Field of the Art

This invention generally relates to methods for identifying mesenchymal stem cell populations particularly useful in different types of cell therapy, differentiating those populations useful in regenerative medicine from those useful in immune system activation. Background of the Invention

Cell therapy with mesenchymal stem cells (MSCs) has been described as a promising clinical strategy for the treatment of Crohn's disease (CD) due to their immunosuppressive and tissue repair properties. Adipose tissue is an important source of this cell type (ASCs, adipose-derived MSCs) with various advantages over its bone marrow homologues (BMSCs, bone marrow-derived MSCs).

ASCs can thus be readily obtained and expanded, and more importantly, ASCs seem to be more potent in terms of immunomodulation compared to their bone marrow homologues. The use of autologous ASCs has been shown to be a promising therapeutic option for treating refractory or perianal Crohn's disease (CD). However, it must be stated that although they are proven to be effective, autologous transplants do not always prevent relapse or improve the disease. In this sense, a randomized, multi-center phase 3 study with 212 patients has recently been published (Panes J et al. 2016 Lancet 388:1281 -90), where allogeneic stem cells isolated from the adipose tissue (from a different donor) are used for treating fistulas in Crohn's disease. Although the results are promising (50% of remission after cell therapy with ASCs v 34% of remission with placebo) given that these are patients who do not respond to any treatment and are far from being optimum. These difficulties are due to differences in the innate immune properties of ASCs as a result of the donor's metabolic phenotype, in the case of autologous transplant, the CD itself would determine said phenotype. It must be pointed out that said differences in the innate immune properties of ASCs can be extrapolated to any type of MSCs of any origin, such as bone marrow or umbilical cord blood, for example. To note, ASCs isolated from other pathological states such as obesity or type 2 diabetes mellitus (T2DM) have also altered the their immunosuppressive properties. In this sense, earlier studies carried out by the authors of the present invention demonstrate that, like macrophages, diversity and plasticity are characteristics of mesenchymal stem cell populations, where mesenchymal stem cell subpopulations in different activation states and/or with single or mixed phenotypes coexist in said populations. In fact, an inflammatory subpopulation which secretes large amounts of interleukin-1 (IL-1 β) (similar to M1 macrophages or macrophages with pro-inflammatory phenotype) and another more immunosuppressive subpopulation which secretes large amounts of transforming growth factor beta (TGFbβ1 ) (similar to M2 macrophages or macrophages with anti-inflammatory phenotype) have been observed in ASCs. This also occurs in other mesenchymal stem cell populations of different origins.

Notably, the first subpopulation presents the activation of inflammasome (a multiprotein complex characteristic of the innate immune system promoting the maturation and secretion of inflammatory cytokines such as I L-1 β).

The identification and characterization of these subpopulations is crucial not only for explaining their true physiological nature, but also invaluable for developing new successful cell therapies.

Brief Description of the Invention

The present invention provides a methodology which allows identifying and characterizing the predominance of different subpopulations existing in a mesenchymal stem cell population, which in turn allows applying this methodology for selecting those MSC populations that are the most useful in different types of cell therapies, prior to the clinical application thereof.

In this sense, the present invention demonstrates that mesenchymal stem cells lose immunosuppressive properties and acquire an inflammatory profile in inflammatory diseases (e.g., obesity or diabetes) (Serena C. et al. 2016 Stem Cells 34:2559 - 73). Furthermore, the immunophenotypic profile of these cells is different according to whether the donor is lean or obese or has Crohn's disease (Serena C. et al. 2017 Stem Cell Reports 9(4):1109-1123). Based on these results, and taking into account the existence of different mesenchymal stem cell subpopulations in a specific population, it is considered that in order to actually improve cell therapy efficacy a pre-implant selection of those stem cells with the best properties for carrying out the cell therapy of interest must be performed, i.e., selecting MSC populations having a predominantly immunomodulatory character in the case of a cell therapy applicable in regenerative medicine, or selecting those MSC populations having a predominantly proinflammatory phenotype in the case of those cell therapies where immune system activation is essential, such as anti-cancer therapy or treatment of infections. Therefore, the present invention provides methods for identifying those mesenchymal stem cell populations that will be useful in cell therapy applicable in regenerative medicine, or selecting those MSC populations having a predominantly pro-inflammatory phenotype, and therefore applicable in those cell therapies where immune system activation is essential. The invention also provides enriched MSC populations in a more immunosuppressive subpopulation which secretes large amounts of transforming growth factor beta (TGFbβ1 ) (similar to M2 macrophages or macrophages with anti-inflammatory phenotype).

Brief Description of the Drawings

Figure 1. CD increases mesenteric ASC proliferation and reduces their adipogenic differentiation capacity. (A) AT-cell number ratio, (B) MTT and (C) BrdU cell proliferation assays were performed as detailed in methods to study the cell proliferation of mesenteric ASCs. (D) Representative images of intracellular lipid enrichment in mature adipocyte (AD) from healthy subjects, active and inactive CD individuals (magnification, x200; scale bar=200 μm). (E) Quantification of Oil Red staining of AD from healthy subjects, active and inactive CD patients. (F) Gene expression of adipogenic markers was analyzed by RT-PCR in AD and undifferentiated ASCs from healthy subjects, active and inactive CD patients. n=5-10 per group as explained in Experimental Procedures. ^* P<0.017 vs healthy ASCs, #P<0.017 as indicated in the figure.

Figure 2. CD triggers an inflammasome-mediated inflammatory response in mesenteric ASCs and increases their metabolic activity. (A) Expression of IL6, TNFA, CCL2, ΙΙ_-1 β, IL10 and adiponectin were analyzed by qPCR in ASCs isolated from VAT of healthy subjects, active and inactive CD patients. (B) Secretion of I L-1 β was analyzed by ELISA from conditioned medium (CM) of ASCs from healthy subjects, active and inactive CD patients. (C) Gene expression of different components of the inflammasome, NLRP1 , NLRP3, and CASP1 , were analyzed by qPCR. (D) Gene expression of glucose and lipid metabolism genes was analyzed by qPCR in VAT-ASCs. (E) Lactate and (F) succinate levels in CM of ASCs isolated from healthy subjects, active and inactive CD patients. n=5-10 per group as explained in Experimental Procedures with the exception of metabolic data (n=4 for all groups). ^* P<0.017 vs healthy ASCs, #P<0.017 as indicated in the figure. Figure 3. CD changes the functional properties of mesenteric ASCs. (A) The migratory capacity of healthy-ASCs, active CD-ASCs and immune cells (Jurkat cells) into 24h-CM of CF of active CD patients or VAT of healthy individuals were assessed in Transwell assays (B) The migratory capacity of basal ASCs isolated from VAT of healthy subjects, active and inactive CD patients, was assessed in Transwell assays. Representative toluidine blue- stained cells are also shown (under panel) (magnification, x200; scale bar=200 μm). (C) CM of VAT from healthy subjects, active and inactive CD patients was tested to ascertain if it promotes the migration of immune cells (monocytes, THP-1 cell line; B lymphocytes, MEC-1 cell line and; T lymphocytes, Jurkat cell line) using the Transwell system. (D) Invasion capacity was studied in ASCs by adding Matrigel to the upper Tanswell chamber. Representative toludine blue-stained cells are also shown (under panel) (magnification, x200; scale bar=200 μm). (E) Zymographic analysis of MMP2/9 activities using gelatin as substrate. Representative zymogram and densitometric analysis is shown. (F) Phagocytosis assay was performed using a rhodamine-based red dye conjugated to E. coli bacteria, which turns bright red upon lysosomal acidification. Phagocytic activity of cells is marked in red, and the cell nucleus is marked in DAPI (blue). Representative images of ASCs from healthy subjects, active and inactive CD patients (magnification, x200; scale bar=200 μm). (G) Phagocytosis was quantified using the Varioskan™ LUX multimode microplate reader. Fluorescence intensity was normalized to total protein content. n=5-10 per group as explained Experimental Procedures with exception of phagocytic data (n=4 for all groups). ^* P<0.05 vs healthy ASCs, #P<0.01 as indicated in the figure.

Figure 4. CD alters the functional properties of subcutaneous-ASCs. (A) Expression of IL1 B, IL6, TNFA and CCL2 were analyzed by qPCR in ASCs isolated from SAT of healthy subjects, active and inactive CD patients. (B) Secretion of I L-1 β was analyzed by ELISA from CM of ASCs from healthy subjects, active and inactive CD patients. (C) Migratory capacity of ASCs isolated from healthy subjects, active and inactive CD patients from SAT was assessed using the Transwell system. (D) CM of SAT from healthy subjects, active and inactive CD patients was tested to ascertain if it promotes the migration of immune cells (monocytes, THP-1 cell line; B lymphocytes, MEC-1 cell line and; T lymphocytes, JURKAT cell line) using the Transwell system. (E) Invasion capacity was studied in ASCs by adding Matrigel to the upper Transwell chamber. (F) MMP2 and MMP9 gene expression was analyzed by qPCR in ASCs from healthy subjects, active and inactive CD patients. (G) Phagocytosis assay was performed using a rhodamine-based red dye conjugated to E. coli bacteria, which turns bright red upon lysosomal acidification. Phagocytic activity of cells is marked in red, and the cell nucleus is marked in DAPI (blue). Representative images of ASCs from healthy subjects, active and inactive CD patients (magnification, x200; scale bar=200 μm). (H) Phagocytosis was quantified using the Varioskan™ LUX multimode microplate reader. Fluorescence intensity was normalized to total protein content. (I) Gene expression of different phagocytic markers, LAMP1 , RAB5A, and RAB7A, was analyzed by qPCR. n=6-10 per group as explained in Experimental Procedures with exception of phagocytic data (n=4 for all groups). ^* P<0.017 vs healthy ASCs, #P<0.017 as indicated in the figure.

Figure 5. CD reduces the immunosuppressive properties of ASCs. (A) ASCs were isolated from SAT of healthy subjects, active and inactive CD patients, and the expression of TGFBIwas analyzed by qPCR. (B) Secretion of TGF-βΙ was analyzed by ELISA in CM of ASCs from healthy subjects, active and inactive CD patients. (C) CM of ASCs from SAT of healthy subjects, active and inactive CD patients, was added to THP-1 PMA-activated macrophage and gene expression of M1/M2 phenotype markers was analyzed by qPCR. (D) Cell proliferation of Jurkat T cells and MEC-1 B cells was measured after adding CM of ASCs from SAT of healthy subjects, active and inactive CD patients. (E) Gene expression of Th1/Th2/Treg markers were studied in naive T lymphocytes that were co-cultured with healthy or active CD-ASCs for 48 h. (F) Secretion of G-CSF was analysed by ELISA in culture supernatant of co-cultured naive T lymphocytes with healthy-ASCs or active CD- ASCs. (G) Gene expression of GCSF in ASCs previously co-cultured with T naive. n=6-10 per group. ^* P<0.017 vs healthy ASCs, #P< 0.017 vs Control (Culture Medium) as indicated in the figure.

Figure 6. Inflammasome inhibition of CD-ASCs reverses the immune-activated phenotype. Invasion capacity was studied in (A) SAT or (B) VAT CD-ASCs treated with 40 ng/ml interleukin 1 receptor antagonist (I LI RA), 20 ng/ml of TGF- β1 , 10 μΜ of YVAD-CHO (caspase 1 inhibitor) or the combined treatments at the same doses described in the figure. MMP2/9 gene expression was analyzed by qPCR in ASCs from (C) SAT and (D) VAT isolated from healthy subjects, active and inactive CD patients. (E) SAT and (F) VAT relative gene expression of inflammasome, phagocytic and metabolic markers was determined in untreated CD-ASCs (basal) or those treated with IL1 RA plus YVAD-CHO. n=4 for all the groups. ^* P<0.017 vs untreated ASCs (basal). #P< 0.017 as indicated in the figure.

Figure 7. Representative scatter plots showing the combination of 2 variables used to calculate the score failing to completely separate between healthy and unhealty patients. A- d) combination of inflammatory cytokines e) combination of inflammatory and antiinflammatory cytokines f) combination of anti-inflammatory cytokines g) combination of phagocytic genes h) combination of invasion markers i) combination of inflammasome markers and k) combination of metabolic genes.

Description of the Invention

Definitions

As they are used herein, the following terms and expressions must have the meanings set forth below. Unless defined otherwise, all the technical and scientific terms used in the present document have the same meaning as is commonly understood by a person skilled in the art to which the present invention belongs.

The articles "a" and "an" refer to one or more than one (i.e., at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

When used in relation to a value, the term "about" refers to the value ± 10%.

"Adipose tissue" indicates any adipose tissue. The adipose tissue can be a brown or white adipose tissue derived, for example, from a subcutaneous, omental/visceral, mammary, gonadal adipose tissue site, or another adipose tissue site. The adipose tissue is preferably a subcutaneous white adipose tissue. The adipose tissue can comprise a primary cell culture or an immortalized cell line. The adipose tissue can be from any organism having adipose tissues. In some embodiments, the adipose tissue is from a mammal, and in additional embodiments the adipose tissue is from a human. Liposuction surgery or lipoaspirate is a suitable adipose tissue source. However, it will be understood that neither the adipose tissue source nor the adipose tissue isolation method is critical for the invention. If cells such as those described in the present document are required for autologous transplant in a subject, the adipose tissue will be isolated from that subject.

The term "constitutively" is understood to mean the expression of a gene without any specific induction.

Throughout the present invention, "substantially pure population" will be understood to be a cell population where the MSCs constitute at least 80% of the total cells in the population, preferably at least 85, 90, 95, 96, 97, 98 or 99% of the total cells in the population. Therefore, the composition of the disclosure which the invention comprises can comprise a cell population in which at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% of the cells are MSCs. In other words, in some embodiments at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% of the cells in the composition are MSCs. The composition of the disclosure which the invention comprises can comprise at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% of MSCs, calculated by number, by weight or by volume of the composition.

Throughout the present invention, "mesenchymal stem cells" (MSCs) will be understood to be a multipotent stromal cell originated from the mesodermal germ layer, which can differentiate into different types of cells, including osteocytes (bone cells), chondrocytes (cartilage cells) and adipocytes (fat cells). The markers expressed by mesenchymal stem cells include CD105 (SH2), CD73 (SH3/4), CD44, CD90 (Thy-1 ), CD71 and Stro-1 , as well as adhesion molecules CD106, CD166, and CD29. The negative markers for MSCs (not expressed) include, among others, hematopoietic markers CD45, CD34, CD14, and costimulatory molecules CD80, CD86 and CD40, as well as adhesion molecule CD31. The MSCs can be obtained, without limitation, from bone marrow, adipose tissue (such as the subcutaneous adipose tissue), liver, spleen, testicles, menstrual blood, amniotic fluid, pancreas, periosteum, synovial membrane, skeletal muscle, dermis, pericytes, trabecular bone, human umbilical cord, lung, dental pulp and peripheral blood. The MSCs according to the invention can be obtained from any of the preceding tissues, such as from bone marrow, subcutaneous adipose tissue or umbilical cord. The MSCs can be isolated from bone marrow by means of methods known by the person skilled in the art. Said methods generally consist of isolating mononuclear cells by means of density gradient centrifugation (Ficoll, Percoll) of bone marrow aspirates, and then seeding the isolated cells on tissue culture plates in medium containing fetal bovine serum. These methods are based on the capacity of MSCs to adhere to plastic, such that while non-adhered cells are removed from the culture, adhered MSCs can be expanded in culture plates. MSCs can also be isolated from a subcutaneous adipose tissue following a similar method known by the person skilled in the art. A method for isolating MSCs from bone marrow or subcutaneous adipose tissue has been previously described (De la Fuente et a/., Exp. Cell Res. 2004, Vol. 297: 313:328). In a particular embodiment of the invention, the mesenchymal stem cells are obtained from umbilical cord, preferably from human umbilical cord.

The terms "comprise" and "comprising" are used in the inclusive, open sense, meaning that additional elements may be included. The term "including" is used herein to mean "including, but not limited to". "Including" and "including, but not limited to" are used interchangeably.

"Marker" refers to a biological molecule the presence, concentration, activity or phosphorylation state of which can be detected and used for identifying the phenotype of a cell.

A "patient", "subject" or "host" to be treated using the target method can mean both a human being and a non-human animal. The term "pharmaceutical composition" refers to a composition envisaged for use in therapy. The compositions of the invention are pharmaceutical compositions envisaged for use in regenerative medicine or in cell therapy for immune system activation. The compositions of the invention can include, in addition to the populations described in the present invention, non-cellular components. Examples of such non-cellular components include, but are not limited to, cell culture media, which can comprise one or more proteins, amino acids, nucleic acids, nucleotides, coenzyme, antioxidants and metals.

The expression "pharmaceutically acceptable" is used herein to refer to those compounds, materials, compositions and/or dosage forms which, within the scope of sound medical judgment, are suitable for use in contact with tissues of human beings and animals without excessive toxicity, irritation, allergic response, or any other problem or complication, proportional to a reasonable risk/benefit ratio.

As it is used herein, the expression "pharmaceutically acceptable vehicle" means a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient or dissolving encapsulation material involved in carrying or transporting the target compound from one organ or part of the body to another organ or part of the body. A vehicle must be "acceptable" in the sense of being compatible with other components of the formulation and not causing harm to the patient.

The term "phenotype" refers to observable characteristics of a cell, such as size, morphology, protein expression, etc.

Description

The present invention provides a methodology which allows identifying and characterizing the predominance of different subpopulations existing in a mesenchymal stem cell population, which in turn allows applying this methodology for selecting those MSC populations that are the most useful in different types of cell therapies, prior to the clinical application thereof. In this sense, the present invention demonstrates that mesenchymal stem cells lose immunosuppressive properties and acquire an inflammatory profile in inflammatory chronic diseases (e.g., obesity or diabetes) (Serena C. et al. 2016 Stem Cells 34:2559 - 73). Furthermore, the immunophenotypic profile of these cells is different according to whether the donor is of a normal or average weight or obese or has Crohn's disease.

Based on these results, and taking into account the existence of different mesenchymal stem cell subpopulations in a specific population, it is considered that in order to actually improve cell therapy efficacy a pre-implant selection of those stem cells with the best properties for carrying out the cell therapy of interest must be performed, i.e., selecting MSC populations having a predominantly immunomodulatory character in the case of a cell therapy applicable in regenerative medicine, or selecting those MSC populations having a predominantly proinflammatory phenotype in the case of those cell therapies where immune system activation is essential, such as anti-cancer therapy or treatment of infections. Given that the donor's phenotype determines the biological properties of the MSCs, this selection would be required in the case of both allogeneic and autologous transplants. This is based on data obtained by the inventors showing that CD, like obesity and diabetes, would alter the immunomodulatory properties of these cells. In addition, not only disease-derived adipose MSCs confer an inflammatory profile and lose its immunosuppressive properties but also MSCs derived from allegedly clinically healthy subjects could also have an inflammatory profile. In that sense, and for the first time, the present invention teaches that prior to any treatment with MSCs, preferably derived from adipose tissue, no matter the subject from which the MSCs have been obtained, either a "healthy" subject or not, such MSCs should be analyzed so as to identify its usefulness or not in cell therapy. This is clearly demonstrated later-on in the present invention, wherein by applying the gene score to adipose MSCs population from seven patients classified clinically as "healthy", five of those seven populations were surprisingly identified as having MSCs with an inflammatory profile; such cell populations would be thus clearly unsuitable for regenerative therapy.

Therefore, the present invention provides methods for identifying those mesenchymal stem cell populations that will be useful in cell therapy applicable in regenerative medicine, or selecting those MSC populations having a predominantly pro-inflammatory phenotype, and therefore applicable in those cell therapies where immune system activation is essential. The invention also provides enriched MSCs populations in a more immunosuppressive subpopulation which secretes significant amounts of transforming growth factor beta (TGFbβ1 ) (similar to M2 macrophages or macrophages with anti-inflammatory phenotype). In this sense, a first aspect of the present invention relates to a method for identifying those substantially pure mesenchymal stem cell populations that are isolated from a biological sample and will be useful in cell therapy applicable in regenerative medicine, where said method comprises: 1 . determining the quantitative gene expression or the product of the gene expression of a selection of different genes involved at least in inflammation, phagocytosis, invasion, inflammasome activation, glycolysis, antigen presentation and immunosuppressive properties, of a substantially pure mesenchymal stem cell population;

2. Determining the differential expression with respect to a calibrator of the said quantitative expression or with respect to a decision point of each of the genes determined in step a);

3. Using the determination of step b) to obtain a score value; wherein a score value higher or lower than a decision point value, that upon calibration, is preferably obtained by using a pruned multivariate decision tree algorithm to maximize success rate while retaining interpretation power for the resulting model, is indicative of said population having or not an immunosuppressive phenotype.

One non limiting manner, in which the method of the first aspect of the invention can be implemented is by facilitating an array capable of providing a score that predicts whether a cell population is useful for regenerative therapy or not. In a preferred embodiment, such array will consist in a selection of different genes involved in inflammation, phagocytosis, invasion, inflammasome activation, glycolysis, antigen presentation and immunosuppressive properties. Quantitative gene expression should be preferably evaluated by Real-time polymerase chain reaction (qPCR). An inter-run calibrator consisting of a mix of RNA samples from cells suitable for regenerative medicine should be use to relativize the samples. The decision points should be the fold increase (fi) respect to the calibrator. Upon calibration, decision points should be computed using a pruned multivariate decision tree algorithm to maximize success rate while retaining interpretation power for the resulting model. Each parameter should be computed using a LOOCV procedure to further avoid overfitting.

With such gene expression array one should be able to ascertain whether a stem cell population is or not adequate for regenerative therapy or need an ex vivo treatment. In a preferred embodiment, such array will consist in a selection of different genes involved in inflammation, phagocytosis, invasion, inflammasome activation, glycolysis, antigen presentation and immunosuppressive properties; wherein:

Specifically, inflammation is determined by the gene expression of pro-inflammatory cytokines, preferably with interleukin 1 beta (IL1 B) (decision point: 3.46-fi; <3.46-fi = suitable and ≥3.46-fi = not suitable); and/or preferably with tumor necrosis factor alpha (TNFA) (decision point: 1 .62-fi; <1 .62-fi = suitable and≥1.62-fi = not suitable); and/or preferably with C-C Motif Chemokine Ligand 2 (CCL2) (decision point: 2.55-fi; <2.55-/Ϊ = suitable and ≥2.55-fi = not suitable); and/or preferably with interleukin 6 (IL6) (decision point: 1 .16-fi; < 1.16-fi = suitable and≥1 .16-fi = not suitable);

Specifically, phagocytosis is determined by the gene expression of phagocytic markers, preferably with member RAS oncogene family (RAB5A) (decision point: 1 .3- fi; < 1.3-/7 = suitable and≥1.3-fi = not suitable); and/or preferably with member RAS oncogene family (RAB7A) (decision point: 1 .12-/7; <1 .12-fi = suitable and≥1 .12-fi = not suitable) and/or preferably with lysosomal-associated membrane protein 1

(LAMP1 ) (decision point: 1 .02-fi; <1.02-fi = suitable and≥1 .02-fi = not suitable);

Specifically, invasion is determined by the gene expression of metalloproteases (MMP) as an example and preferably with MMP2 (decision point: 1 .51 -/7; <1.51 -fi = suitable and ≥1.51 -fi = not suitable); and/or preferably with MMP9 (decision point: 2.17-fi; <2.17-fi = suitable and≥2.17-fi = not suitable);

Specifically, inflammasome activation produces a secretion of I L1 β, and it is determined by the gene expression of critical inflammasome components as an example and preferably with NACHT, LRR and/or PYD domains-containing protein 1 (NLRP1 ) (decision point: 0.91 -fi; <0.91 -fi = suitable and ≥0.91 -fi = not suitable); and/or preferably with NACHT, LRR and PYD domains-containing protein 3 (NLRP3) (decision point: 1 .04-fi; <1.04-fi = suitable and ≥1 .04-// ^' = not suitable) and/or preferably with caspase 1 (CASP1 ) (decision point: 0.95-//; <0.95-// = suitable and

≥0.95-// ^' = not suitable);

Specifically, glycolytic genes produce energy to the cell. As an examples of glycolytic markers and preferably with hexokinase 2 (HK2) (decision point: 1.92-// ^' ; <1.92-// ^' = suitable and ≥1 .92-// ^' = not suitable); and/or preferably with phosphofructokinase M (PFKM) (decision point: 1.17-fi; <1 .17-fi = suitable and >1.17-fi = not suitable); and/or preferably with pyruvate dehydrogenase kinase, isoenyme 4 (PDK4) (decision point: 0.75-fi; <0.75-fi = not suitable and >0.75-fi = suitable); and/or preferably with lactate dehydrogenase b (LDHB) (decision point: 1 .37-// ^' ; < 1.37-fi = suitable and >1.37-fi = not suitable); and/or preferably with succinate dehydrogenase b (SDHB) (decision point: 1 .57-// ^' ; < 1.57-fi = suitable and >1 .57-fi = not suitable) and/or preferably with alpha-ketoglutarate dehydrogenase (OGDH) (decision point: 1.49-// ^' ; < 1.49-fi = suitable and >1.49-fi = not suitable);

Specifically, antigen presentation markers as an example and preferably with CD74 molecule (decision point: 1.87-// ^' ; < 1.87-fi = suitable and >1 .87-fi = not suitable); and/or preferably with class II, major histocompatibility complex, transactivator

(CIITA) (decision point: 1 .95-// ^' ; <1 .95-fi = suitable and >1.95-fi = not suitable); and/or preferably with major histocompatibility complex, class II, DM beta (HLADM) (decision point: 1.78-// ^' ; < 1.78-fi = suitable and >1 .78-fi = not suitable); and/or preferably with major histocompatibility complex, class II, DP beta (HLADPB) (decision point: 2.05-// ^' ; <2.05-// ^' = suitable and≥2.05-fi = not suitable);

Specifically, anti-inflammatory molecules as an example and preferably with interleukin 10 (IL10) (decision point: 0.67-fi; <0.67-fi = not suitable and ≥0.67-fi = suitable); and/or preferably with transforming growth factor beta 1 (TGFB1 ) (decision point: 0.72-fi; <0.72-fi = not suitable and≥0.72-fi = suitable).

It is noted that the term "decision point" in this context refers to a threshold value which indicates whether or not the cells are suitable for regenerative medicine.

Preferably, the variables and the sets of variables to implement the score are as set down below: Pro-inflammatory cytokines:

Anti-inflammatory cytokines:

Phagocytic markers:

Invasion markers:

Inflammasome markers:

Glycolytic genes: Antigen presenting markers:

Formula:

Weight definition of each subscore:

The division between suitable .and non-suitable (suitable is 0.and non-suitable is 1):

The score ranges between 0-7, wherein a higher score value of 3.5 is not considered suitable for use in medicine regenerative purposes.

Surprisingly, such score provides for a predictive power of success of 100% for classifying a MSC, preferably an adipose MSC, population as suitable or not for use in medicine regenerative purposes. In this sense, if we first calculate such score with 6 out of the 7 parameters (pro-inflammatory cytokines, anti-inflammatory cytokines, phagocytic markers, invasion markers, inflammasome markers and glycolytic genes), a predictive power of success of 92.85% for classifying a MSC population as suitable or not for use in medicine regenerative purposes is provided. If we now insert the seven parameters in the score, the success rate goes up to 100%.

It is further noted that as illustrated in figure 7, combinations of 2 parameters in the above score do not result in a correct classification of the cell populations as suitable or not for use in medicine regenerative purposes. That is to say, for said classification to work properly a combination of genes or the product of the expression of these genes must be selected, wherein said combination of genes provides for at least three, four, five, six or preferably seven of the following: one gene or the product of its expression involved in 1 ) inflammation, at least one gene or the product of its expression involved in 2) phagocytosis, at least one gene or the product of its expression involved in 3) invasion, at least one gene or the product of its expression involved in 4) inflammasome activation, at least one gene or the product of its expression involved in 5) glycolysis, at least one gene or the product of its expression involved in 6) antigen presentation, and/or at least one gene or the product of its expression involved in 7) immunosuppressive properties,

In addition, and as already indicated above, not only disease-derived MSCs confer an inflammatory profile and lose its immunosuppressive properties but also MSCs derived from allegedly clinically healthy subjects could also have an inflammatory profile. In that sense, and for the first time, the present invention teaches that prior to any treatment with MSCs, preferably derived from adipose tissue, no matter the subject from which the MSCs have been obtained, either a "healthy" subject or not, such MSCs should be analysed so as to identify its usefulness or not in cell therapy. This is clearly demonstrated below, wherein by applying the gene score algorithm shown below based on the above score, to adipose MSCs population from seven patients classified clinically as "healthy", five of those seven populations were surprisingly identified as having MSCs with an inflammatory profile; such cell populations would be thus clearly unsuitable for regenerative therapy.

It is again noted that from this algorithm five of those seven populations were surprisingly identified as having MSCs with an inflammatory profile. Therefore, prior to any treatment with MSCs derived from adipose tissue, no matter the subject from which the adipose MSCs have been obtained, either a "healthy" subject or not, such MSCs should be analysed so as to identify its usefulness or not in cell therapy.

It must be pointed out that the method of the first aspect of the present invention can also be carried out through a functional characterization of the different cell populations. In this sense, a second aspect of the invention relates to a method for identifying those substantially pure mesenchymal stem cell populations that are isolated from a biological sample and will be useful in cell therapy applicable in regenerative medicine, where said method comprises: a. Determining the migratory capacity of the cell population as well as its invasive capacity and optionally at least one of the following: the expression or secretion of pro-inflammatory cytokines, the expression or secretion of anti-inflammatory cytokines and the inhibition of lymphocyte inhibition;

b. Determining the increase or reduction of each of the functional capacities of step a) with respect to a decision point; and

c. Using the determination of step b) to obtain a score value; wherein a score value higher or lower than a decision point value, that upon calibration, is preferably obtained by using a pruned multivariate decision tree algorithm to maximize success rate while retaining interpretation power for the resulting model, is indicative of said population having or not an immunosuppressive phenotype which secretes large amounts of transforming growth factor beta (TGFbβ1 ) (similar a the M2 macrophages or macrophages with anti-inflammatory phenotype).

In order to implement the second aspect of the present invention and determining whether a cell population of mesenchymal stem cells may be optimal for regenerative therapy, we established a score. Using a mathematical program, we have determined a series of decision points for each functional assay used. Specifically, the decision points have been computed using a pruned multivariate decision tree algorithm to maximize success rate while retaining interpretation power for the resulting model. Each parameter has been computed using a LOOCV procedure to further avoid overfitting. Statistical analysis has been carried out using R software version 3.3.3.

More specifically, the functional studies/assays needed to carry out the method of the second aspect of the invention are based on the following makers (decision points have been computed using a pruned multivariate decision tree algorithm to maximize success rate while retaining interpretation power for the resulting model. Each parameter has been computed using a LOOCV procedure to further avoid overfitting): - Secretion of pro-inflammatory cytokines, preferably I L1 β (for example using an ELISA

Kit) (decision point 140 fg/ml; <140fg/ml = suitable and≥140 ng/ml = not suitable). Predictive power of 93% success (n=28).

Secretion of anti-inflammatory cytokines, preferably ΤGFβ1 (for example using an ELISA Kit) (decision point 580 pg/ml; <580 pg/ml = not suitable and ≥580 pg/ml = suitable). Predictive power of 78% success (n=28).

Migration experiments, preferably using a Transwell system (δ-μm pore polycarbonate membrane) as described previously (Serena C. 2016 Stem Cells 34:2559-73) (decision point <10 cells = not suitable; 10<cells>100 = suitable;≥100 cells = not suitable). Predictive power of 100% success (n=28).

- Invasion experiments, preferably using a Transwell system (δ-μm pore polycarbonate membrane) coated with Matrigel as described previously (Serena C. 2016 Stem Cells 34:2559-73) (decision point <1 cell = suitable; ≥ 1 cell = not suitable). Predictive power of 100% success (n=28).

Inhibition of lymphocyte proliferation, preferably T and B cell proliferation, preferably using the T cell line (Jurkat) or B cell line (MEC-1 ) are assessed using the conditioning media of adipose-derived stem cells and studding the T and B cell proliferation (for example using BrdU cell proliferation assay kit from Millipore, Billerica, USA) as described previously (Serena C. 2016 Stem Cells 34:2559-73) (decision point <1.17 AU (arbitrary units)= suitable;≥ 1 .17 AU = not suitable for T cell proliferation; and decision point <0.85 AU= suitable;≥ 0.85 AU = not suitable for B cell proliferation). Predictive power of 82% and 94% of success (n=28) in T cell and B cell proliferation, respectively.

Score determination

First, we define the variables:

Where Θ(χ , - λ , ) is the function Theta of Heaviside

The score ranges between 0 - 4.

We considered a higher score value of 2.5 not suitable to use for medicine regenerative purposes.

Division between suitable and not suitable is:

Suitable = Θ (score - 2.5)

A third aspect of the present invention relates to a method for identifying those substantially pure mesenchymal stem cell populations that are isolated from a biological sample and will be useful in those cell therapies where immune system activation is essential, where said method comprises: a. determining the quantitative gene expression or the product of the gene expression of a selection of different genes involved at least in inflammation, phagocytosis, invasion, inflammasome activation, glycolysis, antigen presentation and immunosuppressive properties, of a substantially pure mesenchymal stem cell population;

b. Determining the differential expression with respect to a calibrator of the said quantitative expression or with respect to a decision point of each of the genes determined in step a);

c. Using the determination of step b) to obtain a score value; wherein a score value higher or lower than a decision point value, that upon calibration, is preferably obtained by using a pruned multivariate decision tree algorithm to maximize success rate while retaining interpretation power for the resulting model, is indicative of said population having or not a pro-inflammatory phenotype which secretes large amounts of interleukin-1 (IL-1 β) (similar to M1 macrophages or macrophages with pro- inflammatory phenotype) As set forth above, the method of the third aspect of the invention can be implemented by the array and score described in the first aspect of the invention.

It must be pointed out that, like the method of the first aspect of the invention, the method of the third aspect of the present invention can be performed through a functional characterization of the different cell types. In this sense, a fourth aspect of the present invention relates to a method for identifying those substantially pure mesenchymal stem cell populations that are isolated from a biological sample and will be useful in those cell therapies where immune system activation is essential, where said method comprises: a. Determining the migratory capacity of the cell population as well as its invasive capacity and optionally at least one of the following: the expression or secretion of pro-inflammatory cytokines, the expression or secretion of anti-inflammatory cytokines and the inhibition of lymphocyte inhibition ;

b. Determining the increase or reduction of each of the functional capacities of step a) with respect to a decision point; and

c. Using the determination of step b) to obtain a score value; wherein a score value higher or lower than a decision point value, that upon calibration, is preferably obtained by using a pruned multivariate decision tree algorithm to maximize success rate while retaining interpretation power for the resulting model, is indicative of said population having or not an immunosuppressive phenotype which secretes large amounts of transforming growth factor beta (TGFbβ1 ) (similar a the M2 macrophages or macrophages with anti-inflammatory phenotype).

As set forth above, the method of the fourth aspect of the invention can be implemented by the score described in the second aspect of the invention.

The invention further provides in vitro cell culture compositions which can be obtained according to the methods of the invention, and uses of cell populations which can be obtained according to the invention in the production of drugs for use in transplants in a mammal or in the treatment of disorders requiring immune system activation, such as in the treatment of cancer or infectious diseases. Therefore, the substantially pure MSC population expresses the different genes involved in inflammation, phagocytosis, invasion, inflammasome activation, glycolysis, antigen presentation and immunosuppressive properties identified above, below or above significant levels to be considered useful in the production of drugs for use in transplants in a mammal, i.e., in regenerative medicine or to be considered useful in the treatment of disorders requiring immune system activation, such as in the treatment of cancer or infectious diseases.

Therefore, in some cases the stem cell population of the invention is considered useful in the treatment of disorders requiring immune system activation, such as in the treatment of cancer or infectious diseases, if at least about 70% of the cells of the isolated adult stem cell population show a detectable expression of the different genes involved in inflammation, phagocytosis, invasion, inflammasome activation, glycolysis, antigen presentation and immunosuppressive properties as identified in the third aspect of the invention. In other cases, at least about 80%, at least about 90% or at least about 95% or at least about 97% or at least about 98% or at least about 99% or 100% of the cells of the stem cell population must show such detectable expression. The ability to show a detectable expression can be demonstrated by means of using an RT-PCR experiment or FACS. This composition of the disclosure which the invention comprises can therefore comprise at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% (by number of cells, or by weight or volume of the composition) of MSCs expressing such detectable expression of the different genes involved in inflammation, phagocytosis, invasion, inflammasome activation, glycolysis, antigen presentation and immunosuppressive properties as identified in the third aspect of the invention.

Additionally, in some cases the stem cell population of the invention is considered useful in the production of drugs for use in transplants in a mammal, particularly in regenerative medicine, if at least about 70% of the different genes involved in inflammation, phagocytosis, invasion, inflammasome activation, glycolysis, antigen presentation and immunosuppressive properties as identified in the first aspect of the invention. In other cases, at least about 80%, at least about 90% or at least about 95% or at least about 97% or at least about 98% or at least about 99% or 100% of the cells of the stem cell population must show such detectable expression. The ability to show a detectable expression can be demonstrated by means of using an RT-PCR experiment or FACS. This composition of the disclosure which the invention comprises can therefore comprise at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% (by number of cells, or by weight or volume of the composition) of MSCs expressing such detectable expression of the different genes involved in inflammation, phagocytosis, invasion, inflammasome activation, glycolysis, antigen presentation and immunosuppressive properties as identified in the first aspect of the invention.

The term "expressed" is used to describe the presence of a marker inside a cell. To be considered as having been "expressed", a marker must be present at a detectable level. "Detectable level" indicates that the marker can be detected using one of the conventional laboratory methodologies, such as PCR, blotting or FACS analysis. The phenotype characterization of surface markers of an MSC population can be performed using any method known in the art.

Alternatively or additionally, a gene is considered to be expressed by a cell of the population of the invention if the expression can be reasonably detected after 30 PCR cycles, which corresponds with a cell expression level of at least about 100 copies per cell, preferably above a certain detection level or threshold. The terms "express" and "expression" have corresponding meanings. At an expression level below this threshold, a marker is considered to not be expressed. Additionally, the MSC population identified based on the implementation of the methods of the present invention can also be characterized in that the cells do not express a particular selection of markers at a detectable level. As it is defined herein, these markers are said to be negative markers. Additionally, the present invention proposes selecting the MSCs with the best immunomodulatory properties based on a specific panel of immunophenotypic markers identifying the MSC subpopulation with immunosuppressive properties and separating them from those MSCs with inflammatory properties. Furthermore, a fifth aspect of the present invention provides an in vitro method for increasing an MSC subpopulation with immunosuppressive properties in order to diminish the inflammatory properties and improve the immunosuppressive properties of a substantially pure MSC population. In this sense, in order to use the best candidates in terms of immunomodulation before performing stem cell transplant, the present invention provides, in a sixth aspect of the invention, a method comprising the ex vivo treatment of a substantially pure stem cell population with inflammasome component inhibitors, such as caspase 1 inhibitor (YVAD- CHO, Sigma Aldrich) or IL-1 β receptor antagonist (IL1 RA). Therefore, the induction of MSC functionality recovery would be based on the combination of inflammasome pathway inhibitors plus the use of a TGFbetal pathway activator, either with TGFbetal itself or with a receptor agonist thereof.

Potential inflammasome pathway inhibitors useful in the present invention are known to the person skilled in the art (http://www.newsletter-adipogen.com/adi-archive/archive/view /listid- 43/mailid-34-nlrp3-inflammasome-regulation-small-molecules.h tml)

Potential agonists of the receptor of TGFbetal receptor included in the present invention or compounds with TGF-beta mimetic activity can be selected from those indicated in Mol Cancer Ther. 2002 Aug;1 (10):759-68. By way of example, the starting cell population would be treated with the combination of 40 ng/ml of I LI RA combined with 10 μΜ of YVAD-CHO. As shown in Figure 1 , a significant reduction of the inflammatory ASC subpopulation and an increase of the subpopulation with immunomodulatory properties (Figure 1 ) can be seen with this combined treatment. In these particular cases of an increase of a substantially pure MSC population in an MSC subpopulation with immunosuppressive properties, in order to diminish inflammatory properties and improve immunosuppressive properties, the concentration of MSCs in the composition can be at least about 1 x 10 ⁴ cells/ml, at least about 1 x 10 ⁵ cells/ml, at least about 1 x 10 ⁶ cells/ml, at least about 10 x 10 ⁶ cells/ml, or at least about 40 x 10 ⁶ cells/ml. In some cases, at least about 40% (for example, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95% at least about 96%, at least about 97%, at least about 98%, or at least about 99%) of the MSCs in a composition of the invention are prestimulated in order to enhance one or more of their proliferation capacity, migration capacity, survival capacity, therapeutic effect and immunoregulatory properties. In some cases, prestimulation can be achieved by contacting the MSCs with inflammasome pathway inhibitors and a TGFbetal pathway activator, either with TGFbetal itself or with a receptor agonist thereof.

A composition of the invention can contain the MSC progeny identified or obtained by means of any of the aspects of the invention. Such progeny can include later MSC generations, in addition to compromised lineage cells generated by inducing MSC prestimulation described in detail above. Such differentiation can be induced in vitro. It will be understood that progeny cells can be obtained after any number of passes of the parent population. However, in specific embodiments the progeny cells can be obtained after about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10 passes of the parent population.

A composition of the invention identified or obtained by means of any of the aspects of the invention can be provided under sterile conditions and can be free of viruses, bacteria and other pathogens. A composition of the invention can be provided as a pyrogen-free preparation. In one embodiment, a composition of the invention identified or obtained by means of any of the aspects of the invention can be prepared for systemic administration (for example, through the rectal, nasal, oral or vaginal route, by means of an implanted deposit or by means of inhalation).

In another embodiment, a composition of the invention identified or obtained by means of any of the aspects of the invention can be prepared for local administration. A composition of the invention can be administered parenterally. A composition can be administered through the subcutaneous, intracutaneous, intravenous, intramuscular, intraarticular, intrasynovial, intrasternal, intrathecal, intralesional, intralymphatic and intracranial route.

In one case, the cells of the composition of the invention identified or obtained by means of any of the aspects of the invention can be autologous with respect to the subject to be treated. In another embodiment, the cells of the composition of the invention identified or obtained by means of any of the aspects of the invention which are used in the invention can be allogeneic cells, or in another case the cells of the composition of the invention identified or obtained by means of any of the aspects of the invention can be xenogeneic cells with respect to the subject to be treated. Earlier studies have shown that bone marrow-derived allogeneic stromal stem cells and adipose tissue-derived stromal cells do not cause an immune response to lymphocytes when they are contacted with allogeneic lymphocytes in vitro. Accordingly, adipose tissue-derived allogeneic stromal stem cells originating from a donor can be used, in theory, for treating any patient, regardless of the incompatibility of the MHC. In embodiments in which allogeneic stem cells are used, supportive treatment may be required. For example, immunosuppressants can be administered before, during and/or after treatment to prevent graft-versus-host disease (GVHD), according to known methods. The cells can also be modified before administration to suppress an immune reaction in the subject to the cells or vice versa, according to methods known in the art. In one case, the composition of the invention identified or obtained by means of any of the aspects of the invention can be administered by means of injecting or implanting the composition in one or more target sites in the subject to be treated. In another case, the composition of the invention identified or obtained by means of any of the aspects of the invention can be inserted into an administration device which makes introducing the composition into the subject by means of injection or implantation easier. In one embodiment, the administration device can comprise a catheter. In another case, the administration device can comprise a syringe.

The composition of the invention identified or obtained by means of any of the aspects of the invention will generally comprise a pharmaceutically acceptable vehicle and/or diluent. Examples of such vehicles and diluents are widely known in the art and can include: sugars, such as lactose, glucose and sucrose; starches, such as cornstarch and potato starch; cellulose and its derivatives, such as sodium carboxymethylcellulose, ethylcellulose and cellulose acetate; tragacanth powder; malt; gelatin; talc; excipients, such as cocoa butter and suppository wax; oils, such as peanut oil, cotton seed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline solution; Ringer solution; ethyl alcohol; buffered pH solutions; polyesters, polycarbonates and/or polyanhidrides; and other non-toxic compatible substances used in pharmaceutical formulations.

A composition of the invention identified or obtained by means of any of the aspects of the invention can be sterile and fluid to the point that it is readily injectable. Furthermore, the composition can be stable under manufacturing and storage conditions and be preserved from the contaminating action of microorganisms such as bacteria and fungi by means of using, for example, parabens, chlorobutanol, phenol, ascorbic acid and thimerosal.

In one embodiment, the composition of the invention identified or obtained by means of any of the aspects of the invention of the invention can contain one or more (or two or more, or three or more, for example, 1 , 2, 3, 4 or 5) additional therapeutic agents such as a therapeutic agent selected from the following: an analgesic, such as a nonsteroidal antiinflammatory drug, an opioid agonist or a salicylate; an anti-infective agent, such as an antihelminthic, an antianaerobic drug, an antibiotic, an aminoglucoside antibiotic, an antifungal antibiotic, a cephalosporin antibiotic, a macrolide antibiotic, a β-lactam antibiotic, a penicillin antibiotic, a quinolone antibiotic, a sulfonamide antibiotic, a tetracycline antibiotic, an antimycobacterial, an antituberculous antimycobacterial, an antiprotozoal, an antimalarial antiprotozoal, an antiviral agent, an antiretroviral agent, a scabicide, an anti-inflammatory agent, a corticosteroid anti-inflammatory agent, an antipruritic/local anesthetic, a topical anti- infective, an anti-fungal topical anti-infective, an antiviral topical anti-infective; an electrolytic and renal agent, such as an acidifying agent, an alkalizing agent, a diuretic, a diuretic which inhibits carbonic anhydrase, a loop diuretic, an osmotic diuretic, a potassium-sparing diuretic, a thiazide diuretic, electrolyte replacement and an uricosuric agent; an enzyme, such as a pancreatic enzyme and a thrombolytic enzyme; a gastrointestinal agent, such as an antidiarrheal, an antiemetic, a gastrointestinal anti-inflammatory agent, a salicylate gastrointestinal anti-inflammatory agent, an antacid anti-ulcer agent, an anti-ulcer agent which inhibits the gastric acid pump, a gastric mucosa anti-ulcer agent, an H2 blocker antiulcer agent, a cholelitholytic agent, a digestive drug, an emetic, a laxative and stool softener, and a prokinetic agent; a general anesthetic, such as an inhalational anesthetic, a halogenated inhalational anesthetic, an intravenous anesthetic, an intravenous barbiturate anesthetic, an intravenous benzodiazepine anesthetic and an intravenous opioid agonist anesthetic; a hormone or hormone modifier, such as an abortion drug, a suprarenal agent, a corticosteroid suprarenal agent, an androgen, an antiandrogen, an immunobiological agent, such as an immunoglobulin, an immunosuppressant, a toxoid and a vaccine; a local anesthetic, such as a local amide anesthetic and a local ester anesthetic; a musculoskeletal agent, such as an anti-gout anti-inflammatory agent, a corticosteroid anti-inflammatory agent, a gold compound anti-inflammatory agent, a immunosuppressive anti-inflammatory agent, a nonsteroidal anti-inflammatory drug (NSAID), a salicylate anti-inflammatory agent, a mineral; and a vitamin, such as vitamin A, vitamin B, vitamin C, vitamin D, vitamin E and vitamin K.

In another case, the additional therapeutic agent can be a growth factor or another molecule affecting cell proliferation or activation. In another case, said growth factor can induce final differentiation. In another case, the growth factor can be a variant or fragment of a naturally occurring growth factor. Methods for producing such variants are well known in the art and may include, for example, performing conservative amino acid changes, or by means of mutagenesis and assaying the resulting variant for the required functionality.

In one embodiment, the composition of the invention identified or obtained by means of any of the aspects of the invention can be administered to a subject along with one or more (or two or more, or three or more, for example, 1 , 2, 3, 4 or 5) additional therapeutic agents. In some cases, the composition of the invention identified or obtained by means of any of the aspects of the invention and the one or more additional therapeutic agents can be administered to the subject simultaneously. In other cases, the composition of the invention identified or obtained by means of any of the aspects of the invention and the one or more additional therapeutic agents can be administered to the subject sequentially. The one or more additional therapeutic agents can be administered before or after administering the cellular composition.

The dosage of the composition of the invention identified or obtained by means of any of the aspects of the invention and any additional therapeutic agent will vary depending on the patient's symptoms, age and body weight, the nature and severity of the disorder to be treated or prevented, the administration route, and the form of the additional therapeutic agent. The compositions of the invention can be administered in a single dose or in several doses.

The precise time for administration and the amount of any particular agent which will give rise to a more effective treatment in a given patient will depend on the activity, pharmacokinetics and bioavailability of the agent, the physiological condition of the patient (including age, sex, disease type and stage, general physical condition, sensitivity to a given dosage and medication type), the administration route, etc. The information provided herein can be used for optimizing treatment, for example, for determining the optimum time and/or amount for administration, and this will only require routine experimentation, such as monitoring the subject and adjusting the dosage and/or the precise time. While the subject is under treatment, the health of the subject can be monitored by measuring one or more of the relevant indices at predetermined times for a 24-hour period. Treatment guidelines, including dosages, times for administration and formulations, can be optimized according to the results of such monitoring.

Treatment can begin with smaller dosages which are less than the optimum dose. The dosage can be increased in small increments henceforth until the optimum therapeutic effect is obtained.

The combined use of several therapeutic agents can reduce the dosage required for any individual component because the emergence and duration of the effect of the different components may be complementary. In such combined therapy, the different active agents can be administered together or separately, and simultaneously or at different times throughout the day.

In one case, the composition of the invention identified or obtained by means of any of the aspects of the invention can be stably transfected or transiently transduced with a nucleic acid of interest using a plasmid, a viral vector strategy or an alternative. The nucleic acids of interest include, but are not limited to, those encoding gene products which enhance the production of extracellular matrix components found in the type of tissue to be repaired, for example, the intestinal wall or vaginal wall.

The transduction of viral vectors carrying regulator genes into stromal stem cells can be performed with viral vectors including, but not limited to, purified (for example, by means of banding with cesium chloride) adeno-associated viruses, adenoviruses or retroviruses at a multiplicity of infection (viral units:cell) between about 10:1 and 2000:1 . The cells can be exposed to the virus in a medium that may or may not contains serum in the absence or presence of a cationic detergent such as polyethylenimine or Lipofectamine™ for a period of about 1 hour to about 24 hours (Byk T. et al. (1998) Human Gene Therapy 9:2493-2502; Sommer B. et al. (1999) Calcif. Tissue Int. 64:45-49).

Other suitable methods for transferring vectors or plasmids into stem cells include lipid/DNA complexes, such as those described in patent documents US5578475, US5627175, US5705308, US5744335, US5976567, US6020202 and US6051429. Suitable reagents include Lipofectamine, a 3:1 (weight/weight) liposome formulation of the polycationic lipid, 2,3-dioleyloxy-N-[2-(sperminecarboxamido)ethyl]-N,N-dimethyl -1 -propanaminium

trifluoroacetate (DOSPA) (Chemical Abstracts registry name: N-[2-(2,5-bis[(3- aminopropyl)amino]-1 oxpentyl}amino)ethyl]-N,N-dimethyl-2,3-bis(9-octadecenyloxy) -1 - propanaminium trifluoroacetate), and the neutral lipid, dioleoyl phosphatidylethanolamine (DOPE), in membrane-filtered water. An example is the Lipofectamine 2000TM formulation (available from Gibco/Life Technologies as product no. 1 1668019). Other reagents include: FuGENETM 6 transfection reagent (a non-liposomal mixture of lipids and other compounds in 80% ethanol, obtainable from Roche Diagnostics Corp. as product no. 1814443), LipoTAXITM transfection reagent (a lipid formulation of Invitrogen Corp., with product no. 2041 10). The stem cells can be transfected by electroporation, for example, as described in M.L. Roach and J.D. McNeish (2002) Methods in Mol. Biol. 185:1. Viral vector systems suitable for producing stem cells with stable genetic alterations can be based on adenoviruses and retroviruses and can be prepared using commercially available viral components.

The transfection of plasmid vectors carrying regulator genes into the CMM can be achieved in monolayer cultures by means of using calcium phosphate DNA precipitation methods or cationic detergent (Lipofectamine™, DOTAP) or in three-dimensional cultures by means of incorporating the plasmid DNA vectors directly in the biocompatible polymer (Bonadio J. et al. (1999) Nat. Med. 5:753-759). To track and detect the functional proteins encoded by these genes, the viral or plasmid DNA vectors can contain a readily detectable marker gene, such as the green fluorescent protein or beta-galactosidase enzyme, both of which can be tracked by histochemical means. Therefore, as has been described in detail above it is clear that the present invention further provides in vitro cell culture compositions which can be obtained according to the methods of the fifth and sixth aspects of the invention, and the uses of cell populations which can be obtained according to the invention in the production of drugs for use in transplants in a mammal and in the treatment of disorders requiring immune system activation, such as in the treatment of cancer or infectious diseases.

The invention will now be further illustrated by means of the following examples.

Examples

Example 1. Mesenteric ASCs of patients with CD show higher proliferation rates but lower adipogenic capacities than those of healthy donors

ASCs were isolated from VAT of healthy subjects (n=6) and patients with active (n=10) or inactive (n=5) CD. When ASCs were isolated from the same amount of mesenteric AT from patients with CD (CD-ASCs) and from healthy individuals (healthy-ASCs), a greater number of ASCs was obtained from CD patients than from healthy individuals. Correspondingly, the AT-cell number ratio was significantly higher in CD-ASCs, both from active and inactive patients, than in healthy-ASCs (Figure 1A), which suggests an increase in the number of adipocyte precursors in mesenteric AT of CD patients. Moreover, MTT and BrdU incorporation assays revealed a higher proliferation rate in mesenteric CD-ASCs than in healthy-ASCs (Figures 1 B and 1 C). To study the adipogenic potential of ASCs, we cultured them in well-defined adipogenic differentiation medium and evaluated lipid content and gene expression after 14 days. Neutral lipid content, measured by Oil Red O staining, was significantly lower in differentiated CD-ASCs (independent of clinical stage) than in healthy- ASCs, (Figures 1 D and 1 E), concomitant with a decrease in the gene expression of typical adipogenic markers such as leptin, peroxisome proliferator activated receptor gamma (PPARG), fatty acid binding protein 4 (FABP4) and lipoprotein lipase {LPL) (Figure 1 F). Collectively, these data reveal that similar to obesity (Pachon-Pena et al., 2016), CD modifies the plasticity of mesenteric ASCs. Example 2. Mesenteric ASCs from patients with CD present an inflammasome- mediated inflammatory response

It is known that CF exhibits a high level of inflammatory cytokine secretion(Kredel and Siegmund, 2014). Consistent with this, we observed that gene expression of typical inflammatory markers (IL6, TNFA, CCL2 and IL1B) was higher in mesenteric CD-ASCs from active patients than from healthy donors (Figure 2A). Of note, an inflammatory phenotype was also detected in CD-ASCs from inactive patients (Figure 2A). As previously described for mesenteric AT of active CD patients (Zulian et al., 2012), the expression of antiinflammatory markers such as IL10 and adiponectin was also increased in CD-ASCs (Figure 2A). Interestingly, expression of these markers was significantly lower in inactive CD-ASCs than in active CD-ASCs, revealing a decrease in anti-inflammatory mediators but not proinflammatory mediators in the former.

Because of the important role of I L-1 β in host response(Grant and Dixit, 2013), we next examined inflammasome-mediated immune responses in ASCs. In accordance with the gene expression data, I L-1 β protein expression in conditioned medium (CM) of ASCs was significantly higher in CD-ASCs than in healthy-ASCs, and was significantly higher in active CD-ASCs than in inactive CD-ASCs (Figure 2B). Gene expression levels of critical inflammasome components that drive I L-1 β secretion(Grant and Dixit, 2013) were also measured in ASCs from healthy, inactive and active CD subjects. In line with data on I L-1 β secretion, gene expression of NLRP1, NLRP3 and CASP1 was higher in mesenteric CD- ASCs (both active and inactive) than in healthy-ASCs. No significant differences in the gene expression of these inflammasome components were found between ASCs from active and inactive CD patients, with the exception of NLRP1 (Figure 2C).

Given the close link between inflammasome activation and glucose homeostasis (Grant and Dixit, 2013; Serena et al., 2016), we compared the metabolic gene expression profile of ASCs isolated from CD patients and healthy controls (n=4 for all groups). Compared with healthy-ASCs, both active and inactive CD-ASCs displayed a glycolytic phenotype that was characterized by significantly higher expression of SLC2A 1, hexokinase-2 {HK2), phosphofructokinase (PKFM), and lactate dehydrogenase-b (LDHB), and significantly lower expression of pyruvate dehydrogenase kinase-4 (PDK4) (Figure 2D). Interestingly, the level of SLC2A4 mRNA was significantly lower in CD-ASCs from active patients than from inactive patients (Figure 2D), presumably due to the greater systemic inflammation in the former(Papa et al., 1997; Poletto et al., 2015). The glycolytic phenotype of CD-ASCs was accompanied by an increase in the amount of lactate and succinate released into CM (n=4 for all groups) (Figures 2E and 2F), which agreed well with the detected lower and higher mRNA levels of SDHB and OGDH, respectively, in these cells (Figure 2D). CD-ASCs also presented an increase in the expression of the β-oxidation markers CPT1B and SLC25A2 (Figure 2D). Taken together, our data suggest that CD provokes glycolytic and fatty acid oxidation metabolism in mesenteric ASCs, as previously described in some tumor cells(Altundag et al., 2005). For all these experiments, ASCs were isolated from VAT of healthy subjects (n=6), active (n=10) and inactive (n=5).

Example 3. Mesenteric ASCs from patients with CD have an exacerbated macrophage- like phenotype

The inflammatory response, including immune cell migration, is essential both for host defense and tissue repair. We previously showed that ASCs can act as nonprofessional phagocytes, a capacity that is boosted in inflammatory settings such as obesity and type 2 diabetes(Serena et al., 2016). We used ASCs from VAT of healthy subjects (n=6), active (n=10) and inactive (n=5 CD patients for all experiments, with the exception of phagocyte data (n=4 patients for all groups). We first evaluated ASC migration using Transwell assays. Remarkably, the 24h-CM from explants of CF VAT of active CD patients triggered the strong migration of healthy-ASCs, active CD-ASCs and also T Jurkat cells (Figure 3A), which agrees well with our hypothesis that ASCs migrate to CF. Furthermore, the basal migration of ASCs was significantly higher for CD-ASCs, both from active and inactive patients, than for healthy-ASCs (Figure 3B). We also examined the migration of monocytes (THP-1 cells), B (MEC-1 cells) and T (Jurkat cells) lymphocytes to ASC CM placed in the bottom chamber of the Transwell system, and detected that the migration of all cells was significantly greater to CM generated from active CD-ASCs than from healthy-ASCs, whereas CM from inactive CD- ASCs significantly stimulated the migration of B and T lymphocytes only and was moderately less effective than CD-ASC CM in stimulating B cells (Figure 3C). Using Matrigel invasion assays, we found that CD-ASCs exhibited a robust invasion capacity in basal, non-stimulated conditions relative to healthy-ASCs (Figure 3D). Consistent with the observed increase in cell migration/invasion, gene expression of two MMP family proteins, MMP2 and MMP9, was significantly higher in CD-ASCs than in healthy-ASCs (Figure S1A) and correlated with increased MMP2 and MMP9 activity measured by gelatin zymography (Figure 3E). We previously demonstrated that an inflammatory environment such as that found in obesity and type 2 diabetes increases ASC phagocytic capacity(Serena et al., 2016). Since cell migration and invasion are crucial steps for phagocytosis, we hypothesized that ASCs may respond to the bacterial translocation within mesenteric AT described in CD patients(Kruis et al., 2014) by increasing their phagocytic capacity. We therefore studied this parameter in CD-ASCs and healthy-ASCs. Remarkably, the phagocytic cell response against bacteria was significantly higher in CD-ASCs than in healthy-ASCs, as measured by the ingestion of pHrodo-labeled Escherichia coli BioParticles (Figures 3F and 3G). In line with this finding, gene expression of the typical phagocytic/endocytosis markers RAB7A and RAB5A was significantly higher in CD-ASCs than in healthy-ASCs (Figure S1 B). Of note, neither CD- ASCs nor healthy-ASCs were able to phagocytose yeast (data not shown), indicating specific phagocytic capabilities for ASCs. Collectively, these data indicate that mesenteric ASCs isolated from CD patients are immune-activated, even when the disease is in clinical remission.

Example 4. ASCs isolated from subcutaneous fat depots of patients with CD also show an activation of the immune response

To determine whether the proinflammatory effects of CD on ASCs was restricted to visceral fat depots or was a global response, we next examined ASCs isolated from SAT depots. We used ASCs from SAT of healthy subjects (n=6), active (n=10) and inactive (n=6) CD patients for all experiments, with exception of phagocyte data (n=4 patients for all groups). We found that ASCs from SAT, both from active and inactive CD patients, largely recapitulated the phenotype of equivalent VAT-derived ASCs, including a significantly higher AT-cell number ratio and proliferative activity (Figures S2A and 2B), and a reduced adipogenic differentiation capacity (Figures S2C and 2D) as compared with their healthy counterparts. Moreover, gene expression of inflammatory markers {IL1B, IL6, TNFA, and CCL2) was significantly higher in ASCs isolated from SAT of active and inactive CD patients than in cells from healthy donors (Figure 4A). Also, the secretion of I L-1 β into CM was significantly higher for CD-ASCs than for healthy-ASCs (Figure 4B). Overall, these results indicate that ASCs from SAT of CD patients exhibit an inflammatory profile, even during clinical remission of the disease. The migratory properties of SAT-derived ASCs from patients with CD also mirrored those of VAT-derived ASCs with respect to basal migration (Figure 4C), migration of monocytes and B and T lymphocytes to CM (Figure 4D), invasion (Figure 4E) and gene expression of MMP2/9 (Figure 4F). However, contrasting with the results for VAT-derived ASCs, only active CD-ASCs showed a significantly higher phagocytic capacity than their healthy counterparts (Figures 4G and 4H), which was accompanied by significantly higher expression of RAB7A and RAB5A (Figure 4I). Collectively, these findings indicate that while the inflammatory response is also activated in ASCs from the SAT of patients with CD, only those from active CD present a phagocyte-like phenotype. Example 5. Loss of immunosuppressive properties in SAT -ASCs from patients with CD We next explored whether ASCs isolated from SAT of patients with CD retain their typical immunoregulatory properties(Melief et al., 2013). We previously demonstrated that protective immunosuppressive properties, including elevated TGF- β1 secretion, promotion of M2 macrophage polarization and inhibition of T and B cell proliferation, all of which are mainly ascribed to SAT-derived ASCs, are perturbed in ASCs isolated from an obesity-induced inflammatory milieu(Serena et al., 2016). Similar to that observed in obesity, TGFB1 expression was lower both in active (n=10) and inactive (n=6) CD-ASCs than in healthy- ASCs (n=6), and this was significant for levels both in inactive and active CD-ASCs (Figure 5A). Moreover, the level of TGF-D 1 protein in CM was significantly lower from inactive CD- ASCs than from healthy-ASCs (Figure 5B). We also observed that the capacity to promote the M2 macrophage phenotype was blunted in ASCs isolated from CD patients. Accordingly, human THP1 PMA-activated macrophages (M0) were incubated with CM from healthy, active and inactive CD-ASCs (n=6-10 for all groups), and gene expression of typical M2 markers was quantified. Whereas expression of IL10, CD163 and MRC1 in THP1 cells was significantly increased after addition of CM from healthy-ASCs, this increase was less evident when M0 macrophages were cultured with CM from inactive or active CD-ASCs (Figure 5C). We also analyzed the expression of M1 markers and found that CM of healthy- ASCs, but not of CD-ASCs, significantly decreased the expression of a panel of M1 markers (Figure 5C). We next evaluated the capacity of ASCs to inhibit T and B cell proliferation. Whereas CM from healthy-ASCs suppressed the proliferation of T and B cells, CM from CD- ASCs had the opposite effect and significantly increased T and B cell proliferation (Figure 5D). Our results show that the typical immunosuppressive properties of ASCs are blunted in CD patients.

To further examine the inflammatory capacity of ASCs, we performed co-culture experiments with naive T lymphocytes. We found that naive T lymphocytes that were co-cultured with active CD-ASCs presented a significant increase in the gene expression of the Th1 marker TNFA, and a significant decrease of the Th2 marker GATA3 and of the Treg marker (TGFB1), as compared with those co-cultured with healthy-ASCs (Figure 5E). Remarkably, when we studied the secretion of soluble factors, the amount of granulocyte colony- stimulating factor (G-CSF) was significantly lower in the supernatant of naive T lymphocytes co-cultured with active CD-ASCs than with healthy-ASCs (Figure 5F). It is known that G-CSF mediates immune regulation, including the ability to promote regulatory T cells differentiation and has beneficial effects in the prevention of inflammatory bowel diseases in animal models(Rutella et al., 2005). Finally, we separated ASCs and naive T lymphocytes after 48 h of co-culture and analyzed gene expression of GCSF in the two cell types by Q-PCR. We found that GCSF expression was significantly lower in active CD-ASCs than in healthy-ASCs (Figure 5G). This result strongly suggests that healthy-ASCs may produce G-CSF, which promotes T regulatory cell differentiation(Rutella et al., 2005). Conversely, the lower gene expression and production of G-CSF in active CD-ASCs co-cultured with naive T cells may lead to a decrease T regulatory cell differentiation. Thus, the co-culture between naive T cells and ASCs is necessary to secrete G-CSF. Accordingly, CD-ASCs in contact with naive T lymphocytes in vivo may lead to a change in the phenotype of these cells in CD patients and decrease the T regulatory lymphocyte population.

Example 6. Inflammasome-inhibition counters the invasive and glycolytic phenotype of ASCs from CD patients

Both TGF-βΙ and I L-1 β have been previously revealed as key players in the dysfunctional behaviour of ASCs in metabolic diseases linked to inflammation (Barbagallo et al., 2016; Pourgholaminejad et al., 2016; Serena et al., 2016). We therefore attempted to reverse the phenotype of ASCs isolated from CD patients using different strategies to inhibit inflammasome components. To do this, ASCs (n=4 for all groups) were treated with the I L-1 receptor antagonist (IL1 RA), the caspase-1 inhibitor YVAD-CHO or TGF-βΙ , alone or in combination. Only combined treatment of IL1 RA plus YVAD-CHO was effective in significantly reducing the invasion capacity of ASCs isolated from the SAT and mesenteric AT depots of active and inactive CD patients (Figures 6A and 6B), which was accompanied by a significant reduction of MMP2/9 gene expression (Figures 6C and 6D). Moreover, this combination resulted in a decrease in the gene expression of inflammatory and inflammasome markers and also phagocyte markers (Figures 6E and 6F). Surprisingly, the combined treatment of IL1 RA plus YVAD-CHO reversed the effects of the CD environment on glycolytic but not D-oxidation markers (Figures 6E and 6F), suggesting that the inflammasome directs the invasive and glycolytic phenotype of ASCs in the context of CD.

Example 7. Study subjects

Subjects were recruited at the University Hospital Joan XXIII (Tarragona, Spain) and University Hospital Vail d ^' Hebron (Barcelona, Spain) in accordance with the tenets of the Helsinki Declaration. The corresponding hospital ethics committees approved the study, and written informed consent was obtained from all participants before entering in the study. Donors were classified as those in relapse (active) or in remission (inactive) following the Crohn's Disease Activity Index criteria(Best et al., 1976; Van Assche et al., 2010). Healthy subjects (n=6) and inactive CD donors (n=6) were recruited from age- and gender-matched subjects undergoing non-acute surgical interventions such as hernia or cholecystectomy, in a scheduled routine surgery. Active CD patients (n=10) were recruited from those undergoing surgery for symptomatic complications. Visceral adipose tissue (VAT) from mesenteric origin and subcutaneous adipose tissue (SAT) were obtained from the same individual during the surgical procedure, with the exception of one inactive CD patient, in which only SAT was available. Clinical data, anthropometric and biochemical variables from the cohort are presented in Table 1 .

Example 8. ASC isolation and culture

ASCs were isolated as described(Dubois et al., 2008; Gimble and Guilak, 2003). Briefly, SAT and VAT was washed extensively with PBS to remove debris and treated with 0.1 % collagenase in PBS 1 % BSA for 1 h at 37°C with gentle agitation. Digested samples were centrifuged at 300 g at 4°C for 5 min to separate adipocytes from stromal cells. The cell pellet containing the stromal fraction was re-suspended in stromal culture medium consisting of DMEM/F12, 10% FBS and 1 % antibiotic/antimycotic solution. To prevent spontaneous differentiation, primary cultures of ASCs at passage 0 (P0) were grown to 90% confluence and harvested with trypsin-EDTA, and aliquots (1 x10 ⁶ cells) were cryopreserved in liquid nitrogen until required(Pachon-Pena et al., 201 1 ). AT-derived macrophages (ATMs) were also isolated from the stromal vascular fraction of AT biopsies as described(Ceperuelo- Mallafre et al., 2016; Serena et al., 2016; Titos et al., 201 1 ). The AT-cell number ratio was defined as the ratio of the number of cells proliferating at P0 per gram of AT digested.

Example 9. hASC immunophenotyping

ASCs (2*10 ⁵ cells) were incubated with a panel of primary antibodies described in Table S1 (Pachon-Pena et al., 2016). After isolation, the minimal functional and quantitative criteria, established by the International Society of Cell Therapy (ISCT) and the International Federation for Adipose Therapeutics and Science (I FATS), were routinely confirmed by flow cytometry as described(Pachon-Pena et al., 2016; Serena et al., 2016). All experiments were performed in cells at p3-7.

Example 10. Cell migration assay

Basal migratory capacity of ASCs was analyzed using a Transwell system (δ-μm pore polycarbonate membrane) as described(Baek et al., 201 1 ; Corcione et al., 2006; Serena et al., 2016). The migratory capacity of human monocytes (THP-1 cell line) and T and B lymphocytes (Jurkat and MEC-1 cells, respectively) in response to application of 24-h conditioned medium (CM) from undifferentiated ASCs was performed using 5-μηι pore polycarbonate Transwell inserts as described(Serena et al., 2016). A similar experiment was performed to examine the migratory capacity of ASCs and T cells (Jurkat) in response to 24- h CM from VAT explants of active (CF origin) and from healthy individuals (mesenteric origin).

Example 11. Cell invasion assay Invasion capacity was determined as for migration except that the membrane was first coated with Matrigel ^® (Sigma, St. Louis, Missouri, USA) in PBS for 2 h at 37°C. ASCs were added to the upper chamber and incubated for 24 h at 37°C. Cells invading into the lower surface of the Transwell membrane were stained and counted.

Example 12. Zymography

To determine matrix metalloproteinase (MMP-2 and MMP-9) activity, near-confluent ASCs (80%) were serum-deprived for 24 h and the CM was electrophoresed in 8% SDS polyacrylamide gels polymerized with 0.1 % gelatin under non-reducing conditions. Gels were washed with 2.5% Triton X-100 (30 min) to remove SDS, rinsed with substrate buffer (0.2 M NaCI, 5 mM CaCI ₂ , 1 % Triton X-100, 0.02% NaN3, 50 mM Tris pH 7.5) and incubated in this buffer at 37°C overnight to allow protein renaturation and MMP activation. Gels were stained with Coomassie Brilliant Blue (Bio-Rad, Richmond California, USA) to visualize gelatin degradation.

Example 13. Phagocytosis assay

The pHrodo™ Escherichia coli (bacteria) and Zymosan (yeast) BioParticles® Phagocytosis Kits (Invitrogen Molecular Probes, Eugene, Oregon, USA) were used to assess the phagocytic capacity of undifferentiated ASCs (seeded at a density of 20,000 cells/cm ² ). Phagocytic activity was quantified using the Varioskan™ LUX multimode microplate reader (Thermo Fisher Scientific, Waltham, Massachusetts, USA).

Example 14. Cell proliferation assays

MTT assay. Cell proliferation was determined by a standard colorimetric 3-(4,5- dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide (MTT) assay. Briefly, ASCs were seeded in 96-well plates and allowed to attach for 24 h; after which the MTT assay (day+1 ) was performed to count the initial number of cells. After 7 days, a second MTT assay was performed (day+7), and the difference in absorbance between day+7 and day+1 was considered the proliferation of the ASCs.

Example 15. BrdU assay. ASC proliferation was also assessed by incorporation of 5-bromo- 2P-deoxyuridine (BrdU) using the BrdU Cell Proliferation Assay Kit (Millipore, Billerica, Massachusetts, USA). Cells (10,000 cells/well) were cultured in 96-well plates containing DMEM/F12 medium with 10% (v/v) FBS at 37°C in 5% C0 ₂ and allowed to attach for 24 h. BrdU was added to the medium and cells were incubated for a further 18 h. Cells were then fixed and BrdU incorporation was determined with an anti-BrdU specific antibody with detection by spectrophotometry at 450 nm. Proliferation of Jurkat and MEC-1 cells in response to CM from ASCs was performed in the same manner.

Example 16. Co-culture of naive T lymphocytes and ASCs

Co-culture experiments between human naive T lymphocytes and ASCs isolated from healthy subjects or CD patients were performed to measure T cell responses. Briefly, 60,000 ASCs/well from healthy or active CD patients were seeded in 12-well plates and allowed to attach overnight; after which, 800,000 T cells/well were added. After 48 hours, the supernatant was collected to determine T cells subsets (Th1/Th2/Treg) using the Multi- analyte ELISA Array Kit, MEH:003A (Qiagen, Germany) and the gene expression of T cells subset markers were assessed by Q-PCR.

Example 17. Statistical analysis

For in vitro data, experimental results were presented as mean±SD from 4-10 independent experiments (independent donors) performed at least in duplicate. Comparisons among the three groups were performed using the nonparametric Kruskal-Wallis test and between two groups using the Mann-Whitney U Test with Bonferroni adjustment (Significance, P<0.017). Clinical and anthropometrical variables, normally distributed data were expressed as meaniSD and for variables with no Gaussian distribution, values were expressed as median (interquartile range). Statistical analysis was performed with the Statistical Package for the Social Sciences software, version 15 (SPSS, Chicago, Illinois, USA).