JP2010075631A - Method for differentiating and inducing liver from undifferentiated cell - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a technique for generating a liver from an undifferentiated cell, to provide a biological material such as the cell or a tissue which is useful for a purpose, etc., to treat abnormality and disease of a human, etc., caused by the defect or deletion of a gene to be expressed in the liver of an adult, and also to provide an assay system biological material useful for the development of a medicine to prevent and treat the abnormality and disease. <P>SOLUTION: The method for differentiating and inducing the liver from the undifferentiated cell includes processing the undifferentiated cell in a dissociative state with the use of activin. Differentiation and induction are performed by the method so as to obtain the liver, a liver-derived tissue, or liver cells included in the liver and the tissue. In a medicine screening method, the medicine to treat the abnormality and disease of the liver is screened through the use of the liver, the tissue, and the liver cells. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

The present invention relates to a method for inducing differentiation of a liver from undifferentiated cells, a liver induced to differentiate by the method, a tissue derived from the liver, or a hepatocyte contained therein, and a liver abnormality or disease using these. The present invention relates to a method for screening a drug for preventing / treating the disease.

It has been found that the causative gene of cancer that develops in adults is a gene that is expressed only in the fetus and is important for embryogenesis. In response, genes that are expressed specifically in cancer gene therapy and drug development have begun to attract attention.

Xenopus laevis is early in development because of its advantages such as early development, easy judgment of results, easy egg collection, observation of the development process outside the body, and ease of functional analysis of genes. Has played an important role in research. Furthermore, genes that are key to organ development in Xenopus embryos are conserved in all vertebrates, including humans, and because their development is simpler than mammals, their mechanisms are easier to elucidate. Expected to be used as a model organism in gene identification and drug discovery.

So far, methods for producing various organs such as heart, pancreas, eye and kidney from undifferentiated cells of Xenopus have been developed (Non-Patent Documents 1 to 3). However, until now there has been no technique for inducing the liver from undifferentiated cells. The absence of such technology has become a serious obstacle in the process of elucidating the mechanism of liver induction in embryogenesis and developing therapeutic agents.

So far, differentiation induction from mouse embryonic stem cells to hepatocytes has been attempted in a medium supplemented with hepatocyte growth factor (HGF), dexamethasone, and the like (Non-Patent Document 4). Furthermore, induction of differentiation into hepatocytes from rat bone marrow-derived mesenchymal stem cells and mesenchymal stem cells derived from human wisdom wisdom has been attempted (Non-patent Document 5).
Chan TC., Et al., Naturwissenenschaften 86, 224-227 (1999) Moriya N., Et al., Dev. Growth. Differ. 42, 593-602 (2000) Ariizumi T., et al., Int. J. Dev. Biol. 47, 405-410 (2003) Hamazaki T., et al., FEBS Lett., 497, 15-19 (2001) Kiyohito Y., et al., YAKUGAKU ZASSHI 128 (1) 3-9 (2008)

Therefore, the main object of the present invention is to develop a technique for producing a liver from undifferentiated cells. Furthermore, the diagnosis of diseases such as humans that specifically lack or abnormally express proteins involved in liver parenchymal cell formation, the discovery of genes involved in hepatocellular carcinoma, and adults To prevent or treat biological materials such as cells or tissues useful for the purpose of treating abnormalities and diseases such as humans caused by defects or defects in genes expressed in the liver, and those abnormalities and diseases It is to provide biological materials for assay systems useful in the development of drugs.

The present inventor has found that the liver can be induced by treating activin when reassembling dissociated undifferentiated cells, for example, dissociated undifferentiated cells, and the present invention has been completed based on such findings. did.

That is, the present invention relates to the following aspects.
[First Embodiment] A method for inducing differentiation of a liver from undifferentiated cells, comprising treating dissociated undifferentiated cells with activin.
[Second embodiment] A liver induced to differentiate by the method of the present invention, a tissue derived from the liver, or a hepatocyte contained therein.
[Third Aspect] A method for screening a drug for preventing / treating liver abnormalities and diseases using the liver obtained by the present method, the liver-derived tissue, or hepatocytes contained therein.

By using the present invention, it becomes possible to induce the liver to differentiate from undifferentiated cells at a high rate. The liver thus induced has a liver-specific appearance, and is a protein (marker protein) unique to the liver. It was confirmed that genes Hex and transferrin are expressed. Furthermore, the diagnosis of diseases such as humans that specifically lack or abnormally express proteins involved in liver parenchymal cell formation, the discovery of genes involved in hepatocellular carcinoma, and adults Biological materials such as cells and tissues useful for the purpose of treating abnormalities and diseases such as humans caused by defects or defects in genes expressed in the liver, and the prevention and treatment of those abnormalities and diseases Biomaterials for assay systems useful in the development of drugs for the purpose are provided.

A feature of the method of the present invention is that the liver is differentiated by treating dissociated undifferentiated cells with activin. For example, in the step of forming a cell aggregate from dissociated undifferentiated cells, the undifferentiated cells can be treated with activin. In the present specification, the “dissociated state” of undifferentiated cells means that each undifferentiated cell is in a state where it can be substantially subjected to activin treatment. It is not necessary for the cell clumps to dissociate completely into individual cells.

There is no particular limitation on the method of treating dissociated undifferentiated cells with activin, and any method known to those skilled in the art can be used. For example, culturing dissociated undifferentiated cells in a medium containing an appropriate concentration of activin can be mentioned. As shown in the Examples, the concentration of activin in the medium, the treatment time, and the like are expressed in the cell aggregate formed from undifferentiated cells such as the genes Hex and transferrin, which are proteins specific to the liver (marker protein). Appropriate conditions can be set such that differentiation can be induced in liver tissue. Therefore, in the present specification, the term “liver” refers to a cell population having a tissue structure peculiar to the liver and significantly expressing the above-mentioned marker protein expressed specifically in the liver, as shown in Examples. Means the body (tissue).

As the conditions for activin treatment in the step of forming a cell aggregate from dissociated undifferentiated cells, for example, the concentration of activin in the medium is preferably higher than 10 ng / ml, for example, at least 100 ng / ml. The treatment time of undifferentiated cells with activin is preferably at least longer than 5 hours, for example, 24 hours. Usually, reassembly of dissociated cells is completed in about 30 minutes. Therefore, the activin treatment is continued even after dissociated cells reassemble and a cell aggregate is formed. In order to efficiently induce differentiation of the liver from undifferentiated cells, activin treatment such as the number of dissociated undifferentiated cells to be activin treated so that a cell aggregate containing at least about 5,000 cells is formed. It is desirable to appropriately adjust the various conditions and environments in advance.

Although the present invention is not bound by this theory, by treating dissociated undifferentiated cells with activin, each cell can be subjected to activin treatment. It is considered possible to induce differentiation of the liver at a high rate.

In order to prevent the activin to be treated from being adsorbed on the surface of a culture apparatus such as plastic, a serum-free medium containing an appropriate carrier protein such as bovine serum albumin having an appropriate concentration is used as the medium containing activin. It is preferable.

  In this specification, “undifferentiated cells” broadly mean cells in an undifferentiated state. For example, in addition to embryonic stem cells (ES cells), various tissue stem cells such as hematopoietic stem cells, neural stem cells, skin tissue stem cells, etc. It is a concept that includes the like.

  Furthermore, for example, induced pluripotent stem cells (iPS cells: Kazutoshi T. et al.) Obtained by introducing some genes selected from Oct3 / 4, Klf4, Sox2, and c-Myc into somatic cells. , Cell 131, 861-872, Nov., 2007), and cells to which pluripotency is imparted by artificial manipulation by genetic manipulation or the like are also included in “undifferentiated cells”.

There is no particular limitation on the method for preparing dissociated undifferentiated cells. For example, embryonic stem cells obtained by any dissociation method known to those skilled in the art from blastula, which are multicellular embryos resulting from cleavage of fertilized eggs, can be used. Incidentally, blastocysts are subdivided into early, intermediate, and late blastocysts, but late blastocysts are preferred. In particular, undifferentiated cells obtained from a cell layer containing an animal pole of a late blastula are preferred.

One of the processes of growing a fertilized egg into a fetus is a state called a blastocyst. The blastocyst has a spherical shape and is composed of a trophectoderm, which is an outer cell layer, and a blastocoel containing an inner cell mass that will form the body in the future. When undifferentiated cells such as ES cells, iPS cells, and tissue stem cells are cultured in a floating state, a large cell cluster similar to the blastocoel whose peripheral portion is differentiated like an endothelium is formed. In the present invention, this cell cluster is referred to as embryoid bodies (Takahashi et al., 2007; Hamazaki et al., 2001; Yagi et al. 2008). Therefore, undifferentiated cells obtained by dissociating embryoid bodies, which are cell clusters prepared as described above, in the same manner can also be used as undifferentiated cells in the present invention.

The embryoid body can be prepared according to a conventionally known technique. The cells constituting the embryoid body are not particularly limited as long as they are undifferentiated cells that can form embryoid bodies. For example, ES cells can be used as a starting material, and embryoid bodies that are aggregates of ES cells can be prepared by suspension culture in a serum medium or fetal serum-free medium containing fetal calf serum or the like.

For example, undifferentiated cells can be cultured by seeding on feeder cells as necessary. As the feeder cells, cells that have survived but not proliferated by treatment with X-rays, γ-rays, mitomycin C, and the like can be used. For example, mouse fetal fibroblasts treated with mitomycin C can be used as feeder cells for mouse-derived ES cells. Typically, the medium is changed every day, and subculture is performed every 3 days.

The cultured undifferentiated cells are made into a single cell suspension by trypsin treatment, and the obtained suspension is seeded in a culture vessel and cultured in a CO ₂ incubator. By this operation, feeder cells adhere to the culture surface, but undifferentiated cells do not adhere to the culture surface and remain floating. Therefore, undifferentiated cells can be separated from feeder cells by collecting the culture supernatant and centrifuging.

Next, the obtained undifferentiated cells are suspended and cultured overnight in a tilted, low-adhesive culture container, and then the container is placed in a horizontal state and further cultured for 2 to 3 days. To prepare. An embryoid body can also be prepared by culturing undifferentiated cells in a suspended state in droplets formed on mineral oil. As this embryoid body preparation medium, a medium containing fetal calf serum or a serum-free medium can be used.

The obtained embryoid body can be attached to the bottom surface of the culture container by culturing it in a serum medium or serum-free medium using an adhesive container.

The undifferentiated cells that can be used in the present invention are not particularly limited as long as they are cells derived from vertebrates including mammals, but preferably derived from mammals, particularly rodents or primates. Especially preferred are cells derived from humans.

After treatment with activin in this manner, the liver is usually induced to differentiate from undifferentiated cells at a high rate by further culturing the formed cell aggregate for an appropriate period, for example, 1 to 5 days. it can.

An undifferentiated cell forming a cell clump can be dissociated by any method known to those skilled in the art. The culture medium used for culturing the cell agglomeration, dissociation treatment from the cell agglomeration, and culturing the undifferentiated cells in the dissociated state may be appropriately selected from media known to those skilled in the art depending on the type of cells. it can. Since culturing at 25 ° C. or higher increases the possibility of cell death, cells and cell clumps are preferably cultured at a temperature of 25 ° C. or lower, preferably around 20 ° C. In addition, all the steps in the method of the present invention can be performed ex vivo (in vitro).

Activin, also called follicle stimulating hormone secretagogue, is a peptide hormone with a molecular weight of about 27,000. Activin includes activin A which is a homodimer of β chain of inhibin A (β _A β _A ), activin B which is a homodimer of β chain of inhibin B (β _B β _B ), and these There are three types of activin AB which are heterodimers (β _A β _B ). Nine cysteine residues are very well conserved in each activin monomer and belong to the TGF-β superfamily. The amino acid sequences of activins in vertebrates are very homologous, for example, 87% for Xenopus and human activin A and 95% for activin B. Many functions of activin in the living body are known, but the action of inducing differentiation of undifferentiated cells into the liver is not yet known.

The amino acid sequence of activin is known. For example, human-derived activin can refer to the database of each public institution based on the following ID number: NM_002192 (NCBI) Homo Sapiens inhibin, beta A (INHBA), mRNA.

In addition, as activin
(a) a protein having an amino acid sequence in which one or several amino acids are deleted, substituted, inserted or added in the amino acid sequence of natural activin and having the biological activity of activin; or
(b) consisting of an amino acid sequence having a homology of 90% or more, preferably 93% or more, more preferably 95% or more, and further preferably 99% or more with the amino acid sequence of natural activin, and the biological activity of cutivin Similarly, proteins having activity can be used in the present invention. These activins can also be prepared by any genetic engineering technique known to those skilled in the art.

Here, “homology” (or “identity”) is the same in the compatibility relationship between amino acid residues constituting the chain between two chains in the amino acid sequence of the polypeptide. Is the amount (number) of things that can be determined to be, and means the degree of sequence correlation between two polypeptide sequences or two polynucleotide sequences. Homology can be easily calculated. Many methods are known for measuring homology between two polynucleotide or polypeptide sequences, and the term "homology" is well known to those skilled in the art (e.g., Lesk, AM (Ed.), Computational Molecular Biology, Oxford University Press, New York, (1988); Smith, DW (Ed.), Biocomputing: Informatics and Genome Projects, Academic Press, New York, (1993); Grifin, AM & Grifin, HG (Ed. ), Computer Analysis of Sequence Data: Part I, Human Press, New Jersey, (1994); von Heinje, G., Sequence Analysis in Molecular Biology, Academic Press, New York, (1987); Gribskov, M. & Devereux, J. (Ed.), Sequence Analysis Primer, M-Stockton Press, New York, (1991), etc.). Common methods used to measure the homology of two sequences include Martin, J. Bishop (Ed.), Guide to Huge Computers, Academic Press, San Diego, (1994); Carillo, H. & Lipman , D., SIAM J. Applied Math., 48: 1073 (1988), etc., but are not limited thereto.

In the method of the present invention, the activin used may be the same or different species derived from the undifferentiated cells to be treated, but is preferably closely related to each other, in particular, It is preferable that they are derived from the same species.

Furthermore, the present invention also relates to various cells such as the liver thus induced to differentiate, tissues derived from the liver, or hepatocytes (liver parenchymal cells) contained therein. These can be used for, for example, various drug screening methods.

The screening of the present invention can be performed by any method known to those skilled in the art. By the screening method of the present invention, a drug (compound) for preventing / treating a liver abnormality or disease can be identified. The compound may be a substance originally contained in a living body such as a human or a substance synthesized artificially.

The screening method of the present invention can be carried out, for example, by the following steps.
(a) a step of bringing a test compound into contact with the liver or the like,
(b) a step of observing or measuring changes in expression of a liver genetic marker, etc. in the liver, etc., and
(c) selecting the compound that causes such fluctuations.

  In the screening method, the contact in the step (a) is carried out by contacting them by any means known to those skilled in the art, such as adding a test compound to the culture system such as the liver. I can do it. It should be noted that the change in expression of a liver genetic marker or the like can be performed, for example, by measuring the expression of Hex or transferrin, which is a gene of a protein (marker protein) unique to the liver, as shown in the Examples. I can do it.

EXAMPLES Hereinafter, although an Example demonstrates this invention in detail, the technical scope of this invention is limited and is not interpreted at all by description of a following example. Also, unless otherwise noted, the following examples include routine methods in the art, including immunostaining and Real-time PCR methods, and standard methods known to those skilled in the art, such as Sambrook and Maniatis, in Genetic engineering described in Molecular Cloning-A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York, 1989; Ausubel, FM et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, NY, 1995, etc. And performed according to molecular biological techniques. Moreover, the description content of the literature cited as a reference etc. in this specification constitutes a part of the disclosure content of this specification.

According to the method of the present invention, it was possible to induce the liver by forming dissociated reassemblies starting from Xenopus undifferentiated cells, which is a good model for studying early development, (FIG. 1). The liver thus induced was found to be a liver because it has a liver-specific appearance and expresses genes Hex and transferrin, which are proteins specific to the liver (marker protein).

Removal of undifferentiated cells Undifferentiated cells of Xenopus laevis were removed by the method described below. Xenopus embryos that reached late blastocysts were cultured in culture medium (Steinberg's Solution (SS); 58 mM NaCl, 0.67 mM KCl, 0.34 mM Ca (NO ₃ ) ₂ , 0.83 mM MgSO ₄ , 3.0 mM hydroxyethylpiperazinyl ethanesulfonic acid, and 100 mg / l kanamycin sulfate, pH 7.4) and transferred to a petri dish with 3% agarose, and the undifferentiated and multipotent sheet-like cells at the animal pole were placed in a 0. Ten pieces were collected in a 5 mm square.

Dissociation of cells First, 10 sheets of undifferentiated cells (number of cells: about 5,000 cells) taken from Xenopus late blastocysts were dissociated by the following method as a pretreatment for activin treatment. . 100 μL of SS without calcium and magnesium ions (Ca ⁺⁺ , Mg ⁺⁺ Free Steinberg's Solution; 58 mM NaCl, 0.67 mM KCl, 3.0 mM hydroxyethylpiperazinyl ethanesulfonic acid, and 100 mg / l kanamycin sulfate, pH 7.4) The removed five sheet-like undifferentiated cells were transferred to a culture vessel (for example, a 96-well culture dish) and allowed to stand for 20 minutes. This treatment loosened the cell-cell interaction of the sheet-like undifferentiated cells, and each cell dissociated instead of the sheet.

After dissociating the induced cells of the liver, remove SS without calcium and magnesium ions, add 100 ng / ml activin (prepared with SS containing 0.1% BSA), and pipette (eg Pasteur pipette) ) And allowed to stand for 24 hours. This treatment caused the cells to undergo activin treatment while reassembling. When the dissociated reassemblies (cell aggregates) thus treated with activin were transferred to a culture vessel (eg, a 96-well culture dish) filled with SS containing 0.1% BSA, and the culture was continued for 3 days, the liver Highly induced (Figure 2). In this culturing process, the liver was not induced when the sheet-like undifferentiated cells were treated with activin without dissociating them.

Identification of liver The liver thus induced by activin treatment had a unique tissue structure (Fig. 3). In addition, when the expression of various genes when cultured at different activin treatment concentrations was examined by the RT-PCR method, when the activin treatment concentration was 100 ng / ml (lane 4), proteins expressed specifically in the liver It was confirmed that Hex as a (marker protein) was expressed (FIG. 4).

In addition to the liver induction conditions , the activin treatment time was also examined, and in the 5-hour activin treatment, expression of Hex, a liver gene marker, and transferrin, a liver-specific differentiation gene marker, were observed, but also expressed in the heart. Expression of Nkx2.5 and cTnl, which are known, was also observed (FIG. 5). On the other hand, when activin treatment was performed for 24 hours, the expression of heart-specific gene markers Nkx2.5 and cTnl was not observed, and only the expression of liver gene markers Hex and Transferrin was observed (FIG. 6). ). From this, it was found that activin treatment longer than at least 5 hours is necessary to induce only the liver.

The mechanism by which the liver is induced from undifferentiated cells is widely common in amphibians and mammals including humans, and by utilizing the method of the present invention for producing liver cells from undifferentiated and multipotent cells, for example, It can be widely applied to regenerative medicine, drug discovery, therapeutic method development, diagnostic drug development, and drug development drug discovery, including the discovery of cancer markers and the isolation of genes essential for liver development.

Schematic of liver induction from undifferentiated cells. Optical stereomicrograph of the outer shape of the liver derived from undifferentiated cells (untreated dissociated reassemblies (A) and activin-treated dissociated reassemblies (B) containing liver cells, magnification: 25 times) An optical micrograph of a tissue section of liver derived from undifferentiated cells. (Untreated dissociated reassemblies (A) and activin-treated dissociated reassemblies containing liver cells (B) hematoxylin and eosin staining, magnification: 10 times) Examination of activin treatment concentration. (1 to 4 lanes; cultured for 1 day after activin treatment at each concentration. Lane 5; WE is an untreated embryo whose developmental stage has reached 20) Examination of activin treatment time (5 hours). Expression of the liver gene marker Hex was observed 1 hour after activin treatment (lane 3). When the expression of organ-specific markers was examined after 5 days of culture, expression of cardiac markers Nkx2.5 and cTnl was observed in addition to liver marker Transferrin (lane 5). (Lane 1, 2; unprocessed dissociated reassemblies) Examination of activin treatment time (24 hours). Expression of the liver gene marker Hex was observed 3 hours and 5 hours after the activin treatment (lanes 3 and 4). When the expression of organ-specific markers was examined after 5 days of culture, the expression of cardiac markers Nkx2.5 and cTnl was not observed, and only the liver marker Transferrin was observed (lane 5). (Lane 1, 2; unprocessed dissociated reassemblies)

Claims (19)

A method for inducing differentiation of a liver from the undifferentiated cells, comprising treating undifferentiated cells in a dissociated state with activin. The method according to claim 1, comprising treating the undifferentiated cells with activin in the step of forming a cell aggregate from undifferentiated cells in a dissociated state. The method according to claim 2, wherein dissociated undifferentiated cells are cultured in a medium containing activin at a concentration higher than 10 ng / ml to form a cell aggregate. The method according to claim 3, wherein undifferentiated cells in a dissociated state are cultured in a medium containing 100 ng / ml activin to form a cell aggregate. The method according to claim 3 or 4, wherein a serum-free medium containing a carrier protein is used. The method according to any one of claims 1 to 5, wherein dissociated undifferentiated cells are treated with activin for a period longer than at least 5 hours. The method according to any one of claims 1 to 6, wherein the undifferentiated cells in a dissociated state are embryonic stem cells obtained from blastocysts. The method according to claim 7, wherein the blastula is a late blastula. The method according to claim 8, wherein the undifferentiated cells are obtained from a cell layer containing an animal pole of a late blastula. The method according to any one of claims 1 to 6, wherein the undifferentiated cells are obtained from embryoid bodies. The method according to any one of claims 1 to 10, wherein the undifferentiated cells are derived from vertebrates. The method according to claim 11, wherein the undifferentiated cells are derived from a mammal. The method according to claim 12, wherein the undifferentiated cells are derived from a human. The method according to any one of claims 1 to 13, wherein the medium for culturing dissociated undifferentiated cells is a serum-free medium. The method according to any one of claims 1 to 14, wherein the culture of the cell aggregate is continued for 1 to 5 days after the treatment with activin. The method according to any one of claims 1 to 15, wherein the cells are cultured at 20 ° C. 17. The method according to any one of claims 1 to 16, wherein the cell aggregate comprises at least about 5,000 cells. The liver in which differentiation was induced by the method according to any one of claims 1 to 17, the tissue derived from the liver, or hepatocytes contained therein. A method for screening a drug for preventing or treating liver abnormalities or diseases, using the liver according to claim 18, tissue derived from the liver, or hepatocytes contained therein.