JP7829851B2 - Tissue manufacturing methods - Google Patents

Description

This specification discloses methods for producing tissues such as tissues containing chondrocytes and tissues containing hepatocytes.

Pluripotent stem cells, such as embryonic stem cells (ES cells) and induced pluripotent stem cells (iPS cells), can be differentiated into target cells and are expected to have applications in the field of regenerative medicine. Methods for differentiating pluripotent stem cells into various cell types, including cardiomyocytes, retinal pigment epithelial cells, and chondrocytes, have been reported.
Patent documents 1 and 2, and non-patent document 1 describe a method for differentiating pluripotent stem cells into chondrocytes.

Special Publication No. 2015-515825 WO2015/064754

“Directed differentiation of human embryonic stem cells toward chondrocytes.” Nat Biotechnol. 2010 Nov;28(11):1187-94.

Patent Document 1 describes a method for producing cartilage-like tissue, etc., through numerous steps, including identifying a population of paraxial mesoderm cells expressing a predetermined surface antigen, culturing the paraxial mesoderm cell population at a high cell density, culturing the paraxial mesoderm cell population to produce a population of chondrocyte precursors, and culturing the population of chondrocyte precursors to produce cartilage-like tissue, etc.

Patent Document 2 describes a method for producing cartilage cells, which includes the steps of: inducing mesodermal cells by adhering culture of pluripotent stem cells; adhering culture of the cells obtained in the first step in a culture medium containing predetermined components; and suspension culture of the cells after adhering culture in a culture medium containing predetermined components.
Non-patent document 1 describes differentiating pluripotent stem cells into chondrocytes by subculturing them in multiple culture media with different compositions.

The methods described in these documents involve inducing chondrocytes from pluripotent stem cells using multiple culture media with different compositions at each stage, and changing the cell adhesion state (adherent culture or suspension culture) and the type of protein coated on the substrate at each stage, making the procedure extremely complicated.
When attempting to automate the culture process, the complexity of the process becomes a major obstacle, so there is a need for the simplest possible differentiation induction system.

Therefore, this specification discloses a method for producing tissue containing chondrocytes and other cells from embryoid bodies obtained by culturing pluripotent stem cells, without altering the adhesion state, by culturing in a single culture medium.

This disclosure encompasses the following inventions:

(1) A method for producing tissue, comprising culturing embryoid bodies on a cell-adherent substrate in a culture medium containing an animal-derived serum substitute, heregulin β1, and insulin-like growth factor 1 to obtain tissue.
(2) The method according to (1), wherein the tissue is a tissue containing chondrocytes or a tissue containing hepatocytes.
(3) The method according to (1) or (2), wherein the culture medium further comprises ascorbic acid or a salt thereof.
(4) The method according to any one of (1) to (3), wherein the culture medium further comprises basic fibroblast growth factor.
(5) The method according to any one of (1) to (4), further comprising culturing pluripotent stem cells in a culture medium containing an animal-derived serum substitute, heregulin β1, and insulin-like growth factor 1 to obtain the embryoid body.
(6) The method according to (5), wherein the culture medium for culturing the pluripotent stem cells to obtain the embryoid body is the culture medium for culturing the embryoid body to obtain the tissue to which a ROCK inhibitor has been added, or is the same as the culture medium for culturing the embryoid body to obtain the tissue.

According to the tissue manufacturing method described herein, it is possible to produce tissue from embryoid bodies by culturing it in a single culture medium without altering its adhesion state.

Figure 1 shows immunostained images of Edom iPS cell-derived cartilage tissue obtained in Example 1, showing collagen type 2. The upper part of Figure 1 shows an overall view of a single tissue sample, and the lower part shows a magnified view of a region with a high density of collagen type 2 positivity. The darker areas indicate collagen type 2. Figure 2 shows a microscopic image of the Edom iPS cell-derived hepatocyte-containing tissue obtained in Example 4. Figure 3 shows the results of microarray analysis of Edom iPS cell-derived hepatocyte-containing tissue obtained in Example 4.

<1. Embryoid body>
An embryoid body is a three-dimensional cell aggregate formed by culturing pluripotent stem cells, such as embryonic stem cells or induced pluripotent stem cells.
Embryoid bodies are, for example, granular cell aggregates with a long axis length ranging from 100 to 500 μm.
Preferred embodiments of the process for producing embryoid bodies will be described later.

In this specification, embryonic stem cells (ES cells) are preferably mammalian ES cells, and for example, ES cells derived from rodents such as mice or primates such as humans can be used. Particularly preferred are mouse or human-derived ES cells. ES cells refer to stem cell lines produced from the inner cell mass belonging to a part of the embryo at the blastocyst stage, which is the early stage of animal development. They can be proliferated almost indefinitely in vitro while maintaining pluripotency, which theoretically allows them to differentiate into all tissues. As ES cells, for example, cells into which a reporter gene has been introduced near the Pdx1 gene can be used to facilitate confirmation of the degree of differentiation. For example, a 129/Sv-derived ES cell line with the LacZ gene incorporated into the Pdx1 locus or the SK7 ES cell line having a GFP reporter transgene under the control of the Pdx1 promoter can be used. Alternatively, the PH3 ES cell line, which possesses an mRFP1 reporter transgene under the control of an Hnf3β endoderm-specific enhancer fragment and a GFP reporter transgene under the control of the Pdx1 promoter, can be used. Furthermore, the ES cell lines SEES1, SEES2, SEES3, SEES4, SEES5, SEES6, or SEES7, established by the Department of Reproductive and Cellular Medicine at the National Center for Child Health and Development and disclosed in Akutsu H, et al. Regen Ther. 2015;1:18-29, or cell lines obtained by introducing further genes into these ES cell lines, can also be used.

In this specification, induced pluripotent stem cells (iPS cells) are pluripotent cells obtained by reprogramming somatic cells. Several groups, including those led by Professor Shinya Yamanaka of Kyoto University, Rudolf Jaenisch of the Massachusetts Institute of Technology, James Thomson of the University of Wisconsin, and Konrad Hochedlinger of Harvard University, have successfully created induced pluripotent stem cells. For example, International Publication WO2007/069666 describes somatic cell nuclear reprogramming factors containing gene products of Oct family genes, Klf family genes, and Myc family genes, and further describes a method for producing induced pluripotent stem cells by nuclear reprogramming of somatic cells, which includes a step of contacting somatic cells with the above-mentioned nuclear reprogramming factors.

The type of somatic cells used to create iPS cells is not particularly limited, and any somatic cells can be used. Somatic cells encompass all cells that make up a living organism except germ cells, and may be differentiated somatic cells or undifferentiated stem cells. The origin of the somatic cells is not particularly limited and can be any mammal, bird, fish, reptile, or amphibian, but is preferably mammalian (for example, rodents such as mice, or primates such as humans), and particularly preferably mouse or human. Furthermore, when using human somatic cells, they may be from a fetus, newborn, or adult. Specific examples of somatic cells include, for instance, fibroblasts (e.g., skin fibroblasts), epithelial cells (e.g., gastric epithelial cells, hepatic epithelial cells, alveolar epithelial cells), endothelial cells (e.g., blood vessels, lymphatic vessels), nerve cells (e.g., neurons, glial cells), pancreatic cells, blood cells, bone marrow cells, muscle cells (e.g., skeletal muscle cells, smooth muscle cells, cardiomyocytes), hepatocytes, non-hepatocytes, adipocytes, osteoblasts, cells constituting periodontal tissue (e.g., periodontal ligament cells, cementoblasts, gingival fibroblasts, osteoblasts), and cells constituting the kidneys, eyes, and ears.

iPS cells are stem cells that possess the ability to self-renew over a long period under specified culture conditions (for example, under the conditions for culturing ES cells) and have the ability to differentiate into ectoderm, mesoderm, and endoderm under specified differentiation induction conditions. Furthermore, iPS cells may also be stem cells that have the ability to form teratomas when transplanted into test animals such as mice.

To produce iPS cells from somatic cells, at least one type of reprogramming gene must first be introduced into the somatic cells. A reprogramming gene is a gene that codes for a reprogramming factor that has the effect of reprogramming somatic cells into iPS cells. Specific examples of reprogramming gene combinations are listed below, but are not limited to these.
(i) Oct gene, Klf gene, Sox gene, Myc gene (ii) Oct gene, Sox gene, NANOG gene, LIN28 gene (iii) Oct gene, Klf gene, Sox gene, Myc gene, hTERT gene, SV40 largeT gene (iv) Oct gene, Klf gene, Sox gene

<2. Method for producing tissue from embryoid bodies>
A method for producing tissue disclosed herein is characterized by obtaining tissue by culturing embryoid bodies on a cell-adherent substrate in a culture medium containing an animal-derived component-free serum substitute, heregulin β1, and insulin-like growth factor 1.

The tissue manufacturing method described in this disclosure allows for the production of tissue from embryoid bodies using only one type of culture medium and adherent culture on a cell-adherent substrate, thus simplifying the operation. Furthermore, when changing the culture medium during cultivation, only the same medium needs to be replaced. Therefore, when using an automated culture system that automatically performs medium changes, there is no need to clean the system's lines after each medium change, making automation easy.

<2.1. Organization>
In this specification, "tissue" refers to a cellular structure containing numerous cells differentiated from an embryoid body.

Examples of "tissues" include tissues containing chondrocytes and tissues containing hepatocytes. Chondrocytes can be identified by their expression of collagen type 2 (Col2A1). Hepatocytes can be identified by their expression of alpha-fetoprotein (AFP). Since hepatocytes also have a characteristic shape, their shape may also be used as an indicator to identify them.

Tissue containing chondrocytes is useful for transplantation in cartilage reconstruction and as a scaffold for cell culture. After culturing the tissue, it can also be transplanted into non-human animals and further cultured to mature cartilage tissue before being used for its intended purpose.

Tissue containing hepatocytes can be used to manufacture hepatocyte-containing preparations. These preparations can be administered to patients with impaired liver function to replace liver function. Hepatocytes may also be isolated and used from the hepatocyte-containing tissue.
Next, preferred embodiments of the culture medium, substrate, and culture conditions used in the tissue manufacturing method relating to this disclosure will be described below.

<2.2. Culture Medium>
In the tissue production method according to this disclosure, it is preferable to use a culture medium containing an animal-derived component-free serum substitute, heregulin β1, and insulin-like growth factor (IGF1) as the culture medium for embryoid bodies. Surprisingly, the inventors have found that by culturing embryoid bodies in a medium of this composition, tissues such as tissues containing chondrocytes and tissues containing hepatocytes can be induced from embryoid bodies without using a medium of other composition.

The culture medium can be prepared by adding an animal-derived serum substitute, heregulin β1, and IGF1 to a basal medium. Examples of basal media include Knockout DMEM (KDMEM) medium, DMEM medium, EMEM medium, MEM medium, DMEM-F12 medium, BME medium, αMEM medium, IMDM medium, ES medium, DM-160 medium, Fisher medium, F12 medium, WE medium, and RPMI 1640 medium. A basal medium that does not contain animal-derived components is particularly preferred.

An example of an animal-derived component-free serum substitute is KnockOut ^™ serum replacement XenoFree (manufactured by Life Technologies). The amount of the animal-derived component-free serum substitute in the culture medium is not particularly limited; for example, if the animal-derived component-free serum substitute is a liquid formulation, it may be 5 to 30% (v/v) of the total volume of the culture medium.

Heregulin β1 may be, for example, human-derived heregulin β1. The content of heregulin β1 in the culture medium is not particularly limited and may be, for example, 5 to 25 ng/mL relative to the total volume of the culture medium.

IGF1 may be, for example, human-derived IGF1. The IGF1 content in the culture medium is not particularly limited and may be, for example, 50 to 500 ng/mL relative to the total volume of the culture medium.

The culture medium more preferably contains ascorbic acid or a salt thereof and one or more basic fibroblast growth factors, and particularly preferably contains both.

The content of ascorbic acid or its salt in the culture medium is not particularly limited; for example, it may be 10 to 200 μg/mL relative to the total volume of the culture medium.

Basic fibroblast growth factor (FGF basic or bFGF) may be, for example, human-derived basic fibroblast growth factor. The content of basic fibroblast growth factor in the culture medium is not particularly limited and may be, for example, 5 to 100 ng/mL relative to the total volume of the culture medium.

The culture medium preferably further contains amino acids, pyruvate or its salts, antibiotics, etc. The amino acids are preferably one or more selected from non-essential amino acids and L-glutamine or L-glutamine substitutes. GlutaMAX ^™ Supplement (Life Technologies) is a suitable L-glutamine substitute. Sodium pyruvate is an example of a pyruvate salt. Penicillin and streptomycin are examples of antibiotics.

The content of non-essential amino acids in the culture medium is not particularly limited; for example, the total amount of non-essential amino acids relative to the total volume of the culture medium may be 0.01 to 1 mM.

The content of L-glutamine or an L-glutamine substitute in the culture medium is not particularly limited and may be, for example, 0.2 to 10 mM relative to the total volume of the culture medium.

The content of pyruvate or its salt in the culture medium is not particularly limited and may be, for example, 0.1 to 10 mM relative to the total volume of the culture medium.

The culture medium may also contain 1 mM to 20 mM of a ROCK inhibitor (e.g., Y27632).

<2.3. Base material>
The method for producing tissue according to this disclosure is characterized by culturing embryoid bodies on a cell-adherent substrate in the culture medium. The "cell-adherent substrate" used herein can be defined as a substrate to which embryoid bodies to be cultured adhere when seeded on the surface of the substrate in the culture medium. The "cell-adherent substrate" can be any substrate that includes a cell-adherent surface. The cell-adherent substrate is not particularly limited, however. Examples include inorganic materials such as glass, metal, ceramic, and silicon, elastomers, and organic materials represented by plastics (e.g., polystyrene resin, polyester resin, polyethylene resin, polypropylene resin, ABS resin, nylon, acrylic resin, fluororesin, polycarbonate resin, polyurethane resin, methylpentene resin, phenolic resin, melamine resin, epoxy resin, and vinyl chloride resin). A preferred example of a cell-adherent substrate is a substrate that includes polystyrene resin on its surface.

The shape of the substrate is not limited; for example, it can be a flat shape such as a plate, flat membrane, film, or porous membrane, or a three-dimensional shape such as a cylinder, stamp, multiwell plate, or microfluidic channel. A flat surface is preferable for the cell-adhering surface of the substrate.

<2.4. Culture conditions>
A method for producing tissue disclosed herein includes culturing embryoid bodies on a cell-adherent substrate in a culture medium containing an animal-derived component-free serum substitute, heregulin β1, and insulin-like growth factor 1 to obtain tissue.

The seeding density of embryoid bodies on the substrate can be, for example, 9 to 15 cells/ ^cm² . While the culture conditions are not particularly limited, it is preferable to perform adherent culture in a static state at 35 to 38°C in an atmosphere with a carbon dioxide concentration of about 5%. The culture medium can be changed as needed during the culture period. The culture medium can be changed automatically.

When embryoid bodies are cultured on the substrate in the aforementioned culture medium, tissue containing hepatocytes is formed approximately 30 to 40 days after the start of culture. If the target is tissue containing hepatocytes, this tissue can be harvested and used at this point. Further cultivation leads to the formation of tissue containing chondrocytes approximately 70 to 100 days after the start of culture. If the target is tissue containing chondrocytes, this tissue can be harvested and used at this point.

When embryoid bodies are cultured on the substrate in the culture medium, a confluent sheet-like tissue is formed approximately 30 days after the start of culture. Further culture results in the formation of a three-dimensional tissue containing an ECM structure and layered cells, or a three-dimensional granular tissue consisting of suspended and aggregated cells, after 60 to 80 days from the start of culture.

<3. Preparation of Embryoid Body>
A method for producing tissue disclosed herein more preferably further comprises an embryoid body preparation step of culturing pluripotent stem cells in a culture medium containing an animal-derived component-free serum substitute, heregulin β1, and insulin-like growth factor 1 to obtain the embryoid bodies.

Specific examples of pluripotent stem cells used in the embryoid body preparation process are as previously described.

In the embryoid body preparation step, it is preferable to obtain embryoid bodies by suspension culture of pluripotent hepatocytes. Suspension of cells is preferably performed in a container with a U-shaped bottom, such as a plate with multiple U-shaped wells. The duration of the embryoid body preparation step is not particularly limited, but for example, 3 to 5 days is a possible duration. The temperature and carbon dioxide concentration conditions for the embryoid body preparation step can be selected from the same range as those for the tissue preparation step (cultivation of embryoid bodies to obtain tissue).

The preferred embodiment of the "culture medium containing an animal-derived component-free serum substitute, heregulin β1, and insulin-like growth factor 1" in the embryoid body preparation step can be selected from the same range as the preferred embodiment of the culture medium in the tissue preparation step.

The "culture medium containing an animal-derived serum substitute, heregulin β1, and insulin-like growth factor 1" in the embryoid body preparation step may be the same as or different from the "culture medium containing an animal-derived serum substitute, heregulin β1, and insulin-like growth factor 1" in the tissue preparation step. Preferably, the culture medium in the embryoid body preparation step is the same as the culture medium in the tissue preparation step, or the same as the culture medium in the tissue preparation step except that a ROCK inhibitor is added to the culture medium in the tissue preparation step (in this case, the culture medium in the tissue preparation step does not contain a ROCK inhibitor). If the culture medium in the embryoid body preparation step contains a ROCK inhibitor (e.g., Y27632), its concentration may preferably be 1 mM to 20 mM.
The embryoid bodies obtained in the embryoid body preparation step can be used in the tissue preparation step.

The following explanation of this disclosure will refer to specific experimental results, but the scope of this disclosure is not limited to the experimental results.

<Example 1>
(Cell culture)
The National Center for Child Health and Development has established the Edom iPS cell line, a human iPS cell line, by transiently expressing four Yamanaka factors in cells obtained from menstrual blood using a Sendai virus vector (PLoS Genet. 2011 May; 7(5): e1002085. Published online 2011 May 26. doi: 10.1371/journal.pgen.1002085 PMCID: PMC3102737).

Edom iPS cells were pre-grown in a vitronectin-coated cell culture dish (Corning) using StemFit medium (Ajinomoto Co., Ltd.). The grown cells were detached from the dish by treating them with EDTA (Invitrogen) diluted 1/1000 with phosphate-buffered saline (PBS) at 37°C for 10 minutes, and then collected as single cells. The collected Edom iPS cells were seeded at a density of 5.0 × ^10³ cells/well in a Nuncron Sphere 96-well U-bottom plate (Thermo Fisher Scientific). As the culture medium, a medium with the composition shown in the table below (referred to as XF32 medium) with 10 mM Y27632 added was used. On the fourth day of culture in XF32 medium supplemented with Y27632, embryoid bodies (EBs) with a long axis length of approximately 100–500 μm were formed. The EBs were harvested and seeded into 6-well plates at a density of 1.08 × ^10² EBs/well, and the culture was transferred to adherent culture. In adherent culture, the medium was changed every 2–3 days using XF32 medium without Y27632, and the culture was continued in the same medium. After 90 days of culture, Edom iPS cell-derived cartilage tissue was obtained.

The cartilage tissue derived from Edom iPS cells was a soft, granular tissue with a long axis dimension of approximately 30 mm.

(chondrocyte staining)
To confirm the expression of collagen type 2, a chondrocyte marker, in Edom iPS cell-derived cartilage tissue, the tissue was stained with an anti-human collagen type 2 antibody. The Edom iPS cell-derived cartilage tissue was fixed overnight using iPGell (GenoStaff) and 4% paraformaldehyde solution (Fujifilm Wako Pure Chemical Industries) according to the product's protocol. After embedding the fixed tissue in paraffin, tissue sections 4-6 μm thick were prepared. The sections were blocked at room temperature for 30 minutes with PBS containing 1% BSA and 0.1% Triton, and then incubated with (Rabbit + IgG, DAB labeled) anti-human collagen type 2 antibody (abcam, 500-fold dilution) at room temperature for 1 hour. After washing three times with PBS, the sections were mounted on coverslips with mounting medium. Microscopic observation confirmed the presence of collagen type 2-positive cells.

Figure 1 shows immunostained images of Edom iPS cell-derived cartilage tissue for collagen type 2. The upper panel of Figure 1 shows an overall view of a single tissue sample, while the lower panel shows a magnified view of a region with a high density of collagen type 2 positivity.

<Example 2>
Edom iPS cell-derived cartilage tissue was obtained by performing the same procedure as in Example 1, except that the culture medium was automatically changed using a CellKeeper cell culture system with an automated medium exchange function (Lorze Life Sciences).

When collagen type 2 in the obtained Edom iPS cell-derived cartilage tissue was stained and observed using the same procedure as in Example 1, collagen type 2-positive cells were confirmed.

<Example 3>
Except for using SEES2 cells, a human ES cell line established at the Department of Reproductive and Cellular Medicine, National Center for Child Health and Development, and disclosed in Akutsu H, et al. Regen Ther. 2015;1:18-29, instead of Edom iPS cells, the same procedure as in Example 2 was followed, and SEES2 cell-derived cartilage tissue was obtained by culturing for 90 days.

When collagen type 2 in the obtained SEES2 cell-derived cartilage tissue was stained and observed using the same procedure as in Example 1, collagen type 2-positive cells were confirmed.

<Example 4>
Differentiation induction was performed in the same manner as in Example 1, but the culture period, which was 90 days in Example 1, was changed to 35 days. The resulting culture dish was examined under a microscope (Figure 2). Subsequently, tissue samples were taken, suspended in TRIZOL, and stored at -80°C. Microarray analysis was then performed using an Agilent SureScan Microarray Scanner G4900DA at DNA Chip Research Institute, Inc.'s microarray contract analysis service (Figure 3).

In Figure 2, clear cell nuclei and binucleated cells can be observed. This meets the visual criteria for identifying hepatocytes.

In microarray analysis, normalized data was created from the signal intensity obtained using a single-color method, using Agilent's GeneSpringGX11 software and the 75 Percentile Shift normalization algorithm. This method compares RNA expression levels by assuming that the total RNA amount used in the experiment is constant even for different samples, and correcting for experimental errors. The results are shown in Figure 3. In Figure 3, "Hepato1" is the data for samples differentiated using this method, and "pHAES" is the data for the ES cells that served as the raw material for Hepato1, which are used for comparison. As shown in Figure 3, the expression of the liver markers α-fetoprotein (AFP) and albumin (ALB) was confirmed.

Claims (5)

A method for producing tissue containing chondrocytes , comprising culturing embryoid bodies for 70 days or more on a cell-adherent substrate in a culture medium containing an animal-derived serum substitute, heregulin β1, and insulin-like growth factor 1 to obtain tissue containing chondrocytes . The method according to claim 1 , wherein the culture medium further comprises ascorbic acid or a salt thereof. The method according to claim 1 or 2 , wherein the culture medium further comprises basic fibroblast growth factor. The method according to any one of claims 1 to 3 , further comprising culturing pluripotent stem cells in a culture medium containing an animal-derived serum substitute, heregulin β1, and insulin-like growth factor 1 to obtain the embryoid body. The method according to claim 4, wherein the culture medium for culturing the pluripotent stem cells to obtain the embryoid body is a culture medium obtained by adding a ROCK inhibitor to the culture medium for culturing the embryoid body to obtain tissue containing the chondrocytes , or is the same as the culture medium for culturing the embryoid body to obtain tissue containing the chondrocytes .