Medium for producing and / or culturing feline pluripotent stem cells, feline pluripotent stem cells, method for producing the same and method for culturing the same

Abstract

To provide a medium for producing and / or culturing feline animal-derived pluripotent stem cells maintaining an undifferentiated state even after long-term subculture.SOLUTION: A medium for producing and / or culturing feline pluripotent stem cells, which comprises FGF at a concentration of 25ng / mL or higher.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a medium for producing and / or culturing feline-derived pluripotent stem cells. [Background technology]

[0002] In recent years, the number of cats kept as pets has increased, leading to a growing demand for cutting-edge veterinary care for cats. Diabetes, chronic kidney disease, and hereditary diseases, which are common in cats, have low therapeutic efficacy through surgery or medication, and research into transplant treatments has not progressed sufficiently to make them practical. Given this background, there are growing expectations that the establishment of feline stem cells will be applied to regenerative veterinary medicine, such as cell transplantation therapy, and that this will help elucidate the pathology of genetic diseases. Furthermore, if stem cells that differentiate into germ cells can be created, it could be useful for research into the conservation of populations of endangered wild felines through in vitro fertilization, using domestic cats as a model animal. However, to date, no specific tissue stem cells have been established, nor have stable feline pluripotent stem cells that can maintain an undifferentiated state even after long-term passaging. Non-Patent Documents 1 and 2 describe that feline embryonic stem-like cells (ES-like cells) were established, but that the cells could no longer maintain an undifferentiated state after 7 or 13 passages. Furthermore, neither document discloses pluripotent stem cells that can differentiate into all three germ layer cells from which the cells that constitute the body are derived. [Prior art documents] [Non-patent literature]

[0003] [Non-Patent Document 1] X. Yu, 2008, Molecular Reproduction and Development 75:1426-1432 [Non-patent document 2] MC Gomez, 2010, Theriogenology 74(2010) 498-515 Summary of the Invention [Problem to be solved by the invention]

[0004] An objective of the present invention is to provide a medium for producing and / or culturing feline-derived pluripotent stem cells that maintain an undifferentiated state even after long-term subculture.

[0005] Objects of the present invention include providing a medium for producing and / or culturing feline-derived pluripotent stem cells capable of differentiating into three germ layers in vitro and / or in vivo. [Means for solving the problem]

[0006] The present inventors have conducted extensive research in light of the above-mentioned problems and have found that a medium containing FGF at a high concentration of 25 ng / mL or more can solve the above-mentioned problems. Based on this finding, the present inventors have conducted further research and have completed the present invention. Specifically, the present invention encompasses the following aspects.

[0007] Section 1. A medium for producing and / or culturing feline-derived pluripotent stem cells, containing 25 ng / mL or more of fibroblast growth factor (FGF) or a substitute with FGF-like activity equivalent to or greater than 25 ng / mL of FGF. Section 2. Item 1. The medium according to Item 1, wherein the serum content is 1% or less. Section 3. Item 3. The medium according to Item 1 or 2, for use in culture in the presence of feeder cells. Section 4. Item 4. The medium according to any one of Items 1 to 3, wherein the pluripotent stem cells are embryonic stem cells. Section 5. A feline-derived pluripotent stem cell obtained by culturing cells obtained from the inner cell mass of a feline blastocyst-stage embryo and / or feline somatic cells introduced with a reprogramming factor in the medium described in any one of Items 1 to 4. Section 6. Item 6. A feline-derived pluripotent stem cell according to Item 5, which has the ability to differentiate into three germ layers. Section 7. Item 7. A feline-derived pluripotent stem cell according to Item 5 or 6, which has the ability to differentiate into three germ layers in vivo. Section 8. Feline-derived multipotent stem cells with the ability to differentiate into three germ layers. Section 9. Item 9. A feline-derived pluripotent stem cell according to Item 8, which has the ability to differentiate into three germ layers in vivo. Section 10. A method for producing feline-derived pluripotent stem cells, comprising a step of culturing feline-derived pluripotent stem cells, cells obtained from the inner cell mass of feline blastocyst-stage embryos, and / or feline somatic cells introduced with a reprogramming factor in the medium according to any one of Items 1 to 4. Section 11. Item 11. The method of claim 10, further comprising the step of subculturing feline-derived pluripotent stem cells in the medium for 20 or more generations. Section 12. A culture method for maintaining feline-derived pluripotent stem cells in an undifferentiated state, comprising the step of culturing the feline-derived pluripotent stem cells in the medium according to any one of Items 1 to 4. [Effects of the Invention]

[0008] According to the present invention, a medium can be provided for producing and / or culturing feline-derived pluripotent stem cells that maintain an undifferentiated state even after long-term subculture and have the ability to differentiate into three germ layers. [Brief explanation of the drawings]

[0009] [Figure 1] A(i) shows micrographs of a feline blastocyst (A(i)) and a primary colony of feline ES cells (A(ii)) obtained by in vitro fertilization in Example 1. B shows micrographs of colonies of feline ES cells at passage 5 ((i) and (iv)), colonies of feline ES cells at passage 30 ((ii) and (v)), and colonies of feline ES cells after freeze-thawing ((iii) and (vi)). [Figure 2]

[0033] Figure 1 shows the results of alkaline phosphatase staining of feline ES cells in Example 2. Shown are the staining results of feline ES cells at passage 5 ((i) and (iv)), feline ES cells at passage 30 ((ii) and (v)), and feline ES cells after freeze-thawing ((iii) and (vi)). [Figure 3] This shows the results of expression analysis of undifferentiated markers in the feline ES cells of Example 2. A shows the results of quantitative PCR, B shows the results of immunostaining, and C shows the results of flow cytometry. FEF and MEF refer to feeder cells, and NCH01, NCH02, and NCH03 refer to three different lines of feline ES cells prepared in Example 1. [Figure 4] 1 shows the results of the expression analysis of differentiation markers in feline ES cells in Example 3. A shows the results of quantitative PCR, and B shows the results of immunostaining. [Figure 5] The results of the teratoma formation assay in Example 4 are shown. The leftmost column shows photographs of a testis not injected with feline ES cells (left) and an injected testis (right). The right column shows images of teratomas derived from the three germ layers (hair follicles and epidermis: ectoderm-derived, fat: mesoderm-derived, respiratory epithelium: endoderm-derived) observed by HE staining of the ES cell-injected testis. [Figure 6] 1 shows the results of karyotype analysis of feline ES cells in Example 5. (i) to (iii) show photographs of the chromosomes of the NCH01 to 03 strains, respectively. [Figure 7] 1 shows the morphology of feline ES cells when cultured in four types of media with different compositions in Example 6 (bars represent 100 μm (×10) or 20 μm (×40)). DETAILED DESCRIPTION OF THE INVENTION

[0010] In this specification, the expressions "contain" and "comprise" include the concepts of "contain," "comprise," "consist essentially of," and "consist only of."

[0011] 1. Medium for producing and / or culturing feline-derived pluripotent stem cells In one aspect, the present invention relates to a medium for producing and / or culturing feline-derived pluripotent stem cells, which has a high FGF content (also referred to herein as the "medium of the present invention").

[0012] As used herein, pluripotent stem cells refer to undifferentiated cells that have the ability to differentiate into cells of multiple lineages (pluripotency) and the ability to self-replicate. Pluripotent stem cells are preferably stem cells that have the ability to differentiate into tissues of at least two of the three germ layers (endoderm, mesoderm, and ectoderm) and the ability to self-replicate, and are particularly preferably pluripotent stem cells that have the ability to differentiate into tissues of all three germ layers (pluripotency) and the ability to self-replicate. Specific examples of pluripotent stem cells include hematopoietic stem cells and adipose tissue-derived stem cells, and specific examples of pluripotent stem cells include ES cells (embryonic stem cells), iPS cells (induced pluripotent stem cells), EG cells, EC cells, and the like. As used herein, pluripotent stem cells are preferably ES cells or iPS cells.

[0013] Herein, the organism from which feline-derived pluripotent stem cells, which are the target of production and / or culture using the medium of the present invention, are derived is not particularly limited as long as it is a feline, and examples that can be used include domestic cats, tigers, lions, cheetahs, pumas, jaguars, leopards, snow leopards, clouded leopards, Pallas's cat, lynx, caracal, ocelots, sand cats, fishing cats, Bengal leopards, Amur leopards, Bornean leopards, Andean leopards, Malayan leopards, African golden cats, Northern tiger cats, Southern tiger cats, Esperanto lynx, Sundaland clouded leopards, black-footed cats, kodokod, grey cats, marbled cats, Asiatic golden cats, margays, kolo-kolos, rust-spotted cats, Geoffroy's cats, jaguarundi, bobcats, servals, Canadian lynxes, jungle cats, European wildcats, and African wildcats. Among these, those derived from domestic cats, which are widely kept as companion animals and for which there is a growing demand for pluripotent stem cells from the perspective of veterinary regenerative medicine, are preferred.Also preferred are those derived from wild cats such as tigers, Bornean leopards, Andean wildcats, Malayan wildcats, African golden cats, northern tiger cats, southern tiger cats, Iberian lynxes, clouded leopards, Sundaland clouded leopards, snow leopards, leopards, fishing cats, black-footed cats, cheetahs, lions, kodo-kodos, grey cats, and Bengal leopards, whose populations are declining and whose populations are expected to recover using germ cells derived from pluripotent stem cells.

[0014] The medium of the present invention can be used for culturing in the process of producing pluripotent stem cells from cells and / or cell masses used as starting materials, as well as for subculturing pluripotent stem cells. When producing and / or culturing pluripotent stem cells using the medium of the present invention, the methods described in "3. Method for producing feline-derived pluripotent stem cells" and "4. Method for culturing feline-derived pluripotent stem cells" can be used, for example.

[0015] As used herein, fibroblast growth factor (FGF) refers to FGF, FGF-2, FGF-β, or bFGF. The medium of the present invention contains FGF at a high concentration of 25 ng / mL or more. Preferably, the medium of the present invention contains FGF at a concentration of 25 ng / mL or more, 30 ng / mL or more, 35 ng / mL or more, 40 ng / mL or more, 45 ng / mL or more, 50 ng / mL or more, 55 ng / mL or more, 60 ng / mL or more, 65 ng / mL or more, 70 ng / mL or more, 75 ng / mL or more, 80 ng / mL or more, 85 ng / mL or more, 90 ng / mL or more, 95 ng / mL or more, or 100 ng / mL or more.

[0016] The origin of FGF is not particularly limited, but FGF derived from human is preferred. FGF may be commercially available or synthesized according to known information.

[0017] In one embodiment, the medium of the present invention contains a substitute in an amount equivalent to 25 ng / mL of FGF or more. Examples of such substitutes include bFGF-alternative peptides or FGFR1 agonists with FGF-like activity (Yonehara et al., 2016, Journal of Biological Chemistry), or small molecules that act via a pathway other than bFGF. Examples of such small molecules include a combination of ID-8, 1-azakempaullone, and tacrolimus (Yasuda et al., 2018, Nature Biomedical Engineering), trimipramine, or ethhopropazine (Kumagai et al., 2013, Biochemical and Biophysical Research Communications). Other known FGF substitutes can also be used. Whether an FGF substitute has activity equivalent to a given amount of FGF can be determined, for example, by determining whether the proliferation rate is comparable and whether the undifferentiated state is maintained when cells are cultured with FGF and the FGF substitute for a given period of time. Generally, FGFs are expensive, pose safety risks due to the contamination of biological impurities during recombinant protein production, and have greater lot-to-lot performance variability than compounds. The use of FGF substitutes can further reduce lot-to-lot performance variability.

[0018] The culture medium of the present invention may also contain a differentiation inhibitor protein other than FGF, if necessary. If necessary, appropriate components can be selected from these and added to the culture medium to enhance the ability to maintain the undifferentiated state of pluripotent stem cells. The differentiation inhibitor protein is not particularly limited, as long as it is a factor that has the effect of maintaining the undifferentiated state of pluripotent stem cells, or a factor known to be used for maintaining the undifferentiated state of pluripotent stem cells. Examples include LIF (leukemia inhibitory factor), the TGFβ superfamily (activin, Nodal, etc.), and Wnt. The concentration of the differentiation inhibitor protein can be a known concentration used in the culture of pluripotent stem cells. The differentiation inhibitor protein other than FGF may be used alone or in combination of two or more types.

[0019] Generally, these differentiation-inhibiting proteins are very expensive and exhibit greater lot-to-lot variability in performance than compounds. On the other hand, when the medium of the present invention contains 25 ng / mL or more of FGF or an FGF substitute with comparable activity, it can efficiently maintain the undifferentiated state of pluripotent stem cells without the use of differentiation-inhibiting proteins other than FGF. Therefore, from the viewpoint of further reducing the cost of medium preparation or further reducing the variability in performance between medium lots, the medium of the present invention is preferably used for culture without containing differentiation-inhibiting proteins other than FGF or without adding differentiation-inhibiting proteins other than FGF.

[0020] The medium of the present invention is usually prepared by mixing a basal medium with FGF, but a commercially available cell culture medium containing 25 ng / mL or more of FGF may also be used as the medium for producing and / or culturing pluripotent stem cells.

[0021] The basal medium is not particularly limited as long as it can be used as a basal medium for culturing pluripotent stem cells, and typically contains standard inorganic salts such as magnesium, calcium, potassium, zinc, and iron, a buffer, glucose, vitamins, essential amino acids, etc. Specific examples include Dulbecco's Modified Eagle's Medium (DMEM), DMEM / F12, Minimal essential Medium (MEM), Basal Medium Eagle (BME), RPMI1640, F-10, F-12, αMinimal essential Medium (αMEM), Glasgow's Minimal essential Medium (GMEM), Iscove's Modified Dulbecco's Medium (IMDM), StemFit™ AK02N (Ajinomoto), NutriStem (REPROCELL), and mixtures thereof.

[0022] Furthermore, the basal medium may contain, as necessary, a buffer such as HEPES, non-essential amino acids, antioxidants, etc. Examples of non-essential amino acids include L-glutamine, L-alanine, L-asparagine, L-aspartic acid, L-glutamic acid, glycine, L-proline, L-serine, etc., preferably L-glutamine, etc. Examples of antioxidants include ascorbic acid, 2-mercaptoethanol, dithiothreitol, etc., preferably ascorbic acid, etc. The concentrations of these components can be those known to be used in the culture of pluripotent stem cells.

[0023] The medium of the present invention may optionally contain at least one component selected from the group consisting of serum and serum substitutes. If necessary, an appropriate component may be selected from these components and blended into the medium to enhance the ability of pluripotent stem cells to maintain an undifferentiated state.

[0024] The serum is not particularly limited as long as it can be used in culturing pluripotent stem cells. For example, fetal bovine serum (FBS) can be used. The serum concentration can be any known concentration used in culturing pluripotent stem cells. One type of serum may be used alone, or two or more types may be used in combination.

[0025] In general, serum is expensive and there is a large variation in performance between lots. On the other hand, the medium of the present invention can efficiently maintain the undifferentiated state of pluripotent stem cells and further proliferate the cells without using serum. Therefore, from the viewpoint of further reducing the cost of medium preparation or the variation in performance between medium lots, the medium of the present invention preferably has a serum content of 4.5% or less, 4% or less, 3% or less, 2% or less, 1% or less, 0.5% or less, or 0.1% or less. More preferably, the medium of the present invention does not contain serum or is used for culture without adding serum.

[0026] Serum replacement components refer to components that can support the proliferation of pluripotent stem cells when added to a serum-free medium. Specific examples include KnockOut Serum Replacement (KSR™), albumin (e.g., bovine serum albumin), or albumin replacement additives (e.g., bovine pituitary extract, rice hydrolysate, bovine fetal albumin, egg albumin, human serum albumin, bovine embryo extract, AlbuMAX I™, transferrin, insulin, etc.). The serum replacement components can be used at concentrations known to be used in the culture of pluripotent stem cells. Serum replacement components may be used alone or in combination of two or more types. Generally, protein components exhibit significant lot-to-lot variability in performance. Therefore, when using proteins as serum replacement components, it is preferable to use as few different proteins as possible. In one embodiment, the medium of the present invention is preferably used for culture without containing or without adding serum replacement components.

[0027] In addition to the above, the medium of the present invention may contain other known components that can be added to media for pluripotent stem cells. Among these, selenium, ethanolamine, etc. can be mentioned from the viewpoint of enabling more efficient cell proliferation. The concentrations of these components can be any known concentrations used in the culture of pluripotent stem cells.

[0028] The culture medium of the present invention can be prepared in either a solution or dry form. In the case of a solution, it may be provided as a concentrated composition (e.g., 1x to 1000x) and may be diluted appropriately before use. The type of liquid used to dilute or dissolve the composition or culture medium in solution or dry form includes water, an aqueous buffer solution, a physiological saline solution, etc., and can be easily selected as needed.

[0029] When the medium of the present invention is in the form of a solution, the pH is usually adjusted to 7.0 to 8.2, preferably 7.1 to 7.8, and more preferably 7.2 to 7.5, using a pH adjuster such as bicarbonate, and the osmotic pressure is adjusted to 310 to 340 mOsm using a salt such as sodium chloride.

[0030] The medium of the present invention is preferably sterilized to prevent contamination. Sterilization methods include ultraviolet irradiation, heat sterilization, radiation irradiation, filtration, etc.

[0031] The medium of the present invention can be used as is, or with the above-mentioned components added as needed, for culturing pluripotent stem cells (for example, for maintaining pluripotent stem cells, subculturing pluripotent stem stem cells, producing embryonic stem cells (ES cells) and induced pluripotent stem cells (iPS cells), etc.).

[0032] The culture mode using the medium of the present invention may be either adherent culture or suspension culture. In the case of adherent culture, the culture may be in the absence or presence of feeder cells. Preferably, the culture is adherent culture in the presence of feeder cells. In one embodiment, the medium of the present invention may be a feeder-free culture medium that allows stem cells to be cultured in the absence of feeder cells.

[0033] There are no particular limitations on the feeder cells, so long as they provide a scaffold for cell adhesion when co-cultured with stem cells and can supply substances necessary for stem cell survival, proliferation, and maintenance of an undifferentiated state. Suitable feeder cells include, for example, mouse embryonic fibroblasts (MEF) and feline embryonic fibroblasts (FEF). In particular, culturing in the stage of producing pluripotent stem cells from starting cells or the like is preferably carried out in the presence of feeder cells.

[0034] 2. Feline-derived multipotent stem cells In one aspect, the present invention relates to feline-derived multipotent stem cells (sometimes referred to herein as "stem cells of the present invention"). In one embodiment, the present invention relates to feline-derived pluripotent stem cells obtained by culturing cells obtained from the inner cell mass of a feline blastocyst-stage embryo and / or feline somatic cells introduced with reprogramming factors in the medium of the present invention. The pluripotent stem cells and the organisms from which they are derived are the same as those described above in "1. Medium for producing and / or culturing feline-derived pluripotent stem cells."

[0035] A blastocyst-stage embryo is an embryo formed from the inner cell mass (ICM) and trophoblast, and feline fertilized eggs are usually in the blastocyst stage 5 to 7 days after fertilization. From the viewpoint of easy removal of the zona pellucida and efficient yield of the inner cell mass, it is preferable to use an escaped blastocyst, which is an embryo that has further developed and escaped from the zona pellucida. Usually, feline fertilized eggs are in the escaped blastocyst stage 6 to 8 days after fertilization.

[0036] Blastocyst-stage embryos may be either embryos obtained by in vivo fertilization in cats or embryos obtained by in vitro fertilization. Blastocyst-stage embryos obtained by in vivo fertilization in cats can be obtained, for example, by the method described in Non-Patent Document 1. Blastocyst-stage embryos obtained by in vitro fertilization in cats can be obtained, for example, by the method described in Non-Patent Document 2. In one embodiment, from the standpoints of animal ethics, economy, and work efficiency, it is preferable to use embryos obtained by in vitro fertilization as blastocyst-stage embryos. Furthermore, in one embodiment, it is preferable to use blastocyst-stage embryos obtained by in vivo fertilization, from the standpoints that blastocyst-stage embryos grow large and the inner cell mass can be obtained more simply and efficiently.

[0037] The method for isolating the inner cell mass from the blastocyst stage embryo is not particularly limited, and for example, it can be mechanically isolated under a microscope using a syringe needle (31G) or a sharp-tipped glass capillary.

[0038] By culturing the obtained inner cell mass in the medium of the present invention with or without feeder cells, colonies of undifferentiated cells with pluripotency and self-renewal ability are formed. That is, the embryonic stem cells in the inner cell mass added to the medium proliferate by spreading out around the inner cell mass, forming island-like colonies (cell masses). By selecting and isolating the colonies thus formed, feline-derived pluripotent stem cells can be obtained. In one embodiment, the stem cells of the present invention are embryonic stem cells.

[0039] Examples of reprogramming factors include Oct3 / 4, Sox2, Sox1, Sox3, Sox15, Sox17, Klf4, Klf2, c-Myc, N-Myc, L-Myc, Nanog, Lin28, Fbx15, ERas, ECAT15-2, Tcl1, beta-catenin, Lin28b, Sall1, Sall4, Esrrb, Nr5a2, Tbx3, Glis1, etc. These reprogramming factors may be used alone or in combination. Combinations of reprogramming factors may be appropriately selected with reference to known combinations in other mammals (e.g., Nature Protocols; Vol. 4 No. 12 2009, Current Protocols in Stem Cell Biology June 2009: https: / / doi.org / 10.1002 / 9780470151808.sc04a02s9, Whitworth et al. 2012 Stem Cells Dev., Ogorevc et al. 2016 Journal of Animal Science and Biotechnology, etc.). The method for introducing reprogramming factors into somatic cells is not particularly limited, and examples include methods using viral vectors, methods for introducing vectors using electroporation, and transfection. By culturing feline somatic cells introduced with reprogramming factors in the medium of the present invention, feline-derived pluripotent stem cells can be obtained. In one embodiment, the stem cells of the present invention are induced pluripotent stem cells (iPS cells).

[0040] Culture for colony formation of pluripotent stem cells is usually carried out under conditions of 37°C, 5% CO2, and 95% air, with formation beginning after 2 to 5 days. By culturing under these conditions for a few more days, colonies of pluripotent stem cells with an outer diameter of approximately 500 to 1000 μm are formed, and the stem cells of the present invention can be obtained by isolating these.

[0041] Whether the obtained stem cells maintain an undifferentiated state can be confirmed by colony formation, morphological characteristics of the colony-forming cells, or positive alkaline phosphatase staining. Alternatively, it can be determined by confirming the expression of at least one undifferentiation marker, such as OCT3 / 4, SOX2, NANOG, SSEA-1, SSEA-3, SSEA-4, Tra-1-60, or Tra-1-81. The undifferentiation markers may be measured by measuring either gene or protein expression. Known methods can be used as appropriate for measurement. Examples of such methods include quantitative reverse transcription PCR, Northern blotting, in situ hybridization, Western blotting, immunofluorescence staining, immunochemical staining, and flow cytometry analysis. From the perspective of establishing a stable stem cell line, it is preferable that the undifferentiated state be maintained after at least 15 or more passages, 16 or more passages, 17 or more passages, 18 or more passages, 19 or more passages, 20 or more passages, 25 or more passages, or 30 or more passages, or even after repeated freeze-thawing. By culturing cells obtained from the inner cell mass of feline blastocyst-stage embryos and / or feline somatic cells introduced with reprogramming factors in the medium of the present invention, feline-derived pluripotent stem cells that maintain their undifferentiated potential even after 20 or more passages can be obtained.

[0042] The tri-germ differentiation potential (pluripotency) of the obtained stem cells can be determined by confirming the expression of gene and / or protein markers specific to cells of lineages derived from ectoderm, mesoderm, or endoderm. Markers can be appropriately selected from known markers and are not particularly limited. Examples of ectodermal markers include NETSIN, PAX6, β-tubulin (TUBB3), SOX7, Otx-2, SOX1, MAP2, and TUJ1. Examples of mesodermal markers include CD44, αSMA, DESMIN, HAND1, MSX1, and BRACHYURY. Examples of endodermal markers include FOXA2, SOX17, CXCR4, HNF-3β / FoxA2, ​​GATA-4, and GATA-6. Expression of at least one marker for each of the three germ layers among the above markers indicates that the obtained stem cells have tri-germ differentiation potential. Marker measurement methods can be appropriately used. Examples of such methods include quantitative reverse transcription PCR, Northern blotting, in situ hybridization, Western blotting, immunofluorescence staining, immunochemical staining, and flow cytometry analysis. From the perspective of being applicable to regenerative medicine for a wide range of organs and basic research, it is preferable that pluripotent stem cells can differentiate into cells derived from any of the three germ layers. In one embodiment, it is preferable that pluripotent stem cells maintain their ability to differentiate into three germ layers even after long-term passage. By culturing cells obtained from the inner cell mass of feline blastocyst-stage embryos and / or feline somatic cells transfected with reprogramming factors in the medium of the present invention, it is possible to obtain feline-derived pluripotent stem cells that maintain their ability to differentiate into three germ layers even after long-term subculture of 15 or more passages.

[0043] The in vivo tri-germ differentiation potential (pluripotency) of the obtained stem cells can be determined, for example, by a teratoma formation assay in SCID mice or nude mice. From the perspective of clinical application, it is preferable that pluripotent stem cells can differentiate into cells derived from any of the three germ layers even when transplanted into a living body. By culturing cells obtained from the inner cell mass of feline blastocyst-stage embryos and / or feline somatic cells transfected with reprogramming factors in the medium of the present invention, feline-derived pluripotent stem cells that have tri-germ differentiation potential not only during in vitro culture but also when transplanted into a living body can be obtained. In one embodiment, it is preferable that the pluripotent stem cells maintain their in vivo tri-germ differentiation potential even after long-term passage. By culturing cells obtained from the inner cell mass of feline blastocyst-stage embryos and / or feline somatic cells transfected with reprogramming factors in the medium of the present invention, it is possible to obtain feline-derived pluripotent stem cells that maintain their in vivo tri-germ differentiation potential even after long-term subculture of 13 or more passages.

[0044] 3. Method for producing feline-derived pluripotent stem cells In one aspect, the present invention relates to a method for producing feline-derived pluripotent stem cells (sometimes referred to herein as the "production method of the present invention"), which comprises the step of culturing feline-derived pluripotent stem cells, cells obtained from the inner cell mass of a feline blastocyst-stage embryo, and / or feline somatic cells introduced with a reprogramming factor in the medium of the present invention. The process of culturing pluripotent stem cells and cells obtained from the inner cell mass of feline blastocyst-stage embryos and / or feline somatic cells introduced with reprogramming factors is the same as described above in "2. Feline-derived pluripotent stem cells."

[0045] Pluripotent stem cells can be cultured using the medium of the present invention according to standard methods. Representative passaging procedures and culture conditions are as follows: First, the grown pluripotent stem cell colonies are rinsed once or twice with PBS. A sufficient amount of cell detachment solution is then added to cover the cell layer and allowed to stand for several minutes. PBS or a serum-containing basal medium is added, and the cell clumps are separated by pipetting. Cells are then precipitated from this cell suspension, typically by centrifugation. After removing the supernatant, the precipitated cells are resuspended in medium, and a portion of this is seeded on a dish containing feeder cells or a coated dish and cultured at 37°C under 5% CO2. Alternatively, instead of detaching and separating the cells from the dish, the colonies can be cut into small pieces approximately 50 to 200 μm in diameter every 3 to 5 days using a sharp-tipped Pasteur pipette and mechanically passaged.

[0046] Examples of cell detachment solutions that can be used include solutions containing EDTA and solutions containing dispase as an enzyme. From the viewpoint of achieving a higher survival rate of pluripotent stem cells, a solution containing EDTA is preferred. Furthermore, from the viewpoint of simplifying subculture, a solution containing dispase as an enzyme is preferred. The concentrations of EDTA and dispase in the cell detachment solution can be determined according to known concentrations used in cell culture.

[0047] Examples of coating components for the coated dish include gelatin, vitronectin, laminin, etc. Among these, gelatin is preferred.

[0048] In one embodiment, the production method of the present invention further comprises a step of passage-culturing feline-derived pluripotent stem cells in the medium of the present invention for 20 or more generations. According to this production method of the present invention, feline-derived pluripotent stem cells that maintain an undifferentiated state can be obtained even after passage for 20 or more generations.

[0049] 4. Method for culturing feline-derived pluripotent stem cells In one aspect, the present invention relates to a culture method for maintaining the undifferentiated state of feline-derived pluripotent stem cells (sometimes referred to herein as the "culture method of the present invention"), which comprises a step of culturing feline-derived pluripotent cells in the medium of the present invention. The pluripotent stem cells and the culture of the pluripotent stem cells are the same as those described above in "3. Method for producing feline-derived pluripotent stem cells."

[0050] According to the culture method of the present invention, feline-derived pluripotent cells can be maintained in an undifferentiated state. [Example]

[0051] The present invention will be described in detail below based on examples, but the present invention is not limited to these examples.

[0052] Example 1. Generation of feline ES cells Ovaries removed by ovariectomy and epididymis removed by castration from cats were used. Cumulus-oocytes were extracted from the ovaries and matured in vitro. Sperm were collected from the epididymis and cryopreserved. After in vitro fertilization of the cumulus-oocytes and sperm, the fertilized eggs were cultured in 100 μl of IVC I medium for 2 days and then cultured in vitro in 100 μl of IVC II medium under conditions of 5% O2, 5% CO2, and 90% N2. After 6–8 days of culture, inner cell masses (ICMs) were mechanically isolated from blastocyst-stage or eclosed blastocyst-stage embryos using a syringe needle (31G, Dentronics) under a microscope. The isolated ICMs were individually seeded onto gelatin-coated tissue culture dishes previously seeded with inactivated mouse fetal fibroblasts as feeder cells. The cells were cultured in StemFit™ AK02N medium (Ajinomoto) supplemented with 10 μM ROCK inhibitor (Y-27632, Nacalai Tesque). After 2–10 days, sheet-like colonies with distinct margins were formed, similar to ES cell colonies of other species (Figure 1A(ii)). Colony-forming cells had a high nuclear-to-cytoplasmic ratio and distinct nucleoli. These colonies were cultured in StemFit™ medium without ROCK inhibitor and were dissected into small pieces using a sharp-tipped Pasteur pipette every 3–5 days for subculture. Even after repeated subculture and freeze-thawing, colonies with morphological characteristics similar to those of the primary colonies were formed (Fig. 1B). Three ES cell lines, NCH01, NCH02, and NCH03, each derived from a different blastocyst, were obtained.

[0053] Example 2 Verification of the undifferentiated state of feline ES cells 2-1. Alkaline phosphatase staining To confirm the undifferentiated state of the resulting ES cell lines, alkaline phosphatase (AP) staining was performed using the Stemgent™ Alkaline Phosphatase Staining Kit II (REPROCELL). All cell lines, NCH01, NCH02, and NCH03, were AP stained positive after 3 to 5 passages, confirming that they maintained their undifferentiated state (Figures 2i and iv). This characteristic was maintained even after 30 passages (Figures 2ii and v). Furthermore, AP staining remained positive even after repeated freeze-thawing (Figures 2iii and vi).

[0054] 2-2. Quantitative PCR Furthermore, total RNA was isolated from three feline ES cell lines after 19 passages using the FastGene RNA Premium Kit (Nippon Genetics) and reverse transcribed to complementary DNA using random primers and ReverTra Ace (Toyobo). The mRNA expression levels of undifferentiated marker genes (OCT3 / 4, SOX2, and NANOG) were confirmed by qPCR analysis using Taq Pro Universal SYBR qPCR Master Mix (Nanjing Vazyme Biotech) and the StepOnePlus Real-Time PCR System (Thermo Fisher Scientific) and normalized to β-Actin expression. The primer sequences used are listed in Table 1. All cell lines expressed undifferentiated marker genes at the mRNA level. Neither fetal fetal fibroblasts (FEFs) nor mouse fetal fibroblasts (MEFs), used as negative controls, expressed undifferentiated marker genes, demonstrating that the elevated expression levels of undifferentiated marker genes were not due to feeder cell contamination (Figure 3A).

[0055] [Table 1]

[0056] 2-3. Immunostaining Feline ES cells (passage 19) cultured on gelatin-coated 8-well glass slides (AGC Technoglass) were fixed with 4% paraformaldehyde (PFA) for 40 minutes. After permeabilization based on the target protein's localization, the cells were blocked with 10% FBS-supplemented PBS for 35 minutes at room temperature. Primary antibodies were then added and incubated for 16 hours at 4°C. After washing off excess primary antibodies, secondary antibodies were added and incubated for 1 hour at room temperature. The antibodies used are listed in Table 2. After washing the cells, DAPI solution (PBS, 0.2 μg / mL) was added to label DNA and incubated for 2 minutes at room temperature. The cells were then washed and mounted with ProLong Diamond Antifade Mountant (Thermo Fisher Scientific). Stained samples were observed under a confocal laser scanning microscope (FV3000; Olympus). Immunostaining also showed positivity for OCT3 / 4, SOX2, NANOG, and SSEA-4, and some cells were positive for Tra-1-60 (Fig. 3B).

[0057] 2-4. Flow cytometry Feline ES cells after 21 passages were dissociated by incubation with trypsin and then stained with the markers listed in Table 2. For cell surface markers, primary antibody staining was performed in PBS on ice for 30 minutes. If necessary, secondary antibody staining was also performed for 15 minutes. For intracellular proteins, cells were fixed with 4% paraformaldehyde (PFA) in PBS and stained. These cells were permeabilized with 0.1% Triton X-100 in 5% FBS in PBS for 45 minutes at 4°C. Cells were then stained with the primary antibody (Table 2) followed by the secondary antibody (Table 2). Stained cells were analyzed and sorted using an Aria II flow cytometer (BD Biosciences). Flow cytometry confirmed the expression of undifferentiated markers, consistent with the immunostaining results. Cells were positive for OCT3 / 4, SOX2, NANOG, and SSEA-4, and some cells were positive for Tra-1-60 (Figure 3C).

[0058] [Table 2]

[0059] Example 3. Evaluation of pluripotency of feline ES cells Feline ES cells after 21 passages were dissociated into single cells and seeded into Nunclon™ Sphera™ 96-well U-bottom microplates (Thermo Fisher Scientific). They were cultured for 10 days in FBS medium supplemented with 100 ng / ml Activin A (Nacalai Tesque) or without Activin A (Nacalai Tesque) to form multiple embryoid bodies (EBs). RNA extraction, reverse transcription, and qPCR analysis were performed on the resulting EBs as described in Example 2. The primers used are listed in Table 1. qPCR analysis revealed increased expression of germ layer-specific marker genes, including ectoderm (NETSIN, PAX6), endoderm (SOX17), and mesoderm (CD44, αSMA, DESMIN) markers, in these EBs compared to the ES cells from which they were derived (Figure 4A). Immunostaining of feline ES cells after 19 passages was performed in the same manner as in Example 2. The results confirmed the expression of ectoderm (β-tubulin (TUBB3), PAX6, NESTIN), mesoderm (αSMA), and endoderm (FOXA2, SOX17) markers in the EBs (Figure 4B).

[0060] Example 4. Evaluation of in vivo pluripotency (teratoma formation ability) of feline ES cells To evaluate the in vivo pluripotency of feline ES cells after 13 passages, 1 × 10 6 Feline ES cells were suspended in Matrigel diluted 1:1 with DMEM / Nutrient Mixture F-12 Ham and injected into the testes of NOD / SCID mice (n=2). Three months later, all feline ES cell lines formed tumors containing tissues derived from all three germ layers (Figure 5).

[0061] Example 5. Karyotype analysis Three feline ES cell lines were incubated for 2 hours in medium containing 0.04 μg / ml colcemid (Thermo Fisher Scientific), then trypsinized and incubated in 0.075 M KCl at 37°C for 20 minutes. The cells were fixed with a mixture of acetic acid and methanol (1:3). They were then incubated in a solution of quinacrine mustard for 10 minutes. After rinsing with distilled water, they were stained with Hoechst 33258 for 10 minutes. Analysis was performed using a confocal laser scanning microscope (LSM980; Carl Zeiss). All feline ES cell lines had a normal karyotype with 18 autosome pairs and either XX or XY chromosomes (Figure 6). These results indicate that the resulting feline ES cells lack aneuploid chromosomal abnormalities and have a low risk of tumorigenesis, suggesting their safe clinical application.

[0062] Example 6. Examination of medium composition Feline ES cells cultured in StemFit (solution A, solution B, and solution C containing bFGF) were cultured in either (1) StemFit solution A + solution B plus 100 ng / mL bFGF, (2) StemFit solution A + solution B, (3) StemFit (solution A + solution B + solution C) plus 10 nM bFGF inhibitor (PD173074, Selleck), or (4) NutriStem medium (REPROCELL) containing 4 ng / mL bFGF. Cells transferred to (1) StemFit solution A + solution B plus 100 ng / mL bFGF did not differentiate, and colonies persisted even after passaging. However, ES cells transferred to any of (2) to (4) media immediately showed morphological changes and were unable to maintain an undifferentiated state (Figure 7). These results suggest that a high concentration of FGF is required to maintain the undifferentiated state of feline ES cells.

Claims

1. A medium for producing and / or culturing feline-derived pluripotent stem cells, containing 25 ng / mL or more of fibroblast growth factor (FGF) or a substitute with FGF-like activity equivalent to or greater than 25 ng / mL of FGF.

2. 2. The medium according to claim 1, wherein the serum content is 1% or less.

3. The medium according to claim 1, for use in culturing in the presence of feeder cells.

4. The medium of claim 1 , wherein the pluripotent stem cells are embryonic stem cells.

5. A feline-derived pluripotent stem cell obtained by culturing cells obtained from the inner cell mass of a feline blastocyst-stage embryo and / or feline somatic cells introduced with a reprogramming factor in the medium described in any one of claims 1 to 4.

6. The feline-derived multipotent stem cell according to claim 5, which has the ability to differentiate into three germ layers.

7. The feline-derived pluripotent stem cell according to claim 5, which has the ability to differentiate into three germ layers in vivo.

8. Feline-derived multipotent stem cells with the ability to differentiate into three germ layers.

9. The feline-derived pluripotent stem cell according to claim 8, which has the ability to differentiate into three germ layers in vivo.

10. A method for producing feline-derived pluripotent stem cells, comprising the step of culturing feline-derived pluripotent stem cells, cells obtained from the inner cell mass of feline blastocyst-stage embryos, and / or feline somatic cells introduced with a reprogramming factor in the medium described in any one of claims 1 to 4.

11. The method of claim 10, further comprising the step of subculturing feline-derived pluripotent stem cells in the medium for 20 or more generations.

12. A culture method for maintaining feline-derived pluripotent stem cells in an undifferentiated state, comprising the step of culturing the feline-derived pluripotent stem cells in the medium according to any one of claims 1 to 4.

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