KR20090051695A - Method of differentiation into cells producing insulin from islet-neighboring cell - Google Patents

Abstract

To the cell solution containing at least one selected from the surrounding cells of the islet cells (Acinar cell) and pancreatic ductal cells (ductal cell), 15-25% of serum and 15-30 mmol / l of glucose were added based on the final concentration. The method of differentiating the peripheral pancreatic islet cells of the present invention, which is characterized by culturing, into cells capable of secreting insulin, has a high differentiation yield, separates the pancreatic islets and secretes insulin from the remaining cell solution. It is very efficient because it can differentiate.

In addition, the present invention can further reduce the cell death that occurs during the differentiation of the cells that can secrete insulin when the addition of embryonic stem cells or embryonic embryonic stem cells, ultimately can solve the insufficient supply of pancreatic islet cells substantially It has an effect.

Diabetes, Embryonic Stem Cells, Insulin, Pancreatic Islets

Description

Method of differentiation into cells producing insulin from islet-neighboring cell

The present invention is a method of differentiating cells capable of secreting insulin from peripheral cells of the islet, which increases the yield of cells capable of secreting insulin and decreases cell death. It is about.

Diabetes is a disease caused by the lack or absence of insulin, a glucose-regulating hormone secreted by the pancreatic islets, and is classified into type I diabetes (insulin-dependent diabetes) and type II diabetes (insulin-independent diabetes).

Type I diabetes is an autoimmune disease (autoimmune disease) caused by the destruction of the pancreatic islet cells by the immune system is reduced by the secretion of insulin and is difficult to maintain life unless the insulin is supplied from the outside.

Pancreatic islet transplantation is limited to type I diabetic patients who are unable to control their blood sugar with insulin, which can suppress the progression of diabetes, improve the patient's condition and promote ultimate diabetes treatment without using insulin. (Shapiro AM, Lakey JR, Ryan EA, Korbutt GS, Toth E, Warnock GL, Kneteman NM, Rajotte RV. Islet transplantation in seven patients with type 1 diabetes mellitus using a glucocorticoid-free immunosuppressive regimen.N. Engl. J. Med . 343 (2000), pp. 230-238. 2. Markmann JF, Deng S, Desai NM, Huang X, Velidedeoglu E, Frank A, Liu C, Brayman KL, Lian MM, Wolf B, Bell E, Vitamaniuk M, Doliba N, Matschinsky F, Markmann E, Barker CF, Naji A. The use of non-heart-beating donors for isolated pancreatic islet transplantation.Transplantation.2003 May 15; 75 (9): 1423-9.Hering BJ, Kandaswamy R , Ansite JD, Eckman PM, Nakano M, Sawada T, Matsumoto I, Ihm SH, Zhang HJ, Parkey J, Hunter DW, Sutherland DE.Single-d onor, marginal-dose islet transplantation in patients with type 1 diabetes.JAMA. 2005 Feb 16; 293 (7): 830-5.Eratrat in: JAMA. 2005 Apr 6; 293 (13): 1594.).

On the other hand, transplanting the whole pancreas has a higher success rate than transplanting pancreatic islets, but there is a burden of major surgery, while transplanting only pancreatic islets has the advantage that the procedure is easy, safe and repeatable.

However, the supply of pancreatic islet cells using the pancreas is inevitably insufficient compared to the demand, and the development of a new method for increasing the supply of pancreatic islets is required.

Differentiating pancreatic islets from peripheral islet cells as a way to increase the supply of pancreatic islets can be considered as an alternative. For this study, Song KH et al. Ko SH, Ahn YB, Yoo SJ, Chin HM, Kaneto H, Yoon KH, Cha BY, Lee KW, Son HY.In vitro transdifferentiation of adult pancreatic acinar cells into insulin-expressing cells.Biochem Biophys Res Commun. 2004 Apr 16; 316 (4): 1094-100).

However, Song's method was caused by problems of low islet cell differentiation yield and apoptosis (apoptosis).

Accordingly, an object of the present invention is to provide a method for increasing the production yield of cells capable of secreting differentiated insulin from peripheral cells of the islet and preventing cell death.

In order to achieve the above object, the present invention provides a serum solution of 15-25% based on the final concentration in a cell solution containing any one or more selected from the surrounding cells of the islet cells (acinar cell) and the ductal cells (ductal cells), Provided is a method for differentiating islet-secreting peripheral cells of the pancreatic islet, characterized in that after culturing the culture medium by adding glucose 15 ~ 30mmol / l.

In addition, the present invention is an embryonic stem cell or an embryonic stem cell (Embryonic stem cell) in a cell solution containing any one or more selected from the surrounding cells (acinar cells) and pancreatic ductal cells (ductal cells), which are peripheral islets ( Embryoid body) was added, and serum was cultured by adding 15-25% of serum and 15-30 mmol / l of glucose based on the final concentration, followed by culturing peripheral pancreatic islet cells, which are characterized by culturing into cells capable of secreting insulin. It provides a method to make it.

Hereinafter, the present invention will be described in detail by dividing (1) the improvement of the cell differentiation yield capable of secreting insulin and (2) the solution of the cell death caused by apoptosis.

(1) Improvement of cell differentiation yield which can secrete insulin

The present invention provides a cell solution containing any one or more selected from acinar cells and ductal cells, which are peripheral cells of the islet, in order to increase the differentiation yield of cells capable of secreting insulin as a first form. 15 to 25% serum and 15 to 30 mmol / l of glucose are added based on the final concentration. In the paper by Song KH et al., The differentiation yield of the cells capable of secreting insulin is about 5%, whereas the differentiation yield of the cells capable of secreting insulin of the present invention is obtained through Experimental Example 1. It was confirmed that it is 20%.

Serum contains epidermal growth factor (EGF), platelet-derived growth factor (PDGF) and fibroblast growth factor (FGF), and vascular endothelial-derived growth. factor or insulin-like growth factor growth factor; Various growth factors such as IGF-I). These growth factors use the 'PI3kinae pathway' for cell differentiation and the 'MAPKinae pathway' and 'PI3Kinase pathway' for cell growth (Koellensperger E, von Heimburg). D, Markowicz M, Pallua N. Human serum from platelet-poor plasma for the culture of primary human preadipocytes.Stem Cells. 2006 May; 24 (5): 1218-25.Epub 2006 Jan 19.). As in the present invention, the use of a high concentration of serum increases the concentration of growth factors contained in the serum, which greatly affects the differentiation and growth processes of the cells, thereby inducing insulin secretion. Differentiation and growth can be made smoothly.

In the present invention, the serum is added to the culture medium based on the final concentration of 15 to 25%, if less than 15%, the differentiation and growth of cells that can secrete insulin is not made smoothly low yield of cells that can secrete insulin If it exceeds 25%, the efficiency is lowered.

On the other hand, the present invention is a cell solution containing any one or more selected from the surrounding cells (acinar cell) and pancreatic ductal cells (ductal cells), which is the periphery of the islet containing a high concentration of glucose with a final concentration of 15 ~ 30 mmol / l Incubate in the culture, the reason can be divided into two.

First, high concentrations of glucose activate transcription factors such as NF-κB, which promotes cell differentiation and accelerates the differentiation and growth cycle of cells capable of secreting insulin (Iwasaki Y, Kambayashi M). , Asai M, Yoshida M, Nigawara T, Hashimoto K. High glucose alone, as well as in combination with proinflammatory cytokines, stimulates nuclear factor kappa-B- mediated transcription in hepatocytes in vitro.J Diabetes Complications. 2007 Jan-Feb; 21 (1): 56-62.).

Second, the insulin secretion is increased by the pancreatic islets produced by periphery of pancreatic islet cells and the pancreatic duct cells by glucose, and the differentiation and growth of cells caused by 'PI3 Kinase' and 'PI3Kinase' and 'MAPKinase pathway' It can be promoted to increase the yield of cells capable of secreting insulin (Pertseva MN, Shpakov AO, Plesneva SA, Kuznetsova LA.A novel view on the mechanisms of action of insulin and other insulin superfamily peptides: involvement of adenylyl cyclase signaling system Comp Biochem Physiol B Biochem Mol Biol. 2003 Jan; 134 (1): 11-36 .; Amaral ME, Cunha DA, Anhe GF, Ueno M, Carneiro EM, Velloso LA, Bordin S, Boschero AC.Participation of prolactin receptors and phosphatidylinositol 3-kinase and MAP kinase pathways in the increase in pancreatic islet mass and sensitivity to glucose during pregnancy.J Endocrinol. 2004 Dec; 183 (3): 469-76.).

On the other hand, in the present invention, the glucose is added to the culture medium based on the final concentration of 15 ~ 30mmol /, when less than 15mmol /, the differentiation and growth of the apoptotic cells or pancreatic duct cells does not occur smoothly the differentiation of cells that can secrete insulin The yield is lowered, and the efficiency is lowered if it exceeds 30 mmol / l.

On the other hand, the cell solution containing the acinar cells (acinar cell) and the ductal cells (peripheral islet cells) used in the present invention is not limited to those obtained by a specific method, preferably collagen in the extracted pancreas Treating the enzymes capable of degrading the enzyme to break down the collagen, thereby breaking the intercellular binding; After breaking the intercellular binding, isolating islet cells is preferably obtained from a method comprising a, including, the enzyme that can degrade collagen, for example in the past in the case of human collagen (Collagenase), River Laase (Liberase), etc. were used, and now NP-1 is used, and in the case of animals such as mice, collagenase P (collagenase P) and collagenase (collagenase) are mainly used.

On the other hand, the culture medium used in the present invention preferably includes islet cells (islet cells), in this case, cells containing acinar cells (acinar cells), pancreatic cells (ductal cells) and islet cells (islet cells). The solution is most preferably obtained from a method comprising a step of breaking down the intercellular bonds by digesting the collagen by treating an enzyme capable of digesting collagen to the isolated pancreas. In addition, the cell solution including acinar cells, ductal cells and islet cells is most preferably treated with enzymes capable of degrading collagen in the isolated pancreas, thereby degrading collagen. Breaking intercellular binding; After breaking the intercellular binding, isolating and removing some of the islet cells; What is obtained by a method comprising a.

After the islets are isolated from the pancreas, the surplus is discarded. The surplus contains 2-3% of pancreatic islets, ductal cells, and acinar cells. Therefore, the surplus from which the pancreatic islets are partially separated from the pancreas thus removed also contains the islets, the pancreatic duct cells, and the sacrificial cells, which are differentiated using the method used in the present invention.

On the other hand, in the first aspect of the present invention without additionally adding embryonic stem cells or embryonic stem cells, the total total culture period is not necessarily limited to a specific time. Considering differentiation yield and economic aspects, it should be 3-6 days.

(2) solving the problem of cell death due to apoptosis

According to a second aspect of the present invention, an embryonic stem cell or an embryonic stem cell is contained in a cell solution containing any one or more selected from acinar cells and ductal cells. Embryonic stem cells (Embryoid body) is added, 15 ~ 25% serum, 15 ~ 30mmol / l of glucose is added to form a culture medium, and then cultured around the pancreatic islet, characterized in that the secretion of insulin Provided are methods for differentiating cells into cells.

As described above, the use of embryonic stem cells or maploid embryonic stem cells utilizes the trophic effect of embryonic stem cells or embryonic stem cells to allow insulin to secrete insulin from cell solutions including peripheral islet cells. To reduce apoptosis in the process of differentiation.

Tropic effects are those that provide growth factors and nutrients that are important for cell growth so that cells grow well (Lu DP, Chandrakanthan V, Cahana A, Ishii S, O'Neill C. Trophic signals acting via phosphatidylinositol-3 kinase are required for normal pre-implantation mouse embryo development.J Cell Sci. 2004 Mar 15; 117 (Pt 8): 1567-76.).

On the other hand, in the second aspect of the present invention, a cell solution containing acinar cells and ductal cells, which are peripheral cells of the islets, is preferably collagen in the pancreas extracted as in the first embodiment of the present invention. Treating the enzymes capable of degrading the enzyme to break down the collagen, thereby breaking the intercellular binding; After breaking the intercellular binding, the islets obtained from the method comprising; isolating and removing the islet cells.

In addition, in the second aspect of the present invention, the culture medium may preferably be one containing islet cells. At this time, the cell solution including acinar cells, ductal cells and islet cells is most preferably treated with enzymes capable of degrading collagen in the isolated pancreas to decompose collagen. Breaking the intercellular binding; can be used to obtain from the method comprising a. In addition, by treating the extracted pancreas with an enzyme capable of degrading collagen to break down the intercellular binding by degrading collagen; After breaking the intercellular binding, isolating and removing some of the islet cells; What is obtained from the method containing can be used.

On the other hand, in the second aspect of the present invention, during the culturing, it is preferable to sequentially lower the initial concentration from 15 to 30 mmol / l to 10 to 20 mmol / l and 5.5 to 10 mmol / l.

On the other hand, in the second aspect of the present invention, the total culture period is not necessarily limited to a specific period, but preferably 7 to 15 days in consideration of differentiation yield and economic aspects.

On the other hand, in the second aspect of the present invention, the timing of adding embryonic stem cells or embryonic stem cells is not necessarily limited to a specific time period during the culture period, but preferably It is recommended to add at any time within 6 days after the start of the culture from the start of the culture.

As described above, the method of differentiating the peripheral islets of the present invention into cells capable of secreting insulin is insufficient because it can increase the differentiation yield of cells capable of secreting insulin and reduce cell death that occurs during cell differentiation. It is effective to substantially solve the supply of pancreatic islets.

In addition, it is very economical because it is possible to separate the discarded pancreatic islets and differentiate the cells capable of secreting insulin from the remaining excess cell solution.

Hereinafter, the content of the present invention will be described in more detail in the following examples, but the scope of the present invention is not limited only to the following examples, and includes modifications of equivalent technical ideas.

Example 1 Without Embryonic Stem Cells Differentiate cells that can secrete insulin from peripheral islet cells

Since human pancreas is difficult to obtain, it was tested using mouse pancreas.

The mouse pancreas was injected with collagenase P (collagenase P (2mg / ml)) about 3ml, and then the pancreas was detached and activated by enzyme in water bath at 37 ° C. for 12 minutes to 'digestion'. After the density gradient using the 'islet gradient', islet cells (islet; FIG. 3) and peripheral cells (ductal cells, acinar cells; FIG. 4) were separated.

 25 mmol / l glucose (high glucose Dulbeccos modified Eagles medium; DMEM) and 20% serum (fetal bovine serum; FBS; Gibco BRL, Gaithersburg, MD, http://www.gibcobrl.com), 0.55 mM ß-mel In 'bacteria culture dish' with ß-mercaptoethanol, HEPES buffer, 100 units / ml penicillin, 100 μg / ml streptomycin, 100 μg / ml nonessential amino acids Peripheral islet cells were cultured for 4 days.

Four days later, 'dithizone staining' was performed to identify cells capable of secreting differentiated insulin from peripheral islets. 'Dithizone Staining' is a staining method used to check insulin secretion. It reacts with the zinc produced when insulin is secreted and turns red. As a result of the measurement (FIG. 1), it was confirmed that insulin was secreted from the differentiated cells. However, an increase in cell death was observed.

Example 2: Differentiating Cells That Can Secrete Insulin from Peripheral Islet Cells by Addition of Human Maltegrant Embryonic Stem Cells

Since the human pancreas is difficult to obtain, mouse pancreas was used and embryonic stem cells were used to reduce cell death during differentiation into cells capable of secreting insulin from cells around the pancreatic islets. Was used.

Mouse pancreas was injected with collagenase P (collagenaseP (2mg / ml)) about 3ml, and then the pancreas was detached and activated by enzyme in water bath at 37 ° C for 12 minutes to 'digestion'. After the density gradient using the 'islet gradient', islet cells (islet; FIG. 3) and peripheral cells (ductal cells, acinar cells; FIG. 4) were separated.

25 mmol / l glucose (high glucose Dulbeccos modified Eagles medium; DMEM) and 20% serum (fetal bovine serum; FBS; Gibco BRL, Gaithersburg, MD, http://www.gibcobrl.com), 0.55 mM ß-melcapto Around the pancreatic islets in a 'bacteria culture dish' with ethanol (ß-mercaptoethanol), HEPES buffer, 100 units / ml penicillin, 100 μg / ml streptomycin, 100 μg / ml nonessential amino acids The cells were mixed with human embryiod bodies (FIG. 5) and cultured for 5 days. On the third day, 'cell cluster fusion' was established between peripheral cells of the mouse and human embryoid bodies. It was confirmed that this occurred (Fig. 6).

 After 5 days, the maturation period was 3 days with 15.25 mmol / l glucose condition and 2 days with 10.38 mmol / l glucose condition while maintaining a serum condition of 20%.

Experimental Example 1: Analysis of yield of cells capable of secreting insulin

Experimental Example 1 analyzed the yield of cells differentiated in Example 1.

25 mmol / l glucose (high glucose Dulbeccos modified Eagles medium (DMEM)), 20% serum (fetal bovine serum (FBS)), 0.55 mM ß-mercaptoethanol, HEPES buffer, 100 units / ml penicillin ( penicillin), 100 μg / ml streptomycin and 100 μg / ml non-essential amino acids in a 'bacteria culture dish' mixed with surrounding peri-islet cells and human embryonic embryonic stem cells for 4 days , density gradient was performed using ficoll. At this time, about 20% of 'transdifferentiated cells' were separated in the region corresponding to the islet density, and the secretion of insulin was confirmed through 'dithizone staining'.

Yield was measured a total of 10 times, expressed as the total cell volume (cell volume) and the cell volume after separation. Yield measurement results (FIG. 2), the cell volume separated by ficoll in the total volume of 3 ml was 0.6, 0.72, 0.42, 0.66, 0.54, 0.48, 0.6, 0.78, 0.66, 0.54 ml, about 20% The volume corresponding to the degree was measured and this was especially the amount stained by 'dithizone staining'.

This is a significant increase compared to the 5% yield of cells capable of secreting insulin, reported in a paper by Song KH et al.

Experimental Example 2 Insulin Secretion of Differentiated Cells from Peripheral Islet Cells Cultured with Embryonic Stem Cells by RT-PCR

It was confirmed by RT-PCR whether insulin is secreted from the differentiated cells recovered from Example 2 above.

As a result of the measurement (FIG. 7), it was confirmed that Insulin 1 / Preproinsulin 1 (left lane in FIG. 7) and Insulin 2 / Prproinsulin 2 (right lane in FIG. 7) emerge from the differentiated cells.

Experimental Example 3 Transplantation of Cells capable of Secreting Insulin Produced from Peripheral Islet Cells

Diabetic mice were made using streptozotocin (Sigma), a drug that induces diabetes in mice or animals, and the cells capable of secreting insulin differentiated in Example 2 were given to kidneys of nude mice. Transplanted into capsules. As a result of measuring blood glucose of nude mice transplanted with cells capable of secreting insulin and non-transplanted mice (control) (FIG. 8), the control group did not reduce blood glucose and died all over 18 days, but differentiated insulin Nude mice implanted with secretory cells were found to gradually reduce blood glucose levels.

1 is a diagram showing the presence or absence of insulin secretion by 'dithizone Staining' cells that can secrete insulin differentiated from peripheral cells of the pancreatic islets without using embryonic stem cells.

Figure 2 is a measure of the yield of cells capable of secreting insulin.

3 is a photograph of pancreatic islets.

Figure 4 is a photograph of the peripheral cells (ductal cells, acinar cells).

Figure 5 is a photograph of the embryonic embryonic stem cells (embryoid body).

FIG. 6 shows' cells generated between peripheral pancreatic islets and human negative embryonic stem cells on day 3 of culturing mouse ductal cells (acinar cells) and human embryoid bodies together. FIG. It is a photograph showing cluster fusion.

FIG. 7 is a diagram showing that the secretion of insulin from differentiated cells was measured by RT-PCR after culturing together peripheral cells (ductal cells, acinar cells) and embryonic stem cells.

FIG. 8 is a diagram illustrating blood glucose levels of nude mice transplanted with cells capable of secreting insulin generated by culturing peripheral cells (ductal cells, acinar cells) and embryonic stem cells together, and mice (control) without transplantation. .

Claims (10)

To the cell solution containing at least one selected from the surrounding cells of the islet cells (acinar cell) and pancreatic ductal cells (ductal cells), 15-25% of serum and 15-30 mmol / l of glucose are added to the cell solution. After forming the cells, the method of differentiating the surrounding cells of the islet is characterized by culturing into cells capable of secreting insulin Embryonic stem cells or embryoid bodies are added to a cell solution containing any one or more selected from acinar cells and ductal cells, which are peripheral cells of the islet. In addition, 15-25% serum and 15-30 mmol / l of glucose are added based on the final concentration to form a culture solution, followed by culturing the peripheral cells of the pancreatic islet, characterized in that the differentiation into cells capable of secreting insulin. The method according to claim 1 or 2, A cell solution containing acinar cells and ductal cells, which are peripheral cells of the islet, Treating the extracted pancreas with an enzyme capable of degrading collagen to break the binding between cells by degrading collagen; After breaking the intercellular binding, isolating the islet cells is removed; the method of differentiating the peripheral cells of the islet, characterized in that obtained from the method comprising a cell capable of secreting insulin The method according to claim 1 or 2, The culture solution, A method of differentiating peripheral cells of the islet, which is characterized in that it contains islet cells, into cells capable of secreting insulin The method of claim 4, wherein The cell solution containing the acinar cells (ductal cells), pancreatic cells (ductal cells) and islet cells (islet cells), Differentiating the surrounding pancreatic islets into cells capable of secreting insulin, which is obtained from the method comprising a step of breaking down the intercellular binding by treating the extracted pancreas with an enzyme capable of breaking down collagen. How to let The method of claim 4, wherein The cell solution containing the acinar cells (ductal cells), pancreatic cells (ductal cells) and islet cells (islet cells), Treating the extracted pancreas with an enzyme capable of degrading collagen to break down intercellular binding by degrading collagen; After breaking the intercellular binding, isolating and removing some of the islet cells; How to differentiate the peripheral cells of the islets, characterized in that obtained from the method comprising a cell capable of secreting insulin The method of claim 1, The total total incubation period is How to differentiate the peripheral cells of the pancreatic islet, characterized in that it is 3 to 6 days into cells capable of secreting insulin The method of claim 2, Cultivation, Differentiate the peripheral islet cells into cells capable of secreting insulin, characterized in that the glucose concentration in the culture is sequentially lowered from the initial starting concentration of 15-30 mmol / l to 10-20 mmol / l and 5.5-10 mmol / l. How to let The method of claim 8, The total total incubation period is How to differentiate the peripheral cells of the pancreatic islet, characterized in that it is 7-15 days into cells capable of secreting insulin The method of claim 9, Embryonic stem cells or embryonic stem cells (Embryoid body), Method of differentiating peripheral cells of the islet is secreted into cells capable of secreting insulin, characterized in that it is added at any time within 6 days after the start of the culture from the start of the culture

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