Cell culture supplement for producing stem cells, and method for producing stem cells

Abstract

The present invention provides a cell culture supplement for producing stem cells, and a method for producing stem cells.　The cell culture supplement comprises an aqueous solution of a polypeptide represented by formula (1) below.　(1): (Pro-Hyp-Gly)n (In this formula, Hyp represents hydroxyproline, and n represents an integer of 2-1000).

Description

Cell culture supplement for producing stem cells and method for producing stem cells

[0001] [Cross-reference to related applications] This application claims priority based on Japanese Patent Application No. 2021-091262, filed on May 31, 2021, the entire disclosure of which is incorporated herein by reference. The present invention relates to a cell culture supplement for producing stem cells, and a method for producing stem cells using the cell culture supplement.

[0002] Currently, two techniques are used to generate induced pluripotent stem cells from somatic cells. The first, developed by Yamanaka, involves the use of viral vectors to overexpress transcription factor proteins OCT4, SOX2, KLF4, and c-MYC in fibroblasts, resulting in reprogramming (Non-Patent Document 1). The second technique, first reported by Maekawa et al., is the direct reprogramming of somatic cells using chemical compounds (Non-Patent Documents 2, 3, and 4). While iPS cell technology has already been applied in the fields of regenerative medicine and drug discovery in diseased organ models, avoiding the risk of tumorigenesis due to immune rejection or genetic mutations remains a major challenge. Furthermore, reducing the time and cost required for reprogramming iPS cells into target organ cells is an important medical issue. Meanwhile, direct reprogramming using chemical compounds eliminates the risk of tumorigenesis due to genetic mutations and allows organ regeneration to be achieved using only the patient's own somatic cells and a cocktail of chemical compounds, making it promising for personalized regenerative medicine. However, at present, the organ cell types that can be produced using this technology are limited to nerve cells and fat cells, and the development of production methods remains a challenge.

[0003] In recent years, it has become clear that cancers originate from a very small number of cells called cancer stem cells, which form tumors as a heterogeneous population of cells with respect to proliferation and differentiation (Non-Patent Document 5). Similar to pluripotent stem cells present in healthy tissues, cancer stem cells possess the ability to self-renew and differentiate to produce diverse cell types. While many cancer cells can be removed during cancer treatment using anticancer drugs, radiation, cancer immunotherapy, and surgery, it is believed that a very small number of highly treatment-resistant cancer stem cells remain, leading to recurrence and metastasis. Therefore, the development of new drugs, treatments, and diagnostic techniques targeting cancer stem cells has become an important issue.

[0004] To develop new drugs, treatments, and diagnostic methods that target cancer stem cells, it is essential to produce large amounts of cancer stem cells stably as models. This is because the production of cancer stem cells is essential for applications in high-throughput screening, pharmacological testing, and safety testing. However, a major technical barrier has been the need to rely on methods to isolate the very small number of cancer stem cells detected from cancer patient samples.

[0005] Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 2006; 126: 663-676.Maekawa M, Yamaguchi K, Nakamura T, Shibukawa R, Kodanaka I, Ichsaka T, et al. Direct reprogramming of somatic cells is promoted by maternal transcription factor Glis1. Nature 2011; 474:225-229Yang Z, Ting Z, Hongkui D, et al. A XEN-like state bridges somatic cells to Pluripotency during chemical reprogramming. Cell 2015; 163:1678-1691.Takeda Y, Harada Y, Yoshikawa T, Dai P. Chemical compound-based direct reprogramming for future clinical application. Bioscience Reports 2018; 38: 1-19.Reya T, Morrison SJ, Clarke MF, et al. Stem cells, cancer, and cancer stem cells, Nature 2001;414:105-11.

[0006] Induced pluripotent stem (iPS) cells use viral vectors to strongly express transcription factor genes such as SOX2, OCT4, KLF4, and c-MYC in somatic cells such as fibroblasts, which carries the risk of exogenous genes being integrated into the chromosomes of iPS cells, resulting in tumor formation. Furthermore, it takes six months to a year to reprogram fibroblasts to cultivate a large number of iPS cells, and then induces their differentiation into target organ cells to cultivate the required amount for transplantation, which requires significant time and costs.

[0007] Direct reprogramming, which involves adding a cocktail of chemical compounds to the culture medium, is said to be able to directly generate pluripotent stem cells from somatic cells in a culture period of around 60 days. Furthermore, because small molecule compounds are used, there is no risk of foreign genes being integrated into chromosomes or tumor formation. Furthermore, the use of autologous fibroblasts derived from the patient avoids the risk of immune rejection, raising hopes for its practical application in personalized regenerative medicine. However, the organ cell types that can be generated using this technology are currently limited to nerve cells or fat cells, and it has not yet been put to practical use in the fields of medicine and drug discovery.

[0008] On the other hand, while it is known that the presence of cancer stem cells worsens the prognosis of radiation therapy and chemotherapy, a method for isolating these cells has not yet been established, and they have not yet been studied medically. Attempts have been made to extract a small number of cells from cancer patient specimens and then transplant them into immunodeficient NOD mice to create tumor masses (PDOX) using a cell sorter, or to culture them with the addition of multiple recombinant protein factors or hormones, such as bFGF, EGF, and PDGF, but no large-scale, stable method has yet been established. The use of cancer stem cells is essential for high-throughput screening, efficacy evaluation, and safety evaluation of drug candidates.

[0009] An object of the present invention is to provide a novel method for producing stem cells derived from somatic cells.

[0010] To solve the above problems, the inventors conducted extensive research. They investigated cell adhesion and proliferation using a polypeptide containing only repeats of the amino acid sequence Pro-Hyp-Gly, which is a constituent of type I collagen. Contrary to expectations, they found that, despite being part of the amino acid sequence of collagen, cells did not adhere to culture vessels coated with the polypeptide, but instead aggregated in a floating state to form spheroids. Furthermore, they found that using an aqueous solution of the polypeptide as a cell culture supplement could reprogram somatic cells, such as healthy human fibroblasts or mammary epithelial cells, to produce pluripotent stem cells with differentiation potential. They also found that cancer stem cells could be produced from common cultured cancer cell lines (cells lacking stem cell markers).

[0011] The present invention was completed based on these findings and includes the following broad aspects. [Item 1] A cell culture supplement comprising a polypeptide represented by the following formula (1): (Pro-Hyp-Gly)n (1) (wherein Hyp represents hydroxyproline, and n represents an integer of 2 to 1000). [Item 2] The cell culture supplement according to Item 1, which is used for producing stem cells. [Item 3] The cell culture supplement according to Item 2, wherein the stem cells are pluripotent cells or cancer stem cells. [Item 4] The cell culture supplement according to Item 1, which is used for spheroid formation. [Item 5] A spheroid formation agent comprising a polypeptide represented by the following formula (1): (Pro-Hyp-Gly)n (1) (wherein Hyp represents hydroxyproline, and n represents an integer of 2 to 1000). [Item 6] A cell culture substrate, the culture surface of which has been treated with the cell culture supplement according to any one of Items 1 to 4 or the spheroid formation agent according to Item 5. [Item 7] A cell culture system comprising the cell culture supplement according to any one of Items 1 to 4 or the spheroid forming agent according to Item 5, and cultured cells. [Item 8] A method for producing stem cells, comprising a step of culturing cells using the cell culture supplement according to any one of Items 1 to 4 or the spheroid forming agent according to Item 5. [Item 9] The method according to Item 8, wherein the stem cells express at least one stem cell undifferentiation marker gene selected from SOX2, OCT4, and NANOG. [Item 10] The method according to Item 8 or 9, wherein the stem cells are pluripotent stem cells or cancer stem cells. [Item 11] A method for producing tissue using stem cells obtained by the method according to any one of Items 8 to 10.

[0012] The present invention provides a cell culture supplement for producing stem cells. Furthermore, the novel cell culture supplement of the present invention provides a method for producing somatic cell-derived stem cells. By using the stem cell production method of the present invention, the polypeptides of the cell culture supplement exert receptor / ligand activity similar to scaffolding agents or adhesion factors necessary for cultured cells, and further act as signal transduction factors similar to cytokines, chemokines, hormones, etc., making it possible to produce stem cells simply and efficiently. This makes it possible to simply produce autologous stem cells from individual patients with diseases, eliminating the risk of tumor formation due to gene mutations like those in iPS cells, and realizing personalized regenerative medicine, cell therapy, and transplantation therapy that avoids immune rejection.

[0013] Furthermore, by using the cell culture supplement of the present invention, it becomes possible to produce cancer stem cells that express a cancer stem cell marker gene and an undifferentiated stem cell marker gene.

[0014] An example of using a cell culture supplement on a cell culture substrate (semipermeable membrane, hollow fiber). Semipermeable membrane, hollow fiber / microfluidic device. The cell culture supplement of the present invention is coated on the inside of a semipermeable membrane or hollow fiber, or added to the culture medium, and used as a microfluidic device. An example of using a cell culture supplement on a cell culture substrate (capsule). Capsules formed from the culture supplement itself or capsules made of a biodegradable substrate. The cell culture supplement of the present invention is used as a gel-like solid substrate, and cells are embedded therein to form spheroids. An example of using a cell culture supplement on a cell culture substrate (fibrous material, semipermeable membrane, hollow fiber). The cell culture supplement of the present invention is coated on the inner cavity of a biodegradable substrate, semipermeable membrane, hollow fiber, or fibrous material, or added to the culture medium, and spheroids are placed and cultured. An example of using a cell culture supplement on a cell culture substrate (microcarrier / film / embedded carrier based on a fibrous material). The cell culture supplement of the present invention is coated on the inside of a mesh-like, semipermeable, fibrous, or flexible film, and spheroids are cultured by enveloping, sandwiching, or wrapping them. Two-dimensional adherent culture and three-dimensional spheroid culture of healthy human neonatal fibroblasts. (A: Planar 2D culture (day 2 of culture). In a PS culture vessel (flat bottom) without cell culture additives, fibroblasts adhered to the bottom and proliferated. B: 3D culture (day 2 of culture). In a PS culture vessel (flat bottom) treated with cell culture additives, fibroblasts aggregated to form spheroids on day 2 of culture. C: 3D culture (day 20 of culture). In a PS culture vessel (flat bottom) treated with cell culture additives, fibroblasts formed spherical spheroids.) When healthy human neonatal fibroblasts were cultured in a culture vessel (polystyrene; PS / 6-well plate, flat bottom) coated with cell culture additives, the cells self-organized and aggregated over time, forming and growing spheroids from day 2 until day 20. Expression of stem cell undifferentiated marker genes in 3D spheroids of healthy human neonatal fibroblasts. Cell culture supplements were applied to culture vessels (PS / 6-well plate, flat bottom) and healthy human neonatal fibroblasts were cultured for 20 days to form spheroids. The mRNA expression levels of three representative stem cell undifferentiation marker genes (SOX2, OCT4, NANOG) increased, and characteristics of stem cell induction (reprogramming) were detected.3D spheroid culture of healthy human neonatal fibroblasts in a 96-well plate. (A: 96-well (round-bottom) culture (day 0). 40,000 fibroblasts were seeded in a 96-well PS culture vessel (round-bottom) treated with cell culture supplements. B: 96-well (round-bottom) culture (day 2). The seeded fibroblasts began to aggregate and form spheroids. C: 96-well (round-bottom) culture (day 20). A single spheroid formed from day 6 onwards, and remained in good condition even after day 20 of culture.) When human neonatal fibroblasts were cultured in a round-bottom culture vessel (PS / 96-well microplate) coated with cell culture supplements, the cells aggregated over time, forming spheroids and growing from day 2 to day 20. Time-dependent changes in the expression of stem cell undifferentiation marker genes in 3D spheroids of healthy human neonatal fibroblasts. When healthy human neonatal fibroblasts were cultured as spheroids in round-bottom culture vessels (PS / 96-well microplates) coated with cell culture supplements, the mRNA expression levels of three stem cell undifferentiation marker genes (SOX2, OCT4, and NANOG) increased over time and reached a peak on day 20 of culture. Direct reprogramming of somatic fibroblasts was demonstrated without gene transfer (the Yamanaka method for iPS cell generation). Differentiation of healthy human neonatal fibroblast-derived stem cells: Expression of three germ layer differentiation-related genes. When healthy human neonatal fibroblasts were cultured as spheroids in culture vessels coated with cell culture supplements, the expression levels of the mRNAs of differentiation marker genes for the three early germ layers (SOX17; endoderm, GATA6; mesoderm, and MAP2; ectoderm) increased over time and reached a peak on day 20. We found that stem cells could be induced to differentiate into the three germ layers at the initial stage of differentiation into various organs. Expression of stem cell undifferentiation marker proteins in healthy human fibroblast spheroids. Immunostaining using fluorescent dye-labeled antibodies was able to detect the stem cell marker proteins SOX2, OCT4, and NANOG, demonstrating the presence of stem cells among the spheroid-constituting cells. A: Fluorescence detected with NL557-labeled anti-SOX2 antibody. B: Fluorescence detected with NL637-labeled anti-OCT4 antibody. C: Fluorescence detected with NL493-labeled anti-NANOG antibody. NANOG protein-positive stem cells present within the spheroid.Spheroids cultured for 20 days were dispersed with Accutase enzyme reagent and immunostained with fluorescent dye-conjugated anti-NANOG antibody. Nuclear NANOG-positive stem cells were found to be induced in some of the spheroid-forming cells. A: Brightfield image of a portion of cells dispersed from a spheroid. B: NANOG antibody-positive cells (same cell population as A). NANOG protein-positive stem cells were detected in the cell nuclei. C: Nuclear staining with DAPI (same cell population as A). Nuclear DNA was stained blue to show nuclear shape. Two-dimensional and three-dimensional spheroid culture of adult dermal fibroblasts. Using adult dermal fibroblasts, which are minimally invasive and easily collected from the human body, we demonstrated the feasibility of spheroid culture using the cell culture supplement of the present invention. A: Planar two-dimensional culture. Adult dermal fibroblasts adhered to the bottom of a PS culture vessel and proliferated. B: Three-dimensional culture (day 20 of culture). Adult dermal fibroblasts formed spheroids in a 96-well culture vessel treated with cell culture supplement. Expression analysis of undifferentiated stem cell marker genes in 3D spheroid cultures of adult skin fibroblasts. Skin-derived fibroblasts are the least invasive and easiest to sample, essential for personalized regenerative therapy. We demonstrated that stem cells can be induced using the cell culture supplement spheroid culture method. A: SOX2. B: OCT4. C: NANOG. Expression analysis of three germ layer differentiation-related genes in 3D spheroid cultures of adult skin fibroblasts. Skin-derived fibroblasts are the least invasive and easiest to sample, essential for personalized regenerative therapy. We demonstrated that stem cells can be induced using the cell culture supplement spheroid culture method. A: SOX17. B: GATA6. C: HAND1. D: SOX1. 2D adherent culture and 3D spheroid culture of healthy adult human mammary epithelial cells. (A: 2D culture (day 2 of culture). In a PS culture vessel (flat bottom) without cell culture supplements, mammary epithelial cells adhered to the bottom and proliferated. B: 3D culture (day 2 of culture). In a PS culture vessel (flat bottom) treated with cell culture supplements, mammary epithelial cells formed spheroids. C: 3D culture (day 20 of culture). Mammary epithelial cell spheroids on day 20 of culture in a 96-well culture vessel (round bottom) treated with cell culture supplements.When healthy adult human mammary epithelial cells were cultured in a PS culture vessel (flat or round bottom) treated with cell culture supplements, the cells aggregated over time, forming spheroids and growing from day 2 to day 20. This demonstrated that this method is applicable to a wide range of cell types, including adult-derived and neonatal-derived cells, as well as epithelial cells and fibroblasts. Detection of stem cell undifferentiation marker genes in 3D spheroid cultures of healthy adult human mammary epithelial cells. When healthy adult human mammary epithelial cells were cultured in a culture vessel coated with cell culture supplements and allowed to form spheroids, the mRNA expression levels of three representative stem cell undifferentiation markers (SOX2, OCT4, and NANOG) increased, reaching a peak on day 20 of culture. Direct reprogramming of stem cells without gene transfer into somatic epithelial cells was demonstrated. Induction of differentiation of stem cells derived from healthy adult human mammary epithelial cells: Expression of three germ layer-related genes. When healthy human adult mammary epithelial cells were cultured for 20 days in culture vessels coated with cell culture supplements to form spheroids, the mRNA expression levels of the three early germ layer marker genes, SOX17 (endoderm) and MAP2 (ectoderm), reached their peaks on day 20. Meanwhile, FOXA2 (endoderm) expression decreased, and GATA4 (mesoderm) was undetectable. We found that stem cells can be induced to differentiate into the three germ layers at the early stage of differentiation into various organs. Two-dimensional adhesion and three-dimensional spheroid growth of cancer cells with and without cell culture supplements were investigated. When three cancer cell lines (A549 cells (human lung cancer), ES2 cells (human ovarian cancer), and 143B cells (human osteosarcoma)) were cultured in culture vessels (polystyrene; PS / 6-well flat-bottom plates) coated with cell culture supplements, the cells aggregated over time, formed spheroids, and grew by day 15. The relationship between cell culture supplement concentration and spheroid growth is shown. Figure 1 shows the results of spheroid formation in various microplates. Figure 2 shows the quantitative results of apoptosis induction in spheroids produced in U-shaped plates. Figure 3 shows the quantitative results of apoptosis induction in spheroids produced in V-shaped plates. Figure 4 shows the expression levels of cancer stem cell marker genes in A549 human lung cancer cells.When A549 human lung cancer cells were cultured as spheroids in a culture vessel coated with cell culture supplements, the expression levels of three cancer stem cell marker gene mRNAs (CD24, CD44, CD133) increased over time, reaching a peak on the 15th day of culture. We found that it was possible to create cells with the characteristics of lung cancer stem cells. Expression levels of cancer stem cell marker genes in ES2 human ovarian cancer cells. When ES2 human ovarian cancer cells were cultured as spheroids in a culture vessel coated with cell culture supplements, the expression levels of three cancer stem cell marker gene mRNAs (CD24, CD44, CD133) increased over time, reaching a peak on the 15th day of culture. We found that it was possible to create cells with the characteristics of ovarian cancer stem cells. Expression levels of cancer stem cell marker genes in 143B human osteosarcoma cells. When 143B human osteosarcoma cells were cultured as spheroids in culture vessels coated with cell culture supplements, the expression of two of three cancer stem cell marker gene mRNAs (CD44 and CD133) increased over time, reaching a peak on day 15 of culture. CD24 did not increase significantly. We found that it was possible to generate cells with the characteristics of osteosarcoma stem cells. Gene detection in 3D spheroids of A549 human lung cancer cells. When A549 human lung cancer cells were cultured as spheroids in culture vessels coated with cell culture supplements, the mRNA expression levels of three representative stem cell undifferentiation marker genes (SOX2, OCT4, and NANOG) increased over time, reaching a peak on day 15 of culture. Gene detection in 3D spheroids of ES2 human ovarian cancer cells. When ES2 human ovarian cancer cells were cultured as spheroids in a culture vessel coated with cell culture additives, the mRNA expression levels of three undifferentiated stem cell marker genes (SOX2, OCT4, and NANOG) increased over time, reaching a peak on day 15 of culture. Detection of stem cell undifferentiation marker genes in 3D spheroids of 143B human osteosarcoma cells. When 143B human osteosarcoma cells were cultured as spheroids in a culture vessel coated with cell culture additives, the mRNA expression levels of three undifferentiated stem cell marker genes (SOX2, OCT4, and NANOG) increased from day 4 to day 15 of culture.

[0015] As used herein, the singular forms (a, an, the, etc.) are intended to include both the singular and the plural unless otherwise expressly stated herein or clearly contradicted by context.

[0016] <Cell Culture Supplement> The cell culture supplement of the present invention contains a polypeptide represented by the following formula (1): (Pro-Hyp-Gly) n (1) (In the formula, Hyp represents hydroxyproline, and n represents an integer of 2 to 1000.) n represents an integer of 2 to 1000. n is preferably an integer of 10 to 1000, more preferably an integer of 500 to 1000.

[0017] The cell culture supplement of the present invention can be used in producing stem cells. By using the cell culture supplement of the present invention in cell culture, cultured cells form spheroids, allowing stem cells to be produced simply and efficiently.

[0018] As used herein, the term "spheroid" (also referred to as "cell mass") encompasses a plurality of cells that have gathered three-dimensionally and adhered to one another.

[0019] The polypeptide represented by formula (1) can be produced by a known production method. For example, (Pro-Hyp-Gly) ｍ (m is an integer of, for example, about 1 to 10, 1 to 5, 10 to 1000, or 500 to 1000) by condensation reaction.

[0020] The cell culture supplement of the present invention preferably contains an aqueous solution of the polypeptide represented by formula (1). The aqueous solution preferably contains about 0.001 to 0.5% by weight (w / v) of the polypeptide represented by formula (1), and more preferably about 0.01 to 0.25% by weight (w / v). The cell culture supplement of the present invention can be, but is not limited to, an aqueous solution that is substantially free of components other than the polypeptide represented by formula (1).

[0021] As the aqueous solution of the polypeptide represented by the formula (1), a commercially available product such as "PURECOLLA" (manufactured by JNC Corporation) can be used.

[0022] <Use as a Surface Treatment Agent or Additive> In one embodiment, the cell culture supplement of the present invention can be used to treat the surface of the culture surface of a cell culture substrate.

[0023] Such cell culture substrates are not particularly limited, and examples include dishes, petri dishes, microwell plates (flat-bottomed, U-bottomed, V-bottomed, etc.), tubes, films, fibrous materials, semipermeable membranes, hollow membranes, hollow fibers, capsules, microcarriers, etc. Cell culture substrates that can be used include those made from materials such as glass, metal, plastics such as polystyrene (PS), polypropylene (PP), polycarbonite (PC), polyethylene terephthalate (PET), polytetrafluoroethylene (PTFE), polyvinylidene fluoride (PVDF), cycloolefin (COP), acrylic / methacrylic (PMMA), hydrophilic fluorinated resins, polylactic acid (PLA), polybutylene succinate (PBS), and polyhydroxybutyrate / hydroxyhexanoate (PHBH), cellulose membranes, carbon nanotubes, and cellulose nanofibers.

[0024] Examples of the use of cell culture supplements in cell culture substrates are shown in Figures 1 to 4, but are not limited to these.

[0025] Figure 1 shows examples of application to semipermeable membranes and hollow fibers. As shown in Figure 1, the cell culture supplement of the present invention can be used as a microfluidic device by coating the inside of a semipermeable membrane or hollow fiber or adding it to the culture medium. Such microfluidic devices are expected to be applied in fields such as (i) reproducing organ functions such as the kidney and liver, (ii) extracorporeal circulation organ (carrier-type) medical devices for the kidney and liver, (iii) production of recombinant proteins such as monoclonal antibodies, and (iv) drug discovery models / pharmacological evaluation, toxicity evaluation, etc.

[0026] Figure 2 shows an example of application to capsules. As shown in Figure 2, the cell culture supplement of the present invention can be used as a gel-like solid substrate, and cells can be embedded therein to form spheroids. Such capsules are expected to be applied in fields such as (i) organs for regenerative medicine: carriers for transplanting stem cells or differentiation-induced organoids into the body, (ii) transfusion of hematopoietic stem cells, and (iii) production of artificial muscle.

[0027] Figure 3 shows examples of application to fibrous materials, semipermeable membranes, and hollow fibers. As shown in Figure 3, the cell culture supplement of the present invention can be coated on the lumen of a biodegradable substrate, semipermeable membrane, hollow fiber, or fibrous material, or added to a culture medium, and spheroids can be cultured therein. Such fibrous materials, semipermeable membranes, and hollow fibers are expected to be applied in fields such as (i) organ transplantation and regeneration for regenerative medicine: transplantation and regeneration of thin organs such as blood vessels, nerves, intestines, and renal tubules; and (ii) sensor circuits for detecting neural electrical potential, light, odor components, taste components, etc.

[0028] Figure 4 shows an example of application to microcarriers, etc. As shown in Figure 4, the cell culture supplement of the present invention can be coated on the inside of a mesh-like, semipermeable, fibrous, or flexible film, and spheroids can be cultured in a state where they are entrapped, sandwiched, or wrapped around the film. Such macrocarriers are expected to be applied in fields such as transplantation and regeneration of pancreas, kidney, liver, bone marrow, bone, cartilage, muscle, skin, and hair for regenerative medicine.

[0029] When the culture surface of a cell culture substrate is treated with the cell culture supplement of the present invention, it is not necessary to use a culture vessel (e.g., a dish, flask, microfluidic device, cell stack chamber, bag, etc.) with a hydrophilically treated, ultra-low adhesion surface as the cell culture substrate. By using the cell culture supplement of the present invention, spheroids can be easily formed and pluripotent stem cells and cancer stem cells can be produced without using a culture vessel with an ultra-low adhesion surface.

[0030] The culture surface of the cell culture substrate refers to the surface that comes into contact with cells during culture. Part or all of the culture surface can be surface-treated. Surfaces other than the culture surface can also be surface-treated.

[0031] The surface treatment is not particularly limited as long as it is a method capable of immobilizing (e.g., coating) the polypeptide represented by formula (1), which is the active ingredient of the cell culture supplement of the present invention, on the culture surface of the cell culture substrate. Examples include application, immersion, and spraying. When using a cell culture substrate made of plastic, it is preferable to optionally perform a surface treatment such as plasma treatment before surface treatment with the cell culture supplement of the present invention. Furthermore, when using the cell culture supplement of the present invention for surface treatment, known additives may be mixed and used.

[0032] When used for surface treatment of the culture surface of a cell culture substrate, the concentration of the polypeptide represented by formula (1), which is an active ingredient of the cell culture auxiliary of the present invention, is not particularly limited and can be, for example, 0.001 to 0.5% by weight (w / v), preferably 0.01 to 0.25% by weight (w / v).

[0033] When the cell culture substrate is surface-treated with the cell culture supplement of the present invention, the surface can be dried to remove water after the surface treatment. The drying method is not particularly limited, and can be, for example, drying under reduced pressure or using a desiccant.

[0034] Furthermore, after the drying, the cell culture substrate can be sterilized by any means, including, for example, ethylene oxide gas, electron beams, gamma rays, autoclaves, etc.

[0035] In another embodiment, the cell culture supplement of the present invention can be used by adding it to a culture medium.

[0036] The culture media to which the cell culture supplements of the present invention are added can be a wide variety of liquid media, gel-like media, and other media commonly used in conventional cell culture. The media may also contain animal-derived components such as serum / plasma additives (e.g., FBS and FCS) and protein additives (e.g., albumin). Examples of such media include αMEM medium, Neurobasal medium, Neural Progenitor Basal medium, NS-A medium, BME medium, BGJb medium, CMRL 1066 medium, Minimum Essential Medium (MEM), GMEM medium, Eagle MEM medium, Dulbecco's Modified Eagle Medium (DMEM), Glasgow MEM medium, Improved MEM Zinc Option medium, IMDM medium, Medium 199 medium, DMEM / F12 medium, StemPro-34SFM medium, Ham's medium, RPMI 1640 medium, HTF medium, and Fischer's medium. Further, examples of xeno-free or serum-free media include mTeSR medium, Stem Fit medium, Cellartis DEF-CS500 medium, Cellartis MSC-CS500 medium, NutriStem medium, PluriStem Human ES / iPS medium, Fibroblast Growth Medium medium, and Mammary Epithelial Cell Growth medium. Examples of gelatinous media include, but are not limited to, soft agar medium, Matrigel medium, and methylcellulose-based medium. Furthermore, the medium may contain other known additives.

[0037] The amount of the cell culture supplement of the present invention to be added to a culture medium is not particularly limited, and the final concentration of the polypeptide represented by formula (1), which is an active ingredient of the cell culture supplement of the present invention, can be, for example, 0.001 to 0.5% by weight (w / v), preferably 0.01 to 0.25% by weight (w / v).

[0038] Furthermore, a cell culture substrate surface-treated with the cell culture supplement of the present invention and a culture medium containing the cell culture supplement of the present invention can be combined and used for cell culture.

[0039] <Spheroid-forming agent> In another embodiment, the cell culture supplement of the present invention, which contains the polypeptide represented by formula (1), can be used as a spheroid-forming agent.

[0040] The method for forming spheroids can be similar to a conventional method for forming spheroids, except that cell culture is performed using a cell culture instrument having the cell culture supplement of the present invention on its culture surface, or cell culture is performed in a culture medium to which the cell culture supplement of the present invention has been added.

[0041] Common methods for forming spheroids include non-adhesive surface cell culture, hanging drop cell culture, micromolding techniques, and rotary cell culture.

[0042] The cells used for spheroid formation are not particularly limited, and examples include primary cultures of somatic cells derived from various tissues, such as skin-derived fibroblasts, epithelial cells derived from various organs, and somatic cells derived from various tissues, such as the lung, liver, pancreas, kidney, intestine, cartilage, bone, central nervous system, and peripheral nervous system. However, when these primary cultured cells are cultured on a plate, they often lose their inherent functions and undergo abnormal activation, leading to the problem of inducing apoptosis (programmed cell death) known as anoikis. In this context, the formation of spheroids from primary cultured cells using the cell culture supplement of the present invention has been shown to suppress apoptosis, support survival and proliferation, and maintain physiological functions. Therefore, compared with cells cultured on a plate, spheroid-formed cells are expected to maintain higher biological functions and provide models that reflect biological functions. Meanwhile, spheroid formation using established cancer cell lines is also useful. Forming cancer cells into spheroids can reproduce the "malignant tumor functions" present in the patient's body and maintain the activity of enzymes and pump receptors that confer drug resistance to anticancer drugs. For this reason, cancer cell spheroids are expected to be used in drug discovery research as a cancer patient model that can reproduce the pathological condition. Furthermore, when inducing differentiation of iPS cells, a method is often used in which spheroids are first cultured to create embryoid bodies, which are then differentiated into various organs. In this case, it is known that spheroid formation promotes differentiation into the three germ layers (endoderm, mesoderm, and ectoderm), and accelerating the differentiation induction process into organ cells is a major benefit.

[0043] The medium for spheroid formation may be serum-containing or serum-free. Examples of serum-containing media include, but are not limited to, αMEM medium, Neurobasal medium, Neural Progenitor Basal medium, NS-A medium, BME medium, BGJb medium, CMRL 1066 medium, Minimum Essential Medium (MEM), GMEM medium, Eagle MEM medium, Dulbecco's Modified Eagle Medium (DMEM), Glasgow MEM medium, Improved MEM Zinc Option medium, IMDM medium, Medium 199 medium, DMEM / F12 medium, StemPro-34SFM medium, Ham's medium, RPMI 1640 medium, HTF medium, Fischer's medium, and mixtures thereof. The concentration of serum (e.g., fetal bovine serum (FBS), human serum, etc.) added is typically 2-20%, with 5-10% being preferred.

[0044] Serum-free medium (SFM) refers to a medium that does not contain animal-derived components (xeno-free) or raw or untreated serum (serum-free). Examples of SFM include media containing recombinantly produced growth factors (basic FGF, EGF, insulin, etc.). SFM may also contain any serum substitute. Examples of serum substitutes include KnockOut Serum Replacement, albumin (e.g., lipid-rich albumin, recombinant albumin, plant starch, dextran, and protein hydrolysates), transferrin (or other iron transporters), fatty acids, insulin, collagen precursors, trace elements, 2-mercaptoethanol, 3'-thioglycerol, or equivalents thereof. These may be used alone or in combination.

[0045] The medium may contain other known additives. The additives are not particularly limited, and examples thereof include growth factors (e.g., insulin, growth hormone, EGF, FGF, TGF, BMP, Activin, Wnt, LIF, Noggin, etc.), valproic acid, forskolin, retinoic acid, various inhibitors / activators (e.g., A83-01, DZNep, Y27632, SB202190, PD0325901, CHIR99021), polyamines, minerals, sugars (e.g., glucose, etc.), organic acids (e.g., pyruvic acid, lactic acid, etc.), amino acids (e.g., non-essential amino acids (NEAA), L-glutamine, etc.), reducing agents (e.g., 2-mercaptoethanol, etc.), vitamins (e.g., ascorbic acid, d-biotin, etc.), steroids, antibiotics (e.g., streptomycin, penicillin, etc.), surfactants (e.g., Tween-20, Nonidet P-40), buffers (e.g., HEPES, etc.), and nutritional additives (e.g., B27 Examples of additives include: GlutaMax, StemPro-Nutrient Supplement, KnockOut Media, and GlutaMax. These may be used alone or in combination of two or more. Each additive is preferably contained within a known concentration range.

[0046] The culture conditions for spheroid formation are not particularly limited as long as they allow the formation of the desired spheroids. For example, the culture temperature can be 20 to 40°C, preferably 34 to 38°C. Furthermore, the culture can be carried out in a 3 to 10% CO2 atmosphere, preferably 4 to 6% CO2 atmosphere. The culture period for spheroid formation is, for example, about 0.5 hours to 14 days, preferably about 1 to 4 days. Optionally, the medium can be changed one or more times during the culture period.

[0047] The size of the spheroids is usually about 10 to 500 μm in diameter, preferably about 50 to 200 μm. Because nutrient penetration into the interior of spheroids becomes difficult when they become too large, it is desirable to separate the spheroids once they reach an appropriate size. The obtained spheroids can be disaggregated by enzymatic treatment with trypsin and / or collagenase, and then cultured again using the cell culture supplement of the present invention, allowing for repeated spheroid formation.

[0048] The cell concentration in the culture is not particularly limited as long as it allows efficient formation of spheroids. For example, the cell concentration is about 1 × 10 3 ~Approx. 1×10 7 cells / well, preferably about 3 x 10 3 ~Approx. 5×10 6 Examples include:

[0049] In the method for forming spheroids using the cell culture supplement of the present invention, it is believed that the polypeptide of the cell culture supplement of the present invention exerts receptor / ligand activity similar to that of a scaffolding agent or adhesion factor necessary for cultured cells, and further acts as a signal transduction factor similar to cytokines, chemokines, hormones, etc., and promotes the self-organization of cells, thereby promoting the formation of spheroids.

[0050] In another aspect of the present invention, there is provided a cell culture system comprising the cell culture supplement of the present invention and cultured cells. The cultured cells are generally those listed above as cells used for spheroid formation. When producing pluripotent stem cells or cancer stem cells using the culture system, somatic cells or cancer cells, respectively, are preferably used.

[0051] In another aspect, the present invention provides a method for producing stem cells, which comprises the step of culturing cells using the cell culture supplement of the present invention.

[0052] In the present invention, "stem cells" refer to cells that express stem cell undifferentiation marker genes, and examples thereof include pluripotent stem cells, cancer stem cells, somatic stem cells, and mesenchymal stem cells. Among these, the cell culture supplement of the present invention is preferably used for producing pluripotent stem cells and cancer stem cells. The methods for producing pluripotent stem cells and cancer stem cells are described in detail below.

[0053] <Method for producing pluripotent stem cells> In another aspect of the present invention, there is provided a method for producing pluripotent stem cells, which comprises culturing somatic cells using the cell culture supplement of the present invention. Culturing somatic cells using the cell culture supplement of the present invention causes the cultured cells to form spheroids, allowing pluripotent stem cells to be produced simply and efficiently.

[0054] Examples of methods for using the cell culture supplement of the present invention when producing pluripotent stem cells include, but are not limited to, using the cell culture supplement of the present invention to surface treat the culture surface of a cell culture substrate and / or adding the cell culture supplement of the present invention to a culture medium. Preferably, somatic cells are cultured using a cell culture substrate whose culture surface has been surface-treated with the cell culture supplement of the present invention. The use of the cell culture supplement for surface treatment of the culture surface of a cell culture substrate and its addition to a culture medium is the same as described in the section <Use as a surface treatment agent, additive>.

[0055] The somatic cells used in the production of the pluripotent stem cells of the present invention are not particularly limited as long as they are derived from mammals (human, mouse, rat, cow, pig, monkey, sheep, rabbit, dog, etc.), and a wide variety of cells can be used. Among these, humans, monkeys, and mice are preferred, with humans being particularly preferred. The type of somatic cells is also not particularly limited, and a wide variety of cells can be used, including fibroblasts, epithelial cells, chondrocytes, muscle cells, bone cells, hepatocytes, pancreatic cells, intestinal cells, nerve cells, bone marrow cells, and skin cells. Somatic cells derived from both healthy individuals and patients with diseases can be used in the production of the pluripotent stem cells of the present invention.

[0056] As the medium used for producing pluripotent stem cells, a wide range of media can be used, including those listed in the section <Spheroid-forming agent>.

[0057] Furthermore, in the method of producing pluripotent stem cells of the present invention, it is not necessary to add various differentiation and proliferation supplements such as cell growth factors (e.g., PDGF, TGF-β, Activin, BMP, Noggin, LIF, R-Spondin, Wnt-3a, EGF, TPO, SCF, Neuregulin), mesenchymal stem cell maintenance medium, retinoic acid, etc. By using the cell culture supplements of the present invention, it is possible to produce pluripotent stem cells from somatic cells without adding cell growth factors to the medium.

[0058] Pluripotent stem cells can be produced using conventional methods for forming spheroids, except that the cells are cultured using a cell culture substrate having the cell culture supplement of the present invention on its culture surface. Examples include non-adhesive surface cell culture, hanging drop cell culture, micromolding techniques, and rotary cell culture, with non-adhesive surface cell culture being particularly preferred.

[0059] The culture conditions for producing pluripotent stem cells are not particularly limited, but for example, the culture temperature can be 20 to 40°C, preferably 34 to 38°C. Furthermore, the culture can be carried out in an atmosphere of 3 to 10% CO2, preferably 4 to 6% CO2. The culture period is, for example, about 10 to 40 days, preferably about 15 to 30 days. Optionally, the culture medium can be replaced one or more times during the culture period. Optionally, the cell culture substrate can be replaced one or more times during the culture period.

[0060] The size of the spheroids used in the production of pluripotent stem cells is typically about 10 to 500 μm in diameter, preferably about 50 to 200 μm. Because spheroids become difficult to penetrate with nutrients if they become too large, it is desirable to separate the spheroids once they reach an appropriate size. The resulting spheroids can be disaggregated by enzymatic treatment with trypsin and / or collagenase, and then cultured again using the cell culture supplement of the present invention to re-form spheroids that function as pluripotent stem cells.

[0061] The concentration of somatic cells in the culture is not particularly limited as long as it allows efficient production of pluripotent stem cells, but for example, it is about 1 × 10 3 ~Approx. 1×10 7 cells / well, preferably about 3 x 10 3 ~Approx. 5×10 6 Examples include:

[0062] In the method of producing pluripotent stem cells of the present invention, the production of pluripotent stem cells can be confirmed by confirming the expression of stem cell undifferentiation markers, such as OCT4, SOX1, SOX2, SOX3, SOX18, NANOG, Klf2, Klf4, Essrb, Sall4, Tcl1, Tcl3, c-Myc, c-Mycn, LIN28, SSEA-1, SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, AP, Fzd1-10, and TDGF-1.

[0063] Furthermore, the differentiation-inducing ability of pluripotent stem cells obtained by the present invention can be confirmed by examining the expression of genes associated with the three germ layers (endoderm, mesoderm, and ectoderm), such as SOX17, FOXA2, Nestin, Flk1 / KDRAFP, Cripto, Bmp4 / Wnt3, GATA4, GATA6, Brachyury, HAND1, CXCR4, SOX1, Otx-2, Pax2, Pax6, and MAP2.

[0064] Pluripotent stem cells obtained by the production method of the present invention may be a heterogeneous population of cells comprising various differentiation states, such as ES cells, neural stem cells, mesenchymal stem cells, cell fate conversion by transcription factors (iPS cells), cells with phenotypes of stepwise embryonic differentiation such as extraembryonic endoderm cells (XEN-like cells) induced by a chemical cocktail (various cytokines, hormones, agonists, inhibitors, etc.), and cells with characteristics of embryoid bodies obtained by suspension culture of iPS cells.

[0065] The pluripotent stem cell production method of the present invention allows for the production of pluripotent stem cells in a very short period (approximately 10 to 40 days), compared to the six months to a year required for the culture of induced pluripotent stem cells (iPS cells). Furthermore, unlike induced pluripotent stem cells (iPS cells), the lack of exogenous gene integration eliminates the risk of tumor formation upon transplantation. Furthermore, autologous pluripotent stem cells can be easily generated using somatic cells such as fibroblasts, which are easily harvested from human skin tissue, thereby avoiding the risk of immune rejection. Therefore, considering the potential for transplantation therapy, the classification of iPS cells under the Regenerative Medicine Safety Act in Japan may be classified as Type 2 regenerative medicine (medium risk), rather than Type 1 regenerative medicine (high risk).

[0066] Furthermore, the method for producing pluripotent stem cells of the present invention allows for the production of pluripotent stem cells in a shorter time than the production time (approximately 60 days) required by direct reprogramming. Furthermore, because the method for producing pluripotent stem cells of the present invention is a direct reprogramming method using spheroid formation, it has a wider range of applications than direct reprogramming methods using compound cocktails in terms of the selection of tissue materials used for establishment, expansion culture methods, etc.

[0067] The uses of the pluripotent stem cells obtained by the production method of the present invention are not particularly limited, and they can be used for various tests and research, disease treatment, and the like.

[0068] <Method for producing cancer stem cells> In another aspect of the present invention, there is provided a method for producing cancer stem cells, which comprises culturing cancer cells using the cell culture supplement of the present invention. By culturing cancer cells using the cell culture supplement of the present invention, the cells form spheroids, allowing cancer stem cells to be produced simply and efficiently.

[0069] Methods for using the cell culture supplement of the present invention when producing cancer stem cells include, but are not limited to, using the cell culture supplement of the present invention to surface treat the culture surface of a cell culture substrate and / or adding the cell culture supplement of the present invention to a culture medium. Preferably, cancer cells are cultured using a cell culture substrate whose culture surface has been surface-treated with the cell culture supplement of the present invention. The use of the cell culture supplement to surface treat the culture surface of a cell culture substrate and its addition to a culture medium is the same as described in the section <Use as a surface treatment agent, additive>.

[0070] The cancer cells used in the production of cancer stem cells of the present invention are not particularly limited as long as they are derived from mammals (humans, mice, rats, cattle, pigs, monkeys, sheep, rabbits, dogs, etc.), and a wide variety of cancers can be used. Among these, humans, monkeys, and mice are preferred, with humans being particularly preferred. The type of cancer is also not particularly limited, and a wide variety of cancers can be used, including colon cancer, breast cancer, ovarian cancer, cervical cancer, endometrial cancer, prostate cancer, gastric cancer, liver cancer, esophageal cancer, pancreatic cancer, bladder cancer, bile duct cancer, laryngeal cancer, melanoma, lung cancer, chronic lymphocytic leukemia, chronic myeloid leukemia, thyroid cancer, multiple myeloma, and sarcoma. Furthermore, a wide variety of cancers can be used, including established cell lines, primary cultured cells derived from patient specimens, patient specimen-derived, subcultureable cells (PDCC), cancer cells derived from animal transplanted cancer tissues (PDOX), and cancer cells derived from experimental animals.

[0071] As the medium used for producing pluripotent stem cells, a wide range of media can be used, including those listed in the section on "Spheroid-forming agents." Among these, DMEM / F12 medium is preferred.

[0072] Furthermore, in the method of producing cancer stem cells of the present invention, it is not necessary to add various differentiation and proliferation supplements such as cell growth factors (e.g., PDGF, TGF-β, Activin, BMP, Noggin, LIF, R-Spondin, Wnt-3a, EGF, TPO, SCF, Neuregulin), mesenchymal stem cell maintenance medium, retinoic acid, etc. By using the cell culture supplement of the present invention, it is possible to produce cancer stem cells from cancer cells without adding cell growth factors to the medium.

[0073] Cancer stem cells can be produced using conventional methods for forming spheroids, except that the cells are cultured using a cell culture substrate having the cell culture supplement of the present invention on its culture surface. Examples include non-adhesive surface cell culture, hanging drop cell culture, micromolding techniques, and rotary cell culture, with non-adhesive surface cell culture being particularly preferred.

[0074] The culture conditions for producing cancer stem cells are not particularly limited; for example, the culture temperature can be 20-40°C, preferably 34-38°C. Culture can be performed in a 3-10% CO2 atmosphere, preferably 4-6% CO2 atmosphere. The culture period is, for example, about 10-40 days, preferably about 15-30 days. Optionally, the culture medium can be replaced one or more times during the culture period. Optionally, the cell culture substrate can be replaced one or more times during the culture period.

[0075] The size of spheroids used in the production of cancer stem cells is typically about 10 to 500 μm in diameter, preferably about 50 to 200 μm. Because spheroids become difficult to penetrate with nutrients if they grow too large, it is desirable to separate the spheroids once they reach an appropriate size. The resulting spheroids can be disaggregated and then cultured again using the cell culture supplement of the present invention to re-form spheroids that function as cancer stem cells.

[0076] The concentration of cancer cells in the culture is not particularly limited as long as it allows efficient production of cancer stem cells, but for example, it is about 1 × 10 3 ~Approx. 1×10 7 cells / well, preferably about 3 x 10 3 ~Approx. 5×10 6 Examples include:

[0077] In the method of producing cancer stem cells of the present invention, the success of producing cancer stem cells can be confirmed by confirming the expression of cancer cell surface markers, such as CD133, CD44, CD24, ABCB5, EAS, CD34, CD38, CD90, CD117, and CXCR4.

[0078] Cancer stem cells obtained by the production method of the present invention can also be confirmed for the expression of stem cell undifferentiation markers, such as OCT4, SOX1, SOX2, SOX3, SOX18, NANOG, Klf2, Klf4, Essrb, Sall4, Tcl1, Tcl3, c-MYC, c-MYCn, LIN28, SSEA-1, SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, AP, Fzd1-10, and TDGF-1.

[0079] Cancer stem cells obtained by the production method of the present invention may be a heterogeneous population of cancer stem cells containing various cancer stem cells, or may further contain cancer cells.

[0080] The uses of cancer stem cells obtained by the production method of the present invention are not particularly limited, and they can be used in various tests and research, disease treatment, and the like.

[0081] <Induction of Differentiation of Stem Cells> By inducing differentiation of the pluripotent stem cells obtained by the production method of the present invention using known methods, they can be differentiated into cells of endodermal organs (e.g., liver, pancreas, intestinal tract, lung, thyroid, parathyroid, urinary tract, adipose tissue, cartilage tissue, etc.), mesodermal organs (e.g., kidney, ureter, heart, blood, gonads, adrenal cortex, muscle, skeleton, dermis, connective tissue, mesothelium, etc.), and ectodermal organs (e.g., brain, spinal cord, adrenal medulla, epidermis, hair, nails, skin glands, sensory organs, peripheral nerves, lens, etc.). Furthermore, organoids and tissues of the above organs can also be produced using known methods.

[0082] Differentiation induction can be appropriately performed using a differentiation induction method into any cell type, with reference to differentiation treatment methods reported for pluripotent stem cells such as ES cells and iPS cells. For example, differentiation induction into hematopoietic cells can be performed according to the method described in the literature (Niwa A, Heike T, Saito K, et al. A Nobel serum-free monolayer culture for orderly hematopoietic differentiation of human pluripotent cells via mesodermal progenitors. PLos One 2011; Issue 7, Vol. 6,).

[0083] The organoids and tissues obtained by the methods of the present invention can be used to evaluate the efficacy / toxicity of test substances, to elucidate their mechanisms of action, or to analyze the mechanisms of biological phenomena.

[0084] The present invention will be further explained below with reference to examples, but the present invention is not limited thereto.

[0085] Example 1: Spheroid culture of healthy human neonatal fibroblasts and expression of stem cell undifferentiation marker genes (Method) 2 mL of an aqueous solution (0.125%) of the polypeptide represented by formula (1) (cell culture aid) was added to a commercially available 6-well flat-bottom polystyrene culture vessel (CellBIND, CORNING) and allowed to stand at 4°C for 24 hours.

[0086] Afterwards, excess cell culture supplements were removed, and the cells were dried in a dryer at 37°C for 96 hours to complete the cell culture supplement coating process. Healthy human neonatal fibroblasts (ATCC PCS-201-010, Primary Dermal Fibroblasts, Neonatal, hereafter referred to as "fibroblasts") were maintained and cultured in serum-free medium (Promo Cell; Fibroblast Basal Medium, basic-FGF 5 ng / mL, and insulin 2.5 μg / mL). 2 × 10 fibroblasts suspended in 2 mL of the above medium were placed in the cell culture supplement-coated culture vessel. 6 As a control, 2 × 10 fibroblast cells were seeded in the same culture vessel without cell culture supplements. 6 Spheroid formation was observed under a microscope on days 2 and 20 after the start of culture, and photographs were taken (Figure 5).

[0087] Gene mRNA expression analysis was performed using real-time PCR. Fibroblasts were cultured as spheroids using the same method as above. On day 20 (Day 20), the spheroids were collected in a 15 mL centrifuge tube, centrifuged, and then washed with phosphate buffered saline (PBS(-)). For control cells cultured in a culture vessel without cell culture supplements (Day 0), the medium was removed and the cells were washed with PBS(-). The cells were then detached and dispersed from the culture vessel with 0.05% trypsin-EGTA, collected in a 15 mL centrifuge tube, and washed again with PBS(-). RNA was extracted from the collected spheroids and control cells, and the expression of stem cell undifferentiation marker genes was analyzed using real-time PCR.

[0088] Specifically, NucleoZOL (Machley-Nagel) reagent was added to each cell line, and total RNA was extracted according to the manufacturer's recommended protocol. This RNA served as template RNA for stem cell undifferentiation marker detection. Real-time PCR analysis was performed using QuantiFast Probe RT-PCR Kits (QIAGEN; No. 204454) and TaqMan Gene Expression Assay Probes (Thermo Fisher Scientific) PCR probes on a Rotor-Gene Q (QIAGEN) system. Genes detected included cytokines and chemokines expected to be involved in spheroid formation, as well as three transcription factors used in iPS cell generation (Non-Patent Document 1) as probes for detecting stem cell undifferentiation marker gene expression: SOX2 (sex-determining region Y-box 2), OCT4 (octamer binding transcription factor 4), and NANOG (homeodomain transcription factor). β-Actin was also used as an endogenous control gene. For gene expression analysis, relative quantification was performed using the ΔΔCt method (delta-delta Ct method). The results of the ΔΔCt analysis were calculated by setting the gene expression level of the control cells (no cell culture supplements, planar 2D growth on day 0) at a relative value of 1.0, and calculating the fold increase in the expression level (expression level on day 20 of spheroid growth) (Figure 6).

[0089] (Results) Figure 5 shows the results of spheroid culture of fibroblasts using culture vessels treated with cell culture supplements. In the control vessel without cell culture supplements, the fibroblasts adhered to the bottom and proliferated two-dimensionally (day 2 of culture). On the other hand, in the culture vessels coated with cell culture supplements, the cells formed aggregates / spheroids and proliferated three-dimensionally. From day 2 to day 20 of culture, the cells that make up the spheroids proliferated, and the size of the spheroids themselves also increased.

[0090] Figure 6 shows the results of real-time PCR of gene mRNA. We found that the expression levels of the marker proteins SOX2, OCT4, and NANOG, which define stem cell properties, increased in the fibroblast spheroids on day 20 of culture, indicating that the spheroids had acquired stem cell properties. Specifically, the expression levels of SOX2, OCT4, and NANOG in the control fibroblasts cultured without cell culture supplements were set at 1.0, and the expression levels of SOX2, OCT4, and NANOG on day 20 of 3D spheroid culture were compared using the ΔΔCt method. The results showed a 34.4-fold increase in SOX2, a 5.1-fold increase in OCT4, and a 9.1-fold increase in NANOG.

[0091] (Discussion) In Figure 5, when fibroblasts are cultured in a polypropylene cell culture vessel, the bottom of the vessel is generally treated to be hydrophilic or hydrophobic to enhance cell adhesion, so the cells adhere to the bottom of the vessel and proliferate (two-dimensional adhesive proliferation). In contrast, when the culture vessel is coated with an aqueous solution of the polypeptide represented by formula (1), the cells do not adhere to the bottom, but instead move around and aggregate with each other to form spheroids, which are three-dimensional cell aggregates. Because the human body is constructed as a three-dimensional aggregate of cells and performs biological phenomena, three-dimensional cultures better reflect in vivo tissues than two-dimensional cultures, and various physiological functions and gene expression are regulated.

[0092] From the results in Figure 6, we found that when fibroblasts are cultured as spheroids in a culture vessel treated with cell culture additives, stem cells with the characteristics of pluripotent stem cells can be induced without introducing the genes used in conventional iPS cell production methods or using the chemical cocktail used in direct reprogramming with chemical compounds.

[0093] In other words, we have discovered a novel cell culture supplement and spheroid culture method that can induce cells with the characteristics of pluripotent stem cells from somatic fibroblasts using a novel direct-reprogramming mechanism.

[0094] The fold increase in mRNA expression level of stem cell undifferentiation markers is not limited to these values. In addition to SOX2, OCT3 / 4, and NANOG, other known human stem cell undifferentiation markers include Klf4, Essrb, Sall4, Lin28, SSEA, TRA-1-60, and c-MYC, and the present invention is not limited to these three genes in the examples.

[0095] Example 2: Induction of Differentiation of Stem Cells Produced from Healthy Human Neonatal Fibroblasts into the Three Early Germ Layers (Method) To observe spheroid formation and stem cell induction over time, healthy human neonatal fibroblasts were cultured in a 96-well round-bottom culture vessel. A 0.125% aqueous solution of the polypeptide represented by formula (1) (cell culture supplement) was added to a commercially available 96-well round-bottom culture vessel (Nunclon Delta 96U Bottom, Thermo Scientific) at 100 μL / well and allowed to stand at 4°C for 24 hours. The excess cell culture supplement was then removed, and the vessel was dried in a 37°C oven for 96 hours to complete the cell culture supplement coating process. Fibroblasts were maintained and cultured in serum-free medium (Promo Cell; Fibroblast Basal Medium, basic-FGF 5 ng / mL, and insulin 2.5 μg / mL). 4 × 10 fibroblasts suspended in the medium were added to the cell culture supplement-coated culture vessel. 4 The cells were seeded at 100 μL per well and cultured at 37°C and 5% CO2.

[0096] Spheroid formation was observed under a microscope on days 0, 2, and 20 after the start of culture (Figure 7).

[0097] To investigate the mechanism of expression of the stem cell undifferentiation marker genes discovered in Example 1 over time, real-time PCR analysis was performed using TaqMan Gene Expression Assay Probes for three transcription factors: SOX2 (sex-determining region Y-box 2), OCT4 (octamer binding transcription factor 4), and NANOG (homeodomain transcription factor), as well as β-Actin as an endogenous control gene. Expression analysis was performed using relative quantification using the ΔΔCt method (delta-delta Ct method) (Figure 8).

[0098] To investigate whether stem cells derived from fibroblasts have the ability to induce differentiation, we analyzed the expression of three germ layer-related genes, which indicate early differentiation stages. Fibroblasts were cultured in the same manner as above using the same cell culture supplement-treated 96-well culture vessels. Real-time PCR analysis was performed using three germ layer marker genes: SOX17 (sex-determining region Y-box 17; endoderm), GATA6 (GATA family of zinc finger transcription factors; mesoderm), and MAP2 (microtubule associated protein 2; ectoderm), as well as TaqMan Gene Expression Assay Probe for β-Actin as an endogenous control gene. Expression analysis was performed using relative quantification using the delta-delta Ct method (Figure 9).

[0099] (Results) The results of the growth of fibroblast spheroids over time in a 96-well round-bottom culture vessel are shown in Figure 7. By using a 96-well round-bottom culture vessel treated with cell culture supplements, the cells formed single spheroids, allowing for uniform measurement of gene expression and physiological activity for each spheroid.

[0100] The results of real-time PCR of stem cell undifferentiation marker mRNA are shown in Figure 8. As a result, we found that the gene expression levels of stem cell undifferentiation markers SOX2, OCT4, and NANOG increased over time from the fifth day of fibroblast spheroid culture.

[0101] Specifically, the gene expression levels of SOX2, OCT4, and NANOG in fibroblasts cultured in 2D adhesion culture (Day 0) without cell culture supplements (control) were set at 1.0, and the gene expression levels of SOX2, OCT4, and NANOG on Day 5, Day 10, and Day 20 of 3D spheroid culture were compared using ΔΔCt relative quantification. All genes increased over time, reaching their peak on Day 20, with SOX2 increasing 144.0-fold, OCT4 increasing 30.1-fold, and NANOG increasing 35.4-fold.

[0102] Figure 9 shows the results of real-time PCR of the mRNA of three germ layer-related genes. As a result, we found that the gene expression levels of the marker proteins SOX17, GATA6, and MAP2, which define differentiation into the three germ layers, increased from day 5 onwards in spheroid culture of fibroblasts.

[0103] Specifically, the gene expression levels of SOX17, GATA6, and MAP2 in fibroblasts cultured in 2D adhesion culture (Day 0) without cell culture supplements (control) were set at 1.0, and the gene expression levels of SOX17, GATA4, and MAP2 on Day 5 (Day 5), Day 10 (Day 10), and Day 20 (Day 20) of 3D spheroid culture were compared using ΔΔCt relative quantification. All genes increased over time, reaching their peak on Day 20, with SOX17 increasing 359.6-fold, GATA4 increasing 4.2-fold, and MAP2 increasing 24.2-fold.

[0104] (Discussion) Figure 7 shows that stem cells with the characteristics of pluripotent stem cells can be produced (direct reprogramming) from somatic fibroblasts. Furthermore, we demonstrated that this can be achieved in both flat-bottom and round-bottom culture vessels.

[0105] As shown in Figure 8, spheroid culture of fibroblasts in a 96-well culture vessel treated with cell culture supplements produces single, homogeneous spheroids, enabling precise gene expression analysis. As a result, we found that direct reprogramming to pluripotent stem cells with enhanced expression of SOX2, OCT4, and NANOG genes was possible without the introduction of transcription factor genes used in iPS cell generation or the addition of a chemical cocktail used in direct reprogramming. In other words, we have invented a novel direct reprogramming method for generating cells with pluripotent stem cell characteristics from somatic fibroblasts using the cell culture supplement-treated spheroid culture method.

[0106] The results in Figure 9 demonstrate that stem cells derived from fibroblasts can be induced to differentiate into cells with the characteristics of embryoid bodies, which are three-dimensional cell aggregates. Inducing the differentiation of stem cells into mature organ cells generally requires a first step of inducing the stem cells to differentiate into the germ layer of the target organ lineage. This culture method, which uses a mechanism distinct from conventional iPS cell methods or direct reprogramming with chemical compounds, can reprogram somatic cells to stem cells (direct reprogramming) while simultaneously inducing differentiation into embryoid bodies positive for SOX17 (endodermal marker gene), GATA6 (mesoderm marker gene), and MAP2 (ectoderm marker gene). This novel culture method is characterized by a mechanism distinct from conventional iPS cell methods or direct reprogramming with chemical compounds.

[0107] In addition to SOX2, OCT4, and NANOG, other known stem cell undifferentiation marker genes include Klf4, Essrb, Sall4, Lin28, SSEA, and TRA-1-60, and the present invention is not limited to these genes. Similarly, other known three germ layer marker genes include SOX17, GATA6, and MAP2, and the present invention is not limited to these marker genes. Furthermore, the gene expression increase rates of stem cell undifferentiation markers and three germ layer markers are not limited to these numerical values.

[0108] Example 3: Expression of stem cell marker proteins in healthy human neonatal fibroblast spheroids (Method) To confirm the induction of stem cells in fibroblast spheroids, the expression of stem cell marker proteins was examined by immunostaining using fluorescent dye-labeled antibodies. A 0.125% aqueous solution of the polypeptide represented by formula (1) (cell culture supplement) was added to a commercially available 96-well round-bottom polystyrene culture vessel (Nunclon Delta 96U Bottom, Thermo Scientific) at 100 μL / well, and the cell culture supplement was applied in the same manner as in Example 2. Fibroblasts were maintained and cultured in serum-free medium (Fujifilm Wako Pure Chemical Industries, Ltd.; DMEM / F12 medium, human serum albumin recombinant (0.5 mg / mL), human transferrin recombinant (10 μg / mL), basic-FGF (5 ng / mL), and insulin (2.5 μg / mL). 4 x 10 fibroblast cells suspended in the above medium were placed in the above cell culture supplement-coated culture vessel. 4 Cells were seeded at 100 μL per well and cultured at 37°C under 5% CO2 conditions. On day 20 after the start of culture, spheroids were harvested from the 96-well round-bottom culture vessels, fixed with 4% paraformaldehyde, and immunostained. Three fluorescently labeled antibodies for detecting human stem cell marker proteins (manufactured by R&D Systems) were simultaneously reacted: NL557 fluorescent dye-labeled anti-SOX2 antibody (orange fluorescence), NL493 fluorescent dye-labeled anti-NANOG antibody (green fluorescence), and NL637-labeled anti-OCT4 antibody (red fluorescence), and then observed under a fluorescence microscope.

[0109] (Results) Fluorescence microscopy images of immunostained fibroblast spheroids are shown in Figure 10. Fluorescence indicating the stem cell marker proteins SOX2 (orange), OCT4 (red), and NANOG (green) was detected, confirming that stem cells expressing these three stem cell marker proteins were induced in the cells forming the spheroids.

[0110] (Discussion) In Figures 6 and 8, real-time PCR analysis revealed that the cells forming the spheroids expressed stem cell undifferentiation marker genes. Here, it was found that the fibroblast spheroids also expressed marker proteins produced by undifferentiation marker genes, confirming the presence of stem cells in terms of both genes and proteins.

[0111] Example 4: Detection of Stem Cells in Healthy Human Neonatal Fibroblast Spheroids (Method) To detect stem cells induced in fibroblast spheroids, immunostaining was performed using a fluorescent dye-labeled antibody to detect stem cells contained in the spheroid-constituting cells. A 0.125% aqueous solution of the polypeptide represented by formula (1) (cell culture supplement) was added to a commercially available 96-well round-bottom polystyrene culture vessel (Nunclon Delta 96U Bottom, Thermo Scientific) at 100 μL / well, and the cell culture supplement was applied in the same manner as in Example 2. Fibroblasts were maintained and cultured in serum-free medium (Fujifilm Wako Pure Chemical Industries, Ltd.; DMEM / F12 medium, human serum albumin recombinant (0.5 mg / mL), human transferrin recombinant (10 μg / mL), basic-FGF (5 ng / mL), and insulin (2.5 μg / mL). 2 x 10 fibroblast cells suspended in the above medium were placed in the above cell culture supplement-coated culture vessel. 4 Cells were seeded at 100 μL per well and cultured at 37°C under 5% CO2 conditions. On day 20 after the start of culture, spheroids were aseptically harvested from the 96-well round-bottom culture vessel and dispersed with Accutase enzyme reagent. The cells were then transferred to an immunostaining incubator (MilliCell Chamber, Millipore) and cultured in the same medium. After 24 hours, the cells were fixed with 4% paraformaldehyde solution and immunostained. Fluorescently labeled antibodies were used, including NL493 fluorescent dye-conjugated anti-NANOG antibody (green fluorescence) from R&D Systems, and the cells were observed under a fluorescence microscope.

[0112] (Results) Figure 11 shows the presence of NANOG protein-positive stem cells within the cell population obtained by dispersing the spheroids. In Figure 11, the nuclei of the majority of fibroblasts are NANOG-negative, but some cells exhibited strong NANOG antibody-positive green fluorescence in their nuclei, indicating that stem cell marker cells had been induced.

[0113] (Discussion) NANOG protein is a homeobox transcription factor that is produced in the cytoplasm and translocates into the nucleus, where it plays an important role in maintaining the pluripotency of stem cells. Not only were stem cell marker proteins confirmed throughout the spheroids (Figure 10), but we also demonstrated that it is possible to induce stem cells that are positive for NANOG protein in the nucleus at the cellular level.

[0114] Example 5: Preparation of stem cells by spheroid culture of human adult dermal fibroblasts (Method) To induce stem cell induction (direct reprogramming) using healthy human adult dermal fibroblasts, adult dermal fibroblasts were cultured to form spheroids using the same method as in Example 2. An aqueous solution (0.125%) of the polypeptide represented by formula (1) (cell culture supplement) was added to a commercially available polystyrene 96-well round-bottom culture vessel (Nunclon Delta 96U Bottom, Thermo Scientific) and allowed to stand at 4°C for 24 hours. The cell culture supplement was then removed, and the vessel was dried in a dryer at 37°C for 96 hours to complete the cell culture supplement application process. Healthy human adult dermal fibroblasts (Lonza, Normal Human Dermal Fibroblast-Adult, hereafter referred to as "adult dermal fibroblasts") were maintained and cultured in serum-free medium (Fujifilm Wako Pure Chemical Industries, Ltd.; DMEM / F12 medium, human serum albumin recombinant 0.5 mg / mL, human transferrin recombinant 10 μg / mL, basic-FGF 5 ng / mL, and insulin 2.5 μg / mL). 2 × 10 adult dermal fibroblasts suspended in 100 μL of the above medium were cultured in a 96-well round-bottom culture vessel coated with the above cell culture supplements. 4 The cells were seeded at 100 cells / well and cultured at 37°C and 5% CO2.

[0115] To detect stem cell marker genes, mRNA expression was analyzed using real-time PCR. Specifically, on day 20 of culture (Day 20), spheroids were collected in a 15 mL centrifuge tube and centrifuged. The cells were then washed with phosphate buffered saline (PBS). For the control cells cultured in a 2D culture vessel without cell culture supplements (Day 0), the medium was removed and the cells were washed with PBS. After detachment and dispersion, the cells were collected in a 15 mL centrifuge tube and washed again with PBS. NucleoZOL (Machrei-Nagel) reagent was added to the spheroids, and RNA was extracted according to the manufacturer's recommended protocol. Real-time PCR analysis was performed using QuantiFast Probe RT-PCR Kits (QIAGEN; No. 204454) and TaqMan Gene Expression Assay Probes (Thermo Fisher Scientific) PCR probes on a Rotor-Gene Q (QIAGEN) instrument. For gene detection, we used three transcription factors, SOX2 (sex-determining region Y-box 2), OCT4 (octamer binding transcription factor 4), and NANOG (homeodomain transcription factor), as probes to detect the expression of stem cell undifferentiation marker genes, and β-Actin as an endogenous control gene (Figure 13).

[0116] To examine whether spheroid-induced stem cells could differentiate into the three early germ layers, we analyzed the expression of three germ layer marker genes using real-time PCR. Four probes were used: SOX17 (sex-determining region Y-box 17; endoderm), GATA6 (GATA family of zinc finger transcription factors; endoderm), HAND1 (Heart- and neural crest derivatives-expressed protein 1; mesoderm), and SOX1 (HMG-box (high mobility group) DNA-binding domain; ectoderm). A TaqMan Gene Expression Assay Probe for β-Actin was also used as an endogenous control (Figure 14). Gene expression analysis was performed using the delta-delta Ct method for relative quantification. The results of the ΔΔCt analysis were calculated by setting the gene amount expressed by the control cells in the "no cell culture supplements, planar 2D growth on Day 0" group as a relative value of "1.0," and calculating the increase factor of the "expression amount on Day 20 of spheroid growth" for comparison.

[0117] (Results) Figure 12 shows the results of spheroid culture of adult dermal fibroblasts using culture vessels treated with cell culture supplements. In control vessels without cell culture supplements, adult dermal fibroblasts adhered to the bottom and proliferated in two dimensions (culture day 2). On the other hand, in culture vessels coated with cell culture supplements, the cells formed spheroids and proliferated in three dimensions.

[0118] Figure 13 shows the results of real-time PCR of gene mRNA. We found that adult dermal fibroblast spheroids on day 20 of culture had increased expression levels of the stem cell-defining marker proteins SOX2, OCT4, and NANOG, indicating that the spheroids had acquired stem cell properties. Specifically, the expression levels of SOX2, OCT4, and NANOG in control cells cultured in 2D were set at 1.0 relative to the expression levels of SOX2, OCT4, and NANOG in 3D spheroids on day 20 of culture, and were compared using the ΔΔCt method. The results showed a 9.4-fold increase in SOX2, a 13.0-fold increase in OCT4, and a 50.5-fold increase in NANOG.

[0119] Figure 14 shows that the expression levels of the marker proteins SOX17, GATA6, HAND1, and SOX1, which define differentiation into the three germ layers, also increased. The expression levels of SOX17, GATA6, HAND1, and SOX1 in control cells were set at 1.0 relative to the expression levels of SOX17, GATA4, and MAP2 on day 20 (Day 20) of 3D spheroid culture. The expression levels of all genes increased: SOX17 increased 11.4-fold, GATA6 increased 2.2-fold, HAND1 increased 33.9-fold, and SOX1 increased 7.1-fold.

[0120] (Discussion) Previous studies of direct somatic cell reprogramming using compounds have reported stem cell induction experiments using mouse fetal fibroblasts or adult fibroblasts. However, no technology has yet been established for application to human adult fibroblasts. Here, we have discovered a method for stably and reproducibly producing stem cells using the cell culture supplement of the present invention, not only from human neonatal fibroblasts but also from adult skin fibroblasts.

[0121] Since stem cells can be induced using spheroid culture methods using cell culture supplements, using skin-derived fibroblasts, which are the least invasive and easiest to sample from an individual patient, this technology will be useful for personalized regenerative therapy, personalized diagnostic methods, and therapeutic drug screening.

[0122] Example 6: Spheroid culture of healthy human adult mammary epithelial cells and expression of stem cell undifferentiation marker genes (Method) An aqueous solution (0.125%) of the polypeptide represented by formula (1) (cell culture supplement) was added to a commercially available polystyrene 6-well flat-bottom culture vessel (CellBIND, CORNING) or a polystyrene 96-well round-bottom culture vessel (Nunclon Delta 96U Bottom, Thermo Scientific) and allowed to stand at 4°C for 24 hours.

[0123] The cell culture supplements were then removed, and the cells were dried in a dryer at 37°C for 96 hours to complete the cell culture supplement coating process. Healthy human adult mammary epithelial cells (ATCC PCS-600-010, Primary Mammary Epithelial Cells, hereafter referred to as "mammary epithelial cells") were maintained and cultured in serum-free medium (Promo Cell; Mammary Epithelial Cell Growth Medium, EGF 5 ng / mL, and Insulin 2.5 μg / mL). 2 × 10 mammary epithelial cells suspended in 2 mL of the above medium were placed in the cell culture supplement-coated culture vessel. 6 In the case of a 96-well round-bottom culture vessel, 2 × 10 mammary epithelial cells suspended in 100 μL of the above medium were seeded at 1 × 10 cells / well. 4 Spheroid formation was observed under a microscope on days 2 and 20 after the start of culture, and the state of spheroid formation was photographed (Figure 15).

[0124] To investigate whether stem cells can be induced (direct reprogramming) from somatic mammary epithelial cells, mammary epithelial cells were cultured in a 96-well culture vessel treated with cell culture supplements to form spheroids. Gene mRNA expression analysis was performed using real-time PCR.

[0125] Specifically, on day 20 of culture, spheroids were collected in a 15 mL centrifuge tube and centrifuged. The cells were then washed with phosphate buffered saline (PBS). For the control cells cultured in a 2D culture vessel without cell culture supplements (day 0), the medium was removed and washed with PBS. The cells were then detached from the plate using 0.05% trypsin-EGTA, collected in a 15 mL centrifuge tube, and washed again with PBS. NucleoZOL (Machrei-Nagel) reagent was added to the spheroids, and RNA was extracted according to the manufacturer's recommended protocol. Real-time PCR analysis was performed using QuantiFast Probe RT-PCR Kits (QIAGEN; No. 204454) and TaqMan Gene Expression Assay Probes (Thermo Fisher Scientific) PCR probes on a Rotor-Gene Q (QIAGEN) instrument. The genes we attempted to detect were the transcription factors SOX2 (sex-determining region Y-box 2), OCT4 (octamer binding transcription factor 4), and NANOG (homeodomain transcription factor) as probes for detecting the expression of stem cell undifferentiation marker genes, and β-Actin as an endogenous control gene (Figure 16).

[0126] The probes used to detect genes related to early three germ layer differentiation were three germ layer marker genes: SOX17 (sex-determining region Y-box 17; endoderm), GATA4 (GATA family of zinc finger transcription factors; mesoderm), and MAP2 (microtubule associated protein 2; ectoderm), as well as a TaqMan Gene Expression Assay Probe for β-Actin as an endogenous control gene (Figure 17).

[0127] Gene expression analysis was performed using the ΔΔCt method (delta-delta Ct method) for relative quantification. The results of the ΔΔCt analysis were calculated by setting the gene expression level of the control cells (no cell culture supplements, planar 2D growth on day 0) at a relative value of 1.0, and calculating the fold increase in the expression level of the control cells (day 20 of spheroid growth).

[0128] (Results) Figure 15 shows the results of spheroid culture of mammary epithelial cells using culture vessels treated with cell culture supplements. In control vessels without cell culture supplements, mammary epithelial cells adhered to the bottom and showed planar, two-dimensional growth (day 2 of culture). On the other hand, in culture vessels treated with cell culture supplements, the cells self-organized to form spheroids and grew three-dimensionally. From day 2 to day 20 of culture, the cells that made up the spheroids proliferated, and the size of the spheroids themselves also increased.

[0129] Figure 16 shows the results of real-time PCR analysis of mRNA to examine the expression of stem cell undifferentiation marker genes induced (direct-reprogramming) from mammary epithelial cells. The results showed that the gene expression levels of stem cell undifferentiation marker proteins SOX2, OCT4, and NANOG increased on Day 20 (Day 20) of spheroid culture compared to Day 0 in 2D adherent culture of mammary epithelial cells.

[0130] Specifically, the gene expression levels of SOX2, OCT4, and NANOG in mammary epithelial cells cultured in 2D adhesion culture without cell culture supplements (control) were set at 1.0, and the gene expression levels of SOX2, OCT4, and NANOG on day 20 of 3D spheroid culture were compared using ΔΔCt relative quantification. All genes increased on day 20, with SOX2 increasing 24.0-fold, OCT4 1.5-fold, and NANOG 28.7-fold.

[0131] The results of real-time PCR of mRNA for genes related to the early three germ layers are shown in Figure 17. As a result, we found that the expression levels of SOX17 and MAP2, which are marker genes that define differentiation into the three germ layers, increased on day 20 of spheroid culture of mammary epithelial cells.

[0132] Specifically, the gene expression levels of SOX17, GATA4, and MAP2 in mammary epithelial cells cultured in 2D adherent culture (Day 0) without cell culture supplements (control) were set at 1.0 relative to the values ​​of SOX17, GATA4, and MAP2 on Day 20 of 3D spheroid culture. The results showed that SOX17 increased 14.7-fold and MAP2 increased 24.2-fold on Day 20 of culture. However, no expression of the mesodermal marker GATA4 was detected.

[0133] (Discussion) The results in Figure 15 demonstrate that direct reprogramming of adult mammary epithelial cells into stem cells with the characteristics of pluripotent stem cells is possible. Furthermore, we demonstrated that this can be achieved in both flat-bottom and round-bottom culture vessels.

[0134] As shown in Figure 16, mammary epithelial cells cultured as spheroids in a 96-well culture vessel treated with cell culture supplements produced single, homogeneous spheroids, enabling precise gene expression analysis. As a result, we found that direct reprogramming to pluripotent stem cells (PSCs) with enhanced expression of SOX2, OCT4, and NANOG genes was possible without the introduction of transcription factor genes used in iPS cell generation or the addition of a chemical cocktail used in direct reprogramming. In other words, we have invented a novel direct reprogramming method for generating cells with pluripotent stem cell characteristics from somatic mammary epithelial cells using the cell culture supplement-treated spheroid culture method.

[0135] The results in Figure 17 demonstrate that stem cells generated from mammary epithelial cells using this method can be induced to differentiate into three germ layers, the initial stage of differentiation into various organ cell types. Inducing differentiation of stem cells into mature organ cells generally requires a first step of inducing differentiation of the stem cells into the germ layer of the target organ lineage. This culture method, which uses a mechanism distinct from conventional iPS cell methods and chemical-based direct reprogramming, can be described as a novel culture method that simultaneously induces reprogramming of somatic cells into stem cells (direct reprogramming) and simultaneously induces differentiation into embryoid bodies positive for SOX17 (endodermal marker gene) and MAP2 (ectoderm marker gene).

[0136] In addition to SOX2, OCT4, and NANOG, other known stem cell undifferentiation marker genes include Klf4, Essrb, Sall4, and Lin28, and the present invention is not limited to these genes. Similarly, other known early three germ layer marker genes include SOX17, GATA4, and MAP2, and the present invention is not limited to these marker genes. Furthermore, the gene expression increase rates of stem cell markers and early three germ layer markers are not limited to these values.

[0137] Example 7: Two-dimensional adhesive culture and three-dimensional spheroid culture of cancer cells (Method) 2 mL of an aqueous solution (0.125%) of the polypeptide represented by formula (1) (cell culture aid) was added to a commercially available 6-well flat-bottom polystyrene culture vessel (CellBIND, CORNING) and allowed to stand at 4°C for 24 hours.

[0138] The cell culture supplements were then removed, and the plates were dried in a dryer at 37°C for 96 hours to complete the cell culture supplement application process. The human cancer cells used were the common cancer cell lines A549 cells (human lung cancer cells, ATCC CCL-185, carcinoma), ES2 cells (human ovarian cancer cells, ATCC CRL-1978, clear cell carcinoma), and HOS-143 cells (human osteosarcoma cells, ATCC CRL-8303, osteosarcoma). 2 × 10 cells of each type were used. 5The cells were seeded in a culture vessel at a density of 100 μL per well and cultured at 37°C under 5% CO2 conditions. The culture medium used was DMEM supplemented with 10% FBS and penicillin / streptomycin. As a control, 2 × 10 cancer cells were seeded in the same culture vessel without the cell culture supplement. 6 Spheroid formation was observed under a microscope on days 4 and 15 after the start of culture, and the state of spheroid formation was photographed ( FIG. 18 ).

[0139] (Results) The results are shown in Figure 18. In culture vessels without cell culture supplements, cells adhered to the bottom and proliferated (two-dimensional adhesive proliferation). On the other hand, in culture vessels treated with cell culture supplements, cells formed aggregates and formed multiple spheroids. From day 4 to day 15 after the start of culture, the cells that make up the spheroids proliferated, and the size of the spheroids themselves also increased.

[0140] (Discussion) Similar to the results of Examples 1 to 6, it was revealed that cancer cells also form spheroids. Compared to cancer cells cultured in two dimensions, cancer cells cultured in three-dimensional spheroids are said to exhibit characteristics closer to the pathological condition in vivo. In other words, if cancer cells can be cultured in three dimensions, it can be said that a model that reflects the pathological condition of cancer can be created in a culture vessel.

[0141] The polypeptide cell culture supplement represented by formula (1) induced spheroid formation in three cancer cell lines: A549 cells (lung cancer), ES2 (ovarian cancer), and HOS-143B (osteosarcoma). This indicates that the reaction is not limited to these three cell lines, but can provide a broad range of in vitro (in vitro; in a culture vessel) cancer models.

[0142] Example 8: Study on the concentration of cell culture supplement (Method) Dilutions (cell culture supplement) were prepared from a 4-fold to 1024-fold dilution of an aqueous solution (0.5%) of the polypeptide represented by formula (1), and 100 μl of each was added to wells of a commercially available 96-well flat-bottom plate (CellBIND, Corning) and left at 4°C for 16 hours.

[0143] Afterwards, excess cell culture supplements were removed and the plates were dried under reduced pressure at room temperature for 24 hours. HOS-143B (human osteosarcoma) cells were seeded at 3,000 cells / well onto the culture plates coated with cell culture supplements and cultured at 37°C under 5% CO2 conditions. DMEM + 10% FBS medium was used.

[0144] After culturing for 3 days, the cells were observed under a microscope.

[0145] (Results) The results are shown in Figure 19. Spheroid formation was observed on the coated plate with up to 256-fold diluted solutions of the aqueous solution of the polypeptide represented by formula (1), but no spheroid formation was observed with further diluted solutions.

[0146] (Discussion) It was revealed that the spheroid-forming ability of an aqueous solution of the polypeptide represented by formula (1) is concentration-dependent.

[0147] Example 9: Study on plate shape (Method) 200 μl of an aqueous solution (0.125%) of the polypeptide represented by formula (1) (cell culture supplement) was added to each well of a commercially available 96-well flat-bottom plate (Thermofisher), V-bottom plate (Thermofisher), or U-bottom plate (Thermofisher), and left at 4° C. for 16 hours.

[0148] Thereafter, excess cell culture supplements were removed, and the mixture was dried under reduced pressure at room temperature for 24 hours.

[0149] HeLa cells (human cervical epithelioid carcinoma, JCRB9004, National Institutes of Biomedical Innovation, Health and Nutrition) and HOS-143B (human osteosarcoma) were seeded at 2,000 cells / well on culture plates coated with cell culture supplements and cultured at 37°C and 5% CO2. DMEM + 10% FBS medium was used. After two days of culture, the cells were observed under a microscope.

[0150] (Results) The results are shown in Figure 20. In the uncoated wells, cells adhered and spread. On the other hand, one spheroid was formed in the V-bottom plate coated with an aqueous solution of the polypeptide represented by formula (1), and several spheroids were formed in the U-bottom plate. Numerous small spheroids were formed in the flat-bottom plate.

[0151] (Discussion) It was confirmed that spheroids (cell clusters) were formed on plates coated with the polypeptide represented by formula (1), regardless of the shape of the plate.

[0152] Example 10: Study on Inhibition of Apoptosis Induction (Method) Two types of 96-well cell culture plates with different bottom shapes (U-bottom plate #163320; Corning, NY, USA) and V-bottom plate #277143; Thermo Fisher Scientific, MA, USA) were each injected with two different concentrations of aqueous solutions of the polypeptide represented by Formula (1) (4-fold and 8-fold dilutions of a 0.5% solution) at 100 μL / well and 200 μL / well, respectively, and incubated overnight at 4°C. The plates were then dried in a clean bench for approximately 6 hours to complete the cell culture supplement treatment. Two cell lines (canine renal tubule-derived MDCK II cells and HOS-143B cells) were seeded at 3,000 cells / well in 100 μL and cultured for 6 days in an incubator at 37°C in a 5% CO2 atmosphere. Controls were cultured in plates without cell culture supplements. After 3 and 6 days, apoptotic activity was measured in each well using a caspase activity assay kit (Apo-ONE Homogeneous Caspase-3 / 7 Assay kit; Promega, WI, USA). The fluorescent pro-substrate, bis-(N-CBZ-L-aspartyl-L-glutamyl-L-valyl-aspartic acid amide)rhodamine 110 (Z-DEVD-rhodamine 110), was diluted 100-fold in the provided buffer and added to each well in an amount of 50 μL. The plate was gently rocked to avoid disrupting the spheroids and then incubated at room temperature for 1 hour. Then, 100 μL of the culture medium was transferred to a black 96-well cell culture plate (#237105; Thermo Fisher Scientific, MA, USA), and the fluorescence intensity at 590 nm (excitation wavelength 560 nm) was measured using a plate reader. The value obtained by adding only the reagent without adding cells was used as a blank value, and the value obtained by subtracting this value from the measured value was used to obtain the measured value at each culture day. The relative values ​​compared with those of a plate (control) that was not coated with an aqueous solution of the polypeptide represented by formula (1) were blotted.

[0153] (Results) The results are shown in Figures 21 and 22. It was found that apoptosis was inhibited in both MDCK II and HOS-143B cells in a concentration-dependent manner by the polypeptide of formula (1), compared to cells cultured on plates not coated with an aqueous solution of the polypeptide of formula (1). The inhibitory effect on apoptosis was more pronounced on day 6 of culture.

[0154] (Discussion) Previously, major problems in 3D spheroid culture were cell death due to anoikis in primary cultured cells, apoptosis occurring in the central part of spheroid-forming cells, and a decrease in viable cells due to the induction of necrosis. However, the cell culture supplement of the present invention suppresses the induction of apoptosis during spheroid formation, and has been found to be extremely useful not only for stem cell production using spheroids but also for drug discovery research and medical applications.

[0155] Example 11: Detection of Gene Expression of Cancer Stem Cell Markers (Method) Cell culture supplement-treated culture vessels were prepared using the same method as in Example 7, and the same three cancer cell types as in Example 7 were cultured. On days 4 and 15 of culture, the three cancer cell spheroids were collected from each culture vessel into 15 mL centrifuge tubes, centrifuged, and washed with phosphate buffered saline (PBS(-)). For cancer cells cultured on control plates without cell culture supplements, the medium was removed and washed with PBS(-). The cells were detached and dispersed from the plate with 0.05% trypsin-EGTA, collected in 15 mL centrifuge tubes, and washed again with PBS(-). mRNA was extracted from the collected spheroids and control cells, and expression of cancer stem cell marker genes was analyzed by real-time PCR.

[0156] Specifically, NucleoZOL (Machrei-Nagel) reagent was added to each cell line, and RNA was extracted according to the manufacturer's recommended protocol. This RNA served as template RNA for stem cell marker detection. Real-time PCR analysis was performed using QuantiFast Probe RT-PCR Kits (QIAGEN; No. 204454) and TaqMan Gene Expression Assay Probes (Thermo Fisher Scientific) PCR probes on a Rotor-Gene Q (QIAGEN) instrument. Three probes were used to detect the expression of cancer stem cell marker genes: CD24 (mucin-type glycoprotein, ligand for P-selectin, oncogene), CD44 (cell surface glycoprotein involved in cell-cell interaction, adhesion, and migration, cancer stem cell marker), and CD133 (five transmembrane glycoprotein, stem cell marker, and cancer stem cell marker protein). β-Actin was used as an endogenous control gene. The probes used to detect the expression of stem cell undifferentiation marker genes were three transcription factors used in iPS cell production: SOX2 (sex-determining region Y-box 2), OCT4 (octamer binding transcription factor 4), and NANOG (homeodomain transcription factor), along with β-Actin as an endogenous control gene. Gene expression analysis was performed using relative quantification using the delta-delta Ct method. The results of the delta-delta Ct analysis were calculated by setting the gene expression level of the control cells (no cell culture supplements, day 4 of 2D adherent growth) at a relative value of 1.0, and calculating the fold increase in expression level (day 15 of spheroid growth) (Figures 23-25).

[0157] (Results) The results of expression of cancer stem cell marker genes are shown in Figures 23, 24 and 25.

[0158] In Figure 23, it was found that the gene expression levels of cancer stem cell markers CD24, CD44, and CD133 in A549 lung cancer cells increased with the passage of culture time (from day 4 to day 15).

[0159] Specifically, CD24 increased 2.2-fold on day 4 of spheroid culture and 4.2-fold on day 15, CD44 increased 1.6-fold on day 4 and 2.9-fold on day 15, and CD133 increased 3.4-fold on day 4 and 13.5-fold on day 15.

[0160] In Figure 24, the gene expression level of CD24 in ES2 ovarian cancer cells increased 11.6-fold on day 4 of spheroid culture and 63.2-fold on day 15. CD44 increased 1.3-fold on day 4 and 2.9-fold on day 15. CD133 was not detected in adherent cells, and therefore increased 8.5-fold on day 15 of spheroid culture, assuming a control value of 1.0 on day 4.

[0161] In Figure 25, CD24 in HOS-143B osteosarcoma cells increased 1.34-fold on day 4 and 1.2-fold on day 15, CD44 increased 2.3-fold on day 4 and 1.9-fold on day 15, and CD133 decreased 0.3-fold on day 4 but increased 2.1-fold on day 15.

[0162] (Discussion) A549 cells, ES2 cells, and HOS-143 cells are cancer cell lines established as cells that grow adherently. In the cancer cell differentiation hierarchy, they are classified as "differentiated cancer cell lines" that have progressed to a degree of differentiation from cancer stem cells. Therefore, it is known that cancer stem cell marker genes CD24, CD44, and CD133 are not expressed or are expressed at very low levels in cells cultured in conventional 2D adherent culture. When these cancer cells are cultured in the cell culture supplement of the present invention, they form spheroids with a three-dimensional structure and proliferate, and we found that the expression of CD24, CD44, and CD133 genes, which are markers of cancer stem cells, increases from day 4 to day 15 of culture.

[0163] Previously, methods for obtaining cancer stem cells have involved transplanting cancer tissue extracted from cancer patient specimens into mice, or sorting small amounts of cancer stem cells from cancer tissue using a cell sorter and then expanding them over long periods of time in culture media supplemented with many expensive growth factors and hormones. This culture method, however, offers a simple and easy way to produce large quantities of cancer stem cells using only cell culture supplements.

[0164] It should be noted that cancer stem cell markers other than CD24, CD44, and CD133 are known, but the present invention is not limited to these examples.

[0165] Example 12: Detection of gene expression of stem cell undifferentiation marker transcription factors in cancer cells (Method) Culture vessels were coated using the same method as in Examples 7 and 11, and three cancer cell lines, A549 cells, ES2 cells, and 143B cells, were cultured. Three transcription factors, SOX2 (sex-determining region Y-box 2), OCT4 (octamer binding transcription factor 4), and NANOG (homeodomain transcription factor), were used as probes to detect the expression of stem cell undifferentiation marker genes, along with β-Actin as an endogenous control gene. Gene expression analysis was performed using the ΔΔCt method (delta-delta Ct method) for relative quantification. The results of the ΔΔCt analysis were calculated by setting the gene expression level of the control cells (no cell culture supplements, day 4 of 2D adherent growth) at 1.0 relative to the expression level of the control cells (day 15 of spheroid growth) and calculating the fold increase (Figures 26-28).

[0166] (Results) FIG. 26 shows that the gene expression levels of stem cell undifferentiation markers SOX2, OCT4, and NANOG in A549 lung cancer cells increased over time (from day 4 to day 15).

[0167] Specifically, SOX2 increased 5.0-fold on day 4 and 6.0-fold on day 15, OCT4 increased 1.6-fold on day 4 and 3.7-fold on day 15, and NANOG increased 10.6-fold on day 4 and 55.7-fold on day 15.

[0168] In Figure 27, SOX2 in ES2 ovarian cancer cells remained unchanged at 1.1-fold on day 4 and increased 3.4-fold on day 15, OCT4 increased 2.6-fold on day 4 and increased 4.6-fold on day 15, and NANOG increased 3.9-fold on day 4 and increased 10.9-fold on day 15.

[0169] In Figure 28, SOX2 in HOS-143B osteosarcoma cells increased 3.5-fold on day 4 and 1.6-fold on day 15, OCT4 increased 2.5-fold on day 4 and 2.4-fold on day 15, and NANOG increased 1.6-fold on day 4 and 1.4-fold on day 15.

[0170] (Discussion) Stem cell undifferentiation marker genes, such as SOX2, OCT4, and NANOG, are clinically known to be expressed in some cancer tissues. Because SOX2, OCT4, and NANOG transcription factors have traditionally been found to be stem cell undifferentiation markers in ES cells and iPS cells, cancer cells expressing these genes are thought to possess the properties of "cancer stem cells." The A549, ES2, and HOS-143 cells used here are known to express no or very low levels of stem cell undifferentiation marker genes, such as SOX2, OCT4, and NANOG. However, when these three cancer cells were cultured with the cell culture supplement of the present invention, they formed three-dimensional spheroids and proliferated, and the expression of stem cell markers SOX2, OCT4, and NANOG increased from day 4 to day 15 of culture.

[0171] Based on the above, we have invented a method for easily and mass-producing cancer stem cells by culturing cancer cells with this cell culture supplement, which expresses the stem cell undifferentiated marker genes SOX2, OCT4, and NANOG, as well as the cancer stem cell marker genes CD24, CD44, and CD133.

[0172] It should be noted that, although other cancer stem cell undifferentiation markers are known in addition to SOX2, OCT4, and NANOG, the present invention is not limited to these examples.

Claims

1. A somatic cell reprogramming agent comprising a polypeptide represented by the following formula (1): (Pro-Hyp-Gly)n (1) (In the formula, Hyp represents hydroxyproline, and n represents an integer of 2 to 1000.)

2. The somatic cell reprogramming agent of claim 1, wherein the somatic cell is a fibroblast.

3. The somatic cell reprogramming agent of claim 1, wherein the somatic cell is an epithelial cell.

4. The somatic cell reprogramming agent of claim 1, wherein the somatic cell is a neural cell.

5. The somatic cell reprogramming agent of claim 1, wherein the somatic cell is a bone cell.

6. The somatic cell reprogramming agent of claim 1, wherein the somatic cell is a skin cell.

7. A cancer cell reprogramming agent comprising a polypeptide represented by the following formula (1): (Pro-Hyp-Gly) ｎ (1) (In the formula, Hyp represents hydroxyproline, and n represents an integer of 2 to 1000.)

8. A cell culture substrate, the culture surface of which is surface-treated with the somatic cell reprogramming agent according to any one of claims 1 to 6 or the cancer cell reprogramming agent according to claim 7.

9. A cell culture system comprising the somatic cell reprogramming agent according to any one of claims 1 to 6 and cultured somatic cells.

10. A method for producing pluripotent stem cells, comprising a step of culturing somatic cells using the somatic cell reprogramming agent according to any one of claims 1 to 6.

11. The method according to claim 10 , wherein the pluripotent stem cells express a stem cell undifferentiation marker gene.

12. The method of claim 11, wherein the stem cell undifferentiated marker gene is at least one selected from the group consisting of OCT4, SOX1, SOX2, SOX3, SOX18, NANOG, Klf2, Klf4, Essrb, Sall4, Tcl1, Tcl3, c-Myc, c-Mycn, LIN28, SSEA-1, SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, AP, Fzd1-10, and TDGF-1.

13. The manufacturing method described in claim 10, wherein the culture period is 10 days or more.

14. The method of claim 10, wherein the pluripotent stem cells are used for the treatment of a disease.

15. The manufacturing method described in claim 10, comprising adding a somatic cell reprogramming agent described in any one of claims 1 to 6 to a culture medium to culture somatic cells.

16. A method for producing a tissue using pluripotent stem cells obtained by the method according to claim 10.

17. The manufacturing method described in claim 16, wherein the pluripotent stem cells are obtained from somatic cells derived from cows, pigs, or sheep.

18. A cell culture system comprising the cancer cell reprogramming agent described in claim 7 and cancer cells.

19. A method for producing cancer stem cells, comprising a step of culturing cancer cells using the cancer cell reprogramming agent described in claim 7.

20. The manufacturing method described in claim 19, wherein the cancer cell reprogramming agent described in claim 7 is added to a culture medium to culture cancer cells.

21. A method for producing tissue using cancer stem cells obtained by the method described in claim 19.

22. The manufacturing method described in claim 21, wherein the tissue is used for evaluating the efficacy / toxicity of a test substance.