EP3191583A1 - Method for obtaining pancreatic beta cell surrogates by increasing pancreatic and duodenal homeobox 1 (pdx-1) expression - Google Patents

Abstract

Insulin producing cells capable of secreting blood glucose regulating hormones, specially insulin, in response to physiological signals, method of production of said cells and uses thereof.

Description

Method for obtaining pancreatic beta cell surrogates bv increasing pancreatic and duodenal homeobox 1 (PDX-1) expression

FIELD OF THE INVENTION The invention relates to the field of medical treatments and drug screening, more specifically to compounds, compositions and methods useful for treating insulin related diseases and conditions associated with insufficient insulin production, including diabetes and especially type 1 diabetes and type 1 diabetes-related conditions or symptoms thereof. Also relates to the field of regenerative medicine and tissue engineering, and more specifically to the use of insulin- producing cells for preparing a medicament to partially or completely increase, restore or replace the functional activity of a diseased or damaged tissue or organ. Also relates to the use of insulin-producing cells for testing the effect small molecules, biologicals and pharmaceutical compositions.

BACKGROUND OF THE INVENTION

Generation of definitive endoderm from embryonic stem (ES) cells and subsequent differentiation to pancreatic progenitors and pancreatic beta cell surrogates have been reported over the last years(Soria, 2000; Soria 2001 ; Leon Quinto ef al, 2004, Vaca et al., 2006, 2008; D'Amour, Bang et al, 2006; Kroon, Martinson et al., 2008; Zhang, Jiang et al., 2009; Mora-Castilla, Tejedo et al, 2010). The biology of pancreas development might provide clues to improve current differentiation protocols. In this regard, the transcription factor pancreatic and duodenal homeobox 1 (Pdx-1) stands out among the regulators of the pancreatic development and differentiation towards beta cells (Ahlgren, Jonsson ef al, 1996; Stoffers, Zinkin et al., 1997). PDX-1 gene expression is regulated by the interaction of a set of transcription factors with the 6Kb promoter region of the gene. The promoter contains four domains that are conserved in chicken, human and mouse genes and was subdivided based upon this property into areas I (2839 to 2520 bp), II (2252 to 2023 bp), and III (1939 to 1664 bp). A species-specific PDX-1 function was associated with area II due to it is only presence in the mammalian gene (Gerrish, Gannon et al., 2000; Gerrish, Van Velkinburgh et al., 2004). Additionally, it has been suggested that epigenetic mechanisms may be responsible for PDX-1 regulation via changes in DNA methylation, histone modifications or non-canonical chromatin protein binding (Park, Stoffers ef al, 2008).

On the other hand, Polycom b Repressor Complex 2 (PRC2) plays a role in the regulation of key developmental genes in embryonic stem cells (ESC) (Schuetteng ruber, Chourrout et al. 2007; Simon and Kingston 2009). PCR2 catalyzes di-tri methylation of histone H3 Lys 27 residue (H3K27me2/3), which has been associated with the silencing of target genes (Boyer, Plath ef al., 2006; Lee, Jenner et al., 2006; Schuettengruber, Chourrout ef al., 2007; Simon and Kingston 2009). PRC2 recruitment to target genes is dependent on JARID-2, a regulator of histone methyltransferase complexes that plays an essential role in embryonic development. JARID-2 expression is regulated by self-renewal factors, OCT4, SOX2 and NANOG. These factors control the expression of a number of genes involved in embryonic development and are critical for early embryogenesis and for ES cells pluri potency (Peng, Valouev et al., 2009; Landeira, Sauer et al., 2010; Pasini, Cloos ef al., 2010) (Boyer, Lee et al., 2005; Loh, Wu et al., 2006; Kim, Chu et al., 2008). Indeed, PCR2 activity represses Pdx-1 gene expression (Peng, Valouev et al., 2009) and epigenetic changes of Pdx-1 gene have been reported in ES cells during generation of pancreatic endoderm (PE) (Xie, Everett et al. 2013). On the other hand, persistence of both H3Ac and H3K27me3 marks on the silent Pdx-1 gene in hepatoblasts has been reported (Xu, Cole et al., 201 1 ). Acetylation status of regulatory genes is also involved in developmental processes, .for example acetylation of Histones by Histone acetyltransferase p300. P300 acetylase activity has been reported to be involved in histone acetylation of liver target elements and attendant liver gene activation (Xu, Cole et al., 2011). P300 can also act as repressor independently of its histone acetylase activity (Girdwood, Bumpass et al., 2003; Ma, Nguyen ef al., 2005; Chen, Jia ef al., 2013); (Baluchamy, Rajabi et al., 2003). P300 acetylase activity is inhibited by C646: 4-[4-[[5-(4,5-Dimethyl-2-nitrophenyl)-2-furanyl]methylene]-4,5- dihydro-3-methyl-5-oxo-1 H-pyrazol-1 -yljbenzoic acid (C646: p300i).

Nitric oxide (NO) is a regulator of stem cell fate and successful generation of definitive endoderm from ES cells has been achieved by addition of chemical NO donors, such as DETA- NO (Z)-1-[N-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1 ,2-diolate) in differentiation protocols with transient expression of Pdx-1 during the process (Mora-Castilla, Tejedo ef al., 2010).

Type 1 diabetes (previously known as insulin-dependent or childhood-onset diabetes) is characterized by a lack of insulin production. The cause of type 1 diabetes is not known and it is not preventable with current knowledge. This form develops most frequently in children and adolescents, but is being increasingly noted later in life. The incidence of type 1 diabetes and insulin-related conditions or symptoms increased worldwide in the closing decades of the 20th century, especially in childhood. Then, it is necessary to develop new pharmaceutical compositions and treatments for type 1 diabetes and insulin-related conditions. BRIEF DESCRIPTION OF THE INVENTION

A first aspect of the present invention refers to an insulin-producing cell, hereinafter insulin- producing cell of the invention, obtained by a method comprising the addition of a P300 inhibitor to a stem cell culture. This method is referred hereinafter as method of the invention. In a preferred embodiment, the P300 inhibitor is selected from the list consisting on: 2,6-Bis[{3- bromo- -hydroxyphenyl)methylene]cyclohexanone (CAS 932749-62-7), garcinol (Camboginol, CAS 78824-30-3), anacardic acid (CAS 16611 -84-0), curcumin (CAS 458-37-7), demethoxy curcumin (CAS 22608-11 -3) and C646(P300i), or combinations thereof. In a more preferred embodiment, the P300 inhibitor is C646(P300i). In another preferred embodiment, the stem cell is a pluripotent stem cell. More preferably, the stem cell is selected from induced plutipotent stem cell, an hESC obtained whithout the destruction of an embryo, a cell obtained by Somatic Cell Nuclear Transfer (SCNT), a stem cell obtained by parthenogenesis, a cell obtained by transdifferentiation, or an adult pancreatic progenitor stem cell. In another preferred embodiment, the method of the invention also comprises culture the stem cell in presence of a donor of nitric oxide. More preferably, the donor of nitric oxide is selected from the list consisting on: sodium nitroprusside, 3-morpholino-sydnonimine, furoxan CAS 1609, S-nitroso-acetylpenicillamine, S-nitroso-glutathione, sildenafil citrate, nitroprusside, DETA-NO or combinations thereof. In a more preferred embodiment, the donor of nitric oxide is DETA-NO. In another preferred embodiment, the method of the invention also comprises culture the stem cell in presence of inhibitors of histone deacetylases. More preferably the inhibitor is selected from the list consisting on: Valproic acid, vorinostat (SAHA), tefinostat (CHR-3996), benzoic acid derivatives such as givinostat (ITF2357), mocetinostat (MGCD0103) and entinostat (MS-275); cynamic acid derivatives such as panobinostat (LBH589), beiinostat (PXD101) and pracinostat (SB939) and derivatives of pyrimidin-5-carboxoxilic acid CHR-3996 and quisinostat (SB939), or combinations thereof. In a more preferred embodiment, the inhibitor of histone deacetylases is valproic acid.

In another preferred embodiment, the method of the invention also comprises culture the cell in suspension to obtain cell clusters or methods to obtain cellular aggregates. More preferably, the cells are cultured until the expression of the pancreatic markers selected from the list consisting on: PDX-1 , Nkx6.1 , Insulin, Hexokinase IV or Glucokinase, the ATP-dependent potassium channels component Kir6.2 and/or Glut2 (glucose transporter 2). Still more preferably, the cells express all the markers. A second aspect of the present invention refers to an insulin-producing cell population, hereinafter insulin-producing cell population of the invention, comprising at least an insulin- producing cell of the invention.

A third aspect of the present invention refers to a composition comprising an insulin-producing cell of the invention, or an insulin-producing cell population of the invention. More preferably, the composition of the invention is a pharmaceutical composition. Still more preferably, the composition of the invention further comprises a pharmaceutically acceptable carrier.

A fourth aspect of the present invention refers to an tissue or organ comprising an insulin- producing cell of the invention, the insulin-producing cell population of the invention, or the composition of the invention, preferably in combination with a support material or a device.

A fifth aspect of the invention refers to the use of the insulin-producing cell of the invention, the insulin-producing cell population of the invention, the composition of the invention, or an artificial tissue or organ of the invention, for evaluating a pharmacological and/or chemical product.

A sixth aspect of the invention refers to the insulin-producing cell of the invention, the insulin- producing cell population of the invention, the composition of the invention, or an artificial tissue or organ of the invention, for use as a medicament.

A seventh aspect of the invention refers to the insulin-producing cell of the invention, the insulin-producing cell population of the invention, the composition of the invention, or an artificial tissue or organ of the invention, for partially or completely increase, restore or replace the functional activity of a diseased or damaged tissue or organ.

An eighth aspect of the invention refers to the insulin-producing cell of the invention, the insulin-producing cell population of the invention, the composition of the invention, or an artificial tissue or organ of the invention, for use in the treatment, amelioration or prevention of prediabetes or diabetes. A ninth aspect of the invention refers to the insulin-producing cell of the invention, the insulin- producing cell population of the invention, the composition of the invention, or an artificial tissue or organ of the invention, for use in the treatment, amelioration or prevention of type 1 and type 2 diabetes, diabetic ketoacidosis, hyperglycemia, diabetic neuropathy, diabetic cardiopathy, diabetic nefropathy, diabetic rethynopathy and other acute and chronic complications of diabetes like heart disease, stroke, kidney failure, foot ulcers and damage to the eyes.

A tenth aspect of the invention refers to the use of DETA-NO to promote Pdx.1 expression through changes in bivalents marks of histone H3k27me3 and H3K4me3, An eleventh aspect of the invention refers to the use of factors such as DETA-NO, valproic acid and p300 inhibitor to promote pluripotent stem cells differentiation into pancreatic beta cells like.

A twelfth aspect of the invention refers to a method to obtain an insulin-producing cell, previously referred as method of the invention, which promotes PDX-1 expression, beta cell maturation, insulin content and release, further comprising: a. obtaining a sample of stem cell, preferably pluripotent stem cells as defined previously, b. culture of pluripotent stem cells in a culture medium that's induces differentiation by adding or restrinting growth factors and small molecules c. using of DETA-NO to differentiate pluripotent stem cells, d. decreasing fetal bovine serum concentration by 10 to 10%, by 20 to 40%, by 40 to 60 % and by 60 to 99% e. using xeno free culture media f. using resveratrol (3,5,4'-trihydoroxytranstilbene) or other Sirtuin-1 activators at a concentration between 50 to 100 micromolar to induce maturation of insulin producing cells

In a preferred embodiment, the method of the invention also comprises: g. Transplanting these cells into a mammal needing endocrine cell therapy.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1. Nitric oxide increases Pdx1 expression in mouse embryonic stem cells (mESC). A) Relative expression of Pdx1 by real-time polymerase chain reaction (PGR). These values are normalized to the expression values of the β-actin, used as loading control and analyzed using AACt algorithm. It represents the average of three independent experiments. Data are ± SEM. The Y axis corresponds to relative expression level of Pdx1. B) Western blot of PDX1. It shows the results of Western blot of PDX1 in the four culture conditions, β-actin was used as loading control. The blot is representative of 3 independent experiments. C) PDX1 Immunofluorescence. Images show cells cultured in the four conditions and subsequently stained with DAP I (blue), PDX1 (Red) and a-TUBULIN (green). Scale bar, 25 μΜ. The images shown are representative of 3 independent experiments. D) Flow cytometry of PDX1. The green graph show the + LIF conditions and the blue graph show the - LIF. E) Panels A) and E): * P<0,05 statistically significant versus cells cultured in the presence of LIF, ** P<0,05 versus cells cultured in the presence and absence of LIF. +LIF: cells cultured in the presence of LIF; +L1FNO: cells cultured in the presence of LIF plus DETA-NO 500μ ; -LIF: cells cultured without LIF; -LIFNO: cells cultured without LIF plus DETA-NO 500μΜ. In the four conditions cells are cultured for 4 days and with DETA-NO for 19 hours.

Fig. 2. Treatment with NO changes the methylation pattern of Pdx1 promoter. A) Pdx1 promoter scheme. This image shows Pdx1 promoter and gene. Horizontal gray rectangles represent CpG islands and vertical lines are the CpG sites. Vertical gray rectangles represent the transcription start (TSS) and the translation start (+1 ; ATG). The arrows represent the pairs of primers designed to amplify the regions of interest. B) Methylation analysis of distal CpG Island and proximal CpG island by Bisulfite Sequencing PGR. It shows the CpG sites studied (distal CpG island and proximal CpG island). These sites are represented by circles; white circles those CpG sites whose percentage of methylation is very low or even zero, and blacks circles those CpG sites whose methylation level is too high or complete. Among them there is a gradient of methylation that is discussed in the legend of the image. C) Methylation analysis of distal CpG Island and proximal CpG island by Bisulfite Pyrosequencing. This graph represents the percentage of methylation of studied CpG sites. Each line represents a cell culture condition. Blue Line: cells cultured in the presence of leukaemia inhibitory factor (LIF); Purple Line: cells cultured in the presence of LIF plus DETA-NO 500μΜ; Yellow Line: cells cultured in the absence of LIF; Green Line: cells culture without LIF plus DETA-NO 500μΜ; and Red line: INS- 1 cells whose methylation level was used as control.

Fig. 3. Nitric oxide change the H3K27me3 and H3K4me3 occupancy balance but does not change the acetyiated H3 occupancy at Pdx1 promoter. A) Analysis of acetyiated H3 occupancy at Pdx1 by ChlP. This graph represents ChIP analysis for H3 acetyiated represent the relative percent input. B) Analysis of H3K27me3 and H3K4me3 occupancy at Pdx1 promoter by ChlP. This graph shows the H3K27me3 and H3K4me3 occupancy represented as percentage input relativized to pluripotency condition ChlP results. C) Stacked bars for bivalent marks dynamic H3K27me3 and H3K4me3. This graph is represented as a function of the results of the above graph in absolute values and in percent. It represents the average of 3 independent experiments. Data are ± SEM. The Y axis corresponds to the percentage of relative recovered input from control (undifferentiated cells). *P<0,05 versus cells cultured in the presence of LIF. +LIF: cells cultured in the presence of LIF; +LIFNO: cells cultured in the presence of LIF plus 500μΜ DETA-NO; -LIF: cells cultured in the absence of LIF; -LIFNO: cells cultured without LIF plus 500μΜ DETA-NO. In the four conditions cells are cultured for 4 days and with DETA-NO for 19 hours. Fig. 4. After NO treatment PRC2 is released from Pdx1 promoter. A) Study of Polycomb Complex PRC2 and P300 expression by real time PGR. Values are normalized to the expression of β-actin and analyzed using Ct algorithm. It represents the average of 3 independent experiments. Data are means ± SEM. The Y axis corresponds to relative expression level. B) Study of Polycomb Complex PRC2 and P300 expression by Western Blot. The image shown is representative of 3 independent experiments, β-actin, used as loading control. C) Analysis of JARID 2 and P300 occupancy at Pdxl proximal promoter by ChlP. Data are means ± SEM of independent experiments. The Y axis corresponds to relative percent input from undifferentiated cells. D) Co-immunoprecipitation assay of JARID-2, EZH2 and P300. 3% of total co-immunoprecipitated protein was loaded as input. Proteins not retained to complex agarose beads and JARID-2 antibody were loaded as control of immunoprecipitation grade (Flow Through). An immunoprecipitation with IgG antibody was developed as control of nonspecific binding (IgG Control). E) Pdx1 expression analysis by real-time PGR. Data are normalized to the expression values of the β-actin, used as loading control and analyzed using AACt algorithm. Data are means ± SEM of 3 independent experiments. The Y axis corresponds to relative expression level of Pdx1 , * P<0,05 versus cells cultured in the presence of LIF, ** P<0,05 versus cells cultured in the presence and absence of LIF, *^** P<0,05 versus cells cultured in the presence of LIF plus 500 μΜ DETA-NO. +LIF: ceils cultured in the presence of LIF; +LIFNO: cells cultured in the presence of LIF plus DETA-NO 500μΜ; -LIF: cells cultured without LIF; -LIFNO: cells cultured without LIF plus DETA-NO 500μΜ. In the four conditions cells are cultured for 4 days and with DETA-NO for 19 hours.

Fig. 5. Differentiation protocol to generate insulin-producing cells from mES cells. A) Differentiation protocol scheme showing the culture conditions. B) Cell morphology of each step of the differentiation protocol did with phase contrast in a Leica Microscope. C) Qualitative analysis by RT PGR for pancreatic markers for each step of differentiation protocol. Vise: Embryo viscera, Islets: Mouse islets used as control, β-actin was used as loading control cDNA. The image shown is representative of 3-5 independent experiments. D) Quantitative analysis by Real Time PGR for pancreatic markers. These values are normalized to the expression values of the β-actin, used as loading control and analyzed using AACl algorithm. Data shown are mean ± SEM of 3 independent experiments. The Y axis corresponds to relative expression. * P<0,05 statistically significant versus cells on day 0, ** P<0,05 versus cells on day 10, *·* P<0,05 versus cells on day 10 and 11 , *** P<0,05 versus cells on day 11.

Fig. 6. Islet-like clusters immunofluorescence obtained from differentiation protocol of mESC (D3). The left panel shows the immunofluorescence of PDX1 and C-PEPT; DAPI (blue), Pdx1 (green) and C-Pept (red). The panel on the right shows the immunofluorescence of PDX1 and GLUT-2; DAPI (blue), Pdx1 (green) and Glut-2 (red). Bar scale, 25 μΜ. Fig. 7. Overview of the Pdx1 regulation by NO and its contribution to the generation of insulin- producing cells from mESC. A) Pdx1 promoter in untreated mESC and after NO treatment. In untreated mESC, Pdxl gene expression is repressed by PRC2, which leads to a high occupancy of H3k27me3. NO treatment causes the release of PRC2 and P300 from the Pdx1 promoter thus allowing gene expression. Acetyl ati on of H3 remains constant while minor changes in DNA methylation are produced. B) NO treatment modifies the histone marks over Pdx1 promoter. The occupation balance between bivalent marks H3k27me3/H3k4me3 is tipped to H3K27me3 in mESCs. NO treatment equates the occupancy of both modifications. C) Differentiation protocol from mESC into insulin-producing cells. NO treatment releases PRC2 from Pdx1 promoter and Pdx1 is expressed. The expression of endoderm lineage genes (FoxA2, Sox 17 and Ηηίΐβ) is observed after valproic acid treatment. Thus, the addition of valproic acid (HDAC-I inhibitor) allows that chromatin remains opened and helps to differentiate cells into pancreatic lineage. In a later stage the inhibition of P300 stimulates the expression of pancreatic progenitor markers such as: Pdx1 and Pax4. Finally, culture in suspension culture allows the formation of islets-like clusters thus allowing a stables expression beta cells markers Pdx1, Nkx6.1, GM2 and Insulin.

DETAILED DESCRIPTION OF THE INVENTION

In the present invention the inventors show that NO-induced expression of PDX-1 entails the occupancy of Polycomb-Repressive Complex 2 (PRC2) and histone acetyltransferase P300 at its promoter region. These events are accompanied by changes in bivalents marks of histone H3k27me3 and H3K4me3 and in the methylation status of specific CpG islands. The inventors also optimized a differentiation protocol for generating insulin-producing cells by the addition of P300 inhibitor C646 (Ρ300Ί) followed by culture in suspension to obtain cell clusters. This protocol succeeds in obtaining insulin producing cells with sustained expression of pancreatic markers such as Pdx1 , Nkx6.1 , Insulin, Glucokinase, Kir6.2 and Glut2.

INSULIN-PRODUCING CELLS OF THE INVENTION

Based on the knowledge that P300 is a repressor of Pdxl gene and the mechanisms that regulate Pdx1 on mESC, a differentiation protocol was implemented to generate insulin- producing cells. Previously, the inventors reported the generation of definitive endoderm cells with a protocol consisting of sequential exposure to NO, serum withdrawal and treatment with valproic acid (Mora-Castilla, Tejedo et al. 2010). Then, a first aspect of the present invention refers to an insulin-producing cell, hereinafter insulin-producing cell of the invention, obtained by a method comprising the addition of a P300 inhibitor to a stem cell culture. This method is referred hereinafter as method of the invention.

The method of the invention reported here consists of 5 steps (Figure 5A). The first step for murine ESC implies culture of cells for 3 to 5 days in absence of LIF to precondition cells for differentiation. For human pluripotent cells fetal bovine serumis restricted sequentially to 0%, 0,2% and 2%. Additionally, Wnt3a (25 ng/ml) is added the first 2 days and ActvinA (100 ng/ml) is added the first 3-5 days. This step is followed by a "selective step" with 500 μΜ DETA/NO for 19 h. This step leads to a substantial degree of apoptotic death, but surviving cells repress Nanog and express endoderm markers ( ora-Castilla, Tejedo et al, 2010). Nanog repression is required for endoderm differentiation (Segev, Fishman et al. 2004). At this point, Pdx1 gene is expressed at high level, and other markers of pancreatic progenitors are also (Nkx6.1, Glut2, Nkx2.2 and Ptfla). The third step consists of culture with 100 μ valproic acid, (HDAC-I inhibitor) for 6 days. During the last 5 days cells of this period cells are exposed to 2% FBS. Valproic acid succeeds in enhancing definitive endoderm markers such as Sox17, Fox A 2 and ΗηΠβ, but fails to induce Pdx1 expression. It has been reported that histone deacetylase inhibitors modulate the timing and determination of pancreatic cell fates (Ahlgren, Jonsson et al. 1996; Gerrish, Gannon et al. 2000; Hamazaki, Oka et al. 2004). The fourth step consists of exposure to P300 inhibitor (C646) for 20h leading to enhanced expression of Pdx1 and Pax4. The final step consisted of the formation of islets-like clusters by culture in suspension. This strategy allowed to increase the expression of Ptfla, Nkx6.1, GcK, Kir6.2, Glut2 and Ins1 (Figure 5D). Immunofluorescence of cell clusters reveals cells PDX17C-PEPT, PDX17C- PEPT⁺ and that most of cells were PDX17GLUT2* (Figure 6). This simple and cost effective differentiation protocol allows the generation of cells stably expressing beta cells markers such as Pdx1, Nkx6.1, GcK, Kir6.2, Glut-2 and insulin (Figure 7C).

P300 inhibitors are known in the state of the art, for example, but without limiting, the inhibitors described in patents WO2013148114, EP2759295, US7402706, and US6369030.

In a preferred embodiment, the P300 inhibitor P300 inhibitor is selected from the list consisting on: 2,6-Bis[(3-bromo-4-hydroxyphenyl)methylene]cyclohexanone (CAS 932749-62-7), garcinol (Camboginol, CAS 78824-30-3), anacardic acid (CAS 16611 -84-0), curcumin (CAS 458-37-7), demethoxy curcumin (CAS 22608-11 -3) and C646(P300i), or combinations thereof. In a more preferred embodiment, the P300 inhibitor is C646(P300i), that is a cell-permeable, competitive, selective, potent p300/CBP HAT inhibitor.

"C646(P300i)ⁿ or "C646, p300/CBP Inhibitor" is a cell-permeable, competitive, selective, potent p300/CBP histone acetyltransferase inhibitor (K_t = 400 nM). Blocks dynamic acetylation. Is active in vitro and in vivo. His lUPAC name is 4-[4-[[5-(4,5-Dimethyl-2-nitrophenyl)-2- furanyl]methylene]-4,5-dihydro-3-methyl-5-oxo-1 H-pyrazol-1-yl]ben2oic acid (Formula I)

Formula I In another preferred embodiment, the stem cell is a pluripotent stem cell. More preferably, the stem cell is selected from induced plutipotent stem cell, an hESC obtained whithout the destruction of an embryo, a cell obtained by Somatic Cell Nuclear Transfer (SCNT), a stem cell obtained by parthenogenesis, a cell obtained by transdifferentiation, or an adult pancreatic progenitor stem cell. As used herein the phrase "pluripotent stem cells" refers to cells which are capable of differentiating into cells of all three embryonic germ layers (i.e., endoderm, ectoderm and mesoderm). According to some embodiments of the invention, the phrase "pluripotent stem cells" encompasses embryonic stem cells (ESCs) and induced pluripotent stem cells (iPS cells).

The phrase "embryonic stem cells" may comprise cells which are obtained from the embryonic tissue formed after gestation (e.g., blastocyst) before implantation (i.e., a pre-implantation blastocyst), extended blastocyst cells (EBCs) which are obtained from a post-implantation/pre- gastrulation stage blastocyst (see WO2006/040763] and embryonic germ (EG) cells which are obtained from the genital tissue of a fetus any time during gestation, preferably before 10 weeks of gestation. According to some embodiments of the invention, the pluripotent stem cells of the invention are embryonic stem cells, such as from a human or primate (e.g., monkey) origin.

Only in the case of the European Union, in order to avoid the prohibition of EU Directive 98/44 /EC (Biopatent directive), stem cells are not embryonic stem cells, are not human, or are obtained by methods which allow the production of hES cells without destroying an embryo. For example, in 2005, S. Matthew Liao (Am J Bioeth 5(6):8-16) discussed for the first time the "Blastocyst Transfer Method", and in 2006, Klimanskaya et al. (Nature 444.481-485) reported about the successful derivation of hES cells from cells obtained by biopsy of a human blastomere (8-20 cells), which survived this incident. This approach has been termed "Blastomere extraction". These approaches could at least bypass the exclusion set forth by the Biopatent directive under Art 6(c), according to which the use of human embryos for industrial or commercial purposes shall be considered unpatentable.

The embryonic stem cells of the invention can be obtained using well-known cell-culture methods. For example, human embryonic stem cells can be isolated from human blastocysts. Human blastocysts are typically obtained from human in vivo preimplantation embryos or from in vitro fertilized (IVF) embryos. Alternatively, a single cell human embryo can be expanded to the blastocyst stage. For the isolation of human ES cells the zona pellucida is removed from the blastocyst and the inner cell mass (ICM) is isolated by immunosurgery, in which the trophectoderm cells are lysed and removed from the intact ICM by gentle pipetting. The ICM is then plated in a tissue culture flask containing the appropriate medium which enables its outgrowth. Following 9 to 15 days, the ICM derived outgrowth is dissociated into clumps either by a mechanical dissociation or by an enzymatic degradation and the cells are then re- plated on a fresh tissue culture medium. Colonies demonstrating undifferentiated morphology are individually selected by micropipette, mechanically dissociated into clumps, and re-plated. Resulting ES cells are then routinely split every 4-7 days. For further details on methods of preparation human ES cells see Thomson et al. , [U.S. Pat. No. 5,843,780; Science 282: 1 145, 1998; Curr. Top. Dev. Biol. 38: 133, 1998; Proc. Natl. Acad. Sci. USA 92: 7844, 1995]; Bongso et al., [Hum Reprod 4: 706, 989]; and Gardner et al., [Fertil. Steril. 69: 84, 1998].

It will be appreciated that commercially available stem cells can also be used with this aspect of the present invention. Human ES cells can be purchased from the NIH human embryonic stem cells registry (www.escr.nih.gov). Non-limiting examples of commercially available embryonic stem cell lines are BG01 , BG02, BG03, BG04, CY12, CY30, CY92, CY10, TE03, TE04 and TE06.

Extended blastocyst cells (EBCs) can be obtained from a blastocyst of at least nine days post fertilization at a stage prior to gastrulation. Prior to culturing the blastocyst, the zona pellucida is digested [for example by Tyrode's acidic solution (Sigma Aldrich, St Louis, MO, USA)] so as to expose the inner cell mass. The blastocysts are then cultured as whole embryos for at least nine and no more than fourteen days post fertilization (i.e., prior to the gastrulation event) in vitro using standard embryonic stem cell culturing methods. Embryonic germ (EG) cells are prepared from the primordial germ cells obtained from fetuses of about 8-11 weeks of gestation (in the case of a human fetus) using laboratory techniques known to anyone skilled in the arts. The genital ridges are dissociated and cut into small chunks which are thereafter disaggregated into cells by mechanical dissociation. The EG cells are then grown in tissue culture flasks with the appropriate medium. The cells are cultured with daily replacement of medium until a cell morphology consistent with EG cells is observed, typically after 7-30 days or 1 -4 passages. For additional details on methods of preparation human EG cells see Shamblott ei al., [Proc. Natl. Acad. Sci. USA 95: 13726, 1998] and U.S. Pat. No. 6,090,622. The phrase "induced pluri potent stem (iPS) cell" (or embryonic-like stem cell) as used herein refers to a proliferative and pluripotent stem cell which is obtained by de- differentiation of a somatic cell (e.g., an adult somatic cell). IPS cells can be endowed with pluripotency by genetic manipulation which re- program the cell to acquire embryonic stem cells characteristics. For example, the iPS cells of the invention can be generated from somatic cells by induction of expression of Oct-4, Sox2, Kfl4 and c- yc in a somatic cell essentially as described in Takahashi and Yamanaka, 2006, Takahashi et al, 2007, Meissner et al, 2007, and Okita et al, 2007). Additionally or alternatively, the iPS cells of the invention can be generated from somatic cells by induction of expression of Oct4, Sox2, Nanog and Lin28 essentially as described in Yu et al, 2007, and Nakagawa et al, 2008. It should be noted that the genetic manipulation (re-programming) of the somatic cells can be performed using any known method such as using plasmids or viral vectors, or by derivation without any integration to the genome [Yu J, et al., Science. 2009, 324: 797-801]. The iPS cells of the invention can be obtained by inducing de-differentiation of embryonic fibroblasts [Takahashi and Yamanaka, 2006; Meissner et al, 2007], fibroblasts formed from hESCs [Park et al, 2008], Fetal fibroblasts [Yu et al, 2007; Park et al, 2008], foreskin fibroblast [Yu et al, 2007; Park et al, 2008], adult dermal and skin tissues [Hanna et al, 2007; Lowry et al, 2008], b-lymphocytes [Hanna et al 2007] and adult liver and stomach ceils [Aoi et al, 2008]. IPS cell lines are also available via cell banks such as the WiCell bank. Non- limiting examples of commercially available iPS cell lines include the iPS foreskin clone 1 [WiCell Catalogue No. iPS(foreskin)-l-DL-l], the iPSIMR90 clone 1 [WiCell Catalogue No. iPS(IMR90)-l-DL-l], and the iPSIMR90 clone 4 [WiCell Catalogue No. iPS(IMR90)-4-DL-l].

According to some embodiments of the invention, the induced pluripotent stem cells are human induced pluripotent stem cells.

As used herein the phrase "culture medium" refers to a liquid substance used to support the growth of pluripotent stem cells and maintain them in an undifferentiated state. The culture medium used by the invention according to some embodiments can be a water-based medium which includes a combination of substances such as salts, nutrients, minerals, vitamins, amino acids, nucleic acids, proteins such as cytokines, growth factors and hormones, all of which are needed for cell proliferation and are capable of maintaining the pluripotent stem cells in an undifferentiated state. For example, a culture medium according to an aspect of some embodiments of the invention can be a synthetic tissue culture medium such as the KO-DMEM (Gibco- Invitrogen Corporation products, Grand Island, NY, USA), DMEM/F12 (Biological Industries, Biet HaEmek, Israel), Mab ADCB medium (HyClone, Utah, USA) supplemented with the necessary additives as is further described hereinunder. The term "adult stem cell" means a stem cell that is isolated from a tissue or an organ of an animal in a state of post-embryonic growth. Preferably, the stem cells of the invention are isolated on a postnatal state. Preferably they are isolated from a mammal, and most preferably a human, including neonates, juveniles, adolescents and adults. Can be isolated adult stem cells of a variety of tissues and organs such as bone marrow (mesenchymal stem cells, multipotent adult progenitor ceils and hematopoietic stem cells), adipose tissue, cartilage, epidermis, hair follicle, skeletal muscle, cardiac muscle, intestine, liver, neuronal.

An "adult pancreatic progenitor stem cell" is a cell that may proliferate and differentiate into pancreatic cells or a cell that may transdifferentiate into endocrine cells and more specifically into insulin-producing cells.

The invention show that Pdx1 is regulated by NO and its contribution to the generation of insulin-producing cells from mESC. NO treatment releases PRC2 from Pdx1 promoter and Pdx1 is expressed. In another preferred embodiment, the method of the invention also comprises culture the stem cell in presence of a donor of nitric oxide. Donors of nitric oxide are well know in the state of the art.

By "PDX1 " or "Pancreatic and Duodenal Homeobox 1" (also known as GSF; IPF1 ; 1UF1 ; IDX-1 ; ODY4; PDX-1 ; STF-1 ; PAG EN 1 ) is found in chromosome 13(13q12.1). The protein encoded by this gene is a transcriptional activator of several genes, including insulin, somatostatin, glucokinase, islet amyloid polypeptide, and glucose transporter type 2. The encoded nuclear protein is involved in the early development of the pancreas and plays a major role in glucose- dependent regulation of insulin gene expression. Defects in this gene are a cause of pancreatic agenesis, which can lead to early-onset insulin-dependent diabetes mellitus (NIDDM), as well as maturity onset diabetes of the young type 4.

In the context of the present invention, PDX1 is also defined by a nucleotide sequence or polynucleotide, which is the protein coding sequence contained in NP_000200.i, and which comprise different variants from: a) nucleic acid molecules encoding a polypeptide comprising the amino acid sequence of NP_000200.1 , b) nucleic acid molecules whose complementary strand hybrid with the polynucleotide sequence of a), c) nucleic acid molecules whose sequence differs from a) and / or b) due to degeneracy of the genetic code, d) nucleic acid molecules encoding a polypeptide comprising the amino acid sequence with an identity of at least 80%, 90%, 95%, 98% or 99% to NP_000200.1 , and wherein the polypeptide encoded by said nucleic acids having the activity and structural features of the protein PDX1. Such nucleic acid molecules are contained in the sequence of GenBank (NCBI) NM_000209.3.

SEQ ID NO: 1 (NP_000200.1 ):

MNGEEQYYAATQLYKDPCAFQRGPAPEFSASPPACLYMGRQPPPPPPHPFPGALGALEQGS PPDISPYEVPPLADDPAVAHLHHHLPAQLALPHPPAGPFPEGAEPGVLEEPNRVQLPFPWMKS TKAHAWKGQWAGGAYAAEPEENKRTRTAYTRAQLLELEKEFLFNKYISRPRRVELAVMLNLTE RHIKIWFQNRRMKWKKEEDKKRGGGTAVGGGGVAEPEQDCAVTSGEELLALPPPPPPGGAV PPAAPVAAREGRLPPGLSASPQPSSVAPRRPQEPR

SEQ ID NO: 2 (NM_000209.3)

GGGTGGCGCCGGGAGTGGGAACGCCACACAGTGCCAAATCCCCGGCTCCAGCTCCCGAC TCCCGGCTCCCGGCTCCCGGCTCCCGGTGCCCAATCCCGGGCCGCAGCCATGAACGGCG AGGAGCAGTACTACGCGGCCACGCAGCTTTACAAGGACCCATGCGCGTTCCAGCGAGGC CCGGCGCCGGAGTTCAGCGCCAGCCCCCCTGCGTGCCTGTACATGGGCCGCCAGCCCCC GCCGCCGCCGCCGCACCCGTTCCCTGGCGCCCTGGGCGCGCTGGAGCAGGGCAGCCCC CCGGACATCTCCCCGTACGAGGTGCCCCCCCTCGCCGACGACCCCGCGGTGGCGCACCT TCACCACCACCTCCCGGCTCAGCTCGCGCTCCCCCACCCGCCCGCCGGGCCCTTCCCGG AGGGAGCCGAGCCGGGCGTCCTGGAGGAGCCCAACCGCGTCCAGCTGCCTTTCCCATGG ATGAAGTCTACCAAAGCTCACGCGTGGAAAGGCCAGTGGGCAGGCGGCGCCTACGCTGC GGAGCCGGAGGAGAACAAGCGGACGCGCACGGCCTACACGCGCGCACAGCTGCTAGAG CTGGAGAAGGAGTTCCTATTCAACAAGTACATCTCACGGCCGCGCCGGGTGGAGCTGGCT GTCATGTTGAACTTGACCGAGAGACACATCAAGATCTGGTTCCAAAACCGCCGCATGAAGT GGAAAAAGGAGGAGGACAAGAAGCGCGGCGGCGGGACAGCTGTCGGGGGTGGCGGGGT CGCGGAGCCTGAGCAGGACTGCGCCGTGACCTCCGGCGAGGAGCTTCTGGCGCTGCCG CCGCCGCCGCCCCCCGGAGGTGCTGTGCCGCCCGCTGCCCCCGTTGCCGCCCGAGAGG GCCGCCTGCCGCCTGGCCTTAGCGCGTCGCCACAGCCCTCCAGCGTCGCGCCTCGGCG GCCGCAGGAACCACGATGAGAGGCAGGAGCTGCTCCTGGCTGAGGGGCTTCAACCACTC GCCGAGGAGGAGCAGAGGGCCTAGGAGGACCCCGGGCGTGGACCACCCGCCCTGGCAG TTGAATGGGGCGGCAATTGCGGGGCCCACCTTAGACCGAAGGGGAAAACCCGCTCTCTCA GGCGCATGTGCCAGTTGGGGCCCCGCGGGTAGATGCCGGCAGGCCTTCCGGAAGAAAAA GAGCCATTGG I I I I I GTAGTATTGGGGCCCTCTTTTAGTGATACTGGATTGGCGTTGTTTGT GGCTGTTGCGCACATCCCTGCCCTCCTACAGCACTCCACCTTGGGACCTGTTTAGAGAAG CCGGCTCTTCAAAGACAATGGAAACTGTACCATACACATTGGAAGGCTCCCTAACACACAC AGCGGGGAAGCTGGGCCGAGTACCTTAATCTGCCATAAAGCCATTCTTACTCGGGCGACC CCTTTAAGTTTAGAAATAATTGAAAGGAAATGTTTGAGTTTTCAAAGATCCCGTGAAATTGAT GCCAGTGGAATACAGTGAGTCCTCCTCTTCCTCCTCCTCCTCTTCCCCCTCCCCTTCCTCC TCCTCCTCTTCTTTTCCCTCCTCTTCCTCTTCCTCCTGCTCTCCTTTCCTCCCCCTCCTCTTT TCCCTCCTCTTCCTCTTCCTCCTGCTCTCCTTTCCTCCCCCTCCTCTTTCTCCTCCTCCTCC TCTTCTTCCCCCTCCTCTCCCTCCTCCTCTTCTTCCCCCTCCTCTCCCTCCTCCTCTTCTTC TCCCTCCTCTTCCTCTTCCTCCTCTTCCACGTGCTCTCCTTTCCTCCCCCTCCTCTTGCTCC CCTTCTTCCCCGTCCTCTTCCTCCTCCTCCTCTTCTTCTCCCTCCTCTTCCTCCTCCTCTTT CTTCCTGACCTCTTTCTTTCTCCTCCTCCTCCTTCTACCTCCCCTTCTCATCCCTCCTCTTCC TCTTCTCTAGCTGCACACTTCACTACTGCACATCTTATAACTTGCACCCCTTTCTTCTGAGG AAGAGAACATCTTGCAAGGCAGGGCGAGCAGCGGCAGGGCTGGCTTAGGAGCAGTGCAA GAGTCCCTGTGCTCCAGTTCC ACACTGCTG GCAGGGAAGGCAAGGGGGG ACGGGCCTG G ATCTGGGGGTGAGGGAGAAAGATGGACCCCTGGGTGACCACTAAACCAAAGATATTCGGA ACTTTCTATTTAGGATGTGGACGTAATTCCTGTTCCGAGGTAGAGGCTGTGCTGAAGACAA GCACAGTGGCCTGGTGCGCCTTGGAAACCAACAACTATTCACGAGCCAGTATGACCTTCA CATCTTTAGAAATTATGAAAACGTATGTGA7TGGAGGGTTTGGAAAACCAGTTATCTTATTTA ACATTTTAAAAATTACCTAACAGTTATTTACAAACAGGTCTGTGCATCCCAGGTCTGTCTTCT TTTCAAGGTCTGGGCCTTGTGCTCGGGTTATGTTTGTGGGAAATGCTTAATAAATACTGATA ATATGGGAAGAGATGAAAACTGATTCTCCTCACTTTGTTTCAAACCTTTCTGGCAGTGGGAT GATTCGAATTCACTTTTAAAATTAAATTAGCGTGTTTTGTTTTG By "Pdx-1 biological activity" is meant transcriptional regulatory activity, DNA binding activity, specific binding of a Pdx-1 antibody, and/or immunomodulatory activity.

More preferably, the donor of nitric oxide is selected from the list consisting on: sodium nitroprusside, 3-morpholino-sydnonimine, furoxan CAS 1609, S-nitroso-acetylpenicillamine, S- nitroso-glutathione, sildenafil citrate, nitroprusside, DETA-NO or combinations thereof. In a more preferred embodiment, the donor of nitric oxide is DETA-NO.

By "DETA-NO" or "diethylenetriamine/nitric oxide adducf is a compound with CAS number 146724-94-9 and has the lUPAC name 2,2 ' -(Hydroxynitrosohydrazono)bis-ethanimine (Formula II).

Formula II

The expression of endoderm lineage genes (FoxA2, Sox 17 and Ηηίΐβ) is observed after valproic acid treatment. Thus, the addition of histone deacetylase inhibitors, like valproic acid (HDAC-I inhibitor) allows that chromatin remains opened and helps to differentiate cells into pancreatic lineage. Then, in another preferred embodiment, the method of the invention also comprises culture the stem cell in presence of inhibitors of histone deacetylases. More preferably the inhibitor is selected from the list consisting on: Valproic acid, vorinostat (SAHA), tefinostat (CHR-3996), benzoic acid derivatives such as givinostat (ITF2357), mocetinostat (MGCD0103) and entinostat (MS-275); cynamic acid derivatives such as panobinostat (LBH589), belinostat (PXD101 ) and pracinostat (SB939) and derivatives of pyrimidin-5- carboxoxilic acid CHR-3996 and quisinostat (SB939). In a still more preferred embodiment, the histone deacetylase inhibitor is the valproic acid.

The term "culture medium" or "medium" is recognized in the art, and refers generally to any substance or preparation used for the cultivation of living cells. The term "medium", 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. 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". "Defined medium" refers to media that are made of chemically defined (usually purified) components. "Defined media" do not contain poorly characterized biological extracts such as yeast extract and beef broth. "Rich medium" includes media that are designed to support growth of most or all viable forms of a particular species. Rich media often include complex biological extracts. A "medium suitable for growth of a high density culture" is any medium that allows a cell culture to reach an OD600 of 3 or greater when other conditions (such as temperature and oxygen transfer rate) permit such growth. The term "basal medium" refers to a medium which promotes the growth of many types of cells which do not require any special nutrient supplements. Most basal media generally comprise of 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, 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).

The p!uripotent or progenitor stem cells are substantially pure. The term "substantially pure", refers to a population of pluripotent or progenitor stem cells that is at least about 75%, preferably at least about 85%, more preferably at least about 90%, and most preferably at least about 95% pure, with respect to the other cells making up a total cell population.

The cells of the invention could be autologous, allogenic or xenogenic cells, and the biological characteristic of which could be substantially altered as a result of their manipulation for obtaining a therapeutic, diagnostic or preventive effect through metabolic, pharmacological or immunological means. Among then 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.

In another preferred embodiment, the method of the invention also comprises culture the cell in suspension to obtain cell clusters. More preferably, the cells are culture until the expression of the pancreatic markers selected from the list consisting on: PDX-1 , Insulin, Glucokinase, Kir6.2 and/or Glut2. Still more preferably, the cells express all the markers.

In the present invention the term "marker" refers to a protein distinguishing one cell (or set of cells) from another cell (or group of cells). For example, a protein that is expressed on the surface of precursor cells but not other cells of a cell population serves as a marker protein for stem cells.

The cells of the invention are positive for certain phenotypic markers and negative for others. "Positive" means that the cell expressing the marker. To consider that the marker is expressed, must be present at a "detectable". Herein, by "detectable level" is meant that the marker PDX1 detected by one of the standard methods such as PCR, blotting or FACS. It is considered that a gene is expressed by a cell of the invention if it can be reasonably detected after 20 cycles, preferably 25 cycles, and more preferably 30 PCR cycles, which corresponds to a level of expression in the cell of at least 100 copies per cell. It is considered that a marker is not expressed by a cell of the invention, if expression can not be detected at a level of about 10-20 copies per cell. Between these levels of positive / negative, a cell may be weakly positive for a particular marker.

The term "natural expression" or "naturally expresses" means that the cells have not been manipulated by recombinant technology, in any form, that is, for example, that the cells were not artificially induced to express these markers or modulate expression of these markers by introducing into cells of exogenous material, such as the introduction of heterologous promoters, or other sequences operatively linked to any of the endogenous genes, or by introduction of exogenous genes.

CELL POPULATION OF THE INVENTION A second aspect of the present invention refers to an insulin-producing cell population, hereinafter insulin-producing cell population of the invention, comprising at least an insulin- producing cell of the invention.

In a preferred embodiment the cell population of the invention comprises at least 20%, preferably 40%, and even more preferably 50%, 60%, 80%, 90%, 95%, or 99% of insulin- producing cells of the invention.

The cell population of the invention is positive for a particular marker if at least 20% of the cells of the population show detectable expression of the marker, preferably 70%, 80%, 90%, 95%, and more preferably 98%. At times, 99% or 100% of the cells of the invention show detectable expression of the marker. As in the case of the cells of the invention, the expression can be detected, for example but not limited to, PGR techniques, using FACS (fluorescence activated cell sorting), or by immunohistochemistry using specific antibodies. The insulin-producing cells of the invention express the pancreatic markers selected from the list consisting on: PDX-1 , Insulin, Glucokinase, Kir6.2 and/or Glut2. Still more preferably, the cells express all this markers.

ARTIFICIAL TISSUE OR ORGAN OF THE INVENTION

A third aspect of the present invention refers to an artificial tissue or organ, hereinafter artificial tissue or organ of the invention, comprising an insulin-producing cell of the invention, the insulin-producing cell population of the invention, or the composition of the invention, preferably in combination with a support material or included in a device.

"Support" as used herein refers to any device or material that may serve as a foundation or matrix for the maintenance or growth of cells. The support material can be from natural or synthetic origin. For example, but without limiting, the support material from natural origin is selected from the list consisting of: silk, decellularized extracellular matrix collagen based support material, a fibrin based support material, a laminin based support material, a fibronectin based support material and artificial support materials. This list is provided by way of illustration only, and is not intended to be limiting. It will be clear to a person skilled in the art, that any conventional or advanced biomaterial, orthopedic biomaterial or a combination of biomaterials.

"Device" as used herein refers to any constructed tools that may be inserted subcutaneously or in other parts of the body allowing glucose sensing and insulin release. In amore preferred embodiment the device is a hollow fiber that allows implantation of cells in a vascularized bed.

The obtained beta cell like of the invention could be maturating through their implant inside to animal models.

COMPOSITION OF THE INVENTION

A fourth aspect of the present invention refers to composition comprising an insulin-producing cell of the invention, an insulin-producing cell population of the invention and/or the artificial tissue of the invention. More preferably, the composition of the invention is a pharmaceutical composition. In another preferred embodiment of this aspect of the invention, the pharmaceutical composition comprises the insulin-producing cell of the invention, the insulin- producing cell population of the invention and/or the artificial tissue of the invention, and also another active ingredient. Still more preferably, the composition of the invention further comprises a pharmaceutically acceptable excipient or a pharmaceutically acceptable carrier.

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 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 15 070501 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. D EM, 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.

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 a 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.

USES OF THE INSULIN-PRODUCING CELLS OF THE INVENTION IN PHARMACOLOGICAL OR CHEMICAL PRODUCT EVALUATION P2015/070501

The drugs and chemical products must be evaluated before their administration into test animals. To this respect, there are several reports and directives approved by the European Union which aim to restrict or even prohibit animal testing in the sector of cosmetic products (Directive 76 768/EEC of the European Council relating to the approximation of Member States' laws on cosmetic products), and the complete ban is expected to be in force in the next few years. The European Union supports all the measures the main objective of which is the well being of the animals used for testing purposes and for achieving scientific replacement methods to reduce the number of animals used for testing to the minimum (Decision 1999/575/EEC of the Council, dated 23 March 1998, relating to the conclusion by the Community of the European Convention for the protection of vertebrate animals used for experimental and other scientific purposes - Official Record L 222 of 24.08.1999).

Therefore, another aspect of the invention relates to the use of the insulin-producing cell of the invention, the insulin-producing cell population of the invention, the composition of the invention, or an artificial tissue or organ of the invention, for evaluating a pharmacological and/or chemical product.

MEDICAL USES OF THE INVENTION

An infectious, autoimmune, 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 and/or reestablish the normal function of said tissues or organs is important. The damaged tissue or organ can be replaced with new cells, a new tissue or organ which has been produced in the laboratory by means of tissue engineering techniques. The objective of tissue engineering is to construct artificial biological tissues and to use them for medical purposes to restore, replace or increase the functional activities of diseased tissues and organs. In addition, there are many advantages associated with the use of autologous cells or tissues in tissue engineering, which include: (a) a significant reduction of the number of infections from the donor to the recipient by infectious agents; and (b) the absence of host immune graft rejection, therefore the patient does not need to undergo immunosuppressing treatment, side effects and problems associated with immunodepression being prevented.

The insulin-producing cell of the invention, the insulin-producing cell population of the invention, the composition of the invention, or an artificial tissue or organ of the invention can be used to restore, replace or increase the functional activities of diseased tissues and organs, and more specifically, to restore or replace the function of pancreas β beta cells secreting insulin. Then, a sixth aspect of the invention refers to the insulin-producing cell of the invention, the insulin-producing cell population of the invention, the composition of the invention, or an artificial tissue or organ of the invention, for use as a medicament.

The term "medicament", as used in this specification, refers to any substance used for the prevention, diagnosis, relief, treatment or cure of diseases in human beings and animals.

A seventh aspect of the invention refers to the insulin-producing cell of the invention, the insulin-producing cell population of the invention, the composition of the invention, or an artificial tissue or organ of the invention, for partially or completely increase, restore or replace the functional activity of a diseased or damaged tissue or organ. In type 1 diabetes (diabetes mellitus), the pancreatic production of insulin is greatly reduced. Hence type 1 diabetics need regular injections or perfusions of insulin to control their blood glucose to avoid deleterious consequences. Some type 1 diabetics also develop the "dawn syndrome", a state of increased insulin resistance in the early hours of the morning. Type 1 diabetes mellitus Is a form of diabetes mellitus that results from the autoimmune destruction of the insulin-producing beta cells in the pancreas.

Experimental replacement of beta cells (by transplant or from stem cells) is being investigated in several research programs. Islet cell transplantation is less invasive than a pancreas transplant, which is currently the most commonly used approach in humans.

In one variant of this procedure, islet cells are injected into the patient's liver, where they take up residence and begin to produce insulin. The liver is expected to be the most reasonable choice because it is more accessible than the pancreas, blood flow of the liver is part of the portal venous circulation which physiologically collects insulin released from pancreas and islet cells seem to produce insulin well in that environment. The patient's body, however, will treat the new cells just as it would any other introduction of foreign tissue, unless a method is developed to produce them from the patient's own stem cells or an identical twin is available who can donate stem cells. The immune system will attack the cells as it would a bacterial infection or a skin graft. Thus, patients now also need to undergo treatment involving immunosuppressants, which reduce immune system activity.

The insulin-producing cell of the inventions, and more preferably, autologous insulin-producing cells of the invention, can be used to replace the damaged beta cells of the subject.

An eighth aspect of the invention refers to the insulin-producing cell of the invention, the insulin-producing cell population of the invention, the composition of the invention, or an artificial tissue or organ of the invention, for use in the treatment, amelioration or prevention of an insulin 15 070501 related disease or condition. More preferably, the insulin related disease is the pre-diabetes and/or diabetes.

By "amelioration" or "ameliorate" is meant decrease, suppress, attenuate, diminish, arrest, or stabilize the development or progression of a disease. By "disease" is meant any condition or disorder that damages or interferes with the normal function of a cell, tissue, or organ. Examples of diseases include bacterial invasion or colonization of a host cell.

By "pre-diabetes" is meant having a propensity to develop diabetes, or having any symptomatic or pathologic precursor to diabetes. Symptoms of diabetes are known in the art and include alterations in blood glucose levels, increased thirst, increased urination, or an abnormal result in a glucose challenge test. Subject having a propensity to develop diabetes may have a genetic mutation associated with the disease (i.e., a mutation in Pdx-1 ), have a relative diagnosed as having diabetes, have obesity, or otherwise show physiological alterations associated with diabetes. The dosage and the timing of administering the agent depends on various clinical factors including the overall health of the subject and the severity of the symptoms of pre-diabetes or diabetes. In general, once diabetes, pre-diabetes or a propensity to develop diabetes is detected, daily, weekly, or continuous infusion of a purified peptide is used to treat or prevent pre-diabetes or diabetes. Treatment can be continued for a period of time. For example, treatment may be administered indefinitely for as long as the patient shows a propensity to develop pre-diabetes or diabetes (e.g., for days, months, or years).

A ninth aspect of the invention refers to the insulin-producing cell of the invention, the insulin- producing cell population of the invention, the composition of the invention, or an artificial tissue or organ of the invention, for use in the treatment, amelioration or prevention of type 1 diabetes, diabetic ketoacidosis, hyperglycemia, diabetic neuropathy, diabetic cardiopathy, diabetic nefropathy, diabetic rethynopathy and other acute and chronic complications of diabetes like heart disease, stroke, kidney failure, foot ulcers and damage to the eyes.

A tenth aspect of the invention refers to the use of a donor of nitric oxide, and more preferably to the use of DETA-NO, to promotes Pdx1 expression through changes in bivalents marks of histone H3k27me3 and H3K4me3.

An eleventh aspect of the invention refers to the use of factors such as DETA-NO, valproic acid and p300 inhibitor to promote pluripotent stem cells differentiation into pancreatic beta cells like. METHOD OF THE INVENTION

A twelfth aspect of the invention refers to a method to obtain an insulin-producing cell, previously referred as method of the invention, which promotes PDX-1 expression, beta cell maturation, insulin content and release, further comprising: a. obtaining a sample of pluripotent stem cells as defined previously, b. culture of pluripotent stem cells in a culture medium that's induces differentiation by adding or restrinting growth factors and small molecules c. using of DETA-NO to differentiate pluripotent stem cells, d. decreasing fetal bovine serum concentration by 10 to 10%, by 20 to 40%, by 40 to 60 % and by 60 to 99% e. using xeno free culture media f. using resveratrol (3,5,4'-trihydoroxytranstilbene) or other Sirtuin-1 activators at a concentration between 50 to 100 micromolar to induce maturation of insulin producing cells

The first step implies culture of ceils for 3 days in absence of LIF to precondition cells for differentiation. This step is followed by a 'selective step" with 400 to 600, more preferably 450 μ to 550 μΜ, and still more preferably 500 μΜ of a donor of nitric oxide, preferably DETA NO for 16 to 22 h, more preferably 17 to 21 h, still more preferably 18 to 20 h, and particularly 19 h. This step leads to a substantial degree of apoptotic death, but surviving cells repress Nanog and express endoderm markers (Mora-Castilla, Tejedo et al. 2010). Nanog repression is required for endoderm differentiation (Segev, Fishman et al. 2004). At this point, Pdx1 gene is expressed at high level, and other markers of pancreatic progenitors are also (Nkx6.1 , Glut2, Nkx2.2 and Ptfla). The third step consists of culture with 80 μ to 120 μΜ, more preferably 90 μΜ to 1 10 μΜ, and still more preferably 100 μΜ valproic acid, (HDAC-I inhibitor) for 6 days. During the last 5 days cells of this period cells are exposed to 2% FBS. Valproic acid succeeds in enhancing definitive endoderm markers such as Sox17, FoxA2 and ΗηΠ β, but fails to induce Pdx1 expression. It has been reported that histone deacetylase inhibitors modulate the timing and determination of pancreatic cell fates (Ahlgren, Jonsson et al. 1996; Gerrish, Gannon et al. 2000; Hamazaki, Oka et al. 2004). The fourth step consists of exposure to a P300 inhibitor, preferably P300 inhibitor (C646) for 20h leading to enhanced expression of Pdx1 and Pax4. The 5 070501 final step consisted of the formation of islets-like clusters by culture in suspension. This strategy allowed to increase the expression of Pt la, Nkx6.1 , GcK, Kir6.2, Glut2 and Ins1 (Figure 5D). Immunofluorescence of cell clusters reveals cells PDX1 +/C-PEPT-, PDX1+/C-PEPT+ and that most of cells were PDX1+/GLUT2+ (Figure 6). This simple and cost effective differentiation protocol allows the generation of cells stably expressing beta cells markers such as Pdx1, Nkx6.1 , Glucokinase , Kir6.2, Glut-2 and insulin (Figure 7C).

In a preferred embodiment, the method of the invention also comprises: a. Transplanting these cells into a mammal needing endocrine cell therapy.

EXAMPLES OF THE INVENTION

Materials and methods

Cell Culture and treatments. Mouse ES cell lines D3 and R1-E (ATCC, Manassas, USA) were used. INS-1E cells were kindly provided by Dr. P. Maechler, Geneva University. Feeder-free D3 mES cells. mESC D3 and R1/E were maintained at 37° C with 5% C0₂ in Dulbecco's modified Eagle's medium (DMEM) (Gibco, Carlsbad, CA, USA), supplemented with 15% foetal bovine serum (FBS) (Hyclone, Logan, UT, USA), 0.1 mM β-mercaptoethanol (Gibco, Paisley, Scotland, UK), 2mM L-glutamine (Gibco, Paisley, Scotland, UK), 1% MEM nonessential amino acid (Gibco, Paisley, Scotland, UK) and 100 U/ml penicillin: 100 pg/ml streptomycin (Gibco, Paisley, Scotland, UK). Undifferentiated state was maintained by addition of LIF (1000 U/ml) (ESGRO, Chemicon, Charlottesville, VA, USA). INS-1E cells were cultured in RPMI-1640 (LONZA, San Diego, USA), supplemented with 10% FBS, 10mM HEPES (Gibco, Paisley, Scotland, UK), 1 mM sodium pyruvate (Gibco, Paisley, Scotland, UK), 100 U/ml penicillin, 100 pg/ml streptomycin, 2mM L-glutamine and 50 μΜ β-mercaptoethanol (Gibco, Paisley, Scotland, UK). R1/E cells were cultured for 3 days in gelatinized dishes in the presence or absence of LIF and then exposed to 500 μΜ of nitric oxide donor diethylenetriamine nitric oxide adduct (DETA-NO) (Sigma, St. Louis, MO, USA) for 19 hours. Cells were then trypsinized and collected by centrifugation. For differentiation protocols, D3 cells were cultured for 3 days without LIF, 19 hours with 500 μΜ DETA-NO, 6 days with 100 μΜ valproic acid (Sigma) and 20 hours with 50 μΜ Histone Acetyltransferase P300 inhibitor C646 (Millipore, Darmstadt, Germany). Cell aggregates were then gently scraped and transferred to Falcon tubes and allowed to sediment for 10 min. The resulting aggregates were then grown in suspension in 12-24 well plates for 3 days. On the 5^th day of culture FBS was reduced to 2% until the end of the protocol. Culture medium was changed daily. RNA isolation, reverse transcription (RT)-PCR and real-time PGR analysis. Total RNA was extracted using Easy Blue® reagent (Intron Biotechnology, Gyeonggi-do, Korea) and clorophorm/ isopropanol purification procedure. cDNA synthesis was performed with 1 g of total RNA using M-MVL reverse transcriptase (Promega, Madison, Wl, USA) and random primers according to the manufacturer's instructions. For real-time PGR analysis, endogenous mRNA levels were measured by real-time PGR analysis based on SYBR Green (Applied Biosystems, Foster City, CA, USA) detection with the ABI Prism 7500 machine (Applied Biosystems). Results were normalized with fj-Actin expression. Real-time PGR primers used are shown in Supplementary Table 1. Supplementary Table 1

Gene Expression Primers Size (pb) Tm C)

Pdx1 F 5^" - AGCTCCCTTTCCCGTGGATGAAAT - 3^" 112 60

Pdx1 R 5" - TAGGCAGTACGGGTCCTCTTGTTT - 3^*

p300 F 5'- GCATGCGGTCTGTGAACAACATG -3' 191 60

p300 R 5' - AGTACTAGATGGCTGAGCTGCTGT - 3'

Jarid-2 F 5 *- AGAAGGATGATGCCTCCCAAGTGT - 3' 154 60

Jarid-2 R 5' - ATCTTCACTGGAGTTGTGCTGGGA - 3'

EZH2 F 5 '- TCGTAAGTGCAGTTATTCCTTCCA - 3' 151 60

EZH2 R 5 ACG CTC AGC AGT AAG AGC AG - 3"

Pax4 F 5^' - GCAGTGTGAATCAGCTAGGGG - 3^' 103 60

Pax4 R 5' - CAGGGTCGCATCCCTCTTATT - 3^'

Nkx6.1 F 5^' - CTGCACAGTATGGCCGAGATG - 3^' 135 60

Nkx6.1 R 5^' - CCGGGTTATGTGAGCCCAA - 3^'

FoxA2 F 5^' - TCAACGACTGCTTTCTCAAGGTGC - 3^' 97 60

FoxA2 R 5" - TTCTCGAACATGTTGCCCGAGTCT - 3^'

Sox17 F 5^* - TTATGGTGTGGGCCAAAGACGAAC - 3^' 105 60

Sox17 R 5^" - TCAACGCCTTCCAAGACTTGCCTA - 3^'

Nkx2.2 F 5^' - ATCGCTACAAGATGAAACGTGCCC - 3^' 140 60

Nkx2.2 R 5^" - CAGTCGCCCGACCTGAAATTGTTT - 3'

Hnfip F 5^* - ACAGGGCAGAATGTTTGCAACGAG - 3' 150 60

Ηη β R 5^' - TATAGGCATCCATGGCCAGCTTCT - 3^'

Glut2 F 5" - GGCCCTTGTCACAGGCATTCTTATT - 3^' 139 58

Glut2 R 5^' - TGGACAGAAGAGCAGTAGCAGACA - 3^*

Pt la F 5^' - CATTAACTTCCTCAGCGAGCTGGT - 3^' 137 60

Ptfla R 5^' - AGATGATAACCTTCTGGGCCTGGT - 3^'

Insulin 1 F 5' -AACCCCCAGCCCTTAGTGACC - 3^' 124/242 59,5

Insulin 1 R 5' -TTTGGGCTCCCAGAGGGCA - 3^' Glucokinase F 5^' - TGGATGACAGAGCCAGGATGG - 3^" 208 58,8 Glucokinase R 5^' - ACTTCTGAGCCTTCTGGGGTG - 3^*

Kir6.2 F 5'- GGCTCCTAGTGACCTGCACCA -3' 317 61 Ktr6.2 R 5'- CCACAGCCACACTGCGCTT -3'

Glucagon F 5^' - TTACTTTGTGGCTGGATTGCTT - 3^* 148 60

Glucagon R 5^' - AGTGGCGTTTGTCTTCATTCA - 3^'

Somatostatin F 5 -ACCGGGAAACAGGAACTGG-3 ^' 159 60 Somatostatin R 5 '- TTGCTGGGTTCGAGTTGGC -3 '

Amylase 2 F 5^'- TTCGAGTGGCGCTGGGTTGATATT -3^' 214/546 59,5

Amylase 2 F 5^*- TGTTGCACCTTGTCACCATGTCTCT -3^*

b-Actin F 5^'- TCCTGTGGCATCCATGAAACTACA -3^' 93 60 b-Actln R 5" - ACCAGACAGCACTGTGTTGGCATA - 3^'

Pdx1 promoter

Region ChIP primers Size (pb) Tm (°C)

Pdx1 promoter Prox

F 5^' - TGGCGTAGAGAGTCCGCGAGCCACC - 3^" 179 60

Pdx1 promoter Prox

R 5^* - CGC TGA ACT CTG GCA CCG GG - 3"

Pdx1 promoter

Region BSP Primers

Distal CpG F 5^' - GGTAGATTATTTGTGAGGGTTAAT - 3^'

Distal CpG R 5^' - ATACTTTCCCTCTTAAAATCACC - 3^'

Proximall CpG F 5^' - GGAGAGTAGTGGAGAATTGTTAA - 3^'

ProximaH CpG R 5^' - ACTAAC RACCCATATACAAACA - 3^'

Proxtmal2 CpG F 5^' - GGGGTTGTT GGT TTT TAA GT - 3'

Proximal2 CpG R 5^' - TTACCTACCCACTAACCTTTCC - 3^"

Pdx1 promoter

Region Pyrosequencing Primers

Pyro- musPdx1_distal1-F2 5^' - TTTAAGGGGGTGATTAGGTGAAG - 3^'

Bio-Pyro- 5^' - [BtnjATATTAACCCTCACAAATAATCTACCT

musPdx1_distal1-R2 - 3^'

Pyro- 5' - AGGAAGG I I I I l A I TI I TGTTi^'TAA - 3' musPdx1_distal1-

Seq

Pyro- musPdx1_di8tal2-F2 5^' - AGGTAGATTATTTGTGAGGGTTAATAT - 3^*

5^' -

Bio-Pyro- [BtnjATTTCTTCAAAAAAAAAAACCACCTATACC

musPdx1_distal2-R2 - 3^'

Pyro- musPdx1_distal2-

Seq 5^' - ATTATTTGTGAGGGTTAATATT - 3^'

Pyro- muePdx1_proximal1- F2 5^' - TGGGGTG I I t I AGAGTTTATGGTA - 3^'

Bio-Pyro- musPdx1_proximal1- 5^' - (BtnlATACTACTCCTCACTATTCATAATAAC

R2 - 3^'

Pyro- muePdxl jsroximall- Seq 5" - GGAGAGTAGTGGAGAA - 3^*

Pyro- musPdx1_proximat2- 5" - GTTGTTATTATGAATAGTGAGGAGTAGTA - F2 3^'

Bto-Pyro- musPdxl _proximal2- R2 5^' - [BtnJAAAAACTTCCCTACTCCAATAATC - 3^'

Pyro- musPdx1_proximal2- Seq 5' - AATAGTGAGGAGTAGTATT - 3"

Western Blotting. Cells were trypsinized, centrifuged and washed once with cold PBS. Cell pellets were then resuspended and incubated in RIPA buffer (Sigma) supplemented with protease inhibitor (Sigma) and phosphatase inhibitor cocktails (Sigma) for 45 min on ice and sonicated with 4 pulses 10 s each at 10% amplitude in a Branson sonifier (Branson Digital Sonifier, Branson Ultrasonics Corporation, Danbury, CT, USA). After centrifugation, supernatant protein was quantified by Bradford. Proteins were denatured in Laemmli buffer containing 2.5 % β-mercaptoethanol (Sigma) for 10 minutes at 96°C. Proteins were separated by SDS-PAGE and transferred to PVDF membrane. Membranes were then blocked with TBS-Tween containing 5% defatted dried milk for 1 h at room temperature. Then, membranes were probed with anti-Pdxl 1 :1000 (Abeam, Cambridge, UK), anti-P300 (Santa Cruz Biotech, Heidelberg, Germany), anti- Jarid2 (Abeam), anti-Ezh2 (BD Transduction, California, USA) and 1 :10000 anti-p-Actin (Sigma) in TBS-Tween 5% defatted milk overnight at 4C°. Membranes were subsequently washed 3 times with TBS-Tween. Secondary antibodies were used at 1 :20000 anti-rabbit IgG (Sigma) and 1 :40000 anti-mouse IgG (Jackson ImmunoResearch, Suffolk, UK). Proteins were detected by chemiluminescence. Immunocytochemistry:, R1/E cells ( 5x10³) were cultured in wells containing Permanox slides. To analyze Pdx1 expression, cells exposed to DETA-NO for 19h were fixed with 4% paraformaldehyde, permeabilized with cold methanol and blocked with 4% BSA. Then, cells were incubated overnight with 1 :100 anti-Pdxl (Abeam) and 1 :300 anti-a Tubulin (Sigma). For immunofluorescence of islet-like clusters (D3 cells), clusters were fixed with 4% paraformaldehyde in PBS overnight at 4°C, dehydrated with ethanol and xylene, and embedded in paraffin. Sections were cut at a thickness of 5 μιη with a Leica DM6000B microtome. Paraffin sections were dewaxed through a series of xylene and ethanol washes, and incubation in PBS for 5 min and autoclaved in antigen retrieval, 0,01 M solution citrate buffer. Sections were blocked with 3% BSA and 0.2% Triton X-100 in PBS for 1 h at room temperature and incubated overnight at 4°C with 1 :100 anti-Pdxl (Developmental Studies Hybridoma Bank, Iowa, USA), 1 :100 anti C-peptide (Beta Cell Biology, Vanderbilt University) and 1 :100 anti-Glut2 (Chemicon). Finally, cells were washed 3 times with PBS-Tween and detection of primary antibody was developed using 1 :300 anti rabbit alexa fluor 594 (Invitrogen, Carlsbad, USA) and 1 :300 anti- mouse alexa fluor 488 (Invitrogen). DAP I counterstaining was also performed. Fluorescent images were visualized with a Leica DM 5500 microscope (Leica Microsystems, Wetzlar Germany). Images were processed with Adobe Photoshop (Adobe Systems incorporated, San Jose, CA, USA).

Flow Cytometry. Cultured cells were detached by trypsinization and collected following centrifugation. Cells pellets were fixed with 4% paraformaldehyde for 10 min at RT, permeabilized with 0.5% Triton X-100 for 10 min on ice and blocked with 5% FBS at 4°C for 30 min. Cells were then incubated at 4°C for 1 hour with 1 :600 anti-Pdxl (Abeam) or 1 :600 anti rabbit IgG- Phycoerythrin (PE) goat isotype control (Abeam). Then, cells were washed 3 times at 4°C with PBS and exposed to the secondary antibody, 1 :500 anti Rabbit IgG-PE (Abeam) for 30 min a RT. After 3 washes with PBS, cells were resuspended in 500 μΙ PBS and fluorescence readouts were carried out by flow citometry (BD FACSCalibur).

Bisulfite Sequencing PCR (BSP). A region of approximately 2000 base pairs of the Pdx1 promoter was analyzed with the software Methyl Primer Express v1.0. (Applied Biosystems) to identify CpG-rich islands. Primers were designed upon these regions are listed in Supplementary Table 1. Then, genomic DNA from 7.5 x 10⁴ cells was converted with sodium bisulphite using Cells-to-CpG™ Bisulphite Conversion Kit (Applied Biosystems). Converted DNA was amplified by PCR using MyTaq™ HS Red DNA Polymerase, and then PCR products were purified and cloned into pGEM-T to obtain E. coli colonies. 10n colonies per treatment were analyzed by PGR and later sequenced in a DNA analyzer 3730 (Applied Biosystems). The results were analyzed by the BiQ Analyzer Software.

Bisulfite Pyrosequencing. The results obtained by BSP were confirmed by pyrosequencing. Sodium bisulphite modification of genomic DNA of 7.5 x 10" cells was carried out as described above. Converted DNA was eluted in 15 pi and 2 μΙ were used for each polymerase chain reaction (PGR). Primers used to PGR and sequencing were designed using the PyroMark assay design software, version 2.0.01.15. Pyrosequencing primers are shown in Supplementary Table 1 . These primers were designed to hybridize with CpG- free sites to ensure methylation- independent amplification. PGR was performed with biotinylated primers to convert the PGR product to single-stranded DNA templates, using the Vacuum Prep Tool (Biotage, Sweden), according to manufacturer's instructions. Pyrosequencing reactions and methylation quantification were performed in a PyroMark Q24 System version 2.0.6 (Qiagen)

Chromatin Immunoprecipitation Assay. Cells were cross-linked with 1 % (w/v) formaldehyde for 10 min at 37°C. 3x10^e cells were resuspended in lysis buffer containing 10mM NaCI, 10mM TrisHCI (Ph8), 3mM CI₂Mg and 0.5 mM DTT supplemented with proteases inhibitors (Sigma) for 10 min on ice. Cells were then centrifuged for 5 min at 3000rpm at 4°C. Supernatants were discarded and nuclei containing fraction was washed by gentle inversion with buffer containing 10mM Tris HCI (pH 8), 15 mM NaCI and 60 mM KCI. Cell nuclei were subsequently centrifuged for 5 min at 3000 rpm at 4°C and incubated with washing buffer supplemented with 3mM CI₂Ca, protease inhibitors, 0.5 mM DTT and 5-10 pi Micrococcal Nuclease (1 :200 dilution) (New England BioLabs). Cell nuclei were incubated for 20 min at 37°C with orbital shaking. Nuclease activity was halted by addition of 20 pi of 0.5 mM EDTA. Cell nuclei were centrifuged 5 min at 3000 rpm at 4°C and lysed with buffer containing 150mM NaCI, 50mM TrisClH (7.5), 5mM EDTA, 0.5% NP-40, 1 % Triton, 0.01 % SDS and sonicated with 3 pulses of 10 seg each at 10% amplitude in a Branson sonifier. Extracts were then centrifuged 10 min at 10000 rpm at 4°C. Supernatants containing chromatin with an average size of 500 bp were immunoprecipitated with 2-4 pg of antibody. The antibodies used were as follows: anti-P300 (Santa Cruz, Biotech), anti-JARID-2 (Abeam), anti-acetylated H3 (Abeam), anti-trimethyl K4 histone H3 (Abeam) and anti-trimethyl K27 histone H3 (Cell Signaling, Danvers, MA, USA). Rabbit Control IgG (Abeam) and Mouse igG1 Isotype Control mAb (Cell Signalling) were used as ChIP controls. 15 pL of Dynabeads® (Invitrogen, Dynal AS, Oslo, Norway) were used to prepare Ab-beads complexes and incubated for 30min at 4°C under rotation in dilution buffer (0.01 % SDS, 1.1% Triton X-100, 1 .2 mM EDTA, 16.7 mM Tris HCI pH 8.1 , 167 mM NaCI). Then, chromatin was added and incubated for 1 h. Washes of the complex were once with low-salt buffer (0.1 % SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris HCI pH8.1 , 150 mM NaCI), once with high-salt buffer (0.1 % SDS, 1 % Triton X-100, 2 Mm EDTA, 20 mM Tris HCI pH8.1 , 500 mM NaCI), once with LiCI buffer (0.25M LiCI, 1%NP40 (Igepal), 1 % deoxycholate, 1 mM EDTA, 10 mM Tris HCI pH 8), twice with TE buffer (10mM Tris HCI pH 8.1 mM EDTA) and finally eluted with 500 μΙ of 1% SDS, 0,1 M NaHC0₃ solution. DNA was purified was by phenol: chloroform procedure. ChIP analysis was performed by real time-PCR using Sybr Green. Promoter occupancy was determined by percent input method relativized to the pluripotency condition. Primers used are listed in Supplementary table 1.

Immunoprecipitation. Cells were trypsinized from culture dishes, centrifuged and washed once with cold PBS. Cell pellets were then resuspended and incubated in nondenaturing lysis buffer containing 50 mM Tris-HCI, pH 7.4, 300 mM NaCI, 5mM EDTA, 0.1 mM neocuproine 1% Triton X-100, supplemented with protease inhibitor cocktail (Sigma) for 45 min on ice and subsequently sonicated with 4 pulses 10 s each at 10% amplitude. Following centrifugation, supernatant proteins were quantified by Bradford. Samples containing 3 mg of protein were incubated with 3 Mg of anti-JARID-2 (Abeam) for 2 hours stirring at 4°C. Then, 70μΙ of Dynabeads protein A (Life Technologies, Paisley, Scotland) were attached to the complex antibody-proteins overnight at 4°C. Then, samples were washed 3 times with nondenaturing lysis buffer for 5 min stirring at 4°C and one last wash with 200 μΐ Tris 50mM pH8. Proteins were denatured with 50 μΙ of Laemmly buffer 1X supplemented with β-mercaptoethanol, separated by gradient SDS-PAGE (Mini-Protean TGX Gels, 4-15 % acrylamide, Bio Rad, California, USA) and transferred to PVDF membranes. Blockade with TBS-Tween 5% defatted dried milk ensued for 30 min at room temperature and membranes were finally probed with anti-JARID-2 1:1000 (Abeam), anti-P300 1:1000 (Santa Cruz Biotech) and anti-EZH2 1 :500 (BD Transduction). Secondary antibodies were anti-rabbit IgG (Sigma) and anti-mouse IgG (Jackson ImmunoResearch). 100 pg of total protein was loaded as input.

Statistical analyses Comparisons between values were analyzed using one-tailed Student's t test. RESULTS

The present invention provides new method to increase expression of markers such as PDX-1 , glucose transporter 2, Nkx6.1 , Glucokinase, Kir 6.2, Pax4 and Insulin in cells differentiated from pluripotent stem cells. In order to assess the molecular mechanism underlying pancreatic beta cell formation, the inventors focused on methylation of CpG islands, changes in bivalent marks of histones H3K4me3 and H3k27me3 and occupancy of PCR2 and P300 on Pdx1 promoter. DETA-NO Induces Pdx1 Expression

Cells cultured for 3 days in the presence or absence of LIF and 500 μΜ DETA-NO (19h) increase Pdx1 expression, both at mRNA and protein levels (Figure A, B). Moreover, immunofluorescence studies reveal that PDX1 is localized in nuclei (Figure 1C). The population of cells treated with DETA-NO displays a rightward shift in cytometry plots, thus suggesting an enhanced expression of PDX1 protein (Figure 1 D). Quantification of fluorescence intensity shows that PDX1 -positive cells increase their fluorescence significantly following exposure to DETA-NO (Figure 1 E).

Impact of DETA-NO on the Methylation of Pdx 1 promoter To gain insight on the molecular processes involved in the effect of DETA-NO on Pdx1 expression, analysis of epigenetic changes occurring at the Pdxl promoter region was undertaken. 3 CpG islands were detected by Methylprimers Express software: (-2000, -1600), (-350, 650), (1 150, 1650) bp from the translation start. We focused our study on islands dubbed distal CpG (-2000, -1600) and proximal CpG (-350, 650) since it has been reported that the 3 conserved and functionally important subdomains (areas I, II, and III) are localized between - 2800 to -1600 (Gerrish, Van Veikinburgh et al. 2004) and it has been described that methylation of Pdx1 proximal promoter (from -275 to +1 pb) is associated with Pdx1 silencing (Park, Stoffers et al. 2008; Pinney, Jaeckle Santos et al. 201 1 ) (Figure 2A). Bisulfite sequencing PGR analysis shows methylation of 47 CpG sites, belonging to both proximal and distal CpG islands. DETA-NO treatment leads to enhanced methylation of sites and demethylation of site 40. Some of them increase the degree of methylation in the presence or absence of LIF (sites 8, 22 and 46) (Figure 2B). Pyrosequencing study shows similar results, methylation level of the distal CpG island being higher than methylation level of proximal CpG island. Distal CpG island methylation degree of cell cultured in the present of LIF is lower than cells cultured in the absence of LIF and cells treated with NO that show a higher methylation degree, similar to methylation level of INS-1 E. Regarding methylation level of proximal CpG island, cells cultured in the absence of LIF and with NO, presents the most similar methylation pattern with INS-1 E cells. INS- E were used as positive control of Pdx1 expression, (Liu, Liu et al. 20 3) 12 - 16 CpG sites were not analyzed because that region is not conserved between mouse and rat (Figure 2C).

Impact of DETA-NO on Chromatin Remodeling

Histone marks in the proximal region of Pdx1 promoter were studied by ChlP. Occupancy of acetylated H3 at the Pdx1 promoter was studied and the results show that NO does not change significantly the acetylated H3 occupation (Figure 3A). In addition, H3K4me3 and H3K27me3 occupation was studied. ChlP results show that H3K4me3 occupancy is increased after DETA- 5 070501

NO treatment, but not significantly. On the other hand, the H3K27me3 occupancy is decreased in differentiation conditions, but this change is only significantly in conditions without L1F (Figure 3B), All in all the results indicate that exposure to NO tips the balance tips higher occupancy of H3K4me3 and lower occupation of H3k27me3 (Figure 3C). Role of the Repressor Complex 2 in the Regulation of Pdxl expression

To further investigate the mechanism underlying NO-induced H3K27me3 enrichment of Pdxl promoter, expression and function of Polycomb Complex 2 proteins JARID-2 and EZH2 was studied. Results show that while EZH2 does not change its expression following NO treatment, JARID-2 expression is decreased, both mRNA and protein level (Figure 4A and 4B). To ascertain if PRC2 represses directly Pdxl expression, JARID-2 occupancy at Pdxl proximal promoter was studied by ChlP. The result indicates that JARID-2 is released significantly from Pdxl promoter after DETA-NO treatment (Figure 4C).

Furthermore, we studied the contribution of histone acetyltransferase P300 to the regulation of Pdxl expression. Thus, P300 expression was measured and the results indicate that P300 protein is decreased in cells treated with DETA-NO (Figure 4A and 4B). Analysis of P300 occupancy at Pdxl promoter indicates that P300 is released significantly from Pdxl promoter following exposure to NO and in cells cultured in the absence of LIF (Figure 4C). To test the relevance of P300 on Pdxf repression, inhibition studies were performed. The addition of Ρ30Ό inhibitor (P300i) to cells exposed to DETA-NO led to enhanced Pdxl expression, thus suggesting that P300 might act as a repressor of Pdxl (Figure 4E). Furthermore, Chip studies indicate that JARID-2 and P300 occupy the same region at PDX1 promoter. Finally, co- immunoprecipitation reveals that P300 interacts with PRC2 at the Pdxl promoter region (Figure 4D).

Optimized Protocol for Generation of insulin-Producing Cells In view of the relevance of P300 on the regulation of Pd f expression, a modification of a method developed previously was implemented ( ora-Castilla, Tejedo et al. 2010). The modification consisted of the addition of P300i (C646) after treatment with valproic acid, and before growth in suspension for 3 days (Figure 5A). Cell morphology and fate are affected after every step of the protocol. Thus, exposure to DETA-NO leads to substantial cell death and the surviving cells acquire an epithelial morphology. Following exposure to valproic acid, groups of cells form colonies with a round and shiny morphology while others remain scattered and brightless. Addition of P300i provokes considerable cell death, but surviving cells form clusterlike structures. Finally, culture in suspension led to the formation of islets-like clusters (Figure 5B). Analysis of expression markers reveals that DETA-NO treatment leads to an increase in Pdxl, Nkx6.1, Nkx2.2, Ptfla and Glut2, both at the mRNA and protein level (Figure 5C and D). Interestingly, NO increases also pre-mRNA Ins1 level (Figure 5C). Valproic acid treatment enhanced the expression of definitive endoderm genes (Sox17, FoxA2 and Hnflb) and NKx2.2 expression. At day 10, there is decrease in Pdx1 expression which is recovered by exposure to P300i. Moreover, P300i enhances also Pax4 expression. At the end of the differentiation protocol, markers of definitive endoderm (Sox17, Foxa2 and Hnflb) and markers of pancreatic progenitors pancreas genes { Pdx1, Pax4, Nkx6.1, Nkx2.2, Ptfla, KirG.2, GcK, Ins1 and Glut2) are also upregulated (Figure 5D). Cluster formation has been reported to improve long-term maintenance of human islets in vitro (Zhao, Christie et al. 2002) and to increase the percentage of insulin-expressing cells in differentiation protocols (Segev, Fishman et al, 2004). Moreover, suspension culture enables uniformity of cellular composition and pancreatic aggregates can be implanted without disrupting the maturing cellular architecture (Schulz, Young et al. 2012). In the method of the present invention, it is interesting to note that these cells are negative to glucagon, amylase 2 or somatostatin (Figure 5C). Immunofluorescence reveals that most cells express PDX1 and GLUT2 and some of them are positive to C-PEPT (Figure 6). From the results shown in Figure 1 to 4, it can be clearly concluded that a. DETA-NO is great tool to increase the Pdx1 expression in pluriotente stem cells. b. Pdx1 expression after DETA-NO treatment is dependent of PRC2 and P300 release, minor changes in DNA methylation on Pdx 1 promoter. c. DETA-NO induce changes in the occupation balance between bivalent marks H3k27me3/H3k4me3 that is tipped to H3K4me3 in PDX-1 promoter.

From the results shown in Figures 5 and 6, it can be clearly concluded that the combination of DETA-NO, deacetylase inhibitor, P300 inhibitor and suspension culture allowed to increase the expression of beta cell markers.

Bibliography

Ahlgren, U., J. Jonsson, et al. (1996). "The morphogenesis of the pancreatic mesenchyme is uncoupled from that of the pancreatic epithelium in IPF1/PDX1 -deficient mice." Development 122(5): 1409-1416.

Aoi T, Yae K, Nakagawa M, lchisaka T, Okita K, Takahashi K, Chiba T, Yamanaka S. Generation of Pluripotent Stem Cells from Adult Mouse Liver and Stomach Cells. Science. 2008Asano, Y. and M. Trojan owska (2013). "Fli1 represses transcription of the human alpha2(l) collagen gene by recruitment of the HDAC1/p300 complex." PLoS One 8(9): e74930. Baluchamy, S., H. N. Rajabi, et al. (2003). "Repression of c-Myc and inhibition of G1 exit in cells conditionally overexpressing p300 that is not dependent on its histone acetyltransferase activity." Proc Natl Acad Sci U S A 100(16): 9524-9529.

Bernardo, A. S., C. H. Cho, et al. (2009). "Biphasic induction of Pdx1 in mouse and human embryonic stem cells can mimic development of pancreatic beta-cells." Stem Cells 27(2): 341- 351.

Bernstein, B. E., T. S. Mikkelsen, et al. (2006). "A bivalent chromatin structure marks key developmental genes in embryonic stem cells." Cell 125(2): 315-326.

Boyer, L A., T. I. Lee, et al. (2005). "Core transcriptional regulatory circuitry in human embryonic stem ceils." Cell 122(6): 947-956.

Boyer, L. A., . Plath, et al. (2006). "Polycomb complexes repress developmental regulators in murine embryonic stem cells." Nature 441 (7091): 349-353.

Chen, W., W. Jia, et al. (2013). "Distinct roles for CBP and p300 on the RA-mediated expression of the meiosis commitment gene Stra8 in mouse embryonic stem cells." PLoS One 8(6): e66076.

D'Amour, K. A., A. G. Bang, et al. (2006). "Production of pancreatic hormone-expressing endocrine cells from human embryonic stem cells." at Biotechnol 24(11): 1392-1401.

Gerrish, K., M. Gannon, et al. (2000). "Pancreatic beta cell-specific transcription of the pdx-1 gene. The role of conserved upstream control regions and their hepatic nuclear factor 3beta sites." J Biol Chem 275(5): 3485-3492.

Gerrish, K., J. C. Van Velkinburgh, et al. (2004). "Conserved transcriptional regulatory domains of the pdx-1 gene." ol Endocrinol 18(3): 533-548.

Girdwood, D., D. Bumpass, et al. (2003). "P300 transcriptional repression is mediated by SUMO modification." Mol Cell 1 (4): 1043-1054. Hamazaki, T., M. Oka, et al. (2004). "Aggregation of embryonic stem cells induces Nanog repression and primitive endoderm differentiation." J Cell Sci 1 17(Pt 23): 5681-5686.

Hanna J, Wemig M, Markoulaki S, Sun CW, Meissner A, Cassady JP, Beard C, Brambrink T, Wu LC, Townes TM, Jaenisch R. Treatment of sickle cell anemia mouse model with iPS cells generated from autologous skin. Science. 2007, 318(5858): 1920- 1923.

Hmadcha, A., F. J. Bedoya, et al. (1999). "Methylation-dependent gene silencing induced by interleukin 1beta via nitric oxide production." J Exp Med 190(11): 1595-1604. EP2015/070501

Kadowaki, M., H. Yoshioka, et al. (2012). "DNA methy!ation-mediated silencing of nonsteroidal anti-inflammatory drug-activated gene (NAG-1/GDF15) in glioma eel! lines." Int J Cancer 130(2): 267-277.

Kim, J., J. Chu, et al. (2008). "An extended transcriptional network for pluripotency of embryonic stem cells." Cell 132(6): 1049-1061.

Kroon, E., L. A. Martinson, et al. (2008). "Pancreatic endoderm derived from human embryonic stem cells generates glucose-responsive insulin-secreting cells in vivo." Nat Biotechnol 26(4): 443-452.

Landeira, D., S. Sauer, et al. (2010). "Jarid2 is a PRC2 component in embryonic stem cells required for multi-lineage differentiation and recruitment of PRC1 and RNA Polymerase II to developmental regulators." Nat Cell Biol 12(6): 618-624.

Lee, T. I., R. G. Jenner, et al. (2006). "Control of developmental regulators by Polycomb in human embryonic stem cells." Cell 125(2): 301-313.

Leon-Quinto T, Jones J, Skoudy A, Burcin M, Soria B (2004) In vitro directed differentiation of mouse embryonic stem cells into insulin-producing cells. Diabetologia 47(8): 1442-1451

Liu, J., Y. Liu, et al. (2013). "Direct differentiation of hepatic stem-like WB cells into insulin- producing cells using small molecules." Set Rep 3: 1185.

Loh, Y. H., Q. Wu, et al. (2006). 'The Oct4 and Nanog transcription network regulates pluripotency in mouse embryonic stem cells." Nat Genet 38(4): 431-440. Lowry WE, Richter L, Yachechko R, Pyle AD, Tchieu J, Sridharan R, Clark AT, Plath K. Generation of human induced pluripotent stem cells from dermal fibroblasts. Proc Natl Acad Sci U S A, 2008, 105(8):2883-2888.

Ma, H., C. Nguyen, et al. (2005). "Differential roles for the coactivators CBP and p300 on TCF/beta-catenin-mediated survivin gene expression." Oncogene 24(22): 3619-3631. Meissner A, Wemig M, Jaenisch R. Direct reprogramming of genetically unmodified fibroblasts into pluripotent stem cells. Nat Biotechnol. 2007, 25(10): 1177- 1 181.Mora-Castilla, S., J. R. Tejedo, et al. (2010). "Nitric oxide repression of Nanog promotes mouse embryonic stem cell differentiation." Cell Death Differ 17(6): 1025-1033.

Nakagawa M, Koyanagi M, Tanabe K, Takahashi K, lchisaka T, Aoi T, Okita K, Mochiduki Y, Takizawa N, Yamanaka S. Generation of induced pluripotent stem cells without Myc from mouse and human fibroblasts. Nat Biotechnol. 2008, 26(1): 101 -106. Okita K., et al, 2007. Generation of germiine-competent induced pluripotent stem cells. Nature 448: 313-318.

Park, J. H., D. A. Stoffers, et al. (2008). "Development of type 2 diabetes following intrauterine growth retardation in rats is associated with progressive epigenetic silencing of Pdx1." J Clin Invest 118(6): 2316-2324.

Pasini, D., P. A. Cloos, et al. (2010). "JARID2 regulates binding of the Polycom b repressive complex 2 to target genes in ES cells." Nature 464(7286): 306-310.

Peng, J. C, A. Valouev, et al. (2009). "Jarid2/Jumonji coordinates control of PRC2 enzymatic activity and target gene occupancy in pluripotent cells." Cell 139(7): 1290-1302. Pinney, S. E., L. J. Jaeckle Santos, et al. (2011). "Exendin-4 increases histone acetylase activity and reverses epigenetic modifications that silence Pdx1 in the intrauterine growth retarded rat." Diabetologia 54(10): 2606-2614.

Rojas, A., A. Khoo, et al. (2010). "Islet cell development." Adv Exp Med Biol 654: 59-75.

Schuettengruber, B., D. Chourrout, et al. (2007). "Genome regulation by polycomb and trithorax proteins." Cell 128(4): 735-745.

Schulz, T. C, H. Y. Young, et al. (2012). "A scalable system for production of functional pancreatic progenitors from human embryonic stem cells." PLoS One 7(5): e37004.

Segev, H., B. Fishman, et al. (2004). "Differentiation of human embryonic stem cells into insulin- producing clusters." Stem Cells 22(3): 265-274. Simon, J. A. and R. E. Kingston (2009). "Mechanisms of polycomb gene silencing: knowns and unknowns." Nat Rev Mol Cell Biol 10(10): 697-708.

Soria B, Roche E, Berna G, Leon-Guinto T, Reig JA, Martin F (2000) Insulin-secreting cells derived from embryonic stem-cells normalize glycaemia in streptozotocin-induced diabetic mice. Diabetes 49 (2): 157 - 162 Soria B, Andreu E, Berna G, Fuentes E, Gil A, Leon-Quinto T, Martin F, Montanya E, Nadal A, Reig JA, Ripoll C, Roche E, Sanchez-Andres JV, Segura J (2000) Engineering Pancreatic Islets Pflugers Archiv- Eur. J. Physiol. 440 (1): 1-18

Soria B, Skoudy A, Martin F (2001) From stem cells to Π -cells: new strategies in cell therapy of diabetes mellitus. Diabetologia 44 (4): 407- 415 Soria B (2001) ln-vitro differentiation of pancreatic β-cells. Differentiation 68 (4-5): 205-219 Staffers, D. A., N. T. Zinkin, et al. (1997). "Pancreatic agenesis attributable to a single nucleotide deletion in the human IPF1 gene coding sequence." Nat Genet 15(1): 106-110.

Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell. 2006, 126(4):663-676. Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T, Tomoda K, Yamanaka S. Induction of pluripotent stem cells from adult human fibroblasts by defined factors.Cell. 2007, 131 (5):861- 872.

Tejedo, J. R., R. Tapia-Limonchi, et al. (2010). "Low concentrations of nitric oxide delay the differentiation of embryonic stem cells and promote their survival." Cell Death Dis 1 : e80. Vaca P, Martin F, Vegara JM, Rovira JM, Berna G, Soria B (2006) Induction of differentiation of ES cells into islet cells by fetal soluble factors. Stem Cells 24(2): 258- 265

Vaca P, Berna G, Araujo R, Carneiro EM, Bedoya FJ, Soria B, Martin F (2008) Nicotinamide induces differentiation of embryonic stem cells into insulin-secreting cells. Experimental Cell Research 314(5): 969-74 Xie, R., L. J. Everett, et al. (2013). "Dynamic chromatin remodeling mediated by polycomb proteins orchestrates pancreatic differentiation of human embryonic stem cells." Cell Stem Cell 12(2): 224-237.

Xu, C. R., P. A. Cole, et al. (2011). "Chromatin "prepattern" and histone modifiers in a fate choice for liver and pancreas." Science 332(6032): 963-966. Yu J, et al., 2009; Science. 2009, 324: 797-801.

Zhang, D„ W. Jiang, et al. (2009). "Highly efficient differentiation of human ES cells and iPS cells into mature pancreatic insulin-producing cells." Cell Res 19(4): 429-438.

Zhao, M., M. R. Christie, et al. (2002). "Amelioration of streptozotocin-induced diabetes in mice using human islet cells derived from long-term culture in vitro." Transplantation 73(9): 1454- 1460.

Claims

1. An insulin-producing cell obtained by a method comprising the addition of a P300 inhibitor to a stem cell culture.

2. The insulin-producing cell according to claim 1 , wherein the P300 inhibitor is selected from the list consisting on: 2,6-Bis[(3-bromo- -hydroxyphenyl)methylene]cyclohexanone

(CAS 932749-62-7), garcinol (Camboginol, CAS 78824-30-3), anacardic acid (CAS 1661 1-84-0), curcumin (CAS 458-37-7), demethoxy curcumin (CAS 22608-1 1-3) and C646(P300i), or combinations thereof.

3. The insulin-producing cell according to anyone of claims 1 -2, wherein the P300 inhibitor is C646(P300i).

4. The insulin-producing cell according to anyone of claims 1-3, wherein the stem cell is a pluri potent stem cell.

5. The insulin-producing cell according to anyone of claims 1 -4, wherein the stem cell is selected from induced plutipotent stem cell, an hESC obtained whithout the destruction of an embryo, a stem cell obtained by parthenogenesis, a cell obtained by transdifferentiation, or an adult pancreatic progenitor stem cell.

6. The insulin-producing cell according to anyone of claims 1 -5, wherein the method also comprises culture the cell in presence of a donor of nitric oxide.

7. The insulin-producing cell according to anyone of claims 1-6, wherein the donor of nitric oxide is selected from the list consisting on: sodium nitroprusside, 3-morpholino- sydnonimine, furoxan CAS 1609, S-nitroso-acetylpenicillamine, S-nitroso-glutathione, sildenafil citrate, nitroprusside, DETA-NO or combinations thereof.

8. The insulin-producing cell according to anyone of claims 1-7, wherein the donor of nitric oxide is DETA-NO.

9. The insulin-producing cell according to anyone of claims 1 -8, wherein the method also comprises culture the cell in presence of an histone deacetylase inhibitor.

10. The insulin-producing cell according to anyone of claims 1 -9, wherein the histone deacetylase inhibitor is selected from the list consisting on: valproic acid, vorinostat (SAHA), tefinostat (CHR-3996), benzoic acid derivatives such as givinostat (ITF2357), mocetinostat (MGCD0 03) and entinostat ( S-275); cynamic acid derivatives such as panobinostat (LBH589), belinostat (PXD101) and pracinostat (SB939) and derivatives of pyrimidin-5-carboxoxilic acid CHR-3996 and quisinostat (SB939).

11. The insulin-producing cell according to anyone of claims 1 -10, wherein the histone deacetylase inhibitor is valproic acid.

12. The insulin-producing cell according to anyone of claims 1-11 , wherein the method also comprises culture the cell in suspension to obtain cell clusters or cellular aggregates.

13. The insulin-producing cell according to anyone of claims 1 -9, wherein the insulin- producing cell express the pancreatic markers PDX-1 , Nkx6.1 , Insulin, Hexokinase IV or Glucokinase, the ATP-dependent potassium channels component Kir6.2 and/or Glut2 (glucose transporter 2).

14. An insulin-producing cell population comprising at least an insulin-producing cell according to anyone of claims 1 -10.

15. A composition comprising an insulin-producing cell according to anyone of claims 1-10, or an insulin-producing cell population according to claim 11 ,

16. The composition according to claim 12, which is a pharmaceutical composition.

17. The composition according to anyone of claims 12-13, further comprising a pharmaceutically acceptable carrier.

18. An artificial tissue or organ comprising an insulin-producing cell according to anyone of claims 1 -10, the insulin-producing cell population according to claim 1 1 , or the composition according to anyone of claims 2-14, preferably in combination with a support material.

19. Use of the insulin-producing cell according to anyone of claims 1-10, the insulin- producing cell population according to claim 1 1 , the composition according to anyone of claims 12-14, or an artificial tissue or organ according to claim 15, for evaluating a pharmacological and/or chemical product.

20. The insulin-producing cell according to anyone of claims 1-10, the insulin-producing cell population according to claim 11 , the composition according to anyone of claims 12-14, or an artificial tissue or organ according to claim, for use as a medicament.

21. The insulin-producing cell according to anyone of claims 1 -10, the insulin-producing cell population according to claim 1 1 , the composition according to anyone of claims 12-14, or an artificial tissue or organ according to claim 15, for preparing a medicament to partially or completely increase, restore or replace the functional activity of a diseased or damaged tissue or organ

22 The insulin-producing cell according to anyone of claims 1-10, the insulin-producing cell population according to claim 1 1 , the composition according to anyone of claims 12-14, or an artificial tissue or organ according to claim 15, for use in the treatment, amelioration or prevention of an insulin related diseases and conditions associated with insufficient insulin production.

23. The insulin-producing cell according to anyone of claims 1 -10, the insulin-producing cell population according to claim 1 1 , the composition according to anyone of claims 12-14, or an artificial tissue or organ according to claim 15, for use in the treatment, amelioration or prevention of pre-diabetes or diabetes.

24. The insulin-producing cell according to anyone of claims 1-10, the insulin-producing cell population according to claim 11 , the composition according to anyone of claims 12-14, or an artificial tissue or organ according to claim 15, for use in the treatment, amelioration or prevention of type 2 diabetes, diabetic ketoacidosis, hyperglycemia, diabetic neuropathy, neuropathy, diabetic cardiopathy, diabetic nefropathy, diabetic rethynopathy and other acute and chronic complications of diabetes.