WO2022102742A1

WO2022102742A1 - Cell surface marker for high efficiency purification of skeletal muscle lineage cells and skeletal muscle stem cells, and use thereof

Info

Publication number: WO2022102742A1
Application number: PCT/JP2021/041709
Authority: WO
Inventors: 英俊櫻井; ハルチウンミナスナルバンディアン; 明明趙
Original assignee: 国立大学法人京都大学
Priority date: 2020-11-16
Filing date: 2021-11-12
Publication date: 2022-05-19
Also published as: JPWO2022102742A1

Abstract

The present invention provides: a method which is for improving purity of skeletal muscle lineage cells and which is characterized by recovering cells positive for a cell surface marker CDH13 in a cell population; a method which is for producing skeletal muscle lineage cells and which comprises a step for inducing differentiation of pluripotent stem cells into skeletal muscle cells, and a step for recovering cells positive for the cell surface marker CDH13 from a cell population in the process of inducing differentiation; a method which is for improving purity of skeletal muscle lineage cells and which is characterized by recovering cells positive for a cell surface marker FGFR4 in a cell population; and a method which is for producing skeletal muscle stem cells and which comprises a step for inducing differentiation of pluripotent stem cells into skeletal muscle cells, and a step for recovering, from a cell population in the process of inducing differentiation, cells positive for the cell surface marker FGFR4, CDH13-positive cells, or FGFR4-positive and CDH13-positive cells.

Description

Cell surface markers for high-efficiency purification of skeletal muscle lineage cells and skeletal muscle stem cells and their utilization

The present invention relates to a cell surface marker for high-efficiency purification of skeletal muscle lineage cells and skeletal muscle stem cells, and a method for improving the purity of skeletal muscle lineage cells and skeletal muscle stem cells and a method for producing the same.

Skeletal muscle has special flexibility regarding endogenous regeneration ability. Through the regeneration of skeletal muscle, a population of locally resident stem cells called satellite cells plays a fundamental role in tissue maintenance and repair. Satellite cells are dormant under the basement membrane in the homeostatic state, are activated in response to muscle damage, and begin to proliferate. Activated satellite cells migrate to the site of injury, differentiate into myoblasts and myotubes, and later fuse with other fibers to repair the injury. On the other hand, another subset of satellite cells return to dormancy due to self-renewal of the satellite cell pool. Because of these properties, transplantation of healthy satellite cells has been studied as a promising strategy for regenerative medicine in skeletal muscle disease.

However, only a few satellite cells can be isolated, and when they proliferate in vitro, they lose their ability to regenerate and age, making cell therapy less attractive. Human iPS cells, on the other hand, have greater proliferative and differentiating potential and are therefore considered to be a very attractive cell source for generating various cell lines for cell therapy.

Recently, several groups have developed an in vitro differentiation system for obtaining skeletal muscle progenitor cells derived from human iPS cells. Many of these methods artificially overexpress myogenic factors to induce differentiation, but their potential for tumorigenesis reduces clinical safety. The transgene-free method attempts to repeat the developmental stages to induce differentiation of skeletal muscle stem cells from human iPS cells, and is more attractive for clinical application because of the low health-related risks.

Skeletal muscle stem cells produced by a method that does not use a transgene show muscle-forming ability, but they are a cell population with extremely high heterogeneity, and not all cells show muscle-forming ability. Therefore, in order to use skeletal muscle stem cells that have been induced to differentiate from human iPS cells by a method that does not use a gene for cell therapy, skeletal muscle stem cells from a heterogeneous cell population that has been induced to differentiate from human iPS cells to skeletal muscle stem cells. Biomarkers are needed to efficiently select and obtain a highly pure skeletal muscle stem cell population.

Recently, the present inventors have established a method for inducing skeletal muscle stem cells capable of muscle regeneration from human iPS cells (Non-Patent Document 1), focusing on the MYF5 gene known as a satellite cell marker, and MYF5 positive. When cells and MYF5-negative cells were transplanted into the shin muscles of immunodeficient mice, it was suggested that Myf5-positive cells had a higher degree of muscle fiber formation after transplantation and had higher muscle regeneration ability.

The present invention provides a biomarker for obtaining a highly pure skeletal muscle lineage cell population and a skeletal muscle stem cell population, and provides a method for improving the purity of skeletal muscle lineage cells and skeletal muscle stem cells using the biomarker and a method for producing the same. The challenge is to provide.

The present invention includes the following inventions in order to solve the above problems.
[1] A method for improving the purity of skeletal muscle lineage cells in a cell population, which comprises recovering cell surface marker CDH13-positive cells in the cell population.
[2] The method according to the above [1], wherein the cell population is a cell population in the process of inducing differentiation from pluripotent stem cells to skeletal muscle cells.
[3] The method according to the above [2], wherein the pluripotent stem cell is an iPS cell.
[4] A method for improving the purity of skeletal muscle stem cells in a cell population in which the purity of skeletal muscle lineage cells obtained by any of the methods [1] to [3] has been improved. A method characterized by recovering cells positive for the cell surface marker FGFR4.
[5] A method for improving the purity of skeletal muscle stem cells in a cell population, which comprises recovering cell surface marker FGFR4-positive cells in the cell population.
[6] The method according to [5] above, wherein the cell population is a cell population in the process of inducing differentiation of pluripotent stem cells into skeletal muscle cells.
[7] The method according to [6] above, wherein the pluripotent stem cell is an iPS cell.
[8] A method for producing skeletal muscle lineage cells, which are (1) a step of initiating differentiation induction from pluripotent stem cells to skeletal muscle cells, and (2) a cell surface marker CDH13 from a cell population in the process of inducing differentiation. A production method comprising the step of recovering positive cells.
[9] A method for producing skeletal muscle stem cells, which is (1) a step of initiating differentiation induction from pluripotent stem cells to skeletal muscle cells, and (2) positive for the cell surface marker FGFR4 from a cell population in the process of inducing differentiation. A production method comprising the steps of recovering cells, CDH13-positive cells or FGFR4-positive and CDH13-positive cells.
[10] The production method according to the above [9], wherein the step (1) is a step of inducing differentiation using a differentiation medium without introducing an exogenous skeletal muscle inducing gene into pluripotent stem cells.
[11] The production method according to the above [9] or [10], wherein the step (1) is a step of inducing differentiation of skeletal muscle lineage cells from pluripotent stem cells so as to mimic the developmental process of a fetus.
[12] The production method according to any one of [9] to [11] above, wherein the pluripotent stem cell is an iPS cell.
[13] The production method according to any one of [9] to [12] above, wherein the skeletal muscle stem cells are for living-donor transplantation.
[14] A cell surface marker for purifying or detecting skeletal muscle lineage cells consisting of CDH13.
[15] A cell surface marker for purifying or detecting skeletal muscle stem cells consisting of FGFR4.

INDUSTRIAL APPLICABILITY According to the present invention, it is possible to provide a biomarker for acquiring a highly pure skeletal muscle lineage cell population and a skeletal muscle stem cell population. By using the biomarker of the present invention, it is possible to provide a method for improving the purity of skeletal muscle lineage cells and skeletal muscle stem cells and a method for producing them.

It is a figure which shows the result of having analyzed the expression pattern of CDH13 and FGFR4 using the database "Tabula Muris" of mouse single cell RNA sequencing. It is a figure which shows the result of having analyzed the expression pattern of CDH13 and FGFR4 using the gene expression database in the human fetal origin tissue, (A) is the result of CDH13, (B) is the result of FGFR4. It is a figure which shows the result of having analyzed the expression pattern of CDH13 and FGFR4 using the gene expression database in the human adult tissue, (A) is the result of CDH13, (B) is the result of FGFR4. Differentiation into skeletal muscle stem cells was induced using human iPS cells (201B7 and S01), cells were collected on day 84, and Myf5-positive cells and Myf5-negative cells were sorted by FACS, and CDH13 expression level and FGFR4 expression level were obtained. It is a figure which shows the result of having measured, (A) is the result of CDH13, (B) is the result of FGFR4. It is a figure showing the result of inducing differentiation into skeletal muscle stem cells using human iPS cells (CKI), collecting the cells on the 84th day, and confirming the presence of CDH13-positive cells and FGFR4-positive cells by FACS. A) is the result of CDH13, and (B) is the result of FGFR4. Human iPS cells (201B7) were used to induce differentiation into skeletal muscle stem cells, and on day 84, the cells were collected and stained with anti-FGFR4 antibody, and FGFR4 positive cells and FGFR4 negative cells were sorted by FACS, respectively. It is a figure which shows the result of staining with the CDH13 antibody and analyzing the CDH13 positive rate by FACS. Human iPS cells (201B7) were used to induce differentiation into skeletal muscle stem cells, cells were harvested on day 84, CDH13-positive cells and FGFR4-positive cells were sorted by FACS, and CDH13-positive cells, FGFR4-positive cells and non-CDH13-positive cells were sorted. It is a figure which shows the result of having measured the expression level of MYF5, PAX7, MYOD1 in a sorting cell. Differentiation into skeletal muscle stem cells was induced using human iPS cells (CKI), cells were collected on day 84, CDH13-positive cells and FGFR4-positive cells were sorted by FACS, and CDH13-positive cells, FGFR4-positive cells and non-CDH13-positive cells were sorted. It is a figure which shows the result of having measured the expression level of MYF5, PAX7, MYOD1 in a sorting cell. Human iPS cells (DMD) were used to induce differentiation into skeletal muscle stem cells, cells were harvested on day 84, CDH13-positive and FGFR4-positive cells were sorted by FACS, and CDH13-positive, FGFR4-positive and non-CDH13-positive cells were sorted. It is a figure which shows the result of having measured the expression level of MYF5, PAX7, MYOD1 in a sorting cell. Human iPS cells (201B7, CKI and DMD) were used to induce differentiation into skeletal muscle stem cells, and on day 84, the cells were collected and stained with anti-CDH13 antibody, and CDH13-positive cells and CDH13-negative cells were isolated by FACS, respectively. It is a figure which shows the result of having examined the expression of myosin heavy chain (MHC) by sorting and further inducing the differentiation of CDH13 positive cell and CDH13 negative cell into skeletal muscle cells, and (A) is the result of immunostaining MHC (A). B) is a diagram showing the results of calculating the differentiation index (MHC positive cell rate) by image analysis. Human iPS cells (201B7, CKI and DMD) were used to induce differentiation into skeletal muscle stem cells, and on day 84, the cells were collected and stained with anti-FGFR4 antibody, and FGFR4 positive cells and FGFR4 negative cells were separated by FACS, respectively. It is a figure which shows the result of having examined the expression of MHC by sorting and further inducing the differentiation of FGFR4 positive cell and FGFR4 negative cell into skeletal muscle cells, and (A) is the result of immunostaining MHC, and (B) is the differentiation index. It is a figure which shows the result of having calculated (MHC positive cell rate) by image analysis. Human iPS cells (CKI, DMD) were used to induce differentiation into skeletal muscle stem cells, cells were recovered on day 84, and CDH13-positive cells, CDH13-negative cells, FGFR4-positive cells, and FGFR4-negative cells were used using FACS. 4 weeks after transplanting each cell into NOG-mdx mice, the transplantation site was collected, stained with anti-histopeclin antibody, anti-laminin α2 antibody, anti-human nuclear antibody and DAPI, and observed under a confocal microscope. It is a figure which shows the result. It is a figure which shows the result of having measured the number of engraftment cells in the transplantation site collected from the above-mentioned NOG-mdx mouse 4 weeks after transplantation. The above-mentioned transplantation site collected from NOG-mdx mice 4 weeks after transplantation was stained with anti-PAX7 antibody, anti-MYOD antibody, anti-h-LAMIN A / C antibody and DAPI, and observed with a confocal microscope. be. The above-mentioned transplantation site collected from NOG-mdx mice 4 weeks after transplantation was stained with anti-PAX7 antibody, anti-LAMININα2 antibody, anti-h-LAMIN A / C antibody and DAPI, and the results shown by observation with a confocal microscope are shown. be. Human iPS cells (DMD, CKI) were used to induce differentiation into skeletal muscle stem cells, cells were collected on day 84, and CD56-positive cell rate, CD82-positive cell rate, and NGFR-positive cell rate were used using FACS. It is a figure which shows the result of having confirmed the ERBB3-positive cell rate, the CDH13-positive cell rate and the FGFR4-positive cell rate, (A) is the result of DMD, and (B) is the result of CKI. The figure showing the results of measuring the expression levels of MYF5, PAX7, and MYOD1 in the DMD and CKI-derived CD56-positive cells, CD82-positive cells, NGFR-positive cells, ERBB3-positive cells, CDH13-positive cells, and FGFR4-positive cells sorted above. Yes, (A) is the result of DMD, and (B) is the result of CKI. The results of examining the expression of MHC by inducing the differentiation of DMD and CKI-derived CD56-positive cells, CD82-positive cells, NGFR-positive cells, ERBB3-positive cells, CDH13-positive cells and FGFR4-positive cells sorted above into skeletal muscle cells. (A) is the result of immunostaining MHC, and (B) is the result of calculating the differentiation index (MHC positive cell rate) by image analysis.

[Method for improving the purity of skeletal muscle lineage cells]
The present invention provides a method for improving the purity of skeletal muscle lineage cells. Skeletal muscle lineage cells are a broad concept that includes cells capable of skeletal muscle differentiation and cells that have finally differentiated into skeletal muscle, and are skeletal muscle stem cells, myoblasts, myotube cells, mature myotube cells, and skeletal muscle cells ( Includes all muscle fibers). The present inventors have found that skeletal muscle lineage cells express CDH13 on the cell surface, and have found that a high-purity skeletal muscle lineage cell population can be obtained by using the cell surface marker CDH13. Therefore, the method for improving the purity of skeletal muscle lineage cells of the present invention is characterized by using the cell surface marker CDH13. CDH13 is also referred to as cadherin-13, T-cadherin, and H-cadherin.

The method for improving the purity of skeletal muscle lineage cells of the present invention may be any method including a step of recovering cell surface marker CDH13-positive cells from a cell population. The cell population is not particularly limited as long as it is a cell population that may contain skeletal muscle lineage cells. For example, a cell suspension prepared from a surgical specimen containing skeletal muscle can be mentioned. The cell population may be a human cell population or a cell population of a non-human organism. Organisms other than humans are not particularly limited and may be, for example, mammals. Examples of mammals include monkeys, chimpanzees, dogs, cats, cows, horses, pigs, rabbits, mice, rats and the like.

In one embodiment, the cell population may be a cell population in the process of inducing differentiation from pluripotent stem cells to skeletal muscle cells. As a cell population in the process of inducing differentiation from pluripotent stem cells to skeletal muscle cells, a known method for inducing differentiation from pluripotent stem cells to skeletal muscle cells is appropriately selected to initiate differentiation induction, and before the final differentiation is reached. Cell population can be used. As a known method for inducing differentiation of pluripotent stem cells into skeletal muscle cells, for example, the method described in Non-Patent Document 1 (Zhao et al., Stem Cell Reports, Vol. 15 1-15 July 14, 2020.). , International release WO2016 / 108288 The method described in A1, Hicks et al. (Nat Cell Biol. 2018 Jan; 20 (1): 46-57.), Etc. can be mentioned.

The method for recovering CDH13-positive cells from the cell population is not particularly limited, and examples thereof include a method using a cell sorter and a method using affinity chromatography. When a cell sorter is used, for example, a cell suspension of a cell population is prepared, an anti-CDH13 antibody is added to the cell suspension, the anti-CDH13 antibody is bound to the CDH13-positive cells, and the cells are subjected to the cell sorter to obtain the CDH13-positive cells. Can be sorted and collected. When affinity chromatography is used, for example, a cell suspension of a cell population is prepared, and the CDH13-positive cells are bound to the anti-CDH13 antibody by passing the cell suspension through a column filled with a carrier to which the anti-CDH13 antibody is bound. After that, CDH13-positive cells can be recovered by dissociating the binding with the anti-CDH13 antibody. The method for improving the purity of skeletal muscle lineage cells of the present invention reduces the proportion of CDH13-negative cells in the original cell population and increases the proportion of CDH13-positive cells, so that the resulting cell population improves the purity of skeletal muscle lineage cells. It is a cell population.

The pluripotent stem cell that can be used in the present invention is not particularly limited as long as it is a stem cell that has pluripotency capable of differentiating into all cells existing in a living body and also has proliferative ability. For example, embryonic stem (ES) cells, embryonic stem (ntES) cells derived from cloned embryos obtained by nuclear transplantation, sperm stem (GS) cells, embryonic reproductive (EG) cells, artificial pluripotent stem (iPS) cells. , Pluripotent cells (Muse cells) derived from cultured fibroblasts and bone marrow stem cells. Preferred pluripotent stem cells are ES cells, ntES cells, and iPS cells.

(A) Embryonic stem cells ES cells are pluripotent and self-replicating stem cells established from the inner cell mass of early embryos (eg, blastocysts) of mammals such as humans and mice.

ES cells are embryo-derived stem cells derived from the inner cell mass of the scutellum vesicle, which is the embryo after the morula at the 8-cell stage of the fertilized egg, and have the ability to differentiate into all the cells that make up the adult, so-called polymorphism. It has the ability and the ability to proliferate by self-replication. ES cells were discovered in mice in 1981 (M.J. Evans and M.H. Kaufman (1981), Nature 292: 154-156), and then ES cell lines were established in primates such as humans and monkeys (J.A. Thomson et). al. (1998), Science 282: 1145-1147; J.A. Thomson et al. (1995), Proc. Natl. Acad. Sci. USA, 92: 7844-7848; J.A. Thomson et al. (1996), Biol. ., 55: 254-259; J.A. Thomson and V.S. Marshall (1998), Curr. Top. Dev. Biol., 38: 133-165).

For the establishment of ES cells, a method known in this field is used. For example, it can be established by removing the inner cell mass from the blastocyst of a fertilized egg of a target animal and culturing the inner cell mass on a fibroblast feeder. For cell maintenance by subculture, use a culture medium supplemented with substances such as leukemia inhibitory factor (LIF) and basic fibroblast growth factor (bFGF). It can be carried out. For methods of establishing and maintaining ES cells in humans and monkeys, see, for example, USP 5,843,780; Thomson JA, et al. (1995), Proc Natl. Acad. Sci. U S A. 92: 7844-7848; Thomson JA, et al. (1998), Science. 282: 1145-1147; H. Suemori et al. (2006), Biochem. Biophys. Res. Communi., 345: 926-932; M. Ueno et al. (2006), Proc. Natl. Acad. Sci. USA, 103: 9554-9559; H. Suemori et al. (2001), Dev. Dyn., 222: 273-279; H. Kawasaki et al. (2002), Proc. Natl . Acad. Sci. USA, 99: 1580-1585; Klimanskaya I, et al. (2006), Nature. 444: 481-485, etc.

As a culture method for producing ES cells, a method known in the art is used. As the culture medium, use DMEM / F-12 culture medium supplemented with, for example, 0.1 mM 2-mercaptoethanol, 0.1 mM non-essential amino acid, 2 mM L-glutamic acid, 20% KSR (KnockOut Serum Replacement, Invitrogen) and 4 ng / ml bFGF. However, human ES cells can be maintained in a moist atmosphere of 37 ° C, 2% CO ₂ / 98% air (O. Fumitaka et al. (2008), Nat. Biotechnol., 26: 215-224). ES cells may be passaged every 3-4 days, using 0.25% trypsin and 0.1 mg / ml collagenase IV in PBS containing, for example, 1 mM CaCl ₂ and 20% KSR. Can be done.

ES cells can generally be selected by the Real-Time PCR method using the expression of gene markers such as alkaline phosphatase, Oct-3 / 4, and Nanog as an index. In particular, in the selection of human ES cells, the expression of gene markers such as OCT-3 / 4, NANOG, and ECAD can be used as an index (E. Croon et al. (2008), Nat. Biotechnol., 26: 443. -452).

As mouse ES cells, various mouse ES cell lines established by inGenious, RIKEN (RIKEN), etc. can be used. As human ES cells, various human ES cell lines established by the National Institutes of Health (NIH), RIKEN, Kyoto University, and Cellartis can be used. For example, as ES cell lines, NIH CHB-1 to CHB-12 strains, RUES1 strains, RUES2 strains, HUES1 to HUES28 strains, etc., WisCell Research Institute WA01 (H1) strains, WA09 (H9) strains, RIKEN KhES- One strain, KhES-2 strain, KhES-3 strain, KhES-4 strain, KhES-5 strain, SSES1 strain, SSES2 strain, SSES3 strain and the like can be used. The KhES-1, KhES-2, KhES-3 and KthES11 strains are available from the Institute for Frontier Life and Medical Sciences, Kyoto University (Kyoto, Japan).

(B) Sperm stem cells Sperm stem cells are pluripotent stem cells derived from the testis and are the origin cells for spermatogenesis. Similar to ES cells, these cells can be induced to differentiate into various lineages of cells, and have properties such as the ability to produce chimeric mice when transplanted into mouse blastocysts (M. Kanatsu-Shinohara et al. (M. Kanatsu-Shinohara et al.). 2003) Biol. Reprod., 69: 612-616; K. Shinohara et al. (2004), Cell, 119: 1001-1012). It is self-renewable in a culture medium containing a glial cell line-derived neurotrophic factor (GDNF), and sperm by repeating passage under the same culture conditions as ES cells. Stem cells can be obtained (Masanori Takebayashi et al. (2008), Experimental Medicine, Vol. 26, No. 5 (Special Edition), pp. 41-46, Yodosha (Tokyo, Japan)).

(C) Embryonic germ cells Embryonic germ cells are cells with pluripotency similar to ES cells, which are established from primordial germ cells in the embryonic period, such as LIF, bFGF, and stem cell factor. It can be established by culturing primordial germ cells in the presence of the substance of (Y. Matsui et al. (1992), Cell, 70: 841-847; JL Resnick et al. (1992), Nature, 359: 550. -551).

(D) Induced pluripotent stem cells Induced pluripotent stem (iPS) cells are similar to ES cells, which can be produced by introducing specific reprogramming factors into somatic cells in the form of DNA or protein. K. Takahashi and S. Yamanaka (2006) Cell, 126: 663-676; K. Takahashi et al (2007), Cell, 131: 861-872; J. Yu et al. (2007), Science, 318: 1917-1920; Nakagawa, M. et al., Nat. Biotechnol. 26: 101-106 (2008); International release WO 2007/069666). Reprogramming factors are genes that are specifically expressed in ES cells, their gene products or non-cording RNAs, or genes that play an important role in maintaining undifferentiated ES cells, their gene products or non-cording RNAs, or It may be composed of low molecular weight compounds. Genes included in the reprogramming factors include, for example, Oct3 / 4, Sox2, Sox1, Sox3, Sox15, Sox17, Klf4, Klf2, c-Myc, N-Myc, L-Myc, Nanog, Lin28, Fbx15, ERas, ECAT15. -2, Tcl1, beta-catenin, Lin28b, Sall1, Sall4, Esrrb, Nr5a2, Tbx3, Glis1, etc. are exemplified, and these initialization factors may be used alone or in combination. The combinations of initialization factors include WO2007 / 069666, WO2008 / 118820, WO2009 / 007852, WO2009 / 032194, WO2009 / 058413, WO2009 / 057831, WO2009 / 075119, WO2009 / 079007, WO2009 / 091659, WO2009 / 101084, WO2009 / 101407, WO2009 / 102983, WO2009 / 114949, WO2009 / 117439, WO2009 / 126250, WO2009 / 126251, WO2009 / 126655, WO2009 / 157593, WO2010 / 009015, WO2010 / 033906, WO2010 / 033920, WO2010 / 042800, WO2010 / 050626, WO 2010/056831, WO2010 / 068955, WO2010 / 098419, WO2010/102267, WO 2010/111409, WO 2010/111422, WO2010 / 115050, WO2010 / 124290, WO2010 / 147395, WO2010 / 147612, Huangfu D, et al. ( 2008), Nat. Biotechnol., 26: 795-797, Shi Y, et al. (2008), Cell Stem Cell, 2: 525-528, Eminli S, et al. (2008), Stem Cells. 26: 2467 -2474, Huangfu D, et al. (2008), Nat Biotechnol. 26: 1269-1275, Shi Y, et al. (2008), Cell Stem Cell, 3, 568-574, Zhao Y, et al. (2008) ), Cell Stem Cell, 3: 475-479, Marson A, (2008), Cell Stem Cell, 3, 132-135, Feng B, et al. (2009), Nat Cell Biol. 11: 197-203, RL Judson et al., (2009), Nat. Biotech., 27: 459-461, Lyssiotis CA, et al. (2009), Proc Natl Acad Sci U S A. 106: 8912-8 917, Kim JB, et al. (2009), Nature. 461: 649-643, Ichida JK, et al. (2009), Cell Stem Cell. 5: 491-503, Heng JC, et al. (2010), Cell Stem Cell. 6: 167-74, Han J, et al. (2010), Nature. 463: 1096-100, Mali P, et al. (2010), Stem Cells. 28: 713-720, Maekawa M, The combinations described in et al. (2011), Nature. 474: 225-9. Are exemplified.

The reprogramming factors include histone deacetylase (HDAC) inhibitors [eg, small molecule inhibitors such as valproic acid (VPA), tricostatin A, sodium butyrate, MC 1293, M344, siRNA against HDAC and shRNA (eg). , HDAC1 siRNA Smartpool (Millipore), HuSH 29mer shRNA Constructs against HDAC1 (OriGene), etc.), MEK inhibitors (eg PD184352, PD98059, U0126, SL327 and PD0325901), Glycogen synthase kinase- 3 Against small molecule inhibitors such as inhibitors (eg Bio and CHIR99021), DNA methyltransferase inhibitors (eg 5-azacytidine), histone methyltransferase inhibitors (eg BIX-01294), Suv39hl, Suv39h2, SetDBl and G9a Nucleic acid expression inhibitors such as siRNA and shRNA), L-channel calciumagonist (eg Bayk8644), butyric acid, TGFβ inhibitors or ALK5 inhibitors (eg LY364947, SB431542, 616453 and A-83-01), p53 inhibition Agents (eg siRNA and shRNA for p53), ARID3A inhibitors (eg siRNA and shRNA for ARID3A), miRNAs such as miR-291-3p, miR-294, miR-295 and mir-302, Wnt Signaling (eg soluble Wnt3a) ), Nucleic acid peptide Y, prostaglandins (eg, prostaglandins E2 and prostaglandins J2), hTERT, SV40LT, UTF1, IRX6, GLISl, PITX2, DMRTBl, etc. In this specification, the factors used for the purpose of improving the establishment efficiency are not particularly distinguished from the reprogramming factors.

In the case of protein morphology, the reprogramming factor may be introduced into somatic cells by techniques such as lipofection, fusion with cell membrane permeable peptides (eg, HIV-derived TAT and polyarginine), and microinjection.

On the other hand, in the case of DNA morphology, it can be introduced into somatic cells by methods such as viruses, plasmids, vectors such as artificial chromosomes, lipofection, liposomes, and microinjection. Viral vectors include retro viral vectors and lentiviral vectors (above, Cell, 126, pp.663-676, 2006; Cell, 131, pp.861-872, 2007; Science, 318, pp.1917-1920, 2007. ), Adenovirus vector (Science, 322, 945-949, 2008), adeno-associated virus vector, Sendai virus vector (WO2010 / 008054) and the like. Further, the artificial chromosome vector includes, for example, a human artificial chromosome (HAC), a yeast artificial chromosome (YAC), a bacterial artificial chromosome (BAC, PAC) and the like. As the plasmid, a plasmid for mammalian cells can be used (Science, 322: 949-953, 2008). The vector can contain regulatory sequences such as promoters, enhancers, ribosome binding sequences, terminators, polyadenylation sites, etc. so that the nuclear reprogramming substance can be expressed, and further, if necessary, a drug resistance gene (drug resistance gene). For example, canamycin resistance gene, ampicillin resistance gene, puromycin resistance gene, etc.), thymidin kinase gene, diphtheriatoxin gene and other selection marker sequences, green fluorescent protein (GFP), β-glucuronidase (GUS), FLAG and other reporter gene sequences, etc. Can include. In addition, the above vector has LoxP sequences before and after introduction into somatic cells in order to excise both the gene encoding the reprogramming factor or the promoter and the gene encoding the reprogramming factor that binds to the promoter. You may.

In the case of RNA morphology, it may be introduced into somatic cells by a method such as lipofection or microinjection, and in order to suppress degradation, RNA incorporating 5-methylcytidine and pseudouridine (TriLink Biotechnologies) is used. It may be (Warren L, (2010) Cell Stem Cell. 7: 618-630).

Cultures for iPS cell induction include, for example, DMEM, DMEM / F12 or DME cultures containing 10-15% FBS (these cultures also include LIF, penicillin / streptomycin, puromycin, L-glutamine). , Non-essential amino acids, β-mercaptoethanol, etc. can be appropriately contained.) Or mouse ES cell culture medium (TX-WES culture medium, Thrombo X), primate ES cell culture medium (primates). Commercially available cultures such as ES / iPS cell culture medium, Reprocell), serum-free pluripotent stem cell maintenance medium (for example, mTeSR (Stemcell Technology), Essential 8 (Life Technologies), StemFit AK03 (AJINOMOTO)) Illustrated.

As an example of the culture method, for example, in the presence of 5% CO ₂ at 37 ° C., the somatic cells are brought into contact with the reprogramming factor on a DMEM or DMEM / F12 culture medium containing 10% FBS and cultured for about 4 to 7 days. Then, the cells were re-seeded on feeder cells (for example, mitomycin C-treated STO cells, SNL cells, etc.), and about 10 days after the contact between the somatic cells and the reprogramming factor, the culture medium for bFGF-containing primate ES cell culture was used. It can be cultured and give rise to iPS-like colonies about 30-about 45 days or more after the contact.

Alternatively, in the presence of 5% CO ₂ at 37 ° C., DMEM culture medium containing 10% FBS (for example, LIF, penicillin / streptomycin, etc.) on feeder cells (eg, mitomycin C-treated STO cells, SNL cells, etc.). It can appropriately contain puromycin, L-glutamine, non-essential amino acids, β-mercaptoethanol, etc.), and ES-like colonies can be generated after about 25 to about 30 days or more. Desirably, instead of feeder cells, the reprogrammed somatic cells themselves are used (Takahashi K, et al. (2009), PLoS One. 4: e8067 or WO2010 / 137746), or extracellular matrix (eg, Laminin-). 5 (WO2009 / 123349) and Matrigel (BD)) are exemplified.

In addition to this, a method of culturing using a medium containing no serum is also exemplified (Sun N, et al. (2009), Proc Natl Acad Sci U S A. 106: 15720-15725). Furthermore, in order to improve the establishment efficiency, iPS cells may be established under hypoxic conditions (oxygen concentration of 0.1% or more and 15% or less) (Yoshida Y, et al. (2009), Cell Stem Cell. 5: 237. -241 or WO2010 / 013845).

During the above culture, the fresh culture solution and the culture solution are exchanged once a day from the second day after the start of the culture. The number of somatic cells used for nuclear reprogramming is not limited, but ranges from about 5 × 10 ³ to about 5 × 10 ⁶ cells per 100 cm ² culture dish.

The iPS cells can be selected according to the shape of the formed colonies. On the other hand, when a drug resistance gene expressed in conjunction with a gene expressed when somatic cells are reprogrammed (for example, Oct3 / 4, Nanog) is introduced as a marker gene, a culture medium containing the corresponding drug (selection). Established iPS cells can be selected by culturing in a culture medium). In addition, iPS cells are selected by observing with a fluorescence microscope when the marker gene is a fluorescent protein gene, by adding a luminescent substrate when it is a luminescent enzyme gene, and by adding a chromogenic substrate when it is a chromogenic enzyme gene. can do.

As used herein, the term "somatic cell" refers to any animal cell (eg, mammalian cell including human) except germline cells such as eggs, egg mother cells, ES cells or totipotent cells. .. Somatic cells include, but are not limited to, fetal (pup) somatic cells, neonatal (pup) somatic cells, and mature healthy or diseased somatic cells, as well as primary cultured cells. , Passed cells, and established cells are all included. Specifically, the somatic cells include, for example, (1) tissue stem cells (somatic stem cells) such as nerve stem cells, hematopoietic stem cells, mesenchymal stem cells, and dental pulp stem cells, (2) tissue precursor cells, (3) lymphocytes, and epithelium. Cells, endothelial cells, muscle cells, fibroblasts (skin cells, etc.), hair cells, hepatocytes, gastric mucosal cells, intestinal cells, splenocytes, pancreatic cells (pancreatic exocrine cells, etc.), brain cells, lung cells, renal cells And differentiated cells such as fat cells are exemplified.

In addition, when iPS cells and / or cells induced to differentiate from them are used as materials for transplant cells, the HLA genotypes of the transplanted individuals are the same or substantially the same from the viewpoint that rejection does not occur. It is desirable to use cells. Here, "substantially the same HLA type" means that the HLA genotypes match to the extent that the transplanted cells can engraft when the cells are transplanted. For example, the main HLA (HLA-). It is a somatic cell having an HLA type that matches the 3 loci of A, HLA-B and HLA-DR or the 4 loci with HLA-C).

As the induced pluripotent stem cell line, various iPS cell lines established by NIH, RIKEN, Kyoto University, etc. may be used. For example, in the case of human iPS cell lines, RIKEN's HiPS-RIKEN-1A strain, HiPS-RIKEN-2A strain, HiPS-RIKEN-12A strain, Nips-B2 strain, etc., Kyoto University's Ff-WJ-18 strain, Ff -I01s01 shares, Ff-I01s02 shares, Ff-I01s04 shares, Ff-I01s06 shares, Ff-I14s03 shares, Ff-I14s04 shares, QHJI01s01 shares, QHJI01s04 shares, QHJI14s03 shares, QHJI14s04 shares, QHJI14s04 shares, AK5 shares, TkDN-S Examples include 253G1 shares, 201B7 shares, 409B2 shares, 454E2 shares, 606A1 shares, 610B1 shares, 648A1 shares, 1231A3 shares, 1390D4 shares and 1390C1 shares. Alternatively, clinical grade cell lines provided by Kyoto University, Cellular Dynamics International, etc., and research and clinical cell lines prepared using these cell lines may be used.

(E) ES cells (ntES cells) derived from cloned embryos obtained by nuclear transplantation
ntES cells are ES cells derived from cloned embryos produced by nuclear transplantation technology and have almost the same characteristics as ES cells derived from fertilized eggs (T. Wakayama et al. (2001), Science, 292: 740-743; S. Wakayama et al. (2005), Biol. Reprod., 72: 932-936; J. Byrne et al. (2007), Nature, 450: 497-502). That is, ES cells established from the inner cell mass of a blastocyst derived from a cloned embryo obtained by replacing the nucleus of an unfertilized egg with the nucleus of a somatic cell are ntES (nuclear transfer ES) cells. For the production of ntES cells, a combination of nuclear transplantation technology (JB Cibelli et al. (1998), Nature Biotechnol., 16: 642-646) and ES cell production technology (above) is used (Seika Wakayama). Et al. (2008), Experimental Medicine, Vol. 26, No. 5 (Special Edition), pp. 47-52). In nuclear transplantation, somatic cell nuclei can be injected into unenucleated unfertilized eggs of mammals and cultured for several hours to initialize them.

(F) Multilineage-differentiating Stress Enduring cells (Muse cells)
Muse cells are pluripotent stem cells produced by the method described in WO2011 / 007900, specifically fibroblasts or bone marrow stromal cells treated with long-term trypsin, preferably 8 hours or 16 hours. Pluripotent cells obtained by suspension culture after treatment and positive for SSEA-3 and CD105.

[Method for improving the purity of skeletal muscle stem cells]
The present invention provides a method for improving the purity of skeletal muscle stem cells. Skeletal muscle stem cells mean cells that have the ability to selectively differentiate into skeletal muscle cells and have both self-renewal ability and proliferative differentiation ability. Skeletal muscle stem cells in vivo are called satellite cells and usually exist between the basement membrane and plasma membrane of muscle fibers. As used herein, skeletal muscle stem cells include satellite cells.

Pax7, Hesr1, Hesr3, Myf5, CalcR, NGFR, ERBB3, CD56, CD82, etc. are known as marker genes for identifying skeletal muscle stem cells. It was found that FGFR4 (fibroblast growth factor receptor 4) was expressed in FGFR4 (fibroblast growth factor receptor 4), and it was found that a high-purity skeletal muscle stem cell population could be obtained by using the cell surface marker FGFR4. Therefore, the method for improving the purity of skeletal muscle stem cells of the present invention is characterized by using the cell surface marker FGFR4. FGFR4 is also referred to as CD334.

The method for improving the purity of skeletal muscle stem cells of the present invention may be any method including a step of recovering cell surface marker FGFR4-positive cells from a cell population. The cell population is not particularly limited as long as it is a cell population that may contain skeletal muscle stem cells. The cell population used in the method for improving the purity of skeletal muscle lineage cells of the present invention can also be used in the method for improving the purity of skeletal muscle stem cells of the present invention. For example, a cell suspension prepared from a surgical specimen containing skeletal muscle or a cell population in the process of inducing differentiation of pluripotent stem cells into skeletal muscle cells can be used.

In one embodiment, the cell population may be a cell population with improved purity of skeletal muscle lineage cells obtained by the method for improving purity of skeletal muscle lineage cells of the present invention. The cell population with improved purity of skeletal muscle lineage cells is preferably a cell population with improved purity from a cell population in the process of inducing differentiation from pluripotent stem cells to skeletal muscle cells. A cell population containing high-purity CDH13-positive and FGFR4-positive skeletal muscle stem cells is useful as skeletal muscle stem cells for living body transplantation.

The method for recovering FGFR4-positive cells is not particularly limited, and examples thereof include a method using a cell sorter and a method using affinity chromatography. When a cell sorter is used, for example, a cell suspension of a cell population is prepared, an anti-FGFR4 antibody is added to the cell suspension, the anti-FGFR4 antibody is bound to the FGFR4-positive cells, and the cells are subjected to the cell sorter to obtain FGFR4-positive cells. Can be sorted and collected. When affinity chromatography is used, for example, a cell suspension of a cell population is prepared, and the FGFR4 positive cells are bound to the anti-FGFR4 antibody by passing the cell suspension through a column filled with a carrier to which the anti-FGFR4 antibody is bound. After that, FGFR4-positive cells can be recovered by dissociating the binding with the anti-FGFR4 antibody. According to the method for improving the purity of skeletal muscle stem cells of the present invention, the proportion of FGFR4 negative cells in the original cell population decreases and the proportion of FGFR4 positive cells increases. It is a group.

[Manufacturing method of skeletal muscle lineage cells]
The present invention provides a method for producing skeletal muscle lineage cells. The method for producing skeletal muscle lineage cells of the present invention may include the following steps (1) and (2).
(1) Step of initiating differentiation induction from pluripotent stem cells to skeletal muscle cells (2) Step of recovering cell surface marker CDH13-positive cells from cell population in the process of differentiation induction

The method for producing skeletal muscle lineage cells of the present invention can be carried out in the same manner as in the embodiment of the method for producing skeletal muscle stem cells described below. However, in step (2), the time to recover the cell surface marker CDH13-positive cells is not limited to the same time as the method for producing skeletal muscle stem cells described below, and the induction of differentiation from pluripotent stem cells to skeletal muscle cells is not limited to the same time. It may be the time when CDH13-positive cells appear in the developing cell population. For example, when the differentiation induction method from pluripotent stem cells to skeletal muscle cells is induced using the differentiation induction method described in "Experimental Materials and Methods" (2) of the Examples of the present application, 2 weeks to 24 from the start of differentiation induction. CDH13-positive cells can be recovered weekly. When a differentiation-inducing method different from the above is used, preliminary studies can be performed as appropriate to determine the recovery time of CDH13-positive cells.

[Manufacturing method of skeletal muscle stem cells]
The present invention provides a method for producing skeletal muscle stem cells. The method for producing skeletal muscle stem cells of the present invention may include the following steps (1) and (2).
(1) Step of initiating differentiation induction from pluripotent stem cells to skeletal muscle cells (2) Step of recovering cell surface marker FGFR4-positive cells, CDH13-positive cells or FGFR4-positive and CDH13-positive cells from a cell population in the process of inducing differentiation The following description can also be applied to the above-mentioned method for producing skeletal muscle lineage cells.

In step (1), the induction of differentiation from pluripotent stem cells to skeletal muscle cells is started. Examples of pluripotent stem cells include embryonic stem (ES) cells, embryonic stem (ntES) cells derived from cloned embryos obtained by nuclear transplantation, sperm stem (GS) cells, embryonic reproduction (EG) cells, and induced pluripotent cells. Examples include pluripotent stem (iPS) cells, cultured fibroblasts and pluripotent cells (Muse cells) derived from bone marrow stem cells. The pluripotent stem cells used in the present invention may be iPS cells, ES cells, ntES cells, or iPS cells.

The method for inducing differentiation of pluripotent stem cells into skeletal muscle cells is not particularly limited, and can be appropriately selected from known methods for inducing differentiation of pluripotent stem cells into skeletal muscle cells. In one embodiment, the method for inducing differentiation of pluripotent stem cells into skeletal muscle cells is a differentiation medium to which various compounds and physiologically active substances are added without introducing an exogenous skeletal muscle inducing gene into the pluripotent stem cells. It may be a method using. The compounds contained in the differentiation medium include ROCK (Rho-associated coiled-coil forming kinase) inhibitor, GSK3 (Glycogen synthase kinase 3) inhibitor, TGF-β (Transforming Growth Factor-β) inhibitor, and BMP (Bone morphogenetic). protein) Inhibitors and the like. Examples of the physiologically active substance include IGF-1 (Insulin-like Growth Factor 1), bFGF (basic Fibroblast Growth Factor), HGF (Hepatocyte Growth Factor), and retinoic acid.

Furthermore, the method for inducing the differentiation of pluripotent stem cells into skeletal muscle cells is to use a differentiation medium supplemented with the above-mentioned various compounds and physiologically active substances to mature skeletal muscle cells in a manner that mimics the developmental process of the fetus. It may be a method of inducing differentiation of a skeletal muscle lineage cell population including skeletal muscle stem cells. The form that mimics the developmental process of the fetus means a method of inducing differentiation of skeletal muscle lineage cells via paraxial mesoderm cells, somites cells, and cutaneous musculoskeletal cells. Examples of such a differentiation-inducing method include the method described in Non-Patent Document 1 (Zhao et al., Stem Cell Reports, Vol. 15 1-15 July 14, 2020.), And described in International Publication WO2016 / 108288 A1. , And the method described in Hicks et al. (Nat Cell Biol. 2018 Jan; 20 (1): 46-57.).

In step (2), cell surface marker FGFR4-positive cells, CDH13-positive cells or FGFR4-positive and CDH13-positive cells are collected from the cell population in the process of inducing differentiation. The method for recovering each cell surface marker-positive cell is not particularly limited, and examples thereof include a method using a cell sorter and a method using affinity chromatography. When recovering FGFR4 positive cells using a cell sorter, prepare a cell suspension of the cell population, add the anti-FGFR4 antibody to the cell suspension, bind the anti-FGFR4 antibody to the FGFR4 positive cells, and supply the cells to the cell sorter. FGFR4 positive cells can be selected and recovered. When collecting CDH13-positive cells using a cell sorter, prepare a cell suspension of the cell population, add the anti-CDH13 antibody to the cell suspension, bind the anti-CDH13 antibody to the CDH13-positive cells, and supply the cells to the cell sorter. CDH13-positive cells can be selected and recovered. When recovering FGFR4-positive and CDH13-positive cells using a cell sorter, prepare a cell suspension of the cell population, add anti-FGFR4 antibody and anti-CDH13 antibody to the cell suspension, and add anti-FGFR4 antibody to FGFR4-positive cells. By binding anti-CDH13 antibodies to CDH13-positive cells and subjecting them to a cell sorter, FGFR4-positive and CDH13-positive cells can be selected and recovered.

When recovering FGFR4 positive cells using affinity chromatography, prepare a cell suspension of the cell population and pass the cell suspension through a column filled with a carrier to which an anti-FGFR4 antibody is bound to turn the FGFR4 positive cells into an anti-FGFR4 antibody. FGFR4-positive cells can be recovered by binding and then dissociating the binding to the anti-FGFR4 antibody. When recovering CDH13-positive cells using affinity chromatography, prepare a cell suspension of the cell population and pass the cell suspension through a column filled with a carrier bound to the anti-CDH13 antibody to turn the CDH13-positive cells into anti-CDH13 antibody. CDH13-positive cells can be recovered by binding and then dissociating the binding to the anti-CDH13 antibody. When recovering FGFR4-positive and CDH13-positive cells by affinity chromatography, the CDH13-positive cells are recovered by passing the cell suspension of the cell population through a column filled with a carrier to which the anti-CDH13 antibody is bound by the above procedure, and then anti-CDH13. FGFR4-positive and CDH13-positive cells can be recovered through the recovered CDH13-positive cells in a column filled with a carrier to which the FGFR4 antibody is bound. Alternatively, FGFR4-positive cells are collected through a cell suspension of a cell population in a column previously filled with a carrier bound with an anti-FGFR4 antibody, and then the recovered FGFR4-positive cells are passed through a column filled with a carrier bound with an anti-CDH13 antibody. FGFR4-positive and CDH13-positive cells may be recovered.

In step (2), the time to recover the cell surface marker FGFR4-positive cells, CDH13-positive cells or FGFR4-positive and CDH13-positive cell skeletal muscle stem cells from the cell population in the process of inducing differentiation from pluripotent stem cells to skeletal muscle cells is determined. It may be after the time when a part of pluripotent stem cells differentiates into skeletal muscle stem cells. For example, when the differentiation induction method from pluripotent stem cells to skeletal muscle cells is induced using the differentiation induction method described in "Experimental Materials and Methods" (2) of the Examples of the present application, 3 weeks to 24 weeks from the start of differentiation induction. Skeletal muscle stem cells can be recovered by recovering FGFR4-positive cells, CDH13-positive cells or FGFR4-positive and CDH13-positive cells per week. The recovery time may be 10 to 16 weeks, 11 to 13 weeks, or 12 weeks from the start of differentiation induction. In this method for inducing differentiation, the inventors of the present application have confirmed that the skeletal muscle stem cells recovered 12 weeks after the start of the induction of differentiation have a high engraftment rate after transplantation. When a differentiation-inducing method different from the above is used, preliminary studies can be appropriately performed to determine the recovery time of skeletal muscle stem cells.

The skeletal muscle stem cells produced by the method for producing skeletal muscle stem cells of the present invention can be used for living body transplantation. The present inventors transplanted skeletal muscle stem cells (CDH13-positive cells and FGFR4-positive cells) produced by the method for producing skeletal muscle stem cells of the present invention into a low-temperature injured tibialis anterior muscle of a muscular dystrophy model, and as a result, transplanted them into a living body. It has been confirmed that it efficiently differentiates into skeletal muscle cells and engrafts (see Examples). In addition, some of the transplanted cells were positive for PAX7, which is a marker specific to satellite cells, and engrafted in the place where satellite cells exist, inside LAMININ, and it was confirmed that they exist as satellite cells in vivo. ing. This means that the ability to regenerate skeletal muscle is sustained for a long period of time after transplantation, and that the effect of cell therapy may be sustained.

The present invention includes a cell surface marker for purifying or detecting skeletal muscle lineage cells consisting of CDH13. The present invention also includes cell surface markers for purification or detection of skeletal muscle stem cells consisting of FGFR4.

Hereinafter, the present invention will be described in detail by way of examples, but the present invention is not limited thereto.

[Experimental materials and methods]
(1) Pluripotent stem cells Human iPS cell line 201B7 (hereinafter referred to as "201B7") was established by transduction with a retrovirus from commercially available healthy human fibroblasts (Takahashi et al., Cell, 131; 861-872, 2007). The DMD (Duchenne muscular dystrophy) iPS cell line (clone ID: CiRA00111, hereinafter referred to as "DMD") was established by the episomal vector system from the skin fibroblasts of DMD patients lacking the dystrophin gene exxon 44. (Okita et al., Stem Cells, 31; 458-466, 2012). The DMD repair iPS cell line (hereinafter referred to as "CKI") was established by knocking in exon 44 of the dystrophin gene into DMD (Li et al., Stem Cell Reports, 4; 143-154, 2015). The DMD was established with the approval of the Kyoto University Graduate School and the Institutional Review Board of the Faculty of Medicine (approval numbers # R0091 and # G259) and with written consent. The stock iPS cell line Ff-WJ14s01 (hereinafter referred to as "S01") for regenerative medicine was established from the cord blood of a healthy donor with a homozygous HLA refrequency allele in Japan. S01 was established with the approval of the Kyoto University Graduate School and the Institutional Review Board of the Faculty of Medicine (approval numbers # E1762 and # G567) and with written consent.
All human iPS cells were cultured and maintained under feeder-free conditions (Nakagawa et al., Scientific Reports 2014; 4: 3594.). That is, cells were passaged once a week and cultured in iMatrix-511 (Nippi) precoated dish and StemFit AK02N medium (Ajinomoto, hereinafter referred to as "StemFit").

(2) Method for Inducing Differentiation from Human iPS Cells to Skeletal Muscle Stem Cells Induction of differentiation from human iPS cells to skeletal muscle stem cells is described in Non-Patent Document 1 (Zhao et al., Stem Cell Reports, Vol. 15 1-15 July 14). , 2020.). Matrigel precoated 6-well plates were seeded with 1 × 10 ⁴ undifferentiated iPS cells per well. StemFit to which a ROCK inhibitor (10 μM, Y-27632, Sigma) was added was used as the medium. After 2 days, the medium was replaced with StemFit without ROCK inhibitor and cultured for 24 hours. It was then replaced with CDMi medium supplemented with CHIR99021 (Axon MedChem, Tocris) and SB431542 (Sigma). CDMi medium is a 1: 1 mixture of IMDM (Iscove's Modified Dulbecco's Medium, Invitrogen, 12440053) containing L-glutamine, 25 mM HEPES and Ham's F-12 Nutrient Mix (Invitrogen, 11765054) at 1% BSA. (Sigma), 1% penicillin streptomycin mixed solution (Nacalai), 1% CD lipid concentrate (Invitrogen), 1% insulin-transferase-selenium (Invitrogen) and 450 μM 1-thioglycerol (Sigma).

After 7 days, the cells were dissected with Accutase (Nacalai) and passaged into Matrigel precoated 6-well plates (8 x 10 ⁵ cells / well) as single cells. As the medium, CDMi medium supplemented with CHIR99021, SB431542 and a ROCK inhibitor (10 μM) was used. After 1 week (14th day of differentiation), cells were dissociated again with Accutase and passaged with CDMi medium containing Matrigel precoated 6-well plates (8 x 10 ⁵ / well) supplemented with ROCK inhibitor (10 μM). .. After 3 days, 0.2% bovine serum albumin (BSA), 0.1 mM 2-mercaptoethanol (2-ME), 10 ng / ml recombinant human IGF-1 (PeproTech), 10 ng / ml recombinant human bFGF (Oriental yeast) And 10 ng / ml recombinant human HGF (PeproTech) was added to the serum-free medium (SF-O3, Sanko Pure Drug). Medium was exchanged twice a week until the 35th day of differentiation. From day 35 of differentiation, medium was 0.5% penicillin streptomycin mixed solution (Nacalai), 2 mM L-glutamine (Nacalai), 0.1 mM 2-ME, 2% horse serum (HS, Sigma), 5 μM SB431542 and 10 ng / ml. It was changed to DMEM (Invitrogen, 11960069) to which IGF-1 was added. After that, the medium was changed every 2 to 3 days. Cells were cultured in this medium until used in the experiment. Cells on day 84 of differentiation were subjected to analysis.

(3) FACS sorting of living cells On the 84th day of the skeletal muscle stem cell differentiation induction protocol, cells were dissected and sorted by FACS using a surface marker. To dissociate the cells, they were incubated in DMEM medium containing collagenase G (500 μg / mL) and collagenase H (100 μg / mL) (Meiji) for 7 minutes and then incubated in TrypLE (Glibco) for 10 minutes at 37 ° C. The cells were then carefully stripped by pipetting, filtered through a 50 μm mesh and washed twice with HBSS buffer (1000 rpm, 4 ° C., centrifuge for 5 minutes). The cells were then shaded and incubated with the corresponding antibody for 20 minutes on ice. After incubation, cells were washed twice again with HBSS buffer (1000 rpm, 4 ° C., centrifuge for 5 minutes). Finally, cells were resuspended in HBSS buffer containing 1% Hoechst and filtered through a 40 nm mesh. Cells were stored on ice until FACS sorting. FACS sorting and analysis of living cells was performed by Aria II (BD Biosciences).

(4) Quantitative real-time PCR
Cells were harvested and RNA was obtained from the cells using the ReliaPrep RNA Cell Miniprep system (Promega). CDNA was synthesized using the ReverTra Ace qPCR RT kit (TOYOBO). RT-qPCR was performed using the Power SYBR Green Master Mix system (Applied Biosystems) and the StepOne Plus real-time PCR system (Applied Biosystems). Each sample was measured twice or three times. The primer list is shown in Table 1.

(5) Differentiation of FACS sorting cells into skeletal muscle cells and immunocytochemical analysis Fresh cells sorted by FACS were seeded (1 × 10 ⁴ cells / well) on a 48-well plate precoated with iMatrix-511. StemFit containing a ROCK inhibitor was used for the first 48 hours of culture. The cells were then cultured using StemFit without ROCK inhibitors for an additional 3 days until the cells were approximately 80% confluent. On about 5 days, the medium was replaced with a medium containing 2% horse serum (see (1) above) and cultured until it differentiated into skeletal muscle cells (3-5 days). After differentiating into skeletal muscle cells, the cells were fixed with 2% PFA (paraformaldehyde) for 10 minutes and washed 3 times at 4 ° C for 10 minutes using PBS. The cells were then blocked with Blocking one (Nacalai) for 1 hour. Following blocking, cells were incubated overnight with primary antibody diluted in PBS containing 10% Blocking one. The cells were subsequently washed 3 times with PBS (PBS-T) containing 0.2% Triton X100 (Sigma-Aldrich) and the corresponding secondary antibody and DAPI (1: 5000) and cells were incubated for 1 hour at room temperature. .. Cells stained with antibody were observed under a BZ-X700 microscope (Keyence) and analyzed with BZ-X analyzer software (Keyence). The antibodies used are shown in Table 2.

(6) Mouse transplantation experiments 10-16 week old NOG-mdx (muscular dystrophy model, Central Institute for Experimental Animals) male mice were used in all experiments. Fresh cells sorted with FACS for transplantation experiments were counted and resuspended in DMEM medium (3 x 10 ⁵ cells / 50 μL or 1 × 10 ⁵ cells / 50 μL). Cells were stored at 4 ° C until transplantation. Mice were anesthetized with 2% isoflurane and cold-damaged to the tibialis anterior muscle. That is, the tip of the forceps previously cooled with liquid nitrogen was brought into direct contact with the tibialis anterior muscle for 12 seconds three times. After about 2 minutes, the color of the tibialis anterior muscle returned to normal, and then the cells were transplanted. Four weeks after transplantation, mice were euthanized, the tibialis anterior muscle was removed and frozen in liquid nitrogen-cooled isopentane. Frozen muscle was stored at -80 ° C until sections were prepared, and 20 μm sections were prepared using LEICA (CM1950) for histochemical analysis.

(7) Immunohistochemical analysis The frozen sections prepared in (6) were fixed with 4% PFA for 20 minutes and washed with PBS for 10 minutes three times. After washing, the sample was blocked with Blocking one (Nacalai) for 1 hour. Next, the primary antibody diluted with Can get Signal solution B (TOYOBO) and the sample were incubated overnight at 4 ° C. The next day, the sample was washed 3 times with PBS-T for 10 minutes and the corresponding secondary antibody and DAPI (1: 5000) and the sample were incubated for 1 hour at room temperature. After incubation, the samples were washed once with PBS-T for 5 minutes and twice with PBS for 5 minutes. Samples stained with antibody were encapsulated with the encapsulant Aqua-Poly-Mount (Polyscience). Samples were observed using an LSM700 confocal microscope (Carl Zeiss).

(8) RNA Sequencing Total RNA was obtained from cells undergoing RNA sequencing using the ReliaPrep RNA Cell Miniprep system (Promega). RNA quality was assessed using the Agilent 2100 Bioanalyzer System (Agilent). For mRNA sequencing, poly-A mRNA was selected and purified using AMpure XP (Beckman Coulter). Sequence libraries were made from these mRNAs using the TruSeq RNA Sample Prep Kit (Illuminia) according to the manufacturer's protocol. The sequence was performed using the Illumina system (NextSeq 500). For sequence reads, toolkit (version 0.0.14) was used for quality processing, and data with a quality score of over 30 was used for analysis.

(9) Quantification and statistical analysis (9-1) Statistical analysis All statistical analyzes were performed using GraphPad Prism version 8.4.1 (GraphPad Software) for Mac OS X. Differences between the two groups were analyzed by two-sided Student's t-test, and differences between three or more groups were analyzed by analysis of variance by Tukey's multiple comparison range test. When the p-value was 0.05 or less, it was considered that there was a significant difference.

(9-2) Image analysis of differentiation index BZ-X analyzer software (Keyence) was used for image analysis. Five photographs were taken from each well and a total of three independent experiments were performed under each condition to calculate the differentiation index from MYF5-positive cells and PAX7-positive cells to skeletal muscle cells.

(9-3) Engraftment analysis In order to quantify the number of transplanted cells, the tibialis anterior muscle sample was analyzed by immunofluorescence 1 month after transplantation. The tibialis anterior muscle was stained as 20 μm sections. The maximum number of h-SPECTRIN positive fibers detected on one slide was recorded as the number of engrafted fibers.

(9-4) Bioinformatics analysis Human skeletal muscle atlas (https://aprilpylelab.com/), a database of human single-cell RNA sequencing, and Tabula Muris (https: /), a database of mouse single-cell RNA sequencing. /tabula-muris.ds.czbiohub.org/) was used for analysis. FANTOM5 (https://fantom.gsc.riken.jp/data/) was used as the RNA sequence database for human fetuses, and the database provided as accession code: GSE106292 was used for the RNAseq database for human skeletal muscle tissue.

[Example 1: Examination of specific cell surface markers in Myf5-positive cells obtained by inducing differentiation from iPS cells to skeletal muscle stem cells]
Differentiation into skeletal muscle stem cells was induced using 201B7 as human iPS cells, and the cells were collected on day 84 and Myf5 positive cells and Myf5 negative cells were sorted by FACS. RNA-seqing analysis was performed on Myf5-positive cells and Myf5-negative cells, respectively, and CDH13 and FGFR4 were identified as surface markers specifically highly expressed in Myf5-positive cells.

FIG. 1 shows the results of analysis of the expression patterns of CDH13 and FGFR4 using the mouse single-cell RNA sequencing database "Tabula Muris". Both CDH13 and FGFR4 were confirmed to be strongly expressed in the Limb Muscle surrounded by the dotted line.
Although not shown, CDH13 is weak in the results of analysis of the expression patterns of CDH13 and FGFR4 using a database of single-cell RNA sequencing of differentiated skeletal muscle cells from human fetal muscle tissue and human iPS cells. However, it was confirmed that it was strongly expressed from Embryonic to Fetal, FGFR4 was strongly expressed from Embryonic 5 weeks to Fetal 6 weeks, and was also strongly expressed in the iPS cell differentiation system.

FIG. 2 shows the results of analysis of the expression patterns of CDH13 and FGFR4 using a gene expression database in human fetal-derived tissues. (A) is the result of CDH13, and (B) is the result of FGFR4. Both CDH13 and FGFR4 were found to be highly expressed in the tissues of the skeletal muscle system shown in black.
FIG. 3 shows the results of analysis of the expression patterns of CDH13 and FGFR4 using a gene expression database in adult human tissues. (A) is the result of CDH13, and (B) is the result of FGFR4. Both CDH13 and FGFR4 were confirmed to be highly expressed in muscle.

[Example 2: Expression analysis of CDH13 and FGFR4 in cells induced to differentiate from iPS cells to skeletal muscle stem cells]
Differentiation into skeletal muscle stem cells was induced using 201B7 and S01 as human iPS cells, and on day 84, the cells were collected and Myf5-positive cells and Myf5-negative cells were sorted by FACS. Was measured by quantitative real-time PCR.
The results are shown in FIG. (A) is the result of CDH13, and (B) is the result of FGFR4. The expression level was expressed as a relative expression level when the expression level of Myf5-negative cells was 1. Both CDH13 and FGFR4 showed specifically high expression in Myf5-positive cells (*; p <0.05, **; p <0.01, ***; p <0.001).

Differentiation into skeletal muscle stem cells was induced using CKI as human iPS cells, and the cells were collected on day 84 to confirm the presence of CDH13-positive cells and FGFR4-positive cells by FACS.
The results are shown in FIG. (A) is the result of CDH13, and (B) is the result of FGFR4. It was shown that CDH13-positive cells and FGFR4-positive cells were present in a certain proportion in the cells induced to differentiate from CKI to skeletal muscle stem cells.

Using 201B7 as human iPS cells, differentiation into skeletal muscle stem cells was induced, cells were collected on day 84, stained with anti-FGFR4 antibody, and FGFR4 positive cells and FGFR4 negative cells were sorted by FACS, respectively. Next, FGFR4 positive cells and FGFR4 negative cells were stained with anti-CDH13 antibody, respectively, and the CDH13 positive rate was analyzed by FACS. The results are shown in FIG. Approximately 10% of FGR4-positive cells were shown to be almost all CDH13-positive cells. On the other hand, it was shown that about 35% of CDH13-positive cells also exist in FGFR4-negative cells.

[Example 3: Expression analysis of skeletal muscle stem cell-related genes in CDH13-positive cells and FGFR4-positive cells obtained by inducing differentiation from iPS cells to skeletal muscle stem cells]
Differentiation into skeletal muscle stem cells was induced using 201B7, CKI and DMD as human iPS cells, the cells were recovered on day 84, and CDH13-positive cells and FGFR4-positive cells were sorted by FACS. The expression levels of skeletal muscle stem cell-related genes MYF5 and PAX7 and myoblast-related gene MYOD1 were measured by quantitative real-time PCR in CDH13-positive cells, FGFR4-positive cells and non-sorting cells (controls).

Fig. 7 shows the result of 201B7, Fig. 8 shows the result of CKI, and Fig. 9 shows the result of DMD. In each figure, the expression level was expressed as the relative expression level when the expression level of the non-sorting cells (control) was 1. In FIGS. 7, 8 and 9, FGFR4-positive cells highly expressed the skeletal muscle stem cell-related genes MYF5 and PAX7. CDH13-positive cells also had higher expression of MYF5 and PAX7 compared to controls, but lower expression levels than FGFR4-positive cells. In addition, CDH13-positive cells highly expressed the myoblast-related gene MYOD1 (*; p <0.05, **; p <0.01, ***; p <0.001, ****; p <0.0001. ).

[Example 4: Examination of in vitro differentiation potential of CDH13-positive cells and FGFR4-positive cells into skeletal muscle cells obtained by inducing differentiation from iPS cells to skeletal muscle stem cells]
(1) In vitro differentiation ability of CDH13-positive cells into skeletal muscle cells Induction of differentiation into skeletal muscle stem cells using 201B7, CKI and DMD as human iPS cells was performed, and on the 84th day, the cells were recovered and used with anti-CDH13 antibody. Staining was performed and CDH13-positive cells and CDH13-negative cells were sorted by FACS, respectively. The obtained CDH13-positive cells and CDH13-negative cells were further induced to differentiate into skeletal muscle cells, and the ability to differentiate into skeletal muscle cells was examined using the expression of myosin heavy chain (hereinafter referred to as "MHC") as an index.

The results are shown in FIG. (A) is the result of immunostaining MHC, and (B) is the result of calculating the differentiation index (MHC positive cell rate) by image analysis. In 201B7 and CKI, differentiation into MHC-positive skeletal muscle cells was observed only in CDH13-positive cells, and it was shown that almost no MHC-positive skeletal muscle cells differentiated from CDH13-negative cells (***; p. <0.001, ****; p <0.0001).

(2) In vitro differentiation ability of FGFR4-positive cells into skeletal muscle cells Similar to (1) above, differentiation of FGFR4-positive cells into skeletal muscle stem cells was induced using 201B7, CKI and DMD, and the cells were collected on the 84th day. Staining with anti-FGFR4 antibody, FGFR4-positive cells and FGFR4-negative cells were sorted by FACS, respectively. The obtained FGFR4 positive cells and FGFR4 negative cells were further induced to differentiate into skeletal muscle cells, and the ability to differentiate into skeletal muscle cells was examined using the expression of MHC as an index.

The results are shown in FIG. (A) is the result of immunostaining MHC, and (B) is the result of calculating the differentiation index (MHC positive cell rate) by image analysis. Differentiation into MHC-positive skeletal muscle cells was observed with a high efficiency of 80% or more in FGFR4-positive cells, but differentiation of MHC-positive skeletal muscle cells was also observed from FGFR4-negative cells. (**; p <0.01, ***; p <0.001).

[Example 5: Transplantation experiment of CDH13-positive cells and FGFR4-positive cells obtained by inducing differentiation from iPS cells to skeletal muscle stem cells into mice]
(1) Confirmation of engraftment of transplanted cells CKI and DMD were used as human iPS cells to induce differentiation into skeletal muscle stem cells, cells were collected on day 84, and CDH13-positive cells and CDH13-negative cells were used using FACS. , FGFR4 positive cells and FGFR4 negative cells were sorted respectively. Each sorted cell was transplanted into NOG-mdx mice by the method described in (4) of the above [Experimental Materials and Methods], and after 4 weeks, the transplanted site was collected for histochemical analysis and measurement of the number of engrafted cells. Was done.

The transplantation site 4 weeks after the cell transplantation was stained with anti-histopeclin antibody, anti-laminin α2 antibody, anti-human nuclear antibody and DAPI, and the results observed with a confocal microscope are shown in FIG. In the CDH13-positive cell transplantation group and the FGFR4-positive cell transplantation group, a large number of human spectroline-positive muscle fibers were observed, indicating that the transplanted cells differentiated into skeletal muscle cells and engrafted in the living body.

The result of engraftment analysis is shown in FIG. It was shown that the number of engraftment cells in the FGFR4-positive cell transplant group was significantly higher (*; p <0.05, ***; p <0.001, ****; p <0.0001).

(2) Confirmation of differentiation potential in post-transplant muscle tissue In order to confirm the differentiation potential of post-transplant muscle tissue into skeletal muscle lineage cells, the transplanted tissue 4 weeks after transplantation was subjected to anti-PAX7 antibody, anti-MYOD antibody, and anti-h. -Stained with LAMIN A / C antibody and DAPI and observed under a confocal microscope. The results are shown in FIG. Human nucleus-positive and PAX7-positive or MYOD-positive cells were observed from both CDH13-positive cells and FGFR4-positive cells (arrowhead), confirming that the transplanted cells differentiated into skeletal muscle lineage cells in the post-transplant muscle tissue. ..

In order to confirm the ability of post-transplant muscle tissue to differentiate into satellite cells, the transplanted tissue 4 weeks after transplantation was stained with anti-PAX7 antibody, anti-LAMININα2 antibody, anti-h-LAMIN A / C antibody and DAPI, and co-focused. Observed with a microscope. The results are shown in FIG. Human nucleus-positive and PAX7-positive cells located inside laminin were observed from both CDH13-positive cells and FGFR4-positive cells (arrowhead), confirming that the transplanted cells differentiated into satellite cells in the post-transplant muscle tissue. rice field.

[Example 6: Comparison with known skeletal muscle lineage cell surface markers]
(1) FACS analysis Inducing differentiation into skeletal muscle stem cells using DMD and CKI as human iPS cells, and collecting the cells on the 84th day, CD56-positive cell rate, CD82-positive cell rate, and NGFR-positive cell by FACS. The rate, ERBB3-positive cell rate, CDH13-positive cell rate and FGFR4-positive cell rate were confirmed.
The results are shown in FIG. (A) is the result of DMD, and (B) is the result of CKI. It was shown that the proportion of CD56-positive cells and NGFR-positive cells was higher than that of other marker-positive cells.

(2) Expression analysis of skeletal muscle-related genes in each marker-positive cell The skeletons of the DMD and CKI-derived CD56-positive cells, CD82-positive cells, NGFR-positive cells, ERBB3-positive cells, CDH13-positive cells, and FGFR4-positive cells sorted above. The expression levels of MYF5 and PAX7, which are muscle stem cell-related genes, and MYOD1, which is a myoblast-related gene, were measured by quantitative real-time PCR.

The results are shown in Fig. 17. (A) is the result of DMD, and (B) is the result of CKI. In each figure, the expression level was expressed as the relative expression level when the expression level of unsorted cells (not shown) was 1. Expression of MYF5 is high in FGFR4-positive cells. The expression of PAX7 is high in FGFR4-positive cells, and in CKI-derived cells, CDH13-positive cells also have high PAX7 expression. The expression of MYOD1 is high in ERBB3-positive cells and CDH13-positive cells in CKI, suggesting that the fraction contains a large amount of myoblasts. On the other hand, the expression of MYOD1 is low in FGFR4-positive cells, suggesting that the fraction is closer to that of skeletal muscle stem cells (*; p <0.05, **; p <0.01, ***; p <0.001).

(3) In vitro differentiation of each marker-positive cell into skeletal muscle cells Each marker-positive cell was further induced to differentiate into skeletal muscle cells, and the ability to differentiate into skeletal muscle cells was examined using the expression of MHC as an index.
The results are shown in FIG. (A) is the result of immunostaining MHC, and (B) is the result of calculating the differentiation index (MHC positive cell rate) by image analysis. High differentiation potential into MHC-positive skeletal muscle cells was confirmed in CDH13-positive cells and FGFR4-positive cells.

The present invention is not limited to the above-described embodiments and examples, and various modifications can be made within the scope of the claims, and the technical means disclosed in the different embodiments may be appropriately combined. The obtained embodiments are also included in the technical scope of the present invention. In addition, all of the academic and patent documents described in this specification are incorporated herein by reference.

Claims

A method for improving the purity of skeletal muscle lineage cells in a cell population, characterized in that cells positive for the cell surface marker CDH13 in the cell population are recovered.
The method according to claim 1, wherein the cell population is a cell population in the process of inducing differentiation from pluripotent stem cells to skeletal muscle cells.
The method according to claim 2, wherein the pluripotent stem cell is an iPS cell.
A method for improving the purity of skeletal muscle stem cells in a cell population having improved purity of skeletal muscle lineage cells obtained by any of the methods 1 to 3, wherein the cell surface marker FGFR4 positive cells in the cell population are improved. A method characterized by recovering.
A method for improving the purity of skeletal muscle stem cells in a cell population, which comprises recovering cell surface marker FGFR4-positive cells in the cell population.
The method according to claim 5, wherein the cell population is a cell population in the process of inducing differentiation from pluripotent stem cells to skeletal muscle cells.
The method according to claim 6, wherein the pluripotent stem cell is an iPS cell.
A method for producing skeletal muscle lineage cells,
(1) A step of initiating differentiation induction from pluripotent stem cells to skeletal muscle cells, and (2) a step of recovering cell surface marker CDH13-positive cells from a cell population in the process of inducing differentiation.
A manufacturing method characterized by including.
A method for producing skeletal muscle stem cells
(1) A step of initiating differentiation induction from pluripotent stem cells to skeletal muscle cells, and (2) Recovery of cell surface marker FGFR4-positive cells, CDH13-positive cells or FGFR4-positive and CDH13-positive cells from a cell population in the process of differentiation induction. Process to do,
A manufacturing method comprising.
The production method according to claim 9, wherein the step (1) is a step of inducing differentiation using a differentiation medium without introducing an exogenous skeletal muscle inducing gene into pluripotent stem cells.
The production method according to claim 9 or 10, wherein the step (1) is a step of inducing differentiation of skeletal muscle lineage cells from pluripotent stem cells so as to mimic the developmental process of a fetus.
The production method according to any one of claims 9 to 11, wherein the pluripotent stem cell is an iPS cell.
The production method according to any one of claims 9 to 12, wherein the skeletal muscle stem cells are for living-donor transplantation.
Cell surface marker for purification or detection of skeletal muscle lineage cells consisting of CDH13.
Cell surface marker for purification or detection of skeletal muscle stem cells consisting of FGFR4.