Method for culturing canine pluripotent stem cells suitable for inducing differentiation

Abstract

Provided are: a method for culturing canine pluripotent stem cells, the method comprising culturing canine pluripotent stem cells in a culture medium containing a TGF-beta family protein or a Wnt inhibitor, or a TGF-beta family protein and a Wnt inhibitor; and a culture medium for the method.

Description

Culture method for canine pluripotent stem cells suitable for differentiation induction

[0001] The present invention relates to a method for stably culturing canine pluripotent stem cells and a medium for stably culturing canine pluripotent stem cells.

[0002] Dogs are animals that spontaneously develop diseases common to humans (such as cardiovascular diseases, tumors, diabetes, etc.) and are useful as model animals for these diseases. In recent years, with the establishment of canine iPS cells, attempts have been made to apply dogs as a human stem cell disease treatment model to aim for the early realization of human stem cell therapy. However, a human stem cell disease treatment model using dogs has not yet been developed.

[0003] The culture method for canine iPS cells has been developed based on the culture methods used for iPS cells of other animal species including humans, and those that add cell growth factors such as bFGF and LIF to a basal medium containing fetal bovine serum or a serum replacement preparation have been used (Non-Patent Document 1). However, with these methods, long-term passage of canine iPS cells is difficult, and a method that can stably culture them has been demanded. Furthermore, in recent years, a method for culturing canine iPS cells by using the human iPS cell medium "StemFit (registered trademark)" has been reported (Non-Patent Document 2). Since this method can culture canine iPS cells regardless of the cell line, it has been found to have a certain degree of generality compared to the previous methods. However, still, the culture method for canine iPS cells remains at the technical level of applying the culture method for human iPS cells, and a culture method more suitable for canine iPS cells has not been developed.

[0004] Also, although several culture methods for canine iPS cells have been reported as described above, induction of differentiation of functional cells from these cells has not been reported so far.

[0005] Stem Cells and Development, Vol. 22, No. 14, July 2013, p. 2026 - 2035Molecular Reproduction and Development, Vol. 88, Issue 6, June 2021, p. 395 - 404

[0006] The present invention aims to develop a culture method and culture medium more suitable for culturing canine pluripotent stem cells. Furthermore, the present invention aims to develop a method for stably culturing canine pluripotent stem cells, and a method for culturing canine pluripotent stem cells that can stably induce differentiation into functional cells.

[0007] In existing canine iPS cell culture methods, canine iPS cells quickly lose their pluripotency and differentiate upon stimulation such as freezing and thawing. The inventors hypothesized that under existing culture conditions, canine iPS cells maintain pluripotency unevenly, and that some cells with low pluripotency are induced to differentiate upon stimulation. To solve this problem, they diligently conducted research. As a result, they surprisingly discovered that by inhibiting the Wnt signaling pathway, which is important for differentiation induction, and stimulating the activin / transforming growth factor (TGF) β signaling pathway, it is possible to culture highly pluripotent cells uniformly. Thus, the present invention was completed.

[0008] The present invention includes the following embodiments: [1] A method for culturing canine pluripotent stem cells, comprising culturing canine pluripotent stem cells in a medium containing a TGFβ family protein, a Wnt inhibitor, or a TGFβ family protein and a Wnt inhibitor. [2] The method according to [1], wherein the TGFβ family protein is activin. [3] The method according to [1] or [2], wherein the Wnt inhibitor is IWR-1, IWP-2, or XAV939, or a mixture of two or more selected therefrom. [4] The method according to [1], wherein the medium contains a TGFβ family protein and a Wnt inhibitor. [5] The method according to [4], wherein the TGFβ family protein is activin. [6] The method according to [4] or [5], wherein the Wnt inhibitor is IWR-1, IWP-2, or XAV939, or a mixture of two or more selected therefrom. [7] The method according to any one of [1] to [6], which yields canine pluripotent stem cells that can be stably differentiated into functional cells. [8] The method according to any one of [1] to [7], wherein the canine pluripotent stem cells are canine iPS cells. [9] A method for producing cardiomyocytes from canine pluripotent stem cells, comprising: (i) culturing canine pluripotent stem cells in a medium containing a TGFβ family protein or a Wnt inhibitor, or a TGFβ family protein and a Wnt inhibitor; and (ii) inducing differentiation of the cultured canine pluripotent stem cells obtained in (i) into cardiomyocytes by culturing them in a medium containing a differentiation-inducing substance.

[10] The method according to [9], wherein the TGFβ family protein is activin.

[11] The method according to [9] or

[10] , wherein the Wnt inhibitor is IWR-1, IWP-2, or XAV939, or a mixture of two or more selected therefrom.

[12] The method according to any one of [9] to

[11] , wherein the medium in (i) contains a TGFβ family protein and a Wnt inhibitor.

[13] A culture medium for stably culturing canine pluripotent stem cells, comprising a TGFβ family protein or a Wnt inhibitor, or a TGFβ family protein and a Wnt inhibitor.

[14] The culture medium according to

[13] , wherein the TGFβ family protein is activin.

[15] The medium according to

[13] or

[14] , wherein the Wnt inhibitor is IWR-1, IWP-2, or XAV939, or a mixture of two or more selected therefrom.

[16] The medium according to any one of

[13] to

[15] for culturing canine pluripotent stem cells that can be stably differentiated into functional cells.

[0009] According to the method of the present invention, canine pluripotent stem cells can be stably cultured, maintained, and / or proliferated. Furthermore, canine pluripotent stem cells maintained by the culture method of the present invention have higher differentiation potential than those maintained in existing culture media, and can be efficiently differentiated into desired cells, including cardiomyocytes. For example, when canine pluripotent stem cells cultured by the culture method of the present invention were differentiated into cardiomyocytes, it was found that the canine pluripotent stem cells were efficiently differentiated into cardiomyocytes, and beating cardiomyocytes were obtained. The method of the present invention is the only method that can stably induce functional canine pluripotent stem cell-derived cardiomyocytes, and is a highly innovative technology.

[0010] Dogs are useful as a model for human cardiovascular diseases. Therefore, by differentiating functional cells from canine pluripotent stem cells (e.g., canine iPS cells) cultured by the method of the present invention, important insights can be obtained not only in veterinary medicine but also in human medicine. Furthermore, by using the technology of the present invention, it is possible to open up a new industrial field in veterinary regenerative cardiovascular medicine by applying it to veterinary medicine, such as creating iPS cell-derived cardiomyocyte sheets and cardiomyocytes from canine iPS cells, which are already undergoing clinical trials in humans. In addition, it is known that certain dog breeds frequently experience cardiovascular diseases that are infrequent in humans, and it is also possible to establish a therapeutic model using canine iPS cell-derived preparations as a model to verify the therapeutic effect of iPS cell preparations on these diseases.

[0011] While it was previously known that Wnt and Actvin / TGFβ signaling plays important roles in pluripotent stem cells of other animal species, including humans, the fact that these signals can be controlled during the culture of pluripotent stem cells to efficiently and stably maintain them is a remarkable finding discovered for the first time in this invention.

[0012] This is a schematic diagram of a knock-in vector targeting the NANOG gene locus. The results of flow cytometry analysis targeting mClover2-positive cells are shown. "WT" indicates a wild-type canine iPS cell line, and "KI line" indicates a cell line (Nanog gene reporter ciPSC) in which a fluorescent protein (mClover2) expression gene was knocked into the NANOG gene locus of canine iPS cells. The percentage of mClover2-positive cells (%) in the Nanog gene reporter ciPSC cell line after culture in the presence of a small molecule inhibitor is shown. In the figure, "Control" indicates the percentage of mClover2-positive cells (%) in the Nanog gene reporter ciPSC cell line after culture in the absence of a small molecule inhibitor. ****P<0.0001, ***P<0.0005, **P<0.005. This is a micrograph of the Nanog gene reporter ciPSC cell line after culture in the presence of a small molecule inhibitor. In the figure, "w / o inhibitor" indicates the results after culturing in the absence of a small molecule inhibitor. The percentage of mClover2-positive cells (%) in Nanog gene reporter ciPSC strains after culturing in the presence of activin is shown. ****P<0.0001. Micrographs of Nanog gene reporter ciPSC strains after culturing in the absence of activin (0 ng / ml) or in the presence of activin 100 ng / ml. The percentage of mClover2-positive cells (%) in Nanog gene reporter ciPSC strains after culturing in the presence of bFGF is shown. ****P<0.0001, ***P<0.0005, **P<0.005. The percentage of mClover2-positive cells (%) in Nanog gene reporter ciPSC strains after culturing in the presence of a Wnt inhibitor (IWR-1 or IWP-2) is shown. ****P<0.0001, ***P<0.0005. The results of flow cytometry analysis targeting mClover2-positive cells of Nanog gene reporter ciPSC strains cultured in medium containing activin and IWR-1 are shown. The percentage of mClover2-positive cells (%) in Nanog gene reporter ciPSC strains cultured in medium containing activin and IWR-1 is shown. In the figure, "Control" shows the percentage of mClover2-positive cells (%) in Nanog gene reporter ciPSC strains cultured in medium without additives."AR" indicates a culture medium containing activin and IWR-1. ****P<0.0001. This shows the percentage of mClover2-positive cells (%) in Nanog gene reporter ciPSC strains after culturing in a medium containing activin and IWR-1, or activin, IWR-1, and IWP-2. **P<0.005. This is a micrograph of Nanog gene reporter ciPSC strains after culturing in a medium without additives (Control) or in a medium containing activin and IWR-1 (AR). This shows the staining results of mouse tissue samples transplanted with ciPSC strains cultured in AR medium. This shows the relative expression levels of Sox17, Sox1, and T genes after freezing and thawing ciPSCs cultured in AR medium or StemFit. This is a heatmap showing gene groups with different expression patterns in ciPSCs cultured in AR medium or StemFit. The results of flow cytometry analysis of cultured ciPSCs differentiated into endodermal cells are shown. In the figure, "AR" indicates ciPSCs cultured in AR medium, and "SF" indicates ciPSCs cultured in StemFit.

[0013] The present invention provides a method for stably culturing canine pluripotent stem cells, characterized by culturing canine pluripotent stem cells in a culture medium containing TGFβ family proteins and / or a Wnt inhibitor (hereinafter also referred to as "the method of the present invention"). The present invention further provides a culture medium for stably culturing canine pluripotent stem cells, characterized by a culture medium containing TGFβ family proteins and / or a Wnt inhibitor (hereinafter also referred to as "the culture medium of the present invention").

[0014] As used herein, the expression “to culture” cells includes maintaining and proliferating cells. Furthermore, as used herein, the expression “to stably culture” pluripotent stem cells means culturing, maintaining, and proliferating such stem cells while maintaining their pluripotency. Here, “pluripotency” refers to the property of being able to differentiate into all cells that can exist in a living organism.

[0015] In this specification, stem cells refer to immature cells (undifferentiated cells) that possess both self-renewal and differentiation capabilities. In particular, pluripotent stem cells (PSCs) refer to stem cells that are both pluripotent and self-renewal capable. Examples of pluripotent stem cells, though not limited to these, include induced pluripotent stem cells (also called iPS cells or iPSCs), embryonic stem cells (also called ES cells or ESCs), embryonic germ cells (EG cells), Muse cells, expanding potential stem cells (EPSCs), and naive cells. Pluripotent stem cells may be prepared by known methods, or established cell lines may be used.

[0016] 1. Method and Culture Medium of the Present Invention The method of the present invention is characterized by culturing canine pluripotent stem cells in a culture medium containing TGFβ family proteins and / or Wnt inhibitors (i.e., the culture medium of the present invention). Canine pluripotent stem cells cultured by the method of the present invention maintain pluripotency and self-renewal ability, and are therefore suitable for differentiation into various desired functional cells. Furthermore, canine pluripotent stem cells cultured by the method of the present invention can also be cryopreserved and then thawed for use. After thawing, these cells also maintain pluripotency and self-renewal ability and can be differentiated into various desired functional cells.

[0017] The culture medium of the present invention further comprises, in addition to TGFβ family proteins and / or Wnt inhibitors, basic components for culturing stem cells (e.g., sugars, inorganic salts, amino acids, vitamins, and trace elements). Examples of sugars include, but are not limited to, monosaccharides such as glucose, mannose, fructose, and galactose, and disaccharides such as sucrose, maltose, and lactose, with glucose or galactose being preferred. One type of sugar or a combination of two or more sugars may be used. Examples of inorganic salts include calcium salts, magnesium salts, potassium salts, sodium salts, copper salts, iron salts, and zinc salts. Specific examples, but are not limited to, include calcium chloride, calcium nitrate, copper sulfate pentahydrate, iron(III) nitrate notahydrate, magnesium chloride, magnesium sulfate, potassium chloride, sodium bicarbonate, sodium chloride, disodium hydrogen phosphate, sodium dihydrogen phosphate, and zinc sulfate. One type of inorganic salt or a combination of two or more inorganic salts may be used. Examples of amino acids include, but are not limited to, alanine, arginine, asparagine, aspartic acid, cystine, cysteine, glutamine, glutamic acid, glycine, histidine, hydroxyproline, isoleucine, leucine, lysine, methionine, phenylalanine, serine, threonine, tryptophan, tyrosine, valine, etc., or their derivatives, salts, or hydrates, preferably L-amino acids or their derivatives. One amino acid or a combination of two or more amino acids may be used. Examples of vitamins include, but are not limited to, ascorbic acid, biotin, choline, folic acid, inositol, niacin, pyridoxine, riboflavin, thiamine, para-aminobenzoic acid, pantothenic acid, vitamin B12, etc., or their derivatives, salts, or hydrates. One vitamin or a combination of two or more vitamins may be used.Examples of trace components include, but are not limited to, glutathione, hypoxanthine, lipoic acid, linolenic acid, phenol red, putrescine, pyruvate, thymidine, or derivatives, salts, or hydrates thereof, which are commonly used as components of culture media.

[0018] The culture medium of the present invention may further contain serum, or it may be a serum-free medium. Preferably, it is a serum-free medium. In the serum-free medium, Knock-out ＴＭ (Trademark) Serum Replacent (KSR: Thermo Fisher Scientific, Waltham, MA, USA), StemSure (Registered Trademark) Serum Replacent (SSR: FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan), and other serum substitutes, or serum-free supplements such as B27 supplement (Thermo Fisher Scientific, Waltham, MA, USA) may be added. The culture medium of the present invention is preferably a xeno-free medium. The xeno-free medium includes a medium that does not contain xeno-derived components, i.e., a medium that does not contain components from animals other than dogs, and a medium that does not contain any animal-derived components at all. Preferably, a medium that does not contain any animal-derived components at all is used.

[0019] Therefore, the culture medium of the present invention may be a basal medium for stem cells supplemented with TGFβ family proteins and / or Wnt inhibitors. As the basal medium for stem cells, known media or commercially available media can be used. Examples of commercially available basal media include, but are not limited to, StemFit® (Ajinomoto Co., Inc., Tokyo, Japan), StemFlex® Medium (Thermo Fisher Scientific, Waltham, MA, USA), StemPro®-34 (Thermo Fisher Scientific, Waltham, MA, USA), mTeSR® (STEMCELL technologies, Vancouver, Canada), Essential 8 Medium (Thermo Fisher Scientific, Waltham, MA, USA), and others. Examples of known basal culture media include, but are not limited to, MEM (Minimum Essential Medium), BME (Basal Medium Eagle), DMEM (Dulbecco's Modified Eagle Medium), EMEM (Eagle's Minimum Essential Medium), IMDM (Iscove's Modified Dulbecco's Medium), GMEM (Glasgow's MEM), F12 (Ham's F12 Medium), DMEM / F12, and RPMI1640.

[0020] TGFβ family proteins refer to a group of growth factors that have a structure similar to TGFβ and transmit signals via a pathway similar to that of TGFβ. Examples, though not limited to these, include TGFβ, activin, BMP (bone morphogenetic protein), GDF (growth / differentiation factor), nodal, myostatin, etc. Preferably, TGFβ, activin, or BMP are used, more preferably TGFβ or activin. In the present invention, two or more TGFβ family proteins may be used in combination.

[0021] Examples of activins include, but are not limited to, activin A, activin B, activin AB, or mixtures thereof. Preferably, activin A is used. The concentration of activin contained in the culture medium of the present invention is not limited and may be determined as appropriate by those skilled in the art, but for example, it may be about 0.1 to about 300 ng / ml, preferably about 1 to about 100 ng / ml, more preferably about 2 to about 50 ng / ml, even more preferably about 4 to about 30 ng / ml, even more preferably about 10 to about 25 ng / ml, for example, about 20 ng / ml.

[0022] In this specification, Wnt inhibitor refers to a substance that inhibits the Wnt signaling pathway. Examples of Wnt inhibitors, but not limited to, include IWR-1 (4-[(3aR,4S,7R,7aS)-1,3,3a,4,7,7a-hexahydro-1,3-dioxo-4,7-methano-2H-isoindol-2-yl]-N-8-quinolinyl-benzamide) and IWP-2 (N-(6-methyl-2-benzothiazolyl)-2-[(3,4,6,7-tetrahydro-4-oxo-3-phenylthieno[3,2-d]pyrimidin-2-yl)thiol]-acetamide). Examples include WntC59 (4-(2-methyl-4-pyridinyl)-N-(4-(3-pyridinyl)phenyl)0benzeneacetamide), IWP4 (N-(6-methyl-2-benzothiazolyl)-2-[[3,4,6,7-tetrahydro-3-(2-methoxyphenyl)-4-oxothieno[3,2-d]pyrimidin-2-yl]thio]-acetamide), KY2011, and tankyrase inhibitors such as XAV939, WIKI4, TC-E5001, and JW55. The culture medium of the present invention may contain one or more of the above Wnt inhibitors. Preferably, IWR-1, IWP-2, or XAV939, or a mixture of two or more selected therefrom, is used, and more preferably, IWR-1, IWP-2, or a mixture of IWR-1 and IWP-2 is used. The concentration of the Wnt inhibitor contained in the culture medium of the present invention is not limited and can be appropriately determined by those skilled in the art.

[0023] When XAV939 is used as the Wnt inhibitor, the concentration of XAV939 contained in the culture medium of the present invention may be, for example, about 0.2 to about 50 μM, preferably about 0.4 to about 40 μM, more preferably about 1 to about 30 μM, even more preferably about 3 to about 20 μM, and even more preferably about 5 to about 10 μM. When IWR-1 is used as the Wnt inhibitor, the concentration of IWR-1 contained in the culture medium of the present invention may be, for example, about 0.2 to about 50 μM, preferably about 0.4 to about 40 μM, more preferably about 1 to about 30 μM, even more preferably about 3 to about 20 μM, and even more preferably about 5 to about 10 μM. When IWP-2 is used as a Wnt inhibitor, the concentration of IWP-2 contained in the culture medium of the present invention may be, for example, about 1 to about 1000 nM, preferably about 4 to about 900 nM, more preferably about 10 to about 800 nM, even more preferably about 20 to about 600 nM, and even more preferably about 100 to about 500 nM. When a mixture of IWR-1 and IWP-2 is used as the Wnt inhibitor, the culture medium of the present invention may contain, for example, about 0.2 to about 50 μM of IWR-1 and about 1 to about 1000 nM of IWP-2, preferably about 0.4 to about 40 μM of IWR-1 and about 4 to about 900 nM of IWP-2, more preferably about 1 to about 30 μM of IWR-1 and about 10 to about 800 nM of IWP-2, even more preferably about 3 to about 20 μM of IWR-1 and about 20 to about 600 nM of IWP-2, and even more preferably about 5 to about 10 μM of IWR-1 and about 100 to about 500 nM of IWP-2.

[0024] From an ethical standpoint and due to the ease of sample acquisition, canine iPS cells are preferably used as canine pluripotent stem cells. iPS cells are generally produced by reprogramming somatic cells by introducing reprogramming factors in the form of nucleic acids or proteins into somatic cells. Genes used as reprogramming factors for the production of canine iPS cells include, but are not limited to, Oct3 / 4, Sox2, Klf4, c-Myc, Nanog, Lin28A, Sox17, and Sox1. These reprogramming factors may be used individually or in combination. In this specification, canine iPS cells may be produced according to known methods, or established cell lines may be used.

[0025] In the method of the present invention, canine pluripotent stem cells may be cultured on feeder cells or in a feeder-free state. Preferably, they are cultured in a feeder-free state. As feeder cells, those known in the field, such as mouse fibroblasts, can be used.

[0026] The method of the present invention is carried out by adherent culture or suspension culture. In the case of adherent culture, the method of the present invention may also use, for example, but not limited to, extracellular matrix, vitronectin, iMatrix, Matrigel, Geltrex, Laminin-521, Laminin-511, etc. as a scaffold material for cell culture.

[0027] The culture conditions in the method of the present invention, such as culture temperature and culture period, are not particularly limited and can be appropriately determined by those skilled in the art. For example, canine pluripotent stem cells are cultured in the culture medium of the present invention at approximately 36-38°C for 3-6 days at approximately 3-6% CO2. ２ The cells may be cultured under the following conditions. There is no particular limit to the culture period; the culture can be continued until the desired number of cells are obtained, or the culture can be continued until necessary, and subculturing can be performed as needed. Furthermore, canine pluripotent stem cells cultured by the method of the present invention may be cryopreserved for a desired period.

[0028] The method of the present invention allows for the stable culture of canine pluripotent stem cells, resulting in a cell population in which approximately 90% or more maintain pluripotency.

[0029] 2. Differentiation Induction of Cultured Canine Pluripotent Stem Cells Canine pluripotent stem cells cultured by the method of the present invention can be differentiated into desired cells more efficiently than existing culture methods. The cells that can be differentiated are not limited and can be differentiated into any of the endodermal, mesodermal, and ectoderm cells. Differentiation induction into various cell types is carried out by culturing canine pluripotent stem cells in a differentiation induction medium containing an appropriate differentiation induction substance. Differentiation induction substances and differentiation induction media are well known in the art and can be appropriately selected by those skilled in the art. For example, known differentiation induction substances for mesoderm include, but are not limited to, activin, GSK-3β inhibitors (e.g., CHIR99021, etc.), Rock inhibitors (e.g., Y-27632, etc.), BMP (e.g., BMP4, etc.), FGF, FBS, etc. For example, known differentiation-inducing substances for endoderm include, but are not limited to, activin, BMP inhibitors (e.g., LDN193489), GSK-3β inhibitors (e.g., CHIR99021), and Rock inhibitors (e.g., Y-27632). For example, known differentiation-inducing substances for neuroectoderm include, but are not limited to, TGFβ inhibitors (e.g., SB43152), BMP inhibitors (e.g., LDN193489), and Rock inhibitors (e.g., Y-27632). Furthermore, serum-free supplements such as KSR can also be used as differentiation-inducing substances. One or more differentiation-inducing substances may be used in combination. For example, culture may be performed in one step in the presence of one or more differentiation-inducing substances, or culture may be performed in the presence of one or more differentiation-inducing substances, and then the medium may be changed and culture may be performed in the presence of another one or more differentiation-inducing substances.

[0030] Examples of differentiation-inducing substances for cardiomyocytes include activin, GSK-3β inhibitors (e.g., CHIR99021), BMP, Wnt inhibitors, and serum-free supplements (e.g., KSR). One or more differentiation-inducing substances may be used in combination. Preferably, one or more substances selected from the group consisting of activin A, CHIR99021, BMP4, IWR-1, and KSR are used in combination. The differentiation-inducing substances are selected according to the developmental stage, and the differentiation-inducing substances used may be changed at each stage. Thus, according to the method of the present invention, beating cardiomyocytes can be obtained.

[0031] Culture conditions such as culture temperature and culture period are not particularly limited and can be appropriately determined by those skilled in the art. For example, approximately 36-38°C for 3-6 days, approximately 3-6% CO2 ２ You can culture it below.

[0032] Canine pluripotent stem cells cultured by the method of the present invention can be efficiently differentiated into various desired cell types. The differentiation rate in the cell population obtained by differentiation induction of canine pluripotent stem cells cultured by the method of the present invention is high, and differentiation is uniform.

[0033] Terms used herein should be interpreted as having their common meaning in the art unless otherwise specified. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meaning as generally understood by those skilled in the art. Furthermore, the term “approximately” is understood by those skilled in the art, and varies to some extent depending on the context in which it is used, but typically means a number within a range of ±10%, more typically ±5%, more typically ±4%, more typically ±3%, more typically ±2%, and even more typically ±1% of the number to which the term is attached.

[0034] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

[0035] (Materials and Methods) 1. Animals Male NOD / SCID mice aged 4-7 weeks were purchased from Charles River Laboratories Japan, Inc. (Kanagawa, Japan) and used in this example. All experiments were conducted in accordance with the animal care and use committee guidelines of Osaka Metropolitan University.

[0036] 2. Canine iPS cells (ciPSCs: canine-induced pluripotent stem cells) Four female canine iPSC strains (OPUiD05A, OPUiD04B, OPUiD06-UE-2, and OPUiD01-UB-1) were obtained from peripheral blood mononuclear cells (PBMCs) or urine-derived cells (UC) according to previous publications (Kimura et al., 2021, Mol Reprod Dev 88, 395-404; Tsukamoto et al., 2024, J Vet Med Sci 86, 247-257). One female ciPSC line (OPUiD06-UG, also known as SUC0329-5) was established in the laboratory according to a previous report (Tsukamoto et al., 2024). The vectors and cells used in the establishment of each ciPSC line are shown in Table 1. All of these ciPSC lines were induced from cells derived from different donors.

[0037]

[0038] 3. Maintenance of ciPSCs Unless otherwise specified, ciPSCs were basically maintained in StemFit AK02N (Ajinomoto Co., Inc., Tokyo, Japan) on iMatrix-511 silk (Nippi, Incorporated, Tokyo, Japan). For subculturing, confluent ciPSC colonies were dissociated with TrypLE Express (Thermo Fisher Scientific, Waltham, MA, USA) and re-seed on plates containing ciPSC medium supplemented with 10 μM Y-27632 (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan), pre-coated with extracellular matrix. These cells were subculturised regularly every 4 days.

[0039] 4. Quantitative reverse transcription PCR: Total RNA was extracted using NucleoSpin® RNA Plus (MACHEREY-NAGEL, Dueren, Nordrhein-Westfalen, Germany), and cDNA was synthesized using ReverTra Ace® qPCR RT Master Mix (TOYOBO CO., LTD., Osaka, Japan) according to the manufacturer's instructions. qRT-PCR was performed using PowerTrack. ＴＭ The assay was performed using SYBR Green Master Mix for qPCR (Thermo Fisher Scientific, Waltham, MA, USA), and the results were analyzed using comparative CT by QuantStudio 3 (Thermo Fisher Scientific, Waltham, MA, USA).

[0040] 5. Preparation of RNA Sequencing Library The library was prepared using the TruSeq Stranded mRNA Library Prep kit (Illumina, Inc., Research PI, San Diego, CA, USA) according to the manufacturer's instructions. Sequencing was performed in 101-base single-read mode on an Illumina NovaSeq 6000 sequencer (Illumina, Inc., San Diego, CA, USA). HISAT ver. 2-2.1.0 was used in combination with SAMtools ver. 1.11 to map the sequence reads to the canine reference genome sequence (canFam4). FPKM (fragments per kilobase of exon per million mapped fragments) was calculated using Cufflinks version 2.2.1.

[0041] 6. Flow Cytometry Analysis Canine iPSCs were analyzed using an SH800 cell sorter (Sony Corporation, Tokyo, Japan) or a BD Canto II (BD Biosciences, Franklin Lakes, NJ, USA). FACS data were analyzed and visualized using Flowjo software (BD Biosciences, Franklin Lakes, NJ, USA).

[0042] 7. Immunofluorescence Analysis (Immunostaining) Samples were fixed with 4% paraformaldehyde for 10 minutes and permeabilized with 0.1% Triton X-100 for 10 minutes at room temperature. Blocking buffer (MAXblock ＴＭBlocking Medium: Active Motif, Carlsbad, CA, USA), the samples were incubated for 30 minutes and then incubated with the primary antibody at 4°C overnight. Next, the samples were incubated with the fluorescent-conjugated secondary antibody for 1 hour and then mounted using VECTASHIELD Vibrance Antifade Mounting Medium with DAPI (Vector Laboratories, Newark, CA, USA). The specimens were observed and analyzed using EVOS M5000 (Thermo Fisher Scientific, Waltham, MA, USA).

[0043] 8. Chromosome analysis: ciPSCs were incubated with 0.05 mg / mL colcemid (Thermo Fisher Scientific, Waltham, MA, USA), trypsinized, and incubated with 0.075 M KCl. Subsequently, the cells were fixed in acetic acid: methanol (1:3), stained with quinacrine mustard and Hoechst 33258, and observed using confocal laser microscopy (LSM980; Carl Zeiss, Oberkochen, Germany).

[0044] 9. Statistical analysis: Quantitative data were generated using Microsoft Office Excel or GraphPad Prism and presented as mean ± SD. All statistical details of the experiments are described in the figure legends.

[0045] Example 1: Examination of optimal medium conditions: A Nanog gene reporter canine iPS cell line was generated by introducing a fluorescent protein expression gene (mClover2) into the pluripotency gene (NANOG) of canine iPS cells, cultured under various medium conditions, and the optimal medium conditions were selected based on fluorescence expression. That is, cells with low fluorescence emission have lost pluripotency, and cells with high fluorescence emission indicate maintenance of pluripotency. Subsequently, it was confirmed whether canine iPS cells function normally under the selected medium conditions.

[0046] (1) Construction of Nanog gene reporter ciPSC strain To construct a knock-in vector targeting the NANOG gene locus (Figure 1), 5' and 3' homology arms amplified from ciPSC genomic DNA, H2B-IRES-Neo amplified from dClover2-C1 (addgene: 54577), pH2B-mCherry-IRES-neo3 (addgene: 21044), and an MC1-promoter-driven DTA cassette amplified from pDEST-R4R3-MC1DTA (addgene: 139521) were assembled using NEBuilder® HiFi DNA Assembly Master Mix (New England Biolabs, Ipswich, MA, USA). For efficient gene editing, CRISPR (clustered regularly interspaced short palindromic repeats)-related protein 9 (CRISPR / Cas9) and single-stranded guide RNA (sgRNA) were transfected together using a knock-in vector. The sgRNA and Alt-R targeted the NANOG sequence. ＴＭS. p. HiFi Cas9 Nuclease V3 (IDT, Coralville, IA, USA) was used for gene editing. Reverse transfection was performed using Lipofectamine 3000 (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer's instructions. In short, 100 μL of Opti-MEM (Thermo Fisher Scientific, Waltham, MA, USA) containing 50 ng / μl Cas9 protein, 50 ng / μl sgRNAs, a Lipofectamine complex, and a knock-in vector was incubated at room temperature for 15 minutes. After incubation, the mixture was added to 60-80% confluent ciPSCs (ciPSC strain OPUiD05A) in a 12-well plate. After 48 hours, 200 μg / ml of G418 (Sigma-Aldrich, Burlington, MA, USA) was added to the culture medium for selection. After selection, G418-resistant ciPSC colonies were cloned, and the correct insertion of the knock-in construct was confirmed by PCR using primers specific to the insertion sequence.

[0047] (2) Small molecule screening using Nanog gene reporter ciPSC strains Nanog gene reporter ciPSC strains were cultured in the presence of various small molecule inhibitors. Specifically, the cell lines were passaged onto 24-well plates and cultured for 4 days in StemFit supplemented with 10 μM TGFβ inhibitor (SB431542), Wnt inhibitor (100 nM IWP-2, 10 μM IWR-1, or 10 μM XAV939, all obtained from NACALAI TESQUE, INC., Kyoto, Japan), FGF receptor inhibitor (PD173074: Selleck), or retinoic acid receptor (RAR) inhibitor (BMS: MedChemExpress, Monmouth Junction, NJ, USA). As a control, Nanog gene reporter ciPSC strains were similarly cultured in StemFit without the addition of small molecule inhibitors. Furthermore, for comparison, wild-type ciPSC strains without the introduction of the Nanog gene reporter were similarly cultured in StemFit without the addition of small molecule inhibitors. After 4 days of culture, ciPSCs under each condition were observed under a microscope, and the frequency of mClover2-positive cells was further analyzed by flow cytometry. The results are shown in Figures 2-1 to 2-3.

[0048] As is clear from Figure 2-1, no mClover2-positive cells were counted in wild-type ciPSC cells without the introduction of the Nanog gene reporter, whereas approximately 54.5% of mClover2-positive cells were present in the Nanog gene reporter ciPSC cells after culture in the absence of the inhibitor. The percentage of highly mClover2-positive cells after culture in the presence of Wnt inhibitors and RAR inhibitors did not change significantly, whereas the percentage of highly mClover2-positive cells decreased greatly when cultured in the presence of a TGFβ inhibitor, and all cells died when cultured in the presence of an FGF receptor inhibitor (PD173074) (Figure 2-2). Furthermore, under microscopic observation, the morphology and number of cell colonies when cultured in the presence of Wnt inhibitors and RAR inhibitors were almost the same as when cultured in the absence of the inhibitor, whereas the morphology and number of cell colonies when cultured in the presence of a TGFβ inhibitor were smaller and the number of cells was lower compared to when cultured in the absence of the inhibitor (Figure 2-3). Based on these results, we predicted that the substances targeted by the small molecule inhibitors that reduced the mClover2-positive high cell rate—namely TGFβ and FGF—are important for maintaining the pluripotency of ciPS cells.

[0049] (3) Examination of culture medium conditions Based on the results of (2) above, activin A was selected as a TGFβ family member and bFGF (basic fibroblast growth factor) as an FGF to be added to the culture medium. Nanog gene reporter ciPSC strains were cultured for 4 days in StemFit supplemented with 0, 4, 20, or 100 ng / ml of activin A or bFGF. After 4 days of culture, ciPSCs under each condition were analyzed by microscopy and flow cytometry targeting the frequency of mClover2-positive cells. The results are shown in Figures 3-1 to 3-3.

[0050] As a result, adding bFGF to the culture medium had little effect on the percentage of mClover2-positive cells (Figure 3-3), but adding activin at 4 ng / ml significantly increased the percentage of mClover2-positive cells. However, as the amount of activin added increased, the percentage of mClover2-positive cells decreased, and positive cells were no longer detected (Figures 3-1 and 3-2). Therefore, it was found that a culture medium containing activin at an appropriate concentration is suitable for culturing ciPSCs.

[0051] To obtain a more suitable culture medium, the effect of adding a Wnt inhibitor was investigated. IWR-1 and IWP-2 were used as Wnt inhibitors. Nanog gene reporter ciPSC strains were cultured for 4 days in StemFit containing 4, 20, 100, or 500 nM IWP-2, or 0.4, 2, 10, or 50 μM IWR-1. After 4 days of culture, ciPSCs under each condition were analyzed by flow cytometry targeting the frequency of mClover2-positive cells. As a result, it was found that adding a Wnt inhibitor to the culture medium increased the percentage of mClover2-positive cells compared to the control (no Wnt inhibitor added) (Figure 4).

[0052] Furthermore, we investigated combinations of TGFβ family and Wnt inhibitors that were effective in increasing the percentage of mClover2-positive cells. Nanog gene reporter ciPSC strains were cultured for 4 days in StemFit supplemented with 10 μM IWR-1 and 20 ng / ml activin A, or in StemFit supplemented with 2 μM IWR-1, 500 nM IWP-2 and 20 ng / ml activin A. As a control, Nanog gene reporter ciPSC strains were cultured for 4 days in StemFit without additives. After 4 days of culture, ciPSCs under each condition were analyzed by microscopy and flow cytometry targeting the frequency of mClover2-positive cells. As a result, when cultured in a medium supplemented with IWR-1 and activin, the percentage of mClover2-positive cells was 90.8%, a significant increase compared to the control (approximately 50%) (Figures 5-1 and 5-2). Furthermore, similar results were obtained when a mixture of IWR-1 and IWP-2 was used as the Wnt inhibitor (Figure 5-3). In addition, microscopic observation revealed that fibroblast-like cells, as observed in the control, were not observed when cultured in a medium supplemented with IWR-1 and activin (Figure 5-4). Therefore, it was found that using a medium containing a combination of TGFβ family proteins and a Wnt inhibitor is effective in culturing ciPSCs while maintaining their pluripotency.

[0053] Example 2: In vitro differentiation ability of cultured ciPSCs into three germ layers. A ciPSC strain (OPUiD05A) cultured in StemFit medium (hereinafter also referred to as AR medium) supplemented with 10 μM IWR-1 and 20 ng / ml activin A was induced to differentiate into three germ layers. The differentiation induction process was carried out entirely using basal medium aFB27 medium, which consists of 1% B27 supplement (Thermo Fisher Scientific, Waltham, MA, USA), 0.1 mM NEAA, 100 U / ml penicillin - 0.1 mg / ml streptomycin, 0.1% PVA, and 2 mM L-glutamine supplemented in Advanced DMEM / F12 medium (Thermo Fisher Scientific, Waltham, MA, USA). To induce differentiation into the mesendoderm, dissociated ciPSCs were seeded at 200,000 cells / well in 12-well plates on a vitronectin-coated dish and cultured in mesendoderm induction medium for 24–48 hours. The mesendoderm induction medium used was aFB27 medium supplemented with 100 ng / ml activin A, 3 μM CHIR99021 (R&D Systems, Minneapolis, MN, USA), and 10 μM Y-27632. To induce differentiation into the definitive endoderm, after one wash with PBS, the mesendoderm induction medium was replaced with definitive endoderm induction medium, and the cells were cultured for a further 3 days. Modified aFB27 medium supplemented with 100 ng / ml activin A and 0.5 μM BMP inhibitor (LDN193189: Selleck, Houston, TX, USA) was used as the endoderm induction medium. For neuroectoderm (NE) induction, dissociated ciPSCs were seeded at 200,000 cells / well in 12-well plates on a vitronectin-coated dish and cultured in NE induction medium for 72 hours. Modified aFB27 medium supplemented with 10 μM TGFβi (SB43152, NACALAI TESQUE, INC., Kyoto, Japan), 0.5 μM BMP inhibitor, and 10 μM Y-27632 was used as the NE induction medium.

[0054] Cells were immunofluorescently stained after each differentiation induction stage. As a result, the mesoderm marker T, the endoderm marker Foxa2, and the neuroectoderm marker Sox1 were detected at each stage.

[0055] Example 3: In vivo differentiation ability of cultured ciPSCs Five cell lines (OPUiD05A, OPUiD04B, OPUiD06-UE-2, OPUiD01-UB-1, SUC0329-5) were cultured in AR medium. Microscopic observation confirmed that all ciPSC lines maintained uniform cell morphology and proliferated without problems, thus confirming that they could be cultured stably. The ciPSC lines cultured in AR medium were transplanted into immunodeficient mice, and their ability to differentiate into various cell types on the mice was evaluated. Teratoma formation was performed using a known method. In short, approximately 1 x 10 e5 ciPSCs were transplanted subcutaneously or into the testes of NOD / SCID mice. After 2-3 months, the teratomas were dissected and analyzed by hematoxylin and eosin staining. As a result, differentiation into the triploders (ectoderm, mesoderm, and endoderm) was confirmed in all five cell lines tested (OPUiD05A, OPUiD04B, OPUiD06-UE-2, OPUiD01-UB-1, and SUC0329-5) (Figure 6). Since AR medium showed similar results in all five canine iPS cell lines, it was found to be a universally applicable condition for canine iPS cells. Furthermore, even in the cell line (SUC0329-5) that had not previously shown in vivo differentiation ability, in vivo differentiation became possible when cultured in AR medium.

[0056] Example 4: Effects of Freezing and Thawing on Cultured ciPSCs ciPSC strain (OPUiD05A) was cultured in AR medium or StemFit for 4 days, then cryopreserved at -80°C for 60 days. After thawing the cells at 37°C, the expression levels of Sox17 (endoderm marker), Sox1 (ectoderm marker), and T (mesoderm marker) were quantified by quantitative RT-PCR. The thawed cells were also observed under a microscope. The results are shown in Figure 7. As a result, a large number of differentiated cells were observed in the cell group cultured in StemFit after freezing and thawing, whereas no differentiated cells were observed in the cell group cultured in AR medium. Furthermore, quantitative analysis of gene expression showed that the expression of differentiation genes was reduced in cells cultured in AR medium compared to cells cultured in StemFit. Therefore, it was found that ciPSCs cultured in AR medium maintain pluripotency and suppress the appearance of differentiated cells even after freezing and thawing. On the other hand, ciPSCs cultured in conventional culture media were unable to maintain pluripotency, and some cells spontaneously differentiated into each germ layer.

[0057] Example 5: Analysis of gene expression patterns in cultured ciPSCs Gene expression patterns in ciPSC strains (OPUiD05A, OPUiD05A+1 passage, OPUiD06-UG, OPUiD06-UE-2) cultured in AR medium and existing medium (StemFit) were confirmed by bioinformatics analysis. Briefly speaking, for bulk RNA-seq analysis of canine iPSCs, paired sequencing reads were screened for quality using fastQC (v0.11.9), and then aligned using Ensemble ROS Cfam 1.0 dog gene using STAR (v2.7.8a) (Dobin et al., 2013, Bioinformatics 29, 15-21). Raw gene counts were obtained using subread featureCounts (v2.0.0) (Liao et al., 2014, Bioinformatics 30, 923-930), and then imported into DESeq2 workflow for subsequent differential gene expression analysis (Love et al., 2014, Genome Biol 15, 550).

[0058] As a result, cells cultured in AR medium showed a different gene expression pattern than those cultured in StemFit (Figure 8). Furthermore, ciPSCs cultured in AR medium showed a uniform gene expression pattern regardless of the cell line. In addition, it was found that ciPSCs cultured in AR medium had significantly lower expression of differentiation genes.

[0059] Example 6: A ciPSC strain (OPUiD05A) cultured in either StemFit or AR medium was seeded at 100,000 cells / well in a 12-well plate on a vitronectin-coated dish and cultured in aFB27 medium supplemented with 100 ng / ml activin A, 5 μM CHIR99021, and 10 μM Y-27632. After 24 hours, the medium was changed to a medium without Y-27632, and the cells were cultured for a further 5 days. The medium was changed every other day. Differentiated cells were collected daily and subjected to flow cytometry analysis targeting CXCR4, an endodermal cell marker. As a result, ciPSCs cultured in AR medium showed a higher proportion of CXCR4-positive cells (approximately 100%) and achieved more uniform differentiation induction compared to ciPSCs cultured in stemFit (Figure 9).

[0060] Example 7: A ciPSC strain (OPUiD06-UG) cultured in either StemFit or AR medium for differentiation of cultured ciPSCs into cardiomyocytes was passaged onto Geltrex® (Thermo Fisher Scientific, Waltham, MA, USA) and cultured in StemFit medium supplemented with 10 μM Y-27632. After 24 hours, the medium was replaced with basal medium supplemented with 10 μM IWR-1, 20 ng / ml activin A, and 20 ng / ml BMP4 (R&D Systems, Minneapolis, MN, USA). After another 24 hours, the ciPSCs were further cultured in differentiation medium supplemented with 6 μM CHIR99021, 10 ng / ml activin A, and 20 ng / ml BMP4 (Day 0). After 24 hours, CHIR99021 was knocked out at 0.2%. ＴＭCells were cultured for 24 hours in thermoreplacement (KSR: Thermo Fisher Scientific, Waltham, MA, USA) (Day 1). The KSR concentration was changed to 2% and the cells were cultured for another 24 hours (Day 2). On Day 3, differentiated cells were cultured for 48 hours in differentiation medium supplemented with 10 μM IWR1, and then for another 48 hours in medium supplemented with 2% KSR (Days 3-6). On Day 7, the medium was changed to differentiation medium supplemented with 2% KSR and 20 μg / ml insulin, 10 ng / ml activin A, and 20 ng / ml BMP4. Differentiated cells were further cultured until analysis. The differentiation medium consisted of RPMI 1640, insulin-free 1x B27 (Thermo Fisher Scientific, Waltham, MA, USA), 0.1 mM NEAA, 100 U / ml penicillin-0.1 mg / ml streptomycin, sodium pyruvate, and 2 mM Glutamax.

[0061] On day 9 of culture in differentiation medium, the obtained cells were observed using an inverted microscope. The results showed that the cardiomyocyte aggregates obtained by differentiation induction of ciPSCs cultured in AR medium exhibited autonomous pulsation and a homogeneous pulsation rhythm. In contrast, no pulsating cells were observed in differentiation induction from ciPSCs cultured in the existing medium (StemFit).

[0062] Furthermore, the presence or absence of cTnT expression, a cardiac muscle marker, was confirmed by immunohistochemistry in cells obtained on day 9 of culture in differentiation medium. As a result, it was found that almost all of the cardiomyocytes differentiated from ciPSCs cultured in AR medium expressed cTnT. Therefore, it was found that ciPSCs cultured in AR medium differentiate into cardiomyocytes more efficiently than those cultured using existing methods.

[0063] According to the present invention, canine pluripotent stem cells can be stably cultured while maintaining their pluripotency. Canine pluripotent stem cells cultured by the method of the present invention are suitable for differentiation induction into functional cells. Therefore, the technology of the present invention can contribute not only to veterinary medicine but also to the development of human medicine by establishing a human stem cell therapy model using canine iPS cells.

Claims

1. A method for culturing canine pluripotent stem cells, comprising culturing canine pluripotent stem cells in a medium containing a TGFβ family protein, a Wnt inhibitor, or both a TGFβ family protein and a Wnt inhibitor.

2. The method according to claim 1, wherein the TGFβ family protein is activin.

3. The method according to claim 1 or 2, wherein the Wnt inhibitor is IWR-1, IWP-2, or XAV939, or a mixture of two or more selected therefrom.

4. The method according to claim 1, wherein the culture medium comprises a TGFβ family protein and a Wnt inhibitor.

5. The method according to claim 4, wherein the TGFβ family protein is activin.

6. The method according to claim 4 or 5, wherein the Wnt inhibitor is IWR-1, IWP-2, or XAV939, or a mixture of two or more selected therefrom.

7. The method according to any one of claims 1 to 6, which provides canine pluripotent stem cells that can be stably induced to differentiate into functional cells.

8. The method according to any one of claims 1 to 7, wherein the canine pluripotent stem cell is a canine iPS cell.

9. A method for producing cardiomyocytes from canine pluripotent stem cells, comprising: (i) culturing canine pluripotent stem cells in a medium containing a TGFβ family protein or a Wnt inhibitor, or a TGFβ family protein and a Wnt inhibitor; and (ii) inducing differentiation of the cultured canine pluripotent stem cells obtained in (i) into cardiomyocytes by culturing them in a medium containing a differentiation-inducing substance.

10. The method according to claim 9, wherein the TGFβ family protein is activin.

11. The method according to claim 9 or 10, wherein the Wnt inhibitor is IWR-1, IWP-2, or XAV939, or a mixture of two or more selected therefrom.

12. A culture medium for stably culturing canine pluripotent stem cells, comprising a TGFβ family protein, a Wnt inhibitor, or both a TGFβ family protein and a Wnt inhibitor.

Images