Pluripotent stem cell-derived feeder cells

Abstract

To provide culture means that can be used in the culture of the cells that are difficult to culture alone and is free of the problem of mixing of heterologous substance.SOLUTION: The present invention discloses a pluripotent stem cell-derived feeder cell, and a cell culture method that includes the step of culturing the cell as a feeder cell to culture a target cell.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a feeder cell derived from a pluripotent stem cell, and a method for culturing a cell, comprising a step of culturing a cell of interest using the cell as a feeder cell. [Background technology]

[0002] Primary cultured cells obtained from living organisms, pluripotent stem cells such as embryonic stem cells (ES cells) and induced pluripotent stem cells (iPS cells), as well as cells induced to differentiate from pluripotent stem cells, are widely used in fields such as basic research, regenerative medicine, and drug discovery.

[0003] These cells are often difficult to culture alone (especially for long periods of time). For example, the endometrium is the site of implantation of a fertilized egg and is a tissue that plays a role in pregnancy and the maintenance and development of the fetus and placenta. It is desirable to culture the endometrium in vitro for the purposes of regenerative medicine, drug discovery research, and disease elucidation for endometrial-related diseases such as infertility caused by endometrial thinning. However, it has traditionally been difficult to culture endometrial epithelial cells that make up the endometrium.

[0004] In order to culture cells that are difficult to culture, a method is used in which the target cells are cultured on a cell layer (feeder layer) formed by other cells (feeder cells). Animal-derived cells such as mouse embryonic fibroblasts (MEF) are mainly used as feeder cells.

[0005] However, for example, when applying cultured cells to human regenerative medicine or cell therapy, the use of feeder cells derived from a different animal species, such as a mouse, poses the risk of contamination with substances derived from that animal. Summary of the Invention [Problem to be solved by the invention]

[0006] An object of one aspect of the present invention is to provide a culture means capable of supporting the culture of even cells that are difficult to culture alone, and free from the problem of contamination with foreign substances. [Means for solving the problem]

[0007] To achieve the above object, one embodiment of the present invention provides a feeder cell derived from a pluripotent stem cell.

[0008] Another embodiment of the present invention provides a method for culturing cells, comprising the step of culturing cells of interest using pluripotent stem cell-derived feeder cells as feeder cells. Effect of the Invention

[0009] According to one aspect of the present invention, it is possible to culture cells of interest without contamination with substances derived from heterologous cells. [Brief description of the drawings]

[0010] [Figure 1] FIG. 1 shows a method for producing hES cell-derived mesenchymal cells. [Diagram 2] FIG. 1 shows subculture of endometrial epithelial cells without feeder cells. [Diagram 3] FIG. 1 shows cells of passage 1 to passage 5 of the endometrial epithelium. [Figure 4] FIG. 1 shows the results of immunofluorescence staining of endometrial epithelial cells. [Diagram 5] FIG. 1 shows colony area of ​​endometrial epithelial cells. [Figure 6] FIG. 1 shows the number of cell divisions (PDL) of endometrial epithelial cells. [Figure 7] FIG. 1 shows the results of HE staining (left) and Alcian blue staining (right) of chondrocytes. [Figure 8] FIG. 1 shows the proliferation of epidermal cells in the presence or absence of feeder cells. [Figure 9] FIG. 1 shows the morphology of epidermal cells in the presence or absence of feeder cells. [Figure 10] FIG. 1 shows keratinocyte marker expression in epidermal cells. [Figure 11] FIG. 1 shows the culture of hepatocytes in the presence of feeder cells. [Figure 12] hiPS cell culture in the presence of feeder cells (left panel shows the results of co-culture with MEFs, right panel shows the results of co-culture with hES cell-derived mesenchymal cells). [Figure 13] Figure 1 shows the expression of undifferentiated markers and self-renewal markers in hiPS cells, the left panel shows the expression of NANOG, the center panel shows the expression of OCT3 / 4, and the right panel shows the expression of TERT. [Figure 14] 1 shows the expression of pluripotent cell-specific markers in hiPS cells cultured in the presence of MEFs, with results shown from left to right for NANOG, OCT3 / 4, SSEA4, TRA1-60, and SOX2. [Figure 15] 1 shows the expression of pluripotent cell-specific markers in hiPS cells cultured in the presence of hES cell-derived mesenchymal cells, with results for NANOG, OCT3 / 4, SSEA4, TRA1-60, and SOX2 shown from left to right. [Figure 16] FIG. 1 shows the proliferation of hiPS cell-derived hepatocytes. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] [Embodiment 1] One embodiment of the present invention will be described in detail below. Embodiment 1 of the present invention relates to a feeder cell derived from a pluripotent stem cell.

[0012] The term "feeder cells" as used herein refers to cells other than the cells of interest that are cultured together with the cells of interest to support the survival or growth of the cells of interest. Feeder cells may be seeded simultaneously with the cells of interest or before the cells of interest are seeded. In one embodiment, feeder cells are seeded before the cells of interest to form a layer of cells called a feeder layer. The cells of interest may be cultured in contact with feeder cells or without contact with feeder cells. The growth of the cells used as feeder cells may be suppressed or inactivated according to known methods, such as irradiation (e.g., gamma rays), treatment with antibiotics (e.g., mitomycin C), etc.

[0013] The term "pluripotent stem cells" as used herein refers to cells with pluripotency. The term "pluripotency" as used herein refers to the ability of cells to differentiate into all germ layers (i.e., ectoderm, mesoderm and endoderm) that constitute an individual. Examples of pluripotent stem cells include embryonic stem cells (ES cells) and induced pluripotent stem cells (iPS cells), as well as stem cells derived from adult tissues or umbilical cord blood or umbilical cord. Pluripotent stem cells can be derived from any organism. Pluripotent stem cells are derived, for example, from vertebrates, preferably from warm-blooded animals, more preferably from mammals (e.g., mice, rats, guinea pigs, hamsters, rabbits, cats, dogs, sheep, pigs, cows, goats, monkeys, humans), even more preferably from primates, and particularly preferably from humans.

[0014] As used herein, the term "pluripotent stem cell-derived feeder cells" refers to any cells derived from pluripotent stem cells used as feeder cells. Pluripotent stem cell-derived feeder cells are obtained by inducing differentiation of pluripotent stem cells.

[0015] In one embodiment, the pluripotent stem cell-derived feeder cells are obtained by culturing embryoid bodies formed from pluripotent stem cells.

[0016] The term "embryoid body" (also referred to as "EB" herein) as used herein refers to a cell mass formed by culturing pluripotent stem cells. Embryoid bodies are formed by seeding cells in a space such as a well or droplet of a 96-well plate and culturing them in suspension for a certain period of time. Any medium can be used for the culture, for example, EB medium can be used (Ando, ​​Y. et al., Stem Cell. Int., 2017:7541734 (2017)).

[0017] The embryoid bodies formed from pluripotent stem cells can be cultured by any method. For example, the embryoid bodies can be transferred to a culture vessel larger than the one used for their formation and cultured. The culture period can be, for example, 30 days or more, 40 days or more, 50 days or more, or 60 days or more, 100 days or less, 90 days or less, 80 days or less, or 70 days or less. Any medium can be used for the culture, for example, XF32 medium can be used (Ando, ​​Y. et al., supra).

[0018] In one embodiment, the pluripotent stem cell derived feeder cells are mesenchymal cells.

[0019] The term "mesenchymal cells" as used herein refers to cells that constitute non-epithelial mesenchyme and play a role in supporting cells between cells, or cells that have properties similar to such cells. In the present invention, any mesenchymal cells derived from pluripotent stem cells can be used. It has been reported that mesenchymal cells arise during the generation of intestinal organoids from pluripotent stem cells (Uchida, H. et al., JCI Insight, 2:e86492 (2017)). Therefore, mesenchymal cells can be obtained by culturing embryoid bodies in the XF hESC medium (same as the XF32 medium described above) used in this document. Mesenchymal cells can be identified by the expression of mesenchymal cell-specific markers. Thus, mesenchymal cells can be defined as cells that express at least one mesenchymal cell-specific marker. Examples of mesenchymal cell specific markers include CD24, CD320, CD63, CD70, CD99L2, HMMR, ICAM1, IGF2R, ITGA3, MCAM, PDGFRA, PDGFRB, CD29, CD44, CD54, CD59, CD73, CD90, CD105 and HLA-ABC. HLA-DR negativity can also be indicative of mesenchymal cells. Furthermore, expression of cytokine Wnt5A and expression of mesodermal transcription factor Nkx2.5 can be indicative of suitable pluripotent stem cell derived feeder cells.

[0020] However, the pluripotent stem cell-derived feeder cells of the present invention are not limited to the above-mentioned mesenchymal cells, and any pluripotent stem cell-derived cells that can be used as feeder cells can be used in the present invention. For example, like the above-mentioned mesenchymal cells, cells generated when inducing endoderm-derived tissues (e.g., intestine, liver, etc.) or mesoderm-derived tissues (e.g., cartilage, etc.) from pluripotent stem cells can be used as pluripotent stem cell-derived feeder cells in the present invention.

[0021] The cells of the present invention for use as feeder cells are derived from pluripotent stem cells having proliferation ability, and therefore can be easily prepared from pluripotent stem cells even if they do not have high proliferation ability themselves. However, it is desirable to be able to easily proliferate pluripotent stem cell-derived feeder cells for use as feeder cells.

[0022] In one embodiment, the proliferation ability of the pluripotent stem cell-derived feeder cell of the present invention is enhanced. The proliferation ability of the cell can be enhanced by any known method. For example, the proliferation ability of the cell can be enhanced by adding a factor that enhances the proliferation ability of the cell to the medium, or by increasing the expression of a gene that codes for a factor that enhances the proliferation ability of the cell. The expression of the gene can be increased by introducing the gene using a viral vector (e.g., lentiviral vector).

[0023] For example, the proliferation ability of cells can be enhanced by activating the Wnt / β-catenin signaling pathway. Examples of factors used to activate the Wnt / β-catenin signaling pathway include Wnt (e.g., Wnt3A (which may be complexed with afamin (AFM))) and R-spondin.

[0024] Alternatively, the proliferation ability of cells can be enhanced by inhibiting the bone morphogenetic protein (BMP) signaling pathway. An example of a factor used to inhibit the bone morphogenetic protein signaling pathway is noggin.

[0025] For use as feeder cells, it is desirable for pluripotent stem cell-derived feeder cells to have an indefinite proliferation capacity.

[0026] In one embodiment, the feeder cells derived from pluripotent stem cells of the present invention are immortalized. Differentiated cells, such as primary cultured cells isolated from normal tissues, gradually decrease their proliferation ability and eventually lose their proliferation ability. In order to avoid such a decrease or loss of proliferation ability, the term "immortalization" is used herein to allow cells to acquire infinite proliferation ability. Cells can be immortalized by any known method.

[0027] For example, cells can be immortalized by overexpression of telomere reverse transcription protein (TERT). Cells can also be immortalized by overexpression of proteins that control cell cycle. Examples of such proteins include cyclins (e.g., cyclin D1) and cyclin-dependent kinases (CDKs; e.g., CDK4). Furthermore, cells can be immortalized by inactivating tumor suppressor genes (e.g., p53 and Rb). Genes for immortalization can be introduced using viral vectors (e.g., lentiviral vectors) (see Yokoi, T. et al., PLoS ONE. 7(1): e29677 (2012)).

[0028] It is desirable to be able to store pluripotent stem cell-derived feeder cells for use as feeder cells.

[0029] The feeder cells derived from pluripotent stem cells of the present invention can be preserved by any known method. In one embodiment, the cells can be cryopreserved in the presence of a cryopreservation agent. As the cryopreservation agent, for example, dimethyl sulfoxide (DMSO), glycerol, or other commercially available cryopreservation reagents can be used.

[0030] The term "cell of interest" as used herein refers to a cell to be cultured in the presence of the pluripotent stem cell-derived feeder cell of the present invention used as a feeder cell. The cell of interest can be any cell for which it is desired to culture in the presence of a feeder cell. The cell of interest can be derived from any organism. The cell of interest can be derived from, for example, a vertebrate, preferably a warm-blooded animal, more preferably a mammal (e.g., mouse, rat, guinea pig, hamster, rabbit, cat, dog, sheep, pig, cow, goat, monkey, human), even more preferably a primate, and particularly preferably a human.

[0031] The cells that are desired to be cultured in the presence of feeder cells are, for example, cells that require culture in the presence of feeder cells for growth, or cells that are suitable for growth when cultured in the presence of feeder cells. Techniques for culturing without feeder cells (feeder-free) have been developed, and many cells can be cultured under feeder-free conditions. If a cell can be cultured using the pluripotent stem cell-derived feeder cells of the present invention as feeder cells, it can be used as a target cell even if it can be cultured under feeder-free conditions. In one embodiment, the proliferation ability of a target cell is enhanced by culturing it in the presence of the pluripotent stem cell-derived feeder cells of the present invention.

[0032] Examples of cells of interest include, but are not limited to, endometrial epithelial cells, chondrocytes, epidermal cells, hepatocytes, pluripotent stem cells and differentiated cells derived from pluripotent stem cells.

[0033] Endometrial epithelial cells, together with stromal cells, constitute the endometrium. The endometrium is the site of implantation of the fertilized egg and plays a role in pregnancy and the maintenance and development of the fetus and placenta. For the purposes of regenerative medicine, drug discovery research, and disease elucidation for endometrial-related diseases such as infertility caused by endometrial thinning, it is desirable to perform in vitro culture of endometrium. However, it has been difficult to culture endometrial epithelial cells.

[0034] Chondrocytes and cartilage matrix constitute cartilage. It is desirable to prepare a cartilage mass having a size and quality that can be used for treating airway stenosis after tracheotomy in children. Cultured chondrocytes can be used for such a purpose, for example.

[0035] Epidermal cells compose the epidermis of the skin. When skin defects occur due to burns or trauma, autografts are performed, in which normal skin tissue from the patient is transplanted to the damaged area, and cultured epidermis produced by artificially culturing epidermal cells isolated from normal skin tissue is transplanted. Regarding cultured epidermis, autologous cultured epidermis produced from the patient's own epidermal cells is currently being used in Japan as a regenerative medicine product. However, allogeneic cultured epidermis derived from other people has not yet been commercialized, and its practical use is anticipated. For this purpose, the culture of epidermal cells is desired.

[0036] Hepatocytes are cells that are responsible for the main functions of the liver. Cultured hepatocytes are used as experimental systems to elucidate the pathology of liver diseases and to test drug sensitivity. Traditionally, hepatocytes have been cultured under feeder-free conditions or using MEFs as feeder cells. There is a demand for more efficient methods of culturing hepatocytes.

[0037] Pluripotent stem cells have the ability to differentiate into various cells and organs, and are used in fields such as regenerative medicine, drug discovery, and basic research. Pluripotent stem cells have the ability to self-proliferate, but heterologous feeder cells such as MEFs are often used for their culture. In particular, when using pluripotent stem cells for human regenerative medicine or cell therapy, there is a demand for the establishment of a culture method using human-derived feeder cells to avoid contamination with heterologous substances.

[0038] In addition, it is desirable to culture various cells induced to differentiate from pluripotent stem cells, in the same way as primary cells obtained from living organisms.

[0039] The pluripotent stem cell-derived feeder cells of the present invention can be used as feeder cells when culturing these cells.

[0040] [Embodiment 2] The second embodiment of the present invention relates to a method for culturing cells, comprising a step of culturing cells of interest using as feeder cells the pluripotent stem cell-derived feeder cells of the first embodiment of the present invention. For the sake of convenience, the description of the same points as those described in the first embodiment will be omitted.

[0041] As described above, in one aspect, the proliferation ability of a target cell is enhanced by culturing the target cell in the presence of the pluripotent stem cell-derived feeder cell of the present invention. Thus, the method of embodiment 2 of the present invention is a method for enhancing the proliferation ability of a target cell.

[0042] (summary) The embodiments of the present invention are summarized below.

[0043] One embodiment of the present invention relates to pluripotent stem cell-derived feeder cells.

[0044] In one embodiment, the pluripotent stem cells are derived from a primate.

[0045] In one embodiment, the pluripotent stem cells are derived from a human.

[0046] In one embodiment, the pluripotent stem cell-derived feeder cells are obtained by culturing embryoid bodies formed from pluripotent stem cells.

[0047] In one embodiment, the pluripotent stem cell-derived feeder cells are mesenchymal cells.

[0048] In one embodiment, the pluripotent stem cell-derived feeder cells are positive for at least one selected from the group consisting of CD24, CD320, CD63, CD70, CD99L2, HMMR, ICAM1, IGF2R, ITGA3, MCAM, PDGFRA, PDGFRB, CD29, CD44, CD54, CD59, CD73, CD90, CD105 and HLA-ABC, or are negative for HLA-DR.

[0049] In one embodiment, the pluripotent stem cell-derived feeder cells have enhanced expression of Wnt / β-catenin signaling pathway genes.

[0050] In one embodiment, the pluripotent stem cell derived feeder cells are immortalized.

[0051] In one embodiment, the pluripotent stem cell-derived feeder cells are for use as feeder cells in the culture of cells selected from the group consisting of endometrial epithelial cells, chondrocytes, epidermal cells, hepatocytes, pluripotent stem cells, and differentiated cells derived from pluripotent stem cells.

[0052] Another embodiment of the present invention relates to a method for culturing cells, comprising the step of culturing cells of interest using pluripotent stem cell-derived feeder cells as feeder cells.

[0053] In one embodiment, said cells of interest are selected from the group consisting of endometrial epithelial cells, chondrocytes, epidermal cells, hepatocytes, pluripotent stem cells and differentiated cells derived from pluripotent stem cells.

[0054] The present invention is not limited to the above-described embodiments, and various modifications are possible within the scope of the claims. Embodiments obtained by appropriately combining the technical means disclosed in different embodiments are also included in the technical scope of the present invention. EXAMPLES

[0055] [Example 1] Obtaining mesenchymal cells derived from human embryonic stem cells (hES cells) 1-1. Embryoid body formation from hES cells hES cells, SEES2, were exposed to a ROCK inhibitor (Y-27632; 10 μM) and then dissociated into single cells using 0.5 mM EDTA, and plated in a 96-well plate at 5 × 10 cells per well. 3 Cells were seeded at a concentration of 100 mM NaCl.

[0056] The cells were cultured in EB medium (76% KnockOut DMEM, 20% 35 kGy irradiated Xeno-free KnockOut Serum Replacement (XF-KSR, Life Technologies, CA, USA), 2 mM GlutaMAX-I, 0.1 mM non-essential amino acids (NEAA), 50 U / ml penicillin-50 μg / ml streptomycin (Pen-Strep), 50 μg / ml L-ascorbic acid 2-phosphate (Sigma-Aldrich, St. Louis, MO, USA)) for 4 days to form embryoid bodies.

[0057] 1-2. Preparation of hES cell-derived mesenchymal cells from embryoid bodies The embryoid bodies formed in 1-1 were transferred to a T25 flask coated with NMP collagen PS (Nippon Meat Packers Inc.) and cultured for 60 to 70 days in XF32 medium (85% KnockOut DMEM, 15% 35 kGy irradiated XF-KSR, 2 mM GlutaMAX-I, 0.1 mM NEAA, Pen-Strep, 50 μg / ml L-ascorbic acid 2-phosphate, 10 ng / ml heregulin-1β (recombinant human NRG-beta 1 / HRG-beta 1 EGF domain; Wako, Japan), 200 ng / ml recombinant human IGF-1 (LONGR3-IGF-1; Sigma-Aldrich), and 20 ng / ml human bFGF (Kaken Pharmaceutical Co. Ltd.)) to obtain hES cell-derived cells. These cells have mesenchymal cell-like properties, and are hereafter referred to as hES cell-derived mesenchymal cells. The method for producing hES cell-derived mesenchymal cells is shown in FIG.

[0058] The resulting hES cell-derived mesenchymal cells were maintained in α-MEM medium supplemented with 10% FBS (Gibco or HyClone) and 1% Pen-Strep.

[0059] [Example 2] Culture of primary endometrial epithelial cells 2-1. Method Endometrial specimens were collected from the uterus removed from patients with benign gynecological diseases and subjected to enzymatic treatment to dissociate the cells. The dissociated cells were then separated into stromal cells (<40 μm) and endometrial epithelial cells (>40 μm) based on their size, and the endometrial epithelial cells were used in the following experiments.

[0060] A 24-well plate (Thermo Fisher Scientific, 174929) was coated with 0.01% gelatin. 5 × 10 hES cell-derived mesenchymal cells were then placed on the plate as feeder cells. 4 The next day, the endometrial epithelial cells obtained above were seeded at a concentration of 10 × 10 4 The cells were seeded at a density of 1000 cells / well and cultured in modified F medium (Glycotechnica).

[0061] As a control, culture was performed without feeder cells.

[0062] When mouse embryonic fibroblasts (MEFs) or endometrial stromal cells obtained above were used as feeder cells, the experiments were performed under the same conditions, except that these cells were used instead of the hES cell-derived mesenchymal cells.

[0063] 2-2.Results When cultured without feeder cells, only a small proportion of endometrial epithelial cells survived, making subculture difficult (Figure 2).

[0064] On the other hand, when hES cell-derived mesenchymal cells were used as feeder cells, they could be subcultured up to Passage 5, the final culture period being 74 days (FIG. 3).

[0065] After culturing until passage 4, immunofluorescence staining confirmed that expression of pan-cytokeratin, a marker of endometrial epithelial cells, was maintained (Figure 4).

[0066] In passage 2, the total area of ​​colony increased by approximately 2.5 times, and then the colony area decreased (Figure 5).

[0067] Endometrial epithelial cells showed the highest number of cell divisions (PDL) during passage 2. The number of cell divisions decreased during passage 3, but increased again during passage 4 (Figure 6).

[0068] When MEF or endometrial stromal cells were used as feeder cells, they could be subcultured in the same manner as in the case of hES cell-derived mesenchymal cells.

[0069] These results demonstrate that when human endometrial epithelial cells are cultured using hES cell-derived mesenchymal cells as feeder cells, the problem of being unable to culture the cells without feeder cells can be resolved and protein expression on the endometrial epithelial cells is maintained.

[0070] [Example 3] Cultivation of chondrocytes 3-1. Method (i) Simultaneous generation of chondrocytes and mesenchymal cells from hES cells 30 μl of iMatrix-511 silk (MATRIXOME) and 9 ml of PBS were added to a 10 cm dish, and the dish was coated in an incubator for 1 hour. After 1 hour, the solution was removed with an aspirator, and the dish was washed twice with 10 ml of PBS. 9 ml of StemFit® AK02N medium (Ajinomoto Co., Inc.) was added to the coated 10 cm dish.

[0071] Meanwhile, hES cells (SEES2) that had been frozen at -80°C were partially thawed, and 5.5 ml of StemFitAK02N medium and 5.5 μl (1 / 1000 volume) of ROCK inhibitor (final concentration 10 μM) (hereafter referred to as "StemFitAK02N plus medium") were added to suspend the cells. 0.5 ml of the cell suspension was subjected to cell counting, and the remaining 5 ml was centrifuged for 10 minutes. After centrifugation, the supernatant was removed, and the cells were diluted to 1.0 × 106 Cells were suspended in StemFitAK02N plus medium to a concentration of 100000 cells / ml and dissociated into single cells by pipetting. 25 μl of the cell suspension was seeded onto a pre-coated 10 cm dish containing 10 ml of StemFitAK02N plus medium. The medium was replaced with 10 ml of StemFitAK02N medium each day from the following day.

[0072] After seeding, the cells were passaged once, and EBs were prepared using the hES cells on day 8 after passage.

[0073] In detail, the medium was removed, the cells were washed twice with 10 ml of PBS, 1 ml of Triple Select (Gibco) was added, and incubated for 2 min. Triple Select was then removed, the cells were washed twice with 10 ml of PBS, and 1 ml of StemFitAK02N plus medium was added. The cells were detached from the 10 cm dish using a scraper and suspended and dispersed in the medium. The cell suspension was collected, and StemFitAK02N plus medium was added to a total volume of 10.5 ml. 0.5 ml of the obtained cell suspension was subjected to cell counting, and the remaining 10 ml was centrifuged. After centrifugation, the supernatant was removed, and the cells were counted at 1.0 × 10 6 Cells were suspended in StemFitAK02N plus medium to a concentration of 1000 cells / ml and dissociated into single cells by pipetting. 10 μl or 25 μl of the cell suspension was mixed with 10 ml of chondrocyte induction plus medium. 100 μl of the resulting cell suspension was seeded (2000 or 5000 cells / well) into wells of a U-bottom 96-well plate (Corning Incorporated) using an 8-tube pipette to generate EBs.

[0074] (ii) Induction of chondrocytes from EBs 3 ml of collagen hydrochloric acid mixture (collagen:HCl = 1:9 or 1:19) was placed in a T25 flask (TPP) and left to stand in an incubator for 1 hour. The mixture was then removed, and the T25 flask was washed twice with 10 ml of PBS, to which 5 ml of cartilage induction medium was added.

[0075] The EBs obtained in (i) above were collected on the 3rd, 4th or 10th day after seeding. When the EBs were collected on the 10th day, half of the medium was replaced on the 4th day with chondrocyte induction medium (KnockOut DMEM, 67% KnockOut SR Xeno-Free, 1%×100 GlutaMAX, 1%×100 Pen-Strep, 1%×100 NEAA, 1%×100 sodium pyruvate, 0.2% 1μg / ml bFGF, 0.2% 1mg / ml IGF, 0.01% 0.1g / ml heregulin β, 0.1% 50μg / ml ascorbic acid) and 10μl (1 / 1000 volume of medium) of ROCK inhibitor (final concentration 10μM) (hereinafter referred to as "chondrocyte induction plus medium"). The EBs were collected into a reservoir using an 8-tube pipette. 288 EBs collected from three 96-well plates were transferred to a 50 ml tube (FALCON) and left to stand until all EBs had settled. The supernatant was then removed. EBs were seeded onto collagen-coated T25 flasks. 4 ml or 6 ml of chondrocyte induction medium was used for culture, and the medium was replaced with chondrocyte induction medium every two or three days. The culture was continued for 60 or 90 days.

[0076] hES cells (SEES1) frozen at -80℃ were partially thawed, transferred to a 15ml tube containing 9ml of ES Plus medium, and centrifuged for 5 minutes. After centrifugation, the supernatant was removed, and the cells were suspended in 1ml of ES Plus medium, and the entire amount was seeded on the above 10cm dish. From the next day, the medium was replaced with 10ml of ES medium each day.

[0077] On the 8th day after seeding of hES cells, passage was performed as follows: the medium was removed, the cells were washed twice with PBS, and 1 ml of Dissociation Solution (REPROCELL) was added and incubated for 2 minutes. After that, the hES cells were detached by tapping the dish, collected, suspended by pipetting, and seeded on a 10 ml dish seeded with MEFs prepared the day before in the same manner as above.

[0078] After seeding, the cells were passaged once, and EBs were prepared using the hES cells on day 8 after passage.

[0079] In detail, the medium was removed, the cells were washed twice with 10 ml of PBS, 2 ml of ReLeST (STEM CELL) was added, and the cells were incubated for 2 minutes. The 10 cm dish was then tapped approximately 30 times to detach the hES cells from the dish. 5.5 ml of ES medium plus was added to the 10 ml dish, and the cells were suspended and dispersed. 0.5 ml of the resulting cell suspension was subjected to cell counting, and the remaining 5 ml was centrifuged. After centrifugation, the supernatant was removed, and the cells were diluted to 1.0 × 10 6 The cells were suspended in ES medium plus to a concentration of 1000 cells / ml and dissociated into single cells by pipetting. 25 μl of the cell suspension was mixed with 10 ml of chondrocyte induction plus medium. 100 μl of the resulting cell suspension was seeded (5000 cells / well) into the wells of a U-bottom 96-well plate (Corning Incorporated) using an 8-channel pipette to prepare EBs.

[0080] The obtained EBs were used to induce chondrocytes in the same manner as in (ii).

[0081] 3-2.Results The cell sheet formed in (ii) was stained with HE (Figure 7, left). Figure 7 shows that chondrocytes (indicated by the upper arrow) were formed overlapping the mesenchymal cell layer (indicated by the lower arrow).

[0082] Furthermore, Alcian blue staining, which stains cartilage, was performed (Figure 7, right), which demonstrated that the cultured cells were chondrocytes.

[0083] These results demonstrate that chondrocytes can be cultured using hES cell-derived mesenchymal cells as feeder cells.

[0084] [Example 4] Cultivation of epidermal cells 4-1. Method Yub2459keratino, a cell line established from epidermis derived from a polydactyly patient, was subcultured in epidermal induction medium (Defined keratinocyte SFM + ROCK inhibitor (Y-27632; 10 μM)) at 37°C and 5% CO2. 0.1% gelatin was placed in a 10 cm dish and left to stand for 15 minutes or more to coat it. Next, irradiated (30 Gy) hES cell-derived mesenchymal cells as feeder cells were cultured in epidermal induction medium at 1 × 10 per dish. 6 The next day, epidermal cells were seeded at 0.5 × 10 cells per dish. 6 Cells were seeded.

[0085] As controls, cultures were performed using MEFs instead of hES cell-derived mesenchymal cells or without feeder cells.

[0086] 4-2.Results Epidermal cells were cultured for 157 days, from Passage 5 to Passage 22. When cultured using hES cell-derived mesenchymal cells or MEFs as feeder cells, the epidermal cells were able to maintain their proliferation ability. On the other hand, when cultured without feeder cells, the cell proliferation ability gradually decreased with repeated passages, and the cells hardly proliferated at all (Figure 8).

[0087] Furthermore, it was confirmed that epidermal cells cultured on MEF and hES cell-derived mesenchymal cells maintained high cell density even after repeated passages and maintained the cobblestone-like cell morphology characteristic of epidermal cells (Figure 9).

[0088] Next, the expression of keratinocyte markers in the epidermal cells at Passage 22 was observed by immunostaining. The expression of keratin 14 and keratin 10 was confirmed using antibodies against them (mouse IgG3 (ab7800) from Abcam and rabbit IgG (905403) from BioLegend, respectively). The expression of keratin 14 was observed on epidermal cells cultured with MEFs and hES cell-derived mesenchymal cells (Figure 10). In epidermal cells cultured with MEFs, the expression of keratin 10 was observed in the center of the colony (indicated by the dotted line) where the cells were aggregated and layered (Figure 10, center). In epidermal cells cultured with hES cell-derived mesenchymal cells, the expression of keratin 10 was confirmed in the center of the cells (indicated by the dotted line), although not as much as in the culture with MEFs (Figure 10, right).

[0089] These results demonstrate that epidermal cells can be cultured using hES cell-derived mesenchymal cells as feeder cells.

[0090] [Example 5] Cultivation of hepatocytes 5-1. Method Primary hepatocytes, Hep2007 (Passage 15), were subcultured in modified F medium at 37°C under 5% CO2. 0.1% gelatin was placed in a 6 cm dish and left to stand for 15 minutes or more to coat it. Next, irradiated (30 Gy) hES cell-derived mesenchymal cells as feeder cells were cultured in modified F medium at 0.5 × 10 per dish. 6 The next day, hepatocytes Hep2007 were seeded at 1.0–2.0 × 10 per dish. 6 Cells were seeded.

[0091] As controls, cultures were performed using MEFs instead of hES cell-derived mesenchymal cells or without feeder cells.

[0092] 5-2.Results The results are shown in Figure 11. Both of the two photographs in Figure 11 show hepatocytes when hES cell-derived mesenchymal cells were used as feeder cells. When hES cell-derived mesenchymal cells were used as feeder cells, four passages were possible, demonstrating that proliferation ability could be maintained better than in the other two conditions (MEF, no feeder cells).

[0093] [Example 6] Cultivation of human induced pluripotent stem cells (hiPS cells) 6-1. Co-culture of hiPS cells with hES cell-derived mesenchymal cells or MEFs Irradiated (30 Gy) hES cell-derived stromal cells were placed in a 10 cm dish at 2.0 × 10 cells per dish. 6 The next day, menstrual blood-derived hiPS cells, Edom22iPS #s31, were seeded in 10 cm dishes at 8 × 10 per dish. 4 The cells were seeded in the XF32 medium (Nishiwaki, M. et al., bioRxiv preprint doi: https: / / doi.org / 10.1101 / 729525).

[0094] As a control, culture was performed using MEFs instead of hES cell-derived mesenchymal cells.

[0095] The morphology of hiPS cells cultured on MEFs was similar to that of hiPS cells cultured on hES cell-derived mesenchymal cells (Figure 12). The left side of Figure 12 shows the results of co-culture with MEFs, and the right side of Figure 12 shows the results of co-culture with hES cell-derived mesenchymal cells.

[0096] 6-2. Expression of undifferentiated markers and self-renewal markers in hiPS cells The gene expression levels of NANOG and OCT3 / 4, which are undifferentiated markers, and TERT, which is a marker for evaluating self-renewal ability, in hiPS cells cultured on MEF or hES cell-derived mesenchymal cells were compared using qRT-PCR. The nucleotide sequences of the forward and reverse primers for amplifying NANOG, the forward and reverse primers for amplifying OCT3 / 4, and the forward and reverse primers for amplifying TERT are shown in SEQ ID NOs: 1 to 6, respectively.

[0097] The results are shown in Figure 13 (left: NANOG, center: OCT3 / 4, right: TERT). The expression levels of OCT3 / 4 and TERT were approximately the same in both cases. On the other hand, the expression level of NANOG was higher when co-cultured with MEFs than when co-cultured with hES cell-derived mesenchymal cells. However, a certain degree of expression was observed even when cultured on hES cell-derived mesenchymal cells, suggesting that the undifferentiated state was maintained.

[0098] 6-3. Expression of pluripotent cell-specific markers in hiPS cells We examined whether hiPS cells maintained pluripotency on hES cell-derived mesenchymal cells by immunohistochemical staining. In detail, the expression of pluripotent cell specific markers NANOG, OCT3 / 4, SSEA4, TRA1-60, and SOX2 was compared with that in co-culture with MEFs. The results of culture in the presence of MEFs or hES cell-derived mesenchymal cells are shown in Figures 14 and 15, respectively. In Figures 14 and 15, the results for NANOG, OCT3 / 4, SSEA4, TRA1-60, and SOX2 are shown from left to right. The antibodies used were as follows: NANOG: Repro CELL #RCAB0003P; OCT3 / 4: Santa Cruz #sc-5279; SSEA4: MILLIPORE #MAB4304; TRA1-60: MILLIPORE #MAB4360; SOX2: MILLIPORE #AB5603.

[0099] The staining patterns in both cases were similar, indicating that pluripotency was maintained in both cases.

[0100] These results demonstrate that when hES cell-derived mesenchymal cells are used as feeder cells, hiPS cells can be cultured while maintaining their undifferentiated state.

[0101] [Example 7] Cultivation of differentiated cells derived from pluripotent stem cells iPS cell-derived hepatocytes (cellartis-Hep#O) were induced to differentiate from iPS cells (cell#O) using a differentiation kit (cellartis, Takara Bio), and were subcultured as cell sheets when they reached confluence after differentiation induction. In the subculture, modified F medium was used as the medium, and MEF or human ES cell-derived mesenchymal cells were used as feeder cells at 1 × 10 per well. 5 The cells were seeded at a concentration of 0.05%.

[0102] The results are shown in Figure 16. When MEF or human ES cell-derived mesenchymal cells were used as feeder cells, the proliferation ability of iPS cell-derived hepatocytes (cellartis-Hep#O) could be maintained in almost the same manner.

[0103] As described above, by using cells derived from pluripotent stem cells such as hES cell-derived mesenchymal cells as feeder cells, it was possible to culture endometrial epithelial cells, chondrocytes, epidermal cells, hepatocytes, pluripotent stem cells, and differentiated cells derived from pluripotent stem cells. Thus, unlike the conventional culture using MEF, which is a heterologous cell, a culture method using allogeneic cells has been established. Furthermore, the pluripotent stem cell-derived feeder cells of the present invention can be cryopreserved, and can be easily used as feeder cells when culturing various cells. [Industrial Applicability]

[0104] The feeder cells provided by the present invention can be used in regenerative medicine, drug discovery research, and disease elucidation.

Claims

1. The method includes a step of culturing a cell of interest using, as a feeder cell, a pluripotent stem cell-derived feeder cell, which is a mesenchymal cell obtained by culturing an embryoid body formed from a pluripotent stem cell; the target cell is selected from the group consisting of endometrial epithelial cells, chondrocytes, epidermal cells, and hepatic cells; The feeder cells are seeded simultaneously with the cells of interest, or seeded prior to seeding the cells of interest and cultured together with the cells of interest, or When the target cells are chondrocytes, the feeder cells are generated when inducing chondrocytes from the embryoid bodies and are cultured together with the target cells. Cell culture methods.

2. The culture method according to claim 1 , wherein the pluripotent stem cells are derived from a primate.

3. The culture method according to claim 2 , wherein the pluripotent stem cells are derived from a human.

4. The culture method according to any one of claims 1 to 3, wherein the pluripotent stem cell-derived feeder cells are immortalized.

5. The culture method according to claim 1 , wherein the hepatocytes are hepatocytes derived from pluripotent stem cells.

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