise to the yolk sac), as well as to the extraembryonic (like the placenta). That is, they can form all cell types and give rise to a complete organism. Pluripotent stem cells can form any type of cell corresponding to the three embryonic lineages (endoderm, ectoderm and mesoderm), as well as the germinal and yolk sac. They can, therefore, form cell lineages but from them a whole organism cannot be formed. Multipotent stem cells are those that can only generate cells of the same embryonic layer or lineage of origin. In the context of the present invention, the stem cell is not a human embryonic stem cell. In a preferred embodiment, the stem cell of the first method of the invention is an adult stem cell.

The term "adult stem cell" means that the stem cell is isolated from a tissue or organ of an animal in a state of growth after the embryonic state. Preferably, the stem cells of the invention have been isolated in a postnatal state. Preferably they have been isolated from a mammal, and more preferably from a human, including neonates, juveniles, adolescents and adults. Adult stem cells can be assimilated from a wide variety of tissues and organs, such as bone marrow (mesenchymal stem cells, multipotent adult progenitor cells and hematopoietic stem cells), adipose tissue, cartilage, epidermis, hair follicle, skeletal muscle, heart muscle, intestine , liver, neuronal, etc. In a preferred embodiment, the stem cell of the first method of the invention is a mesenchymal stem cell.

As used in the present invention, the term "mesenchymal stem cell" (in English, "mesenchymal stem cell" or "MSC") refers to a multipotent somatic stem cell derived from mesoderm, which has the ability to regenerate itself and to differentiate itself to produce descendant cells with a wide phenotypic variety, including connective tissues, bone marrow stroma, adipocytes, dermis and muscle, among others. In general, MSCs have an expression profile of cellular markers characterized in that they are negative for markers CD19, CD45, CD14 and HLA-DR, and are positive for markers CD105, CD106, CD90 and CD73.

In a preferred embodiment, the MSCs originate from bone marrow or subcutaneous adipose tissue. Bone marrow MSCs can be isolated by procedures known to those skilled in the art. In general, these methods consist of isolating Mononuclear cells by density gradient centrifugation (Ficoll, Percoll) of bone marrow aspirates, and subsequently sow isolated cells in tissue culture plates in medium containing bovine fetal serum. These methods are based on the ability of MSCs to adhere to plastic, so that while non-adherent cells are removed from the culture, adhered MSCs can expand into culture plates. MSCs can also be isolated from subcutaneous adipose tissue following a similar procedure, known to the person skilled in the art. A method for isolating MSC from bone marrow or subcutaneous adipose tissue has been previously described (De la Fuente et al, Exp. Cell Res. 2004, Vol. 297: 313: 328).

The first method of the invention allows to determine the immunoregulatory power or capacity of a stem cell. "Immunoregulatory capacity or potency" means the property that stem cells have to suppress the immune response by inhibiting the maturation of dendritic cells and suppressing the function of T and B lymphocytes and Natural Killer cells in autoimmune diseases. and inflammatory.

The immunoregulatory capacity or potency of the stem cells of the first method of the invention can be analyzed in vitro by a lymphocyte proliferation inhibition assay (Example 3.1.). The immunoregulatory capacity of said cells can also be analyzed in vivo by an endotoxemia induction assay by intraperitoneal injection of LPS (Example 3.2.).

A stem cell with a greater capacity or immunoregulatory power also has a greater therapeutic potential, both when used for the treatment of diseases in which there is an unwanted activity of the immune system (autoimmune or inflammatory diseases), since they have a greater therapeutic effect, as for the treatment of diseases that require tissue repair (since tissue repair has an inflammatory component and a greater immunosuppressive capacity allows to prevent the host from rejecting the cells when they are of allogeneic or xenogenic origin). The therapeutic potential of a stem cell is the result of a set of properties of said cell, such as its proliferation and migration capacity, its differentiation potential and its immunoregulatory capacity. The proliferation and migration capacity and the differentiation potential of a stem cell correlate negatively with the senescence of said stem cell. The term "senescence", as used in the present invention, refers to the inability of the cell to divide after reaching a certain number of cell divisions. According to the first method of the invention, stem cells that have reduced expression levels of miR-335 have a greater immunoregulatory capacity, a lower senescence and a greater therapeutic potency.

The term "microRNA" (miRNA or miRNA) is a single-stranded RNA, between 21 and 25 nucleotides in length, which has the ability to regulate the expression of other genes through various processes, using the path of ribointerference. MicroRNAs post-translationally regulate gene expression by repressing the translation of a target messenger RNA. A microRNA is complementary to a region of one or more messenger RNAs (mRNAs).

The term "miR-335" as used in the present invention refers to the human microRNA as defined in the microRNA base "miRBase: Sequences" of the Wellcome Trust Sanger Institute (http: //microrna.sanger .ac.uk / sequences / index.shtml). miR-335 maps on the human chromosome 7q32.2 and is located within the second intron of the MEST / PEG1 gene that is one of the few human coding genes that have a maternal imprint. The expression of miR-335 therefore depends on the expression of MEST / PEG1. In the context of the present invention, the term miR-335 includes both the premature form thereof, mir-335 {Accession number MI0000816, and the mature forms miR-335-5p {Accession number MIMAT0000765) and miR-335-5p { Accession number MIMAT0004703), as defined in the "miRBase: Sequences" database of the Wellcome Trust Sanger Institute (http://microrna.sanger.ac.uk/sequences/index.shtml), Relay 18 (November 2011 ).

The first method of the invention involves, in a first step, determining in a stem cell or stem cell population the levels of expression and / or activity of miR-335.

To determine the expression levels of miR-335, it is necessary to obtain RNA from stem cells whose immunosuppressive capacity is to be analyzed. Total RNA can be purified from the stem cells by homogenization in the presence of a nucleic acid extraction buffer, followed by centrifugation. Acids Nuclei are precipitated and the DNA is removed by DNase treatment and precipitation. Nucleic acids, specifically RNA and specifically miRNA, can be isolated by any technique known to those skilled in the art. There are two main methods to isolate RNA: (i) phenol-based extraction and (ii) silica matrix or glass fiber filter (GFF) binding. Phenol-based reagents contain a combination of denaturing and RNAse inhibitors for the breakdown of cells and tissues and the subsequent separation of RNA from contaminants. Phenol-based isolation procedures can recover RNA species in the range of 10-200 nucleotides for example, miRNA, ribosomal RNA (rRNA) and small nuclear RNA (RNAs). If a sample of total RNA was purified by the GFF procedure or conventional silica matrix column, small-sized RNAs may have been lost. However, extraction procedures such as those using Trizol or TriReagent will purify all RNAs, large and small, and are the recommended methods to isolate total RNA from biological samples that will contain miRNA. Any method required for the treatment of a sample before quantification of the expression level of miR-335 is within the scope of the present invention.

Once an RNA preparation of the stem cells to be analyzed is available, the method of the invention requires determining the levels of miR-335 expression in the RNA isolated from stem cells. Methods for determining microRNA expression levels in cells or biological samples include generic methods for the detection and quantification of nucleic acids, especially RNA, optimized methods for the detection and quantification of small RNA species, since both mature microRNAs and precursors fall within this category, as well as methods specially designed for the detection and quantification of microRNA. Illustrative, non-limiting examples of methods that can be employed to determine the levels of one or more microRNAs include:

1. Methods based on hybridization, such as Northern blot analysis and in situ hybridization.

2. Real-time multiplex and / or singleplex RT-PCR (reagents available from, for example, Applied Biosystems and System Biosciences (SBI)), including PCR with Quantitative real-time reverse transcriptase (qRT-PCR) as described in US 5,928,907 and US 6,015,674;

3. Detection of individual molecules as described by Neely, et al, Nat.

Methods 3 (l): 41-6 (2006) and in US 6,355,420; US 6,916,661 and US 6,632,526;

4. Pearl flow cytometry methods as described by Lu, et al, Nature 435: 7043 (2005) and in US 6,524,793; Y

5. Assays using nucleic acid matrices as described by Nelson, et al, Nat. Methods 1 (2): 155-61 (2004); Wu, et al, RNA 13 (1): 151-9 (2007) and in US 6,057,134; US 6,891,032; US 7,122,303; US 6,458,583; US 6,465,183; US 6,461,816; US 6,458,583; US 7,026,124; US 7,052,841; US 7,060,809; US 6,436,640 and US 7,060,809.

In a particular embodiment, the expression level of miR-335 is determined by real-time quantitative RT-PCR (qRT-PCR), a modification of the polymerase chain reaction (PCR) used to rapidly measure the amount of a PCR product. This is preferably done in real time, therefore it is an indirect method to quantitatively measure starting quantities of DNA, complementary DNA or RNA. This is commonly used to determine if a genetic sequence is present or not, and if the number of copies in the sample is present. Like other forms of PCR, the procedure is based on the amplification of DNA samples, using thermal cycles and a thermostable DNA polymerase. The three commonly used quantitative PCR methods are: by agarose gel electrophoresis, by using SYBR Green (a double stranded DNA dye) and by a fluorescent indicator probe. The last two methods can be analyzed in real time, thus constituting real-time PCR methods.

The fluorescent indicator probe method is the most accurate and the most reliable of the methods. It uses a sequence-specific nucleic acid-based probe, so that it only quantifies the sequence that hybridizes with the probe and not all double stranded DNA. Said probe, which has at its 3 'end a fluorophore and at its 5' end a molecule that blocks its flourescence emission (quencher or quencher), hybridizes specifically in the central part of the PCR product to be obtained. Thus, when PCR is performed with the probe plus the pair of specific primers, the probe amplicon hybrid but, due to the proximity of the fluorophore to the damper, flourescence is not emitted; when the polymerase begins to synthesize the complementary chain for the single-stranded template DNA primed, as the polymerization progresses, it reaches the probe attached to its complementary sequence, so that the polymerase hydrolyzes the probe through its 5'-3 'exonuclease activity , thereby separating the fluorescent indicator and the switch. This results in an increase in the fluorescence that is detected. During the thermal cycles of the real-time PCR reaction, the increase in fluorescence is monitored as it is released from the double-labeled probe hydrolyzed in each PCR cycle, allowing accurate determination of the final DNA amounts, and also initials.

Any PCR method that allows to determine the expression of miR-335 is within the scope of the present invention. In a particular embodiment, miR-335 is detected by real-time PCR using the TaqMan (Applied Biosystems) probe as a double-labeled probe.

The levels of miR-335 can be quantified by comparison with an internal standard, for example, the level of messenger RNA (mRNA) of a maintenance or housekeeping gene present in the same sample or the level of a non-coding RNA of maintenance. MRNA that can be determined in accordance with the present invention include, but are not limited to, myosin, glyceraldehyde-3-phosphate dehydrogenase (GADPH) microglobulin β-2, ubiquitin, ribosomal protein 18S, cyclophilin, IP08, HPRT, PSMB4, tubulin , β-actin. Non-coding RNAs that can be used as an internal standard include, without limitation, the RNAs U6, RNU19, RNU24, RNU38B, RNU43, RNU44, RNU48, RNU49, RNU58A, RNU58B, RNU66, RNU6B, RPL21, snoRNA135, snoRNA132, snoRNA202, snoRNA235 , snoRNA251, U6, U18, U47, U54, U75, U87, Z30, HY3, 5S rRNA and 4.5S RNA

In one embodiment, the first method of the invention comprises determining the activity levels of miR-335. In the context of the present invention, "miR-335 activity" refers to the ability of miR-335 to inhibit the expression of a target gene. Therefore, miR-335 activity can be detected by assays in which the expression levels of its target genes are quantified. By "miR-335 target genes" are understood all those genes whose expression is directly regulated by miR-335. Thus, according to a form of embodiment of the first method of the invention, when the expression of miR-335 target genes is increased in a stem cell, this implies that miR-335 activity levels are reduced in said stem cell. According to another embodiment of the first method of the invention, when the expression of miR-335 target genes is decreased in a stem cell, this implies that miR-335 activity levels are high in said stem cell.

Preferably, the miR-335 target genes are those that have in their coding region or in their 5 'or 3' UTR regions at least one miR-335 binding site. Illustrative non-limiting examples of miR-335 target genes are those listed in Table 1.

Table 1

Illustrative examples of miR-335 target genes

Symbol of «e 11 (.1 * 11

AIG1 Androgen-induced 1

AMOT Angiomotin

APLN Apelin

ARHGAP24 Rho GTPase activating protein 24

ARID5B AT rich interactive domain 5B (MRF l-like)

ARMC8 Armadillo repeat containing 8

C14orf43 Chromosome 14 open reading frame 43

CA5B Carbonic anhydrase VB, mitochondrial

CCDC1 15 Coiled-coil domain containing 1 15

CCDC28A Coiled-coil domain containing 28A

CHN1 Chimerin (chimaerin) 1

COL16A1 Collagen, type XVI, alpha 1

DAAM1 Dishevelled associated activator of morphogenesis 1

EF B 1 Ephrin-B 1

EGR2 Early growth response 2 (Krox-20 homolog, Drosophila)

EGR3 Early growth response 3

ENAH Enabled homolog (Drosophila)

EPHA3 EPH A3 receiver

FILIP1L Filamin A interacting protein 1-like

F BP1L Formin binding protein 1-like

FOXP1 Forkhead box Pl

GAS1 Growth arrest-specific 1

GLI3 GLI family zinc finger 3

IRF2BP2 Interferon regulatory factor 2 binding protein 2 Gene gene symbol

ITGB8 Integrin, beta 8

KIAA0355 KIAA0355

LBH Limb bud and heart development homolog (mouse)

LYPD6 LY6 / PLAUR domain containing 6

MAF V-maf musculoaponeurotic fibrosarcoma oncogene homolog (avian)

MAN1C1 Mannosidase, alpha, class 1C, member 1

MYLK Myosin light chain kinase

NFATC1 Nuclear factor of activated T-cells, cytoplasmic, calcineurin-dependent 1

Nuclear factor of kappa light polypeptide gene enhancer in B-cells

NFKBIA

inhibitor alpha

NFYB Nuclear transcription factor Y, beta

NHS Nance-Horan syndrome (congenital cataracts and dental anomalies)

PARP14 Poly (ADP-ribose) polymerase family, member 14

PARP9 Poly (ADP-ribose) polymerase family, member 9

PIGK Phosphatidylinositol glycan anchor biosynthesis, class K

P IB Protein kinase (camp-dependent, catalytic) beta inhibitor

PRKD1 Protein Kinase DI

PRR5L Proline rich 5 like

PSD3 Pleckstrin and Sec7 domain containing 3

PURB Purine-rich element binding protein B

RBM12 RNA binding motif protein 12

RUNX2 Runt-related transcription factor 2

SALL2 Sal-like 2 (Drosophila)

SDC1 Syndecan 1

Sema domain, immunoglobulin domain (Ig), transmembrane domain

SEMA4F

(TM) and short cytoplasmic domain, (semaphorin) 4F

Sema domain, transmembrane domain (TM), and cytoplasmic domain,

SEMA6D

(semaphorin) 6D

SH3BP5 SH3 -domain binding protein 5 (BTK-associated)

SLC6A6 Solute carrier family 6 (neurotransmitter transporter, taurine), member 6

Signal transducer and activator of transcription 3 (acute-phase response

STAT3

factor)

TBL1X Transduction (beta) -like lX-linked

TBL1XR1 Transduction (beta) -like 1 X-linked receiver 1

THOC7 THO complex 7 homolog (Drosophila)

TMEM35 Transmembrane protein 35

TPD52L1 Tumor protein D52-like 1

TPM1 Tropomyosin 1 (alpha)

TPST1 Tyrosylprotein sulfotransferase 1

TRIB2 Tribbles homolog 2 (Drosophila)

WSB1 WD repeat and SOCS box-containing 1 Gene symbol (in

ZSCAN2 Zinc f inge r anc SCA] ST d ⁽ ) m <lin com taining 2

In a preferred embodiment, the miR-335 target gene is RUNX2. The term "RUNX2", as understood in the present invention, refers to a transcriptional factor involved in osteogenesis. There are three transcriptional variants of RUNX2, (GenBank Accession Numbers M_001015051, M_004348), and these variants have a miR-335 binding site in their 3 'UTR region. In this way, the expression of RUNX2 is negatively regulated by miR-335.

In another preferred embodiment of the second method of the invention, the miR-335 target gene is "SOX4". The term "SOX4", as understood in the present invention, refers to a transcription factor involved in the regulation of progenitor cell development and migration (GeneBank Accession Number: M_003107.2).

To determine the activity of miR-335 according to the first method of the invention, the expression levels of the miR-335 target genes can be determined by analyzing the nucleic acid levels of said genes or by analyzing the levels of the encoded protein by said genes, using methods known in the art. For example, to analyze the nucleic acid levels of the miR-335 target genes, for example, the RUNX2 and SOX4 genes, conventional techniques can be used to determine the expression levels of a given gene in a given cell, such as RT -PCR, Northern blot and the like to determine mRNA expression. In this case, the first method of the invention can additionally include carrying out an extraction step in order to obtain the total RNA, which can be done by conventional techniques. Virtually any conventional method can be used within the framework of the invention to detect and quantify mRNA levels encoded by the miR-335 target genes, (e.g., the RUNX2 and SOX4 genes) and their corresponding complementary DNA (cDNA) . By way of illustration, not limitation, the levels of mRNA encoded by said genes can be quantified by the use of conventional methods, for example, methods comprising amplification of mRNA and quantification of the product of amplification of said mRNA, such as electrophoresis. and staining, or alternatively, by Southern blotting and using probes appropriate, Northern blot and use of specific probes of the mRNA of the genes of interest or their corresponding cDNA, mapping with the nuclease SI, RT-PCR, hybridization, microarrays, etc., preferably, by quantitative real-time PCR using sets of appropriate probes and primers. Similarly, the level of cDNA corresponding to the mRNA encoded by the miR-335 target genes (for example, the RUNX2 and SOX4 genes) can also be quantified using conventional techniques; in this case, the method of the invention includes a step of synthesis of the corresponding cDNA by reverse transcription (RT) of the corresponding mRNA followed by amplification and quantification of the amplification product of said cDNA. Conventional methods for quantifying expression levels can be found, for example, in Sambrook et al, 2001, "Molecular cloning: a Laboratory Manual", 3rd ed., Cold Spring Harbor Laboratory Press, NY, Vol. 1-3. In a preferred embodiment of the invention, the quantification of the expression level of the miR-335 target genes (for example, the RUNX2 and SOX4 genes) is performed by a polymerase chain reaction (PCR), in any of its variants.

The expression of miR-335 target genes can also be determined at the protein level, that is, by measuring the level of polypeptides encoded by said target genes. Such methods are well known in the art and include, but are not limited to, for example, Western Blot, ELISA, RIA, immunofluorescence, flow cytometry, etc., which use antibodies against proteins encoded by genes. The expression level of the miR-335 target genes (for example, the RUNX2 and SOX4 genes) can be quantified by any conventional method that allows detecting and quantifying said proteins in a sample of a subject. By way of illustration, not limitation, the levels of miR-335 target genes, for example, the RUNX2 and SOX4 genes) can be quantified, for example, by the use of antibodies capable of binding to the proteins encoded by said genes and subsequent quantification. of the complexes formed. The antibodies used in these assays may or may not be labeled. Illustrative examples of markers that can be used include radioactive isotopes, enzymes, fluorophores, chemiluminescent reagents, enzyme substrates or cofactors, enzyme inhibitors, particles, dyes, etc. There is a wide variety of known assays that can be used in the present invention, which use unlabeled antibodies (primary antibody) and labeled antibodies. (secondary antibody); These techniques include Western blotting or Western blotting, ELISA (enzyme-linked immunosorbent assay), RIA (radioimmunoassay), competitive EIA (competitive enzyme immunoassay), DAS-ELISA (ELISA sandwich with double antibody), immunocytochemical and immunohistochemical techniques , techniques based on the use of biochips or microarrays of proteins that include specific antibodies or tests based on colloidal precipitation in formats such as dipsticks. Other ways to detect and quantify said miR-335 target gene proteins (eg, RUNX2 and SOX4 genes), include affinity chromatography techniques, ligand binding assays, etc.

In a particular embodiment, the quantification of the levels of the protein encoded by the miR-335 target genes is performed by western blot, ELISA, immunohistochemistry or an array of proteins.

For the detection of miR-335 target gene expression, reporter genes can also be used, that is, a DNA construct comprising the promoter of the gene under study operatively coupled to a reporter gene can be employed. Thus, in one embodiment, the alteration of the levels of the miR-335 target genes (for example, the RUNX2 and SOX4 genes) is an increase and, consequently, the change in expression in the reporter gene is indicative that miR-335 activity is being inhibited. Reporter genes suitable for use in the present invention include luciferase.

The first method of the invention requires comparing the levels of expression and / or activity of miR-335 in a stem cell with a reference value. The term "reference value", as used in the present invention, refers to a value that derives from a collection of samples consisting of stem cells of the same type as stem cells whose immunoregulatory capacity is being tested by The first method of the invention. Said collection of samples comes from a subject, preferably from two or more subjects, which is known to not have a disease that can be treated by administration of stem cells or, alternatively, of the general population. The reference value can be an expression value or an activity value of miR-335. The expression reference value of miR-335 is determined by techniques well known in the state of the art, for example, by isolating RNA from each sample of stem cells in the collection, determining miR-335 expression levels in each isolated RNA and calculating the average miR-335 expression levels determined in each sample of stem cells. Alternatively, the reference value could be determined by measuring miR-335 expression levels in an RNA sample obtained by mixing equal amounts of RNA from each of the stem cell samples of the aforementioned collection. The activity reference value of miR-335 can be determined by previously described techniques, that is, by determining the expression levels of miR-335 target genes (eg, RUNX2 and SOX4 genes) in each sample of stem cells of the collection and the calculation of the average of all values. Alternatively, the reference value of miR-335 activity could be determined by measuring the expression levels of miR-335 target genes in a sample obtained by mixing equal amounts of each of the stem cell samples from the aforementioned collection. The collection of stem cell samples to be analyzed to calculate the reference value is preferably derived from a population of two or more subjects; for example, the population may comprise 3, 4, 5, 10, 15, 20, 30, 40, 50 or more subjects.

Alternatively, the reference value may correspond to the levels of an RNA that is constitutively expressed in the cell and that does not show variations in its expression levels in high power stem cells or immunregulatory capacity with respect to stem cells that do not have High power or regulatory capacity. Said reference RNA includes, for example, messenger RNA (mRNA) of a maintenance gene or "housekeeping" present in the same sample or the level of a non-coding maintenance RNA. MRNA that can be determined in accordance with the present invention include, but are not limited to, myosin, glyceraldehyde-3-phosphate dehydrogenase (GADPH) microglobulin β-2, ubiquitin, ribosomal protein 18S, cyclophilin, IP08, HPRT, PSMB4, tubulin , β-actin. The reference RNA may also be non-coding RNAs such as, for example, RNAs U6, RNU19, RNU24, RNU38B, RNU43, RNU44, RNU48, RNU49, RNU58A, RNU58B, RNU66, RNU6B, RPL21, snoRNA142, snoRNA14, snoRNA2N, snoRNA2N2 snoRNA234, snoRNA251, U6, U18, U47, U54, U75, U87, Z30, HY3, 5S rRNA and 4.5S RNA. The term "constitutive expression gene" or "housekeeping gene", as used in the present invention, refers to a gene

A "subject", as used herein, refers to a mammal, human or non-human, preferably a human being. The subject can be any subject, a subject predisposed to a disease (for example, a disease capable of being treated with stem cells) or a subject suffering from said disease.

The term "disease that can be treated with a stem cell" includes, but is not limited to, an autoimmune disease, an inflammatory disease, graft versus host disease, a disease that requires induction of transplant tolerance or a disease that requires repair and / or tissue regeneration.

Once the reference value is established, the first method of the invention comprises comparing the levels of expression and / or activity of miR-335 in a stem cell with said reference value, so that high levels with respect to the reference value are indicative. that said stem cell has a low immunoregulatory capacity, while decreased levels with respect to the reference value are indicative that said stem cell has a high immunoregulatory capacity.

In the context of the present invention, "high levels with respect to the reference value" means any variation in the levels of expression and / or activity of miR-335 above the reference level. A variation of the level of expression and / or activity of miR-335 above the reference value may be at least 1, 1 times, 1.5 times, 5 times, 10 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times, 100 times or even more compared to the reference value.

On the other hand, in the context of the present invention, "decreased levels with respect to the reference value" means any variation in the levels of expression and / or activity of miR-335 below the reference value. A variation of the expression level of miR-335 below the reference value may be at least 0.9 times, 0.75 times, 0.2 times, 0.1 times, 0.05 times, 0.025 times, 0 , 02 times, 0.01 times, 0.005 times or even less compared to the reference value.

Once said comparison has been made, the first method of the invention allows to determine whether the stem cell whose expression levels and / or miR-335 activity have been analyzed has a low or high capacity or immunoregulatory power. Additionally, the first method of the invention also allows determining whether said stem cell has a low or high therapeutic power.

In a particular embodiment, when the levels of expression and / or activity of miR-335 in a stem cell are high relative to the reference value, it is indicative that said stem cell has a low capacity or low immunoregulatory power. In another embodiment of the invention, when the levels of expression and / or activity of miR-335 in a stem cell are high with respect to the reference value, it is indicative that said stem cell has a low therapeutic power.

In a particular embodiment, when the levels of expression and / or activity of miR-335 in a stem cell are decreased with respect to the reference value, it is indicative that said stem cell has a high immunoregulatory capacity or potency. In another embodiment of the invention, when the levels of expression and / or activity of miR-335 in a stem cell are decreased with respect to the reference value, it is indicative that said stem cell has a high therapeutic power.

The terms "high" or "low", as used herein, referring to therapeutic potency and / or immunoregulatory capacity, refer to the suitability of a stem cell to be effective in the treatment of a certain disease. .

Methods for selecting a batch of stem cells for therapeutic applications related to immunoregulatory activity

In another aspect, the invention relates to a method for selecting a batch of stem cells for therapeutic applications related to the immunoregulatory activity of said stem cells, wherein said method comprises determining in the stem cells of said batch the expression levels and / or activity of miR-335, where if the levels of expression and / or activity of miR-335 are lower than a reference value, the batch of stem cells is selected for therapeutic applications related to the immunoregulatory activity of said stem cells. The terms and expressions "stem cells", "miR-335", "miR-335 activity", "miR-335 levels", "high levels and / or expression" have been described in detail in relation to the first method of the invention and are equally applicable to the method of batch selection of stem cells.

The term "therapeutic applications related to immunoregulatory activity" refers to all indications in which stem cells are used by virtue of their effects on the immune system, including, without limitation, graft versus host disease (GVHD), autoimmune diseases, inflammation, etc.

In a first step, the methods for characterizing batches of stem cells of the invention comprise determining in the stem cells of said batch the levels of expression and / or activity of miR-335. Suitable methods for carrying out said determination have been described in detail in the context of the first method of the invention and include, without limitation, methods based on the quantification of the miR-335 or methods based on the determination of the activity of the miR-335 , thereby determining the levels of gene expression whose expression is regulated by miR-335. Such genes have been described in detail in Table 1 of the present invention.

In a second stage, the level of expression and / or activity of the miR-355 obtained in the first stage is compared with a reference value, wherein said reference value is a predetermined value or the value of the expression level and / or miR-355 activity in the stem cells of a control batch previously selected as suitable for therapeutic applications related to immunoregulatory activity.

The term "stem cells of a control batch previously selected as suitable for therapeutic applications related to immunoregulatory activity", as used in the present invention, refers to cells that have been identified as suitable for use in activity related applications. immunoregulatory, using either the determination of the levels or activity of miR-335 according to the first method of the invention or by any of the potency tests commonly used to release batches of stem cells for use in therapy. Thus, the cells in the control lot are cells that have one or more of the following properties:

They are cells that have reduced levels of activity and / or expression of miR-335, according to the present invention.

Other criteria that can be used to determine the potency of a batch of cells are any of those contained in the article "Reflection Paper on stem cell-based medicinal products" of the European Medicines Agency (EMA / CAT / 571134/2009) which includes :

- Expression of relevant products (cytokines, growth factors, enzymes, etc.)

Capacity of formation of matrices and cellular and extracellular structures Capacity of interaction with other cells such as, for example, cells of the immune system)

Differentiation, proliferation and migration capacity.

Other potency assays that can be used to select a batch of control cells include, for example, the assays mentioned by Bieback et al. (Transfus. Med. Hemother. 2008; 35: 286-294) including:

- Clongenicity test as described by Luna et al. (Transfusion 1971; 11: 345-349).

- Differentiation to osteogenic, chondrogenic and adipogenic lineages, using standard methods such as those described by Delorme B et al, (Regen Med 2006; 1: 497-509) - Immunomodulatory capacity test, determining parameters such as expression of HLA class I molecules. Thyl (CD90), vascular adhesion molecules (VCAM, CD106), intracellular adhesion molecules 1 and 2 (ICAM-1 and ICAM-2) , cell adhesion molecule of activated leukocytes (ALCAM, CD166), functional antigen of lymphocytes 3 and different integrins. Other assays related to immunoregulatory capacity include, without limitation, the determination of inhibition of T-cell allooreactivity in mixed lymphocyte cultures or in mitogen-induced lymphocyte proliferation (PHA or concanavalin A).

- Regulation of hematopoiesis, determining parameters such as increased expansion of hematopoietic stem cells as a result of the contact of stem cells with CD34 + cells

Other tests to determine stem cell potency described in the potency test guide published by the US Food and Drug Administration. Alternatively, it is possible to use the test described by Hall and Rich (Bioluminescence assays for assessing potency of cellular therapeutic producís, In: Cellular Therapy: Principies, Methods and Regulations, 2009, Areman EM & Loper K, 581-591. AABB. ISBN 978 -1-56395-296-8 Bethesda, MD) based on determining the proliferation of stem cells contacted with a mixture of growth factors.

In a preferred embodiment, the cells that are selected as a control lot as suitable for therapeutic applications related to the immunoregulatory activity are autologous, syngeneic, allogeneic or xenogeneic with respect to the cells of the batch to be selected.

In another embodiment, the potency assay according to the present invention is used as a potency assay within the regulatoryly accepted release criteria of batches of stem cells for therapeutic applications.

In another preferred embodiment, the batch stem cell is an adult stem cell. In an even more preferred embodiment, the adult stem cell is an adult stem cell of mesenchymal origin. In an even more preferred embodiment, the adult mesenchymal stem cell is derived from bone marrow or subcutaneous adipose tissue. Invention kit

In another aspect, the invention relates to a kit, hereafter kit of the invention, to determine the immunoregulatory capacity of a stem cell comprising

(i) suitable reagents to determine the expression level of miR-335 and

(ii) suitable reagents to determine the level of expression of a reference RNA.

By "suitable reagents for determining the expression level of miR-335" is meant any reagent necessary to specifically detect the expression of miR-335 by the detection methods previously described in the context of the first method of the invention. In a particular embodiment, the reagents suitable for determining the level of miR-335 expression are one or more pairs of oligonucleotides specifically designed to amplify miR-335 using the methods of the invention in an RT-PCR assay, preferably a real-time RT-PCR assay.

By "suitable reagent to determine the level of expression of a reference RNA is meant any reagent necessary to specifically detect the expression of an RNA by any of the detection methods previously described in the context of the first method of the invention. In a particular embodiment, the reagents suitable for determining the level of expression of a reference RNA are one or more pairs of oligonucleotides specifically designed to amplify said RNA or probes that specifically hybridize with said RNA. In a preferred embodiment , the determination of RNA levels is carried out by an RT-PCR assay, preferably a real-time RT-PCR assay.

In a preferred embodiment, the reference RNA is the messenger RNA (mRNA) of a housekeeping or maintenance gene. MRNA that can be determined in accordance with the present invention include, but are not limited to, myosin, glyceraldehyde-3-phosphate dehydrogenase (GADPH) microglobulin β-2, ubiquitin, ribosomal protein 18S, cyclophilin, IP08, HPRT, PSMB4, tubulin , β-actin. In another preferred embodiment, the reference RNA may also be a non-coding RNA such as, for example, RNAs U6, RNU19, RNU24, RNU38B, RNU43, RNU44, RNU48, RNU49, RNU58A, RNU58B, RNU66, RNU6B, RPL21 , snoRNA135, snoRNA142, snoRNA202, snoRNA234, snoRNA251, U6, U18, U47, U54, U75, U87, Z30, HY3, 5S rRNA and 4.5S RNA.

Stem cells, specifically MSCs, are considered immunologically privileged due to their reduced expression of the major histocompatibility complex (MHC) of class I and II, as well as the absence of surface expression of CD40, CD80 and CD86, molecules costimulators required for the activation of T lymphocytes. Thus, in a particular embodiment, the kit of the invention additionally comprises reagents suitable for determining the expression and / or activity levels of a marker of the immunoregulatory capacity and / or a senescence marker in said stem cell.

Markers of the immunoregulatory capacity of a stem cell are selected from the group consisting of MHC class I, MHC class II, CD40, CD80 (B71), CD86 (B72), B7-DC (PD-L2), B7H1 (PD- L1), B7H2 (CD275 / ICOSL), B7H3 (CD276), B7H4 (VTCN1), B7H5, B7H6, B7H7. In a preferred embodiment, the immunoregulatory capacity marker of a stem cell is B7H1.

In another embodiment, the senescence marker is selected from the group consisting of β-galactosidase, Ki67, p21 (CDKN1A), γΗ2ΑΧ, SAHF, p53 and, Lamin B.

In a preferred embodiment, the senescence marker of a stem cell is β-galactosidase. The use of β-galactosidase as a marker of cellular senescence is based on the detection of β-galactosidase activity at suboptimal pH. Lysosomal β-galactosidase is active at pH 4, but in senescent cells, due to the characteristic increase in lysosome mass in that state, there is an increase in β-galactosidase activity that allows its detection at pH 6. Suitable reagents To measure β-galactosidase activity are those that allow the detection of β-galactosidase activity at pH 6, and are known to the person skilled in the art. These reagents can include, but are not limited to, fixation solutions, staining solutions and artificial substrates of the enzyme which, after being hydrolyzed by β-galactosidase, release a chromogenic substance that can be observed through microscopy techniques. These compounds are generally derivatives of galactopyranosides. Examples of compounds that are artificial substrates of beta-galactosidase and that may be part of the kit of the invention include, but are not limited to: nitro-phenyl-B-Dgalactopyranoside (O PG), red chlorophenyl BD-galactopyranoside (CPRG), bromine -chloroindolyl BD-galactopyranoside (X-gal), fluoresein di-BD-galactopyranoside (FDG) and the galacton substrate. In a preferred embodiment, the kit of the invention comprises X-gal as a suitable reagent to determine the levels of expression and / or activity of β-galactosidase.

In another aspect, the invention relates to a kit according to the invention to determine the immunoregulatory capacity or potency of a stem cell or to select a batch of stem cells for therapeutic applications related to the immunoregulatory activity of said stem cells.

In vitro method to increase the immunoregulatory capacity of a stem cell

In a second aspect, the invention relates to an in vitro method, hereinafter second method of the invention, to increase the immunoregulatory capacity of a stem cell, which comprises inhibiting in said stem cell the expression and / or activity of miR -335.

The second method of the invention comprises inhibiting the expression and / or activity of miR-335 in a stem cell. To inhibit the expression and / or activity of miR-335, different methods known to those skilled in the art can be used, for example, the stem cell in question can be treated with a miR-335 inhibitor. The term "miR-335 inhibitor", as used herein, refers to any agent or small molecule that inhibits, reduces or decreases the expression and / or activity of miR-335. In one embodiment, the miR-335 hybrid inhibitor with miR-335 and thus inhibits its activity. Illustrative non-limiting examples of miR-335 inhibitors include: antisense oligonucleotides, interference RNA (RNAi), ribozymes, DNA enzymes, natural or synthetic small compounds, preferably small organic compounds, etc.

Antisense oligonucleotides are simple strands of DNA or RNA that are complementary to a chosen sequence. Antisense RNAs prevent translation of proteins by joining their messenger RNAs. Antisense DNAs can bind to a specific and complementary sequence of RNA (coding or non-coding), resulting in a DNA / RNA hybrid that can be degraded by the enzyme RNase H. For use in the present invention, a construct which comprises an antisense oligonucleotide can be distributed, for example, as an expression plasmid which, when transcribed in the cell, produces RNA that is complementary at least in part to the miR-335 sequence. Alternatively, the antisense construct can be an oligonucleotide probe that is generated ex vivo and that, when introduced into the cell, hybridizes with miR-335 preventing its function. Such oligonucleotide probes are preferably modified oligonucleotides, which are resistant to endogenous nucleases, for example, exonucleases and / or endonucleases, and which are therefore stable in vivo. Illustrative nucleic acid molecules for use as antisense oligonucleotides include phosphoramidate, phosphothionate and methylphosphonate DNA analogs (see, for example, US5176996, US5264564 and US5256775). Additionally, for a review of the general approaches to construct oligomers useful in antisense therapy see, for example, Van der Krol et al., BioTechniques 6: 958-976, 1988; and Stein et al, Cancer Res 48: 2659-2668, 1988.

Preferably, in vitro studies should be performed to quantify the ability of antisense oligonucleotides to inhibit the function of miR-335. Advantageously, said studies will use controls that distinguish between antisense gene inhibition and non-specific biological effects of oligonucleotides. It is also preferred that these studies compare the levels of the target RNA or protein with those of an internal control of RNA or protein. The results obtained using the antisense oligonucleotides can be compared with those obtained using a control oligonucleotide. It is preferred that the control oligonucleotide be approximately the same length as the oligonucleotide to be tested and that the oligonucleotide sequence differs from the antisense sequence no more than is necessary to prevent specific hybridization to the target sequence.

The antisense oligonucleotides can be DNA or RNA or chimeric mixtures or derivatives or modified versions thereof, single chain or double chain. The oligonucleotide can be modified in the base, in the sugar or in the phosphate skeleton, for example, to improve the stability of the molecule, its hybridization capacity etc. The oligonucleotide may include other bound groups, such as peptides (for example, to direct them to host cell receptors) or agents to facilitate transport across the cell membrane (Letsinger et al, Proc. Nati. Acad. Sci. USA 86 : 6553-6556, 1989; Lemaitre et al, Proc. Nati. Acad. Sci. 84: 648-652, 1987; WO88 / 09810) or the blood brain barrier (WO89 / 10134), intercalating agents (Zon, Pharm. Res. 1988. 5: 539-549). For this purpose, the oligonucleotide may be conjugated to another molecule, for example, a peptide, a transport agent, a hybridization triggered cutting agent, etc.

In some cases, it may be difficult to achieve intracellular concentrations of the antisense oligonucleotide sufficient to suppress the function of endogenous miR-335. Thus, a preferred approach uses a recombinant DNA construct in which the antisense oligonucleotide is placed under the control of a strong pol III or pol II promoter.

The mir-335 inhibitor can be an interference RNA. An interference RNA (RNAi) is an RNA that modulates the expression of a gene through the mechanism of RNA interference. In one embodiment, the RNAi is a small RNAi. Small interference RNAs or siRNAs (siRNAs) are agents capable of inhibiting the expression of a target gene by RNA interference. An siRNA can be chemically synthesized, or, alternatively, it can be obtained by in vitro transcription or it can be synthesized in vivo in the target cell. Typically, siRNAs consist of a double strand of RNA between 15 and 40 nucleotides in length, which may contain a 3 'and / or 5' protruding region of 1 to 6 nucleotides. The length of the protuberant region is independent of the total length of the siRNA molecule. SiRNAs act by degradation or post-transcriptional silencing of the target messenger.

The siRNAs can be called shRNA (short hairpin RNA), characterized in that the antiparallel chains that form the siRNA are connected by a loop or hairpin region. The shRNAs may be encoded by plasmids or viruses, particularly retroviruses, and be under the control of promoters such as the U6 promoter of RNA polymerase III.

In a particular embodiment, the siRNAs that can be used in the present invention are substantially homologous to miR-335. By "substantially homologs "are understood to have a sequence that is sufficiently complementary or similar to the miR-335 sequence, such that the siRNA is capable of causing degradation by RNA interference. Suitable siRNAs to cause such interference include formed siRNAs. by RNA, as well as siRNA that contain different chemical modifications such as:

SiRNA in which the bonds between nucleotides are different from those that appear in nature, such as phosphorothioate bonds;

RNA chain conjugates with a functional reagent, such as a fluorophore;

modifications of the ends of the RNA chains, in particular the 3 'end by modification with different functional groups of the hydroxyl in 2' position;

nucleotides with modified sugars such as 2'-0-methylribose or 2'-0-fluorosibose O-alkylated moieties;

nucleotides with modified bases such as halogenated bases (for example 5-bromouracil and 5-iodouracil), alkylated bases (for example 7-methylguanosine).

The siRNAs and siRNAs that can be used in the present invention can be obtained using a series of techniques known to the person skilled in the art.

In another embodiment, the miR-335 inhibitor is a ribozyme specifically designed to catalytically cut the miR-335 sequence. Ribozymes are enzymatic RNA molecules capable of catalyzing the specific cut of RNA [for a review see Rossi, 1994. Current Biology 4: 469-471]. The sequence of ribozyme molecules preferably includes one or more sequences complementary to the target RNA, this is miR-335, and the well-known sequence responsible for RNA cutting or a functionally equivalent sequence [see, for example, US5093246]. Ribozymes may be composed of modified oligonucleotides (for example, to improve stability, targeting, etc.) and should be distributed to cells expressing the target gene in vivo. A preferred method of distribution involves using a DNA construct that "encodes" the ribozyme under the control of a strong constitutive promoter of pol III or pol II, so that the transfected cells will produce sufficient amounts of the ribozyme to destroy the endogenous target messengers and inhibit translation. Since ribozymes, contrary to other antisense molecules, are catalytic, a lower intracellular concentration is required to be effective.

In another embodiment, the miR-335 inhibitor is a DNA enzyme. DNA enzymes incorporate some of the mechanistic characteristics of both antisense oligonucleotide technologies and ribozyme technologies. DNA enzymes are designed to recognize a particular nucleic acid target sequence (in this case, the miR-335 sequence), similar to the antisense oligonucleotide; however, similar to ribozyme, they are catalytic and specifically cut the target nucleic acid.

In a preferred embodiment, the miR-335 inhibitor is a nucleic acid, for example, an antisense oligonucleotide or an RNAi. In a more preferred embodiment, the miR-335 inhibitor is single chain polynucleotide that has the ability to specifically hybridize with miR-335 preventing its function. By "preventing its function" is meant to block at least partially the activity of miR-335.

The miR-335 inhibitor can be introduced into the target cell using any suitable protocol. When the miR-335 inhibitor is a nucleic acid, such as an antisense oligonucleotide or an RNAi, it can be introduced into the target cell using any known technique of transferring nucleic acids to cells in vitro. Such techniques include, but are not limited to, electroporation, nucleofection, lipofection, calcium phosphate mediated transfection, magnetofection, or viral infection (transduction). Usually, the transfer procedure includes the transfer of a selectable marker to the cells. The cells are then screened to isolate the cells that have incorporated and express the transferred gene. The transfer of the miR-335 inhibitor nucleic acid to the target cell can be transient or stable. In a preferred embodiment, the transfer is stable, so that the nucleic acid is expressible by the cell and preferably inheritable and expressible by its cellular progeny. In a preferred embodiment, the miR-335 inhibitor nucleic acid is transferred to the stem cell by lipofection with a reagent such as Lipofectamine 2000 (Invitrogen).

In a particular embodiment of the second method of the invention, the stem cell whose immunoregulatory capacity is to be increased is an adult stem cell, preferably a mesenchymal stem cell, more preferably a mesenchymal stem cell from bone marrow or subcutaneous adipose tissue.

Methods for detecting miR-335 expression and / or activity levels have been previously exposed, in the context of the first method of the invention.

The terms "immunoregulatory capacity", "stem cell", "adult stem cell", "mesenchymal stem cell" and "miR-335" have been described in the context of the first method of the invention.

Therapeutic methods of the invention

The use of stem cells, specifically the use of MSC, in the treatment of diseases is being studied, among other reasons, based on their anti-inflammatory properties in vitro, their efficacy in animal models and some isolated results achieved in clinical trials in humans. Phase I / II clinical trials are being developed to study its use in the treatment of Crohn's disease and multiple sclerosis and it is planned to begin them in the treatment of systemic lupus erythematosus, systemic sclerosis, systemic vasculitis, type 1 diabetes and others. Many immune based diseases.

The ability of MSCs to interact with cells of the immune system to control an immune response is also known from the state of the art, which in the case of autoimmune diseases, is responsible for the destruction of different tissues or specific cells causing their deterioration. In these cases, the use of MSC manages to energize the T, B and NK lymphocytes, achieving an asymptomatic state free of immunosuppressive medications.

Therefore, in another aspect, the present invention relates to a stem cell characterized in that it exhibits reduced levels of expression and / or activity of miR-335 for use in medicine.

In another aspect, the present invention also relates to a stem cell characterized in that it has reduced levels of expression and / or activity of miR-335 for use in the treatment of an autoimmune disease, of an inflammatory disease, of graft versus disease. host, in induction of transplant tolerance or in tissue repair and regeneration. In another aspect, the invention also relates to a method, hereinafter the therapeutic method of the invention, for the treatment of an autoimmune disease, an inflammatory disease, graft versus host disease, type 1 diabetes, diabetes type 2, of a cardiovascular disease, for the induction of transplant tolerance, or for the repair and regeneration of tissues in a subject, which comprises administering to said subject a therapeutically effective amount of a stem cell characterized in that it has reduced levels of expression and / or activity of miR-335.

In another aspect, the present invention also relates to a use of a stem cell characterized in that it has reduced levels of expression and / or activity of miR-335 with respect to a reference value for preparing a medicament for the treatment of a disease selected from the group formed by an autoimmune disease, an inflammatory disease and graft versus host disease.

The term "inflammatory disease" includes any disease caused by uncontrolled and continued activation of inflammatory responses that cause tissue damage; Said inflammatory response can be triggered by infectious agents, physical agents, chemical agents, tumors and cell death. "Autoimmune diseases", to the extent that they also have an inflammatory component, fall within the term "inflammatory diseases" as used herein. Illustrative, non-limiting examples of inflammatory diseases and autoimmune diseases include, Addison's disease, alopecia areata, ankylosing spondylitis, hemolytic anemia, pernicious anemia, thrush, aphthous stomatitis, arthritis, arteriosclerosis, osteoarthritis, rheumatoid arthritis, aspermiogenesis, bronchial asthma autoimmune asthma, autoimmune hemolysis, Bechet's disease, Boeck's disease, inflammatory bowel disease, Burkitt's lymphoma, Crohn's disease, chorioiditis, ulcerative colitis, celiac disease, cryoglobulinemia, dermatitis herpetiformis, dermatomyositis, insulin-dependent diabetes, juvenile diabetes, diseases autoimmune demielinizers, Dupuytren's contracture, encephalomyelitis, allergic encephalomyelitis, endophthalmia, allergic enteritis, autoimmune enteropathy syndrome, leprosy nodal erythema, idiomatic facial paralysis, chronic fatigue syndrome, rheumatic fever, glomerulonephritis, Goodpasture syndrome, syn Graves drome, Harnman-Rich disease, Hashimoto's disease, sudden loss of hearing, chronic hepatitis, Hodgkin's disease, paroxysmal hemoglobinuria, hypogonadism, regional ileitis, iritis, leukopenia, disseminated lupus erythematosus, systemic lupus erythematosus, cutaneous lupus erythematosus, lymphogranuloma, infectious mononucleosis, myastheniasis gravis, primary myelosterosis, primary myelitis, gravis myelitis sympathetic ophthalmia, granulomatous orchitis, pancreatitis, pemphigus vulgaris, polyarteritis nodosa, chronic polyarthritis, polymyositis, acute polyradicultis, psoriasis, purpura, pyoderma gangrenosum, Reiter's syndrome, sarcoidosis, ataxic sclerosis, systemic sclerosis, progressive sclerosis, multiple sclerosis, progressive sclerosis, multiple sclerosis disseminated, infertility due to anti-sperm antibodies, thrombocytopenia, thymoma, acute anterior uveitis, vitiligo, diseases associated with AIDS, SCID and Epstein Barr virus such as Sjorgren's syndrome, B-cell lymphoma associated with AIDS or Epstein's virus -Barr, sickness Parasitic s such as leishmaniasis and immunosuppressed states such as viral infections after transplants, AIDS, cancer, chronic active hepatitis, transplant rejection as a result of tissue or organ transplantation and graft versus host disease that may result from bone marrow transplantation bone or hematopoietic stem cells.

The term "graft versus host disease" or "GVHD" refers to a syndrome observed after an allogeneic hematopoietic stem cell transplant and presumably transmitted by the donor's T lymphocytes when reacting against the recipient's tissues. EICH can be acute or chronic.

Certain non-inflammatory diseases that have an inflammatory component, such as type 1 diabetes, type 2 diabetes and cardiovascular disease, also fall within the scope of the therapeutic methods and uses of the invention. The term "type 1 diabetes", also called "type I diabetes mellitus" or "juvenile diabetes" or "insulin dependent diabetes mellitus", as used in the context of the present invention, is a metabolic disease characterized by destruction Selective beta cells of the pancreas causing an absolute insulin deficiency.-It differs from type 2 diabetes because it is a type of diabetes characterized by occurring early in life, usually before age 30. Only 1 in 20 people with diabetes have type I diabetes, which occurs most frequently in young people and children. Insulin administration in these patients is essential. Type 1 diabetes is classified into autoimmune cases — the most common form — and in idiopathic cases. The susceptibility to type 1 diabetes mellitus seems to be associated with multiple genetic factors, although only 15-20% of patients have a positive family history.

The term "type 2 diabetes", as used in the present invention, refers to a disease characterized by an inappropriate elevation of blood glucose levels that causes chronic complications due to the involvement of large and small vessels and nerves. The underlying alteration in this disease is the difficulty for the action of insulin (such as a loss of tissue sensitivity to this hormone) that is called insulin resistance and an inadequate secretion of insulin by the cells responsible for its production in the pancreas. In addition to increasing the concentration of glucose, the deficient action of insulin often results in elevated cholesterol and / or triglyceride levels.

The term "cardiovascular disease", as used herein, refers to any disease or dysfunction or alteration of the heart or of the rest of the cardiovascular or blood system.

The immunomodulatory capacity of the stem cells not only has importance in the treatment of inflammatory, autoimmune diseases and in the treatment of graft versus host disease, but they are also outlined as an indispensable treatment element to promote tolerance towards solid organs such like the heart, lung and kidney; By way of illustration, co-infusion of stem cells is possible at the time of transplantation.

Therefore, in another aspect, the present invention also relates to a use of a stem cell characterized in that it exhibits reduced levels of expression and / or activity of miR-335 to prepare a medicament to induce transplant tolerance.

Once the stem cells enter the bloodstream, they are able to detect molecules that are secreted by damaged or dying tissues in what is known as the "homing" or nesting phenomenon. Once it is in close proximity to the tissue damaged, the stem cell adheres to the surface of the organ through molecular receptors that it expresses on the surface of the cell membrane.This starts a series of events that allow the stem cell to integrate into the damaged organ and begin to secrete factors of growth that stimulate locally Resident stem cells of the affected organ itself, in addition to changing the inflammatory microenvironment to give rise to a microenvironment permissible for cell regeneration, where a process of cell fusion or differentiation begins then becoming a physiologically mature cell, in addition to promoting the formation of New blood vessels Therefore, in another aspect, the invention relates to a use of a stem cell characterized in that it has reduced levels of expression and / or activity of miR-335 with respect to a reference value for preparing a medicament for tissue repair and regeneration. . The stem cell according to the therapeutic uses and methods of the invention is preferably an adult stem cell, more preferably a mesenchymal stem cell, even more preferably a mesenchymal stem cell of bone marrow or subcutaneous adipose tissue.

In a particular embodiment, the stem cell is modified by a miR-335 inhibitor. The term "miR-335 inhibitor" as well as the methods for incorporating said inhibitor into the stem cell have been previously described in the context of the second method of the invention. The miR-335 inhibitor is preferably a single chain polynucleotide that specifically hybridizes with miR-335 preventing its function. More preferably, the miR-335 inhibitor contains the sequence SEQ ID No: 1 or a clearly related sequence. The term "clearly related sequence" includes functionally equivalent sequences, that is, sequences having an identity with the sequence SEQ ID NO: 1 of at least 85%, typically at least 90%, advantageously at least 95%, preferably at least 99%, and maintain the ability to hybridize with miR-335 and prevent its function. Illustrative tests to detect the levels of expression and / or activity of miR-335 have been previously exposed so that the person skilled in the art could easily identify if a sequence is clearly related to the sequence shown in SEQ ID NO: 1, or is functionally equivalent to it.

The cells according to the therapeutic uses and methods of the invention have reduced levels of expression and / or activity of miR-335. In the context of the present invention, "reduced levels of expression and / or activity of miR-335" refer to reduced levels of expression and / or activity of said miR-335 with respect to a value of reference. "Reduced levels of expression and / or activity with respect to the reference value" means any variation in the levels of expression and / or activity of miR-335 below the reference value.

A variation of the level of expression and / or activity of miR-335 below the reference value may be at least 0.9 times, 0.75 times, 0.2 times, 0, 1 times, 0.05 times, 0.025 times, 0.02 times, 0.01 times, 0.005 times or even less compared to the reference value.

The stem cell according to the therapeutic uses and methods of the invention can be autologous, allogeneic or xenogeneic with respect to the subject to be treated. The term "autologous," as used herein, means that the donor and the recipient of the stem cell is the same subject. The term "allogeneic" means that the donor and the recipient of the stem cell are different subjects. The term "xenogeneic" means that the donor and the recipient of the stem cell are subjects of different species.

The term subject has been previously described.

The therapeutic method of the invention comprises administering to a subject a therapeutically effective amount of a stem cell characterized in that it has reduced levels of expression and / or activity of miR-335 with respect to a reference value. By "therapeutically effective amount of a stem cell" in the context of the therapeutic method of the invention, is meant an amount of a stem cell, or a population of substantially homogeneous stem cells, which is capable of producing the desired therapeutic effect, and in In general, it will be determined, among other factors, taking into account the characteristics of the subject, the severity of the disease, the form of administration, etc. For this reason, the doses mentioned in this invention should be taken into account only as a guide for the person skilled in the art, who should adjust that dose depending on the factors described above. By way of illustration, not limitation, the stem cells according to the uses and therapeutic methods of the invention can be administered as a single dose, containing from about lxl 0 ⁵ to about 10x10 ⁶ stem cells of the invention per kilogram (kg) of body weight of the recipient, preferably between about 5xl0 ⁵ and about 5xl0 ⁶ stem cells of the invention per kg of body weight of the recipient, more preferably between about lxl O ⁶ and about 2x10 ⁶ stem cells of the invention per kg of body weight of the receptor, depending on the factors described above. The dose of stem cells according to the therapeutic uses and methods of the invention can be repeated, depending on the state and evolution of the subject, in time intervals of days, weeks or months that the specialist must establish in each case.

Administration of the stem cell according to the therapeutic uses and methods of the invention to the subject will be carried out by conventional means. For example, said stem cell can be administered to said subject intravenously using suitable devices, such as syringes, catheters (a standard peripheral intravenous catheter, a central venous catheter or a pulmonary arterial catheter, etc.), trocars, cannulas, etc. The flow of the cells can be controlled by inflating and deflating in series of distal and proximal balloons located within the vasculature of the subject, thus creating temporary areas without flow that promote cellular therapeutic action. In all cases, the stem cell will be administered using the equipment, apparatus and devices suitable for the administration of cellular compositions and known to those skilled in the art.

As the person skilled in the art understands, sometimes the direct administration of the stem cell according to the therapeutic uses and methods of the invention to the site that is intended to benefit can be advantageous. Thus, direct administration of said stem cells to the desired organ or tissue can be achieved by direct administration (eg, by injection, etc.) on the external surface of the affected organ or tissue by insertion of a suitable device, eg , an appropriate cannula, by arterial or venous perfusion (including retrograde flow mechanisms) or by other means known in the art.

Stem cells according to the therapeutic uses and methods of the invention, if desired, can be stored until the moment of their application by conventional procedures known to those skilled in the art. For short-term storage (less than 6 hours), said cells can be stored at or below room temperature in a sealed container, complementing it or not with a nutrient solution. Medium-term storage (less than 48 hours) is preferably carried out at 2-8 ° C, in an iso-osmotic solution and buffered in a container composed of, or coated with, a material that prevents cell adhesion. Longer term storage is preferably carried out by means of Proper cryopreservation and storage under conditions that promote retention of cellular function.

Stem cells according to the therapeutic uses and methods of the invention can be used in a combination therapy with other additional compounds that may be useful for the treatment of the disease to be treated. Said additional compounds may be administered together with said stem cells as part of the same composition or, alternatively, they may be administered in the form of a separate composition for simultaneous or successive administration (sequential in time) with respect to the administration of the cells mother according to the therapeutic uses and methods of the invention.

In vitro method for the generation of regulatory T cells

Mesenchlear stem cells when co-cultured with peripheral blood mononuclear cells are able to increase the proportion of subpopulations of T lymphocytes with regulatory cell phenotypes such as (D i · CD25aUo, CD4 + / CTLA- +, CD4 ÷ / CD25 ÷ / CTLA-4 ÷ Therefore, in another aspect, the invention relates to an in vitro method, hereinafter third method of the invention, for generating regulatory T cells, which comprises contacting a population of cells containing T cells with a population of stem cells characterized in that they have reduced levels of expression and / or activity of miR-335 under conditions suitable for the generation of regulatory T cells.

The term "regulatory T cells", as used in the present invention, refers to T cells whose main function is to control inflammation and maintain self-tolerance by expanding and controlling the activation of autoreactive CD4 + effector T cells. Regulatory T cells are primarily between the subpopulation of CD4 + T cells that exhibit high levels of CD25 expression and Foxp3 transcription factor. Specifically, the subpopulations CD4 + CD25 + ^alt0 Foxp3 + (Treg cells), CD4 + IL10 + Foxp3- (Trl cells) and CD4 + TGF-p + (Th3 cells) are considered regulatory cells. However, there are also subpopulations of CD8 + T cells, such as subpopulations CD8 + CD25 +, CD8 + CD28- and CD8 + IL-10 +, which are also capable of repressing activation and lymphocyte proliferation, and therefore are included within the definition of regulatory T cells according to the third method of the invention.

The third method of the invention comprises contacting a population containing T cells with stem cells. In a particular embodiment, the population containing T cells is a population of peripheral blood mononuclear cells. "Peripheral blood mononuclear cells" or "PBMC" include lymphocytes, monocytes and macrophages. Methods for isolating these PBMCs from a blood sample are well known in the art. In another particular embodiment, the population containing T cells is a population of PBMC that has been enriched in CD4 + lymphocytes, either in CD8 + lymphocytes or in both types of CD4 + and CD8 + lymphocytes.

Stem cells exhibiting reduced levels of expression and / or activity of miR-335 have been previously described, in the context of the therapeutic uses and methods of the invention. Said stem cells are preferably adult stem cells, more preferably mesenchymal stem cells.

According to the third method of the invention, the population of T cells and the stem cells must be co-cultured under conditions suitable for the generation of regulatory T cells. "Suitable conditions for the generation of regulatory T cells" are those that allow obtaining T cells capable of carrying out the functions of regulatory T cells, such as inhibition of mixed lymphocyte reaction (MLR), and are known by the skilled.

The "mixed lymphocyte reaction" or "MLR" is an in vitro method to analyze the proliferation of helper T cells. Said method consists in the co-culture of allogeneic lymphocytes, which causes the expansion of the helper T cell population. Regulatory T cells, when added to a culture of an MLR, are capable of inhibiting the proliferation of said helper T lymphocytes. According to the third method of the invention, when a population containing T cells with a population of stem cells characterized by having reduced levels of expression and / or activity of miR-335 is co-cultivated, regulatory T cells are obtained which, when added to an MLR are able to inhibit the proliferation of helper T lymphocytes. Said inhibition of T helper lymphocyte proliferation can be at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at minus 70%, at least 80%, at least 90%, at least 95% or even 100%.

In a particular embodiment, suitable conditions for the generation of regulatory T cells consist of co-culture of stem cells, preferably MSC, with a population containing T cells, preferably a population of PBMC, for a period of time of 1 to 5 days, preferably 4 days. Co-cultures can be carried out using different proportions of MSC and T cells, for example, 1: 2, 1: 20, 1: 200, 1: 2000 and 1: 20000. In a preferred embodiment, the ratio of MSC and T cells is 1: 2000.

In a particular embodiment of the third method of the invention, agents that stimulate the activation of regulatory T cells can be added during the co-culture of the stem cell population with the population containing T cells, in addition to the activation signals provided by the stem cells. Agents that stimulate T cell activation may be antibodies, chemical stimuli of T cell function and inhibitors of T cell inhibition. In another particular embodiment, co-culture of the stem cell population with The population that contains T cells can be made with an antigen so that the expansion of antigen-specific regulatory T cells is induced. The antigen can be an autoantigen.

According to the third method of the invention, the population of stem cells may be autologous or allogeneic with respect to the population containing T cells. The term "autologous", according to the third method of the invention, refers to the population of cells mother and the population that contains T cells are derived from the same subject. The term "allogeneic", according to the third method of the invention, refers to the fact that the population of stem cells and the population containing T cells are derived from different subjects. The term subject has been previously described.

The specific examples provided below serve to illustrate the nature of the present invention. These examples are included for illustrative purposes only and should not be construed as limitations on the invention claimed herein. Therefore, the examples described below illustrate the invention without limiting its scope of application. EXAMPLES

EXAMPLE 1

Relationship of the level of expression of miR-335 in hMSCs with the age of the donor.

objective

To study whether there is a significant correlation between the endogenous levels of miR-335 in human mesenchymal stem cells (hMSC) and the age of the donor.

Method

Bone marrow and subcutaneous adipose tissue hMSC were isolated from human donors of different ages (between 18 and 55 years). After being cultured ex vivo under standard conditions (low glucose DMEM, 10% fetal bovine serum, 5 mM glutamine, at 37 ° C in an incubator with 5% C0 ₂ and 95% humidity) for one week, they were collected and were used for the isolation of total RNA by the miRNAeasy kit (Qiagen), following the manufacturer's instructions. The relative expression levels of miR-335 were quantified in the different RNA samples by real-time PCR using TaqMan (Applied Biosystems) probes. As endogenous control, U6 was used.

Results

Figure 1 shows the relative expression levels of miR-335 in hMSC from donors of different ages. It can be clearly seen that the hMSCs of older donors have significantly higher endogenous levels of miR-335 than the hMSCs of younger donors. This suggests that miR-335 could be a positive marker of cellular senescence in hMSC, and therefore a negative marker of the therapeutic capacity of said cells.

EXAMPLE 2

Correlation of the expression level of miR-335 in hMSC with cell senescence in culture. 2.1. Levels of cellular senescence in hMSC that overexpress miR-335.

objective

To determine if the exogenous overexpression of miR-335 affects the levels of cellular senescence in hMSC cultures.

Method

Cellular senescence in culture is usually measured by the expression of β-galactosidase. A higher percentage of positive cells for said expression indicates a higher level of cell senescence. Overexpression of miR-335 was induced in two different bone marrow hMSC isolates by transduction with a lentiviral vector encoding said miRNA. The same hMSC transduced with the same lentiviral vector but without coding miR-335 was used as a negative control. After isolating the transduced cells by flow cytometry, β-galactosidase expression levels were measured by specific staining using a commercial kit (Stemgent). The number of β-galactosidase positive cells was quantified by direct observation under a microscope.

Results

As shown in Figure 2A, the exogenous overexpression of miR-335 induces a significantly higher level of cellular senescence in bone marrow hMSC.

2.2. MiR-335 levels in senescent hMSC.

objective

To determine if the levels of endogenous expression of miR-335 correlate positively with the levels of cellular senescence in hMSC.

Method

Cellular senescence was induced in bone marrow hMSC cultures by gamma irradiation (10 Gy). After ten days in culture, it was verified by staining with β-galactosidase (see Example 2.1) that more than 85% of the cells were senescent. TO from said cells, total RNA was isolated and the relative expression of miR-335 was quantified following the method described above, compared to non-irradiated cells from the same donors.

Results

The results (Figure 2B) show that in the hMSC treated with gamma radiation (senescent) they significantly increase their endogenous levels of miR-335.

2.2. Relationship of miR-335 expression levels with telomeric length in hMSC.

objective

Check whether there is a correlation between the expression levels of miR-335 and the telomeric length in hMSC, since said telomeric length is generally considered as a negative marker of cellular senescence.

Method

Bone marrow hMSC was transduced with a lentiviral vector encoding the catalytic subunit of human telomerase (hTERT). With this, it was achieved that said cells possess telomerase activity (which they lack under physiological conditions). The telomeric length in said transduced cells and in control cells was measured by a specific quantitative PCR assay, as well as the relative expression of miR-335 by RT-PCR.

Results

The results (Figure 3) showed that hMSCs with longer telomeres have lower endogenous levels of miR-335 compared to control cells from the same donor and cultured ex vivo during the same time.

2.3. Effect of p02 on the expression of miR-335.

objective For some time now, it has been known that mammalian cells grown at a concentration of 0 ₂ of 3% show a significantly greater proliferative capacity and chromosomal stability than those grown under standard culture conditions (20% 0 ₂ ) (Samper et al, 2003; Estrada et al, 2011). The following experiment aims to determine if there is a correlation between the expression levels of miR-335 in hMSC and the tension of 0 ₂ at which the culture is performed.

Method

Bone marrow hMSC were cultured under standard conditions (20% 0 ₂ ) or 3% of 0 ₂ , for 10 days. Next, the RNA from both cultures was isolated and the relative level of miR-335 was quantified by the method already described.

Results

As can be clearly seen in Figure 4, miR-335 expression levels decrease in hMSC grown at 3% of 0 ₂ , compared to those grown at 20%) of 0 ₂ . This result also confirms that miR-335 can be considered as a positive marker of cellular senescence.

EXAMPLE 3

Correlation of the expression level of miR-335 in hMSC with its activity

immunoregulatory

objective

Check that overexpression of miR-335 in hMSC affects its immunoregulatory activity.

3.1. In vitro immunoregulatory activity.

Method

A lymphocyte proliferation inhibition assay was performed using the following protocol. Human peripheral blood mononuclear cells (PBMCs) were isolated and cultured in triplicate (10 ⁵ cells / well) with complete culture medium in presence of phytohemagglutinin (1 μg / ml), in the presence or absence of bone marrow hMSC (2x10 ³ to 5x10 ⁴ cells / well), in 96-well concave-bottom plates. After 96 h of culture, cell proliferation was evaluated using a colorimetric BrDU incorporation assay (Roche). HMSCs transduced with a lentiviral vector encoding miR-335 and control hMSCs were tested in parallel (transduced with a similar lentiviral vector that does not encode said miRNA).

Results

The results (Figure 5) show that hMSCs that overexpress miR-335 have a capacity to inhibit the proliferation of human lymphocytes significantly less than control cells. This result suggests that miR-335 can be considered a negative marker of the immunoregulatory capacity of hMSC.

3.2. Immunoregulatory activity in vivo.

Method

Experimental endotoxemia was induced in 10-week BALB / c male mice by intraperitoneal injection of 400 Dg / mouse of LPS. 30 minutes later, the mice received by the same route 10 ⁶ hMSC or physiological serum (negative control). One group of animals received hMSC transduced with a lentiviral vector encoding miR-335 and another control hMSC group (transduced with a similar lentiviral vector that does not encode said miRNA). Experimental groups consisting of 10 animals each were used. Subsequently, the survival of the animals was monitored during the 96 hours following the administration of the LPS.

Results

96 h after administration of the LPS, the mice that received the control cells showed a survival greater than 80%, compared to a mortality of 100% in the mice that did not receive cells. In contrast, the animals that received the hMSC that expressed miR-335 showed significantly lower survival (less than 40%, Figure 6). This result also points to the value of miR-335 as a negative marker of the immunoregulatory capacity of hMSC.

Claims

1. A method for determining the immunoregulatory capacity or potency of a stem cell, which comprises determining in said stem cell the levels of expression and / or activity of miR-335, where elevated levels with respect to a reference value are indicative of that said stem cell has a low capacity or immunoregulatory power, or where decreased levels with respect to a reference value are indicative that said stem cell has a high capacity or immunoregulatory power.

2. A method for selecting a batch of stem cells for therapeutic applications related to the immunoregulatory activity of said stem cells comprising determining in the stem cells of said batch the expression and / or activity levels of miR-335, wherein miR-335 expression and / or activity levels are lower than a reference value, the batch of stem cells is selected for therapeutic applications related to the immunoregulatory activity of said stem cells.

3. A method for selecting a batch of stem cells for therapeutic applications related to the immunoregulatory activity of said stem cells comprising determining the levels of expression and / or activity of miR335 in stem cells of said batch and in stem cells of a batch control previously selected as suitable for therapeutic applications related to immunoregulatory activity, where if the levels of expression and / or activity of miR335 in the stem cells are similar to the levels of expression and / or activity of miR335 in the cells of the control lot , then the batch of stem cells is selected for therapeutic applications related to the immunoregulatory activity of said stem cells.

4. Method according to claim 3 wherein the stem cells of the control batch previously selected as suitable for therapeutic applications related to the immunoregulatory activity are autologous, syngeneic, allogeneic or xenogeneic with respect to the cells of the batch to be selected.

5. Method according to any of claims 1 to 4 for use as a potency test within the regulatoryly accepted release criteria of batches of stem cells for therapeutic applications.

6. Method according to any one of claims 1 to 5, wherein the stem cell is an adult stem cell.

7. Method according to claim 6, wherein the stem cell is a mesenchymal adult stem cell.

8. A method according to claim 7, wherein the adult mesenchymal stem cell is derived from bone marrow or subcutaneous adipose tissue.

9. A kit for determining the immunoregulatory capacity or potency of a stem cell or for selecting a batch of stem cells for therapeutic applications related to the immunoregulatory activity of said cells comprising

(i) suitable reagents to determine the expression level of miR-335 and

(ii) suitable reagents to determine the level of expression of a reference RNA.

10. Kit according to claim 9 wherein the reference RNA is selected from the group consisting of RNU19, RNU24, RNU38B, RNU43, RNU44, RNU48, RNU49, RNU58A, RNU58B, RNU66, RNU6B, RPL21, snoRNA135, snoRNA202, snoRNA202, snoRNA202, snoRNA202 , snoRNA251, U6, U18, U47, U54, U75, U87, Z30, HY3, 5S rRNA and 4.5S RNA.

11. Kit according to claims 9 or 10, further comprising suitable reagents for determining the level of expression and / or activity of at least one additional marker of the immunoregulatory capacity and / or of a senescence marker in said stem cell.

12. Kit according to claim 11, wherein the additional marker of the immunoregulatory capacity or potency is selected from the group consisting of MHC class I, MHC class II, CD40, CD80, CD86, B7-DC (PD-L2), B7H1 (PD-L1), B7H2 (CD275 / ICOSL), B7H3 (CD276), B7H4 (VTCN1), B7H5, B7H6, B7H7.

13. Kit according to any of claims 9 or 10, wherein the senescence marker is selected from the group consisting of beta-galactosidase, Ki67, p21 (CDKN1A), γΗ2ΑΧ, SAHF, p53 and Lamin B.

14. Use of a kit according to any of claims 10 to 13 to determine the immunoregulatory capacity or potency of a stem cell or to select a batch of stem cells for therapeutic applications related to the immunoregulatory activity of said stem cells.

15. An in vitro method for increasing the immunoregulatory capacity or potency of a stem cell, which comprises inhibiting the expression and / or activity of miR-335 in said stem cell.

16. Method according to claim 15, wherein said stem cell is an adult stem cell.

17. Method according to claim 16, wherein said adult stem cell is a mesenchymal stem cell.

18. The method of claim 17, wherein said adult mesenchymal stem cell is derived from bone marrow or subcutaneous adipose tissue.

19. A stem cell characterized in that it has reduced levels of expression and / or activity of miR-335 for use in the treatment of a disease selected from the group consisting of an autoimmune disease, an inflammatory disease, graft versus host disease, diabetes type 1, type 2 diabetes and cardiovascular disease.

20. A stem cell characterized in that it has reduced levels of expression and / or activity of miR-335 for use in inducing transplant tolerance.

21. A stem cell characterized in that it has reduced levels of expression and / or activity of miR-335 for use in tissue repair and regeneration.

22. A stem cell for use according to any of claims 19 to 21 wherein said stem cell is modified by a miR-335 inhibitor.

23. A stem cell for use according to claim 22, wherein the miR-335 inhibitor is a single chain polynucleotide that specifically hybridizes with miR-335 preventing its function.

24. A stem cell for use according to claim 23, wherein the single chain polynucleotide that specifically hybridizes with miR-335 preventing its function contains the sequence shown in SEQ ID NO: 1, or a clearly related sequence.

25. A stem cell for use according to any one of claims 19 to 24, wherein the stem cell is an adult stem cell.

26. A stem cell for use according to claim 25, wherein the adult stem cell is a mesenchymal adult stem cell.

27. A stem cell for use according to claim 26, wherein the adult mesenchymal stem cell is derived from bone marrow or subcutaneous adipose tissue.

28. A stem cell for use according to any one of claims 19 to 27, wherein the stem cell is autologous, allogeneic or xenogeneic with respect to the subject to be treated.

29. An in vitro method for generating regulatory T cells, comprising contacting a population of cells containing T cells with a population of stem cells characterized in that they have reduced levels of expression and / or activity of miR-335 under conditions suitable for the generation of regulatory T cells.

30. In vitro method according to claim 29, wherein the population containing T cells is a population of peripheral blood mononuclear cells (PBMC).

31. In vitro method according to claims 29 or 30, wherein the population of stem cells is a population of adult stem cells.

32. In vitro method according to claim 31, wherein the adult stem cell population is a population of mesenchymal adult stem cells.

33. In vitro method according to any of claims 29 to 32, wherein the population of stem cells is autologous or allogeneic with respect to the population containing T cells.