JP6464529B2 - Bone differentiation inducing method and ossification promoting / suppressing molecule screening method - Google Patents

Description

The present invention relates to a method for screening a molecule that promotes or suppresses ossification using cells that differentiate into osteoblasts, and in particular, bone using cells from an individual suffering from progressive ossifying fibrodysplasia. The present invention relates to a method for screening a molecule that promotes or suppresses crystallization. The invention also relates to a method for inducing bone differentiation of such cells.

Bone is a dynamic tissue in which bone formation by osteoblasts and bone resorption by osteoclasts are constantly repeated. It is known that when an abnormality occurs in the functional balance between osteoblasts and osteoclasts, this dynamic equilibrium state is broken and various bone metabolic disorders are caused.
In recent years, research has been conducted on the use of bone regeneration ability in bone and cartilage damage and dental treatment. In bone formation, undifferentiated mesenchymal system aggregates and differentiates into chondroblasts, endochondral bone formation in which cartilage is formed as a template for forming bone, and mesenchymal cells aggregate and bone from inside There is membranous bone formation that differentiates into blasts, but in both cases it is said that osteoblasts are responsible for bone formation

Runx2 (CBFA1, AML3) is known as a transcription factor for inducing differentiation from stem cells to osteoblasts, and osterix is known as a transcription factor for promoting differentiation. In addition, TGFβ family growth factors such as bone morphogenetic protein (BMP) have already been found as regulatory factors for these transcription factors. BMP is a bone-inducing cytokine, and its signal is transmitted into cells by type I and type II serine / threonine kinase receptors, and further into the nucleus by phosphorylation of the transcriptional regulator Smad by type I receptors. Is done. Lack of BMP signaling causes brachydactyly and achondroplasia, whereas excess BMP signals progressive ossificans line維異forming diseases; may cause such (Fibrodysplasia ossificans progressiva FOP) has become clear (Non-Patent Document 1).

  FOP is one of the intractable diseases in which the soft tissues of the whole body become ossified, and it has not been established a treatment method that develops by 10 years old and dies by restrictive breathing disorder by about 40 years old ( Non-patent documents 1 to 3). It has been found that this is caused by an excessive hyperactivity abnormality caused by a point mutation of the ALK2 kinase gene, which is a BMP1 type receptor (Non-Patent Documents 3 to 9). The most common mutation is R206H, which is thought to alter ALK2 kinase activity, resulting in a constitutive increase in ALK2 kinase activity. For example, several other mutations such as G356D in ALK2 have been reported with FOP phenotypic mutations, which have also been reported to affect kinase activity and cause constitutive activation of ALK2. (Non-Patent Document 10). It has been shown that the kinase activity of ALK2 (G356D) is weaker than that of ALK2 (R206H), and it is suggested that clinical changes are caused by differences in biological activity in ALK2 mutants (Non-patent Document 11). .

  Under such circumstances, a low molecular weight compound that inhibits bone differentiation by inhibiting BMP signaling was sought, and dorsomorphin and its derivative LDN-193189 were found. (Non-patent document 12, Non-patent document 13). LDN-193189, a specific inhibitor of BMPI receptor kinase, reduces ectopic bone formation and dysfunction, which are the main symptoms of progressive ossification fibrodysplasia, in experiments using pathological model mice It is known to do (Non-Patent Document 14).

  Conventionally, many studies have been conducted on osteoblast differentiation systems using human normal mesenchymal cells. However, there are few studies using disease-derived mesenchymal cells. Regarding FOP, as a previous study, there has been one report that mesenchymal cells were isolated from the teeth of FOP patients, and the cells were stimulated with BMP2 and BMP4 to induce differentiation into osteoblasts (Non-patent literature). 4). However, cells must be isolated from the patient's teeth, which is problematic as a general technique.

  Thus, a method for efficiently differentiating into osteoblasts using ES cells derived from an individual has been tried, but has not yet been developed. Therefore, it is possible to establish an efficient differentiation induction method for osteoblasts by differentiating ES cells into mesenchymal stem cells, then purifying and proliferating mesenchymal stem cells, and then differentiating them into osteoblasts. Attempts have been made (Non-Patent Documents 15 and 16).

  Under such circumstances, a tool that can directly and easily search for a compound that controls (suppresses or promotes) ossification has been desired. That is, a method for screening a compound involved in the efficient control of ossification in vitro has been desired.

F. S. Kaplan et al., Methods Enzymol 484, 357 (2010). E. M. Shore, F. S. Kaplan, Bone 43, 427 (2008). T. Fukuda et al., J Biol Chem 284, 7149 (2009). P. C. Billings et al., J bone Miner Res 23, 305 (2008) J. L. Fiori, P. C. Billings, L. S. de la Pena, F. S. Kaplan, E. M. Shore, J Bone Miner Res 21, 902 (2006). F. S. Kaplan et al., Hum Mutat 30, 379 (2009). A. B. Shafritz et al., N Engl J Med 335, 555 (1996). Q. Shen et al., J Clin Invest 119, 3462 (2009). E. M. Shore et al., Nat Genet 38, 525 (2006). H. Furuya et al., Am J Med Genet A 146A, 459 (2008). T. Fukuda et al., Biochem Biophys Res Commun 377, 905 (2008). Yu PB et al., Nat Chem Biol, 4, 33-41 (2008). Cuny et al, Bioorg Med Chem Lett, 18, 4388-4392 (2008). Yu PB et al., Nat Med. Dec; 14 (12): 1363-9 (2008). T. Barberi et al., PLOS Medicine, June 2005, Vol. 2, Issue 6, e161, 0554-0560. C. Xu et al., Stem Cells, 2004; 22: 972-980. M. Hamasaki et al., Stem Cells, 2012; 30: 2439-2449.

  An object of the present invention is to provide a relatively simple method for screening a molecule involved in the control (promotion or suppression) of ossification in vitro (cell level). Another object of the present invention is to provide a cell and a differentiation induction method that can be used in such a screening method.

  As a result of intensive studies to solve the above-mentioned problems, the present inventors have found that fibroblasts derived from patients with progressive ossifying fibrodysplasia have four factors (Oct4, Sox2, KLF4, and c) as reprogramming genes. When the cells into which -Myc) were introduced were cultured in the presence of BMP-6, it was found that the promotion of ossification, which is a clinical finding of FOP, can be easily reproduced in vitro, and the present invention was completed.

That is, the aspects of the present invention are as follows.
1. somatic or cells derived from affected individuals initialization gene introduced progressive ossificans line維異forming diseases for creating iPS cells passaged cells of bone morphogenetic protein BMP-6 A method for inducing differentiation of somatic cells or cells derived therefrom into osteoblasts, comprising culturing in the presence.
2. The method according to the 1, wherein the somatic cell is a skin line fibroblasts.
3. 3. The method according to 2 above, wherein the cell is a human cell.
4). The cell has the following gene:
1) a gene encoding an amino acid sequence in which the 206th arginine is mutated to histidine in the amino acid sequence shown in SEQ ID NO: 2, and / or 2) the 356th glycine in the amino acid sequence shown in SEQ ID NO: 2 is aspartic acid A gene encoding an amino acid sequence mutated into
The method according to any one of 1 to 3 above, comprising:
5. 5. The method according to any one of 1 to 4, wherein the reprogramming gene includes at least an Oct3 / 4 gene, a Klf4 gene, and a Sox2 gene.
6). 6. The method according to 5 above, wherein the reprogramming gene further comprises a Myc gene.
7). The method according to any one of 1 to 5, wherein the cell culture is performed in a medium containing BMP-6 at a concentration of 10 to 100 ng / ml.

8). The following steps:
a) the early gene for creation of iPS cells into somatic cells from an individual suffering from progressive ossificans line維異forming diseases were passaged in cells or cell-derived cells transduced osteoinductive factor Culturing in the presence of BMP-6 and the test substance; and b) selecting test substances that inhibit or promote ossification by evaluating ossification of the cells:
Screening method for ossification control (promoting or suppressing ossification) molecules.
9. The method according to the 8 the somatic cell is a skin line fibroblasts.
10. 10. The method according to 9 above, wherein the cell is a human cell.
11. The cell has the following gene:
1) a gene encoding an amino acid sequence in which the 206th arginine is mutated to histidine in the amino acid sequence shown in SEQ ID NO: 2, and / or 2) the 356th glycine in the amino acid sequence shown in SEQ ID NO: 2 is aspartic acid A gene encoding an amino acid sequence mutated into
The method according to any one of 8 to 10 above, comprising:
12 The method according to any one of 8 to 11, wherein the reprogramming gene includes at least an Oct3 / 4 gene, a Klf4 gene, and a Sox2 gene.
13. 13. The method according to 12 above, wherein the reprogramming gene further comprises a Myc gene.
14 The method according to any one of 8 to 13, wherein the cells are cultured in a medium containing BMP-6 at a concentration of 10 to 100 ng / ml.

15. The following genes:
1) Oct3 / 4 gene, Klf4 gene, and Sox2 gene, which are reprogramming genes for generating iPS cells, and
2) A gene encoding an amino acid sequence in which the 206th arginine is mutated to histidine in the amino acid sequence shown in SEQ ID NO: 2 and / or the 356th glycine in the amino acid sequence shown in SEQ ID NO: 2 is mutated to aspartic acid An ALK2 mutant gene comprising a gene encoding the determined amino acid sequence,
From the introduced mammalian cells (somatic cells (preferably skinline fibroblasts) or iPS cells), which comprises culturing in the presence of osteoinductive factors BMP-6, of the cells into osteoblasts Differentiation induction method.
16. 16. The differentiation induction method according to 15, wherein the cell derived from the mammal is further introduced with a Myc gene as an initialization gene.
17. 17. The method according to 15 or 16 above, wherein the mammal-derived cell is a human cell.
18. The method according to any one of 15 to 17, wherein the cells are cultured in a medium containing BMP-6 at a concentration of 10 to 100 ng / ml.
19. The following genes:
1) Oct3 / 4 gene, Klf4 gene, and Sox2 gene, which are reprogramming genes for generation of iPS cells, and
2) A gene encoding an amino acid sequence in which the 206th arginine is mutated to histidine in the amino acid sequence shown in SEQ ID NO: 2 and / or the 356th glycine in the amino acid sequence shown in SEQ ID NO: 2 is mutated to aspartic acid An ALK2 mutant gene comprising a gene encoding the determined amino acid sequence,
A the introduced cells from mammals (somatic cells (preferably skinline fibroblasts) or iPS cells),
Cells that differentiate into osteoblasts by culturing in the presence of osteoinductive factor BMP-6 (preferably at a concentration of 10-100 ng / ml, more preferably about 50 ng / ml).
20. 20. The cell according to 19, wherein the cell derived from the mammal is further introduced with a Myc gene as an reprogramming gene.
21. 21. The cell according to the above 19 or 20, wherein the cell is a human cell.

22. The following steps:
a) culturing the cell according to any one of 19 to 21 in the presence of an osteoinductive factor BMP-6 and a test substance; and b) evaluating ossification of the cell by evaluating ossification. Screen for test substances that suppress or promote ossification:
Screening method for ossification control (promoting or suppressing ossification) substance.
23. 23. The screening method according to 22 above, wherein the culture is performed in a medium containing BMP-6 at a concentration of 10 to 100 ng / ml.
24. A kit for screening a substance for controlling ossification (promoting or inhibiting ossification), the cell according to any one of the above 19 to 21, the bone inducing factor BMP-6, and a culture for culturing the cell A kit containing a medium.

  According to the present invention, molecules involved in the control (promotion or suppression) of ossification can be screened with a relatively simple in vitro (cell level) screening system. The present invention also provides a screening method that can be used as a drug screening system that can easily and efficiently obtain drug candidates related to bone diseases.

The effect of BMP-4 and BMP-7 for iPS cell formation (and cellular reprogramming) from healthy individuals skin line fibroblasts, is the result of examination in the presence and absence of LDN193189 a ALK2 kinase inhibitor . (A) shows the experimental design. The lower left photograph is the result of observing the state of cells with a microscope (FIG. 1B). The lower right photo shows the number and ratio of typical and atypical iPS cell colonies (FIG. 1C). It is a result of studying the effect of BMP-6 for iPS cell formation (and cellular reprogramming) from healthy individuals skin line fibroblasts. The top row shows the experimental design. It is the result of having added BMP-6 to the skin fibroblast derived from a healthy person and a FOP patient (F206H mutation patient and G356D mutation patient), and having tested about the osteoblast induction. The photo on the left is one week later, and the photo on the right is two weeks later. It is the result of having tested about the effect | action of LDN193189 and Dorsomorphin which are ALK2 kinase inhibitors with respect to the osteoblast induction of the skin fibroblast derived from a FOP patient in presence and absence of BMP-6. It is the result observed with the microscope after 2 weeks of culture. It is the result of having tested about the effect of LDN193189 and Dorsomorphin which are ALK2 kinase inhibitors with respect to the osteoblast induction of the skin fibroblast derived from a FOP patient (F206H mutation patient and G356D mutation patient) in the presence or absence of BMP-6. . It is the result of detecting osteoblasts by ALP staining after 2 weeks of culture. A healthy person and the BMP (BMP-4, 6 and 7) for FOP from patients skin lines fibroblasts and measuring the effect of ALP activity by the processing. Control is the APL activity in BMP non addition in healthy human skin line fibroblasts. As a positive control for measuring ALP activity, iPS cells of a healthy person were used.

The present invention, early genes for creation of iPS cells were passaged in transfected cells (or cells derived from the skin line fibroblasts from an individual suffering from progressive ossificans line維異formed Diseases Cell) is cultured in the presence of osteoinductive factor BMP-6 and a test substance, and ossification of the cell is evaluated, thereby selecting a test substance that suppresses or promotes ossification. The present invention relates to a method for screening a substance for controlling ossification (promoting or suppressing ossification).

  In this specification, “differentiation” means that a certain cell becomes a specialized cell. In the present specification, “ossification” or “bone differentiation” means that the cells become specialized cells having the characteristics of bone, for example, osteoblasts. Osteoblasts can be detected by, for example, alkaline phosphatase (ALP) staining.

Skin fibroblasts of patients with advanced ossificans line維異forming disease (FOP) used in one embodiment of the present invention, the present inventors that iPS cell establishment is difficult report found previously ( Non-patent document 17). Here, iPS cell establishment is difficult even if a reprogramming gene described later is introduced into a patient-derived cell (somatic cell) and induced into an induced pluripotent stem cell (iPS cell) by a conventional technique. This means that iPS cells cannot be produced, or induced pluripotent stem cells appear only with extremely low efficiency. More specifically, even if induced pluripotent stem cells are induced by introducing reprogramming genes into cells derived from patients with the disease and culturing in various iPS media, iPS cell colonies are completely absent on the media. It does not appear, or it means that the formation frequency of iPS cell colonies on the medium is very low compared to the case where iPS cells are induced from cells derived from healthy solids. More specifically, reprogramming genes (in particular, Oct3 / 4 gene, Klf4 gene, Sox2 gene, and c-Myc gene) are introduced into cells derived from the patient with the disease, and 1-2 × per 100 mm dish. When iPS cells are induced by seeding 10 ⁵ cells, the number of colonies formed is smaller than that when iPS cells are induced from cells derived from healthy solids. This means that the number of iPS cell colonies to be performed is, for example, 50 or less.

  It is known that the responsible gene of FOP is the ALK2 gene (ACVR1). The nucleotide sequence of human ALK2 gene mRNA (Homo sapiens activin A receptor, type I (ACVR1), transcript variant 1, mRNA) is published as NCBI accession number: NM_001105. The base sequence of the CDS region is shown in SEQ ID NO: 1, and the amino acid sequence is shown in SEQ ID NO: 2. When comparing the ALK2 gene of healthy subjects and FOP patients, the 617th base in the nucleotide sequence of the CDS region is guanine (G), whereas in FOP patients, the base is mutated to adenine (A). It is known that this base mutation causes the 206th arginine to be mutated to histidine (R206H) in the amino acid sequence of the protein encoded by the ALK2 gene. Furthermore, as a rare example of mutation of the ALK2 gene in FOP patients, the 1067th base in the nucleotide sequence of the CDS region was mutated from guanine (G) to adenine (A), and the amino acid sequence from the 356th glycine. A case of mutating to asparagine (G356D) is known. ALK2 kinase functions as a single transmembrane receptor of BMP (Bone morphogenetic protein) known as a growth factor that induces bone tissue. When activated by binding to BMP in the extracellular region, ALK2 kinase activates and generates a bone formation signal in the cell. introduce. In healthy individuals, ALK2 kinase is inactivated “off” in the absence of BMP binding, whereas in FOP patients ALK2 kinase is activated “on” even in the absence of BMP binding. In FOP, since ALK2 kinase is always in an activated state, it is thought that ectopic bone formation proceeds by transmitting a signal that promotes bone formation. .

  In the present invention, “individual” means a mammal, preferably a human. In the present invention, the somatic cell includes all cells other than the inner germ cells of the cells constituting the living body, and may be a differentiated somatic cell or an undifferentiated stem cell. In addition, in the present invention, “derived from” means that a cell collected from an individual may be used as it is in the method of the present invention to introduce the reprogramming gene, or a cultured cell line established by culturing the collected cell Means that an reprogramming gene may be introduced into the method of the present invention. When the cells are cryopreserved, they may be cryopreserved before introducing the reprogramming gene, or may be cryopreserved after introducing the reprogramming gene. Specific examples of the cells in the present invention include dermal fibroblasts obtained by culturing skin collected from an individual suffering from a disease. Furthermore, as a cell in the present invention, a cell containing a gene encoding ALK2 kinase having the mutation of R206H and / or G356D described above can be used. Specifically, a human cell containing a gene encoding ALK2 kinase having a mutation of R206H and / or G356D in the amino acid sequence shown in SEQ ID NO: 2 can be used. A gene encoding ALK2 kinase having these mutations can be introduced into any cell by a conventional method, and such a cell can be used in the same manner. By introducing the reprogramming gene into these cells, it can be used in the method of the present invention.

  In the present invention, the “cell into which the reprogramming gene has been introduced or the cell from which the cell has been passaged” refers to the above-described cell into which the reprogramming gene has been introduced as it is for the screening method of the present invention. Alternatively, a cultured cell (or a cultured cell line) obtained by introducing a reprogramming gene and then culturing cells and subcultured may be used in the method of the present invention. Alternatively, a cell into which the reprogramming gene has been introduced or a cell in which the reprogramming gene has been introduced may be cryopreserved, and the cells may be thawed and cultured if necessary and used in the method of the present invention. In such a case, the iPS cell can be used in the method of the present invention after culturing the cell into which the reprogramming gene has been introduced under conditions capable of generating an iPS cell as described later, and then producing the iPS cell.

In the method of the present invention, first, at least one reprogramming gene is introduced into a cell. The reprogramming gene is a gene encoding a reprogramming factor that has the action of reprogramming somatic cells to become iPS cells. Specific examples of the combination of reprogramming genes include the following combinations, but are not limited thereto.
(I) Oct gene, Klf gene, Sox gene, Myc gene (ii) Oct gene, Sox gene, NANOG gene, LIN28 gene (iii) Oct gene, Klf gene, Sox gene, Myc gene, hTERT gene, SV40 large T gene (Iv) Oct gene, Klf gene, Sox gene

  Each of the Oct gene, Klf gene, Sox gene and Myc gene includes a plurality of family genes. As specific examples of each family gene, those described in pages 11 to 13 of the specification of International Publication No. WO2007 / 069666 can be used. Specifically, it is as follows.

  Specific examples of genes belonging to the Oct gene include Oct3 / 4 (NM_002701), Oct1A (NM_002697), and Oct6 (NM_002699) (in parentheses indicate NCBI accession numbers of human genes). Oct3 / 4 is preferable. Oct3 / 4 is a transcription factor belonging to the POU family, is known as an undifferentiated marker, and has been reported to be involved in maintaining pluripotency.

  Specific examples of genes belonging to the Klf gene include Klf1 (NM_006563), Klf2 (NM_016270), Klf4 (NM_004235), and Klf5 (NM_001730) (in parentheses indicate NCBI accession numbers of human genes) ). Klf4 is preferred. Klf4 (Kruppel like factor-4) has been reported as a tumor suppressor.

  Specific examples of genes belonging to the Sox gene include, for example, Sox1 (NM_005986), Sox2 (NM_003106), Sox3 (NM_005634), Sox7 (NM_031439), Sox15 (NM_006942), Sox17 (NM_0022454), and Sox18 (NM_018419). (In parentheses indicate the NCBI accession number of the human gene). Preferably it is Sox2. Sox2 is a gene that is expressed during early development and encodes a transcription factor.

  Specific examples of genes belonging to the Myc gene include c-Myc (NM_002467), N-Myc (NM_005378), and L-Myc (NM_005376) (in parentheses are the NCBI accession numbers of human genes). Show). Preferably, it is c-Myc. c-Myc is a transcriptional regulator involved in cell differentiation and proliferation, and has been reported to be involved in maintaining pluripotency.

  The genes described above are genes that exist in common in mammals including humans, and genes derived from any mammal (eg, derived from mammals such as humans, mice, rats, monkeys) can be used in the present invention. . In addition, several (for example, 1 to 30, preferably 1 to 20, more preferably 1 to 10, more preferably 1 to 5, particularly preferably 1 to 3) of wild type genes. It is also possible to use a mutated gene having a base substitution, insertion and / or deletion and having a function similar to that of a wild-type gene.

  In the present invention, it is particularly preferable to use a combination of the Oct3 / 4 gene, the Klf4 gene, and the Sox2 gene as the reprogramming gene, and more preferably a combination of these three and the c-Myc gene.

The method for introducing the reprogramming gene into the cell is not particularly limited as long as the introduced reprogramming gene is expressed to achieve cell reprogramming. For example, the reprogramming gene can be introduced into a cell using an expression vector containing at least one or more reprogramming genes. When two or more kinds of reprogramming genes are introduced into a cell using a vector, the expression vector may be introduced into a cell by incorporating two or more kinds of reprogramming genes into one expression vector. Two or more types of expression vectors into which various types of reprogramming genes are incorporated may be prepared and introduced into cells.
The type of expression vector is not particularly limited, and may be a viral vector or a plasmid vector, but a viral vector is preferred. Examples of virus vectors that can be used in the present invention include retrovirus vectors (including lentivirus vectors), adenovirus vectors, adeno-associated virus vectors, Sendai virus vectors, and the like. Of these, Sendai virus vectors are preferred.

  In the method of the present invention, cells into which the reprogramming gene has been introduced are cultured together with BMP-6 in the presence of a test substance. In this step, BMP- is added to any medium that can be used for inducing or maintaining osteoblasts. 6 and the test substance may be added to culture the cells. Specifically, media capable of maintaining the undifferentiation and pluripotency of ES cells and induced pluripotent stem cells are known in the art, and can be used in combination with appropriate media. That is, as a medium that can be used for culturing cells into which an reprogramming gene has been introduced in the method of the present invention in the presence of a test substance together with BMP-6, ES medium, and 10 ng / ml FGF-2 in ES medium are used. DMEM supplemented with MEF-conditioned ES medium (hereinafter MEF-conditioned ES medium), a predetermined amount of KnockOut Serum Replacement (KSR) (Invitrogen) and / or bFGF, which is a supernatant obtained by culturing mouse embryonic fibroblasts for 24 hours after the addition A medium etc. can be raised. In the present invention, the medium for culturing the cells into which the reprogramming gene has been introduced includes various growth factors, cytokines, hormones and the like (eg, FGF-2, TGFb-1, activin A, Nanoggin, BDNF, Ingredients involved in the growth and maintenance of human ES cells such as NGF, NT-1, NT-2 and NT-3 may be added.

  BMP-6 used in the method of the present invention is a naturally occurring BMP-6 protein (including BMP-6 protein and a naturally-provided variant), and an effect of inducing bone differentiation of cells which is the object of the present invention. As long as it has a modified BMP-6 protein. The modified BMP-6 protein includes not only those in which a part of amino acids in the polypeptide is replaced, but also those in which deletion or addition is made, and those in which some residues of the peptide are modified. Including. Furthermore, a polypeptide consisting of a part of the peptide sequence and a chimeric protein are also included. BMP-6 DNA and protein sequences and methods for producing them are described in Japanese Patent Application Laid-Open No. 2004-073208 and US Pat. No. 5,187,076. Which is incorporated herein by reference and is part of this specification.

  The concentration of BMP-6 added to the culture medium in the method of the present invention may be any concentration as long as it induces bone differentiation induction of cells, which is the object of the present invention, but is 10 to 100 ng / ml, preferably about 50 ng. Can be used at a concentration of / ml.

  In the present invention, when the cells into which the reprogramming gene has been introduced are cultured together with BMP-6 in the presence of a test substance, appropriate support cells may be used. The feeder cells used in the present invention are not particularly limited as long as they can generally induce and maintain pluripotent stem cells. Specific examples of feeder cells include MEF cells. In addition, as the supporting cells, those in which cell growth has been lost by mitomycin C treatment or irradiation can be used. MEF cells (from ICR mice) are registered with ATCC as catalog number ATCC # SCRC-1046. MEF cells can be obtained according to the description in the literature (Nagy A, et al. Manipulating The Mouse Embryo: A Laboratory Manual. Third Edition Cold Spring Harbor Press; 2003).

The cells into which the reprogramming gene used in the present invention has been introduced are cultured under conditions that give rise to iPS cells, then the medium is changed, unnecessary components are removed from the medium, and then cultured in the presence of a test substance together with BMP-6. may be, conditions that iPS cells is not particularly limited as long as the conditions from the initialization gene is introduced cells can generate iPS cells used in the present invention, use a line fibroblast cells from FOP patients In the presence of dorsomorphin and / or LDN-193189. When dorsomorphin is used, the concentration is, for example, 1 to 4 μM. When LDN-193189 is used, the concentration is, for example, 200 nM to 1 μM.

  Any substance can be used as the test substance used in the present invention. The kind of the test substance is not particularly limited, and may be a low molecular compound, a compound present in a natural product extract, or a synthetic peptide. Alternatively, the test substance may also be a compound library, a phage display library or a combinatorial library. The test substance is preferably a low molecular compound, and may be a compound library of low molecular compounds. The construction of a compound library is known to those skilled in the art, and a commercially available compound library can also be used.

  As described above, since the cells into which the initial gene used in the present invention has been introduced are cultured with BMP-6, ossification of the cells (bone differentiation) occurs. By culturing the cells in the presence of the test substance, A test substance that controls ossification (suppresses or promotes ossification) can be selected. To determine whether to control ossification, for example, compare the negative control group in which cells were cultured in a medium to which no test substance was added and the test sample group in which cells were cultured in a medium to which the test substance was added. Then, it can be performed by confirming the ossification (bone differentiation) of the cells. Confirmation of ossification can be performed, for example, by performing alkaline phosphatase (ALP) staining and confirming the state of cells with a microscope, but the method is not particularly limited, and other methods such as ossification or As an index of bone differentiation, there are alizarin red staining, calcium quantification, and measurement of osteocalcin protein concentration in the culture supernatant by ELISA.

According to yet another aspect of the present invention, passage of the body cells or cells derived from the initialization gene to an individual suffering from progressive ossificans line維異forming diseases introduced for the creation of iPS cells There is provided a method for inducing differentiation of somatic cells or cells derived therefrom into osteoblasts, comprising culturing the resulting cells in the presence of the osteoinductive factor BMP-6.

  “Differentiation”, “individual”, “derived”, “somatic cell”, “passaged cell derived from that cell”, “reprogramming gene”, used in the method for inducing differentiation into osteoblasts of the present invention, Each term of “BMP-6” is as described above with respect to the screening method for the ossification (bone differentiation) regulator of the present invention. The method for inducing differentiation into osteoblasts comprises the reprogramming gene of the present invention. It can be carried out by culturing the cells derived from the introduced progressive ossifying fibrodysplasia individual with BMP-6. As the culture medium for differentiation induction, confirmation of ossification or bone differentiation, and other conditions and methods, the conditions and methods described above with respect to the screening method for ossification (bone differentiation) regulator of the present invention can be used.

  According to another aspect of the present invention, the following genes: (1) reprogramming genes for generating iPS cells (for example, Oct3 / 4 gene, Klf4 gene, and Sox2 gene (optionally further c-Myc And (2) a gene encoding an amino acid sequence in which the 206th arginine is mutated to histidine in the amino acid sequence shown in SEQ ID NO: 2, and / or the 356th amino acid in the amino acid sequence shown in SEQ ID NO: 2 Osteoblasts of cells comprising culturing a cell derived from a mammal into which an ALK2 mutant gene containing a gene encoding an amino acid sequence in which the second glycine has been mutated to aspartic acid is introduced in the presence of the osteoinductive factor BMP-6 A differentiation induction method is provided.

As shown in the Examples below, cells from FOP patients have the mutations described above for the ALK2 gene. Therefore, cells having the above-described reprogramming gene and the above-described mutation ALK2 gene can be induced to differentiate into osteoblasts by culturing in the presence of the osteoinductive factor BMP-6.
The cell having the above reprogramming gene and ALK2 mutant gene is not particularly limited as long as it is a cell derived from a mammal and can introduce these genes and express those genes, and any cell can be used. Can do. Mammals are not particularly limited, and examples include humans, mice, rats, monkeys, and the like, and humans are particularly preferable. Further, examples of the cells, for example, a line fibroblast cells collected from a mammalian individual, stem cells, established cultured cells, and the like can be mentioned, there preferably skin line fibroblasts, in mouse embryonic lines fibroblasts , particularly preferably human skin line fibroblasts. Moreover, when the cell to be used already has an reprogramming gene (for example, established iPS cell), it can be used as the cell of the present invention by introducing the ALK2 mutant gene into the cell. Specifically, after preparing cells having these genes, the cells are cultured in the presence of BMP-6 to confirm that the cells differentiate into osteoblasts, and can be used in the present invention.

  As a method for introducing the reprogramming gene into cells, the gene introduction methods described for the screening method for ossification regulators can be used. As a method for introducing the ALK2 mutant gene, a conventional method can be used. For example, it can be introduced by the lipofectin method. Furthermore, the reprogramming gene and the ALK2 mutant gene can be simultaneously introduced into cells.

  In the method for inducing differentiation of cells into osteoblasts using cells having the reprogramming gene and the ALK2 mutant gene of the present invention, the terms “differentiation”, “BMP-6”, and “reprogramming gene” The screening method for the ossification (bone differentiation) regulator of the invention is as described above, and the differentiation induction method for osteoblasts is performed by introducing a cell into which the reprogramming gene of the present invention and the ALK2 mutant gene have been introduced into BMP- 6 and culturing together. As the culture medium for differentiation induction, confirmation of ossification or bone differentiation, and other conditions and methods, those described above for the screening method for the ossification (bone differentiation) controlling substance of the present invention can be used.

  Furthermore, according to another aspect of the present invention, the following genes: (1) Reprogramming genes for generating iPS cells (eg, Oct3 / 4 gene, Klf4 gene, and Sox2 gene (optionally further c-Myc And (2) a gene encoding an amino acid sequence in which the 206th arginine is mutated to histidine in the amino acid sequence shown in SEQ ID NO: 2, and / or the 356th amino acid in the amino acid sequence shown in SEQ ID NO: 2 A cell derived from a mammal into which an ALK2 mutant gene containing a gene encoding an amino acid sequence in which the second glycine has been mutated to aspartic acid is introduced, and is cultured in the presence of the osteoinductive factor BMP-6 to become an osteoblast. Differentiating cells are provided.

  In the cells having the reprogramming gene of the present invention and the ALK2 mutant gene, the terms “differentiation”, “mammal”, “cell”, “BMP-6”, and “reprogramming gene” As described above with respect to the method for inducing differentiation of cells into osteoblasts using cells having a gene and an ALK2 mutant gene, the cells into which the reprogramming gene and the ALK2 mutant gene of the present invention have been introduced are used together with BMP-6. Differentiate into osteoblasts by culturing. The above-described conditions and methods can be used as a gene introduction method, a culture medium for differentiation induction, confirmation of ossification or bone differentiation, and other conditions and methods.

  Still further, according to another aspect of the present invention, the following genes: (1) reprogramming genes for generating iPS cells (for example, Oct3 / 4 gene, Klf4 gene, and Sox2 gene (optionally further c- And (2) a gene encoding an amino acid sequence in which the 206th arginine is mutated to histidine in the amino acid sequence shown in SEQ ID NO: 2, and / or in the amino acid sequence shown in SEQ ID NO: 2. A cell derived from a mammal into which an ALK2 mutant gene containing a gene coding for an amino acid sequence in which the 356th glycine is mutated to aspartic acid is introduced, is cultured in the presence of an osteoinductive factor BMP-6 and a test substance, Bossification control (promoting or suppressing ossification) substance comprising screening test substance that suppresses ossification or promotes ossification by evaluating ossification Screening methods are provided.

  In the screening method for ossification control materials using cells having the reprogramming gene and ALK2 mutant gene of the present invention, “differentiation”, “mammal”, “cell”, “BMP-6”, and “reprogramming gene” Are as described above with respect to the method for inducing differentiation of cells into osteoblasts using cells having the reprogramming gene of the present invention and the ALK2 mutant gene, and the reprogramming gene and the ALK2 mutant gene of the present invention. Can be performed by culturing the cells into which BMP-6 is introduced together with BMP-6 and the test substance. The above-described conditions and methods can be used as a gene introduction method, a culture medium for differentiation induction, confirmation of ossification or bone differentiation, and other conditions and methods.

  The present invention will be described more specifically with reference to the following examples, but the present invention is not particularly limited by the following examples.

(A) Materials and methods (1) Generation of skin-derived fibroblasts According to a protocol approved by the Ethics Committee, fibroblasts from explants of skin biopsies of FOP patients and healthy individuals under informed consent Made. Skin samples from patients and healthy subjects were minced and cultured in DMEM medium supplemented with 10% fetal bovine serum (FBS). After confirming the appearance of fibroblasts, fibroblasts were grown to introduce the reprogramming gene, then placed in a frozen solution consisting of 10% DMSO + 90% FBS and stored frozen.

(2) Maintenance and generation of iPS cells 20% KNOCKOUT ™ serum replacement (KSR, Invitrogen), 2 mM L-glutamine, 1 × 10 ⁻⁴ M non-essential amino acids (NEAA, Sigma), 1 × DMEM supplemented with 10 ⁻⁴ M 2-mercaptoethanol (Sigma), 0.5% penicillin and streptomycin (Nacalai Tesque, Japan), and 5 ng / mL basic fibroblast growth factor (bFGF, Wako, Japan) Human iPS cells were maintained on mitomycin C (MMC) treated MEF feeder cells in human iPS medium containing / F12 (Sigma).

N. Fusaki, H. Ban, A. Nishiyama, K. Saeki, M. Hasegawa, Proc. Jpn. Acad. Ser., B. Phys. Biol. Eci., 85, 348 (2009) IPS cells were generated from human-derived fibroblasts. One day before infection, 5 × 10 ⁵ human fibroblasts were seeded per well in a 6-well plate, and then the following Sendai virus (SeV) vector was used at multiplicity of infection (MOI) 3. Infected with. Seven days after infection, fibroblasts infected with trypsin were collected, and MEF treated with MMC of 5.4 × 10 ⁴ cells per 60 mm dish or 1-2 × 10 ⁵ cells per 100 mm dish Seeded on feeder cells. On the next day, the culture medium was replaced with human iPS cell medium, and the culture was continued until 30 days after infection, and colonies were observed.
The effect of bone morphogenetic proteins (BMP-4, 6, and 7) on iPS cell generation was observed in BMP-4 (10 ng / ml), BMP-6 ( 50 ng / ml) and a medium supplemented with BMP-7 (10 ng / ml) were used, and culture was performed until the 30th day. In some experiments, LDN-193189 (STEMGENT; stemgent), an ALK2 kinase inhibitor, was added to the human iPS medium at a concentration of 200 nM.

(3) Construction and detection of Sendai virus (SeV) vector SeV vectors containing Oct3 / 4 gene, Sox2 gene, K1f4 gene and c-Myc gene are N. Fusaki, H. Ban, A. Nishiyama, K. Saeki, M. Hasegawa, Proc. Jpn. Acad. Ser., B. Phys. Biol. Eci., 85, 348 (2009). Nested PCR was performed to detect the SeV genome. One microgram of total RNA extracted from dermal fibroblasts and iPS cells was reverse transcribed into cDNA. Subsequently, cDNA was amplified using a pair of SeV-specific primers, and 1/10 volume of these PCR products was further amplified using a pair of nested primers. Table 1 shows primer sequences and amplification conditions.

(4) was conducted in the same manner as FOP patient-derived skin lines fibroblasts differentiation inducing the above into osteoblasts (2). Skin lines fibroblasts derived from FOP patients or healthy individuals, recovery Oct3 / 4 gene, Sox2 gene, were infected with SeV vector containing the K1f4 gene and c-Myc gene, the infected lines fibroblast cells after infection 7 days And seeded on MEF feeder cells. The next day, the medium was replaced with human iPS cell medium supplemented with 50 ng / ml of BMP-6, and the culture was continued until 30 days after infection. In some experiments, human iPS medium supplemented with 200 nM and 1 μM of LDN-193189 (STEMGENT) and dorsomorphin (Sigma), which are ALK2 kinase inhibitors, was used, respectively.

(5) Alkaline phosphatase staining and immunohistochemistry Alkaline phosphatase staining was performed using a leukocyte Alkaline Phosphatase kit (Sigma). For immunocytochemistry, cells were fixed with PBS containing 4% paraformaldehyde for 30 minutes at 4 ° C. For molecules localized in the nucleus, the sample was treated with 0.2% Triton X-100 for 15 minutes at room temperature. Cells were washed 3 times with PBS containing 2% FBS and then incubated overnight at 4 ° C. in PBS containing 2% FBS with primary antibody. Primary antibodies include SSEA4 (1: 500, Millipore), TRA-160 (1: 500, Millipore), Nanog (1: 1000, R & D Systems) and Oct3 / 4 (1: 500, Santa-Cruz). . As the secondary antibody, goat anti-mouse IgG bound to Alexa 488 (1: 1000, Invitrogen) and donkey anti-goat IgG bound to Alexa 488 (1: 1000, Invitrogen) were used. For staining of differentiation markers, Sox17 (1: 200, R & D Systems), Foxa2 (1: 200, R & D Systems) and Brachyury (1: 200, R & D Systems) were used. Nuclei were stained with 1 μg / mL Hoechst 33258 (Invitrogen).

(5) DNA isolation and sequencing The mutation of the ALK2 gene (R206H: 617G> A and G356D: 1067G> A) in the FOP-derived iPS cell line was confirmed by sequencing the DNA. IPS cell lines in lysis buffer containing 50 mM Tris-HCl (pH 7.5), 20 mM EDTA (pH 8.0), 0.1 M NaCl, 1% SDS and 0.15 mg / mL proteinase K. Incubate overnight at 55 ° C. Genomic DNA was extracted with phenol / chloroform / isoamyl alcohol. Then 100 ng of genomic DNA was amplified by PCR. The base sequence of the obtained PCR product was determined by ABI PRISM (trademark) 310 Genetic Analyzer (BigDye (trademark) terminator v1.1 cycle sequencing kit, Applied Biosystems). Table 2 shows the primer sequences used to determine the base sequence.

(B) Results (1) Information on FOP patients and healthy subjects According to the above materials and methods, the skin of FOP patients (F1, F2, F3, F4) and healthy subjects (N1, N2, N3) by Sendai virus (SeV) method produced from derived lines fibroblasts of iPS cells was confirmed mutations ALK2 genes. The following shows information on patients and normals from which the cells used in this example are derived.

(2) Effect of bone morphogenetic protein on iPS cell generation In accordance with the materials and methods described above, an attempt was made to generate iPS cells from the skin-derived fibroblasts of healthy persons (N1) by the Sendai virus (SeV) method. The effects of ALK2 kinase inhibitor LDN-193189 (200 nM added) and bone morphogenetic proteins BMP-4 (10 ng / ml added) and BMP-7 (10 ng / ml added) on iPS cell generation were examined. The results are shown in FIG. (A) shows the experimental design. The lower left photograph is the result of observing the state of the cells cultured under the presence and absence of LDN-193189, BMP-4 and BMP-7 with a microscope on the 30th day after infection (FIG. 1B). . The lower right photo shows the number and ratio of typical and atypical iPS cell colonies 30 days after infection (FIG. 1C).
Moreover, the influence of BMP-6 (50 ng / ml addition) in the production | generation of the iPS cell from the fibroblast derived from the skin of a healthy subject (N1) was examined. The results are shown in FIG. The upper graph shows the experimental design, the lower left photo shows the results when BMP-6 is not added, and the right photo shows the results when BMP-6 is added.
When BMP-4 is added, the number of colonies of iPS cells is reduced only by a small amount, and differentiated colonies increase, whereas when BMP-6 is added, iPS cell colonies themselves are drastically reduced. This result suggests that the downstream signals of BMP-4 and BMP-6 are different. Therefore, considering that 1) the cause of FOP is a BMP receptor abnormality and 2) it is difficult to establish iPS cells from FOP, the abnormality of FOP is similar to the state of adding BMP-6 to normal cells. It is possible that

(3) In accordance with the differentiation inducing the materials and methods to osteoblasts FOP patients skin-derived lines fibroblasts from healthy individuals by Sendai virus (SeV) method (N1) and FOP patients (F206H variant patients (F2) and G356D mutation The reprogramming gene was introduced into the fibroblasts derived from the skin of the patient (F4) and cultured in the presence and absence of BMP-6 (50 ng / ml added). The appearance of osteoblasts was confirmed by alkaline phosphatase staining after 1 and 2 weeks of culture after conversion to iPS cell medium. The results are shown in FIG.
It was confirmed that skin fibroblasts derived from healthy people hardly induce osteoblasts, whereas skin fibroblasts derived from FOP patients can efficiently induce differentiation into osteoblasts in the presence of BMP-6. It was done.

(4) Screening of bone differentiation control substance using the FOP patient-derived skin line fibroblasts
In the above method using the skin line fibroblasts, normal whereas in (healthy subjects fibroblasts) little differentiation not induced, FOP patient-derived skin line blasts easy differentiation into osteoblasts To be enhanced. So according to the above method, the induction of differentiation into osteoblast cells using FOP patients (F2) from the skin line fibroblasts was confirmed the effect of LDN-193189 and Dorsomorphin a ALK2 kinase inhibitor. The addition amounts of LDN-193189, Dorsomorphin, and BMP-6 are 200 nM, 1 μM, and 50 ng / ml, respectively. FIG. 4 shows the results of confirming the appearance of osteoblasts by alkaline phosphatase staining after 2 weeks of culture after conversion to iPS cell culture medium. When LDN-193189 or dorsomorphin was added, differentiation into osteoblasts could be suppressed preferentially in FOP-derived skin fibroblasts.
Similarly, using the F206H variant FOP patients (F2) and G356D mutation FOP patients (F4) from the skin line fibroblasts was confirmed the effect of LDN-193189 and dorsomorphin. The addition amounts of LDN-193189, dorsomorphin and BMP-6 are the same as described above. FIG. 5 shows the results of confirming the appearance of osteoblasts by alkaline phosphatase staining after 2 weeks of culture after conversion to iPS cell culture medium. Also in skin lines fibroblasts of FOP patients from any of the mutations, the ALK2 kinase inhibitors (LDN-193189 and dorsomorphin), the differentiation into osteoblasts is inhibited was confirmed.

(5) in accordance with a healthy person and influence the materials and methods of the bone morphogenetic protein to the skin line fibroblast cells from FOP patients, healthy individuals by Sendai virus (SeV) method from skin (N1) and the patient FOP (F2) fibrosis Reprogramming genes are introduced into blast cells and cultured in the presence and absence of BMP-4 (added 10 ng / ml), BMP-6 (added 50 ng / ml) or BMP-7 (added 10 ng / ml) It was. Alkaline phosphatase activity was measured after 1 week and 2 weeks of culture after conversion to iPS cell medium. Alkaline phosphatase activity was measured using Lab Assay ^™ ALP (Wako Pure Chemical Industries) according to the manufacturer's protocol. The results are shown in FIG.

  The above detailed description is merely illustrative of the objects and objects of the invention and is not intended to limit the scope of the appended claims. Various changes and substitutions to the described embodiments will be apparent to those skilled in the art from the teachings described herein without departing from the scope of the appended claims.

  It was revealed that bone differentiation of cells can be induced by culturing cells in which reprogramming genes have been introduced into fibroblasts derived from the skin of FOP patients in the presence of BMP-6. Using this bone differentiation induction system, molecules involved in the control (promotion or suppression) of bone differentiation can be screened with a relatively simple in vitro (cell level) screening system.

Claims (10)

Oct3 / 4 gene, Klf4 gene, in vitro using a linear fibroblast cells derived from human suffering initialization gene introduced progressive ossificans line維異forming diseases including Sox2 gene and c-Myc gene A method for inducing the promotion of ossification comprising the following steps:
a) were infected with Sendai virus vector comprising said four genes lines fibroblast cells derived from human suffering from progressive ossificans line維異forming diseases, then the cells of dorsomorphin and / or LDN-193,189 culturing in the presence;
b) recovering the cells; and c) culturing the recovered cells in a medium containing 10-50 ng / ml of the osteoinductive factor BMP-6 and neither of dorsomorphin and LDN-193189, Inducing the promotion of ossification of the cells,
A guidance method comprising: The method according to claim 1, wherein the culture of the cells in the presence of BMP-6 in the step c) is performed in a medium containing BMP-6 at a concentration of 50 ng / ml. The line fibroblasts is a skin line fibroblasts, the method according to claim 1 or 2. The line fibroblast cells, the following genes:
1) a gene encoding an amino acid sequence in which the 206th arginine is mutated to histidine in the amino acid sequence shown in SEQ ID NO: 2, and / or 2) the 356th glycine in the amino acid sequence shown in SEQ ID NO: 2 is aspartic acid A gene encoding an amino acid sequence mutated into
The a method according to any one of claims 1-3.   The method according to claim 1, wherein the reprogramming gene further comprises a Myc gene. A ossification control (ossification promoting or ossification inhibition) Screening method for a substance, the following steps: in a) a line fibroblast cells derived from human suffering from progressive ossificans line維異forming diseases, Oct3 / 4 gene, Klf4 gene, were infected with Sendai virus vector having an initialization gene containing the Sox2 gene and c-Myc gene, then culturing the cells in the presence of dorsomorphin and / or LDN-193189;
b) the step of recovering the cells; and c) the presence of the test substance in a medium containing 10-50 ng / ml of the osteoinductive factor BMP-6 and neither of the dorsomorphin and LDN-193189. were cultured under, then the step of selecting the test substance that promotes inhibit or ossification ossification by evaluating the ossification of the cells after the culture,
A screening method comprising: The method according to claim 6, wherein the cell culture in the presence of BMP-6 in the step c) is performed in a medium containing BMP-6 at a concentration of 50 ng / ml. The line fibroblasts is a skin line fibroblasts The method according to claim 6 or 7. The line fibroblast cells, the following genes:
1) a gene encoding an amino acid sequence in which the 206th arginine is mutated to histidine in the amino acid sequence shown in SEQ ID NO: 2, and / or 2) the 356th glycine in the amino acid sequence shown in SEQ ID NO: 2 is aspartic acid A gene encoding an amino acid sequence mutated into
The method according to claim 6 , comprising: The method of claim 6 , wherein the reprogramming gene further comprises a Myc gene.

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