JP6864335B2 - How to make chondrocytes - Google Patents

Description

The present invention broadly relates to a method for producing paraxial mesoderm or progenitor cells thereof, for example, paraxial mesoderm-derived cells and / or lateral plate mesoderm-derived cells, and producing chondrocytes and the like from the paraxial mesoderm and / or lateral plate mesoderm cells. Regarding how to do it.

Known cartilage diseases include osteoarthritis in the elderly, articular cartilage defect due to trauma, skeletal dysplasia in children, hypotension, nasal deformity due to cleft lip and palate, osteochondritis dissecans, and traumatic injury. .. In particular, the number of patients with knee osteoarthritis is extremely high at 25.3 million in Japan alone and 7.8 million with symptoms, so there is a great need for regenerative medicine for articular cartilage. It can be said that it is an organization. However, since chondrocytes have poor proliferative capacity, in humans, there is a problem that once the cartilage is damaged, it hardly regenerates, and there is no established regenerative medicine for treating cartilage disease at this time. It is the actual situation.

In order to treat traumatic cartilage deficiency and osteochondritis dissecans, autologous cartilage transplantation has been attempted in the affected area, but arthroscopic surgery is required to collect cartilage tissue. , There is a limit to the amount of cartilage that can be cultured, and there are drawbacks such as fibrocartilage-like regeneration and not normal vitreous cartilage regeneration.

Cartilage regenerative medicine using mesenchymal stem cells has also been attempted, but the amount of cells that can be cultured is still limited. Due to such quantitative restrictions, conventional cartilage regenerative medicine cannot be applied to osteoarthritis, which requires a huge number of cells.

In this respect, pluripotent stem cells are considered as promising candidates for cartilage regenerative medicine because the amount of cells that can be cultured is unlimited.

So far, Oldershaw et al. (Non-Patent Document 1) using human ES cells and Yamashita et al. (Non-Patent Document 1) using human iPS cells have been reported as the main methods for inducing chondrocyte differentiation using pluripotent stem cells. Patent Document 2) and the like.

The method of Oldershaw et al. Induces differentiation from human ES cells via mesoderm to chondrocytes over a period of about 2 weeks using multiple types of cytokines in stages. Although an increase in cartilage markers is finally shown, its verification is in vitro and no further tissue formation is mentioned. When the present inventors applied the method of Oldershaw et al. To human iPS cells, the chondrocyte markers were certainly elevated well, but after that, they were three-dimensionally cultured and transplanted into the knee joint of immunodeficient mice. As a result, about 7 The development of teratomas was observed in% (Saito T et al. Biomed Res. 36: 179-86, 2015). In other words, it is possible that a small part of undifferentiated cells remained even after the induction of differentiation.

Both methods use cytokines stepwise to induce differentiation of pluripotent stem cells into chondrocytes over a period of about 2 weeks, but there are many types of cytokines used. Due to the high cost of cytokines, these methods, which require a large number of cytokines, may be costly for clinical application. In addition, since cytokines are proteins, their quality tends to vary depending on the manufacturing process and the subsequent storage state, which may affect the differentiation induction efficiency itself.

Nat Biotechnol. 28: 1187-94, 2010 Stem Cell Rports. 4:404-18, 2015

In this respect, the low molecular weight compound can be produced at a very low cost when mass-produced, and there is almost no variation in quality between lots. In addition, it is generally known that low-molecular-weight compounds can induce differentiation of pluripotent stem cells with better differentiation-inducing efficiency by culturing for a shorter period of time as compared with the differentiation-inducing method using cytokines. In view of such circumstances, it is an object of the present invention to provide a method for producing paraxial mesoderm cells and / or lateral plate mesoderm cells using a low molecular weight compound and inducing differentiation into chondrocytes and the like. ..

As a result of diligent studies by the present inventors, pluripotent stem cells were cultured by adding a glycogen synthase kinase kinase 3β (GSK-3β) inhibitor, which is an activator of Wnt / β-catenin signal, to a predetermined concentration medium. However, surprisingly, they have found that differentiation is induced into chondrocytes via mesoderm, axial mesoderm and / or lateral plate mesoderm, and have completed the present invention.

That is, the present application includes the following inventions.
[1] A method for producing chondrocytes or progenitor cells thereof, which comprises a step of culturing pluripotent stem cells in a medium containing a glycogen synthase kinase kinase 3β (GSK-3β) inhibitor having a concentration of more than 3 μM.
[2] The method according to [1], wherein the addition of the GSK-3β inhibitor to the medium is discontinued with an increase in the middle endoderm marker as an index.
[3] The method according to [1] or [2], wherein the period of the culture step is 1 to 5 days, preferably 2 days.
[4] The method according to any one of [1] to [3], wherein the medium further contains a retinoic acid receptor (RAR) agonist.
[5] Medium containing GSK-3β inhibitor and RAR agonist for 1 to 3 days, preferably 2 days, medium containing RAR agonist and not containing GSK-3β inhibitor for 1 day or longer, preferably 2 to 12 days. The method according to [4], which comprises a step of culturing.
[6] GSK-3β inhibitors are CHIR-99021, CHIR-98014, 3F8, A 1070722, AR-A 014418, BIO, BIO-acetoxime, 10Z-Hymenialdisine, Indirubin-3'-oxime, Kenpaullone, L803, L803- The compound according to any one of [1] to [5], which is a compound selected from the group consisting of mts, MeBIO, NSC 693868, SB 216763, SB 415286, TC-G 24, TCS 2002, TCS 21311 and TWS 119. Production method.
[7] The production method according to any one of [4] to [6], wherein the concentration of the GSK-3β inhibitor in the medium is 3 to 20 μM, preferably 10 μM.
[8] RAR agonists are TTNPB, AM580, AM80, 9-cis retinoic acid, all-trans retinoic acid (ATRA), LGD1550, E6060, AGN193312, AM555S, CD2314, AGN193174, LE540, CD437, CD666, CD2325, SR11254, SR11363. , SR11364, AGN193078, TTNN (Ro19-0645), CD270, CD271, CD2665, SR3985, AGN193273, Ch55, 2AGN190521, CD2366, AGN193109 and Re80. The method described.
[9] The method according to any one of claims [4] to [8], wherein the concentration of the RAR agonist in the medium is 1 nM to 10 μM, preferably 100 nM.
[10] The method according to any one of [1] to [9], wherein the progenitor cells are paraxial mesoderm and / or lateral plate mesoderm cells.
[11] A kit for producing chondrocytes or progenitor cells thereof, which comprises a GSK-3β inhibitor and optionally a RAR agonist.

According to the present invention, pluripotent stem cells are more simply and efficiently converted into chondrocytes or progenitor cells thereof, for example, axial mesoderm-derived cells and / or side plates, as compared with conventional differentiation induction methods using cytokines. It can induce differentiation into the mesoderm. In particular, the present invention can realize autonomous and stepwise differentiation induction from pluripotent stem cells to axial mesoderm and / or lateral plate mesoderm, and thus to chondrocytes via mesoderm. Therefore, it can be applied not only to regenerative medicine but also to basic research on cartilage development process and drug discovery research for cartilage disease.

In particular, when a GSK-3β inhibitor and a RAR agonist are combined as a low molecular weight compound, the induction of differentiation of pluripotent stem cells into chondrocytes or their progenitor cells can be further promoted, and the result is retinoic acid receptor. It is surprising considering the report that body γ is involved in the suppression of chondrocyte differentiation (Shimono K et al. Nat Med 2011).

Japanese Patent Application Laid-Open No. 2015-500630 discloses a method for producing intermediate mesoderm cells, which comprises a step of culturing human pluripotent stem cells in a medium containing a GSK-3β inhibitor and a retinoic acid derivative. .. This publication is common to the present invention in that it uses a GSK-3β inhibitor and a retinoic acid derivative, but the upper limit of the concentration of CHIR-99021 used in the examples as a GSK-3β inhibitor is "3 μM". It is different from the present invention in that the final product is "retrorenal cells" and does not pass through the progenitor cells of chondrocytes, such as paraxial mesoderm, on the way.

FIG. 1 shows the results of examining the concentration and administration period of CHIR99021 suitable for inducing differentiation of pluripotent stem cells (RAR agonist is not used). T and MIXL1 were used as mesoderm markers. The expression level on the vertical axis is a value standardized by β-actin. FIG. 2 shows the results of examining the concentration of TTNPB suitable for inducing differentiation of pluripotent stem cells into chondrocytes (CHIR was used at 10 μM × 2 days). COL2A1, COL11A2, SOX5,9 were used as cartilage markers. The expression level on the vertical axis is a value standardized by β-actin. FIG. 3 shows the results of examining the concentration of TTNPB suitable for inducing differentiation of pluripotent stem cells (CHIR was used at 10 μM × 2 days). T and MIXL1 were used as mesoderm markers. The expression level on the vertical axis is a value standardized by β-actin. FIG. 4 shows the results of examining the administration period of TTNPB suitable for inducing differentiation of pluripotent stem cells into chondrocytes (CHIR was used at 10 μM × 2 days). The start time of TTNPB administration was divided between days 0 and 3. COL2A1, COL11A2 and SOX9 were used as cartilage markers. The expression level on the vertical axis is a value standardized by β-actin. FIG. 5 shows the time course of the expression level of cartilage markers (COL2A1, COL11A2, SOX5, 9) when CHIR was administered at 10 μM for 2 days and TTNPB was administered at 100 nM every day. The expression level on the vertical axis is a value standardized by β-actin. Samples collected daily were analyzed. FIG. 6 shows the time course of the expression level of cartilage markers (SOX6, ACAN) when CHIR was administered at 10 μM for 2 days and TTNPB was administered at 100 nM every day. The expression level on the vertical axis is a value standardized by β-actin. Samples collected daily were analyzed. FIG. 7 shows the time course of the expression level of the hypertrophy marker (COL10A1) when CHIR was administered at 10 μM for 2 days and TTNPB was administered at 100 nM every day. The expression level on the vertical axis is a value standardized by β-actin. Samples collected daily were analyzed. FIG. 8 shows the time course of the expression level of the mesoendoderm marker (T and MIXL1) when CHIR was administered at 10 μM for 2 days and TTNPB was administered at 100 nM every day. The expression level on the vertical axis is a value standardized by β-actin. Samples collected daily were analyzed. FIG. 9 shows changes over time in the expression levels of mesoderm markers (TBX6, MEOX1 and HAND1) when CHIR was administered at 10 μM for 2 days and TTNPB was administered at 100 nM daily. The expression level on the vertical axis is a value standardized by β-actin. Samples collected daily were analyzed. FIG. 10 shows the results of examining the concentration of CHIR99021 suitable for inducing differentiation of pluripotent stem cells into chondrocytes (TTNPB was administered at 100 nM daily). COL2A1, COL11A2, SOX5,9 were used as cartilage markers. The expression level on the vertical axis is a value standardized by β-actin. FIG. 11 shows the results of examining the concentration of CHIR99021 suitable for inducing differentiation of pluripotent stem cells into chondrocytes (TTNPB was administered at 100 nM daily). T and MIXL1 were used as mesoderm markers. The expression level on the vertical axis is a value standardized by β-actin. FIG. 12 shows the results of examining the concentration of CHIR99021 suitable for inducing differentiation of pluripotent stem cells into chondrocytes (TTNPB was administered at 1 μM daily). COL2A1, COL11A2, SOX5,9 were used as cartilage markers. The expression level on the vertical axis is a value standardized by β-actin. FIG. 13 is a diagram showing the relationship between the concentration of CHIR99021 and the expression level of OSR1. CHIR was administered at the prescribed concentration for 2 days, and TTNBP was administered at 100 nM daily. The expression level on the vertical axis is a value standardized by β-actin. OSR1 is an intermediate mesoderm marker used in Japanese Patent Publication No. 2015-500630 (above). FIG. 14 shows the results of examining the concentration of CHIR99021 suitable for inducing differentiation of pluripotent stem cells into chondrocytes (TTNPB was administered at 100 nM daily). COL2A1, COL11A2, SOX5,9 were used as cartilage markers. The expression level on the vertical axis is a value standardized by β-actin. FIG. 15 is a diagram comparing the differentiation induction protocol of the present invention with respect to the expression level of cartilage markers (SOX6, ACAN) and the protocols described in Non-Patent Document 1 (Oldershaw et al.) And Non-Patent Document 2 (Yamashita et al.). Is. In the differentiation induction group using either protocol, iPS cells of the same passage number derived from the same clone and maintained and cultured under the same feeder-free conditions using the Essential 8® Medium Kit are pluripotent. Used as stem cells. The expression level on the vertical axis is a value standardized by β-actin. FIG. 16 is a diagram comparing the differentiation induction protocol of the present invention with respect to the expression level of the ossification markers (COL1A1, RUNX2) and the protocols described in Non-Patent Document 1 (Oldershaw et al.) And Non-Patent Document 2 (Yamashita et al.). Is. In the differentiation induction group using either protocol, iPS cells of the same passage number derived from the same clone and maintained and cultured under the same feeder-free conditions using the Essential 8® Medium Kit are pluripotent. Used as stem cells. The expression level on the vertical axis is a value standardized by β-actin. FIG. 17 shows the differentiation induction protocol of the present invention and the protocols described in Non-Patent Document 1 (Oldershaw et al.) And Non-Patent Document 2 (Yamashita et al.) Regarding the expression levels of markers of other strains other than cartilage markers and ossification markers. It is a figure comparing with. In the differentiation induction group using either protocol, iPS cells of the same passage number derived from the same clone and maintained and cultured under the same feeder-free conditions using the Essential 8® Medium Kit are pluripotent. Used as stem cells. The expression level on the vertical axis is a value standardized by β-actin. FIG. 18 shows the results of FACS analysis of chondrocytes obtained according to the protocol of Non-Patent Document 1 (Oldershaw et al.). The same feeder derived from the same clone and using the Essential 8® Medium Kit in the differentiation induction group (iPS cells before initiating the differentiation induction protocol) using either protocol and in the control group. IPS cells of the same passage number maintained and cultured under free conditions were used as pluripotent stem cells. In addition, each antibody used was labeled with Alexa Fluor (registered trademark) 647. FIG. 19 shows the results of FACS analysis of chondrocytes obtained according to the protocol of Non-Patent Document 2 (Yamashita et al.). The same feeder derived from the same clone and using the Essential 8® Medium Kit in the differentiation induction group (iPS cells before initiating the differentiation induction protocol) using either protocol and in the control group. IPS cells of the same passage number maintained and cultured under free conditions were used as pluripotent stem cells. In addition, each antibody used was labeled with Alexa Fluor (registered trademark) 647. FIG. 20 shows the results of FACS analysis of chondrocytes obtained according to the protocol of Non-Patent Document 2 (Yamashita et al.). The same feeder derived from the same clone and using the Essential 8® Medium Kit in the differentiation induction group (iPS cells before initiating the differentiation induction protocol) using either protocol and in the control group. IPS cells of the same passage number maintained and cultured under free conditions were used as pluripotent stem cells. In addition, each antibody used was labeled with Alexa Fluor (registered trademark) 647. FIG. 21 is a diagram comparing TTNPB with other retinoic acid receptor (RAR) agonists for the expression levels of cartilage markers (COL2A1, COL11A2, SOX5,9). CHIR was administered at 10 μM for 2 days, and TTNBP was administered at 100 nM daily. The expression level on the vertical axis is a value standardized by β-actin. FIG. 22 is an immunostaining photograph of a SCID (Sevefe Combined ImmunoDeficiency) mouse knee joint into which chondrocytes obtained according to the present invention have been transplanted. In the photo on the left, the hyaline cartilage of the femoral patellar femoral joint surface (sagittal cut) 8 weeks after transplantation is stained red with safranin O (the part surrounded by the central square), and this is enlarged on the right side. In the photograph, human-derived chondrocytes believed to have been obtained by the present invention are stained green with vimentin. The cells stained in blue are the cell nuclei stained with DAPI, and both human and mouse cells are stained.

The method for producing chondrocytes or progenitor cells thereof according to the present invention includes a step of culturing pluripotent stem cells in a medium containing a GSK-3β inhibitor having a concentration of more than 3 μM.

Chondrocytes are cells that produce extracellular matrix that constitutes cartilage, such as collagen and proteoglycans. In another embodiment, chondrocytes are also defined as cells expressing cartilage markers selected from the group consisting of COL2A1 (type II collagen), COL11A2 (type XI collagen), SOX5,6,9 and ACAN (aggrecan). Will be done. As used herein, the term "progenitor cell" of a chondrocyte means a cell derived from a pluripotent stem cell that is capable of differentiating into a chondrocyte. Such a population of progenitor cells can include cells derived from the mesoderm, such as paraxial mesoderm and / or lateral plate mesoderm, preferably paraxial mesoderm.

Paraxial mesoderm cells are mesoderm cells present on both sides of the primitive streak that differentiate into skeletal muscle, bone, and cartilage. The paraxial mesoderm segmentes to form a segment and then differentiates into the ventromedial vertebral plate and the dorsolateral skin muscle plate. Cartilage is a further differentiated vertebral plate. The mesoderm can be classified into paraxial mesoderm, spinal mesoderm, intermediate mesoderm, lateral plate mesoderm, and the like.

The chondrocytes produced in the present invention or progenitor cells thereof can be prepared using pluripotent stem cells as a starting material. Examples of pluripotent stem cells used in the present invention include not only iPS cells and ES cells, but also embryonic reproductive stem cells (EG cells) and somatic cell-derived ES (ntES) cells. However, any pluripotent stem cell can be used in the present invention as long as it can differentiate into chondrocytes or progenitor cells thereof, such as paraxial mesoderm, in a medium containing a GSK-3β inhibitor and optionally a RAR agonist. Can be used.

Glycogen synthase 3, a type of serine-threonine kinase, has two isoforms, α and β. As used herein, "GSK-3β inhibitor" means a substance that inhibits the kinase activity of the GSK-3β protein. Multiple GSK-3β inhibitors may be used as long as the effects of the present invention are not impaired. As GSK-3β inhibitors, for example, CHIR (CHIR99021 or CHIR98014), 3F8, A 1070722, AR-A 014418, BIO, BIO-acetoxime, 10Z-Hymenialdisine, Indirubin-3'-oxime, Kenpaullone, L803, L803-mts , MeBIO, NSC 693868, SB 216763, SB 415286, TC-G 24, TCS 2002, TCS 21311, TWS 119 and the like. Of these, CHIR99021 is preferable.

The GSK-3β inhibitor is added to the medium to a final concentration greater than 3 μM, such as 3-20 μM, preferably 10 μM. Pluripotent stem cells can be induced to differentiate even at 20 μM or more, but the viability of the cells can be reduced. In addition, at a concentration of 30 μM or higher, no increase in expression may be observed depending on the type of marker.

The medium used in the present invention is not particularly limited as long as the effects of the present invention are not impaired, and for example, a basal medium used for culturing animal cells can be used. The basal medium includes, for example, Dulbecco's modified Eagle's Medium (DMEM) medium, Eagle's Minimum Essential Medium (EMEM) medium, Ham's F12 medium, RPMI 1640 medium, Fischer's medium, Neurobasal Medium (Life Technologies), or a mixture of these. Be done. The medium may contain serum or may be serum-free. If desired, the medium may be, for example, albumin, insulin, transferase, selenium, fatty acids, trace elements, β-mercaptoethanol, thiolglycerol, lipids, amino acids, L-glutamine, non-essential amino acids, vitamins, growth factors, GSK- It may contain one or more substances such as low molecular weight compounds other than 3β inhibitors and RAR agonists, antibiotics, antioxidants, buffers, inorganic salts, cytokines and the like. The medium can be replaced at an appropriate frequency, eg, daily after day 2 of culture, with medium prepared to the desired concentration of GSK-3β inhibitor or RAR agonist. At that time, the culture may be washed with PBS or the like.

In the present invention, feeder cells can be used to acclimate the medium or to support the proliferation of pluripotent stem cells in culture. In another embodiment, the culture in the present invention may be carried out under feeder-free conditions. The culture can be carried out by either a plane (monolayer) culture or a three-dimensional culture, but the culture method can be appropriately changed depending on the type of target cells. For example, chondrocytes may be changed to three-dimensional culture in the middle because the ability to produce substrates such as extracellular matrix is reduced by long-term planar culture, or they become dedifferentiated chondrocytes.

Pluripotent stem cells are induced to differentiate into chondrocytes or progenitor cells thereof by culturing in a medium containing a GSK-3β inhibitor for a predetermined period of time. Although it depends on the culture conditions, pluripotent stem cells can be induced to differentiate into mesoderm, paraxial mesoderm, and lateral plate mesoderm by culturing for about 1 to 5 days. Various conditions such as temperature in cell culture are not particularly limited, and may be general conditions (for example, 37 ° C., _{under 5% CO 2} ), or may be appropriately changed according to the purpose. Although not intended to be limited, the induction of differentiation from pluripotent stem cells can be confirmed using the expression of marker genes as listed below as an index.
・ Undifferentiated markers: OCT3 / 4, NANOG
・ Medium endoderm marker: T, MIXL1
・ Mesoderm markers: TBX6, MEOX1, HAND1, OSR1
・ Cartilage markers: COL2A1, COL11A2, SOX5,6,9, ACAN
・ Hypertrophy marker: COL10A1
・ Ossification markers: COL1A1, RUNX2
・ Neuroectoderm marker: SOX1
・ Endoderm marker: SOX17
・ Myocardial marker: NKX2-5
・ Blood cell hemangioblast marker: VEGFα

For example, when pluripotent stem cells are cultured in the presence of a GSK-3β inhibitor, changes in the expression of mesoderm markers and mesoderm markers should be monitored after a lapse of a predetermined period from the start of culture, for example, within a few days. Therefore, it can be confirmed that the induction of differentiation into cartilage progenitor cells occurs over time. For example, although it depends on the culture conditions, when pluripotent stem cells are cultured in the presence of a GSK-3β inhibitor, the expression of the mesoderm marker increases around the 1st to 2nd day of the culture, and the 3rd to 4th day of the culture. The expression of the mesoderm marker may decrease and the expression of the mesoderm marker may increase around the time. Therefore, when it is desired to induce differentiation into mesoderm-derived cells, for example, chondrocytes, the addition of the GSK-3β inhibitor to the medium can be stopped using the increase in the mesoderm marker as an index.

In addition to the GSK-3β inhibitor, the above-mentioned basal medium may further contain other low-molecular-weight compounds for achieving desired differentiation induction and the like, preferably a retinoic acid receptor (RAR) agonist. Other components to be included in the medium can be appropriately determined by those skilled in the art.

Here, retinoic acid (RA) is known to bind to intracellular nuclear receptors and participate in various physiological actions. For example, retinoic acid nuclear receptors include retinoic acid receptor (RAR) and retinoid X receptor (RXR), each of which has α, β, and γ subtypes. However, it has been reported that RARγ-selective agonists suppress the differentiation of mesenchymal stem cells into cartilage cells (Shimono K et al. Nat Med 2011 (above)). On the other hand, in the present invention, the combined use of a GSK-3β inhibitor and a RAR agonist can further promote the induction of differentiation of pluripotent stem cells into chondrocytes or progenitor cells thereof. The RAR agonist can be added to the medium to a final concentration of 1 nM to 10 μM, preferably 10 to 100 nM, more preferably 100 nM. However, these concentrations are merely examples, and the concentration of the low molecular weight compound can be appropriately adjusted by those skilled in the art depending on the desired level of differentiation induction.

When a GSK-3β inhibitor and a RAR agonist are used in combination, it is preferable that both are added to the medium from the beginning of the culture. When pluripotent stem cells are differentiated into chondrocytes, they are cultured in a medium containing a GSK-3β inhibitor and a RAR agonist for a predetermined period of time, for example, 1 to 3 days, preferably 2 days, followed by GSK containing a RAR agonist. It may be further cultured in a medium containing no -3β inhibitor for a predetermined period of time, for example, for 1 day or longer, preferably 2 to 12 days, and more preferably 3 to 7 days.

As used herein, "RAR agonist" means a compound having agonistic activity on any or all of RARα, RARβ or RARγ. Although not intended to be limited, RAR agonists include, for example, TTNPB, AM580, AM80, 9-cis retinoic acid, all-trans retinoic acid (ATRA), LGD1550, E6060, AGN193312, AM555S, CD2314, Selected from the group consisting of AGN193174, LE540, CD437, CD666, CD2325, SR11254, SR11363, SR11364, AGN193078, TTNN (Ro19-0645), CD270, CD271, CD2665, SR3985, AGN193273, Ch55, 2AGN190521, CD2366, AGN193109 and Re80. obtain. Among these low molecular weight compounds, TTNPB, AM580, AM80, ATRA and 9-cis-Retinoic Acid are preferable as RAR agonists.

Cells that have been induced to differentiate with a GSK-3β inhibitor or the like may be subjected to an additional culture step, for example, to enhance chondrocyte differentiation, induce chondrocyte proliferation, or to promote further differentiation, proliferation, and maturation. It is also intended to be cultured in the presence of compounds that increase chondrocyte matrix production.

The chondrocytes produced according to the present invention can be used for repair and reconstruction of cartilage tissue. Examples of such cartilage tissue include articular cartilage, costal cartilage, thyroid cartilage, tracheal cartilage, articular meniscus, articular disc, intervertebral disc, pubic symphysis, laryngeal cartilage, external auditory canal cartilage, and ear cartilage. The chondrocytes produced by the present invention have various cartilage diseases such as osteoarthritis in the elderly, articular cartilage defect due to trauma, skeletal dysplasia in children, hypotension, nasal deformity due to lip cleft, and osteochondritis. It is intended to be used for the treatment of osteochondritis, traumatic injuries, etc. In addition, by using pluripotent stem cells derived from a patient, the chondrocytes obtained by the present invention can be autologously transplanted into the affected area.

In addition, the resulting chondrocytes may be included in pharmaceutical compositions that enhance chondrogenesis or increase cartilage substrate production. Such a pharmaceutical composition can be used for the prevention or treatment of the cartilage disease. In addition to chondrocytes, pharmaceutical compositions include carriers, excipients, disintegrants, buffers, emulsifiers, suspending agents, soothing agents, stabilizers, preservatives, preservatives, cytoprotectants (eg, dimethylsulfoxide). (DMSO) and serum albumin), antibiotics, pH regulators, various components (vitamins, cytokines, growth factors, steroids, bone inducers (BMP), etc.) for the purpose of cell activation, proliferation or differentiation induction, etc. ) May be included. The target test animals are mammals such as humans, cows, horses, pigs, sheep, goats, cats, dogs, rabbits, rats, and mice.

In another embodiment, the invention further comprises culturing pluripotent stem cells in a medium containing a glycogen synthase kinase kinase 3β (GSK-3β) inhibitor at a concentration greater than 3 μM. Alternatively, a method for producing muscle cells is provided. Those skilled in the art can appropriately determine the components to be included in the medium.

The present invention further provides a method for treating a disease by administering the obtained cells to a subject who needs treatment for a disease related to chondrocytes, osteoocytes and / or muscle cells. The method of administration to the target site is not particularly limited, but it is intended, for example, to inject the required cells into the affected area such as a tissue defect.

In yet another aspect, the invention provides a kit for producing chondrocytes or progenitor cells thereof, comprising a GSK-3β inhibitor and optionally a RAR agonist. The kits of the invention can also further include media and other low molecular weight compounds. The GSK-3β inhibitor and optionally the RAR agonist are preferably stored in separate containers, but may be stored in the same container. Each container may pre-fill with medium along with the low molecular weight compounds. The kit according to the present invention may further have instructions or indications describing the differentiation-inducing method according to the present disclosure. Such kits are intended primarily provided as reagents.

The present invention further provides an agent for inducing differentiation into chondrocytes or progenitor cells thereof, which comprises a GSK-3β inhibitor and optionally a RAR agonist.

Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited thereto.

(Establishment of human iPS cells)
Human iPS cells were established by introducing the four genes of human OCT4, SOX2, KLF4, and MYC into neonatal epidermal fibroblasts (Lonza Japan) using a pMXs retroviral vector (Takahashi et al., Cell 2007) (Takahashi et al., Cell 2007). Clone name; 7F3955).

(Maintenance of human iPS cells and adaptation to feeder-free culture)
Human iPS cells established as described above are StemSure® D- on feeder cells prepared by treating mouse embryo-derived fibroblasts (MEF) with mitomycin C (Wako, 134-07911). Based on MEM (High Glucose) with Phenol Red and Sodium Pyruvate (Wako, 197-16275), 15% StemSure® Serum Replacement (Wako, 197-16775), 2 mM L-Glutamine (Gibco, 25030-081) ), 1% Non-essential amino acid (Gibco, 11140-050), 50 U / ml Penicillin and 50 ug / ml Streptomycin (Sigma, P4333), 0.1 mM β-mercaptoethanol (Gibco, 21985-023), 5 ng / It was maintained in a medium consisting of ml human FGF2 (Oriental yeast, 47079000).

Human iPS cells maintained on these feeders are sprinkled on a culture dish coated with Matrigel® Basement Membrane Matrix (Corning, 356234) and Essential 8® Medium Kit (Gibco, A1517001) or Essential 8 Feeder-free culture was performed in a medium containing 50 U / ml Penicillin and 50 ug / ml Streptomycin (Sigma, P4333) to Flex Medium Kit (Gibco, A2858501), and the same was performed by 5-10 passages. Sufficiently acclimatized to feeder-free culture under conditions.

(Protocol for inducing differentiation into chondrocytes)
The protocol was initiated when the number of cells on a 35 mm dish ^{reached 1.0-2.0 × 10 6.}

The basal medium for the differentiation induction protocol is based on DMEM / F-12, GlutaMAX® supplement (Gibco, 10565-018), 2% B27 supplement (Gibco, 17504-044), 1% ITS-X (Gibco). , 51500-056), 1% Non-essential amino acid (Gibco, 11140-050), 50 ug / ml L (+)-Ascorbic Acid (Nacalai Tesque, 03420-52), 50 U / ml Penicillin and 50 ug / It consisted of a composition containing ml Streptomycin (Sigma, P4333), 90 uM β-mercaptoethanol (Gibco, 21985-023).

CHIR99021 (Cayman, 13122), TTNPB (Santa Cruz, 203303), AM580 (Santa Cruz, 203505), AM80 (Cayman, 71770), ATRA (Cayman, 11017), 9-cis-Retinoic Acid (Cayman) , 14587) were added at different dose periods and concentrations to examine the ability to induce differentiation into paraxial mesoderm and / or lateral plate mesoderm and chondrocytes.

Real-time RT-PCR and Fluorescence Activated Cell Sorting (FACS) were used for the analysis of each marker in this example. TRNA in real-time RT-PCR was recovered using TRI Reagent (Cosmo Bio, TR118) and purified using Tissue Total RNA Mini Kit (Chiyoda Science, FATRK 001). 1.0 μg of tRNA was reverse transcribed by ReverTra Ace qPCR RT Master Mix with gDNA Remover (Toyobo, FSQ-301) to obtain single-stranded cDNA. Quantitative PCR was performed using THUNDERBIRD SYBR qPCR Mix (Toyobo, QPS-201) and various primers.

For FACS, target cell samples were treated according to the protocol using the BD Pharmingen® Transcription Factor Buffer Set (BD Pharmingen, 562574), Alexa Fluor 647 Mouse Anti-Sox9 (BD Pharmingen, 565493), Alexa Fluor. Antibody treatment was performed on 647 Mouse anti-Oct3 / 4 (BD Pharmingen, 560329) and Alexa Fluor 647 Mouse anti-Human Nanog (BD Pharmingen, 561300) at the concentration and reaction time recommended by the manufacturer. Subsequently, FACSAria (registered trademark) Fusion cell sorter (BD Biosciences) was used to perform FACS of 10,000 cells × 3 sets for each marker, and the positive cell rate was calculated.

Example 1: Examining the optimum concentration of the GSK-3β inhibitor alone The effect of the GSK-3β inhibitor on the differentiation potential of pluripotent stem cells was investigated using the endoderm markers T and MIXL1. Planar culture of human iPS cells (7F3955) acclimatized to feeder-free culture was performed in a medium in which a GSK-3β inhibitor was added to a basal medium of the above differentiation induction protocol to a predetermined concentration. Hereinafter, unless otherwise specified, 7F3955 is used for human iPS cells. The medium was replaced daily with 2 ml of the above basal medium added so that the final concentration of the GSK-3β inhibitor was the same as the initial concentration. After culturing for 1-4 days under these conditions, the medium endoderm marker increased in a concentration-dependent manner when the concentration of CHIR99021 was 5-10 μM (Fig. 1). However, at CHIR99021 20 μM and above, cell viability decreased, and at 30 μM, no increase in markers was observed, and cells were almost killed (results not shown).

Example 2: Examination of the combined effect of a GSK-3β inhibitor and a RAR agonist In Japanese Patent Publication No. 2015-500630 (above), CHIR99021 (3 μM) as a GSK-3β inhibitor and TTNPB as a RAR agonist ( It is described that human iPS cells differentiated into intermediate mesoderm cells in the presence of 1 μM). In this example, the effect of the combined use of a GSK-3β inhibitor and a RAR agonist on the differentiation potential of pluripotent stem cells was investigated.

2-1: Examination of optimum concentration of RAR agonist
Human iPS cells were cultured by the same method as in Example 1 except that the concentration of CHIR99021 was 10 μM and a different concentration of TTNPB (1 nM to 10 μM) was further added as a low molecular weight compound. One day after culturing, the medium was replaced with one prepared so that the final concentration of each low molecular weight compound was the same as the initial concentration. Two days after culturing, the culture was washed twice with PBS, and thereafter, without adding CHIR99021, it was replaced daily with the basal medium added so that the final concentration of TTNPB was the same as the initial concentration. The cells were cultured for a total of 5 days.

The expression level of the marker in the culture was analyzed. As a result, it was clarified that TTNPB increased cartilage markers in combination with CHIR99021 at any concentration (Fig. 2). The increase in cartilage markers due to the combined use of these low molecular weight compounds was particularly remarkable when the concentration of TTNPB was 10 to 100 nM. However, at higher concentrations, some cartilage markers showed a decrease in value. In addition, the endoderm marker value after 2 days of culturing was decreased in a concentration-dependent manner when TTNPB was administered as compared with the case where CHIR99021 was administered alone (Fig. 3). Although not intended to be bound by theory, the results show that when TTNPB is used in combination compared to CHIR99021 alone, the cells are ahead of the endoderm at 2 days of culture. It is also considered to indicate that it is differentiated into stages.

From the above results, it is considered important to adjust the concentration of RAR agonist combined with GSK-3β inhibitor in order to ensure the induction of differentiation into chondrocytes.

2-2: Examination of timing of administration of RAR agonist
The timing of administration of TTNPB was investigated under the condition that CHIR99021 was fixed at 10 μM and the concentration of TTNPB was fixed at 100 nM. The sample was obtained by adding TTNPB at the same concentration every day from the 0th day of culture as in 2-1 and adding TTNPB for the first time on the 2nd day of culture and then adding at the same concentration every day. Three kinds of the obtained cultures and the cultures obtained by adding TTNPB for the first time on the third day of the culture and then adding the same concentration every day were prepared. In all cultures, CHIR99021 was administered at the timing of medium replacement on the second day of culture, as in 2-1. When each sample was compared, the increase of cartilage markers was better when TTNPB was added from the beginning of the culture (Fig. 4).

The change over time of the cartilage marker in the above sample to which 10 μM CHIR99021 was added to the medium from the first day of the culture to the timing of the medium change on the second day of the culture and 100 nM of TTNBP was added daily was examined. Samples cultured for 14 days were collected at the same timing every day, and changes in various markers were analyzed for each. As a result, the values of cartilage markers showed a sharp increase 4 to 5 days after the culture (Fig. 5 and FIG. 5). 6). On the other hand, no increase in the value of the hypertrophy marker indicating cartilage hypertrophy differentiation was observed during the plane culture (Fig. 7). Prior to that, the mesoderm markers increased from 1 to 2 days of culture, and the paraxial mesoderm and mesoderm markers increased from 2 to 4 days of culture (FIGS. 8 and 9, respectively). From this result, it is considered that the stepwise differentiation induction is progressing over time under the culture conditions of the present invention.

2-3: Examination of the optimum concentration of GSK-3β inhibitor Human iPS cells were cultured by the same method as in 2-1 except that CHIR99021 and 100 nM TTNPB were added at different concentrations. One day after culturing, the medium was replaced with one prepared so that the final concentration of each low molecular weight compound was the same as the initial concentration. Two days after culturing, the culture was washed twice with PBS, and thereafter, without adding CHIR99021, it was replaced daily with the basal medium added so that the final concentration of TTNPB was the same as the initial concentration. The cells were cultured for a total of 5 days. As a result, it was clarified that CHIR99021 raises cartilage markers in combination with TTNPB at a concentration of more than 3 μM, especially 5 to 10 μM. At a concentration of 20 μM, some cartilage markers showed a decrease in value. The results after culturing for 5 days are shown in FIG.

In addition, CHIR99021 increased the mesoendoderm marker from 1 μM to 20 μM in a concentration-dependent manner (Fig. 11).

The elevation of cartilage markers was particularly pronounced at concentrations of 5-10 μM (Fig. 12). Here, the conditions when CHIR99021 shown in FIG. 12 has a concentration of 3 μM are completely in agreement with the concentration described in the examples of Japanese Patent Publication No. 2015-500630 (above). In addition, 3 μM CHIR99021 markedly increased the intermediate mesoderm marker OSR1 as compared with the concentrations before and after that (Fig. 13). From these results, pluripotent stem cells are induced to differentiate into cartilage cells when the concentration of CHIR99021 is more than 3 μM, and when the concentration is less than this concentration, it is published in JP-A-2015-500630 (above). As described, it is considered that differentiation is induced into the intermediate mesoderm.

2-4: Examination of the optimal period for administration of GSK-3β inhibitor
The optimal duration of administration of CHIR99021 was investigated under the condition that CHIR99021 was fixed at 10 μM and the concentration of TTNPB was fixed at 100 nM. In this experiment, the administration period of CHIR99021 was divided into 0 to 5 days, and each cell medium was administered. At the end of CHIR99021 administration, the culture was washed twice with PBS, and thereafter, without adding CHIR99021, the basal medium added so that the final concentration of TTNPB was 100 nM was replaced daily for a total of 5 days. It was cultured. The results are shown in FIG.

As is clear from the results shown in FIG. 14, the elevation of cartilage markers was the best in the sample to which CHIR99021 was administered for 2 days.

From the above results, it is considered that the optimum condition for inducing the differentiation of human iPS cells into chondrocytes is that CHIR 10 μM is administered for 2 days after culturing and TTNPB 100 nM is administered daily for 4 days or more. Similar trends were confirmed in other human iPS cell clones and mouse ES cells. For the other confirmed human iPS cell clones, the four genes of human OCT4, SOX2, KLF4, and MYC were introduced into adult peripheral blood collected from volunteers who obtained consent using the Sendai virus vector (Masaki et al). ., Development 2015) It was established by (clone name; PB001, PB004). Mouse ES cells were maintained according to the following method and acclimated to feeder-free culture.

StemSure® D-MEM (High Glucose) with Phenol Red and Sodium Pyruvate (Wako, 197-16275) was applied on feeder cells prepared by treating mouse embryonic fibroblasts (MEF) with mitomycin C. Based on 15% StemSure® Serum Replacement (Wako, 197-16775), 2 mM L-Glutamine (Gibco, 25030-081), 1% Non-essential amino acid (Gibco, 11140-050), 50 U It was maintained in medium containing / ml Penicillin and 50 ug / ml Streptomycin (Sigma, P4333), 0.1 mM β-mercaptoethanol (Gibco, 21985-023), 10 ³ units / ml mouse LIF (Wako, 199-16051).

Mouse ES cells maintained on these feeders were sprinkled on a gelatin-coated culture dish in DMEM / F-12, GlutaMAX® supplement (Gibco, 10565-018) and Neurobasal Medium (Gibco, 21103-049). ) 1: 1 mixed medium, 0.5% N2 supplement (Gibco, 17502-048), 1% B27 supplement (Gibco, 17504-044), 25 ug / ml BSA fraction V (Wako, 015-23295), 50 U / ml Penicillin and 50 ug / ml Streptomycin (Sigma, P4333), 10 ³ units / ml Mouse LIF (Wako, 199-16051), 1 uM PD0325901 (Wako, 163-24001), and 3 uM CHIR99021 (Cayman, Feeder-free culture was performed in a medium supplemented with 13122), and 5-10 passages were sufficiently acclimatized to the feeder-free culture under the same conditions (Silva J et al., PLoS Biol 2008. and Ying). QL et al., Nature 2008).

Example 3: Comparison with differentiation induction method using cytokines 3-1: Comparison by real-time RT-PCR The differentiation induction method according to the present invention is described in Non-Patent Document 1 (Oldershaw) and Non-Patent Document 2 (Yamashita). It was compared with the conventional differentiation induction method using cytokines as described above. For the present invention, the optimum differentiation-inducing conditions determined in Example 2 were used. Culturing was carried out for a maximum of 9 days. On the other hand, with respect to the prior art, differentiation induction was performed according to the protocol described in each non-patent document. Oldershaw samples were cultured for 13 days, and Yamashita samples were cultured for 14 days. In order to make the conditions of each group as similar as possible, in each protocol, the same number of passages derived from the same clone and maintained and cultured under the same feeder-free conditions using the Essential 8® Medium Kit. A comparative experiment was performed using iPS cells. The results of real-time RT-PCR of each sample are shown in FIGS. 15 and 16. As shown in FIG. 15, the differentiation-inducing method according to the present invention had a better elevation of cartilage markers than the conventional protocol. On the other hand, a marked increase in ossification markers was confirmed in the samples obtained by the protocol of Yamashita et al. (Fig. 16). Further, when other markers were also analyzed, the value of the differentiation induction method of the present invention was generally lower than that of the prior art (FIG. 17).

Markers unfavorable for chondrocyte differentiation, such as hypertrophy markers, neuroectoderm markers, and endoderm markers, were sufficiently lower than normal tissues (results not shown).

3-2: Comparison by FACS As a result of applying each of the above samples to FACS, undifferentiated marker-positive cells such as OCT3 / 4 and NANOG remained in the prior art sample at the end of planar culture (Fig. 18 and FIG. 19). This indicates that uniform differentiation induction has not been achieved with the samples of the prior art. When such cells are used for transplantation as they are, the risk of tumorigenesis cannot be ruled out. However, Yamashita et al. Reported that transplantation was performed after suspension culture was performed for a longer period of time after the completion of planar culture, and as far as this was concerned, there were no transplant cases that caused tumorigenesis.

On the other hand, in the differentiation induction protocol of the present invention, at the end of 9-day planar culture, the SOX9-positive cell rate was 97.9%, which was very efficient in inducing differentiation into chondrocytes, and OCT3 / 4, NANOG, etc. were not yet available. No residual differentiation marker-positive cells were observed (Fig. 20).

Example 4: Examination of other RAR agonists
The combined effect of RAR agonists other than TTNPB with GSK-3β inhibitors was also confirmed. AM580 (Santa Cruz, 203505), AM80 (Cayman, 71770), ATRA (Cayman, 11017), 9-cis-Retinoic Acid (Cayman, 14587) were used as RAR agonists. The concentration of each RAR agonist was 100 nM, and the other optimal differentiation-inducing conditions determined in Example 2 were used. As a result, an increase in cartilage markers comparable to TTNPB was observed regardless of which RAR agonist was used (Fig. 21).

Example 5: SCID (Severe Combined ImmunoDeficiency) Mouse Knee Joint Transplantation and Histological Examination After Transplantation Cell dissociation was performed on day 9 of the protocol determined to be optimal in Example 2, and 75 mm Transwell (registration). ^{1.7 × 10 6} cells were suspended in protocol basal medium in a 3.4 mm inner diameter cloning ring (IWAKI, RING-05) placed on 0.4um Pore Polycarbonate Membrane Insert, Sterile (Corning, 3419) with 0.4um Pore Polycarbonate Membrane Insert, Sterile (Corning, 3419). The suspension was added. As it was, it was cultured in the protocol basal medium for 1 week to form a disk (Saito et al., Biomed Res 2015. was partially modified).

The formed disc was transplanted into the femoral patellofemoral joint of SCID mice. The results of staining the articular surface (sagittal section) with safranin O, which stains hyaline cartilage red, 8 weeks after transplantation are shown on the left side of FIG. 22. The right side of FIG. 22 is an enlarged photograph of the transplantation site (the part covered by the square in the left figure) double-stained with anti-human vimentin / DAPI. These results indicate that the safranin O-staining positive region was clearly derived from human cells, and that the transplanted human cells were engrafted. No teratoma or other tumorigenesis was observed in the individuals who underwent the transplantation experiment. In short, according to the present invention, pluripotent stem cells become chondrocytes or progenitor cells thereof with better differentiation induction efficiency in a shorter period of culture as compared with conventional differentiation induction methods using cytokines and the like. Not only can it induce differentiation, but it also makes it possible to provide chondrocytes with extremely low risk of tumorigenesis and high engraftment.

Claims (7)

1) A step of culturing pluripotent stem cells in a medium containing a glycogen synthase kinase kinase 3β (GSK-3β) inhibitor at a concentration of more than 3 μM to 20 μM and a retinoic acid receptor (RAR) agonist.
2) Chondrocytes or cells comprising a step of further culturing the cultured cells in a medium containing a RAR agonist and not containing a GSK-3β inhibitor after the elevation of the endoderm marker of pluripotent stem cells is confirmed. A method for producing the progenitor cell. Wherein 1) is a period of the culturing process of up to three days, the duration of the culturing step of the two) is up to 12 days The method of claim 1. The method according to claim 2, wherein the period of the culturing step of 1) is 1 to 3 days, and the period of the culturing step of 2) is 1 to 12 days. GSK-3β inhibitors are CHIR-99021, CHIR-98014, 3F8, A 1070722, AR-A 014418, BIO, BIO-acetoxime, 10Z-Hymenialdisine, Indirubin-3'-oxime, Kenpaullone, L803, L803-mts, MeBIO , NSC 693868, SB 216763, which is SB 415286, TC-G 24, TCS 2002, 1 or more compounds selected from the group consisting of TCS 21311 and TWS 119, according to any one of claims 1 to 3 Manufacturing method. RAR agonists are TTNPB, AM580, AM80, 9-cis retinoic acid, all-trans retinoic acid (ATRA), LGD1550, E6060, AGN193312, AM555S, CD2314, AGN193174, LE540, CD437, CD666, CD2325, SR11254, SR11363, SR11364, The method according to any one of claims 1 to 4 , which is selected from the group consisting of AGN193078, TTNN (Ro19-0645), CD270, CD271, CD2665, SR3985, AGN193273, Ch55, 2AGN190521, CD2366, AGN193109 and Re80. .. The method according to any one of claims 1 to 5 , wherein the concentration of the RAR agonist in the medium is 1 nM to 10 μM. The method according to any one of claims 1 to 6 , wherein the progenitor cells are paraxial mesoderm and / or lateral plate mesoderm cells.

Images