This invention relates to the general field of biotechnology, and specifically relates to the field of regenerative medicine, cell based therapies and the manufacturing of Advanced Therapy Medicinal Products (ATMPs). In particular, the present application provides an optimized medium for growth of mammalian progenitors cells, in particular provides culture media and methods for expanding populations of progenitors cells. The cell culture medium is characterized by the presence of at least a nitric oxide donor.

Background of the invention

Stem cell-based strategies have been widely used as a promising therapy to treat diverse types of diseases such as cardiomyopathies, neuronal illness or metabolic diseases including diabetes and its complications. hPCs (human progenitor cells) demonstrate regenerative properties and multipotentiality and due to the current clinical limitations of using Embryonic Stem Cells (ESC) and induced Pluripotent Stem Cells (iPSC), these cells have been proposed as a potential candidate for cell therapy and tissue engineering. Until 2014, there were more than 1300 registered cell-based therapy clinical trials in different clinical trial phases aimed at evaluating the potential of hPCs cell-based therapy worldwide (T. RJ Eathman, Nienow AW, McCall MJ, Coopman K, Kara B, Hewitt CJ. The translation of cell-based therapies: clinical landscape and manufacturing challenges. Regen. Med. (2025). 10(1 ), 49-64). Thanks to these studies, to date hPCs have been shown to be effective in the treatment of many diseases, including both immune diseases and non-immune diseases (Karussis et al., 2010; Le Blanc et al., 2008; Skrahin et al., 2014). However, there is a still a need to establish standards for cell expansion protocols, product quality, and safety controls in order to enable translational activities and ultimately facilitate comparison of clinical trial results.

In this sense, for clinical use, efficient ex-vivo expansion of hPCs is a challenging requirement for large scale production of clinical grade cells. Cost-effective, robust, scalable culture methods using chemically defined materials need to be developed in order to address this need. Previously reported efforts to develop defined media for hPCs such as hMSC (human mesenchymal stem cells) culture only resulted in slow or limited proliferation, and were unsuccessful in expanding these cells from primary cultures. In addition, these media were dependent on basic fibroblast growth factors (bFGF) and transforming growth factors (TGF)- betal , thus increasing large scale production costs (Jung et al., 2010). Therefore a major step forward would be the identification of defined, preferably serum-free culture conditions capable of supporting both the isolation and rapid expansion of hPCs in a medium which could in turn be use to replace the current mediums comprising expensive growth factors. Therefore, a well formulated chemically defined medium for hPCs isolation and expansion would greatly contribute to the achievement of enhanced culture protocols to meet efficacy endpoints in upcoming clinical studies.

Brief description of the invention

The invention provides new culture media and methods for hPCs ^' isolation and expansion using said media, which provide significant advantages over known culture media and isolation and expansion methods. These culture media are characterized by the presence of a nitric oxide donor. In addition the culture media of the invention are preferably serum supplemented or serum-free.

The examples of the invention show that after successive passages in the culture media of the invention, the cells (hPCs) display a good expression profile measured by flow cytometry of positive immunological markers which clearly correlates with the maintenance of their multipotent characteristics. In addition, as shown in Figure 9, these cells, the hPCs obtained after culture in the media of the invention, have a very low differentiation degree since these lack expression of CD45 (pan-leukocyte marker), CD34 (primitive hematopoietic progenitors) and HLA class II markers. Therefore, the new culture media presented herein are useful for the maintenance and expansion of human Progenitors Cells, and the cells obtained therefrom preserve their potential to differentiate into different tissues such as osteocytes, chondrocytes and adipocytes (Figure 10).

Brief description of the drawings

Figure 1. Cell proliferation measured by Bromodeoxyuridine (BrdU) incorporation of cells cultured in DMEM low glucose medium supplemented with 2.5, 5 and 7.5% of fetal bovine serum (FBS). Low concentrations of nitric oxide donor DETA/NO (2, 5 and 10μΜ) were evaluated for each serum concentration. DMEM low glucose medium supplemented with 10% of FBS was used as a control. It represents the average of five independent experiments. Data are ± SEM. From these results DMEM low glucose with 5% of FBS supplemented with 5μΜ DETA NO was the most appropriate composition for the low serum culture medium of the invention.

Figure 2. Ki67 expression analyzed by immunoflorescence microscopy for cells cultured in DMEM with 5% FBS and DMEM with 5% FBS supplemented with 5μΜ DETA/NO. a-Tubulin antibody and DAPI was used as counterstaining. The image is representative of three independent experiments. The quantification was performed by choosing seven fields per sample and quantifying all the Ki67 positive cells. Data are ± SEM.

Figure 3. This figure shows the immunological phenotype of cells cultured in DMEM with 10% FBS and DMEM with 5% FBS supplemented with 5μΜ DETA NO. Cells were Trypsinized and washed with DPBS. Then, approximately 1 x10 ⁵ cells were incubated directly with 3uL of the respective antibody for 30 minutes. The cells were washed with DPBS and re-suspended in 400uL DPBS and quantification determined by Flow Cytometry.

Figure 4. Differentiation potential of cells cultured in DMEM with 5% FBS supplemented with 5μΜ DETA NO. Differentiation was induced following manufacturer recommendations. Figure 5. This figure illustrates the cell morphology and the cell density at the end of each cell passing. It can be observed that the morphology of the cells cultured in the chemically defined medium is similar to the morphology of those cells cultured in the control medium, and the cell density was higher in the plates of cells cultured with the chemically defined medium.

Figure 6. Figure 6 represents the cell proliferation at the end of each cell passing. It can be observed that the proliferation of the cells cultured in the chemically defined medium increases respect to the cells cultured in the control medium.

Figure 7. Figure 7 illustrates Bone Marrow human mesenchymal stem cells cultured in the control medium and in the chemically defined medium, and later stainned with DAPI, Ki-67 (red) and a-Tubulin (green). It could be proved that the expression of Ki-67 protein was similar in the cells cultured with both media.

Figure 8. Figure 8 illustrates the expression of pluripotent and mesenchymal phenotypic characteristic genes by cualitative PCR. The image shows that the cells cultured in chemically defined medium maintain pluripotency expressig Nanog and Oct4, express the mesenchymal stem cells surface markers Nestin and Integrin, and express the positive markers CD105, CD90 y CD73.

Figure 9. Analysis of cell potential differentiation of Adipose Tissue human Mesenchymal Stem cells cultured in a a serum-free medium with 75% of bFGF supplemented with 2 μΜ of NO

Description of the invention CUL TURE MEDIA OF THE INVENTION

A first aspect of the invention refers to a culture medium, hereinafter culture medium of the invention, comprising: a) a nitric oxide donor, and

b) a basal medium which promotes the growth of microorganisms and mammalian cells and comprises four basic chemical groups: amino acids, carbohydrates, inorganic salts, and vitamins. As used herein, the term "culture" refers to any growth of cells, organisms, multicellular entities, or tissue in a medium. The term "culturing" refers to any method of achieving such growth, and may comprise multiple steps.

As used herein, the term "cell culture" refers to a growth of cells in vitro. In such a culture, the cells proliferate, but they do not organize into tissue per se.

As used herein, the term "culture medium" or "medium" or "media" is recognized in the art, and refers generally to any substance or preparation used for the cultivation of living cells. The term "medium" or "media", as used in reference to a cell culture, includes the components of the environment surrounding the cells. Media may be solid, liquid, gaseous or a mixture of phases and materials. Media include liquid growth media as well as liquid media that do not sustain cell growth. Media also include gelatinous media such as agar, agarose, gelatin and collagen matrices. Exemplary gaseous media include the gaseous phase that cells growing on a petri dish or other solid or semisolid support are exposed to.

As used herein, the term "medium" also refers to material that is intended for use in a cell culture, even if it has not yet been contacted with cells. In other words, a nutrient rich liquid prepared for bacterial culture is a medium. Similarly, a powder mixture that when mixed with water or other liquid becomes suitable for cell culture may be termed a "powdered medium".

As used herein, the term "basal medium" refers to a medium which promotes the growth of many types of microorganisms and mammalian cells which do not require any special nutrient supplements. Most basal media generally comprise four basic chemical groups: amino acids, carbohydrates, inorganic salts, and vitamins. A basal medium generally serves as the basis for a more complex medium, to which supplements such as serum, buffers, growth factors, lipids, and the like are added. Examples of basal media include, but are not limited to, Eagles Basal Medium, Minimum Essential Medium, Dulbecco's Modified Eagle's Medium (DMEM), Medium 199, Nutrient Mixtures Ham's F-10 and Ham's F-12, Mc Coy's 5A, Dulbecco's MEM/F-12, RPMI 1640, and Iscove's Modified Dulbecco's Medium (IMDM).

Preferably, the culture medium of the invention is manufacture complying with good manufacturing practices (GMP).

Preferably, the nitric oxide donor is selected from the list consisting of: S-Nitrosoglutathione (GSNO) (CAS 57564-91 -7), (±)-S-Nitroso-N-acetylpenicillamine (SNAP) (CAS 79032-48-7), Spermine NONOate (CAS 136587-13-8), Sodium Nitroprusside, Dihydrate (CAS 13755-38-9), NOR-1 (CAS 163032-70-0) and Diethylenetriamine/nitric oxide adduct (DETA/NO) (CAS 146724-94-9) or any combination thereof. In a more preferred embodiment, the nitric oxide donor is DETA NO. In another more preferred embodiment the nitric oxide donor, DETA/NO, is in a concentration range from about 1 μΜ to about 50 μΜ, and still more preferably is in a concentration range from about 2 μΜ to 10 μΜ.

Preferably, the basal medium is selected from the list consisting of Eagles Basal Medium, Minimum Essential Medium, Dulbecco's Modified Eagle's Medium, Medium 199, Nutrient Mixtures Ham's F-10 and Ham's F-12, Mc Coy's 5A, Dulbecco's MEM/F-I 2, RPMI 1640, and Iscove's Modified Dulbecco's Medium (IMDM).

Low serum culture medium

As shown in the examples, the addition of nitric oxide donors to a basal medium allows the reduction of the percentage of fetal bovine serum (FBS) thus obtaining a low serum culture medium. Therefore, in a preferred embodiment of the first aspect of the invention, the culture medium of the invention is only supplemented with fetal bovine serum (FBS) in an amount of 10%, preferably 7.5%, more preferably 5%, still more preferably 2.5% by weight over the total weigh of the culture medium.

In another preferred embodiment, the culture medium of the invention is only supplemented with Human AB serum. More preferably in an amount of 10 %, preferably 7,5 %, more preferably 5 %, still more preferably 2,5 % by weight over the total weigh of the culture medium.

In another preferred embodiment, the culture medium of the invention is only supplemented with thrombin-activated platelet-rich plasma. More preferably in an amount of 10 %, preferably 7,5 %, more preferably 5 %, still more preferably 2,5 % by weight over the total weigh of the culture medium. In another preferred embodiment, the amino acids of the basal medium are selected from the list consisting of Glycine, L-Alanyl-Glutamine, L-Arginine hydrochloride. L-Cystine, L-Histidine hydrochloride-H20, L-lsoleucine, L-Leucine, L-Lysine hydrochloride, L-Methionine, L- Phenylalanine, L-Serine. L-Threonine, L-Tryptophan, L-Tyrosine, L-Valine, or any combinations thereof. In another preferred embodiment, the vitamins of the basal medium are selected from the list consisting of Choline chloride, D-Calcium pantothenate, Folic Acid, Niacinamide, Pyridoxine hydrochloride, Riboflavin, Thiamine hydrochloride, i-lnositol, or any combinations thereof. In another preferred embodiment, the Inorganic Salts of the basal medium are selected from the list consisting of Calcium Chloride (CaCI2-2H20), Ferric Nitrate (Fe(N03)3"9H20), Magnesium Sulfate (MgS04-7H20), Potassium Chloride (KCI), Sodium Bicarbonate (NaHC03), Sodium Chloride (NaCI), Sodium Phosphate monobasic (NaH2P04-2H20), or any combinations thereof.

Free serum culture medium

In another preferred embodiment of the first aspect of the invention, the culture medium of the invention, of the first aspect of the invention, is a free serum culture medium, hereinafter free serum culture medium of the invention, wherein the basal medium comprises a mixture of Dulbecco's modified Eagle's medium (DMEM) and Ham ^' s F12 nutrient mixture (DMEM/F12).

In a preferred embodiment, the basal medium of the free serum culture medium of the invention is prepared by mixing 50% of volume for each medium (Dulbecco's modified Eagle's medium (DMEM) and Ham ^' s F12 nutrient mixture). In another preferred embodiment, the DMEM basal

In another preferred embodiment, the free serum culture medium or any of the other media as defined in the first aspect, further comprises insulin, preferably recombinant Human Insulin (rHu), and more preferably, said insulin is at a concentration range of about 10 mg/L to about 30 mg/L, and more preferably of about 20 mg/L.

In another preferred embodiment, the free serum culture medium or any of the other media as defined in the first aspect, further comprises transferrin, preferably human holo-Transferrin solubilised in PBS, in final concentration in the culture medium of about 5 mg/L to about 15 mg/L, and more preferably of about 10 mg/L.

In another preferred embodiment, the free serum culture medium or any of the other media as defined in the first aspect, further comprises ascorbic acid, more preferably L-ascorbic acid 2- phosphate sesquimagnesium salt hydrate, and more preferably, said Ascorbic acid is at a concentration range of about 50 mg/L to about 70 mg/L, and more preferably of about 60 mg/L.

In another preferred embodiment, the free serum culture medium or any of the other media as defined in the first aspect, further comprises sodium selenite and more preferably, said sodium selenite is at a concentration range of about 0.0005 mg/L to about 0.0030 mg/L, and more preferably of about 0.0015 mg/L. In another preferred embodiment, the free serum culture medium or any of the other media as defined in the first aspect, further comprises Basic Fibroblast Growth Factor (bFGF), more preferably Recombinant Human Fibroblast Growth Factor-basic, and more preferably, said Basic Fibroblast Growth Factor is at a concentration range of about 50 μg L to about 150 μg L, and more preferably of about 100 μg L.

In another preferred embodiment, the final concentration of bFGF are decreased in at least 25%, more preferably at least 50%, and still more preferably in at least 75% regarding with media containing 100 μg L.

In another preferred embodiment, the free serum culture medium or any of the other media as defined in the first aspect, further comprises Transforming Growth Factor (TGF-β), more preferably Recombinant Transforming Growth Factor (TGF-β), and more preferably, said Transforming Growth Factor (TGF-β) is at a concentration range of about 0.001 mg/L to about 0.010 mg/L, and more preferably of about 0.002 mg/L.

In another preferred embodiment, the free serum culture medium or any of the other media as defined in the first aspect, further comprises Hydrocortisone, and more preferably, said Hydrocortisone is at a concentration range of about 50 nM to about 200 nM, and more preferably of about 150 nM. In another preferred embodiment, the free serum culture medium or any of the other media as defined in the first aspect, further comprises other additives selected from the group consisting of: sodium pyruvate, glutamine, non-essential aminoacids, and combinations thereof in order to stimulate the cellular growth and proliferation.

METHOD OF EXPANSION AND MAINTAINING PROGENITORS CELLS OF THE INVENTION

A second aspect of the invention refers to a method of expanding and maintaining stem cells, preferably isolated stem cells, and more preferably isolated Progenitor cells, in an adequate potency state, hereinafter the method of expanding and maintaining progenitor cells of the invention, comprising culturing the progenitor cells in a culture medium of the invention (any of the cell culture media detailed in the first aspect of the invention), thereby expanding and maintaining the progenitor cells in an undifferentiated state.

Progenitor cells have been found in adult tissue. By "adult" it is meant that the stem cells are not embryonic. In one embodiment, "adult" means post-embryonic or "post-natal". With respect to the stem cells of the present invention, the term "adult stem cell" means that the stem cell is isolated from a tissue or organ of an animal at a stage of growth later than the embryonic stage. In one aspect, the stem cells of the invention may be isolated at the post-natal stage. The cells may be isolated from a mammal, such as a rat, mouse, pig or human. Adult stem cells are unlike embryonic stem cells, which are defined by their origin, the inner cell mass of the blastocyst. Adult stem cells according to the invention may be isolated from any non-embryonic tissue, and will include neonates, juveniles, adolescents and adult subjects. Generally the stem cell of the present invention will be isolated from a non-neonate mammal, and for example from a non-neonate human, rat, mouse or pig. Preferably, the stem cells of the present invention are isolated from a human.

The term "isolated" indicates that the cell or cell population to which it refers is not within its natural environment. The cell or cell population has been substantially separated from surrounding tissue. In some embodiments, the cell or cell population is substantially separated from surrounding tissue if the sample contains at least about 75%, in some embodiments at least about 85%, in some embodiments at least about 90%, and in some embodiments at least about 95% adult stem cells. In other words, the sample is substantially separated from the surrounding tissue if the sample contains less than about 25%, in some embodiments less than about 15%, and in some embodiments less than about 5% of materials other than the adult stem cells. Such percentage values refer to percentage by weight or by cell number. The term encompasses cells which have been removed from the organism from which they originated, and exist in culture. The term also encompasses cells which have been removed from the organism from which they originated, and subsequently re-inserted into an organism. The organism which contains the re-inserted cells may be the same organism from which the cells were removed, or it may be a different organism, i.e. a different individual of the same species. The term "progenitor cells" refers to early descendants of stem cells that can differentiate to form one or more kinds of cells, but cannot divide and reproduce indefinitely. Specifically a "progenitor cell" is often more limited than a stem cell in the kinds of cells it can become.

The term "multipotent" refers to a cell which is capable of giving rise to multiple different types of cell. Specifically, the term refers to a cell which is able to differentiate into cell types of mesodermal, endodermal and ectodermal origin.

The term "differentiation" refers to the formation of cells expressing markers known to be associated with cells that are more specialized and closer to becoming terminally differentiated cells that are incapable of further division or differentiation. For example, in a pancreatic context, differentiation might be seen in the production of islet-like cell clusters containing an increased proportion of beta epithelial cells that produce increased amounts of insulin. The terms "further" or "greater" differentiation refers to cells that are more specialized and closer to becoming terminally differentiated cells incapable of further division or differentiation than the cells from which they were cultured. The term "final differentiation" refers to cells that have become terminally differentiated cells incapable of further division or differentiation. In another preferred embodiment the progenitor cells are mammalian multipotent stem cells. More preferably the mammalian multipotent cells are human multipotent cells (hMC). In another preferred embodiment, the multipotent cells are mesenchymal stem cells (MSCs), more preferably are mammalian mesenchymal stem cells and more preferably, human mesenchymal stem cells (hMSCs). In a still more preferably embodiment, the hMSCs are adipose-derived hMSC.

Mesenchymal stem cells (MSCs) are a heterogeneous population of stromal cells isolated from multiple species, residing in most connective tissues including bone marrow, adipose, umbilical cord, placenta, amniotic fluid and perivascular tissues. MSCs can differentiate into cells of the mesenchymal lineage, such as bone, cartilage and fat but, under certain circumstances, have been reported to acquire the phenotype of cells of the endodermal and neuroectodermal lineage, suggesting some potential for "transdifferentiation". Within the bone marrow these cells are tightly intermingled with and support hematopoiesis and the survival of hematopoietic stem cells in acquiescent state (Meuleman N et al., Stem Cells Dev. 18(9): 1247-52, 2009). The term "mesenchymal stem cell" or "MSC" is used interchangeably for adult cells which are not terminally differentiated, which can divide to yield cells that are either stem cells, or which, irreversibly differentiate to give rise to cells of a mesenchymal cell lineage, e.g., adipose, osseous, cartilaginous, elastic and fibrous connective tissues, myoblasts) as well as to tissues other than those originating in the embryonic mesoderm (e.g., neural cells) depending upon various influences from bioactive factors such as cytokines.

Methods of isolating, purifying and expanding mesenchymal stem cells (MSCs) are known in the arts. Mesenchymal stem cells may be isolated from various tissues including but not limited to bone marrow, peripheral blood, blood, placenta (both chorionic and/or amniotic), cord blood, umbilical cord, amniotic fluid and from adipose tissue. A method of isolating mesenchymal stem cells from peripheral blood is described by Kassis et al [Bone Marrow Transplant. 2006 May; 37(10):967-76]. A method of isolating mesenchymal stem cells from placental tissue is described by Zhang et al [Chinese Medical Journal, 2004, 1 17 (6):882-887]. Methods of isolating and culturing adipose tissue, placental and cord blood mesenchymal stem cells are described by Kern et al [Stem Cells, 2006; 24: 1294-1301]. For example, adipose tissue-derived MSCs can be obtained by liposuction and mononuclear cells can be isolated manually by removal of the fat and fat cells, or using the Celution System (Cytori Therapeutics)

Preferably the MSCs are at least 50 % purified, more preferably at least 75 % purified and even more preferably at least 90 % purified. In another preferred embodiment, the serum free media are accurate to culture and expansion of MSCs genetically modified.

MSCs may be genetically modified to correct a genetic disease, to express a protein which induces the differentiation thereof. Alternatively, MSCs may be genetically modified to express a polynucleotide agent e.g. an siRNA or miRNA that down-regulates expression of a particular protein or polynucleotide agent in order to induce differentiation.

To express such agents in mesencyhymal stem cells, a polynucleotide sequence encoding the agent is preferably ligated into a nucleic acid construct suitable for mesenchymal stem cell expression. Such a nucleic acid construct includes a promoter sequence for directing transcription of the polynucleotide sequence in the cell in a constitutive or inducible manner.

Constitutive promoters suitable for use with some embodiments of the invention are promoter sequences which are active under most environmental conditions and most types of cells such as the cytomegalovirus (CMV) and Rous sarcoma virus (RSV). Inducible promoters suitable for use with some embodiments of the invention include for example tetracycline-inducible promoter (Zabala M, et al., Cancer Res. 2004, 64(8): 2799-804).

Eukaryotic promoters typically contain two types of recognition sequences, the TATA box and upstream promoter elements. The TATA box, located 25-30 base pairs upstream of the transcription initiation site, is thought to be involved in directing RNA polymerase to begin RNA synthesis. The other upstream promoter elements determine the rate at which transcription is initiated.

Preferably, the promoter utilized by the nucleic acid construct of some embodiments of the invention is active in the specific cell population transformed - i.e. mesenchymal stem cells.

Enhancer elements can stimulate transcription up to 1 ,000 fold from linked homologous or heterologous promoters. Enhancers are active when placed downstream or upstream from the transcription initiation site. Many enhancer elements derived from viruses have a broad host range and are active in a variety of tissues. For example, the SV40 early gene enhancer is suitable for many cell types. Other enhancer/promoter combinations that are suitable for some embodiments of the invention include those derived from polyoma virus, human or murine cytomegalovirus (CMV), the long term repeat from various retroviruses such as murine leukemia virus, murine or Rous sarcoma virus and HIV. Other possibilities are known in the state of the art.

In the construction of the expression vector, the promoter is preferably positioned approximately the same distance from the heterologous transcription start site as it is from the transcription start site in its natural setting. As is known in the art, however, some variation in this distance can be accommodated without loss of promoter function. In addition to the elements already described, the expression vector of some embodiments of the invention may typically contain other specialized elements intended to increase the level of expression of cloned nucleic acids or to facilitate the identification of cells that carry the recombinant DNA. For example, a number of animal viruses contain DNA sequences that promote the extra chromosomal replication of the viral genome in permissive cell types. Plasmids bearing these viral replicons are replicated episomally as long as the appropriate factors are provided by genes either carried on the plasmid or with the genome of the host cell.

The vector may or may not include a eukaryotic replicon. If a eukaryotic replicon is present, then the vector is amplifiable in eukaryotic cells using the appropriate selectable marker. If the vector does not comprise a eukaryotic replicon, no episomal amplification is possible. Instead, the recombinant DNA integrates into the genome of the engineered cell, where the promoter directs expression of the desired nucleic acid.

Examples for mammalian expression vectors include, but are not limited to, pcDNA3, pcDNA3.1 (+/-), pGL3, pZeoSV2(+/-), pSecTag2, pDisplay, pEF/myc/cyto, pCMV/myc/cyto, pCR3.1 , pSinRep5, DH26S, DHBB, pNMTI, pNMT41 , pNMT81 , which are available from Invitrogen, pCI which is available from Promega, pMbac, pPbac, pBK-RSV and pBK-CMV which are available from Strategene, pTRES which is available from Clontech, and their derivatives.

Expression vectors containing regulatory elements from eukaryotic viruses such as retroviruses can be also used. SV40 vectors include pSVT7 and pMT2. Vectors derived from bovine papilloma virus include pBV-IMTHA, and vectors derived from Epstein Bar virus include pHEBO, and p205. Other exemplary vectors include pMSG, pAV009/A <+>, ρΜΤΟ 0/Α <+>, pMAMneo- 5, baculovirus pDSVE, and any other vector allowing expression of proteins under the direction of the SV-40 early promoter, SV-40 later promoter, metallothionein promoter, murine mammary tumor virus promoter, Rous sarcoma virus promoter, polyhedrin promoter, or other promoters shown effective for expression in eukaryotic cells.

Viruses are very specialized infectious agents that have evolved, in many cases, to elude host defense mechanisms. Typically, viruses infect and propagate in specific cell types. The targeting specificity of viral vectors utilizes its natural specificity to specifically target predetermined cell types and thereby introduce a recombinant gene into the infected cell. Thus, the type of vector used by some embodiments of the invention will depend on the cell type transformed. The ability to select suitable vectors according to the cell type transformed is well within the capabilities of the ordinary skilled artisan and as such no general description of selection consideration is provided herein. For example, bone marrow cells can be targeted using the human T cell leukemia virus type I (HTLV-I) and kidney cells may be targeted using the heterologous promoter present in the baculovirus Autographa calif ornica nucleopolyhedro virus (AcMNPV) as described in Liang CY et al., 2004 (Arch Virol. 149: 51 -60).

In some embodiments, a lentiviral vector is used to transfect the mesenchymal stem cells.

Various methods can be used to introduce the expression vector of some embodiments of the invention into mesenchymal stem cells. Such methods are generally described in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Springs Harbor Laboratory, New York (1989, 1992), in Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Md. (1989), Chang et al., Somatic Gene Therapy, CRC Press, Ann Arbor, Mich. (1995), Vega et al., Gene Targeting, CRC Press, Ann Arbor Mich. (1995), Vectors: A Survey of Molecular Cloning Vectors and Their Uses, Butterworths, Boston Mass. (1988) and Gilboa et at. [Biotechniques 4 (6): 504-512, 1986] and include, for example, stable or transient transfection, lipofection, electroporation and infection with recombinant viral vectors.

Introduction of nucleic acids by viral infection offers several advantages over other methods such as lipofection and electroporation, since higher transfection efficiency can be obtained due to the infectious nature of viruses.

Other vectors can be used that are non- viral, such as cationic lipids, polylysine, and dendrimers. Nanoparticles are also contemplated.

Other modes of transfection that do not involved integration include the use of minicircle DNA vectors or the use of PiggyBac transposon that allows the transfection of genes that can be later removed from the genome. According to a particular embodiment, the mesenchymal stem cells are genetically modified to express a miRNA (or group of miRNAs) in order to induce differentiation. Alternatively, or additionally, the mesenchymal stem cells are genetically modified to express a polynucleotide agent that down regulates a miRNA (or group of miRNAs) in order to induce differentiation. The term "microRNA", "miRNA", and "miR" are synonymous and refer to a collection of non- coding single- stranded RNA molecules of about 19-28 nucleotides in length, which regulate gene expression. miRNAs are found in a wide range of organisms and have been shown to play a role in development, homeostasis, and disease etiology.

Tumorigenic activity Advantageously, the cells of the invention lack in vivo tumorigenic activity. Thus, said cells are characterized in that they do not present tumorigenic activity, i.e., they do not present an altered behavior or proliferative phenotype which gives rise to a tumor cell. The maintenance conditions of the cells of the invention can also contain cellular factors that allow cells to remain in an undifferentiated form. It is apparent to those skilled in the art that prior to differentiation; supplements that inhibit cell differentiation must be removed from the culture medium. It is also apparent that not all cells will require these factors. In fact, these factors may elicit unwanted effects, depending on the cell type.

MULTIPOTENT STEM CELL OF THE INVENTION

In a third aspect, the invention relates to an isolated hPC, preferably a multipotent stem cell, obtainable by the method of expanding and maintaining multipotent stem cells of the invention, hereinafter multipotent stem cell of the invention.

The isolated adult stem cell of the invention is characterized in that it has a distinctive expression level for certain markers.

The examples of the invention show that the culture media of the invention are useful for the maintenance and expansion of progenitor cells, such as for example human Mesenchymal Stem Cells. After three successive passages the cells display a good expression profile measured by flow cytometry of positive immunological markers CD90, CD105, CD73 that is a distinctive sign of the maintenance of their multipotent characteristics. Meanwhile, these cells have a very low differentiation degree since these lack expression of CD45 (pan-leukocyte marker), CD34 (primitive hematopoietic progenitors) and HLA class II markers. Therefore, in a preferred embodiment of the third aspect of the invention, the multipotent stem cells cultured in any of the culture media of the first aspect of the invention, exhibit a good expression profile as measured by flow cytometry of positive immunological markers CD90, CD105, CD73 following at least 2 passages, and more preferably, following at least 3 passages.

In another preferred embodiment, the multipotent stem cells cultured in said culture medium lack expression of CD45 (pan-leukocyte marker), CD34 (primitive hematopoietic progenitors) and HLA class II markers.

As shown in the examples, the cells cultured with 2μΜ of DETA NO show a higher number of cells positive for Ki67 than cells cultured without nitric oxide donors, a cellular marker for proliferation (Scholzen T, Gerdes J: The Ki-67 protein: from the known and the unknown. J Cell Physiol 2000, 182:31 1 -322).

Cell-surface markers can be identified by any suitable conventional technique, usually based on a positive/negative selection; for example, antibodies can be used, preferably monoclonal antibodies against cell-surface markers, whose presence/absence in the cells has to be confirmed, can be used with the aim of characterizing the stem cells of the invention on the basis of their immunocytochemical profile, although other conventional techniques known by a person skilled in the art can also be used, for example, RT-PCR.

"Marker" refers to a biological molecule whose presence, concentration, activity, or phosphorylation state may be detected and used to identify the phenotype of a cell.

The term "expressed" is used to describe the presence of a marker within a cell. In order to be considered as being expressed, a marker must be present at a detectable level.

By "detectable level" is meant that the marker can be detected using one of the standard laboratory methodologies such as PCR, blotting or FACS analysis. A gene is considered to be expressed by a cell of the population of the invention if expression can be reasonably detected after 30 PCR cycles, which corresponds to an expression level in the cell of at least about 100 copies per cell. The terms "express" and "expression" have corresponding meanings. At an expression level below this threshold, a marker is considered not to be expressed. The comparison between the expression level of a marker in an adult stem cell of the invention, and the expression level of the same marker in another cell, such as for example an embryonic stem cell, may preferably be conducted by comparing the two cell types that have been isolated from the same species. Preferably this species is a mammal, and more preferably this species is human.

Such comparison may conveniently be conducted using a reverse transcriptase polymerase chain reaction (RT-PCR) experiment.

"Fluorescence activated cell sorting (FACS)" is a method of cell purification based on the use of fluorescent labeled antibodies. The antibodies are directed to a marker on the cell surface, and therefore bind to the cells of interest. The cells are then separated based upon the fluorescent emission peak of the cells. "Natural expression" refers to the endogenous expression of one or more genes in a cell. For expression to be considered natural, the cell will express the gene without the need for any recombinant manipulation to introduce the gene or any of its regulatory elements into the cell or to modulate these genes' expression by introduction of exogenous genetic material. The term "recombinant manipulation" refers to any sort of manipulation of the genetic material contained within the cell, wherein genetic material is combined with other genetic material with which it is not naturally associated. This includes, by way of example only, gene insertion, gene deletion, and insertion of a heterologous (non-natural) or stronger promoter or other regulatory element operably linked to either the endogenous gene or an exogenously introduced version of the gene, including insertion of an exogenous promoter or regulatory element, or insertion of an endogenous promoter or regulatory element at a position at which it would not be expected to occur. The naturally expressed gene will not contain or be associated with any heterologous sequences, and in particular, will not contain any retroviral sequences, whether promoter sequences, regulatory sequences, or otherwise.

Natural expression is from genomic DNA within the cells, and so each gene that is naturally expressed may include introns between the exons within its coding sequence. The naturally expressed gene will show an intron-exon structure which is identical to that found within a non- manipulated cell. Natural expression is not from cDNA. Natural expression can if necessary be proven by any one of various methods, such as sequencing out from within the reading frame of the gene to check that no extraneous heterogenous sequence is present. The copy number of the gene can also be checked as the natural copy number, for example, using a technique such as fluorescence in situ hybridisation. The gene will thus be present in the genome in its natural genome context, and the histone condensation state will be such as to allow appropriate expression of the gene. The terms "naturally expressing", "naturally expresses", and "naturally expresses" have their corresponding meanings.

In another preferred embodiment of the third aspect of the invention, the isolated multipotent stem cells of the invention are mammalian multipotent stem cells. More preferably the mammalian multipotent cells are human multipotent cells (hMC). In another preferred embodiment, the multipotent cells are mesenchymal stem cells (MSCs), more preferably are mammalian mesenchymal stem cells and more preferably, human mesenchymal stem cells (hMSCs). In a still more preferably embodiment, the hMSCs are adipose-derived hMSC.

In a fourth aspect, the invention relates to an isolated cell population, hereinafter isolated cell population of the invention, comprising at least an isolated progenitor cell of the invention.

In another preferred embodiment the isolated progenitor cells of the invention are mammalian progenitor cells. More preferably the mammalian multipotent cells are human multipotent stem cells (hMSC).

More preferably, the isolated cell population of the invention comprises at least a 60%, and still more preferably at least a 70%, 80%, 90%, 95%, 98% and particularly a 99% of multipotent stem cells of the invention. The multipotent stem cells of the invention are characterized in that express the markers described above.

The adult stem cell population of the invention is considered to express a marker if at least about 70% of the cells of the population show detectable expression of the marker. In other aspects, at least about 80%, at least about 90% or at least about 95% or at least about 97% or at least about 98% or more of the cells of the population show detectable expression of the marker. In certain aspects, at least about 99% or 100% of the cells of the population show detectable expression of the markers. Expression may be detected through the use of an RT- PCR experiment or through fluorescence activated cell sorting (FACS). It should be appreciated that this list is provided by way of example only, and is not intended to be limiting.

If desired, the stem cells of the invention can be expanded clonally using a method that is suitable for cloning cell populations. Alternatively, a population of stem cells of the invention can be collected and the cells can be plated on separate plates (or in the wells of a multiwell plate). In another alternative embodiment, said stem cells can be subcloned on a multiwell plate in a random relation to facilitate the operation of placing a single cell in each well. Of course, the stem cells of the invention can be cloned at low density (e.g. in a Petri dish) and can be isolated from other cells using suitable devices (e.g. cloning rings). The clonal population can be expanded in a suitable culture medium.

The stem cells as well as the cells present in the cell population of the invention can be cells of autologous, allogeneic or xenogeneic origin. In a particular embodiment, said cells are of autologous origin and are isolated from the adipose tissue of the subject to whom they will be administered, thus reducing potential complications associated with antigenic and/or immunogenic responses to said cells.

CELL CULTURE SUPERNATANT OR MEDIA

Further, fractions of media conditioned by multipotent stem cells or the cell culture supernatant are a source of various factors that either alone or in combination with one another have shown an ability to affect other cells. Accordingly, the conditioned media has been shown to be an excellent source of material useful for producing, concentrating, and isolating a broad spectrum combination of compounds in relative percentages and forms that cause a therapeutic effect when administered to an individual suffering from a disease.

The stem cell conditioned media or the cell culture supernatant of the invention is produced by a method comprising the steps of culturing at least one stem cell in a cell culture medium of the invention, and collecting a quantity of the cell culture medium or the cell culture supernatant after a culture duration.

Consequently, a fifth aspect of the invention relates to a cell culture supernatant or media, hereinafter cell culture supernatant of the invention, obtainable from the culture of progenitor cells in the culture medium of the invention. In a preferred embodiment, the cell culture supernatant is concentrated to a concentration selected from the group of at least 50-fold, between 50-fold and 100-fold, at least 100-fold, between 100-fold and 250-fold, and at least 250-fold. In another embodiment, the cell culture supernatant is fractionated to remove substances less than a specified kDa range selected from the group consisting of less than about 5 kDa, less than about 10 kDa, less than about 20 kDa, less than about 30 kDa, less than about 40 kDa, less than about 50 kDa, and greater than about 50 kDa. In yet another embodiment, the cell culture supernatant is acted upon using a process selected from centrifugation, concentration, fractionation, lyophilization, freeze-drying, or combinations thereof.

COMPOSITION OF THE INVENTION

In a sixth aspect, the invention relates to a composition, hereinafter a composition of the invention, comprising an isolated multipotent stem cell of the invention, the isolated cell population of the invention, the culture media of the invention after expanding and maintaining multipotent stem cells, and/or the cell culture supernatant of the invention.

In a preferred embodiment the composition of the invention also comprises a pharmaceutically acceptable carrier or excipient. In another preferred embodiment the composition of the invention also comprises another active ingredient. In another preferred embodiment the composition of the invention is a pharmaceutical composition. As used herein, the term "active ingredient", "active substance", "pharmaceutically active substance", "active ingredient" or "pharmaceutically active ingredient" means any component which potentially provides a pharmacological activity or another different effect in diagnosing, curing, mitigating, treating, or preventing a disease, or which affects the structure or function of the human body or body of other animals. Examples of active ingredients of biological origin include growth factors, hormones, and cytokines. A variety of therapeutic agents are known in the art and may be identified by their effects. Certain therapeutic agents are capable of regulating cell proliferation and differentiation. Examples include chemotherapeutic nucleotides, drugs, hormones, non-specific (non-antibody) proteins, oligonucleotides (e.g., antisense oligonucleotides that bind to a target nucleic acid sequence (e.g., mRNA sequence)), peptides, and peptidomimetics.

The pharmaceutical compositions of the present invention can be used in a treatment method in an isolated manner or together with other pharmaceutical compounds.

The term "pharmaceutically acceptable excipient" as used here refers to the fact that it must be approved by a regulatory agency of the federal government or a national government or one listed in the United States Pharmacopoeia or the European Pharmacopoeia, or some other pharmacopoeia generally recognized for use in animals and in humans. The term "vehicle" relates to a diluent, excipient, carrier or adjuvant with which the stem cells, progenitor cells or differentiated cells of the invention, the immortalized cells of the invention, as well as the cells of the cell population of the invention, must be administered; obviously, said vehicle must be compatible with the cells. Illustrative, non-limiting examples of said vehicle include any physiologically compatible vehicle, for example isotonic solutions (e.g. sterile saline solution (0.9% NaCI), phosphate -buffered saline solution (PBS), Ringer-lactate solution, etc.), optionally supplemented with serum, preferably with autologous serum; culture media (e.g. DMEM, RPMI, McCoy, etc.); or, preferably, a solid, semisolid, gelatinous or viscous support medium, such as collagen, collagen-glycosamine-glycan, fibrin, polyvinyl chloride, poly-amino acids, such as polylysine, or polyornithine, hydrogels, agarose, dextran sulphate silicone. Moreover, if desired, the support medium can, in special embodiments, contain growth factors or other agents. If the support is solid, semisolid, or gelatinous, the cells can be introduced in a liquid phase of the vehicle that is treated subsequently so that it is converted into a more solid phase. In some embodiments of the invention in which the vehicle has a solid structure, said vehicle can be configured according to the form of the lesion.

In a more preferred embodiment, the vehicle is the culture medium of the invention.

The pharmaceutical composition of the invention can, if desired, also contain, when necessary, additives for increasing and/or controlling the desired therapeutic effect of the cells, e.g. buffering agents, surface-active agents, preservatives, etc. The pharmaceutically acceptable carrier may comprise a cell culture medium which supports the cells' viability. The medium will generally be serum-free in order to avoid provoking an immune response in the recipient. The carrier will generally be buffered and/or pyrogen free. Also, for stabilizing the cellular suspension, it is possible to add chelating agents of metals. The stability of the cells in the liquid medium of the pharmaceutical composition of the invention can be improved by adding additional substances, such as, for example, aspartic acid, glutamic acid, etc. Said pharmaceutically acceptable substances that can be used in the pharmaceutical composition of the invention are generally known by a person skilled in the art and are normally used in the production of cellular compositions. Examples of suitable pharmaceutical vehicles are described in "Remington's Pharmaceutical Sciences" by E.W. Martin. Additional information on said vehicles can be found in any manual of pharmaceutical technology (that is, galenical pharmacy).

The pharmaceutical composition of the invention will be administered in a suitable pharmaceutical form of administration. For this, the pharmaceutical composition of the invention will be formulated according to the chosen form of administration. The formulation will be adapted to the method of administration. In a special embodiment, the pharmaceutical composition is prepared in a liquid, solid or semisolid dosage form, e.g. in the form of suspension, in order to be administered by implanting, injection or infusion to the subject needing treatment. Illustrative, non- limiting examples include formulation of the pharmaceutical composition of the invention in a sterile suspension with a pharmaceutically acceptable excipient, e.g. an isotonic solution, for example, phosphate-buffered saline solution (PBS), or any other suitable, pharmaceutically acceptable vehicle, for administration to a subject parenterally, although other routes of administration can also be used.

The administration of the pharmaceutical composition of the invention to the subject who needs it will be carried out using conventional means. In a particular embodiment, said pharmaceutical composition of the invention can be administered to the subject parenterally using suitable devices such as syringes, catheters, trocars, cannulas, etc. In all cases, the pharmaceutical composition of the invention will be administered using equipment, apparatus and devices suitable for the administration of cellular compositions and known by a person skilled in the art. In another embodiment, direct administration of the pharmaceutical composition of the invention to the site that is intended to benefit may be advantageous. In this method, direct administration of the pharmaceutical composition of the invention to the desired organ or tissue can be achieved by direct administration (e.g. by injection, etc.) on the external surface of the affected organ or tissue by inserting a suitable device, e.g. a suitable cannula, by infusion (including reverse flow mechanisms) or by other means described in this patent or known in the art.

The pharmaceutical composition of the invention can be stored until the moment of its application by the conventional methods known by a person skilled in the art. For short-term storage (less than 6 hours), the pharmaceutical composition of the invention can be stored at or below room temperature in a sealed container, supplemented or not with a nutrient solution. Medium-term storage (less than 48 hours) is preferably carried out at 2-8[deg.]C, and the pharmaceutical composition of the invention includes, in addition, an iso-osmotic, buffered solution in a container made of or lined with a material that prevents cellular adhesion. Longer- term storage is preferably carried out by means of suitable cryopreservation and storage in conditions that promote the retention of cellular function. In a concrete embodiment, the pharmaceutical composition of the invention can be used in combination therapy. Said additional medicinal products can form part of the same pharmaceutical composition or can, alternatively, be supplied in the form of a separate composition for simultaneous or successive (sequential in time) administration relative to the administration of the pharmaceutical composition of the invention. THERAPEUTIC USES OF THE INVENTION

In a seventh aspect, the invention relates to the isolated multipotent stem cell of the invention, the isolated cell population of the invention, the cell culture supernatant of the invention and/or the composition of the invention for use in medicine.

Said medicament is a medicament for somatic cell therapy. "Somatic cell therapy" is understood as the use of living, autologous, allogenic or xenogenic somatic cells, the biological characteristic of which have been substantially altered as a result of their manipulation for obtaining a therapeutic, diagnostic or preventive effect through metabolic, pharmacological or immunological means. Among the medicaments for somatic cell therapy are, for example, but not limited to: cells manipulated to modify their immunological, metabolic or other type of functional properties in qualitative and quantitative aspects; sorted, selected and manipulated cells which are subsequently subjected to a manufacturing process for the purpose of obtaining the end product; cells manipulated and combined with non-cellular components (for example, biological or inert matrices or medical devices) performing the principle intended action in the finished product; autologous cell derivatives expressed ex vivo (in vitro) under specific culture conditions; cells which are genetically modified or are subjected to another type of manipulation to express homologous or non-homologous functional properties not expressed before.

An infectious, inflammatory, genetic or degenerative disease, physical or chemical damage, or blood flow interruption, can cause cell loss from a tissue or organ. This cell loss would lead to an alteration of the normal function of said tissue or organ; and consequently lead to the development of diseases or physical consequences reducing the person's quality of life. Therefore, attempting to regenerate or and reestablish the normal function of said tissues or organs is important. The damaged tissue or organ can be replaced by a new tissue or organ which has been produced in the laboratory by means of tissue engineering techniques.

Then, in a eighth aspect, the invention relates to the isolated progenitor cell of the invention, the isolated cell population of the invention, the cell culture supernatant of the invention and/or the composition of the invention for use to partially or completely increase, restore or replace the functional activity of a diseased or damaged tissue or organ.

The cells may also be transplanted to a healthy region of the tissue. In some cases the exact location of the damaged tissue area may be unknown and the cells may be inadvertently transplanted to a healthy region. In other cases, it may be preferable to administer the cells to a healthy region, thereby avoiding any further damage to that region. Whatever the case, following transplantation, the cells preferably migrate to the damaged area. For transplanting, the cell suspension is drawn up into the syringe and administered to anesthetized transplantation recipients. Multiple injections may be made using this procedure. MSCs possess remarkable immunosuppressive properties and can inhibit the proliferation and function of the major immune cell populations, including T cells, B cells and natural killer (NK) cells; modulate the activities of dendritic cells (DCs); and induce regulatory T cells both in vivo and in vitro. These unique properties make MSCs ideal candidates for clinical application as immunosuppressants. The immunomodulatory effect of MSCs is mediated by a non-specific anti-proliferative action of these cells, which is dependent on cell-cell contact or secreted soluble factors such as indoleamine 2,3-dioxygenase (IDO), prostaglandin E2 (PGE2), nitric oxide (NO), histocompatibility leucocyte antigen-G (HLA-G), transforming growth factor (TGF)-3, interferon (IFN)-y and interleukin (IL)-1 β (Shi et al., Clin Exp Immunol. 201 1 Apr; 164(1 ): 1-8).

Then, in an ninth aspect the present invention relates to the isolated progenitor cell of the invention, the isolated cell population of the invention, the cell culture supernatant of the invention and/or the composition of the invention for use in the treatment, prevention or amelioration of metabolic disease, degenerative diseases, infective diseases and immune diseases.

In another preferred embodiment the metabolic disease is selected from the list consisting of diabetes type 1 and type 2 andmetabolic syndrome. In another preferred embodiment the degenerative diseases selected from the list consisting of Acute coronary syndrome, Congestive heart failure, Cardiomyopathy, Angina, Congenital disease, Peripheral vascular disease, Cardiac hypertrophy

In another preferred embodiment the infective diseases selected from the list consisting of sepsis associated acute kidney injury, acute lung injury.

In a preferred embodiment the immune disease is selected from the list consisting of graft- versus-host disease or solid organ transplant rejection.

In another preferred embodiment the autoimmune disease is selected from the list consisting of rheumatoid arthritis, multiple sclerosis, Type I diabetes, Crohn's disease, Guillain-Barre syndrome, lupus erythematosus, myasthenia gravis, optic neuritis, psoriasis, Graves' disease, Hashimoto's disease, Ord's thyroiditis, aplastic anemia, Reiter's syndrome, autoimmune hepatitis, primary biliary cirrhosis, antiphospholipid antibody syndrome, opsoclonus myoclonus syndrome, temporal arteritis, acute disseminated encephalomyelitis, Goodpasture's syndrome, Wegener's granulomatosis, coeliac disease, pemphigus, polyarthritis, warm autoimmune hemolytic anemia, and scleroderma, or combinations thereof.

In another aspect, the invention relates to a method of preventing, treating, or ameliorating one or more symptoms associated with autoimmune diseases, inflammatory disorders, or immunologically mediated diseases, in a subject suffering from any of said disorders or diseases, which comprises administering to said subject in need of such treatment of a prophylactically or therapeutically effective amount of said cell population.

As used in the specification and the appended claims the term "autoimmune diseases" is understood as diseases resulting from an immune response to self-components.

The term "immunoregulatory agent" refers to an agent that inhibits or reduces one or more biological activities of the immune system. An immunoregulatory agent is an agent that inhibits or reduces one or more biological activities (e.g., the proliferation, differentiation, priming, effector function, production of cytokines or expression of antigens) of one or more immune cells (e.g., T cells).

The term "immune disease" refers to a condition in a subject characterized by cellular, tissue and/or organ injury caused by an immunological reaction of the subject. The term "autoimmune disease" refers to a condition in a subject characterized by cellular, tissue and/or organ injury caused by an immunological reaction of the subject to its own cells, tissues and/or organs. Illustrative, non-limiting examples of autoimmune diseases which can be treated with the immunomodulatory cells of the invention include alopecia areata, ankylosing spondylitis, antiphospholipid syndrome, autoimmune Addison's disease, autoimmune diseases of the adrenal gland, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune oophoritis and orchitis, autoimmune thrombocytopenia, Behcet's disease, bullous pemphigoid, cardiomyopathy, celiac sprue-dermatitis, chronic fatigue immune dysfunction syndrome (CF1 DS), chronic inflammatory demyelinating polyneuropathy, Churg- Strauss syndrome, cicatrical pemphigoid, CREST syndrome, cold agglutinin disease, discoid lupus, essential mixed cryoglobulinemia, fibromyalgia-fibromyositis, glomerulonephritis, Graves' disease, Guillain- Barre, Hashimoto's thyroiditis, idiopathic pulmonary fibrosis, idiopathic thrombocytopenia purpura (ITP), IgA neuropathy, juvenile arthritis, lichen planus, Meniere's disease, mixed connective tissue disease, multiple sclerosis, type 1 or immune-mediated diabetes mellitus, myasthenia gravis, pemphigus vulgaris, pernicious anemia, polyarteritis nodosa, polychondritis, polyglandular syndromes, polymyalgia rheumatica, polymyositis and dermatomyositis, primary agammaglobulinemia, primary biliary cirrhosis, psoriasis, psoriatic arthritis, Raynauld's phenomenon, Reiter's syndrome, sarcoidosis, scleroderma, progressive systemic sclerosis, Sj ogren's syndrome, Good pasture's syndrome, stiff-man syndrome, systemic lupus erythematosus, lupus erythematosus, takayasu arteritis, temporal arteristis/giant cell arteritis, ulcerative colitis, uveitis, vasculitides such as dermatitis herpetiformis vasculitis, vitiligo, Wegener's granulomatosis, Anti-Glomerular Basement Membrane Disease, Antiphospholipid Syndrome, Autoimmune Diseases of the Nervous System, Familial Mediterranean Fever, Lambert-Eaton Myasthenic Syndrome, Sympathetic Ophthalmia, Polyendocrinopathies, Psoriasis, etc.

The term "Immune mediated inflammatory disease" shall be taken to mean any disease characterized by chronic or acute inflammation, resulting from, associated with or triggered by, a dysregulation of the normal immune response e.g. Crohn's disease, type 1 diabetes mellitus, rheumatoid arthritis, inflammatory bowel disease, psoriasis, psoriatic arthritis, ankylosing spondylitis, systemic lupus erythematosus, Hashimoto's disease, graft-versus-host disease, Sjogren's syndrome, pernicious anemia, Addison disease, scleroderma, Goodpasture's syndrome, ulcerative colitis, autoimmune hemolytic anemia, sterility, myasthenia gravis, multiple sclerosis, Basedow's disease, thrombopenia purpura, Guillain-Barre syndrome, allergy, asthma, atopic disease, arteriosclerosis, myocarditis, cardiomyopathy, glomerular nephritis, hypoplastic anemia, and rejection after organ transplantation.

The term "inflammatory disease" refers to a condition in a subject characterized by inflammation, e.g., chronic inflammation. Illustrative, non-limiting examples of inflammatory disorders include, but are not limited to, Celiac Disease, rheumatoid arthritis (RA), Inflammatory Bowel Disease (IBD), asthma, encephalitis, chronic obstructive pulmonary disease (COPD), inflammatory osteolysis, allergic disorders, septic shock, pulmonary fibrosis (e.g. , idiopathic pulmonary fibrosis), inflammatory vacultides (e.g. , polyarteritis nodosa, Wegner's granulomatosis, Takayasu's arteritis, temporal arteritis, and lymphomatoid granulomatosus), post-traumatic vascular angioplasty (e.g. , restenosis after angioplasty), undifferentiated spondyloarthropathy, undifferentiated arthropathy, arthritis, inflammatory osteolysis, chronic hepatitis, and chronic inflammation resulting from chronic viral or bacterial infections.

As used herein, the terms "treat", "treatment" and "treating" refer to the amelioration of one or more symptoms associated with a disorder including, but not limited to, an inflammatory disorder, an autoimmune disease or an immunologically mediated disease including rejection of transplanted organs and tissues, that results from the administration of the cell population of the invention,

METHOD OF GENERATING LINEAGE-SPECIFIC CELLS The invention contemplates that once stem cells have been established in culture, their ability to serve as progenitors for mature cells or cell lines can be maintained, for example, by regular passage to fresh medium as the cell culture reaches an appropriate density or percentage of confluency, or by treatment with an appropriate growth factors, or by modification of the culture medium or culture protocol, or by some combination of the above. The cells of the invention present the capacity to proliferate and be differentiated into at least two, more preferably three, four, five, six, seven or more cell lineages. Illustrative, non-limiting examples of cell lineages in which the cells of the invention can be differentiated include osteocytes, adipocytes, chondrocytes, tenocytes, myocytes, cardiomyocytes, hematopoietic-supporting stromal cells, endothelial cells, neurons, astrocytes, and hepatocytes. Cells of the invention can proliferate and differentiate into cells of other lineages by conventional methods. Methods of identifying and subsequently isolating differentiated cells from their undifferentiated counterparts can be also carried out by methods well known in the art. The cells of the invention are also capable of being expanded ex vivo.

Then, in a tenth aspect, the present invention relates to a method of generating lineage- specific cells from progenitor cells, the method comprising: (a) culturing the progenitorcells of the invention, to thereby obtain expanded, progenitorcells that maintain their potency;

(b) subjecting said expanded, progenitor cells to culturing conditions suitable for differentiating and/or expanding lineage specific cells;ln another preferred embodiment the isolated progenitor cells of the invention are mammalian progenitor cells. More preferably the mammalian multipotent cells are human multipotent stem cells (hMSC)..

The term "lineage specific cell" refers to a cell that is no longer mutipotent but is committed towards a particular cell type. The lineage specific cell may be a progenitor cell or a fully differentiated cell. Selection of particular lineage specific cells towards which the PCs are differentiated is dependent on the required qualification. Thus, for example, if the MSCs are being qualified as being suitable for the autologous treatment of a brain disease, the lineage specific cell is selected according to the brain disease for which the MSCs are intended to treat.

In an eleventh aspect, the present invention relates to a method for preparing a culture medium of the invention, including the addition/mixing of an effective amount of at least one nitric oxide donor to a culture medium as defined previously.

In an twelfth aspect, the present invention relates to a method of production of peptides and protein of biological interest comprising culture progenitor cells in the culture medium of the invention.

Examples of the invention

Example 1. Analysis of Cell Proliferation in a medium with low FBS

The pluripotency of Adipose Tissue (PCS50001 ) human mesenchimal stem cells cultured in media with low concentrations of FBS and suplemented with NO was studied by BrdU incorporation, and compared with the the cells cultured in control medium (10% FBS). Figure 1 represents the cell pluripotency of each cultured condition. It can be observed that in all studied conditions have proliferation levels similar to the control, and that the most optimal condition is %% of FBS suplemented with 5 μΜ of DETA-NO.

Example 2. Analysis of Cell Proliferation by Ki-67 expression in a medium with low FBS

To confirm the viability of the medium with low FBS, the maintenance of proliferation was studied by analyzing Ki-67 expression by an inmunofluorescence assay. Figure 2 represents Adipose Tissue mesenchymal stem cells cultured in the control medium and in the low FBS medium supplemented with 5 μΜ of DETA-NO, and later stainned with DAPI, Ki-67 (red) and o Tubulin (green). Figure 2 demonstrates that the expression of Ki-67 protein is higher in the optimized medium (medium supplemented with 5 μΜ of DETA-NO).

Example 3. Analysis of Phenotipic Characteristics of Adipose Tissue mesenchymal stem cells cultured in a medium with low FBS suplemented with a nitric oxide donor.

To confirm the viability of the low FBS medium supplemented with 5 μΜ of DETA-NO, we analyzed the maintenance of the phenotypic characteristics of Adipose Tissue mesenchymal stem cells. Figure 3 represents the percentage of total cells that express each antibody by flow citometry. Figure 3 shows that cells cultured in the optimized medium present a phenotypic patron similar to the control.

Example 4. Analysis of Potential of Differentiation of Adipose Tissue human mesenchymal stem cells in a medium with low FBS suplemented with a nitric oxide donor

The capacity of Adipose Tissue mesenchymal stem cells to differentiate toward the germinal lawyer after being cultured in low FBS medium supplemented with 5 μΜ of DETA-NO was determined. Figure 4 shows that these cells maintained the potential to differentiate toward Adipocytes, Osteocytes and Chondrocites.

Example 5. Free-serum culture medium. Analysis of cell morphology.

The morphology of Bone marrow (PCS500012) and Adipose Tissue (PCS50001 ) human mesenchimal stem cells cultured in the optimized chemically defined medium was studied taking photos wih a microscope, and compared with the morphology of cells cultured in control medium. A culture protocol was performed during 42 days, passing cells each 7 days. Figure 5 illustrates the cell morphology and the cell density at the end of each cell passing. It can be observed that the morphology of the cells cultured in the chemically defined medium is similar to the morphology of those cells cultured in the control medium, and the cell density was higher in the plates of cells cultured with the chemically defined medium.

Example 6. Analysis of Cell Proliferation in continuous culture protocol

The proliferation of Bone marrow (PCS500012) and Adipose Tissue (PCS50001 ) human mesenchimal stem cells cultured in the optimized chemically defined medium was studied by counting number of total cells, and compared with the proliferation of the cells cultured in control medium. A continuous culture protocol was performed during 42 days, passing cells each 7 days, an counting them at this point. Figure 6 represents the cell proliferation at the end of each cell passing. It can be observed that the proliferation of the cells cultured in the chemically defined medium increases respect to the cells cultured in the control medium, this constitutes a very considerable increased in Bone Marrow mesenchymal stem cells. Example 7. Analysis of Cell Proliferation

To confirm the viability of the optimized chemically defined medium, the maintenance of proliferation was studied by analyzing Ki-67 expression by an inmunofluorescence assay. Figure 7 illustrates Bone Marrow human mesenchymal stem cells cultured in the control medium and in the chemically defined medium, and later stainned with DAPI, Ki-67 (red) and o Tubulin (green). It could be proved that the expression of Ki-67 protein was similar in the cells cultured with both media.

Example 8. Analysis of Phenotipic Characteristic

To confirm the viability of the optimized chemically defined medium, the pluripotency and the maintenance of the phenotypic characteristic in Bone Marrow mesesnchymal stem cells was analyzed. Figure 8 illustrates the expression of pluripotent and mesenchymal phenotypic characteristic genes by cualitative PCR. The image shows that the cells cultured in chemically defined medium maintain pluripotency expressig Nanog and Oct4, express the mesenchymal stem cells surface markers Nestin and Integrin, and express the positive markers CD105, CD90 y CD73.

Example 9. Analysis of the Potential of Differentiation of Adipose Tissue human mesenchymal stem cellsina serum-free medium with 75% of bFGF supplemented with 2 uM of NO

The capacity of Adipose Tissue mesenchymal stem cells to differentiate toward the germinal lawyer after being cultured in a serum-free medium with 75% of bFGF supplemented with 2 μΜ of NOwas determined, and it was proved they maintained the potential to differentiate toward Adipocytes, Osteocytes and Condrocites. This is shown in figure 9.