JP2019103393A - Method for induction of skeletal muscle stem cell - Google Patents

Abstract

To provide a method for the induction of a skeletal muscle stem cell.SOLUTION: A method for the induction of a skeletal muscle stem cell has a step of introducing (i) Pax3 gene or Pax7 gene and (ii) a gene encoding an effector transcription factor of Notch signal path into a somatic cell.SELECTED DRAWING: None

Description

  The present invention mainly relates to a method for inducing skeletal muscle stem cells.

  Intractable myopathies such as muscular dystrophies, although various studies have been developed, there is still no universal treatment.

  Studies using a model mouse of Duchenne muscular dystrophy (DMD) due to dystrophin deficiency are expected to be an effective therapeutic method for skeletal muscle lineage stem cell transplantation (Non-patent Document 1). However, in humans, it is difficult to control skeletal muscle stem cells of sufficient quality and quantity for treatment, and there is a need for coordinated development of skeletal muscle stem cells.

Sakai H, Sato T, Sakura H, Yamamoto T, Hanaoka K, et al. (2013) Fetal Skeletal Muscle Progenitors Have Reactive Capacity after Intramuscular Engraftment in Dystrophin Deficit Mice. PLoS ONE 8 (5): e63016.

  An object of the present invention is to provide a method for inducing skeletal muscle stem cells.

  The inventors of the present invention conducted intensive studies to solve the above-mentioned problems, and introduced the following steps into a somatic cell: (i) Pax3 gene or Pax7 gene, and (ii) a gene encoding an effector transcription factor of Notch signal pathway. Were found to be capable of inducing skeletal muscle stem cells. The present invention has been completed by further studies on such findings.

That is, the present invention includes the following aspects:
Item 1. A method for inducing skeletal muscle stem cells, comprising the steps of: (i) introducing a Pax3 gene and / or a Pax7 gene, and (ii) a gene encoding an effector transcription factor of at least one Notch signal pathway into a somatic cell.

  Item 2. The method according to Item 1, wherein a gene encoding a transcription factor that promotes at least one growth is further introduced.

  Item 3. The method according to Item 2, wherein Pax3 gene and / or Pax7 gene, Heyl gene, and Klf4 gene are introduced.

  Item 4, any of items 1 to 3 including a step of causing a somatic cell into which a gene is to be introduced to transiently express a MyoD1 family gene, or a step of causing a MyoD1 family gene to be transiently expressed after introducing the gene. Method according to paragraph 1.

  Item 5. The method according to any one of Items 1 to 4, wherein the somatic cell is a mesodermal cell.

  Item 6. The method according to Item 5, wherein the mesodermal cells are derived from pluripotent stem cells.

  Item 7. A therapeutic agent for muscle disease, which comprises the skeletal muscle stem cell according to any one of items 1 to 6.

  The present invention provides a method of inducing skeletal muscle stem cells. Since the skeletal muscle stem cells obtained by the method of the present invention can be easily derived from the somatic cells of the transplanted individual, problems such as immunological rejection do not occur even when the obtained skeletal muscle stem cells are transplanted. It is possible to prepare skeletal muscle stem cells of sufficient quality and quantity, and can be used for the treatment of refractory muscle disease.

GFP positive cells were collected from mouse embryonic stage (Fetus), young age (infant) and adult stage respectively. Principal component analysis results of Pax3 positive cells by comprehensive gene expression analysis using a microarray method. 1) mouse embryonic stem cells (ESC), 2) mouse germ stem cells (GSC), 3) mouse fibroblasts (MEF), 4) mouse neural stem cells (NSC), 5) mouse iPS cells (iPS), and 6) Pax3 positive skeletal muscle stem cells (Pax3-GFP). Common transcription factor group 108 gene. 18 types of transcription factors extracted. Genetically modified mouse information for detecting skeletal muscle stem cells. (DM; dermomyotome, M; myotome, DML; dorsomedial lip, VLL; ventolateral lip) Conceptual diagram of differentiation induction to skeletal muscle stem cells. A method of recovering genetically modified mouse-derived fibroblasts. Changes in GFP and RFP expression over time when the muscle regulatory factor MyoD is introduced into genetically modified fibroblasts. Analysis results of Pax3-GFP expression. Schematic representation of GFP gene knock-in to human PAX3 locus and inducible MYOD gene. Immunostaining diagram of PAX3 expression. Appearance of MYOD expression induction (red) and myocyte differentiation by addition of doxycycline (DOX). GFP expression (PAX3) and mCherry (DOX-MYOD) expression upon addition and addition of DOX. Fig. 3D Transcript analysis in GFP positive cells under conditions. Cell morphology (lower panel) and GFP expression (upper panel) during differentiation induction from human iPS cells to mesodermal lineage cells. The mesodermal induction day 7 GFP expression plot (upper panel) and the ratio of ectoderm (neural) and mesoderm (PSM; presomitic mesoderm, somite) (lower panel). Gene expression changes in GFP-positive cells during mesoderm induction. Summary of the method. A cell population in which GFP expression was detected under skeletal muscle stem cell culture conditions for one week after DOX treatment was performed for 72 hours after PAX3, HEYL, KLF4 gene introduction into mesodermal cells. The ratio of GFP-positive expression in the DOX-treated population in which the DOX-treated cell population and the PAX3, HEYL, and KLF4 genes were introduced into mesodermal cells for 72 hours. Intracellular gene expression comparison in FIG. 4C. Differentiation induction result of skeletal muscle. Outline of transplantation experiment. Skeletal muscle cross-sectional view after transplantation. Immunostaining results of dystrophin (DYS), human nucleus (hNUC), general nucleus (DAPI), laminin a2 chain (LAMA2). 3 Results of selection of GFP positive cells (+ GFP + cells) and GFP negative cells (+ GFP-cells) in a cell population cultured for 1 week after DOX treatment after gene transfer, and results of transplantation of the same number of cells (left panel), and Quantitative graph (right panel). The immunostaining figure (left panel) and its quantification graph (right panel) of skeletal muscle stem cell marker PAX7 expression in the same conditions as FIG. 5C.

  The present invention relates to a method of inducing skeletal muscle stem cells.

Skeletal muscle stem cells Skeletal muscle stem cells (SC) are tissue stem cells present in skeletal muscle and are cells that contribute to regeneration upon muscle damage. Skeletal muscle stem cells are mononuclear cells, and exist in vivo under the basement membrane of multinucleated myofibers. Skeletal muscle stem cells are considered to be derived from muscle precursor cells in the developmental / growing stage as cells expressing the transcription factors Pax3 and / or Pax7.

Somatic Cell The method of the present invention for inducing the musculoskeletal stem cell of the present invention comprises the step of introducing a specific gene into somatic cells as a raw material.

  As a somatic cell of a raw material, a thing derived from a mammal is used. Mammals include non-human mammals such as mice, rats, hamsters, dogs, cats, monkeys, rabbits, cows, horses, pigs and humans, and humans.

  The somatic cells from which skeletal muscle stem cells are derived are not particularly limited. For example, fibroblasts, keratinocytes, oral mucosal epithelial cells, nasal mucosal epithelial cells, airway mucosal epithelial cells, gastric mucosal epithelial cells, intestinal mucosal epithelial cells, vascular endothelial cells, smooth muscle cells, adipocytes, gingival cells Somatic cells such as blast cells, gingival epithelial cells), dental pulp cells, periodontal ligament cells, bone marrow cells, bone marrow-derived stromal cells, white blood cells, lymphocytes, myocytes, conjunctival epithelial cells, and osteoclasts can be used.

  In addition, pluripotent stem cells such as embryonic stem cells (Embryonic stem cells: ES cells) and induced pluripotent stem cells (iPS cells); endoderm, mesoderm, external derived from pluripotent stem cells Also, somatic cells derived from pluripotent stem cells can be used.

  Using somatic stem cells such as mesenchymal stem cells (Mesenchymal stem cells: MSCs), neural stem cells, hepatic stem cells, enteral stem cells, skin stem cells, hair follicle stem cells, pigment cell stem cells, etc. You can also.

  These somatic cells can be obtained by techniques known to those skilled in the art.

  As a preferable example of the somatic cell of a raw material, human iPS cell 201 B7 strain is mentioned. A method for producing human iPS cell 201B7 strain is described in the literature: Takahashi et al., Cell (2007) 131, 861-872.

  In a preferred embodiment of the present invention, mesodermal cells differentiated from pluripotent stem cells are used as somatic cells of the raw material.

  Mesodermal cells include mesothelial mesodermal cells, paraxial mesodermal cells, and lateral plate mesodermal cells. Among them, paraxial mesodermal cells are preferred.

  As mesodermal cells, it is also possible to use medial mesodermal cells, paraxial mesodermal cells or cells obtained by further differentiating lateral plate mesodermal cells. For example, segment cells induced to differentiate from paraxial mesodermal cells can be used.

  The induction of differentiation of mesodermal cells from pluripotent stem cells can be carried out, for example, by the method described in the literature: Loh et al., Cell (2017) 166, 451-467.

In particular,
(1) induction of differentiation from pluripotent stem cells to paraxial mesodermal cells;
(2) Compound CHIR 990 21 (6-[[4- (2,4-Dichlorophenyl) -5- (5-methyl-1H-imidazol-2), which is then a combination of GSK-3 inhibitor and ALK inhibitor -yl) -2-pyrimidinyl] amino] ethyl] amino] -3-pyridinecarbonitrile, GSK-3 inhibitor) and LDN 193189 (4- (6- (4- (piperazin-1-yl) phenyl) pyrazolo [1,5 -a] Pyrimidin-3-yl) quinoline hydrochloride, ALK2 and ALK3 inhibitors, and growth factor FGF2 to induce differentiation into somatic cells, differentiation of pluripotent stem cells to mesodermal cells It can do induction.

The method of inducing skeletal muscle stem cells according to the present invention
(I) Pax3 gene or Pax7 gene, and
(Ii) introducing a gene encoding an effector transcription factor of the Notch signal pathway into somatic cells of the raw material.

(I) Pax3 gene or Pax7 gene
Pax3 gene or Pax7 gene is a known gene of transcription factor belonging to the paired box (Pax) family.

(Ii) A gene encoding an effector transcription factor of the Notch signaling pathway When a ligand binds to a Notch receptor present on the cell membrane, the Notch intracellular domain translocates into the nucleus, and the effector transcription factor present in the nucleus Association occurs to activate the target gene. A gene encoding an effector transcription factor of the Notch signaling pathway is a gene encoding an effector transcription factor that functions downstream of such a Notch receptor. Examples of a gene encoding an effector transcription factor of the Notch signal pathway include Heyl gene, Hey1 gene, Hes1 gene and the like. The gene encoding the effector transcription factor of the Notch signal pathway can introduce one or more kinds.

(Iii) A gene encoding a transcription factor that promotes proliferation In the induction method of the present invention, in addition to the genes of (i) and (ii) above, introducing a gene encoding a transcription factor that promotes proliferation, It is preferable from the induction efficiency of skeletal muscle stem cells. Genes encoding transcription factors that promote proliferation include Klf family genes. Klf family genes are known to encode transcription factors that promote proliferation. And Klf genes include Klf4 gene, Klf2 gene, Klf5 gene and the like. One or more types of genes encoding transcription factors that promote growth can be introduced.

  Other further introductions can be made as long as the effects of the present invention are not impaired. Examples of such a gene include, but are not limited to, Egr2 (Krox 20) gene, Mef 2 c gene, Pitx 2 gene and the like.

  In the induction method of the present invention, preferred examples of the gene to be introduced into the somatic cells of the raw material include a combination of Pax3 gene and Heyl gene; a combination of Pax7 gene and Heyl gene; a combination of Pax3 gene, Heyl gene, and Klf4 gene; A combination of the gene, the Heyl gene, and the Klf4 gene is exemplified. A combination of Pax3 gene, Heyl gene, and Klf4 gene; combination of Pax7 gene, Heyl gene, and Klf4 gene is preferable, and a combination of Pax3 gene, Heyl gene, and Klf4 gene is particularly preferable.

The nucleotide sequences of the above genes and the amino acid sequences of the proteins encoded by the respective genes are known. The cDNA nucleotide sequence of the gene that can be used in the present invention and the amino acid sequence of the protein encoded by the gene are registered under the accession number registered in GenBank provided by the National Center for Biotechnology Information (NCBI; National Center for Biotechnology Information). The following is an example (in the case where multiple revisions are registered, it is understood to point to the latest revision):
Human Pax3 gene cDNA sequence: NM_000438, NM_001127366, NM_018427, NM_181457, NM_181458, NM_181459, NM_181460, NM_181461 (e.g., NM_000438.5, NM_001127366.2, NM_013942.4, NM_181457.3, NM_181458.3, NM_181459.3, NM_181460. 3, NM_181461.3),
Human Pax3 protein amino acid coordination sequence: NP_000429, NP_001120838, NP_852230, NP_852123, NP_852124, NP_852125, NP_852126 (e.g., NP_000429.2, NP_001120838.1, NP_039230.1, NP_852122.1, NP_852123.1, NP_852124.1, NP_852125.1, NP_852126.1).

Human Pax7 gene cDNA sequence: NM_001135254, NM_002584, NM_013945 (for example, NM_001135254.1, NM_002584.2, NM_013945),
Human Pax7 protein amino acid coordination sequence: NP_001128726, NP_002575, NP_039236 (e.g., NP_001128726.1, NP_002575.1, NP_039236.1).

Human Heyl gene cDNA sequence: NM_014571 (eg, NM_014571.3),
Human Heyl protein amino acid coordination sequence: NP_055386 (eg, NP_055386.1).

Human Hey1 gene cDNA sequences: NM_001040708, NM_001282851, NM_012258 (eg, NM_001040708.1, NM_001282851.1, NM_012258. 3),
Human Hey1 protein amino acid coordination sequence: NP — 001035798, NP — 001269780, NP — 036390 (eg, NP — 001035798.1, NP — 001269780.1, NP — 036390.3).

Human Hes1 gene cDNA sequence: NM_005524 (eg, NM_005524.3),
Human Hes1 protein amino acid coordination sequence: NP_005515 (eg, NP_005515.1).

Human Klf4 gene cDNA sequence: NM_001314052, NM_004235 (for example, NM_001314052.1, NM_004235.5),
Human Klf4 protein amino acid coordination sequence: NP_001300981, NP_004226 (for example, NP_001300981.1, NP_004226.3).

Human Klf2 gene cDNA sequence: NM — 01627 (eg, NM — 016270.3),
Human Klf2 protein amino acid coordination sequence: NP_057354 (eg, NP_057354.1).

Human Klf5 gene cDNA sequence: NM_001286818, NM_001730 (eg, NM_001286818.1, NM_001730.4),
Human Klf5 protein amino acid coordination sequence: NP — 001273747, NP — 001721 (eg, NP — 001273747.1, NP — 001721.2).

Human Egr2 gene cDNA sequence: NM_000399, NM_0011361177, NM_001136178, NM_001136179, NM_001321037 (for example, NM_000399.4, NM_001136177.2, NM_001136178.1, NM_001136179.2, NM_001321037.1),
Human Egr2 protein amino acid coordination sequence: NP — 000390, NP — 001129649, NP — 001129650, NP — 001129651, NP — 0201397966 (eg, NP — 000390.2, NP — 001129649.1, NP — 00011296500.1, NP — 0001129651.1, NP — 001307966.1).

Human Mef2c gene cDNA sequence: NM_001131005, NM_0011933348, NM_001193348, NM_001193349, NM_001193350, NM_001308002, NM_002397 (eg, NM_001131005.2, NM_001193347.1, NM_001193348.1, NM_001193349.1, NM_001193350.1, NM_001308002.1, NM_0023974) ,
Amino acid coordination sequence of human Mef2c protein: NP_001124277, NP_001180276, NP_001180277, NP_001180278, NP_001180279, NP_001294931, NP_0023947.1, NP_0011240277.1, NP_001180277.1, NP_001180277.1, NP_001180279.1, NP_001280279.1, NP_001280437.1, 2).

Human Pitx2 gene cDNA sequence: NM_000325, NM_0100204397, NM_0100204398, NM_001204399, NM_153426, NM_153427 (e.g.
Human Pitx2 protein amino acid coordination sequence: NP_000316, NP_001191326, NP_001191328, NP_001191328, NP_700475, NP_700476 (for example, NP_000316.2, NP_001191326.1, NP_001191327.1, NP_001191328.1, NP_700475.1, NP_700476.1).

  In the method of the present invention, the means for introducing the above-mentioned gene into somatic cells can be performed by means widely used in the method of differentiation induction by gene transfer. Specifically, it is preferable to incorporate the target gene into one or more expression vectors, introduce the expression vector into a target somatic cell, and express it in cells.

  Methods of gene transfer include methods of infecting viral vectors such as retrovirus vectors, adenovirus vectors, lentivirus vectors, adeno-associated virus vectors, herpes virus vectors, Sendai virus vectors and the like. In addition, non-viral vectors such as cationic liposomes, cationic polymers, and electroporation may be used to transfect plasmid vectors, episomal vectors, and gene expression products (mRNA, proteins).

Transient expression of the MyoD1 family gene In the method of the present invention, the somatic cell into which the gene is to be introduced is a somatic cell in which the MyoD1 family gene is expressed transiently, or the MyoD1 family gene is transiently introduced after gene introduction. Preferably, the method further comprises the step of expressing. In the present specification, "transiently expressed" may be expressed as "primed".

MyoD family genes are involved in the control of muscle differentiation bHLH (basic helix loop helix
A group of genes encoding type transcription factors. Specifically, MyoD1, Myf5, myogenin and MRF4 can be mentioned, with preference given to MyoD1.

  The means for transient expression of the MyoD family gene is not particularly limited as long as transient expression of the gene is achieved. For example, the method described in JP-A-2014-533491 can be used. Above all, a preferable embodiment is a means for realizing transient expression of MyoD family gene by introducing a vector having MyoD family gene under control of a tetracycline responsive promoter into cells and culturing in the presence of doxycycline. Can be mentioned as

  The period of transient expression of the MyoD family gene can be, for example, 1 week (7 days) or less, preferably about 3 to 5 days, and more preferably about 3 days.

  The cDNA nucleotide sequence of the human (Homo sapiens) MyoD1 gene and the amino acid sequence of the protein encoded thereby are registered under the following accession number (when multiple revisions are registered, the latest revision Understood to point):

Human MyoD gene cDNA sequence: NM_002478 (eg, NM_002478.4),
Human MyoD protein amino acid coordination sequence: NP_002469 (eg, NP_002469.2).

Culturing In the method of the present invention, it is preferable to culture a differentiated somatic cell of a mammal in a medium after introduction of a gene.

Culturing can be performed in a suitable container for storing cells and media. As a suitable culture | cultivation method, although the culture | cultivation method about conditions about about 37 degreeC and a carbon dioxide density | concentration about 5% is illustrated, it is not limited to this. The culture under the above conditions can be performed, for example, using a known CO ₂ incubator.

  The period of culture is not particularly limited as long as the effects of the present invention are not impaired. For example, it can be about 12 hours to about 1 month, about 1 day to about 3 weeks, about 3 days to about 2 weeks. Media exchange can be performed as needed. The culture conditions are preferably according to a conventional method.

  Passage can be performed as necessary in culture. When passaging, cells are harvested before or immediately after reaching confluence and the cells are seeded in fresh medium. In addition, in the culture of the present invention, the culture medium can also be replaced as appropriate.

Medium The medium used in the method of the present invention is not particularly limited. Normal liquid media such as DMEM (Dulbecco's Modified Eagle's Medium), EMEM (Eagle's minimal essential medium), and alpha MEM (alpha Modified Minimum Essential Medium) can be used. If necessary, components such as serum components (Fetal Bovine Serum (FBS), Human Serum (HS)), antibacterial agents such as streptomycin and penicillin, and Non-Essential Amino Acids (NEAA) can be added.

Preparation Thus, skeletal muscle stem cells are derived from somatic cells.

  In one embodiment, the prepared skeletal muscle stem cells have the introduced gene as a foreign gene. Here, “foreign” is mainly an aspect of the gene introduced as a result of the above-mentioned introduction means or an expression product thereof, which is different from the natural aspect. For example, a gene whose expression is controlled by a promoter other than a natural promoter, a position on a non-natural chromosome, or an embodiment of a gene existing extrachromosomally can be mentioned.

  Skeletal muscle stem cells may be obtained as a mixture with cells other than skeletal muscle stem cells (eg, original somatic cells). In such a case, skeletal muscle stem cells and cells other than skeletal muscle stem cells can be separated as necessary. The means for separation is not particularly limited. For example, cell sorters or magnetic beads can be used for separation using Pax3 protein and / or Pax7 protein expression as an indicator.

Therapeutic Use The skeletal muscle stem cells obtained by the present invention can be used to treat various diseases. In this case, the skeletal muscle stem cells are a therapeutic agent and can be provided in the form of a graft material. An implant material is introduced into a living body for repair and reconstruction of muscle tissue (particularly, muscle fibers).

  Examples of the disease to be treated include, but are not limited to, muscle damage due to trauma and injury, refractory muscle disease such as muscular dystrophy and muscle atrophy due to aging and disuse.

  As used herein, and unless otherwise indicated, the term "treatment" intends treatment to be performed while the patient suffers from a particular disease or disorder, whereby the severity of the disease or disorder, or one or more It means that the symptoms thereof are alleviated or the progression of the disease or disorder is delayed or slowed down. In the present specification, "treatment" includes "prevention".

  Although an example is shown below, the present invention is not limited to this example.

<Materials and Methods>
Experimental mouse
Pax3 ^{GFP / +} (Relaix et al. 2005, Nature) and BAC MyoD-Cre; Rosa26 ^{CAG-LSL-tdTomato / +} (Sato et al. 2014, Nat Commun) is used for skeletal muscle stem cell collection, Dmd, ^{-/ y} (Kudoh et al. 2000, BBRC) and NSG (Charles River Japan) were prepared respectively for use in preparing an immunodeficient muscular dystrophy model mouse for cell transplantation.

The harvested cell transplantation experiments are as follows. Dmd- ^{/ y} : Contains 5.0 x 10 ⁵ cells for the lower tibialis anterior muscle of NSG 8-week-old male mice, and 20 μL of 10% matrigel (BD), 2% horse serum (SIGMA) and 10 μM Y-27632 (Nacalai) The suspension was suspended in HBSS solution (GIBCO) and injected intramuscularly. After 4 weeks, analysis was performed by immunostaining.

Cell sorting method FACSJazz (BD) was used to sort the target living cells from mouse embryos, adult mouse skeletal muscle tissue and human iPS cell-derived cell populations. Skeletal muscle tissues were isolated from mouse embryos, mouse hindlimbs, abdominal muscles and diaphragm tissues, and reacted with 0.2% collagenase solution (Worthington) for several hours at 37 ° C. to dissociate the tissues. In order to recover single cells from the reaction solution, filtration (BD; cell strainer 35 μm) and centrifugation at 1000 rpm for 5 minutes were performed. Resuspend the precipitated cell population in cell sorting solution (1% BSA in PBS), add 1 μg / mL Propidium iodide (Molecular Probes) to remove dead cells, and sort live cells and fluorescent cells The APC-labeled anti-DLL1 antibody (R & D, 1/200 dilution) was used for sorting of mesodermal lineage cells.

Cell Culture Method Mouse primary fibroblasts were seeded onto a culture dish coated with 0.1% gelatin solution in PBS, and cultured in 10% FBS in DMEM (Nacalai). For forced gene expression by pMXs vector, forced expression of each gene is performed by virus infection in a 70% confluent state, and then 5 ng / mL basic to skeletal muscle stem cell culture solution (DMEM / F12 (Nacalai) containing 20% FBS basic Expansion culture was performed for two weeks with FGF (WAKO).

  The maintenance culture method of human iPS cells was performed according to Takahashi et al. 2007 Cell and Nakagawa et al. 2014 Scientific Reports. Myogenic differentiation by MYOD forced expression using constant Doxycycline (DOX: Tocris) treatment was performed according to Tanaka et al 2013 PLoS One. The induction of human iPS cells into mesodermal lineage cells was performed by modifying Loh et al. 2016 Cell. The specific method is as follows.

Single-cell human iPS cells were seeded on culture dishes coated with Geltrex (GIBCO) solution (50000 cells / 6 wells) and then added with 10 μM Y-27632 (Nacalai) in mTeSR solution (STEMCELL Technologies) Incubate. After 3 days, 1% BSA (SIGMA), 1% CD lipid concentrate (GIBCO), 1% Insulin-Transferrin-Selenium (GIBCO), 20 μM 1 to mesodermal differentiation medium (IMDM / F12: 50: 50 (GIBCO) -Change the medium to Thioglycerol (SIGMA), 10 μM SB431542 (StemGent), 10 μM CHIR99021 (StemGent), 2 μM DMH1 (TOCRIS), 20 ng / mL basic FGF (WAKO), change the culture medium every two days, and culture for 1 week. The obtained cell population is detached using TrypLE Select (GIBCO), and the DLL1-positive cells containing PAX3-GFP-positive are isolated and recovered. The obtained cells are scaled up, and Geltrex-coated culture dishes 5.0 × 10 ⁵ Culture is carried out in mesoderm induction medium for 3 days at cells / 6 cm.The detached cells are recovered, and preparations for introducing plasmid DNA containing the target gene into cells using an electroporator (NEPA21; Nepagene) are prepared. the .DNA5μg to do, recovered 1.0 × 10 ⁶ mixed with cells in 125volt to a method according to Tanaka et al 2013 PLoS One Gene transfer is carried out and seeded on culture dishes coated with Geltrex from the next day 1 μg / mL DOX is added to skeletal muscle stem cell culture solution from the next day, and cultured for 72 hours. Replace the culture solution every two days for at least one week, confirm GFP-positive cells, isolate and collect by FACS, then observe and statistically process using FACsJAZZ and fluorescence microscope BZX-700 (Keyence) Did.

Microarray analysis and real-time PCR transcript analysis
Total RNA was extracted from the recovered cells using RNeasy micro kit (QIAGEN), hybridization was performed using Mouse Gene 1.0 ST Array (Affymetrix), and experiments were performed according to the Affymetrix protocol. The gene expression profile in adult mouse Pax3 positive skeletal muscle stem cells was analyzed from the database obtained from Montarras et al. 2005 Science and Sakai et al. 2013 PLoS One. In addition, for realtime PCR analysis using extracted RNA, RNA is reverse transcribed to cDNA using SuperScript VILO kit (Invitrogen), and the synthetic cDNA is used as a template for Thunder Bird SYBR qPCR mix (TOYOBO) and Thermal Cycler Dice Realtime System (TAKARA) Real-time PCR analysis was performed using. At that time, Rpl13a encoding ribosomal protein in both mouse and human products was used as an mRNA expression internal standard.

Plasmid DNA preparation Total RNA was extracted from adult mouse adult skeletal muscle stem cells and human iPS cell-derived teratoma, cDNA reverse transcription was performed using SuperScript 3 reverse transcriptase (Invitrogen) and oligo dT (Invitrogen), and synthetic cDNA was registered as a template The primer oligo was designed to contain the coding sequence of each gene, RT-PCR was performed, and cloning was performed on pENTR1A vector (Invitrogen). In order to generate a plasmid for intracellular gene forced expression, recombination was performed on pMXs gateway vector (Takahashi et al. 2006 Cell) and pPiggyBac EF1a gateway vector (Takana et al. 2013 PLoS One) by LR cloning (Invitrogen).

Immunostained cells and muscle tissue were immersed in 4% paraformaldehyde solution (WAKO) and fixed at 4 ° C. for 10 minutes, and sectioned muscle tissue was embedded in Frozen Section Compound (Leica) and used for cryosection. The immobilized sample is reacted with 0.1% Triron solution, reacted with Blocking One solution (Nacalai Tesque) for 30 minutes at room temperature, and then primary antibody is added to PBST (PBS containing 0.1% Tween 20) containing 0.2% BlockingOne solution. The reaction was allowed to proceed overnight at 4 ° C. The antibodies used were PAX3 (DSHB concentrate, 1/100 dilution), PAX7 (DSHB concentrate, 1/50 dilution), MYOG (Santacruz biotech, 1/200 dilution), hNUC (Millipore, 1/200 dilution), LAMA2 (Millipore) , 1/200 dilution), DYS (Abcam, 1/200 dilution).

  The next day, after washing three times with PBST, the secondary antibody was added to 0.2% BlockingOne solution in PBST, and allowed to react at room temperature for 1 hour. The antibodies used are Alexa 488 labeled anti-mouse IgG antibody, Alexa 594 labeled anti-rabbit IgG alternation, and Alexa 647 labeled anti-rat IgG antibody (Molecular Probes, 1/500 dilution). After washing three times with PBST, they were mounted using Slowfade Diamond Antifade Mountant with DAPI (Molecular Probes), and fluorescence observation and statistical processing were performed using BZX-700.

<Result>
Transcription factors strongly expressed in mouse Pax3-GFP positive skeletal muscle stem cells (Figure 1)
As shown in FIG. 1A, GFP positive cells (Pax3 positive cells) were collected from each of mouse embryonic development (Fetus), weak-age (infant) and adult.

  Total RNA was extracted from each of the collected Pax3 positive cells, and comprehensive gene expression analysis was performed using a microarray method. The results of principal component analysis of gene expression analysis are shown in FIG. 1B. As shown in FIG. 1C, among gene expression in Pax3 positive cells obtained at each developmental growth stage, there were 108 common transcription factor groups. FIG. 1D shows 18 extracted transcription factors in a group of common transcription factors. Common transcription factors included skeletal muscle stem cell markers Pax 3 and Pax 7; muscle regulators Myod 1, Myf 5 and Myog.

The documents cited in this example are as follows.
Relaix et al., Nature (2005) 435: 948-953
Sato et al., Nat Commun (2014) 5: 4597
Kudoh et al., BBRC (2005) 328: 507-516
Nakagawa et al., Scientific Reports (2014) 4: 3594
Tanaka et al, PLoS One (2013) 8: e61540
Montarras et al. 2005 Science (2005) 309: 2064-2067
Sakai et al., PLoS One (2013) 8: e63016.

Transfer of candidate genes into mouse fibroblasts (Figure 2)
FIG. 2A shows the expression of GFP and RFP in mice with Pax3 ^{GFP / +} ; BAC MyoD-Cre; Rosa26 ^{CAG-LSL-tdTomato / +} (Pax3-GFP; MyoD-RFP) gene complex-introduced mice used for analysis.

  FIG. 2B shows a conceptual diagram of differentiation induction to skeletal muscle stem cells.

  As shown in FIG. 2C, each negative fraction is recovered by FACS to remove GFP and RFP positive cells, and genetically modified fibroblasts in which GFP and RFP expression is negative for use in gene transfer experiments are shown. Obtained.

  FIG. 2D shows changes in GFP and RFP expression over time when the muscle regulatory factor MyoD is introduced into genetically modified fibroblasts. In the gene transfer of MyoD alone, Pax3-GFP expression was not observed until 14 days after the transfer. On the other hand, FIG. 2E shows gene transfer when eight types of transcription factors (+8 transcription factors; Egr2, Heyl, Klf4, Mef2c, MyoD, Pax3, Pax7, Pitx2) including MyoD are introduced into genetically modified fibroblasts. The results of expression analysis of Pax3-GFP after 2 weeks are shown. The expression of Pax3-GFP was confirmed, suggesting that differentiation induction to skeletal muscle stem cells is taking place.

Induction of differentiation from PAX3-GFP transgenic human iPS cells (Figure 3)
FIG. 3A schematically shows the GFP gene knockin to the human PAX3 locus and the inducible MYOD gene.

  In FIG. 3B, human iPS cells into which the above gene was introduced were prepared. The state of GFP expression in teratoma (EB) produced using the said gene transfer human iPS cell and the immunostaining figure of PAX3 expression in each of a GFP positive cell and a GFP negative cell are shown.

  In transgenic human iPS cells, MYOD was forcibly expressed by constant Doxycycline (DOX) treatment. As a result, differentiation into myocytes could be confirmed. FIG. 3C shows the appearance of MYOD expression induction and differentiation into myocytes by the addition of doxycycline (DOX) in transgenic human iPS cells.

  FIG. 3D shows the analysis results of GFP expression (PAX3 expression) and mCherry (DOX-MYOD expression) without and with DOX added. In the case of continuous DOX treatment (continuous DOX), almost no GFP expression was observed. On the other hand, in the case of 72 hours of DOX treatment (DOX for 72 hours), the proportion of GFP-positive cells was significantly increased.

  FIG. 3E shows the analysis results of transcripts in GFP-positive cells with and without DOX addition. Elevated expression of central nervous marker SOX1 and neural crest marker SOX10 was observed in PAX3-GFP positive cells with and without DOX addition.

  FIG. 3F shows cell morphology (lower panel) and GFP expression (upper panel) at the time of induction of differentiation from human iPS cells to mesodermal lineage cells. It showed a morphology similar to the epithelial-like structure similar to that of the mouse somatic segment.

  FIG. 3G shows a GFP expression plot at day 7 of mesoderm induction (upper panel) and the proportions of ectoderm (neural) and mesoderm (PSM; presomitic mesoderm, somite) (lower panel).

  FIG. 3H shows gene expression changes in GFP-positive cells upon mesoderm induction. Compared with the cell population treated with DOX 72 hours without mesoderm induction, neural crest cell induction (+ 5 μM CHIR99021), GFP-positive cells (MDC for 1wks, 2wks) during mesoderm induction have expression of SOX1 and SOX10 neural markers It did not rise, and expression of the segmental marker DMRT2 was high.

Induction of differentiation into adult skeletal muscle stem cells by gene transfer into mesodermal derived cells (Fig. 4)
Mesodermal derived cells
(A) DOX treatment (MYOD-primed) for 72 hours after gene introduction (cDNA introduction) from iPS cell-derived mesoderm-derived cells using specific cells (DLL1 + cells), skeletal muscle stem cell culture conditions (20% FBS in DMEM / F12 + 5 ng / ml bFGF) The flow of the method of obtaining a GFP positive cell.

  FIG. 4B shows an example of a cell population in which GFP expression was detected under skeletal muscle stem cell culture conditions for 1 week after mesodermal cells were subjected to DOX treatment for 72 hours after PAX3, HEYL and KLF4 gene introduction.

  FIG. 4C shows the expression ratio of GFP-positive cells in each of the cell population treated with DOX for 72 hours in mesoderm cells and the population treated with PAX3, HEYL and KLF4 genes and treated with DOX. The GFP positive rate is increased in the case where three genes have been introduced by DOX treatment alone.

  FIG. 4D shows a comparison of intracellular gene expression in FIG. 4C. The adult skeletal muscle stem cell markers PAX7 and CALCR, which were almost undetectable by DOX treatment at the time of mesoderm induction, were treated with two gene transfer of HEYL and KLF4 and three genes of PAX3, HEYL, and KLF4 for DOX treatment An increase in expression was observed in GFP positive cells.

  FIG. 4E shows the results of skeletal muscle differentiation in low-serum condition (2% horse serum) test tube culture using GFP-positive cells (3F + DOX) in which 3 genes of PAX3, HEYL and KLF4 were introduced and treated with DOX. .

Results of muscle transplantation experiment using PAX3-GFP positive cells obtained after gene transfer (FIG. 5)
FIG. 5A shows an outline of the transplantation experiment. Cells treated with mesodermal induction were treated with DOX for 72 hours after 3 gene introductions of PAX3, HEYL, KLF4 and continued to culture for 1 week. Using GFP positive 500000 cells obtained, muscular dystrophy model (Dmd- ^{/ y} ; NSG, 8 weeks old) was injected intramuscularly into the lower tibialis anterior muscle.

  FIG. 5B shows an example of a skeletal muscle cross-sectional view after transplantation. Immunostaining was performed for dystrophin (DYS), human nucleus (hNUC), whole nucleus (DAPI), and laminin a2 chain (LAMA2). Dystrophin expression was observed specifically at the site where human nuclei are present.

After sorting the cells into a cell population of DOX-treated and cultured for one week after gene transfer into GFP-positive cells (+ GFP + cells) and GFP-negative cells (+ GFP- cells), the same number of cells were transferred to a muscular dystrophy model.
FIG. 5C shows an example (left panel) of skeletal muscle cross-sectional views after transplantation in each case and quantitative results of dystrophin positive muscle fibers (right panel). In GFP positive cells (day 7) obtained at mesoderm induction, no expression of dystrophin was observed after transplantation. In addition, when GFP-positive cells after gene transfer are transplanted with cells cultured in a test tube (+ cultured GFP +), dystrophin expression is significantly greater than when transplanted immediately after recovery of GFP-positive cells. Decreased to

  FIG. 5D shows the immunostaining diagram (left panel) of skeletal muscle stem cell marker PAX7 expression and the quantification results (right panel) of PAX7 positive cells under the same conditions as FIG. 5C. Only when transplanted GFP-positive cells after gene transfer, PAX7-positive cells derived from the transplanted cells were observed in the transplanted muscle tissue.

Claims (7)

A method for inducing skeletal muscle stem cells, comprising the steps of: (i) introducing a Pax3 gene and / or a Pax7 gene, and (ii) a gene encoding an effector transcription factor of at least one Notch signaling pathway into a somatic cell.   The method according to claim 1, further introducing a gene encoding a transcription factor that promotes at least one growth.   The method according to claim 2, wherein Pax3 gene and / or Pax7 gene, Heyl gene, and Klf4 gene are introduced.   The method according to any one of claims 1 to 3, wherein the somatic cell into which the gene is to be introduced is a somatic cell in which the MyoD1 family gene is transiently expressed, or the step of transiently expressing the MyoD1 family gene after gene introduction. Method described in Section.   The method according to any one of claims 1 to 4, wherein the somatic cell is a mesodermal cell.   6. The method of claim 5, wherein the mesodermal cells are derived from pluripotent stem cells.   A therapeutic agent for muscle disease, comprising the skeletal muscle stem cell according to any one of claims 1 to 6.

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