COMPOSITION FOR INDUCING DIFFERENTIATION INTO INSULIN-PRODUCING CELLS, AND USE THEREOF

Abstract

The present invention relates to a composition for inducing differentiation into insulin-producing cells, and a method for inducing differentiation into insulin-producing cells. By using a differentiation inducing composition according to an exemplary embodiment or a differentiation inducing method according to an exemplary embodiment, insulin-producing cells can be prepared in a short period by effectively inducing the differentiation of various types of stem cells into insulin-producing cells, and can be mass-produced in a relatively simple manner, and thus a pharmaceutical composition for preventing or treating diabetes mellitus, comprising insulin-producing cells and/or insulin produced thereby, can be provided.

Claims

1. A composition for inducing differentiation into insulin-producing cells, comprising putrescine, or one or more selected from the group consisting of putrescine, glucosamine, and nicotinamide.

2. The composition of claim 1, wherein the composition further comprises a STAT3 inhibitor.

3. The composition of claim 2, wherein the STAT3 inhibitor is one or more selected from the group consisting of JSI-124, BP-1-102, and CPT.

4. The composition of claim 1, wherein the putrescine is comprised at a concentration of 1 to 20 mM.

5. The composition of claim 1, wherein the glucosamine is comprised at a concentration of 1 to 20 mM.

6. The composition of claim 1, wherein the nicotinamide is comprised at a concentration of 5 to 20 mM.

7. The composition of claim 2, wherein the STAT3 inhibitor is comprised at a concentration of 0.1 to 50 μM.

8. The composition of claim 1, wherein the composition is capable of inducing differentiation of adult stem cells, embryonic stem cells, and a combination thereof into insulin-producing cells.

9. The composition of any one of claims 1 to 8, wherein the composition further comprises one or more culture media selected from the group consisting of CMRL1066, a Dulbecco's Modified Eagle's Medium (DMEM), a minimal essential medium (MEM), Basal Medium Eagle (BME), RPMI1640, F-10, F-12, an α minimal essential medium (αMEM), a Glasgow's minimal essential medium (GMEM), McCoy's 5A, a 199 medium, and endothelial growth medium MV2.

10. A culture medium composition for inducing differentiation into insulin-producing cells, comprising the composition for inducing differentiation into insulin-producing cells according to any one of claims 1 to 8, and a culture medium.

11. The culture medium composition of claim 10, wherein the culture medium is one or more culture media selected from the group consisting of CMRL1066, a Dulbecco's Modified Eagle's Medium (DMEM), a minimal essential medium (MEM), Basal Medium Eagle (BME), RPMI1640, F10, F-12, an α minimal essential medium (αMEM), a Glasgow's minimal essential medium (GMEM), McCoy's 5A, a 199 medium, and endothelial growth medium MV2.

12. A method for inducing differentiation into insulin-producing cells, the method comprising culturing isolated cells in the culture medium composition according to claim 10.

13. The method of claim 12, wherein the culturing step comprises culturing isolated cells under conditions of a temperature of 35 to 38° C. and 5% CO.sub.2 in the culture medium composition for inducing differentiation for 3 days to 6 days.

14. The method of claim 12, wherein the culture medium further comprises one or more culture media selected from the group consisting of CMRL1066, a Dulbecco's Modified Eagle's Medium (DMEM), a minimal essential medium (MEM), Basal Medium Eagle (BME), RPMI1640, F10, F-12, an α minimal essential medium (αMEM), a Glasgow's minimal essential medium (GMEM), McCoy's 5A, a 199 medium, and endothelial growth medium MV2.

15. The method of claim 12, wherein the method comprises culturing cells by adding a STAT3 inhibitor to the culture medium composition on day 3 to day 5 after cell culture.

16. The method of claim 12, wherein the STAT3 inhibitor is one or more selected from the group consisting of JSI-124, BP-1-102, and CPT.

17. The method of claim 12, wherein the isolated cells are adult stem cells, embryonic stem cells, or a combination thereof.

18. A method for preventing or treating diabetes mellitus, the method comprising administering a composition comprising the composition for inducing differentiation into insulin-producing cells according to any one of claims 1 to 8 and a mixture of patient-derived blood as an active ingredient to a subject in need.

19. A method for preventing or treating diabetes mellitus, the method comprising administering a composition comprising insulin-producing cells differentiated by the method according to claim 12 as an active ingredient to a subject thereof.

Description

DESCRIPTION OF DRAWINGS

[0112] FIG. 1A illustrates a schematic view of the experimental procedure for transplantation of bone marrow cells treated (or primed) with putrescine into diabetes mellitus mouse models prepared by streptozotocin (STZ) administration, and FIG. 1B illustrates the results of measuring random blood glucose (upper graph) and body weight (lower graph) of the diabetes mellitus mouse models. In FIG. 1B, the control indicates normal mice in which diabetes mellitus has not been induced, (STZ) indicates diabetes mellitus mouse models, (STZ+control cells) indicates diabetes mellitus mouse models transplanted with bone marrow cells in which differentiation has not been induced, and (STZ+Put-primed cells) indicates diabetes mellitus mouse models transplanted with bone marrow cells primed with putrescine.

[0113] FIG. 2A illustrates the mRNA expression levels of insulin (INS) and factors that promote the differentiation of insulin-producing beta cells (MAFA, PDX-1, and NEUROG3) in human bone marrow cells that have been induced to differentiate by adding putrescine, and FIG. 2B illustrates the mRNA expression levels of insulin (INS) and factors that promote the differentiation of insulin-producing beta cells (MAFA, NEUROG3) in umbilical cord blood cells that have been induced to differentiate by adding putrescine. In FIG. 2A, * means p<0.05; and ** means p<0.01, and the same applies to the following drawings.

[0114] FIG. 3 illustrates the mRNA expression levels of insulin (INS) and beta cell differentiation-related genes (MAFA, PDX1, or NEUROG3) in cells, in which mouse bone marrow cells were treated with glucosamine and/or putrescine to induce differentiation. In FIG. 3, ‘GlcN’ means glucosamine, ‘Put’ means putrescine, ‘−’ means that the corresponding component is not included, and ‘+’ means that the corresponding component is included. The same applies to other drawings.

[0115] FIG. 4 illustrates the mRNA expression levels of insulin (INS) and beta cell differentiation-related genes (MAFA, PDX1, or NEUROG3) in cells, in which umbilical cord blood cells were treated with glucosamine, putrescine, and/or nicotinamide to induce differentiation. In FIG. 4, ‘GlcN’ means glucosamine, ‘Put’ means putrescine, NAD means nicotinamide, and the same applies to other drawings.

[0116] FIG. 5 illustrates the mRNA expression levels of insulin (INS) and a beta cell differentiation-related gene (PDX1) in cells, in which mouse bone marrow cells were treated with glucosamine, putrescine, and/or nicotinamide to induce differentiation.

[0117] FIG. 6A and FIG. 6B illustrate the mRNA expression levels of insulin (INS) and beta cell differentiation-related genes (MAFA, PDX1, NEUROG3, NEUROD1, or NKX6.1) in cells, in which umbilical cord blood cells were treated with glucosamine, putrescine, and nicotinamide in a DMEM or CMRL1066 culture medium to induce differentiation. Specifically, FIG. 6A illustrates the results of RT-PCR, and FIG. 6B illustrates the results of quantitative real-time PCR for each gene. In FIGS. 6A and 6B, NIT-1 means an insulin-producing cell line (beta cell line; insulinoma) in which differentiation has been completed, and the same applies to the following drawings. In FIGS. 6A and 6B, ‘−’ means the results for cells cultured in a culture medium which does not include glucosamine, putrescine, or nicotinamide, and ‘P+G+N’ or ‘P/G/N’ means the results in cells in which differentiation has been induced in a culture medium including glucosamine, putrescine, and nicotinamide.

[0118] FIG. 7A illustrates a method of inducing differentiation into insulin-producing cells by co-treating human embryonic mesenchymal stem cells with glucosamine, putrescine, and nicotinamide. FIG. 7B illustrates the results (RT-PCR) of comparing the expression levels of insulin and beta cell differentiation-related genes (PDX1, NEUROG3, NEUROD1, or NKX6.1) for cells in which embryonic mesenchymal stem cells have been treated with glucosamine, putrescine, and nicotinamide together to induce differentiation. In FIG. 7B, CTL means the control cultured in CMRL 1066 culture medium which does not include glucosamine, putrescine, or nicotinamide, and the numbers 1, 2, and 3 represent the results of repeated experiments under the same conditions, respectively.

[0119] FIG. 8A illustrates a schematic view of a method of inducing differentiation into insulin-producing cells by treating mouse bone marrow cells with glucosamine, putrescine, and nicotinamide together to culture cells, and treating the cells with a STAT3 inhibitor JSI-124 on day 0 (at the start of culture), day 2 or day 4 after the culture. FIGS. 8B and 8C illustrate the results of comparing the expression levels of insulin and beta cell differentiation-related genes (PDX1, NEUROG3, or NEUROD1) for cells that have been induced to differentiate into insulin-producing cells by treating bone marrow cells with glucosamine, putrescine, and nicotinamide together to prime cells and treating the cells with a STAT3 inhibitor JSI-124 on day 0, 2, or 4 after the culture. Specifically, FIG. 8B illustrates RT-PCR results, FIG. 8C illustrates quantitative real-time PCR results, and in FIG. 8C, ‘-’ means a group which is treated with only putrescine, glucosamine, and nicotinamide, and is not treated with a STAT3 inhibitor.

[0120] FIG. 9A illustrates a schematic view of a method of inducing differentiation into insulin-producing cells by treating umbilical cord blood cells with glucosamine, putrescine, and nicotinamide together to culture cells and further treating the cells with various types of STAT3 inhibitors (JSI-124, CPT, or BP-1-102) on day 4 after the culture. FIG. 9B illustrates the results of comparing the expression levels of insulin and beta cell differentiation-related genes (MAFA, PDX1, NEUROG3, NEUROD1, or NKX6.1) for cells that have been induced to differentiate into insulin-producing cells by treating umbilical cord blood cells with glucosamine, putrescine, and nicotinamide together to culture cells and treating the cells with various types of STAT3 inhibitors (JSI-124, CPT, or BP-1-102) on day 4 after the culture (FIG. 9B does not illustrate the results for CPT).

[0121] FIG. 10A illustrates the results of a method of inducing differentiation into insulin-producing cells by treating umbilical cord blood cells with glucosamine, putrescine, and nicotinamide together to culture cells, treating the cells with BP-1-102 on day 4 after the culture, and then maintaining the cells in suspension for 6 days to induce differentiation into insulin-producing cells. Subsequently, the cells were washed and re-suspended in CMRL 1066 culture medium, and then cultured for an additional 3 days. FIG. 10B illustrates the results of measuring the insulin secretion in cells that have been induced to differentiate by the method described in FIG. 10A.

[0122] FIG. 11A illustrates the efficacy of inducing differentiation into insulin-producing cells by the different concentrations of putrescine in the composition for inducing differentiation. FIG. 11B illustrates the cell viability (%) by the different concentrations of putrescine in the composition for inducing differentiation.

[0123] FIG. 12A illustrates a schematic view of a method of transplanting bone marrow cells primed with putrescine, glucosamine, nicotinamide and a STAT3 inhibitor into a kidney capsule of mice on day 7 after STZ induction to generate a diabetes mellitus mouse model. In FIG. 12A, BMNCs indicate bone marrow-derived mononuclear cells (referred to as BMNCs or bone marrow cells herein). FIGS. 12B to 12D illustrate the results of measuring food and water intake, random blood glucose, and body weight of the diabetes mellitus mouse models transplanted with the primed BMNCs for 42 days after STZ induction. FIG. 12E illustrates the results of glucose tolerance test. The mice were fasted for 16 hours and intraperitoneally injected with a bolus of glucose (1 mg/g body weight) and the blood glucose levels were measured at the indicated time points. FIG. 12F illustrates the results of in vivo glucose-stimulated insulin secretion that measures the insulin levels in blood after glucose stimulation of 2 mg/g body weight in a diabetes mellitus mouse model transplanted with the primed BMNCs.

[0124] FIG. 13A illustrates the results of observing the protein expression for insulin and PDX1 by immunofluorescence staining in diabetes mellitus mouse models transplanted with BMNCs primed with putrescine, glucosamine, nicotinamide and a STAT3 inhibitor. FIG. 13B illustrates the results of observing the expression of insulin protein through immunohistochemistry with anti-insulin antibody after isolating pancreatic tissue from a diabetes mellitus mouse model transplanted with BMNCs-derived insulin-producing cells.

MODES OF THE INVENTION

[0125] The present invention will be described in more detail with reference to the following examples, but the scope of rights is not intended to be limited to the following examples.

Example 1. Induction of Differentiation into Insulin-Producing Cells by Putrescine

Example 1.1 Effect of Reducing Blood Glucose in a Diabetes Mellitus Mouse Model

[0126] In the present example, it was intended to investigate whether the diabetes mellitus mouse models transplanted with bone marrow cells primed with putrescine had the ability to regulate blood glucose levels.

[0127] Bone marrow cells were prepared by flushing the femurs and tibias of 8-week-old male C57B/6 mice (Seoul National University Institute of Laboratory Animal Resources). Whole bone marrow cells were suspended in a lysis solution (BD Pharmingen) to remove red blood cells, washed, and re-suspended in the standard culture medium (Dulbecco's Modified Eagle's Medium (DMEM)) supplemented with 5.5 mM glucose, 10% FBS, and 1% antibiotics. Bone marrow cells (5×10.sup.6 cells/well) were seeded in a 12-well non-coated plate, treated with putrescine to a final concentration of 10 mM, and cultured for 6 days in suspension on a shaking (30-60 rpm) platform in a cell culture incubator (37° C., 5% CO.sub.2, and 90 to 95% humidity).

[0128] Matrigel grafts were prepared by mixing bone marrow cells primed with putrescine (2×10.sup.6 cells) with the same volume of Matrigel (BD), and transplanted into the subcutaneous space on the back of mice. Diabetes mellitus was induced in 8-week-old male C57BL/6 mice (Orientbio Inc.) by a single intraperitoneal injection of 140 mg/kg streptozotocin (STZ, Sigma) and the mice with random blood glucose level at 400 mg/dl or more were considered as STZ-induced diabetes mellitus mouse models.

[0129] In the diabetes mellitus mouse model, the cells primed with putrescine (2×10.sup.6 cells) as described above were transplanted into the subcutaneous space twice on days 3 and 14 after STZ administration. Random blood glucose levels and body weight of diabetes mellitus mouse models transplanted with the primed cells were measured for 28 days, which are illustrated in FIG. 1B.

[0130] As illustrated in FIG. 1B, random blood glucose levels were increased and body weights were decreased in all of the STZ-administered groups, compared to the control group. However, an increase in blood glucose levels was inhibited and a decrease in body weight caused by diabetes mellitus was suppressed in a group that was transplanted with the cells primed with putrescine (Put-primed cells).

Example 1.2 Differentiation of Human Bone Marrow Cells into Insulin-Producing Cells

[0131] In the present example, it was verified whether human-derived stem cells were induced to differentiate into insulin-producing cells by putrescine. Human bone marrow cells derived from white male donors were purchased from KOMABIOTECH Inc.

[0132] Human-derived bone marrow cells (5×10.sup.6 cells/well) were seeded into 12-well non-coated plates and primed for 6 days in a DMEM culture medium containing 5.5 mM glucose, 10% FBS, and 1% antibiotics, supplemented with putrescine to a concentration of 5 mM. The cells were cultured in suspension on a shaking (30 to 60 rpm) platform in a cell culture incubator (37° C., 5% CO.sub.2).

[0133] To investigate whether or not insulin-producing cells appeared, the gene expression levels for insulin (INS) and factors (MAFA, PDX-1, and NEUROG3) that promote the differentiation of insulin-producing beta cells were measured using quantitative real-time PCR after harvesting the cells on day 6 after culture. The results are illustrated in FIG. 2A.

[0134] Specifically, total RNA was extracted by collecting cells on day 6 after culture. The RNA concentration/quality was evaluated using a NanoDrop spectrophotometer (NanoDrop Technology, Wilmington, Del., USA). The same amount (1 μg) of RNA was reverse-transcribed into cDNA using a reverse transcription kit (Enzynomics, Daejeon, Korea), and quantitative real-time PCR (ABI PRISM 7900, Applied Biosystems) was performed using SYBR Green PCR Master Mix (Applied Biosystems, Foster City, Calif., USA). The gene expressions for insulin (INS), MAFA, PDX-1, NEUROG3 and the like were measured by performing a cycle consisting of 95° C. for 10 minutes, 95° C. for 10 seconds and 60° C. for 30 seconds by repeating the cycle 40 times. The relative mRNA expression of each sample was standardized by GAPDH (control gene) and the statistical analysis was performed using the Student's unpaired t-test. The base sequences of the primers used for amplification are shown in the following Table 1.

TABLE-US-00001 TABLE 1 Base sequence Primer (5′->3′) SEQ ID NO INS Forward CTG CAT CAG AAG  SEQ ID NO: 1 AGG CCA TCA AG Reverse GGG TGT GTA GAA  SEQ ID NO: 2 GAA GCC TCG MAFA Forward CGC ACG CTC AAG  SEQ ID NO: 3 AAC CG Reverse GCC AGC TTC TCG  SEQ ID NO: 4 TAT TTC TCC TTGT PDX-1 Forward TTC ACG AGC CAG  SEQ ID NO: 5 TAT GAC CTT CAC Reverse GAA GAC AGA CCT  SEQ ID NO: 6 GGG ATG CAC A NEUROG3 Forward CTA AGA GCG AGT  SEQ ID NO: 7 TGG CAC TG Reverse CCG AGT TGA GGT  SEQ ID NO: 8 TGT GCA TT GAPDH Forward CTG CAC CAC CAA  SEQ ID NO: 9 CTG CTT AG Reverse AGG CAG GGA TGA  SEQ ID NO: 10 TGT TCT GG

[0135] As illustrated in FIG. 2A, it was confirmed that the mRNA expression of insulin (INS), MAFA, PDX1, and NEUROG3 was significantly increased in human-derived bone marrow cells in which putrescine was added to induce differentiation, compared to a control (indicated as ‘-’ in FIG. 2A) cultured in a culture medium composition which did not include putrescine.

Example 1.3 Differentiation of Human Umbilical Cord Blood Stem Cells into Insulin-Producing Cells

[0136] As another source of human-derived stem cells, umbilical cord blood-derived mononuclear cells (or umbilical cord blood cells herein), which are relatively easily supplied, were treated with putrescine to induce differentiation into insulin-producing cells.

[0137] Umbilical cord blood-derived mononuclear cells were freshly isolated using Ficoll-gradient protocol and suspended in a lysis solution (BD Pharmingen) to remove red blood cells.

[0138] Differentiation was induced by treating (priming) with putrescine in the same manner as in the method of Example 1.2, except that putrescine was added to a concentration of 1 mM or 5 mM, and the gene expression levels for insulin (INS) and factors (MAFA, NEUROG3) that promote the differentiation into insulin-producing beta cells were measured by performing quantitative real time PCR in the same manner as in the method of Example 1.2. As illustrated in FIG. 2B, the mRNA expression of beta cell-specific genes (INS, MAFA, and NEUROG3) was significantly increased in human-derived umbilical cord blood cells to which 5 mM putrescine was added for differentiation, compared to control cells (indicated as ‘0’ in FIG. 2B) cultured in a culture medium composition which did not include putrescine.

Example 1.4 Effect on Differentiation into Insulin-Producing Cells by Different Concentrations of Putrescine

Example 1.4.1 Effect on Differentiation into Insulin-Producing Cells by Different Concentrations of Putrescine

[0139] Human umbilical cord blood cells were cultured in a culture medium (putrescine 0 mM) which did not include putrescine or a culture medium including putrescine at various concentrations of 0.1 to 20 mM in the same manner as in Example 1.3. After being cultured for 6 days, cells were collected and subjected to quantitative real-time PCR to measure the gene expression levels for insulin (INS) and factors (MAFA, PDX-1, NEUROG3, or NKX6.1) that promote the differentiation into insulin-producing beta cells, and the gene expression levels are illustrated in FIG. 11A (the primers used to measure the gene expression levels are shown in Tables 1 and 2).

[0140] As illustrated in FIG. 11A, the gene expression levels for insulin and the factors that promote the beta cell differentiation were significantly increased in the cells primed with putrescine at a concentration of 5 mM or more.

Example 1.4.2 Analysis of Cell Viability by Different Concentrations of Putrescine

[0141] Human umbilical cord blood cells were cultured in a culture medium (putrescine 0 mM) which did not include putrescine or a culture medium including putrescine at a concentration of 5 to 20 mM in the same manner as in Example 1.3. After being cultured for 6 days, cells were collected, and live and dead cells were stained with acridine orange (AO, live cell stain) and propidium iodide (PI, dead cell stain), respectively, and then the cell number and the cell viability (%) were acquired using a Cellometer Fluorescent Viability Cell Counter K2, as shown in FIG. 11B.

[0142] As illustrated in FIG. 11B, the number of cells in the culture medium composition containing 20 mM putrescine was sharply decreased. Based on this finding, putrescine concentrations above 20 mM appear to affect the cell viability.

Example 2. Differentiation into Insulin-Producing Cells by Co-Treatment of Putrescine and Glucosamine

[0143] Mouse bone marrow cells were isolated from 8-week-old male C57BL/6 mice (Orientbio Inc.) in the same manner as in Example 1.1. Bone marrow cells in DMEM culture medium (Dulbecco's Modified Eagle's Medium) (standard medium) containing 5.5 mM glucose, 10% FBS, and 1% antibiotics were seeded at 5×10.sup.6 cells/ml into a 12-well non-coated plate, treated with glucosamine (GlcN) and putrescine (Put) to a concentration of 10 mM each or together, and cultured for 6 days in suspension for differentiation.

[0144] To investigate whether or not insulin-producing cells appeared, the gene expression levels of insulin (INS), MAFA, PDX-1, and NEUROG3 were measured by a quantitative real-time PCR method, as in the method of Example 1.2, which are shown in FIG. 3.

[0145] As illustrated in FIG. 3, when cells were treated with glucosamine or putrescine compared to the control, the gene expression of insulin and factors that promote the differentiation into insulin-producing cells was changed, and the mRNA expression of insulin (INS), MAFA, PDX-1 and NEUROG3 was all synergistically increased in the group co-treated with putrescine and glucosamine compared to the group treated with putrescine or glucosamine alone. Particularly, the insulin expression was synergistically increased when differentiation was induced by the co-treatment with glucosamine and putrescine (26-fold compared to the control), rather than when differentiation was induced by the treatment with glucosamine (2-fold compared to the control) or putrescine (4.3-fold compared to the control) alone.

Example 3. Optimization for Differentiation into Insulin-Producing Cells

[0146] In the present example, it was intended to investigate the effect of a composition containing putrescine, glucosamine, and nicotinamide on differentiation into insulin-producing cells.

[0147] Samples were divided into (1) putrescine alone-treated group (5 mM), (2) putrescine (5 mM) and glucosamine (5 mM)-co-treated group, (3) putrescine (5 mM), glucosamine (5 mM), and nicotinamide (10 mM)-co-treated group, and (4) control (non-treated group). Umbilical cord blood cells (prepared in the same manner as in Example 1.3) and mouse bone marrow cells (prepared in the same manner as in Example 1.1) were cultured in suspension to induce the differentiation into insulin-producing cells.

[0148] On day 6 after priming the umbilical cord blood cells or mouse bone marrow cells for differentiation, total RNA was extracted to perform quantitative real-time PCR, similarly to the method of Example 1.2, and the mRNA expression of insulin and beta cell differentiation-related genes (MAFA, PDX1, or NEUROG3) was determined, as shown in FIGS. 4 (in primed umbilical cord blood cells) and 5 (in primed mouse bone marrow cells).

[0149] As illustrated in FIG. 4, the mRNA expression of insulin and beta cell differentiation-related genes was synergistically increased in the co-treated group of putrescine, glucosamine, and nicotinamide, compared to the co-treated group of putrescine and glucosamine. In the co-treated group of putrescine, glucosamine, and nicotinamide, mRNA expression of insulin was increased about 142.5-fold compared to the control, and about 14-fold or more, compared to the co-treated group of glucosamine and putrescine (the expression level of insulin was increased 10-fold compared to the control).

[0150] As illustrated in FIG. 5, the expression of a beta cell differentiation-related gene (PDX1) was synergistically significantly increased in the group, in which mouse bone marrow cells were co-treated with putrescine, glucosamine, and nicotinamide, compared to each group in which differentiation was induced by treating mouse bone marrow cells individually with putrescine, glucosamine, or nicotinamide.

Example 4. Efficacy of Inducing Differentiation into Insulin-Producing Cells by Different Cell Culture Media

[0151] Umbilical cord blood cells were isolated in the same manner as in Example 1.3, and the differentiation effect on culture medium was determined. Umbilical cord blood mononuclear cells were cultured for 6 days in suspension under conditions similar to those in Example 1.1, in a standard culture medium (DMEM) or CMRL1066 culture medium, supplemented with glucosamine, putrescine, and nicotinamide, such that the concentrations of glucosamine, putrescine, and nicotinamide were 5 mM, 5 mM, and 10 mM, respectively. On day 6 after the culture, the expression levels of genes were compared in the cells in which differentiation was induced in the DMEM or CMRL1066 culture medium, respectively, using RT-PCR and quantitative real-time PCR. The sequences of primers used for RT-PCR and real-time PCR are shown in Tables 1 and 2. The conditions for quantitative real-time PCR were the same as in Example 1.2, and RT-PCR was performed with the following steps; 1) initial denaturation of 94° C. for 5 minutes; 2) amplification of 94° C. for 30 seconds, 60° C. for 30 seconds, and 72° C. for 1 minute by repeating the cycle 30 times; 3) final extension of 72° C. for 10 minutes.

TABLE-US-00002 TABLE 2 Base sequence Primer (5′->3′) SEQ ID NO NEUROD1 Forward TAA GAC GCA GAA  SEQ ID NO: 11 GCT GTC CA Reverse GTC CGA GGA TTG  SEQ ID NO: 12 AGT TGC AG NKX6.1 Forward GAA CCG CCG GAC  SEQ ID NO: 13 CAA GT Reverse GTC GTC CGA GTT  SEQ ID NO: 14 GGG ATC CAG GAPDH Forward CTG CAC CAC CAA  SEQ ID NO: 9 CTG CTT AG Reverse AGG CAG GGA TGA  SEQ ID NO: 10 TGT TCT GG

[0152] FIG. 6A illustrates the results of RT-PCR, and FIG. 6B illustrates the results of quantitative real-time PCR. As shown in FIGS. 6A and 6B, the mRNA expression of insulin and beta cell differentiation-related genes was significantly increased when cells were cultured in a CMRL 1066 culture medium to which glucosamine, putrescine, and nicotinamide were added.

Example 5. Verification of Ability to Differentiate into Insulin-Producing Cells Using Human Embryonic Stem Cell-Derived Mesenchymal Stem Cells (E-MSCs)

[0153] Human embryonic stem cell-derived mesenchymal stem cells (E-MSCs) (provided by Seoul National University Hospital) were cultured adherently in a CMRL 1066 culture medium containing 10% FBS and 1% antibiotics in a surface treated culture dish under the conditions of 37° C. and 5% CO.sub.2, and primed with glucosamine (5 mM), putrescine (5 mM), and nicotinamide (10 mM). Cells were collected on day 3 after priming, and total RNA was extracted to perform RT-PCR on the gene expression for insulin and beta cell differentiation-related genes (PDX1, NEUROG3, NEUROD1, or NKX6.1), similarly to the method of Example 4, and the results are shown in FIG. 7B. In FIG. 7B, NIT-1 is an insulin-producing cell line (beta cell line; insulinoma), in which differentiation was completed, and CTL means a control which was cultured in a CMRL 1066 culture medium in the absence of glucosamine, putrescine, or nicotinamide.

[0154] As illustrated in FIG. 7B, it was observed that when differentiation was induced by treating human E-MSCs with a combination of glucosamine, putrescine, and nicotinamide, the mRNA expression of insulin and beta cell differentiation-related genes was significantly increased just on day 3 after the treatment. Therefore, it was confirmed that the method for inducing differentiation into insulin-producing cells (composition for inducing differentiation) could be applied not only to adult stem cells, but also to embryonic stem cell-derived mesenchymal stem cells (embryonic stem cells).

Example 6. Efficacy of Differentiation into Insulin-Producing Cells Relative to the Treatment Time of a STAT3 Inhibitor

[0155] After mouse bone marrow cells were cultured under conditions similar to those in Example 1.1 by treating glucosamine, putrescine, and nicotinamide together in a CMRL 1066 culture medium, the cells were further treated with a STAT3 inhibitor JSI-124 (Calbiochem, DongNam Chemical) on day 0, 2, or 4 after the culture, such that the concentration of JSI-124 in the culture medium was 100 nM (0.1 μM), and then the cells were collected on day 6. In order to confirm the appearance of insulin-producing cells, total RNA was extracted from the collected cells similarly to the method in Example 1.2, and the expression levels of insulin (INS) and beta cell differentiation-related genes (PDX1, NEUROG3, or NEUROD1) were confirmed by RT-PCR and real-time PCR, which are illustrated in FIGS. 8B and 8C. FIGS. 8B and 8C show RT-PCR and real-time PCR results, respectively, and in FIG. 8C, ‘−’ means a group which is treated with only putrescine, glucosamine, and nicotinamide, excluding a STAT3 inhibitor.

[0156] As illustrated in FIGS. 8B and 8C, it could be confirmed that in the experimental group to which the STAT3 inhibitor was added, the expression levels of genes promoting differentiation into insulin-producing cells were increased, and in particular, when JSI-124 was added on day 4 after the culture, the mRNA expression levels of insulin (INS) and beta cell differentiation-related genes (PDX1, NEUROG3, or NEUROD1) were significantly increased compared to those when treated with JSI-124 at other time points.

Example 7. Efficacy of Differentiation into Insulin-Producing Cells by Different Types of STAT3 Inhibitors

[0157] In order to investigate the differentiation effect depending on the type of STAT3 inhibitor, human umbilical cord blood cells isolated in the same manner as in Example 1.3 were cultured in a CMRL1066 culture medium to which glucosamine, putrescine, and nicotinamide (5 mM putrescine, 5 mM glucosamine, and 10 mM nicotinamide) were added together, and further treated with various types of STAT3 inhibitors (JSI-124 (0.1 μM; Calbiochem, DongNam Chemical), CPT (1 μM; Sigma, DongNam Chemical), or BP-1-102 (10 μM; Calbiochem, DongNam Chemical)) at a suitable concentration at which each STAT3 has an inhibitory activity. Cells were collected on day 6 after the culture to confirm the appearance of insulin-producing cells. Total RNA was extracted from the collected cells similarly to the method in Example 1.2, and the mRNA expression levels of insulin (INS) and beta cell differentiation-related genes (NEUROG3, NEUROD1, or NKX6.1) were confirmed by RT-PCR and real-time PCR, which are illustrated in FIG. 9B.

[0158] It could be seen that among the STAT3 inhibitors, the mRNA expression levels of insulin and beta cell differentiation-related genes were significantly increased in the order of CPT <JSI-124<BP-1-102, when each inhibitor was additionally treated to cells supplemented with the composition comprising glucosamine, putrescine, and nicotinamide (the results for CPT are not illustrated in FIG. 9B). In particular, it was confirmed that when cells were further treated with BP-1-102 on day 4, the mRNA expression of insulin was increased about 3-fold or more compared to when cells were treated only with the three factors of glucosamine, putrescine, and nicotinamide.

Example 8. Assessment of Insulin Secretion Capability by Treatment with a STAT3 Inhibitor

[0159] As in Example 7, umbilical cord blood cells were primed in the CMRL1066 culture medium containing glucosamine, putrescine, and nicotinamide in suspension, and BP-1-102 was further added on day 4 to facilitate the differentiation into insulin-producing cells. The primed cells were collected on day 6, washed with PBS to remove the remaining differentiation-inducing factors (glucosamine, putrescine, nicotinamide, and BP-1-102), re-suspended in a CMRL1066 culture medium containing 11 mM glucose, along with 10% FBS and 1% antibiotics and then seeded onto a 6-well surface treated culture plate. After 3 days, the supernatants were collected, and insulin levels were measured using an insulin ELISA kit (Ultrasensitive Insulin ELISA, Catalog #80-INSHUU-E01.1; ALPCO, Genomicsone Co., Ltd.), as described in FIG. 10A.

[0160] As shown in FIG. 10B, it was confirmed that umbilical cord blood cells differentiated into insulin-producing cells secreted insulin extracellularly. It was also confirmed that when differentiation was induced by treatment together with a STAT3 inhibitor BP-1-102 on day 4 after the culture, the insulin secretion was increased 10-fold or more compared to when only three differentiation-inducing factors (glucosamine, putrescine, and nicotinamide) were added.

Example 9. Safety and Validation of Efficacy of Transplantation of Differentiated Mouse Bone Marrow Cells in Diabetes Mellitus Mouse Models

[0161] In the present example, it was intended to investigate whether mouse bone marrow cells primed with a composition comprising putrescine, glucosamine, nicotinamide and a STAT3 inhibitor had the capability to regulate blood glucose levels in diabetes mellitus mouse models.

[0162] Mouse bone marrow cells (referred to BMNCs herein in FIG. 12A) were isolated from 8-week-old male C57BL/6 mice (Seoul National University Institute of Laboratory Animal Resources), in the same manner as described in Example 1.1. BMNCs were seeded into a 12-well non-coated plate at 5×10.sup.6 cells/ml in a CMRL1066 medium containing 10% FBS and 1% antibiotics, and primed for 6 days with putrescine, glucosamine, nicotinamide and a STAT3 inhibitor. Primed or non-primed BMNCs (1×10.sup.6 cells/mouse) were resuspended in the same volume of Matrigel (Corning Life Sciences) for kidney capsule transplantation. Diabetes mellitus was induced in male C57BL/6 mice aged 8 weeks (Seoul National University Institute of Laboratory Animal Resources) by a single intraperitoneal injection of 150 mg/kg of streptozotocin (STZ, Sigma). Three-day post STZ stimulation, animals with random blood glucose levels 400 mg/dL for three consecutive days were considered as STZ-induced diabetes mellitus mice. As illustrated in FIG. 12A, the primed cell-containing Matrigel solution was transplanted into a kidney capsule after the kidney was exposed through a small lumbar incision on day 7 after STZ administration. Random blood glucose levels, body weight, and food and water intake were measured every 3-4 day for 42 days. As shown in FIGS. 12B to 12D, the STZ-treated control mice implanted with non-primed BMNCs exhibited metabolic parameters including a significant increase in both food and water intake, blood glucose levels and persistent weight loss. However, the primed cell-implanted mice showed decreased blood glucose levels as early as day 18 following transplantation. Abnormally increased food and water intake decreased after transplantation. The mice also maintained their body weight. Diabetes mellitus mouse models transplanted with the primed BMNCs showed significantly lower fasting blood glucose levels and improved glucose tolerance following intraperitoneal glucose challenge at a concentration of 1 g/kg body weight on day 27 after grafting, compared to the matched controls, as illustrated in FIG. 12E.

[0163] To confirm the capability of insulin secretion in vivo following glucose loading, mice were fasted in the same manner as above on day 34 after transplantation, and blood samples were obtained via tail vein at baseline insulin levels (0 min) before and 15, 30 minutes after a bolus of glucose at 2 g/kg body weight. Plasma insulin levels were measured using the Mouse Ultrasensitive Insulin ELISA (ALPCO). In vivo glucose-stimulated insulin secretion from the primed cell-transplanted mice displayed significantly increased levels of plasma insulin at 15 min post-glucose injection compared to the corresponding controls in STZ-induced mice, as illustrated in FIG. 12F. These results suggested that BMNC-derived insulin-producing cells are functional in vivo and capable of lowering hyperglycemia in diabetes mellitus mouse models.

Example 10. The Presence of Insulin- and PDX1-Expressing Cells in Primed Cell-Transplanted Kidney

[0164] In order to confirm whether the primed cells transplanted under the kidney capsule were involved in the amelioration of hyperglycemia, the kidney sections from the nephrectomized mice were subjected to immunofluorescence staining using anti-insulin and anti-PDX1 antibodies.

[0165] As illustrated in FIG. 13A, immunofluorescence staining confirmed the presence of insulin- and PDX1-expressing cells in the primed cell-transplanted kidney; however, the expression of insulin and PDX1 was not observed in the non-primed cell-transplanted kidney. In FIG. 13B, to rule out the possibility of endogenous pancreatic beta cell regeneration, immunohistochemistry with anti-insulin antibody from the pancreatic tissues was performed on day 42 from the STZ-induced mice transplanted with primed cells.

[0166] As illustrated in FIG. 13B, the pancreas from the mice grafted with primed cells showed near complete loss of pancreatic islets, which is comparable to the diabetic controls, and no evidence of endogenous beta cell regeneration. As an additional assessment, the pancreatic insulin content in these mice was quantitatively determined. Pancreatic tissues cut in half were placed into acid-ethanol buffer containing 1.5% HCl in 70% EtOH solution overnight at −20° C. for insulin extraction. The tissues were then homogenized and centrifuged, and the supernatants were neutralized with 1 M Tris with a pH of 7.5 to measure the insulin content by the Mouse Insulin ELISA kit (ALPCO). Similar to the results of immunohistochemistry, no significant difference between the groups (control vs. primed cells) was observed. Thus, the primed cells respond to glucose challenge in vivo by releasing insulin.

INDUSTRIAL APPLICABILITY

[0167] The composition for inducing differentiation into insulin-producing cells in the present invention can produce insulin-producing cells using various types of stem cells in a short period of time by a simple method, and the insulin-producing cells differentiated by the method of the present invention or insulin produced therefrom can be effectively used for the treatment of diabetes mellitus. In the current situation, where there is no clear treatment method for diabetes mellitus other than insulin injection therapy, the present invention is expected to be an innovative technology for the treatment of diabetes mellitus, in that insulin-producing cells can be easily produced by priming the various types of adult or embryonic stem cells with the composition comprising putrescine, glucosamine, nicotinamide and a STAT3 inhibitor for a short period of time. Further, the composition provided in the present invention can be free from debates on stability and ethics, minimizes an in vitro manipulation stage, and easily mass-produce insulin-producing cells in a short period of time without using an expensive differentiation-inducing factor, gene manipulation or the like, and thus the composition is expected to be commercialized and applicable in various fields.