WO2011016485A1 - METHOD FOR INDUCING DIFFERENTIATION OF iPS CELLS INTO HEPATIC PARENCHYMAL CELLS - Google Patents

Abstract

Provided is a method for inducing differentiation of iPS cells into hepatic parenchymal cells. Said method yields sufficiently functional, safe hepatic parenchymal cells and can produce large quantities. The provided method includes: (a) a step in which iPS cells are cultured in a suspension culture and embryoid bodies are formed; (b) a step in which 10 to 15 embryoid bodies per well are adhered to a gelatin-covered plate and cultured, inducing embryonic endoderm cells; and (c) a step in which HFG and DMSO are added and the embryonic endoderm cells are cultured on a basement membrane substrate, inducing differentiation into hepatic parenchymal cells.

Description

Method for inducing differentiation from iPS cells to hepatocytes

The present invention relates to a method for inducing differentiation from iPS cells (induced pluripotent stem cells) to functional hepatocytes and hepatocytes differentiated thereby.

As a conventional technique, several reports have been made on methods for inducing differentiation of hepatocytes from embryonic stem (ES) cells.

For example, in the reports on the induction of hepatocyte differentiation in ES cells, the mainstream is that the embryoid body formation period is 2 to 2.5 days (Non-Patent Documents 1 to 3).

On the other hand, no specific technique for inducing differentiation of hepatocytes from iPS cells has been reported at present.

ES cells have pluripotency and can differentiate into any of the three germ layer cells. Moreover, ES cells have infinite proliferation ability in an experimental system. Therefore, it is possible to treat patients with liver diseases by inducing differentiation of many stem cells from ES cells and using them as a material for bioartificial liver. However, when cell therapy is performed with ES cell-derived hepatocytes, long-term use of immunosuppressive agents is indispensable, and complications such as infections may occur. In addition, since embryos of fertilized eggs are required to obtain ES cells, ethical problems are involved in the establishment and operation of ES cells.

Since iPS cells do not require embryos of fertilized eggs, and because it is possible to use the patient's own somatic cells, it is expected to be a promising cell source for both ethical issues and rejection. Yes.

However, the current state of research on iPS cell differentiation induction is not yet sufficient. In addition, in the differentiation induction of iPS cells, it is difficult to select the cell density and culture medium at the time of culturing, and iPS cells can be transferred to definitive endoderm which is a precursor tissue of hepatic parenchymal cells compared to ES cells. There are problems that differentiation efficiency is low, and further, the function of the obtained hepatocytes is not sufficient.

Therefore, development of a technique for inducing differentiation of functional hepatocytes from iPS cells is eagerly desired.

On the other hand, the basement membrane is composed of proteins such as laminin, entactin, type IV collagen, and extracellular matrix (ECM) such as proteoglycan, which are directly below the basal basal surface, the inner lower surface of epithelial tissue, and contain cells. It is a structure of 50 to 100 nm, and is considered essential for immature epithelial cells to proliferate and differentiate into mature cells to express their original form and function. For the basement membrane, epithelial cells have been cultured on a collagen matrix in which fibroblasts are embedded, or on a collagen fiber substrate to which Matrigel (Matrigel (registered trademark), Becton Dickinson) has been added. In vitro production methods such as a culture method have been developed, and examples of use as a culture substrate and production of artificial tissue have been reported (Patent Documents 1 to 3).

JP 2003-93053 A JP 2003-169846 A JP 2003-93050 A Table 2004-085606

Basma H, Soto-Gutieerrez A, Yannam GR, Liu L, Ito R, Yamamoto T, Ellis E, Carson SD, Sato S, Chen Y, Muirhead D, Navarro-Alvarez N, Wong RJlatyJL , Mercer DF, Miller JD, Strom SC, Kobayashi N, Fox IJ. Differentiation and transplantation of human embryonic stem cell-derived hepatocytes. Gastroenterology 2009; 136: 990-999. Rambhatla L, Chiu CP, Kundu P, Peng Y, Carpenter MK. Generation of hepatocyte-like cells from human embryonic stem cells. Cell Transplant 2003; 12: 1-11. Soto-Gutieerrez A, Kobayashi N, Rivas-Carrillo JD, Navarro-Alvarez N, Zhao D, Okitsu T, Noguchi H, Basma H, Tabata Y, Chen Y, Tanaka K, Narushima M, U , Yoon JW, Lebkowski J, Tanaka N, Fox IJ. Reversal of mouse hepatic failure using an implanted liver-assist device containing ES cell-derived hepatocytes. Nat Biotechnol 2006; 24: 1412-1419.

An object of the present invention is to provide a method for efficiently inducing differentiation of functionally superior liver parenchymal cells from iPS cells, and functional liver as a bioartificial liver material without causing ethical problems. Providing parenchymal cells.

Therefore, we succeeded in inducing differentiation of hepatocytes from iPS cells by devising and combining the following points.

The present invention is a method for inducing differentiation from iPS cells to hepatocytes,
(A) a step of suspension culture of iPS cells to form an embryoid body;
(B) inducing definitive endoderm by adhering and culturing 10-15 embryoid bodies on a plate using gelatin as a coating agent per well, and (c) adding HGF and DMSO, The present invention relates to a method comprising inducing hepatocyte differentiation by culturing definitive endoderm on a basement membrane substrate.

In the method of the present invention, the suspension culture period in step (a) is preferably 5 days.

The basement membrane matrix is preferably derived from alveolar type II epithelial cells or liver parenchymal cells.

According to the differentiation inducing method of the present invention, functionally superior hepatocytes can be obtained. In addition, definitive endoderm can be stably obtained, and differentiation into hepatocytes can be efficiently induced. Since the differentiated hepatocytes of the present invention are hepatocytes based on iPS cells, ethical problems associated with the establishment and operation of ES cells can be avoided. Furthermore, by using individual-derived iPS cells, individual-specific hepatocytes that do not require immunosuppressive agents can be obtained. Therefore, hepatocytes derived from the iPS cells of the present invention are more suitable as a material for bioartificial liver.

It is a figure which shows the differentiation process from an iPS cell to a hepatocyte. A is a scheme showing the passage of days and the differentiation process. B to E are (B) iPS cells, (C) embryoid bodies (EB), (D) definitive endoderm (DE), and (E to G) iPS in the process of differentiation from iPS cells to hepatocytes, respectively. 2 is a phase contrast micrograph of cell-derived hepatocytes (iPS-Heps). (E) shows iPS-Heps in Comparative Example 1, (F) in Example 1 and (G) in Example 2. It is a microscope image which shows the expression of albumin by immunohistochemical staining. Example 4, Comparative Example 1, undifferentiated iPS cells, and T3-Alb7-sBM substrate-only sample were used as controls. Microscopic image Alb was expressed using rabbit anti-mouse albumin antibody (Cat. No. 64560, MP biomedicals) as the primary antibody and DyLight54-labeled sheep anti-rabbit antibody (Catalog No. STAR36D549, MorphoSys UK) as the secondary antibody. ) Is dyed. The microscopic image DAPI shows a cell nucleus immunostained with DAPI (Catalog No. H-1200, manufactured by Vector Laboratories Inc.). MERGE indicates a superposition of two microscopic images of Alb and DAPI. It is a graph which shows the ammonia removal rate of the iPS cell origin hepatocyte of this invention. It is a graph which shows the urea production amount of the iPS cell origin hepatocyte of this invention. It is a graph which shows the albumin production amount of the iPS cell origin hepatocyte of this invention. 2 is a phase contrast micrograph (AC) and an electron micrograph (DF) of hepatic parenchymal cells induced to differentiate from iPS cells according to Comparative Example 1. FIG. In the strongly magnified image of the phase contrast microscope observation (FIG. 6B), polygonal cells having an abundant circular nucleus and abundant granules in the cytoplasm are clearly observed. It is positive for PAS staining, indicating that intracellular granules are glycogen (FIG. 6C). In addition, observation by an electron microscope also shows a glycogen field (FIG. 6E), which is a site where glycogen is gathered, and a luminal formation image with microvilli (FIG. 6F), indicating a cell structure consistent with liver parenchymal cells. FIG. 3 is a diagram showing expression of various genes in cells at each stage of the differentiation induction process of Comparative Example 1. The genes examined were alphafetoprotein (AFP), albumin (Alb), transferrin (Trf), phosphoenolpyruvate carboxykinase 1 (PCK1), carbamyl phosphate synthetase (CPS), sex determination gene box 17 (Sex determining region Y , Box 17) (Sox17) and β-actin gene. Lanes 1-4 show iPS cells, embryoid bodies, definitive endoderm and iPS-Heps, respectively. The relative mRNA level in each differentiation process of the comparative example 1 of alpha fetoprotein (AFP), albumin gene (Alb), transferrin (Trf), phosphoenolpyruvate carboxylase 1 (PCK1), and carbamyl phosphate synthetase (CPS) is shown. It is a graph.

The present invention relates to a method for inducing differentiation of iPS cells into functional hepatocytes.

The method of inducing differentiation of hepatocytes from the iPS cells of the present invention comprises: (a) a step of suspension-culturing iPS cells to form embryoid bodies; (b) transferring embryoid bodies to a plate using gelatin as a coating agent. A step of inducing definitive endoderm by adhering and culturing as 10-15 per well, and (c) hepatic parenchyma by adding HGF and DMSO and culturing definitive endoderm on a basement membrane substrate Inducing cell differentiation.

In the present specification, when “per well” is described, it means one well of a 12-well culture plate, and the bottom area of one well means 3.6 cm ² .

Next, an embodiment of the differentiation induction method of the present invention is shown.

1) The iPS cells were preferably suspended in culture for 5 days to form embryoid bodies. In the previous reports on induction of hepatocyte differentiation in ES cells, the mainstream is that the embryoid body formation period is 2 to 2.5 days. However, iPS cells have a lower efficiency of differentiation into definitive endoderm, which is a precursor tissue of hepatic parenchymal cells, compared to ES cells, and there is a tendency that sufficient definitive endoderm cannot be obtained in the case of definitive body formation for 2 to 2.5 days. is there. Therefore, for the formation of embryoid bodies, suspension culture for 5 days is preferable in order to increase the efficiency of differentiation into definitive endoderm.

2) After transferring the embryoid body to a gelatin-coated plate, add activin A 100 ng / ml and basic fibroblast growth factor (bFGF) 100 ng / ml, low concentration serum replacement (serumsereplacement) for 3 days Cultured and induced definitive endoderm. Embryoid bodies formed over 5 days have a lower ability to adhere to the plate than those formed over 2 to 2.5 days, and thus adhere to the plate and start to differentiate without a coating. I can't. Therefore, we obtained good adhesion by using gelatin as a coating agent.

3) Embryoid bodies were cultured in 12-well gelatin-coated plates, and 10 to 15 embryoid bodies per well were induced to start definitive endoderm induction. In the differentiation induction process, cell growth starts from around the embryoid body and these differentiate into hepatocytes, so it is important to transfer the embryoid body to the plate at an appropriate density. When the density of embryoid bodies is low (for example, less than 10 per well), fewer hepatocytes are obtained at the end of differentiation induction. On the other hand, when the density of embryoid bodies is high (for example, when there are more than 15 per well), differentiation into cardiomyocytes is likely to be induced, so that the purity of the obtained hepatocytes decreases. In addition, cells tend to be killed due to lack of medium. Therefore, it was important to start definitive endoderm induction with 10 to 15 embryoid bodies per well.

4) In a series of differentiation induction processes, a serum-free medium, that is, knockout DMEM and knockout serum substitute (KSR) was used. Conventionally, a serum medium has been used in the differentiation induction process of ES cells. However, in the culture and differentiation induction of iPS cells, the serum-free medium showed higher cell proliferation ability than the serum medium. In the differentiation induction, hepatocyte growth factor (HGF) and dimethyl sulfoxide (DMSO) are added, and the definitive endoderm is cultured on the basement membrane substrate, thereby metabolizing ammonia and urea. We succeeded in inducing differentiation of functional hepatocytes that can be produced.

1) Suspension culture of iPS cells preferably for 5 days to form embryoid bodies 2) Use gelatin as a coating agent to adhere embryoid bodies to plates 3) Embryoid bodies per well Initiate definitive endoderm induction as 10-15, 4) preferably using serum-free medium, ie knockout DMEM and KSR, add HGF and DMSO, and culture definitive endoderm on basement membrane substrate Thus, the above-described steps 1) to 4), which start differentiation into hepatocytes, and combinations thereof are the features of the present invention.

According to the present invention, hepatocytes can be induced from iPS cells effectively and stably.

IPS cells are preferably derived from mammals, and examples of mammals include humans, primates such as cynomolgus monkeys, and mice.

As a method for producing iPS cells, OkitaOK, Ichisaka T, Yamanaka S., Generation of germline-competent induced-pluripotent stem cells. Nature 2007; 448: 313-317 can be referred to. For example, mouse iPS cells are available from the RIKEN Cell Bank.

In the present invention, if necessary, iPS cells are passaged, maintained and proliferated under non-differentiating conditions (hereinafter, this step may be referred to as a preparation step), and a part thereof is used as necessary. Induces differentiation into hepatocytes.

In the preparation step, it is preferable to use a container coated with feeder cells as a container for culturing iPS cells from the viewpoint of proliferation ability. Examples of feeder cells include mouse embryonic fibroblasts irradiated with γ rays. In the case of mouse-derived iPS cells, it is preferable to use a culture vessel coated with feeder cells.

As the culture conditions in the preparation step, the same conditions as for normal ES cells can be employed. The main points of the preparation process include changing the medium every 24 hours to prevent nutrient depletion, and subculturing before the cell density increases and the colonies adhere to each other. The culture temperature is preferably in the range of 36 to 37 ° C., and the pH is preferably in the range of 7.3 to 7.4. In the process of preparing mouse iPS cells, leukemia inhibitory factor (LIF) is generally used to suppress differentiation.

The hepatic parenchymal cells induced to differentiate from iPS cells by the method of the present invention can be confirmed by observation of morphological characteristics and reverse transcription polymerase chain reaction (RT-PCR).

The morphological confirmation method is specific to liver parenchymal cells, such as cells with multiple nuclei observed with a phase contrast microscope and abundant granules in the cytoplasm observed with an electron microscope, especially glycogen granules. Specific morphological features.

In addition, the expression of induced hepatic parenchymal cells can be evaluated from the expression of the gene by RT-PCR. If it can be confirmed by RT-PCR or the like that the albumin gene is expressed and that the undifferentiated α-fetoprotein is not expressed, it can be said that differentiation is induced. In addition, as an index indicating the maturity of hepatocytes, albumin, carbamyl phosphate synthetase (CPS), an enzyme related to urea synthesis, phosphoenolpyruvate carboxykinase 1 (PCK1), transferrin (Trf), an enzyme that regulates gluconeogenesis It is preferable that gene expression such as is observed.

Furthermore, whether or not it can actually function as a hepatocyte is largely determined by whether it can be metabolized by adding ammonia, lidocaine, diazepam, or the like to the medium. Moreover, if albumin and urea are produced in the medium, it can be said that they have differentiated into sufficiently functional hepatocytes.

As the basement membrane substrate used in the present invention, various substrates can be used and are not particularly limited. For example, the basement membrane obtained by culturing cells having the ability to form basement membranes in the presence of Matrigel described in JP-A-2003-93053, etc., from the point that the function of the obtained hepatic parenchymal cells has been sufficiently confirmed. By culturing cells having basement membrane-forming ability on collagen fibers coated with a MAST polymer (maleic 結合 anhydride-styrene copolymer) bound to a substrate or sugar chain β-GlcNAc described in JP-A-2003-93050, etc. The resulting basement membrane substrate is preferred. Matrigel is a basement membrane preparation extracted from the Engelbreth-Holm-Swarm tumor matrix (J. Exp. Med. 145, 204-220, 1977) and may affect extracellular matrix synthesis. In addition to various cytokines, laminin-111, entactin, type IV collagen, perlecan and the like are included (Exp. Cell Res. 202, 1-8, 1992).

Various cells such as epithelial cells, vascular endothelial cells, and glia can be used as cells having the ability to form a basement membrane. From the viewpoint of induction of differentiation into the liver, for example, cells derived from lung such as alveolar type II epithelial cells, cells derived from liver such as liver parenchymal cells, or 293 cells are genetically modified to stabilize hLN-511. It is preferable to use rLN-10 cells that are expressed and secreted in large quantities extracellularly, and most preferably hepatocytes. It is preferable to use established cells (cancerous and immortalized cell lines) because a large amount of stable quality basement membrane substrate is obtained, and in view of safety, it is preferable to use immortalized cell lines. More preferred.

Laminin, the main glycoprotein of the basement membrane, is a complex composed of three types of α, β, and γ subunits, and there are various isoforms such as laminin-111 (α1β1γ1) and laminin-511 (α5β1γ1). .

As a method for producing such a basement membrane substrate, for example, the technique described in Japanese Patent Application Laid-Open No. 2003-93050 or Japanese Patent Application Laid-Open No. 2003-93053 can be used.

Also, by co-culturing various cells together with definitive endoderm on the basement membrane matrix, the function of the obtained hepatic parenchymal cells can be improved. For co-culture, hepatic sinusoidal endothelial cells, bile duct epithelial cells, hepatic stellate cells, and the like can be used, and human hepatic sinusoidal endothelial cell lines are preferred.

Therefore, a preferred embodiment of the present invention is a method for inducing differentiation from iPS cells to hepatocytes, wherein (a) the iPS cells are cultured in suspension to form embryoid bodies, (b) as a coating agent A step of inducing definitive endoderm by attaching 10-15 embryoid bodies to a plate using gelatin and culturing; and (c) adding HGF and DMSO on a basement membrane substrate The present invention relates to a method comprising a step of inducing hepatocyte differentiation by co-culturing definitive endoderm with hepatic sinusoidal endothelial cells.

In the present invention, iPS cells are differentiated into definitive endoderm, pooled to the required number of cells, and then differentiated into hepatocytes as necessary, so that a large number of safe hepatocytes can be produced without causing ethical problems. Can supply. The development of a bioartificial liver that can benefit everyone by using this as the cell source for bioartificial liver is greatly expected.

Therefore, another aspect of the present invention relates to the use of liver parenchymal cells induced to differentiate from iPS cells. These hepatocytes can be used as a cell source for bioartificial liver.

A bioartificial liver is an artificial liver device that mimics the liver in the living body, in which liver parenchymal cells are incorporated into a carrier, fixed, and made into a reactor. The patient's blood is guided into the device, and the metabolic capacity of liver parenchymal cells is used. Thus, the apparatus can remove toxins in blood and supply physiologically active substances such as coagulation factors derived from liver cells.

Examples of such a bioartificial liver include a hybrid type artificial liver combining a hollow fiber type reactor (device) and separated / cultured cells. There are three types of bioartificial livers: those that are attached outside the body and connected to blood vessels, those that are placed in the body and connected to blood vessels, and those that are placed in the abdominal cavity without being connected to blood vessels. The hepatocytes of the present invention can be used in any form of bioartificial liver, but are preferably in vitro from the viewpoint of avoiding the risks associated with cell transfer.

Bioreactors used in bioartificial livers include Cedars-Sinai Medical Center (Los Angeles, California, USA) with the support of Circe Biomedical Inc. (Lexington, Massachusetts, USA). HepatAssist (Hui T, と し た Rozga J, Demetriou AA. J Hepatobiliary Pancreat Surg 2001; 8: 1-15) for the treatment of bioartificial liver using porcine liver parenchymal cells centered on Demetriou et al. .) And German MERLach et al.'S MELS (Modular Extracorporporeal Liver System) using porcine liver parenchymal cells are known. Since these reactors do not have a scaffold for adhering hepatocytes, the cells tend to be in a floating state simply by filling the space inside the hollow fiber or the space outside the hollow fiber. Liver parenchymal cells tend to have insufficient differentiation function in a floating state, and collide with surrounding cells and are susceptible to stress stimulation. Therefore, a reactor composed of a hollow fiber and a non-woven fabric is more preferably used so as to provide a scaffold for hepatocytes (Naoya Kobayashi, et al., “Hybrid Artificial Liver”, Hepatobiliary Pancreas, 2003, 46, p381-393) .

Furthermore, hepatocytes, particularly human hepatocytes, induced to differentiate from iPS cells by the method of the present invention are useful as cells for drug metabolism tests. That is, it is useful as a test cell in new drug development, safety test, toxicity test and the like.

Liver parenchymal cells are the center of various drug metabolisms, and in order to test the toxicity and safety of drugs, it is necessary to administer drugs to hepatic parenchymal cells and examine how they are metabolized. And since there is a big difference between humans and experimental animals in substance metabolism, the final test is indispensable using human hepatocytes. In this respect, the present invention that can provide a large amount of human hepatocytes of uniform quality is useful. As a device used for such a metabolic test, a device having the same structure as the bioartificial liver can be used.

Furthermore, hepatocytes, particularly human hepatocytes, induced to differentiate from iPS cells by the method of the present invention can be used for the production of physiologically active substances. Examples of such physiologically active substances include albumin and various blood coagulation factors. As a device for producing such a physiologically active substance, one having the same structure as that of the bioartificial liver can be used.

Hereinafter, the present invention will be described with reference to examples using mouse-derived iPS cells, but the present invention is not limited thereto.

Examples 1 to 4
Induction of differentiation from iPS cells to hepatocytes (1) Cultivation of undifferentiated iPS cells Mouse iPS cells (Cell no. APS0001, Cell name iPS-MEF-Ng) obtained from the RIKEN BioResource Center through the Ministry of Education, Culture, Sports, Science and Technology's National BioResource Project -20D-17, Lot no. 006). Mouse iPS cells are feeders of mouse embryo fibroblasts (MEF) (Dainippon Sumitomo Pharma Co., Ltd.) inactivated by mitomycin C (Catalog No. GR-311, Biomol, Enzo Life Sciences International). Cells were cultured on gelatin (catalog number ES-006-B, Specialty media, Chemicon international) coated plates at 37 ° C. with a CO ₂ concentration of 5%. The medium was Complete ES cell medium (catalog number SF001-500P, Millipore, Chemicon International), and leukemia inhibitory factor (LIF, catalog number LIF2010, Millipore, Chemicon International) 1000 U / ml was added. . Mouse iPS cells with passage number 5-20 (FIG. 1 (B)) were used for differentiation induction experiments.

(2) Stage 1: Embryoid body (EB) formation (Day 0 to Day 4)
IPS cells cultured on feeder cells were separated from the plate using trypsin-EDTA (Catalog No. T4049, Sigma-Aldrich Japan), and then centrifuged at 800 rpm for 3 minutes and collected as a pellet. Thereafter, knockout serum replacement (KSR, catalog number 10828, GIBCO, Invitrogen) 15%, non-essential amino acids (catalog number 1681049, MP biomedicals) 1%, 2- Mercaptoethanol (Catalog No. 21985-023, Gibco, Invitrogen) 1%, Penicillin / Streptomycin (Catalog No. ES-101-B, Specialty Media, Chemicon International) 1%, L-Glutamine (Catalog No. DSM202, DS Pharma Biomedical ( DS pharma biomedical)) was suspended in a knockout DMEM (catalog number 10829, Gibco, Invitrogen) supplemented with 1%. This cell suspension was transferred to a low-adhesion 6-well plate (Catalog No. 3471, Costar, Corning), suspended in suspension at a density of 2 × 10 ⁵ cells / 2 ml / well, and embryoid bodies Began to form. The medium was changed in half the amount every day.

After the start of embryoid body formation, the cells began to aggregate, and a day later, spherical cell clusters were formed. These cell agglomerates were further combined, and after 5 days, they became a cluster-like agglomeration (FIG. 1 (C)).

(3) Stage 2: Induction of definitive endoderm (DE) (Day 5 to Day 7)
On day 5, the medium containing embryoid bodies was collected from one well and centrifuged at 1,000 rpm for 3 minutes to collect the embryoid bodies as pellets. The embryoid body was then penicillin / streptomycin 1%, L-glutamine 1%, activin A (catalog number 338-AC, R & D Systems) 100 ng / ml, bFGF (catalog number 100-18B, Peprotech) 100 ng Suspended in 12 ml of knockout DMEM to which / ml was added. Thereafter, a gelatin-coated 12-well plate (catalog number 353503, Nippon Becton Dickinson) was prepared, and 10 to 15 embryoid bodies were transferred per well. In order to form definitive endoderm, the cells were further cultured for 3 days. KSR was added at 0.2% on the 6th day and 2% on the 7th day. The medium was changed in half the amount every day.

After being transferred to the gelatin-coated plate, the embryoid body adhered to the plate, and cell growth started from around the embryoid body (FIG. 1 (D)).

(4) Preparation of basement membrane substrate a) Preparation of mLN-111 isoform basement membrane (mLN111-sBM) substrate Rat immortalized alveolar type II epithelial cells (SV40-T2 cells: Dr. A. Clement, Hopital Armand Trousseau) , Paris (see Reference 6) alveolar type II epithelial cells (collected from rats transfected with SV40-large T antigen gene) according to the method described in Reference 7 (Catalog No. 356234, By culturing in the presence of Nippon Becton Decktonson, a mLN-111 isoform-type basement membrane (mLN111-sBM) structure was prepared on a 12-well culture plate insert (Catalog No. 353494, Nippon Becton Deckonson). And a mixture of protease inhibitors (PIC, manufactured by Peptide Laboratories) At the same time as dissolving and eluting the lipid component of epithelial cells using 2 ml of 0.1% Triton X-100 (surfactant) in a phosphate buffer (pH 7.2; PBS (−)) After repeating the operation of dissolving the protein remaining on the surface of the basement membrane structure with the coexisting 50 mM NH ₃ twice (the protein of the basement membrane structure must not be dissolved), the PBS (−) solution containing PIC again The base material was washed with surfactant and alkali to remove the alveolar type II epithelial cell layer and the basement membrane structure was exposed to reveal the mLN-111 isoform-type basement membrane (mLN111-sBM) substrate Was made.

b) Preparation of T3-Alb7 isoform-type basement membrane (T3-Alb7-sBM) substrate Human immortalized liver parenchymal cells (T3-Alb7 cells, drug-inducible Cre recombinase expressing immortalized human hepatocyte cell line TTNT-16-T3 (Depositing organization: IPOD, National Institute of Advanced Industrial Science and Technology, Patent Biological Depositary Center, Address: 1-chome, East 1-chome, Tsukuba, Ibaraki, Japan, Chuo 6 (postal code 305-8666), Date of deposit: October 2003 1 day, accession number: FERM BP-08501, Totsugawa T, et al., Survival of liver failure pigs by transplantation of reversibly immortalized human hepatocytes with Tamoxifen-mediated self-recombination. See J. Hepatol. 47: 74-82, 2007. MAST polymer (maleic anhydride-s) to which a sugar chain β-GlcNAc is bonded according to the method described in JP-A-2003-93050) A basement membrane structure was formed by seeding on collagen fibers coated with tyrene copolymer (see Patent Document 4) and culturing for about 2 weeks. After formation of the basement membrane structure, T3-Alb7 cells were treated with a D-PBS (−) solution containing 50 mM NH ₃ , 0.1% Triton X-100, and protease inhibitor cocktail (PIC, manufactured by Peptide Laboratories). Thus, only the cells were removed, and the basement membrane structure formed by the T3-Alb7 cells was exposed without damaging, thereby preparing a T3-Alb7 isoform-type basement membrane (T3-Alb7-sBM) substrate.

(5) Third stage: differentiation into iPS cell-derived hepatocytes (iPS-Heps) (from day 8 to day 16)
On the 8th day, definitive endoderm was separated from the plate using trypsin-EDTA (catalog number T4049, Sigma-Aldrich Japan), then centrifuged at 800 rpm for 3 minutes and collected as a pellet. KSR 10%, non-essential amino acid 1%, L-glutamine 1%, dimethyl sulfoxide (DMSO, catalog number D4540-100ML, Sigma-Aldrich) 1%, HGF (catalog number 100-39, Peprotech) 100 ng / ml The added knockout DMEM was prepared as a medium, and suspended in 0.5 ml of the medium collected from the pellet collected from one well. This suspension was dropped into the insert containing mLN111-sBM substrate or T3-Alb7-sBM substrate prepared in (4) above.

1.5 ml of medium was added to one well of an insert-compatible 12-well culture plate (catalog number 353503, Nippon Becton Dickinson). This was designated Example 1. In addition, 5.0 × 10 ⁵ human immortalized hepatic sinusoidal endothelial cell line TMNT-1 (deposited organization: National Institute of Advanced Industrial Science and Technology, Patent Organism Depositary) , Address: 1-chome, Higashi 1-chome, Tsukuba, Ibaraki, Japan, Central 6 (postal code 305-8656), deposit date: April 16, 2002, deposit number: FERM BP-8017, see reference 5, 10,000 Cultivated in DMEM (DMEM high glucose) containing 4.5 g / L glucose and supplemented with 10% FBS (fetal bovine serum) and 1% penicillin and streptomycin. Add 1.5 ml of the medium to the well, and place the insert containing the definitive endoderm, mLN111-sBM substrate and 0.5 ml of the medium. This was designated Example 2. On the 10th day, the medium was replaced with HGF-free medium, that is, knockout DMEM supplemented with 10% KSR, 1% non-essential amino acid, 1% L-glutamine, and 1% DMSO. Exchanged. Differentiation induction was completed on the 16th day. Compared to Comparative Example 1 in which no basement membrane substrate was used, in Examples 1 and 2, a greater number of iPS cell-derived hepatocytes were obtained (FIG. 1 (EG)).

1.5 ml of medium was added to one well of a 12-well culture plate corresponding to insert, and an insert containing definitive endoderm, T3-Alb7-sBM substrate and 0.5 ml of medium was placed on the plate and cultured. On the bottom of the 12-well culture plate for inserts, 5.0 × 10 ⁵ TMNK-1s are seeded beforehand and cultured for 1 day or more, 1.5 ml of medium is added to the wells, definitive endoderm, T3- An insert containing Alb7-sBM substrate and 0.5 ml of medium was placed and cultured, and this was designated as Example 4. On the 10th day, the medium was replaced with HGF-free medium, ie, knockout DMEM supplemented with 1% KSR, 1% non-essential amino acid, 1% L-glutamine, and 1% DMSO, and thereafter the entire medium was replaced every 2 days. Exchanged. Differentiation induction was completed on the 16th day. Compared to Comparative Example 1 in which no basement membrane substrate was used, in Examples 3 and 4, a greater number of iPS cell-derived hepatic parenchymal cells were obtained (FIGS. 2 and 5).

Test example 1
Functional Evaluation of iPS Cell-Derived Liver Parenchymal Cells (A) Ammonia Metabolism Ability According to Undifferentiated iPS Cells, iPS Cell-Derived Liver Parenchymal Cells Differentiated According to the Methods of Example 1 and Example 2, 0.56 mM ammonia sulfate (catalog number 02619-15, Sigma-Aldrich) was added to iPS cell-derived hepatocytes derived from differentiation, and the culture supernatant was collected after 24 hours to measure ammonia metabolic capacity. did. The ammonia concentration was measured using Ammonia Test-Wako (catalog number 277-14401, manufactured by Wako Pure Chemical Industries).

The results are shown in FIG. In undifferentiated iPS cells, the ammonia concentration rather increased, and the removal rate was −42.9 ± 10.4%. The iPS cell-derived hepatic parenchymal cells of the present invention metabolize 63.7 ± 7.3% of ammonia added to the culture medium in Example 1, and 31.9 ± 5.3% in Example 2. did. This showed an excellent metabolic capacity compared to 18.1 ± 5.9% of the iPS cell-derived hepatic parenchymal cells of Comparative Example 1.

(B) Production of urea Undifferentiated iPS cells, iPS cell-derived liver parenchymal cells induced to differentiate according to the methods of Examples 1 and 2, and iPS cell-derived liver parenchymal cells induced to differentiate according to the method of Comparative Example 1 described later In order to evaluate each urea production ability, the culture solution was collected 24 hours after the medium exchange, and the production amount of urea was measured (consigned to SRL). The results are shown in FIG. Undifferentiated iPS cells did not show urea-producing ability. The iPS cell-derived hepatic parenchymal cells of the present invention exhibited 133 ± 153 ng urea production ability per well in Example 1, and 167 ± 153 ng urea production ability per well in Example 2. This showed a tendency for the amount of urea production to be large compared to 33.3 ± 57.7 ng of the iPS cell-derived liver parenchymal cells of Comparative Example 1.

(C) Albumin production Each of undifferentiated iPS cells, iPS cell-derived liver parenchymal cells induced to differentiate according to the methods of Examples 2 to 4, and iPS cell-derived liver parenchymal cells induced to differentiate according to the method of Comparative Example 1 described later. In order to evaluate the amount of albumin produced, the culture solution was collected 24 hours after the medium exchange, and the amount of albumin was measured using an AssayMax mouse albumin ELISA kit (catalog number EMA3201-1, manufactured by Gentaur). The results are shown in FIG. Undifferentiated iPS cells did not show albumin producing ability. The iPS cell-derived hepatocytes of Comparative Example 1 showed an albumin producing ability of 53 ± 38 pg. In Example 1, albumin production was not shown, and in Example 2, albumin production of 514 ± 890 pg was observed. Compared to these, albumin production was significantly higher at 2320 ± 64 pg in Example 3 and 2635 ± 111 pg in Example 4.

(D) Positive rate of albumin expression of undifferentiated iPS cells, iPS cell-derived liver parenchymal cells induced to differentiate according to the method of Example 4, and iPS cell-derived liver parenchymal cells induced to differentiate according to the method of Comparative Example 1 described later Rabbit anti-mouse albumin antibody (Cat. No. 64560, manufactured by MP biomedicals) is used as the primary antibody, and DyLight54-labeled sheep anti-rabbit antibody (Cat. # STAR36D549, manufactured by MorphoSys UK) is used as the secondary antibody to evaluate the positive rate of each albumin expression. The expressed albumin (Alb) was stained. In addition, the total number of cells and the number of Alb-expressing cells were measured by immunostaining the cell nuclei with DAPI (catalog number H-1200, manufactured by Vector Laboratories Inc.), and the number of nuclei stained with Alb-positive cells / DAPI. The positive rate of Alb expression was calculated. The results are shown in FIG. In the sample of undifferentiated iPS cells and the control T3-Alb7-sBM substrate alone, the Alb positive rate was 0%. The iPS cell-derived liver parenchymal cells of Comparative Example 1 showed an Alb positive rate of 20.6%. On the other hand, in Example 4, a significantly high Alb positive rate of 31.8% was observed.

Comparative Example 1
The same procedures as in Example 1 (1) to (3) were performed to obtain definitive endoderm.

(4a) Third stage: differentiation into iPS cell-derived liver parenchymal cells (iPS-Heps) (Days 8 to 16)
On day 8, the medium was replaced with knockout DMEM supplemented with 10% KSR, 1% non-essential amino acids, 1% L-glutamine, 1% DMSO, 100 ng / ml HGF (Catalog No. 100-39, Peprotech). The medium was changed in half the amount every day. Differentiation induction was completed on the 16th day. Morphologically, at the final stage of differentiation, mouse iPS cells showed a morphology similar to that of hepatocytes. That is, it became a polygonal cell with abundant granules in the cytoplasm (FIG. 1E, FIG. 6A, FIG. 6B).

Reference example 1
Cell morphology of iPS cell-derived liver parenchymal cells (iPS-Heps) in Comparative Example 1 The cell morphology of iPS cell-derived liver parenchymal cells in Comparative Example 1 was observed using a phase contrast microscope and an electron microscope. In the strongly magnified image of the phase contrast microscope observation (FIG. 6B), polygonal cells having an abundant circular nucleus and abundant granules in the cytoplasm are clearly observed. It is positive for PAS staining, indicating that intracellular granules are glycogen (FIG. 6C). In addition, observation by an electron microscope also shows a glycogen field (FIG. 6E), which is a site where glycogen is gathered, and a luminal formation image with microvilli (FIG. 6F), indicating a cell structure consistent with liver parenchymal cells.

Reference example 2
Gene expression in the process of inducing differentiation from iPS cells to liver parenchymal cells RT-PCR was performed at each stage of the process of inducing differentiation in Comparative Example 1. RNA was extracted using TRI zol (catalog number 15596-026, Invitrogen). cDNA was prepared from 2 μg of RNA using MuLV reverse transcriptase (Catalog No. N8080018, Applied Biosystems) and RNase inhibitor (Catalog No. N808119, Applied Biosystems) using a Takara Thermal Cycler. Created. RT-PCR was performed using AmpliTaq Gold DNA polymerase, GeneAmp PCR Gold Buffer, MgCl ₂ solution (Catalog No. 606080, Applied Biosystems). The genes include alphafetoprotein (AFP), albumin (Alb), transferrin (Trf), phosphoenolpyruvate carboxykinase 1 (PCK1), carbamyl phosphate synthetase (CPS), sex-determining gene box 17 (Sox17) and endogenous The β-actin gene as a control was examined. The primers used in RT-PCR are shown in Table 1. PCR products were electrophoresed on 2.5% agarose gel and visualized using ethidium bromide (FIG. 7A). In FIG. 7A, lanes 1 to 4 are iPS cells, EB, DE, and iPS-Heps, respectively.

Real-time RT-PCR was performed using the Light Cycler 1.5 (Roche Applied Science) kit using the Light Cycler Fast DNA Master SYBR Green I (Catalog No. 03003230001, Roche Applied Science, IN) kit. . The genes include alphafetoprotein (AFP), albumin (Alb), transferrin (Trf), phosphoenolpyruvate carboxykinase 1 (PCK1), carbamyl phosphate synthetase (CPS) and the endogenous control β-actin gene. Examined. Table 2 shows the primers used in real-time RT-PCR.

In the results of RT-PCR and real-time RT-PCR, the gene expression of albumin, which is an indicator of mature liver parenchymal cells, gradually increased with the differentiation process. The genes that are abundant in liver parenchymal cells such as carbamyl phosphate synthetase (CPS), an enzyme related to urea synthesis, phosphoenolpyruvate carboxykinase 1 (PCK1), an enzyme that regulates gluconeogenesis, and so on are also analyzed over time. Expression was enhanced (FIG. 7B).

Reference example 3
The effect of embryoid body formation period on the definitive endoderm differentiation efficiency was investigated.

EBs formed over 5 days as in the first stage of Example 1 were subjected to endoderm differentiation induction over 5 days under the same conditions as in the second stage of Example 1 (Sample 1). EBs formed in the same manner as in the first stage of Example 1 except that the formation period was 2 days were subjected to endoderm differentiation induction over 5 days under the same conditions as in the second stage of Example 1 (Sample 2). ). The gene expression of the obtained cells was analyzed by real-time PCR as in Example 2. The genes examined were sex-determining gene box 17 (Sox17), chemokine (C—X—C motif) receptor 4 (chemokine (CXC motif) receptor 4) (Cxcr4) and forkhead box A2 (forkhead box A2) (Foxa2) Table 3 shows the primers used.

In sample 1, compared to sample 2, the expression of panendoderm markers Sox17 and Foxa2 is 1.4 and 2.8-fold, respectively, and the expression of definitive endoderm marker Cxcr4 is 1.1-fold. Met.

From the above, it was shown that the differentiation efficiency of the definitive endoderm is better when the embryoid body is formed for 5 days than for 2 days.

[References]
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SEQ ID NO: 1: PCR forward primer for detecting AFP gene SEQ ID NO: 2: PCR reverse primer for detecting AFP gene SEQ ID NO: 3: PCR forward primer for detecting Alb gene SEQ ID NO: 4: Alb PCR reverse primer for detecting gene SEQ ID NO: 5: PCR forward primer for detecting Trf gene SEQ ID NO: 6: PCR reverse primer for detecting Trf gene SEQ ID NO: 7: for detecting PCK1 gene PCR forward primer SEQ ID NO: 8: PCR reverse primer for detecting PCK1 gene SEQ ID NO: 9: PCR forward primer for detecting CPS gene SEQ ID NO: 10: PCR reverse primer for detecting CPS gene Limer SEQ ID NO: 11: PCR forward primer for detecting the Sox17 gene SEQ ID NO: 12: PCR reverse primer for detecting the Sox17 gene SEQ ID NO: 13: PCR forward primer SEQ ID NO: for detecting the β-actin gene 14: Reverse primer for PCR for detecting β-actin gene SEQ ID NO: 15: Forward primer for real-time RT-PCR for detecting AFP gene SEQ ID NO: 16: Reverse for real-time RT-PCR for detecting AFP gene Primer SEQ ID NO: 17: Real-time RT-PCR forward primer for detecting Alb gene SEQ ID NO: 18: Real-time RT-PCR reverse primer for detecting Alb gene SEQ ID NO: 19 Real-time PCR forward primer for detecting the ox17 gene SEQ ID NO: 20: Real-time PCR reverse primer for detecting the Sox17 gene SEQ ID NO: 21: Real-time PCR forward primer for detecting the Cxcr4 gene SEQ ID NO: 22: Cxcr4 gene Reverse primer for real-time PCR for detecting NO SEQ ID NO: 23: Forward primer for real-time PCR for detecting Foxa2 gene SEQ ID NO: 24: Reverse primer for real-time PCR for detecting Foxa2 gene

Claims (6)

A method for inducing differentiation from iPS cells to hepatocytes,
(A) a step of suspension culture of iPS cells to form an embryoid body,
(B) inducing definitive endoderm by adhering and culturing 10-15 embryoid bodies on a plate using gelatin as a coating agent per well, and (c) adding HGF and DMSO, A method comprising inducing hepatocyte differentiation by culturing definitive endoderm on a basement membrane substrate. The method according to claim 1, wherein the suspension culture period of (a) is 5 days. The method according to claim 1, wherein the basement membrane matrix is derived from alveolar type II epithelial cells or hepatocytes. The method according to claim 1, wherein the differentiation induction of (c) is further performed by co-culture with non-parenchymal cells of the liver. The method according to claim 4, wherein the nonparenchymal cells of the liver are hepatic sinusoidal endothelial cells. Liver parenchymal cells induced by the method of claim 1.

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