WO2005095589A1

WO2005095589A1 - Pancreatic precursor cell line transdifferentiated from pancreatic acinar cell

Info

Publication number: WO2005095589A1
Application number: PCT/KR2004/002669
Authority: WO
Inventors: Si-Young Song; Ji-Eun Lee; Han-Soo Kim; Jing Wen
Original assignee: Yonsei University
Priority date: 2004-04-02
Filing date: 2004-10-18
Publication date: 2005-10-13
Also published as: KR100660473B1; KR20050097619A

Abstract

Disclosed are pancreatic precursor cells transdifferentiated from pancreatic acinar cells, which express both a pancreatic ductal cell marker gene and a gene participating in the pancreatic development, and a method of preparing such pancreatic precursor cells, comprising (1) isolating the pancreatic acinar cells from an adult, (2) in vitro culturing of the pancreatic acinar cells in a medium for mammalian cell culture and (3) isolating the pancreatic precursor cells expressing both a pancreatic ductal cell marker gene and a gene participating in the pancreatic development during the in vitro culturing. In addition, the present invention discloses pancreatic islet cells that are transdifferentiated from the pancreatic precursor cells and express a pancreatic islet cell marker gene, and a method of preparing such pancreatic islet cells, comprising (1) contacting the pancreatic precursor cells, prepared as described above, with a growth factor, (2) culturing the pancreatic precursor cells in a medium for mammalian cell culture and (3) isolating the pancreatic islet cells expressing a pancreatic islet cell marker gene during the culturing.

Description

PANCREATIC PRECURSOR CELL LINE TRANSDIFFERENTIATED FROM PANCREATIC ACINAR CELL

Technical Field

The present invention relates to a pancreatic precursor cell line transdifferentiated from pancreatic acinar cells. More particularly, the present invention relates to a pancreatic precursor cell line transdifferentiated from pancreatic acinar cells, which expresses both a pancreatic ductal cell marker gene and a gene participating in the pancreatic development, and a preparation method thereof. Also, the present invention is concerned with pancreatic islet cells transdifferentiated from the pancreatic precursor cell line and expressing a pancreatic islet cell marker gene, and a preparation method thereof.

Background Art

The mammalian pancreas consists of (1) the ductal tree, (2) the exocrine acini that produce digestive enzymes and (3) the endocrine tissue that produces hormones. Pancreatic ductal epithelial cells, acinar cells and islet cells all are derived from the primitive ductal structure formed during the protodifferentiated state of the early pancreatic development (J. Slack, Development, 1995, vol.121, pp.1569-1580). The more advanced stage of pancreatic morphogenesis is achieved by the expression of insulin promoter factor (IPF) as well as the interaction among ductal cells, islet cells and acinar cells. After this embryonic development, ductal cells become remarkably reduced in their capacity to differentiate into acinar cells (M. Eisner et al. Diabetologia, 2000, vol.43, pp.1528-1533). However, ductal cells of the adult pancreas can differentiate into islet cells under certain conditions suitable for intercellular interaction (S. Bonner- Weir et al. Diabetes, 1993, vol.42, pp.1715-1720; L. Rosenberg, Cell Transplant, 1995, vol.4, pp.371-383). The differentiation of pancreatic ductal cells into endocrine cells producing hormones is similar to the ontogeny of the pancreatic development. Various pancreatic islet cells have been prepared using the differentiation capacity of pancreatic ductal cells. For example, U.S. Patent No. 5,888,705 discloses a method of inducing the differentiation of isolated human mature pancreatic cells into islet cells using hepatocyte growth factor/scatter factor (HGF/SF). PCT Publication No. WO97/15310 discloses a method of inducing mature pancreatic ductal cells to differentiate into islet cells including culturing the mature pancreatic ductal cells in a serum-containing, low-glucose medium and then culturing the cultured cells in a medium with higher glucose content. U.S. Patent No. 5,834,308 discloses a method of producing islet cells, including isolating precursor cells from prediabetic adults and culturing the precursor cells in a medium that stimulates the growth of functional islet cells. However, all of the above "precursor cells" are differentiated only into islet cells. Pancreatic precursor cells of the aforementioned studies do not have the capacity to differentiate into both endocrine and exocrine cell types. The ideal population of pancreatic precursor cells should be able to differentiate into endocrine cells (i.e., islet-α, islet-β, islet-δ and islet-PP cells) and exocrine cells (i.e., acinar cells), as well as ductal cells. This is because these precursor cells are clinically useful. To solve these problems, PCT Publication No. WO 01/77300 discloses a population of pancreatic epithelial precursor cells that are capable of differentiating into functional endocrine and exocrine cells, methods of isolating the pancreatic epithelial precursor cells, characterization of pancreatic epithelial precursor cells, and application of the pancreatic epithelial precursor cells. However, since the pancreatic epithelial precursor cells are isolated from the human fetal pancreatic tissue, this patent is problematic in that the raw material is difficult to obtain. In addition, with high pluripotency, these embryonic cells are highly apt to differentiate into unwanted cells so that they may be transformed to cancer cells. Recently, acinar cells have been reported to participate in the histogenesis of ductal adenocarcinoma. When pancreatic acinar cells are chemically induced to cause carcinogenesis, they dedifferentiate and thus produce ductal-like cells (A. Krapp et al. Genes Dev. 1998, vol.12, pp.3752-3763).

Disclosure of the Invention

Leading to the present invention, the intensive and thorough research based on that pluripotential precursor cells capable of differentiating into all kinds of pancreatic cells may be present during the transdifferentiaiton of pancreatic acinar cells into ductal cells, was conducted by the present inventors, resulted in the finding of pancreatic precursor cells that have characteristics similar to ductal cells and express genes participating in the pancreatic development during the transdifferentiation of acinar cells. Therefore, in one aspect, the present invention provides pancreatic precursor cells transdifferentiated from pancreatic acinar cells, which express both a pancreatic ductal cell marker gene and a gene participating in the pancreatic development. In another aspect, the present invention provides a method of preparing pancreatic precursor cells transdifferentiated from pancreatic acinar cells, comprising (1) isolating the pancreatic acinar cells from an adult, (2) in vitro culturing of the pancreatic acinar cells in a medium for mammalian cell culture and (3) isolating the pancreatic precursor cells expressing both a pancreatic ductal cell marker gene and a gene participating in the pancreatic development during the in vitro culturing. In a further aspect, the present invention provides pancreatic islet cells that are transdifferentiated from the pancreatic precursor cells and express a pancreatic islet cell marker gene. In yet another aspect, the present invention provides a method of preparing pancreatic islet cells transdifferentiated from the pancreatic precursor cells, comprising (1) contacting the pancreatic precursor cells, prepared as described above, with a growth factor, (2) culturing the pancreatic precursor cells in a medium for mammalian cell culture and (3) isolating the pancreatic islet cells expressing a pancreatic islet cell marker gene during the culturing.

Brief Description of the Drawings The above objects of the present invention will be more clearly understood from the following detailed description taken in conjunction with the accompanying drawings, in which: Fig. 1 photographically shows the morphological changes of pancreatic acinar cells according to culture time {A-F: χ200; G-H: lOO; after culturing, day 0 (A), day 1 (B), day 3 (C), day 5 (D), day 10 (E), day 15 (F); YGIC4 (G), YGIC5 (H)}; Fig. 2 photographically shows the morphological changes of pancreatic acinar cells according to culture time {x 10400; after culturing, day 0 (A), day 1 (B-C), day 3 (D), day 10 (E-F), day 15 (G); YGIC4 (H), YGIC5 (I)} Fig. 3 is a graph showing amylase activity of pancreatic acinar cells according to culture time; Fig. 4 photographically shows the morphological changes of pancreatic acinar cells upon three-dimensional culture according to culture time {after culturing in collagen, day 0 (A), day 3 (B), day 5 (C); after culturing in matrigel, day 0 (D), day 3 (E), day 5 (F)}; Fig. 5 shows the results of BrdU incorporation assay for measuring the proliferative ability of pancreatic precursor cells obtained in the present invention; Fig. 6 shows the results of Western blotting for expression markers of YGIC4 and YGIC5 cell lines according to the present invention; and Fig. 7 shows the results of Western blotting for expression markers of pancreatic islet cells transdifferentiated from YGIC4 and YGIC5 cell lines according to the present invention.

Best Mode for Carrying Out the Invention The term "pancreatic precursor cells", as used herein, refer to pluripotential cells obtained by the transdifferentiation of pancreatic acinar cells, which lose characteristics of pancreatic acinar cells and come to have characteristics of pancreatic ductal cells and characteristics of pancreatic precursor cells capable of differentiating into all types of pancreatic cells (that is, acinar cells, ductal cells and islet cells). To more clearly describe the characteristics of the pancreatic precursor cells according to the present invention, it is necessary to plainly define the terms used in the present specification and the characteristics of differentiated cells constituting the pancreas. Of course, the characteristics of differentiated pancreatic cells are known in the art, but are defined to plainly describe the scope of the present invention, as follows. The term "characteristics", as used herein, is intended to include innate morphologies and phenotypes specific for certain cells. The morphology means an innate shape and color of a corresponding cell, and a phenotype means a marker gene expressed specifically in the corresponding cell. "Pancreatic acinar cells" are cells that secrete various digestive enzymes participating in the degradation of proteins, lipids and polysaccharides. They contain 1 to 3 prominent nucleoli and well-developed endoplasmic reticulum and Golgi apparatuses as expression organelles, and are abundant in zymogen granules containing various digestive enzymes. Pancreatic acinar cell marker genes include, but are not limited to, amylase and p48. "Pancreatic islet cells" may be classified into four cell types according to their functions: (1) α cells that produce glucagon; (2) β cells that produce insulin; (3) δ cells that produce somatostatin; and (4) pp cells that produce pancreatic polypeptides. That is, pancreatic islet cells, ovoid in shape and about 75 μm to 175 μm in size (long axis), produce the pancreas-derived hormones and secrete the hormones into the blood stream. Pancreatic islet cell marker genes include the aforementioned various hormones. "Pancreatic ductal cells" are cuboid epithelial cells which are about 10 μm in diameter, tightly dense in appearance and relatively small and rounded compared to acinar cells. Pancreatic ductal cell marker genes include cytokeratins, such as CK 7, CK 8, CK 18 and CK 19, CFTR, mucin MUC1, carbonic anhydrase II and carbohydrate antibody 19.9 (syalyl-Lewis-a). On the other hand, the term "transdifferentiation", as used herein, typically means that cells in a differentiated state are converted to ones in another differentiated state. Since transdifferentiation employs completely differentiated cells as a starting material, it differs from stem cell-derived neogenesis. During transdifferentiation, numerous genes switch on and off, and thus a dramatic change may be obtained in morphologies and phenotypes of cells. In the present invention, the

"transdifferentiation" has two meanings: one involves a change of pancreatic acinar cells to pancreatic precursor cells; and the other involves a change of the pancreatic precursor cells obtained as above to pancreatic islet cells. Thus, the "pancreatic precursor cells" according to the present invention are characterized in that expression of an acinar cell marker gene (e.g., amylase, p48) decreases, the endoplasmic reticulum and Golgi apparatus in the cytoplasm degenerate and zymogen granules are reduced, whereas a pancreatic ductal cell marker gene (e.g., CK 19, CFTR) is expressed, and the pancreatic precursor cells have the morphology of pancreatic ductal cells. In addition, the pancreatic precursor cells express a gene participating in the pancreatic development, and thus possess a multipotency to differentiate into all types of cells constituting the pancreas. The "gene participating in the pancreatic development", expressed in the pancreatic precursor cell according to the present invention, refers to a gene that is originally expressed in pancreatic precursor cells and induces their differentiation into specific pancreatic cells. Non-limiting examples of the gene include Notch- 1, Jagged- 1, Wnt-4, β-catenin, neurogenin 3, Neuro D, Pax 4, Pax 6, Pdx-1 , PTFl/p48 and Tcf-4. The pancreatic precursor cell of the present invention may express a marker gene specifically expressed only in stem cells with limited Differentiation, such as hematopoietic stem cells, nerve stem cells and hepatic stem cells. Examples of such a marker gene include SCF/c-kit and vimentin. Pancreatic acinar cells, as a starting material for obtaining the pancreatic precursor cells of the present invention, are preferably isolated from the adult mammalian pancreas. In particular, since the pancreatic precursor cells possess the multipotency to differentiate into several types of cells constituting the pancreas, it may be used for the treatment of various pancreatic diseases, which requires the transplantation of the cells, tissue or organ of the pancreas. Thus, when the pancreatic precursor cells of the present invention are used for such transplantation, the pancreatic precursor cells are more preferably prepared using pancreatic acinar cells isolated from the individual into who it will be transplanted, in order to prevent the graft from being rejected. In an embodiment of the present invention, pancreatic acinar cells were isolated from rats and transdifferentiated to produce two pancreatic precursor cell lines, which were designated as "YGIC4" and "YGIC5", respectively. The "YGIC5" was deposited at an international depositary authority, Korean Cell Line Research Foundation (KCLRF; Cancer Research Institute, Seoul National University College of Medicine, Yungon- dong, Chongno-gu, Seoul, Republic of Korea) on January 29, 2004, and was assigned accession number KCLRF-BP-00093. The transdifferentiation for preparing the pancreatic precursor cells according to the present invention may be achieved by a method known in the art. In the present invention, transdifferentiation of pancreatic acinar cells into precursor cells may be induced by in vitro culturing of pancreatic tissue. Compared to other tissues, for acinar cells derived from the pancreas, intercellular interaction is more important for the maintenance and growth of the cells. Thus, only in vitro culturing may satisfy requirements for transdifferentiation of the acinar cells. In this regard, the pancreatic precursor cells according to the present invention may be prepared by a method comprising (1) isolating the pancreatic acinar cells from an adult, (2) in vitro culturing of the pancreatic acinar cells in a medium for mammalian cell culture and (3) isolating the pancreatic precursor cells expressing both a pancreatic ductal cell marker gene and a gene participating in the pancreatic development during the in vitro culture. (1) Isolation of pancreatic acinar cells First, pancreas tissue is isolated from the adult pancreas and microdissected. The purpose of microdissection is to separate structures containing acinar cells from non- pancreatic tissues such as fat, membranes or connective tissue. In one aspect, the pancreatic tissue may be microdissected by a physical means using, for example, a homogenizer, mortar and pestle, a blender, scalpels, syringes, forceps or an ultrasonic device. In another aspect, the pancreatic tissue may be microdissected by enzymatic digestion. Non-limiting examples of available enzymes include neutral proteases, serine proteases, including trypsin, chymotrypsin, elastase and collagenase.

Alternatively, the physical means may be used in combination with enzyme treatment. To be maintained stably, the pancreas tissue microdissected as described above is preferably preserved in a buffer solution, for example, containing bovine serum albumin (BSA), pyruvate or a trypsin inhibitor. By the microdissection, some cells of the pancreas tissue are isolated in a single cell form, and others are isolated in cell aggregates. From the pancreas tissue microdissected into a desired size, acinar cells may be isolated using a density gradient. Substances that can be used to create a density gradient include, but are not limited to, serum (BSA), ovalbumin, nonionic synthetic polymers of sucrose (Ficoll™), colloidal polyvinylpyrrolidone-coated silica (Percoll™), polyvinylpyrrolidone or PVP, and methylcellulose. In a preferred embodiment, the microdissected pancreas tissue cells are is centrifuged, and the resulting cell suspension passes through a filter to give desired pancreatic acinar cells. (2) In vitro culture of adult pancreatic acinar cells The isolated pancreatic acinar cells may be cultured in vitro in a typical medium for mammalian cell culture. After three to five days, transdifferentiated cells emerge, and at this time, pancreatic precursor cells may be isolated by a single cell isolation method. Media useful for the in vitro culture according to the present invention are commercially available, or may be prepared, for example, according to components and their ratio described in the catalogue from the American Type Culture Collection (ATCC). Examples of available media include Ham medium, IMDM Iscove's medium, Leibovitz L15 medium, Mac Coy 5A medium, M199 medium, Melnick's medium, MEM Eagle's Minimum Essential medium, NCTN medium, Puck's medium, RPMI medium, Swim S77 medium, Trowell T8 medium, Waymouth medium and Williams E medium. In particular, Waymouth medium is preferred in the present invention. In addition, the

Waymouth medium is preferably supplemented with an about 0.1-1% antibiotic and about 1-10% FBS. In addition, preferably, a substance capable of suppressing acute differentiation of cells is introduced at the early stage of the in vitro culture. The differentiation suppressor useful in the present invention includes steroid substances known in the art, whereas dexamethasone and its derivatives are preferred. Dexamethasone is used in a suitable amount of 5 to 15 μg/ml. Also, after the pancreatic acinar cells clearly display the characteristics of pancreatic ductal cells during in vitro culture (12 to 16 days after the culture starts), the cells are preferably cultured in a medium not containing the differentiation suppressor. In addition, for the in vitro culture according to the present method, it is important to determine a time point suitable for subculturing. Preferably, subculturing is carried out at a point in time when expression of an acinar cell marker gene and a differentiation-associated factor, pdx-1 or c-kit, decreases. This phenomenon occurs after about 8 to 12 days of primary culture. In the present invention, the pancreatic acinar cells may be induced to transdifferentiate into pancreatic precursor cells by secondary culture (for example, in plates, flasks, roller bottles and petri dishes) as well as tertiary culture. The tertiary culture, is not limited to, but may use matrigel or collagen. Matrigel, a mixture of extracellular basement membrane components, is preferably used for cells that are unable to survive as single cells or for identifying the three-dimensional structure of cells in vitro (example, formation of cystic structure). Collagen serves as an intercellular connecting agent, and is suitable for identifying intercellular interaction. (3) Evaluation of the differentiated degree of cells The differentiated degree of cells may be determined by morphology or expression of marker genes or a combination thereof. The morphology and marker genes of various cells constituting the pancreas are the same as described above. Changes in morphology during transdifferentiation may be easily observed using an inverted light microscope or electron microscope. Change in phenotype, is not limited to, but may be detected by RT-PCR_. Western blotting, immunohistochemistry and ELISA. As noted above, the pancreatic precursor cell line obtained as described above is capable of differentiating into all types of cells constituting the pancreas. Thus, the present invention provides a pancreatic islet cell that is transdifferentiated from the pancreatic precursor cell according to the present invention and expresses a pancreatic islet cell marker gene. Examples of the pancreatic islet cell marker gene are the same as described above. The pancreatic islet cells transdifferentiated from the pancreatic precursor cells may be prepared by a method comprising (1) contacting the pancreatic precursor cells, prepared as described above, with a growth factor, (2) culturing the pancreatic precursor cells in a medium for mammalian cell culture and (3) isolating the pancreatic islet cells expressing a pancreatic islet cell marker gene during the culturing. The growth factor that can be used in step (1) of the above method includes, but is not limited to, activin A, Betacellulin and GLP-1. At step (1) of the above method according to the present invention, the pancreatic precursor cells transdifferentiated from pancreatic acinar cells are directly treated with a growth factor, or are transfected with a vector expressing a growth factor. The vector may be introduced into the pancreatic precursor cells by various methods including calcium phosphate transfection, DEAE-dextran mediated transfection, microinjection, cationic lipid-mediated transfection, electroporation, transduction, scrape loading, ballistic introduction and infection. The medium for mammalian cell culture, used in steps (2) and (3) of the above method, and the detection of the differentiation of the pancreatic precursor cells into pancreatic islet cells and the degree of the differentiation may be obtained and achieved according to the aforementioned method of preparing pancreatic precursor cells transdifferentiated from pancreatic acinar cells. A better understanding of the present invention may be obtained through the following examples which are set forth to illustrate, but are not to be construed as the limit of the present invention.

EXAMPLE 1 : Primary culture

The pancreas was isolated from male Sprague-Dawley rats with an initial weight of about 100-150 g (Jackson Laboratory, Bar Harbor, Maine, USA), homogenized in a spinner flask, and washed twice with a pancreas solution A (4 g glucose, 16.36 g NaCl, 1 M KC1, 1 M MgCl , 1 M CaCl ) containing 500 mg bovine serum albumin, 500 mg pyruvate and 600 mg trypsin inhibitor. Subsequently, the tissue was treated with collagenase type IN (Sigma, St. Louis, MO, USA) at a 37°C water bath for 6-8 min, and centrifuged at 1000 rpm for 10 sec. The resulting cell suspension passed through a 200- μm nylon mesh filter. The isolated acinar cells were washed and then cultured in Waymouth medium supplemented with 10% FBS (HyClone, Logan, Utah, USA) and 10 μg/ml dexamethasone (Sigma) at 37°C in a 5% CO2/95% air humidified incubator.

During the culturing, the cells were observed under an inverted light microscopy to monitor their morphological changes. Identical cells were captured as images with the passage of time (Fig. 1), and collected on days 0, 1, 3, 5, 10, 15 and 20 after starting the primary culture. During the culturing, morphologically identical cells were selected from single cells and cloned using cloning cylinders. As shown in Fig. 1 , on day 2 and day 3 of the primary culture, aggregates of the pancreatic acinar cells, attached to the bottom of plates, were detected, and from day 4 to and day 5, they were expanded. For the above five days of the primary culture, the pancreatic cells displayed the morphology of acinar cells, but, from day 6 to day 8, the cells morphology were gradually changed to that of cuboid like ductal cells.

EXAMPLE 2: Structural analysis of cultured cells using electron microscope

The cultured cells prepared in Example 1 were analyzed for their structure. In detail, the cells were washed with PBS (pH 7.4) twice and fixed in PBS containing 1% glutaraldehyde. Then, the cells were postfixed in osmium tetroxide (OsO₄), and samples were observed under a transmission electron microscope. As a result, on day 0, the cells isolated from the pancreas were at a state of cytodifferentiation of acinar cells. As a secretory organelle prominent in acinar cells, granules filled with high-density zymogens were observed, which were located in the apical region of cells and connected to the juxtanuclear Golgi and rough endoplasmic reticulum, positioned at the basal region of cells (Fig. 2). On day 2, acinar cells displayed major morphological changes, and in particular, high-density granules were observed at the edge of cells. On day 2 to day 4, the morphology of exocrine acinar cells observed on day 0 was not observed any more. These transitional cells with a decrease in the secretory organelle and zymogen granules indicate that the cells are derived from the acinar cell lineage. This transdifferentiation of acinar cells occurred simultaneously with the increase of non-differentiated cells in a culture, and this change was continued until day 3 to day 5. In addition, for this period, acinar cells continuously underwent degranulation (the D panel of Fig. 2). Further, after reconstruction of intercellular skeleton, primitive microvilli were developed around cell aggregates (the E panel of Fig. 2). The overall morphological appearance of the transdifferentiated ductal cells was fairly similar to the profile of non-differentiated ductal cells.

EXAMPLE 3: Amylase activity assay

Amylase activity was determined by measuring the amount of degraded starch when a culture supernatant was reacted in a substrate buffer solution containing starch for a predetermined period of time, wherein the amount of degraded starch was calculated from the difference between initial starch and residual starch (Fig. 3). 1 unit of Caraway (Asan Pharm. Co., Hwa-Sung, Korea), used in this assay, refers to the amount of an enzyme capable of digesting 100 mg starch at 37°C for 30 min. As shown in Fig. 3, when acinar cells underwent transdifferentiation during in vitro culturing, amylase expression levels decreased, and amylase activity was thus reduced.

EXAMPLE 4: Three-dimensional cell culture in matrigel or collagen

Matrigel (Becton Dickinson, Franklin lakes, NJ, USA) was thawed according to the manufacturer's protocol and sprayed into each well of culture plates placed on ice. The plates were warmed to room temperature for several minutes, and a suitable density of cells was mixed with matrigel and cultured. The cells were cultured in a new medium, Waymouth medium supplemented with 10% FBS, and this medium was replaced with a fresh one twice per week. Separately, another cell substrate, collagen I gel, was prepared and suitably mixed with cells, and the cells were cultured. The cells were observed under an inverted light microscope to monitor moφhological changes of the cells. Day 1, day 5 and day 10 after the overlay of matrigel or collagen gel, images for identical cells were captured. In the three-dimensional cell culture in matrigel or collagen, acinar cells were also found to transdifferentiate into ductal cells (Fig. 4). In the collagen substrate, the pancreatic acinar cells started to form the ductal structure on day 5 after the primary culture (the A and B panels of Fig. 4), and on day 10, most cells formed a cystic structure

(the C panel of Fig. 4). In the primary culture of acinar cells in matrigel, cell aggregates started to form and become flat but roughly spherical on day 3. On day 3 to day 5, acinar cells completed morphological changes to ductal cells (the D and E panels of Fig. 4). On day 10, a cystic structure was continuously expanded (the F panel of Fig. 4).

EXAMPLE 5: Analysis of BrdU incorporation

To investigate proliferative ability of cells, cells were cultured in 96-well plates at 37°C for 0, 1, 3, 5, 7, 10, 12 and 15 days. Subsequently, using a BrdU labeling kit

(Roche Inc., Mannheim, Germany), BrdU was added to the cells, and the cells were further re-cultured for 24 hrs. After a culture medium was removed, the cells were fixed and treated with FixDenat to denature DNA. The produced immune complexes were detected by an additional substrate reaction. The reaction product was quantitatively analyzed by measuring absorbance at 370 nm using a scanning multi-well spectrophotometer (ELISA reader, Molecular Devices, Sunnyvale, CA, USA) (Fig. 5). As shown in Fig. 5, on day 5 to day 10 for which the utmost expression of ductal cells occurred, no cell proliferation was observed. These results indicate that the transdifferentiation of pancreatic acinar cells into pancreatic precursor cells with the characteristics of ductal cells is not induced by proliferation of existing ductal cells.

EXAMPLE 6: Evaluation of proliferation and tumor-forming ability and Western blotting for pancreatic cell markers of YGIC4 and YGIC5 cell lines

Cells during the above culturing were cloned into two morphologically identical cell lines using cloning cylinders, which were designated as "YGIC4" and "YGIC5", respectively. The "YGIC5" was deposited at an international depositary authority, Korean Cell Line Research Foundation (KCLRF; Cancer Research Institute, Seoul National University College of Medicine, Yungon-dong, Chongno-gu, Seoul, Republic of Korea) on January 29, 2004, and was assigned accession number KCLRF-BP-00093. A. Evaluation of cell proliferation YGIC4 and YGIC5 cells were individually plated at a density of 5x10⁴ cells/ml and reached confluence after 4.5 days. Thus, YGIC4 and YGIC5 cells were found to have doubling times of 35 hrs and 32 hrs, respectively. On the other hand, YGIC4 and YGIC5 cells were grown without a feeder after twelve passages. When YGIC cells were grown in monolayers, they exhibited the typical irregular and cobblestone-like morphology of fusiform epithelial cells. In addition, these cells did not contain any zymogen granules, and primitive microvilli emerged around cell aggregates. YGIC cells were stored in liquid nitrogen upon rounds of subculturing, and were regenerated by thawing for use in experiments. These cells were found to maintain the initial morphology even after fifty passages. B. Evaluation of tumor-forming ability of YGIC4 and YGIC5 cells To determine the ability of the YGIC4 and YGIC5 cell lines to form tumors, 4χl0⁶ cells in 200 μl PBS were subcutaneously injected into six-week-old ICR/nude mice. YGIC cells were found not to proliferate when subcutaneously injected into ICR/nude mice. This result indicates that the cell lines are normal cells not forming tumors. In addition, the cell lines were found not to possess a K-ras codon 12 mutation (found in 90% of pancreatic cancer). The YGIC cells obtained according to the present invention were found to have immortal morphological features and growth properties and not to form tumors. C. Western blotting Western blotting was carried out to determine cell types with the following primary antibodies: mouse monoclonal anti-cytokeratine 19 (1 :1000, DAKO), rabbit polyclonal anti-amylase (1 :2000, Sigma), rabbit polyclonal anti-insulin, rabbit polyclonal anti-48, rabbit polyclonal anti-Pdxl (1 :1000), mouse monoclonal anti-CFTR, mouse monoclonal anti-vimentin, rabbit polyclonal anti-Notch- 1, rabbit polyclonal anti-Tcf-4, rabbit polyclonal anti-Wnt, rabbit polyclonal anti-β-catenin (1 :1000, Santa Cruz Biotechnology Inc. USA), rabbit polyclonal anti-Jagg- 1 and rabbit polyclonal anti-SCF. The YGIC cells were found to express a pancreatic ductal cell marker CFTR and cytokeratine 19, while not expressing exocrine cell markers, amylase and p48, and an endocrine beta cell marker, insulin. Also, the YGIC cells were found to express genes participating in the pancreatic development, Notch- 1, Jagged- 1, Wnt-4, β-catenin, Pdx-1 and Tcf-4. Further, the YGIC cells expressed SCF/c-kit and vimentin.

EXAMPLE 7: Transdifferentiation of YGIC4 YGIC5 cells into pancreatic islet cells The YGIC cell lines prepared in Example 6 were cultured in Waymouth medium supplemented with an antibiotic (penicillin, streptomycin or amphotericin B) and 10% FBS in a 5% CO2/95% air humidified incubator. The cultured YGIC cell lines were transfected with pBX322/GLP-l (10 nmol/L) using Lipofectamine. The transfected YGIC cell lines were evaluated with regards to the expression of stem cell factor (SCF), c-kit, PDX-1, Pax-4, Pax-6, Ngn-3, NeuroD, insulin, glucagons and CFTR by Western blotting, RT-PCR and ELISA (Fig. 7). As shown in Fig. 7, by treatment of the GLP-1, the cells displayed no change in expression levels of factors participating in the embryonic pancreatic development, Hes-1, Notch- 1, neuroD and sonic hedgehog (SHH), but exhibited reduced expression levels of neurogenin-3 (ngn-3). Also, the GLP-1 resulted in increased expression of PDX-1, a regulator of the pancreatic β-islet cell development, whereas resulting in decreased expression of Pax-6. Further, the GLP-1 resulted in increased expression of stem cell markers SCF and c-kit, whereas resulting in decreased expression of a pancreatic ductal cell marker CFTR. GLP-1 promoted the expression of insulin and glucagon, but did not affect the expression of Glut-2 and glucokinase.

Industrial Applicability As described hereinbefore, the pancreatic precursor cells derived from pancreatic acinar cells according to the present invention are capable of differentiating into all types of cells constituting the pancreas, and are thus useful for treating pancreatic diseases requiring organ or tissue transplantation. INDICATIONS RELATING TO DEPOSITED MICROORGANISM OR OTHER BIOLOGICAL MATERIAL

(PCT Rule 13bιs)

A. The indications made below relate to the deposited microorganism or other biological material referred to in the description on page 8, line 4-14

B. IDENTIFICATION OF DEPOSIT Further deposits are on an additional sheetD Name of depositary institution Korea Cell Line Research Foundation KCLRF) Address of depositary i titυtion(including postal code and country) Cancer Research Institute, Seoul National University, College of Medicine 28 Vongon-dong, Chongno-gu, SEOUL 110-744, Republic of Korea Date of deposit Accession Number 29/01/2004 KCLRF-BP-00093 C.ADDITIONAL INDICATIONSfZeαve blank if not applicable) This information is continued on an additional sheet D

D.DESIGNATED STATES FOR WHICH INDICATIONS ARE MAOEfif the indications are not for all designated States)

E.SEPARATE FURNISHING OF INDICATIONS(7eave blank if not applicable) The indications listed below will be submitted to the International Bureau later(-_->ec /_μ the general nature of the indications e.g., "Accession Number of Deposit")

For receiving Office use only For international Bureau use only D This sheet was received with the international D This sheet was received by the International Bureau application on:

Authorized officer Authorized officer

Form PCT RO/134(July 1998)

Claims

1. A pancreatic precursor cell transdifferentiated from a pancreatic acinar cell, which expresses both a pancreatic ductal cell marker gene and a gene participating in pancreatic development.

2. The pancreatic precursor cell transdifferentiated from the pancreatic acinar cell according to claim 1 , wherein the pancreatic precursor cell has the morphology of a pancreatic ductal cell.

3. The pancreatic precursor cell transdifferentiated from the pancreatic acinar cell according to claim 1 , wherein the pancreatic ductal cell marker gene is CK 7, CK 8, CK 18, CK 19, CFTR, mucin MUCl, carbonic anhydrase II, or carbohydrate antibody 19.9 (cyalyl-Lewis-a).

4. The pancreatic precursor cell transdifferentiated from the pancreatic acinar cell according to claim 1 , wherein the gene participating in pancreatic development is Notch-1, Jagged-1, Wnt-4, β-catenin, neurogenin 3, Neuro D, Pax 4, Pax 6, Pdx-1, PTFl/p48, or Tcf-4.

5. The pancreatic precursor cell transdifferentiated from the pancreatic acinar cell according to any one of claims 1 to 4, further expressing SCF/c-kit or vimentin.

6. The pancreatic precursor cell transdifferentiated from the pancreatic acinar cell according to claim 1 , wherein the pancreatic acinar cell is derived from a mammal.

7. The pancreatic precursor cell transdifferentiated from the pancreatic acinar cell according to claim 6, wherein the mammal is a rat.

8. The pancreatic precursor cell transdifferentiated from the pancreatic acinar cell according to claim 1, wherein the pancreatic precursor cell has accession number KCLRF-BP-00093.

9. A method of preparing a pancreatic precursor cell transdifferentiated from a pancreatic acinar cell, comprising: (1) isolating the pancreatic acinar cell from an adult; (2) in vitro culturing the pancreatic acinar cell in a medium for mammalian cell culture; and (3) isolating the pancreatic precursor cell expressing both a pancreatic ductal cell marker gene and a gene participating in the pancreatic development during the in vitro culturing.

10. The method of preparing the pancreatic precursor cell transdifferentiated from the pancreatic acinar cell according to claim 9, wherein the cell is cultured in a dexamethasone-containing medium for mammalian cell culture until day 12 to day 16 of the in vitro culturing.

11. The method of preparing the pancreatic precursor cell transdifferentiated from the pancreatic acinar cell according to claim 10, wherein the dexamethasone is contained in the medium in an amount of 5 to 15 μg/ml.

12. The method of preparing the pancreatic precursor cell transdifferentiated from the pancreatic acinar cell according to claim 9 or 10, wherein the medium for mammalian cell culture is a Waymouth medium.

13. The method of preparing the pancreatic precursor cell transdifferentiated from the pancreatic acinar cell according to claim 9, wherein the pancreatic precursor cell of step (3) has the morphology of a ductal cell.

14. A pancreatic islet cell which is transdifferentiated from the pancreatic precursor cell according to any one of claims 1 to 8 and expresses a pancreatic islet cell marker gene.

15. The pancreatic islet cell according to claim 14, wherein the pancreatic islet cell marker gene is insulin, glucagon, somatostatin, or a pancreatic polypeptide.

16. A method of preparing a pancreatic islet cell transdifferentiated from a pancreatic precursor cell, comprising: (1) contacting the pancreatic precursor cell according to any one of claims 1 to 8 with a growth factor; (2) culturing the pancreatic precursor cell in a medium for mammalian cell culture; and (3) isolating the pancreatic islet cell expressing a pancreatic islet cell marker gene during the culturing.

17. The method of preparing the pancreatic islet cell transdifferentiated from the pancreatic precursor cell according to claim 16, wherein the pancreatic precursor cell transdifferentiated from the pancreatic acinar cell is, at step (1), directly treated with a growth factor.

18. The method of preparing the pancreatic islet cell transdifferentiated from the pancreatic precursor cell according to claim 16, wherein the pancreatic precursor cell transdifferentiated from the pancreatic acinar cell is, at step (1), transfected with a vector expressing a growth factor.

19. The method of preparing the pancreatic islet cell transdifferentiated from the pancreatic precursor cell according to any one of claims 16 to 18, wherein the growth factor is activin A, Betacellulin or GLP-1.