JP2024076356A - Cell reprogramming agent for producing stem cells and method for producing stem cells - Google Patents

Abstract

[Problem] To provide a cell culture auxiliary for producing stem cells, and a method for producing stem cells. [Solution] A cell reprogramming agent comprising an aqueous solution of a polypeptide represented by the following formula (1): (Pro-Hyp-Gly)n (1) (wherein Hyp represents hydroxyproline, and n represents an integer between 100 and 1000) [Selected Figure] None

Description

The present invention relates to a cell reprogramming agent for producing stem cells, and a method for producing stem cells using the cell reprogramming agent.

Currently, there are two main techniques for producing induced pluripotent stem cells from somatic cells. The first is related to induced pluripotent stem cells (iPS cells) created by Yamanaka, which uses a viral vector to overexpress transcription factor proteins OCT4, SOX2, KLF4, and c-MYC in fibroblasts and reprogram them (Non-Patent Document 1). The second is a direct somatic cell reprogramming technique using chemical compounds, which was first reported by Maekawa et al. (Non-Patent Documents 2, 3, 4). iPS cell technology has already been put to practical use in the field of regenerative medicine and in the application of drug discovery in diseased organ models, but the risk of tumor formation due to immune rejection or genetic mutation is a major issue. In addition, reducing the time and cost required for reprogramming iPS cells from the establishment of iPS cells to target organ cells is an important medical issue. On the other hand, chemical direct reprogramming technology does not require the worry of tumor formation due to genetic mutation, and organ regeneration can be achieved using only the patient's own somatic cells and a cocktail of chemical compounds, so it is expected to be applied to personalized regenerative medicine. However, at present, the types of organ cells that can be produced using this technology are limited to nerve cells and fat cells, and developing production methods remains a challenge.

In addition, it has been discovered in recent years that cancers originate from a very small number of cells called cancer stem cells, and that tumors form as a heterogeneous cell population in terms of proliferation and differentiation functions (Non-Patent Document 5). Like pluripotent stem cells present in healthy biological tissues, cancer stem cells are characterized by their ability to self-replicate and to differentiate into a variety of cells. During cancer treatment, many cancer cells can be removed by anticancer drugs, radiation, cancer immunotherapy, and surgery, but it is believed that recurrence and metastasis occur when a very small number of cancer stem cells that are highly resistant to treatment remain. Therefore, the development of new drugs, treatments, and diagnostic techniques that target cancer stem cells has become an important issue.

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

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; 474:225-229 Yang 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.

Induced pluripotent stem cells (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 foreign genes being incorporated into the chromosomes of iPS cells and causing tumor formation. In addition, it takes six months to a year to initialize fibroblasts to culture a large number of iPS cells, and then induces their differentiation into the cells of the desired organ and cultures the amount required for transplantation, which requires a lot of time and costs a lot of money.

Direct reprogramming, in which a cocktail of chemical compounds is added to the culture medium, is said to be able to directly produce pluripotent stem cells from somatic cells in a culture period of around 60 days. In addition, because small molecule compounds are used, there is no risk of foreign genes being incorporated into chromosomes or of tumor formation. Furthermore, by using autologous fibroblasts derived from the patient, the risk of immune rejection can be avoided, and hopes are high that this technology can be put to practical use in personalized regenerative medicine. However, the organ cell types that can be produced using this technology are still limited to nerve cells or fat cells, and it has not yet been put to practical use in the fields of medicine or drug discovery.

On the other hand, it is known that the presence of cancer stem cells worsens the prognosis of radiation therapy and chemotherapy treatment, but a method for isolating these cells has not yet been established, and they have not been medically examined. Methods have been attempted in which a small number of cells are extracted from a cancer patient specimen and then transplanted into an immunodeficient NOD mouse to create a tumor mass (PDOX), and then the cells are isolated using a cell sorter. Alternatively, culture methods have been attempted in which multiple recombinant protein factors or hormones, such as bFGF, EGF, and PDGF, are added, but a large-scale, stable method has not yet been established. The use of cancer stem cells is essential for high-throughput screening, efficacy evaluation, and safety evaluation of drug candidates.

The objective of the present invention is to provide a novel method for producing stem cells derived from somatic cells, etc.

In order to solve the above problems, the inventors have conducted extensive research. They have found that when cells are cultured using a chemically synthesized polypeptide that has a repeat sequence of only Pro-Hyp-Gly, an amino acid sequence that constitutes type I collagen, spheroids are formed. They have also found that by using the polypeptide as a cell reprogramming agent, somatic cells such as healthy human fibroblasts or mammary epithelial cells can be initialized (reprogrammed) to produce pluripotent stem cells with differentiation capabilities. They have also found that cancer stem cells can be produced from general cultured cancer cell lines (cells that do not have stem cell markers).

The present invention has been completed based on these findings, and includes the following broad aspects.
[Item 1]
A 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 100 to 1000.)
[Item 2]
Item 2. The cell reprogramming agent according to Item 1, which is used in the production of stem cells.
[Item 3]
Item 3. The cell reprogramming agent according to Item 2, wherein the stem cell is a pluripotent cell or a cancer stem cell.
[Item 4]
Item 2. The cell reprogramming agent according to Item 1, which is used for forming a spheroid.
[Item 5]
A spheroid-forming 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 100 to 1000.)
[Item 6]
Item 6. A cell culture substrate, the culture surface of which has been treated with the cell reprogramming agent according to any one of Items 1 to 4 or the spheroid forming agent according to Item 5.
[Item 7]
A cell culture system comprising the cell reprogramming agent 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 reprogramming agent according to any one of items 1 to 4 or the spheroid forming agent according to item 5.
[Item 9]
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]
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 an organoid or tissue using stem cells obtained by the method according to any one of items 8 to 10.

The present invention provides a cell reprogramming agent for producing stem cells. In addition, the novel cell reprogramming agent of the present invention provides a method for producing stem cells derived from somatic cells, etc. By using the stem cell production method of the present invention, the polypeptide of the cell reprogramming agent exerts a receptor/ligand action like 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 induces the action as a so-called stem cell niche factor, 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, and realizes personalized regenerative medicine, cell therapy, and transplantation therapy that does not involve the risk of tumor formation due to gene mutations like iPS cells and avoids immune rejection.

In addition, by using the cell reprogramming agent of the present invention, it becomes possible to produce cancer stem cells that express cancer stem cell marker genes and stem cell undifferentiation marker genes.

An example of using a cell reprogramming agent on a cell culture substrate (semipermeable membrane, hollow fiber). Semipermeable membrane, hollow fiber/microfluidic device. The cell reprogramming agent 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 reprogramming agent in a cell culture substrate (capsule). A capsule formed from the reprogramming agent itself or a capsule made from a biodegradable substrate. The cell reprogramming agent 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 reprogramming agent on a cell culture substrate (fibrous material, semipermeable membrane, hollow fiber). The cell reprogramming agent 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 medium, and spheroids are placed and cultured. An example of using a cell reprogramming agent on a cell culture substrate (embedding carrier based on a microcarrier/film/fibrous material). The cell reprogramming agent of the present invention is coated on the inside of a mesh-like, semipermeable, fibrous, or flexible film, and spheroids are cultured in a state of being wrapped, sandwiched, or wrapped. 2D adhesion culture and 3D spheroid culture of healthy human neonatal fibroblasts. (A: Planar 2D culture (culture day 2). In PS culture vessels (flat bottom) without a cell reprogramming agent, fibroblasts adhered to the bottom and proliferated. B: 3D culture (culture day 2). In PS culture vessels (flat bottom) treated with a cell reprogramming agent, fibroblasts aggregated to form spheroids on the second day of culture. C: 3D culture (culture day 20). In PS culture vessels (flat bottom) treated with a cell reprogramming agent, fibroblasts formed spherical spheroids.) When healthy human neonatal fibroblasts were cultured in a culture vessel (polystyrene; PS/6-well plate, flat bottom) coated with a cell reprogramming agent, the cells self-organized and aggregated over time, forming and growing spheroids from the second day until the 20th day. Expression of stem cell undifferentiation marker genes in healthy human neonatal fibroblast 3D spheroids. A cell reprogramming agent was applied to a culture vessel (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). A PS 96-well culture vessel (round bottom) was treated with a cell reprogramming agent and 40,000 fibroblasts were seeded. 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 was formed from day 6 onwards, and the cells maintained a good condition even after day 20 of culture.) A cell reprogramming agent was applied to a round-bottom culture vessel (PS/96-well microplate) and human neonatal fibroblasts were cultured as spheroids. The cells aggregated over time, forming spheroids that grew from day 2 to day 20. Time course of expression of stem cell undifferentiation marker genes in healthy human neonatal fibroblast 3D spheroids. When a cell reprogramming agent was applied to a round-bottom culture vessel (PS/96-well microplate) and healthy human neonatal fibroblast cells were cultured as spheroids, the mRNA expression levels of three stem cell undifferentiation marker genes (SOX2, OCT4, NANOG) increased over time, reaching a peak on the 20th day of culture. We found that it is possible to induce (reprogram) stem cells without introducing genes into somatic fibroblast cells (the Yamanaka method for producing iPS cells). Induction of differentiation of healthy human neonatal fibroblast-derived stem cells: Expression of genes related to differentiation into three germ layers. When healthy human neonatal fibroblasts were cultured as spheroids in a culture vessel coated with a cell reprogramming agent, the expression levels of the mRNAs of differentiation marker genes into the early three germ layers (SOX17; endoderm, HAND1; mesoderm, MAP2; ectoderm) increased over time and reached a peak on the 20th day. We found that stem cells can be induced to differentiate into the three germ layers at the early 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 stem cell marker proteins SOX2, OCT4, and NANOG, indicating the presence of stem cells in the spheroid-constituting cells. A: Fluorescence detected by NL557-labeled anti-SOX2 antibody. B: Fluorescence detected by NL637-labeled anti-OCT4 antibody. C: Fluorescence detected by NL493-labeled anti-NANOG antibody. NANOG protein-positive stem cells present within spheroids. Spheroids cultured for 20 days were dispersed with Accutase enzyme reagent and immunostained with fluorescent dye-labeled anti-NANOG antibody. Stem cells with intranuclear NANOG-positive were found to be induced in some of the spheroid-forming cells. A: Bright field image of some of the cells dispersed from the 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), comparison image showing intranuclear DNA stained blue to show the shape of the nuclei. 2D and 3D spheroid culture of adult skin fibroblasts. Using adult skin fibroblasts, which are minimally invasive and easy to collect from the human body, we demonstrated that spheroid culture is possible using the cell reprogramming agent of the present invention. A: Planar 2D culture, adult skin fibroblasts adhered to the bottom of a PS culture vessel and proliferated. B: 3D culture (20th day of culture), adult skin fibroblasts formed spheroids in a 96-well culture vessel treated with a cell reprogramming agent. Expression analysis of undifferentiated stem cell marker genes in 3D spheroid culture of adult skin fibroblasts. We demonstrated that stem cells can be induced by cell reprogramming agent spheroid culture method using skin-derived fibroblasts, which are the least invasive and easiest to collect samples, essential for implementing personalized regenerative therapy. A: SOX2. B: OCT4. C: NANOG. Expression analysis of three germ layer differentiation-related genes in three-dimensional spheroid culture of adult skin fibroblasts. We demonstrated that stem cells can be induced by cell reprogramming agent spheroid culture method using skin-derived fibroblasts, which are the least invasive and easiest to collect samples, essential for implementing personalized regenerative therapy. A: SOX17. B: GATA6. C: HAND1. D: SOX1. 2D adhesion culture and 3D spheroid culture of healthy human adult mammary epithelial cells. (A: 2D culture (culture day 2). In PS culture vessels (flat bottom) without cell reprogramming agents, mammary epithelial cells adhered to the bottom and proliferated. B: 3D culture (culture day 2). In PS culture vessels (flat bottom) treated with cell reprogramming agents, mammary epithelial cells formed spheroids. C: 3D culture (culture day 20). Mammary epithelial cell spheroids on culture day 20 in a 96-well culture vessel (round bottom) treated with a cell reprogramming agent.) When healthy human adult mammary epithelial cells were cultured in PS culture vessels (flat or round bottom) treated with a cell reprogramming agent, the cells aggregated over time and formed spheroids and grew from day 2 to day 20. This demonstrated that this method can be used with a wide variety of cells, including adult-derived and neonatal-derived cells, as well as epithelial cells and fibroblasts. Detection of stem cell undifferentiation marker genes in 3D spheroid culture of healthy adult human mammary epithelial cells. When healthy adult human mammary epithelial cells were cultured in a culture vessel coated with a cell reprogramming agent to form spheroids, the mRNA expression levels of three representative stem cell undifferentiation markers (SOX2, OCT4, NANOG) increased and reached a maximum value on the 20th day of culture. We found that it is possible to induce stem cells (direct-reprogramming) without introducing genes into somatic epithelial cells. Induction of differentiation of stem cells derived from healthy adult human mammary epithelial cells: Expression of genes related to the three germ layers. When healthy adult human mammary epithelial cells were cultured for 20 days in a culture vessel coated with a cell reprogramming agent to form spheroids, the mRNA expression levels of SOX17 (endoderm) and MAP2 (ectoderm), among the early three germ layer marker genes, reached their highest levels on the 20th day. Meanwhile, FOXA2 (endoderm) 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. 2D adhesion and 3D spheroid growth of cancer cells with and without cell reprogramming agents. When three types of cancer cells (A549 cells; human lung cancer, ES2 cells; human ovarian cancer, 143B; human osteosarcoma) were cultured in a culture vessel (polystyrene; PS/6-well plate, flat bottom) coated with a cell reprogramming agent, the cells aggregated over time and formed spheroids and grew by the 15th day. The relationship between cell reprogramming agent concentration and spheroids is shown. The results of spheroid formation in various microplates are shown. The quantitative results of apoptosis induction in spheroids prepared in U-shaped plates are shown. The quantitative results of apoptosis induction in spheroids prepared using V-shaped plates are shown. 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 a cell reprogramming agent, 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 a cell reprogramming agent, 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. It was 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 a culture vessel coated with a cell reprogramming agent, the expression of two of the three cancer stem cell marker gene mRNAs (CD44, CD133) increased over time, reaching a peak on the 15th day of culture. CD24 did not increase significantly. We found that it was possible to create 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 a culture vessel coated with a cell reprogramming agent, the mRNA expression levels of three representative stem cell undifferentiation marker genes (SOX2, OCT4, NANOG) increased over time, reaching a peak on the 15th day 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 a cell reprogramming agent, the mRNA expression levels of three undifferentiated stem cell undifferentiation marker genes (SOX2, OCT4, NANOG) increased over time, reaching a peak on the 15th day 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 a cell reprogramming agent, the mRNA expression levels of three stem cell undifferentiation marker genes (SOX2, OCT4, NANOG) increased from the 4th to 15th days of culture. Electrophoresis results of polypeptide fractions in Example 13. Spheroid formation activity of ultrafiltration fraction polypeptide in Example 13. No coating: Cell culture without applying polypeptide to plate. Sample (A): Cell culture with ultrafiltration fraction sample (A) applied to plate. Sample (B): Cell culture with ultrafiltration fraction sample (B) applied to plate. Sample (C): Cell culture with ultrafiltration fraction sample (C) applied to plate. Sample (E): Cell culture with ultrafiltration fraction sample (E) applied to plate. Sample (F): Cell culture with ultrafiltration fraction sample (F) applied to plate.

<Cell reprogramming agents>
The cell reprogramming agent of the present invention comprises 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 100 to 1000.)
n represents an integer of 100 to 1000. The lower limit of n is preferably 150, more preferably 200, even more preferably 250, particularly preferably 300, and most preferably 350. The upper limit of n is preferably 900, more preferably 800, even more preferably 700, particularly preferably 600, and most preferably 500. When n is 50 or less, particularly 20 or less, reprogramming is not substantially induced.
It is known that the polypeptide forms a complex and assumes a triple helical structure. Whether or not the polypeptide assumes a triple helical structure can be confirmed by measuring the circular dichroism spectrum of the polypeptide solution. Specifically, if the polypeptide exhibits a positive Cotton effect at wavelengths of 220-230 nm and a negative Cotton effect at wavelengths of 195-205 nm, it is considered that the polypeptide assumes a triple helical structure.

The cell reprogramming agent of the present invention can function as a stem cell niche factor involved in cell proliferation, differentiation, reprogramming (initialization), and apoptosis control. The niche factor activity of the cell reprogramming agent of the present invention can be reproduced not only in vivo but also in vitro (in a test tube), and can induce spheroid formation by self-organization in cultured somatic cells, bind as a ligand to multiple types of cell membrane receptors, and induce signal transduction and gene expression. In this specification, the term "stem cell niche factor" refers to a factor involved in a one-to-one reaction between proteins (e.g., FGF, EGF, Activin, BMP, Noggin, TGF-β, Wnt, R-Spondin, etc.) that maintain the pluripotency of tissue stem cells, ES cells, and iPS cells, induce differentiation, etc., and their receptors; a microenvironment surrounded by extracellular matrix such as collagen and laminin, and factors that constitute them, etc.

In one embodiment, the cell reprogramming agent of the present invention can be used when producing stem cells from somatic cells, etc. By using the cell reprogramming agent of the present invention in cell culture, cultured cells (somatic cells, etc.) form spheroids, making it possible to simply and efficiently produce stem cells.

In this specification, the term "spheroid" (also called a cell mass) refers to a three-dimensional collection of multiple cells that are adhered to each other.

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, 1 to 5, 1 to 10, or 1 to 20)
The peptide oligomer represented by the following formula (1) can be produced by subjecting the peptide oligomer represented by the following formula (1) to a condensation reaction.

The cell reprogramming agent 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 contains about 0.01 to 0.25% by weight (w/v). The cell reprogramming agent 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).

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

<Use as a surface treatment agent or additive>
In one embodiment, the cell reprogramming agent of the present invention can be used for surface treatment of the culture surface of a cell culture substrate.

Such cell culture substrates are not particularly limited, and examples thereof 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. Examples of cell culture substrates that can be used include glass, metals, plastics such as polystyrene (PS), polypropylene (PP), polycarbonite (PC), polyethylene terephthalate (PET), polytetrafluoroethylene (PTFE), polyvinylidene fluoride (PVDF), cycloolefin (COP), acrylic/methacrylic (PMMA), hydrophilic fluoride resins, polylactic acid (PLA), polybutylene succinate (PBS), polyhydroxybutyrate/hydroxyhexanoate (PHBH), cellulose membranes, carbon nanotubes, cellulose nanofibers, etc.

Examples of the use of a cell reprogramming agent on a cell culture substrate are shown in Figures 1 to 4, but are not limited to these.

Figure 1 shows an example of application to a semipermeable membrane and hollow fibers. As shown in Figure 1, the cell reprogramming agent of the present invention can be used as a microfluidic device by coating the inside of a semipermeable membrane or hollow fibers or adding it to the culture medium. Such a microfluidic device is expected to be applied in fields such as (i) reproduction of organ functions such as kidney and liver, (ii) extracorporeal circulation organ (carrier type) medical devices such as kidney and liver, (iii) production of genetically modified proteins such as monoclonal antibodies, and (iv) drug discovery model/pharmacological evaluation, toxicity evaluation, etc.

Figure 2 shows an example of application to capsules. As shown in Figure 2, the cell reprogramming agent of the present invention can be used as a gel-like solid substrate, into which cells can be embedded 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 differentiated organoids into the body, (ii) transfusion of hematopoietic stem cells, and (iii) manufacturing of artificial muscles.

Figure 3 shows examples of application to fibrous materials, semipermeable membranes, and hollow fibers. As shown in Figure 3, the cell reprogramming agent of the present invention can be coated on the inner cavity of a biodegradable substrate, semipermeable membrane, hollow fiber, or fibrous material, or added to the medium, and spheroids can be placed and cultured. Such fibrous materials, semipermeable membranes, and hollow fibers are expected to be applied in fields such as (i) organs for regenerative medicine: transplantation and regeneration of thin organs such as blood vessels, nerves, intestines, and renal tubules, and (ii) sensor circuits for nerve-related electrical potentials, light, odor components, taste components, etc.

Figure 4 shows an example of application to microcarriers, etc. As shown in Figure 4, the cell reprogramming agent 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 in which they are wrapped, sandwiched, or wrapped. Such macrocarriers, etc. are expected to be applied in the field of transplantation and regeneration of pancreas, kidney, liver, bone marrow, bone, cartilage, muscle, skin, hair, etc. for regenerative medicine.

When the culture surface of a cell culture substrate is treated with the cell reprogramming agent 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 reprogramming agent 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.

The culture surface of the cell culture substrate refers to the surface that comes into contact with the cells during culture. A part or all of the culture surface can be surface-treated. In addition, surfaces other than the culture surface can also be surface-treated.

The surface treatment is not particularly limited as long as it is a means capable of immobilizing (e.g., coating) the polypeptide represented by formula (1), which is the active ingredient of the cell reprogramming agent of the present invention, on the culture surface of the cell culture substrate. Examples include application, immersion, spraying, etc. 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 reprogramming agent of the present invention. In addition, when using the cell reprogramming agent of the present invention for surface treatment, known additives may be mixed and used.

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 the active ingredient of the cell reprogramming agent 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).

When a cell culture substrate is surface-treated using the cell reprogramming agent of the present invention, the substrate can be dried to remove water after the surface treatment. There are no particular limitations on the means for drying, and drying can be performed, for example, under reduced pressure or using a desiccant.

Furthermore, after the drying, the cell culture substrate can be sterilized. The means for sterilization are not particularly limited, and can be, for example, ethylene oxide gas, electron beam, gamma ray, autoclave, etc.

In another embodiment, the cell reprogramming agent of the present invention can be added to a culture medium for use.

As the culture medium to which the cell reprogramming agent of the present invention is added, liquid media, gel media, etc. used in normal cell culture can be widely used. The medium may contain serum/plasma additives such as FBS and FCS, which are animal-derived components, and protein additives such as albumin. Examples 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, Fischer's medium, etc. Furthermore, examples of xeno-free or serum-free culture 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, Mammary Epithelial Cell Growth medium, etc. Examples of gel-like hydrogel culture media include, but are not limited to, soft agar medium, Matrigel medium, and Methylcellulose-based medium. In addition, the culture media may contain other known additives.

The amount of the cell reprogramming agent of the present invention added to the culture medium is not particularly limited, and the final concentration of the polypeptide represented by formula (1), which is the active ingredient of the cell reprogramming agent 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).

In addition, a cell culture substrate surface-treated with the cell reprogramming agent of the present invention can be combined with a culture medium to which the cell reprogramming agent of the present invention has been added and used for cell culture.

<Spheroid forming agent>
In another embodiment, the cell reprogramming agent of the present invention, which comprises the polypeptide represented by formula (1), can be used as a spheroid-forming agent.

As a method for forming spheroids, cells can be cultured using a cell culture instrument having the cell reprogramming agent of the present invention on its culture surface, or cells can be cultured in a culture medium to which the cell reprogramming agent of the present invention has been added. Note that the methods described in "<Use as a surface treatment agent and additive>" can be preferably used as a method for obtaining a cell culture instrument having the cell reprogramming agent of the present invention on its culture surface, and a method for obtaining a culture medium to which the cell reprogramming agent of the present invention has been added.

The cells used for spheroid formation are not particularly limited, and examples include primary cultured cells of somatic cells derived from various tissues such as fibroblasts derived from skin tissues, 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, their original functions are lost and abnormal activation is often induced, and apoptosis (programmed cell death) called anoikis is also induced, which is a problem. In this situation, it has been confirmed that when spheroids are formed from primary cultured cells using the cell reprogramming agent of the present invention, the occurrence of apoptosis is suppressed, and the physiological functions are maintained while supporting survival and proliferation. For this reason, it is expected that spheroid-formed cells can maintain their biological functions to a high degree compared to cells cultured on a plate, and a model reflecting biological functions can be provided. On the other hand, it is also useful to form spheroids from established cancer cells. It is known that when cancer cells are cultured in spheroids, the "malignant tumor functions" when they exist in the patient's body can be reproduced, and the activity of enzymes, pump receptors, etc. that cause drug resistance to anticancer drugs can be maintained. For this reason, cancer cell spheroids are expected to be used in drug discovery research as a cancer patient model that can reproduce the pathology. Furthermore, in the case of differentiation induction of iPS cells, a method is often used in which spheroids are first cultured to create embryoid bodies, and then differentiated into each organ. In this case, it is known that the formation of spheroids promotes differentiation into the three germ layers (endoderm, mesoderm, and ectoderm), and the promotion of the differentiation induction process into organ cells is a major benefit.

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 mixed media thereof. The concentration of serum (e.g., fetal bovine serum (FBS), human serum, etc.) to be added is 2 to 20%, but 5 to 10% is preferably used.

Serum-free medium (SFM) refers to a medium that does not contain animal-derived components (xeno-free) or raw or unpurified serum (serum-free), and includes media containing recombinantly produced growth factors (basic-FGF, EGF, insulin, etc.). SFM may contain any serum substitute. Examples of serum substitutes include KnockOut Serum Replacement, albumin (e.g., albumin substitutes such as lipid-rich albumin, recombinant albumin, vegetable 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 of two or more.

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.), nutritional additives (e.g., B27 supplement, N2 supplement, StemPro-Nutrient Supplement, KnockOut Media, GlutaMax, etc. These may be used alone or in combination of two or more. Each additive is preferably contained within a known concentration range.

The culture conditions for spheroid formation are not particularly limited as long as the desired spheroids are formed. For example, the culture temperature can be 30 to 45°C, preferably 34 to 38°C. The culture can be performed in an atmosphere of 0.05 to 10% _CO2 , preferably 3 to 10% _CO2 , more preferably 4 to 6% _CO2 . The culture period for forming spheroids is, for example, about 0.5 to 100 hours, preferably about 0.5 hours to 14 days, more preferably about 1 day to 4 days. Optionally, the medium can be replaced once or more during the culture period.

The size of the spheroids is usually about 10 to 500 μm in diameter, preferably about 50 to 200 μm. If the spheroids become too large, it becomes difficult for nutrients to penetrate into the interior, so it is desirable to separate the spheroids when 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 reprogramming agent of the present invention, allowing spheroids to be repeatedly formed.

The cell concentration in culture is not particularly limited as long as spheroids are efficiently formed, but examples include about 1 × 10 ³ to about 1 × 10 ⁷ cells/well, and preferably about 3 × 10 ³ to about 5 × 10 ⁶ cells/well.

In the method for forming spheroids using the cell reprogramming agent of the present invention, it is believed that the polypeptide of the cell reprogramming agent of the present invention exerts receptor/ligand action like 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.

In another aspect of the present invention, a cell culture system is provided that includes the cell reprogramming agent of the present invention and cultured cells. As the cultured cells, those listed above as cells used for spheroid formation are widely used. When producing pluripotent stem cells or cancer stem cells using the culture system, somatic cells or cancer cells, respectively, are preferably used.

In another aspect of the present invention, a method for producing stem cells is provided, which comprises a step of culturing somatic cells or the like using the cell reprogramming agent of the present invention.

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, mesenchymal stem cells, etc. Among these, the cell reprogramming agent of the present invention is preferably used for producing pluripotent stem cells and cancer stem cells. The method for producing pluripotent stem cells and cancer stem cells is described in detail below.

<Method for producing pluripotent stem cells>
In another aspect of the present invention, there is provided a method for producing pluripotent stem cells, comprising culturing somatic cells with the cell reprogramming agent of the present invention. By culturing somatic cells with the cell reprogramming agent of the present invention, the cultured cells form spheroids, and pluripotent stem cells can be produced simply and efficiently.

Methods of using the cell reprogramming agent of the present invention when producing pluripotent stem cells include, but are not limited to, using the cell reprogramming agent of the present invention to surface treat the culture surface of a cell culture substrate and/or adding the cell reprogramming agent 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 reprogramming agent of the present invention. The use of the cell reprogramming agent to surface treat the culture surface of a cell culture substrate and the use of the cell reprogramming agent by adding it to a culture medium are the same as those described in the section <Use as a surface treatment agent and additive>.

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 (humans, mice, rats, cows, pigs, monkeys, sheep, rabbits, dogs, etc.), and can be widely used. Among these, humans, monkeys, and mice are preferred, and humans are particularly preferred. The type of somatic cells is also not particularly limited, and a wide range of cells can be used, including fibroblasts, epithelial cells, chondrocytes, muscle cells, bone cells, liver cells, pancreatic cells, intestinal cells, nerve cells, bone marrow cells, and skin cells. In addition, somatic cells derived from healthy individuals and somatic cells derived from patients with diseases can be used in the production of the pluripotent stem cells of the present invention.

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.

Furthermore, in the method of producing pluripotent stem cells of the present invention, there is no need 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, and retinoic acid. By using the cell reprogramming agent of the present invention, it is possible to produce pluripotent stem cells from somatic cells without adding cell growth factors to the medium.

The method for producing pluripotent stem cells can be carried out in accordance with the usual method for forming spheroids, except that the cells are cultured using a cell culture substrate having the cell reprogramming agent of the present invention on its culture surface. Examples of the method include the non-adhesive surface cell culture method, the hanging drop cell culture method, the use of micromolding techniques, and the rotary cell culture method, with the non-adhesive surface cell culture method being particularly preferred.

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. The culture can be performed 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 medium can be replaced at least once during the culture period. Optionally, the cell culture substrate can be replaced at least once during the culture period.

The size of the spheroids used in the production of pluripotent stem cells is usually about 10 to 500 μm in diameter, preferably about 50 to 200 μm. If the spheroids become too large, it becomes difficult for nutrients to penetrate into the interior, so it is desirable to separate the spheroids when 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 reprogramming agent of the present invention to form spheroids that function as pluripotent stem cells again.

The concentration of somatic cells in the culture is not particularly limited as long as pluripotent stem cells are efficiently produced, but examples include about 1 x ¹⁰³ to about 1 x ¹⁰⁷ cells/well, preferably about 3 x ¹⁰³ to about 5 x ¹⁰⁶ cells/well.

In the method for producing pluripotent stem cells of the present invention, it is possible to confirm that pluripotent stem cells have been produced by confirming the expression of stem cell undifferentiation markers. Examples of stem cell undifferentiation markers include 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.

Furthermore, whether the pluripotent stem cells obtained by the present invention have the ability to induce differentiation can be confirmed by confirming the three germ layers-related genes. Examples of genes related to the three germ layers (endoderm, mesoderm, and ectoderm) include SOX17, FOXA2, Nestin, Flk1/KDRAFP, Cripto, Bmp4/Wnt3, GATA4, GATA6, Brachyury, HAND1, CXCR4, SOX1, Otx-2, Pax2, Pax6, and MAP2.

The pluripotent stem cells obtained by the manufacturing method of the present invention may be a heterogeneous population of cells including 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) produced by a chemical cocktail (various cytokines, hormones, agonists, inhibitors, etc.), and even cells with characteristics of embryoid bodies obtained by suspension culture of iPS cells.

The method for producing pluripotent stem cells in the present invention allows pluripotent stem cells to be produced in a very short period of time (approximately 10 to 40 days) compared to the six months to a year required for culturing induced pluripotent stem cells (iPS cells). In addition, since there is no incorporation of foreign genes, as is known with induced pluripotent stem cells (iPS cells), there is no risk of tumor formation during transplantation. In addition, it is possible to easily produce autologous pluripotent stem cells using somatic cells such as fibroblasts, which are easy to collect even from human skin tissue, and therefore the problem of immune rejection can be avoided. Therefore, when considering the application of iPS cells in transplantation therapy, it is considered that the classification of iPS cells under the Regenerative Medicine Safety Act in Japan will be Type 2 regenerative medicine (medium risk) rather than Type 1 regenerative medicine (high risk).

Furthermore, the method for producing pluripotent stem cells in the present invention allows pluripotent stem cells to be produced in a shorter time than the production period (approximately 60 days) required by the direct reprogramming method. In addition, since the method for producing pluripotent stem cells in the present invention is a direct reprogramming method using spheroid formation, it has a wider range of applications in terms of the selection of tissue materials used for establishment, expansion culture methods, etc., than the direct reprogramming method using a compound cocktail.

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

<Method of producing cancer stem cells>
In another aspect of the present invention, there is provided a method for producing cancer stem cells, comprising culturing cancer cells using the cell reprogramming agent of the present invention. By culturing cancer cells using the cell reprogramming agent of the present invention, the cells form spheroids, and cancer stem cells can be produced simply and efficiently.

Methods of using the cell reprogramming agent of the present invention when producing cancer stem cells include, but are not limited to, using the cell reprogramming agent of the present invention to surface treat the culture surface of a cell culture substrate and/or adding the cell reprogramming agent 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 reprogramming agent of the present invention. The use of the cell reprogramming agent to surface treat the culture surface of a cell culture substrate and the use of the cell reprogramming agent by adding it to a culture medium are the same as those described in the section <Use as a surface treatment agent and additive>.

The cancer cells used in the production of the cancer stem cells of the present invention are not particularly limited as long as they are derived from mammals (humans, mice, rats, cows, pigs, monkeys, sheep, rabbits, dogs, etc.), and can be widely used. Among them, humans, monkeys, and mice are preferred, and humans are particularly preferred. The type of cancer is also not particularly limited, and a wide range of cancers can be used, including, for example, colon cancer, breast cancer, ovarian cancer, cervical cancer, endometrial cancer, prostate cancer, stomach 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. In addition, a wide range of cancers can be used, including established cell lines, primary cultured cells derived from patient specimens, patient specimen-derived passaging-capable cells (PDCC), cancer cells derived from animal transplanted cancer tissues (PDOX), and cancer cells derived from experimental animals.

As a medium 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.

In addition, in the method for producing cancer stem cells of the present invention, there is no need to add various differentiation and proliferation auxiliary agents 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, and retinoic acid. By using the cell reprogramming agent of the present invention, it is possible to produce cancer stem cells from cancer cells without adding cell growth factors to the medium.

The method for producing cancer stem cells can be carried out in accordance with the usual method for forming spheroids, except that the cells are cultured using a cell culture substrate having the cell reprogramming agent of the present invention on the culture surface. Examples include the non-adhesive surface cell culture method, the hanging drop cell culture method, the use of micromolding techniques, and the rotary cell culture method, with the non-adhesive surface cell culture method being particularly preferred.

The culture conditions for producing cancer stem cells are not particularly limited, but for example, the culture temperature can be 20 to 40°C, preferably 34 to 38°C. The culture can be performed 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 medium can be replaced at least once during the culture period. Optionally, the cell culture substrate can be replaced at least once during the culture period.

The size of the spheroids used in the production of cancer stem cells is usually about 10 to 500 μm in diameter, preferably about 50 to 200 μm. If the spheroids become too large, it becomes difficult for nutrients to penetrate into the interior, so it is desirable to separate the spheroids when they reach an appropriate size. The obtained spheroids can be disaggregated and then cultured again using the cell reprogramming agent of the present invention to form spheroids that once again function as cancer stem cells.

The concentration of cancer cells in the culture is not particularly limited as long as cancer stem cells are efficiently produced, but examples include about 1 x ¹⁰³ to about 1 x ¹⁰⁷ cells/well, preferably about 3 x ¹⁰³ to about 5 x ¹⁰⁶ cells/well.

In the method for producing cancer stem cells of the present invention, it is possible to confirm that cancer stem cells have been produced by confirming the expression of cancer cell surface markers. Examples of cancer cell surface markers include CD133, CD44, CD24, ABCB5, EAS, CD34, CD38, CD90, CD117, and CXCR4.

In addition, the expression of stem cell undifferentiation markers can be confirmed in the cancer stem cells obtained by the manufacturing method of the present invention. Examples of stem cell undifferentiation markers include 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.

The 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, and may further contain cancer cells.

The uses of the cancer stem cells obtained by the manufacturing method of the present invention are not particularly limited, and they can be used for various tests and research, disease treatment, etc.

<Induction of stem cell differentiation>
By inducing differentiation of the pluripotent stem cells obtained by the production method of the present invention using a known method, 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.

Differentiation induction can be appropriately performed using any cell differentiation induction method with reference to the 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; issue7,Vol.6, ).

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

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

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) (UNIX Corporation, PURECOLLA) (cell reprogramming agent) was added to a commercially available polystyrene 6-well flat-bottom culture vessel (CellBIND, Corning) and left to stand at 4°C for 24 hours.

After that, excess cell reprogramming agent was removed, and the cells were dried in a dryer at 37 °C for 96 hours to complete the cell reprogramming agent application process. Healthy human neonatal fibroblasts (Lonza: Normal Human Dermal Fibroblasts-Neonatal, hereinafter 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). Fibroblasts suspended in 2 mL of the above medium were seeded in the above cell reprogramming agent-coated culture vessel at 2 × ^{10 6 cells} /well and cultured under 37 °C and 5% CO ₂ conditions. As a control, fibroblasts were seeded in the same type of culture vessel without the cell reprogramming agent. Microscopic observation was performed on the 2nd and 20th days after the start of culture, and the spheroid formation status was photographed (Figure 5).

Gene mRNA expression analysis was performed using real-time PCR. Fibroblasts were cultured as spheroids using the same method as above, and on day 20 (Day 20), the spheroids were collected in a 15 mL centrifuge tube and centrifuged, and the cells were washed and collected with phosphate buffer PBS(-). For the control cells cultured in a culture vessel without a cell reprogramming agent (Day 0), the medium was removed and the cells were washed with PBS(-), 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 expression analysis of stem cell undifferentiation marker genes was performed using real-time PCR.

Specifically, NucleoZOL (Machrei-Nagel) reagent was added to each cell, and total-RNA was extracted according to the protocol recommended by the reagent manufacturer, and used as template RNA for stem cell undifferentiation marker detection. For real-time PCR analysis, QuantiFast Probe RT-PCR Kits (QIAGEN; No. 204454) and TaqMan Gene Expression Assay Probe (Thermo Fisher Scientific) PCR probes were used, and PCR analysis was performed using a Rotor-Gene Q (QIAGEN) device. The genes that were detected included cytokines and chemokines that are expected to be involved in spheroid formation, as well as three types of transcription factors used in iPS cell production (Non-Patent Document 1) as probes for detecting the expression of stem cell undifferentiation marker genes: SOX2 (sex-determining region Y-box 2), OCT4 (octamer binding transcription factor 4), and NANOG (homeodomain transcription factor), and β-Actin was used as an endogenous control gene. In gene expression analysis, relative quantification was performed using the ΔΔCt method (delta delta Ct method). The results of the ΔΔCt method analysis were calculated by setting the amount of genes expressed by the control cells in "no cell reprogramming agent, planar 2D growth day 0" at a relative value of "1.0," and calculating the increase rate of the "expression amount on day 20 of spheroid growth" (Figure 6).

(result)
Figure 5 shows the results of spheroid culture of fibroblasts using culture vessels treated with a cell reprogramming agent. In a control vessel without a cell reprogramming agent, fibroblasts adhered to the bottom and proliferated two-dimensionally (culture day 2). On the other hand, in a culture vessel coated with a cell reprogramming agent, 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.

Figure 6 shows the results of real-time PCR of gene mRNA. In fibroblast spheroids on the 20th day of culture, the gene expression levels of marker proteins SOX2, OCT4, and NANOG, which define the properties of stem cells, increased, and it was found that the fibroblast spheroids had acquired the properties of stem cells. Specifically, the gene expression levels of SOX2, OCT4, and NANOG in fibroblasts cultured without a cell reprogramming agent (control) were set at 1.0, and the gene expression levels of SOX2, OCT4, and NANOG on the 20th day of 3D spheroid culture were compared relatively using the ΔΔCt method. As a result, SOX2 increased 34.4-fold, OCT4 increased 5.1-fold, and NANOG increased 9.1-fold.

(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 adhesion to cells, so the cells adhere to the bottom of the vessel and grow (two-dimensional adhesion and growth). In contrast, when the vessel is coated with an aqueous solution of the polypeptide represented by formula (1), the cells do not adhere to the bottom, but move around among themselves and aggregate with each other to form spheroids, which are three-dimensional cell aggregates. Since the human body is constructed as a three-dimensional aggregate of cells and life phenomena are carried out, three-dimensional cultures reflect in vivo tissues better than two-dimensional cultured cells, and various physiological functions and gene expression are controlled.

The results in Figure 6 show that when fibroblasts are cultured as spheroids in a culture vessel treated with a cell reprogramming agent, stem cells with the characteristics of pluripotent stem cells can be induced without the introduction of genes used in conventional iPS cell production methods, and without the use of chemical cocktails used in direct-reprogramming with chemicals.

In other words, we have discovered a new cell reprogramming agent spheroid culture method that can induce cells with the characteristics of pluripotent stem cells from somatic fibroblast cells using a novel direct-reprogramming mechanism.

The increase in mRNA expression level of the stem cell undifferentiation marker is not limited to this value. In addition, other than 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.

Example 2: Induction of differentiation of stem cells generated from healthy human neonatal fibroblasts into the three early germ layers (Method)
To observe the spheroid formation and stem cell induction of healthy human neonatal fibroblasts over time, culture was performed using a 96-well round-bottom culture vessel. An aqueous solution (0.125%) of the polypeptide represented by formula (1) (Unix Corporation, PURECOLLA) (cell reprogramming agent) was added at 100μL/well to a commercially available polystyrene 96-well round-bottom culture vessel (Nunclon Delta 96U Bottom, Thermo Scientific) and left at 4℃ for 24 hours. After that, excess cell reprogramming agent was removed, and the cells were dried in a dryer at 37℃ for 96 hours to complete the cell reprogramming agent application process. Fibroblasts were maintained and cultured in serum-free medium (Promo Cell; Fibroblast Basal Medium, basic-FGF 5 ng/mL and Insulin 2.5μg/mL). Fibroblasts suspended in the above-mentioned medium were seeded at 4 x ¹⁰⁴ cells/well/100 µL into the cell reprogramming agent-coated culture vessel and cultured under conditions of 37°C and 5% _CO2 .

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

To investigate the mechanism of expression of the stem cell undifferentiation marker genes found 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).

To investigate whether stem cells induced from fibroblasts have the ability to induce differentiation, we performed expression analysis of three germ layer-related genes that indicate the early stage of differentiation. Fibroblasts were cultured in the same manner as above using a 96-well culture vessel treated with a cell reprogramming agent. The probes for detecting the expression of the three germ layer-related genes were three germ layer marker genes: SOX17 (sex-determining region Y-box 17; endoderm), HAND1 (heart-and neural crest derivatives-expressed protein-1; 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, and real-time PCR analysis was performed. In the expression analysis, relative quantification was performed using the ΔΔCt method (delta delta Ct method) (Figure 9).

(result)
The results are shown in Figure 7. The growth of fibroblast spheroids over time in a 96-well round-bottom culture vessel. By using a 96-well round-bottom culture vessel treated with a cell reprogramming agent, the cells formed a single spheroid, allowing the gene expression and physiological activity of each spheroid to be measured uniformly.

Figure 8 shows the results of real-time PCR of stem cell undifferentiation marker mRNA. As a result, it was found that the gene expression levels of stem cell undifferentiation markers SOX2, OCT4, and NANOG increased over time from the fifth day of culturing fibroblast spheroids.

Specifically, the gene expression levels of SOX2, OCT4, and NANOG in fibroblasts cultured in 2D adhesion (Day 0) without the cell reprogramming agent as a control were set at 1.0 relative to each other, and the gene expression levels of SOX2, OCT4, and NANOG on Day 5 (Day 5), Day 10 (Day 10), and Day 20 (Day 20) after 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.

Figure 9 shows the results of real-time PCR of the mRNA of genes related to the three germ layers. As a result, it was found that the gene expression levels of the marker proteins SOX17, HAND1, and MAP2, which define differentiation into the three germ layers, increased from the fifth day onwards when fibroblast spheroid culture was started.

Specifically, the gene expression levels of SOX17, GATA6, and MAP2 in fibroblasts cultured in 2D adhesion (Day 0) without the cell reprogramming agent as a control were set at a relative value of 1.0, and the gene expression levels of SOX17, HAND1, and MAP2 on Day 5 (Day 5), Day 10 (Day 10), and Day 20 (Day 20) in 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, HAND1 increasing 4.2-fold, and MAP2 increasing 28.6-fold.

(Discussion)
In Figure 7, we showed that it is possible to directly reprogram stem cells with the characteristics of pluripotent stem cells from somatic fibroblasts. Furthermore, we showed that this can be achieved in both flat-bottom and round-bottom culture vessels.

In Figure 8, when fibroblasts are cultured as spheroids in a 96-well culture vessel treated with a cell reprogramming agent, a single homogenous spheroid can be produced, enabling precise gene expression analysis. As a result, we found that direct reprogramming is possible to initialize pluripotent stem cells with enhanced expression of SOX2, OCT4, and NANOG genes without using the method of introducing transcription factor genes used in iPS cell production, and without adding to the culture medium a compound cocktail used in direct reprogramming with compounds. In other words, we have invented a new direct reprogramming method to produce cells with the characteristics of pluripotent stem cells from somatic fibroblast cells using a spheroid culture method with a cell reprogramming agent.

The results in Figure 9 show that stem cells created from fibroblasts can be induced to differentiate into cells with the characteristics of embryoid bodies, which are three-dimensional cell clusters. In general, in order to induce differentiation from stem cells into mature organ cells, a process is required in which the stem cells are first induced to differentiate into the germ layer of the target organ lineage. This culture method uses a mechanism different from the conventional iPS cell method or direct-reprogramming method using chemical compounds, and can be said to be a new culture method that brings about the initialization (direct-reprogramming) of somatic cells into stem cells and at the same time realizes the induction of differentiation into embryoid bodies that are positive for SOX17 (endodermal marker gene), HAND1 (mesoderm marker gene), and MAP2 (ectoderm marker gene).

Note that, 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, HAND1, and MAP2, and the present invention is not limited to these marker genes. Furthermore, the gene expression increase rates of the stem cell undifferentiation markers and the three germ layer markers are not limited to these values.

Example 3: Expression of stem cell marker proteins in healthy human neonatal fibroblast spheroids (Methods)
To confirm whether stem cells were induced in the fibroblast spheroids, the expression of stem cell marker proteins was examined by immunostaining using fluorescent dye-labeled antibodies. An aqueous solution (0.125%) of a polypeptide represented by formula (1) (Unix Corporation, PURECOLLA) (cell reprogramming agent) was added at 100 μL/well to a commercially available 96-well round-bottom culture vessel made of polystyrene (Nunclon Delta 96U Bottom, Thermo Scientific), and the cell reprogramming agent coating process was completed 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). Fibroblasts ( ⁴ x 104 cells/well/100μL) suspended in the above medium were seeded on the cell reprogramming agent-coated culture vessel and cultured under 37℃ and 5% _CO2 conditions. On the 20th day after the start of culture, spheroids were collected from the 96-well round-bottom culture vessel, fixed with 4% paraformaldehyde solution, and then immunostained. Three types of fluorescently labeled antibodies, 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), for detecting human stem cell marker proteins manufactured by R&D System, were reacted simultaneously and observed under a fluorescent microscope.

(result)
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 the three stem cell marker proteins were induced in the cells forming the spheroids.

(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 marker protein made from the undifferentiation marker gene was also expressed in the fibroblast spheroids, confirming the presence of stem cells in terms of both genes and proteins.

Example 4: Detection of stem cells in healthy human neonatal fibroblast spheroids (Methods)
To detect stem cells induced into fibroblast spheroids, stem cells contained in the spheroid-constituting cells were detected by immunostaining using fluorescent dye-labeled antibodies. A solution (0.125%) of a polypeptide represented by formula (1) (PURECOLLA, manufactured by Unix Corporation) (cell reprogramming agent) was added at 100 μL/well to a commercially available 96-well round-bottom culture vessel made of polystyrene (Nunclon Delta 96U Bottom, Thermo Scientific Co., Ltd.), and the cell reprogramming agent coating process was completed in the same manner as in Example 2 above. 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). Fibroblasts (2 x ¹⁰⁴ cells/well/100μL) suspended in the above medium were seeded on the cell reprogramming agent-coated culture vessel and cultured under 37℃ and 5% _CO2 conditions. On the 20th day after the start of culture, spheroids were aseptically collected from the 96-well round-bottom culture vessel, dispersed with Accutase enzyme reagent, and 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. NL493 fluorescent dye-labeled anti-NANOG antibody (green fluorescence) manufactured by R&D system was used as the fluorescent-labeled antibody, and observation was performed under a fluorescent microscope.

(result)
Figure 11 shows the presence of NANOG protein-positive stem cells in the cell population obtained by dispersing spheroids. In Figure 11, the nuclei of the majority of fibroblasts are NANOG negative, but some cells show strong NANOG antibody-positive green fluorescence in the nuclei, indicating that stem cell marker cells have been induced.

(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. In addition to confirming stem cell marker proteins throughout the spheroids (Figure 10), we also demonstrated that it is possible to induce stem cells that are positive for NANOG protein in the nucleus at the cellular level.

Example 5: Preparation of stem cells by spheroid culture of human adult skin fibroblasts (Method)
In order to induce stem cells (direct-reprogramming) in healthy adult human dermal fibroblasts, adult dermal fibroblasts were cultured to form spheroids in the same manner as in Example 2. An aqueous solution (0.125%) of a polypeptide represented by formula (1) (Unix Corporation, PURECOLLA) (cell reprogramming agent) 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 reprogramming agent was then removed, and the vessel was dried in a dryer at 37°C for 96 hours to complete the cell reprogramming agent 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 0.5mg/mL, human transferrin recombinant 10μg/mL, basic-FGF 5ng/mL, and insulin 2.5μg/mL). Using the above-mentioned cell reprogramming agent-coated 96-well round-bottom culture vessel, 2× ¹⁰⁴ adult dermal fibroblasts suspended in 100μL of the above-mentioned medium were seeded/well and cultured at 37℃ and 5% _CO2 .

To detect stem cell marker genes, mRNA expression analysis was performed using real-time PCR. Specifically, on the 20th day after the start of culture (Day 20), spheroids were collected in a 15 mL centrifuge tube and centrifuged, and the cells were washed and collected with phosphate buffer PBS (-). For the control cells cultured in a 2D culture vessel without a cell reprogramming agent (Day 0), the medium was removed and the cells were washed with PBS (-), detached and dispersed from the plate with 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 protocol recommended by the reagent manufacturer to use as template RNA. For real-time PCR analysis, QuantiFast Probe RT-PCR Kits (QIAGEN; No. 204454) and the PCR probe of TaqMan Gene Expression Assay Probe (Thermo Fisher Scientific) were used, and PCR analysis was performed using a Rotor-Gene Q (QIAGEN) device. To detect the genes, we used three types of 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 undifferentiated marker genes, and β-Actin as an endogenous control gene (Figure 13).

To examine whether the spheroid-induced stem cells could differentiate into the three early germ layers, we examined the expression of three germ layer marker genes by real-time PCR. The detection probes used were the four three germ layer marker genes 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), as well as the TaqMan Gene Expression Assay Probe for β-Actin as an endogenous control gene (Figure 14).
In gene expression analysis, relative quantification was performed using the ΔΔCt method (delta delta Ct method). The results of the ΔΔCt method analysis were calculated by setting the amount of genes expressed by the control cells in the "no cell reprogramming agent, planar 2D growth day 0" as "1.0" relative to the "expression amount on the 20th day of spheroid growth" for comparison.

(result)
The results of spheroid culture of adult dermal fibroblasts using culture vessels treated with a cell reprogramming agent are shown in Figure 12. In the control vessel without the cell reprogramming agent, adult dermal fibroblasts adhered to the bottom and proliferated in two dimensions (culture day 2). On the other hand, in the culture vessels coated with the cell reprogramming agent, the cells formed spheroids and proliferated in three dimensions.

FIG. 13 shows the results of real-time PCR of gene mRNA.
In adult dermal fibroblast spheroids on the 20th day of culture, the gene expression levels of marker proteins SOX2, OCT4, and NANOG, which define the properties of stem cells, increased, and it was found that the cells had acquired the properties of stem cells. Specifically, the gene expression levels of SOX2, OCT4, and NANOG in the control cells cultured in 2D were set at 1.0 relative to each other, and the gene expression levels of SOX2, OCT4, and NANOG on the 20th day of 3D spheroid culture were compared using the ΔΔCt method. As a result, SOX2 increased 9.4-fold, OCT4 increased 13.0-fold, and NANOG increased 50.5-fold.

Figure 14 shows that the expression levels of marker proteins SOX17, GATA6, HAND1, and SOX1, which define differentiation into the three germ layers, increased. The gene expression levels of SOX17, GATA6, HAND1, and SOX1 in control cells were set at 1.0 relative to each other, and the gene expression levels of SOX17, GATA6, and MAP2 on day 20 (Day 20) of 3D spheroid culture were compared using ΔΔCt relative quantification. The expression levels of all genes increased, with SOX17 increasing 11.4-fold, GATA6 increasing 2.2-fold, HAND1 increasing 33.9-fold, and SOX1 increasing 7.1-fold.

(Discussion)
So far, experiments on stem cell induction using mouse fetal fibroblasts or adult fibroblasts have been reported for somatic cell direct reprogramming technology using chemical compounds. However, the technology for application to human adult fibroblasts has not yet been established. Here, it can be said that a method for stably and reproducibly producing stem cells using the cell reprogramming agent of the present invention has been found for human neonatal fibroblasts as well as adult skin fibroblasts.

Since stem cells can be induced by cell reprogramming agent spheroid culture using skin-derived fibroblasts, which are the least invasive and easiest to collect samples from individual patients, this technology will be useful for personalized regenerative therapy, personalized diagnostic methods, and therapeutic drug screening.

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) (PURECOLLA, manufactured by Unix Corporation) (cell reprogramming agent) was added to a commercially available polystyrene 6-well flat-bottom culture vessel (CellBIND, manufactured by Corning) or a polystyrene 96-well round-bottom culture vessel (Nunclon Delta 96U Bottom, manufactured by Thermo Scientific) and left to stand at 4°C for 24 hours.

Thereafter, the cell reprogramming agent was removed, and the cells were dried in a dryer at 37°C for 96 hours to complete the cell reprogramming agent application process. Healthy human adult mammary epithelial cells (Lonza: Human Mammary Epithelial Cells, hereinafter 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 2.5μg/mL). In the case of a 6-well flat-bottom culture vessel, 2×10 ⁶ cells/well of mammary epithelial cells suspended in 2mL of the above medium were seeded in the above cell reprogramming agent-coated culture vessel, and in the case of a 96-well round-bottom culture vessel, 2×10 ⁴ cells/well of mammary epithelial cells suspended in 100μL of the above medium were seeded, and each was cultured under 37°C and 5% CO2 conditions. As a control, mammary epithelial cells were seeded under the same conditions in the same type of culture vessel without the cell reprogramming agent. On days 2 and 20 after the start of culture, microscopic observation was performed, and the state of spheroid formation was photographed ( FIG. 15 ).

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 a cell reprogramming agent and allowed to form spheroids. Gene mRNA expression analysis was performed using real-time PCR.

Specifically, on the 20th day after the start of culture (Day 20), the spheroids were collected in a 15 mL centrifuge tube and centrifuged, and the cells were washed and collected with phosphate buffer PBS(-). For the control cells cultured in 2D culture vessels without cell reprogramming agents (Day 0), the medium was removed and washed with PBS(-), the cells were detached and dispersed from the plate with 0.05% trypsin-EGTA, collected in a 15 mL centrifuge tube, and washed again with PBS(-). NucleoZOL (Machler-Nagel) reagent was added to the spheroids, and RNA was extracted according to the protocol recommended by the reagent manufacturer to serve as template RNA. For real-time PCR analysis, QuantiFast Probe RT-PCR Kits (QIAGEN; No. 204454) and the PCR probe of TaqMan Gene Expression Assay Probe (Thermo Fisher Scientific) were used, and PCR analysis was performed using a Rotor-Gene Q (QIAGEN) device. The genes we attempted to detect were the three 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 undifferentiated marker genes, and β-Actin as an endogenous control gene (Figure 16).

The probes used to detect genes related to early three germ layer differentiation were the 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 the TaqMan Gene Expression Assay Probe for β-Actin as an endogenous control gene (Figure 17).

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 amount of genes expressed by the control cells in the "no cell reprogramming agent, planar 2D growth day 0" to a relative value of "1.0," and calculating the increase fold of the "expression amount on day 20 of spheroid growth."

(result)
Figure 15 shows the results of spheroid culture of mammary epithelial cells using a culture vessel treated with a cell reprogramming agent. In a control vessel without a cell reprogramming agent, mammary epithelial cells adhered to the bottom and showed planar two-dimensional proliferation (2 days of culture). On the other hand, in a culture vessel coated with a cell reprogramming agent, the cells self-organized to form spheroids and proliferated in 3D. From the 2nd to 20th day of culture, the cells that make up the spheroids proliferated, and the size of the spheroids themselves also increased.

Figure 16 shows the results of real-time PCR analysis of mRNA to investigate the expression of stem cell undifferentiation marker genes induced (direct-reprogramming) from mammary epithelial cells. As a result, it was found that the gene expression levels of stem cell undifferentiation marker proteins SOX2, OCT4, and NANOG increased on the 20th day (Day 20) from the start of spheroid culture compared to 2D adherent culture of mammary epithelial cells (Day 0).

Specifically, the gene expression levels of SOX2, OCT4, and NANOG in mammary epithelial cells cultured in 2D adhesion without cell reprogramming agents as a control were set at a relative value of 1.0, and the gene expression levels of SOX2, OCT4, and NANOG on day 20 (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.

Figure 17 shows the results of real-time PCR of mRNA of genes related to the early three germ layers. As a result, it was found that the gene expression levels of SOX17 and MAP2, which are marker genes that define differentiation into the three germ layers, increased on the 20th day after the start of spheroid culture of mammary epithelial cells.

Specifically, the gene expression levels of SOX17, GATA4, and MAP2 in mammary epithelial cells cultured in 2D adhesion (Day 0) without cell reprogramming agents as a control were set at 1.0 relative to each other, and the gene expression levels of SOX17, GATA6, and MAP2 on Day 20 of 3D spheroid culture were compared using ΔΔCt relative quantification. As a result, on Day 20 of culture, SOX17 increased 124.7-fold and MAP2 increased 2.7-fold. On the other hand, no expression of the mesodermal marker GATA6 gene was detected.

(Discussion)
The results in Figure 15 show that it is possible to directly reprogram adult mammary epithelial cells to produce stem cells with the characteristics of pluripotent stem cells. Furthermore, it was shown that this can be achieved in both flat-bottom and round-bottom culture vessels.

In Figure 16, when mammary epithelial cells are cultured as spheroids in a 96-well culture vessel treated with a cell reprogramming agent, a single homogenous spheroid can be produced, enabling precise gene expression analysis. As a result, it was found that direct reprogramming to pluripotent stem cells with enhanced expression of SOX2, OCT4, and NANOG genes is possible without using the method of introducing transcription factor genes used in iPS cell production, and without adding to the culture medium a compound cocktail used in direct reprogramming with compounds. In other words, it can be said that a new direct reprogramming method has been invented to produce cells with the characteristics of pluripotent stem cells from somatic mammary epithelial cells using a spheroid culture method with a cell reprogramming agent.

The results in Figure 17 show that stem cells created from mammary epithelial cells using this method can be induced to differentiate into triploid bodies, which are the early stage of differentiation into various organ cells. In general, in order to induce differentiation of stem cells into mature organ cells, a process is required in which the stem cells are first induced to differentiate into the germ layer of the target organ lineage. This culture method uses a mechanism different from the conventional iPS cell method and direct reprogramming methods using chemical compounds, and can be said to be a new culture method that brings about the initialization (direct reprogramming) of somatic cells into stem cells and at the same time realizes the induction of differentiation into embryoid bodies that are positive for SOX17 (endodermal marker gene) and MAP2 (ectoderm marker gene).

Note that, in addition to SOX2, OCT4, and NANOG, other known stem cell undifferentiation marker genes include Klf4, Essrb, Sall4, Lin28, etc., 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.

Example 7: 2D adhesion culture and 3D spheroid culture of cancer cells (Methods)
2 mL of an aqueous solution (0.125%) of the polypeptide represented by formula (1) (UNIX Corporation, PURECOLLA) (cell reprogramming agent) was added to a commercially available polystyrene 6-well flat-bottom culture vessel (CellBIND, Corning) and left to stand at 4°C for 24 hours.

After that, the cell reprogramming agent was removed, and the cells were dried in a dryer at 37°C for 96 hours to complete the cell reprogramming agent application process. As human cancer cells, 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), which are common cancer cell lines, were used. Each cancer cell was seeded in a culture vessel at 2× ¹⁰⁵ cells/well/100μL and cultured at 37°C and 5% CO2. The medium used was DMEM medium supplemented with 10% FBS and penicillin/streptomycin. As a control, 2× ¹⁰⁶ cancer cells/well were seeded in the same type of culture vessel without the cell reprogramming agent. Microscopic observation was performed on the 4th and 15th days after the start of culture, and the spheroid formation status was photographed (Figure 18).

(result)
The results are shown in Figure 18. In culture vessels without a cell reprogramming agent, the cells adhered to the bottom and proliferated (two-dimensional adhesion and proliferation). On the other hand, in culture vessels treated with a cell reprogramming agent, the cells formed aggregates and formed multiple spheroids. From the 4th to 15th day of culture, the cells constituting the spheroids proliferated, and the size of the spheroids themselves increased.

(Discussion)
As with the results of Examples 1 to 6, it was revealed that cancer cells also form spheroids. It is said that cancer cells cultured in 3D spheroids exhibit characteristics closer to the pathology in vivo than cancer cells cultured in 2D. In other words, if cancer cells can be cultured in 3D, it can be said that a model that reflects the pathology of cancer can be created in a culture vessel.

The polypeptide cell reprogramming agent represented by formula (1) induced spheroid formation in three cancer cell lines, A549 cells (lung cancer), ES2 (ovarian cancer), and HOS-143B (osteosarcoma), in a similar manner, suggesting that this method is not limited to these three cell types, but can provide a common in vitro (in vitro; in a culture vessel) cancer model.

Example 8: Examination of the concentration of cell reprogramming agents (Method)
A 4-fold to 1024-fold dilution solution (cell reprogramming agent) of an aqueous solution (0.5%) of the polypeptide represented by formula (1) (PURECOLLA, manufactured by Unix Corporation) was prepared, and 100 μl of each solution was added to wells of a commercially available 96-well flat-bottom plate (CellBIND, Corning) and left at 4°C for 16 hours.

Thereafter, excess cell reprogramming agent was removed, and the cells were dried under reduced pressure at room temperature for 24 hours.
HOS-143B (human osteosarcoma) was seeded at 3,000 cells/well on a culture plate coated with a cell reprogramming agent and cultured at 37°C under 5% _CO2 conditions. DMEM + 10% FBS was used as the medium.

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

(result)
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.

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

Example 9: Study on plate shape (method)
An aqueous solution (0.125%) of the polypeptide represented by formula (1) (PURECOLLA, manufactured by Unix Corporation) (cell reprogramming agent) was added in an amount of 200 μl per 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.

Then, excess cell reprogramming agent was removed and the cells were dried under reduced pressure at room temperature for 24 hours.

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 a culture plate coated with a cell reprogramming agent and cultured at 37°C and 5% _CO2 . DMEM + 10% FBS was used as the medium. After 2 days of culture, the cells were observed under a microscope.

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

(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.

Example 10: Study on the Induction and Inhibition of Apoptosis (Method)
Two types of 96-well cell culture plates with different bottom shapes (U-bottom plate #163320; Corning, NY, USA, V-bottom plate #277143; Thermo Fisher Scientific, MA, USA) were filled with two different concentrations of the polypeptide represented by formula (1) (PURECOLLA, Unix Corporation) at 100μL/well and 200μL/well, respectively, and left to stand overnight at 4℃. After that, the excess cell reprogramming agent was removed, and the cells were dried in a clean bench for about 6 hours to complete the cell reprogramming agent treatment. Two types of cell lines (canine renal tubule-derived MDCK II cells and HOS-143B cells) were seeded at 3,000 cells/well/100μL, and cultured in an incubator at 37℃ in a 5% _CO2 atmosphere for 6 days. The control was cultured in a plate without the cell reprogramming agent. After 3 and 6 days, apoptosis activity in each well was measured using a caspase activity measurement kit (Apo-ONE Homogeneous Caspase-3/7 Assay kit; Promega, WI, USA). A fluorescent precursor substrate, bis-(N-CBZ-L-aspartyl-L-glutamyl-L-valyl-aspartic acid amide)rhodamine 110 (Z-DEVD-rhodamine 110), was diluted 100-fold with the included buffer and added to each well in an amount of 50 μL. The plate was gently rocked to avoid disrupting the spheroids in the wells, and then the plate was left to stand 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 value at 590 nm (excitation light 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 this value was subtracted from the measured value 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.

(result)
The results are shown in Figures 21 and 22. It was found that apoptosis was suppressed in both MDCK II and HOS-143B cells depending on the concentration of the polypeptide represented by formula (1), compared to cells cultured on a plate not coated with an aqueous solution of the polypeptide represented by formula (1). The inhibitory effect on apoptosis was more significant on the sixth day of culture.

(Discussion)
In the past, 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 reprogramming agent of the present invention suppresses the induction of apoptosis during cell spheroid formation, and we found that it is extremely useful not only for stem cell production using spheroids, but also for drug discovery research and medical applications.

Example 11: Detection of gene expression of cancer stem cell markers (method)
A cell reprogramming agent-treated culture vessel was completed in the same manner as in Example 7, and the same three types of cancer cells as in Example 7 were cultured. On the 4th and 15th days after the start of culture, the three types of cancer cell spheroids were collected from each culture vessel into a 15 mL centrifuge tube, centrifuged, and the cells were washed with phosphate buffer PBS(-). After removing the medium, the cancer cells cultured on the control plate without the cell reprogramming agent were washed with PBS(-), detached and dispersed from the plate with 0.05% trypsin-EGTA, collected in a 15 mL centrifuge tube, and washed again with PBS(-). mRNA was extracted from the collected spheroids and control cells, and expression analysis of cancer stem cell marker genes was performed by real-time PCR.

Specifically, NucleoZOL (Machrei-Nagel) reagent was added to each cell, and RNA was extracted according to the protocol recommended by the reagent manufacturer, and used as template RNA for stem cell marker detection. For real-time PCR analysis, QuantiFast Probe RT-PCR Kits (QIAGEN; No. 204454) and TaqMan Gene Expression Assay Probe (Thermo Fisher Scientific) PCR probes were used, and PCR analysis was performed using a Rotor-Gene Q (QIAGEN) device. Three types of 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 cell-cell interaction, adhesion and migration, cancer stem cell marker), and CD133 (five transmembrane glycoprotein, stem cell marker and cancer stem cell marker protein) PCR probes. 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), as well as β-Actin 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 method analysis were calculated by setting the amount of genes expressed by the control cells "without cell reprogramming agent, 2D adhesion growth on day 4" as "1.0" relatively, and calculating the increase fold of the "expression amount on day 15 of spheroid growth" (Figures 23-25).

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

In Figure 23, we found that the gene expression levels of cancer stem cell markers CD24, CD44, and CD133 in A549 lung cancer cells increased over the course of culture time (from day 4 to day 15).

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.

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 proliferating cells, and increased 8.5-fold on day 15, assuming a control value of 1.0 on day 4 of spheroid culture.

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.

(Discussion)
A549 cells, ES2 cells, and HOS-143 cells are cancer cell lines established as cells that grow by adhesion. In the cancer cell differentiation hierarchy, they are classified as "differentiated cancer cell lines" that have progressed in the 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 a normal 2D adhesion culture. When these cancer cells are cultured with the cell reprogramming agent of the present invention, they form spheroids with a three-dimensional structure and grow, and it has been found that the expression of CD24, CD44, and CD133 genes, which are indicators of cancer stem cells, increases from the 4th day of culture to the 15th day.

Conventionally, cancer stem cells have been obtained by transplanting cancer tissue extracted from cancer patient specimens into mice, or by extracting minute amounts of cancer stem cells from cancer tissue using a cell sorter and multiplying them over a long period of time in culture medium containing many expensive growth factors and hormones. In this context, this culture method can be said to provide a simple method for producing large amounts of cancer stem cells by simply culturing them with cell reprogramming agents.

Note that cancer stem cell markers other than CD24, CD44, and CD133 are also known, but the present invention is not limited to these examples.

Example 12: Detection of gene expression of stem cell undifferentiation marker transcription factor in cancer cells (method)
Coating of the culture vessel was completed in the same manner as in Examples 7 and 11, and three types of cancer cells, A549 cells, ES2 cells, and 143B cells, were cultured. Three types of 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 undifferentiated marker genes, and β-Actin was used as an endogenous control gene. In gene expression analysis, relative quantification was performed using the ΔΔCt method (delta delta Ct method). The results of the ΔΔCt method analysis were calculated by setting the gene amount expressed by the control cells "without cell reprogramming agent, 2D adhesion growth on the 4th day" to "1.0" relatively, and calculating the increase fold of the "expression amount on the 15th day of spheroid growth" to be compared (Figures 26 to 28).

(result)
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).

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.

In Figure 27, SOX2 in ES2 ovarian cancer cells was 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.

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.

(Discussion)
It is clinically known that stem cell undifferentiation marker genes such as SOX2, OCT4, and NANOG are expressed in some cancer tissues. Conventionally, SOX2, OCT4, and NANOG transcription factors are stem cell undifferentiation markers possessed by ES cells and iPS cells, so cancer cells with expression of these genes are considered to have the characteristics of "cancer stem cells". It is known that the stem cell undifferentiation marker genes such as SOX2, OCT4, and NANOG are not expressed or are expressed at a very low level in the A549 cells, ES2 cells, and HOS-143 cells used here. Under these circumstances, when these three types of cancer cells are cultured with the cell reprogramming agent of the present invention, they form spheroids with a three-dimensional structure and grow, and it was found that the expression of the stem cell markers SOX2, OCT4, and NANOG genes increased from the 4th day of culture to the 15th day.

Based on the above, the researchers have invented a method for easily and mass-produced cancer stem cells that express the undifferentiated stem cell marker genes SOX2, OCT4, and NANOG, as well as the cancer stem cell marker genes CD24, CD44, and CD133, by culturing cancer cells with this cell reprogramming agent.
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.

Example 13: Examination of the molecular weight of the polypeptide (1) Fractionation of the polypeptide component by ultrafiltration (method)
The polypeptide represented by formula (1) (PURECOLLA, manufactured by Unix Corporation) was fractionated using three types of ultrafiltration membranes with different molecular weight cutoffs, and the spheroid formation ability of the polypeptide samples with different molecular weights was examined. In the experiment, three types of centrifuge tubes (Amicon centrifugal ultrafiltration filter units) equipped with ultrafiltration membranes were used to fractionate the polypeptide dissolved in the cell reprogramming agent raw material solution based on its molecular weight. The molecular weight cutoff ranges of the ultrafiltration membranes used were three types: 100,000 or more, 50,000 or more, and 10,000 or more.
For the cell reprogramming agent polypeptide, the sample before ultrafiltration was labeled with a fluorescent dye using the FluoroDY-495 Protein Labeling kit reagent, and after fractionation, the polypeptide was visualized fluorescently by electrophoresis. Electrophoresis was performed using a 5% to 25% polyacrylamide gradient gel at 200 mV for 30 minutes. The gel after electrophoresis was analyzed using a fluorescent imaging device (Bio-Rad, Gel Doc). The results are shown in Figure 29.

(Results) By ultrafiltration, the cell reprogramming agent polypeptide was separated into the following four molecular weight fractions (Figure 29; lanes (B), (C), (E), and (F)). Note that (M) in Figure 29 indicates a molecular weight marker.
(A): Cell reprogramming agent polypeptide source (including a wide range of molecular weight polypeptides)
(B): Molecular weight of 100,000 or more
(C): Molecular weight less than 100,000
(E): Molecular weight less than 50,000
(F): Molecular weight of 10,000 or less Gel lane (A) is the polypeptide raw material, and a wide range of peptides with molecular weights was detected, from the white band with a molecular weight of 100,000 or more visible at the top of the gel to small peptides with a molecular weight of 14,000 or less. Fraction (B) was detected as a dark band with a molecular weight of 100,000 or more. Fraction (C) was detected as a broad mixture with a molecular weight of 100,000 or less and including molecules smaller than 14,000. Fraction (E) was detected as a broad mixture with a molecular weight ranging from 50,000 to 14,000 or less, and fraction (F) was detected as a mixture with a molecular weight of 10,000 or less. Since the polypeptide represented by the formula (Pro-Hyp-Gly)n is known to have a triple helical structure, the above molecular weights are considered to be the molecular weights of a triple helical structure complex.

(2) Spheroid-forming activity of fractionated components (Method)
The ultrafiltration samples (A), (B), (C), (E), and (F) obtained in (1) were each applied to a 96-well polystyrene plate in the same manner as in Example 2, and the spheroid formation activity of the cells was examined. The results are shown in Figure 30.

(result)
Samples (A) and (B) showed spheroid formation activity, while the other samples (C), (E), and (F) did not show spheroid formation and showed adhesion and proliferation to the bottom. From the above, it is considered that the spheroid formation activity shown by this cell reprogramming agent polypeptide is brought about by the polypeptide with a molecular weight of 100,000 or more.

(Discussion)
It was found that the molecular weight of the active molecule of the polypeptide that serves as a cell reprogramming agent is 100,000 or more. Since the polypeptide represented by the formula (Pro-Hyp-Gly)n is known to have a triple helical structure, the molecular weight is considered to be the molecular weight in the triple helical structure complex. It is considered that this active molecule induces receptor/ligand binding, and induces spheroid formation activity, apoptosis suppression, and stem cell induction.

Claims (17)

A 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 100 to 1000.) The cell reprogramming agent according to claim 1, which is used in the production of stem cells. The cell reprogramming agent according to claim 2, wherein the stem cells are pluripotent cells or cancer stem cells. The cell reprogramming agent according to claim 1, which is used for forming spheroids. A spheroid-forming 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 100 to 1000.) A cell culture substrate, the culture surface of which is treated with the cell reprogramming agent according to any one of claims 1 to 4 or the spheroid forming agent according to claim 5. A cell culture system comprising a cell reprogramming agent according to any one of claims 1 to 4 or a spheroid forming agent according to claim 5, and cultured cells. A method for producing stem cells, comprising a step of culturing cells using the cell reprogramming agent according to any one of claims 1 to 4 or the spheroid forming agent according to claim 5. The method of claim 8, wherein the stem cells express at least one stem cell undifferentiation marker gene selected from SOX2, OCT4, and NANOG. The method according to claim 8, wherein the stem cells are pluripotent stem cells or cancer stem cells. A method for producing an organoid or tissue using stem cells obtained by the method according to claim 8. An apoptosis inhibitor 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 100 to 1000.) The apoptosis inhibitor according to claim 12, which inhibits apoptosis in spheroids. A cell culture auxiliary 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 100 to 1000.) The cell culture supplement according to claim 14, which is used in the production of stem cells. The cell culture supplement according to claim 15, wherein the stem cells are pluripotent cells or cancer stem cells. The cell culture supplement according to claim 14, which is used for forming spheroids.