WO2015190522A1 - Method for producing endodermal stem cells - Google Patents

Abstract

The present invention addresses the problem of providing: a method for producing endodermal stem cells efficiently; and others. A method for producing endodermal stem cells, which comprises: (A) a step of expressing a gene in liver cells, wherein the gene is a gene that encodes a protein having an activity of allowing the phase of the cells to be transitioned to S phase through G0 or G1 phase; (B) a step of culturing the cells obtained in step (A) in the presence of an extracellular growth factor; and (C) a step of selecting cells in each of which the expression of a molecular marker AFP is negative among from the cells obtained in step (B).

Description

Method for producing endoderm stem cells

The present invention relates to a method for producing endoderm stem cells and a technique using the same.

Diabetes is a serious disease that is spreading worldwide. The global diabetic population estimated by WHO is over 150 million in 2000 and is expected to reach about 300 million in 2025. In such a situation, for example, islet (insulin producing cell) transplantation is attracting attention as an effective treatment for patients with type I diabetes. However, the shortage of donors providing transplantable islets is a serious problem. Yes. In addition, if the HLA antigen suitability is low, it is necessary to use an immunosuppressive agent for the recipient in order to control the rejection reaction, and side effects due to the immunosuppressive agent also become a problem.

As one of the new treatments for type I diabetes, research on treatment by regenerative medicine in which islet cells are regenerated and transplanted from their own cells is underway. This method is expected to have many advantages such as elimination of the problem of rejection by using its own cells, and is highly expected.

In recent years, vigorous research has been conducted on embryonic stem cells (ES cells) and stem cells artificially prepared by gene transfer (artificial pluripotent stem cells: iPS cells). It is expected that it can be utilized.
However, although methods for producing islet cells from iPS cells have been rapidly developed in recent years, problems such as the risk of tumor formation after transplantation still remain (Non-patent Document 1). In addition, although a method for isolating pancreatic stem cells from a patient's pancreatic tissue and inducing differentiation into pancreatic islet cells has been reported (Non-Patent Document 2), it is difficult to obtain islet cells sufficient for transplantation, and it is practically used. The current situation is not.

In order to carry out islet transplantation treatment for diabetic patients under the above-described circumstances, a technique for constructing a system capable of stably supplying sufficient donors and islet cells (particularly insulin-producing cells) is required. . Therefore, an object of the present invention is to provide a technique capable of stably supplying stem cells that can also be used for islet transplantation.

As a result of intensive studies in view of the above problems, the present inventors have determined not only hepatocytes from liver-derived cell groups (primary cultured hepatocytes) but also organs derived from other endoderm (for example, pancreas). It was found that cells that can differentiate into constituent cells (ie, endoderm stem cells) can be obtained. The present inventors have made further studies and improvements based on such knowledge, and have completed the present invention.
Exemplary inventions include the following.
Item 1.
(A) In a liver cell group, a step of expressing a gene encoding a protein having an activity of passing through the G0 phase or G1 phase and shifting to the S phase;
(B) a step of culturing the cells obtained in step (A) in the presence of an extracellular growth factor; and (C) a cell in which expression of the molecular marker AFP is negative from the cells obtained in step (B). Process of selecting;
A method for producing an endoderm stem cell, comprising:
Item 2.
Item 2. The method according to Item 1, wherein the endoderm stem cell is a stem cell having an ability to differentiate into an insulin-producing cell.
Item 3.
Item 3. The method according to Item 1 or 2, wherein the protein is a cyclin-dependent kinase.
Item 4.
Item 4. The method according to Item 3, wherein the cyclin-dependent kinase is cyclin-dependent kinase 4 or cyclin-dependent kinase 6.
Item 5.
Item 5. The method according to any one of Items 1 to 4, wherein the steps (A) and (B) are repeated at least twice.
Item 6.
Item 6. The method according to any one of Items 1 to 5, wherein the extracellular growth factor is a hepatocyte growth factor.
Item 7.
In the presence of at least two or more selected from the group consisting of the culture supernatant of animal serum, epidermal growth factor, nicotinamide, dimethyl sulfoxide and HuS-E / 2 cells (FERM ABP-10908). Item 7. The method according to any one of Items 1 to 6, which is performed.
Item 8.
Item 8. The method according to any one of Items 1 to 7, wherein the culture is continued for 7 days or more.
Item 9.
Item 9. The method according to any one of Items 1 to 8, wherein the expression is transient expression.
Item 10.
An endoderm stem cell obtainable by the method according to any one of Items 1 to 9.
Item 11.
Item 11. The endoderm stem cell according to Item 10, into which an exogenous gene has been introduced.
Item 12.
Item 10. A method for producing insulin-producing cells, comprising a step of culturing endoderm stem cells obtained by the method according to any one of Items 1 to 9 in a medium suitable for differentiation into insulin-producing cells.
Item 13.
Item 14. The method according to Item 12 or 13, wherein the insulin-producing cells have glucose reactivity.
Item 14.
Item 14. An insulin-producing cell obtainable by the method according to item 12 or 13.

Human primary hepatocytes (for example, cells derived from liver tissue that have become incompatible with organ transplantation for various reasons) have already been established for their isolation and cryopreservation, and are commercially available. Therefore, the present invention using human primary hepatocytes as a raw material makes it possible to more stably provide cells that constitute an endoderm-derived organ including insulin-producing cells (or islet cells). Therefore, the possibility of constructing a large-scale insulin producing cell bank is expanded by utilizing the present invention. These insulin-producing cell banks not only provide highly tissue-compatible transplant-producing insulin-producing cells to diabetics around the world, but also supply many donor-derived insulin-producing cells for research studies such as regenerative medicine Make it possible. In addition, it becomes possible to prepare hepatocytes collected in small quantities by a liver biopsy from a diabetic patient in a medical institution as a starting material, and process (cultivate) the cells to prepare insulin-producing cells for autotransplantation. Furthermore, the present invention can also be used for the treatment of diseases relating to organs derived from the endoderm other than the pancreas.

FIG. 1 shows the results of examining gene markers expressed in proliferated cells by introducing and expressing the CDK4 gene or CDK6 gene in the primary hepatocyte group. FIG. 2 shows a scheme for inducing differentiation of endoderm stem cells into insulin-producing cells. FIG. 3 shows changes in the expression of the Pdx1 gene, which is a pancreatic differentiation marker associated with the induction of differentiation of endoderm stem cells into insulin-producing cells. FIG. 4 shows changes in the expression of an insulin gene, which is a pancreatic β cell marker, with the induction of differentiation of endoderm stem cells into insulin-producing cells. FIG. 5 shows changes in the expression of a glucokinase gene, which is a pancreatic differentiation marker accompanying the induction of differentiation of endoderm stem cells into insulin-producing cells. FIG. 6 shows changes in the expression of the glucagon gene, which is a marker for pancreatic α cells, with the induction of differentiation of endoderm stem cells into insulin-producing cells. FIG. 7 shows changes in the expression of the somastatin gene, which is a pancreatic Δ cell marker, with the induction of differentiation of endoderm stem cells into insulin-producing cells. FIG. 8 shows changes in expression of the Glut2 gene and the Nkx6-1 gene accompanying the induction of differentiation of endoderm stem cells into insulin-producing cells. FIG. 9 shows changes in the expression of the insulin gene and the somatostatin gene before and after induction of differentiation into insulin-producing cells. FIG. 10 shows the results of a glucose stimulation (glucose responsiveness) test in differentiation-induced insulin-producing cells. FIG. 11 shows changes in expression of various gene markers accompanying the induction of differentiation of endoderm stem cells into hepatocytes.

The endoderm stem cells are obtained through the following steps (A) to (C).
(A) A step of expressing a gene encoding a protein having an activity of passing through the G0 phase or G1 phase and transferring to the S phase in the liver cell group (B) Extracellular growth of the cells obtained in the step (A) A step of culturing in the presence of a factor (C) A step of selecting a cell in which the expression of the molecular marker AFP is negative from the cells obtained in the step (B)

1. Step (A)
A liver cell group means a cell group prepared from liver tissue. Liver cells are usually composed of a plurality of cells, mainly liver parenchymal cells, but may also include other cells such as sinusoidal endothelial cells, stellate cells, and Kupffer cells. As the liver cell group, a commercially available product may be used, or a sample collected from a living body may be used. For example, when human liver cells are used, commercially available frozen human liver cells sold by XenoTech, In Vitro Technologies, etc. can be used. When using commercially available frozen human liver cells, it is preferable to use a cell separation kit or the like to remove dead cells by centrifugation. When collecting a liver cell group from a living body, liver tissue collected using a biopsy needle or the like can be prepared by digesting collagenase according to a conventional method to separate hepatocytes and removing dead cells.

The liver cell group preferably has high viability. For example, the viability is 50% or more, more preferably 60% or more, still more preferably 70% or more, still more preferably 80% or more, particularly preferably. 90% or more. Cell viability can be measured using a commercially available analyzer. The liver cell group preferably has a high adhesion rate (for example, 70% or more) to a plate coated with collagen or the like. Viability can be measured according to a known method, for example, by treating a group of cells with trypan blue dye and measuring the proportion of dead cells stained blue using a microscope or the like. Can do.

“A protein having an activity of passing through the G0 phase or G1 phase and transferring to the S phase” (in this specification, sometimes referred to as “cell cycle reactivation protein”) passes through the G0 phase or the G1 phase. There is no limitation as long as it has an activity to shift to the S phase. “Transfer to the S phase by passing the G0 phase or the G1 phase” means (1) acting on the cells in the G0 phase and being in a dormant state by deviating (escaping) from the cell cycle. It means to enter the cell cycle again by shifting to (2), or to act on the cells in the G1 phase and shift the cell cycle from the G1 phase to the S phase.

The means for expressing the cell cycle reactivation protein gene is not limited as long as the cell cycle reactivation protein can be expressed. For example, transient expression may be used, or stable expression may be used. Transient expression means that a gene is introduced into a cell by a DNA transfection method or the like and expressed transiently. Transient usually refers to a period of hours to days. In contrast, stable expression means that a gene to be expressed is stably expressed in a chromosome. From the viewpoint that the endoderm stem cells produced by the method of the present invention have the same or possible similar structures and properties as endoderm stem cells in vivo, the gene of the cell cycle reactivation protein is: It is preferably expressed transiently.

Transient expression is not particularly limited, and can be performed, for example, by introducing an expression vector having a target gene downstream of an expression promoter into a cell and expressing the gene from this expression vector. In this case, as an expression promoter, for example, CMV promoter, SV40 promoter and the like can be used, but are not limited thereto. Examples of expression vectors include, but are not limited to, plasmid vectors and liposomes as non-viral vectors, and adenovirus vectors and retrovirus vectors as virus vectors. It is preferable to use a non-viral vector from the viewpoint of safety when using the cells to be produced for pharmaceutical purposes and ensuring that the gene to be introduced is transiently expressed, and in particular, the origin of replication in the host cell. Non-viral vectors that do not contain are preferred. In order to carry out transient expression more reliably, it is possible to add a step of confirming that the introduced cell is not incorporated into the chromosome. Examples of plasmid vectors that can be used from such a viewpoint include pcDNA and pSVL. As a method for introducing an expression vector into a cell, for example, a lipofection method, an electroporation method, a method in which a gene is incorporated into a viral vector and infected, and the like can be used, but not limited thereto.

Although stable expression is not particularly limited, it can be performed, for example, by the following method. An expression vector having a target gene and a dominant selection marker downstream of the expression promoter is introduced into the cell, and a strain in which the target gene is integrated into the chromosome is established. In this established strain, stable expression is performed. In this case, as an expression promoter, for example, CMV promoter, SV40 promoter and the like can be used, but are not limited thereto. As a dominant selection marker, for example, various drug resistance genes can be used, but not limited thereto. When a drug resistance gene is used as a dominant selection marker, only cell lines that stably express the drug resistance gene can be selected by continuing cell culture in the presence of a drug exhibiting resistance. . Usually, in such a cell line, the target gene is considered to be stably expressed as well. Whether or not the target gene is actually stably expressed can be clarified by analyzing the base sequence of the chromosome by a DNA sequence or the like. Moreover, as a method for introducing an expression vector into a cell, for example, a lipofection method, an electroporation method, or the like can be used, but is not limited thereto. When a viral vector is used, a method of incorporating a gene into a viral vector and infecting it can also be used.

When a gene is introduced into liver cells, it is preferable to use a transfection reagent that has relatively low cytotoxicity. In order to improve transfection efficiency, for example, it is preferable to perform transfection on cells in good condition. In the case of human liver cells, the cell state is relatively good within 2 to 3 days after seeding on a culture plate. Before performing the step (A), the liver cell group can be cultured in advance in a medium capable of maintaining the original function of the cells. For example, a commercially available dedicated medium (Human Hepatocyte Serum Free Medium, Toyobo) suitable for the characteristics of liver cells can be preferably used. When frozen human liver cells are thawed and seeded on a plate, it is desirable to add about 5 to 10% by volume of fetal calf serum to the medium in order to promote adhesion. Other culture conditions such as culture temperature can be appropriately set according to a conventionally known method.

2. Process (B)
Step (B) is a step of culturing the cells obtained in step (A) while giving growth stimulation with extracellular growth factor. By the action of the cell cycle reactivating protein introduced from the outside in the step (A), the cell cycle shifts from the G0 phase or the G1 phase to the S phase, and further to the M phase (mitotic phase), and then to the G1 phase again Proceed with At the same time, the action of the cell cycle reactivation protein is activated by the action of the extracellular growth factor present in the medium. As a result, stem cells proliferate predominantly and include endoderm stem cells.

The extracellular growth factor used in the step (B) is not particularly limited as long as it has a function of externally supporting the growth of liver-derived somatic stem cells. Examples of extracellular growth factors include cell growth factors and hormones that stimulate cell growth. Examples of the cell growth factor include epidermal growth factor (EGF), hepatocyte growth factor (Hepatocyte Growth Factor; HGF), platelet-derived growth factor (PDGF), insulin-like growth factor (IGF), vascular endothelial cells. Examples include growth factors (Vascular Endothelial Growth Factor; VEGF) and fibroblast growth factors (FGF). Of these, EGF and HGF are preferred. These may be used alone or in combination of two or more. For example, by using at least one cell growth factor selected from the group consisting of EGF and HGF in combination with at least one cell growth factor selected from the group consisting of VEGF and FGF, Proliferation can be improved synergistically. A preferred extracellular growth factor is hepatocyte growth factor. The concentration of the extracellular growth factor added in the medium is not particularly limited. For example, it is preferably 0.1 to 200 ng / ml, more preferably 1.0 to 100 ng / ml, and 5 to 50 ng / ml. Further preferred. In the step (B), the medium may be exchanged at an appropriate interval. Although not particularly limited, the medium may be changed once every two or three days. The medium may be replaced with a medium containing the same concentration of extracellular growth factor, or may be replaced with a medium containing a different concentration of extracellular growth factor. The medium may be replaced with a medium containing the same extracellular growth factor, or may be replaced with a medium containing a different extracellular growth factor.

The culture in the step (B) is preferably performed using a medium suitable for obtaining embryonic stem cells. As such a medium, in addition to the above-mentioned extracellular growth factor, animal serum, epidermal growth factor, nicotinamide, dimethyl sulfoxide and HuS-E / 2 cells (FERM ABP-10908) culture supernatant It is preferable to include one or more selected, preferably two or more, more preferably three or more, still more preferably four or more, and still more preferably all.

Animal serum is not particularly limited, and examples include human serum and fetal bovine serum. These are preferably added to the medium in the range of 0.1 to 20% by volume. When nicotinamide is added to the medium, the amount added is preferably 1 to 100 mM. When dimethyl sulfoxide is added to the medium, the addition amount is preferably 0.1 to 2% by volume. When the culture supernatant of HuS-E / 2 cells is added to the medium, the amount added is preferably 10 to 90% by volume.

Steps (A) and (B) may be performed once each, or may be performed a plurality of times as necessary. Alternatively, these may be set as a set and repeated a plurality of times. When it is performed a plurality of times, it can be performed preferably 2 to 10 times, more preferably 3 to 8 times, and even more preferably 3 to 5 times. It is considered that repeating steps (A) and / or (B) in this way is preferable in order to transiently express a gene encoding a cell cycle reactivation protein and shift stem cells to a proliferative state. It is done.

Steps (A) and (B) can be continued until a stem cell containing the required amount of endoderm stem cells is obtained. The culture period is not particularly limited and can be, for example, several days to several weeks. In one embodiment, the culture is preferably performed for 7 days or more. When the step (B) is performed only once, for example, if the step (B) is continued for a period until the colony formation by the stem cells is sufficiently performed, a group of stem cells including endoderm stem cells can be easily collected. The end point of the step (B) is preferably a time point at which a stem cell colony can be confirmed with a microscope or the naked eye, or a time point at which a stem cell colony consisting of 10 to 10,000 cell groups is formed, and more preferably 100 to 100- This is the time when a stem cell colony consisting of 1000 cell groups is formed. Many cells other than stem cells are killed by the end of the step (B), or even if they are alive, the growth is stopped and the cell morphology is clearly different. For this reason, a highly pure stem cell group can be acquired easily.

3. Process (C)
By collecting the colonies obtained by the step (B) and selecting cells that are negative for the cell surface molecular marker AFP, endoderm stem cells can be obtained. Colony recovery can be performed by a conventionally known method, for example, a limiting dilution method or a method using a micropipette under a microscope. In addition to being negative for the molecular marker AFP, the endoderm stem cells are preferably albumin negative, C-Met positive, EpCam positive, Thy1 positive, and CD34 negative. The presence or absence of the expression of the molecular marker or gene marker can be confirmed by any technique, and can be performed using, for example, a commercially available kit.

Judgment of being an endoderm stem cell can also be performed by other methods (for example, examining whether or not to differentiate into cells of a plurality of endoderm-derived organs). For example, the differentiation of the obtained cells into pancreatic cells is performed, and the presence or absence of gene markers (Pdx1, insulin, glucokinase, etc.) of the pancreatic cells is confirmed to confirm whether the cells can be divided into pancreatic cells. Can do. In addition, hepatocyte differentiation can be induced on the obtained cells and the presence or absence of hepatocyte gene markers (albumin, drug metabolizing enzyme genes, etc.) can be confirmed. .

According to the present invention, a stem cell group preferably containing 50% or more endoderm stem cells in terms of the number of cells, more preferably a cell group containing 80% or more endoderm stem cells can be prepared. According to the present invention, it is possible to produce a group of stem cells, more preferably substantially composed of only endoderm stem cells, and more preferably an isolated endoderm stem cell group.

Endoderm stem cells are somatic stem cells that have the ability to differentiate into cells that constitute an organ derived from the endoderm (ie, multipotent) and self-proliferating ability. Examples of organs derived from the endoderm include, but are not limited to, the esophagus, stomach, small intestine, large intestine, lung, thyroid gland, pancreas, and liver. As organs constituted by cells from which endoderm stem cells can differentiate, pancreas and liver are preferable. In one embodiment, the endoderm stem cells preferably have the ability to differentiate into insulin-producing cells, and stably supply a high-purity population of insulin-producing cells having properties equivalent to those of insulin-producing cells in vivo. Can be used to The insulin-producing cells thus obtained can be finally used in clinical applications such as cell preparations, or in various research and development such as new drug development or disease research. The endoderm stem cell may further contain an exogenous gene as necessary.

Induction of differentiation of endoderm stem cells into insulin-producing cells can be performed by culturing endoderm stem cells in a medium suitable for differentiation into insulin-producing cells and other conditions. A medium and other culture conditions suitable for differentiation can be set by appropriately selecting from known conditions. For example, Zhang D. Can be used to differentiate from ES cells to insulin-producing cells (Cell Research 19 (4), 429-38, 2009). That is, it is a method of inducing differentiation stepwise by changing the medium (culture environment) such as specialization, cell proliferation, and cell maturation from endoderm stem cells toward pancreatic differentiation. The insulin-producing cells thus induced to differentiate preferably have glucose responsiveness.

Hereinafter, although an example and a test example are shown and the present invention is explained in detail, the present invention is not limited to these examples.

Example 1. Preparation of endoderm stem cells (1) Culture of primary human hepatocytes After thawing frozen primary human hepatocytes (XenoTech), Ficoll isolation using Hepatocyte Isolation Kit (XenoTech) to separate live and dead cells As a result, a high viability hepatocyte suspension was obtained. These cells were suspended in Human Hepatocyte Serum Free Medium (Toyobo Co., Ltd.) supplemented with fetal bovine serum at a rate of 10% by volume, and about 5 × 10 ⁵ cells / cell-coated 12-well cell culture plate (AGC Techno Glass) / It seed | inoculated so that it might become the cell density of a well. The seeded cells were cultured in a CO ₂ incubator for 48 hours under conditions of 37 ° C. and 5% CO ₂ , and hepatocytes were sufficiently adhered to the plate.

(2) Transfection The commercially available protein expression plasmid pcDNA3 (Life Technologies) was used for transfection. A human CDK4 gene introduction plasmid was prepared by inserting the human CDK4 gene into the cloning site between EcoRI and XbaI of the plasmid. Further, a human CDK6 gene introduction plasmid was prepared by inserting the human CDK6 gene into the cloning site between HindIII and BamHI of the plasmid. DNAs encoding human CDK4 and human CDK6 are designed based on the nucleotide sequence registered in NCBI (National Center for Biotechnology Information) (accession number of CDK4 gene: CAG47043, accession number of CDK6 gene: NP_001138778). RT-PCR using total RNA purified from HuS-E / 2 cells (human hepatocyte-derived cells, deposited at the National Institute of Advanced Industrial Science and Technology Patent Biological Depositary: FERM ABP-10908) as a template Obtained. As the primer, a forward primer consisting of the base sequence shown in SEQ ID NO: 1 and a reverse primer consisting of the base sequence shown in SEQ ID NO: 2 were used. The base sequence of SEQ ID NO: 1 includes a sequence corresponding to Flag Tag.

The plasmid (pcDNA-FLAG-CDK4 or pcDNA-FLAG-CDK6) having Flag Tag introduced into the N-terminal side of the open reading frame of DNA encoding CDK4 or CDK6 obtained in this way was 0.3 μg per well. It was added to the medium together with Effectene transfection reagent (Qiagen), and the CDK4 or CDK6 gene was introduced into the hepatocyte group. Thereafter, transfection was repeated 5 times at a frequency of once every 5 days. As a negative control, a similar transfection operation was performed using a plasmid not containing the CDK gene.

(3) Culture of transfected cells From the time of transfection, the HuS-E / 2 cells are cultured in a DMEM-based culture solution containing 5% human serum and 5% fetal bovine serum. A DMEM-based culture medium in which the prepared conditioned medium was added and mixed was used. In addition, DMEM-based cultures include 35.8 mM sodium bicarbonate, 0.34 μg / ml insulin, 4.1 μg / ml transferrin, 4.1 ng / ml EGF (epidermal growth factor), 20 ng / ml. HGF (hepatocyte growth factor), 8.3 mM nicotinamide, 0.81 vol% dimethyl sulfoxide were added.

(4) endodermal stem pcDNA-FLAG-CDK4 to obtain 14 days of culture cells, or cells with pcDNA-FLAG-CDK6 and transfection that forms several small colonies in the petri dish was observed . This colony was cloned by a limiting dilution method to obtain clonal cells. FIG. 1 shows the results of examining the gene expression related to hepatocytes for each of the obtained clones. Although some of them showed the properties of hepatic stem cells, there were clonal cells that were negative for alpha fetoprotein (hereinafter, AFP), which is one of the markers of hepatic stem cells (FIG. 1). As described later, it was confirmed that the cells having a negative AFP marker can also differentiate into cells constituting an organ (pancreas) derived from the endoderm other than the liver. Therefore, cells that are negative for the AFP marker were obtained as endoderm stem cells (ie, stem cells that can differentiate into endoderm-derived organs). Endodermal stem cells were confirmed to have self-replicating ability because of continued proliferation while maintaining the above-mentioned differentiation ability over one year after establishment.

Example 2 Confirmation of differentiation ability into endoderm-derived organ (1) Induction of differentiation into insulin-producing cells A test was conducted to differentiate the endoderm stem cells obtained in Example 1 into insulin-producing cells using a known technique. Specifically, the following three-stage differentiation induction procedure shown in FIG. 2 was performed. First, 0.5 mass / volume% bovine serum albumin, 0.5 volume% ITS solution (Life Technologies), 0.5 times B27 solution (Life Technologies), 2 μM retinoic acid (Sigma), The culture was performed for 4 days in a 1: 1 mixed medium of F12 medium and IMDM medium supplemented with 20 ng / ml FGF7 (Peprotech) and 50 ng / ml NOGGIN (Peprotech). Next, 0.5 mass / volume% bovine serum albumin, 1 volume% ITS solution (Life Technologies), 1-fold N2 solution (Life Technologies), 50 ng / ml EGF (Sigma) were added. Cultivated in DMEM medium for 5 days. Thereafter, 1% by volume ITS solution (Life Technologies), 10 ng / ml bFGF (Peprotech), 10 mM nicotinamide (Sigma), 50 ng / ml Exendin4 (Peprotech), and 10 ng / ml BMP4 ( The cells were cultured for 4 days in DF12 medium supplemented with Peprotech.

(2) examined the expression variation of various marker genes for cells before and after performing the differentiation induction of marker expression above in cells after differentiation induction. As a result, Pdx1, insulin, glucokinase, glucagon, and somatostatin, which are pancreatic differentiation markers, were all greatly expressed during the maturation period from the 9th day to the 13th day after the start of differentiation induction. It was also found that expression of glucagon and somatostatin was also greatly induced during maturation (FIGS. 3 to 7). For Glut2 and Nkx6-1, expression was induced to a level close to that of human islet tissue after differentiation induction (FIG. 8). Insulin and somatostatin are known to be produced in islet β cells and islet δ cells, respectively. When the same cells were stained simultaneously by the indirect fluorescent antibody method using an anti-insulin antibody and an anti-somatostatin antibody before and after differentiation induction, it was confirmed that these two proteins were produced in one cell (FIG. 9). ).

(3) Insulin production ability and glucose-responsive differentiation induced cells are secreted into the culture medium, and the reactivity of the cells to high-concentration glucose is determined according to the C peptide in the culture supernatant (cleavage of insulin Product) was used as an index and examined using an ELISA kit (Mercodia). As a result, a detectable amount of C peptide was present in the culture solution. Moreover, it was found that when the glucose concentration of the culture solution was increased to 10 times and exposed for 1 hour, the amount of C peptide was induced twice (FIG. 10). From the above, it was found that differentiation-induced cells are cells that have the ability to produce and secrete insulin, react to a high concentration of glucose, and induce secretion.

Example 3 Induction of differentiation into hepatocytes Human hepatic stem cells having the properties of endoderm stem cells obtained in Example 1 were confirmed to have the ability to differentiate into hepatocytes. That is, human hepatic stem cells having the properties of endoderm stem cells are cultured in a medium in which 30 ng / ml FGF4 and 20 ng / ml BMP2 are added to Hepatic basal medium (Lonza) for 5 days, and then Human Hepatocyte Serum-free Medium. (Toyobo) was cultured for about one month. During this time, as a result of investigating gene markers expressed in the cells over time, AFP turned positive on the 5th day of culture and further showed albumin positive after about 1 month of culture (FIG. 11). This result indicates that human hepatic stem cells having the properties of endoderm stem cells are differentiated into hepatocytes.

Claims (14)

1. (A) In a liver cell group, a step of expressing a gene encoding a protein having an activity of passing through the G0 phase or G1 phase and shifting to the S phase;
   (B) a step of culturing the cells obtained in step (A) in the presence of an extracellular growth factor; and (C) a cell in which expression of the molecular marker AFP is negative from the cells obtained in step (B). Process of selecting;
   A method for producing an endoderm stem cell, comprising:
2. The method according to claim 1, wherein the endoderm stem cell is a stem cell having an ability to differentiate into an insulin-producing cell.
3. The method according to claim 1 or 2, wherein the protein is a cyclin-dependent kinase.
4. The method according to claim 3, wherein the cyclin-dependent kinase is cyclin-dependent kinase 4 or cyclin-dependent kinase 6.
5. The method according to any one of claims 1 to 4, wherein the steps (A) and (B) are repeated at least twice.
6. The method according to any one of claims 1 to 5, wherein the extracellular growth factor is a hepatocyte growth factor.
7. In the presence of at least two or more selected from the group consisting of the culture supernatant of animal serum, epidermal growth factor, nicotinamide, dimethyl sulfoxide and HuS-E / 2 cells (FERM ABP-10908). The method according to any one of claims 1 to 6, which is carried out.
8. The method according to any one of claims 1 to 7, wherein the culture is continued for 7 days or more.
9. The method according to any one of claims 1 to 8, wherein the expression is transient expression.
10. An endoderm stem cell obtainable by the method according to any one of claims 1 to 9.
11. The endoderm stem cell according to claim 10, into which an exogenous gene has been introduced.
12. A method for producing insulin-producing cells, comprising a step of culturing endoderm stem cells obtained by the method according to any one of claims 1 to 9 in a medium suitable for differentiation into insulin-producing cells.
13. The method according to claim 12 or 13, wherein the insulin-producing cells have glucose reactivity.
14. Insulin-producing cells obtainable by the method according to claim 12 or 13.
    

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