JP2007014273A - Hepatic tissue/organ and method for preparation thereof - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for efficiently preparing a hepatic tissue/organ having high hepatic function in high probability to enable the differentiation induction of not only a hepatic parenchymal cell but also non-hepatic parenchymal cell such as vascular cell and bile duct cell from single embryonic stem cell without carrying out the administration of a drug and the transfer of exogenote to an embryonic stem cell and provide a hepatic tissue/organ prepared by the method. <P>SOLUTION: The method for the preparation of a hepatic tissue/organ contains a subclone separation step to separate subclone by the recloning of an embryonic stem cell, a screening step to form a plurality of embryoids derived from the subclone simultaneously under the same conditions, carry out the adhesion culture of the embryoid and select a subclone characterized in that the number of embryoids pulsating after 48 hr from the start of the adhesion culture is ≥70% of the total number of the embryoids, and a subclone culture step to culture the screened subclone. The invention further provides the hepatic tissue/organ prepared by the method. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a method for producing liver tissue / organ from embryonic stem cells, and a liver tissue / organ produced by the method.

Conventionally, treatment with an organ transplant or an artificial organ has been performed on a biological tissue or organ that has been damaged by a disease or an accident and has suffered a serious dysfunction or dysfunction.
In particular, the liver is an organ that performs many chemical reactions essential for the maintenance of life, such as metabolic reactions and synthesis reactions of substances necessary for life, and detoxification reactions of toxic substances. It is extremely difficult to substitute with. In addition, organ transplantation inevitably causes rejection of the transplanted liver, and has a problem of an absolute donor shortage in addition to side effects due to immunosuppression for suppressing the rejection.

  In order to solve these problems, an artificial liver using a combination of living hepatocytes has been studied, and a hybrid type artificial liver (biohybrid type artificial liver) composed of a combination of the hepatocytes and an artificial device has been proposed. Specifically, a bioreactor in which living body-derived hepatocytes are held in contact with a hollow fiber in a hollow fiber module is known as an artificial liver (see, for example, Patent Documents 1 and 2). Attempts have also been made for clinical applications using such bioreactors.

However, in the hollow fiber module of the bioreactor, there is a structural limitation that oxygen and nutrients can be supplied only to hepatocytes in contact with the hollow fiber, and there is a problem that the upper limit value of the cell density is limited. .
Furthermore, since the hepatocytes derived from living organisms used for the hybrid type artificial liver are mainly hepatocytes isolated from the liver, they are poorly proliferative, have a short life span, and are difficult to cultivate for a long time. There is a problem that the function as a hepatocyte is significantly reduced. For this reason, the hybrid-type artificial liver can only replace a part of the original liver function for a short period of time due to the structural limitations of the hollow fiber module and the performance of the hepatocytes used. Yes.

On the other hand, in recent years, embryonic stem cells having the ability to differentiate into various cells and the ability to proliferate are known as regenerative medicine and cell medicine techniques for regenerating and supplementing lost cells, tissues, and organs. A method using an ES cell has attracted attention.
The embryonic stem cell is obtained from an undifferentiated cell mass in a blastocyst, can be repeatedly expanded and passaged in vitro, and selectively induces differentiation of a cell having a target function Therefore, application to various diseases is expected.

However, as a liver regeneration technique using the embryonic stem cells, only differentiation of hepatocytes is induced using various differentiation factors (see Non-Patent Documents 1 to 5). Even when hepatocytes obtained from the embryonic stem cells are applied to the hybrid type artificial liver, liver functions are not sufficient with hepatocytes alone, and there are structural limitations on the modules described above. Therefore, there is a problem that an artificial liver satisfying the functions of the original liver tissue / organ cannot be obtained.
Therefore, at present, regeneration of liver tissues / organs having an original three-dimensional configuration and function has not been achieved yet.

JP-A-9-56814 JP 2004-166717 A K. Abe, H .; Niwa, et al. Exp Cell Res. , 229 (1), 27-34 (1996) M.M. Schuldiner, O.M. Yanuka, et al. Proc Natl Acad Sci USA. , 97 (21), 11307-11212 (2000) T. T. et al. Hamasaki, Y. et al. Iiboshi, et al. FEBS Lett. 497 (1), 15-19 (2001) N. Lumelsky, O .; Blondel, et al. Science. , 292 (5520), 1389-1394 (2001) S. Yamada, H .; Kojima, et al. Am J Physiol Gastrointest Liver Physiol. , 281 (1), 229-36 (2001)

An object of the present invention is to solve the conventional problems and achieve the following objects.
That is, the present invention is based on the versatility and embryological knowledge of embryonic stem cells, and it is possible to develop embryonic stem cells from embryonic stem cells without developing drugs or introducing foreign genes. Based on this, it is possible to induce differentiation of liver parenchymal cells together with non-hepatic parenchymal cells such as vascular cells and bile duct cells as well as actual liver tissues and organs and liver tissues / organisms with high liver function It is an object of the present invention to provide a method for producing an organ with high probability and embryologically efficiently, and a liver tissue / organ produced by the method.

In order to solve the above-mentioned problems, the present inventors have conducted extensive studies, and as a result, recloned parental cells of embryonic stem cells, and subclones with high differentiation ability based on the pulsation rate of the obtained subclones. And the subclone was used to obtain a new finding that mature liver tissue having a liver-specific function can be differentiated with high probability.
It is known that the signal from the heart primordium is important for the development of the liver, but in the induction of liver tissue differentiation from embryonic stem cells, by using subclones screened based on the pulsation rate In addition, a technique capable of efficiently producing liver tissues / organs only from embryonic stem cells is not known, which is a new finding of the present inventor. The present invention is based on such findings by the present inventors, and means for solving the problems are as follows. That is,
<1> a subclone separation step of recloning embryonic stem cells to separate subclones;
Among the subclones, a plurality of embryoid bodies derived from the subclones are simultaneously formed under the same conditions, and then the embryoid bodies are adhered and cultured, and the embryo is beating 48 hours after the start of the adhesion culture. A screening step of selecting a subclone in which the number of bodies is 70% by number or more with respect to the number of all embryoid bodies;
And a subclone culturing step for culturing the screened subclone. In the method for producing a liver tissue / organ described in <1>, a subclone of the embryonic stem cell is separated by the subclone separation step, and a plurality of embryoid bodies derived from the subclone are simultaneously obtained by the screening step. After forming under the same conditions, the embryoid bodies are adherent cultured, and the number of the embryoid bodies pulsating 48 hours after the start of the adhesion culture is equal to the number of all the embryoid bodies. A subclone that is 70% by number or more is selected, and the selected subclone is cultured in the subclone culture step. As a result, liver tissue and organs are efficiently developed embryologically.
<2> The method for producing a liver tissue / organ according to <1>, wherein, in the screening step, 96 to 480 embryoid bodies derived from subclones are simultaneously formed under the same conditions, and adhesion culture is performed. In the method for producing a liver tissue / organ described in <2>, the number% of the beating embryoid bodies in 96 to 480 embryoid bodies is determined. As a result, screening is performed reliably.
<3> In the screening step, a plurality of embryoid bodies derived from subclones are simultaneously formed under the same conditions, and then the embryoid bodies are adhered and cultured, and the pulsating embryo 24 hours after the start of the adhesion culture The method for producing a liver tissue / organ according to any one of <1> to <2>, wherein a subclone having a number of striatum of 50% by number or more with respect to the number of all embryoid bodies is selected. is there. In the method for producing a liver tissue / organ described in <3>, a plurality of embryoid bodies derived from the subclone are simultaneously formed under the same conditions, and then the embryoid bodies are adhered and cultured. A subclone is selected in which the number of embryoid bodies pulsating after 24 hours is 50% by number or more based on the number of all embryoid bodies. As a result, liver tissue and organs are efficiently produced with high probability.
<4> The liver according to any one of <1> to <3>, wherein in the subclone culture step, the subclone is differentiated into cardiomyocytes, liver parenchymal cells, and liver non-parenchymal cells to form liver tissue / organ. This is a tissue / organ production method. In the method for producing a liver tissue / organ described in <4>, the subclone is differentiated into hepatic parenchymal cells and hepatic non-parenchymal cells. As a result, unlike the conventional artificial liver consisting only of liver parenchymal cells, the liver tissue / organ equivalent to the normal liver in the living body is produced.
<5> The method for producing a liver tissue / organ according to <4>, wherein the liver non-parenchymal cells are vascular endothelial cells. In the method for producing a liver tissue / organ described in <5>, the subclone is differentiated into vascular endothelial cells together with hepatocytes. As a result, unlike the conventional artificial liver consisting only of liver parenchymal cells, the long-lived and highly functional liver tissue / organ is produced.
<6> Recloning in the subclone separation step involves culturing embryonic stem cells in a medium containing LIF (leukemia inhibitory factor), obtaining a clone derived from the formed colony, and isolating the colony obtained by culturing the clone The method for producing a liver tissue / organ according to any one of <1> to <5>. In the method for producing a liver tissue / organ according to <6>, the subclone is isolated from a colony derived from a colony formed by culturing the embryonic stem cell in a medium containing LIF (leukemia inhibitory factor). Is done. As a result, a subclone maintaining an undifferentiated state is obtained.
<7> The method for producing a liver tissue / organ according to any one of <1> to <6>, wherein the foreign gene is not substantially introduced into the subclone in the subclone culture step. In the method for producing a liver tissue / organ described in <7>, the subclone is cultured without substantially introducing a foreign gene. As a result, a highly safe liver tissue / organ is efficiently produced.
<8> The method for producing a liver tissue / organ according to any one of <1> to <7>, wherein 96 to 480 embryoid bodies derived from the subclone are mixed and cultured in the subclone culture step. .
<9> A liver tissue / organ produced by the method according to any one of <1> to <8> and derived from a single cell of an embryonic stem cell subclone.
<10> The liver tissue / organ according to <9>, which is used as an artificial liver.
<11> A drug screening method comprising administering a test substance to the liver tissue / organ described in <9>. According to the drug screening method described in <11>, a test substance is administered to the liver tissue / organ and the target substance is efficiently screened.
<12> The drug screening method according to <11>, wherein the test substance is any one of a therapeutic drug for liver dysfunction, a functional food, and an active ingredient thereof.
<13> The drug screening method according to any one of <11> to <12>, wherein a search for any one of a cholesterol degradation promoting substance and a cholesterol biosynthesis inhibiting substance is performed.

  According to the present invention, non-liver parenchyma such as vascular cells and bile duct cells as well as hepatic parenchymal cells can be obtained from a single embryonic stem cell without substantially administering a drug or introducing a foreign gene into the embryonic stem cell. It is possible to provide a method for efficiently producing a liver tissue / organ having high liver function with high probability, and a liver tissue / organ produced by the method capable of inducing differentiation in combination with cells. .

(Manufacturing method of liver tissue / organ)
The method for producing a liver tissue / organ of the present invention includes a subclone separation step, a screening step, and a subclone culture step, and further includes other steps appropriately selected as necessary.

The specific cell culture operation in the method for producing hepatocytes / organs of the present invention can be performed according to a conventional method in the technical field, and the culture condition is 37 ° C. in the presence of 5% CO _2. Is preferred.
In addition, in the method for producing hepatocytes / organs of the present invention, the instrument / device used for cell culture can be performed using instruments / devices generally used in the technical field.

<Subclone separation process>
The subclone separation step comprises a step of recloning embryonic stem cells to separate subclones. The recloning refers to further cloning a parent cell clone obtained by cloning a parent cell. The subclone refers to a clone obtained by cloning from the parent cell clone.

The embryonic stem cell is not particularly limited as long as it is a cell having multipotency that can be cultured in vitro, is established by culturing an early embryo, and can differentiate into various cells constituting an adult. It can be appropriately selected according to the purpose. For example, ES (Embionic stem) cells established from the inner cell mass in the early embryo before implantation, EG (Embionic germ) cells established from germ cells, and cells obtained by culturing the embryonic stem cells, etc. These may be cell lines that have already been established.
As the embryonic hepatocytes, cells derived from various animals such as mammals, birds, and reptiles can be used. Among these, mice, rats, guinea pigs, hamsters, rabbits, cats, dogs, sheep, pigs can be used. Embryonic stem cells derived from mammals such as cattle, goats, monkeys, and humans are preferred.

The embryonic stem cells were recloned by culturing the embryonic stem cells in a medium containing LIF (leukemia inhibitory factor), obtaining single cells derived from the formed colonies, and culturing the single cells. This is done by separating the colonies.
Specifically, after growing the embryonic stem cells in an LIF (leukemia inhibitory factor) -containing medium while maintaining an undifferentiated state, a single cell state in which cells do not adhere to each other using an enzyme or the like is used. An undifferentiated embryonic stem cell clone is obtained from the embryonic stem cell in a single cell state, and the embryonic stem cell clone is cultured and proliferated while maintaining the undifferentiated state to form a colony. The method of obtaining a clone is mentioned.
Examples of the method for bringing the embryonic stem cell into a single cell state include a conventional operation method. Specifically, after removing the embryonic stem cell culture medium from the culture vessel in which the embryonic stem cells proliferated confluently, using phosphate buffered saline (PBS), washed several times, The trypsin-EDTA solution is administered to the culture vessel and left for 5 to 20 minutes. Thereafter, trypsin-EDTA solution is removed, the embryonic stem cells are suspended in the embryonic stem cell culture medium, and the embryonic stem cells are precipitated by centrifugation, and then suspended again in the embryonic stem cell culture medium. A method is mentioned.

  Examples of the embryonic stem cell culture medium include a medium obtained by adding serum, LIF (leukemia inhibitory factor), L-glutamine, 2-mercaptoethanol and the like to a minimum medium for cell culture. For example, DMEM (Dulbecco's modified Eagle medium) includes a medium comprising FBS (fetal bovine serum) 20%, sodium pyruvate 1 mM, non-essential amino acid 0.1 mM, 2-mercaptoethanol 0.1 mM, and LIF 1000 U / L. .

The method for culturing the embryonic stem cell or the embryonic stem cell clone is not particularly limited as long as it is a method capable of maintaining the characteristics of undifferentiated embryonic stem cells, and can be appropriately selected according to the purpose. For example, a method of co-culture with supporting cells using the above-mentioned embryonic stem cell culture medium includes, for example, mouse fetal fibroblasts treated with mitomycin C, STO cells, NHL7 cells, etc. Can be mentioned.
The cell density of the embryonic stem cells or the embryonic stem cell clones seeded in the culture vessel is preferably 10,000 to 80,000 cells / cm ² .

In the culture of the embryonic stem cell clone, in order to facilitate the acquisition of the subclones, a drug resistance gene is introduced into the embryonic stem cell clone and cultured on a drug-added medium to control the number of colonies formed. May be.
Examples of the drug resistance gene include a neomycin resistance gene, a puromycin resistance gene, and a hygromycin resistance gene.
Examples of the method for introducing the drug resistance gene include an electroporation method and a retrovirus vector method.

As a method for separating the subclones, for example, the embryonic stem cell clones are cultured in the embryonic stem cell culture medium for 4 to 7 days, and the formed colonies are aspirated using a pipette or the like under a stereomicroscope. And then suspending the colony in a single cell state in the embryonic stem cell culture medium.
As the colony, those having relatively fast growth are preferable, and those having a morphological feature such that, when observed under a stereomicroscope, the swell is high and flat cells are not present in the periphery, such as shown in FIG. Such forms of colonies are preferred.

<Screening process>
In the screening step, a plurality of embryoid bodies derived from the subclone are simultaneously formed under the same conditions, and then the embryoid bodies are cultured in adhesion, and the embryoid-like pulsation occurs 48 hours after the start of the adhesion culture. The method comprises a step of selecting a subclone whose body number is 70% by number or more with respect to the number of all embryoid bodies.

The embryoid body to be formed in the screening step is a structure similar to an embryo in which 500 to 2,000 subclones in a single cell state are aggregated, and the subclone is used in a medium having the same composition. However, those formed by suspension culture at the same temperature under the same conditions are preferred.
As a method for producing an embryoid body derived from the subclone, for example, the subclone in a single cell state is suspended in a medium for embryoid body formation not containing LIF so as to be 10 to 25 cells / μL. And suspension culture.
Examples of the suspension culture method include a hanging drop method in which a culture container in which a cell suspension is dropped is inverted to form a suspended drop and cultured.
As the culture vessel for the embryoid body, for example, a multiwell plate or the like is preferable. A 96-well plate is preferable as the multiwell plate.

  Examples of the embryoid body-forming medium include a medium substantially free from LIF, which is obtained by adding serum, amino acids, 2-mercaptoethanol, and the like to a minimal medium for cell culture. For example, IMDM ( A medium obtained by adding 20% FBS (fetal bovine serum), 1 mM sodium pyruvate, 0.1 mM non-essential amino acid, and 0.1 mM 2-mercaptoethanol to Iskov's modified Dulbecco medium).

Here, the pulsating embryoid body refers to an embryoid body that continuously contracts at a constant rhythm. Specifically, it is an embryoid body in which the contraction is observed continuously and regularly (for example, the interval between contraction and contraction is 0.1 to 10 seconds) when observed under a stereomicroscope.
The ratio of the number of embryoid bodies that are beating to the number of all embryoid bodies (hereinafter referred to as “beating rate”) is, for example, that the embryoid bodies are observed under a stereomicroscope and beaten. From the number of embryoid bodies that are present, it can be obtained by the following formula (1).
In the measurement of the pulsation rate, the total number of embryoid bodies is preferably 50 or more, and more preferably 100 or more.
Beating rate (number%) = [(number of beating embryoid bodies) / (total number of embryoid bodies)] × 100
Formula (1)

As the subclone, the pulsation rate of the embryoid body derived from the subclone is preferably 70% or more, more preferably 95% or more after 48 hours from the start of the adhesion culture, and the adhesion culture start. In particular, it is particularly preferably 50% or more after 24 hours.
When the pulsation rate of the embryoid body derived from the subclone is less than 70% after 48 hours from the start of the adhesion culture, the liver tissue / organ may not be obtained from the subclone.

<Subclone culture process>
The subclone culturing step comprises a step of differentiating the subclones screened in the screening step into cardiomyocytes, liver parenchymal cells, and liver non-parenchymal cells to form liver tissues / organs. Further, in the subclone culture step, a foreign gene is not substantially introduced into the subclone. For this reason, the said liver tissue and organ which are highly safe efficiently can be manufactured.
The subclones screened in the screening step are subcultured while maintaining an undifferentiated state using the embryonic stem cell culture medium until they are used for production of the liver tissue / organ. In addition, it is preferable that the 6th generation is the upper limit.

  The subclone screened in the screening step is suspended in the embryoid body-forming medium as a single cell state, and suspended in culture to form the embryoid body. Subsequently, the obtained liver-like tissue / organ can be produced by collecting 96-480 embryoid bodies obtained, seeding them in an adhesion culture container using a differentiation medium, and performing adhesion culture.

  Examples of the differentiation medium include a medium which is obtained by adding serum, amino acids, 2-mercaptoethanol, LIF and the like to a minimal medium for cell culture, and does not substantially contain LIF. For example, Iscove's Examples include a medium obtained by adding 20% FBS (fetal bovine serum), 1 mM sodium pyruvate, 0.1 mM non-essential amino acid, and 2-mercaptoethanol to modified DMEM (Iscob modified Dulbecco-Eagle medium).

The adhesion culture vessel is not particularly limited as long as it is a cell culture incubator capable of adhesion culture of the embryoid body, and can be appropriately selected according to the purpose. For example, a multiplate, a microwell plate , Petri dishes, flasks, dishes, bags, and hollow fiber devices. In the adhesion culture, a surface treatment may be performed in order to control adhesion between the embryoid body and the adhesion culture container.
Examples of the surface treatment include gelatin coating, collagen coating, fibronectin coating, laminin coating, and matrigel coating.

The recloned embryonic stem cell subclone is differentiated into cardiomyocytes, liver parenchymal cells, and liver non-parenchymal cells by the method for producing liver tissues / organs of the present invention. -18 days later, it has characteristics of normal liver tissue / organ in vivo both morphologically and histologically, and can be differentiated into the liver tissue / organ composed of liver parenchymal cells and liver non-parenchymal cells. it can.
Examples of the morphological characteristics include characteristics such as having a binuclear morphology and being positive for albumin staining.
Examples of the histological characteristics include having ammonia-degrading ability, protein (albumin) production ability, and testosterone hydroxylation activity.

<Cardiomyocytes>
The differentiation from embryonic stem cells to liver tissue has a correlation with the appearance of cardiomyocytes. From developmental findings, the endoderm is differentiated into the foregut by the signal from the heart primordia and pulsates. It is known to differentiate into liver diverticulum and liver bud by signals from cells and into liver tissue.
As a method for confirming the expression of the cardiomyocytes, it can be confirmed by starting the pulsation of the embryoid body colony, and gene expression of a cardiomyocyte-specific marker such as ANP, Nkx2.5, GATA4, or the myocardium This can be confirmed by expression of a cell-specific marker protein.

<Liver parenchymal cells>
It is known that the hepatocytes will emerge developmentally following the appearance of the cardiomyocytes in the embryoid body. Experimentally, it is known that the expression of hepatocytes is observed in the vicinity of the cardiomyocytes differentiated from embryoid bodies.
As a method for confirming differentiation into hepatocytes and differentiation into hepatocytes,
Transthyretin (TTR), α-fetoprotein (AFP) fetal liver cells, a1-antitrypsin (AAT), tyrosine aminotransferase (TAT), tryptophan oxygenase (TO), tyrosine aminotransferase, asialoglycoprotein receptor (ASGR), It can be confirmed by expression of a hepatocyte (fetal hepatocyte) -specific marker gene such as albumin, expression of a hepatocyte-specific marker protein, or immunostaining with an anti-albumin antibody.
In addition, since many cells having two nuclei are confirmed in mouse hepatocytes, it can be confirmed by morphologically confirming the cell nuclei.

<Non-liver cells>
Examples of the hepatic non-parenchymal cells include sinusoidal endothelial cells, Kupffer cells, and bile duct cells, and are known to be developmentally expressed almost simultaneously with the expression of the hepatic parenchymal cells. It is known that differentiation into cells is important.
As a method for confirming the differentiation ability into the non-hepatic parenchymal cells and the differentiation into the non-hepatic parenchymal cells, blood vessels such as CD31 / PECAM1, vascular endothelial growth factors VEGFR-1 and VEGFR-2, and angiogenesis regulating factor VEGF are used. It can be confirmed by endothelium-specific marker gene expression, vascular endothelium-specific marker protein expression, or immunostaining with CD31 / PECAM1 antibody.

The method for detecting the expression of the specific marker gene is not particularly limited and may be appropriately selected from known methods. Examples thereof include RT-PCR, in situ hybridization, and microarray.
There is no restriction | limiting in particular as the detection method of the said protein expression, It can select suitably from a well-known method, For example, ELISA method, Western blotting method, immunohistochemistry method etc. are mentioned.

  The method for producing a liver tissue / organ of the present invention is not limited to hepatic parenchymal cells but also vascular cells, bile duct cells, etc., from a single embryonic stem cell without administering a drug or introducing a foreign gene into the embryonic stem cell. Therefore, it can be suitably used for the production of liver tissues and organs described later.

(Liver tissue / organ)
The liver tissue / organ of the present invention is a liver tissue / organ produced by the above-described method for producing a liver tissue / organ of the present invention and derived from a single cell of an embryonic stem cell subclone.

The use of the liver tissue / organ is not particularly limited as long as the liver function is required, and can be appropriately selected according to the purpose. For example, it can be used as an artificial liver. Examples include bioartificial livers in fields such as regenerative medicine and cell medicine, extracorporeal circulation type artificial livers in hybrid type artificial liver devices, and bioreactors.
As the liver tissue / organ, it is preferable to use the liver tissue / organ on the 10th to 18th day from the start of the adhesion culture of the embryoid body, and the liver tissue / organ on the 15th to 18th day is used. Is more preferable.

  Examples of the bioartificial liver include those in which the liver tissue / organ is transplanted into the liver, spleen, or abdominal cavity and the liver is reconstructed in the body.

As the extracorporeal circulation type artificial liver, for example, a known dialysis apparatus or a blood circulation circuit in plasma separation is provided with a module filled with the liver tissue / organ, and a body fluid such as blood to be treated and the liver tissue・ Those that metabolize or remove harmful substances by bringing them into contact with organs.
The module is not particularly limited as long as it can circulate the liquid to be treated and can culture the liver tissue / organ, and can be appropriately selected according to the purpose. Examples include yarn modules and bioreactors.

  Examples of the bioreactor include those that produce a biologically active substance or useful substance derived from the liver using the reactor including the liver tissue / organ, such as a bioreactor for blood coagulation factor production.

  Since the liver tissue / organ of the present invention contains liver parenchymal cells and liver non-parenchymal cells, it is advantageous in that it has a high liver function as compared with a conventional artificial liver composed of only liver parenchymal cells.

(Drug screening method)
The drug screening method includes administering a test substance to the liver tissue / organ of the present invention, and further includes other steps appropriately selected as necessary.
As a specific method of the drug screening method, for example, a test substance is added to the liver tissue / organ culture medium of the present invention, and after incubation for a certain time, the culture medium and the liver tissue / organ are constituted. Examples thereof include a method for analyzing a metabolite contained in at least one of cells.

There is no restriction | limiting in particular as said test substance, According to the objective, it can select suitably, For example, hepatic dysfunction therapeutic agent, functional food, and these active ingredients are mentioned.
There is no restriction | limiting in particular as said hepatic dysfunction therapeutic agent, According to the objective, it can select suitably, Any of a novel therapeutic agent and the existing therapeutic agent may be sufficient. The screening of the new therapeutic agent is suitable for evaluating the effectiveness of the therapeutic agent, and the screening of the existing therapeutic agent is suitable for evaluating the effectiveness and suitability of the therapeutic agent.
There is no restriction | limiting in particular as said functional food, According to the objective, it can select suitably, For example, the said functional food etc. with which the liver function improvement effect is anticipated are mentioned. Screening using the functional food as a test substance is suitable for searching and evaluating useful substances and toxic substances in the functional food.

Examples of the drug screening include screening for components useful for improving liver function, screening for harmful components that damage the liver, and the like.
Examples of screening for components useful for improving liver function include searching for cholesterol degradation promoting substances and cholesterol biosynthesis inhibitors.

  Hereinafter, although the Example of this invention is described, this invention is not limited to this Example at all.

(Test example)
<Induction of embryonic stem cell differentiation>
-Culture of embryonic stem cells (parent cells)-
129 / Ola mouse-derived embryonic stem cells E14.1 ((P.O. Seglen, Methods Cell Biol., 13, 29-83 (1976), R. Kuhn, K. Rajewsky et al., Science., 254 (5032) 707-710 (1991)); a generous gift of Dr. Yoichiro Iwakura and Dr. Nobuaki Yoshida of the Institute of Medical Science, The University of Tokyo) was co-cultured with mitomycin C-treated mouse embryonic fibroblasts as feeder cells. The embryonic stem cell E14-1 is Dulbecco's modified Eagle medium (D-MEM; manufactured by Gibco), fetal bovine serum (FBS; manufactured by Hyclone) 20%, sodium pyruvate (Invitrogen) 1 mM, non-essential amino acid Using a medium containing 100 μM solution (Gibco), 100 μM M2-mercaptoethanol (Sigma) and 10 ³ U / ml LIF (Chemicon), the cells were cultured while maintaining undifferentiated characters.
The medium was changed every day, and the E14.1 cells were grown for 3 days until they reached confluence.

-Production of embryoid bodies-
The E14.1 cells that reached confluence were treated with PBS containing 0.05% trypsin, 1.0% chicken serum, and 1 mM EDTA to obtain a single cell state. Next, the E14.1 cells were placed in Iskov modified Dulbecco medium (IMDM; manufactured by Gibco) with 20% fetal bovine serum (FBS; manufactured by Hyclone), 1 mM sodium pyruvate (Invitrogen), a non-essential amino acid solution (Gibco). Suspended in a medium for embryoid body formation added with 100 μM and 100 μM of M2-mercaptoethanol (manufactured by Sigma) so that the cell suspension contains 1,000 E14-1 cells per 50 μL. Prepared.
Each well of the main body of a 96-well plate (IWAKI) was filled with 350 μL of mineral oil (manufactured by SIGMA) in advance, and the E14.1 cell suspension was placed in a site corresponding to each well of the inner part of the lid of the 96-well plate. 50 μL of the suspension was added dropwise. The lid on which the E14.1 cell suspension was mounted was inverted and combined on the 96-well plate body, and culture was started (hereinafter referred to as the hanging drop method). By the hanging drop method, the E14.1 cells were cultured at 37 ° C. for 5 days in an incubator aerated with 5% CO ₂ to obtain embryoid bodies.

-Differentiation induction-
A total of 20 embryoid bodies in one well of the 96-well plate were seeded in one well of a gelatin-coated 6 cm plate (manufactured by IWAKI) as an adhesion culture container. In the adhesion culture vessel, 4 mL of a differentiation medium (20% FCS, IMDM, 100 × non-essential amino acid, 100 × sodium pyruvate, mercaptoethanol) was added, and the above-mentioned in an incubator aerated with 5% CO ₂ at 37 ° C. Adhesive culture of embryoid bodies was started.
The adhesion culture vessels were divided into 4 groups, and growth factors were added to the differentiation medium based on the following conditions, followed by culturing for 18 days.
* 1: Fibroblast growth factor (Invitrogen) 100 ng / mL
* 2: Hepatocyte growth factor (Genzyme) 20 ng / ml
* 3: Mouse Oncostatin M (Genzyme) 10 ng / mL
In addition to the above, dexamethasone (manufactured by ISN Biomedical) 100 nM, ITS (composition: insulin 10 μg, transferrin 5 μg, selenium 5 ng / mL; manufactured by Invitrogen) 10 μg / mL, and nicotinamide (manufactured by Nacalai Tesque) 10 mM 6 Added on the day.

  The embryoid bodies adhered to the gelatin in the adhesion culture vessel and proliferated to form colonies. The embryoid body colonies on the 10th day from the start of the adhesion culture were observed under a stereomicroscope, and the embryoid body colonies in which pulsations were observed and the embryoid body colonies in which no pulsations were observed were collected. About the colony, the expression of the liver specific marker and the atrial specific marker was observed by RT-PCR method shown below. As a positive control for liver-specific markers, fetal hepatocytes and primary cultured adult hepatocytes isolated from mouse liver at 15 days of gestation and adult (10 weeks old) mouse liver by the method shown below, As a positive control for an atrial specific marker, fetal cardiomyocytes isolated by the method described below from a mouse heart at 15 days of gestation were used. Moreover, E14.1 cells before differentiation induction were used as negative subjects.

-Preparation of positive control cells-
(1) Fetal hepatocytes, fetal cardiomyocytes Liver and heart excised from 15-day-old mice were cut into small pieces, treated with Hanks solution supplemented with collagenase II, and treated in a conventional manner to release the cells. . Dulbecco's modified Eagle medium (Gibco) supplemented with 10% fetal bovine serum, 100 μM non-essential amino acid solution, penicillin 1000 U / ml, streptomycin-glutamine 1000 μg / mL, seeded on 6 cm dishes coated with gelatin. Was added and allowed to stand for several hours, and then the medium was changed. The prepared fetal liver cells (FL) and fetal heart cells (FH) were cultured at 37 ° C. while changing the medium daily in an incubator aerated with 5% CO ₂ .
(2) Primary cultured adult hepatocytes Hepatocytes from the liver of 129 / SvJ male mice (10 weeks old) by the collagenase perfusion method (PO Seglen, Methods Cell Biol., 13, 29-83 (1976)). A suspension was obtained. From the hepatocyte suspension, hepatocytes were recovered by 50% Percoll density gradient centrifugation. The obtained hepatocytes were seeded in a gelatin-coated dish and Dulbecco's modified Eagle medium (Gibco) supplemented with 10% fetal bovine serum, 100 μM non-essential amino acid solution, 1000 U / ml penicillin, and 1000 μg / ml streptomycin-glutamine. The product was allowed to stand for several hours and then the medium was changed to obtain primary cultured adult hepatocytes. The prepared primary cultured adult hepatocytes (AL) were cultured at 37 ° C. while changing the medium every day in an incubator aerated with 5% CO ₂ .

-Analysis by RT-PCR method-
About each embryoid body colony cultured under the conditions A to D shown in Table 1 above, pulsation is observed on the 10th day (240 hours later) after seeding in an adhesion culture vessel and starting the adhesion culture. And those without pulsation were selected. From the total 8 types of embryoid body colonies collected, MagEXtractor mRNA kit (manufactured by Toyobo Co., Ltd.) was used to prepare 2 μg of total RNA according to the attached manual, and superscript II first strand synthesis primer oligo ligogen in vitro Each cDNA was synthesized using the following method.
Here, the presence or absence of pulsation of the embryoid body colony was determined by observing the embryoid body colony under a stereomicroscope and determining a colony where continuous and regular contraction was observed as having pulsation.

  2 μg of total RNA was prepared from each of the fetal liver cells, fetal cardiomyocytes, primary cultured adult hepatocytes, and E14.1 cells in the same manner as the 8 embryoid body colonies, and each cDNA was synthesized. .

10 mmol / L Tris-HCl (pH 8.3), 50 mmol / L KCl, 1.5 mmol / L MgCl ₂ , 0.2 mmol / L dNTP, 5U using a solution prepared by adjusting each cDNA to 0.1 μg / μL. A reaction solution was prepared comprising / μL Ex Taq DNA polymerase (manufactured by Takara Bio Inc.) and 0.2 μmol / L of each primer shown below.

The primer is an albumin-specific primer (SEQ ID NO: 5′-GCTACGGGCACAGTGCTTG-3 ′, SEQ ID NO: 5′-CAGGATTGCAGACAGATATAGTC-3 ′, T. Hamazaki to evaluate the gene expression level of albumin as a hepatocyte marker. , Y. Iiboshi et al., FEBS Lett, 497 (1), 15-19 (2001)), and an ANP-specific primer (SEQ ID NO: SEQ ID NO :) for evaluating the gene expression level of atrial sodium peptide (ANP) as a cardiomyocyte marker 3: 5′-ATGGGCTCTCTTCATCAC-3 ′, SEQ ID NO: 5′-TGTTGCAGCCTAGTCCCACTC-3 ′).
Further, hypoxanthine phosphoribosyltransferase (hprt) specific primers (SEQ ID NO: 5: 5′-GTTGGATACAGGCCCAGACTTTGTTG-3 ′, SEQ ID NO: 6: 5′-GAGGGGTAGGCTGGCCCTATAGGGCT-3 ′) were used as controls.

  The reaction solution was reacted at 94 ° C. for 4 minutes for 1 cycle, then at 94 ° C. for 1 minute, 50 ° C. for 1 minute, 72 ° C. for 1 minute, 2 cycles, 94 ° C. for 1 minute, and 55 ° C. for 1 minute. The PCR reaction was performed under the conditions of 27 cycles of 1 minute at 72 ° C, 1 cycle at 94 ° C, 1 minute at 55 ° C, 1 minute at 55 ° C, and 10 minutes at 72 ° C. The solution after the PCR reaction was subjected to agarose gel electrophoresis, and the expression of specific genes corresponding to each primer was evaluated from the appearing bands. The results are shown in FIG.

++: expression was detected strongly +: expression was detected ±: expression was detected only slightly −: expression was not detected

  From FIG. 1 and Table 2, in the embryoid body colonies in which pulsations were observed, the expression of albumin gene was strongly observed regardless of the presence or absence of growth factor addition during culture. This demonstrates that cardiomyocytes and hepatocytes can be induced to differentiate from embryonic stem cells without the addition of growth factors, and in particular, the possibility of hepatocytes being induced by the presence of beating cells. It was.

Example 1
<Establishment of subclones by recloning>
The E14.1 cells were grown to confluence while maintaining an undifferentiated character as in the test example. The embryonic stem cell culture medium was removed, and the cultured E14.1 cells were washed twice with PBS (−), then separated with trypsin-EDTA medium, suspended in the embryonic stem cell culture medium, Cell state.
The gelatin-coated 10 cm dish (manufactured by IWAKI) was seeded with the E14.1 clone cells in a single cell state, and cultured for 5 days in an incubator aerated with 5% CO ₂ at 37 ° C. while changing the medium. The E14.1 clonal cells did not aggregate with each other, adhered to the inner surface of the dish, and repeatedly divided and proliferated to form colonies.

  The colonies were selected under a stereomicroscope, and 40 to 80 colonies were picked up using a glass pipette having a pore diameter of about 0.5 mm.

Each colony was treated with PBS containing 0.05% trypsin, 1.0% chicken serum, and 1 mM EDTA to form a single cell, and then the embryonic stem cell culture medium (Dulbecco's modified Eagle medium ( D-MEM (Gibco), fetal bovine serum (FBS; Hyclone) 20%, sodium pyruvate (Invitrogen) 1 mM, non-essential amino acid solution (Gibco) 100 μM, M2-mercaptoethanol (Sigma) 100 μ, LIF (Chemicon) 10 ³ U / ml), maintaining the undifferentiated character, and establishing My-1, Ab-3, and My-5 lines as subclones did.

The subclones of My-1, Ab-3, and My-5 were cultured in the same manner as in the test examples to prepare embryoid bodies, and the embryoid bodies for 10 96-well plates were obtained.
Subsequently, the embryoid bodies in one well of the 96-well plate were seeded on a gelatin-coated 96-well plate (manufactured by IWAKI) as an adhesion culture container, and a total of three were produced. In the adhesion culture vessel, 0.3 mL of the differentiation medium was added, and adhesion culture of the embryoid body was started in an incubator in which 5% CO ₂ was aerated at 37 ° C.
Every day (24 hours) from the start of culture in the adhesion culture container, the embryoid body colonies in the adhesion culture container were observed with a stereomicroscope to confirm the presence or absence of pulsation. The presence or absence of pulsation of the embryoid body colony was determined in the same manner as in the test example. The ratio of the number of colonies in which pulsation was observed among all the observed colonies is shown in FIG.

<Subclone differentiation>
The culture of each of the obtained My-1, Ab-3, and My-5 subclones was continued, and an embryoid body colony was collected 48 hours after seeding in an adhesion culture vessel, in the same manner as in the test example, The expression of albumin gene was evaluated by RT-PCR method. However, the embryoid body colonies were collected without sorting based on the presence or absence of pulsation.
As a result of agarose gel electrophoresis, bands showing the expression of the albumin gene were observed in My-1 and Ab-3, but a band showing the expression of the albumin gene of My-5 could not be visually confirmed. The results are shown in Table 3.

*: Pulsation rate 48 hours after seeding in an adherent culture vessel +: Expression was detected-: Expression was not detected

(Example 2)
<Induction of differentiation of recloned subclones>
(1) Differentiation evaluation of subclone -analysis by RT-PCR method-
Using the subclone Ab-3 obtained in Example 1 (the pulsation rate after 48 hours from the start of adhesion culture is 80%), the same as the condition D of the test example in which no growth factor was added to the differentiation medium. To obtain embryoid body colonies. Embryoid bodies on the 3rd and 5th days from the start of culture, and embryoid body colonies on the 2nd, 4th, 6th, and 8th days after seeding on the tissue culture plate were collected, respectively, and total RNA was obtained in the same manner as in the test examples. 2 μg of each was prepared, and each cDNA was synthesized. Further, 2 μg of total RNA was prepared from the fetal hepatocytes, the primary cultured adult hepatocytes, and the E14.1 cells in the same manner as in the test examples, and each cDNA was synthesized.

10 mmol / L Tris-HCl (pH 8.3), 50 mmol / L KCl, 1.5 mmol / L MgCl ₂ , 0.2 mmol / L dNTP, mol using a solution prepared by adjusting each cDNA to 0.1 μg / μL. / L, 5U / μL Ex Taq DNA polymerase (manufactured by Takara Bio Inc.) and 0.2 μmol / L of each primer shown below were prepared.

  The primer is a transthyretin (TTR) -specific primer (SEQ ID NO: 7: 5′-CTCACCACAGATGAGAAG-3 ′, SEQ ID NO: 8: 5) for evaluating the gene expression level of transthyretin as an endoderm-specific marker. ′ -GGCTGAGGTCTCCAATTC-3 ′, T. Hamazaki, Y. Iiboshi et al., FEBS Lett, 497 (1), 15-19 (2001)), α-fetoprotein (AFP) to evaluate the gene expression level of α-fetoprotein Specific primers (SEQ ID NO: 9: 5′-TCGTATTCCAACAGGAGGG-3 ′, SEQ ID NO: 10: 5′-AGGCTTTTGCTCACCAG-3 ′, T. Hamazaki, Y. Iiboshi et al., FEBS Lett, 497 (1), 15 19 (2001)), a1-antitrypsin (AAT) specific primer (SEQ ID NO: 11: 5′-AATGGAAGAAGCCATTCGAT-3 ′, SEQ ID NO: 12: 5′-AAAGACTGTTAGCTGCTGCAGC) to evaluate the gene expression level of a1-antitrypsin -3 ′, T. Hamazaki, Y. Iiboshi et al., FEBS Lett, 497 (1), 15-19 (2001)), and the albumin-specific primer used in the test examples were used as hepatocyte markers. In addition, as an undifferentiated cell marker, Oct-3 / 4 specific primer (SEQ ID NO: 13: 5′-AGCACGAGTGGAAAGCACT-3 ′, SEQ ID NO: 14: 5) was used to evaluate the gene expression level of the transcription factor Oct-3 / 4. '-CTCATTGTTGTCGGCCTTCCT-3') and the hprt-specific primer used in the test example was used as a control.

  The reaction solution was reacted at 94 ° C. for 4 minutes for 1 cycle, then at 94 ° C. for 1 minute, 50 ° C. for 1 minute, 72 ° C. for 1 minute, 2 cycles, 94 ° C. for 1 minute, and 55 ° C. for 1 minute. The PCR reaction was performed under the conditions of 27 cycles of 1 minute at 72 ° C, 1 cycle at 94 ° C, 1 minute at 55 ° C, 1 minute at 55 ° C, and 10 minutes at 72 ° C. The solution after the PCR reaction was subjected to agarose gel electrophoresis, and the expression of specific genes corresponding to each primer was evaluated from the appearing bands. The results are shown in FIG.

++: Expression was strongly detected.
+: Expression was detected.
±: Slight expression was detected.
-: Expression was not detected.

  From FIG. 3 and Table 4, expression of hepatocyte-specific genes was confirmed in cells in the embryoid body colony of the subclone Ab-3, and the subclone obtained by recloning was differentiated into hepatocytes. It was found to have the ability.

-Analysis by Western blotting-
The subclone Ab-3 obtained in Example 1 was used and cultured in the same manner as in condition D of the test example in which no growth factor was added to the differentiation medium to obtain embryoid body colonies. Embryoid body colonies at 4, 6, 8, 12, 16, and 18 days after seeding on the tissue culture plate were collected, and a cell lysate was prepared by the following method. In addition, cell lysates were prepared from the primary cultured adult hepatocytes, STO cells, and E14.1 cells in the same manner as the embryoid body colonies. The fetal hepatocytes and the primary cultured adult hepatocytes were prepared in the same manner as in the test examples.

Each of the cells was trypsinized, centrifuged and washed with PBS, and the cell residue was treated with 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Nonidet P-40, 0.1% SDS, 1% sodium deoxycholate, A cell lysate was prepared by dissolving in a buffer containing 2 mM EDTA, 1 mM PMSF (phenylmethylsulfonyl fluoride), 2 μg / mL aprotinin, 10 μg / mL leupeptin 5 μg / mL pepstatin.
The cell lysate is centrifuged at 4 ° C. and 12,000 rpm for 10 minutes, the supernatant is recovered, the amount of protein is measured using a BCA protein assay kit (Pierce), and the protein concentration in each solution is 10 μg. The sample solution was obtained by adjusting to be / mL.
15 μL of the sample solution was added on each lane of 8% polyacrylamide gel, and electrophoresis was performed at 120 mA for 1.5 hours at room temperature. The migrated protein was transferred from the polyacrylamide gel onto a polyvinylidene difluoride (PVDF) membrane (manufactured by Bio-Rad) at 0.3 V for 1 hour. After blocking the PVDF membrane on which the protein was transferred with 5% skim milk dissolved in PBS for 1 hour at room temperature,
The mixture was incubated with a peroxidase-labeled anti-rabbit sheep antibody (manufactured by Amersham Pharmacia Biotech) diluted with a 0.1% Tween 20-containing TBS-T solution supplemented with 0.1% BSA at room temperature for 1 hour. After the PVDF membrane is washed with a TBS-T solution, light is emitted using chemiluminescence (ECL, manufactured by Amersham Pharmacia Biotech), an X-ray film (RX-U, manufactured by Fuji Photo Film Co., Ltd.) is exposed, and the protein is removed. Detected. The results are shown in FIG.

  FIG. 4 confirmed that hepatocyte-specific albumin and cardiomyocyte-specific actin were produced from the cells in the embryoid body colony of the subclone Ab-3 obtained by recloning. From this, it was found that liver-like cells producing albumin can be obtained from an embryonic stem cell subclone obtained by recloning without adding a growth factor to the differentiation medium. Moreover, since the amount of albumin detected increased with time, it was found that the proportion of liver-like cells contained in the embryoid body colony increased with time, and differentiation was promoted.

(2) Production of growth factor In (1) above, since the embryoid body differentiated without adding a growth factor to the differentiation medium, the embryoid body itself may produce a growth factor. Investigated about.
As primers, HGF (hepatocyte growth factor) specific primer (SEQ ID NO: 15: 5'-AGACACCACACCCGCACAGT-3 ', SEQ ID NO: 16: 5'-ATAGGGGCAATAATCCCAAGG-3'), FGF (fibroblast growth factor) specific primer Gene expression was evaluated by the RT-PCR method in the same manner as in (1) above except that (SEQ ID NO: 17: 5′-ACCGAGAGGTTCAACCTGCC-3 ′, SEQ ID NO: 18: 5′-GCCATAGTGAGTCCGAGGACC-3 ′) was used. The results are shown in FIG.

  From FIG. 5, it was found that the embryoid bodies themselves produced growth factors after seeding on the tissue culture plate.

(Example 3)
<Involvement of growth factors in embryonic stem cell differentiation>
(1) Effect of growth factor addition at the early stage of differentiation Using the subclone Ab-3 obtained in Example 1, a group in which a growth factor was added to the differentiation medium in the same manner as in the condition C of the test example, and a test In the same manner as in the condition D of the example, the culture was divided into groups in which no growth factor was added to the differentiation medium, and embryoid body colonies were obtained respectively. Embryoid bodies colonies on the 10th day after seeding were collected on the tissue culture plate, 2 μg of total mRNA was prepared in the same manner as in the test examples, and cDNA was synthesized. In the same manner as in the test examples, 2 μg of total RNA was prepared from the fetal hepatocytes, the primary cultured adult hepatocytes, and the E14.1 cells, and each cDNA was synthesized.

10 mmol / L Tris-HCl (pH 8.3), 50 mmol / L KCl, 1.5 mmol / L MgCl ₂ , 0.2 mmol / L dNTP, 5U using a solution prepared by adjusting each cDNA to 0.1 μg / μL. A reaction solution was prepared comprising / μL Ex Taq DNA polymerase (manufactured by Takara Bio Inc.) and 0.2 μmol / L of each primer shown below.

  The primer is a tyrosine aminotransferase (TAT) specific primer (SEQ ID NO: 19: 5′-) for evaluating the gene expression level of tyrosine aminotransferase (TAT) as a mature hepatocyte (liver parenchymal cell) specific marker. ACCTTCAATCCCATCCGA-3 ', SEQ ID NO: 20: 5'-TCCCGAACTGGATAGGTAG-3', T. Hamazzi, Y. Iiboshi et al., FEBS Lett, 497 (1), 15-19 (2001)), and tryptophan oxygenase (TO) gene In order to evaluate the expression level, tryptophan 2,3-dioxykinase-specific primer (SEQ ID NO: 21: 5′-TGCGCAAGAAACTTCAGAGGTGA-3 ′, SEQ ID NO: 22: 5′-TGCGCAAGAACT) As the TCAGAGGTGA-3 ′) and hepatocyte marker, the albumin-specific primer used in the test example was used. Moreover, the hprt specific primer used in the test example was used as a control.

  The reaction solution was reacted at 94 ° C. for 4 minutes for 1 cycle, then at 94 ° C. for 1 minute, 50 ° C. for 1 minute, 72 ° C. for 1 minute, 2 cycles, 94 ° C. for 1 minute, and 55 ° C. for 1 minute. The PCR reaction was performed under the conditions of 27 cycles of 1 minute at 72 ° C, 1 cycle at 94 ° C, 1 minute at 55 ° C, 1 minute at 55 ° C, and 10 minutes at 72 ° C. The solution after the PCR reaction was subjected to agarose gel electrophoresis, and the expression of specific genes corresponding to each primer was evaluated from the appearing bands. The results are shown in FIG.

(2) Effect of growth factor addition in late differentiation stage Using the subclone Ab-3 obtained in Example 1, a group in which a growth factor was added to the differentiation medium in the same manner as in Condition C of the test example, and test In the same manner as in the condition D of the example, the culture was divided into groups in which no growth factor was added to the differentiation medium, and embryoid body colonies were obtained respectively. Embryoid bodies colonies on the 18th day after seeding on the tissue culture plate were collected, 2 μg of total mRNA was prepared in the same manner as in the test examples, and cDNA was synthesized. Further, in the same manner as in the test examples, 2 μg of total RNA was prepared from the primary cultured adult hepatocytes and E14.1 cells, and each cDNA was synthesized.

10 mmol / L Tris-HCl (pH 8.3), 50 mmol / L KCl, 1.5 mmol / L MgCl ₂ , 0.2 mmol / L dNTP, mol using a solution prepared by adjusting each cDNA to 0.1 μg / μL. / L, 5U / μL Ex Taq DNA polymerase (manufactured by Takara Bio Inc.) and 0.2 μmol / L of each primer shown below were prepared.

The primer expresses the gene expression level of the tyrosine aminotransferase specific primer, the tryptophan 2,3-dioxykinase specific primer, and the asialoglycoprotein receptor (ASGR) as a mature hepatocyte (hepatocyte) specific marker. To investigate, an ASGR-1 specific primer (SEQ ID NO: 23: 5′-GCTGGAAAAACAGCCAGAAGG-3 ′, SEQ ID NO: 24: 5′-CGTTCTCTCCCACCCATTC-3 ′), and an ASGR-2 specific primer (SEQ ID NO: 25: 5 ′ -CGGACCCTGAAAGAAACCTT-3 ', SEQ ID NO: 26: 5'-ATGAAACTGGCTCCTGTGCT-3'), and the albumin-specific primer used in the test example was used as a hepatocyte marker .
Furthermore, as a biliary epithelial cell marker, in order to evaluate the gene expression level of liver-specific organic anion transporter (LST-1), an LST-1 specific primer (SEQ ID NO: 27: 5′-TGCGCAAGAAACTTCAGAGGTGA-3 ′, SEQ ID NO: 28: 5'-TGAGTTGGACCCCTTTTCAC-3 ') was used. As a control, the hprt-specific primer used in the test example was used.

  The reaction solution was reacted at 94 ° C. for 4 minutes for 1 cycle, then at 94 ° C. for 1 minute, 50 ° C. for 1 minute, 72 ° C. for 1 minute, 2 cycles, 94 ° C. for 1 minute, and 55 ° C. for 1 minute. The PCR reaction was performed under the conditions of 27 cycles of 1 minute at 72 ° C, 1 cycle at 94 ° C, 1 minute at 55 ° C, 1 minute at 55 ° C, and 10 minutes at 72 ° C. The solution after the PCR reaction was subjected to agarose gel electrophoresis, and the expression of specific genes corresponding to each primer was evaluated from the appearing bands. The results are shown in FIG.

  From FIG. 6, in the embryoid body colony on the 10th day after seeding on the tissue culture plate, the embryoid body cultured with the growth factor added to the differentiation medium is the same as the embryoid body cultured without addition. In comparison, the proportion of cells differentiated into hepatocytes was high, and it was found that the addition of growth factors accelerates differentiation into hepatocytes. However, from FIG. 7, in the embryoid body colony 18 days after seeding on the tissue culture plate, the embryoid body cultured with the growth factor added to the differentiation medium and the embryoid body cultured without the addition. There was no significant difference in the expression of hepatocyte-specific cells from the body, indicating that the addition of growth factors was not essential for embryoid body differentiation. In particular, the expression of LST-1 was observed only in the embryoid body cultured without adding a growth factor, and thus it was revealed that the differentiation into liver tissue may be inhibited by the addition of the growth factor. .

Example 4
<Histomorphological observation>
The subclone Ab-3 was cultured in the same manner as in Example 2 without adding a growth factor to the differentiation medium. The embryoid body colonies on the 10th day after seeding in the adhesion culture vessel and the embryoid body colonies on the 18th day were immunostained with an albumin antibody by the method shown below, and albumin producing cells were observed.

  The medium in the adhesion culture vessel was removed, PBS supplemented with 4% paraformaldehyde was added, and the embryoid body colonies were fixed for 30 minutes. Next, 0.1% triton X was added and treated at room temperature for 1 hour to increase the permeability of the cell membrane. The fixed embryoid body colony was treated with a blocking buffer (Jacson ImmunoResearch) supplemented with 4% donkey serum for 10 minutes at room temperature, and then diluted with a rabbit anti-mouse albumin antibody (Cappel) diluted 250-fold as a primary antibody. And a FITC-labeled donkey anti-goat IgG antibody (manufactured by Jackson ImmunoResearch) diluted 100 times as a fluorescent secondary antibody, reacted at room temperature for 1 hour, and fluorescence microscope ( BX60: manufactured by Olympus Corporation). FIG. 8B shows a photograph of the embryoid body colony on the 10th day after seeding in the adhesion culture vessel in which the albumin-producing cells are immunostained, and FIGS. 9A and 9B show photographs of the embryoid body colony on the 18th day. Moreover, the photograph which observed the embryoid body colony of the 10th day under white light is shown to FIG. 8A.

8A and 8B show that the albumin-producing cells form island clusters around the beating cardiomyocyte region (CA).
Further, FIG. 9A shows that the proportion of cells differentiated into albumin-producing cells is high in the embryoid body colonies on day 18, and from FIG. 9B, the albumin-producing cells are binuclear cells. I understand. Since cells having two nuclei are morphological characteristics of mature hepatocytes in normal mouse individuals, it was confirmed that albumin producing cells in embryoid body colonies were also morphologically hepatocytes.

(Example 5)
<Functional evaluation of embryoid body-derived hepatocytes>
Using the subclone Ab-3, the embryoid body was prepared in the same manner as in Example 2. A group of 30 embryoid bodies collected and a group of 50 embryoid bodies were seeded in the adhesion culture vessel and cultured for adhesion. The differentiation medium was cultured without adding growth factors to obtain embryoid body colonies on the 10th day and embryoid body colonies on the 18th day.
A serum-free medium (IMDM) supplemented with 2 mM NH ₄ Cl is added to each of the embryoid body colonies, the embryonic stem cells E14.1, the mouse primary cultured adult hepatocytes, and the mouse hepatocyte cell line HePa1-6. added and incubated at 37 ° C., _NH 4 Cl added after 6 hours, 12 hours, and after 24 hours, the _NH 4 Cl amount present in the serum-free medium was measured by an indophenol method, increased or decreased relative to the addition amount Evaluated. The results are shown in FIG.
In FIG. 10, “□” indicates the mouse primary cultured adult hepatocytes, “■” indicates 50 subclone Ab-3 embryoid colonies on the 18th day, and “◯” indicates the subclone Ab− on the 18th day. 3 embryoid body colonies 30; "●" is the result of the 10th day subclone Ab-3 embryoid body colony 30; "▼" is the result of HePa1-6 and "▲" is the result of E14.1 cells Respectively. In either case, genomic DNA was extracted from the used cells, the number of cells was measured, and the results were converted to the resolution per 100 cells.

  From FIG. 10, the embryoid body colonies differentiated from 30 embryoid bodies or 50 embryoid bodies of the subclone Ab-3 show high ammonia resolution, and colonies obtained by collecting and culturing a large number of embryoid bodies Was found to have differentiated into liver tissue exhibiting high liver function.

(Example 6)
<Differentiation into liver tissue>
-Analysis by RT-PCR method-
The subclone Ab-3 obtained in Example 1 was used and cultured in the same manner as in Condition D of Test Example in which no growth factor was added to the differentiation medium to obtain embryoid body colonies. Embryoid bodies on the 3rd and 5th days from the start of culture, and embryoid body colonies on the 2nd, 4th, 6th, and 8th days after seeding on the tissue culture plate were collected, respectively, and total RNA was obtained in the same manner as in the test examples. 2 μg of each was prepared, and each cDNA was synthesized. Further, 2 μg of total RNA was prepared from the fetal liver cells and E14.1 cells in the same manner as in the test examples, and each cDNA was synthesized.

10 mmol / L Tris-HCl (pH 8.3), 50 mmol / L KCl, 1.5 mmol / L MgCl ₂ , 0.2 mmol / L dNTP, 5U using a solution prepared by adjusting each cDNA to 0.1 μg / μL. A reaction solution was prepared comprising / μL Ex Taq DNA polymerase (manufactured by Takara Bio Inc.) and 0.2 μmol / L of each primer shown below.

  The primer is a PECAM-1-specific primer (SEQ ID NO: 29: 5′-GTCATGGCCATGGTCAGTAG-3 ′, SEQ ID NO: 30: 5′-) as a vascular endothelial cell-specific marker for examining the gene expression level of CD31 / PECAM1. AGCAGGACAGGTCCCAACAAC-3 ′), VEGFR-1 specific primer (SEQ ID NO: 31: 5′-TGTGGAGAACTACTGGTGACTCT-3 ′, SEQ ID NO: 32: 5′-TGGAGAACAGCAGGACTCCTT-3 ′) in order to examine the gene expression level of vascular endothelial growth factor And VEGFR-2 specific primers (SEQ ID NO: 33: 5'-TCTGTGGTTCTGCGTGGAGA-3 ', SEQ ID NO: 34: 5'-GTATCATTTCCAACCACCC-3') and blood vessels To examine the gene expression levels of endogenous regulatory factors, VEGF-specific primers (SEQ ID NO 35: 5'-CAGGCTGCTGTAACGATGAA-3', SEQ ID NO 36: 5'-AATGCTTTCTCCGCTCTGAA-3') was used. Moreover, the Oct-3 / 4 specific primer used in Example 2 was used as an undifferentiated cell marker, and the hprt specific primer used in the test example was used as a control.

  The reaction solution was reacted at 94 ° C. for 4 minutes for 1 cycle, then at 94 ° C. for 1 minute, 50 ° C. for 1 minute, 72 ° C. for 1 minute, 2 cycles, 94 ° C. for 1 minute, and 55 ° C. for 1 minute. The PCR reaction was performed under the conditions of 27 cycles of 1 minute at 72 ° C, 1 cycle at 94 ° C, 1 minute at 55 ° C, 1 minute at 55 ° C, and 10 minutes at 72 ° C. The solution after the PCR reaction was subjected to agarose gel electrophoresis, and the expression of a specific gene corresponding to each primer was evaluated from the appeared bands. The results are shown in FIG.

++: Expression was strongly detected.
+: Expression was detected.
±: Slight expression was detected.
-: Expression was not detected.

  From FIG. 11 and Table 5, it was found that the embryoid body differentiated from the subclone Ab-3 was not only differentiated into hepatocytes but also differentiated into vascular endothelial cells. From this, it was revealed that the embryoid body differentiates at the gene level in the same manner as the ontogenetic time axis, and has the possibility of constructing liver tissues and organs.

-Morphological analysis by immunostaining-
From the subclone Ab-3, embryoid bodies were prepared in the same manner as in Example 2. Twenty embryoid bodies were collected and seeded in the adhesion culture vessel. The differentiation medium was cultured without adding growth factors. The embryoid body colonies (hereinafter referred to as A10) on the 10th day after seeding in the adhesion culture vessel and the embryoid body colonies (hereinafter referred to as A18) on the 18th day were immunostained. Albumin producing cells were stained in the same manner as in Example 4 to develop a red color. For vascular endothelial cells, goat IgG anti-mouse PECAM-1 antibody (manufactured by Santa cruz Biotechnology) diluted 250-fold as a primary antibody and TRITC-labeled porcine anti-rabbit antibody (DAKO A / S) diluted 60-fold as a secondary antibody. The product was dyed and colored green in the same manner as in Example 4 except that the product was used. Stained embryoid body colonies were observed using a fluorescence microscope and a confocal microscope. The results are shown in FIGS.

FIG. 12 is a photograph in which only the A10 vascular endothelial cells are stained. From FIG. 12, it was found that vascular endothelial cells were detected in a mesh pattern and formed a blood vessel-like structure.
FIG. 13 is a photograph of the albumin cells and vascular endothelial cells of A10. From FIG. 13, it was found that the vascular endothelial cells extended and contacted the region of the albumin producing cell group. FIG. 14 is a photograph of the albumin cells and vascular endothelial cells of A18. Compared with A10 in FIG. 13, it was found that the vascular endothelial cells proliferated while building a network structure in parallel with the albumin producing cell group, and formed a network with the albumin producing cells. From these facts, it was revealed that the subclone differentiates not only into hepatocytes but also into vascular endothelial cells, and as a result, liver tissue is formed.

(Example 7)
<Influence of vascular endothelial cells on liver tissue formation>
-Morphological analysis by immunostaining-
The subclone Ab-3 was cultured in the same manner as in Example 2 without adding growth factors to the differentiation medium to obtain embryoid body colonies. Further, N- (2,6-dioxo-3-piperidyl) phthalimide (thalidomide, manufactured by Tocris Cookson) was dissolved in dimethyl sulfoxide (DMSO) at concentrations of 25 μg / mL and 100 μg / mL in the differentiation medium. The culture was carried out in the same manner as in Example 2 except that the added one was added to obtain embryoid body colonies.
For each embryoid body, the pulsation rate on the third day after seeding in the adhesion culture vessel was observed. The results are shown in Table 6.

Table 6 revealed that the addition of thalidomide to the differentiation medium decreased the pulsation rate of the embryoid body and inhibited differentiation into cardiomyocytes.

  Embryoid body colonies (A18) cultured with 100 μg / mL of thalidomide added to the differentiation medium were immunostained for albumin-producing cells and vascular endothelial cells in the same manner as in Example 4, and fluorescence microscopy and confocal Observation was performed using a microscope. The results are shown in FIG. FIG. 16 shows the results of embryoid body colonies obtained by culturing without adding thalidomide.

  Moreover, about each obtained embryoid body colony, the area of the expression area | region of the vascular endothelial cell was measured using the image analysis software (Scion image Beta 4.0.2), and the comparison was performed. The measurement was performed by selecting 15 regions from 3 types of each colony, obtaining the average value and standard deviation, and analyzing by Student's t-test. The significance level was P <0.001. The results are shown in FIG.

  From FIG. 15 and FIG. 16, it was found from tissue observation that the expression of vascular endothelial cells and the expression of albumin producing cells were also suppressed by adding thalidomide to the differentiation medium. FIG. 17 also shows that the area of the vascular endothelial cell expression region was reduced to about 20% by adding thalidomide to the differentiation medium.

-Analysis by RT-PCR method-
The subclone Ab-3 obtained in Example 1 was used in the same manner as in the test examples except that the conditions shown in Table 7 below were used, to obtain embryoid body colonies. Embryoid body colonies (A18) on the 18th day after seeding on the tissue culture plate were collected, 2 μg of total RNA was prepared in the same manner as in the test examples, and each cDNA was synthesized. Further, 2 μg of total RNA was prepared from the E14.1 cells, the fetal cardiomyocytes, the fetal hepatocytes, and the primary cultured living hepatocytes in the same manner as in the test examples, and each cDNA was synthesized.
* 1: N- (2,6-dioxo-3-piperidyl) phthalimide (thalidomide, manufactured by Tocris Cookson)
* 2: Same as condition C in the test example

10 mmol / L Tris-HCl (pH 8.3), 50 mmol / L KCl, 1.5 mmol / L MgCl ₂ , 0.2 mmol / L dNTP, 5U using a solution prepared by adjusting each cDNA to 0.1 μg / μL. A reaction solution was prepared comprising / μL Ex Taq DNA polymerase (manufactured by Takara Bio Inc.) and 0.2 μmol / L of each primer shown below.

  The primers include PECAM-1 specific primers (SEQ ID NOs: 29 and 30), VEGFR-1 specific primers (SEQ ID NOs: 31 and 32), and VEGFR-2 specific primers (SEQ ID NOs: 33 and 34) used in Example 6. ), And the albumin-specific primer (SEQ ID NO: 1 and 2) used in the test example and the tyrosine aminotransferase (TAT) -specific primer (SEQ ID NO: 19 and 20) used in Example 1. As a control, the hprt-specific primer (SEQ ID NOs: 5 and 6) used in the test examples was used.

  The reaction solution was reacted at 94 ° C. for 4 minutes for 1 cycle, then at 94 ° C. for 1 minute, 50 ° C. for 1 minute, 72 ° C. for 1 minute, 2 cycles, 94 ° C. for 1 minute, and 55 ° C. for 1 minute. The PCR reaction was performed under the conditions of 27 cycles of 1 minute at 72 ° C, 1 cycle at 94 ° C, 1 minute at 55 ° C, 1 minute at 55 ° C, and 10 minutes at 72 ° C. The solution after the PCR reaction was subjected to agarose gel electrophoresis, and the expression of a specific gene corresponding to each primer was evaluated from the appearing band. The results are shown in FIG.

++: Expression was strongly detected.
+: Expression was detected.
±: Slight expression was detected.
-: Expression was not detected.

  From FIG. 18 and Table 8, it was found that the embryoid body differentiated from the subclone Ab-3 was suppressed in the expression of vascular endothelial cells at the gene level in a concentration-dependent manner by the addition of thalidomide. In addition, since the expression of albumin is also suppressed, the presence of cells differentiated into vascular endothelial cells in the induction and proliferation of hepatocytes in vitro derived from embryonic stem cells, similar to the findings in vivo Was found to be necessary.

The method for producing liver tissue / organ of the present invention differentiates not only single hepatocytes but also non-hepatocytes such as vascular cells and bile duct cells without using differentiation factors for embryonic stem cells. It can be induced and can efficiently produce liver tissues / organs with high liver function with high probability, so in the production of bioartificial liver in fields such as regenerative medicine and cell medicine, The recloning method in the method for producing a liver tissue / organ of the present invention can be applied to the production of a chimeric mouse.
In addition, the liver tissue / organ of the present invention produced by the production method can be suitably used as a bioartificial liver or an artificial liver for transplantation. It can be suitably used as an evaluation device.

FIG. 1 is an electrophoretogram showing the results of a test example. FIG. 2 is a graph showing the ratio of the number of colonies in which pulsation was observed in Example 1. FIG. 3 is an electrophoresis photograph showing the results of RT-PCR in Example 2. 4 is a photograph showing the results of Western blot analysis (protein migration) in Example 2. FIG. FIG. 5 is an electrophoresis photograph showing the results of RT-PCR in Example 2. FIG. 6 is an electrophoresis photograph showing the results of RT-PCR in Example 3 (1). FIG. 7 is an electrophoresis photograph showing the results of RT-PCR in Example 3 (2). FIG. 8A is a photograph of an embryoid body colony observed 10 days after seeding in an adherent culture vessel in Example 4. FIG. 8B is a photograph of an embryoid body colony 10 days after seeding of an adhesion culture vessel immunostained in Example 4. FIG. 9A is a photograph of an embryoid body colony 18 days after seeding of an adhesion culture vessel immunostained in Example 4. FIG. 9B is a photograph of an embryoid body colony 18 days after seeding of an adhesion culture vessel immunostained in Example 4. The position of the nucleus is indicated by a white circle. FIG. 10 is a graph showing the evaluation results of ammonia resolution in Example 5. FIG. 11 is an electrophoresis photograph showing the results of RT-PCR in Example 6. FIG. 12 is a photograph showing the results of tissue immunostaining of embryoid body colonies of Example 6. FIG. 13 is a photograph showing the results of tissue immunostaining of embryoid body colonies of Example 6. FIG. 14 is a photograph showing the results of tissue immunostaining of embryoid body colonies of Example 6. FIG. 15 is a photograph showing the results of tissue immunostaining in Example 7. FIG. 16 is a photograph showing the results of tissue immunostaining in Example 7. FIG. 17 is a graph showing vascular endothelial cell expression regions of Example 7. FIG. 18 is an electrophoretogram showing the RT-PCR result of Example 7. FIG. 19 is a photograph showing an example of a preferred subclone form for separation in the subclone separation step of the method for producing liver tissue / organ of the present invention.

Claims (13)

A subclone isolation step of recloning embryonic stem cells to isolate subclones;
Among the subclones, a plurality of embryoid bodies derived from the subclones are simultaneously formed under the same conditions, and then the embryoid bodies are adhered and cultured, and the embryo is beating 48 hours after the start of the adhesion culture. A screening step of selecting a subclone in which the number of bodies is 70% by number or more with respect to the number of all embryoid bodies;
And a subclone culturing step for culturing the screened subclone.   The method for producing a liver tissue / organ according to claim 1, wherein in the screening step, 96 to 480 embryoid bodies derived from subclones are simultaneously formed under the same conditions, and adhesion culture is performed.   In the screening step, a plurality of embryoid bodies derived from subclones are simultaneously formed under the same conditions, and then the embryoid bodies are adhered and cultured, and the embryoid bodies pulsating 24 hours after the start of the adhesion culture The method for producing a liver tissue / organ according to any one of claims 1 to 2, wherein a subclone having a number of 50% by number or more with respect to the number of all the embryoid bodies is selected.   The method for producing a liver tissue / organ according to any one of claims 1 to 3, wherein in the subclone culture step, the subclone is differentiated into cardiomyocytes, liver parenchymal cells, and liver non-parenchymal cells to form liver tissues / organs. .   The method for producing a liver tissue / organ according to claim 4, wherein the liver non-parenchymal cells are vascular endothelial cells.   Recloning in the subclone separation step is performed by culturing embryonic stem cells in a medium containing LIF (leukemia inhibitory factor), obtaining a clone derived from the formed colony, and culturing the clone to isolate the colony obtained. The method for producing a liver tissue / organ according to any one of claims 1 to 5.   The method for producing a liver tissue / organ according to any one of claims 1 to 6, wherein the foreign gene is not substantially introduced into the subclone in the subclone culture step.   The method for producing a liver tissue / organ according to any one of claims 1 to 7, wherein in the subclone culture step, 96 to 480 embryoid bodies derived from subclones are mixed and cultured.   A liver tissue / organ produced by the method according to claim 1 and derived from a single cell of an embryonic stem cell subclone.   The liver tissue / organ according to claim 9, which is used as an artificial liver.   A drug screening method comprising administering a test substance to the liver tissue / organ according to claim 9.   The drug screening method according to claim 11, wherein the test substance is any one of a therapeutic drug for liver dysfunction, a functional food, and an active ingredient thereof. The drug screening method according to any one of claims 11 to 12, wherein either one of a cholesterol degradation promoting substance and a cholesterol biosynthesis inhibiting substance is searched.