RU2430158C1 - Method for differentiation of adult human stem cells in insulin-secreting cells - Google Patents

Abstract

FIELD: medicine. ^ SUBSTANCE: stem cells of an adult human recovered from subcutaneous fat eye tissues are induced to differentiate in insulin-secreting cells in a medium containing cytokines and growth factors, including the B27 additive, fibroblast-2 growth factor, epidermal growth factor, nicotinamide, glucagon-like peptide-1, active A, insulin-like growth factor and betacelluline. For a cultivation process, high-concentrated glucose is replaced for low-concentrated glucose. ^ EFFECT: invention enables producing cells synthesising considerable amounts of insulin and C-peptide. ^ 3 cl, 8 dwg, 2 tbl, 9 ex

Description

BACKGROUND OF THE INVENTION

1. Field of invention

The present invention relates to the induction of differentiation of adult stem cells into insulin secreting cells.

2. Background of the invention

Type 1 diabetes, also called insulin-dependent diabetes mellitus, is characterized by the loss of insulin-producing beta cells, which is usually caused by an autoimmune attack of the pancreatic islets of Langerhans and leads to insulin deficiency. In the body, insulin plays a key role in blood sugar homeostasis. When insulin is absent or insufficiently produced, there is an increase in blood sugar, which leads to complications in various organs such as kidneys, nerves, retina, etc. Basically, treatment for type 1 diabetes is based on the use of insulin; since injections are required throughout the patient’s life, the use of insulin is not the main treatment for type 1 diabetes. On the other hand, transplantation of exogenous pancreas or beta cells makes it possible to treat type 1 diabetes mellitus. However, many histological limitations are imposed on this measure, which are associated with the surgical transplantation of exogenous pancreas. For example, transplanted cells can be destroyed by autoimmune reactions of the recipient. Recently, many studies have focused on developing methods for treating diabetes using stem cells, the typical representatives of which are embryonic and adult stem cells. Studies have been reported in which embryonic stem cells differentiated into ex vivo insulin-producing cells that can be used to treat hyperglycemia, as revealed by transplantation in mice with diabetes [Fujikawa T, Oh SH, Pi L, Hatch HM, Shupe T, Petersen BE (2005) Teratoma formation leads to failure of treatment for type I diabetes using embryonic stem cell-derived insulin-producing cells. Am J Pathol 166, 1781-1791; D'Amour KA, Bang AG, Eliazer S, Kelly OG, Agulnick AD, Smart NG, Moorman MA, Kroon E, Carpenter MK, Baetge EE (2006) Production of pancreatic hormone-expressing endocrine cells from human embryonic stem cells. Nat Biotechnol 24, 1392-1401]. However, the use of embryonic stem cells in the clinic leads to significant problems, since they cause the formation of cancer during transplantation [Kroon E, Martinson LA, Kadoya K, Bang AG, Kelly OG, Eliazer S, Young H, Richardson M, Smart NG, Cunningham J, Agulnick AD, D'Amour KA, Carpenter MK, Baetge EE (2008) Pancreatic endoderm derived from human embryonic stem cells generates glucose-responsive insulin-secreting cells in vivo. Nat Biotechnol . 26, 397-398].

It has been reported that ectodermal progenitor cells can differentiate into insulin-producing cells [Hori Y, Gu X, Xie X, and Kim SK. (2005) Differentiation of insulin-producing cells from human neural progenitor cells. PLOS Med . 2, 103] and that the epithelial cells of the pancreatic duct can differentiate into structures similar to the structure of the islets of Langerhans of the pancreas [Bonner-Weir S, Taneja M, Weir GC, Tatarkiewicz K, Song KH, Sharma A and O'Neil JJ (2000) . In vitro cultivation of human islets from expanded ductal tissue. Proc Natl Acad Sci USA. 97, 7999-8004]. The latter is not favorable since insulin-producing cells were obtained from embryonic brain cells that can cause tumor formation in the recipient [Amariglio N, Hirshberg A, Scheithauer BW, Cohen Y, Loewenthal R, Trakhtenbrot L, Paz N, Koren-Michowitz M, Waldman D, Leider-Trejo L, Toren A, Constantini S, Rechavi G. Donor-derived brain tumor following neural stem cell transplantation in an ataxia telangiectasia patient. PLoS Med. 2009 Feb 17; 6 (2): e1000029]. The latter is not favorable, since only a very limited number of stem cells can be obtained from duct tissues. Moreover, it was reported that when differentiation of embryonic stem cells is induced in a medium containing insulin, the insulin secreted by differentiated cells is actually insulin taken from the medium, as demonstrated by the fact that no propeptide is secreted that breaks down into insulin and C peptide [Rajagopal J, Anderson WJ, Kume S, Martinez OI, and Melton DA (2003) Insulin staining of ES cell progeny from insulin uptake. Science . 299, 363; Hansson M, Tonning A, Frandsen U, Petri A, Rajagopal J, Englund MC, Heller RS, Hakansson J, Fleckner J, Skold HN, melton K, Semb H, and Serup P (2004) Artifactual insulin release from differentiated embryonic stem cells . Diabetes . 53, 2603-2609]. Thus, in recent years, many attempts have been made to induce differentiation of embryonic stem cells in the absence of insulin. Differentiation of stem cells into insulin-producing cells in the presence of insulin-like growth factors or betacelluline has been reported; these data are summarized below in table 1. Ethical problems and the above cancer formation make it difficult to use embryonic stem cells in the treatment of diabetes. In addition, as a result of differentiation from pluripotent stem cells derived from human skin fibroblasts or mesenchymal stem cells, only surprisingly low levels of insulin and C-peptide can be achieved.

Table 1
Examination of differentiation of stem cells into insulin-producing cells ex vivo Cell Insulin secretion C peptide secretion Literary source Embryonic stem cell - 5 ng / μg DNA Jiang et al., 2007 ¹⁾ Fibroblast skin 0.15 ng / μg DNA Tateishi et al., 2008 ²⁾ Mesenchymal stem cell 1.291 honey / l 163 ng / l Li et al., 2008 ^{3) 1)} Jiang J, Au M, Lu K, Eshpeter A, Korbutt G, Fisk G, Majumdar AS (2007) Generation of insulin-producing islet-like clusters from human embryonic stem cells. Stem cells 25, 1940-1953.

²⁾ Tateishi K, He J, Taranova O, Liang G, D'Alessio AC, Zhang Y (2008) Generation of insulin-secreting islet-like clusters from human skin fibroblasts. J Biol Chem. 283, 31601-31607.
³⁾ Li L, Li F, Qi H, Feng G, Yuan K, Deng H, Zhou H. (2008) Coexpression of Pdx1 and betacellulin in mesenchymal stem cells could promote the differentiation of nestin-positive epithelium-like progenitors and pancreatic islet -like spheroids. Stem Cells Dev. 17, 815-823.

SUMMARY OF THE INVENTION

The authors of the present invention conducted a thorough and comprehensive study of the cell therapy of type 1 diabetes mellitus, which resulted in the present invention. In this study, it was found that incubation of adult stem cells, if it is performed in an environment containing various cytokines and growth factors, which are known to be effective in differentiating into beta cells, with a change in glucose concentration in the medium from a high level to low level, allows you to differentiate adult stem cells into highly effective insulin-producing cells.

Therefore, an object of the present invention is to provide a method for differentiating adult stem cells into insulin secreting cells.

BRIEF DESCRIPTION OF THE DRAWINGS

The above and other objects, features and other advantages of the present invention will become more apparent from the following detailed description in conjunction with the accompanying drawings, in which:

Figure 1 is a light micrograph showing the morphology of the cells after, under various conditions for three weeks, the differentiation of stem cells obtained from the subcutaneous layer around the eye was induced into insulin-secreting cells;

Figure 2 is a histogram showing the amount of secreted insulin measured using an enzyme-linked immunosorbent assay after the differentiation of stem cells obtained from the subcutaneous layer around the eye, under the conditions of three weeks, into insulin-secreting cells and in they were exposed to 25 mM glucose for two hours;

Figure 3 is a histogram showing the amount of C-peptide secreted measured by enzyme-linked immunosorbent assay after differentiation of stem cells obtained from the subcutaneous layer around the eye for three weeks was induced into insulin-secreting cells , and subjected them to 25 mM glucose for two hours;

Figure 4 is a light micrograph showing the expression of insulin, proinsulin, CK19 and C-peptide measured by immunocytochemical analysis after differentiation of stem cells obtained from the subcutaneous layer around the eye was induced for three weeks under various conditions. insulin secreting cells;

Figure 5 is a photograph of an agarose gel showing the expression of genes characterizing beta cells isolated from cells obtained after three weeks of differentiation between stem cells obtained from the subcutaneous layer around the eye and differentiating them to insulin secreting cells;

6 is a photomicrograph showing staining of cells obtained after, within three weeks, cells obtained from the subcutaneous layer around the eye differentiate into insulin-secreting cells using dithizone, which is specific for insulin-producing beta- cells;

Fig. 7 is a histogram showing the amounts of secreted insulin measured using an enzyme-linked immunosorbent assay after different conditions were induced for three weeks to differentiate stem cells obtained from the subcutaneous layer around the eye into insulin-secreting cells and they were exposed to 25 mM glucose for 2 hours; and

Fig. 8 is a histogram showing the amount of secreted C-peptide measured using an enzyme-linked immunosorbent assay after different conditions were induced for three weeks to differentiate stem cells obtained from the subcutaneous layer around the eye into insulin-secreting cells, and they were exposed to 25 mM glucose for 2 hours.

DESCRIPTION OF PREFERRED EMBODIMENTS

To a large extent, the present invention was carried out using two experiments: in one of them, the effect of insulin-like growth factors on differentiation was tested, and in the other, differentiation was performed in the presence of beta-celluline instead of activin A.

In accordance with one of their aspects, the present invention relates to a method for differentiation of adult stem cells ex vivo into cells secreting insulin, which comprises: cultivating adult stem cells for 4-10 days in an environment containing 20-30 mm glucose, with the addition of 5-20% fetal calf serum, 1-10 nM activin A, 5-20 nM glucagon-like peptide-1 and 10-30 ng / ml fibroblast growth factor-2, and then for 10-20 days in a medium containing 4- 7 mm glucose, with the addition of 5-20% fetal calf serum, 5-20 mm nicotine ida, 1-10 nM activin A, 5-20 nM glucagon-like peptide-1 and 10-100 ng / ml insulin-like growth factor.

More specifically, differentiation of adult stem cells isolated from subcutaneous adipose tissue around the eyes was induced into insulin secreting cells for the treatment of type 1 diabetes in a medium containing cytokines and growth factors, including fibroblast growth factor-2, nicotinamide, glucagon-like peptide-1, activin A, insulin-like growth factor, etc., while the high glucose content in the first culture was replaced by the low glucose content in the second culture.

The two-stage cultivation method was applied to three groups. The control was exposed to a medium containing 20-30 mm glucose in the presence of nicotinamide, activin A and glucagon-like peptide-1 (NAG) for 4-10 days, followed by incubation for 10-20 days in a medium containing 4-7 mm glucose, in the presence of the same cytokines [NAG group], as described in Korean Patent Application No. 2007-44663, which was filed by the present inventors earlier in May 2007. The second group was first exposed to an environment containing 20 for 4-10 days -30 mM glucose, in combination with fetal calf serum, activin A, fibroblast-2 growth factor and glucagon-like peptide-1, followed by a second incubation for 10-20 days in a medium containing 4-7 mm glucose, in combination with fetal calf serum, nicotinamide, activin A, glucagon-like peptide - 1 and insulin-like growth factor-1 [NAGI1 group]. The differentiation of the third group was induced in the same way that was applied to the second group, except that in the second stage, instead of insulin-like growth factor-1, insulin-like growth factor-2 was used.

In accordance with another aspect, the present invention relates to a method for differentiating adult stem cells ex vivo into insulin secreting cells, which is distinguished by the use of betacellululin (known to be an effective tool that promotes differentiation) and the addition of B27 (known as a serum supplement) to provide conditions for differentiation and highly effective secretion of insulin (example 7).

More specifically, in order to differentiate adult stem cells, incubation was performed for 1-7 days in a medium containing 20-30 mM glucose (high glucose medium), supplemented with B27, fibroblast growth factor-2 and epidermal growth factor, and then for 1-7 days in an environment with the same concentration of glucose, with the addition of fetal calf serum, activin A, fibroblast growth factor-2 and glucagon-like peptide-1.

After this, the cells were cultured for 1-7 days in a medium containing 4-7 mm glucose (low glucose medium), in the presence of cytokines and growth factors, including fetal calf serum, activin A, fibroblast growth factor-2, glucagon-like peptide -1, and finally, for 10-20 days in a medium containing 4-7 mm glucose, with the addition of fetal calf serum, beta-celluline, nicotinamide and glucagon-like peptide-1.

In the present invention, DMEM (Dulbecco's Modified Eagle's Medium) or DMEM / F12 (1: 1 Dulbecco's Modified Eagle's Medium: Nutrient F-12 Mixture) can be used.

A detailed description of the induction of differentiation of adult stem cells into cells secreting insulin is given below.

Adipose tissue obtained surgically from the subcutaneous layers around the eye or in the cheek was treated with type I collagenase at 37 ° C for 30 minutes and then centrifuged. The cell pellet thus obtained was cultured in medium supplemented with fetal calf serum to obtain adult stem cells.

To differentiate stem cells into insulin secreting cells, they were cultured for 15-30 days in a medium inducing differentiation. First, they were incubated for 4-10 days in collagen-coated culture dishes in a high glucose medium supplemented with 5-20% fetal calf serum, 5-20 nM glucagon-like peptide-1, 1-10 nM activin A and 10 -30 ng / ml fibroblast growth factor-2. After that, they were transferred to a low glucose medium supplemented with 5-20% fetal calf serum, 5-20 mM nicotinamide, 5-20 nM glucagon-like peptide-1, 1-10 nM activin A and 10-100 ng / ml insulin-like growth factor, followed by incubation for 10-20 days.

Upon completion of the incubation for 15-30 days, before exposing the cells to a high glucose medium for 2 hours, they were treated with bovine serum albumin (BSA) in a low glucose medium for 10-15 hours. Finally, in the culture thus obtained, a quantitative analysis of the formation of insulin and C-peptide was performed using enzyme-linked immunosorbent assay. It was found that the differentiation conditions established in the present invention induce the secretion of increased amounts of insulin and C-peptide compared to the undifferentiated state (negative control). Whereas between the NAGI1 group (with the addition of the insulin-like peptide-1) and the NAG group there were no differences in the secretion of insulin and the C-peptide, the use of the insulin-like peptide-2 (the NAGI2 group) made it possible to increase the secretion of insulin by eight times and the C-peptide by six times compared with the use of insulin-like peptide-1 (group NAGI1). Genetic analysis of cells differentiated in the presence of an insulin-like peptide-2 showed that ND (neuro D1), PC (prohormone convertase) 1/3, PC2, NGN3, Glut (glucose transporter) 1, Glut 2, pax4, pdx1 - it is known that all these genes are specific for pancreatic beta cells - started to be expressed when their differentiation was induced in the culture medium in the first stage, and that the Nkx6.1 gene and the insulin gene were expressed during incubation in the culture medium from the second stage. Also, expression of CK19, proinsulin, insulin and C-peptide, which was measured by immunochemical staining, and strong staining with dithizone, a paint that is specific for pancreatic beta cells, were observed in the cells.

Thus, the method for differentiating adult stem cells into insulin secreting cells in accordance with the present invention essentially consisted of a two-stage cultivation process in which differentiation of adult stem cells was first induced in a high glucose medium in the presence of fibroblast-2 growth factor, activin A and glucagon-like peptide-1, and then in a low glucose medium in the presence of nicotinamide, activin A, glucagon-like peptide-1 and insulin-like factor growth-2, which has the ability to cause increased insulin secretion with respect to the agent used in the NAG group, as described in Korean Patent Application No. 2007-44663, which was filed in May 2007.

Alternatively, adult stem cells can be differentiated into cells that efficiently secrete insulin, in accordance with the present invention as follows.

Adult stem cells were cultured for 1-7 days in a high glucose medium that contained 20-30 mM glucose, 0.5 × -4 × B27 supplement, 10-30 ng / ml fibroblast growth factor-2 and 10-30 ng / ml epidermal growth factor, then for 1-7 days in a high glucose medium that contained 20-30 mM glucose, 5-20% fetal calf serum, 1-10 nM activin A, 5-20 nM glucagon-like peptide -1 and 10-30 ng / ml fibroblast growth factor-2, and then for 1-7 days in a medium with a low glucose content, which contained 4-7 mm glucose, 5 -20% fetal calf serum, 1-10 nM activin A, 5-15 nM glucagon-like peptide-1 and 10-30 ng / ml fibroblast growth factor-2, and, finally, for 10-20 days in a medium with a low content glucose, which contained 5-20% fetal calf serum, 5-20 mM nicotinamide, 1-10 ng / ml betacelluline and 5-20 nM glucagon-like peptide-1.

It was found that after differentiation in the presence of betacelluline, differentiated cells (BTC) produce both insulin and C-peptide, each in six times the amount compared to the NAG group.

Therefore, cells differentiated in the presence of B27 and betacelluline supplementation instead of fetal calf serum and activin A, respectively, can secrete insulin more efficiently than NAG cells.

A better understanding of the present invention can be achieved using the following examples, which are given in order to illustrate the present invention, and should not be construed as limiting it.

Example 1: Stem Cell Isolation

Adipose tissue, with the permission of patients, surgically removed from the subcutaneous layer around the eye in a plastic surgery clinic, was treated with a 0.075% type 1 collagenase solution at 37 ° C for 30 minutes and then with the same volume of medium supplemented with fetal calf serum as the collagenase solution to suppress enzyme activity. After centrifugation, the cell pellet thus formed was washed twice with culture medium and incubated in DMEM-LG supplemented with 10% fetal calf serum.

Example 2: Stem Cell Culture

The resulting stem cells were cultured for 21 for in a medium inducing differentiation. For this, the cells were first seeded at a density of 5 × 10 ³ cells / well on 48-well collagen-coated plates, into which medium containing 25 mM glucose (DMEM-HG) was added before incubation for 7 days with 10% fetal calf serum, 4 nM activin A, 10 nM glucagon-like peptide-1 and 20 ng / ml fibroblast growth factor-2. After this, the cells were further cultured for another 14 days in medium containing 5.5 mM glucose (DMEM-LG), supplemented with 10% fetal calf serum, 10 mM nicotinamide, 4 nM activin A, 10 nM glucagon-like peptide-1 and 50 ng / ml insulin-like growth factor-1 or insulin-like growth factor-2.

During incubation, the old medium was regularly replaced with fresh with an interval of 3-4 days. After a total incubation of 3 weeks, the differentiation of the cells was investigated as follows.

Example 3: Morphological changes in cells after differentiation

Stem cells were isolated from subcutaneous fat around the eye, cultured and their morphological changes were observed. During incubation in a medium that does not contain growth factors and cytokines, the cell morphology did not change, while under the influence of growth factors and / or cytokines, such as nicotinamide, activin A and glucagon-like peptide-1 or insulin-like growth factor-1 or -2, observed that the stem cells underwent morphological changes and took a rounded shape (figure 1). At 3 weeks after the induction of differentiation, the cells acquired a rounded shape and formed clusters of cells.

Example 4: Study of the secretion of insulin and C-peptide

After completion of cultivation for 3 weeks, the cells were exposed for 12 hours to a low glucose medium (DMEM-LG) supplemented with 0.5% bovine serum albumin. Then the cells were exposed to DMEM-HG for 2 hours, and secreted insulin was quantified using enzyme-linked immunosorbent assay (Fig.2). Also, the amount of C-peptide coupled to insulin in proinsulin was also measured using enzyme-linked immunosorbent assay (Fig.3). For enzyme-linked immunosorbent assay of insulin and C-peptide, commercially available kits from Mercodia, Sweden were used. Standard solutions with previously known concentrations and previously obtained cultures in the amount of 25 μl per well were plated on 96-well microplates coated with human insulin or C-peptide, after which antibodies to insulin or C-peptide were added to the plates and incubated for 1 hour. Then, incubation was carried out for 30 min in a TMB substrate solution and the optical density was measured. Neither insulin nor the C-peptide was secreted by cells in which differentiation was not induced, while it was found that groups that were cultured using the two-step method of the present invention secreted insulin and C-peptide. In particular, when measuring insulin and the C peptide, the group that was cultured in the presence of the insulin-like peptide-2 (NAGI2 group) showed an eight-fold level of insulin secretion and a six-fold level of C-peptide secretion compared to the corresponding levels of the NAG group.

Example 5: Expression of Proteins Specific for Insulin Secreting Cells

After stem cells obtained from subcutaneous fat around the eye were induced to differentiate in the presence of an insulin-like peptide-2, for which the highest efficiency was shown by enzyme-linked immunosorbent assay, the expression of insulin-secreting cells was analyzed for the expression of CK19, insulin, C- peptide and proinsulin (which was not cleaved before extracellular secretion) using immunocytochemical analysis (figure 4). After differentiation, which lasted for 3 weeks, for 2 hours, the cells were exposed to a high glucose concentration, fixed at 4 ° C for 90 min in 4% paraformaldehyde and washed three times with phosphate-buffered saline. To remove endogenous peroxidase, the cells were treated for 10 min in 3% hydrogen peroxide and then incubated at room temperature for 60 min in PBS with 2% bovine serum albumin added to block background signals. At 4 ° C, cell cultures were reacted for 12 hours with PBS diluted primary human antibodies against CK19 (1: 400), C-peptide (1: 500), insulin (1: 500) and proinsulin (1: 500). As a negative control, PBS was used that did not contain primary antibodies. For 20 min, they were treated with each secondary antibody conjugated with biotin and streptavidin conjugated with hydrogen peroxide, and then stained with 3,3'-diaminobenzidine (DAB). Compared to the control, in which differentiation was not induced, significantly increased levels of expression of CK19, C-peptide, insulin and proinsulin were observed in groups that were treated with a combination of nicotinamide, activin A and a glucagon-like peptide (Fig. 4).

Example 6: Expression of genes involved in insulin secretion

After the differentiation of stem cells obtained from the subcutaneous layer around the eye was induced, the cells were taken on days 7, 14, and 21, and the expression of genes characteristic of insulin-secreting cells was examined by reverse transcriptase polymerase chain reaction (RT-PCR).

First, total RNA was isolated from differentiated cells. One ml of Tri Reagent was added to the cell tube and mixed thoroughly with 200 μl of chloroform and then incubated at room temperature for 15 minutes. After centrifugation at 14,000 rpm for 15 minutes, the supernatant thus obtained was transferred to a new tube and mixed with isopropanol. After centrifugation at 14,000 rpm for 10 min, a precipitate formed, to which 75% ethanol was then added. This suspension was again centrifuged at 14,000 rpm for 10 minutes. The supernatant was removed and the remaining 75% ethanol was evaporated, after which the residue was dissolved in sterilized, deionized water and incubated at 65 ° C for 5 min to elute the RNA. Quantitative analysis of this RNA was carried out by determining the optical density at 260 nm in a UV spectrophotometer. A total of 7.5 μg RNA was mixed with 1 × reaction buffer, 1 mM dNTP, 0.5 mg / ml oligo-dT, 20 IU RNase inhibitor and 20 IU M-MuLV reverse transcriptase and sterile deionized water was added to a final volume of 50 μl before incubation at 42 ° C for 60 minutes

PCR was carried out in a mixed solution containing 1 × Taq buffer, 2 mM MgCl ₂ , 0.25 U Taq polymerase and 10 pmol primers, each cDNA sequence of which is complementary to each gene shown in table 2.

PCR began with pre-denaturation at 94 ° C for 5 min, followed by 35 cycles of denaturation at 94 ° C for 30 sec, annealing at the appropriate temperature for 30 sec and elongation at 72 ° C for 30 sec, and the final elongation of fragments at 72 ° C for 5 minutes 15 μl of each PCR product thus obtained was loaded with 6 × application buffer (0.25% bromphenol blue, 0.25% xylene cyanol, 40% sucrose) on a 2% agarose gel and electrophoresis was performed at an voltage of 100 V for 30 minutes. After electrophoresis, the gels were stained with ethidium bromide and examined in a UV transilluminator (Vilber loumat, FRANCE). The level of expression of each gene was determined and compared with the level of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a control. Data for comparative analysis was obtained on the basis of three experiments.

The sequence of primers used in RT-PCR for amplification of the corresponding genes that are characteristic of cells secreting insulin, their names and sizes of PCR products are listed below in table 2.

TABLE 2 Gene Primer Size (bp) Annealing temperature (° C) Mark GAPDH 5'-acaactttggtatcgtggaa-3 '
(SEQ ID NO: 1)
5'-aaattcgttgtcataccagg-3 '
(SEQ ID NO: 2) 456 53 NM_002046 Insulin 5'-aaccaacacctgtgcggctc-3 '
(SEQ ID NO: 3)
5'-aagggctttattccatctctctctcg-3 '
(SEQ ID NO: 4) 320 59 J00265 ngn3 5'-cgtgaactccttgaactgagcag-3 '
(SEQ ID NO: 5)
5'-tggcactcctgggacgagtttc-3 '
(SEQ ID NO: 6) 211 61 AF234829 pdx1 5'-cccatggatgaagtctacg-3 '
(SEQ ID NO: 7)
5'-gtcctcctcctttttccac-3 '
(SEQ ID NO: 8) 262 54 NM_000209 Gk 5'-gaataccccccagagaccttttc-3 '
(SEQ ID NO: 9)
5'-ggtttcttcctgagccagcg-3 '
(SEQ ID NO: 10) 182 61 M90299 Isl1 5'-agcatcaatgtcctctcaacttcc-3 '
(SEQ ID NO: 11)
5'-tgtttggcaaggcaatgacc-3 '
(SEQ ID NO: 12) 493 57 NM_002202 Nkx6.1 5'-acacgagacccactttttcccc-3 '
(SEQ ID NO: 13)
5'-tgctggacttgtgcttcttcaac-3 '
(SEQ ID NO: 14) 336 59 NM_006168 Glut 2 5'-agctttgcagttggtggaat-3 '
(SEQ ID NO: 15)
5'-aataagaatgcccgtgacga-3 '
(SEQ ID NO: 16) 300 57 NM_000340 Nd 5'-cagaaccaggacatgccc-3 '
(SEQ ID NO: 17)
5'-atcaaaggaagggctggtg-3 '
(SEQ ID NO: 18) 216 60 NM_002500 Glucagon 5'-atgaacgaggacaagcgc- 3 '
(SEQ ID NO: 19)
5'-gtaacgaaccgaccactt-3 '
(SEQ ID NO: 20) 236 57 NM_002054 Sst 5'-ggctgcgctgtccatcgtcctg-3 '
(SEQ ID NO: 21)
5'-ttgcgttctcggggtgccatagc-3 '
(SEQ ID NO: 22) 276 63 NM_001048 PP 5'-ctctgttactacagccactg-3 '
(SEQ ID NO: 23)
5'-agtcgtaggagacagaaggt-3 '
(SEQ ID NO: 24) 261 55 NM_002722.3 PC1 / 3 5'-ttggctgaaagagaacgggatacatct-3 '
(SEQ ID NO: 25)
5'-acttcttttttgattgctttggcggtg-3 '
(SEQ ID NO: 26) 457 60 NM_000493.3 PC2 5'-gcatcaagcacagacctacactcg-3 '
(SEQ ID NO: 27)
5'-gagacacaaccaccctctcatccttc-3 '
(SEQ ID NO: 28) 309 63 NM 003594.2 GLUT1 5'-ctcactgctcaagaagacatgg-3 '
(SEQ ID NO: 29)
5'-ctgggtaacagggatcaaacag-3 '
(SEQ ID NO: 30) 366 60 NM 006516.1 GLP-1R 5'-gttcccctgctgtttgttgt-3 '
(SEQ ID NO: 31)
5'-cttggcaagtctgcatttga-3 '
(SEQ ID NO: 32) 228 55 NM002062.2 PAX4 5'-cacctctctgcctgaggacacggtgag-3 '
(SEQ ID NO: 33)
5'-ctgcctcattccaagccatacagtagtg-3 '
(SEQ ID NO: 34) 444 63 NM 006193.1

As follows from the data in Fig. 5, in cells in which differentiation was not induced, neuro D and islet 1 were expressed, which are known to play important roles in the development of the pancreas, and glucagon, SST (somatostatin) and PP (pancreatic polypeptide) secreted by cells other than pancreatic endocrine beta cells, but when differentiation was induced, cells began to express PC2, PC1 / 3, Glp1R, neurogenin 3, Glut2, Glut1, pax4 and pdx1 on day 7 and Nkx6.1 and insulin on day 14, and their expression levels increased significantly for 21 days.

Example 7: Dithizone Staining

It is known that insulin forms a complex with zinc ion (Zn ⁺⁺ ) with the formation of hexamers. It is known that dithizone is a zinc chelating agent that selectively stains pancreatic beta cells due to the high zinc content in these cells. For this, cells that differentiated in the presence of an insulin-like peptide-2 were stained with dithizone. For this, 50 mg of dithizone powder was dissolved in 5 ml of dimethyl sulfoxide (DMSO) and 10 μl of dithizol solution was diluted in 1 ml of culture medium. After filtering through 0.2 μm, a nylon filter staining the dithizone solution was dripped onto the culture plate and incubated at 37 ° C for 15 minutes. Cells were washed three times in PBS and examined under an optical microscope. As can be seen in Fig.6, the differentiated cells were clearly stained, while the cell clusters had a saturated color.

Briefly, as described above, when stem cells derived from subcutaneous fat around the eye were first exposed to high concentrations of glucose in combination with activin A, fibroblast-2 growth factor and glucagon-like peptide-1, and then exposed to low concentrations of glucose in the presence of nicotinamide, activin A, glucagon-like peptide-1 and insulin-like growth factor-2, they differentiated into insulin-secreting cells in which the formation of insulin, C-peptide and proinsulin in large The number. In addition, they expressed genes characteristic of pancreatic beta cells, and were also clearly stained with dithizone. Thus, the established method of the present invention enables the effective differentiation of adult stem cells into insulin-secreting beta cells.

Example 8: Stem Cell Culture

The resulting stem cells were cultured for 21 for in a medium inducing differentiation . For this, the cells were first seeded at a density of 5 × 10 ³ cells / well on 48-well collagen-coated plates, into which medium containing 25 mM glucose (DMEM-HG) was added for 3 days before incubation with 1 × B27 , 20 ng / ml fibroblast growth factor-2; and 20 ng / ml epidermal growth factor. After this, the cells were additionally incubated for another 4 days in a medium containing 25 mM glucose, with the addition of 10% fetal calf serum, 4 nM activin A, 20 ng / ml fibroblast growth factor-2 and 10 nM glucagon-like peptide-1. Subsequently, the cells were cultured for 3 days in a medium containing 5.5 mM glucose (DMEM-LG), supplemented with 10% fetal calf serum, 4 nM activin A, 20 ng / ml fibroblast growth factor-2 and 10 nM glucagon-like peptide - 1, and then, before incubation for an additional 10 days, it was transferred to a new medium containing 5.5 mM glucose (DMEM-LG), with the addition of 10 mM nicotinamide, 10 nM glucagon-like peptide and 10 ng / ml betacelluline.

During the incubation process, the old medium was periodically replaced with fresh at intervals of 3-4 days. After incubation, in General, for 3 weeks, the differentiation of cells was investigated as follows.

Example 9: Investigation of the secretion of insulin and C-peptide

After completion of the 3-week cultivation, the cells were exposed for 12 hours to a low glucose medium (DMEM-LG) supplemented with 0.5% bovine serum albumin. Then, the cells were exposed to DMEM-HG for 2 hours and a quantitative analysis of secreted insulin was performed using enzyme-linked immunosorbent assay (Fig.7). Also, using the immunocytochemical analysis, the amount of C-peptide coupled with insulin in proinsulin was measured (Fig. 8). For enzyme-linked immunosorbent assay of insulin and C-peptide, commercially available kits from Mercodia, Sweden were used. Standard solutions with already known concentrations and previously obtained cultures were plated in the amount of 25 μl per well in 96-well plates coated with human insulin or C-peptide, after which antibodies to insulin or C-peptide were added to the wells and incubated for 1 hour. After that, 200 μl of TMB substrate solution was added to the wells. After incubation for 30 minutes, their optical densities were measured. Cells that did not induce differentiation did not secrete either insulin or C-peptide, and it was found that groups that were cultured using the two-step method of the present invention form insulin and C-peptide. In particular, the measurements showed that the group that was cultivated in the same manner as in Example 8 secreted about 6 times more insulin and about 6 times more C-peptide than the NAG group.

By synthesizing insulin and C-peptide in amounts of 7-8 times the amounts synthesized by the NAG group, it was expected that according to the disclosure of Korean Patent Application No. 2007-44663, filed in May 2007, insulin-secreting cells differentiated from adult stem cells in the culture media of the present invention, as described previously, are an excellent treatment for type 1 diabetes.

Although preferred embodiments of the present invention are disclosed for illustrative purposes, those skilled in the art will recognize that various modifications, additions, and substitutions are possible without departing from the scope and spirit of the invention disclosed in the appended claims.

Claims (3)

1. The method of differentiation of adult stem cells ex vivo into cells secreting insulin, which contains:
cultivation of adult stem cells for 4-10 days in a medium containing 20-30 mm glucose, with the addition of 5-20% fetal calf serum, 1-10 nm activin A, 5-20 nm glucagon-like peptide-1 and 10-30 ng / ml fibroblast growth factor-2; and
cultivation of cultured stem cells for 10-20 days in a medium containing 4-7 mm glucose, with the addition of 5-20% fetal calf serum, 5-20 mm nicotinamide, 1-10 nm activin A, 5-20 nm glucagon-like peptide - 1 and 10-100 ng / ml insulin-like growth factor. 2. The method according to claim 1, where the insulin-like growth factor is an insulin-like growth factor-2. 3. The method of differentiation of adult stem cells from an adult ex vivo into cells secreting insulin, which contains the sequential cultivation of adult stem cells:
for 1-7 days in a medium containing 20-30 mm glucose, with the addition of B27, 10-30 ng / ml fibroblast growth factor-2 and 10-30 ng / ml epidermal growth factor;
for 1-7 days in a medium containing 20-30 mm glucose, with the addition of 5-20% fetal calf serum, 1-10 nm activin A, 5-20 nm glucagon-like peptide-1 and 10-30 ng / ml growth factor fibroblasts-2;
for 1-7 days in a medium containing 4-7 mm glucose, with the addition of 5-20% fetal calf serum, 1-10 nm activin A, 5-20 nm glucagon-like peptide-1 and 10-30 ng / ml growth factor fibroblasts-2; and
for 10-20 days in a medium containing 4-7 mm glucose, with the addition of 5-20% fetal calf serum, 5-20 mm nicotinamide, 1-10 ng / ml betacelluline and 5-15 nm glucagon-like peptide.

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