WO2019053295A1 - Procédés pour améliorer l'efficacité d'une thérapie cellulaire avec des populations de cellules souches mésenchymateuses - Google Patents

Procédés pour améliorer l'efficacité d'une thérapie cellulaire avec des populations de cellules souches mésenchymateuses Download PDF

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WO2019053295A1
WO2019053295A1 PCT/EP2018/075248 EP2018075248W WO2019053295A1 WO 2019053295 A1 WO2019053295 A1 WO 2019053295A1 EP 2018075248 W EP2018075248 W EP 2018075248W WO 2019053295 A1 WO2019053295 A1 WO 2019053295A1
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determined
gene expression
expression
ascs
inflammatory
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Sonia FERNANDEZ VELEDO
Carolina SERENA PERELLÓ
Joan Josep VENDRELL ORTEGA
Antonio Zorzano Olarte
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Fundació Institut D'investigació Sanitària Pere Virgili
Consorcio Centro de Investigación Biomédica en Red, M.P.
Universitat Rovira I Virgili
Universitat De Barcelona
Fundació Institut De Recerca Biomédica (Irb Barcelona)
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Publication of WO2019053295A1 publication Critical patent/WO2019053295A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6881—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for tissue or cell typing, e.g. human leukocyte antigen [HLA] probes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • 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.
  • 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).
  • CD perianal Crohn's disease
  • 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.
  • ASCs can be extrapolated to any type of MSCs of any origin, such as bone marrow or umbilical cord blood, for example.
  • ASCs isolated from other pathological states such as obesity or type 2 diabetes mellitus (T2DM) have also altered the their immunosuppressive properties.
  • T2DM type 2 diabetes mellitus
  • an inflammatory subpopulation which secretes large amounts of interleukin-1 (IL-1 ⁇ ) (similar to M1 macrophages or macrophages with pro-inflammatory phenotype) and another more immunosuppressive subpopulation which secretes large amounts of transforming growth factor beta (TGFb ⁇ 1 ) (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.
  • IL-1 ⁇ interleukin-1
  • TGFb ⁇ 1 transforming growth factor beta
  • 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 ⁇ ).
  • 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.
  • 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).
  • inflammatory diseases e.g., obesity or diabetes
  • 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 (TGFb ⁇ 1 ) (similar to M2 macrophages or macrophages with anti-inflammatory phenotype).
  • TGFb ⁇ 1 transforming growth factor beta
  • FIG. 1 CD increases mesenteric ASC proliferation and reduces their adipogenic differentiation capacity.
  • A AT-cell number ratio
  • B MTT
  • C BrdU cell proliferation assays were performed as detailed in methods to study the cell proliferation of mesenteric ASCs.
  • E Quantification of Oil Red staining of AD from healthy subjects, active and inactive CD patients.
  • CD triggers an inflammasome-mediated inflammatory response in mesenteric ASCs and increases their metabolic activity.
  • A Expression of IL6, TNFA, CCL2, ⁇ _-1 ⁇ , 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 ⁇ was analyzed by ELISA from conditioned medium (CM) of ASCs from healthy subjects, active and inactive CD patients.
  • CM conditioned medium
  • 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.
  • 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.
  • E Zymographic analysis of MMP2/9 activities using gelatin as substrate.
  • 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 ⁇ 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.
  • 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.
  • 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).
  • Phagocytosis was quantified using the VarioskanTM LUX multimode microplate reader. Fluorescence intensity was normalized to total protein content.
  • 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- ⁇ 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.
  • FIG. 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- ⁇ 1 , 10 ⁇ 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.
  • I LI RA interleukin 1 receptor antagonist
  • 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.
  • an element means one element or more than one element.
  • the 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • mesenchymal stem cells 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 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).
  • the mesenchymal stem cells are obtained from umbilical cord, preferably from human umbilical cord.
  • 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.
  • 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.
  • pharmaceutically acceptable refers 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.
  • 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.
  • phenotype refers to observable characteristics of a cell, such as size, morphology, protein expression, etc.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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 (TGFb ⁇ 1 ) (similar to M2 macrophages or macrophages with anti-inflammatory phenotype).
  • TGFb ⁇ 1 transforming growth factor beta
  • 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 .
  • step b) 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.
  • 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.
  • decision points 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.
  • 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.
  • such array will consist in a selection of different genes involved in inflammation, phagocytosis, invasion, inflammasome activation, glycolysis, antigen presentation and immunosuppressive properties; wherein:
  • IL1 B interleukin 1 beta
  • TNFA tumor necrosis factor alpha
  • CCL2 C-C Motif Chem
  • RAS oncogene family RAS oncogene family
  • MMP2 metalloproteases
  • glycolytic genes produce energy to the cell.
  • IL10 interleukin 10
  • TGFB1 transforming growth factor beta 1
  • the variables and the sets of variables to implement the score are as set down below:
  • Pro-inflammatory cytokines are as set down below:
  • Anti-inflammatory cytokines
  • Glycolytic genes Antigen presenting markers:
  • the score ranges between 0-7, wherein a higher score value of 3.5 is not considered suitable for use in medicine regenerative purposes.
  • 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.
  • 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.
  • the 7 parameters pro-inflammatory cytokines, anti-inflammatory cytokines, phagocytic markers, invasion markers, inflammasome markers and glycolytic genes
  • 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,
  • MSCs preferably derived from adipose tissue
  • MSCs preferably derived from adipose tissue
  • 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;
  • step b Determining the increase or reduction of each of the functional capacities of step a) with respect to a decision point;
  • step b) 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 (TGFb ⁇ 1 ) (similar a the M2 macrophages or macrophages with anti-inflammatory phenotype).
  • TGFb ⁇ 1 transforming growth factor beta
  • a score 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.
  • 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 ⁇ (for example using an ELISA
  • Predictive power of 82% and 94% of success (n 28) in T cell and B cell proliferation, respectively.
  • the score ranges between 0 - 4.
  • 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;
  • step b) 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 ⁇ ) (similar to M1 macrophages or macrophages with pro- inflammatory phenotype)
  • IL-1 ⁇ interleukin-1
  • 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.
  • 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 ;
  • step b Determining the increase or reduction of each of the functional capacities of step a) with respect to a decision point;
  • step b) 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 (TGFb ⁇ 1 ) (similar a the M2 macrophages or macrophages with anti-inflammatory phenotype).
  • TGFb ⁇ 1 transforming growth factor beta
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • a marker is considered to not be expressed.
  • 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.
  • 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.
  • 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 ⁇ 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.
  • inflammasome component inhibitors such as caspase 1 inhibitor (YVAD- CHO, Sigma Aldrich) or IL-1 ⁇ receptor antagonist (IL1 RA).
  • 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.
  • the starting cell population would be treated with the combination of 40 ng/ml of I LI RA combined with 10 ⁇ of YVAD-CHO.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • compositions 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.
  • 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.
  • 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.
  • Previously 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.
  • 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.
  • supportive treatment may be required.
  • immunosuppressants can be administered before, during and/or after treatment to prevent graft-versus-host disease (GVHD), according to known methods.
  • GVHD graft-versus-host disease
  • 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.
  • 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.
  • 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.
  • the administration device can comprise a catheter.
  • the administration device can comprise a syringe.
  • 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.
  • 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
  • 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.
  • 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 ⁇ -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 antivasive agent,
  • the additional therapeutic agent can be a growth factor or another molecule affecting cell proliferation or activation.
  • said growth factor can induce final differentiation.
  • 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.
  • 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.
  • 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.
  • 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.
  • compositions 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.
  • the different active agents can be administered together or separately, and simultaneously or at different times throughout the day.
  • 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.
  • 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 LipofectamineTM 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).
  • a cationic detergent such as polyethylenimine or LipofectamineTM
  • 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
  • DOSPA trifluoroacetate
  • DOPE dioleoyl phosphatidylethanolamine
  • An example is the Lipofectamine 2000TM formulation (available from Gibco/Life Technologies as product no. 1 1668019).
  • 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 (LipofectamineTM, 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).
  • 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.
  • 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.
  • Example 1 Mesenteric ASCs of patients with CD show higher proliferation rates but lower adipogenic capacities than those of healthy donors
  • CD-ASCs patients with CD
  • health-ASCs healthy individuals
  • a greater number of ASCs was obtained from CD patients than from healthy individuals.
  • 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.
  • I L-1 ⁇ 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 ⁇ secretion(Grant and Dixit, 2013) were also measured in ASCs from healthy, inactive and active CD subjects.
  • Example 3 Mesenteric ASCs from patients with CD have an exacerbated macrophage- like phenotype
  • 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).
  • Example 4 ASCs isolated from subcutaneous fat depots of patients with CD also show an activation of the immune response
  • 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.
  • 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).
  • G-CSF granulocyte colony- stimulating factor
  • 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).
  • 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.
  • 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).
  • 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
  • TGF- ⁇ and I L-1 ⁇ 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- ⁇ , alone or in combination.
  • VAT Visceral adipose tissue
  • SAT subcutaneous adipose tissue
  • 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.
  • AT-derived macrophages 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.
  • 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.
  • ISCT International Society of Cell Therapy
  • I FATS Adipose Therapeutics and Science
  • Basal migratory capacity of ASCs was analyzed using a Transwell system ( ⁇ - ⁇ m 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- ⁇ pore polycarbonate Transwell inserts as described(Serena et al., 2016).
  • CM conditioned medium
  • 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.
  • Matrigel ® Sigma, St. Louis, Missouri, USA
  • MMP-2 and MMP-9 matrix metalloproteinase 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.
  • substrate buffer 0.2 M NaCI, 5 mM CaCI 2 , 1 % Triton X-100, 0.02% NaN3, 50 mM Tris pH 7.5
  • the pHrodoTM Escherichia coli (bacteria) and Zymosan (yeast) BioParticles® Phagocytosis Kits 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 VarioskanTM LUX multimode microplate reader (Thermo Fisher Scientific, Waltham, Massachusetts, USA).
  • 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.
  • MTT assay 4,5- dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide
  • 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.
  • BrdU 5-bromo- 2P-deoxyuridine
  • Example 16 Co-culture of naive T lymphocytes and ASCs

Abstract

La présente invention concerne des procédés d'identification de populations de cellules souches mésenchymateuses qui sont utiles dans la thérapie cellulaire applicable dans la médecine régénérative, ou de sélection de populations de CSM ayant un phénotype principalement pro-inflammatoire, et donc applicables dans des thérapies cellulaires où l'activation du système immunitaire est essentielle. L'invention concerne également des populations de CSM enrichies dans une sous-population plus immunosuppressive qui sécrète de grandes quantités de facteur de croissance transformant bêta (TGFbß1) (similaire à des macrophages M2 ou à des macrophages ayant un phénotype anti-inflammatoire).
PCT/EP2018/075248 2017-09-18 2018-09-18 Procédés pour améliorer l'efficacité d'une thérapie cellulaire avec des populations de cellules souches mésenchymateuses WO2019053295A1 (fr)

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CN113881707A (zh) * 2021-10-25 2022-01-04 中国人民解放军军事科学院军事医学研究院 调控脐带间充质干细胞免疫抑制作用的产品、方法及用途

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CN113633775A (zh) * 2021-09-01 2021-11-12 吉林大学 过表达肌型磷酸果糖激酶的试剂在制备延缓细胞衰老的药物中的应用
CN113881707A (zh) * 2021-10-25 2022-01-04 中国人民解放军军事科学院军事医学研究院 调控脐带间充质干细胞免疫抑制作用的产品、方法及用途

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