Patent Application
20190062699
The present invention relates to a highly functional hepatocyte induced from an iPS cell, and use thereof.
Even in these days with advanced medical techniques including those for acute stage management, there are still never-ceasing deaths by liver failure in Japan. In particular, fulminant hepatitis, of which mortality rate is as extremely high as 70 to 90%, is a disease that gives bad prognosis. Although liver transplantation provides the highest curative effect as the treatment of liver failure, there are many cases where liver transplantation cannot be performed, because of shortage of donors or drastic progress of acute hepatic failure.
Hybrid artificial liver support systems (HALSSs) are expected as systems used for assisting the functions of the liver in the period before liver transplantation or until regeneration of the patient's own liver. Although primarily cultured swine hepatocytes are preferred as hepatocytes used for the hybrid artificial livers because of favorable growth thereof, it is concerned that reactivation of endogenous retrovirus cannot be completely denied. Furthermore, hepatocytes extracted from brain-dead donors' livers are limited in obtainable quantity. Therefore, hepatocytes that can be proliferated an vitro are required.
Several methods are known for inducing hepatocytes by differentiation from pluripotent hepatocytes. For example, Patent document 1 discloses a method for differentiating, an embryonic stem cell to induce a hepatocyte, wherein embryonic stem cells are cultured in the presence of deletion type hepatocyte growth factor, bFGF, DMSO and dexamethasone. Since it is known that cells can be placed under conditions closer to the environment in the living body to improve cell functions by three-dimensionally culturing the cells on a scaffold, this patent document proposes to place the ES cells in a reactor after the embryoid body formation, and perform three dimensional induction by differentiation with dHGF, and describes that the ammonia-metabolizing ability, which is the highly developed function specific to the hepatocytes, and so forth are thereby markedly enhanced compared with those obtainable by plate culture. Patent document 2 discloses a method for preparing a liver tissue or organ, which comprises the subclone separation step of re-cloning embryonic stem cells and separating subclones, the screening step of allowing simultaneous formation of embryoid bodies derived from subclones among the aforementioned subclones under the same conditions, then performing adhesion culture of the embryoid bodies, and selecting a subclone that gives a number of embryoid bodies that are pulsing at the point 48 hours after the start of the culture not lower than 70% in terms of embryoid body number, and the subclone culture step of culturing the retrieved subclone. Patent document 2 also describes that the obtained cell has morphological and histological characteristics of normal liver cell or organ, specifically, for example, it has the binucleated morphology, it is positive to albumin staining, and it has ammonia decomposition ability, protein (albumin) production ability, and testosterone hydroxylation activity. Patent document 3 discloses a method for inducing differentiation into the liver. This method comprises the following steps: (i) the step of providing a group of artificial pluripotent stein (iPS) cells, (ii) the step of culturing the cells of the group in an endoderm induction medium to form a group of anterior definitive endoderm (ADE) cells (the endoderm induction medium mentioned above mentioned above is a predetermined chemical medium having the following properties, i.e., it has a fibroblast growth factor activity, stimulates the signaling pathway comprising SMAD2 and SMAD3, and the signaling pathway comprising SMAD1, SMAD5, and SMAD9, and inhibits phosphatidylinositol 3-kinase (PI3K) and glycogen synthase kinase 3β (GSK3β)), and (iii) the step of culturing the ADE cells of the aforementioned group in a hepatic induction medium to form a group of hepatic precursor cells (the hepatic induction medium mentioned above is a predetermined chemical medium that stimulates the signaling pathway comprising SMAD2 and SMAD3). Patent document 4 discloses a method for producing a highly functional hepatocyte using a pluripotent stem cell, which comprises obtaining a pluripotent stem cell-derived primitive endoderm from a pluripotent stem cell by a method comprising the steps (A) and (B), obtaining a liver precursor cell from the pluripotent stem cell-derived primitive endoderm by a method comprising the step (C), and obtaining a highly functional hepatocyte from the liver precursor cell by a method comprising the step (D): (A) the step of performing culture in a serum-free and feeder-free environment, (B) the step of performing culture in the presence of albumin and at least one kind of cytokine, (C) the step of performing culture in the presence of SHH or SHH agonist, and at least one kind of cytokine, and (D) the step of allowing maturation by performing culture in the presence of at least one kind of cytokine.
Patent document 4 also describes that, in a preferred embodiment, an ES cell or iPS cell is used as the pluripotent stem cell, and more preferably, an iPS cell established by using a Sendai virus vector is used as the iPS cell.
Although hybrid artificial livers are expected to serve as a bridge to liver transplantation or regeneration of patient's own liver, about 1×1010 to 1011 hepatocytes are required per one artificial liver module. The important functions such as ammonia-metabolizing ability of the artificial hepatocytes examined so far to date have not been clarified, or it cannot be said that the functions of such hepatocytes are sufficient. The inventors of the present invention conducted various researches for obtaining highly functional hepatocytes that are theoretically able to infinitely proliferate from human iPS cells, and as a result, accomplished the present invention.
The present invention provides the followings.
the differentiation induction step 1 of culturing an iPS cell in a differentiation induction medium I containing activin A,
the differentiation induction step 2 of culturing a cell obtained in the differentiation induction step 1 in a differentiation induction medium II containing bone morphogenetic protein 4 (BMP4) and fibroblast growth factor 2 (FGF2): and
the differentiation induction step 3 of culturing a cell obtained in the differentiation induction step 2 in a differentiation induction medium III containing hepatocyte growth factor (HGF), oncostatin M, dexamethasone and N,N′-(methylenebis)(4,1-phenylene)diacetamide (FH1) and/or 2-([N-(5-chloro-2-methylphenyl)(methylsulfonamido)-N-(2,6-difluorophenyl)acetamide (FPH1) to obtain an artificial hepatocyte.
According to the present invention, a highly functional hepatocyte can be induced from an iPS cell by differentiation. An artificial liver tissue produced by the induction method, and a hybrid artificial liver using the tissue can also be provided.
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Numerical value ranges indicated as “X to Y” include the values of X and Y as the maximum and minimum values, unless especially indicated. The expression “A and/or B” means at least one of A and B, unless especially indicated.
The present invention provides an artificial hepatocyte having a high ammonia-metabolizing function and able to constitute a mesh structure.
The expression “able to constitute a mesh structure” used fix the characteristics of the cell means that when the objective cell is cultured under appropriate conditions, a predetermined mesh structure is constituted. The mesh structure is constituted if the cells are inoculated in a culture vessel, which may be coated with an extracellular matrix (ECM) such as Matrigel (registered trademark), hyaluronic acid, heparin, fibronectin, laminin, vitronectin, or other ECM as required, at an appropriate density, and cultured for several days using an appropriate medium. The mesh structure comprises string portions having a thickness corresponding to a width of at least one cell to 1000 μm (more specifically, a width of several cells to 500 μm, further specifically 20 to 250 μm), and voids having a circular shape, elliptical shape, or the like. The diameters of the voids are typically 100 to 2000 μm, more specifically 200 to 1000 μm, further specifically 300 to 1000 μm. The mesh structure may have a thickness of 10 to 60 μm corresponding to the width of 1 to 3 cells.
The term ammonia-metabolizing function referred to in the present invention means ammonia-metabolizing unction observed when the objective cell is cultured under appropriate conditions, unless especially indicated. Methods for measuring ammonia-metabolizing function are well known to those skilled in the art. It is preferable to perform adhesion culture using a culture vessel coated with EMC as required and the compound mentioned later (FH1) without using a feeder cell (HUVECs, MSCs, and the like). As for more detailed conditions, the methods described in the section of Examples of the present specification can be referred to.
The artificial hepatocyte obtained by the present invention has a high ammonia-metabolizing function. Specifically, the artificial hepatocyte of the present invention has an ammonia-metabolizing ability not lower than 100 μg/dl/24 h, preferably not lower than 120 μg/dl/24 h, more preferably that comparable to the metabolizing ability of the primarily cultured hepatocyte (160 to 200μg/dl/24 h), i.e., 160 μg/dl/24 h, still more preferably 180 μg/dl/24 h.
Culture in Serum-Free and/or Feeder-Free Environment
As the medium for culturing artificial hepatocyte of the present invention, the various existing media developed for culturing hepatocytes can be used. Examples of usable medium include the differentiation induction medium III mentioned later, HCM™ BulletKit™ Medium (Lonza Walkersville, Inc), and HBM™ Basal Medium (Lonza Walkersville, Inc).
The artificial hepatocyte of the present invention can be cultured in a serum-free and/or feeder-free environment, preferably a serum-free and feeder-free environment. To culture in a serum-free and feeder-free environment means to perform culture by using a medium containing neither animal serum nor human serum in an environment where any cells other than the objective hepatocyte, such as mouse fetal fibroblast, a feeder cell (human umbilical vein endothelial cell (HUVEC)), or mesenchymal stem cell (MSC), do not coexist.
For the culture of the artificial hepatocyte of the present invention, various kinds of existing culture vessels can be used. Although material of the culture vessel can be appropriately chosen by those skilled in the art, it is preferable to use a culture vessel coated with EMC, since such a culture vessel provides favorable growth and maintains high functionality of the hepatocyte. Preferred examples of ECM include Matrigel (registered trademark), hyaluronic acid, heparin, fibronectin, laminin vitronectin, proteoglycan (chondroitin sulfate proteoglycan, heparan sulphate proteoglycan, keratan sulfate proteoglycan, dermatan sulfate proteoglyan), various collagens, gelatin, tenascin, entactin, elastin, fibrillin, and so forth.
It has been confirmed that the artificial hepatocyte of the present invention can be continuously cultured for about ten days by using the differentiation induction medium III mentioned later. It is considered that the hepatocyte can be subcultured and proliferated by inoculating it at an appropriate density. The appropriate density is, for example, such a density that the artificial hepatocytes exactly contact with one another. A density lower than such a density provides bad proliferation, and a density higher than such a density may induce marked cell death in a region where three-dimensional construction is observed.
The artificial hepatocyte of the present invention can be produced by the method comprising the following steps:
the differentiation induction step 1 of culturing an iPS cell in the differentiation induction medium I containing activin A,
the differentiation induction step 2 of culturing a cell obtained in the differentiation induction step 1 in the differentiation induction medium II containing bone morphogenetic protein 4 (BMP4) and fibroblast growth factor 2 (FGF2); and
the differentiation induction step 3 of culturing a cell obtained in the differentiation induction step 2 in the differentiation induction medium III containing hepatocyte growth factor (HGF), oncostatin M, dexamethasone, and a compound having N-phenyl-2-(N-phenylmethylsulfonamido)acetamide structure to obtain an artificial hepatocyte.
In the differentiation induction step 1, an iPS cell is cultured in the differentiation induction medium I containing at least activin A.
In the present invention, an iPS cell derived from human or nonhuman animal is used as the starting cell. Methods for obtaining and establishing an iPS cell are well known to known those skilled in the art. Human iPS cells can be purchased or provided from Institute of Physical and Chemical Research (Riken) BioResource Research Center, Kyoto University, or National Center for Child Health and Development. Examples of iPS cells that are available and can be used for the present invention include, for example, those of the human artificial pluripotent stein (iPS) cell strain HiPS-RIKEN-1A (Institute of Physical and Chemical Research, HPS0003), which was established by introducing four factors (Oct3/4, Sox2, Klf4, and c-Myc) into an umbilical cord-derived fibroblast (RCB0436 HUC-F2) using a retrovirus vector, human iPS cell strain HiPS-RIKEN-2A (Institute of Physical and Chemical Research, HPS0009), which was established by introducing four factors (Oct3/4 Sox2, Klf4, and c-Myc) into an umbilical cord-derived fibroblast (RCB0197 HUC-Fm) using, a retrovirus vector, human iPS cell strain HiPS-RIKEN-12A (Institute of Physical and Chemical Research, HPS0029), which can be obtained by introducing three factors (Oct3/4, Sox2, and Klf4) into an umbilical cord-derived fibroblast using a retrovirus vector, human iPS cell strain Nips-B2 (Institute of Physical and Chemical Research, HPS0223), which was established by introducing four factors (Oct3/4, Sox2, Klf4, and c-Myc) using a Sendai virus vector, and so forth.
When an iPS cell is established, it is preferable to use a Sendai virus vector as the vector for introducing the factors. Retrovirus vectors enter into cell nuclei, and induce gene expression as DNA, and therefore it is concerned that the vectors may positively enter into patient's chromosomes, or cause genetic recombination with a chromosomal DNA, when the obtained iPS cell is used for the patient, even though such events may very rarely occur. On the other hand, Sendai virus vectors do not enter into cell nuclei, but replicate genomes thereof within the cytoplasm, and produce a lot of proteins. These genomes are made of RNA, and they materially differ from DNAs of the patient's chromosomes. Therefore, it is considered that there is theoretically no risk of modification of chromosomes of the patient in the nucleus by a Sendai virus vector.
Methods for establishing an iPS cell using a Sendai virus (SeV) vector are well known to those skilled in the art, and it can be established by, for example, culturing a commercial human fibroblast or the like in a medium containing a Sendai virus vector carrying a reprogramming factor expression unit. Examples of such a Sendai virus vector carrying a reprogramming factor expression unit include, for example, CytoTune-iPS (DNAVEC Corporation), and so forth.
The differentiation induction medium I used in this step contains a cytokine such as Wnt3A or activin A. It is preferred that at least activin A is contained. As for the amount of the cytokine, amount of Wnt3A is, for example, about 10 to 50 ng/mL, preferably about 25 ng/ml, and amount of activin A is, for example, about 10 to 100 ng/ml, preferably about 100 ng/ml.
The differentiation induction medium I is free from serum, and for example, a medium having a basic composition such as RPMI1640 to which the aforementioned cytokine and serum substitute are added can be used. Examples of the serum substitute include B27 (registered trademark, Life Technologies), Knockout (registered trademark) Serum Replacement (Life Technologies), and so forth. A specific example it the composition of the differentiation induction medium I is that mentioned in the section of Examples in this specification. As for the composition of B27, G J. Brewer et al., Optimized Survival of Hippocampal Neurons in B27-Supplemented Neurobasal™, a New Serum-free Medium Combination, Journal of Neuroscience Research 35567476 (1993) can be referred to.
This step is performed by culturing iPS cells at 37° C. for several days in a 5% CO2 incubator using the differentiation induction medium I. The culture period of the differentiation induction step 1 is specifically 4 to 10 days, preferably 6 to 8 days, more preferably 7 days. The differentiation induction step 1 can be peiformed until the cells come to exhibit morphological characteristics of endomere.
In the differentiation induction step 2, the cells obtained by the differentiation induction step 1 are cultured in the differentiation induction medium II.
The differentiation induction medium II used in this step contains various cytokines, for example, fibroblast growth factor 2 (FGF2), bone morphogenetic protein 4 (BMP4), and hepatocyte growth factor (HGF). As for the amounts of the cytokines, amounts of FGF2, HGF, and BMP4 are, for example, 5 to 50 ng/ml, 5 to 50 ng/ml, and 5 to 50 ng/ml, respectively, preferably about 10 ng/ml, about 20 ng/ml, and about 20 ng/ml, respectively. The differentiation induction medium II contains at least the bone morphogenetic protein 4 (BMP4) and the fibroblast growth factor 2 (FGF2). The differentiation induction medium II can also be serum-free, and for example, a medium having a basic composition such as RPMI1640 to which the aforementioned cytokines and serum substitute are added can be used. A specific example of the composition of the differentiation induction medium II is that mentioned in the section of Examples in this specification.
This step can be performed by culturing the cells obtained in the step 1 at 37° C. in a 5% CO2 incubator using the differentiation induction medium II. The culture period of the differentiation induction step 2 is specifically 1 to 6 days, preferably 2 to 5 days, more preferably 3 to 4 days.
This step is a step of culturing the cells obtained in the differentiation induction step 2 in the differentiation induction medium III to obtain artificial hepatocytes.
Examples of the cytokine and hormone used in this step include oncostatin M (OSM), dexamethasone, and so forth. Amounts to be used of OSM and dexamethasone are, for example, 10 to 50 ng/ml, and 0.05 to 0.5 μM, respectively, preferably about 25 ng/ml, and about 0.1 μM, respectively. The differentiation induction medium III contains at least hepatocyte growth factor (HGF), oncostatin M, dexamethasone, and N,N′-(methylenebis)(4,1-phenylene)diacetamide (FH1) and/or 2-(N-(5-chloro-2-methylphenyl)(methylsulfonamido-N-(2,6-difluorophenyl)acetamide (FPH1). Specific examples of the composition of the differentiation induction medium II are those mentioned in the section of Examples in this specification.
The differentiation induction medium III contains N,N′-(methylenebis)(4,1-phenylene)diacetamide (FH1) and/or 2-N-(5-chloro-2-methylphenyl)(methylsulfonamido)-N-(2,6-difluorophenyl)acetamide (FPH1) represented by the following formulas.
In a preferred embodiment, the differentiation induction medium III contains FH1 and FPH1.
This step can be performed by culturing the cells obtained in the differentiation induction step 2 in the differentiation induction medium III for about several days to several weeks. In this culture period, it is preferable to exchange the medium at a frequency of at least every 3 days, preferably everyday.
All of the differentiation induction steps 1 to 3 can he performed under a serum-free and/or feeder-free environment, preferably serum-free and feeder-free environment. They can also be performed by using a culture vessel coated with ECM, as required.
It is known that hepatic parenchymal cells embryologically appear following the appearance of the cardiac muscle cells in the embryoid body. It is experimentally known that expression of hepatic parenchymal cells is observed near the cardiac muscle cells differentiated from the embryoid bodies.
As for the method for confirming the differentiation of the cells undergone the differentiation induction steps 1 to 3 into hepatocytes, the differentiation can be confirmed on the basis of expression of a hepatocyte (fetal hepatocyte)-specific marker gene or expression of a hepatocyte-specific marker protein such as transthyretin (TTR), α-fetoprotein (AFP) fetal hepatocyte, a1-antitrypsin (AAT), tyrosine aminotransferase (TAT), tryptophan oxygenase (TO), tyrosine aminotransferase, asialoglycoprotein receptor (ASGR), and albumin, or by immunostaining using an anti-albumin antibody. Further, since many binucleated cells are observed among mouse hepatocytes, the differentiation can be confirmed by confirming morphology of the cell nuclei.
The confirmation is preferably performed on die basis of criteria that they can constitute a mesh structure, and they have a high ammonia-metabolizing ability, as described in the section of the artificial hepatocyte mentioned above.
The present invention provides a hybrid artificial liver and a method for producing the same. It is preferred that the hybrid artificial liver uses at least 109 of the artificial hepatocytes of the present invention.
Hybrid artificial livers are classified into three forms, i.e., those equipped outside the body and connected to a blood vessel, those retained inside the body and connected to a blood vessel, and those retained in the abdominal cavity without connecting to a blood vessel. Since the hybrid artificial liver of the present invention uses hepatocytes derived from iPS cells, it is considered that it is preferably of the extracorporeal type in order to avoid the risk concerning cell transfer, and so forth.
In the development of a hybrid artificial liver, design and development of a reactor are also important elements. As bioreactors, there are known those of various types, such as HepatAssist (Hui T, Rozga J, Demetrios A A, J. Hepatobiliary Pancreat. Surg., 2001, 8:1-15), which is for hybrid artificial liver treatment using swine hepatocytes, and MELS (Modular Extracorporeal Liver System) using swine hepatocytes. These reactors can also be used for the present invention. Hepatocytes in a floating state tend not to show sufficient differentiation function, collide with surrounding cells, and easily receive stress stimuli. Further, it is considered that a reactor comprising a scaffold of hollow fibers and nonwoven fabric etc. is preferred so that a scaffold can be provided to the hepatocytes, since it has been found that the artificial hepatocytes of the present invention provide higher ammonia-metabolizing ability when they are cultured as adhesion culture according to the researches conducted by the inventors of the present invention.
Any hollow fiber membrane can be used so long as the cells can adhere to the membrane surface, and substance exchange is not disturbed, and specifically, commercial products conventionally used for medical uses, for example, polysulfone membrane, saponified ethylene-vinyl acetate random copolymer membrane (for example, Eval (trade name), Kuraray Medical. Inc., etc.), and so forth are preferred. As for pore sizes of commercial hollow fiber membranes, there are those having various pore sizes depending on uses thereof such as dialysis membrane (5 nm or smaller), plasma segregation membrane (20 to 30 nm), and plasma skimming membrane (30 to 200 nm). From the viewpoint of substance permeability; a plasma skimming membrane (30 to 200 nm) is preferred. In order to avoid the risk of rejection, the pore size is most preferably 30 to 100 nm, so that immunocompetent cells and immunoglobulin in the blood flowing through the inside of the hollow fibers should not directly contact with the cells filled on the scaffold of nonwoven fabric or the like outside the hollow fibers.
As the nonwoven fabric, those processed and modified so that cells can adhere to them are preferred. Examples of the fibers of the nonwoven fabric include those of polytetrafluoroethylene (PTFE), and so forth. Those consisting of polytetrafluoroethylene (PTFE) treated with polyamino acid methane (PAU) are particularly preferred, because of ease of processing thereof.
The artificial hepatocytes obtained according to the present invention can be used for a test for evaluating toxicity of drugs. When they are used for a toxicity test, an objective drug is added to the artificial hepatocytes, the cells are cultured, for example, at 37° C. in a 5% CO2 incubator, and the toxicity can be evaluated on the basis of change of ammonia-metabolizing ability with time, or presence or absence, or degree of cell death. Examples of techniques for measuring cell death include detection of fragmentation of chromosomal DNA by electrophoresis, detection of chromosomal DNA-reduced cells using flow cytometry, detection of cells causing apoptosis using annexin V, detection of dead cells using dead cell staining agent such as propidium iodide (PI), ATP assay, MTT assay, intracellular glutathione assay, LDH assay, and so forth.
The artificial hepatocytes obtained according to the present invention can be used for drug screening. Specific examples of the method for drug screening include, for example, a method of adding a test substance to a culture medium of a tissue comprising the artificial hepatocytes of the present invention or artificial hepatocytes, incubating them for a predetermined time, and then analyzing a metabolic product contained in at least either one of the culture medium and the cells constituting the liver tissue or organ, or the like, and so forth.
The test substance is not particularly limited, and can be appropriately chosen depending on the purpose, and examples include, for example, drugs, diagnostic agents, and functional foods relating to liver functions, as well as active ingredients of these. Drug screening can also be performed as screening for ingredients useful for improvement of liver functions, or screening for harmful ingredients that damage the liver. Examples of the screening for ingredients useful for improvement of the liver functions include, for example, searches for a cholesterol decomposition-promoting substance, a cholesterol biosynthesis inhibitor, and so forth.
iPS cells were prepared by the following method.
The following procedure was performed to confirm acquisition of iPS cells.
During the culture performed as described later, morphology was periodically observed with the all-in-one fluorescence microscope BZ-9000 (KEYENCE).
Confirmation of Elimination of SeV Vector (CytoTune iPS2.0)
The results of the immunostaining and flow cytometry are shown in
According to the following method, iPS cells for inducing iPS-derived highly functional hepatocytes (CiPS-Hep) were prepared. In the confirmation of the aforementioned prepared iPS cells such as iPS cell colony observation, the methods described in this section were performed.
2.5 μl
According to the following method, CiPS cells were differentiated to induce highly functional hepatocytes
When floating culture is performed, loosen the cell pellet by pipetting using the medium III, and inoculate the cells onto an MPC treatment plate (MD6 with Lid Low-Cell Binding, Nalge Nunc International, Japan).
When HUVEC and MSC are used as feeder cells, perform co-culture as required as adhesion culture (refer to Non-patent document 1 cited below).
Acquisition of iPS cells was confirmed by carrying out the following procedure.
Photographs of the obtained iPS-REP cells are shown in
The obtained results of the ammonia metabolism test and RT-PCR are shown in
5 mM)
o
10 μg/ml)
Biotec
10 μg/ml)
10 μg/ml)
25 mM)
12.86 mM)
indicates data missing or illegible when filed
The highly functional artificial hepatocyte induced from an iPS cell obtained by the present invention can be favorably used as a hybrid artificial liver. It can also be used for drug screening, toxicity test based on ammonia metabolism or the like as an index, and so forth.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2016-037518 | Feb 2016 | JP | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/JP2017/007025 | 2/24/2017 | WO | 00 |
