Human permanent chondrocytes and methods for preparing them, transplant materials and compositions for the treatment of cartilage diseases

Abstract

This invention provides permanent cartilage in which ossification is suppressed when transplanted into the living body. [Solution] A method for preparing GDF5-positive permanent chondrocytes, comprising culturing human chondrocytes in a medium containing a MEK inhibitor.

Description

Technical Field

[0001] The present invention relates to human permanent cartilage progenitor cells, a method for preparing the same, a transplantation material, and a composition for treating cartilage diseases.

Background Art

[0002] Osteoarthritis (OA), which is a typical degenerative and destructive change of articular cartilage, has an extremely large number of patients (7.8 million in Japan), but there is still no drug that can suppress the progression of the disease itself, and the administration of analgesics as symptomatic treatment is the main treatment method. As the disease worsens, arthroplasty, which replaces the joint with a metal artificial joint, is performed, but there are risks such as a service life limit (15 - 20 years), infection, pulmonary embolism secondary to thrombosis, and dislocation. The regeneration of cartilage tissue for damage to limb joint cartilage has been a long-standing problem in the field of plastic surgery. Currently, transplantation of autologous mesenchymal stem cells / chondrocytes (Non-Patent Document 1) and allogeneic chondrocytes (Non-Patent Document 2) has been attempted, but problems such as high cost, invasiveness, limitations in the amount of collection, and individual differences in the purity / quality control of the cell source have become issues for obtaining stable and high-quality clinical results.

[0003] In addition, in the fields of plastic surgery and orthopedic surgery, attempts have been made to tissue-engineer human cartilage tissue from cultured autologous chondrocytes or chondrocytes derived from human iPS cells and transplant it for the removal of malignant tumors that occur on the face, trauma such as traffic accidents, cleft lip and palate with nasal deformity, or microtia.

[0004] Furthermore, Patent Document 1 discloses a method for obtaining human cartilage progenitor cells from human iPS cells via lateral plate mesoderm.

[0005] Currently, although cartilage regeneration technology in the field of cartilage regenerative medicine has been developing, ossification after transplantation remains a major problem in autologous cartilage and cartilage cells / tissues derived from human iPS cells (Non-Patent Document 3).

[0006] Cartilage-based reconstruction is used not only in orthopedic surgery but also in plastic surgery for ear and nasal morphology correction, and reconstruction of eyelids and orbital floors, and in pediatric surgery for conditions such as laryngotracheal stenosis, using autologous costal cartilage as a substitute tissue. However, these also present the problem of ossification. Although many cartilage regeneration techniques have been disclosed, a technique for creating permanent cartilage tissue that does not ossify has not yet been established. [Prior art documents] [Patent Documents]

[0007] [Patent Document 1] WO2021 / 54449 [Non-patent literature]

[0008] [Non-Patent Document 1] J Bone Joint Surg Br 84-B: 571-578, 2002 [Non-Patent Document 2] npj Regen Med 7:71, 2022 [Non-Patent Document 3] J. Cel. Biol. 1987, 104(5), 1435-1441 [Non-Patent Document 4] Br. J. Plast Surg. 1993, 46(5), 416-420 [Non-Patent Document 5] Tissue Eng Part A: 1355-367 2021 [Overview of the Initiative] [Problems that the invention aims to solve]

[0009] The present invention aims to provide permanent cartilage in which ossification is suppressed when transplanted into the living body. [Means for solving the problem]

[0010] In order to solve the above problems, the inventors of this invention conducted extensive research and found that by culturing human chondrocytes in a culture medium containing a MEK inhibitor, permanent chondrocytes can be obtained, and when these permanent chondrocytes are transplanted into the body, permanent cartilage with suppressed ossification can be obtained.

[0011] The present invention provides the following: a method for preparing human permanent chondrocytes, human permanent chondrocytes, transplantation material, and composition for the treatment of cartilage diseases. [1] A method for preparing GDF5-positive human permanent chondrocytes, comprising culturing human chondrocytes in a medium containing a MEK inhibitor. [2] The preparation method described in [1], wherein the concentration of the MEK inhibitor in the culture medium is 0.001 to 10 μM. [3] Human permanent chondrocytes that are GDF5-positive and form permanent cartilage with suppressed ossification during in vivo transplantation. [4] A transplant material comprising human permanent chondrocytes described in [3] or permanent chondrocytes derived therefrom. [5] A composition for the treatment of cartilage disease, comprising the human permanent chondrocytes and scaffold described in [3]. [6] A paste-like composition for treating cartilage diseases as described in [5]. [Effects of the Invention]

[0012] Human chondrogenic progenitor cells derived from human pluripotent stem cells represent a unique technology that allows us to study the branching points that determine cartilage fate. Through the use of GDF5-positive cartilage tissue containing human pluripotent stem cell-derived chondrogenic progenitor cells, we have developed a culture method that determines the fate of these cells towards permanent cartilage, successfully developing permanent chondrogenic progenitor cells with suppressed ossification. Furthermore, this technology is expected to have a ripple effect, potentially providing a clue to solving the problem of ossification in autologous and allogeneic cartilage transplantation.

[0013] In addition, the timing of administration, dosage, and administration interval described above can be changed as appropriate without being limited to the examples.

[0014] The features of the present invention are shown in the following (1) to (5). (1) By adding a MEK inhibitor during chondrogenesis using human chondrocyte progenitor cells derived from human pluripotent stem cells, we successfully induced GDF5-positive permanent chondrocyte progenitor cells that are the origin of permanent cartilage. (2) GDF5-positive permanent chondrocyte progenitor cells were negative for growth plate cartilage markers and highly expressed permanent cartilage markers. (3) By transplanting GDF5-positive permanent chondrocyte progenitor cells subcutaneously into immunodeficient animals, a permanent cartilage tissue mass that did not ossify for at least 3 months could be obtained. (4) By transplanting GDF5-positive permanent chondrocyte progenitor cells into the knee cartilage defect of immunodeficient animals, engraftment of PRG4-positive cartilage tissue mass was observed even after 3 months. (5) By newly developing a method for producing permanent cartilage with suppressed ossification, it is also expected to be applicable to a method for producing cartilage used for cartilage regeneration in regenerative medicine.

Brief Description of Drawings

[0015] [Figure 1] It is a design diagram of a GDF5-tdTomato reporter. A figure of the PCR image when knocked into human iPS cells is also shown. [Figure 2] It is a microscopic photograph and a flow cytometry analysis diagram of CD140b and CD90 for evaluating chondrogenic ability forward during induction of chondrocyte progenitor cells from GDF5-tdTomato reporter-introduced human iPS cells (Example 1). [Figure 3] It is a microscopic photograph and a flow cytometry analysis diagram during the search for induction of a GDF5-expressing cartilage tissue mass using GDF5-tdTomato reporter chondrocyte progenitor cells (Example 2). [Figure 4] It is a schematic diagram until the induction of a GDF5-expressing cartilage tissue mass and a figure of the RT-qPCR results during the induction of a GDF5-expressing tissue mass (Example 2). [Figure 5] It is a heat map diagram of the Bulk RNA-seq results during the induction of a GDF5-expressing cartilage tissue mass (Example 2). [Figure 6] This figure shows the results of special staining and immunofluorescence staining during induction of GDF5-expressing cartilage tissue (Example 2). [Figure 7] This figure shows the results of GDF5-expressing cartilage tissue transplantation (subcutaneous transplantation) (Example 3). [Figure 8] This figure shows the results of GDF5-expressing cartilage tissue transplantation (transplantation into a cartilage defect in the knee joint) (Example 3). [Modes for carrying out the invention]

[0016] In this specification, "ossification" means that cartilage calcifies, the calcified cartilage is absorbed by osteoclasts, and replaced by osteocytes formed by osteoblasts.

[0017] In this specification, "suppression of ossification" broadly encompasses not only the complete absence of ossification but also the observation of suppression of ossification compared to cases where the material is transplanted into the body without undergoing the process of the present invention. Whether or not ossification has been suppressed can be confirmed, for example, by the methods described in the examples below, using safranin O staining, Kossa staining, etc.

[0018] In the present invention, permanent chondrocytes in which ossification is suppressed for a long period after transplantation into vivo can be obtained by culturing human chondrocytes in a medium containing a MEK inhibitor. The medium used to induce permanent chondrocytes from human chondrocytes is not particularly limited other than containing a MEK inhibitor. In a preferred embodiment, a medium obtained by adding a MEK inhibitor to a known chondrocyte induction medium can be used. Specifically, the medium may contain BMP4, TGFβ1, L-ascorbic acid, ITS, or other medium components such as DMEM medium, IMDM medium, or F12 medium in addition to the MEK inhibitor, and may optionally contain antibacterial agents such as streptomycin and penicillin, non-essential amino acids (NEAA), ROCK inhibitors (e.g., Y-27362), etc.

[0019] The human permanent chondrocytes of the present invention encompass a cell population containing human permanent chondrocytes, or a cartilage tissue containing human permanent chondrocytes.

[0020] The permanent chondrocytes prepared by this invention, when transplanted into the body, form permanent cartilage with suppressed ossification, making them excellent transplant materials for treating cartilage-related diseases. Examples of cartilage-related diseases include type II collagen disorders, relapsing polychondritis, osteoarthritis, rheumatoid arthritis, and achondroplasia.

[0021] Human chondrogenic cells, which serve as raw materials for obtaining permanent chondrogenic cells with suppressed ossification, can be induced from pluripotent stem cells such as human iPS cells and human ES cells. Preferred human chondrogenic cells are those disclosed in Patent Document 1. When human chondrogenic cells are induced into cartilage using an induction medium that does not contain MEK inhibitors, for example, a medium containing BMP4 and TGFβ, they become hypertrophic cartilage such as growth plate cartilage. Since ossification is not completely suppressed in this hypertrophic cartilage, it gradually ossifies when transplanted into the body. The greatest feature of the present invention is the suppression of cartilage ossification in vivo. In one preferred embodiment, after transplantation into the body, for example, no cartilage ossification is observed even after more than 3 months have passed since transplantation.

[0022] The permanent chondrocytes of the present invention are negative for growth plate cartilage markers and highly express permanent cartilage markers. On the other hand, when human chondrocytes are cultured in a medium that does not contain MEK inhibitors, they develop into hypertrophic cartilage such as growth plate cartilage that is positive for growth plate cartilage markers and negative for permanent cartilage markers. When this hypertrophic cartilage is transplanted into the body, it gradually ossifies. Growth plate cartilage markers and permanent cartilage markers can be confirmed by bulk RNA-seq.

[0023] In order to obtain cartilage with suppressed ossification in vivo, the inventors tested numerous substances other than MEK inhibitors as additives to the culture medium for guiding human chondrocytes to permanent chondrocytes. These included SAG21K (HH activator), Vismodegib (HH inhibitor), ATRA (RA signaling activator), CHIR99021 (GSK-3 inhibitor), C59 (PORCN or WNT inhibitor), XAV939 (tankyrase 1 inhibitor), SF1670 (PTEN inhibitor), MK-206 (AKT inhibitor), VoHPIC (PTEN inhibitor), MYH1485 (mTOR activator), DAPT (γ-secretase or Notch inhibitor), LDN-19189 (ALK2 / 3 inhibitor), A-83-01 (ALK4 / 5 / 7 inhibitor), Rapamycin (mTOR inhibitor), 10% FBS, {100 ng / mL}. The TGFb1+300 ng / mL BMP4+10%FBS culture did not have an ossification-inhibiting effect when transplanted in vivo. Permanent chondroprogenitor cells with suppressed ossification were obtained only when a MEK inhibitor was used. These ossification-inhibited permanent chondroprogenitor cells were GDF5-positive, while chondroprogenitor cells that produced ossification, obtained using the other 17 additives and culture media without additives, were all GDF5-negative. From these results, it became clear that GDF5 positivity is important for suppressing ossification in chondroprogenitor cells.

[0024] Non-exclusive examples of MEK inhibitors include binimetinib, CI-1040, cobimetinib, pimacertib, PD318088, PD325901, PD334581, PD98059, refametinib, selumetinib, trametinib, GSK1120212, MEK162, RDEA119, AZD6244, AZD8330, E6201, TAK-733, GDC-0623, PD035901, PD184352, WX-554, U0126-EtOH, PD98059, BIX 02188, BIX 02189, honokiol, SL-327, GDC-0623, and APS-2-79 HCl, with PD325901 being preferred. MEK inhibitors were difficult to use in cell culture because they suppress cell proliferation and, in large quantities, can kill cells. However, the fact that these MEK inhibitors suppress the ossification of permanent chondrocytes was an unexpected result for the inventors.

[0025] The concentration of the MEK inhibitor in the culture medium is preferably 0.001 to 10 μM, more preferably 0.01 to 10 μM, and even more preferably 0.1 to 10 μM.

[0026] When inducing permanent chondrocytes with suppressed ossification from human chondrocyte progenitor cells, the culture temperature is preferably in the range of 36-38°C. The pH of the culture medium is preferably 7.3-7.6. The culture period can be appropriately set within the range of 1 to 1000 hours.

[0027] In a preferred embodiment of the present invention, the composition for treating cartilage diseases may include a scaffold. The scaffold is used to attach or embed cells on its surface and / or inside, thereby maintaining the physical integrity of the graft and providing strength. The scaffold is not particularly limited as long as it is a material that can connect cells at least physically (mechanically), and may be a cell-derived material or a non-cell-derived material. Examples of scaffolds include, but are not limited to, films made of amniotic membrane, PVDF, polytetrafluoroethylene (PTFE), polyurethane, polypropylene, polyester, vinyl chloride, polycarbonate, acrylic, silicone, 2-methacryloyloxyethyl phosphorylcholine (MPC), polylactic acid, polyglycolic acid, lactic acid-glycolic acid copolymer (PLGA), etc.; temperature-responsive gels (commercial name: Mebiol Gel) made by crosslinking fibrin, gelatin, collagen sodium alginate, poly(N-isopropylacrylamide) with polyethylene glycol; hyaluronic acid, glycosaminoglycans, proteoglycans, chondroitin, cellulose, agarose, carboxymethylcellulose, chitin, chitosan, gelatin, atelocollagen, elastin, fibronectin, pronectin, laminin, tenascin, fibroin, entactin, thrombospongin, retronectin, dextrin, trehalose, etc.

[0028] In another embodiment of the present invention, the composition for treating cartilage diseases of the present invention may contain photopolymerizable monomers such as methacrylic acid and acrylic acid, and permanent chondrocytes. In this case, the composition can be injected into the affected area, such as a cartilage defect, using a syringe or the like, and the photopolymerizable monomers can be polymerized by irradiating them with light.

[0029] In one preferred embodiment, the implant material of the present invention can be used to treat bone or cartilage-related diseases and is also suitable for use in plastic surgery or cosmetic surgery. For example, the shape of the nose is determined by the greater alar cartilage, nasal septum cartilage, lateral nasal cartilage, etc., and the shape of the ear is determined by the auricular cartilage. In cases of trauma such as traffic accidents, congenital nasal bone defects, microtia, etc., it becomes necessary to reshape or reconstruct the nose or ear, and this can be done by implanting the implant material of the present invention. Even when cartilage of the desired shape is implanted in a location other than the nose or ear where cartilage does not naturally exist, cosmetic effects can be expected. For example, a cartilage plate or cartilage block can be prepared and then shaped to the desired form to create an implant material of the desired shape. [Examples]

[0030] The present invention will be further described below with reference to examples, but the present invention is not limited thereto.

[0031] Manufacturing Example 1: Preparation of GDF5-tdTomato Reporter Plasmid We created a reporter that can visualize cells in which the GDF5 gene is activated. The reporter was created by introducing IRES-tdTomato-PGK-Neo® (registered trademark) into the 3'UTR side of GDF5, resulting in a GDF5-tdTomato reporter. (Figure 1)

[0032] Example 1: Production of GDF5-tdTomato reporter iPS cell line and culture of chondrogenic cells suitable for large-scale culture. First, the above-mentioned GDF5-tdTomato reporter was introduced into human iPS cells using electroporation, and after single cloning, it was established (= GDF5 reporter iPS cells).

[0033] GDF5 reporter iPS cells were induced into LBMs via the lateral plate mesoderm expressing PRRX1, and CD140b+ / CD90+ LBMs that maintain high PRRX1 expression and prospectively regulate chondrogenesis were cultured as expandable LBMs (ExpLBM = chondrogenic progenitor cells) (Figure 2).

[0034] Example 2: Induction of permanent chondrocarcinoma cells Our previous culture method for inducing chondroprogenitor cells, based on bulk RNA-seq results, revealed that the resulting cells contained both growth plate cartilage and permanent cartilage markers. Since this method failed to induce chondroprogenitor cells with suppressed ossification, we screened whether ossification of human chondroprogenitor cells could be suppressed using culture media supplemented with or without inhibitors or agonists of bone and cartilage-related signals (18 substances (single substances or compositions)). The substances were added for two weeks during the cartilage induction period after cell aggregation (Step 3). When the 19 types of chondroprogenitor cells (#0~#18) were transplanted into vivo, ossification of cartilage was suppressed when the MEK inhibitor #9 was added to the culture medium, while ossification was not suppressed in the other 17 types of chondroprogenitor cells (including the unadded medium). Furthermore, when tdTomato expression was analyzed using fluorescence microscopy and flow cytometry in chondrocytes obtained after 2 weeks of culture with MEK inhibitors #0 to #18 added to the culture medium, tdTomato expression was observed only with MEK inhibitor #9 (Figure 3).

[0035] These results suggest that MEK inhibitors induce permanent chondrocytes with suppressed ossification by making chondrocytes GDF5-positive.

[0036] When qPCR was used to examine chondrocytes from a MEK inhibitor-treated group (GDF5-expressing chondrocytes, #9) and an induction group strongly expressing growth plate cartilage markers (GDF5-non-expressing chondrocytes, #0-#8, #10-#18), the GDF5-expressing chondrocytes showed high expression of articular cartilage-related markers GDF5 and PRG4, while growth plate cartilage-related markers IHH, COL10A1, and RUNX2 were expressed at low levels (Figure 4).

[0037] Furthermore, bulk RNA-seq was performed using these RNAs. It was revealed that various markers, which were mixed in with chondrocyte progenitor cells produced using previous methods, were specifically expressed as articular cartilage-related markers in GDF5-expressing permanent chondrocyte progenitor cells, similar to the results of qPCR. Moreover, in GDF5-non-expressing chondrocyte progenitor cells, only growth plate cartilage-related markers were expressed (Figure 5).

[0038] When these chondrogenic cells were immunofluoresced with various cartilage-related markers, the MEK inhibitor-treated group showed high expression of PRG4, COL2, ACAN, and COL1, and low expression of RUNX2 and COLX (Figure 6).

[0039] Example 3: GDF5-expressing chondrocytes In vitro-induced GDF5-expressing chondroprogenitor cells and GDF5-non-expressing chondroprogenitor cells were transplanted subcutaneously into immunodeficient animals, and changes in the chondroprogenitor cells were detected for 3 months. After 3 months, GDF5-expressing chondroprogenitor cells showed positive for Safranin O staining and negative for vonKossa staining. On the other hand, in GDF5-non-expressing chondroprogenitor cells, some vonKossa-positive areas were observed from 1 month after transplantation, and after 3 months, approximately half showed vonKossa positivity (Figure 7).

[0040] Furthermore, GDF5-expressing chondrogenic cells were transplanted into knee joint cartilage defects in immunodeficient animals, and their progress was observed. After 3 months, Safranin O-positive permanent cartilage was observed to have engrafted in the articular cartilage site, and it was positive for hVimenntin antibodies that recognize human-specific antigens. Even more interestingly, it was revealed that strongly PRG4-positive permanent cartilage was formed at the transplanted site (Figure 8).

Claims

1. A method for preparing GDF5-positive human permanent chondrocytes, comprising culturing human chondrocytes in a medium containing a MEK inhibitor.

2. The preparation method according to claim 1, wherein the concentration of the MEK inhibitor in the culture medium is 0.001 to 10 μM.

3. Human permanent chondrocytes that are GDF5-positive and form permanent cartilage with suppressed ossification during in vivo transplantation.

4. A transplant material comprising human permanent chondrocytes or permanent chondrocytes derived therefrom as described in claim 3.

5. A composition for treating cartilage disease, comprising human permanent chondrocytes and a scaffold as described in claim 3.

6. The composition for treating cartilage disease according to claim 5, which is in the form of a paste.

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