WO2004104195A1 - Insulin-producing cells and method of constructing the same - Google Patents

Abstract

It is intended to provide insulin-producing cells, which can be constructed with the use of liver-origin cells obtained from a normal liver tissue as the starting material, and a method of constructing the same. By culturing small-sized hepatocytes which are undifferentiated stem cells under definite conditions, these cells can be differentiated into insulin-producing cells. Further, the stimulus-response extracellular insulin secretion system of the cells can be improved by transferring a Pdx-1 gene into the cells. The insulin-producing/secreting cells thus obtained are physiologically closely similar to islet β cells and usable in treating or studying diabetes, etc. In particular, it is expected that these cells would be usable as donor cells in cell transplantation therapy for diabetes.

Description

 Description Insulin producing cells and methods for producing them

 The present invention relates to an insulin producing cell and a method for producing the same. INDUSTRIAL APPLICABILITY The present invention is particularly useful for the treatment of diabetes, and can be used for the fundamental treatment of diabetes by cell transplantation, and for the field of cell production therefor. Background art

 In recent years, transplantation treatment of insulin-producing cells and humans incorporating them has been seen as a promising treatment for radical diabetes, and the induction of insulin from ES cells (embryonic stem cells) derived from fertilized eggs to insulin-producing cells is promising. Some successful cases have been reported. However, the application of ES cells to medicine has many difficulties to solve, including ethical issues, and its clinical application is not very realistic at present.

 On the other hand, some methods for artificially inducing the insulin production function using adult stem cells or the like instead of ES cells have already been proposed. For example, U.S. Patent No. 6,448,045 discloses that the expression of the PDX-1 (Pdx-1) gene in cultured cells and the stimulation of the insulin gene with the GLP-1 receptor agonist are further described in U.S. Pat. Methods for inducing expression have been disclosed. However, in consideration of cell transplantation treatment for diabetic patients, a method of inducing insulin production from liver-derived cells, which are easily available from patients and the like, is more desirable than the method using 腌 cells.

Ferder S. et al. (2000) Nature Medicine Vol. 6, No. 5, pp. 568-572 shows that the PDX-1 gene was introduced into mice using a viral vector, and that the expression of the insulin gene could be induced in the liver. Has been reported. However, in this way, the gene was directly introduced into the individual by the virus vector without isolating the liver cells, It is difficult to clinically apply diabetes treatment by ectopic expression of insulin. Also, Yang L. et al. (2002) PNAS Vol. 99, No. 12, pp. 8078-8083 discloses a method for imparting an insulin producing function by culturing a hepatic oval cell. I have. However, hepatic round cells do not exist in the normal liver, and appear only when they are damaged by drugs or the like, making them difficult to use in transplantation medicine and the like.

 As described above, a method of inducing insulin production by transdifferentiation (transd ferentiation) in vitro using liver-derived cells, particularly liver-derived undifferent stem cells obtained from healthy liver tissue, has been a successful example. Not reported.

 The liver is an organ that is developed and differentiated from the foregut, which is the same as the knee, and studies on differentiation induction using adult stem cells have shown that insulin production from undifferentiated stem cells derived from adult healthy liver tissue has been reported. This suggests the possibility of inducing differentiation into cells, that is, the possibility of producing insulin-producing cells from liver-derived cells. If a method for producing such insulin-producing cells is established, it will be possible to provide a material that is extremely useful for treating diabetes by transplantation. In addition, for clinical application, it is desirable that the physiological functions related to insulin secretion of transplantation cells be as close to normal islet β cells as possible. Disclosure of the invention

 The present invention has been made in view of the above-mentioned problems, and has as its object to provide liver-derived cells obtained from normal liver tissue, which are insulin-producing cells that can be used for the treatment of diabetes or its research. To provide an insulin-producing cell that can be produced using the method, and a method for producing the same.

'The present inventors have focused on small hepatocytes (hereinafter abbreviated as “SHC”), which can be separated from adult healthy liver tissue and have the properties of hepatic progenitor cells. And proceeded with intensive research. As a result, (1) this small hepatocyte (2) to enhance the stimulus-responsive insulin extracellular secretion mechanism of cells by introducing the Pdx-1 gene into the cells by culturing them under certain conditions. (3) The introduction of the Pdx-1 gene induces the expression of synabtotagmin 1 involved in the transport of secretory granules by producing Pdx-1 gene. It was found that the present invention was completed.

 That is, the present invention includes the following inventions A) to Q) as medically and industrially useful products and methods.

 A) Insulin producing cells obtained by culturing liver-derived cells. Here, the liver-derived cell means a cell obtained from a healthy liver tissue. Therefore, cells that appear only when damaged by drugs such as liver round cells and are not suitable for transplantation treatment are not included in the liver-derived cells mentioned here.

 B) The insulin producing cell according to A) above, which is obtained by culturing small hepatocytes as liver-derived cells.

 C) The insulin-producing cell according to A) or B) above, which is responsive to stimulation by introducing a transcription factor Pdx-1 (Pancreatic duodenal homeobox-l) gene or a gene of a protein that positively regulates its expression. Insulin producing cells with improved extrinsic extracellular secretory function.

 D) The insulin-producing cell according to A) or B) above, wherein a stimulus-responsive insulin extracellular secretory function is improved by inducing expression of a synaptotagmin or a membrane protein for extracellular transport of other hormonal substances. Insulin producing cells.

 E) The insulin-producing cell according to any one of the above A) to D), which is a cell derived from a rat, a mouse, or a human.

F) Insulin obtained from the insulin-producing cell according to any one of A) to E). G) A method for producing insulin-producing cells, which comprises inducing insulin-producing functions by culturing liver-derived cells.

 H) A method for producing insulin-producing cells as described in G) above, wherein small hepatocytes are cultured as liver-derived cells.

 I) It is characterized in that after culturing cells under the first culture condition containing serum in the medium, further cell culture is performed under the second culture condition in which the serum concentration in the medium is lower than the first culture condition. The method for producing an insulin-producing cell according to the above G) or H).

 J) The method for producing an insulin-producing cell according to the above I), wherein the serum concentration in the medium under the second culture condition is adjusted to about 0.1 to 0.5%.

K) The method for producing insulin-producing cells according to I) or J) above, wherein a mixed medium of DMEM and ham _{F12K is used in} the second culture condition.ώ L) Second culture condition The method for producing insulin-producing cells according to any one of the above (I) to (K), wherein the Pdx- 導入 gene is introduced into the cells during or before the cultivation under the following conditions.

 M) The method for producing insulin-producing cells according to L) above, wherein an adenovirus vector is used for introducing the Pdx-1 gene into cells.

 N) The method for producing an insulin-producing cell according to any of the above G) to M), wherein the method is produced from a cell derived from a rat, a mouse, or a human liver.

 O) Insulin producing cells obtained by culturing differentiated stem cells.

 Ii) A method for producing an endocrine hormone-producing cell, comprising inducing glucagon and / or somatostatin production by culturing liver-derived cells and other undifferentiated stem cells.

Q) An endocrine hormone obtained from a cell produced by the method described in the above item Ρ). Further objects, features, and advantages of the present invention will be more fully understood from the following description. Also, the advantages of the present invention will become apparent in the following description with reference to the accompanying drawings. BRIEF DESCRIPTION OF THE FIGURES

 FIG. 1 is a flowchart schematically showing a process for producing insulin-producing cells from small hepatocytes.

 FIG. 2 is a diagram schematically illustrating the manufacturing process of FIG.

 (A) to (c) of FIG. 3 show the results of examining the presence or absence of insulin production in the cells obtained by the above-described production process by immunofluorescent staining.

 FIG. 4 is a diagram showing the results of examining the expression of genes such as transcription factors involved in development and differentiation by RT-PCR at each stage of the production process.

 FIG. 5 is a graph showing the results of examining the amount of insulin production in the cells obtained by the above-described production steps.

 FIG. 6 is a graph showing the results obtained by examining how the amount of insulin secreted by cells changes in response to various stimuli for the cells obtained by the above-described production process. FIG. 7 is a diagram illustrating the mechanism of insulin secretion in knee island β cells in response to various stimuli.

 FIG. 8 is a graph showing the results of measuring the amount of residual insulin in cells after the stimulation test.

 FIG. 9 is a diagram showing the results of gene expression of a protein contributing to insulin secretion measured by RT-PCR.

 FIG. 10 is a diagram showing, as a model, the interrelationship between Pdx-1, Syt1, NeuroD, Pax6, and HNF-3fi in Tengshima β cells.

 FIG. 11 is a diagram showing each primer sequence used in the RT-PCR analysis of FIG. '

 FIG. 12 is a diagram showing the respective primer sequences used in the RT-PCR analysis of FIG. 4, as in FIG.

Fig. 13 shows the cell (+ Pdx-1 and -Pdx-1) cells prepared by the method of Fig. 1. FIG. 4 shows the results of examining the gene expression of glucagon and somatostatin by RT-PCR. BEST MODE FOR CARRYING OUT THE INVENTION

 Hereinafter, specific embodiments, technical ranges, and the like of the present invention will be described in detail.

 (1) Method for producing insulin-producing cells from liver-derived cells

 The present inventors have focused on small hepatocytes (SHCs), which have the ability to proliferate in vitro for a long time and differentiate into liver parenchymal cells, among liver-derived cells that can be separated from normal liver tissue. Various conditions for inducing the function to be produced were examined. As a result, they successfully transdifferentiated small hepatocytes into insulin-producing cells by culturing them in a safe manner without using a method that would be toxic to living organisms. Furthermore, transfection and forced expression of the transcription factor Pdx-1 gene in these cells enabled the secretion of insulin hormone in response to various stimuli, similar to the physiological function of knee island β cells in vivo. .

 The details of each experimental method and the results of the experiments performed by the present inventors will be described in Examples below.However, in the following, referring to FIG. 1, a method for actually inducing insulin-producing and secreting cells from small hepatocytes will be described. explain.

 First, in step 1, cells containing small hepatocytes (SHC) were isolated from normal liver tissue of rats using collagenase hepatic perfusion. Specifically, a normal rat (Sague-Dawley breed, male) weighing 100-150 g was laparotomized, and a force urge was introduced from the lower part of the portal vein to the base of the liver, and Hepes was injected according to the usual method. Liver perfusion was performed with -buffered salt solution, and non-real liver cells (ιιοη-parenchymal cells: NPC) containing SHC were isolated by digestion with collagenase. After further purifying the cells by centrifugation, the cells were divided into three groups.

Next, in step 2, the three groups of SHC are each cultured in HGM medium for 5 days. Specifically, inoculate 2-5X10 ⁴ viable isolated cells / cm ² into Petri dishes for cell culture. Was. At this time, an HGM (hepatocyte growth medium) medium based on DMEM (Dulbecco's Modified Eagle's Medium) to which the following components were added was used as a medium (culture medium).

10% FBS (Fetal bovine serum)

20 mM HEPES

25 mM NaHCO ₃

30 mg / L L-prohne

 10 mg / L insulin-transferrin-sodium selenite (ITS)

20 ng / mL EGF (epidermal growth factor)

0.004 g / L trace metals (ZnCl 2, ZnS0, CuS0 4, MnS0 4)

10 mM nicotinamide

ImM ascorbic acid 2 -phosphate

10 " ⁷ M Dexamethasone

5.5 mM D-glucose

Antibiotics (100 U / ml penicillin, 100 g / ml streptomycin, 0.25 g / ml Fungizone) after cells were cultured for 2 days in an incubator at _{37 ° C, 5% C0 2} -95% air, with a new medium The cells were replaced and cultured for another 3 days. As described above, in step 2, the cells were cultured in the HGM medium for 53 times, but the period may be about 3 to 7 days as described later.

 In the next step 3, 1% dimethylsulfoxide (DMSO) is added to the above HGM medium and cultured under the same conditions for 2 days. Thereafter, two of the three groups of cultured SHC cells were subjected to the following procedures 4 and 5 as an experimental group. On the other hand, the remaining one group of cells was used as a control group, and the culture conditions of this step 3 were continued until the 15th day.

In step 4, two groups of SHC cells were cultured for two days under low serum culture conditions different from the medium in step 3. Specifically, as in steps 2 and 3, the serum type used was fetal calf serum (FBS), and the concentration in the medium (culture solution) was set to a low concentration of 0.2%. As a medium, a medium (DMEM / F12K) in which DMEM and ham F12K (Kaigh's modification of Ham's F12) were mixed at a ratio of about 1: 1 was used, and the following components were added.

0.2% FBS

20 ng / mL HGF (hepatocytes growth factor

(10 ng / mL EGF, epiaermal growth factor)

25 mM NaHCO ₃

30 mg / L L-proline

 10 mg / L insulin-transferrin-sodium selenite (ITS)

10 mM nicotinamide

10-7 M Dexamethasone

Antibiotics (Same conditions as in steps 2 and 3)

 The conditions in the incubator were set to the same conditions as in steps 2 and 3. After the culturing in step 4, in step 5, the Pdx-1 IRES-GE gene is used in one of the two groups of SHC cells and the GFP gene is used in the other cell using the adenovirus vector. (For convenience, the former cells are sometimes referred to as “+ Pdx-l” and the latter cells are sometimes called “-Pdx-1”). Specifically, based on an E1-deficient adenovirus vector, an expression vector is prepared in which the Pdx-1-IRES-GFP gene or the GFP gene is downstream of the chicken beta-actin (AG) promoter. The Pdx-1-IRES-GFP gene contains the gene sequence encoding the mouse transcription factor Pdx-1 (Pancreatic duodenal homeobox-1), the coding sequence of IRES (internal ribosome entry site), which is a ribosome binding site in mRNA, and And a sequence encoding the green fluorescent protein GFP. GFP is for confirming the presence or absence of gene introduction.

The adenovirus expression vector described above was infected (transfection) to the two groups of cells after step 4 under conditions of a duplicate infection rate (MOI) of 30-50. (+ Pdx-1) forcibly expressed Pdx-1 and GFP, and the other cell (-Pdx-) forcibly expressed only GFP. In step 5, after the gene was introduced into the cells, culturing was continued for another 6 days in the same medium and culture conditions as in step 4.

 Thus, in this method, three groups of cells were prepared: the experimental group (+ Pdx-1), the experimental group (-Pdx-1), and the sealed group. Then, on days 9, 12, and 15, the amount of insulin produced by each cell was measured. The results are shown in the graph of FIG. In the graph, “Group 1” represents the experimental group, and “Group 2” represents the control group. As shown in the graph, on the 15th day, the amount of insulin production in the experimental group (+ Pdx-1 and -Pdx-1) was significantly increased as compared with the control group. In the cells of this experimental group (+ Pdx-1 and -Pdx-1), not only insulin but also production of glucagon and somatostatin were observed (see FIG. 13).

 In addition, when intracellular insulin was detected using an anti-insulin antibody, it was observed that insulin was produced intracellularly in the cells of the experimental group (+ Pdx-1) on day 15 ( (See the left panel in Figure 3 (b)). In contrast, in the cells of the control group on the 15th day, almost no signal was detected, and almost no insulin production was observed (see the left panel in FIG. 3 (c)).

In addition, we examined how the amount of insulin secreted by each cell changes in response to various stimuli. The results are shown in the upper graphs of FIG. The graph on the left shows Krebs-Ringer bicarbonate buffer (KRBB) containing 0.2% FBS, ダ ル concentration dalcos (25 mM) and 10 iiM glucagon-Iike peptide 1 (GLP-1 [7-37]). The middle graph shows the results of stimulation with 45 mM KCl and 0.2 mM tolbutamide added to a low concentration of darcos (5 mM) KRBB solution, and the right graph shows the control. And the measurement results using only the low concentration glucose (5 mM) KRBB solution as the basal buffer. In each graph, “Group 1” represents the experimental group, and “Group 2” represents the control group. From these results, in particular, the experimental group (+ Pdx-1) in which Pdx-1 was forcibly expressed had a higher stimulus responsiveness than the experimental group (-Pdx-1). Surin extracellular secretion was shown to be promoted. This was also confirmed from the result of examining the amount of insulin remaining in the cells 30 minutes after the stimulation test. That is, the amount of residual insulin in the cells of the experimental group (+ Pdx-1) was remarkably smaller than that of the experimental group (-Pdx-1), and the stimulus-responsive insulin extracellular secretory function was higher in the experimental group (+ Pdx-1). Was improved (see Figure 8).

 As a result of RT-PCR analysis, in the experimental groups (+ Pdxl and -Pdxl), the expression of intracellular transport membrane proteins (such as SNAP25) contributing to hormone secretion was observed. In particular, in the experimental group in which Pdx-1 was forcibly expressed (+ Pdx-1), induction of expression of synaptotagmin 1 (Syt 1) was observed (see FIG. 9). This synabtotagmin 1 binds to secretory granules and cell membranes, and is thought to play an important role in the secretion of hormones from inside to outside of cells. Therefore, it is considered that the expression of synaptotagmin 1 was induced by forcibly expressing Pdx-1, and thus, the insulin secretion function of the experimental group (+ Pdx-1) in response to various stimuli was improved.

 The details of each experimental method will be described in Examples below.

 As described above, in the cells of the experimental group (+ Pdx-1 and -Pdx-1), the amount of insulin production was significantly increased as compared with the control group. Producing cells could be induced. Furthermore, by forcibly expressing Pdx-1, the stimulus-responsive insulin secretion function could be improved. As a result, it was possible to construct insulin-producing secretory cells having physiological functions close to those of knee island β cells in vivo, which regulate the amount of insulin secreted in response to various stimuli. Furthermore, by transplanting these cells into drug-induced diabetic nude mice, the effect of reducing the increase in blood glucose level was obtained.

 (2) Modifications of the present invention

 Of course, the present invention is not limited to the above-described method and the insulin-producing cells produced by this method, and it goes without saying that various modifications are possible.

For example, the above method uses small hepatocytes (SHC) as a material and Although it is one that induces the surin-producing function, other liver-derived cells can be isolated from normal liver tissue, as long as they have the properties of undifferentiated stem cells. It is considered that insulin production function can be induced by similar culture induction.

 Furthermore, it is considered that undifferentiated stem cells other than liver-derived cells can be induced to differentiate into insulin-producing cells by the production method of the present invention. Such undifferentiated stem cells include: (1) adult-derived undifferentiated stem cells, for example, hematopoietic stem cells, which are isolated from peripheral blood or bone marrow components, and isolated from hepatocyte-like cells. And (2) cells induced to differentiate into ES cells into hepatocyte-like cells.

 In order to induce differentiation into insulin-producing cells, it is considered important to suppress the proliferation of undifferent stem cells originally intended to differentiate into certain cells. Therefore, in the method described in _h, the medium used was changed from a medium with a high serum concentration to a medium with a low serum concentration during the culture. Specifically, in steps 2 and 3, a medium containing 10% fetal calf serum (FBS) was used, and in subsequent steps 4 and 5, the proliferation of small hepatocytes to be differentiated into liver parenchymal cells was performed. To control, a medium with a low serum concentration of 0.2% is used.

 In other words, after culturing the cells under the first culture condition containing serum in the medium, further culturing the cells under the second culture condition in which the serum concentration in the medium is lower than the first culture condition. Is thought to be important for induction of insulin production function. At this time, the serum concentration in the medium under the second culture condition is preferably about 0.1 to 0.5% in order to suppress the growth, although it depends on the kind of serum used and the like. It is more preferable to set it to about 0.2 to 0.3%. On the other hand, the serum concentration in the medium under the first culture condition is preferably about 5 to 20%, more preferably about 7 to 15%.

The type of serum used for the first and second culture conditions is not particularly limited, and examples thereof include serum of mammals other than seaweed and seaweed (such as seaweed chicken). Examples of serum include fetal serum (Fetal ca serum: FCS or Fetal bovine serum: FBS), newborn serum (Newborn Bovine Serum: NBS), calf serum (Calf serum: CS), and the like. May include fetal bovine serum (03 or 83). Further, it is preferable to use the same serum type throughout the first and second culture conditions.

 The medium (culture medium) to be used is not particularly limited. However, in the first culture condition, it is preferable to use an HGM medium based on DMEM from the viewpoint of promoting the growth of hepatocytes. Under the second culture conditions, it is preferable to use a medium other than the HGM medium from the viewpoint of once suppressing the growth of cells. In the experiments performed by the present inventors, the basal medium component under the second culture condition was higher in DMEM and ham F1 than in a mixed medium (DMEM / F12) of DMEM and Ham's F12, which is widely used. Better results were obtained using a mixed medium (DMEM / F12K) with 2K (Kaigh's modification of Ham's F12). Good results are obtained when the ratio of DMEM to ham F12K is approximately 1: 1, and the glucose (D-glucose) concentration in the medium is around 5 mM (1 to: 1.3 g / L). was gotten.

 The medium may be prepared from reagents, but since various media including DMEM and ham F12K are currently on the market, these may be prepared using commercially available products.

As the medium additive in the second culture condition, epidermal growth factor (EGF), nicotinamide (Nicotiamide), and liver growth factor (HGF) are preferable. It is desirable that EGF and dycotinamide be added to help the induction of insulin-producing cells, and that HGF be added to help the survival of liver cells that are vulnerable to the severe conditions of low serum concentration. The concentration of each added substance is not particularly limited, but for example, the concentration of HGF is in the range of 10-25 ng / mL. In addition, Dexamethasone and ITS, cell growth factors (fibroblast growth factor, etc.), hypothalamus and pituitary gland extracts are also preferred medium additives. Other commonly used nutrients, antibiotics, etc. may be added to the medium.

 The cell culture period is also not particularly limited, and may be appropriately set in relation to other conditions.For example, Step 2 is about 3 to 7 days, Step 3 is about 1 to 4 days, and 4 is preferably set to about 2 to 8 days. If the Pdx-1 gene is not forcibly expressed, step 5 is unnecessary, and step 4 may be extended for an appropriate period.

 The method for introducing the Pdx-1 gene into cells is not particularly limited, and various gene transfer methods such as a phosphate canolescence method, a lipofection method, a microinjection method, a DEAE dextran method, a virus method, and an electoral poration method can be used. Since a method is known, an appropriate method may be selected from known gene introduction methods. Among them, the virus method using a virus vector is preferable because gene transfer can be performed with high efficiency, and various known virus vectors can be used in addition to a general adenovirus vector. In the above method performed by the present inventors, an adenovirus vector in which a coding sequence of IRES is connected downstream of the Pdx-1 gene, which is the target gene, and placed downstream of the chicken betaactin (AG) promoter is used. However, highly efficient gene transfer was obtained by transfection using this vector.

 The introduction of the Pdx-1 gene into cells is preferably performed during the culture under the second culture condition, but may be performed before the culture. In the above method, the mouse Pdx-1 gene was introduced so as to be distinguishable from the expression of endogenous rat Pdx-1, but a gene derived from the same species as the host cell may be introduced.

 It is considered that insulin-producing cells can be produced from cells derived from mouse and human liver by similar culture induction. When human liver-derived cells are used, a part of the liver may be excised by a known method performed in living liver transplantation or the like, and liver-derived cells may be prepared from the tissue.

Also, as described above, forced expression of Pdx-1 induces the expression of synaptotagmin 1, which significantly enhances the stimulus-responsive insulin-secreting function of cells. It is thought that it was done. Therefore, in addition to the forced expression of Pdx-1, the expression of membrane proteins for extracellular transport of hormonal substances, in particular, synabtotagmin 1 (or other synaptotagmins), is induced by other methods. The stimulus-responsive insulin secretion function may be improved. Thus synaptotagmin 1 (or other extracellular transport membrane protein SNAP25, GB _Y, TGase etc. 2) Another method of inducing expression of, for example, transcription factors Neuro D, the expression of Pdxl upstream For example, a method of forcibly expressing a series of transcription factors and genes that promote extracellular transport of hormones and neurotransmitters, which are controlled by the enzyme, may be considered.

 (3) Field of application (utility) of the present invention

 The present invention is particularly useful for treating or researching diabetes. By the production method of the present invention, it is possible to synthesize insulin in the undifferentiated stem cells derived from the liver, and by forcibly expressing the transcription factor Pdx-1, the cells can stimulate the synthesized insulin in response to stimulation. Acquired the ability to secrete efficiently. Even in normal knee β cells, insulin is always stored in the cells and released into the blood promptly in response to stimulation, enabling strict glycemic control. In light of this, the insulin-producing cells of the present invention are considered to be cells having physiologically similar functions to knee island β cells. '

 Furthermore, in the process of inducing the differentiation of liver-derived cells into insulin-producing cells by the production method of the present invention, the expression of genes that play an important role in controlling the development and differentiation of the original Tengjima β-cells was examined over time. An expression pattern was shown that linked to mimic the pattern of normal occurrence (see Examples below).

Therefore, the insulin-producing cell and the method for producing the same of the present invention can be used as a donor cell used in cell transplantation therapy, which is expected as a fundamental treatment for diabetes, and in the field of cell production therefor. For example, undifferentiated stem cells can be isolated from the liver of a diabetic patient, and insulin-producing cells produced based on the cells can be used as donor cells for autologous transplantation, or can be produced based on liver-derived cells obtained. Surin producing cells may be used as donor cells for allogeneic transplantation.

 In addition, the method for producing the insulin-producing cells of the present invention can be used not only in direct treatment of diabetes but also in basic research, animal tests, clinical tests, etc. for the development of therapeutic methods as a precedent step. Available.

 Furthermore, the insulin synthesized and secreted by the insulin-producing cells of the present invention can be used as pharmaceuticals (insulin preparations), reagents, experimental materials, and the like. Included in the invention. Insulin isolated and purified from rat and mouse cells may be derived from the insulin 1 gene or the insulin 2 gene. A known method may be used for isolating and purifying insulin.

 Further, according to the method of the present invention, it is possible to induce production of not only insulin but also glucagon and Z or somatostatin (see FIG. 13). The endocrine hormone-producing cells thus obtained and endocrine hormones isolated and purified from the cells are also industrially useful and included in the present invention.

 Hereinafter, the present invention will be described more specifically with reference to examples, but the present invention is not limited to these examples.

 [Example 1: Production of insulin-producing cells from small hepatocytes]

 Insulin-producing cells were produced from small hepatocytes by the method shown in FIGS. The manufacturing method is as described above, and the description is omitted here. In FIG. 2, “Group 1” indicates the experimental group, and “Group 2” indicates the control group. The following figures (the same applies hereafter).

Fig. 3 (a) shows the results of observation of cells on day 7 of culture using a phase contrast microscope, and Figs. 3 (b) and (c) show the results of detection of insulin using an anti-insulin antibody by immunofluorescence staining. is there. The left panel in (b) shows the results of fluorescent staining of insulin in the cells of the experimental group (+ Pdx-1) on day 15, and the right panel shows the results obtained by DAPI fluorescence of the cell nuclei in these cells. The result is superimposed on the result of staining. D API is a dye substance for nuclear staining of cells. The left panel in (c) shows the results of the fluorescent staining of insulin in the cells of the control group on day 15, and the right panel shows the results and the results of the fluorescent staining of DAPI in the same cells superimposed. . In the cells of the 15-day experimental group (+ Pdx-1), insulin was observed to be produced intracellularly (left panel in (b)). Showed almost no insulin production (left panel in (c)).

 In addition, the amount of insulin production of each cell on day 9, day 12, and day 13 of culture was measured. For the quantification of insulin, the cells were collected and immersed in ethanol (containing 0.1N hydrochloric acid) at minus 20 ° C for 12 to 24 hours to extract insulin according to the usual method. . This exudate was measured and quantified by ELISA. The total protein content in the sample was quantified by the BCA method, and the extraction efficiency between samples was corrected. The results are shown in the graph of FIG. As shown in the graph, on the 15th day, the amount of insulin production of the experimental group (+ Pdx-1 and -Pdx-1) was significantly increased as compared with the control group.

 [Example 2: Examination of gene expression during culture process]

 At each stage of the culture method shown in Fig. 1, the expression of genes such as transcription factors involved in development was examined by RT-PCR. In the RT-PCR method, RNA is extracted from cultured cells according to the conventional method, reverse transcription is performed using Superscript-II Reverse Transcriptase, converted to complementary DNA, and then PCH reaction is performed using Taq polymerase. I got it. The primers used in each PCR reaction were as shown in FIGS. 11 and 12, and nested PCR was performed as necessary. The results were confirmed by agarose gel electrophoresis. Figure 4 shows the results.

On the 0th day of culture, SHC expresses albumin, a marker of mature hepatocytes, c-Met expressed on mature hepatocytes and β-cells, and neurogenin 3 (ngn 3), a marker of knee island ancestral cells. Expressed. On day 7 of the culture, the expression of albumin and -eurogenin 3 disappeared, and the expression of nestin was observed (lanes 1 and 2). This The results indicate that the nestin-positive SHCs proliferated by the cultures in steps 2 and 3 above.

 On day 15 of the culture, the expression of insulin 1 was detected only in the cells of the experimental group (+ Pdx-1 and -Pdx-1). These cells also expressed β-cell markers, GLUT2 and ΙΑΡΡ, indicating that SHC had been differentiated into β-cells.

 Insulin 2 expression was observed only in the experimental group (+ Pdx-1), but not in the experimental group (-Pdx-1). Forced expression of mouse mPdx-1 induced expression of endogenous rat rPdx-1 and NeuroD. It is considered that the expression of Pdx-1 and neuron or Neuro D is important for the expression of insulin 2. Mature β-cell markers Isll and Pax6 were detected after culture 9. In contrast, expression of the exocrine cell marker PTF1 was not observed in any of the cells analyzed.

 Mutations in HNF-la (hepatocytes nuclear factor-la), HNF-lb, and HNF-4a cause diabetes, but there was no change in their expression among the cells examined in this study.

 [Example 3: Insulin hormone secretion in response to various stimuli]

 We investigated how the amount of insulin secreted by each cell changes in response to various stimuli. The results are shown in the upper graphs of FIG. The graph on the left shows the Krebs-Ringer bicarbonate buffer (KRBB) solution containing 0.2% FBS to which high-concentration glucose (25 mM) and 10 nM glucagon-like peptide 1 (GLP-1 [7-37]) were added. The middle graph shows the results of stimulation with 45 mM KC1 and 0.2 mM tolbutamide added to KRBB solution with low concentration of glucose (5 mM) .The right graph is for control, and the basal buffer This is the result of measurement using only the KRBB solution with a low concentration of dalcose (5 mM). The insulin secretion quantification was performed in the same manner as in the experiment in FIG.

As shown in FIG. 7, glucose promotes insulin secretion in vivo, When glucose is transported intracellularly, adenosine triphosphate (ATP) is generated through the glycolytic crepes cycle, and the influx of Ca ²⁺ is increased, and then the Ca stored in the cell is increased. ²⁺ is released into the cytoplasm. This increase in intracellular Ca ²⁺ concentration leads to phosphorylation reactions involving protein kinase A (PKA) and protein kinase C (PKC), which promote insulin secretion. GLP-1 is an activator of the cAMP-PKA pathway, and K + and tolbutamide enhance Ca2 ⁺ entry into cells, and both stimulate insulin secretion.

As shown in the upper graphs in Fig. 6, the experimental group in which Pdx-1 was forcibly expressed (+ Pdx-1) had a higher stimulus-responsive insulin secretion function than the experimental group (-Pdx-1). Was. This was also confirmed from the result of examining the amount of insulin remaining in the cells 30 minutes after the stimulation test. That is, the amount of residual insulin in the cells of the experimental group (+ Pdx-1) was remarkably smaller than that of the experimental group (-Pdx-1), and the stimulus-responsive insulin extracellular secretory cells in the experimental group (+ Pd _X -l). It was confirmed that the function was improved (see Fig. 8). When the time change of insulin secretion in the cells of the experimental group (+ Pdx-1) was examined, the time change was similar to the time change of insulin secretion from knee island β-cells due to glucose stimulation (Fig. 6). See lower graph).

 [Example 4: Induction of synabtotagmin 1 expression by forced expression of Pdx-1]

 RT—As a result of PCR analysis, expression of other proteins that contribute to the secretion of insulin hormone, SNAP25, GBy, and TGase2, were determined in the experimental group on day 15 of culture (both + Pdx-1) and -Pdx-1. In the same way. In particular, in the experimental group (+ Pdx-1) in which Pdx-1 was forcibly expressed, induction of synaptotagmin 1 (Sytl) expression was observed. (See Figure 9).

The above results indicate that synaptotagmin 1 (Syt 1) is considered to be a direct or indirect target substance of the transcription factor Pdx-1, and that Pdx-1 positively regulates the transcription of Syt 1. From the experimental results so far, the model shown in Fig. 10 suggests that the interaction between Pdx-1, Syt1, Neuro D, Pax6, and HNF-3B in knee island β-cells is considered. Thus, it is thought that Neuro D and the like also positively regulate the transcription of Sytl.

 It should be noted that the specific embodiments or examples made in the section of the best mode for carrying out the invention merely clarify the technical contents of the present invention, and such specific examples The present invention is not to be construed as being limited to only the above, and various modifications can be made within the scope of the following claims. Industrial potential

 INDUSTRIAL APPLICABILITY As described above, the present invention relates to insulin-producing cells obtained by culturing liver-derived cells and a method for producing the same, as described above. It can be used in the field of therapy and cell production therefor, and has various other usefulness.

Claims

The scope of the claims 1. Insulin producing cells obtained by culturing liver-derived cells.  2. The insulin-producing cell according to claim 1, which is obtained by culturing small hepatocytes as liver-derived cells.  3. The insulin-producing cell according to claim 1 or 2, wherein the stimulus-responsive insulin extracellular secretory function is improved by introducing a Pdx-1 gene or a gene of a protein that positively regulates its expression. Producing cells.  4. The insulin-producing cell according to claim 1 or 2, wherein the stimulation-responsive insulin-phosphorus extracellular secretory function is enhanced by inducing expression of a membrane protein for extracellular transport of synabtotagmin or another hormonal substance. Insulin producing cells. 5. The insulin-producing cell according to any one of claims 1 to 4, which is a cell derived from a rat, a mouse, or a human.  6. Insulin obtained from the insulin-producing cell according to any one of claims 1 to 5.  7. A method for producing insulin-producing cells, which comprises inducing an insulin-producing function by culturing liver-derived cells.  8. The method for producing insulin-producing cells according to claim 7, wherein small hepatocytes are cultured as liver-derived cells. 9. After culturing the cells under the first culture condition containing serum in the medium, further culturing the cells under the second culture condition in which the serum concentration in the medium is lower than the first culture condition. Item 9. The method for producing an insulin producing cell according to Item 7 or 8. 10. The method for producing insulin-producing cells according to claim 9, wherein the serum concentration in the medium under the second culture condition is about 0.1 to 0.5%. 11. The method for producing insulin-producing cells according to claim 9 or 10, wherein a mixed medium of DMEM and ham F12K is used in the second culture condition. Law. The insulin-producing cell according to any one of claims 9 to 11, wherein the Pdx-1 gene is introduced into the cell during or before the culture under the second culture condition. Production method. 13. The method for producing an insulin-producing cell according to claim 12, wherein an adenovirus vector is used for introducing the Pdx-1 gene into the cell. The method for producing insulin-producing cells according to any one of claims 7 to 13, wherein the method is prepared from cells derived from rat, mouse, or human liver. Insulin producing cells obtained by culturing undifferentiated stem cells. A method for producing endocrine hormone-producing cells, which comprises inducing glucagon and / or somatostatin production by culturing liver-derived cells and other undifferentiated stem cells. An endocrine hormone obtained from a cell produced by the method according to claim 16.