KR101496804B1 - The production and application of insulin producing cells - Google Patents

Abstract

The present invention relates to a medium composition for inducing the differentiation into insulin producing cells containing glucosamine, a method for inducing the differentiation into insulin producing cells using the medium composition, and a cell therapy agent for treating diabetes, containing insulin producing cells prepared by the method. The glucosamine according to the present invention can effectively induce the differentiation into insulin producing cells through glucose metabolism regulation. The induction of the differentiation into insulin producing cells using glucosamine as described above is safe since gene manipulation is excluded; is cheaper since high-priced compounds or growth factors, which have unknown mechanisms, are not needed; and can minimize cell contamination and deformation which are problematic at the time of in vitro manipulation since the differentiation occurs for a short time.

Description

[0001] The present invention relates to insulin producing cells,

The present invention relates to a medium composition for inducing differentiation into an insulin producing cell comprising glucosamine, a method for inducing differentiation into insulin producing cells using the medium composition, and a cell therapy agent for treating diabetes comprising insulin producing cells produced by the method .

Diabetes mellitus is a disease characterized by chronic hyperglycemia as the most important feature. It prolongs wound healing caused by microvascular damage, neurological disease, kidney failure, cardiac disease, retinal disease and so on, which increases social and economic burden. There are no treatments that can cure diabetes so far, and insulin-enriched therapies that repeatedly administer insufficient insulin in diabetic patients have been used in clinical practice for a long time, but the inconvenience of repeated administration several times a day can not prevent diabetic complications It is a reality.

There are two major types of diabetes (type 1 and type 2). In the case of type 1 diabetes, it occurs because the immune cells destroy insulin-producing cells and diabetes mellitus can not regulate blood glucose due to lack of insulin-producing cells. In the case of type 2 diabetes, the organ / tissue / cell in the body is caused by resistance to insulin due to various causes. In the end, the function of insulin-producing cells due to hyperglycemia is reduced or eliminated, As a result, diabetes mellitus with hyperglycemia accompanies complications.

Although islet or cell-derived islet cell transplantation studies have been conducted as a means of treating diabetes mellitus resulting from the loss of insulin-producing cells, there is an inconvenience in that patients receiving islet transplantation have to take an immunosuppressive agent for a lifetime, In 62.5% of patients, the number of donors is insufficient compared to the number of patients requiring islet transplantation.

As an alternative to insufficient islet donors, studies are under way to treat diabetes by transplanting into a patient differentiation / proliferation of insulin-producing cells from stem cells, but the results are insignificant. For the purpose of replacing the insufficient pancreatic islets, studies have been actively carried out to treat diabetes by differentiating into cells that produce insulin by using various cells such as embryonic stem cells and degenerated stem cells in vitro, and then transplanting them. However, Considering the fact that proliferation does not occur in the case of still vague and differentiated cells, although it is expected that astronomical costs will be required to obtain the amount of cells required for the treatment of diabetes, the efficiency is very low and the clinical application is impossible due to safety concerns (Zhang D et al. 2009; Thatava T et al. 2011).

On the other hand, adult stem cells have been proposed as a means to eliminate concerns about safety. In addition to bone marrow aspiration, bone marrow cells can be easily obtained from the peripheral blood by freeing the bone marrow cell mobilization factors such as G-CSF and AMD3100 (Yeoh JS et al. 2007) (1986), and the results of the study are summarized as follows: (1) The results of the study are as follows.

Accordingly, the inventors of the present invention have made efforts to develop a novel therapeutic agent for diabetes, and have found that glucosamine can effectively induce differentiation into insulin-producing cells through the regulation of glucose metabolism.

It is an object of the present invention to provide a medium composition for inducing differentiation into an insulin producing cell comprising glucosamine.

It is still another object of the present invention to provide a method for inducing differentiation into an insulin producing cell using the above-mentioned culture medium composition.

It is still another object of the present invention to provide a cell therapy agent for treating diabetes comprising insulin-producing cells produced by the above method, or a pharmaceutical composition for preventing or treating diabetes.

In order to solve the above problems, the present invention provides a medium composition for inducing differentiation into insulin-producing cells comprising glucosamine.

The present invention also provides a method for inducing differentiation into an insulin producing cell, comprising culturing the cells in the above-mentioned culture medium composition.

The present invention also provides a cell therapy agent for treating diabetes comprising insulin-producing cells produced by the above method, or a pharmaceutical composition for preventing or treating diabetes.

Glucosamine according to the present invention can effectively induce differentiation into insulin-producing cells through regulation of glucose metabolism. As described above, induction of differentiation into insulin-producing cells using glucosamine is safe because it excludes genetic manipulation, does not require expensive compounds or growth factors that are unclear in mechanism, It is possible to minimize contamination and degeneration of cells which are problematic in in vitro manipulation.

FIG. 1 shows the results of immunohistochemical staining for differentiation of bone marrow cells into insulin-producing cells.
FIG. 2 is a graph showing the results of real-time PCR for the differentiation of gluconeamine-induced bone marrow cells into insulin-producing cells in vitro.
FIG. 3 is a graph showing the results of confirming the differentiation of glucosamine-stimulated bone marrow cells into insulin-producing cells through immunofluorescence staining.
FIG. 4 is a graph showing the results of immunohistochemical staining for differentiation of peripheral blood-derived bone marrow cells into insulin-producing cells by glucosamine.
FIG. 5 is a graph showing blood glucose control effect upon bone marrow cell transplantation, which is differentiated by glucosamine in a diabetic animal model.

Hereinafter, the present invention will be described in detail.

The present invention provides a medium composition for inducing differentiation into an insulin producing cell comprising glucosamine.

In the present invention, the term " culture medium "means a medium capable of supporting the induction and survival of differentiation into insulin-producing cells in vitro, and includes all of conventional culture media suitable for culturing cells The medium to be used for culturing is preferably a cell culture minimum medium (CCMM), which generally contains a carbon source, a nitrogen source and a trace element component. Examples of such cell culture medium include DMEM (Dulbecco's Modified Eagle's Medium), MEM (Minimal Essential Medium), BME (Basal Medium Eagle), RPMI1640, F-10, F-12, ), GMEM (Glasgow's Minimal Essential Medium), Iscove's Modified Dulbecco's Medium, etc. The medium may also be penicillin, streptomycin, gentamicin gentamicin, and the like.

In addition to glucosamine, the medium may further include at least one substance inducing differentiation into one or more insulin-producing cells known in the art.

Glucosamine according to the present invention can effectively induce differentiation into insulin-producing cells through regulation of glucose metabolism. As described above, induction of differentiation into insulin-producing cells using glucosamine is safe because it excludes genetic manipulation, does not require expensive compounds or growth factors that are unclear in mechanism, It is possible to minimize contamination and degeneration of cells which are problematic in in vitro manipulation.

The present invention also provides a method for inducing differentiation into an insulin producing cell, comprising culturing the cells in the above-mentioned culture medium composition.

Such cells include, but are not limited to, bone marrow cells or adult stem cells.

The adult stem cells include those derived from one or more selected from the group consisting of bone marrow, umbilical cord blood, blood, skin, fat, and placenta. Preferably, the adult stem cells are derived from bone marrow or cord blood.

In addition, the present invention provides a cell therapy agent for treating diabetes comprising insulin-producing cells produced by the above method.

In addition, the present invention provides a pharmaceutical composition for preventing or treating diabetes comprising insulin-producing cells produced by the above method.

The diabetes includes type 1 diabetes or type 2 diabetes.

In the present invention, "cell therapeutic agent" is a pharmaceutical (US FDA regulation) used for the purpose of treatment, diagnosis and prevention of cells and tissues produced by separation, culture and special works from human, Refers to a drug used for therapeutic, diagnostic and prophylactic purposes through a series of actions such as alive, homologous, or xenogeneic cell propagation, screening, or otherwise altering the biological characteristics of a cell to restore it.

The compositions of the present invention may further comprise suitable carriers, excipients and diluents conventionally used in the manufacture of pharmaceutical compositions. The pharmaceutical composition according to the present invention may be formulated in the form of oral, granule, tablet, capsule, suspension, emulsion, syrup, aerosol or other oral formulations, external preparation, suppository and sterilized injection solution, . Suitable formulations known in the art are preferably those disclosed in Remington ' s Pharmaceutical Sciences, Mack Publishing Company, Easton PA.

Examples of carriers, excipients and diluents that can be included in the pharmaceutical composition of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, Cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used. Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like. These solid preparations are prepared by mixing at least one excipient such as starch, calcium carbonate, sucrose, lactose, It is prepared. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral administration include suspensions, solutions, emulsions, syrups and the like. Various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included in addition to water and liquid paraffin, which are simple diluents commonly used. have. Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the non-aqueous solvent and suspension include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. Examples of the suppository base include witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin and the like.

The term "administering" as used herein is meant to provide any desired composition of the invention to a subject in any suitable manner.

The preferred dosage of the pharmaceutical composition of the present invention varies depending on the condition and the weight of the individual, the degree of disease, the type of drug, the route of administration and the period of time, but can be appropriately selected by those skilled in the art. The administration may be carried out once a day or divided into several times. The dose is not intended to limit the scope of the invention in any way. The dosage may vary depending on factors such as the type of disease of the individual, the severity, the activity of the drug, the sensitivity to the drug, the time of administration, the route of administration and the rate of release, the duration of treatment, Can be determined.

Hereinafter, the present invention will be described in more detail by way of examples. It should be noted, however, that the following examples are provided to further illustrate the present invention and are not intended to limit the scope of the present invention.

Example 1 Identification of Insulin-Producing Cells from Bone Marrow Cells

In order to confirm whether the bone marrow cells can differentiate into insulin-producing cells, chimera mice were prepared. More specifically, donor bone marrow cells were transfected with a transgenic mouse (B6-tg (Ins2-luc / EGFP / TK) 300 kauf / J) expressing various genes (EGFP, Luciferase, etc.) under the control of insulin promoter. Normal C57BL / 6 mice were used. The femur and tibia of the transgenic mice at 8 to 12 weeks of age were separated and flashed with PBS to obtain bone marrow cells. Red blood cells were then removed using a hemolytic solution (BD Pharmingen) and prepared in PBS at a concentration of 5 × 10 ⁷ / ml. Transplantation was completed by injecting 0.1 ml of the prepared bone marrow cells through the above procedure through the tail vein of C57BL / 6 mice irradiated with 1,000 cGy of radiation 18 hours before transplantation. Four weeks later, 150 mg / kg of streptozotocin (STZ, Sigma) was intraperitoneally administered to the chimeric mouse transplanted with bone marrow derived from the transgenic mouse to induce diabetes mellitus. After 7 days of STZ administration, blood glucose levels were measured, and individuals showing blood glucose levels of 450 mg / dl were classified into diabetic mice. Three days later, autopsy was performed to obtain pancreas and adipose tissue. The presence of EGFP expressing cells in the above tissues was confirmed by immunohistochemistry. The results are shown in Fig.

As shown in FIG. 1, EGFP-expressing cells were not observed in pancreatic and adipose tissues of normal chimeric mice not receiving STZ, but EGFP was found in pancreatic and adipose tissues of diabetic mice induced by STZ administration Expressing cells were observed. On the other hand, after 4 weeks of observation, no decrease in blood glucose was observed, confirming that bone marrow cells could be differentiated into insulin-producing cells and that the cause was hyperglycemia.

Example 2: Whole cell differentiation of insulin-producing cells by controlling glucose metabolism in bone marrow cells in vitro

Bone marrow cells were prepared from normal C57BL / 6 mice or transgenic mice in the same manner as in Example 1. The bone marrow cells were suspended in DMEM medium (standard medium) containing 5.5 mM glucose, 2% FBS and 1% antibiotic, and the bone marrow cells were suspended at 5 x 10 ⁶ cells / ml and dispensed into 6 well plates in which cell attachment was inhibited. (5, 10, and 15 mM), fructose (5, 10, and 15 mM), and xylose (5, 10, and 20 mM) The cells were treated with xylitol (5, 10, 15 mM) or glucosamine (5, 10, 15 mM) and then cultured in a cell culture incubator for 9 days to evaluate whether insulin producing cells appeared. For this purpose, the shaker attached to the cell culture incubator for suspension culture was allowed to shake continuously at 30 to 60 rpm. The presence of insulin-producing cells was assessed by real-time PCR methods (neurogenin 3, PDX-1, MafA, Ptf1a) for the factors promoting insulin-producing beta cell differentiation in cultured 3, And were confirmed by fluorescence staining (insulin, PDX-1). The results are shown in Fig.

As shown in FIG. 2, in the experimental group to which various concentrations of glucose, fructose, pyruvate, xylose or xylitol was added, it was not possible to identify changes in the expression of factors promoting differentiation into insulin producing cells, The appearance of the cells could not be confirmed. On the other hand, in the experimental group in which glucosamine was added and suspended, the expression of neurogenin 3, PDX-1, MafA, and Ptf1a, which promote insulin-producing cell differentiation, was increased with the passage of time.

Example 3. Insulin-producing cell differentiation of glucosamine-sensitized bone marrow cells

Based on the results of Example 2, in order to confirm whether the cells cultured in suspension by addition of glucosamine can be differentiated into insulin-producing cells, normal C57BL / 6 mice or transgenic mice To prepare bone marrow cells. The bone marrow cells were suspended at 5 x 10 < ⁶ > cells / ml using a standard medium supplemented with 10 mM glucosamine, and cultured for 9 days in the same manner as in the floating culture method described in Example 2 above. Cells were harvested on days 3, 6, and 9, washed with PBS to remove residual glucosamine, resuspended in RPMI 1640 medium containing 11 mM glucose, 10% FBS, 1% antibiotic, Well plates and further cultured for 3 days. The presence of insulin-producing cells was confirmed by immunofluorescence staining (insulin, PDX-1) and EGFP expression. In order to confirm whether the adherent cells secrete insulin, the insulin present in the culture supernatant obtained after adherent culture was quantitated by ELISA. The results are shown in Fig.

As shown in FIG. 3, when bone marrow cells suspended in glucosamine for 3 days or more were adhered and cultured, it was confirmed that bone marrow cells were completely differentiated into insulin-producing cells. From the above results, it was confirmed that glucosamine plays a role in controlling the differentiation of bone marrow cells to regulate glucose metabolism and to differentiate into cells capable of producing insulin.

Example 4 Differentiation of Peripheral Blood-Derived Bone Marrow Cells into Insulin-Producing Cells

In order to confirm whether the bone marrow-derived cells obtained from the peripheral blood can be differentiated into insulin-producing cells, the differentiation of bone marrow cells was evaluated in the same manner as in Example 3 above. First, to obtain bone marrow cells mobilized with peripheral blood, normal C57BL / 6 mice were subcutaneously injected with 250 μg / kg of G-CSF once daily for 4 days, and after the last G-CSF injection, Blood was obtained. Peripheral blood was collected and suspended in a standard medium supplemented with 10 mM glucosamine for 3 days, and then recovered. After washing with PBS, the residual glucosamine was removed, and the cells were treated with 11 mM glucose, 10% FBS, 1% After resuspending in RPMI1640 medium containing antibiotics, the cells were transferred to a 6-well plate for attachment culture and further cultured for 3 days. The presence of insulin-producing cells was confirmed by immunofluorescence staining (insulin, PDX-1) and EGFP expression in the same manner as in Example 3. The results are shown in Fig. 4

As shown in Fig. 4, it was confirmed that the bone marrow cells, which were obtained from the peripheral blood, were completely differentiated into insulin-producing cells as in the case of Example 3 above.

Example 5 Induction of Complete Differentiation and Glycemic Control through Whole Bone Marrow Cell Transplantation

Based on the results of Examples 3 and 4, the bone marrow cells were transplanted into diabetic model mice in order to examine whether the bone marrow cells that were completely differentiated using glucosamine could be completely differentiated in vivo. First, the differentiated bone marrow cells were cultured for 3 days using a standard medium containing 10 mM glucosamine, and then washed with PBS. Diabetes was induced in the mice in the same manner as in Example 1, and after 3 days, 2 x 10 < ⁶ > fully differentiated cells per individual were transplanted through the vein. Blood glucose was monitored in diabetic mice transplanted with cells for 30 days after transplantation. The results are shown in Fig.

As shown in Fig. 5, in the control group transplanted with the cells prepared without glucosamine, blood glucose decline due to transplantation of the cells was not observed throughout the experiment, but the test cells that had been transplanted with glucosamine- , It was confirmed that blood glucose decreased until day 12 from 5 days after transplantation. From the above results, it was confirmed that the implanted fully differentiated cells were completely differentiated in vivo to regulate blood glucose.

Claims (8)

A medium composition for inducing differentiation into an insulin producing cell comprising glucosamine. A method for inducing differentiation into an insulin producing cell, comprising culturing the cells in the medium composition of claim 1. 3. The method according to claim 2, wherein the cell is a bone marrow cell or an adult stem cell. 4. The method according to claim 3, wherein the adult stem cells are derived from at least one selected from the group consisting of bone marrow, umbilical cord blood, blood, skin, fat and placenta. delete delete delete delete

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