Cells that secrete Noggin protein and method for producing organoids using them

Abstract

To provide a pluripotent stem cell which secretes only Noggin as a protein required for guiding various organoid from human and mouse tissues, the cell capable of secreting Noggin that can improve the induction efficiency of organoid.SOLUTION: Provided is a Noggin-secreting cell that has a nucleic acid encoding Noggin.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to cells that secrete Noggin protein and a method for producing organoids using the same.

Background Art

[0002] In life science research, generally, two-dimensional cell culture is used. However, living tissues and organs actually have a three-dimensional structure. In two-dimensional culture, cells are cultured on a plastic plate, so the cells are stretched flat and subjected to strong tension. Moreover, cell adhesion and information transmission are also in the two-dimensional direction, so a strict physiological state cannot be reproduced. Therefore, three-dimensional culture is closer to the original physiological state and can reproduce a reaction closer to that of a living body. For example, two-dimensional culture is often used to examine the effects of drugs such as anticancer agents on cancer cells. However, in two-dimensional culture, the drug reaches the cells in a planar manner, so the effect appears high even at low concentrations, and the effect of the drug may not be observed at the concentration assumed to be administered to patients. On the other hand, in three-dimensional culture, drug delivery and information transmission are also three-dimensional, and the drug concentration is different inside and outside the actual cell mass, so it is possible to reproduce an environment closer to that of a living body and examine the effect of the drug. In three-dimensional culture, spheroids formed by cell aggregation have been common until now, but recently, in addition to spheroids, technology for producing organoids from stem cells has been established. Organoids can be produced from patient-derived organs, tissues, and iPS cells, and are very useful for elucidating disease states and are also expected to be applied to regenerative medicine. However, three-dimensional culture is more costly than two-dimensional culture and is not a widely adopted method. In particular, organoid culture requires high technology and costs because proliferation and differentiation induction are performed using various inductive factors and inhibitors from a small number of stem cells or iPS cells present in living tissues.

[0003] When culturing organoids, it is necessary to purchase and add recombinant proteins such as Noggin, R-spondin, Wnt-3a, or Activin, or to add the culture supernatant of cells that secrete these proteins. The type and amount of additives added must be adjusted depending on the type of organoid and the stage of culture. In particular, when culturing intestinal cell organoids, at least three proteins are required: noggin, spongin, and winth. Cells that secrete only spongin and winth are commercially available. On the other hand, cells that secrete only noggin are not commercially available. Therefore, the only options were to extract noggin from cells that secrete multiple types of proteins including noggin, or to use noggin protein while ignoring the presence of other secreted proteins. Furthermore, depending on the type of organoid, spongin and winth may not be necessary, in which case the only option was to purchase and add recombinant noggin protein, which was costly. Therefore, from a cost perspective, the establishment of cells that secrete only noggin was desired.

[0004] Patent Document 1 discloses an invention relating to dorsal tissue-acting factors and compositions, and in particular discloses an expression vector for expressing human noggin, which has nerve tissue induction-promoting activity, in eukaryotic host cells. Furthermore, Patent Document 1 describes the successful expression of bioactive noggin in two mammalian cell lines (COS cells and mouse 293 cells). Patent Document 2 discloses a recombinant vector that contains nucleic acid encoding noggin as an axon elongation promoter and can express noggin in cells. However, there are no reports on the provision of noggin-producing cells suitable for organoid production. [Prior art documents] [Patent Documents]

[0005] [Patent Document 1] Patent No. 3431153 [Patent Document 2] Japanese Patent Publication No. 2008-255071 [Overview of the Initiative] [Problems that the invention aims to solve]

[0006] In light of the above issues, the objective is to provide cells that secrete noggin, a protein necessary for inducing various organoids from pluripotent stem cells, human, mouse, dog, and other animal tissues, and that can secrete noggin capable of improving the organoid induction efficiency. [Means for solving the problem]

[0007] As a result of diligent research, the inventors succeeded in establishing cells that secrete noggin. Furthermore, they confirmed that using the noggin secreted by these cells improves the organoid induction efficiency, leading to the completion of the present invention. That is, the present invention includes the following aspects: One aspect of the present invention is, [1] relating to nogging-secreting cells having nucleic acids that encode nogging. Here, in one embodiment, the noggin-secreting cells of the present invention are [2] Noggin-secreting cells as described in [1] above, The secreted nogging is characterized by having one of the following amino acid sequences (1) to (3), and the nogging is for organoid induction: (1) Amino acid sequence shown in Sequence ID No. 1 (2) An amino acid sequence that is 85% or more identical to the amino acid sequence shown in Sequence ID No. 1 and has organoid-inducing ability. (3) An amino acid sequence in which one or more amino acids are added, deleted, or substituted in the amino acid sequence shown in Sequence ID No. 1, and which has organoid-inducing ability. Furthermore, in one embodiment, the noggin-secreting cells of the present invention are [3] Noggin-secreting cells as described in [1] or [2] above, The secreted noggin is characterized by having a purification tag attached to it. Furthermore, in one embodiment, the noggin-secreting cells of the present invention are [4] Noggin-secreting cells as described in [1] or [3] above, Noggin-secreting cells are cells in which the secreted noggin forms dimers. Furthermore, in one embodiment, the noggin-secreting cells of the present invention are [5] Noggin-secreting cells as described in any of [1] to [4] above, The noggin-secreting cells are characterized by being HEK293 cells having nucleic acids that encode noggin. Furthermore, in one embodiment, the noggin-secreting cells of the present invention are [6] Noggin-secreting cells as described in any of [1] to [5] above, It is characterized by the absence of R-spondin or Wnt-3a secretion. Furthermore, in one embodiment, the noggin-secreting cells of the present invention are [7] Noggin-secreting cells as described in any of [1] to [6] above, It is characterized by being deposited under the accession number NITE P-03381. Another aspect of the present invention is: [8] A method for producing a culture containing noggin, The present invention relates to a manufacturing method comprising the step of culturing noggin-secreting cells as described in any of [1] to [7] above. Another aspect of the present invention is: [9] The present invention relates to a noggin-containing composition comprising a culture or a purified product thereof, recovered from a culture medium in which noggin-secreting cells described in any of [1] to [8] above were cultured. Another aspect of the present invention is:

[10] A method for producing organoids, The present invention relates to a method for producing organoids, comprising the step of establishing organoids from cells using the noggin-containing composition described in [9] above. [Effects of the Invention]

[0008] By using the Noggin-secreting cells provided by the present invention, it becomes unnecessary to purchase expensive recombinant Noggin proteins, and it is possible to reduce the costs of various tests. In addition, in the production of organoids, it becomes possible to administer the Noggin protein in the required amount at the required culture timing, or to freely combine other proteins and administer the Noggin protein, similar to the administration of a single protein such as spondin or Wnt.

Brief Description of the Drawings

[0009] [Figure 1] Figure 1 shows an image taken under an optical microscope on the 7th day of culture after transfection of Noggin-expressing cells transfected with the Noggin-Fc tag protein expression vector or the Noggin-STII tag protein expression vector prepared in the following examples. [Figure 2] Figure 2 shows images taken under an optical microscope (lower left image) and a fluorescence microscope (upper left image) on the 7th day of culture after transfection of Noggin-expressing cells transfected with the pPBP-3×EGFPK expression vector prepared in the following examples, as well as their composite image (upper right image). [Figure 3] Figure 3 shows an image taken under an optical microscope on the 14th day of culture after transfection of Noggin-expressing cells transfected with the Noggin-Fc tag protein expression vector or the Noggin-STII tag protein expression vector prepared in the following examples. [Figure 4] Figure 4 shows images taken under an optical microscope (lower left image) and a fluorescence microscope (upper left image) on the 14th day of culture after transfection of Noggin-expressing cells transfected with the pPBP-3×EGFPK expression vector prepared in the following examples, as well as their composite image (upper right image). [Figure 5]Figure 5 shows a schematic diagram and the number of amino acid residues of the polypeptides produced from each of the pPBP-3×EGFPK expression vector, pPBP-3×Noggin-Fc expression vector, and pPBP-3×Noggin-STII expression vector used in the following examples. In the figure, Fl indicates the FLAG (registered trademark) tag. [Figure 6] Figure 6 shows a schematic diagram and the number of amino acid residues after signal peptide cleavage in the polypeptides produced from each of the pPBP-3×Noggin-FC expression vector and pPBP-3×Noggin-STII expression vector used in the following examples. In the figure, FI indicates the FLAG (registered trademark) tag. [Figure 7] Figure 7 shows the results of SDS-PAGE of the Fc-tagged Noggin protein recovered from the culture supernatant of Noggin-expressing cells prepared in the following examples, and Western blotting using CBB staining (left figure) and POD-labeled anti-human IgG antibody (right figure). Lane 1 shows the non-denatured elution sample, and lane 2 shows the sample treated with SDS buffer for denaturation. [Figure 8] Figure 8 shows the results of Western blotting using a POD-labeled anti-Strep Tag II antibody for SDS-PAGE of the STII-tagged Noggin protein recovered from the culture supernatant of Noggin-expressing cells prepared in the following examples. Lane 1 shows the sample subjected to SDS-PAGE without affinity purification, lane 2 shows the sample subjected to SDS-PAGE after affinity purification with Protein G beads, and lane 3 shows the sample subjected to SDS-PAGE after purification with StrepTactin (registered trademark) beads. The arrow in the figure indicates a band of approximately kDa. [Figure 9] Figure shows images of the cell state on the 6th day of culture when mouse intestinal organoids were induced using the Noggin-containing composition of the present invention, taken under an optical microscope. The upper left figure shows the induction result using commercially available recombinant Noggin (100 ng / ml), and the other figures show the induction results when the Noggin-containing composition of the present invention was added to the induction medium at a concentration of 0% to 30% (vol / vol). [Figure 10] Figure 10 shows the results of Western blot analysis to confirm whether or not expressed noggin forms dimers within cells. The results of immunoblotting with POD-labeled anti-human IgG antibody after SDS-PAGE are shown for each undenatured and denatured sample. [Modes for carrying out the invention]

[0010] One aspect of the present invention provides nogging-secreting cells having nucleic acids encoding nogging. The origin of the noggin protein secreted by the noggin-secreting cells of the present invention is not particularly limited, and the noggin-secreting cells can suitably produce noggin proteins of various organisms. Preferably, it is a noggin protein derived from mammals. Examples of mammals include humans, mice, rats, dogs, cattle, pigs, etc., and preferably humans or mice. In one embodiment, the nucleic acid encoding noggin can be represented as a nucleic acid encoding any of the following amino acid sequences (1) to (3): (1) Amino acid sequence shown in Sequence ID No. 1 (2) An amino acid sequence that is 85% or more identical to the amino acid sequence shown in Sequence ID No. 1 and has organoid-inducing ability. (3) An amino acid sequence in which one or more amino acids are added, deleted, or substituted in the amino acid sequence shown in Sequence ID No. 1, and which has organoid-inducing ability.

[0011] As described above, noggin in this specification also includes polypeptides comprising amino acid sequences that have 85% or more identity with the amino acid sequence shown in SEQ ID NO: 1 and that have organoid-inducing ability. In preferred embodiments, the polypeptide is a polypeptide comprising amino acid sequences that have 90%, 95%, 96%, 97%, 98%, or 99% or more identity with the amino acid sequence shown in SEQ ID NO: 1.

[0012] In this specification, "one or more amino acids deleted, substituted or added" means that a number of amino acids (preferably 22 or fewer, 20 or fewer, 15 or fewer, 10 or fewer, more preferably 7 or fewer, and even more preferably 5 or fewer) that can be deleted, substituted or added by known mutant peptide production methods such as site-directed mutagenesis. Furthermore, when there are mutations such as additions, deletions, or substitutions in the amino acid sequence shown in Sequence ID No. 1, it is preferable that the mutations occur on the N-terminal side of the amino acid sequence. It is even more preferable that the mutations occur in amino acids other than cysteine ​​that form the disulfide (SS) bond on the C-terminal side of the amino acid sequence.

[0013] It is preferable to add nucleic acids encoding secretory signal peptides for secreting noggin outside the cell, and tags for protein purification, to the "nucleic acid encoding noggin." The secretory signal peptide can be expressed by attaching it to the noggin protein, which is the protein to be expressed, in the form of a fusion protein in which the C-terminal side of the signal peptide is bound to the N-terminal side of the signal peptide. The secretory signal peptide is not limited as long as it can secrete noggin from the host cell into the extracellular space, and any known secretory signal peptide can be used. Examples of secretory signal peptides are, but are not limited to, those described in International Publication No. 2015 / 133074 and International Publication No. 2011 / 078351. Those skilled in the art can appropriately select a preferred secretory signal peptide depending on the host cell, culture conditions, etc. The protein purification tag may be attached to either the C-terminus or N-terminus of the protein to be expressed, but the C-terminus is preferable considering post-translational modifications. The tag should be attached to the protein to be expressed in the form of a fusion protein in which the N-terminus of the protein purification tag is bound. The protein purification tag is not limited to any known tag, as long as the nogging secreted into the culture medium can be recovered. The protein purification tag is not limited to the following, but can be appropriately selected depending on the purpose. Examples include Fc tags such as the partial human IgG heavy chain Fc tag described in SEQ ID NO: 2, the Strep tag (Strept-tag® II / STII) consisting of 8 amino acid residues that bind to streptavidin as described in SEQ ID NO: 3, histidine tags, cysteine ​​tags, FLAG® tags, and HA tags. Those skilled in the art can select a preferred tag suitable for purification depending on the host cells and culture conditions. The nogging and the protein purification tag may be linked by a linker, for example, as shown in SEQ ID NO: 4.

[0014] The noggin protein expressed by noggin protein-expressing cells may be a fragment of the noggin protein, or it may contain amino acid sequences other than the amino acid sequence of the noggin protein, as long as it has organoid-inducing ability. The amino acid sequences other than the amino acid sequence of the noggin protein are not particularly limited, but examples include the addition of the amino acid sequence of the protein purification tag mentioned above. Furthermore, the amino acid sequence of the noggin protein does not need to be exactly the same as the amino acid sequence obtained from a database such as GenBank, and may be substantially the same as the amino acid sequence obtained from the database, as long as it has organoid-inducing ability. Substantially identical amino acid sequences include those that are at least 85% identical, more preferably at least 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence obtained from the database.

[0015] In this specification, "having organoid-inducing ability" means that when used in culture in the organoid production method described herein or in known organoid production methods, it can exert its function as a noggin protein to induce target cells into organoids. The induction of target cells into organoids can be confirmed, for example, by adding the noggin-containing composition of the present invention along with Wnt-3a and R-spondin to the culture medium and culturing the target cells, as described in the examples below.

[0016] The cells from which the noggin-secreting cells are derived are not limited to any known cultured cells that can be cultured to express and secrete noggin, as long as the noggin gene can be introduced into them. Cells that have established gene introduction conditions and culture conditions and are easy to handle are more preferable, and are not limited to the following, but examples include HEK293 cells, COS7 cells, and CHO cells.

[0017] In one embodiment of the present invention, the noggin-secreting cells do not secrete R-spondin or Wnt-3a. R-spondin and Wnt-3a are important factors in organoid formation, similar to noggin. By using noggin-secreting cells that secrete only noggin and not R-spondin or Wnt-3a, which are useful for organoid induction, it is possible to use only noggin protein for organoid induction at the desired concentration and timing.

[0018] Furthermore, in one embodiment, the noggin-secreting cells of the present invention are cells deposited with the National Institute of Technology and Evaluation (NITE) Patent Organism Depositary Center (2-5-8 Higashi-Kazusa-Kamatari, Kisarazu City, Chiba Prefecture, Japan) on February 10, 2021, under accession number NITE P-03381.

[0019] Cells expressing noggin protein can be produced using known genetic engineering techniques. Specifically, noggin protein-expressing cells can be produced by inserting the DNA encoding the desired noggin protein into a known expression vector and introducing the resulting expression vector into a suitable host cell. Cells that stably express the noggin protein or cells that transiently express the noggin protein can both be suitably used as noggin-expressing cells according to the present invention. The amino acid sequence of the noggin protein and the nucleotide sequence of the gene encoding the noggin protein can be obtained from known databases such as GenBank. The GenBank accession number for the amino acid sequence of human noggin protein is AAA83259, the GenBank accession number for the nucleotide sequence encoding human noggin protein is U31202, the GenBank accession number for the amino acid sequence of mouse noggin protein is EDL15873, and the GenBank accession number for the nucleotide sequence of mouse noggin protein is NM_008711.2.

[0020] One aspect of the present invention provides a method for producing a culture containing noggin, comprising the step of culturing the noggin-secreting cells described above. The culture medium used for culturing Noggin protein-expressing cells is not particularly limited, and an appropriate medium can be selected from known media (e.g., DMEM high glucose w / L-glutamine (WAKO#044-29765)) depending on the type of cell. The culture period is also not particularly limited, and a period during which the cells can be cultured without changing the medium should be appropriately selected. The serum is not particularly limited as long as it is serum prepared for cell culture, but bovine serum is preferred. Examples of bovine serum include fetal bovine serum (FBS), newborn calf serum, and calf serum, all of which can be suitably used. The amount of serum added is not particularly limited, and it should be added to the medium to a concentration recommended for culturing the Noggin protein-expressing cells being used. In one embodiment, however, FBS can be added to a known medium at a concentration of 10% (vol / vol).

[0021] By culturing Noggin protein-expressing cells in a culture medium, a culture containing the Noggin protein expressed by the Noggin protein-expressing cells can be obtained. Examples of cultures include culture supernatant, a mixture of culture supernatant and cells, and a mixture of culture supernatant and cell disruptors, which can be prepared by known methods. Noggin protein is secreted into the culture medium upon addition of a signal peptide. Therefore, it is preferable that the culture contains culture supernatant, and more preferably culture supernatant from which cells have been removed.

[0022] Another aspect of the present invention provides a noggin-containing composition comprising a culture recovered from a culture medium in which the noggin-secreting cells described above were cultured, or a purified product thereof. The noggin-containing composition may, for example, use the culture supernatant of the culture medium in which the noggin-expressing cells were cultured as is, or it may be used as a composition containing the purified product after purification. The purified product can be prepared by expressing the noggin protein as a tag-fusion noggin protein to which a protein purification tag has been attached, and then recovering it by affinity purification using the tag. The purification method can be selected from known optimal systems depending on the tag used. When using an Fc tag, for example, Protein G beads can be used, and when using a Strep tag, Strep-Tactin® beads can be used. The prepared noggin-containing composition is preferably sterilized by a known method such as a vacuum filter and stored frozen in aliquots until use.

[0023] The present invention will be described more specifically below with reference to examples, but the technical scope of the present invention is not limited to these examples. [Examples]

[0024] (1. Preparation of Nogin protein expression vector) Using a plasmid (pCDNA3 mNog-hFc: obtained from Keio University) containing cDNA encoding mouse noggin ORF and partial human IgG heavy chain (Fc) as a template, cDNA containing mouse noggin-encoding cDNA (SEQ ID NO: 5) and human IgG heavy chain (Fc)-encoding cDNA (SEQ ID NO: 6) was amplified using High Fidelity PCR enzyme. The resulting amplified cDNA was ligated to restriction enzyme KpnI and PmeI-treated sites of plasmid pPBP-3×EGFPK (an EGFP expression vector driven by a CMV enhance / promoter, where 3× indicates a sequence of three consecutive Flag tag sequences, and K indicates the presence of a restriction enzyme KpnI site) using the In-fusion HD Cloning Kit to construct a Noggin-Fc-tagged protein expression vector (pPBP-3×Flag-Nogging-Fc). The nucleotide sequence encoding the 3×FLAG tag is described in SEQ ID NO: 7. Similarly, using the plasmid pCDNA3 mNog-hFc (obtained from Keio University) as a template, cDNA containing the mouse noggin encoding cDNA (SEQ ID NO: 5) was amplified using primers containing the nucleotide sequence encoding Strep-TagII and a High Fidelity PCR enzyme. The obtained mouse noggin and Strep-TagII encoding cDNAs were ligated to restriction enzyme KpnI and PmeI-treated sites of plasmid pPBP-3×EGFPK using the In-fusion HD Cloning Kit to construct a Noggin-STII tag protein expression vector (pPBP-3×Flag-Nogging-STII). The nucleotide sequence encoding STII is described in SEQ ID NO: 8. The obtained plasmid was transformed into E. coli DH5α-competent cells according to a standard procedure. After transformation, the plasmid was isolated from the colonized DH5α cells, and the restriction enzyme sites and sizes were confirmed by restriction enzyme digestion and gel electrophoresis. The correctness of the base sequence was also confirmed by sequencing. For the gene transfer into HEK293 cells described below, a vector obtained by extraction and purification from the DH5α colonies prepared in this example was used.

[0025] (2. Generation of Nogin-expressing cells) Human fetal kidney-derived HEK293 cells were transfected with a pPBP-3×EGFPK expression vector (vector control), a Noggin-Fc tagged protein expression vector, or a Noggin-STII tagged protein expression vector using the PiggyBac Transposon Vector System (System Biosciences). Specifically, transfection was performed as follows. First, seed HEK293 cells into a 6-well plate (2 × 10⁻¹⁶ cells). 5 Cells were cultured (day 1). The following day, gene transfection was attempted using the transfection reagent XtremeGENE9 (Roche Diagnostics) (day 2 of culture). Noggin-Fc tagged protein expression vector or Noggin-STII tagged protein expression vector was transfected with piggyBac tramposase expression vector pCMV-HyPBase in a 1:1 ratio. In addition, pPBP-3×EGFPK (7121bp) without Noggin cDNA insertion was also used for transfection as a control to confirm transfection efficiency.

[0026] 72 hours after transfection (day 5 of culture), HEK293 cells were treated with trypsin and re-seeded in 10 cm dishes. Figures 1 and 2 show images of HEK293 cells with each expression vector introduced, taken under a light microscope or fluorescence microscope, on day 7 of culture. On day 8 of culture, puromycin (Cat#ant-pr-1; Final concentration: 1 μg / mL: Invivogen) was added as a selective agent to screen for cells expressing the noggin protein, and these cells were established. On day 11 of culture, the culture medium was replaced with fresh medium and culture was continued. Figures 3 and 4 show images of HEK293 cells with each expression vector introduced, taken under a light microscope or fluorescence microscope, on day 14 of culture. On day 15 of culture, cells expressing the noggin protein were collected by filter filtration. The collected cells were cryopreserved as a cell stock using Cellmenity (Waken B-Tech), a serum-free cell cryopreservation solution. Furthermore, one cell line of HEK293 cells into which the Noggin-Fc tag protein expression vector was introduced was deposited with the National Institute of Technology and Evaluation (NITE) Patent Microorganism Depository Center (NITE Patent Microorganism Depository Center: 2-5-8 Kazusa-Kamatari, Kisarazu City, Chiba Prefecture, Japan) on February 10, 2021, under accession number NITE P-03381.

[0027] (3. Culture of Nogin-expressing cells and preparation of culture supernatant) In this example, the secretion of noggin into the culture medium was confirmed by culturing the noggin-expressing cells prepared as described above. 3-1. Composition of the culture medium The composition of the culture medium (10% FBS, antibiotic-containing DMEM medium) used in this example is as follows: [Table 1]

[0028] 3-2. Thawing cryopreserved cells The cryopreserved Noggin-expressing cells (NITE P-03381) were thawed using the following method: 5 mL of culture medium was poured into a 15 mL tube, and another 10 mL was poured into a 10 cm dish. Both were warmed in a 37°C incubator. The cryopreserved solution containing the Noggin-expressing cells was removed from the vial deep freezer and quickly heated in a 37°C bath for 1-2 minutes until thawed, leaving only small ice fragments. The thawed Noggin-expressing cell solution was transferred to a 15 mL tube containing culture medium using a pipette and suspended. The solution was then centrifuged at 1,000 rpm for 5 minutes. After centrifugation, the supernatant was removed, and 1 mL of fresh culture medium was added. The entire volume of culture medium in the 15 mL tube was transferred to a 10 cm dish containing culture medium. Subsequently, the cells were cultured at 37°C and 5% CO2. The next day, the culture medium was replaced with fresh medium. Three days after waking the Noggin-expressing cells, puromycin was added to the culture medium to a final concentration of 1 μg / mL.

[0029] 3-3. Preparation of culture supernatant After thawing, Noggin-expressing cells were cultured until they reached 90% confluence, and then subcultured as follows: The culture medium was removed by aspiration. The cells were washed with 5-10 mL of PBS. After washing, 1 mL of 0.25% Trypsin / 1 mM EDTA was added and incubated at 37°C for 2 minutes. 5 mL of culture medium was added, the cells were harvested and transferred to a 15 mL tube. Then, the tubes were centrifuged at 1,000 rpm for 5 minutes. After centrifugation, the supernatant was removed. Fresh selective medium (10% FBS, antibiotic, puromycin-containing DMEM medium) was added, and a new T175cm tube was added. 2 The solution was dispensed into flasks (1:4 dilution; 25 mL). The cells were then cultured at 37°C for 3-5 days until confluence. Once the cells reached confluence, they were treated with trypsin and incubated at T175cm. 2 The cells were transferred to a flask and 25 mL of 10% (vol / vol) FBS-containing DMEM (Puromycin-free) was added (1:5 dilution). The cells were then incubated at 37°C for 3-5 days until confluence. Once the cells were again confluent, they were treated with trypsin and incubated at T175cm. 2The cells were transferred to a flask and cultured using 25 mL of 10% (vol / vol) FBS-containing DMEM medium (Puromycin-free) (1:2 dilution). The cells were then incubated at 37°C for 1 week until confluence was reached. These subculturing steps prevented the introduction of Puromycin into the medium. After 1 week of culture, the culture supernatant was collected and 25 mL of 10% (vol / vol) FBS-containing DMEM medium was added. After another 2 days of culture, the culture supernatant was collected again and 25 mL of 10% (vol / vol) FBS-containing DMEM medium was added. The culture supernatant can be collected 3-4 times. The collected culture supernatant was centrifuged at 300g for 5 minutes, and then sterilized using a vacuum filter. The sterilized culture supernatant was dispensed in fixed amounts into 15mL cryopreservation vials and stored frozen at -20°C until use.

[0030] 3-4. Electrophoresis and Western Blotting Figure 5 shows schematic diagrams and amino acid residue counts of polypeptides produced from the pPBP-3×EGFPK, pPBP-3×Noggin-FC, and pPBP-3×Noggin-StII vectors used in the above examples. Figure 6 shows schematic diagrams and amino acid residue counts of polypeptides produced from the pPBP-3×Noggin-FC and pPBP-3×Noggin-StII vectors after signal peptide cleavage. Noggin-Fc tagged protein was recovered from the culture supernatant of cells into which a Noggin-Fc tagged protein expression vector had been introduced by affinity purification using Protein G beads. The recovered Fc tagged fused Noggin protein was subjected to SDS-PAGE, followed by CBB staining and Western blotting using POD-labeled anti-human IgG antibody (POD Goat anti-human IgG, Cat# 109-035-003: Jackson). As a result, as shown in Figure 7, a band of approximately 57 kDa representing a monomer consisting of Noggin and the Fc tag was confirmed. Furthermore, Noggin-STII tagged protein was recovered from the culture supernatant of cells into which the Noggin-STII tagged protein expression vector had been introduced by affinity purification using Strep Tactin beads. The recovered Strep-tag fused Noggin protein was subjected to SDS-PAGE, and Western blotting was performed using POD-labeled anti-Strep Tag II antibody (Anti-Strep-tag II antibody, mouse mono, Cat# M211-3: MBL). As a result, as shown in Figure 8, a band of approximately 27 kDa representing a monomer consisting of Noggin and the STII tag was confirmed.

[0031] (4. Creation of organoids) As described above, it was confirmed that the culture supernatant of cells into which the Noggin-Fc tag protein expression vector was introduced contains noggin. Therefore, we investigated whether organoids could be prepared using this culture supernatant as a noggin-containing composition.

[0032] Organoids were produced by adding the culture supernatant (0, 10, 20, 30%) of commercially available recombinant mouse noggin (final concentration 100 ng / ml: Peprotech) or a transformant (accession number NITE P-03381) into which the above-mentioned Noggin-Fc tag protein expression vector was introduced, to DMEM-F12 medium containing Wnt-3a and R-spondin, and culturing mouse intestinal tissue-derived cells at 37°C for 6 days. Micrographs of the cultured cells are shown in Figure 9. As shown in Figure 9, it was confirmed that organoids were formed when the culture supernatant of a transformant into which the Noggin-Fc tag protein expression vector was introduced, as well as when commercially available recombinant mouse noggin was added. Furthermore, since organoids were formed by using the culture supernatant, it was confirmed that noggin is secreted extracellularly, and that it is possible to use the culture supernatant as a noggin-containing composition without disrupting the cells.

[0033] (5. Dimerization of noggin proteins within cells) Noggin is known to form dimers via disulfide bonds (Jay Groppe et al., Nature Vol.420, 12 December 2002, 636-642(2002)). Therefore, we investigated whether the expressed noggin formed dimers within the cells.

[0034] Nogging cell extracts were prepared using the RIPA buffer shown in Table 2. First, the culture medium was aspirated and washed twice with 4 mL of chilled PBS. Next, 80 μL of RIPA Buffer was added, and the precipitate was collected with a cell scraper and transferred to a 1.5 mL tube. After mixing, the tube was placed on ice for 10 minutes, vortexed every 3 minutes, and the cells were destroyed by sonication. The cell supernatant was centrifuged at 14,000 rpm for 40 minutes at 4°C, and the supernatant was stored at -80°C until use. For Western blot analysis, an equal volume of 2×SDS-sample buffer or undenatured sample buffer, as described below, was added to prepare the Nogging cell extract.

[0035] [Table 2]

[0036] Next, the culture supernatant (Noggin cell medium) of cells into which the Noggin-Fc tag protein expression vector had been introduced, and the Noggin cell extract described above, were treated in the following three groups. (a) Unpurified with Protein G / Undenatured (No SDS buffer treatment) (b) Purified with Protein G / Undenatured (no SDS buffer treatment) (c) Purified with Protein G / Denatured (treated with SDS buffer) Western blot analysis was then performed. The results are shown in Figure 10. Samples (a) to (c) above were applied to lanes 1 to 3, respectively. The denaturation conditions (with SDS buffer treatment) were as follows: Equal volumes of 2×SDS-sample buffer (composition: (1) 0.125 mol / l Tris-HCl, pH 6.8 (2) 4 w / v% SDS, (3) 20 w / v% Glycerol, (4) 0.002 w / v% BPB, (5) 10 vol% 2-mercaptoethanol) were added, mixed, and heated at 95°C for 5 minutes. Furthermore, for the undenatured condition (without SDS buffer treatment), an equal volume of the undenatured condition sample buffer (composition: (1) 0.125 mol / l Tris-HCl, pH 6.8, (3) 20 w / v% Glycerol, (4) 0.002 w / v% BPB, (5) 10 vol% 2-mercaptoethanol) was added and mixed (without heating).

[0037] As is clear from Figure 10, both the nogging cell extract and the nogging cell medium showed a band around 110 kDa in lane 2 (undenatured cells) and a band around 57 kDa in lane 3 (denatured cells). Therefore, it was confirmed that nogging forms dimers within the cells, and these dimers are secreted extracellularly.

Claims

1. A noggin-secreting cell having nucleic acid encoding noggin, The secreted noggin (1) The amino acid sequence shown in Sequence ID No. 1 or (2) An amino acid sequence that is 95% or more identical to the amino acid sequence shown in Sequence ID No. 1 and has organoid-inducing ability. And, The aforementioned noggin is for organoid induction, It does not secrete R-spondin and Wnt-3a, and The nogging-secreting cell, wherein the nucleic acid encoding nogging is a nucleic acid encoding the amino acid sequence of (1) or (2) above.

2. Noggin-secreting cells according to claim 1, Nogging-secreting cells to which a purification tag is attached to the secreted nogging.

3. Noggin-secreting cells according to claim 1 or 2, Noggin-secreting cells are cells in which the secreted noggin forms dimers.

4. Noggin-secreting cells according to any one of claims 1 to 3, The noggin-secreting cells are HEK293 cells having nucleic acids that encode noggin.

5. Noggin-secreting cells according to any one of claims 1 to 4, Noggin-secreting cells deposited under accession number NITE P-03381.

6. A method for producing a culture containing noggin, A method for manufacturing a cell culture of noggin-secreting cells according to any one of claims 1 to 5.

7. A noggin-containing composition comprising a culture recovered from a culture medium in which noggin-secreting cells according to any one of claims 1 to 5 are cultured, or a purified product thereof.

8. A method for creating organoids, A method for producing organoids, comprising the step of establishing organoids from cells using a composition containing noggin obtained by the production method described in claim 6.

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