Cell population of human urine-derived cells, and cell population of myotubes induced from the cell population, and production method for the cell population of myotubes

Abstract

To provide a cell population of human urine-derived cells with high muscle differentiation potential as well as a cell population of myotubes induced from the cell population of human urine-derived cells with high muscle differentiation potential and a production method thereof.SOLUTION: Disclosed are a cell population of human urine-derived cells positive for CD90, a cell population of myotubes induced from the cell population of the human urine-derived cells positive for CD90, and a method for producing myotubes derived from human urine-derived cells, comprising a step of separating the human urine-derived cells positive for CD90.SELECTED DRAWING: Figure 4

Description

[Technical field]

[0001] The present disclosure relates to cell populations of CD90 positive human urine-derived cells, populations of myotubes derived from the cell populations, and methods for producing the populations of myotubes. [Background technology]

[0002] Human urine derived cells (UDCs) are primary cultured cells derived from the upper urinary tract that can be collected from human urine. It is known that a cell population of human urine derived cells is a heterogeneous cell population containing cells of various morphologies and origins, such as renal epithelial cells and urinary tract epithelial cells. In addition, human urine derived cells, although being primary cultured cells derived from humans, have very good proliferation ability and can be collected from urine, a specimen that can be obtained non-invasively. Therefore, efforts are being made to induce human urine derived cells into various cells and use them as medical and biological tools (Non-Patent Document 1).

[0003] One of the cells that have been studied for induction from human urine-derived cells is myotubes. Myotubes (also called "myotube cells") are immature muscle cells. Since collection of myotubes from human subjects is highly invasive, attempts have been made to induce myotubes from human urine-derived cells for the purpose of non-invasively obtaining myotubes for use in drug evaluation for muscle diseases, research on muscle diseases, biopsies, etc. (Non-Patent Document 2).

[0004] The present inventors have developed a method for producing myotubes (myotube cells) from human urine-derived cells (urinary cells), which comprises the steps of introducing MyoD1 (myoblast determination protein 1), a type of muscle regulatory factor (MRF), into human urine-derived cells (urinary cells) and exposing the urine-derived cells to at least one epigenetics-regulating compound (Patent Document 1). [Prior art documents] [Patent documents]

[0005] [Patent Document 1] International Publication No. 2020 / 136696 [Non-patent literature]

[0006] [Non-Patent Document 1] MariaSofia Falzarano and Alessandra Ferlini, "Urinary Stem Cells as Tools toStudy Genetic Disease: Overview of the Literature", J. Clin. Med. 8(5),627 (2019). [Non-Patent Document 2] EllisY. Kim et al., "Direct reprogramming of urine-derived cells with inducibleMyoD for modeling human muscle disease", Skeletal Muscle volume 6:32(2016). [Non-Patent Document 3] RicardoMondragon-Gonzalez and Rita CR Perlingeiro, "Recapitulating muscledisease phenotypes with myotonic dystrophy 1 induced pluripotent stem cells: atool for disease modeling and drug discovery", Dis. Model Mech. 11(7): dmm034728(2018). [Non-Patent Document 4] TingZhou et al., "Generation of human induced pluripotent stem cells from urine samples", Nature Protocols 7, 2080-2089 (2012). Summary of the Invention [Problem to be solved by the invention]

[0007] Since the cell population of human urine-derived cells is a heterogeneous cell population, it is believed that it contains cells that are easily differentiated into myotubes (high muscle differentiation potential) and cells that are not easily differentiated into myotubes (low muscle differentiation potential). Therefore, if a population of cells with high muscle differentiation potential can be selected from the cell population of human urine-derived cells, it is believed that myotubes induced from human urine-derived cells can be efficiently produced.

[0008] It is also known that some muscle disease-specific genes or proteins show increased expression levels when muscle differentiation progresses and more mature myotubes are formed (Non-Patent Document 3). Therefore, when using myotubes induced from human urine-derived cells for drug evaluation against muscle diseases, or for research or biopsy of muscle diseases, it is considered effective to select a population of human urine-derived cells with high muscle differentiation potential.

[0009] To date, no cell surface markers specific to human urine-derived cells with high muscle differentiation potential have been identified.

[0010] An object of the present disclosure is to provide a cell population of human urine-derived cells with high muscle differentiation potential. Also, an object of the present disclosure is to provide a myotube cell population induced from the cell population of human urine-derived cells with high muscle differentiation potential, and a method for producing the same. [Means for solving the problem]

[0011] The present inventors discovered that CD90 (Cluster of Differentiation 90, also known as THY1), a mesenchymal stem cell marker, is a cell surface marker specific to human urine-derived cells with high muscle differentiation potential, and completed the present disclosure.

[0012] The present disclosure relates, for example, to the following: [1] A population of CD90-positive human urine-derived cells. [2] The cell population described in [1] for use in inducing myotubes. [3] The cell population according to [1] or [2], wherein the CD90-positive human urine-derived cells are derived from a patient with a muscle disease or muscular dystrophy. [4] A method for producing myotubes derived from human urine-derived cells, comprising the step of isolating CD90-positive human urine-derived cells. [5] The method according to [4], wherein the step of isolating CD90-positive human urine-derived cells is a step of isolating CD90-positive human urine-derived cells from human urine-derived cells prepared from urine of a patient with a muscle disease or muscular dystrophy. [6] The method according to [4] or [5], further comprising the step of inducing myotubes from the separated CD90-positive human urine-derived cells after the separation step. [7] The method according to [6], wherein the step of inducing myotubes comprises introducing the MYOD1 gene into the CD90-positive human urine-derived cells. [8] The method according to [6] or [7], wherein the step of inducing myotubes does not include producing induced pluripotent stem cells from the CD90-positive human urine-derived cells. [9] A population of myotubes induced from a population of CD90-positive human urine-derived cells.

[10] The myotube cell population described in [9], wherein the CD90-positive human urine-derived cells are derived from a patient with a muscle disease or muscular dystrophy.

[11] A method for isolating human urine-derived cells with high muscle differentiation potential, the method comprising a step of isolating human urine-derived cells that are positive for CD90.

[12] The method according to

[11] , wherein the human urine-derived cells are human urine-derived cells derived from a patient with a muscle disease or muscular dystrophy.

[13] An anti-CD90 antibody or an antigen-binding fragment thereof, and The package insert describes that human urine-derived cells are evaluated for CD90 expression, and those that are evaluated to have high CD90 expression are isolated as human urine-derived cells with high muscle differentiation potential. Kit including:

[14] A method for evaluating the muscle differentiation potential of human urine-derived cells, comprising a step of evaluating the expression of CD90 in the human urine-derived cells.

[15] The method according to

[14] , wherein the human urine-derived cells are human urine-derived cells derived from a patient with a muscle disease or muscular dystrophy.

[16] An anti-CD90 antibody or an antigen-binding fragment thereof, and The package insert states that the expression of CD90 in human urine-derived cells is evaluated, and that human urine-derived cells that are evaluated to have a high level of CD90 expression are evaluated as human urine-derived cells with high muscle differentiation potential. Kit including: Effect of the Invention

[0013] According to one embodiment of the present disclosure, a cell population of human urine-derived cells with high muscle differentiation potential can be provided.

[0014] According to one embodiment of the present disclosure, it is possible to provide a myotube cell population induced from a cell population of human urine-derived cells with high muscle differentiation potential, and a method for producing the same.

[0015] According to an embodiment of the present disclosure, a cell population of myotubes can be produced with high efficiency using a cell population of human urine-derived cells.

[0016] According to a cell population of human urine-derived cells according to an embodiment of the present disclosure, a myotube cell population can be produced while suppressing the influence of heterogeneity of human urine-derived cells for each subject from which urine was collected.

[0017] According to one embodiment of the present disclosure, a cell population of human urine-derived cells can be provided that can be induced to become myotubes without producing induced pluripotent stem cells from the human urine-derived cells.

[0018] According to one embodiment of the present disclosure, a population of myotubes induced from a cell population of human urine-derived cells can be produced without generating induced pluripotent stem cells from the human urine-derived cells.

[0019] According to one embodiment of the present disclosure, a cell population of human urine-derived cells is provided that can be induced into myotubes without exposure to epigenetic regulatory compounds.

[0020] According to one embodiment of the present disclosure, a cell population of induced myotubes can be produced from a cell population of human urine-derived cells without exposure to epigenetic regulatory compounds. [Brief description of the drawings]

[0021] [Figure 1] FIG. 1 is a schematic diagram showing the design of the retroviral vector (MYOD1 viral vector) used to introduce the MYOD1 gene into human urine-derived cells in experimental method 2. [Diagram 2] FIG. 1 shows the results of single-cell RNAseq analysis of a cell population with high muscle differentiation potential (Good UDCs) and a cell population with low muscle differentiation potential (Bad UDCs) before and after muscle differentiation induction in Example 1. [Diagram 3] FIG. 1 shows the distribution of fluorescence intensity when a cell population of CD90-positive human urine-derived cells was separated by flow cytometry in Example 2. [Figure 4] FIG. 13 shows fluorescent images of cell populations obtained by inducing muscle differentiation from cell populations of CD90-negative or CD90-positive human urine-derived cells in Example 3. [Diagram 5] FIG. 13 shows the fusion index of cell populations obtained by inducing muscle differentiation from cell populations of CD90-negative or CD90-positive human urine-derived cells in Example 3. [Figure 6] Figure 6(A) shows the results of Western blotting of cell populations obtained by inducing muscle differentiation from cell populations of CD90-negative or CD90-positive human urine-derived cells in Example 3. Figures 6(B) and 6(C) show the band intensities of MYOD1 and MYHC, respectively, normalized with the band intensity of α-tubulin (α-Tub) in Figure 6(A). [Figure 7] Figure 7(A) shows the results of Western blotting when an exon skipping drug was added to a myotube cell population induced from a cell population of CD90-positive human urine-derived cells derived from a DMD patient in Example 4. Figure 7(B) shows the band intensity of DYS1 normalized by the band intensity of α-tubulin (α-Tub) in Figure 7(A). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0022] Although embodiments of the present disclosure will be described below, the present disclosure is not limited to the following embodiments.

[0023] A first aspect of the present disclosure is a cell population of CD90 positive human urine-derived cells.

[0024] Human urine derived cells (UDCs) are primary cultured cells derived from the upper urinary tract that can be collected from human urine. It is known that a cell population of human urine derived cells is a heterogeneous cell population containing cells of various morphologies and origins, such as renal epithelial cells and urinary tract epithelial cells. The human urine derived cells according to the present disclosure may be, for example, cells obtained by isolating urine collected from a human subject or a culture thereof, or cells obtained by culturing cells obtained by isolating urine collected from a human subject. In this case, the method for isolating the cells is not particularly limited as long as it is a method that can isolate the cells from urine containing the cells or a culture thereof, and may be, for example, centrifugation or filtration, and is preferably centrifugation.

[0025] Methods for preparing human urine-derived cells are known in the art (e.g., Non-Patent Document 4) and are not particularly limited, and can be prepared, for example, by the method shown below. Urine collected from a human subject is centrifuged to remove the supernatant, and the pellet is mixed with an initial medium and incubated at about 37°C, and then cultured in a growth medium, and cells that form colonies about several days to two weeks after the start of culture are selected. The cells obtained in this way become stable cell lines with similar characteristics even after multiple subcultures (Non-Patent Document 4).

[0026] The human urine-derived cells according to the present disclosure may be derived from urine collected from a human subject, for example, from urine collected from a healthy individual, from urine collected from a patient with a muscle disease or a patient with muscular dystrophy (e.g., a patient with Duchenne muscular dystrophy), or may be a mixture of cells derived from both, which can be freely selected by those skilled in the art according to the purpose. Furthermore, the human urine-derived cells according to the present disclosure may be cells derived from urine obtained from a single collection from a human subject, or may be a mixture of cells derived from urine obtained from multiple collections from a human subject.

[0027] Examples of urine collected from muscular dystrophy patients include urine collected from patients with congenital muscular dystrophy such as Duchenne muscular dystrophy, Becker muscular dystrophy, Fukuyama muscular dystrophy, merosin deficiency, Ullrich syndrome, etc. Examples of urine collected from patients with muscle diseases include urine collected from patients with neuromuscular junction diseases such as myopathy, inflammatory muscle diseases, and myasthenic syndrome, neurodegenerative diseases such as amyotrophic lateral sclerosis, peripheral neuropathy such as spinal muscular atrophy, diseases that cause disuse muscle atrophy including sequelae of stroke, sarcopenia, or cancer cachexia.

[0028] In the present disclosure, a cell population means a group (assembly, population) of multiple cells. That is, the cell population according to the present disclosure is not limited to a physical aggregate of cells such as a colony or a spheroid, but also includes a group of multiple cells suspended in a solution. The cell population according to the present disclosure may be, for example, a group of multiple cells suspended in a single liquid that exists in a spatially continuous manner, or a group of multiple cells existing inside a single physically partitioned container (such as a test tube, a tube, a cell culture container, or a culture dish). The number of cells constituting the cell population according to the present disclosure may be, for example, 2 or more, 10 or more, 100 or more, 1000 or more, or 10000 or more.

[0029] The cell population according to the first aspect of the present disclosure is a cell population of CD90-positive human urine-derived cells.

[0030] CD90 (Cluster of Differentiation 90), also known as THY1, is a cell surface protein. In humans, it is known to be expressed in a wide range of organs, including muscle, brain, and kidney. CD90 is also known as one of the mesenchymal stem cell markers.

[0031] In the present disclosure, CD90-positive human urine-derived cells are human urine-derived cells with a high expression level of CD90. It can also be said that CD90-positive human urine-derived cells are human urine-derived cells that highly express CD90. The expression level or amount of CD90 in these may be the expression level or amount evaluated according to a method that is usually used by those skilled in the art when evaluating the expression level or amount of a cell surface protein. Examples of the evaluation method used for such evaluation include immunostaining using a labeled anti-CD90 antibody, Western blotting, and quantitative PCR (q-PCR), and from the viewpoint of directly detecting the protein, immunostaining using a labeled anti-CD90 antibody or Western blotting is preferable. More specifically, the expression level or amount of CD90 may be evaluated, for example, using the fluorescence intensity at a wavelength corresponding to the fluorescence wavelength of a fluorescent dye in the cells when an anti-CD90 antibody labeled with a fluorescent dye is contacted with the cells as an index.

[0032] Specifically, CD90-positive human urine-derived cells may be, for example, human urine-derived cells that meet at least one of the following (a) to (e). In a preferred embodiment, the expression level or amount of CD90 in the following (a) to (e) may be the fluorescence intensity at a wavelength corresponding to the fluorescence wavelength of a fluorescent dye when contacted with an anti-CD90 antibody labeled with the fluorescent dye. (a) Human urine-derived cells having an expression level or amount of CD90 that is at least 3-fold, at least 5-fold, or at least 10-fold the average expression level or amount of CD90 in human urine-derived cells prepared by a similar method from the same human subject. (b) Human urine-derived cells having a higher CD90 expression level or amount than the lower limit of the 80% confidence interval, 85% confidence interval, 90% confidence interval, 95% confidence interval, 98% confidence interval or 99% confidence interval in the distribution with the higher mean expression level or amount, when the expression level or amount in human urine-derived cells prepared by a similar method from the same human subject is fitted as a superposition of two normal distributions. (c) Human urine-derived cells, whose CD90 expression level or amount is in the top 10%, top 15%, top 20%, top 25%, top 30%, or top 50% of the CD90 expression level or amount in human urine-derived cells prepared by a similar method from the same human subject. (d) Human urine-derived cells, wherein the expression level or amount of CD90 is 3 times or more, 5 times or more, or 10 times or more the average expression level or amount of CD90 in cells obtained by knocking down or knocking out CD90 in the human urine-derived cells. (e) Human urine-derived cells having a higher expression level or amount of CD90 than the upper limit of the 80% confidence interval, 85% confidence interval, 90% confidence interval, 95% confidence interval, 98% confidence interval, or 99% confidence interval when the distribution of the expression level or amount of CD90 in an established cultured cell line with low CD90 expression (e.g., HT-29 cells, Caco-2 cells, HepG2 cells, or MDA-MB-231 cells) is fitted as a normal distribution.

[0033] In a preferred embodiment, the CD90-positive human urine-derived cells may be human urine-derived cells that meet at least one of the following (a') to (e'): In the following (a') to (e'), "CD90 staining intensity" refers to the fluorescence intensity at a wavelength corresponding to the fluorescence wavelength of a fluorescent dye when contacted with an anti-CD90 antibody labeled with the fluorescent dye, and the fluorescence intensity can be measured, for example, by a flow cytometer. (a') Human urine-derived cells with CD90 staining intensity 5-fold or greater than the mean CD90 staining intensity in human urine-derived cells prepared by a similar method from the same human subject. (b') Human urine-derived cells with CD90 staining intensity higher than the lower limit of the 95% confidence interval of the distribution with the higher CD90 staining intensity, when the CD90 staining intensities of human urine-derived cells prepared by the same method from the same human subject were fitted as a superposition of two normal distributions. (c') Human urine-derived cells with CD90 staining intensity that is in the upper 25% of CD90 staining intensity in human urine-derived cells prepared by a similar method from the same human subject. (d') Human urine-derived cells, having a CD90 staining intensity that is 5-fold or more higher than the average CD90 staining intensity in cells obtained by knocking out CD90 in the human urine-derived cells. (e') Human urine-derived cells with CD90 staining intensity higher than the upper limit of the 95% confidence interval when the distribution of CD90 staining intensity in HT-29 cells is fitted as a normal distribution.

[0034] The percentage of CD90-positive human urine-derived cells in cells contained in the cell population of CD90-positive human urine-derived cells according to the first aspect of the present disclosure may be, for example, 50% or more, 60% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more, or 100%. In a preferred embodiment, the percentage of CD90-positive human urine-derived cells in cells contained in the cell population of CD90-positive human urine-derived cells may be 90% or more. The cell population according to the first aspect of the present disclosure may be a cell population obtained by sorting human urine-derived cells so that the percentage of CD90-positive human urine-derived cells is as described above.

[0035] The cell population of CD90-positive human urine-derived cells according to the first aspect of the present disclosure has high muscle differentiation potential. In other words, the cell population of CD90-positive human urine-derived cells according to the first aspect of the present disclosure is easily differentiated (easily induced) into myotubes. Thus, in one embodiment, the cell population of CD90-positive human urine-derived cells according to the first aspect of the present disclosure may be a cell population for use in inducing myotubes.

[0036] Myotubes (also called "myotube cells") are immature muscle cells. Myotubes are so-called multinucleated cells having two or more nuclei, and myofibrils are formed around myotubes. Therefore, whether a certain cell is a myotube or whether a certain cell has been induced to be a myotube can be evaluated based on the characteristic morphology of such a myotube. Specifically, for example, whether a certain cell is a myotube can be evaluated based on the cell having two or more nuclei after staining the cell nucleus with a nuclear staining reagent such as DAPI or Hoechst. Also, for example, whether a certain cell is a myotube can be evaluated based on the observation of a fluorescent image derived from myofibrils when the cell is stained with an antibody against a protein highly expressed in myofibrils, such as a labeled anti-myosin (MYHC) antibody, or the observation of strong fluorescence when the cell is stained with an antibody against a muscle-specific transcription factor, such as a labeled anti-myogenin antibody. For example, whether a certain cell is a myotube can be evaluated based on a high ratio of cell nuclei contained within myotubes among the cell nuclei within the field of view of plate-cultured cells (fusion index).

[0037] According to the cell population according to the first aspect of the present disclosure, a cell population of human urine-derived cells having high muscle differentiation potential can be provided. Thus, according to the cell population according to the first aspect of the present disclosure, a cell population of myotubes can be efficiently produced. If a cell population of myotubes can be efficiently produced, it is useful, for example, for high-throughput screening of therapeutic and preventive agents targeting myotubes. Furthermore, according to the cell population according to the first aspect of the present disclosure, human urine-derived cells can be induced to become myotubes without producing induced pluripotent stem cells from human urine-derived cells. Furthermore, for example, according to the cell population according to the first aspect of the present disclosure, human urine-derived cells can be induced to become myotubes without exposure to an epigenetics control compound.

[0038] Furthermore, according to the cell population of the first aspect of the present disclosure, human urine-derived cells can be induced to become myotubes while suppressing the influence of variations in muscle differentiation potential of urine collected from a specific human subject from one collection to another, thereby obtaining a myotube cell population. In this way, for example, a myotube cell population can be obtained that is induced from human urine-derived cells prepared from urine collected at multiple time points from a single patient with a muscle disease or muscular dystrophy, and these myotube cell populations are useful for monitoring the progression of a disease or for studying a disease by obtaining a genetic profile and phenotype at each time point.

[0039] Furthermore, according to the cell population of the first aspect of the present disclosure, human urine-derived cells can be induced to become myotubes while suppressing the influence of variations in muscle differentiation potential among human subjects, and a myotube cell population can be obtained. In this way, for example, a myotube cell population can be obtained from human urine-derived cells of an individual patient with a muscle disease or muscular dystrophy, and these myotube cell populations are useful, for example, for testing therapeutic and preventive agents that are useful in individual patients without the actual administration (so-called companion diagnostics), or for disease research by obtaining genetic profiles and phenotypes in myotubes derived from individual patients.

[0040] A specific example of the application can be the testing and companion diagnosis of exon skipping drugs in human urine-derived cells from muscular dystrophy patients, and more specifically, the testing and companion diagnosis of exon skipping drugs in human urine-derived cells from Duchenne muscular dystrophy patients. Duchenne muscular dystrophy (DMD) is a chronic progressive muscle disease in which dystrophin is lost from the muscle cell membrane due to a mutation in the responsible gene DMD, resulting in degeneration of muscle fibers. Exon skipping therapy is known as a treatment method for Duchenne muscular dystrophy. Exon skipping therapy is a treatment that targets mRNA precursors and adjusts splicing with an antisense nucleic acid drug to make frameshift mutations in-frame, thereby restoring expression of truncated dystrophin protein. In 2020, the present inventors achieved practical application of viltolarsen (exon 53 skipping drug), an antisense nucleic acid drug for Duchenne muscular dystrophy caused by exon 53 deletion mutation. On the other hand, in the process of expanding the number of targeted deletion mutations, it is becoming clear that the exon skip induction effect in the patient body to which the drug is administered cannot necessarily be predicted only from the patient's genomic DNA mutation pattern. Therefore, according to the cell population according to the first aspect of the present disclosure, a myotube cell population can be obtained from human urine-derived cells of individual Duchenne muscular dystrophy patients, so that an individualized profile of a drug expected to be effective in each patient can be obtained (so-called companion diagnosis), and it is expected that it will be possible to assist in providing individualized medical care for each patient (personalized medicine, precision medicine).

[0041] A second aspect of the present disclosure is a method for producing myotubes derived from human urine-derived cells, comprising a step of separating CD90-positive human urine-derived cells (separation step). As the CD90-positive human urine-derived cells in this aspect, CD90-positive human urine-derived cells in the cell population of CD90-positive human urine-derived cells according to the first aspect of the present disclosure can be used. That is, in the separation step, the CD90-positive human urine-derived cells described in the first aspect of the present disclosure are separated.

[0042] As the human urine-derived cells from which the cells are separated in the separation step (i.e., the human urine-derived cells before separation), those described in the first aspect can be used, for example, those prepared by the preparation method described in the first aspect can be used. In one embodiment of the second aspect, the separation step may be a step of separating CD90-positive human urine-derived cells from human urine-derived cells prepared from urine of a patient with a muscle disease or muscular dystrophy.

[0043] The method of cell sorting in the sorting step is not particularly limited as long as it is a method capable of sorting CD90-positive human urine-derived cells. For example, a method (so-called flow cytometry method) can be mentioned in which human urine-derived cells are contacted with an anti-CD90 antibody labeled with a fluorescent dye, and then cells with high fluorescence intensity are sorted as CD90-positive human urine-derived cells using a flow cytometer. Another method can also be mentioned in which human urine-derived cells are contacted with an anti-CD90 antibody labeled with an affinity tag such as biotin or His tag, and then contacted with beads, columns, culture dishes, etc. whose surfaces are modified with a molecule that recognizes the affinity tag, such as streptavidin or anti-His tag antibody, to trap CD90-positive human urine-derived cells on the surface of the beads, columns, culture dishes, etc., and then biotin or His tag or a derivative thereof is added to release the CD90-positive human urine-derived cells from the surface, and the cells are sorted (so-called affinity tag purification method or affinity chromatography method). In a preferred embodiment, the method for sorting the cells in the sorting step may be a flow cytometry method.

[0044] In these separation methods, the conditions for contacting the human urine-derived cells with the labeled anti-CD90 antibody are not particularly limited, and can be performed under conditions commonly used by those skilled in the art. For example, a commercially available solution of the labeled anti-CD90 antibody is diluted about 11 to 10,001 times with phosphate buffer solution (PBS) containing 2% fetal bovine serum to prepare a solution, and the human urine-derived cells are exposed to the solution, and then incubated in the dark at a temperature condition of about ice-cooling to room temperature for about 1 minute to 6 hours.

[0045] In one embodiment, the production method according to the second aspect of the present disclosure comprises, after the sorting step, a step of inducing myotubes from the CD90-positive human urine-derived cells sorted in the sorting step (induction step).

[0046] The method for inducing human urine-derived cells into myotubes in the induction step is not particularly limited as long as the method is capable of inducing myotubes.

[0047] In a preferred embodiment, the induction step includes forcibly expressing MYOD1 in CD90-positive human urine-derived cells. The method of forced expression is not particularly limited as long as it is a method commonly used by those skilled in the art, and may be, for example, introducing the MYOD1 gene, a pre-mRNA (pre-mRNA) which is a transcription product of the MYOD1 gene, or an mRNA (messenger RNA) obtained by processing the pre-mRNA into the cells.

[0048] In a more preferred embodiment, the induction step includes introducing the MYOD1 gene into CD90-positive human urine-derived cells. MYOD1 (myoblast determination protein 1) is a muscle-specific transcription factor that is listed as one of the muscle control factors and belongs to the MYOD family. It is known that the introduction of the MYOD1 gene into fibroblasts and the like can induce differentiation into myotubes. It is also known that the introduction of the MYOD1 gene into human urine-derived cells can induce differentiation of the human urine-derived cells into myotubes, albeit with low efficiency and insufficiency (Non-Patent Document 2).

[0049] The MYOD1 gene is well known in the art, and although not particularly limited, the human MYOD1 gene is preferably used. The sequence of the MYOD1 gene, for example, the sequence of the human MYOD1 gene, is registered in GenBank (National Center for Biotechnology Information, NCBI) under the accession number NM_002478.4.

[0050] The introduction of the MYOD1 gene into human urine-derived cells can be performed by a method known in the art, for example, by the method described in Non-Patent Document 2 or Patent Document 1. For example, the MYOD1 gene is cloned and incorporated into an appropriate expression vector (for example, a retroviral vector). In addition to the MYOD1 gene, a promoter, an enhancer, a selection marker gene, and the like may be inserted into the expression vector. The promoter can be appropriately selected, and it is preferable to use an inducible promoter. When MYOD1 is expressed and muscle differentiation begins in human urine-derived cells, the proliferation ability is significantly reduced. However, by using an inducible promoter, it is possible to control cell proliferation by suppressing muscle differentiation and induction of differentiation into myotubes. Specifically, the MYOD1 gene is introduced into human urine-derived cells using, for example, the TRE3GS promoter as an inducible promoter, and then the human urine-derived cells into which the MYOD1 gene has been introduced are grown, and then doxycycline (Dox) is added to the medium to activate the promoter, and the MYOD1 gene is expressed to induce differentiation into myotubes. Although the selection marker gene is not essential, it is preferable to incorporate it into the expression vector since it allows easy selection of human urine-derived cells into which the MYOD1 gene has been introduced. Examples of the selection marker gene include puromycin resistance gene, neomycin resistance gene, zeocin resistance gene, hygromycin resistance gene, and blasticidin resistance gene. Such an expression vector is introduced into human urine-derived cells using a method known in the art, for example, using a commercially available transfection reagent. Selection of the introduced cells is also known in the art, and for example, when a puromycin resistance gene is inserted into an expression vector, cells that are resistant to puromycin are selected.

[0051] The conditions for introducing the MYOD1 gene in the induction step are not particularly limited, and examples of the medium include a proliferation medium (a mixture of equal amounts of REGM Bullet Kit (Lonza; CC-3190) and high-glucose DMEM, supplemented with 15% tetracycline-free fetal bovine serum, 0.5% Glutamax (Thermo Fisher Scientific; 35050-061), 0.5% non-essential amino acids (Thermo Fisher Scientific; 11140-050), 2.5 ng / mL fibroblast growth factor-basic (bFGF) (Sigma, St Louis, USA; F0291), PDGF-AB (Peprotech, Rocky Hill, NJ; 100-00AB), EGF (Peprotech; AF-100-15), 1% penicillin / streptomycin, and 0.5 μg / mL amphotericin B), a differentiation medium (high-glucose-containing DMEM with GlutaMAX-I (Thermo Fisher Scientific; 10569-010), 5% horse serum, ITS Liquid Media Supplement (Sigma; I3146), 1 μg / mL doxycycline). For example, the culture temperature may be a temperature suitable for culturing human urine-derived cells, for example, 30 to 40°C, preferably about 37°C, and the pH is maintained, for example, near neutral. The culture period may be 1 hour to 4 weeks, preferably about 1 day to 2 weeks.

[0052] In one embodiment, the induction step may include exposing CD90-positive human urine-derived cells to an epigenetics-controlling compound. Introduction of the MYOD1 gene and exposure to an epigenetics-controlling compound efficiently induces muscle differentiation in human urine-derived cells (Patent Document 1). Thus, in one embodiment, when the induction step includes exposure to an epigenetics-controlling compound, human urine-derived cells can be more efficiently induced to become myotubes. In this case, introduction of the MYOD1 gene and exposure to the epigenetics-controlling compound may be performed simultaneously, or introduction of the MYOD1 gene may be performed after exposure to the epigenetics-controlling compound.

[0053] Epigenetic control refers to the control of gene expression by chromosomal changes without changes in DNA base sequence. Such chromosomal changes include chemical modifications such as methylation of DNA in nucleosomes, acetylation and methylation of histones, and such chemical modifications of DNA and histones control gene expression. Therefore, as an inhibitor of an enzyme involved in such epigenetic control, the epigenetic control compound according to the present disclosure may be an inhibitor of histone methyltransferase (HMT), histone demethylase (histone demethylase), histone deacetylase (HDAC), SIRT2 (Sirtuin 2), or PARP (poly ADP ribose polymerase).

[0054] As specific epigenetics controlling compounds and conditions for exposing human urine-derived cells to the epigenetics controlling compounds, those described in Patent Document 1 can be used.

[0055] In one embodiment, the induction step may not include exposing CD90-positive human urine-derived cells to an epigenetics-regulating compound. The present inventors have found that CD90-positive human urine-derived cells have high muscle differentiation potential. Thus, according to the production method according to one embodiment of the second aspect of the present disclosure, even if the induction step does not include exposing CD90-positive human urine-derived cells to an epigenetics-regulating compound, human urine-derived cells can be efficiently induced to become myotubes and myotubes can be produced. More specifically, in one embodiment, the induction step may not include exposing CD90-positive human urine-derived cells to an inhibitor listed as an epigenetics-regulating compound in Patent Document 1.

[0056] In one embodiment, the induction step may not include the production of induced pluripotent stem cells (iPS cells) from CD90-positive human urine-derived cells. That is, in one embodiment, the induction step may be a step of directly inducing CD90-positive human urine-derived cells into myotubes (so-called a step of performing direct reprogramming). When producing another somatic cell from a differentiated somatic cell, it is also possible to first produce iPS cells from the differentiated somatic cell and then differentiate the iPS cells into another somatic cell, and examples of producing iPS cells from human urine-derived cells have also been reported. However, since CD90-positive human urine-derived cells have enhanced muscle differentiation potential, according to the method according to the second aspect of the present disclosure, CD90-positive human urine-derived cells can be induced into myotubes by a simple method that does not include the production of iPS cells from CD90-positive human urine-derived cells, that is, does not go through iPS cells once.

[0057] In one embodiment, the induction step may include producing induced pluripotent stem cells (iPS cells) from CD90-positive human urine-derived cells. In one embodiment, the induction step may include producing induced pluripotent stem cells (iPS cells) from CD90-positive human urine-derived cells and creating myotubes from the induced pluripotent stem cells (so-called indirect reprogramming step).

[0058] The production of myotubes by the production method according to the second aspect of the present disclosure can be evaluated, for example, by staining cell nuclei with a nuclear staining reagent such as DAPI or Hoechst and finding that the cells have two or more nuclei. For example, the production of myotubes can be evaluated based on the observation of a fluorescent image derived from myofibrils when the cells are stained with an antibody against a protein highly expressed in myofibrils, such as a labeled anti-myosin (MYHC) antibody, or the observation of strong fluorescence when the cells are stained with an antibody against a muscle-specific transcription factor, such as a labeled anti-myogenin antibody. For example, the production of myotubes can be evaluated based on the high ratio of cell nuclei contained in myotubes (fusion index) among cell nuclei in the field of view in plate-cultured cells (for example, the fusion index is 2 times or more, 3 times or more, or 5 times or more than before muscle differentiation induction, or the fusion index is 0.5 or more, 0.6 or more, or 0.7 or more).

[0059] The production method according to the second aspect of the present disclosure can be used, for example, for the same applications as the cell population of CD90-positive human urine-derived cells according to the first aspect of the present disclosure.

[0060] A third aspect of the present disclosure is a myotube cell population induced from a cell population of CD90-positive human urine-derived cells. In other words, the third aspect of the present disclosure is a myotube cell population produced from a cell population of human urine-derived cells by the production method according to the second aspect. The cell population according to one embodiment of the third aspect of the present disclosure may be a myotube cell population induced from a cell population of CD90-positive human urine-derived cells derived from a patient with a muscle disease or muscular dystrophy.

[0061] The fourth aspect of the present disclosure is a method for isolating human urine-derived cells with high muscle differentiation potential, which includes a step of isolating CD90-positive human urine-derived cells. The human urine-derived cells with high muscle differentiation potential according to the fourth aspect of the present disclosure refer to cells that are evaluated as having high muscle differentiation potential in a cell population of CD90-positive human urine-derived cells according to the first aspect of the present disclosure, and may be cells that have a high ratio of cell nuclei contained in myotubes (fusion index) among cell nuclei in a field of view when muscle differentiation is induced under plate culture (for example, a fusion index of 2 times or more, 3 times or more, or 5 times or more than that before muscle differentiation induction, or a fusion index of 0.5 or more, 0.6 or more, or 0.7 or more). The step of isolating CD90-positive human urine-derived cells can be performed by the same method as the separation step in the production method according to the second aspect of the present disclosure, that is, for example, by flow cytometry or affinity purification or affinity chromatography. In one embodiment of the fourth aspect of the present disclosure, the human urine-derived cells may be human urine-derived cells derived from a patient with a muscle disease or a patient with muscular dystrophy.

[0062] A fifth aspect of the present disclosure is a kit including an anti-CD90 antibody or an antigen-binding fragment thereof, and a package insert that describes separating CD90-positive human urine-derived cells as human urine-derived cells with high muscle differentiation potential. The kit according to the fifth aspect of the present disclosure can be used to perform the method of separating human urine-derived cells with high muscle differentiation potential according to the fourth aspect of the present disclosure. That is, in one embodiment, the fifth aspect of the present disclosure may be a kit for use in the method of separating human urine-derived cells with high muscle differentiation potential according to the fourth aspect of the present disclosure. In addition, in one embodiment, the fifth aspect of the present disclosure may be a kit for use in the method of separating human urine-derived cells with high muscle differentiation potential according to the second aspect of the present disclosure.

[0063] In the present disclosure, an antigen-binding fragment of an anti-CD90 antibody refers to a portion of the antibody that recognizes and binds to CD90, and includes the variable domain of the antibody or at least the antigen-binding region. Examples of antigen-binding fragments include Fab fragments, Fab' fragments, F(ab') 2Examples of such fragments include Fv fragments and Fv fragments, which may be prepared by methods commonly used by those skilled in the art.

[0064] The anti-CD90 antibody or its antigen-binding fragment contained in the kit according to one embodiment of the fifth aspect of the present disclosure may be labeled, and is preferably labeled. When the anti-CD90 antibody or its antigen-binding fragment is labeled, CD90-positive human urine-derived cells can be separated based on the label used for labeling. The label in the anti-CD90 antibody or its antigen-binding fragment may be, for example, labeling with a fluorescent dye or labeling with an affinity tag. Examples of affinity tags include biotin and His tags. When the anti-CD90 antibody or its antigen-binding fragment is labeled with a fluorescent dye, CD90-positive human urine-derived cells can be separated, for example, by the flow cytometry method described in the second aspect of the present disclosure. When the anti-CD90 antibody or its antigen-binding fragment is labeled with an affinity tag, CD90-positive human urine-derived cells can be separated, for example, by the affinity purification method or affinity chromatography method described in the second aspect of the present disclosure.

[0065] The sixth aspect of the present disclosure is a method for evaluating the muscle differentiation potential of human urine-derived cells, comprising a step of evaluating the expression of CD90 in the human urine-derived cells. The expression of CD90 in human urine-derived cells can be evaluated by the same method as the evaluation method for evaluating cells contained in a cell population of CD90-positive human urine-derived cells according to the first aspect of the present disclosure as having high muscle differentiation potential. For example, when muscle differentiation is induced under plate culture, the expression of myofibrils and / or the cell nuclei becoming multinucleated may be used as an indicator. In this case, the method for evaluating the expression of CD90 in human urine-derived cells is not particularly limited as long as it is a method capable of evaluating the expression of cell surface proteins, and can be freely selected by those skilled in the art. For example, the method may be performed by contacting human urine-derived cells with a fluorescently labeled anti-CD90 antibody, or contacting human urine-derived cells with an anti-CD90 antibody as a primary antibody and a fluorescently labeled secondary antibody, and then evaluating the fluorescence of the human urine-derived cells with a fluorescent microscope, a flow cytometer, a microwell plate reader, a fluorometer, or the like, and the fluorescence intensity may be used as an indicator. In one embodiment of the sixth aspect of the present disclosure, the human urine-derived cells may be human urine-derived cells from a muscle disease or muscular dystrophy patient.

[0066] The seventh aspect of the present disclosure is a kit including an anti-CD90 antibody or an antigen-binding fragment thereof, and a package insert stating that expression of CD90 in human urine-derived cells is evaluated, and human urine-derived cells evaluated to have a high CD90 expression level are evaluated as human urine-derived cells with high muscle differentiation potential. The kit according to the seventh aspect of the present disclosure can be used to carry out the method of evaluating the muscle differentiation potential of human urine-derived cells according to the sixth aspect of the present disclosure. That is, in one embodiment, the seventh aspect of the present disclosure may be a kit for use in the method of evaluating the muscle differentiation potential of human urine-derived cells according to the sixth aspect of the present disclosure. In addition, in one embodiment, the seventh aspect of the present disclosure may be a kit for use in the method of separating human urine-derived cells with high muscle differentiation potential according to the second aspect of the present disclosure. As the anti-CD90 antibody or antigen-binding fragment thereof according to the seventh aspect of the present disclosure, those described in the fifth aspect of the present disclosure can be used. EXAMPLES

[0067] The present disclosure will be described more specifically below using examples, but the present disclosure is not limited to the following examples.

[0068] All experiments shown in the Examples were performed with the approval of the National Center of Neurology and Psychiatry (NCNP). In addition, the provision of spot urine samples was performed after written consent was obtained from the individual or their legal representative.

[0069] [Experimental method 1: Preparation of human urine-derived cells] Human urine-derived cells used in the examples of the present disclosure were prepared according to the following protocol. Urine was collected by having a human subject urinate into a sterile plastic bottle (Corning Incorporated, NY, USA; 430281). Primary cells contained in the collected urine were cultured within a few hours after collection using a method slightly modified from that described in Non-Patent Document 4.

[0070] Specifically, the collected urine was dispensed into multiple 50 mL conical tubes and centrifuged at 400 × g for 10 minutes at room temperature, and the supernatant was removed. The pellet was then suspended in PBS and collected in one conical tube, and 10 mL of washing solution (Ca 2+ and Mg 2+The cells were then centrifuged at 200 × g for 10 minutes at room temperature, and the supernatant was removed. The pellet was suspended in 1.5 mL of initial medium (equal parts of high glucose DMEM (GE Healthcare, Logan, UT; SH30022.FS) and Ham's F-12 Nutrient Mix (Thermo Fisher Scientific; 11765-054) supplemented with REGM SingleQuots (Lonza, Basel, Switzerland; CC-4127), 10% tetracycline-free fetal bovine serum (Clontech; 631106), 1% penicillin / streptomycin, and 0.5 μg / mL amphotericin B) and incubated on a gelatin-coated 6-well plate (IWAKI, Shizuoka, Japan; 4810-020) in 5% CO. 2The cells were cultured in an incubator at 37°C. The initial medium was added at a rate of 1.5 mL every day, and on the fourth day of culture, it was replaced with 2 mL of growth medium (a mixture of equal volumes of REGM Bullet Kit (Lonza; CC-3190) and high-glucose DMEM, supplemented with 15% tetracycline-free fetal bovine serum, 0.5% Glutamax (Thermo Fisher Scientific; 35050-061), 0.5% non-essential amino acids (Thermo Fisher Scientific; 11140-050), 2.5 ng / mL fibroblast growth factor-basic (bFGF) (Sigma, St Louis, USA; F0291), PDGF-AB (Peprotech, Rocky Hill, NJ; 100-00AB), EGF (Peprotech; AF-100-15), 1% penicillin / streptomycin, and 0.5 μg / mL amphotericin B. The amphotericin B / gentamicin in the REGM Bullet Kit was omitted). The human urine-derived cells formed colonies within a few days to about two weeks after the start of culture. The human urine-derived cells obtained by the above-mentioned procedures were used in the examples of the present disclosure.

[0071] [Experimental method 2: Introduction of the MYOD1 gene into human urine-derived cells] In the examples of the present disclosure, the MYOD1 gene was introduced into human urine-derived cells according to the following protocol. The retroviral vector described in Patent Document 1 (hereinafter referred to as "MYOD1 viral vector"; a schematic diagram is shown in Figure 1) was used to introduce the MYOD1 gene into human urine-derived cells. In the MYOD1 viral vector shown in Figure 1, the MYOD1 gene is under the control of the TRE3GS promoter, so expression of the MYOD1 gene can be induced by doxycycline (Dox). In addition, the MYOD1 viral vector contains a puromycin resistance gene as a selection marker.

[0072] The MYOD1 viral vector was prepared according to the method described in Patent Document 1. Human urine-derived cells were seeded on a culture dish or plate (3,000 to 5,000 cells / cm).2 ), MYOD1 was introduced into human urine-derived cells by infecting them with the MYOD1 viral vector using polybrene after overnight culture in growth medium. Two days after infection, puromycin was added to the medium and cultured for 7 days to select human urine-derived cells transfected with the MYOD1 gene.

[0073] [Example 1: Identification of cell surface markers specific to human urine-derived cells with high muscle differentiation potential by single-cell RNA sequencing analysis of human urine-derived cells] According to Experimental Method 1, urine was collected from one healthy subject multiple times, and human urine-derived cells were established for each collection, to obtain a cell population of human urine-derived cells for each collection. The MYOD1 gene was introduced into each cell population according to Experimental Method 2 to induce muscle differentiation. Each obtained cell population was immunostained with mouse anti-MYOGENIN antibody (1:200, SantaCruz; sc-12732) and anti-MYHC antibody (1:200; eBioscience, #14-6503-82), and the expression levels of MYOGENIN and MYHC and the Fusion Index were evaluated. The degree of muscle differentiation was evaluated based on the expression levels of MYOGENIN and MYHC and the Fusion Index, and cell populations with high muscle differentiation potential (Good UDCs) and cell populations with low muscle differentiation potential (Bad UDCs) were selected.

[0074] Next, single-cell RNAseq analysis was performed on Good UDCs and Bad UDCs before muscle differentiation, and on the muscle-differentiated cell population 30 days after the introduction of the MYOD1 gene into Good UDCs and Bad UDCs. Specifically, a cDNA library was prepared for each cell from the obtained cell population using a 10xGenomics kit, and sequencing was performed on an Illumina HiSeq 4000. Using the Seurat package (v.4.0.1), the gene expression profile of each cell was analyzed based on the unique molecular identifier (UMI) added during library preparation, and cells with similar profiles were classified as the same cluster. The clustering results were dimensionally compressed using the UMAP method (Uniform Manifold Approximation and Projection method) and displayed in two dimensions. As a result, Good UDCs and Bad UDCs before muscle differentiation, and the muscle-differentiated cell population 30 days after the introduction of the MYOD1 gene into Good UDCs and Bad UDCs were depicted as different clusters in terms of gene expression profile.

[0075] The results are shown in Figure 2. As shown in Figure 2, when comparing the clusters of Good UDCs and Bad UDCs, clusters expressing CD90 were found only in the Good UDCs. This suggests that CD90 may be a marker specific to human urine-derived cells with high muscle differentiation potential.

[0076] [Example 2: Separation of a cell population of CD90-positive human urine-derived cells] Using the expression of CD90, a candidate marker specific to human urine-derived cells with high muscle differentiation potential found in Example 1, as an index, human urine-derived cells were separated by flow cytometry. Human urine-derived cells obtained from urine collected from one healthy subject according to Experimental Method 1 were exposed to a trypsin-EDTA solution to be detached from the culture dish, and then suspended in a growth medium. The obtained cell suspension was centrifuged at 350×g for 5 minutes, the supernatant was removed, and the cells were resuspended in phosphate buffered saline (PBS) containing 2% fetal bovine serum. A solution of fluorescently labeled anti-CD90 antibody (FITC anti-human CD90 (Thy1) Antibody, BioLegend; 328107) was added thereto at a 21-fold dilution, and the cells were incubated in the dark under ice cooling for 20 minutes. The solution containing the human urine-derived cells labeled with fluorescently labeled anti-CD90 antibody was centrifuged at 350×g for 5 minutes, the supernatant was removed, and the cells were resuspended in phosphate buffered saline (PBS) containing 2% fetal bovine serum, and then filtered through a 70 μm filter (Falcon). The resulting labeled human urine-derived cells were sorted using a flow cytometer (FACSAria Fusion (BD Biosciences, San Jose, CA)) with the fluorescence intensity of the dye (FITC) modified on the anti-CD90 antibody as an index. The data obtained by the flow cytometer was analyzed using FlowJo (v10, BD Biosciences).

[0077] The results of the analysis using FlowJo are shown in Figure 3. In the figure, the horizontal axis indicates the fluorescence intensity of the dye (FITC) modified on the anti-CD90 antibody, and the vertical axis indicates the number of cells. According to Figure 3, the human urine-derived cells contained a cell population that was well labeled with the anti-CD90 antibody, i.e., a CD90-positive cell population (Positive), and a cell population that was not well labeled with the anti-CD90 antibody, i.e., a CD90-negative cell population (Negative). Therefore, as shown in Figure 3, a total of two cell populations, the CD90-positive cell population and the CD90-negative cell population, were separated and used in the following Examples.

[0078] [Example 3: Induction of muscle differentiation in CD90 positive or negative cell populations] The MYOD1 gene was introduced into the CD90-positive cell population and the CD90-negative cell population sorted in Example 2 according to Experimental Method 2. Each cell population was incubated at 37° C., 5% CO 2 The cells were expanded in a growth medium under the conditions of (a) to the number of cells required for differentiation induction, and muscle differentiation was induced. The degree of muscle differentiation induction in the cultured cell population was evaluated by immunofluorescence staining and Western blotting.

[0079] Immunofluorescence staining was performed according to the following protocol. Cells in the cell population were washed with PBS, fixed with 4% paraformaldehyde, and incubated with 0.1% Triton-X for 10 min at room temperature. Anti-myosin heavy chain antibody (1:50, R&D, Minneapolis, USA; MAB4470) was used as the primary antibody, and Alexa Fluor 546 goat anti-mouse IgG(H+L) (1:300, Invitrogen; A11003) was used as the secondary antibody. DAPI was used for nuclear staining. Images were captured using a fluorescence microscope (BZ-9000 or BZ-X800, KEYENCE, Osaka, Japan), and analyzed using a BZ-X Analyzer (KEYENCE).

[0080] Western blotting was performed according to the following protocol: Cells in the cell population were lysed using RIPA buffer (Thermo Fisher Scientific; 89900) containing protease inhibitors (Roche, Indianapolis, IN, USA; 04693116001), and the supernatant was collected by centrifugation at 14,000 × g for 15 min at 4 °C. Total protein concentration was measured using a BCA protein assay kit (Thermo Fisher Scientific; 23227), and the protein was denatured with NuPAGE® LDS Sample Buffer (Thermo Fisher Scientific; NP0007) and then subjected to SDS-PAGE on NuPAGE® Novex Tris-Acetate Gel 3-8% (Invitrogen; EA03785BOX) and transferred to a PVDF membrane (Millipore, Billerica, MA, USA; IPVH304F0). Antibody reactions were performed using anti-MYOD1 antibody (1:200; Santa Cruz, sc-32758), mouse anti-MYHC antibody (1:200, eBioscience, #14-6503-82), and mouse anti-α-tubulin antibody (1:1000, Sigma; T6199) as primary antibodies, and anti-mouse horseradish peroxidase-conjugated secondary antibodies (1:50000, Cell Signaling Technology) as secondary antibodies. After antibody reactions, the bands of interest were detected using ECL Prime Western Blotting Detection Reagent (GE Healthcare, UK; RPN2232).

[0081] Fluorescence images in immunofluorescence staining are shown in FIG. 4. Fusion index (the ratio of cell nuclei contained in myotubes, shown as MYHC, to cell nuclei contained in the image, shown as DAPI) in FIG. 4 is shown in FIG. 5. The results in FIG. 5 are shown as mean ± standard deviation (Mean ± SD), and 0.0021 indicates that the P value in Student's T-test was 0.0021. The results of band detection in Western blotting are shown in FIG. 6(A). The band intensities of MYOD1 and MYHC normalized by the band intensity of α-tubulin (α-Tub) in FIG. 6(A) are shown in FIG. 6(B) and FIG. 6(C), respectively. The results in FIG. 6(B) and (C) are shown as mean ± standard deviation (Mean ± SD), and 0.2742 and <0.0001 indicate that the P values ​​in Student's T-test were less than 0.2742 and 0.0001, respectively.

[0082] First, the results of Figures 4 and 5 show that many of the human urine-derived cells in the CD90-positive human urine-derived cell population (CD90-positive) were induced to become myotubes, whereas the CD90-negative human urine-derived cell population (CD90-negative) showed almost no induction to become myotubes, showing a significant difference in the rate of muscle differentiation between the two groups. This was also confirmed by the expression levels of MYHC shown in Figures 6(A) and 6(C).

[0083] On the other hand, according to the results in Figures 6(A)-(C), there was no significant difference in the expression level of MYOD1 between CD90 positive / negative mice, and only a significant difference was observed in the expression level of MYHC. Therefore, it was revealed that the difference in the proportion of cells induced to differentiate into muscle cells between the two groups was due to the difference in the muscle differentiation induction ability between the two groups, not due to the difference in the efficiency of MYOD1 gene introduction or expression of MYOD1.

[0084] These results indicate that the CD90-positive human urine-derived cell population has high muscle differentiation potential, that is, CD90 is a cell surface marker specific to human urine-derived cells with high muscle differentiation potential.

[0085] As a comparative example, when cell sorting similar to that in Example 2 was performed using a fluorescently labeled anti-CD24 antibody (B&D, 560991) instead of a labeled anti-CD90 antibody, and differentiation was induced by the same method as in Example 3, no clear difference in muscle differentiation potential was observed between the CD24-positive and CD24-negative populations. Furthermore, when cell sorting similar to that in Example 2 was performed using a fluorescently labeled anti-CD133 antibody (abcam, ab253259) instead of a labeled anti-CD90 antibody, and differentiation was induced by the same method as in Example 3, no clear difference in muscle differentiation potential was observed between the CD133-positive and CD133-negative populations. Therefore, based on these results, it has become clear that, among various mesenchymal stem cell markers, CD90 in particular is a cell surface marker specific to human urine-derived cells with high muscle differentiation potential.

[0086] [Example 4: Evaluation of dystrophin restoration levels by exon skipping drugs using myotube cell populations induced from a cell population of CD90-positive human urine-derived cells from DMD patients] We investigated whether myotube cell populations induced from a cell population of CD90-positive human urine-derived cells reflect abnormalities in protein expression in muscle cells of muscle disease patients, and whether it is possible to evaluate drugs by exon skipping, which is a drug that targets such abnormalities in protein expression. Specifically, we investigated whether the abnormal expression of dystrophin protein in DMD patients can be improved by exposing myotubes produced from the urine of DMD patients to exon skipping drugs.

[0087] Urine was collected from one Duchenne muscular dystrophy patient (DMD patient) with exon 45 deletion in the DMD gene, and human urine-derived cells were established and muscle differentiation was induced according to the procedures described in Methods 1 and 2. In the CD90(-) and CD90(+) groups, CD90-negative and CD90-positive cell populations were isolated by the same method as in Example 2. Seven days after muscle differentiation induction, the exon skipping drug group was changed to a differentiation medium (containing high glucose-containing DMEM with GlutaMAX-I (Thermo Fisher Scientific; 10569-010), 5% horse serum, ITS Liquid Media Supplement (Sigma; I3146), and 1 μg / mL doxycycline) containing the exon skipping drug AON and 6 μM endoporter (Gene Tools, Philomath, OR, USA). In the exon skipping drug-free group, the differentiation medium was changed instead. After 3 days, the medium was changed to differentiation medium alone, and the cells were collected 14 days after muscle differentiation induction. Details of the AON used were as described in Wilton, SD et al. Mol Ther 15, 1288-1296 (2007).

[0088] The expression of dystrophin protein in the collected cells was evaluated by Western blotting, which was performed in the same manner as in Example 3, except that mouse anti-dystrophin antibody (1:20, Leica) and mouse anti-α-tubulin antibody (1:1000, Sigma; T6199) were used as primary antibodies.

[0089] The results of band detection by Western blotting are shown in Figure 7(A). The band intensity of dystrophin 1 (DYS1) normalized by the band intensity of α-tubulin (α-Tub) in Figure 7(A) is shown in Figure 7(B). In Figures 7(A) and (B), Mix indicates the results of cell populations in which muscle differentiation was induced from human urine-derived cells without sorting using CD90 as an indicator, while CD90(-) and CD90(+) indicate the results of cell populations in which muscle differentiation was induced from CD90-negative and CD90-positive human urine-derived cell populations, respectively. The results in Figure 7(B) are shown as mean ± standard deviation (Mean ± SD), and PMO 10 μM indicates the group to which differentiation medium containing 10 μM of exon skipping drug was added.

[0090] As shown in Figure 7 (A) and (B), in the myotube cell population obtained by inducing muscle differentiation from a cell population of CD90-positive human urine-derived cells, the increase in the expression level of dystrophin 1 protein by exon skipping drugs was confirmed as a band, and the degree of increase was greater than when cells were not selected using CD90 as an indicator. This demonstrated that the myotube cell population obtained by inducing muscle differentiation from a cell population of CD90-positive human urine-derived cells can be used to sensitively evaluate the level of dystrophin restoration by exon skipping drugs.

Claims

1. A cell population of CD90-positive human urine-derived cells.

2. The cell population of claim 1 for use in inducing myotubes.

3. The cell population according to claim 1 or 2, wherein the CD90-positive human urine-derived cells are derived from a patient with a muscle disease or muscular dystrophy.

4. A method for producing myotubes derived from human urine-derived cells, comprising a step of isolating CD90-positive human urine-derived cells.

5. The method according to claim 4, wherein the step of isolating CD90-positive human urine-derived cells is a step of isolating CD90-positive human urine-derived cells from human urine-derived cells prepared from urine of a patient with a muscle disease or a patient with muscular dystrophy.

6. The method according to claim 4 or 5, further comprising, after the separating step, a step of inducing myotubes from the separated CD90-positive human urine-derived cells.

7. The method of claim 6, wherein the step of inducing myotubes comprises introducing a MYOD1 gene into the CD90-positive human urine-derived cells.

8. The method of claim 6, wherein the step of inducing myotubes does not include generating induced pluripotent stem cells from the CD90-positive human urine-derived cells.

9. A myotube cell population derived from a CD90 positive human urine-derived cell population.

10. The myotube cell population of claim 9 , wherein the CD90-positive human urine-derived cells are derived from a patient with a muscle disease or muscular dystrophy.

11. A method for isolating human urine-derived cells having high muscle differentiation potential, the method comprising a step of isolating CD90-positive human urine-derived cells.

12. The method of claim 11 , wherein the human urine-derived cells are human urine-derived cells from a patient with a muscle disease or muscular dystrophy.

13. An anti-CD90 antibody or an antigen-binding fragment thereof, and A package insert stating that the expression of CD90 in human urine-derived cells is evaluated, and human urine-derived cells evaluated to have a high level of CD90 expression are isolated as human urine-derived cells with high muscle differentiation potential. Kit including:

14. 1. A method for assessing the muscle differentiation potential of human urine-derived cells, comprising assessing expression of CD90 in the human urine-derived cells.

15. The method of claim 14, wherein the human urine-derived cells are human urine-derived cells from a patient with a muscle disease or muscular dystrophy.

16. An anti-CD90 antibody or an antigen-binding fragment thereof, and A package insert stating that the expression of CD90 in human urine-derived cells is evaluated, and human urine-derived cells evaluated to have a high level of CD90 expression are evaluated as human urine-derived cells with high muscle differentiation potential. Kit including:

Images