KR100660473B1 - Pancreatic Precursor Cell Line Transdifferentiated from Pancreatic Acinar Cell - Google Patents

Abstract

The present invention provides a method for separating pancreatic progenitor cells and (1) adult pancreatic progenitor cells from pancreatic progenitor cells that simultaneously express marker genes of pancreatic islet cells and genes involved in pancreatic development, and (2) the pancreatic progenitor cells. Transducing differentiated pancreatic progenitor cells from pancreatic progenitor cells, which comprises culturing the cells in vitro and (3) isolating pancreatic progenitor cells which simultaneously express marker genes of pancreatic islet cells and genes involved in pancreatic development during culture. It relates to a manufacturing method of.

In addition, the present invention is a pancreatic islet cell characterized in that the transition is differentiated from the pancreatic progenitor cells and express the marker gene of pancreatic islet cells and (1) contacting the growth factor with the pancreatic progenitor cells obtained above, (2) 1) culturing the pancreatic progenitor cells and (3) isolating pancreatic islet cells expressing a marker gene of pancreatic islet cells during the culturing.

Transitional differentiation, pancreatic progenitor cells, pancreatic progenitor cells, pancreatic islets cells, pancreatic islets cells

Description

Pancreatic Precursor Cell Line Transdifferentiated from Pancreatic Acinar Cell}

1 is a photograph showing the morphological changes according to the incubation time of pancreatic progenitor cells {A-F x200; G-H x100; Day 0 (A), Day 1 (B), Day 3 (C), Day 5 (D), Day 10 (E), and Day 15 (F) after initiation of culture; YGIC4 (G), YGIC5 (H)}.

Figure 2 is a photograph showing the morphological changes according to the incubation time of pancreatic progenitor cells {x10400; Day 0 (A), Day 1 (B)-(C), Day 3 (D), Day 10 (E)-(F), and Day 15 (G) after incubation; YGIC4 (H), YGIC5 (I)}.

Figure 3 shows the results of measuring the activity of amylase according to the incubation time of pancreatic progenitor cells.

Figure 4 is a photograph showing the morphological changes according to the culture time in three-dimensional culture of pancreatic progenitor cells {day 0 (A), day 3 (B), day 5 (C) after the start of culture in collagen; Day 0 (D), Day 3 (E), Day 5 (F)} after incubation in Matrigel, respectively.

Figure 5 shows the results of BrdU integration analysis to confirm the proliferative capacity of the pancreatic progenitor cells obtained in the present invention.

Figure 6 is a photograph showing the results of Western blotting to confirm the expression markers of YGIC4 and YGIC5 cell line according to the present invention.

Figure 7 is a photograph showing the results of Western blotting to identify the expression markers of pancreatic islet cells differentiated from the YGIC4 or YGIC5 cell line according to the present invention.

The present invention relates to a pancreatic progenitor cell line differentiated from pancreatic progenitor cells, and more particularly, to the pancreatic progenitor cell differentiated from pancreatic progenitor cells characterized by simultaneously expressing a marker gene of pancreatic islet cells and a gene involved in pancreatic development. It relates to a cell and a method for producing the same. In addition, the present invention relates to pancreatic islet cells expressing the marker gene of pancreatic islet cells and transducing differentiation in said pancreatic progenitor cells and a method for producing the same.

The pancreas of a mammal consists of (1) the ductal tree, (2) the exocrine acini producing digestive enzymes, and (3) the endocrine tissue producing hormones. Pancreatic ductal epithelial cells, acinar cells and islet cells all derive from primitive tubular structures formed in the protodifferentiated state of early pancreatic development (J Slack, Development, 1995, 121, 1569-1580). Further morphogenesis of the pancreas is achieved by the expression of insulin-promoter-factor (IPF) as well as the interaction between islets, islets and progenitor cells.

 After the development of this fetus, the differentiation capacity of pancreatic islets into progenitor cells is markedly reduced (M. Elsner et al. Diabetologia, 2000, 43, 1528-1533). However, pancreatic islets in adult pancreas can differentiate into islet cells given the conditions under which appropriate intercellular interactions can occur (S. Bonner-Weir et al. Diabetes, 1993, 42, 1715-1720; L. Rosenberg, Cell Transplant, 1995, 4, 371-83). Differentiation of pancreatic pancreatic islets into endocrine-producing cells is similar to ontogeny of pancreatic development.

Various pancreatic islet cells have been prepared using the differentiation ability of these pancreatic islets. US Pat. No. 5,888,705 describes a method for isolating mature pancreatic cells from humans and then differentiating the pancreatic cells into islet cells using hepatocyte growth factor / scattering factor (HGF / SF). WO 97/15310 discloses a method in which mature pancreatic islet cells are first cultured in low glucose concentration medium containing serum and then transferred to culture medium of higher concentration of glucose component to differentiate into islet cells. . US Pat. No. 5,834,308 discloses a method for isolating progenitor cells from early diabetic adults and culturing them in a medium that promotes the growth of functional islets.

But all these 'progenitor cells' only differentiate into islet cells. The pancreatic progenitor cells in the aforementioned literature do not have the ability to differentiate into any form of endocrine or exocrine cells. An ideal pancreatic progenitor cell group should be capable of differentiating into not only pancreatic islets but also endocrine cells (ie islet-α, -β, -δ, -PP cells) and exocrine cells (i.e., progenitor cells, pancreatic islets). This is because such progenitor cells are particularly useful for clinical applications.

In order to solve these problems, WO 01/77300 discloses a group of pancreatic epithelial progenitor cells capable of differentiating into endocrine and exocrine cells, a method for separating pancreatic epithelial progenitor cells, and characteristics of pancreatic epithelial progenitor cells. Determination and use of pancreatic epithelial progenitor cells were disclosed. However, since the pancreatic epithelial progenitor cells are obtained from fetuses, it is difficult to obtain starting materials. In addition, such embryonic cells may be transformed into cancer cells because of their high pluripotency and high potential for differentiation into unwanted cells.

Recently, ovarian cells have been known to be involved in the histogenesis of ductal adenocarcinoma. When pancreatic progenitor cells were chemically induced to cause carcinogenesis, they were found to dedifferentiate to produce vascular-like cells (A. Krapp et al. Genes Dev. 1998, 12, 3752-3763). ).

Accordingly, the present inventors pay attention to the fact that during the transitional differentiation of pancreatic progenitor cells into pancreatic islets, there may be precursor cells having pluripotency capable of differentiating into all of the pancreatic cells. At the same time, pancreatic progenitor cells expressing genes involved in the development of the pancreas were identified and the present invention was completed.

Accordingly, the present invention provides pancreatic progenitor cells differentiated from pancreatic progenitor cells which simultaneously express marker genes of pancreatic islets and genes involved in pancreatic development.

As another aspect, (1) isolating pancreatic progenitor cells of an adult, (2) ex vivo culture of the pancreatic progenitor cells in a medium for culturing mammalian cells, and (3) pancreatic pancreatic islet marker genes and pancreas. It provides a method for producing transitional differentiated pancreatic progenitor cells from pancreatic progenitor cells comprising the step of isolating pancreatic progenitor cells that simultaneously express genes involved in development.

As another aspect, there is provided pancreatic islet cells expressing transitional differentiation in the pancreatic progenitor cells and expressing marker genes of pancreatic islet cells.

As another aspect, (1) contacting the previously obtained pancreatic progenitor cells with growth factors, (2) culturing in a mammalian culture medium, and (3) pancreas expressing marker genes of pancreatic islet cells during culture. It provides a method for producing pancreatic islet cells differentiated from pancreatic progenitor cells, including islet cells.

"Pancreatic precursor cell" of the present invention is a pluripotent cell obtained by transdifferentiating pancreatic progenitor cells, the characteristic of the pancreatic progenitor cells disappears, and all of the features of the pancreatic islet cells and the pancreas constitute the pancreas. It has the characteristics as a pancreatic progenitor cell capable of differentiating into cells (i.e., progenitor cells, islets cells and islets cells).

In order to more clearly describe the characteristics of the pancreatic progenitor cells according to the present invention, it is necessary to clarify the terms used in the specification of the present invention and the characteristics of the differentiated cells constituting the pancreas. Of course, the characteristics of differentiated pancreatic cells are known in the art, but are defined as follows to clarify the scope of the present invention.

"Characteristics" in the specification of the present invention includes the morphology and phenotype unique to a specific cell, the shape means the shape and color unique to the cell alone. , Genotype refers to a marker gene that is uniquely expressed in a given cell.

"Pancreatic acinar cells" are cells that secrete various digestive enzymes involved in the breakdown of proteins, fats, and polysaccharides, which have one to three large nucleoli and vesicles that are expressed organelles. And the Golgi apparatus is remarkably developed and rich in zymogen granules containing various digestive enzymes. Marker genes of pancreatic progenitor cells include, but are not limited to, amylase or p48.

"Pancreatic islet cells" can be classified into four cells according to their function as follows: (1) α-cells: glucagon production; (2) β-cells: insulin production; (3) δ-cells: somatostatin production and (4) pp-cells: pancreatic polypeptide production. In other words, pancreatic islets are cells that produce and secrete the pancreas-derived hormones into the bloodstream and have an ovate size of about 75 μm to 175 μm. Marker genes of pancreatic islet cells include various hormones described above.

"Pancreatic ductal cells" are smaller, rounder, about 10 μm in diameter, appear tightly packed, and are cubic epithelial cells compared to the cells of the chambers. Marker genes of pancreatic pancreatic islet cells include cytokeratins CK 7, CK 8, CK 18, CK 19, CFTR, mucin MUC1, carbonic anhydrase II, and carbohydrate antibody 19.9 (sial-Lewis-a). Included.

On the other hand, "transdifferentiation" in the context of the present invention usually means that cells in one differentiation state change to another differentiation state. Transitional differentiation is different from neogenesis derived from stem cells because the starting material is cells that have already been differentiated. During this transdifferentiation process, many genes are switched on and off, resulting in dramatic changes in morphology and phenotype. In the present specification, "differentiation" has two meanings, one of which is to change from pancreatic progenitor cells to pancreatic progenitor cells, and the other is to change from pancreatic progenitor cells obtained earlier to pancreatic islet cells.

Thus, the "pancreatic progenitor cells" according to the present invention are the markers of pancreatic islet cells, while the expression of marker cells (eg amylase, p48) of the progenitor cells is reduced, the endoplasmic reticulum and Golgi apparatus are degraded, and the enzyme source granules are reduced. Genes (for example CK 10, CFTR) are expressed and take the form of pancreatic islets. In addition, the pancreatic progenitor cells express genes involved in pancreatic development, thereby retaining pluripotency (multipotency) that can be differentiated into all cells of the pancreas.

The "gene involved in pancreatic development" expressed in pancreatic progenitor cells according to the present invention means a gene that is originally expressed in pancreatic progenitor cells and induces differentiation into specific pancreatic cells, but is not limited thereto. Notch-1, Jagged-1, Wnt-4, β-catenin, neurogenin 3, Neuro D, Pax 4, Pax 6, Pdx-1, PTF1 / p48, Tcf-4 and the like.

The pancreatic progenitor cells of the present invention may also express marker genes that are specifically expressed only in tissue-restricted stem cells, such as hematopoietic stem cells, neural stem cells, and liver stem cells. Such marker genes include, for example, SCF / c-kit or vimetin.

Pancreatic progenitor cells, which are starting materials for obtaining pancreatic progenitor cells according to the present invention, are preferably those isolated from adult pancreas. In particular, since the pancreatic progenitor cells have pluripotency to differentiate into various kinds of cells constituting the pancreas, they can be used for the treatment of various pancreatic diseases requiring transplantation of pancreatic cells, tissues or organs. Therefore, if the pancreatic progenitor cells of the present invention are used for transplantation, it is more preferable to use pancreatic progenitor cells isolated from the individual to be transplanted in order to prevent rejection.

In one aspect of the present invention, pancreatic progenitor cells isolated from rats were transferred and differentiated to obtain pancreatic progenitor cell lines, which were named YGIC4 and YGIC5, respectively, and "YGIC5" was the Korean Cell Line Research Foundation, an international depository institution, on January 29, 2004. It was deposited with KCLRF-BP-00093 (KCLRF, Cancer Research Center, Seoul National University College of Medicine, Yongun-dong, Jongno-gu, Seoul, Korea).

Transitional differentiation for obtaining pancreatic progenitor cells according to the present invention can be achieved using methods known in the art.

In the present invention, in vitro culture of pancreatic tissue can induce transitional differentiation from pancreatic progenitor cells to progenitor cells. Compared to other tissues, the pancreatic progenitor cells can meet the requirements of transdifferentiation by culturing in vitro because intercellular interactions are important for cell maintenance and growth. Therefore, the pancreatic progenitor cell according to the present invention comprises (1) isolating pancreatic progenitor cells from adults, (2) ex vivo culture of the pancreatic progenitor cells in a mammalian cell culture medium, and (3) pancreatic islets during culture. The pancreatic progenitor cells may be prepared using a method for producing transitional differentiated pancreatic progenitor cells from pancreatic progenitor cells, including separating pancreatic progenitor cells expressing a marker gene of a cell and a gene involved in pancreatic development.

(1) Isolation of Pancreatic Progenitor Cells

First, the pancreatic tissue is separated from the adult pancreas and finely cut. The purpose of fine cleavage is to separate structures containing progenitor cells from non-pancreatic tissue such as fat, membrane structure and connective tissue. In one embodiment, pancreatic tissue can be finely cut by physical means using homogenizers, mortars, blenders, surgical masses, syringes, forceps or ultrasound devices. In another embodiment, enzymes may be used, examples of which may include, but are not limited to, sepin proteases, including elutase and collagenase, including neutral proteases, trypsin, chymotrypsin and thermolysine. Do not. As another aspect, the physical means and the enzyme treatment may be combined.

In order to stably maintain the pancreatic tissue obtained through this process, it is preferable to preserve it in a buffer solution containing, for example, bovine serum albumin (BSA), pyruvate, or trysin inhibitor.

By microdissection of pancreatic tissue, some are separated into single cells, and some are separated into cell aggregates. Cells finely cut to the desired size can be separated using a concentration gradient. Materials which may be to make use of a concentration gradient of serum (BSA), ovalbumin, polyvinyl fatigue of the synthetic polymer (FicollTM) a nonionic sucrose, gelatinous pyrrolidone-coated anhydrous silicon (Percoll ^TM), polyvinylpyrrolidone or PVP, and Methylcellulose, including but not limited to. In the present invention, the desired pancreatic progenitor cells can be obtained by centrifuging the finely cut cells in one embodiment and then filtration using filter paper.

(2) Ex vivo culture of adult pancreatic progenitor cells

The isolated pancreatic progenitor cells may be cultured in vitro in a conventional mammalian cell culture medium. After 3-5 days from the start of the culture, cells of transitional differentiation type can be observed, and pancreatic progenitor cells can be isolated by single cell separation method at this time.

The medium usable in the ex vivo culture according to the present invention can be obtained from commercial suppliers, for example, according to the components and ratios thereof described in the catalog of the American Type Culture Collection. Examples of media that can be used are Ham badge, IMDM Iscove's badge, Leibovitz L15 badge, Mac Coy 5A badge, M199 badge, Melnick's badge, MEM Eagle's Minimum Essential badge, NCTN badge, Puck's badge, RPMI badge, Swim S77 badge, Trowell T8 badge , Waymouth badge, Williams E badge and the like. Particular preference is given to using Waymouth medium in the present invention. In addition, it is preferable to add about 0.1 to 1% antibiotic and about 1 to 10% FBS to the Waymouth medium.

In addition, at the beginning of the extracellular culture, it is preferable to treat a substance capable of suppressing rapid differentiation of cells. Differentiation inhibitors usable in the present invention include steroidal substances known in the art, but it is more preferred to use dexamethasone and analogs thereof. When using dexamethasone, the amount thereof is suitably 5 to 15 µg / ml. In addition, it may be preferable to culture in a medium that does not contain a differentiation inhibitor after the time point at which the characteristics of the pancreatic duct cells become prominent during the ex vivo culture of the pancreatic progenitor cells (about 14 to 16 days after the start of culture).

In addition, it is important to establish a suitable time point for subculture during the ex vivo culture of the production method according to the present invention. In the present invention, it is preferable to decrease the expression of pdx-1 or c-kit, which is a marker gene of progenitor cells and a factor involved in differentiation. This phenomenon usually occurs about 8-12 days after the start of the primary culture.

In the present invention, transduction differentiation into pancreatic progenitor cells can be induced not only through secondary culture (eg, plate, flask, roller bottle, petri dish, etc.) but also by tertiary culture. In the third culture, but not limited thereto, matrigel or collagen may be used. Matrigel is a mixture of substances that form an extracellular basal layer, and is preferably used for cells that do not survive only a single cell or to determine the three-dimensional structure of cells (for example, pancreatic ducts of the pancreas) in vitro. Collagen is a substance that connects cells to each other and is suitable for use when checking the interaction between cells.

(3) Confirmation of differentiation stage

The degree of differentiation of cells can be determined by the expression of the form or marker gene or by a combination thereof. The types and marker genes of the various cells constituting the pancreas are as described above.

Changes in morphology during transitional division can be easily observed using an inverted light microscope or electron microscope, and changes in phenotype are not limited to RT-PCR and Western blotting. (western blotting), immunohistochemical staining (immunohistochemistry), ELISA method can be confirmed.

The pancreatic progenitor cell line of the present invention thus obtained can be differentiated into all the cells constituting the pancreas as noted above. Accordingly, the present invention provides pancreatic islet cells characterized by transdifferentiating from pancreatic progenitor cells according to the present invention and expressing marker genes of pancreatic islet cells. Marker genes of the pancreatic islet cells are as described above.

The pancreatic islet cells differentiated from the pancreatic progenitor cells according to the present invention are (1) contacting the pancreatic progenitor cell line with growth factors, (2) culturing in a mammalian culture medium, and (3) pancreatic islets during culture. It can be prepared through the step of isolating pancreatic islet cells expressing the marker gene of the cell.

Growth factors available in step (1) of the preparation method include, but are not limited to, activin A, Betacellulin or GLP-1.

Step (1) in the production method according to the present invention is to directly process the growth factor in the pancreatic progenitor cells that are differentiated from the pancreatic progenitor cells, or to introduce a vector containing the growth factor into the pancreatic progenitor cells that are transduced from the pancreatic progenitor cells It includes. Methods available for introducing vectors into pancreatic progenitor cells include calcium phosphate transfection, DEAE-dextran mediated transfection, transvection, microinjection, cations Cationic lipid-mediated transfection, electroporation, transduction, scrape loading, ballistic introduction or infection, and the like.

Hereinafter, the examples are only for illustrating the present invention in more detail, and the scope of the present invention is not limited by these examples in accordance with the gist of the present invention, those skilled in the art. Will be self-evident.

<Example 1>

Primary culture

Pancreas were isolated from approximately 100-150 g of Sprague Dawley male rats (Jackson Laboratory, Bar Harbor, Maine, USA), crushed in a spinner flask, and then 500 mg bovine serum albumin, 500 g pyruvate and Pancreatic solution A (600 g glucose, 16.36 g NaCl, 1 M KCl, 1 M MgCl ₂ , 1 M CaCl ₂ ) containing 600 mg trypsin inhibitor was washed twice. The tissue was then treated with collagenase type IV (Sigma, St. Louis, MO, USA) for 6-8 minutes in a 37 ° C. water bath and centrifuged for 10 seconds at 1000 rpm. The cell suspension thus produced was filtered using a 200 탆 pore nylon mesh filter paper. The isolated progenitor cells were washed and then cultured in a 37 ° C., 5% CO ₂ wet incubator in Waymouth medium containing 10% fetal bovine serum (HyClone, Logan, Utah, USA) and 10 μg / ml dexamethasone (Sigma). Conformation was monitored at the same time using inverted light microscopy. The same cells were taken sequentially (FIG. 1) and were collected on days 0, 1, 3, 5, 10, 15 and 20 since the start of primary culture. During incubation, single cells were cloned by selecting morphologically identical cells using a cloning cylinder.

As shown in FIG. 1, after 2-3 days of incubation, the aggregates of the pancreatic progenitor cells adhered to the plate surface were confirmed and began to expand from the 4th to 5th days. During this period, the pancreatic cells showed the morphology of the progenitor cells, but gradually changed in morphology from 6 to 8 days after the start of the culture, thereby taking the cubic form as the pancreatic islets.

<Example 2>

Structural Analysis of Cultured Cells by Electron Microscopy

The structure of the cultured cells obtained in Example 1 was analyzed. Specifically, cells were washed twice with phosphate buffer solution (PBS, pH 7.4) and fixed in PBS containing 1% glutaraldehyde. The cells were then postfixed with osmium tetraoxide (OsO ₄ ) and samples were treated under transmission electron microscopy.

Observed by transmission electron microscopy, cells isolated from the pancreas on day 0 showed cytodifferentiation as progenitor cells. Particularly noticeable secretory organs in the progenitor cells are the high-density enzyme source located in the apical region of the cell, connected to the juxtanuclear Golgi and the rough endoplasmic reticulum located in the basal region. Filled granules could be seen (FIG. 2). On day 2 after incubation, the progenitor cells began to show major morphological changes, especially high-density granules around the cells.

On days 2 to 4, the morphology of the exocrine progenitor cells that were seen on the start of culture could no longer be observed. As described above, the transitional cells showing a decrease in secretory organ and enzyme source granules show that the cells are lineages of the progenitor cells. Transitional differentiation of these progenitor cells occurred in parallel with an increase in the number of undifferentiated cells in culture, and this change persisted on the following 3-5 days. In addition, even during this period, degranulation of the cell granules continued (FIG. 2D). In addition, after reconstitution of the cytoskeleton, primitive microvilli developed around the cell aggregates (FIG. 2E). The overall morphological appearance of the transitional differentiated progenitor cells was significantly similar to the profile of undifferentiated islet cells (FIGS. 2F-2G).

<Example 3>

Assay of Amylase Activity

The activity analysis of amylase is a measure of the amount of starch that has been degraded as a result of reacting the supernatant of cells with substrate buffer containing soluble starch for a period of time. Measurement of the activity of amylase was analyzed (FIG. 3). One unit of Caraway (Asan Pharm. Co., Hwa-Sung, Korea) used in the assay refers to the amount of enzyme capable of digesting 100 mg of starch at 37 ° C. for 30 minutes.

As shown in FIG. 3, as the in vitro culture progresses, the expression of amylase decreases as the progenitor cells migrate and differentiate, thereby confirming that the activity decreases.

<Example 4>

Three-Dimensional Cultures on Matrigel or Collagen

Matrigel (Becton Dickinson, Franklin lakes, NJ, USA) was thawed according to the instructions provided by the manufacturer, sprayed into a cell culture well placed on ice and then warmed to room temperature for several minutes before And incubated with appropriate mixing. Waymouth medium containing 10% fetal bovine serum was used as fresh medium, which was changed twice a week. Another cell substrate was prepared with a gel containing collagen I, which was appropriately mixed with cells and then subjected to tertiary culture. Backlight microscopy was used to monitor morphological changes. The same cells were taken continuously on day 1, 5 and 10 after overlay of Matrigel or collagen.

In three-dimensional culture in Matrigel or collagen, the progenitor cells also showed a transitional differentiation to pancreatic islets (FIG. 4). Pancreatic progenitor cells in collagen matrix began to form tubular structures on day 5 of primary culture (FIGS. 4A and 4B), and most of the cells formed on day 10 formed cystic ducts (FIG. 4C). Under Matrigel culture conditions, primary cultures of senescent cells began to form aggregates of cells and became flat globules on day 3. Complete morphological changes from progenitor cells to pancreatic islet cells were observed on days 3-5 (FIGS. 4D and 4E) and on day 10 the cystic structures continued to expand (FIG. 4F).

Example 5

BrdU Integration Analysis

Cells were cultured in 96-spheres at 37 ° C. for 0, 1, 3, 5, 7, 10, 12 and 15 days to determine the proliferative capacity of the cells. BrdU (BrdU labeling kit, Roche Inc., Mannheim, Germany) was then added to the cells and the cells were cultured for 24 hours. After removing the culture medium, the cells were fixed and the DNA was denatured by adding FixDenat. The resulting immune complexes were detected by further substrate response. Reaction products were quantified by measuring absorbance at 370 nm using a scanning multi-well spectrometer (ELISA reader, Molecular Devices, Sunnyvale, Calif., USA) (FIG. 5).

As shown in FIG. 5, it was confirmed that there is no proliferation of cells at 5-10 days when the expression of pancreatic islets is the highest. That is, it was found that the transitional differentiation into pancreatic progenitor cells having the characteristics of the islets is not due to the proliferation of the existing islets.

<Example 6>

Western blotting analysis and analysis of cell proliferation and tumorigenesis of pancreatic cell markers in YGIC4 and YGIC5 cell lines

As mentioned above, the cells in culture were cloned into two morphologically identical cell lines using cloning cylinders and named YGIC4 and YGIC5, respectively.

A. Analysis of Cell Proliferation

YGIC4 and YGIC5 reached confluence in 4.5 days when plated at a density of 5 × 10 ⁴ cells / ml. Thus, the doubling time of YGIC4 and YGIC5 was found to be 35 and 32 hours, respectively.

On the other hand, YGIC4 and YGIC5 were cultured without a feeder after the 12th passage. YGIC cells, when cultured in monolayer, exhibited irregular and ulcerous (cobblestone-like) shapes in the form of fusiform epithelial cell types. In addition, these cells did not contain any enzyme source granules in the cytoplasm and showed primitive microvilli around the cell aggregates. YGIC cells were stored in liquid nitrogen at various passages and regenerated by thawing. These cells were found to retain their initial morphology after 50 passages.

B. Identify Tumor

Tumorogenesis of cell lines YGIC4 and YGIC5 was analyzed by intradermal injection of 200 μl PBS containing 4 × 10 ⁶ cells into 6 week old ICR / nude mice.

YGIC cells did not proliferate even when injected intradermal into ICR / nude mice. That is, the cell lines were confirmed to be normal cells that do not form a tumor. In addition, genetic sequencing of the cell lines confirmed that they did not carry mutated codons 12K-ras (90% of pancreatic cancers show 12K-ras mutations). The morphological and growth characteristics of the YGIC cells obtained according to the present invention were found to be immortalized but do not form tumors.

C. Western Blotting

Western blotting was performed to identify various cell types using the following primary antibodies: mouse monoclonal anti-cytokeratin 19 (1: 1000, DAKO), rabbit polyclonal anti-amylase (1: 2000, Sigma) , Rabbit polyclonal anti-Pdx1 (1: 1000), mouse monoclonal anti-CFTR, mouse monoclonal anti-vimetin, rabbit polyclonal anti-Notch-1, rabbit polyclonal anti-Tcf-4, rabbit Polyclonal anti-Wnt and rabbit polyclonal anti-β-catenin (1: 1000, Santa Cruz Biotechnology Inc. USA) (FIG. 6).

The YGIC cells express CFTR and cytokeratin 19, which are pancreatic islet cell markers, but did not express amylase, which is a marker of exocrine cells, and insulin, which is a marker of p48 and endocrine beta cells. Furthermore, the inventors were able to identify the expression of Notch-1, Jagged-1, Wnt-4, β-catenin and Tcf-4 genes involved in pancreatic development.

<Example 7>

Confirmation of Transitional Differentiation into Pancreatic Islet Cells in YGIC4 and YGIC5 Cell Lines

The YGIC cell line obtained in the above examples was cultured in an incubator with 5% CO ₂ , saturated humidity with Waymouth medium supplemented with antibiotics (penicillin, streptomycin or amphotericin B) and 10% FBS. The cultured YGIC cell line was transformed with pBX322 / GLP-1 (10 nmol / L) using lipofectamine. These transformed YGIC cell lines were stem cell factor (SCF), c-kit, PDX-1, Pax-4, Pax-6, Ngn-3 using Western blotting, RT-PCR and ELISA methods. , NeuroD, insulin, glucagon and CFTR expression was identified (Fig. 7).

As shown in FIG. 7, the expression levels of Hes-1, Notch-1, neuroD, and sonic hedgehog (SHH), which are factors involved in pancreatic development of the embryo, were not changed by GLP-1 treatment, but neurogenin-3 ( The expression of ngn-3) was decreased. In addition, the expression of PDX-1, a developmental regulator of pancreatic β-islet cells, was increased by GLP-1, whereas the expression of Pax-6 was decreased. In addition, the expression of SCF and c-kit, which are stem cell markers, was increased by GLP-1, but the expression of CFTR, a marker of islet cells, was decreased. In addition, GLP-1 promoted the expression of insulin and glucagon, but had no effect on the expression of Glut-2 and glucokinase.

Pancreatic progenitor cells derived from pancreatic progenitor cells according to the present invention can differentiate into all types of cells constituting the pancreas, and thus can be used for the treatment of pancreatic diseases requiring transplantation of organs or tissues.

Claims (19)

Marker genes of pancreatic islet cells selected from the group consisting of CK 7, CK 8, CK 18, CK 19, CFTR, mucin MUC1, carbonic anhydrase II and carbohydrate antibody 19.9 (sial-Lewis-a); Genes involved in pancreatic development selected from the group consisting of Notch-1, Jagged-1, Wnt-4, β-catenin, neurogenin 3, Neuro D, Pax 4, Pax 6, Pdx-1, PTF1 / p48 and Tcf-4 Pancreatic progenitor cells differentiated from pancreatic progenitor cells, characterized in that the expression at the same time. 2. The pancreatic progenitor cell of claim 1, wherein the pancreatic progenitor cells have a form of pancreatic islet cells. delete delete The pancreatic progenitor cell of claim 1 or 2, further expressing SCF / c-kit or vimetin. 2. The pancreatic progenitor cell of claim 1, wherein said pancreatic progenitor cells are derived from a mammal. 7. The pancreatic progenitor cell of claim 6, wherein said mammal is a rat. 2. The pancreatic progenitor cell of claim 1, wherein said pancreatic progenitor cell is Accession No. KCLRF-BP-00093. (1) isolating pancreatic progenitor cells from the moody pancreatic tissue, (2) ex vivo culture of the pancreatic progenitor cells in a mammalian cell culture medium, and (3) CK 7, CK 8, CK during culture. 18, CK 19, CFTR, mucin MUC1, carbonic anhydrase II, and carbohydrate antibody 19.9 (sialyl-Lewis-a), marker genes of pancreatic islet cells selected from the group consisting of Notch-1, Jagged-1 Pancreatic progenitor cells expressing genes involved in pancreatic development selected from the group consisting of Wnt-4, β-catenin, neurogenin 3, Neuro D, Pax 4, Pax 6, Pdx-1, PTF1 / p48 and Tcf-4 Method for producing a transitional pancreatic progenitor cells from the pancreatic progenitor cells comprising the step of separating. 10. The method for producing pancreatic progenitor cells differentiated from pancreatic progenitor cells according to claim 9, wherein the method is cultured in a mammalian culture medium containing dexamethasone until 14 to 16 days after the start of ex vivo culture. The method of claim 10, wherein the dexamethasone is contained in an amount of 5 to 15 µg / ml. The method for producing pancreatic progenitor cells differentiated from pancreatic progenitor cells according to claim 9 or 10, wherein the mammalian cell culture medium is a Waymouth medium. 10. The method for producing pancreatic progenitor cells differentiated from pancreatic progenitor cells according to claim 9, wherein the pancreatic progenitor cells of step (3) have a form as pancreatic islet cells. Pancreatic islets cells characterized in that they are transfected from the pancreatic progenitor cells according to claim 1 or 2 and express the marker gene of pancreatic islet cells selected from the group consisting of insulin, glucagon, somatostatin and pancreatic polypeptide. delete (1) contacting the transitional pancreatic progenitor cells in the pancreatic progenitor cells according to (1) or (2) with a growth factor selected from the group consisting of activin A, Betacellulin and GLP-1, (2) for mammalian culture Culturing in pancreatic progenitor cells comprising culturing in the medium and (3) isolating pancreatic islet cells expressing marker genes of pancreatic islet cells selected from the group consisting of insulin, glucagon, somatostatin and pancreatic polypeptide during culture. Method for producing pancreatic islet cells. 17. The method of claim 16, wherein in step (1), the pancreatic progenitor cells are directly treated with growth factors selected from the group consisting of activin A, Betacellulin, and GLP-1. Method for producing differentiated pancreatic islet cells. 17. The pancreatic progenitor of claim 16, wherein in step (1), a pancreatic progenitor is introduced into the pancreatic progenitor cells differentiated from the pancreatic progenitor cells, comprising a growth factor selected from the group consisting of activin A, Betacellulin, and GLP-1. Method for producing pancreatic islet cells differentiated from cells. delete

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