JP7848994B2 - Cells that secrete Noggin protein and method for producing organoids using them - Google Patents

Description

NPMD NITE P-03381

This invention relates to cells that secrete the noggin protein and a method for producing organoids using the same.

In life science research, two-dimensional cell culture is commonly used, but 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. Furthermore, cell adhesion and communication are also in a two-dimensional direction, making it impossible to reproduce the exact physiological state. For this reason, three-dimensional culture is closer to the original physiological state and can reproduce reactions closer to those of living organisms. For example, two-dimensional culture is often used to investigate the effects of drugs such as anticancer drugs on cancer cells, but in two-dimensional culture, the drug reaches the cells in a two-dimensional manner, so the effect is high even at low concentrations, and the drug's effect may not be observed at concentrations that would be expected to be administered to patients. On the other hand, in three-dimensional culture, the drug's reach and communication are also three-dimensional, and the drug concentration differs inside and outside the actual cell aggregate, allowing for the reproduction of an environment closer to that of living organisms to investigate the drug's effect. In three-dimensional culture, spheroids, which are aggregates of cells that form clumps, have been common until now, but recently, in addition to spheroids, the technology for organoids made from stem cells has been established. Organoids can be created from patient-derived organs and tissues, as well as iPS cells, making them extremely useful for elucidating disease mechanisms and promising applications in 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 advanced technology and expense because it involves inducing proliferation and differentiation from a small number of stem cells or iPS cells present in living tissue using various inducing factors and inhibitors.

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.

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.

Patent No. 3431153 Japanese Patent Publication No. 2008-255071

In light of the above challenges, 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 in a way that improves the efficiency of organoid induction.

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 encoding 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) The 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 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 not secreting R-spondin or Wnt-3a.
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 that includes a 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 an organoid,
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.

By using the noggin-secreting cells provided by this invention, the need to purchase expensive recombinant noggin proteins is eliminated, thereby reducing the costs of various tests. Furthermore, in organoid production, it becomes possible to administer noggin proteins at the required culture timing, in the required amount, or in combination with other proteins, similar to the administration of individual proteins such as spongin or winth.

Figure 1 shows images taken under a light microscope on day 7 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 example described below. Figure 2 shows images of Noggin-expressing cells transfected with the pPBP-3×EGFPK expression vector prepared in the example below, taken under a light microscope (lower left panel) and a fluorescence microscope (upper left panel) on day 7 of culture after transfection, as well as a composite image of these images (upper right panel). Figure 3 shows images taken under a light microscope on day 14 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 example described below. Figure 4 shows images of Noggin-expressing cells transfected with the pPBP-3×EGFPK expression vector prepared in the example below, taken under a light microscope (lower left panel) and a fluorescence microscope (upper left panel) on day 14 of culture after transfection, as well as a composite image of these images (upper right panel). Figure 5 shows schematic diagrams and amino acid residue counts of polypeptides produced from 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® tag. Figure 6 shows schematic diagrams and amino acid residue counts after signal peptide cleavage of polypeptides produced from the pPBP-3×Noggin-FC expression vector and the pPBP-3×Noggin-STII expression vector used in the following examples. In the figure, FI indicates the FLAG® tag. Figure 7 shows the results of Western blotting using CBB staining (left) and POD-labeled anti-human IgG antibody (right) on Fc-tagged Noggin protein recovered from the culture supernatant of Noggin-expressing cells prepared in the following examples, subjected to SDS-PAGE. Lane 1 shows the undenatured eluted samples, and lane 2 shows the samples denatured using SDS buffer. Figure 8 shows the results of Western blotting using a POD-labeled anti-StrepTag II antibody on STII-tagged Noggin protein recovered from the culture supernatant of Noggin-expressing cells prepared in the following examples, subjected to SDS-PAGE. 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® beads. The arrows in the figure indicate bands of approximately 27 kDa. Figure 9 shows images of the state of cells on day 6 of culture when mouse intestinal organoids were induced using the noggin-containing composition of the present invention, as captured under a light microscope. The upper left figure shows the induction results using commercially available recombinant noggin (100 ng/ml), while the other figures show the induction results when the noggin-containing composition of the present invention was added to the induction medium at concentrations of 0% to 30% (vol/vol). Figure 10 shows the results of Western blot analysis to confirm whether or not expressed noggin forms dimers within the cells. The results of immunoblotting with POD-labeled anti-human IgG antibody after SDS-PAGE are shown for each undenatured and denatured sample.

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) The 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 has organoid-inducing ability.

As described above, the 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 possess 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.

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 (S-S) bond on the C-terminal side of the amino acid sequence.

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 in the form of a fusion protein in which the C-terminal side of the signal peptide is attached to the N-terminal side of noggin, the protein to be expressed. 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 not limited to those listed below, but include 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 consisting of eight amino acid residues that bind to streptavidin described in SEQ ID NO: 3 (Strept-tag® II/STII), histidine tag, cysteine tag, FLAG® tag, and HA tag. Those skilled in the art can select a preferred tag suitable for purification depending on the host cell 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.

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 noggin protein's amino acid sequence, as long as it has organoid-inducing ability. The amino acid sequences other than the noggin protein's amino acid sequence 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 databases such as GenBank; as long as it has organoid-inducing ability, it may be substantially identical to the amino acid sequence obtained from the database. A substantially identical amino acid sequence includes sequences 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.

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 together with Wnt-3a and R-spondin to the culture medium and culturing the target cells, as described in the examples below.

The cells from which noggin-secreting cells are derived are not limited to any known cultured cells that can be cultured and express and secrete noggin, as long as they can be introduced with the noggin gene. Cells with established gene introduction and culture conditions and that are easy to handle are more preferable. Examples, though not limited to the following, include HEK293 cells, COS7 cells, and CHO cells.

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.

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.

Cells expressing noggin protein can be produced using known genetic recombination 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.

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 (for example, DMEM high glutase 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).

By culturing Noggin protein-expressing cells in a culture medium, a culture containing the Noggin protein expressed by the Noggin protein-expressing cells is obtained. Examples of cultures include culture supernatant, a mixture of culture supernatant and cells, and a mixture of culture supernatant and cell disruption, all of 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.

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.

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.

(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 a High Fidelity PCR enzyme. The amplified cDNA obtained was ligated to the restriction enzyme KpnI and PmeI treatment sites of plasmid pPBP-3×EGGFPK (an EGFP expression vector driven by CMV enhance/promote, 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 tag protein expression vector (pPBP-3×Flag-Nogging-Fc). The nucleotide sequence encoding the 3×FLAG tag is described in Sequence ID No. 7.
Similarly, using the plasmid pCDNA3 mNog-hFc (obtained from Keio University) as a template, a 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 the 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 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.

(2. Generation of Noggin-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, HEK293 cells were seeded on a 6-well plate (2 × ^10⁵ cells/well) (Day 1 of culture). The following day, gene transfection was attempted using the transfection reagent XtremeGENE9 (Roche Diagnostics) (Day 2 of culture). Noggin-Fc tag protein expression vector or Noggin-STII tag 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.

Seventy-two 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 recovered cells were cryopreserved as a cell stock using Cellmenity (manufactured by 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 (2-5-8 Kazusa-Kamatari, Kisarazu City, Chiba Prefecture, Japan) on February 10, 2021, under accession number NITE P-03381.

(3. Culture of Noggin-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:

3-2. Thawing of 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 small ice fragments remained. The thawed Noggin-expressing cell solution was transferred to a 15 mL tube containing culture medium using a pipette and suspended. Next, the solution was 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 inducing noggin-expressing cells, puromycin was added to the culture medium to a final concentration of 1 μg/mL.

3-3. Preparation of Culture Supernatant After thawing, Noggin-expressing cells were cultured until 90% confluence was reached, 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 collected and transferred to a 15 mL tube. The cells were then centrifuged at 1,000 rpm for 5 minutes. The supernatant was removed after centrifugation. Fresh selective medium (DMEM medium containing 10% FBS, antibiotic, and puromycin) was added and distributed into a new T175 ^cm² flask (1:4 dilution; 25 mL). The cells were then cultured at 37°C for 3-5 days until confluence was reached. Once the cells reached confluence, they were trypsin-treated, transferred to a T175 ^cm² 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. Again, once the cells reached confluence, they were trypsin-treated, transferred to a T175 ^cm² flask, and cultured in 25 mL of 10% (vol/vol) FBS-containing DMEM (Puromycin-free) medium (1:2 dilution). The cells were then incubated at 37°C for 1 week until confluence. These subculturings prevented the introduction of Puromycin into the culture medium. After 1 week of culture, the culture supernatant was collected, and 25 mL of fresh 10% (vol/vol) FBS-containing DMEM medium was added. After another two days of incubation, the culture supernatant was collected and 25 mL of fresh 10% (vol/vol) FBS-containing DMEM medium was added. The culture supernatant can be collected 3 to 4 times.
The collected culture supernatant was centrifuged at 300 g for 5 minutes, and then sterilized using a vacuum filter. The sterilized culture supernatant was dispensed in fixed amounts into 15 mL cryopreservation vials and stored frozen at -20°C until use.

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 Noggin protein was subjected to SDS-PAGE, and CBB staining and Western blotting with POD-labeled anti-human IgG antibody (POD Goat anti-human IgG, Cat# 109-035-003: Jackson) were performed. 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, the Noggin-STII tag protein was recovered from the culture supernatant of cells into which the Noggin-STII tag 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.

(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.

To DMEM-F12 medium containing Wnt-3a and R-spondin, culture supernatant (0, 10, 20, 30%) of commercially available recombinant mouse noggin (final concentration 100 ng/ml: Peprotech) or the culture supernatant (0, 10, 20, 30%) of a transformant (accession number NITE P-03381) into which the Noggin-Fc tag protein expression vector obtained above was introduced was added. Mouse intestinal tissue-derived cells were cultured at 37°C for 6 days to produce organoids. Micrographs of the cultured cells are shown in Figure 9. As shown in Figure 9, organoid formation was confirmed when the culture supernatant of a transformant (introduced with the Noggin-Fc tag protein expression vector) was added, similar to the case when commercially available recombinant mouse noggin was added. Furthermore, since organoids were formed using the culture supernatant, it was confirmed that noggin is secreted extracellularly, and that the culture supernatant can be used as a noggin-containing composition without disrupting the cells.

(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.

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 mixture 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 either 2×SDS-sample buffer or undenatured sample buffer (described later) was added to prepare the Nogging cell extract.

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).

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 (8)

A noggin-secreting cell having nucleic acid encoding noggin,
The secreted noggin is (1) an amino acid sequence shown in SEQ ID NO: 1 or (2) an amino acid sequence that is 95% or more identical to the amino acid sequence shown in SEQ ID NO: 1 and has organoid-inducing ability.
The aforementioned noggin is for organoid induction,
The nogging-secreting cell, which does not secrete R-spondin and Wnt-3a, and in which the nucleic acid encoding nogging is a nucleic acid encoding the amino acid sequence of (1) or (2) above. Noggin-secreting cells according to claim 1,
Nogging-secreting cells to which a purification tag is attached to the secreted nogging. Noggin-secreting cells according to claim 1 or 2,
Noggin-secreting cells are cells in which the secreted noggin forms dimers. 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. Noggin-secreting cells according to any one of claims 1 to 4,
Noggin-secreting cells deposited under accession number NITE P-03381. 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. A noggin-containing composition comprising a culture or purified product thereof, recovered from a culture medium in which noggin-secreting cells according to any one of claims 1 to 5 are cultured. 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.