WO2019093047A1 - Method for producing functional exocrine gland in vitro, and exocrine gland produced thereby - Google Patents

Abstract

[Problem] To provide a method for inducing a functional exocrine gland from pluripotent stem cells. [Solution] A method for producing a functional exocrine gland in vitro, said method comprising: (i) a step for culturing an oral ectoderm-containing cellular aggregate under such conditions that the expression of Sox9 and Foxc1 is induced; and (ii) a step for further culturing all or part of the cellular aggregate, that has been cultured in step (i), in a medium containing FGF7 and FGF10.

Description

Method of producing functional exocrine glands in vitro and exocrine glands produced by said method

The present invention relates to a method of inducing functional exocrine glands from pluripotent stem cells, and exocrine glands induced by the method.

The ultimate goal of regenerative medicine is replacement regenerative medicine, which replaces the diseased or damaged organ or tissue that has become dysfunctional with age, with a fully functional regenerated organ or tissue. However, to date, regeneration of organs or tissues having the same function and structure as organs or tissues in a living body has not been achieved.

Recent development of stem cell technology has established a technique for inducing cells that constitute various tissues of a living body from stem cells. However, since organs of a living body take on a three-dimensional arrangement of multiple types of functional cells and their development progresses through the interaction of each cell, it is necessary to compare specific organs in comparison with the case of inducing each cell. It is very difficult to artificially guide. With current regenerative medicine technology, construction techniques for rapidly transplantable regenerative organs have not yet been developed.

The present inventors artificially reconstruct an organ primordia such as a hair follicle primordial or the like using epithelial cells and mesenchymal cells derived from organ primordia in a living body ("Organ primordia Law) was established (Non-Patent Document 1). Furthermore, the present inventors use the above-described organ primordia method to prepare a primordia of a secretory gland such as a salivary gland and a lacrimal gland, insert a guide into the primordia, and culture the living body. Also shown is a method of manufacturing an organ primordia having a conduit connectable with the conduit (US Pat. However, the methods described in these documents use epithelial cells and / or mesenchymal cells derived from a tissue of a living body at least in part as a source of regenerated organ primordia. Therefore, as a more versatile method for producing organs, development of a method for producing organs in vitro, which is completely derived from pluripotent stem cells, is required.

Until now, by using the serum-free aggregation suspension culture method (SFEBq method), suspension culture of aggregates of pluripotent stem cells in a serum-free medium, eye cup, anterior segment tissue, pituitary body, etc. in vitro It has been reported that it can be induced (Non-patent document 2 for eye cup, Non-patent document 3 for anterior segment tissue, and non-patent document 4 for pituitary).

WO 2012/108069

Nakao, K. , Et al. Nat Methods 4, 227-30 (2007) M. Eiraku et al. , Nature 472, 51 (Apr 07, 2011) T. Nakano et al. , Cell stem cell 10, 771 (Jun 14, 2012) H. Suga et al. , Nature 480, 57 (Nov 09, 2011)

As described above, research has been actively conducted on methods for reconstituting tissues from pluripotent stem cells. However, the molecular mechanism of the developmental process of exocrine gland tissues such as salivary glands is extremely complicated, and there are still many unknown points, and the method for inducing differentiation of functional exocrine glands in vitro derived from pluripotent stem cells has been described so far. Not reported

The present inventors diligently studied about a method of inducing differentiation of functional exocrine gland derived from pluripotent stem cells. As a result, it has been surprisingly found that, by adding predetermined steps to oral ectoderm derived from pluripotent stem cells, functional exocrine glands (eg, salivary glands) can be produced in vitro. , Came to complete the present invention.

Thus, in one embodiment, the present invention is a method of producing a functional exocrine gland in vitro, comprising
(I): culturing cell aggregates containing oral ectoderm under conditions that induce the expression of Sox9 and Foxc1;
(Ii): further culturing all or part of the cell aggregate cultured in the above step (i) in a medium containing FGF7 and FGF10,
On the way, including.

In one embodiment of the present invention, the oral ectoderm is characterized by being derived from ES cells or iPS cells.

In one embodiment of the present invention, the exocrine gland is a salivary gland.

In one embodiment of the present invention, the “condition for inducing the expression of Sox9 and Foxc1” in the step (i) is
(A) A condition that induces the expression of Sox9 and Foxc1 by introducing the Sox9 expression gene and the Foxc1 expression gene into the cell aggregate containing the above-mentioned oral ectoderm, or
(B) conditions for inducing the expression of the introduced Sox9 gene and Foxc1 gene in a cell aggregate containing oral ectoderm into which Sox9 expression gene and Foxc1 expression gene have been introduced in advance;
It is characterized by being.

In one embodiment of the present invention, the gene transfer is characterized by the transfer of a gene by a viral vector, the transfer of a gene by a transfection reagent, or the transfer of a gene by electroporation.

In one embodiment, the virus vector is an adenovirus vector, an adeno-associated virus vector, a lentivirus vector, a retrovirus vector, or a Sendai virus vector.

In one embodiment of the present invention, the step (ii) is a step of further culturing a part of the outer layer of the cell aggregate cultured in the step (i) in a medium containing FGF7 and FGF10. It is characterized by

In one embodiment of the present invention, in the step (ii), the concentration of FGF7 in the medium is 1 to 1000 ng / ml, and the concentration of FGF10 in the medium is 1 to 1000 ng / ml. .

In one embodiment of the present invention, the step (i) is characterized in that it is carried out using a growth factor-free medium.

In one embodiment of the present invention, the culture period of the step (i) is 0 to 7 days, and the culture period of the step (ii) is 10 to 32 days.

Another embodiment of the present invention relates to a pharmaceutical composition for use in the treatment of a disease accompanied by tissue disorder or dysfunction of salivary gland, wherein said pharmaceutical composition is regenerated by any of the above methods It is characterized by containing a salivary gland.

In one embodiment of the present invention, the pharmaceutical composition comprises a complex comprising a cell assembly substantially composed of mesenchymal cells, and a regenerating salivary gland produced by any of the above methods.
It is characterized by including.

In one embodiment of the present invention, in the complex, a filamentous structure is inserted so as to penetrate the cell assembly substantially composed of the mesenchymal cells and the regenerated salivary gland. It features.

In one embodiment of the present invention, all or part of the regenerated salivary gland or all or part of the complex is characterized in being embedded in a cell culture scaffold material.

In one embodiment of the present invention, the scaffold material is a collagen gel.

In one embodiment of the present invention, the disease is Sjogren's syndrome, litholithiasis, LADDO (Lacrimo-auriculo-dento-digital syndrome), ALSG (aplasia of lacrimal and salivary glands), Mikulicz disease (IgG4 associated sclerosability) Disease), other salivary gland inflammatory diseases, or salivary gland dysfunction due to the side effect of radiation treatment.

Another embodiment of the present invention relates to the use of the regenerated salivary gland produced by any of the above methods for the production of a pharmaceutical composition for use in the treatment of a disease associated with tissue disorder or dysfunction of the salivary gland.

Another embodiment of the present invention relates to a method of treating a disease associated with tissue disorder or dysfunction of salivary gland, wherein the method comprises regenerating salivary gland produced by the above method, or the pharmaceutical composition as described above. Applying to a subject in need of treatment of a disease.

In one embodiment of the present invention, the subject is a mammal other than human.

The invention which arbitrarily combines one or more features of the present invention mentioned above is also included in the scope of the present invention.

FIG. 1 shows the procedure of an embodiment of the present invention. The abbreviations in the figure mean the following. ES: Embryonic Stem Cell (embryonic stem cells) ME: Mesendoderm (embryonic stem cells) DE: Definitive Ectoderm (embryonic ectoderm) NNE: Non-neural Ectoderm (non-neural ectoderm) NE: Neural Ectoderm (neural ectoderm) EPI : Epidermis (epidermis) OE: Oral Ectoderm (oral ectoderm) OESG: Oral Epithelial Salivary Gland (oral epithelial salivary gland) SG: Salivary Gland (salivary gland) FIG. 2 is a diagram showing that Pan-cytokeratin (PK) -positive primordial oral mucosa (oral ectoderm) positive is formed in the outer layer of the aggregate on the 8th day after the induction of differentiation induction. FIG. 3 shows that Sox9-expressing gene and Foxc1-expressing gene are introduced into the aggregate on the 8th day of differentiation induction initiation using an adenovirus vector, then the outer layer of the aggregate is separated and further cultured in the presence of FGF7 and FGF10 It is the figure which showed that. FIG. 4 is a diagram showing that a budding-like structure similar to the embryonic salivary gland primordia is formed from the clumps on day 23 of differentiation induction, and branching morphogenesis is observed on day 28 of differentiation induction. FIG. 5 shows that ES cell-derived salivary glands on day 23 and 28 of differentiation induction are composed of Pan-cytokeratin (PK) -positive epithelial cells, and Keratin 18 (K18) -positive ductal epithelium is placed on the mesial side, AQP5 It is a figure which shows that the positive acinar cell and the (alpha)-SMA positive myoepithelial cell are arrange | positioned at the centrifugal side. The arrowhead "e" in the figure indicates an acini (epithelial bud), and the arrow "d" in the figure indicates a duct (duct). FIG. 6 is a diagram showing that, as a result of component analysis of gene expression profile by RNA-sequence, gene expression of ES cell-derived salivary gland (i-SG) is similar to that of salivary gland source at 15-18 days of embryonic day . FIG. 7 shows that ES cell-derived salivary glands show an increase in intracellular Ca 2+ concentration by carbachol stimulation, and that the action is suppressed by atropine. FIG. 8 left is a stereomicroscopic image of ES cell-derived salivary glands 30 days after transplantation. In FIG. 8 (GFP / DAPI stained image) and FIG. 8 right (PAS stained image), the excretory duct and ES of the recipient are obtained by transplanting the ES cell-derived salivary gland (i-SG) into the parotid ablated mouse. FIG. 5 is a diagram showing that cell-derived salivary gland structures are connected and engrafted mature acinar cells having mucus-producing ability. FIG. 9A is a diagram showing an outline of a method of transplanting ES cell-derived salivary glands in the present example. FIG. 9B shows a histologic image of the case where fetal derived salivary gland mesenchymal tissue is added to ES cell-derived salivary gland and transplanted (FIG. 9B left: stereomicroscopic image of ES cell-derived salivary gland 30 days after transplantation, in FIG. 9B: H & E) Stained image, right in FIG. 9B: PAS stained image). FIG. 10 is a diagram showing that the recipient-derived nerve invades into the transplanted ES cell-derived salivary gland structure. Arrowheads in the figure represent nerve fibers, arrows represent calponin-positive myoepithelial cells, and myoepithelial cells are arranged to wrap ES cell-derived acinar cells (A, B, C), and nerve fibers close to myoepithelial cells. Is oriented. FIG. 11A is a diagram showing that the amount of salivary secretion is increased by taste stimulation by citric acid to a recipient transplanted with ES cell-derived salivary gland obtained by laminating salivary gland mesenchymal tissue of 13.5 days of gestational age. FIG. 11B is a diagram showing an increase in the amount of saliva secretion by stimulating the transplanted ES cell-derived salivary gland with the parasympathomimetic agent pilocarpic acid.

In the present invention, “pluripotent stem cell” refers to a cell having both pluripotency capable of differentiating into any cell of a living body and self-replication ability capable of maintaining pluripotency even after differentiation and proliferation. Cells and iPS cells.

In the present invention, "ES cells (Embryonic Stem cells)" refers to stem cell lines produced from an inner cell mass belonging to a part of blastocyst stage embryo which is an early developmental stage of an animal, and very many And pluripotency capable of differentiating into cells, and self-replication ability capable of maintaining pluripotency even after division and proliferation.

The origin of ES cells that can be used in the present invention is not particularly limited, and ES cells derived from the inner cell mass of any animal can be used. For example, ES cells derived from the inner cell mass of human, mouse, rat, dog, cat, rabbit, cow, horse, sheep, goat, pig and monkey can be used as a source of ES cells.

In general, “iPS cells (induced Pluripotent Stem cells)” are pluripotent capable of differentiating into so many cells like ES cells by introducing several types of genes and / or drugs into somatic cells, for example. Refers to cells that have sex and self-replication ability to maintain pluripotency even after division and proliferation. However, the present invention is not limited to the above description, and broadly includes cells that those skilled in the art recognize as "iPS cells".

The origin of the iPS cells that can be used in the present invention is not particularly limited, and any animal-derived iPS cells can be used. For example, as a source of iPS cells, iPS cells derived from human, mouse, rat, dog, cat, rabbit, cow, horse, sheep, goat, pig and monkey can be used. Further, somatic cells from which iPS cells can be derived which can be used in the present invention are not particularly limited, and iPS cells derived from cells derived from any tissue can be used. Furthermore, the method for inducing iPS cells that can be used in the present invention is not particularly limited, and iPS cells derived using any method can be used as long as they can induce iPS cells from somatic cells. be able to.

In the present invention, the method for culturing pluripotent stem cells without differentiation is not particularly limited, and a person skilled in the art can appropriately select a known culture environment or culture medium. For example, adhesion culture may be performed using feeder cells or the like, or suspension culture may be performed without using feeder cells or the like. In addition, as a medium for culturing pluripotent stem cells, a medium generally used for culturing pluripotent stem cells can be used, and the composition thereof is not particularly limited, and a person skilled in the art is an expert in the field. It can be suitably prepared based on common sense.

The "exocrine gland" in the present invention refers to a gland in which secretory granules from exocrine cells are released directly or through a conduit to the surface of the body surface or lumenal epithelium. Examples of the "exocrine gland" in the present invention include lacrimal gland, salivary gland, cardiac gland, pyloric gland, gastric gland, intestinal crypt, prostate, sweat gland, sebaceous gland and the like, preferably salivary gland. In addition, when manufacturing a salivary gland by the method of this invention, the confirmation that salivary gland was formed can use suitably the method which can be utilized for persons skilled in the art, such as analysis by a tissue image and functional analysis by stimulation.

The "oral ectoderm" in the present invention refers to a single-layer cell sheet that forms the mucous membrane epithelium of the oral cavity during embryonic development. The oral ectoderm develops from the ectoderm adjacent to the interbrain in the early embryo, and a portion of it forms the ratoke sac underlying the pituitary. The presence of oral ectoderm in tissues can be confirmed, for example, by expression of pan-cytokeartin (PK).

The buccal ectoderm used in the methods of the present invention may be derived from pluripotent stem cells (eg, ES cells or iPS cells). The method for inducing buccal ectoderm from pluripotent stem cells is not limited, and methods known to those skilled in the art can be used. For example, the induction of buccal ectoderm from ES cells in the examples of the present invention is described by K. K. et al. R. Koehler, A .; M. Mikosz, A .; I. Molosh D. Patel, E., et al. Reference is made to the method described in Hashino, Nature 500, 217 (Aug 08, 2013). Suga et al. , Nature 480, 57 (Nov 09, 2011), and Ochiai H et al. , Stem Cell Research 15: 290-298, 2015, and the like.

In some of the methods listed above, SFEBq method (serum agglutination suspension culture: S erum-free F loating culture of E mbryoid B ody-like aggregates with q uick reaggregation) utilize. In the SFEBq method, pluripotent stem cells are single-celled with an enzyme and reaggregated into a cluster of about 3000 cells is used as a material for differentiation culture. In the case of culturing this cell aggregate, in a method of adhering cells to a culture petri dish and conducting culture as in the usual cell culture, three-dimensional tissue formation is impaired, and a clean structure can not be formed. Therefore, by coating the culture vessel with non-cell-adhesive polymer to prevent cells and tissues from adhering to the vessel, three-dimensional tissue formation is enabled by the culture method in which the cell mass is suspended in the culture solution. . In the SFEBq method, the cells are cultured for several days while suspended in a special culture solution that does not contain any component having a neural differentiation inhibitory effect such as serum or transcription factor. This method makes it possible to differentiate some cells into ectodermal cells. For the specific procedure of the SFEBq method, the description of WO2009 / 148170 can be referred to.

The “cell aggregate containing buccal ectoderm” in the present invention may be a cell clump containing the number of buccal ectoderm required for induction to exocrine glands, and can be prepared, for example, by the method described above.

In the first culture step of the present invention, cell aggregates containing oral ectoderm are cultured under conditions that induce the expression of Sox9 and Foxc1. The culture in the first step of the present invention may preferably be suspension culture. Here, the “conditions for inducing the expression of Sox9 and Foxc1”, for example, induces the expression of Sox9 and Foxc1 by introducing the Sox9 expression gene and the Foxc1 expression gene into the cell aggregate containing the oral ectoderm. It may be a condition. Specifically, the Sox9 expression gene and the Foxc1 expression gene are introduced into a cell aggregate containing oral ectoderm by gene transfer with a viral vector, gene transfer with a transfection reagent, or gene transfer with electroporation. You may When a gene is introduced by a viral vector, for example, an adenovirus vector, an adeno-associated virus vector, a lentivirus vector, a retrovirus vector, a Sendai virus vector, etc. can be used.

As another “condition for inducing the expression of Sox9 and Foxc1”, for example, in the cell aggregate containing the oral ectoderm in which Sox9 expression gene and Foxc1 expression gene have been introduced in advance, the Sox9 gene and the Foxc1 gene Conditions may be mentioned which induce (start) expression. Specifically, for example, by using a Tet expression induction system, expression is induced by inducing Sox9 expression gene and Foxc1 expression gene introduced at the ES cell stage with a drug in the first culture step of the present invention. It can be started. In addition, it is not limited to the above-mentioned example, but various gene expression induction systems available to those skilled in the art can be used in the present invention.

In the process of the present invention, the “condition for inducing the expression of Sox9” and the “condition for inducing the expression of Foxc1” may be the same or different. The respective conditions can be appropriately selected by those skilled in the art based on the common technical knowledge in this field.

The first culture step of the present invention may be carried out for 0 to 7 days (preferably 0 to 3 days, more preferably 0 to 1 day). The first step on day 0 means, for example, starting the second step of the present invention the day the Sox 9 expression gene and the Foxc 1 expression gene were introduced into a cell aggregate containing oral ectoderm.

The medium used in the first culture step of the present invention is a medium substantially free of growth factor (eg, growth-factor-free Chemically Defined Medium (gfCDM medium) used in the SFEBq method) However, various other media may be used as long as they do not cause unintended differentiation induction in oral ectoderm. It should be noted that substances generally added to the cell culture medium (for example, supplements such as amino acids and transferrin, antibiotics, etc.) may be added to the culture medium as long as they do not cause unintended differentiation induction in oral ectoderm. Good.

In the second culture step of the present invention, all or part of the cell aggregate prepared in the first step is further cultured in a medium containing FGF7 and FGF10. The culture in the second step of the present invention may preferably be suspension culture. The concentration of FGF7 in the culture medium and FGF10 may be contained at a concentration necessary for inducing exocrine glands (eg, salivary glands) from oral ectoderm, and can be appropriately adjusted by those skilled in the art. For example, the concentration of FGF7 in the medium may be in the range of 1 to 1000 ng / ml, preferably 5 to 750 ng / ml, 10 to 500 ng / ml, 20 to 400 ng / ml, 30 to 300 ng / ml, 40 It may be in the range of ̃200 ng / ml or 50 ̃150 ng / ml. Also, for example, the concentration of FGF10 in the medium may be in the range of 1 to 1000 ng / ml, preferably 5 to 750 ng / ml, 10 to 600 ng / ml, 30 to 500 ng / ml, 50 to 400 ng / ml , 100-300 ng / ml, or 150-250 ng / ml.

As the medium used in the second culture step of the present invention, various media can be used as long as they do not cause unintended differentiation induction in oral ectoderm. For example, BME medium, BGJb medium, CMRL 1066 medium, Glasgow MEM medium, Improved MEM Zinc Option medium, IMDM medium, Medium 199 medium, Eagle MEM medium, αMEM medium, αMEM medium, DMEM medium, advanced DMEM / F12 medium, Ham medium, Ham ' s F-12 medium, RPMI 1640 medium, Fischer's medium, Neurobasal medium, mixed medium thereof and the like can be mentioned. In addition, substances (eg, supplements such as amino acids and transferrin, antibiotics, etc.) generally added to the culture medium for cell culture may be added to the culture medium as long as they do not cause differentiation induction to the oral ectoderm. .

In the second culture step of the present invention, the cell aggregate cultured in the first culture step may be used as it is, but using only a part of the cell aggregate cultured in the first culture step It is also good. For example, a part of the outer layer of the cell aggregate cultured in the first culture step can be isolated and applied to the second culture step to increase the induction efficiency of exocrine glands (eg, salivary glands) . The method of isolating a part of the outer layer of the cell aggregate is not limited, and may be mechanically isolated using, for example, a pipette, an injection needle, a medical scalpel or the like.

The second culture step of the present invention is carried out for 10 to 32 days (preferably 12 to 20 days, more preferably 15 to 18 days).

Other general cell culture conditions can be appropriately prepared based on the common technical knowledge of those skilled in the art.

The regenerated salivary glands produced by the method of the present invention can be used, for example, in the treatment of diseases involving tissue damage or dysfunction of the salivary glands. Non-limiting examples of such diseases include Sjogren's syndrome, litholithiasis, Lacrimo-auriculo-dento-digital syndrome, ALSG (aplasia of lacrimal and salivary glands), Mikulicz's disease (IgG4 related sclerosability) Disease), other salivary gland inflammatory diseases, or salivary gland dysfunction due to the side effect of radiation treatment.

The treatment of diseases utilizing the present invention can be used for any animal, for example, mammals (mouse, rat, dog, cat, rabbit, cow, horse, sheep, goat, pig, monkey) , Humans, etc.). In addition, when it is not preferable, humans can be excluded from the subject.

When the regenerated salivary glands produced by the method of the present invention are used to treat a disease, it is preferable to transplant the regenerated salivary glands produced by the method of the present invention to a subject in need of treatment. The method for transplanting the regenerating salivary gland to the subject is not limited, but for example, referring to WO 2012/108069, after inserting a guide (filamentary structure) for promoting the formation of the duct of the secretory gland into the regenerating salivary gland, It can be ported. By inserting a guide into the regenerating salivary gland and transplanting it into the subject, it is possible to direct the formation of the ducts of the secretory gland in the tissue to be transplanted.

The regenerated salivary gland produced by the method of the present invention may be transplanted to a subject alone, but when it is adhered to a cell assembly substantially composed of mesenchymal cells and transplanted as a complex, The growth efficiency of salivary gland tissue at the transplantation destination is improved. The origin of the mesenchymal cells used in the present invention is not limited, and may be, for example, mesenchymal cells derived from pluripotent stem cells (eg, ES cells, iPS cells) by a known method, or a living tissue It may be a mesenchymal cell of origin (eg, a fetal mesenchymal cell).

In addition, the whole or part of the regenerated salivary gland produced by the method of the present invention or the complex of the regenerated salivary gland and the mesenchymal cells is embedded in a scaffold for cell culture and then transplanted to the subject. And increase the survival rate in the target tissue. Examples of scaffolds for cell culture which can be used in the present invention include collagen gels (more specifically, type I collagen gel, type III collagen gel, type IV collagen gel, matrigel, etc.) .

The material of the thread-like structure used as a guide for promoting the formation of the conduit of regenerated salivary gland in the present invention is not limited, for example, fibers made of synthetic or natural bioabsorbable polymers, metal fibers such as stainless steel, Chemical fibers such as carbon fibers and glass fibers, natural animal fibers, plant fibers and the like can be mentioned, and more specifically, nylon yarn, stainless steel wire and the like can be mentioned. Also, the guide may have various shapes as long as it is thread-like, for example, it may be in the form of hollow fiber. In addition, it is preferable to use a bioabsorbable one when the filamentous structure and the regenerated salivary gland are buried under the skin of the recipient after transplantation.

The diameter and length of the thread-like structure used as a guide for promoting the formation of the conduit of the regenerating salivary gland can be appropriately designed according to the subject. For example, the diameter may be 5 to 100 μm, more preferably 10 to 70 μm, and still more preferably 20 to 50 μm. In addition, the length of the guide may be, for example, 1 mm to 10 mm, more preferably 2 to 8 mm, and still more preferably 2 mm to 5 mm.

The method of inserting the filamentous structure into the regenerating salivary gland is not limited as long as it is inserted so as not to destroy the tissue of the regenerating salivary gland. In addition, when the regenerating salivary gland and the aggregate of mesenchymal cells are adhered and transplanted as a complex, the filamentous structure is inserted so as to penetrate the regenerating salivary gland and the aggregate of mesenchymal cells. Preferably.

Moreover, after inserting the filamentous structure into the regenerated salivary gland, the regenerated salivary gland may be cultured for a certain period of time with the filamentous structure inserted, and then subjected to transplantation. The culture period after guide insertion can be set appropriately depending on the subject, but can be cultured, for example, for 1 to 7 days.

The terms used herein, except as specifically defined, are used to describe particular embodiments and are not intended to limit the invention.

In addition, the term "including" as used herein is intended to include the described items (members, steps, elements, numbers, etc.) unless clearly understood in context. It does not exclude the existence of other matters (members, steps, elements, numbers, etc.).

Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terms used herein should be construed as having a meaning consistent with that in the present specification and related art, unless a different definition is explicitly stated, and are idealized or excessively formalized. It should not be interpreted in the sense of meaning.

In the following, the invention is described in more detail with reference to the examples. However, the present invention can be embodied in various aspects and should not be construed as limited to the embodiments set forth herein.

FIG. 1 shows an outline of the procedure of this embodiment. Among the procedures shown in FIG. 1, the procedure for inducing from the oral ectoderm (OE) to the salivary gland (SG) is the subject of the present invention.

Materials and Methods Mouse ES cells are dispersed into single cells by trypsinization, and clumps are formed according to the SFEBq method (Nakano et al, Cell Stem Cell, 10 (6): 771-785, 2012) to induce differentiation Flocculation culture for 37 ° C. in the presence of 5% CO 2. The specific procedure is shown below.

(1) Induction of ES Cells into Oral Ectoderm The dispersed 3,000 mouse ES cells were seeded in each well of a low cell adsorptive surface-coated round bottom 96 well plate. The culture medium for differentiation induction is a growth factor-free chemically-defined medium (gfCDM; Wataya et al, Proc Natl Acad Sci USA, 105 (33): 11796-11801, 2008). Using.

One day after ES cell seeding (one day after induction of differentiation induction), Growth factor free matrigel at a final concentration of 1% was added. BMP4 (10 ng / ml) and SB-431542 (1 μM) were added 3 days after the induction of differentiation induction, and 5 days FGF2 (25 ng / ml) and LDN-193189 (1 μM) were added.

In addition, the induction from ES cells to oral ectoderm in this example is as described in K. et al. R. Koehler, A .; M. Mikosz, A .; I. Molosh D. Patel, E., et al. It implemented with reference to the method as described in Hashino, Nature 500, 217 (Aug 08, 2013). Because BMP4 induces stem cells into ectoderm, SB-431542 (TGF-β inhibitor) suppresses differentiation into mesendoderm, and FGF2 thickens the epithelium of the outer layer of cell mass, so LDN-193189 ( BMP inhibitors are added respectively to suppress differentiation into the epidermis (see FIG. 1).

(2) Induction of oral ectoderm into salivary gland: First culture step On day 8, human Sox9 and mouse Foxc1 were overexpressed in the outer layer of cell aggregates using an adenovirus vector. In addition, the culture medium in this process used gfCDM culture medium.

(3) Derivation from oral ectoderm to salivary gland: Second culture step The outer layer of the aggregate was mechanically isolated under a stereomicroscope and transferred to a 24-well plate coated with a low cell adsorptive surface. The medium used was one obtained by adding 1% matrigel to a medium (advanced DMEM / F12 supplemented with N2 supplement, GlutaMax, penicillin, streptomycin) to which FGF7 (100 ng / ml) and FGF10 (200 ng / ml) were added. Medium replacement was continued once every two days.

(4) Analysis of induced salivary glands As analysis of mouse ES cell-derived salivary glands, histological analysis, preparation of gene expression profile using RNA-seq, measurement of intracellular Ca ²⁺ concentration by carbacol stimulation, parotid gland Histological analysis and salivation measurement by transplantation experiment to a resected model mouse were performed.

(5) Results At 8 days after initiation of differentiation induction of ES cells, cell aggregates including Pan-cytokeratin (PK) positive primary oral mucosa (oral ectoderm) were induced in the outermost layer of aggregates (FIG. 2).

Eight days after initiation of differentiation induction, genes expressing two types of transcription factors (Sox9, Foxc1) were introduced into cell aggregates containing the induced oral ectoderm using an adenovirus vector. Thereafter, after the outer layer of the cell aggregate was surgically isolated, suspension culture was continued in the presence of FGF7 and FGF10 (FIG. 3). At 23 days after initiation of differentiation induction from ES cells (15 days after introduction of transcription factor), budding-like morphological changes similar to mouse salivary gland protozoa are observed in the surface layer of aggregates, and 28 days (introduction of transcription factor) After 20 days, three-dimensional branching morphogenesis was observed (FIG. 4).

This structure was composed of Pan-cytokeratin-positive epithelial cells, and Keratin 18-positive ductal epithelium, AQP5-positive acinar cells, and α-SMA-positive myoepithelial cells were arranged with polarity (FIG. 5). Moreover, the result of gene expression profile by RNA-sequence was also similar to the gene expression profile in the salivary gland source at 15 to 18 days of gestation (FIG. 6).

Furthermore, in this ES cell-derived salivary gland, an increase in intracellular Ca ²⁺ concentration was observed by carbachol stimulation (FIG. 7, left, middle), and its action was suppressed by atropine (FIG. 7, right).

In addition, when ES cell-derived salivary glands were transplanted into mice from which parotid glands had been excised, they were joined with existing excretory ducts, and production of saliva containing salivary secretory proteins was observed (Fig. 8 left: ES cell-derived 30 days after transplantation) Stereomicroscopic image of salivary gland, in FIG. 8: GFP / DAPI stained image, FIG. 8 right: PAS stained image). The transplantation of regenerated salivary gland was performed according to the method described in WO 2012/108069. That is, the isolated regenerated salivary gland was embedded in a scaffold material (collagen gel), and a guide (a filamentous structure) for promoting formation of a duct of a secretory gland was inserted, and then transplanted to a mouse (FIG. 9A).

When fetal fetal salivary gland mesenchymal tissue was added to ES cell-derived salivary gland and transplanted in the same procedure as above, it was also joined with the existing excretory duct, and saliva production including salivary secretory protein was observed (FIG. 9B) Left: Stereomicroscopic image of ES cell-derived salivary gland 30 days after transplantation, in FIG. 9B: H & E stained image, FIG. 9B right: PAS stained image).

As a result of histological analysis, the transplanted ES cell-derived salivary gland contained nerves and blood vessels from the recipient (FIG. 10). That is, it has been shown that the regenerated salivary glands produced by the method of the present invention properly engraft the recipient's tissue and cooperate with the surrounding tissue.

In addition, even when only the regenerating salivary gland was embedded in the scaffolding material and engrafted, engraftment of functional salivary gland was observed at the transplantation destination, but mesenchymal cells were added to the regenerating salivary gland and encased in the scaffolding material When buried and transplanted, the growth of regenerated salivary glands at the transplant destination was even better.

The ES cell-derived salivary glands in which the 13.5-day-old salivary gland mesenchymal tissues were adhered and transplanted, also exhibited salivation in response to taste stimulation with citric acid (FIG. 11A). In addition, when the transplanted ES cell-derived salivary gland was stimulated with the parasympathomimetic agent pilocarpic acid, an increase in salivary secretion was observed (FIG. 11B). That is, it has been shown that the regenerated salivary glands produced by the method of the present invention properly engraft and function in recipient tissues.

Claims (19)

A method of producing a functional exocrine gland in vitro, comprising
(I): culturing cell aggregates containing oral ectoderm under conditions that induce the expression of Sox9 and Foxc1;
(Ii): further culturing all or part of the cell aggregate cultured in the above step (i) in a medium containing FGF7 and FGF10,
including,
Method. The method according to claim 1, wherein
The oral ectoderm is characterized by being an oral ectoderm derived from ES cells or iPS cells,
Method. The method according to claim 1 or 2, wherein
The exocrine gland is a salivary gland,
Method. A method according to any one of the preceding claims, wherein
The “condition for inducing the expression of Sox9 and Foxc1” in the step (i) is
(A) A condition that induces the expression of Sox9 and Foxc1 by introducing the Sox9 expression gene and the Foxc1 expression gene into the cell aggregate containing the above-mentioned oral ectoderm, or
(B) conditions for inducing the expression of the introduced Sox9 gene and Foxc1 gene in a cell aggregate containing oral ectoderm into which Sox9 expression gene and Foxc1 expression gene have been introduced in advance;
Is characterized by
Method. The method according to claim 4, wherein
The introduction of the gene is characterized in that the introduction of the gene by a viral vector, the introduction of the gene by a transfection reagent, or the introduction of the gene by electroporation is carried out.
Method. The method according to claim 5, wherein
The viral vector is an adenoviral vector, an adeno-associated viral vector, a lentiviral vector, a retroviral vector, or a Sendai virus vector,
Method. The method according to any one of claims 1 to 6, wherein
Characterized in that the step (ii) is a step of further culturing a part of the outer layer of the cell aggregate cultured in the step (i) in a medium containing FGF7 and FGF10.
Method. A method according to any one of the preceding claims, wherein
In the step (ii), the concentration of FGF7 in the medium is 1 to 1000 ng / ml, and the concentration of FGF10 in the medium is 1 to 1000 ng / ml,
Method. A method according to any one of the preceding claims, wherein
The step (i) is characterized in that it is carried out using a growth factor-free medium.
Method. A method according to any one of the preceding claims, wherein
The culture period of the step (i) is 0 to 7 days,
The culture period of the step (ii) is 10 to 32 days,
Method. A pharmaceutical composition for use in the treatment of a disease associated with tissue damage or dysfunction of salivary gland,
The pharmaceutical composition is characterized in that it comprises regenerated salivary glands produced by the method according to any one of claims 1-10.
Pharmaceutical composition. The pharmaceutical composition according to claim 11, which is
The pharmaceutical composition comprises a complex comprising a cell assembly substantially comprising mesenchymal cells, and a regenerating salivary gland produced by the method according to any one of claims 1 to 10.
Characterized in that,
Pharmaceutical composition. A pharmaceutical composition according to claim 12, which is:
In the complex, a thread-like structure is inserted so as to penetrate the cell assembly substantially composed of the mesenchymal cells and the regenerated salivary gland.
Pharmaceutical composition. The pharmaceutical composition according to any one of claims 11 to 13, wherein all or part of the regenerated salivary gland or all or part of the complex is embedded in a scaffold for cell culture. Are characterized by
Pharmaceutical composition. The pharmaceutical composition according to claim 14, which is
The scaffold material is a collagen gel,
Pharmaceutical composition. The pharmaceutical composition according to any one of claims 11 to 15, wherein
Said diseases include Sjogren's syndrome, lipolithiasis, Lacrimo-auriculo-dento-digital syndrome, ASLIA (aplasia of lacrimal and salivary glands), Mikulicz's disease, other salivary gland inflammatory diseases, or side effects of radiation therapy Characterized by salivary gland dysfunction due to
Pharmaceutical composition. The use of a regenerated salivary gland produced by the method according to any one of claims 1 to 10 for the production of a pharmaceutical composition for use in the treatment of a disease associated with tissue disorder or dysfunction of salivary gland. A method of treating a disease associated with tissue damage or dysfunction of salivary gland,
A regenerated salivary gland produced by the method according to any one of claims 1 to 10, or a pharmaceutical composition according to any one of claims 11 to 16 to a subject in need of the treatment of the disease. Applying, including
Method. Method according to claim 18, wherein
The subject is a non-human mammal,
Method.

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