Method for producing neural crest cells

Abstract

The present invention addresses the problem of providing a simple and efficient method for inducing differentiation of pluripotent stem cells into neural crest cells.　Provided is a method for producing neural crest cells, said method comprising a step for culturing stem cells for at least 6 days under conditions in which the expression or activity of a novel protein kinase C (nPKC) and / or an atypical protein kinase C (aPKC) is suppressed or inhibited.

Description

Method for producing neural crest cells

[0001] This invention relates to a method for producing neural crest cells, and a method for inducing the differentiation of neural crest cells, etc.

[0002] When inducing the differentiation of human pluripotent stem cells into specific differentiated cells for use in cell therapy, it is necessary to induce intermediate differentiation stages, such as neural crest cells, before inducing final differentiation.

[0003] Neural crest cells are cells that arise at the boundary between the neuroectoderm and epidermal ectoderm during vertebrate embryonic development. During the formation of the neural tube, they migrate from its dorsal periphery to various tissues throughout the body. Once neural crest cells reach their destination tissue, they differentiate into specific cell types, giving rise to skull, cartilage, smooth muscle, and pigment cells, as well as neurons and glial cells of the peripheral nervous system.

[0004] Methods for inducing differentiation from pluripotent stem cells into neural crest cells have been developed in the past using fibroblast growth factor (FGF), transforming growth factor β (TGF-β) inhibitors, and Wnt signaling activators.

[0005] Non-patent document 1 describes a method for inducing neural crest cells from human iPS cells and further inducing mesenchymal stromal cells, and discloses the use of a TGF-β inhibitor, epidermal growth factor (EGF), and basic fibroblast growth factor (bFGF) for the maintenance culture of neural crest cells.

[0006] Non-patent document 2 describes a method for inducing neural crest cells from human embryonic stem cells and human iPS cells, and further inducing smooth muscle cells, etc. It discloses the use of GSK3β inhibitors and TGFβ inhibitors for the maintenance culture of neural crest cells.

[0007] Non-patent document 3 describes a method for inducing neural crest cells from human iPS cells and maintaining them using a GSK3β inhibitor, a TGFβ inhibitor, EGF, and bFGF.

[0008] There is a need for an efficient method to differentiate human iPS cells into neural crest cells for use in cell therapy for intractable or progressive peripheral nerve diseases. However, the methods described above involve a complex differentiation induction process and are not sufficiently efficient. Furthermore, they require the use of multiple growth factors such as FGF, making them far from simple.

[0009] Fukuta M. et al., PLoS One, 2014, 9(12):e112291.Menendez L. et al., Proc. Natl. Acad. Sci., 2011, 108(48):19240-5.Horikiri T. et al., PLoS One, 2017, 12(1):e0170342.

[0010] The objective is to provide a simple and efficient method for inducing differentiation from pluripotent stem cells into neural crest cells.

[0011] To solve the above problems, the inventors cultured human iPS cells in the presence of various PKC inhibitors and evaluated their activity in inducing differentiation into neural crest cells. As a result, they found that when using inhibitors that inhibit conventional PKC (cPKC) and novel PKC (nPKC), the efficiency of differentiation into neural crest cells was significantly increased compared to inhibitors that inhibit only cPKC. Furthermore, they found a surprising effect when using a pan-PKC inhibitor that inhibits all of cPKC, nPKC, and atypical PKC (aPKC), that the majority of cells differentiated into neural crest cells.

[0012] The present invention is based on the above findings and provides the following: (1) A method for producing neural crest cells, comprising the step of culturing pluripotent stem cells for 6 days or more under conditions in which the expression or activity of novel protein kinase C (nPKC) and / or atypical protein kinase C (aPKC) is suppressed or inhibited. (2) The method according to (1), wherein the culture is adherent culture. (3) The method according to (1) or (2), wherein the culture is further carried out under conditions in which the expression or activity of conventional protein kinase C (cPKC) is suppressed or inhibited. (4) The method according to (3), wherein the culture is carried out in the presence of one or more PKC inhibitors selected from the group consisting of sotrastaurin, Ro 32-0432, enzastaurin, Ro 31-8220, bisindolylmaleimide VIII, and staurosporine. (5) The method for producing a cell according to any one of (1) to (4), wherein the culture is carried out under conditions in which the expression or activity of nPKC, aPKC, and cPKC is suppressed or inhibited. (6) The method for producing a cell according to (5), wherein the culture is carried out in the presence of a pan-PKC inhibitor. (7) The method for producing a cell according to (6), wherein the pan-PKC inhibitor is Go 6983 or Lottrelin. (8) The method for producing a cell according to any one of (1) to (7), wherein the culture is carried out in a basic medium or a serum-free medium that does not contain basic fibroblast growth factor (bFGF) and / or transforming growth factor β (TGF-β). (9) The method for producing a cell according to (8), wherein the serum-free medium does not contain any non-human components. (10) The method for producing a cell according to any one of (1) to (9), wherein the pluripotent stem cells are induced pluripotent stem cells (iPS cells). (11) The method for producing a cell according to any one of (1) to (10), wherein embryoid bodies of the pluripotent stem cells are not formed before the culture step. (12) A method for inducing differentiation of neural crest cells, comprising the step of culturing stem cells for 6 days or more under conditions in which the expression or activity of novel protein kinase C (nPKC) and / or atypical protein kinase C (aPKC) is suppressed or inhibited. This specification includes the disclosures of Japanese Patent Application No. 2024-182222, which forms the basis of the priority of this application.

[0013] The present invention provides a simple and efficient method for inducing differentiation from pluripotent stem cells into neural crest cells.

[0014] Figure 1 shows an overview of the culture method for inducing differentiation of human iPS cells into neural crest cells. Figure 2 shows the results of evaluating the positive rate of CD271, a neural crest cell marker, by flow cytometry after inducing differentiation of the WTC11 cell line as human iPS cells into neural crest cells. Results for undifferentiated iPS cells and results after culturing iPS cells in a medium without PKC inhibitors or in a medium containing various PKC inhibitors are shown. Figure 3 shows the results of evaluating the positive rate of CD271, a neural crest cell marker, by flow cytometry after inducing differentiation of the 1231A3 cell line as human iPS cells into neural crest cells. Figure 4 shows the results of evaluating the positive rate of CD271, a neural crest cell marker, by flow cytometry after inducing differentiation of the HiPS-NB1RGB cell line as human iPS cells into neural crest cells. Figure 5 shows the area ratio of regions positive for each marker after immunostaining evaluation of the neural crest cell marker SOX10 and the neural progenitor cell marker PAX6 following 10 days of culture of the human iPS cell line HiPS-NB1RGB in or without Go 6983. Figure 6 shows the results of flow cytometry evaluation of the positive rate of the neural crest cell marker CD271 after differentiation of the human iPS cell line HiPS-NB1RGB into neural crest cells. Results for undifferentiated iPS cells and results after culturing iPS cells in a medium without PKC inhibitors or in a medium containing various PKC inhibitors are shown.

[0015] 1. Method for Producing Neural Crest Cells 1-1. Overview The first aspect of the present invention is a method for producing neural crest cells. The production method in this aspect includes a step of culturing pluripotent stem cells under conditions in which the expression or activity of novel protein kinase C (nPKC) and / or atypical protein kinase C (aPKC) is suppressed or inhibited, and neural crest cells can be produced simply and efficiently.

[0016] 1-2. Definitions of Terms The terms frequently used in this specification are defined below.

[0017] In this specification, "pluripotency" is synonymous with multipotency and refers to the property of a cell that can differentiate into multiple cell lineages through differentiation. In particular, it refers to the property of being able to differentiate into all of the endoderm, mesoderm, and ectoderm, but does not refer to the possibility of differentiation into extraembryonic tissues such as the placenta.

[0018] In this specification, "pluripotent stem cells" refers to stem cells that possess the pluripotency described above. Specific examples of pluripotent stem cells include induced pluripotent stem cells and embryonic stem cells.

[0019] In this specification, "induced pluripotent stem cell (iPSC)" refers to cells obtained from somatic cells through induction treatment that possess totipotency similar to that of embryonic stem cells. Typically, iPS cells possess pluripotency, allowing them to differentiate into any type of cell in the body except extraembryonic tissues, and proliferative capacity, enabling them to grow almost indefinitely in culture. While iPS cells can be obtained from various types of cells by various methods, they are usually produced by introducing four reprogramming factors—OCT3 / 4, SOX2, KLF4, and C-MYC—into somatic cells.

[0020] In this specification, "embryonic stem cell (ESC)" refers to cells derived from an early embryo that are capable of proliferating while maintaining their undifferentiated and pluripotent state. Embryonic stem cells can be obtained by culturing cells from the inner cell mass, which are undifferentiated stem cells present inside the blastocyst of an early embryo.

[0021] In this specification, "somatic cells" refers to cells other than germ cells that make up an animal organism. In this specification, somatic cells are not limited to cells that can acquire pluripotency through reprogramming induction. Furthermore, the animal species from which the somatic cells originate is not limited. The animal species from which the somatic cells originate is, for example, mammals. For example, it may be any mammalian species such as mouse, rat, rabbit, cattle, cynomolgus monkey, marmoset, or human, but human is preferred. The tissues or organs from which the somatic cells originate are not particularly limited, but those that are easy to collect and can be efficiently reprogrammed are preferred. For example, they may be skin, organs such as the liver, blood, dental pulp cells, etc. Furthermore, somatic cells may be differentiated or undifferentiated cells, and may be cell lines or primary cultured cells isolated from tissue. Differentiated cells are preferred. Specific examples of somatic cells in this specification include human fibroblasts, human epithelial cells, human hepatocytes, human hematopoietic cells, mesenchymal cells, nerve cells, muscle cells, etc.

[0022] In this specification, "reprogramming" refers to the operation or process of changing a somatic cell into another cell type. Generally, it refers to dedifferentiating a differentiated cell to change it into an undifferentiated cell. In this specification, unless otherwise specified, it refers to the operation or process of changing a somatic cell into an iPS cell.

[0023] In this specification, "reprogramming factor (initialization factor)" means a factor that, when introduced into somatic cells, either alone or in combination with other factors, can induce reprogramming of somatic cells. In this specification, when the term "reprogramming factor" is used without specifying whether it is a protein or a gene, it means either the protein to which the reprogramming factor belongs, the nucleic acid encoding the protein, or a gene expression vector containing the nucleic acid. Examples of reprogramming factors include, for example, any of the four factors OCT3 / 4, SOX2, KLF (e.g., KLF1, KLF2, KLF4, or KLF5), and C-MYC (sometimes referred to as the "four initialization factors" in this specification), as well as related factors of any of the four initialization factors.

[0024] In this specification, "neural crest cells" refer to a group of cells characteristic of vertebrates that develop from the neural ectoderm and epidermal ectoderm at the boundary when the neural tube is formed from the neural plate during early development. Neural crest cells are known to differentiate into various tissues and organs, such as pigment cells, peripheral nerves, endocrine cells, and connective tissue of the head. CD271 and SOX10 are known markers for neural crest cells.

[0025] In this specification, "protein kinase C (PKC)" refers to a type of protein kinase that has the activity to phosphorylate the hydroxyl groups of serine and / or threonine residues in proteins. PKC is primarily derived from Ca2+, which contributes to its kinase activity. 2+ And conventional PKCs (cPKCs) that require binding with diacylglycerol (DAG), and kinase activity with Ca 2+ Novel PKCs (nPKCs) that do not require binding to Ca, and kinase activity with Ca 2+ PKCs are classified into three groups: conventional PKCs, atypical PKCs (aPKCs), and atypical PKCs (also called atypical PKCs), which do not require binding to DAGs. Isozymes belonging to each group include PKCα, PKCβI, PKCβII, and PKCγ for conventional PKCs; PKCδ, PKCε, PKCη, and PKCθ for novel PKCs; and PKCζ and PKCι for atypical PKCs.

[0026] In this specification, "protein kinase C inhibitor (PKC inhibitor)" refers to a substance that has the activity to suppress or inhibit the kinase activity of any PKC. Generally, PKC inhibitors can be classified based on their inhibitory activity against PKCs belonging to the three groups described above.

[0027] In this specification, "cPKC inhibitor" means an inhibitor having the activity to suppress or inhibit at least one cPKC isozyme (one or more of PKCα, PKCβI, PKCβII, or PKCγ). Unless otherwise specified, it means an inhibitor that does not substantially have the activity to suppress or inhibit the kinase activity of PKC other than cPKC.

[0028] In this specification, "nPKC inhibitor" means an inhibitor having the activity to suppress or inhibit at least one nPKC isozyme (one or more of PKCδ, PKCε, PKCη, or PKCθ). Unless otherwise specified, it means an inhibitor that does not substantially have the activity to suppress or inhibit the kinase activity of PKCs other than nPKC.

[0029] In this specification, "aPKC inhibitor" means an inhibitor having the activity to suppress or inhibit at least one aPKC isozyme (one or more of PKCζ or PKCι). Unless otherwise specified, it means an inhibitor that does not substantially have the activity to suppress or inhibit the kinase activity of PKCs other than aPKC.

[0030] In this specification, "pan-PKC inhibitor" means an inhibitor having activity to suppress or inhibit all of at least one cPKC isozyme, at least one nPKC isozyme, and at least one aPKC isozyme. A pan-PKC inhibitor is preferably one that can suppress or inhibit substantially all PKC isozymes. In this specification, "substantially suppressing or inhibiting all PKC isozymes" means suppressing or inhibiting 80% or more, preferably 90% or more, or 95% or more of all PKC isozymes.

[0031] In this specification, "expression or activity of PKC is suppressed or inhibited" means that the expression or activity of PKC is reduced compared to the expression or activity of PKC in pluripotent stem cells maintained in an undifferentiated state under normal culture conditions in the art. The degree to which the expression or activity of PKC is reduced is not particularly limited, as long as neural crest cells can differentiate from pluripotent stem cells in the culture of this process. For example, the expression or activity of PKC may be suppressed by 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, 99% or more, or 100% or more compared to PKC in pluripotent stem cells maintained in an undifferentiated state.

[0032] In this specification, "gene knockdown" refers to an operation that reduces the expression level of a target gene. For example, the antisense method in which mRNA of the target gene is degraded by RNaseH using antisense DNA capable of hybridizing to RNA transcribed from the target gene, and the RNA interference method (RNAi) in which gene expression is suppressed post-transcriptionally by degrading the target mRNA using siRNA or shRNA, etc. are mentioned. For example, methods such as introducing an expression vector of antisense nucleic acid or shRNA into cells, and using antisense nucleic acid or shRNA containing artificial nucleic acid are mentioned.

[0033] In this specification, "gene knockout" refers to destroying the function of a gene by adding an insertion or deletion to the target gene on the chromosome. For gene knockout, gene targeting methods that modify endogenous genes using homologous recombination, artificial DNA cleavage enzymes such as ZFN and TALEN, and genome editing techniques using site-specific nucleases such as CRISPR / Cas can be used.

[0034] In this specification, "culturing" refers to maintaining, proliferating, and / or differentiating cells in vitro. For example, culturing cells outside the tissue or outside the body using a culture container such as a cell culture dish or flask is mentioned.

[0035] In this specification, "adherent culture" means culturing cells in a state where the cells are attached to a container, for example, in a state where the cells are attached to a sterile plastic or a coated plastic cell culture dish or flask.

[0036] In this specification, "suspension culture" means culturing cells in a state where they are dispersed as single cells or cell aggregates in a medium without attaching them to a container.

[0037] In this specification, "basic medium" or "minimal medium" refers to a solution containing components necessary for cell survival, such as inorganic salts, essential amino acids, vitamins, and buffers. Examples of basic media include, but are not limited to, EMEM medium (also referred to as αMEM medium), DMEM medium, IMDM medium, GMEM medium, HAM F10 medium, HAM F12 medium, RPMI1640 medium, Essential 6 medium, and combinations thereof. Mediums to which specific factors have been added to a basic medium are collectively referred to as CDM medium (Chemically defined medium).

[0038] In this specification, "serum-free medium" refers to a medium that contains components necessary for cell survival and proliferation but does not contain unprocessed or unpurified serum. Components necessary for cell survival and proliferation that may be included in serum-free medium include hormones and growth factors, as well as serum albumin, transferrin, fatty acids, collagen precursors, trace elements, 2-mercaptoethanol, or 3'-thiolglycerol. Furthermore, serum-free medium may contain purified blood-derived components or animal tissue-derived components. Examples of serum-free media include, but are not limited to, MesenCult, StemPro MSC, BMN211, StemMACS, MSC NutriStem XF, Xuri NSC, PRIME-XV, StemXVivo, Human Mesenchymal-XF Expansion Medium, stemgro, STK2, PLTMax, ProculAD, Mesenchymal Stem Cell Growth Medium XF, Mesenchymal Stem Cell Growth Medium 2, MesenGro, StemFit, MSC-T4, CiMS-BM, MSC-Brew GMP Medium, StemXVivo Serum-Free Human MSC Expansion Media, and combinations thereof. Media containing artificial serum are considered to be included in serum-containing media.

[0039] In this specification, "a plurality" means, for example, 2 to 40, 2 to 30, 2 to 20, 2 to 15, 2 to 10, 2 to 7, 2 to 5, 2 to 4, 2 to 3, or 2.

[0040] 1-3. Method The manufacturing method of this aspect includes, as an essential step, a culturing step of culturing pluripotent stem cells, and as optional steps, an initialization step, an iPS cell selection step, a pre-culturing step, and / or a recovery step. Hereinafter, the specific configuration of each step will be described.

[0041] (Culturing step) In the manufacturing method of this aspect, the "culturing step" is a step of culturing pluripotent stem cells under conditions where the expression or activity of novel PKC (nPKC) and / or atypical PKC (aPKC) is suppressed or inhibited. By culturing in this step, the differentiation of pluripotent stem cells into neural crest cells is induced.

[0042] The culturing of pluripotent stem cells in this step is characterized by being performed under conditions where the expression or activity of nPKC and / or aPKC is suppressed or inhibited. In this specification, "the expression or activity of nPKC and / or aPKC is suppressed or inhibited" means that the expression or activity of at least one nPKC isozyme and / or at least one aPKC isozyme is suppressed or inhibited. For example, among PKCδ, PKCε, PKCη, and PKCθ classified as nPKC, the expression or activity of one or more, two or more, or three or more, or all of them is suppressed or inhibited, and / or among PKCζ and PKCι classified as aPKC, the expression or activity of one or two or all of them is suppressed or inhibited. Those among nPKC and / or aPKC whose expression or activity is suppressed or inhibited are not particularly limited. For example, the expression or activity of PKCζ belonging to aPKC may be suppressed or inhibited.

[0043] In this step, as a specific method for suppressing or inhibiting the expression or activity of nPKC and / or aPKC, any method known in the art can be used. Without limitation, the expression or activity of nPKC and / or aPKC can be suppressed or inhibited by a PKC inhibitor, gene knockdown, or gene knockout, or any combination thereof.

[0044] In one embodiment, the culture in this step is carried out in the presence of at least one nPKC inhibitor and / or at least one aPKC inhibitor, or in the presence of a PKC inhibitor that inhibits at least one nPKC and at least one aPKC. Specific examples of nPKC inhibitors that can be used in this embodiment include VTX-27 and PKC-theta inhibitors (e.g., PKC-theta inhibitor 1 and PKC-theta inhibitor 2). Specific examples of aPKC inhibitors include ζ-Stat (NSC37044) and ZIP (myristoylated peptide). Furthermore, specific examples of PKC inhibitors that inhibit both nPKC and aPKC include any combination of the above-mentioned nPKC inhibitors and aPKC inhibitors.

[0045] In this process, the concentration of the PKC inhibitor used is not limited, as long as neural crest cells can differentiate from pluripotent stem cells by reducing PKC expression or activity. The concentration of the inhibitor in the culture medium used in this process is, for example, 0.1 μM or higher, 1 μM or higher, 5 μM or higher, or 10 μM or higher. The upper limit of the concentration is not particularly limited, but may be, for example, 1 mM or less, 100 μM or less, 50 μM or less, 20 μM or less, or 15 μM or less. Examples of ranges include 0.1 μM to 1 mM, 1 μM to 100 μM, 5 μM to 50 μM, or 10 μM to 20 μM. The same applies to the concentrations of other PKC inhibitors used, as described later.

[0046] In one embodiment, the culture in this step is carried out under conditions in which the expression levels of the nPKC gene and / or the aPKC gene are reduced by gene knockdown. For example, the culture in this step can be carried out in the presence of antisense DNA, siRNA, or shRNA against the nPKC gene and / or the aPKC gene.

[0047] In further embodiments, the culture in this step is carried out under conditions in which the expression or activity of cPKC is further suppressed or inhibited. "Conditions in which the expression or activity of cPKC is further suppressed or inhibited" means conditions in which the expression or activity of nPKC and cPKC, or aPKC and cPKC, preferably nPKC and cPKC, is suppressed or inhibited, or conditions in which the expression or activity of nPKC, aPKC, and cPKC is suppressed or inhibited. For example, with respect to cPKC, the expression or activity of one or more, two or more, three or more, or all of the PKC isozymes classified as cPKC—PKCα, PKCβI, PKCβII, and PKCγ—may be suppressed or inhibited. The method for suppressing or inhibiting each PKC in this embodiment can be a PKC inhibitor, gene knockdown, gene knockout, or any combination thereof, as described above. For example, in this embodiment, culture under conditions in which the expression or activity of nPKC and cPKC is suppressed or inhibited is carried out in the presence of inhibitors that inhibit nPKC and cPKC, or in the presence of a combination of an nPKC inhibitor and a cPKC inhibitor. Examples of PKC inhibitors that have activity to inhibit nPKC and cPKC include sotrastaurin, Ro 32-0432 (sometimes called Bisindolylmaleimide XI), enzastaurin, Ro 31-8220 (sometimes called Bisindolylmaleimide IX), Bisindolylmaleimide VIII, and staurosporine. Salts of these compounds, such as Bisindolylmaleimide XI hydrochloride, Bisindolylmaleimide IX mesylate, and Bisindolylmaleimide VIII acetate, are also examples.Furthermore, when using a combination of nPKC inhibitors and cPKC inhibitors, the nPKC inhibitors are as specified above, and specific examples of cPKC inhibitors include ruboxystaurin (sometimes called LY333531, and its salt, Rubocistaurin (LY333531) HCl), GF 109203X (Bisindolylmaleimide I), and Go 6976. Any combination of these can be used.

[0048] In further embodiments, the culturing in this step is carried out under conditions in which the expression or activity of nPKC, aPKC, and cPKC is suppressed or inhibited. In this specification, "the expression or activity of nPKC, aPKC, and cPKC is suppressed or inhibited" means that the expression or activity of at least one nPKC isozyme, at least one aPKC isozyme, and at least one cPKC isozyme is suppressed or inhibited. For example, the expression or activity of one or more, two or more, three or more, or all of the isozymes classified as nPKC, namely PKCδ, PKCε, PKCη, and PKCθ, is suppressed or inhibited; the expression or activity of one or two or all of the isozymes classified as aPKC, namely PKCζ and PKCι, is suppressed or inhibited; and the expression or activity of one or more, two or more, three or more, or all of the isozymes classified as cPKC, namely PKCα, PKCβI, PKCβII, and PKCγ, is suppressed or inhibited. The method for suppressing or inhibiting each PKC in this embodiment can be a PKC inhibitor, gene knockdown, gene knockout, or any combination thereof, as described above. For example, the culture in this embodiment is carried out in the presence of a pan-PKC inhibitor that inhibits nPKC, aPKC, and cPKC, or in the presence of a combination of an nPKC inhibitor, an aPKC inhibitor, and a cPKC inhibitor. Examples of pan-PKC inhibitors having activity to inhibit nPKC, aPKC, and cPKC include Go 6983 and Lottrelin, with Go 6983 being particularly preferred. Salts of these compounds can also be used. When using combinations of nPKC inhibitors, aPKC inhibitors, and cPKC inhibitors, specific examples of each inhibitor are as described above, and any combination of these can be used. In this embodiment, combinations of PKC inhibitors having activity to inhibit nPKC and cPKC (e.g., sotrastaurin, Ro 32-0432, enzastaurin, Ro 31-8220, bisindolylmaleimide VIII, and staurosporine) and aPKC inhibitors can also be used.

[0049] The culture period in this step is not limited as long as neural crest cells can differentiate from pluripotent stem cells. The culture period is generally 6 days or more, for example, 7 days or more, 8 days or more, 9 days or more, or 10 days or more, and / or 30 days or less, 25 days or less, 20 days or less, or 15 days or less, preferably 10 days to 15 days, 11 days to 14 days, or 12 days to 13 days.

[0050] In this step, the culture conditions such as CO2 concentration and medium exchange frequency are not limited. For example, the culture temperature can be about 30 ° C to about 40 ° C (for example, 37 ° C), the CO2 concentration can be about 2% to about 10% (for example, 5% CO2), and static culture can be carried out, and the medium can be exchanged every 2 days. Also, the culture mode is not limited, and it can be, for example, adherent culture, suspension culture, floating culture, etc., but adherent culture is preferably used. Also, the seeding density of the pluripotent stem cells used in this step is not particularly limited, for example, 1.0×10 2 cells / cm 2 ~1.0×10 5 cells / cm 2 or 1.0×10 3 cells / cm 2 ~1.0×10 4 cells / cm 2 may be used.

[0051] In one embodiment, the culture in this step can be carried out using a basal medium. The basal medium may be, for example, Essential 6. Alternatively, the culture in this step can also be carried out using a serum-free medium. In one embodiment, the serum-free medium may be a medium that does not contain fibroblast growth factors (FGF) such as basic fibroblast growth factor (bFGF) and / or transforming growth factor β (TGF-β). For example, the serum-free medium can be one that does not contain basic fibroblast growth factor (bFGF). Also, the serum-free medium can be a medium that does not contain factors such as Wnt activators (eg, GSK-3 inhibitors such as CHIR-99021 and BIO) and epidermal growth factor (EGF).

[0052] If serum-free culture is used in this process, the serum-free culture medium may not contain non-human-derived components. Culture under conditions that do not contain non-human-derived components is sometimes referred to as "xeno-free" in this technical field.

[0053] The neural crest cells obtained in this process may be maintained or proliferated by performing expansion culture or subculture as appropriate after this process.

[0054] (Initialization step) In the manufacturing method of this embodiment, the "initialization step" is a step in which somatic cells are reprogrammed by introducing a reprogramming factor (initialization factor) into somatic cells. This step is an optional step and may be performed as needed.

[0055] In this process, four factors (four reprogramming factors), such as OCT3 / 4, SOX2, KLF (e.g., KLF1, KLF2, KLF4, or KLF5), and C-MYC, are introduced into somatic cells. These factors can be introduced into somatic cells as proteins or their peptide fragments, nucleic acids encoding them, or gene expression vectors containing such nucleic acids in an expressible state. The specific introduction method should be appropriately selected depending on the type of protein, plasmid DNA, mRNA, viral vector, etc. For example, reprogramming factors can be introduced into somatic cells by viral infection, lipofection, liposomes, electroporation, calcium phosphate, DEAE-Dextran, microinjection, or electroporation. In addition, known gene transfer methods (transformation methods) in this field, such as those described in Green & Sambrook, 2012, Molecular Cloning: A Laboratory Manual Fourth Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, can also be used.

[0056] iPS cells reprogrammed in this process can be proliferated by culturing them using feeder cells or the like. Feeder cells are not limited to those mentioned above, but may include cells whose proliferation has been stopped by irradiation or antibiotic treatment (e.g., mouse embryonic fibroblasts (MEFs), human embryo-derived cells, or fibroblasts). If feeder cells are not used, methods using culture dishes coated with basement membrane matrix, laminin, and vitronectin, or methods using a culture medium containing basement membrane matrix, laminin, and vitronectin can be used.

[0057] For culturing iPS cells, any known cell culture medium can be appropriately selected and used. For example, commercially available mammalian cell basal media such as DMEM with added serum or serum replacement may be used. As an example of serum replacement, for example, KnockOut® Serum Replacement: KSR (ThermoFisher, SCIENTIFIC) may be used. Alternatively, commercially available primate ES cell medium or primate ES / iPS cell medium may be used. These media may contain known additives suitable for culturing pluripotent stem cells such as ES cells or iPS cells, such as N2 supplement, B27(R) supplement, insulin, bFGF, activin A, heparin, ROCK (Rho-associated coiled-coil forming kinase / Rho-binding kinase) inhibitor, and / or GSK-3 inhibitor. Furthermore, in culturing iPS cells, there are no limitations on culture conditions such as temperature, CO2 concentration, culture period, and frequency of medium changes. For example, the culture may be incubated statically at 37°C and 5% CO2, with half the culture medium replaced every two days, for 2 to 40 days depending on the colony formation stage.

[0058] (iPS cell selection step) In the manufacturing method of this embodiment, the "iPS cell selection step" is a step of selecting iPS cells induced after the reprogramming step described above. This step is a selection step and may be performed as needed. The method of selecting iPS cells in this step is not limited. For example, a method of selection using the expression of an iPS cell marker gene as an indicator, a method of selection by a selection marker gene, or a method of selection by a reporter gene can be used. Examples of iPS cell marker genes are not limited to genes that are not expressed in somatic cells targeted for reprogramming but are expressed in iPS cells, and include Oct3 / 4, Sox2, Nanog, ERas, Esg1, TRA1-60, or TRA-1-85 genes, and endogenous alkaline phosphatase genes. Examples of selection marker genes include drug resistance genes such as ampicillin resistance genes, kanamycin resistance genes, tetracycline resistance genes, chloramphenicol resistance genes, neomycin resistance genes, puromycin resistance genes, and hygromycin resistance genes. Other examples of reporter genes include genes that encode fluorescent proteins such as GFP and RFP, and luciferase genes.

[0059] (Pre-culture step) In the manufacturing method of this embodiment, the "pre-culture step" is a step of pre-culturing iPS cells or pluripotent stem cells after the iPS cell selection step described above and / or before the culture step described above. This step is a selection step and may be performed as needed. In the pre-culture in this step, for example, the cells may be cultured statically at 37°C and 5% CO2, with half the amount of culture medium replaced every two days, and cultured for 2 to 40 days depending on the colony formation state. In addition, an undifferentiated maintenance medium such as StemFit AK02N medium may be used in this step. In this step, the iPS cells or pluripotent stem cells are cultured with adhesion to form colonies, and these can be subjected to the culture step described above to induce them to become neural crest cells.

[0060] In one embodiment, the manufacturing method of this embodiment does not involve the formation of embryoid bodies of pluripotent stem cells before the culture step. For example, the pre-culture step is not suspension culture, and does not involve the formation of embryoid bodies of pluripotent stem cells. In this specification, "embryonic body" means a pseudo-embryo that can be formed by suspension culture of pluripotent stem cells. Embryoid body formation is widely used when differentiating pluripotent stem cells into cardiomyocytes, nerve cells, etc., but in the embodiments described later, it has become clear that pluripotent stem cells can be differentiated into neural crest cells with high efficiency without the need for embryoid body formation.

[0061] (Recovery step) In the manufacturing method of this embodiment, the "recovery step" is a step of recovering neural crest cells obtained by differentiation from pluripotent stem cells from the culture vessel after the culture step described above. This step is an optional step and may be performed as needed.

[0062] The method for recovering neural crest cells from the culture vessel in this process is not limited. For example, neural crest cells may be detached from the culture vessel by enzymatic detachment, chemical detachment, and / or physical detachment, and then the cell suspension containing neural crest cells may be recovered by aspiration or decantation. In this process, neural crest cells can also be selectively recovered by separating them from iPS cells or neural progenitor cells that did not differentiate into neural crest cells.

[0063] The neural crest cells recovered in this process may be differentiated into various tissues and organs, such as pigment cells, peripheral nerves, endocrine cells, and connective tissue of the head, as needed.

[0064] 1-4. Effects: The neural crest cell production method of the present invention allows for the efficient and simple production of neural crest cells from pluripotent stem cells such as iPS cells, without relying on growth factors such as FGF and TGFβ.

[0065] Furthermore, the manufacturing method of the present invention makes it possible to ensure high safety in the production of neural crest cells by using a xeno-free minimal medium or a basic medium such as CDM medium.

[0066] 2. Method for Inducing Differentiation of Neural Crest Cells A second aspect of the present invention is a method for inducing differentiation of neural crest cells. The differentiation induction method of this aspect includes a step of culturing pluripotent stem cells under conditions in which the expression or activity of novel protein kinase C (nPKC) and / or atypical protein kinase C (aPKC) is suppressed or inhibited, and can easily and efficiently induce pluripotent stem cells into neural crest cells. The differentiation induction method of this aspect includes a culture step of culturing pluripotent stem cells as an essential step, and includes a reprogramming step, an iPS cell selection step, a pre-culture step, and / or a harvesting step as selective steps. The specific configuration of each step is the same as the configuration described in the first aspect, so a description is omitted here.

[0067] The present invention will be described in more detail below using examples. However, the technical scope of the present invention is not limited to these examples.

[0068] <Example 1: Induction of neural crest cell differentiation using PKC inhibitors> (Objective) To evaluate the activity of various PKC inhibitors in inducing differentiation from human iPS cells into neural crest cells.

[0069] (Methods) (1) Three cell lines were used as cultured human iPS cells (hereinafter sometimes referred to as "hiPSCs"): WTC11 cell line (Coriell Institute, GM25256), 1231A3 cell line (RIKEN BioResource Research Center, Cell Materials Development Laboratory, HPS0381), and HiPS-NB1RGB cell line (RIKEN BioResource Research Center, Cell Materials Development Laboratory, HPS5067). Each iPS cell was cultured at 0.25 μg / cm³. 2 iPS cells were seeded in StemFit AK02N medium (Ajinomoto) supplemented with iMatrix-511 silk (Matrixome) and 10 μM Y-27632 (Wako), and cultured for 4 days to form colonies (Figure 1, Day -4 to Day 0). The seeding density of iPS cells was 1.8 × 10⁶. 3 cells / cm 2 or 3.6 × 10 3 cells / cm 2During the 4-day culture period, the culture medium was changed twice using StemFit AK02N medium. The culture medium used in this study did not contain any PKC inhibitors.

[0070] Next, the culture medium was replaced with Essential 6 medium (Thermo Fisher Scientific), a minimal medium supplemented with various PKC inhibitors as shown in Table 1, and cultured for 10 days (Figure 1, Day 0 to Day 10). During the 10-day culture period, the culture medium was changed every two days. Furthermore, the culture medium used in this culture did not contain fibroblast growth factor (FGF), TGF-β, or Wnt activator.

[0071]

[0072] GF109203X is an inhibitor that inhibits conventional PKC (cPKC), and has the effect of inhibiting the protein kinase activity of PKCα, PKCβI, PKCβII, and PKCγ (Toullec D., et al., J Biol Chem., 1991, 25;266(24):15771-81).

[0073] Ro 32-0432 hydrochloride (hereinafter abbreviated as "Ro 32-0432") is an inhibitor that inhibits conventional PKC (cPKC) and novel PKC (nPKC), and has the effect of inhibiting the protein kinase activity of PKCα, PKCβI, PKCβII, PKCγ, PKCδ, PKCε, PKCη, and PKCθ (Wilkinson SE, Biochem J. 1993,294 (Pt 2)(Pt 2):335-7).

[0074] Sotrastaurin is an inhibitor that inhibits both conventional PKC (cPKC) and novel PKC (nPKC), and has the effect of inhibiting the protein kinase activity of PKCα, PKCβI, PKCβII, PKCγ, PKCδ, PKCε, PKCη, and PKCθ (Wagner J., J. Med. Chem., 2009, 52(20):6193-6196.; Skvara, H., et al., J. Clin. Investig. 2008, 118(9):3151-3159.).

[0075] Go 6983 is an inhibitor that inhibits all types of PKC, including conventional PKC (cPKC), novel PKC (nPKC), and atypical PKC (aPKC), and has the effect of inhibiting the protein kinase activity of PKCα, PKCβI, PKCβII, PKCγ, PKCδ, PKCε, PKCη, PKCθ, PKCζ, and PKCι (Gschwendt M., et al., FEBS Lett., 1996, 392(2):77-80.; Young LH, et al., Cardiovascular Drug Reviews, 2005, 23(3):255-272.; Kawano T., et al., Pharmaceutica, 2021, 13(11):1748.).

[0076] (2) Flow cytometry analysis Cells cultured for 10 days in the presence or absence of various PKC inhibitors (Figure 1, Day 10) and undifferentiated iPS cells were detached / dispersed using Accutase (nacalai-tesque) and suspended in 0.1% BSA (WAKO) / 0.5 mM EDTA (nacalai-tesque) in PBS (nacalai-tesque). BV421-labeled anti-CD271 antibody (BD Bioscience) was added to the resulting cell suspension and reacted on ice in the dark for 1 hour. After the reaction, the cells were washed with 0.1% BSA / 0.5 mM EDTA in PBS, and the cell clamps were filtered off using a cell strainer (Corning). Next, the percentage of cells showing positivity for CD271, a neural crest cell marker, was measured using a SONY SH800S (SONY).

[0077] (Results) The flow cytometry results are shown in Figure 2 (WTC11 cell line), Figure 3 (1231A3 cells), and Figure 4 (HiPS-NB1RGB cells).

[0078] For the WTC11 cell line, the percentage of neural crest cells (CD271-positive cells) after culture in the presence of GF109203X was 9.2%, while the percentages after culture in the presence of Ro 32-0432 and Sotrastaurin were 50% and 63%, respectively, indicating a 5-6-fold or greater increase in the proportion of CD271-positive cells. Furthermore, after culture in the presence of Go 6983, the percentage of neural crest cells (CD271-positive cells) was 85%, representing a 9-fold or greater increase in the proportion of CD271-positive cells (Figure 2).

[0079] For the 1231A3 cell line, the percentage of neural crest cells (CD271-positive cells) after culture in the presence of GF109203X was 19.6%, while the percentages after culture in the presence of Ro 32-0432 and Sotrastaurin were 43% and 59%, respectively, indicating an approximately 2-3-fold increase in the proportion of CD271-positive cells. Furthermore, after culture in the presence of Go 6983, the percentage of neural crest cells (CD271-positive cells) was 87%, representing a more than 4-fold increase in the proportion of CD271-positive cells (Figure 3).

[0080] For the HiPS-NB1RGB cell line, the percentage of neural crest cells (CD271-positive cells) after culture in the presence of GF109203X was 37%, while the percentages after culture in the presence of Ro 32-0432 and Sotrastaurin were 82% and 69%, respectively. The percentage of neural crest cells (CD271-positive cells) after culture in the presence of Go 6983 was 89%, indicating an approximately 2-3-fold increase in the proportion of CD271-positive cells (Figure 3).

[0081] The results above clearly show that in the presence of Ro 32-0432 and Sotrastaurin, which are inhibitors that inhibit both cPKC and nPKC, the efficiency of differentiation induction into neural crest cells increased by approximately 2 to 6 times compared to the presence of GF109203X, which inhibits only cPKC, indicating a significant increase in differentiation efficiency. Furthermore, in the presence of Go 6983 (10 μM), a pan-PKC inhibitor that inhibits all cPKC, nPKC, and aPKC, a remarkable effect was revealed: more than 85% differentiated into neural crest cells.

[0082] <Example 2: Evaluation of neural crest cell marker SOX10 expression> (Objective) To evaluate the expression of SOX10, a neural crest cell marker different from CD271, which was used in Example 1.

[0083] (Method) For the HiPS-NB1RGB cell line, which is a human iPS cell, colonies were formed by culturing for 4 days using the same method as in "(1) Culture" of Example 1. Then, the cells were cultured for 10 days in either a minimal medium (Essential 6 medium) supplemented with Go 6983, a pan-PKC inhibitor that inhibits all cPKC, nPKC, and aPKC, or a minimal medium (Essential 6 medium) without Go 6983.

[0084] The cultured cells described above were fixed by treating them with 4% PFA in PBS (nacalai-tesque) for 10 minutes, and then permeabilized with 0.1% TritonX-100 (nacalai-tesque) in PBS for 10 minutes. Subsequently, they were blocked with 0.1% BSA (WAKO) in PBS (nacalai-tesque) for 30 minutes, after which anti-SOX10 antibody (R&D systems) and anti-PAX6 antibody (MBL) were added and the cells were reacted overnight at 4°C. After washing the cells with PBS, fluorescently labeled secondary antibodies, AF488-labeled anti-Goat IgG antibody (ThermoFisher) and DyLight™ 649-labeled Rabbit IgG antibody (Biolegend), were added and the cells were reacted on ice for 2 hours under light protection. After washing the cells with PBS, DAPI-containing anti-bleeding cell culture solution (nacalai-tesque) was added. Observation and imaging were performed using a fluorescence microscope (Keyence). The obtained images were analyzed using ImageJ to quantify the areas positive for SOX10, PAX6, and DAPI. SOX10 was used as a neural crest cell marker, and PAX6 was used as a neural progenitor cell marker.

[0085] (Results) The quantitative results are shown in Figure 5. Under conditions of culturing for 10 days in the absence of Go 6983, the area of ​​SOX10-positive cells corresponding to neural crest cells was 10.7%, while the area of ​​PAX6-positive cells corresponding to neural progenitor cells was 77.5%. This is because it is known that most cells differentiate into neurons under conditions that inhibit both BMP signaling and TGFβ signaling (dual SMAD inhibition), and it is thought that these signals do not enter the iPS cells in the Essential 6 medium used for culture in this example.

[0086] On the other hand, when Go 6983 was cultured in Essential 6 medium for 10 days, the area of ​​SOX10-positive cells, which correspond to neural crest cells, was 83.2%, while the area of ​​PAX6-positive cells, which correspond to neural progenitor cells, was only 0.3% (Figure 5).

[0087] These results suggest that cells that differentiate into neural progenitor cells in the absence of Go 6983 differentiated into neural crest cells in the presence of Go 6983, revealing that the differentiation of iPS cells can be dramatically altered by using Go 6983.

[0088] <Example 3: Comparison of Pan-PKC Inhibitors and PKCβ Inhibitors> (Objective) To compare the activity of pan-PKC inhibitors and PKCβ inhibitors in inducing differentiation from human iPS cells to neural crest cells.

[0089] (Method) HiPS-NB1RGB cell lines were cultured for 10 days in the presence of a PKC inhibitor using the same method as in Example 1. In this example, Go 6983 (a pan-PKC inhibitor) or LY333531 (a PKCβ inhibitor) was used as the PKC inhibitor. As described above, Go 6983 is a pan-PKC inhibitor that inhibits all cPKC, nPKC, and aPKC. LY333531 has the effect of inhibiting PKCβI and PKCβII among conventional PKCs (cPKC) (Jirousek MR, et al., J Med Chem., 1996, 39 (14):2664-2671). In this example, each inhibitor was used at a concentration of 5 μM.

[0090] Flow cytometry analysis was performed on cells cultured for 10 days in the presence or absence of various PKC inhibitors, as well as on undifferentiated iPS cells, using the same method as in Example 1.

[0091] (Results) The flow cytometry results are shown in Figure 6. After culturing in the presence of LY333531, the percentage of neural crest cells (CD271-positive cells) was 1.6%, which was similar to the percentage after culturing in the absence of a PKC inhibitor (1.8%). In contrast, in the presence of Go 6983, a remarkable effect was observed, with 58.6% differentiating into neural crest cells. All publications, patents, and patent applications cited herein are incorporated herein by direct reference.

Claims

1. A method for producing neural crest cells, comprising the step of culturing pluripotent stem cells for 6 days or more under conditions in which the expression or activity of novel protein kinase C (nPKC) and / or atypical protein kinase C (aPKC) is suppressed or inhibited.

2. The manufacturing method according to claim 1, wherein the culture is an adherent culture.

3. The production method according to claim 1, wherein the culture is carried out under conditions in which the expression or activity of conventional protein kinase C (cPKC) is further suppressed or inhibited.

4. The method for producing a drug according to claim 3, wherein the culture is carried out in the presence of one or more PKC inhibitors selected from the group consisting of sotrastaurin, Ro 32-0432, enzastaurin, Ro 31-8220, bisindolylmaleimide VIII, and staurosporine.

5. The manufacturing method according to claim 1, wherein the culture is carried out under conditions in which the expression or activity of nPKC, aPKC, and cPKC is suppressed or inhibited.

6. The manufacturing method according to claim 5, wherein the culture is carried out in the presence of a pan-PKC inhibitor.

7. The manufacturing method according to claim 6, wherein the pan-PKC inhibitor is Go 6983 or Lottrelin.

8. The method for producing the product according to claim 1, wherein the culture is carried out in a basic medium or a serum-free medium that does not contain basic fibroblast growth factor (bFGF) and / or transforming growth factor β (TGF-β).

9. The manufacturing method according to claim 8, wherein the serum-free medium does not contain non-human derived components.

10. The method for producing a pluripotent stem cell according to claim 1, wherein the pluripotent stem cell is an induced pluripotent stem cell (iPS cell).

11. The manufacturing method according to claim 1, wherein the embryoid body of the pluripotent stem cells is not formed before the culturing step.

12. A method for inducing differentiation of neural crest cells, comprising the step of culturing pluripotent stem cells for 6 days or more under conditions in which the expression or activity of novel protein kinase C (nPKC) and / or atypical protein kinase C (aPKC) is suppressed or inhibited.

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