Glomus cell-like cells and methods for producing them

Abstract

To provide glomus cell-like cells capable of reproducing the hypoxia response of the carotid body.SOLUTION: Provided is a method for producing glomus cell-like cells, the method including: (a) a step for inducing differentiation of pluripotent stem cells into autonomic progenitor cells; and (b) a step for culturing the autonomic progenitor cells obtained by step (a) in the presence of an FGF signaling pathway activator, an EGF signaling pathway activator and an IGF-1 signaling pathway activator.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to glomus cell-like cells and a method for producing the same. [Background technology]

[0002] The carotid body (CB) is a peripheral chemoreceptor located at the bifurcation of the carotid artery. CBs are composed of nerve-like glomus cells (also called type I cells) and glial-like supporting cells (also called type II cells), with glomus cells being the chemoreceptors. When glomus cells sense hypoxia, hypercapnia, or acidosis, they release neurotransmitters such as ATP and dopamine, stimulating the medulla oblongata via the glossopharyngeal nerve, which in turn induces an increase in respiratory rate and heart rate. Therefore, compounds that modulate the sensitivity of glomus cells or their neurotransmitter release activity could be potential therapeutic agents for respiratory failure. Furthermore, recent studies have revealed that glomus cells sense insulin and glucose and are involved in maintaining energy homeostasis, making them a promising new therapeutic target for diabetes.

[0003] Traditionally, non-human animal models have been used in CB research. However, testing numerous drug candidates requires a considerable number of animals, resulting in significant time and economic costs. Furthermore, in recent years, there has been a strong demand for alternative testing systems that do not use animals, from an ethical standpoint, such as animal welfare and protection. In addition, considering species differences, there is a need for testing systems that closely resemble human models, and the development of an in vitro CB model using human glomus cells is desired.

[0004] On the other hand, supporting cells of CB are precursor cells of glomus cells, and it has been reported that glomus cells can be produced from supporting cells in vitro (Patent Document 1, Non-Patent Document 1). However, since supporting cells must be collected from animals in order to produce glomus cells, there are difficulties in large-scale culture of human glomus cells in particular. No method for producing glomus cells or supporting cells from human pluripotent stem cells has been reported to date. [Prior art documents] [Patent Documents]

[0005] [Patent Document 1] International Publication No. 2009 / 016262 [Non-patent literature]

[0006] [Non-Patent Document 1] Pardal,R.et al.,Cell,2007;131(2):364-377 [Overview of the Initiative] [Problems that the invention aims to solve]

[0007] The present invention aims to provide glomus cell-like cells that can reproduce the hypoxic response of CB in vitro, and a simple and efficient method for preparing them. [Means for solving the problem]

[0008] The inventors have previously developed a technique for efficiently inducing autonomic nerve cells or their precursor cells from human pluripotent stem cells (e.g., International Publication No. 2016 / 194522). By adding specific culture conditions to the above method, the inventors have succeeded in producing glomus cell-like cells that exhibit a hypoxic response from pluripotent stem cells.

[0009] In other words, according to one embodiment, the present invention provides a method for producing glomus cell-like cells, comprising the steps of (a) inducing the differentiation of pluripotent stem cells into autonomic progenitor cells, and (b) culturing the autonomic progenitor cells obtained by step (a) in the presence of an FGF signaling pathway activator, an EGF signaling pathway activator, and an IGF-1 signaling pathway activator.

[0010] Preferably, the method further includes, before step (a), a step of introducing an exogenous nucleic acid encoding endothelial PAS domain-containing protein 1 (EPAS1) into the pluripotent stem cell.

[0011] Preferably, the FGF signal pathway activator is bFGF.

[0012] Preferably, the EGF signal pathway activator is EGF.

[0013] Preferably, the IGF-1 signal pathway activator is IGF-1.

[0014] Preferably, the pluripotent stem cells are of human origin.

[0015] Moreover, according to one embodiment, the present invention provides glomus cell-like cells produced by the above method.

[0016] Moreover, according to one embodiment, the present invention provides glomus cell-like cells comprising an exogenous nucleic acid encoding EPAS1 and expressing tyrosine hydroxylase, potassium channel subfamily K member 3, and olfactory receptor 51E2.

[0017] Preferably, the above glomus cell-like cells have an increased production amount of ATP, dopamine, or its metabolite in a hypoxic state.

[0018] Moreover, according to one embodiment, the present invention provides a method for screening a compound that regulates the activity of glomus cells, comprising: (1) a step of contacting a candidate compound with the above glomus cell-like cells; and (2) a step of measuring ATP released from the above glomus cell-like cells.

Advantages of the Invention

[0019] According to the method of the present invention, glomus cell-like cells can be produced with high efficiency and easily. Further, the glomus cell-like cells according to the present invention are excellent in hypoxic responsiveness and are useful for the development of an in vitro CB model, the search for compounds that regulate the activity of CB, and the development of therapeutic agents for CB-related diseases.

Brief Description of the Drawings

[0020] [Figure 1] Figure 1 is a schematic diagram showing the production schedule of glomus cell-like cells. [Figure 2] Figure 2 is a diagram showing microscopic images (bright field) of embryoid bodies (EBs) on day 0 of induction, cells on day 13 of induction (autonomic nerve progenitor cells), and cells on day 16 of induction (glomus cell-like cells). [Figure 3] Figure 3 is a heat map showing the results of RNA-seq analysis of iPS cells on day -3 of induction (denoted as "hiPSC" in the figure), cells on day 13 of induction (denoted as "Progenitors"), and cells on day 16 of induction (denoted as "Glomus cells"). [Figure 4] Figure 4 is a graph showing the expression levels of the OR51E2 gene in EBs on day 0 of induction, cells on day 13 of induction (autonomic nerve progenitor cells), and cells on day 18 of induction (glomus cell-like cells). [Figure 5] Figure 5 is a graph showing the ATP production of glomus cell-like cells in response to hypoxic stimulation. [Figure 6] Figure 6 is a graph showing the epinephrine production of glomus cell-like cells in response to hypoxic stimulation. [Figure 7] Figure 7 is a graph showing the dopamine (DA) production of glomus cell-like cells in response to hypoxic stimulation. [Figure 8] Figure 8 is a graph showing the production of 3,4-dihydroxyphenylacetic acid (DOPAC) by glomus cell-like cells in response to hypoxic stimulation. [Figure 9] Figure 9 is a graph showing the expression levels of the TH gene in glomus cell-like cells in response to hypoxic stimulation. <亮 [Figure 10]Figure 10 is a graph showing the expression level of the KCNK3 gene in glomus cell-like cells in response to hypoxic stimulation. [Figure 11] Figure 11 is a graph showing the effect of tetraethylammonium chloride (TEA) on promoting ATP production in glomus cell-like cells in response to hypoxic stimulation. [Figure 12] Figure 12 is a graph showing the inhibitory effect of nifedipine (Nif.) on ATP production in glomus cell-like cells in response to hypoxic stimulation. [Figure 13] Figure 13 is a graph showing the inhibitory effect of lidocaine (Lid.) on ATP production in glomus cell-like cells in response to hypoxic stimulation. [Figure 14] Figure 14 is a graph showing the expression level of the EPAS1 gene in iPS cells into which the exogenous EPAS1 gene has been introduced. [Figure 15] Figure 15 is a graph showing the expression level of the EPAS1 gene in cells prepared from iPS cells into which the exogenous EPAS1 gene was introduced, on day 17 of induction. [Figure 16] Figure 16 is a graph showing the expression levels of the TH gene in EPAS1-expressing iPS cells (undifferentiated, induction day 3, labeled "hiPSC" in the figure), autonomic progenitor cells induced from EPAS1-expressing iPS cells (induction day 13, labeled "Progenitors"), and EPAS1-overexpressing glomus cell-like cells (induction day 17, labeled "Glomus cells"). [Figure 17] Figure 17 is a graph showing the expression levels of the KCNK3 gene in EPAS1-expressing iPS cells (undifferentiated, induction day 3), autonomic progenitor cells induced from EPAS1-expressing iPS cells (induction day 13), and EPAS1-overexpressing glomus cell-like cells (induction day 17). [Figure 18] Figure 18 is a graph showing the expression levels of the OR51E2 gene in EPAS1-expressing iPS cells (undifferentiated, induction day 3), autonomic progenitor cells induced from EPAS1-expressing iPS cells (induction day 13), and EPAS1-overexpressing glomus cell-like cells (induction day 17). [Figure 19]Figure 19 is a graph showing ATP production in EPAS1-overexpressing glomus cell-like cells under normal oxygen or hypoxic conditions. [Modes for carrying out the invention]

[0021] The present invention will be described in detail below, but the present invention is not limited to the embodiments described herein.

[0022] According to a first embodiment, the present invention is a method for producing glomus cell-like cells, comprising the steps of (a) inducing differentiation of pluripotent stem cells into autonomic progenitor cells, and (b) culturing the autonomic progenitor cells obtained by step (a) in the presence of an FGF signaling pathway activator, an EGF signaling pathway activator, and an IGF-1 signaling pathway activator.

[0023] In the method of this embodiment, autonomic nervous system progenitor cells are induced from pluripotent stem cells. Examples of "pluripotent stem cells" include, but are not limited to, embryonic stem (ES) cells, induced pluripotent stem (iPS) cells, embryonic germ (EG) cells, pluripotent germ stem (mGS) cells, and Muse cells. In the method of this embodiment, any type of pluripotent stem cell may be used, but it is preferable to use ES cells or iPS cells, and more preferable to use iPS cells.

[0024] The pluripotent stem cells in this embodiment may be derived from any vertebrate, preferably from mammals such as mice, rats, rabbits, sheep, goats, pigs, cattle, monkeys, and humans, and particularly preferably from humans.

[0025] Methods for preparing pluripotent stem cells are well-established (for example, for iPS cells, Cell, 2007;131(5):861-872, doi:10.1016 / j.cell.2007.11.019), and pluripotent stem cells can be prepared from any tissue or cell according to methods known in this field. Alternatively, already established iPS cell lines or ES cell lines can be obtained from sources such as the Kyoto University iPS Cell Research Foundation (CiRA_F), the RIKEN BioResource Research Center (RIKEN BRC), or the American Type Culture Collection (ATCC).

[0026] In this embodiment, "autonomic progenitor cells" refer to neural crest cells or cells that have undergone more advanced differentiation into autonomic nervous system cells and that have the ability to differentiate into sympathetic and parasympathetic nerve cells. In this embodiment, autonomic progenitor cells can be defined, for example, based on the expression of SOX10 (neural crest cell marker gene) and PHOX2B (autonomic nerve cell marker gene).

[0027] Methods for inducing pluripotent stem cells into autonomic progenitor cells are already well-established (e.g., International Publication No. 2020 / 040286, International Publication No. 2016 / 194522), and autonomic progenitor cells can be prepared according to methods known in this field. Specifically, autonomic progenitor cells can be prepared by culturing pluripotent stem cells under conditions in which BMP signaling pathway inhibitors such as dolsomorphin, TGF signaling pathway inhibitors such as SB431542, Wnt signaling pathway activators such as CHIR99021, and FGF signaling pathway activators such as basic fibroblast growth factor (bFGF) are appropriately added to a basic medium such as DMEM / HAM's F-12 medium, human stem cell (hES) medium, N2 medium, or a mixture thereof. In this case, it is preferable that the pluripotent stem cells are cultured for 2 to 3 days in a medium containing a Rho kinase (ROCK) inhibitor such as Y-27632 before the above differentiation induction.

[0028] Autonomic progenitor cells can be stably subcultured while maintaining their differentiation potential by using a medium supplemented with EGF or basic fibroblast growth factor (bFGF) until differentiation into glomus cell-like cells is induced (Fukuta et al., PLoS ONE, 9(12):e112291, 2014).

[0029] Next, autonomic progenitor cells are cultured in the presence of FGF signaling pathway activators, EGF signaling pathway activators, and IGF-1 signaling pathway activators. This allows the autonomic progenitor cells to be induced into glomus cell-like cells.

[0030] The "FGF signaling pathway activator" in this embodiment may be any known compound that activates the FGF (fibroblast growth factor) receptor and its downstream signaling pathway. Examples include, but are not limited to, the FGF1 family, such as acid fibroblast growth factor (FGF1, aFGF), basic fibroblast growth factor (FGF2, bFGF), the recombinant FGF2 FGF-G3™, and fibroblast growth factor chimeras (FGFC). The FGF signaling pathway activator in this embodiment is preferably bFGF, and the concentration of bFGF in the culture medium can preferably be 5 to 50 ng / mL.

[0031] The "EGF signaling pathway activator" in this embodiment may be any known compound that activates the EGF (epidermal growth factor) receptor and its downstream signaling pathway, such as EGF, TGF-β, amphiregulin (AREG), heparin-binding EGF-like growth factor (HB-EGF), betacellulin (BTC), epigen (EPG), and epiregulin (EPR), but is not limited to these. The EGF signaling pathway activator in this embodiment is preferably EGF, and the concentration of EGF in the culture medium can preferably be 5 to 50 ng / mL.

[0032] The "IGF-1 signaling pathway activator" in this embodiment may be any known compound that activates the IGF-1 (insulin-like growth factor 1) receptor and its downstream signaling pathway, such as IGF-1, IGF-2, and insulin, but is not limited to these. The IGF-1 signaling pathway activator in this embodiment is preferably IGF-1, and the concentration of IGF-1 in the culture medium can preferably be 5 to 50 ng / mL.

[0033] The culture medium used here may be, for example, DMEM / HAM's F-12 medium as the base medium, with fetal bovine serum (FBS), penicillin / streptomycin, non-essential amino acid solution, N2 supplement, B-27 supplement, etc., added as appropriate. Any equivalent substitute can be used instead of FBS, and such substitutes include, but are not limited to, KnockOut Serum Replacement (KSR) (Thermo Fisher Scientific: 10828028), StemSure® Serum Replacement (SSR) (Fujifilm Wako Pure Chemical Industries: 191-18375), and XF212 XerumFree (TNC BIO BV: XF212-0100-1s). FBS or its substitute may preferably be added at a concentration of 10-20%. Autonomic progenitor cells are, for example, 1 × 10⁶ 6 ~1 × 10 8 Seedling may be seeded at a concentration range of cells / mL. The culture period in the presence of FGF signaling pathway activators, EGF signaling pathway activators, and IGF-1 signaling pathway activators may be, for example, 3 to 50 days, preferably 3 to 7 days. The culture conditions are preferably 37°C and 5% CO2 conditions, for example, when using mammalian autonomic progenitor cells.

[0034] In the method of this embodiment, it is preferable to introduce an exogenous nucleic acid encoding a factor related to the development and / or function of glomus cells (hereinafter referred to as "glomus cell-related factor"). This makes it possible to produce glomus cell-like cells with improved function. Examples of glomus cell-related factors include, but are not limited to, endothelial PAS domain-containing protein 1 (EPAS1, also known as HIF-2α), brain-derived neurotrophic factor (BDNF), paired homeobox protein 2B (PHOX2B), SRY-BOX transcription factor 4 (SOX4), SRY-BOX transcription factor 11 (SOX11), and hypoxia-inducible factor 1A (HIF1A). In the method of this embodiment, it is particularly preferable to introduce an exogenous nucleic acid encoding EPAS1.

[0035] Exogenous nucleic acids encoding glomus cell-related factors may be introduced into cells at any point in time. For example, exogenous nucleic acids encoding glomus cell-related factors may be introduced into pluripotent stem cells before differentiation induction, into autonomic progenitor cells obtained by differentiation induction, or into autonomic progenitor cells that are differentiating into glomus cells. In the method of this embodiment, it is preferable that the exogenous nucleic acids encoding glomus cell-related factors be introduced into pluripotent stem cells before differentiation induction.

[0036] The exogenous nucleic acids encoding glomus cell-related factors in this embodiment may be derived from any vertebrate, preferably from mammals such as mice, rats, rabbits, sheep, goats, pigs, cattle, monkeys, and humans, and particularly preferably from humans. The genes encoding glomus cell-related factors have already been cloned, and their nucleic acid sequence information can be obtained from a predetermined database. For example, NM_001430.5 for the human EPAS1 gene, NM_001143805.1 for the human BDNF gene, NM_003924.4 for the human PHOX2B gene, NM_003107.3 for the human SOX4 gene, NM_003108.4 for the human SOX11 gene, and NM_00124308.2 for the human HIF1A gene (all NCBI RefSeq IDs) are available.

[0037] The glomus cell-related factors in this embodiment may include variants and homologs of those factors having equivalent activity. In other words, the glomus cell-related factors in this embodiment may include proteins consisting of amino acid sequences that have 80% or more, preferably 90% or more, and more preferably about 95% or more, identity with the amino acid sequences registered in the database, provided that their physiological activity is maintained. The identity of the amino acid sequences can be calculated using sequence analysis software or using programs commonly used in this field (such as FASTA or BLAST). Furthermore, the glomus cell-related factors in this embodiment may include proteins consisting of amino acid sequences in which one to several amino acids are substituted, deleted, inserted, and / or added to the amino acid sequences registered in the database, provided that their physiological activity is maintained. Here, "one to several" may be, for example, 1 to 30, preferably 1 to 10, and particularly preferably 1 to 5.

[0038] Exogenous nucleic acids encoding glomus cell-related factors can be introduced into cells by methods well known in this field. For example, these nucleic acids can be cloned into expression vectors and then introduced into cells. Expression vectors can include, but are not limited to, viral vectors such as retroviruses, lentiviruses, adenoviruses, and Sendai viruses, as well as plasmid vectors such as pCMV.

[0039] In the method of this embodiment, the expression vector can be introduced into cells by methods well known in the art, depending on its type. If it is a non-viral vector, it can be introduced by, for example, lipofection, electroporation, or microinjection. If it is a viral vector, it can be introduced by infecting cells with an appropriate titer or multiple degree of infection (MOI).

[0040] According to the method of this embodiment, it is possible to produce glomus cell-like cells.

[0041] According to a second embodiment of the present invention, the present invention relates to glomus cell-like cells produced by the above method.

[0042] Here, "glomus cell-like cells" refer to cells that express marker genes expressed in glomus cells, namely tyrosine hydroxylase (TH), potassium channel subfamily K member 3 (KCNK3), and olfactory receptor 51E2 (OR51E2), and can mimic the hypoxic response of glomus cells. Therefore, it is preferable that the glomus cell-like cells of this embodiment express EPAS1 in addition to the above markers. It is even more preferable that the glomus cell-like cells of this embodiment further express ubiquitin C-terminal hydrolase L1 (UCHL1), heme oxygenase (HO-2), maxi-K channel (Maxi-K), class III β-tubulin (TUBB3), hypoxia-inducible factor 1A (HIF1A), and / or dopamine receptor (DRD2).

[0043] In other words, according to the third embodiment of the present invention, the present invention provides a glomus cell-like cell comprising an exogenous nucleic acid encoding endothelial PAS domain-containing protein 1 (EPAS1), and expressing tyrosine hydroxylase (TH), potassium channel subfamily K member 3 (KCNK3), and olfactory receptor 51E2 (OR51E2). The "exogenous nucleic acid" in this embodiment is the same as defined in the first embodiment.

[0044] In this embodiment, it is preferable that the glomus cell-like cells overexpress EPAS1. "Overexpressing EPAS1" means that the cells express EPAS1 at a level exceeding that of glomus cell-like cells prepared without introducing the exogenous nucleic acid encoding EPAS1.

[0045] The expression of markers and EPAS1 can be analyzed using known methods such as RT-PCR, Western blotting, and flow cytometry.

[0046] In this embodiment, glomus cell-like cells may be defined not only based on the expression of markers and EPAS1, but also on hypoxic responsiveness, for example, based on increased production of ATP or dopamine or its metabolites under hypoxic conditions. Examples of dopamine metabolites include, but are not limited to, tyrosine, L-DOPA, 3,4-dihydroxyphenylacetic acid (DOPAC), epinephrine, and norepinephrine. ATP or dopamine or its metabolites may be measured by methods well known in the art, for example, by liquid chromatography-mass spectrometry (LC-MS / MS) or ELISA.

[0047] Here, "hypoxic state" refers to a condition in which the oxygen supply to cells falls below physiological levels. Experimentally, a hypoxic state can be induced by culturing cells in an atmosphere with oxygen concentrations of, for example, 10%, 5%, 3%, 2%, or lower. On the other hand, "normal oxygen state" refers to a condition in which the oxygen supply to cells is at physiological levels, and experimentally, this means culturing cells in an atmosphere with an oxygen concentration similar to that of the atmosphere (approximately 21%).

[0048] The glomus cell-like cells of the second and third embodiments can reproduce the hypoxic response of glomus cells in vitro and are useful for creating in vitro CB models.

[0049] According to a fourth embodiment of the present invention, the present invention is a method for screening compounds that modulate glomus cell activity, comprising the steps of (1) contacting a candidate compound with the glomus cell-like cells and (2) measuring the ATP released from the glomus cell-like cells.

[0050] In this embodiment, the "candidate compound" may be a small molecule compound, nucleic acid, protein, peptide, antibody, lipid, etc., or a mixture thereof (e.g., an extract from a cell or tissue, a culture supernatant from a cell or tissue, etc.). Furthermore, these candidate compounds may be novel or known. In the method of this embodiment, commercially available compound libraries may be used, and preferred compound libraries include, for example, the Standard Compound Library (RIKEN NPDepo), the Osaka University Original Compound Library (Osaka University), InhibitorSelect® Libraries (Merck), and the SCREEN-WELL® Compound Library (ENZO LIFE SCIENCES, INC.).

[0051] To bring a candidate compound into contact with glomus cell-like cells, the glomus cell-like cells can be cultured in a medium containing the candidate compound for a certain period of time. The concentration of the candidate compound to be added will vary depending on the type of candidate compound, but for example, if it is a low molecular weight compound, it can be appropriately selected within the range of 1 pM to 100 mM. The culture period may be, for example, 1 second to 72 hours.

[0052] Next, the ATP released from the glomus cell-like cells is measured. ATP can be measured by methods well known in this field, such as liquid chromatography-mass spectrometry (LC-MS / MS) or ELISA. Furthermore, commercially available ATP measurement kits can be used in the method of this embodiment. For example, the ATP Determination Kit (Thermo Fisher Scientific) is a preferred commercially available product.

[0053] To determine whether ATP production has changed due to the addition of a candidate compound, the cultures without the candidate compound may be analyzed and compared in parallel, or the results may be compared with those of previously performed analyses of cultures without the candidate compound. In the method of this embodiment, if the ATP release from glomus cell-like cells in the culture medium with the candidate compound is significantly increased or decreased compared to glomus cell-like cells in the culture medium without the candidate compound, the candidate compound can be evaluated as a promising compound for regulating glomus cell activity. Compounds that promote glomus cell activity may be useful for treating conditions such as respiratory failure, hypotension, hypoglycemia, and dysfunction due to infection. Compounds that suppress glomus cell activity may be useful for treating conditions such as diabetes, hypertension, altitude sickness, hyperactivity due to infection, hyperventilation, and carotid body tumors (also known as paragangliomas or glomus tumors). [Examples]

[0054] The present invention will be further explained below with reference to examples. However, these examples do not limit the present invention in any way.

[0055] <1. Differentiation induction from iPS cells to glomus cell-like cells> (1-1) Reagents The reagent information (reagent name, product number, manufacturer, abbreviation, etc.) used in this embodiment is as follows. • mTeSR1-cGMP (STEMCELL Technologies: ST-85850G) (hereinafter referred to as "mTeSR1") • DMEM / Ham's F-12 (Fujifilm Wako Pure Chemical Industries: 048-29785) • DMEM (high-glucose) (Fujifilm Wako Pure Chemical Industries: 043-30085) ·Opti-MEM (Thermo Fisher Scientific: 31985062) • Stem Cell Banker (STEM-CELLBANKER(trademark) GMP grade) (ZNQ: CB045) • Fetal Bovine Serum (Biowest, Nuaille, France) (hereinafter referred to as "FBS") • Knockout Serum Replacement (Thermo Fisher Scientific: 10828-028) (hereinafter referred to as "KSR") • N2 supplement with transferrin (APO) (Fujifilm Wako Pure Chemical Industries: 141-09041) supplement (hereinafter referred to as "N2") • MEM non-essential amino acids solution (Fujifilm Wako Pure Chemical Industries: 139-15651) (hereinafter referred to as "NEAA") • B-27 supplement (Thermo Fisher Scientific: 17504044) (hereinafter referred to as "B27") Monothioglycerol solution (Fujifilm Wako Pure Chemical Industries: 195-15791) Penicillin-streptomycin solution (Fujifilm Wako Pure Chemical Industries: 168-23191) (hereinafter referred to as "P / S") • Ethanol (99.5%) (Fujifilm Wako Pure Chemical Industries: 057-00456) • Y-27632 (Fujifilm Wako Pure Chemical Industries: 036-24023) • Forskolin (Fujifilm Wako Pure Chemical Industries: 067-02191) (hereinafter referred to as "FSK") • Dorsomorphin (Sigma-Aldrich: P5499-5MG) (hereinafter referred to as "DM") • SB431542 hydrate (Sigma-Aldrich: S4317-5MG) (hereinafter referred to as "SB") • CHIR99021 (Cayman Chemical Company: 13122) (Hereinafter referred to as "CHIR") ·IWR-1 (Sigma-Aldrich: I0161-5MG) ·SANT1 (Sigma-Aldrich: S4572-5MG) • Bone morphogenetic factor 4 (truncated), human, recombinant (Fujifilm Wako Pure Chemical Industries: 022-17071) (hereinafter referred to as "BMP4") • Fibroblast growth factor (basic) (basic FGF), human, recombinant (Fujifilm Wako Pure Chemical Industries: 064-04541) (hereinafter referred to as "bFGF") • Epidermal Growth Factor (EGF) (Fujifilm Wako Pure Chemical Industries: 059-07873) • Insulin-like growth factor-I (IGF-1) (Fujifilm Wako Pure Chemical Industries: 096-05741) • iMatrix-511 (Nippi: 892012) Lipidure (NOF Corporation: CM5206) ·Accutase (Thermo Fisher Scientific: A11105-01) ·TrypLE express (Thermo Fisher Scientific: 12604-013) Ultratrapure distilled water (Thermo Fisher Scientific: 10977-015) (hereinafter referred to as "DW") • D-PBS(-) (Fujifilm Wako Pure Chemical Industries: 045-29795) (Hereinafter referred to as "PBS") • Tris Buffer Powder, pH 7.4 (Takara Bio: T9153) • Hydrochloric acid (Fujifilm Wako Pure Chemical Industries: 080-01066) Albumin, from Bovine Serum (Fujifilm Wako Pure Chemical Industries: 017-23294) (hereinafter referred to as "BSA") • Dimethyl sulfoxide (Fujifilm Wako Pure Chemical Industries: 046-21981) (hereinafter referred to as "DMSO")

[0056] (1-2) Stock solution • FSK: Prepared to 10 mM using DMSO. • DM: Prepared to 1 mM using DMSO. • SB: Prepared to 10 mM using DMSO. • CHIR: Prepared to 3 mM using DMSO. • IWR-1: Prepared to 10 mM using DMSO. • SANT1: Prepared to 250 μM using DMSO. • BMP4: Prepared to 100 μg / mL using a 4 mM hydrochloric acid solution with 0.1% BSA added. bFGF: Prepared to 500 μg / mL with 1 mM Tris buffer (pH 7.4), then adjusted to 10 μg / mL using DMEM. • EGF and IGF-1: Prepared to 20 μg / mL using PBS. • Ascorbic acid: Prepared to 50 mg / mL using PBS. • Y-27632: Prepared to 10 mM using Opti-MEM. Lipidure: Prepared to 0.5% using ethanol (99.5%).

[0057] (1-3) Culture medium Human stem cell medium (hESM): DMEM / Ham's F-12 supplemented with 20% KSR, 1% NEAA, 1% Monothioglycerol solution, and 1% P / S. • N2 medium: DMEM / Ham's F-12 medium supplemented with 1% N2, 1% NEAA, and 1% P / S. • Glomus cell differentiation medium (GDM): DMEM / Ham's F-12 supplemented with 15% FBS, 1% N2, 2% B27, 20 ng / mL bFGF, 20 ng / mL EGF, 20 ng / mL IGF-1, and 1% P / S.

[0058] (1-4) Differentiation induction from iPS cells to glomus cell-like cells • Formation of embryoid bodies (-3 days): Human iPS cells (201B7 strain) were obtained from RIKEN BRC. 6-well plates were coated with Lipidure and washed with PBS. Human iPS cells (201B7 strain) were refracted using mTeSR1 supplemented with 10 μM Y-27632 at a rate of 1 × 10⁶ 6 Cells were seeded at a cell / well concentration and cultured on an orbital shaker (Waken B-Tech: WB-101SRC) at 95 rpm. At 24 and 48 hours after seeding, 2 mL / well of mTeSR1 supplemented with 10 μM Y-27632 was added. Cells were cultured for 3 days until embryoid bodies (EBs) were formed. • Differentiation step 1 (Day 0): The culture medium was replaced with hESM supplemented with 2 μM DM, 10 μM SB, and 10 ng / ml bFGF (4 mL / well), and the cells were incubated on an orbital shaker for 2 days. • Differentiation step 2 (day 2): The culture medium was replaced with hESM supplemented with 3 μM CHIR, 20 μM SB, and 10 ng / ml bFGF (6 mL / well), and the cells were incubated on an orbital shaker for 3 days. • Differentiation step 3 (day 5): The culture medium was replaced with hESM:N2 (3:1) mixed medium supplemented with 3 μM CHIR and 10 ng / ml bFGF (4 mL / well), and the cells were incubated on an orbital shaker for 2 days. • Differentiation step 4 (day 7): The culture medium was replaced with hESM:N2 (1:1) mixed medium supplemented with 10 μM IWR-1, 250 nM SANT1, 25 ng / ml BMP4, and 10 ng / ml bFGF (3 mL / well), and the cells were incubated on an orbital shaker for 2 days. • Differentiation step 5 (day 9): The culture medium was replaced with hESM:N2 (1:3) mixed medium supplemented with 10 μM IWR-1, 250 nM SANT1, 25 ng / ml BMP4, and 10 ng / ml bFGF (3 mL / well), and incubated on an orbital shaker for 3 days. The medium was then replaced with fresh hESM:N2 (1:3) mixed medium and incubated for a further 24 hours. Differentiation step 6 (day 13): The culture medium was replaced with GDM (3 mL / well), and the cells were incubated on an orbital shaker for 3-4 days.

[0059] Figure 1 shows an outline of the schedule for generating glomus cell-like cells. Figure 2 shows microscopic images (bright-field) of embryoid bodies (EBs) on day 0 of induction, cells (autonomic progenitor cells) on day 13 of induction, and cells (glomus cell-like cells) on day 16 of induction.

[0060] <2. Gene expression in glomus cell-like cells> Total RNA was extracted from iPS cells (undifferentiated, induction day 3), autonomic progenitor cells (induction day 13), and glomus cell-like cells (induction day 16) using NucleoSpin RNA. A library for RNA-seq was prepared using TruSeq stranded mRNA (Illumina). Sequencing was performed using NovaSeq 6000 (Illumina). Mapping and quantification were performed using STAR (2.7.1a) and RSEM (1.3.1). hg38 was used as the reference genome, and Ensembl GRCh38 was used for gene annotation. Differentially expressed genes were analyzed using the edgeR package (version 3.24.3) of the statistical analysis software R (version 3.5.1).

[0061] The results are shown in Figure 3. In glomus cell-like cells, increased expression was observed in genes related to hypoxic and immune responses, including the glomus cell marker TH, as well as in nerve growth factor and receptor genes that have been shown to be expressed in mouse or rat glomus cells.

[0062] Next, the expression of OR51E2 in normally oxygenated iPS cells, autonomic progenitor cells, and glomus cell-like cells was analyzed by qPCR. qPCR was performed using the same procedure as described in (3-3) below.

[0063] The results are shown in Figure 4. In the figure, "hiPSC" refers to iPS cells (undifferentiated, induction day 3), "Progenitors" refers to autonomic progenitor cells (induction day 13), and "Glomus cells" refers to glomus cell-like cells (induction day 16). Increased expression of OR51E2 was confirmed with differentiation (*P<0.05, n=3, Student's t-test).

[0064] <3. Hypoxic response of glomus cell-like cells> (3-1) ATP production Glomus cell-like cells induced 16-19 days prior were used. Glomus cell-like cells were washed twice with KRB (Krebslinger buffer: 120mM NaCl, 5mM KCl, 25mM NaHCO3, 2.5mM CaCl2, 1.1mM MgCl2, 0.1% bovine serum albumin, 2.8mM glucose, pH 7.2), then KRB was added and the cells were incubated in a hypoxic CO2 incubator (2% O2) for 10-30 minutes. The supernatant was collected, and ATP was measured using the ATP Determination Kit (Thermo Fisher Scientific: A22066). Cells were lysed in RIPA buffer (Fujifilm Wako Pure Chemical Industries: 182-02451), and total protein content was measured using the Pierce® BCA Protein Assay Kit (Thermo Fisher Scientific: 23225). ATP measurement results were corrected for the total protein content.

[0065] The results are shown in Figure 5. It was confirmed that ATP production from glomus cell-like cells increased in response to hypoxic stimulation (*P<0.05, n=3, Student's t-test).

[0066] (3-2) Production of catecholamines Glomus cell-like cells induced 16-19 days prior were used. Hypoxia stimulation was performed using the same procedure as described in (3-1) above. After removing KRB, the cells were suspended in a buffer (10 mM phosphate buffer, pH 5.0) used as the mobile phase for HPLC, and the cells were disrupted by sonication (3 sets of 10 seconds each). The cells were centrifuged at 12,000 × g at 4°C for 5 minutes, and the supernatant was passed through a protein concentrator to remove proteins from the solution. The resulting solution was subjected to HPLC, and epinephrine, dopamine (DA), and 3,4-dihydroxyphenylacetic acid (DOPAC) were measured.

[0067] The results of epinephrine measurements are shown in Figure 6, DA measurements in Figure 7, and DOPAC measurements in Figure 8. It was confirmed that the production of epinephrine, DA, and DOPAC increased in response to hypoxic stimulation (*P<0.05, n=3, Student's t-test).

[0068] (3-3) Gene expression Gene expression in glomus cell-like cells was analyzed by qPCR. Hypoxia stimulation was performed using the same procedure as in (3-1) above. Total RNA was extracted using NucleoSpin RNA (Takara Bio, U0955B). cDNA was prepared using ReverTra Ace® qPCR RT Master Mix with gDNA Remover (Toyobo, FSQ-301). qPCR was performed using THUNDERBIRD® SYBR® qPCR Mix (Toyobo, QPS-201). Housekeeping gene 36B4 was used as an internal control. The reaction and analysis were performed in triple replication.

[0069] Table 1. Primer sets used for qPCR [Table 1]

[0070] The expression level of TH is shown in Fig. 9, and the expression level of KCNK3 is shown in Fig. 10. It was confirmed that the expression levels of both TH and KCNK3 increased upon hypoxia stimulation (*P<0.05, n = 3, Student's t-test).

[0071] (3 - 4) Changes in the hypoxic response due to ion channel inhibition K + Using KRB containing the K channel inhibitor tetraethylammonium chloride (FUJIFILM Wako Pure Chemical Industries: 206 - 04501) (final concentration: 5 mM), Ca 2+ Using the Ca channel inhibitor nifedipine (FUJIFILM Wako Pure Chemical Industries: 141 - 05783) (final concentration: 5 μM), or Na + Using KRB containing the Na channel inhibitor lidocaine (FUJIFILM Wako Pure Chemical Industries: 120 - 02691) (final concentration: 1 μM), cells were stimulated with hypoxia according to the procedure in (3 - 1) above, and the ATP production was analyzed. As a control, the ATP production was analyzed in the same manner using KRB without an ion channel inhibitor.

[0072] The results are shown in Figs. 11 - 13 (*P<0.05, n = 3, Student's t-test). Inhibiting the K + channel increased ATP production in response to hypoxia stimulation (Fig. 11). No ATP was detected when the ATPase apyrase (Sigma - Aldrich: A6132 - 200UN) (final concentration: 2 U / mL) was added (Fig. 11). On the other hand, inhibiting the Ca 2+ channel or the Na + channel decreased ATP production in response to hypoxia stimulation (Figs. 12, 13). ATP production in response to hypoxia stimulation also decreased when using KRB without Ca 2+ These results confirmed that the glomus cell-like cells prepared by the procedure in (1 - 4) above showed a hypoxic response by a molecular mechanism similar to that of glomus cells.

[0073] <​​We commissioned VectorBuilder to synthesize a plasmid containing the EPAS1 gene (NCBI RefSeq ID: NM_001430.5) (pLV-hEPAS1, vector ID: VB11202-1497fcy).

[0074] HEK293T cells (1.5 × 10⁶) were placed in a 6-well plate coated with 0.1 w / v% gelatin. 6 HEK293T cells (2 wells) were seeded. DMEM containing 10% FBS and 1% non-essential amino acids (NEAAs) was used as the culture medium. pLV-hEPAS1 plasmid (4 μg), package plasmid psPAX2 (addgene, #12260) (2 μg), and envelope plasmid (pMD2.G) (addgene, #12259) (2 μg) were transfected into 500 μl of Opti-MEM (Thermo Fisher Scientific) and polyethyleneimine (Polysciences inc.) in two wells. After 20-24 hours, the entire medium was replaced with DMEM containing 10% FBS, 1% non-essential amino acids (NEAAs), and 1% penicillin-streptomycin. After another 24 hours and 48 hours, the entire medium was replaced with fresh medium, and the culture supernatant was collected. The culture supernatant was filtered through a PVDF syringe filter (0.45 μm), then Lenti-X Concentrator (Clontec) was added, and the mixture was centrifuged to obtain a virus pellet. The pellet was then suspended in the culture medium to be used, and the virus suspension was stored at -80°C.

[0075] Human iPS cells (201B7 strain) were placed in a 6-well plate coated with iMatrix using mTeSR1 containing 10 μM Y-27632, resulting in 6 × 10⁶ cells. 4Cells were seeded at a concentration of cells / well (Day 0). The following day, the culture medium was replaced with fresh mTeSR1 (2 mL / well), and 200 μL / well of virus suspension was added (Day 1). Subsequently, the culture medium was replaced with fresh mTeSR1 daily, and 4 days after virus addition, it was replaced with mTeSR1 containing 0.3 μg / mL of puromycin (Day 5). Puromycin selection was performed by culturing cells in mTeSR1 containing 0.3 μg / mL of puromycin for more than 10 days after virus addition. EPAS1 expression in selected iPS cells (under normal oxygen conditions) was analyzed by qPCR. Furthermore, selected iPS cells were induced into glomus cell-like cells using the procedure described in (1-4) above, and EPAS1 expression in cells (under normal oxygen conditions) on day 17 of induction was analyzed by qPCR. qPCR was performed using the same procedure as described in (3-3) above.

[0076] The results are shown in Figures 14 and 15. In the figures, "Control" refers to wild-type iPS cells. It was confirmed that iPS cells into which EPAS1 was introduced strongly expressed EPAS1 (Figure 14, *P<0.05, n=3, Student's t-test). Based on this result, iPS cells into which EPAS1 was introduced will be referred to as "EPAS1-expressing iPS cells" below. Glomus cell-like cells induced from EPAS1-expressing iPS cells also strongly expressed EPAS1 (Figure 15, *P<0.05, n=3, Student's t-test). Based on this result, glomus cell-like cells induced from EPAS1-expressing iPS cells will be referred to as "EPAS1-overexpressing glomus cell-like cells" below.

[0077] <5. Gene expression in glomus cell-like cells prepared from EPAS1-expressing iPS cells> The expression of the glomus cell marker gene TH and glomus cell-related factors KCNK3 and OR51E2 in EPAS1-expressing iPS cells (undifferentiated, induction day 3), autonomic progenitor cells induced from EPAS1-expressing iPS cells (induction day 13), and EPAS1-overexpressing glomus cell-like cells (induction day 17) was analyzed by qPCR. qPCR was performed using the same procedure as described in (3-3) above.

[0078] The results are shown in Figures 16-18. In the figures, "hiPSC" refers to EPAS1-expressing iPS cells, "Progenitors" refers to autonomic progenitor cells induced from EPAS1-expressing iPS cells (13 days after induction), and "Glomus cells" refers to EPAS1-overexpressing glomus cell-like cells (17 days after induction). Increased expression of TH, KCNK3, and OR51E2 was confirmed with differentiation (*P<0.05, n=3, Student's t-test).

[0079] <6. Hypoxic response of glomus cell-like cells prepared from EPAS1-expressing iPS cells> (6-1) ATP production Following the procedure described in (3-1) above, ATP production in EPAS1-overexpressing glomus cell-like cells (17 days after induction) in response to hypoxic stimulation was analyzed. EPAS1-overexpressing glomus cell-like cells (17 days after induction) under normal oxygen conditions (20% O2) were used as a control.

[0080] The results are shown in Figure 19 (*P<0.05, n=3, Student's t-test). EPAS1-overexpressing glomus cell-like cells showed high ATP production capacity in response to hypoxic stimulation, and their production was more than 10 times higher than that of glomus cell-like cells prepared from iPS cells without EPAS1 (Figure 4). These results confirm that EPAS1-overexpressing glomus cell-like cells are highly sensitive to hypoxic conditions and possess high hypoxic responsiveness.

[0081] <7. Screening of compounds that regulate glomus cell activity> Screening was performed on the activity-modulating ability of glomus cells for 87 commercially available compounds shown in Table 2 below. Glomus cell-like cell spheres induced 16-19 days after preparation according to the procedure in (1) above were washed twice with KRB and added together with KRB containing the compound in a 4-5 sphere / well (96-well plate) configuration. ATP production was analyzed according to the procedure in (3-1) above. As a control, KRB containing DMSO (used as a solvent for the compound) was used.

[0082] Table 2. Screened compounds [Table 2A] [Table 2B] [Table 2C]

[0083] As a result, 19 compounds were found that increased ATP production by more than 1.5 times compared to the control, and 19 compounds were found that decreased it to 0.5 times or less (data omitted). Nifedipine was among the compounds that decreased ATP production. Nifedipine has already been reported to suppress the hypoxic response of rat glomus cells (Buttigieg J. and Nurse CA., Biochem. Biophys. Res. Commun., 2004;322(1):82-7). Therefore, the above results support the fact that this screening method can accurately select compounds that regulate glomus cell activity.

Claims

1. (a) A step of inducing differentiation of pluripotent stem cells into autonomic progenitor cells, and (b) A step of culturing the autonomic progenitor cells obtained in step (a) in the presence of an FGF signaling pathway activator, an EGF signaling pathway activator, and an IGF-1 signaling pathway activator. A method for producing glomus cell-like cells, including [specific cells / symptoms].

2. (c) A step prior to step (a) of introducing an exogenous nucleic acid encoding endothelial PAS domain-containing protein 1 into the pluripotent stem cells. The method according to claim 1, further comprising:

3. The method according to claim 1, wherein the FGF signaling pathway activator is bFGF.

4. The method according to claim 1, wherein the EGF signaling pathway activator is EGF.

5. The method according to claim 1, wherein the IGF-1 signaling pathway activator is IGF-1.

6. The method according to claim 1, wherein the pluripotent stem cells are of human origin.

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