KR20190001297A - Method for preparing intestinal organoid from embryonic stem cells - Google Patents

Abstract

The present invention relates to a method for preparing a small intestinal organoid from embryonic stem cells and a small intestinal organoid prepared by the method. The small intestinal organoid according to the present invention is capable of controlling production and differentiation at each step, therby facilitating diverse treatments and efficient production. By using pluripotent stem cells, mass production of the small intestinal organoid is possible, which can be used for identification for a development mechanism of various diseases through disease modeling, drug efficacy testing, and treatment models for organ replacement. The embryonic stem cells, with no immunological rejection, are expected to be useful for studies on novel regenerative medicine, development of organ transplantation technology, major factors involved in development of intestine and on identifying a development mechanism of the same.

Description

Technical Field [0001] The present invention relates to a method for preparing a small intestinal organoid from embryonic stem cells,

The present invention relates to a method for producing a small intestine organogenesis from embryonic stem cells and a small intestine organogenesis produced by the above production method.

An organoid is a long-term analogue, meaning a small culture that reproduces both the morphology and function of tissues or organs that have been re-agglutinated and recombined through 3D culture of cells isolated from stem cells or organs. Such organoids include several specific cell groups constituting organs or tissues, and are similar in shape and structural organization to actual tissues or organs, and can reproduce the specific functions of each organ. The organoid is formed by a common sequence of processes, in which cells with the same function are clustered together and placed in the proper position, and after the division of the cells, more detailed differentiation occurs. This is called lineage specification, and the precursor is differentiated into complete adult cells to perform actual function.

  The development of effective organotypic culture technology can be applied to a variety of applications such as disease modeling, drug efficacy testing, and organ replacement. Organogens are a powerful model of personalized medicine that are expected to be more effective at testing the safety and efficacy of new drugs than those made in two dimensions and helping to select the most appropriate drug for each patient or individual have. It can also be used for therapeutic purposes to improve the condition by implanting organoids in organs damaged or poorly developed. Such organogenesis technology is theoretically known to be able to produce almost all kinds of organs by stem cells alone, and thus it is expected to be applicable to various diseases, and recent studies in the field of regenerative medicine are becoming more active.

On the other hand, the epithelial cells of the small intestine that simultaneously treat various functions function to secrete digestive enzymes to digest food, absorb nutrients from food, and protect small intestine from life-threatening microorganisms or carcinogens. The stem cells present in the lower crypt of the small intestine are differentiated into transit-amplifying cells (TA cells), and the differentiated cells move up to the villi and are further differentiated into different types of cells, This process occurs within 5 days. It is known that in the human intestines, an average of 1011 epithelial cells die each day and are replenished with new epithelial cells derived from stem cells. Thus, the ability to maintain the ability of autologous proliferation of pluripotent stem cells to differentiate at the same time can be a good research subject for regenerative medicine, and understanding these abilities is an important first step in the development of future organ transplantation techniques and treatment of diseases. .

The existing zyganoids were first successfully obtained from the Hans Clevers laboratory in 2009 for the first time to cultivate the stem cells in a three-dimensional matrix using matrigel. The zygotes were obtained from the stem cells in a similar manner to the in vivo intestinal structure, , And it has been reported that the zygonoids thus formed can retain their properties for several years during which they are cultured. These organoids are produced using adult stem cells, which are stem cells in the intestine. The organoids derived from adult stem cells mimic the niche environment of stem cells when the tissue is self-replicating or recovering from damage, Can be formed. In particular, the Wnt signal is very important for the normal role of epithelial stem cells. Therefore, Wnt3a, R-spondin, etc., which are active substances of Wnt signal, are included as main elements in adult stem cell culture protocol, and Lgr5 + cells always appear in this culture environment. Here, Lgr5 is a target gene of the Wnt signal and a factor that acts as a receptor for R-spondin to amplify the Wnt signal, and is also an indicator of the activity of adult stem cells. Lgr5 + cells are very important adult stem cells observed in organogenesis from most tissues and organs. However, since there is a limit to the number of cells that can be obtained, there is a limit to efficient production and research, so a new model is needed for research on development, differentiation and maturation of intestines.

Therefore, when pluripotent stem cells are used instead of adult stem cells in the production of enteric organisms, it is possible to mass-proliferate, differentiate into almost all the body cells, and have immunity rejection, which is advantageous in transplanting to other people and other species. However, since it is difficult to control the differentiation, it may be transformed into a cancer cell. Therefore, it is necessary to precisely control the differentiation control technique.

On the other hand, during the development of the welfare animal, the major part of the digestive system or the respiratory system develops from the innermost embryoid endoderm. In the endoderm, specialization of the system begins with a focus on the expression of Cdx2, one of the homeobox genes. Up to now, a suitable culture method has been required in the presence of mutations in cancer cells due to an artificial growth factor treatment in forming pluripotent stem cells using the pluripotent stem cells. Therefore, if the developmental process of the welfare animal can be imitated in vitro to produce a zyganogen without mutation into a cancer cell, this is an important starting point that can not only present a new model but also extend the range of research .

In order to overcome the above conventional problems, the present inventors prepared a small intestine organotypes expressing Cdx2 by culturing the cultures obtained from mouse embryonic stem cells three-dimensionally and specializing them as endoderm, It has been confirmed experimentally that it has the characteristics of small intestine. Thus, the present invention has been completed by developing a method for producing small intestinal organoid by inducing differentiation into endoderm without treatment of an artificial growth factor from pluripotent stem cells capable of mass proliferation instead of adult stem cells.

Accordingly, it is an object of the present invention to provide a method for producing small intestinal organotypes from embryonic stem cells and a small intestine organotypes produced by the method.

However, the technical problem to be solved by the present invention is not limited to the above-mentioned problems, and other matters not mentioned can be clearly understood by those skilled in the art from the following description.

In order to achieve the object of the present invention, the present invention provides a method for producing a small intestinal organoid from embryonic stem cells, comprising the following steps.

(a) preparing a three-dimensional embryoid body from embryonic stem cells;

(b) characterizing said cultures as endoderm lines;

(c) inducing the expression of the Cdk2 gene in the endoderm to be specialized in the small intestine; And

(d) culturing the small intestine organs in which the Cdk2 expression is induced.

In one embodiment of the present invention, the embryonic stem cells may be mouse embryonic stem cells.

In another embodiment of the present invention, the culture of step (a) can be produced by culturing mouse embryonic stem cells for 3 to 10 days.

In another embodiment of the present invention, the step (b) may be carried out by treating the cultured body with an activin A and culturing it.

In another embodiment of the present invention, the endoderm can be one that expresses the Foxa2 gene.

In another embodiment of the present invention, in step (c), the expression of the Cdk2 gene is selected from the group consisting of a Wnt / beta-catenin signaling pathway activator and a Notch signaling inhibitor Lt; / RTI >

In another embodiment of the present invention, the Wnt / beta-catenin signaling pathway activator is selected from the group consisting of glycogen synthase kinase-3 beta (GSK-3 beta) inhibitor 6- Bromoindubin-3 ' -oxime (BIO). ≪ / RTI >

In another embodiment of the present invention, the Notch signaling inhibitor is a gamma-secretase inhibitor N- [N- (3,5-difluorophenacetyl) -L (3,5-difluorophenacetyl) -L-alanyl] -S-phenylglycine t-butyl ester (DAPT).

In another embodiment of the present invention, the BIO and DAPT may be treated at concentrations of 0.5 to 10 uM and 1 to 20 uM, respectively.

The present invention also provides the small intestine organs produced by the above production method.

In one embodiment of the present invention, the small intestinal organoid may exhibit the following characteristics.

(a) there is a paneth cell specific granule present in the small intestine mucosa;

(b) small intestinal stem cell markers Msi1 and Lgr5; Differentiation markers Lysozyme, Muc2, and ChgA; And Cck, a long-acting hormone; And

(c) Small intestinal paranuclear cells and stem cells are present.

The present invention induces mouse embryonic stem cells to differentiate into endoderm lines, thereby realizing small intestinal organogens having the characteristics of small intestine. Therefore, the small intestine organs produced by the method according to the present invention can efficiently regulate the production time and production amount at each stage of cell differentiation, and it is easy to mass-proliferate as a characteristic of pluripotent stem cells, Mass culture can be secured. Through this system, it can be used as a model system for regeneration treatment through transplantation of various small intestinal lesions, monitoring the efficacy of therapeutic drugs, drug response test for personalized medical treatment, and identification of the cause of disease. Observation of reaction changes and the like are possible. In addition, it can be used as a model system for developing original technology for avoiding immune rejection reaction in organ transplantation.

FIG. 1 shows the process of producing a culture from mouse embryonic stem cells and morphological changes of mouse embryonic stem cells according to the above process.
FIG. 2 is a graph showing the results of three-dimensional culturing of cultures of spermatogonial stem cells and RT-PCR for comparing the mRNA levels of Foxa2, an endoderm-specific gene with or without Actin A treatment Results.
FIGS. 3A to 3C are graphs showing the expression levels of Cdx2 in endoderm differentiated from mouse embryonic stem cells, and FIG. 3a and FIG. 3b show activation of the endothelial / (Fig. 3A) and protein (Fig. 3A) by performing the RT-PCR and Western blotting together with the GSK-3? Inhibitor, a GSK-3? Inhibitor, and DAPT, a gamma- secretase inhibitor that inhibits the Notch signaling pathway (FIG. 3b). FIG. 3c shows the results of immunohistochemical staining for comparing the expression level of Cdx2 protein according to the treatment concentration of BIO and DAPT.
FIGS. 4A to 4C are the results of verifying whether the small intestinal organogenesis differentiated from mouse embryonic stem cells has substantially the characteristics of small intestine. FIG. 4A shows the crypt derived-organoid and structural characteristics (Msi1 and Lgr5), differentiation markers (Lysozyme, Muc2, ChgA), and the expression of Cck mRNA, which is a long-term secretory hormone, by small intestine specific genes, RT-PCR FIG. 4c shows results of immunohistochemical staining of Paneth cells and stem cells, which are specific for small intestine.
FIG. 5 is a diagram illustrating an overall process of the method for preparing a small-cell organoid according to the present invention.

The present invention relates to a method for producing a small intestine organogenesis from embryonic stem cells and a small intestine organogenesis produced by the above production method. The present inventors have completed the present invention by developing a method for producing small intestinal organotypes by inducing differentiation into endoderm without processing artificial growth factors from embryonic stem cells, pluripotent stem cells capable of mass proliferation instead of adult stem cells.

Accordingly, the present invention provides a method for producing a small intestinal organoid from embryonic stem cells comprising the following steps.

(a) preparing a three-dimensional embryoid body from embryonic stem cells;

(b) characterizing said cultures as endoderm lines;

(c) inducing the expression of the Cdk2 gene in the endoderm to be specialized in the small intestine; And

(d) culturing the small intestine organs in which the Cdk2 expression is induced.

Hereinafter, each step will be described in detail.

As used herein, the term " embryonic stem cell (ESC) " is a cell extracted from an embryo before 14 days after fertilization and can be differentiated into all tissues of the body. Also referred to as undifferentiated undifferentiated cells. Unlike adult stem cells, embryonic stem cells are capable of infinite proliferation, and thus mass production is possible when organogens are manufactured using them. In the present invention, mouse-derived embryonic stem cells are used, but the present invention is not limited thereto.

The step (a) may be carried out by dividing mouse embryonic stem cells into a plate treated with a substance having low adhesiveness and culturing for 3 days to 10 days, more preferably 5 days to 7 days, further preferably 6 days Can be obtained. The cultures can be formed by gravity as agglomerated cell clusters of mouse embryonic stem cells.

In the step (b), the cultured medium may be cultured in a matrigel to be three-dimensionally cultured, and then may be specialized as an endoderm by actin A treatment. Activin A activates nodal signaling, thereby specializing the cultures as endoderm. At this time, knockout serum replacement (KSR) is used instead of FBS in the culture to prevent the decrease of activity of actin A Can be used. In this way, the specialized endoderm expresses the Foxa2 gene.

In step (c) above, specialization into the intestinal tract may be achieved by inducing a gut-specific gene through signal transduction regulation. Specifically, the expression of Cdk2 can be induced to be specialized in the small intestine by controlling the Wnt / β-catenin signaling pathway and the notch signaling pathway in the special endemic endoderm. More specifically, 6-bromoindirubin (6-bromoindubin-3-phosphate) inhibitor, a glycogen synthase kinase-3? Inhibitor, was used as a winter / (3,5-difluorophenylacetyl) -L-alanyl] - 3'-oxime (BIO) and the notch signal transduction pathway inhibitor γ- S-phenylglycine t-butyl ester (DAPT) was added at a concentration of 0.5 to 10 uM and 1 to 20 uM, respectively, , More preferably 1 to 5 uM, and 2 to 10 uM, but the present invention is not limited thereto, and any substance capable of regulating the signal transduction pathway can be appropriately selected and used by those skilled in the art.

The step (d) may be performed by culturing the small intestine organs in which the Cdk2 expression has been induced in the same culture medium as that of the organogen culture medium, which is induced into the adult stem cells without treatment of a separate growth factor.

The present invention has verified through the examples that the small intestine organogens prepared by the above method have substantially the characteristics of small intestine.

In one embodiment of the present invention, the structural characteristics of the crypt derived-organoids prepared from the small intestine organs and the enteral glands according to the present invention were compared. As a result, (Msi1 and Lgr5), and differentiation markers (Lysozyme, Muc2, ChgA, and Muc2) were measured by measuring the mRNA levels of small intestine specific genes, ), And Cck, a long-acting hormone, were all expressed. In addition, it was confirmed that the small intestine organs according to the present invention exhibited the small intestinal characteristics by confirming the presence of parathyroid cells and stem cells present in the small intestine (see Example 4).

Accordingly, the present invention provides a small-molecule organotin produced by the above-described method.

Hereinafter, preferred embodiments of the present invention will be described in order to facilitate understanding of the present invention. However, the following examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited by the following examples.

[Example]

Example 1: Three-dimensional spherical culture using mouse embryonic stem cells

In order to make an embryoid-body (EB), which is an early stage spheroid of three-dimensional tissue culture that can develop into all germ layers using mouse embryonic stem cells, a 96-well plate in mouse embryonic stem cells to 10 ³ cells / well each division and DMEM high glucose base culture medium (15% FBS, 0.1 mM NEAA , 0.1 mM beta-mercaptoethanol (beta-mercaptoethanol), 0.1 mg / ml penicillin / streptomycin ( penicillin / streptomycin) at 37 < 0 > C. As a result, as shown in Fig. 1, after culturing for about 6 days, it was confirmed that a culture product was effectively formed between mouse embryonic stem cells by gravity.

Example 2: Three-dimensional culture of cultures and specialization into endoderm

According to the method of Example 1, a culture made from mouse embryonic stem cells was planted in a matrigel to start three-dimensional culture. In the next step, to prepare endogenous endogenous endogenous gland organoids, actin A (Activin A), which is one of TGF-beta superfamily, was treated at a concentration of 100 ng / ml to activate the nodal signal transduction pathway, To be specialized as endoderm. At this time, 15% knockout serum replacement (KSR) was added instead of FBS, since fetal bovine serum (FBS) contained in the culture broth of the cultures may reduce the effect of theactin A, and 0.1 mM NEAA , And 0.1 mg / ml penicillin / streptomycin in DMEM high glucose medium.

RT-PCR was performed to measure the mRNA level of Foxa2 , an endoderm-specific gene, in order to examine whether or not the endothelial cells were cultured in the same manner as described above. As a result, as shown in FIG. 2, the expression of the gene was increased when Actin A was treated and cultured, whereas the expression of T, which is a mesodermal-specific gene, disappeared. These results indicate that the pluripotent mouse embryonic stem cells, which are essential steps of intestinal development, are well differentiated into Foxa2 + endoderm.

Example 3: Cdx2 induction, a long-development-specific gene

During the development of the intestine, the systematic specialization of the intestines begins, focusing on the expression of Cdx2 , one of the homeobox genes. Thus, in order to induce the expression of Cdx2 , the embryonic stem cell cultures were specialized as endoderm lines according to the methods of Examples 1 and 2 above. More specifically, expression of the gene was induced by controlling the Wnt / beta-catenin signaling pathway and / or Notch signaling pathway.

First, treatment with BIO (6-bromoindirubin-3'-oxime), which is a glycogen synthase kinase-3? Inhibitor, activates the mutant / beta-catenin signaling pathway (B and -BD IOs). As a result, as shown in FIGS. 3A and 3B, the mRNA and protein expression of the gene was not significantly increased, whereas the gamma-secretase inhibitor The expression of Cdx2 mRNA and protein was markedly increased when treated with BDP + (BD and + BD IOs) simultaneously with DAPT (N- [3,5- difluorophenacetyl) -L-alanyl] -S-phenylglycine t- Respectively. Furthermore, in order to determine the optimal treatment concentration, the BIO and DAPT were treated with 1 uM / 2 uM or 5 uM / 10 uM, respectively, and immunohistochemistry was performed to compare the protein levels of Cdx2 . As a result, it was confirmed that Cdx2 protein was highly expressed when BIO and DAPT were treated at 5 uM and 10 uM, respectively.

Example 4: Culture of small intestine organs

The intestinal organogenesis induced by cdx2 expression produced according to the methods of Examples 1 to 3 was carried out under the same conditions as those of the organogenic culture medium derived from existing adult stem cells without any other growth factors, Factor (EGF), Noggin, and R-spondin 1. Thereafter, it was verified that the organotypes cultured by the above method had substantially small intestinal characteristics.

First, the structure of the organoid according to the present invention was compared with that of a crypt derived-organoid prepared from the intestine. As a result, as shown in FIG. 4A, Paneth cell specific granules existing in the small intestine mucosa were observed in the same manner as in the comparative group. In addition, RT-PCR was performed to measure mRNA levels of small intestine specific genes. As a result, small intestinal specific cell markers ( Msi1 and Lgr5 ), differentiation markers ( Lysozyme , Muc2 , ChgA ) And secretion hormone Cck were all expressed. Further, as shown in FIG. 4C, it was confirmed by immunohistochemical staining that paranuclear cells and stem cells present in the small intestine were present in the organotypes of the present invention. From the above results, it was found that small intestine organs were successfully prepared from mouse embryonic stem cells according to the manufacturing method of the present invention. The overall process of the method for preparing the small intestine organoid of the present invention is illustrated in FIG.

It will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the spirit or scope of the invention. There will be. It is therefore to be understood that the above-described embodiments are illustrative in all aspects and not restrictive.

Claims (11)

A method for producing a small intestine organs from embryonic stem cells, comprising the steps of:
(a) preparing a three-dimensional embryoid body from embryonic stem cells;
(b) characterizing said cultures as endoderm lines;
(c) inducing the expression of the Cdk2 gene in the endoderm to be specialized in the small intestine; And
(d) culturing the small intestine organs in which the Cdk2 expression is induced.
The method according to claim 1,
Wherein said embryonic stem cells are mouse embryonic stem cells.
The method according to claim 1,
Wherein said culturing of said step (a) is carried out by culturing mouse embryonic stem cells for 3 to 10 days.
The method according to claim 1,
Wherein the step (b) is carried out by treating the cultured body with Actin A (Actin A) and culturing.
The method according to claim 1,
Wherein the endoderm expresses the Foxa2 gene.
The method according to claim 1,
In the step (c), the expression of the Cdk2 gene is characterized by simultaneous treatment of a Wnt / β-catenin signaling pathway activator and a Notch signaling pathway inhibitor Lt; / RTI >
The method according to claim 6,
The Wnt / beta-catenin signaling pathway activator may be selected from the group consisting of 6-bromoindirubin-3'-oxime (GSK-3beta) inhibitor, glycogen synthase kinase- (6-bromoindirubin-3'-oxime; BIO).
The method according to claim 6,
The notch signaling inhibitor may be selected from the group consisting of a gamma-secretase inhibitor N- [N- (3,5-difluorophenacetyl) -L-alanyl] -S-phenylglycine tert-butyl ester (DAPT), which is characterized in that it is N- [N- (3,5-difluorophenacetyl) -L-alanyl] -S-phenylglycine t-butyl ester.
9. The method according to claim 7 or 8,
Wherein said BIO and DAPT are each treated at a concentration of 0.5 to 10 uM and 1 to 20 uM.
A small intestine organs produced by the method of any one of claims 1 to 8.
11. The method of claim 10,
Wherein the small intestine organs have the following characteristics:
(a) there is a paneth cell specific granule present in the small intestine mucosa;
(b) small intestinal stem cell markers Msi1 and Lgr5; Differentiation markers Lysozyme, Muc2, and ChgA; And Cck, a long-acting hormone; And
(c) Small intestinal paranuclear cells and stem cells are present.

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