Satellite cells and compositions, and methods for producing the same.

Abstract

To provide satellite cells and compositions and methods for producing the same.SOLUTION: Disclosed herein are methods for producing satellite cells and compositions including satellite cells. In one aspect, the disclosure provides a non-naturally occurring satellite cell. In another aspect, the non-naturally occurring satellite cell exhibits a morphology that resembles the morphology of an endogenous satellite cell. In another aspect, the non-naturally occurring satellite cell expresses Pax7. In another aspect, the non-naturally occurring cell exhibits a response to a muscle injury. In another aspect, the non-naturally occurring satellite cell initiates muscle regeneration following transplantation.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Related applications This application claims the benefits of U.S. Provisional Application No. 62 / 510,617, filed on 24 May 2017. All teachings of the said application are incorporated herein by reference. [Background technology]

[0002] Background of the Invention Skeletal muscle possesses remarkable regenerative capacity. This regenerative capacity is attributed to satellite cells (skeletal muscle stem cells) (Collins et al., 2005; Kuang et al., 2007; Mauro, 1961). In response to injury or disease, satellite cells break their quiescent state and proliferate, giving rise to a migration and amplification population of precursor cells called myoblasts (Yin et al., 2013). When cultured in vitro, satellite cells become myoblasts and lose the ability to rearrange the stem cell niche after in vivo transplantation (Montarras et al., 2005). To date, there is no evidence to demonstrate that myoblasts cultured in vitro can dedifferentiate and revert back to satellite cells. [Overview of the project] [Means for solving the problem]

[0003] Among other uses, there is a particular need for a method or protocol to generate sufficient quantities of satellite cells useful for cell therapy and screening. The 3D culture system described herein (e.g., skeletal muscle organoids) creates a suitable niche for in vitro cultured myoblasts to dedifferentiate into satellite cells. The creation of this niche allows myoblasts to return to a quiescent state, adopt the transcriptional signature of satellite cells, and begin expressing key glycoproteins present only in the satellite cell niche.

[0004] In some embodiments, the present disclosure provides satellite cells that do not exist naturally (i.e., non-native).

[0005] In some embodiments, non-naturally occurring satellite cells exhibit a morphology similar to that of endogenous or naturally occurring satellite cells. In some embodiments, non-naturally occurring satellite cells express Pax7 and / or Myf5, and in some embodiments, this expression is at a level consistent with the expression levels of each gene in endogenous or naturally occurring satellite cells. In some embodiments, non-naturally occurring cells exhibit a response to muscle injury. For example, non-naturally occurring satellite cells may initiate or promote muscle regeneration after transplantation. In some embodiments, non-naturally occurring satellite cells express at least one quiescence-related gene. Quiescence-related genes may be selected from the group consisting of, for example, Spry1, Nm1, Nfia, Fos, and Dusp1. In some embodiments, non-naturally occurring satellite cells express at least one Notch signaling pathway gene. Notch signaling pathway genes may be selected from the group consisting of Notch1, Notch2, Notch3, HeyL, Hey2, and Hes. In some embodiments, naturally occurring satellite cells express Pax7, Myf5, at least one quiescence-related gene, and at least one Notch signaling pathway gene. In some embodiments, naturally occurring satellite cells express at least one, at least two, or at least three of the genes identified in Figures 8A and 8C herein. In some embodiments, naturally occurring satellite cells express at least one, at least two, or at least three of the genes identified in Figure 8C herein. Naturally occurring satellite cells do not express MyoD (a gene whose expression is associated with myoblasts).

[0006] In some embodiments, satellite cells that do not exist naturally are dedifferentiated in vitro from myoblasts. Satellite cells that do not exist naturally may be derived from dedifferentiated myoblasts. Satellite cells that do not exist naturally may be human. In some embodiments, satellite cells that do not exist naturally are not genetically modified, but in other embodiments, they may be genetically modified. For example, satellite cells that do not exist naturally may be modified to express or express at increased levels a protein of interest (e.g., insulin) that may be beneficial to the cell recipient. Satellite cells that do not exist naturally may have the ability to rearrange satellite cell niches and / or may be expandable in culture.

[0007] In some embodiments, the disclosure provides cell lines comprising satellite cells that do not exist in nature as described herein.

[0008] In some embodiments, the present disclosure provides compositions comprising satellite cells that do not exist in nature, wherein the satellite cells that do not exist in nature are derived from dedifferentiated myoblasts; the satellite cells that do not exist in nature are capable of expanding and growing in culture; the satellite cells that do not exist in nature have the ability to rearrange satellite cell niches; and the satellite cells that do not exist in nature promote muscle regeneration after transplantation.

[0009] In some embodiments, the studies described herein provide a method for generating non-naturally occurring satellite cells from a population of myoblasts, comprising exposing a cell population containing myoblasts in spin culture to at least one culture medium for a sufficient time to induce dedifferentiation of at least one myoblast in spin culture to non-naturally occurring satellite cells. In some embodiments, the at least one culture medium is myoblast medium, spin medium, and / or differentiation medium.

[0010] In some embodiments, the Disclosure provides a method for generating non-natural satellite cells from a population of myoblasts, comprising: contacting a cell population containing myoblasts with a myoblast culture medium to form an expanded myoblast population; contacting the expanded myoblast population in spin culture with a spin culture medium to form at least one skeletal muscle organoid containing differentiated cells and proliferative cells; and contacting the at least one skeletal muscle organoid in spin culture with a differentiation medium to induce dedifferentiation of at least one proliferative cell of the skeletal muscle organoid into non-natural satellite cells.

[0011] In some embodiments, the cell population is maintained in a suspension culture on spin medium for a period of time sufficient to induce in vitro maturation of at least one (typically at least several) myoblasts in the cell population into at least one skeletal muscle organoid containing differentiated and proliferative cells. In some embodiments, the period includes at least 10 days, 10 to 30 days, or 10 to 20 days. In some embodiments, the period is 20 days.

[0012] In some embodiments, at least one skeletal muscle organoid is maintained in a suspension culture for a period sufficient to induce in vitro maturation of at least one proliferative cell of the skeletal muscle organoid into at least one non-spontaneous satellite cell. In some embodiments, the period includes at least 10 days, 10 to 30 days, or 10 to 20 days; in some embodiments, the period is 10 days. In some embodiments, at least 1% of myoblasts in the cell population are induced to dedifferentiate into non-spontaneous satellite cells. Thus, non-spontaneous satellite cells described herein may be derived from dedifferentiated myoblasts.

[0013] In some embodiments, the in vitro generation of satellite cells that do not exist naturally is scalable. Satellite cells that do not exist naturally may be able to expand and proliferate in culture. In some embodiments, satellite cells that do not exist naturally have the ability to rearrange the satellite cell niche.

[0014] In some embodiments, the present disclosure provides isolated populations of naturally occurring satellite cells produced according to the methods described herein.

[0015] In some embodiments, the Disclosure provides microcapsules containing an isolated population of non-naturally occurring satellite cells described herein, encapsulated therein.

[0016] In some embodiments, the present disclosure provides compositions comprising a population of satellite cells that do not exist in nature, produced according to the methods described herein.

[0017] In some embodiments, the disclosure provides assays for identifying one or more candidate agents that promote the dedifferentiation of at least one myoblast into at least one non-naturally occurring satellite cell.

[0018] In some embodiments, the Disclosure provides a method for treating a subject in need of treatment, comprising administering to the subject a composition comprising a composition comprising a non-naturally occurring satellite cell (e.g., an isolated population of non-naturally occurring satellite cells) produced according to a method described herein. In some embodiments, the satellite cells are encapsulated in microcapsules. The non-naturally occurring satellite cells may be produced from a population of myoblasts obtained from the same subject to which the non-naturally occurring satellite cells are administered. In some embodiments, the subject has or is at increased risk of developing a muscle degeneration disorder, muscle trauma, or age-related muscle weakness (e.g., sarcopenia).

[0019] In some embodiments, the disclosure provides the use of isolated populations of non-naturally occurring satellite cells, produced, for example, by the methods described herein, for administration to subjects requiring such administration. Isolated populations of non-naturally occurring satellite cells may be encapsulated in microcapsules and administered to subjects. In some embodiments, subjects have or are at increased risk of developing muscle degeneration disorders, muscle trauma, or age-related muscle weakness (e.g., sarcopenia).

[0020] Unless otherwise specified, the implementation of this invention typically utilizes prior arts of cell biology, cell culture, molecular biology, recombinant biology, microbiology, recombinant nucleic acid (e.g., DNA) technology, immunology, and RNA interference (RNAi), which are within the scope of the skills of the art. A non-limiting description of these specific techniques is found in the following publication: Ausubel, F et al., (eds.), Current Protocols in Molecular Biology, Current Protocols in Immunology, Current Protocols in Protein Science, and Current Protocols in Cell Biology, all John Wiley&Sons,NY,edition as of December 2008;Sambrook,Russell,and Sambrook,Molecular Cloning:A Laboratory Manual,3rd ed.,Cold Spring Harbor Laboratory Press,Cold Spring Harbor,2001;Harlow,E.and Lane,D.,Antibodies-A Laboratory Manual,Cold Spring Harbor Laboratory Press,Cold Spring See Harbor, 1988; Freshney, RI, “Culture of Animal Cells, A Manual of Basic Technique”, 5th ed., John Wiley & Sons, Hoboken, NJ, 2005. Non-limiting information regarding therapeutic agents and human diseases can be found in Goodman and See Gilman's The Pharmacological Basis of Therapeutics, 11th Ed., McGraw Hill, 2005, Katzung, B. (ed.) Basic and Clinical Pharmacology, McGraw-Hill / Appleton & Lange; 10th ed. (2006) or 11th edition (July 2009). Non-limiting information on genes and genetic disorders can be found in McKusick, VA: Mendelian Inheritance in Man. A Catalog of Human Genes and Genetic Disorders. Baltimore: Johns Hopkins University Press, 1998 (12th edition) or in the more recent online database: Online Mendelian Inheritance in Man, OMIM (trademark). McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University (Baltimore, MD) and National Center for Biotechnology Information, National Library of Medicine (Bethesda, MD), as of May 1, 2010, World Wide. Web URL: ncbi.nlm.nih.gov / omim / and Online Mendelian Inheritance in Animals (OMIA), a database of genes, inherited disorders and traits in animal species (other than human and mouse), can be found at omia.angis.org.au / contact.shtml. All patents, patent applications and other publications (e.g., scientific papers, books, websites and databases) referred to herein are incorporated by reference in their entirety. In case of any conflict between this specification and any of the incorporated references, this specification (including any corrections thereof that may be based on the incorporated references) shall prevail. Unless otherwise indicated, standard meanings recognized in the art for terms are used herein. Standard abbreviations for various terms are used herein.

[0021] The patent or application file contains at least one drawing created in color. Copies of this patent or patent application publication that include color drawings will be provided by the office upon request and payment of the necessary fees. In certain embodiments, for example, the following items are provided. (Item 1) Non-naturally occurring satellite cells. (Item 2) The non-naturally occurring satellite cells according to Item 1, which exhibit a morphology similar to that of endogenous satellite cells. (Item 3) The non-naturally occurring satellite cells according to Item 1, which express Pax7. (Item 4) The non-naturally occurring cells according to Item 1, wherein the non-naturally occurring satellite cells exhibit a response to muscle injury. (Item 5) The non-naturally occurring cells according to Item 1, wherein the non-naturally occurring satellite cells cause muscle regeneration after transplantation. (Item 6) The naturally occurring satellite cells described in item 1 express at least one quiescence-related gene. (Item 7) A cell that does not exist naturally, as described in item 6, wherein the aforementioned quiescence-related gene is selected from the group consisting of Spry1, Nm1, Nfia, Fos, and Dusp1. (Item 8) The aforementioned naturally occurring satellite cells express at least one Notch signaling pathway gene, as described in item 1. (Item 9) Cells that do not exist naturally, as described in item 8, in which the Notch signaling pathway genes are selected from the group consisting of Notch1, Notch2, Notch3, HeyL, Hey2, and Hes. (Item 10) The aforementioned satellite cells, which do not exist naturally, express Myf5, as described in item 1. (Item 11) The aforementioned non-naturally occurring satellite cells express Pax7, Myf5, at least one quiescence-related gene, and at least one Notch signaling pathway gene, as described in item 1. (Item 12) The aforementioned non-naturally occurring satellite cells express at least one gene identified in Figure 8A and / or Figure 8C, as described in item 1. (Item 13) The aforementioned non-naturally occurring satellite cells express at least two genes identified in Figure 8A and / or Figure 8C, as described in Item 1. (Item 14) The aforementioned non-naturally occurring satellite cells express at least three genes identified in Figure 8A and / or Figure 8C, as described in Item 1. (Item 15) The aforementioned naturally occurring satellite cells express at least one gene identified in Figure 8C, as described in item 1. (Item 16) The aforementioned naturally occurring satellite cells express at least two genes identified in Figure 8C, as described in item 1. (Item 17) The aforementioned naturally occurring satellite cells express at least three genes identified in Figure 8C, as described in item 1. (Item 18) The aforementioned naturally occurring satellite cells do not express MyoD, and are the naturally occurring cells described in item 1. (Item 19) The aforementioned satellite cells, which do not exist naturally, are produced by in vitro dedifferentiation from a population of myoblasts, as described in item 1. (Item 20) The aforementioned satellite cells, which do not exist naturally, are derived from dedifferentiated myoblasts, and are the cells described in item 1 that do not exist naturally. (Item 21) The aforementioned satellite cells, which do not exist in nature, are capable of expanding and proliferating in culture, as described in item 1. (Item 22) The naturally occurring satellite cells described in item 1, which have the ability to rearrange the satellite cell niche. (Item 23) The aforementioned satellite cells, which do not exist naturally, are human cells, as described in item 1. (Item 24) The aforementioned naturally occurring satellite cells are genetically modified cells as described in item 1 that do not exist naturally. (Item 25) The aforementioned naturally occurring satellite cells are genetically modified cells, as described in item 1, that do not exist naturally. (Item 26) A cell line containing cells described in any one of items 1 through 25. (Item 27) A composition containing satellite cells that do not exist in nature, a. The aforementioned satellite cells, which do not exist naturally, are derived from dedifferentiated myoblasts; b. The aforementioned satellite cells, which do not exist naturally, are capable of expanding and proliferating in culture; c. The naturally non-existent satellite cells have the ability to rearrange the satellite cell niche; or d. A composition in which the aforementioned naturally occurring satellite cells induce muscle regeneration after transplantation. (Item 28) The composition according to item 27, wherein the naturally occurring satellite cells express at least one quiescence-related gene. (Item 29) The composition according to item 28, wherein the quiescent gene is selected from the group consisting of Spry1, Nm1, Nfia, Fos, and Dusp1. (Item 30) The composition according to item 27, wherein the naturally occurring satellite cells express at least one Notch signaling pathway gene. (Item 31) The composition according to item 30, wherein the Notch signaling pathway gene is selected from the group consisting of Notch1, Notch2, Notch3, HeyL, Hey2, and Hes. (Item 32) The composition according to item 27, wherein the naturally occurring satellite cells express Myf5. (Item 33) The composition according to item 27, wherein the naturally occurring satellite cells express at least one gene identified in Figure 8A and / or Figure 8C. (Item 34) The composition according to item 27, wherein the naturally occurring satellite cells express at least two genes identified in Figure 8A and / or Figure 8C. (Item 35) The composition according to item 27, wherein the naturally occurring satellite cells express at least three genes identified in Figure 8A and / or Figure 8C. (Item 36) The composition according to item 27, wherein the naturally occurring satellite cells express at least one gene identified in Figure 8C. (Item 37) The composition according to item 27, wherein the naturally occurring satellite cells express at least two genes identified in Figure 8C. (Item 38) The composition according to item 27, wherein the naturally occurring satellite cells express at least three genes identified in Figure 8C. (Item 39) The composition according to item 27, wherein the naturally occurring satellite cells do not express MyoD. (Item 40) The composition according to item 27, wherein the cells that do not exist in nature express Pax7. (Item 41) A method for generating non-natural satellite cells from a population of myoblasts, comprising: contacting a cell population containing myoblasts in spin culture with at least one culture medium for a sufficient time to induce dedifferentiation of at least one myoblast in spin culture into non-natural satellite cells. (Item 42) The method according to item 41, wherein at least one of the culture media is myoblast culture medium. (Item 43) The method according to item 41, wherein at least one of the culture media is a spin medium. (Item 44) The method according to item 41, wherein at least one of the culture media is a differentiation medium. (Item 45) A method for generating naturally occurring satellite cells from myoblasts, The process of bringing a cell population containing myoblasts into contact with myoblast culture medium to form an expanded and proliferated myoblast population; To bring a population of myoblasts that has expanded and proliferated during spin culture into contact with a spin culture medium to form at least one skeletal muscle organoid containing differentiated cells and proliferative cells; The at least one skeletal muscle organoid in spin culture is brought into contact with the differentiation medium for a sufficient amount of time to induce the dedifferentiation of at least one proliferative cell of the skeletal muscle organoid into satellite cells that do not exist in nature. Methods that include... (Item 46) The method according to item 45, wherein the cell population is maintained in a suspension culture in spin medium for a sufficient period of time to induce in vitro conversion of the myoblasts in the cell population to skeletal muscle organoids containing differentiated cells and proliferative cells. (Item 47) The method described in item 46, wherein the aforementioned period includes at least 10 days. (Item 48) The method described in item 46, wherein the aforementioned period includes a period of 10 to 30 days. (Item 49) The method described in item 46, wherein the aforementioned period includes 10 to 20 days. (Item 50) The method described in item 46, wherein the aforementioned period includes 20 days. (Item 51) The method according to item 45, wherein the at least one skeletal muscle organoid is maintained in a suspension culture for a period of time sufficient to induce in vitro dedifferentiation of at least one proliferative cell of the skeletal muscle organoid into at least one non-native satellite cell. (Item 52) The method described in item 51, wherein the aforementioned period includes at least 10 days. (Item 53) The method described in item 51, wherein the aforementioned period includes a period of 10 to 30 days. (Item 54) The method described in item 51, wherein the aforementioned period includes 10 to 20 days. (Item 55) The method described in item 51, wherein the aforementioned period includes 10 days. (Item 56) The method according to item 45, wherein at least 1% of the myoblasts in the cell population are induced to dedifferentiate into satellite cells that do not exist in nature. (Item 57) The method according to item 45, wherein the naturally occurring satellite cells express Pax7, Myf5, at least one quiescence-related gene, and at least one Notch signaling pathway gene. (Item 58) The method according to item 45, wherein the naturally occurring satellite cells express at least one gene identified in Figure 8A and / or Figure 8C. (Item 59) The method according to item 45, wherein the naturally occurring satellite cells express at least two genes identified in Figure 8A and / or Figure 8C. (Item 60) The method according to item 45, wherein the naturally occurring satellite cells express at least three genes identified in Figure 8A and / or Figure 8C. (Item 61) The method according to item 45, wherein the naturally occurring satellite cells express at least one gene identified in Figure 8C. (Item 62) The method according to item 45, wherein the naturally occurring satellite cells express at least two genes identified in Figure 8C. (Item 63) The method according to item 45, wherein the aforementioned naturally occurring satellite cells express at least three genes identified in Figure 8C. (Item 64) The method according to item 45, wherein the aforementioned naturally occurring satellite cells exhibit a response to muscle injury. (Item 65) The method described in item 45, wherein satellite cells that do not exist naturally promote muscle regeneration after transplantation. (Item 66) The method described in item 45, wherein the naturally occurring satellite cells are derived from dedifferentiated myoblasts. (Item 67) The method according to item 45, wherein the naturally non-existent satellite cells have the ability to rearrange the satellite cell niche. (Item 68) The method according to item 45, wherein the naturally occurring satellite cells include human cells. (Item 69) The method described in item 45 allows for the in vitro generation of satellite cells that do not exist in nature, with the ability to scale them up or down. (Item 70) The method according to item 45, wherein the aforementioned satellite cells, which do not exist in nature, can be expanded and proliferated in culture. (Item 71) An isolated population of naturally occurring satellite cells produced according to the method described in any one of items 45-70. (Item 72) Microcapsules containing an isolated population of naturally occurring satellite cells as described in item 71. (Item 73) A composition comprising a population of naturally occurring satellite cells produced in accordance with the method described in any one of items 45 to 70. (Item 74) An assay comprising an isolated population of naturally occurring satellite cells produced according to the method described in any one of items 45-70. (Item 75) The assay described in item 74 for use in identifying one or more candidate agents that promote the dedifferentiation of at least one myoblast into at least one satellite cell that does not exist in nature. (Item 76) A method for treating a subject requiring treatment, comprising administering to the subject a composition comprising an isolated population of satellite cells produced in accordance with the method described in any one of items 45 to 70. (Item 77) The method according to item 76, wherein the satellite cells are encapsulated in microcapsules. (Item 78) The method according to item 76, wherein the naturally occurring satellite cells are produced from a population of myoblasts obtained from the same subject to whom the naturally occurring satellite cells are administered. (Item 79) The method according to item 76, wherein the subject has a muscle degeneration disorder or has an increased risk of developing a muscle degeneration disorder. (Item 80) The method according to item 76, wherein the subject has a muscle injury or has an increased risk of developing a muscle injury. (Item 81) The method according to item 76, wherein the subject has sarcopenia or has an increased risk of developing sarcopenia. (Item 82) Use of isolated populations of naturally occurring satellite cells produced by any of the methods described in items 45-70 for administration to subjects requiring such administration. (Item 83) The use described in item 82, wherein an isolated population of satellite cells not naturally occurring is encapsulated in microcapsules and administered to the subject. (Item 84) The use described in item 82, wherein the subject has a muscle degeneration disorder or is at increased risk of developing a muscle degeneration disorder. (Item 85) The use described in item 82, wherein the subject has a muscle injury or is at increased risk of developing a muscle injury. (Item 86) The use described in item 82, wherein the subject has a muscle injury or is at increased risk of developing a muscle injury. [Brief explanation of the drawing]

[0022] [Figure 1A-B]Figures 1A-1D show the culture of three-dimensional skeletal muscle organoids. Figure 1A shows a schematic diagram outlining the isolation of myoblasts and the expansion, growth, and culture in a spin flask to form three-dimensional skeletal muscle organoids. Figure 1B shows mouse three-dimensional skeletal muscle organoids 10, 20, and 30 days after seeding in a spin flask. Immunofluorescence imaging with antibodies against Pax7 (green) and MyHC (red). Nuclei are counterstained with Hoechst. Scale bar indicates 100 μm. Figure 1C shows human three-dimensional skeletal muscle organoids 10 days after seeding in a spheroid plate. On day 2, the skeletal muscle organoids were transferred from the spheroid plate to a low-adhesion 10 cm plate for a further 8 days of culture on an orbital shaker. Figure 1D shows human three-dimensional skeletal muscle organoids 10 days after seeding in a spheroid plate. Immunofluorescence imaging with antibodies against Pax7 (green) and MyHC (red). Nuclei are counterstained with Hoechst. The scale bar indicates 100 μm. [Figure 1C-D] Figures 1A-1D show the culture of three-dimensional skeletal muscle organoids. Figure 1A shows a schematic diagram outlining the isolation of myoblasts and the expansion, growth, and culture in a spin flask to form three-dimensional skeletal muscle organoids. Figure 1B shows mouse three-dimensional skeletal muscle organoids 10, 20, and 30 days after seeding in a spin flask. Immunofluorescence imaging with antibodies against Pax7 (green) and MyHC (red). Nuclei are counterstained with Hoechst. Scale bar indicates 100 μm. Figure 1C shows human three-dimensional skeletal muscle organoids 10 days after seeding in a spheroid plate. On day 2, the skeletal muscle organoids were transferred from the spheroid plate to a low-adhesion 10 cm plate for a further 8 days of culture on an orbital shaker. Figure 1D shows human three-dimensional skeletal muscle organoids 10 days after seeding in a spheroid plate. Immunofluorescence imaging with antibodies against Pax7 (green) and MyHC (red). Nuclei are counterstained with Hoechst. The scale bar indicates 100 μm. [Figure 2A]Figures 2A–2B show transcriptional profiling of satellite-like cells derived from skeletal muscle organoids. Figure 2A provides a heatmap showing the gene profiles of satellite cells and cultured myoblasts. Red indicates upregulated genes, and blue indicates downregulated genes between myoblasts and satellite cells. Figure 2B shows qPCR validation from skeletal muscle organoid-derived nGFP+ cells after 30 days of 3D culture. The experiment was performed in three biological replicates (n=3) using separate myoblast cell lines derived from individual Pax7nGFP mice. Results are presented as logarithmic changes relative to proliferating myoblasts. [Figure 2B] Figures 2A–2B show transcriptional profiling of satellite-like cells derived from skeletal muscle organoids. Figure 2A provides a heatmap showing the gene profiles of satellite cells and cultured myoblasts. Red indicates upregulated genes, and blue indicates downregulated genes between myoblasts and satellite cells. Figure 2B shows qPCR validation from skeletal muscle organoid-derived nGFP+ cells after 30 days of 3D culture. The experiment was performed in three biological replicates (n=3) using separate myoblast cell lines derived from individual Pax7nGFP mice. Results are presented as logarithmic changes relative to proliferating myoblasts. [Figure 3A-B]Figures 3A–3D demonstrate that satellite-like cells derived from skeletal muscle organoids express proteoglycans unique to the satellite cell niche. Figure 3A provides a heatmap showing a subset of satellite cell-specific proteoglycan genes compared to expression on cultured myoblasts. Red indicates upregulated genes, and blue indicates downregulated genes between myoblasts and satellite cells. Figure 3B shows qPCR validation of proteoglycan genes from skeletal muscle organoid-derived nGFP+ cells after 30 days of 3D culture. The experiment was performed in three biological replicates (n=3) using separate myoblast cell lines derived from individual Pax7nGFP mice. Results are shown as logarithmic changes relative to proliferating myoblasts. Figure 3C provides a schematic diagram of skeletal muscle organoid culture and differentiation for proteoglycan detection. Figure 3D shows frozen section skeletal muscle organoids differentiated at day 20 or day 10. Immunofluorescence imaging using antibodies against Pax7 (green) and Bgn (top red) or Tgfbr3 (bottom red). Nuclei are counterstained with Hoechst. Scale bar indicates 100 μm. [Figure 3C-D]Figures 3A–3D demonstrate that satellite-like cells derived from skeletal muscle organoids express proteoglycans unique to the satellite cell niche. Figure 3A provides a heatmap showing a subset of satellite cell-specific proteoglycan genes compared to expression on cultured myoblasts. Red indicates upregulated genes, and blue indicates downregulated genes between myoblasts and satellite cells. Figure 3B shows qPCR validation of proteoglycan genes from skeletal muscle organoid-derived nGFP+ cells after 30 days of 3D culture. The experiment was performed in three biological replicates (n=3) using separate myoblast cell lines derived from individual Pax7nGFP mice. Results are shown as logarithmic changes relative to proliferating myoblasts. Figure 3C provides a schematic diagram of skeletal muscle organoid culture and differentiation for proteoglycan detection. Figure 3D shows frozen section skeletal muscle organoids differentiated at day 20 or day 10. Immunofluorescence imaging using antibodies against Pax7 (green) and Bgn (top red) or Tgfbr3 (bottom red). Nuclei are counterstained with Hoechst. Scale bar indicates 100 μm. [Figure 4A]Figures 4A–4E illustrate the method for generating skeletal muscle organoids. Figure 4A provides a schematic diagram outlining the formation of skeletal muscle organoid (SkMO) cells. SkMO cells generally encompass SkMO 30D growth cells and SkMO 30D Diff cells. SkMO 30D growth cells are skeletal muscle organoid cells grown for 30 days in spin culture medium. SkMO 30D Diff cells are skeletal muscle organoid cells grown for 20 days in spin culture medium and then for 10 days in differentiation medium (referred to herein as satellite cells, which do not exist in nature). Figure 4B shows SkMO 30D growth cells and SkMO 30D Diff cells. Figure 4C shows that SkMO cells express Pax7 (an important transcription factor for endogenous satellite cells). The transcriptional profiles of SkMOs are examined by RNA-seq, and their ability to cluster in state space is evaluated using the multidimensional scaling plot shown in Figure 4D. SkMO GFP+ cells (green and blue) approximate endogenous satellite cells (red) in terms of transcriptional signature. SkMO D30 growth cells (blue) are shown to be more proliferative than SkMO D30 Diff cells (green), and SkMO D30 Diff cells cluster near endogenous satellite cells (red). Figure 4E provides a global heatmap and dendrogram that further support the data provided in Figure 4D. [Figure 4B-C]Figures 4A–4E illustrate the method for generating skeletal muscle organoids. Figure 4A provides a schematic diagram outlining the formation of skeletal muscle organoid (SkMO) cells. SkMO cells generally encompass SkMO 30D growth cells and SkMO 30D Diff cells. SkMO 30D growth cells are skeletal muscle organoid cells grown for 30 days in spin culture medium. SkMO 30D Diff cells are skeletal muscle organoid cells grown for 20 days in spin culture medium and then for 10 days in differentiation medium (referred to herein as satellite cells, which do not exist in nature). Figure 4B shows SkMO 30D growth cells and SkMO 30D Diff cells. Figure 4C shows that SkMO cells express Pax7 (an important transcription factor for endogenous satellite cells). The transcriptional profiles of SkMOs are examined by RNA-seq, and their ability to cluster in state space is evaluated using the multidimensional scaling plot shown in Figure 4D. SkMO GFP+ cells (green and blue) approximate endogenous satellite cells (red) in terms of transcriptional signature. SkMO D30 growth cells (blue) are shown to be more proliferative than SkMO D30 Diff cells (green), and SkMO D30 Diff cells cluster near endogenous satellite cells (red). Figure 4E provides a global heatmap and dendrogram that further support the data provided in Figure 4D. [Figure 4D]Figures 4A–4E illustrate the method for generating skeletal muscle organoids. Figure 4A provides a schematic diagram outlining the formation of skeletal muscle organoid (SkMO) cells. SkMO cells generally encompass SkMO 30D growth cells and SkMO 30D Diff cells. SkMO 30D growth cells are skeletal muscle organoid cells grown for 30 days in spin culture medium. SkMO 30D Diff cells are skeletal muscle organoid cells grown for 20 days in spin culture medium and then for 10 days in differentiation medium (referred to herein as satellite cells, which do not exist in nature). Figure 4B shows SkMO 30D growth cells and SkMO 30D Diff cells. Figure 4C shows that SkMO cells express Pax7 (an important transcription factor for endogenous satellite cells). The transcriptional profiles of SkMOs are examined by RNA-seq, and their ability to cluster in state space is evaluated using the multidimensional scaling plot shown in Figure 4D. SkMO GFP+ cells (green and blue) approximate endogenous satellite cells (red) in terms of transcriptional signature. SkMO D30 growth cells (blue) are shown to be more proliferative than SkMO D30 Diff cells (green), and SkMO D30 Diff cells cluster near endogenous satellite cells (red). Figure 4E provides a global heatmap and dendrogram that further support the data provided in Figure 4D. [Figure 4E]Figures 4A–4E illustrate the method for generating skeletal muscle organoids. Figure 4A provides a schematic diagram outlining the formation of skeletal muscle organoid (SkMO) cells. SkMO cells generally encompass SkMO 30D growth cells and SkMO 30D Diff cells. SkMO 30D growth cells are skeletal muscle organoid cells grown for 30 days in spin culture medium. SkMO 30D Diff cells are skeletal muscle organoid cells grown for 20 days in spin culture medium and then for 10 days in differentiation medium (referred to herein as satellite cells, which do not exist in nature). Figure 4B shows SkMO 30D growth cells and SkMO 30D Diff cells. Figure 4C shows that SkMO cells express Pax7 (an important transcription factor for endogenous satellite cells). The transcriptional profiles of SkMOs are examined by RNA-seq, and their ability to cluster in state space is evaluated using the multidimensional scaling plot shown in Figure 4D. SkMO GFP+ cells (green and blue) approximate endogenous satellite cells (red) in terms of transcriptional signature. SkMO D30 growth cells (blue) are shown to be more proliferative than SkMO D30 Diff cells (green), and SkMO D30 Diff cells cluster near endogenous satellite cells (red). Figure 4E provides a global heatmap and dendrogram that further support the data provided in Figure 4D. [Figure 5A-B]Figures 5A–5D provide biomarker and phenotypic data demonstrating the similarity of skeletal muscle organoid (SkMO)-derived cells to endogenous satellite cells. Figure 5A shows the results of an EdU uptake assay (48-hour treatment) showing that myoblasts divide and take up EdU, while endogenous satellite cells do not. SkMO-derived cells are considerably more quiescent than myoblasts and are very similar to endogenous satellite cells (purple) (SkMO D30 Diff conditions (red)) (Figure 5B). Figure 5C provides an in vitro assay to predict satellite cell function. Satellite cells cultured as single cells in a 96-well suspension plate form clones, whereas myoblasts do not. This is similar to the classic cloning assay of adult stem cells. Figure 5D shows the size of each clone derived from a given cell type. SkMO-derived cells are more similar to endogenous satellite cells. [Figure 5C-D] Figures 5A–5D provide biomarker and phenotypic data demonstrating the similarity of skeletal muscle organoid (SkMO)-derived cells to endogenous satellite cells. Figure 5A shows the results of an EdU uptake assay (48-hour treatment) showing that myoblasts divide and take up EdU, while endogenous satellite cells do not. SkMO-derived cells are considerably more quiescent than myoblasts and are very similar to endogenous satellite cells (purple) (SkMO D30 Diff conditions (red)) (Figure 5B). Figure 5C provides an in vitro assay to predict satellite cell function. Satellite cells cultured as single cells in a 96-well suspension plate form clones, whereas myoblasts do not. This is similar to the classic cloning assay of adult stem cells. Figure 5D shows the size of each clone derived from a given cell type. SkMO-derived cells are more similar to endogenous satellite cells. [Figure 6A]Figures 6A–6F provide in vivo data demonstrating that skeletal muscle organoid (SkMO) cells engraft and rearrange after transplantation into injured skeletal muscle. Figure 6A provides a schematic diagram showing a method for engrafting and rearranging SkMO cells after transplantation. Figures 6B–6C show that bioluminescence (BLI) signals originate from SkMO-treated legs, whereas cells in myoblast-treated legs do not provide sufficient BLI signals (Figure 6B). This was quantified over a 21-day time course (D2, D7, D14, and D21), and statistics are included (Figure 6C). Figure 6D shows fibers derived from myoblasts and donor-derived SkMO D30 Diff cells. Figure 6E provides quantification of engraftment shown in Figure 6D. Figure 6F shows rearrangement of satellite cell niches by SkMO-derived cells after transplantation compared to myoblasts. Figure 6G provides quantification of the rearrangement assay (*Note: SkMO cells are not currently significant). [Figure 6B-C] Figures 6A–6F provide in vivo data demonstrating that skeletal muscle organoid (SkMO) cells engraft and rearrange after transplantation into injured skeletal muscle. Figure 6A provides a schematic diagram showing a method for engrafting and rearranging SkMO cells after transplantation. Figures 6B–6C show that bioluminescence (BLI) signals originate from SkMO-treated legs, whereas cells in myoblast-treated legs do not provide sufficient BLI signals (Figure 6B). This was quantified over a 21-day time course (D2, D7, D14, and D21), and statistics are included (Figure 6C). Figure 6D shows fibers derived from myoblasts and donor-derived SkMO D30 Diff cells. Figure 6E provides quantification of engraftment shown in Figure 6D. Figure 6F shows rearrangement of satellite cell niches by SkMO-derived cells after transplantation compared to myoblasts. Figure 6G provides quantification of the rearrangement assay (*Note: SkMO cells are not currently significant). [Figure 6D-G]Figures 6A–6F provide in vivo data demonstrating that skeletal muscle organoid (SkMO) cells engraft and rearrange after transplantation into injured skeletal muscle. Figure 6A provides a schematic diagram showing a method for engrafting and rearranging SkMO cells after transplantation. Figures 6B–6C show that bioluminescence (BLI) signals originate from SkMO-treated legs, whereas cells in myoblast-treated legs do not provide sufficient BLI signals (Figure 6B). This was quantified over a 21-day time course (D2, D7, D14, and D21), and statistics are included (Figure 6C). Figure 6D shows fibers derived from myoblasts and donor-derived SkMO D30 Diff cells. Figure 6E provides quantification of engraftment shown in Figure 6D. Figure 6F shows rearrangement of satellite cell niches by SkMO-derived cells after transplantation compared to myoblasts. Figure 6G provides quantification of the rearrangement assay (*Note: SkMO cells are not currently significant). [Figure 7A-B] Figures 7A–7C provide human data demonstrating that human myoblasts form SkMO cells. SkMO cells formed from human myoblasts are Pax7 positive (Figure 7A). Figure 7B provides a schematic diagram showing a method for generating SkMO cells from human myoblasts and transplanting those cells. Figure 7C provides BLI quantification of human myoblasts. [Figure 7C] Figures 7A–7C provide human data demonstrating that human myoblasts form SkMO cells. SkMO cells formed from human myoblasts are Pax7 positive (Figure 7A). Figure 7B provides a schematic diagram showing a method for generating SkMO cells from human myoblasts and transplanting those cells. Figure 7C provides BLI quantification of human myoblasts. [Figure 8A]Figures 8A–8E identify genes that are upregulated and downregulated in endogenous satellite cells and SkMO cells. Multiplex RNA sequencing of cells was performed to identify genes that are upregulated and downregulated in endogenous satellite cells and SkMO cells compared to myoblasts. Many genes were identified that are >2 times upregulated (Figure 8A) and downregulated (Figure 8B) in endogenous satellite cells, SkMO D30 growth cells, and SkMO D30 Diff cells compared to myoblasts. These genes can be used to broadly define endogenous satellite cells. Genes involved in the standard pathway are highlighted in green, and genes involved in the p53 pathway are highlighted in yellow. (Note: Pax7 and Tgfbr3 were not identified by multiplex RNA sequencing and are therefore identified in red, but are known to be present in satellite cells and SkMO cells based on previously obtained data). In addition, many genes have been identified that are >2-fold upregulated (Figure 8C) and downregulated (Figure 8D) in SkMO D30 growth cells and SkMO D30 Diff cells compared to myoblasts, but are not present in endogenous satellite cells. These genes may be used to distinguish SkMO cells from endogenous satellite cells. Genes unique to SkMO cells compared to endogenous satellite cells may be involved in various pathways, including inhibition of Wnt signaling (highlighted in red) and / or proteasome activation (highlighted in purple). Figure 8E provides a Venn diagram demonstrating the overlap of genes that are upregulated and downregulated in endogenous satellite cells, SkMO D30 growth cells, and SkMO D30 Diff cells. Further identified pathways involved (Notch activation, Wnt inhibition, proteasome activation) have been identified. [Figure 8B]Figures 8A–8E identify genes that are upregulated and downregulated in endogenous satellite cells and SkMO cells. Multiplex RNA sequencing of cells was performed to identify genes that are upregulated and downregulated in endogenous satellite cells and SkMO cells compared to myoblasts. Many genes were identified that are >2 times upregulated (Figure 8A) and downregulated (Figure 8B) in endogenous satellite cells, SkMO D30 growth cells, and SkMO D30 Diff cells compared to myoblasts. These genes can be used to broadly define endogenous satellite cells. Genes involved in the standard pathway are highlighted in green, and genes involved in the p53 pathway are highlighted in yellow. (Note: Pax7 and Tgfbr3 were not identified by multiplex RNA sequencing and are therefore identified in red, but are known to be present in satellite cells and SkMO cells based on previously obtained data). In addition, many genes have been identified that are >2-fold upregulated (Figure 8C) and downregulated (Figure 8D) in SkMO D30 growth cells and SkMO D30 Diff cells compared to myoblasts, but are not present in endogenous satellite cells. These genes may be used to distinguish SkMO cells from endogenous satellite cells. Genes unique to SkMO cells compared to endogenous satellite cells may be involved in various pathways, including inhibition of Wnt signaling (highlighted in red) and / or proteasome activation (highlighted in purple). Figure 8E provides a Venn diagram demonstrating the overlap of genes that are upregulated and downregulated in endogenous satellite cells, SkMO D30 growth cells, and SkMO D30 Diff cells. Further identified pathways involved (Notch activation, Wnt inhibition, proteasome activation) have been identified. [Figure 8C]Figures 8A–8E identify genes that are upregulated and downregulated in endogenous satellite cells and SkMO cells. Multiplex RNA sequencing of cells was performed to identify genes that are upregulated and downregulated in endogenous satellite cells and SkMO cells compared to myoblasts. Many genes were identified that are >2 times upregulated (Figure 8A) and downregulated (Figure 8B) in endogenous satellite cells, SkMO D30 growth cells, and SkMO D30 Diff cells compared to myoblasts. These genes can be used to broadly define endogenous satellite cells. Genes involved in the standard pathway are highlighted in green, and genes involved in the p53 pathway are highlighted in yellow. (Note: Pax7 and Tgfbr3 were not identified by multiplex RNA sequencing and are therefore identified in red, but are known to be present in satellite cells and SkMO cells based on previously obtained data). In addition, many genes have been identified that are >2-fold upregulated (Figure 8C) and downregulated (Figure 8D) in SkMO D30 growth cells and SkMO D30 Diff cells compared to myoblasts, but are not present in endogenous satellite cells. These genes may be used to distinguish SkMO cells from endogenous satellite cells. Genes unique to SkMO cells compared to endogenous satellite cells may be involved in various pathways, including inhibition of Wnt signaling (highlighted in red) and / or proteasome activation (highlighted in purple). Figure 8E provides a Venn diagram demonstrating the overlap of genes that are upregulated and downregulated in endogenous satellite cells, SkMO D30 growth cells, and SkMO D30 Diff cells. Further identified pathways involved (Notch activation, Wnt inhibition, proteasome activation) have been identified. [Figure 8D]Figures 8A–8E identify genes that are upregulated and downregulated in endogenous satellite cells and SkMO cells. Multiplex RNA sequencing of cells was performed to identify genes that are upregulated and downregulated in endogenous satellite cells and SkMO cells compared to myoblasts. Many genes were identified that are >2 times upregulated (Figure 8A) and downregulated (Figure 8B) in endogenous satellite cells, SkMO D30 growth cells, and SkMO D30 Diff cells compared to myoblasts. These genes can be used to broadly define endogenous satellite cells. Genes involved in the standard pathway are highlighted in green, and genes involved in the p53 pathway are highlighted in yellow. (Note: Pax7 and Tgfbr3 were not identified by multiplex RNA sequencing and are therefore identified in red, but are known to be present in satellite cells and SkMO cells based on previously obtained data). In addition, many genes have been identified that are >2-fold upregulated (Figure 8C) and downregulated (Figure 8D) in SkMO D30 growth cells and SkMO D30 Diff cells compared to myoblasts, but are not present in endogenous satellite cells. These genes may be used to distinguish SkMO cells from endogenous satellite cells. Genes unique to SkMO cells compared to endogenous satellite cells may be involved in various pathways, including inhibition of Wnt signaling (highlighted in red) and / or proteasome activation (highlighted in purple). Figure 8E provides a Venn diagram demonstrating the overlap of genes that are upregulated and downregulated in endogenous satellite cells, SkMO D30 growth cells, and SkMO D30 Diff cells. Further identified pathways involved (Notch activation, Wnt inhibition, proteasome activation) have been identified. [Figure 8E]Figures 8A–8E identify genes that are upregulated and downregulated in endogenous satellite cells and SkMO cells. Multiplex RNA sequencing of cells was performed to identify genes that are upregulated and downregulated in endogenous satellite cells and SkMO cells compared to myoblasts. Many genes were identified that are >2 times upregulated (Figure 8A) and downregulated (Figure 8B) in endogenous satellite cells, SkMO D30 growth cells, and SkMO D30 Diff cells compared to myoblasts. These genes can be used to broadly define endogenous satellite cells. Genes involved in the standard pathway are highlighted in green, and genes involved in the p53 pathway are highlighted in yellow. (Note: Pax7 and Tgfbr3 were not identified by multiplex RNA sequencing and are therefore identified in red, but are known to be present in satellite cells and SkMO cells based on previously obtained data). In addition, many genes have been identified that are >2-fold upregulated (Figure 8C) and downregulated (Figure 8D) in SkMO D30 growth cells and SkMO D30 Diff cells compared to myoblasts, but are not present in endogenous satellite cells. These genes may be used to distinguish SkMO cells from endogenous satellite cells. Genes unique to SkMO cells compared to endogenous satellite cells may be involved in various pathways, including inhibition of Wnt signaling (highlighted in red) and / or proteasome activation (highlighted in purple). Figure 8E provides a Venn diagram demonstrating the overlap of genes that are upregulated and downregulated in endogenous satellite cells, SkMO D30 growth cells, and SkMO D30 Diff cells. Further identified pathways involved (Notch activation, Wnt inhibition, proteasome activation) have been identified. [Modes for carrying out the invention]

[0023] Detailed description of the invention Aspects of this disclosure relate to compositions, methods, kits and agents for generating satellite cells (hereinafter referred to as satellite cells not naturally occurring or non-native satellite cells) from at least one myoblast, and to satellite cells generated by said compositions, methods, kits and agents for use in cell therapies, assays and various treatment methods.

[0024] In vivo-produced satellite cells generated according to the methods described herein exhibit numerous advantages; for example, they possess the ability to rearrange satellite cell niches to enhance muscle regeneration. In addition, the generated satellite cells may provide a novel platform for cell therapy (e.g., transplantation into subjects requiring additional and / or functional satellite cells) and research.

[0025] definition For convenience, certain terms used herein, i.e., in this specification, the examples, and the appended claims, are grouped together herein. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the invention pertains.

[0026] As used herein, the term “somatic cell” refers to any cell that makes up the body of an organism, as opposed to germline cells. In mammals, germline cells (also known as “gametes”) are sperm and eggs that fuse during fertilization to produce a cell called a zygote (from which the entire mammalian embryo develops). All other cell types in the body of a mammal (except sperm and eggs, the cells from which they are made (germ cells), and undifferentiated stem cells) are somatic cell types; viscera, skin, bone, blood, and connective tissue are all composed of somatic cells. In some embodiments, somatic cells are “non-embryonic somatic cells,” meaning somatic cells that are not present in or obtained from an embryo and do not arise in vitro from the proliferation of such cells. In some embodiments, somatic cells are “adult somatic cells,” meaning cells that are present in or obtained from an organism or fetus other than an embryo, or arise in vitro from the proliferation of such cells.

[0027] As used herein, the term “adult cells” refers to cells found throughout the body after embryonic development.

[0028] The terms “precursor” or “progenitor” cells are used interchangeably herein and refer to cells that have a cellular phenotype that is more primitive than cells that can be produced by differentiation (i.e., they are in an earlier stage of developmental pathway or progression than fully differentiated cells). Often, progenitor cells also have significant or very high proliferative capacity. Depending on the developmental pathway and the environment in which the cells develop and differentiate, progenitor cells may give rise to multiple different differentiated cell types or a single differentiated cell type.

[0029] The term "phenotype" refers to one or more total biological characteristics that define a cell or organism under a particular set of environmental conditions and factors, regardless of the actual genotype.

[0030] As used herein, the term “pluripotency” refers to cells that have the ability to differentiate into one or more differentiated cell types, preferably into cell types characteristic of all three germ layers. Pluripotent cells are primarily characterized by their ability to differentiate into one or more cell types, preferably into all three germ layers, for example, using a nude mouse teratoma formation assay. Pluripotency can also be demonstrated by the expression of embryonic stem (ES) cell markers, but the preferred pluripotency test is the demonstration of the ability to differentiate into cells of each of the three germ layers. It should be noted that simply culturing such cells does not mean they will spontaneously become pluripotent. Reprogrammed pluripotent cells (e.g., iPS cells, as the term is defined herein) also feature long-term passage capability without loss of growth capacity, compared to primary cell parents, which generally have a very limited number of mitotic capacity in culture.

[0031] As used herein, the term “stem cell” refers to an undifferentiated cell capable of proliferation that gives rise to more progenitor cells capable of producing a large number of mother cells, and that gives rise to differentiated daughter cells or differentiateable daughter cells. The daughter cells themselves may be induced to proliferate and produce offspring that subsequently differentiate into one or more mature cell types, while also retaining one or more cells with parental developmental capacity. The term “stem cell” refers to a subset of precursors that, under certain conditions, has the ability or potential to differentiate into a more specialized or differentiated phenotype, and which, under certain conditions, retains the ability to proliferate substantially without differentiation. In one embodiment, the term stem cell generally refers to a naturally occurring mother cell whose offspring cells (offspring) often specialize in different directions by differentiation, for example by acquiring completely distinct characteristics, as they occur in the progressive diversification of embryonic cells and tissues. Cell differentiation is a complex process that typically occurs through many cell divisions. Differentiated cells may originate from pluripotent cells that themselves originate from pluripotent cells, etc. Each of these pluripotent cells can be considered a stem cell, but the range of cell types can vary considerably. Some differentiated cells also have the ability to produce cells with higher developmental potential. Such ability may be naturally occurring or can be artificially induced by treatment with various factors. In many biological cases, stem cells are also “multipotent” because they can produce offspring of more than one different cell type, but this does not require “stem status.” Self-renewal is another classic part of the stem cell definition, and when used herein, it is essential. Theoretically, self-renewal can occur by one of two main mechanisms. Stem cells may divide asymmetrically, with one daughter retaining stem status and the other daughter expressing some other different specific function and phenotype. Alternatively, some stem cells in a population may divide symmetrically into two stems, thus maintaining some stem cells in the population as a whole, while other cells in the population produce only differentiated offspring.Formally, cells that begin as stem cells can progress to a differentiated phenotype, but can also "revert" to the stem cell phenotype and re-express the stem cell phenotype (a term often referred to by those skilled in the art as "dedifferentiation," "reprogramming," or "retrodifferentiation"). As used herein, the term "pluripotent stem cells" includes embryonic stem cells, induced pluripotent stem cells, placental stem cells, and the like.

[0032] The terms “endogenous myosatellite cells” or “endogenous satellite cells” are used herein to refer to small mononuclear progenitor cells in mature muscle that are substantially devoid of cytoplasm. They are found sandwiched between the basement membrane and the myofibrous membrane (cell membrane) of individual muscle fibers and may be difficult to distinguish from the submyofibrous nucleus of the fiber. Endogenous satellite cells can differentiate and fuse to enhance existing muscle fibers and form new fibers. These cells correspond to the oldest known adult stem cell niche and are involved in normal muscle growth and regeneration after injury or disease. In uninjured muscle, the majority of endogenous satellite cells are quiescent; they do not differentiate and do not undergo cell division. In response to mechanical strain, endogenous satellite cells are activated. Activated endogenous satellite cells first proliferate as skeletal myoblasts before undergoing myogenic differentiation. Endogenous satellite cells are distinguishable from naturally occurring satellite cells of the present invention by many of the features described herein.

[0033] As used herein, “satellite cells,” “satellite cells not found in nature,” “non-native satellite cells,” and “SkMO 30D Diff cells” all refer to satellite cells produced by the dedifferentiation of myoblasts. Satellite cells may exhibit one or more features shared with endogenous satellite cells, including, but not limited to, the ability to rearrange satellite cell niches, the ability to promote muscle regeneration and exhibit appropriate expression of genetic markers, appropriate expression of glycoproteins, and the ability to expand in culture. However, satellite cells not found in nature are not identical to and are distinguishable from endogenous satellite cells as described herein (including distinctions based on gene expression).

[0034] As used herein, the term “proliferation” means the growth and division of cells. In some embodiments, as used herein with respect to cells, the term “proliferation” refers to a group of cells whose number can increase over a period of time.

[0035] As used herein, “induction,” “enhancement,” or “increase” of satellite cell proliferation means that satellite cells replicate at a faster rate and / or more frequently. In some embodiments of this and other embodiments described herein, satellite cell proliferation is increased by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 1x, 1.1x, 1.5x, 2x, 3x, 4x, 5x, 10x, 50x, 100x or more compared to an untreated control. The percentage or multiplier of satellite cell proliferation may be determined by measuring the number of replicated satellite cells during contact with the compounds described herein, compared to a control in which satellite cells have not been in contact with the compounds. The increase in proliferation may also be based on the ratio of replicated cells to the total number of cells in each treated and untreated control. In some embodiments, the total number of cells in the treated and untreated controls is used to determine the proliferation. Satellite cell proliferation can be determined using the BrdU incorporation method described in U.S. Patent Application Publication No. 2009 / 0136481 (the contents of which are incorporated herein by reference).

[0036] In the context of cell-on-organ development, the adjectives “differentiated” or “in the process of differentiation” are relative terms, meaning that a “differentiated cell” is a cell that has progressed further along the developmental pathway than the cell being compared. Thus, stem cells can differentiate into lineage-restricting progenitor cells (e.g., ectoderm stem cells), which can then further differentiate into other types of progenitor cells (e.g., neuroectoderm cells), and subsequently into final-stage differentiated cells (which may or may not retain the ability to proliferate further and play a characteristic role in a particular tissue type).

[0037] The term “differentiated cell” means any primary cell that is not pluripotent in its native form, as the term is defined herein. In other words, the term “differentiated cell” refers to a more specialized cell type derived from a less specialized cell type (e.g., stem cells, e.g., induced pluripotent stem cells) in the cell differentiation process.

[0038] In contrast, the adjectives “dedifferentiated” or “dedifferentiated” are relative terms meaning that a “dedifferentiated cell” is a cell that has reverted to an earlier version of the cell in its developmental pathway. The term “dedifferentiated cell” means any cell that is a less specialized cell type derived from a more specialized cell type. For example, myoblasts can dedifferentiate into satellite cells.

[0039] As used herein, the term “cell culture medium” (also referred to herein as “culture medium” or “culture medium”) refers to a medium for culturing cells that contains nutrients that maintain cell viability and support cell growth. A cell culture medium may contain, in appropriate combinations, any of the following: salts, buffers, amino acids, glucose or other sugars, antibiotics, serum or serum substitutes, and other components, such as peptide growth factors. Cell culture media commonly used for specific cell types are known to those skilled in the art.

[0040] The term "nutrient medium" refers to a culture medium for cells that contains nutrients that promote growth. Nutrient media may contain, in appropriate combinations, any of the following: isotonic saline, buffer, amino acids, antibiotics, serum or serum substitutes, and exogenous additives. "Conditioned medium" is prepared by culturing a first cell population in the medium and then harvesting the medium. The conditioning medium can then be used (along with what the cells have secreted into the medium) to support the growth of a second cell population.

[0041] Various culture media referred to herein include myoblast medium, spin medium, and differentiation medium. The term "differentiation medium" refers to a medium for both the differentiation and dedifferentiation of cells.

[0042] As used herein, the term “drug” means any compound or substance, e.g., small molecules, nucleic acids, polypeptides, peptides, drugs, ions, etc. “Drug” can be any chemical substance, entity or part, e.g., synthetic and naturally occurring proteinaceous and non-proteinaceous entities, e.g., synthetic and naturally occurring proteinaceous and non-proteinaceous entities. In some embodiments, the drug is nucleic acid, nucleic acid analogue, protein, antibody, peptide, aptamer, nucleic acid oligomer, amino acid or carbohydrate, e.g., proteins, oligonucleotides, ribozymes, DNAzymes, glycoproteins, siRNA, lipoproteins, aptamers, and their modifications and combinations, e.g., proteins, oligonucleotides, ribozymes, DNAzymes, glycoproteins, siRNA, lipoproteins, aptamers, and their modifications and combinations. In certain embodiments, the drug is a small molecule having a chemical moiety. For example, the chemical moiety includes substituted or unsubstituted alkyl, aromatic or heterocyclyl moieties, e.g., macrolides, leptomycin and related natural products or analogues. The compound may be known to have the desired activity and / or properties, or may be selected from a library of various compounds.

[0043] As used herein, the term “contact” (i.e., contact between at least one embryoid body or its precursor and a differentiation medium or drug) is intended to include incubating the differentiation medium and / or drug and the cells together in vitro (e.g., adding the differentiation medium or drug to cells in culture). In some embodiments, the term “contact” is not intended to include in vivo exposure of cells to compounds disclosed herein that may be naturally present in the subject (i.e., exposure that may occur as a result of natural physiological processes). The step of contacting at least one myoblast or its precursor with a differentiation medium or drug, as in embodiments relating to the production of satellite cells, can be carried out in any suitable manner. For example, the cells may be treated in adherent culture or suspension culture. In some embodiments, the cells are treated under conditions that promote the formation of skeletal muscle organoids (also referred to herein as myosspheres). This disclosure intends any conditions that promote the formation of skeletal muscle organoids. Examples of conditions that promote the formation of skeletal muscle organoids include, but are not limited to, suspension cultures in low-adhesion tissue culture plates, spinner flasks, and Aggrewell plates. In some embodiments, the inventors observed that skeletal muscle organoids remained stable in a culture medium containing 20% ​​serum (e.g., thermoinactivated fetal bovine serum).

[0044] Furthermore, it is understood that cells that have been in contact with a differentiation medium and / or drug may be stabilized or further differentiated by being simultaneously or subsequently in contact with another drug, such as another differentiation drug or environment.

[0045] The term "exogenous" refers to a substance present in a cell or organism other than its natural source. For example, the terms "exogenous nucleic acid" or "exogenous protein" refer to a nucleic acid or protein introduced into a biological system (e.g., a cell or organism) through a human-involved process, either not normally present or present in smaller quantities. If a substance is introduced into a cell, or into the cellular ancestor from which it is inherited, it would be considered exogenous. In contrast, the term "endogenous" refers to a substance that is native to the biological system.

[0046] The term “expression” refers to cellular processes that involve the production of RNA and proteins, and, if necessary, the secretion of proteins, such as transcription, translation, folding, modification, and processing, where applicable. “Expression products” include RNA transcribed from genes and polypeptides obtained by the translation of mRNA transcribed from genes.

[0047] As used herein, the terms “isolated” or “partially purified” refer, in the case of nucleic acids or polynucleotides, to nucleic acids or polynucleotides isolated from at least one other component (e.g., nucleic acids or polynucleotides) that would be present with the nucleic acid or polynucleotide in its natural source and / or, in the case of secreted polypeptides, if expressed or secreted by cells. Chemically synthesized nucleic acids or polynucleotides, or those synthesized using in vitro transcription / translation, are considered “isolated.”

[0048] As used herein, the term “isolated cells” refers to cells or offspring of such cells that have been removed from the organism in which they are normally found. Where necessary, the cells are cultured in vitro, for example, in the presence of other cells. Where necessary, the cells are then introduced into a second organism or reintroduced into the organism from which they (or cells derived therefrom) were isolated.

[0049] As used herein, the term “isolated population” in relation to an isolated population of cells refers to a population of cells that have been taken out and separated from a mixed or heterogeneous population of cells. In some embodiments, the isolated population is a substantially pure population of cells compared to the heterogeneous population from which the cells were isolated or concentrated.

[0050] The terms “concentrated” and “concentrated” are used interchangeably herein and mean that the yield (percentage) of one type of cell is increased by at least 10% compared to the percentage of that type of cell in the initial culture or preparation.

[0051] The term “modulate” is used in accordance with its use in the art, meaning to cause or facilitate a qualitative or quantitative change, alteration or modification of a process, pathway, or phenomenon of interest. Such changes may include, but are not limited to, an increase, decrease, or alteration of the relative strength or activity of different components or branches of the process, pathway, or phenomenon. A “modulator” is a drug that causes or facilitates a qualitative or quantitative change, alteration or modification of a process, pathway, or phenomenon of interest.

[0052] As used herein, the term "DNA" is defined as deoxyribonucleic acid.

[0053] As used herein, “marker” is used to describe the characteristics and / or phenotype of a cell. A marker may be used to select cells containing the characteristics of interest. Markers will vary from cell to cell. Markers are characteristic regardless of the morphological, functional, or biochemical (enzymatic) characteristics of cells of a particular cell type or molecules expressed by the cell type. Preferably, such markers are proteins, and more preferably, have epitopes to antibodies or other binding molecules available in the art. However, markers may consist of any molecule found in a cell, for example, but not limited to proteins (peptides and polypeptides), lipids, polysaccharides, nucleic acids, and steroids. Examples of morphological characteristics or traits include, but not limited to, shape, size, and nucleus-to-cytoplasmic ratio. Examples of functional characteristics or traits include, but not limited to, the ability to adhere to a particular substrate, the ability to take up or expel a particular dye, the ability to migrate under particular conditions, and the ability to differentiate or dedifferentiate along a particular lineage. Markers may be detected by any method available to those skilled in the art. Markers may also be the absence of morphological characteristics, or the absence of proteins, lipids, etc. A marker can be a combination of a unique panel of features, such as the presence or absence of polypeptides and other morphological features.

[0054] The term “selection marker” refers to a gene, RNA, or protein that, when expressed, confers to a cell the expression of a specific protein, which can be used as a basis for a selective phenotype, such as resistance to cytotoxic or cell growth inhibitors (e.g., antibiotic resistance), trophotrophy, or distinguishing cells that express a protein from those that do not. Proteins whose expression can be easily detected, such as fluorescent or luminescent proteins, or enzymes that act on a substrate to produce a colored, fluorescent, or luminescent substance (“detectable markers”), constitute a subset of selection markers. The presence of selection markers linked to native expression regulatory elements of genes that are normally selectively or exclusively expressed in pluripotent cells makes it possible to identify and select somatic cells that have been reprogrammed to a pluripotent state. Various selection marker genes may be used, such as the neomycin resistance gene (neo), the puromycin resistance gene (puro), guanine phosphoribosyltransferase (gpt), dihydrofolate reductase (DHFR), adenosine diaminase (ada), puromycin-N-acetyltransferase (PAC), hygromycin resistance gene (hyg), multidrug resistance gene (mdr), thymidine kinase (TK), hypoxanthine-guanine phosphoribosyltransferase (HPRT), and hisD gene. Detectable markers include green fluorescent protein (GFP), blue, sapphire, yellow, red, orange, and cyan fluorescent proteins, and any variant thereof. Luminescent proteins, such as luciferases (e.g., firefly or sea urchin luciferase), are also useful. As will be apparent to those skilled in the art, as used herein, the term “selection marker” may refer to a gene or a gene expression product, such as an encoded protein.

[0055] In some embodiments, a selection marker confers a proliferation and / or survival advantage to cells expressing it compared to cells that do not express it or express it at significantly low levels. Such a proliferation and / or survival advantage typically arises when the cells are maintained under specific conditions, i.e., “selective conditions.” To ensure effective selection, the cell population may be maintained under conditions and for a sufficient period of time such that cells that do not express the marker do not proliferate and / or survive and are eliminated from the population or reduced to a negligible number. The process of selecting cells that express a marker conferring a proliferation and / or survival advantage by maintaining a cell population under selective conditions to almost completely eliminate cells that do not express the marker is referred to herein as “positive selection,” and the marker is said to be “useful for positive selection.” Negative selection and markers useful for negative selection are also of interest to certain methods described herein. The expression of such markers confers a disadvantage in proliferation and / or survival to cells that express them compared to cells that do not express them or express them at significantly low levels (or, in other words, cells that do not express the markers are thought to have an advantage in proliferation and / or survival compared to cells that express them). Therefore, cells that express the markers are largely or completely eliminated from the cell population when maintained under selection conditions for a sufficient period of time.

[0056] The terms “subject” and “individual” are used interchangeably herein and refer to an animal from which cells can be obtained and / or to which treatment with cells described herein (including prophylactic treatment) is provided, e.g., human. In treatment of an infection, condition or disease condition specific to a particular animal, e.g., human subject, the term subject refers to that particular animal. Where used interchangeably herein, “non-human animal” and “non-human mammal” include mammals, e.g., rats, mice, rabbits, sheep, cats, dogs, cattle, pigs, and non-human primates. The term “subject” also encompasses any vertebrate, e.g., mammals, reptiles, amphibians, and fish, but not limited to mammals. However, advantageously, subject is a mammal, e.g., human or other mammals, e.g., domestic mammals, e.g., dogs, cats, horses, etc., or production mammals, e.g., cattle, sheep, pigs, etc.

[0057] When applied to isolated cells, terms such as “to treat,” “to treat,” and “treatment” include subjecting cells to any kind of process or condition, or performing any kind of operation or procedure on cells. When applied to subjects, terms such as “to treat,” “to treat,” and “treatment” refer to providing medical or surgical attention, care, or management to an individual. Typically, an individual is in a diseased or injured state, or has an increased disease risk compared to the average member of the population, and requires such attention, care, or management. It may include administering an effective amount of composition to a subject so that the subject exhibits a reduction in at least one symptom of a disease or improvement of the disease, e.g., a beneficial or desired clinical outcome. For the purposes of the present invention, beneficial or desired clinical outcomes include, but are not limited to, relief of one or more symptoms, whether detectable or undetectable; attenuation of the degree of disease; stabilization of the disease state (i.e., no worsening); delay or slowing of disease progression; improvement or reduction and remission (whether partial or complete) of the disease state. Treatment may refer to extending survival compared to the survival expected without treatment. Therefore, those skilled in the art will understand that treatment may improve a diseased condition but may not cure the disease. The term “treatment” includes prevention. Those who require treatment include those already diagnosed with a condition (e.g., muscle disorder or disease) and those who are likely to develop the condition due to genetic susceptibility or other factors.

[0058] The term "tissue" refers to a group or layer of specialized cells that perform specific functions together. The term "tissue-specific" refers to a source of cells that originate from a particular tissue.

[0059] The terms “decrease,” “decrease,” “decrease,” “decrease,” or “inhibit” are all used herein to generally mean a decrease of a statistically significant amount. However, to avoid misunderstanding, “decrease,” “decrease,” “decrease,” or “inhibit” means a decrease of at least 10% compared to a reference level, for example, a decrease of at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least 60%, at least about 70%, at least about 80%, or at least about 90% compared to a reference level, or a decrease up to 100% (i.e., a level of non-existence compared to a reference sample), or any decrease between 10% and 100%.

[0060] The terms “increased,” “enhance,” “boost,” or “activate” are all used herein to mean an increase of a statistically significant amount; to avoid any misunderstanding, the terms “increased,” “enhance,” “boost,” or “activate” mean an increase of at least 10% compared to a reference level, for example, an increase of at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% compared to a reference level, or an increase up to 100%, or any increase between 10% and 100%, or an increase of at least about 2 times, at least about 3 times, at least about 4 times, at least about 5 times, or at least about 10 times compared to a reference level, or any increase between 2 times and 10 times or more.

[0061] The terms "statistically significant" or "significantly significant" refer to statistical significance, generally meaning that the concentration of a marker is two standard deviations (2SD) lower or less than the normal concentration. The aforementioned terms refer to statistical evidence that there is a difference. It is defined as the probability of making a decision to reject the null hypothesis if the null hypothesis is indeed true. The decision is often made using a p-value.

[0062] As used herein, the terms “contains” or “includes” are used in reference to compositions, methods and their components that are essential to the present invention, but include the inclusion of unspecified elements, whether essential or not.

[0063] As used herein, the term “essentially derived from” refers to elements required for a given embodiment. The term allows for the presence of further elements that do not substantially affect the basic, novel, or functional features of that embodiment of the invention.

[0064] The term "consisting of" refers to the compositions, methods, and their respective components described herein, and is exclusive of any elements not listed in the description of the embodiments.

[0065] Where used herein and in the appended claims, the singular forms “a,” “an,” and “the” include multiple references unless otherwise clearly indicated in the context. Thus, for example, a reference to “method” includes one or more methods and / or processes of the kinds described herein, and / or this would be apparent to those skilled in the art by reading this disclosure, etc.

[0066] Cloning and cell culture Exemplary molecular genetics and genetic engineering methods that may be used in the techniques described herein can be found, for example, in the latest editions of Molecular Cloning: A Laboratory Manual (Sambrook et al., Cold Spring Harbor); Gene Transfer Vectors for Mammalian Cells (Miller & Calos eds.); and Current Protocols in Molecular Biology (FMAusubel et al., eds., Wiley & Sons). Cell biology, protein chemistry, and antibody techniques can be found, for example, in Current Protocols in Protein Science (JE Colligan et al., eds., Wiley & Sons); Current Protocols in Cell Biology (JS Bonifacino et al., Wiley & Sons); and Current Protocols in Immunology (JE Colligan et al., eds., Wiley & Sons). Exemplary reagents, cloning vectors, and kits for genetic manipulation can be commercially available, for example, from BioRad, Stratagene, Invitrogen, ClonTech, and Sigma-Aldrich Co.

[0067] Appropriate cell culture methods include, for example, Culture of Animal Cells: A Manual of Basic Technique (RIFreshney). ed.,Wiley&Sons);General Techniques of Cell Culture(MAHarrison&I.F.Rae,Cambridge Univ.Press) and Embryonic Stem Cells:Methods and Protocols(K.Turksen ed.,Humana This can be found in the latest edition of the Press. Suitable tissue culture supplies and reagents are commercially available from, for example, Gibco / BRL, Nalgene-Nunc International, Sigma Chemical Co., and ICN Biomedicals.

[0068] Pluripotent stem cells can be continuously cultured by those skilled in the art using culture conditions that promote proliferation without promoting differentiation. An exemplary serum-containing ES medium consists of 80% DMEM (e.g., Knock-Out DMEM, Gibco), 20% either standard fetal bovine serum (FBS, Hyclone) or a serum substitute (International Publication No. 98 / 30679), 1% non-essential amino acids, 1 mM L-glutamine, and 0.1 mM β-mercaptoethanol. Immediately before use, human bFGF is added at 4 ng / mL (International Publication No. 99 / 20741, Geron Corp.). Traditionally, ES cells are cultured on a layer of feeder cells, typically fibroblasts derived from embryonic or fetal tissue.

[0069] Alternatively, pluripotent SCs can be maintained in an undifferentiated state without the use of feeder cells. The feeder-free culture environment includes a suitable cell substrate, particularly an extracellular matrix, such as MATRIGEL® (a gelatinous protein mixture) or laminin. Typically, enzymatic digestion is stopped before the cells are completely dispersed (approximately 5 minutes with collagenase IV). Then, clumps of approximately 10 to 2,000 cells are plated directly onto the substrate without further dispersion.

[0070] Generation of satellite cells Aspects of this disclosure relate to the generation of satellite cells (e.g., skeletal muscle cells). Generally, satellite cells produced according to the methods disclosed herein, but not limited to, exhibit several functional satellite cell characteristics, including the ability to rearrange satellite cell niches, the ability to promote muscle regeneration, and the ability to show appropriate expression of genetic markers, and are capable of large-scale proliferation in culture.

[0071] Satellite cells can be produced according to any suitable culture protocol or set of culture protocols for dedifferentiating myoblasts to a desired dedifferentiation stage. In some embodiments, satellite cells are produced by culturing at least one myoblast for a certain period of time under conditions suitable for dedifferentiation into satellite cells. In some embodiments, satellite cells are a substantially pure population of satellite cells.

[0072] In certain embodiments, myoblasts are cultured in myoblast medium. Myoblasts may be cultured on a microplate (e.g., a collagen-coated dish) in the medium. In some embodiments, the myoblast medium contains a nutrient medium (e.g., Ham F10), fetal bovine serum (e.g., 20% heat-inactivated fetal bovine serum), basic fibroblast growth factor, non-essential amino acids, and glutamax. Myoblasts may be cultured on a microplate for 1–30 days, 1–15 days, 1–10 days, 1–5 days, or about 4 days. In some embodiments, myoblasts are cultured on a microplate until they reach 50–100 confluence, 75–90 confluence, or about 80 confluence.

[0073] In some embodiments, myoblasts are isolated from the skeletal muscle of a subject. For example, myoblasts may be isolated from the skeletal muscle of humans or non-human animals, such as rats, mice, rabbits, sheep, cats, dogs, cattle, pigs, and non-human primates. Alternatively, myoblasts may be embryonic muscle cells.

[0074] In certain embodiments, myoblasts are cultured in myoblast medium until they reach about 80 confluence, and then seeded in a spinner flask, spheroid plate, etc. In some embodiments, the spinner flask is seeded at a density of about 500,000 cells / ml to about 1,500,000 cells / ml, or about 750,000 cells / ml to about 1,250,000 cells / ml, or about 1,000,000 cells / ml. The myoblasts are cultured in a spinner flask in spin medium. In some embodiments, the spin medium contains a basal medium (e.g., DMEM:F12), fetal bovine serum (e.g., 20% heat-inactivated fetal bovine serum), basic fibroblast growth factor, non-essential amino acids, and glutamax. The myoblasts may be cultured in the spinner flask for at least 10 days, at least 20 days, at least 30 days, 10–30 days, 10–20 days, or in some embodiments, 20 days. The resulting skeletal muscle organoids or myosspheres are composed of a combination of Pax7+ and MyHC+ cells.

[0075] Myoblasts cultured in a spinner flask in spin medium form three-dimensional spheres (e.g., myospheres or skeletal muscle organoids). In some embodiments, the skeletal muscle organoids contain differentiated skeletal muscle cells and proliferative myofiocytes. Once the skeletal muscle organoids are formed, the medium may be changed to a differentiation medium. In some embodiments, the differentiation medium is a serum-free medium. The differentiation medium may contain a nutrient medium (e.g., DMEM), non-essential amino acids, and glutamax. In some embodiments, the skeletal muscle organoids are cultured in the differentiation medium for at least 1 day, at least 5 days, at least 10 days, 1–10 days, 1–30 days, 10–30 days, 10–20 days, or 10 days in some embodiments. During culture in the differentiation medium, the proliferative myofiocytes present in the skeletal muscle organoids become quiescent and revert to a satellite cell state. The satellite cells can then be isolated from the medium.

[0076] In some embodiments, satellite cells are capable of expanding and growing in culture. In some embodiments, the generated satellite cells are contacted with compounds to increase satellite cell proliferation. Methods for inducing, enhancing, or increasing satellite cell proliferation include contacting satellite cells with compounds selected from the group consisting of kinase inhibitors, G protein-coupled receptor (GPCR) modulators, epigenetic modifiers, histone deacetylase (HDAC) modulators, Hedgehog signaling pathway modulators, neuropeptides, dopamine receptor modulators, serotonin receptor modulators, histamine receptor modulators, adenosine receptor agonists, ionophores, ion channel modulators, gamma-secretase modulators, corticosteroids, and any combination thereof. Examples of growth enhancers, but not limited to, those described in International Patent Application PCT / US2017 / 016099 (which is incorporated herein by reference).

[0077] Satellite cells In some embodiments, the present disclosure provides functional satellite cells. In some embodiments, the methods of the present invention exhibit one or more features, including, but not limited to, the ability to rearrange satellite cell niches, the ability to promote muscle regeneration, the appropriate expression of genetic markers, the appropriate expression of glycoproteins, and enable the generation of satellite cells that can be expanded and proliferated in culture.

[0078] In some embodiments, the presence of satellite cells produced by the dedifferentiation of myoblasts by exposure to at least one differentiation medium can be confirmed using any means common to those skilled in the art. In some embodiments, such satellite cells can be identified by selective gene expression markers. In some embodiments, the method may include detecting the positive expression (e.g., presence) of the marker in the satellite cells. In some embodiments, the marker can be detected using a reagent. The reagent for the marker may be, for example, an antibody against the marker, or a primer for an RT-PCR or PCR reaction, such as a semi-quantitative or quantitative RT-PCR or PCR reaction. Such markers can be used to assess whether satellite cells have been produced. Antibodies or other detection reagents may be linked to labels for use in detection, such as radiolabeling, fluorescent (e.g., GFP) labeling, or colorimetric labeling. If the detection reagent is a primer, it may be supplied as a dry preparation, such as a lyophilized preparation, or as a solution.

[0079] The progression of at least one myoblast or its precursor to satellite cells can be monitored by determining the expression of markers characteristic of satellite cells. In some processes, the expression of a particular marker is determined by detecting its presence or absence. Alternatively, the expression of a particular marker can be determined by measuring the level at which the marker is present in cells in a cell culture or cell population. In certain processes, the expression of markers characteristic of satellite cells, as well as the absence of significant expression of markers characteristic of myoblasts or their precursors, can be determined.

[0080] As described with respect to monitoring the production of satellite cells from myoblasts, qualitative or semi-quantitative techniques, such as blot transcription and immunocytochemistry, may be used to measure marker expression using methods generally known to those skilled in the art. Alternatively, marker expression may be precisely quantified using techniques such as quantitative PCR by methods commonly known in the art.

[0081] In some embodiments, the generated satellite cells express one or more gene expression markers selected from the core myogenic genes Pax7 and Myf5. Alternatively, the generated satellite cells do not express the master transcription factor of skeletal muscle MyoD.

[0082] In some embodiments, or in addition, satellite cells may be characterized by expressing one or more markers selected from the quiescence-related genes Spry1, Nm1, Nfia, Fos, Dusp1, or any combination thereof. In some embodiments, satellite cells of a skeletal muscle organoid culture may be characterized by expressing one or more markers selected from the Notch signaling pathway Notch1, Notch2, Notch3, HeyL, Hey2, Hes, or any combination thereof. Satellite cells may express Pax7, Myf5, at least one quiescence-related gene, and at least one Notch signaling pathway gene.

[0083] In some embodiments, satellite cells express glycoproteins (for example, glycoproteins are typically present only in the satellite cell niche). Possible glycoprotein markers expressed by satellite cells include Bgn, TgfbrIII, Dag1, Dcn, and Gpc3.

[0084] In some embodiments, satellite cells express at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least fifteen, at least 20, at least 25, or at least 30 of the following genes: Dcn, Dpt, Heyl, Nr4a1, Col4a1, Pax7, Tgfbr3, Fos, Egr1, Dag1, Apoe, Spry1, Kat2b, Cav1, Cd82, Igfbp4, Thbs2, Igfbp 7, Serping1, Fosb, Zfp36, Nrep, Sepp1, Junb, Ier2, Col15a1, Adh1, Cp, Ramp2, Sparcl1, P2ry14, Pmp22, Igf1, S1pr3, Klf9, Col6a1, Txnip, Glul, Col6a 2, Ncald, Cc2d2a, Gpm6b, Timp3, Prkcdbp, Nfia, Gpr124, Psmb11, Ccdc80, Malat1, Zfp36l1, Gsn, Sdpr, Slc16a2, Olfml3, 1810026B05Rik, Adamts1, Olf ml2b, Gng11, Cd200, Fcgrt, Nav1, Lamc1, Atp2b4, Tagln, Mkl2, Col4a2, Htra3, Fxyd1, Ogn, Tgfb3, Tcp11l2, Spats2l, Capn6, Fgfr1, Mt2, Cdkn2c, Tln2, Ndrg2, Bhlhe40, Fstl1, Tcf4, Igsf3, Tmem123, Col3a1, Emp2, Itm2a, Xbp1, Crip1, Sparc, Cd9, Itgb5, Sdc1, Vcan, Bgn, Igfbp5, Col5a1, Zcchc24, Lum, P ostn, Sfrp4, Spon2, Col8a1, Wisp2, Adamts2, Colec12, Dnm3os, Plat, 2310010J17Rik, Ctsa, Ank, Dkk2, Fap, Mmp3, Dmp1, Col6a3, Pdgfrb, Col1a1, Mgp, Tmem100, Ctsc, Nr2f1, Cfh, Pdgfra, Dio2, Htra1, Dkk3, Islr, Foxs1, Tgfb2, Tgm2, Thbd, Uba7, Agtr2, Cyp1b1, Tspan15, Osr2, Fam129a, Plekha6, Mmp2,Arrdc3, Matn2, Mab21l1, Pvrl2, Adamtsl4, Cd248, Gulp1, Mfap2, Idua, Cspg4, Spock1, Mfap4, Hexb, Cilp, Pold4, Crebrf, Col14 a1, Tmem42, Nedd9, Rcn3, Sept8, Pcolce, Edil3, Nupr1, Phf17, Plin3, Aebp1, Hmgcs1, P4ha1, Lama2, Csrp2, Rnf167, H1f0, Fn1, Mm p14, Cpq, Mrc2, Ifngr1, St3gal5, Fbn1, Sdc2, Adcy7, Ckm, Hexa, Capg, Klhl13, Loxl1, Fdft1, Ifi27l1, Rbp1, Nr2f2, Cst3, Pxdn, Ecm1, Mybpc1, Igf2r, Serpinh1, Mt1, Lpar1, Atraid, Scd2, Dap, Ppic, App, Lamp2, Col5a2, Gpc1, Hspa5, Pmepa1, Laptm4a, and Lrp1. In some embodiments, the expressed genes were upregulated at least 1.5 times, at least 2 times, at least 2.5 times, or at least 3 times compared to myoblasts.

[0085] In some embodiments, satellite cells express at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least fifteen, at least 20, at least 25, or at least 30 of the following genes: Dcn, Dpt, Heyl, Nr4a1, Col4a1, Pax7, Tgfbr3, Fos, Egr1, Dag1, Apoe, Spry1, Kat2b, Cav1, Cd82, Igfbp4, Thbs2, Igfbp7, Serping1, Fosb, Zfp36, Nrep, Sepp1, Junb, Ier2, Col15a1, Adh1, Cp, Ramp2, Sparcl1, P2ry14, Pmp22, Igf1, S1pr3, Klf9, C ol6a1, Txnip, Glul, Col6a2, Ncald, Cc2d2a, Gpm6b, Timp3, Prkcdbp, Nfia, Gpr124, Psmb11, Ccdc80, Malat 1, Zfp36l1, Gsn, Sdpr, Slc16a2, Olfml3, 1810026B05Rik, Adamts1, Olfml2b, Gng11, Cd200, Fcgrt, Nav1, L amc1, Atp2b4, Tagln, Mkl2, Col4a2, Htra3, Fxyd1, Ogn, Tgfb3, Tcp11l2, Spats2l, Capn6, Fgfr1, Mt2, Cdkn2c, Tln2, Ndrg2, Bhlhe40, Fstl1, Tcf4, Igsf3, Tmem123, Col3a1, Emp2, Itm2a, Xbp1, Crip1, Sparc, and Cd9. In some embodiments, the expressed genes are upregulated at least 1.5 times, at least 2 times, at least 2.5 times, or at least 3 times compared to myoblasts.

[0086] In some embodiments, satellite cells express at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least fifteen, at least 20, at least 25, or at least 30 of the following genes: Itgb5, Sdc1, Vcan, Bgn, Igfbp5, Col5a1, Zcchc24, Lum, Postn, Sfrp4, Spon2, Col 8a1, Wisp2, Adamts2, Colec12, Dnm3os, Plat, 2310010J17Rik, Ctsa, Ank, Dkk2, Fap, Mmp3, Dmp1, Col6a3, Pdgfrb, Col1a1, Mgp, Tmem 100, Ctsc, Nr2f1, Cfh, Pdgfra, Dio2, Htra1, Dkk3, Islr, Foxs1, Tgfb2, Tgm2, Thbd, Uba7, Agtr2, Cyp1b1, Tspan15, Osr2, Fam129a, Pl ekha6, Mmp2, Arrdc3, Matn2, Mab21l1, Pvrl2, Adamtsl4, Cd248, Gulp1, Mfap2, Idua, Cspg4, Spock1, Mfap4, Hexb, Cilp, Pold4, Creb rf, Col14a1, Tmem42, Nedd9, Rcn3, Sept8, Pcolce, Edil3, Nupr1, Phf17, Plin3, Aebp1, Hmgcs1, P4ha1, Lama2, Csrp2, Rnf167, H1f0, F n1, Mmp14, Cpq, Mrc2, Ifngr1, St3gal5, Fbn1, Sdc2, Adcy7, Ckm, Hexa, Capg, Klhl13, Loxl1, Fdft1, Ifi27l1, Rbp1, Nr2f2, Cst3, Pxdn, Ecm1, Mybpc1, Igf2r, Serpinh1, Mt1, Lpar1, Atraid, Scd2, Dap, Ppic, App, Lamp2, Col5a2, Gpc1, Hspa5, Pmepa1, Laptm4a, and Lrp1. In some embodiments, the expressed genes are upregulated at least 1.5 times, at least 2 times, at least 2.5 times, or at least 3 times compared to myoblasts.

[0087] In some embodiments, satellite cells express at least one of the following genes: Pax7, Tgfbr3, Fos, Spry1, Cav1, and Cd82. The expressed genes are at least 2-fold upregulated compared to myoblasts.

[0088] In some embodiments, satellite cells express one or more genes involved in Wnt pathway inhibition. In some embodiments, the genes involved in Wnt pathway inhibition are selected from the group consisting of Sfrp4, Wisp2, Dkk2, and Dkk3. In some embodiments, satellite cells express one or more genes involved in proteasome activation. In some embodiments, the genes involved in proteasome activation are selected from the group consisting of Ctsa, Ctsc, Idua, Hexb, Hexa, Igf2r, Lamp2, and Laptm4a.

[0089] In some embodiments, satellite cells downregulate the expression of at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least fifteen, at least 20, at least 25, or at least 30 of the following genes: Tspan9, Sema3e, Hspd1, Tipin, Rasa1, Sct, Mdm2, Csf1, Ms tn, Alox5, Ckap2, Camk1g, Mcpt8, Ccng1, Tuba1c, Rps27l, Hmga2, Ccnb1, Itga6, Nasp, Dynap, Myof, Epha2, Gtse1, Lce1g, Cd24a , Lgals3, Steap1, Ankrd1, Hes6, Cox6b2, Gm15772, S100a6, Col18a1, Steap2, Smyd4, Tmem171, Cttn, Car3, Rrm2, Asns, Chrna1, U gcg, Cdh2, Myod1, Tpm1, Pa2g4, Fabp5, Mcm4, Rrm1, Ftl1, Aaas, Tmsb10, Lrrn1, Ddx21, Odc1, Tmem55a, Prdx6, Sox11, Cited2, Dd x39, Slc44a2, Bax, Gal, Rbmxl1, Mak16, Nap1l1, Dctpp1, Nme1, Ccnd1, Cks1b, Myl9, Prrc2c, Set, Hnrnpf, Ppp1r14b, Ran, Tuba1 b, Rbm3, Hsp90aa1, Nme2, Hspe1, Snrpg, Npm1, Reln, Msln, Sema6d, Itgb4, Ldhb, Nop58, Dkc1, Rpl12, Gchfr, Mgmt, Milr1, Fnbp1l, Anxa3, Tek, 2010204K13Rik, Selp, Efnb2, Cirh1a, Fgf21, Stmn2, Gpatch4, Peg3, Snhg8, Fbl, Ahcy, Ebna1bp2, Nop56, and Pcna. In some embodiments, expression is downregulated to at most 1 / 1.5, at most 1 / 2, at most 1 / 2.5, or at most 1 / 3 compared to myoblasts.

[0090] In some embodiments, satellite cells downregulate the expression of at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least fifteen, at least 20, at least 25, or at least 30 of the following genes: Tspan9, Sema3e, Hspd1, Tipin, Rasa1, Sct, Mdm2, Csf1, Mstn, Alox5, Ckap2, Camk1g, Mcpt8, Ccng1, Tuba1c, Rps27l, Hmga2, Ccnb1, Itga6, Nasp, Dynap, Myof, Epha2, Gtse1, Lce1g, Cd24a, Lgals3, Steap1, Ankrd 1, Hes6, Cox6b2, Gm15772, S100a6, Col18a1, Steap2, Smyd4, Tmem171, Cttn, Car3, Rrm2, Asns, Chrna1 , Ugcg, Cdh2, Myod1, Tpm1, Pa2g4, Fabp5, Mcm4, Rrm1, Ftl1, Aaas, Tmsb10, Lrrn1, Ddx21, Odc1, Tmem55 a, Prdx6, Sox11, Cited2, Ddx39, Slc44a2, Bax, Gal, Rbmxl1, Mak16, Nap1l1, Dctpp1, Nme1, Ccnd1, Cks1b, Myl9, Prrc2c, Set, Hnrnpf, Ppp1r14b, Ran, Tuba1b, Rbm3, Hsp90aa1, Nme2, Hspe1, Snrpg, and Npm1. In some embodiments, expression is downregulated to at most 1 / 1.5, at most 1 / 2, at most 1 / 2.5, or at most 1 / 3 compared to myoblasts.

[0091] In some embodiments, satellite cells downregulate the expression of at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least fifteen, at least 20, or at least 25 of the following genes: Reln, Msln, Sema6d, Itgb4, Ldhb, Nop58, Dkc1, Rpl12, Gchfr, Mgmt, Milr1, Fnbp1l, Anxa3, Tek, 2010204K13Rik, Selp, Efnb2, Cirh1a, Fgf21, Stmn2, Gpatch4, Peg3, Snhg8, Fbl, Ahcy, Ebna1bp2, Nop56, and Pcna. In some embodiments, expression is downregulated to at most 1 / 1.5, at most 1 / 2, at most 1 / 2.5, or at most 1 / 3 compared to myoblasts.

[0092] The present invention is not limited to the markers listed herein as satellite cell markers, and is understood to also encompass other markers, such as cell surface markers, antigens, and other gene products including ESTs, RNA (including microRNA and antisense RNA), DNA (including genes and cDNA) and parts thereof.

[0093] Markers characteristic of satellite cells include the expression of cell surface proteins or coding genes, the expression of intracellular proteins or coding genes, and cellular morphological features. Those skilled in the art will recognize that known immunofluorescence, immunochemistry, polymerase chain reaction, in-situ hybridization, Northern blot analysis, and chemical, radiochemical, or biological methods can readily confirm the presence or absence of satellite cell-specific features.

[0094] Aspects of this disclosure relate to isolated populations of satellite cells produced according to the methods described herein. Isolated populations of satellite cells can be obtained by dedifferentiating at least some myoblasts in a population into satellite cells.

[0095] Aspects of this disclosure include microcapsules containing isolated populations of cells described herein (e.g., satellite cells). Microcapsules are well known in the art. Appropriate examples of microcapsules are described in the literature (e.g., Olive et al., “Application of cell encapsulation for controlled delivery of biological therapeutics”, Advanced Drug Delivery Reviews (2013), dx.doi.org / 10.1016 / j.addr.2013.07.009; Hernandez et al., “Microcapsules and microcarriers for in situ cell delivery”, Advanced Drug Delivery Reviews 2010;62:711-730; Murua et al., “Cell microencapsulation technology: Towards clinical application”, Journal of Controlled Release 2008;132:76-83; and Zanin et al., “The development of encapsulated cell technologies as therapies for neurological and sensory diseases”, Journal of Controlled Release 2012;160:3-13). Microcapsules can be formulated in various ways. An exemplary microcapsule comprises an alginate core surrounded by a polycation layer covered by an alginate outer membrane. The polycation membrane forms a semipermeable membrane that provides stability and biocompatibility. Examples of polycations include, but are not limited to, poly-L-lysine, poly-L-ornithine, chitosan, lactose-modified chitosan, and photopolymerized biomaterials. In some embodiments, the alginate core is modified to produce a scaffold containing an alginate core having a covalently conjugated oligopeptide having an RGD sequence (arginine, glycine, aspartic acid), for example.In some embodiments, the alginate core is modified to produce covalently enhanced microcapsules having, for example, chemoenzymatically modified alginates with enhanced stability. In some embodiments, the alginate core is modified to produce membrane-mimicking films assembled by, for example, in-situ polymerization of acrylate-functionalized phospholipids. In some embodiments, the microcapsules consist of alginates enzymatically modified using epimerase. In some embodiments, the microcapsules include covalent linkages between adjacent layers of the microcapsule membrane. In some embodiments, the microcapsules include subsieve-sized capsules containing alginates coupled with a phenol moiety. In some embodiments, the microcapsules include a scaffold containing alginate-agarose. In some embodiments, satellite cells are modified with PEG before being encapsulated in the alginate. In some embodiments, isolated populations of cells, e.g., satellite cells, are encapsulated in photoreactive liposomes and alginates. It should be recognized that the alginates used in microcapsules can be replaced with, but are not limited to, PEG, chitosan, PES hollow fibers, collagen, hyaluronic acid, dextran with RGD, EHD and PEGDA, PMBV and PVA, PGSAS, agarose, agarose with gelatin, PLGA, and other suitable biomaterials including multilayer embodiments thereof.

[0096] Confirmation of the presence and identification of satellite cells The presence of satellite cells, such as non-naturally occurring satellite cells produced by the dedifferentiation of myoblasts as described herein, can be confirmed by any means common to those skilled in the art.

[0097] In some embodiments, the presence of satellite cell markers, such as chemically induced satellite cells, may be detected by detecting the presence or absence of one or more markers indicating endogenous satellite cells. In some embodiments, the method may include detecting the positive expression (e.g., presence) of satellite cell markers. In some embodiments, markers may be detected using reagents for detecting, for example, Pax7 and Myf5. Further markers to be detected include the Notch signaling pathway markers Notch1, Notch2, Notch3, HeyL, Hey2, and Hes1, as well as quiescence-related genes or markers Nm1, Nfia, Fos, Spry1, and Dusp1. Other markers to detect include Dcn, Dpt, Heyl, Nr4a1, Col4a1, Pax7, Tgfbr3, Fos, Egr1, Dag1, Apoe, Spry1, Kat2b, Cav1, Cd82, Igfbp4, Thbs2, Igfbp7, Serping1, Fosb, Zfp36, Nrep, Sepp1, Junb, Ier2, Col15a1, and Ad h1, Cp, Ramp2, Sparcl1, P2ry14, Pmp22, Igf1, S1pr3, Klf9, Col6a1, Txnip, Glul, Col6a2, Ncald, Cc2 d2a, Gpm6b, Timp3, Prkcdbp, Nfia, Gpr124, Psmb11, Ccdc80, Malat1, Zfp36l1, Gsn, Sdpr, Slc16a2, Ol fml3, 1810026B05Rik, Adamts1, Olfml2b, Gng11, Cd200, Fcgrt, Nav1, Lamc1, Atp2b4, Tagln, Mkl2, C ol4a2, Htra3, Fxyd1, Ogn, Tgfb3, Tcp11l2, Spats2l, Capn6, Fgfr1, Mt2, Cdkn2c, Tln2, Ndrg2, Bhlhe 40, Fstl1, Tcf4, Igsf3, Tmem123, Col3a1, Emp2, Itm2a, Xbp1, Crip1, Sparc, Cd9, Itgb5, Sdc1, Vcan, Bgn, Igfbp5, Col5a1, Zcchc24, Lum, Postn, Sfrp4, Spon2, Col8a1, Wisp2, Adamts2, Colec12, Dnm3os,Plat, 2310010J17Rik, Ctsa, Ank, Dkk2, Fap, Mmp3, Dmp1, Col6a3, Pdgfrb, Col1a1, Mgp, Tmem100 , Ctsc, Nr2f1, Cfh, Pdgfra, Dio2, Htra1, Dkk3, Islr, Foxs1, Tgfb2, Tgm2, Thbd, Uba7, Agtr2, Cyp 1b1, Tspan15, Osr2, Fam129a, Plekha6, Mmp2, Arrdc3, Matn2, Mab21l1, Pvrl2, Adamtsl4, Cd248 , Gulp1, Mfap2, Idua, Cspg4, Spock1, Mfap4, Hexb, Cilp, Pold4, Crebrf, Col14a1, Tmem42, Nedd9 , Rcn3, Sept8, Pcolce, Edil3, Nupr1, Phf17, Plin3, Aebp1, Hmgcs1, P4ha1, Lama2, Csrp2, Rnf16 7, H1f0, Fn1, Mmp14, Cpq, Mrc2, Ifngr1, St3gal5, Fbn1, Sdc2, Adcy7, Ckm, Hexa, Capg, Klhl13, Lo The group can be selected from xl1, Fdft1, Ifi27l1, Rbp1, Nr2f2, Cst3, Pxdn, Ecm1, Mybpc1, Igf2r, Serpinh1, Mt1, Lpar1, Atraid, Scd2, Dap, Ppic, App, Lamp2, Col5a2, Gpc1, Hspa5, Pmepa1, Laptm4a, and Lrp1.

[0098] The reagent for the marker may be, for example, an antibody against the marker, or a primer for an RT-PCR or PCR reaction, such as a semi-quantitative or quantitative RT-PCR or PCR reaction. Such markers may be used to assess whether satellite cells have been produced. Antibodies or other detection reagents may be linked to labels for use in detection, such as radiolabeling, fluorescent (e.g., GFP) labeling, or colorimetric labeling. If the detection reagent is a primer, it may be supplied as a dry preparation, such as a lyophilized preparation, or in solution.

[0099] In some embodiments, the presence of satellite cell markers may be determined by detecting the presence or absence of one or more markers indicating satellite cells. For example, satellite cells may be identified from endogenous satellite cells. In some embodiments, the method may include detecting the positive expression (e.g., presence) of satellite cell markers. The markers are Itgb5, Sdc1, Vcan, Bgn, Igfbp5, Col5a1, Zcchc24, Lum, Postn, Sfrp4, Spon2, Col8a1, Wisp2, Adamts2, Colec12, Dnm3os, Plat, 2310010J17Rik, Ctsa, Ank, Dkk2, Fap, Mmp3, Dmp1, Col6a3, Pdgfrb, Col1a1, Mgp, Tmem100 , Ctsc, Nr2f1, Cfh, Pdgfra, Dio2, Htra1, Dkk3, Islr, Foxs1, Tgfb2, Tgm2, Thbd, Uba7, Agtr2, Cyp1b1, Tspan15, O sr2, Fam129a, Plekha6, Mmp2, Arrdc3, Matn2, Mab21l1, Pvrl2, Adamtsl4, Cd248, Gulp1, Mfap2, Idua, Cspg4, Spo ck1, Mfap4, Hexb, Cilp, Pold4, Crebrf, Col14a1, Tmem42, Nedd9, Rcn3, Sept8, Pcolce, Edil3, Nupr1, Phf17, Pli n3, Aebp1, Hmgcs1, P4ha1, Lama2, Csrp2, Rnf167, H1f0, Fn1, Mmp14, Cpq, Mrc2, Ifngr1, St3gal5, Fbn1, Sdc2, Adc The group may be selected from y7, Ckm, Hexa, Capg, Klhl13, Loxl1, Fdft1, Ifi27l1, Rbp1, Nr2f2, Cst3, Pxdn, Ecm1, Mybpc1, Igf2r, Serpinh1, Mt1, Lpar1, Atraid, Scd2, Dap, Ppic, App, Lamp2, Col5a2, Gpc1, Hspa5, Pmepa1, Laptm4a, and Lrp1.

[0100] The progression of at least one myoblast or its precursor to satellite cells can be monitored by determining the expression of markers characteristic of satellite cells. In some processes, the expression of a particular marker is determined by detecting its presence or absence. Alternatively, the expression of a particular marker can be determined by measuring the level at which the marker is present in cells of a cell culture or cell population. In certain processes, the expression of markers characteristic of satellite cells and the absence of significant expression of markers characteristic of the myoblast or its precursor from which it originates are determined.

[0101] As described with respect to monitoring the production of satellite cells from myoblasts, quantitative or semi-quantitative techniques, such as blot transcription and immunocytochemistry, may be used to measure marker expression using methods generally known to those skilled in the art. Alternatively, marker expression may be accurately quantified by using techniques such as quantitative PCR using methods generally known in the art. Such techniques for measuring extracellular marker content, such as ELISA, may be used.

[0102] The present invention is not limited to the markers listed herein as satellite cell markers, and it should be understood that the present invention also includes markers such as cell surface markers (e.g., CD56, EGFR, and β1-integrin), antigens and other gene products, e.g., ESTs, RNA (e.g., microRNA and antisense RNA), DNA (e.g., genes and cDNA), and parts thereof.

[0103] Concentration, isolation, and purification of satellite cells Another aspect of the present invention relates to the isolation of a population of satellite cells from a heterogeneous population of cells, e.g., a mixed population of cells including satellite cells, myoblasts, differentiated skeletal muscle cells, and proliferative myofiocytes. The population of satellite cells produced by any of the above processes can be concentrated, isolated, and / or purified by using any cell surface marker present in the satellite cells but not in the myoblasts or their precursors from which the satellite cells are obtained. Such cell surface markers are also referred to as satellite cell-specific affinity tags. Examples of satellite cell-specific affinity tags are marker molecules present on the cell surface of satellite cells but substantially absent in other cell types (e.g., myoblasts), e.g., polypeptide-specific antibodies, ligands, or other binders. In some processes, antibodies that bind to cell surface antigens on satellite cells are used as affinity tags for concentration, isolation, or purification of chemically induced satellite cells (e.g., by contact with at least one differentiation medium as described herein) produced by the methods described herein. Such antibodies are known and commercially available.

[0104] Those skilled in the art will readily understand the process for using antibodies for the enrichment, isolation, and / or purification of satellite cells. For example, in some embodiments, a reagent such as an antibody is incubated with a cell population containing satellite cells that has been treated to reduce intercellular and substrate adhesion. The cell population is then washed, centrifuged, and resuspended. In some embodiments, if the antibody is not already labeled, the cell suspension is incubated with a secondary antibody capable of binding to the primary antibody, such as a FITC-conjugated antibody. The satellite cells are then washed, centrifuged, and resuspended in buffer. The suspension of satellite cells is then analyzed and sorted using a fluorescence-activated cell sorter (FACS). Antibody-bound fluorescence-reprogrammed cells are collected separately from unbound non-fluorescent cells, thereby isolating the satellite cells from other cells present in the cell suspension, such as myoblasts, differentiated skeletal muscle cells, or proliferative myofibrillators.

[0105] In another embodiment of the process described herein, the isolated cell composition containing satellite cells may be further purified by using another affinity-based method or by a further sorting round using the same or different markers specific to satellite cells. For example, in some embodiments, FACS sorting is used to first isolate satellite cells expressing Myf5, either alone, with Pax7 expression, or with the satellite cell markers disclosed herein, from cells in the cell population that do not express one of the markers (e.g., negative cells). A second FACS sorting, for example, sorting of positive cells using FACS again to isolate cells positive for a different marker than in the first sorting, enriches the cell population for reprogrammed cells.

[0106] In an alternative embodiment, FACS sorting is used to isolate cells by negative sorting for markers that are present in most myoblasts but not in satellite cells.

[0107] In some embodiments of the processes described herein, satellite cells are fluorescently labeled without the use of antibodies and then isolated from unlabeled cells by using a fluorescence-activated cell sorter (FACS). In such embodiments, nucleic acids encoding GFP, YFP, or another nucleic acid encoding an expressible fluorescent marker gene, such as a gene encoding luciferase, are used to label the reprogrammed cells using the above method.

[0108] In addition to the approaches described above, chemically induced satellite cells can also be isolated by other techniques for cell isolation. Furthermore, satellite cells can also be enriched or isolated by serial subculturing methods under growth conditions that promote selective survival or selective proliferation of satellite cells. Such methods are known to those skilled in the art.

[0109] In some embodiments, isolated satellite cells are capable of expanding in culture. In some embodiments, isolated satellite cells are contacted with compounds to increase satellite cell proliferation. Methods for inducing, enhancing, or increasing satellite cell proliferation include contacting satellite cells with compounds selected from the group consisting of kinase inhibitors, G protein-coupled receptor (GPCR) modulators, epigenic modifiers, histone deacetylase (HDAC) modulators, Hedgehog signaling pathway modulators, neuropeptides, dopamine receptor modulators, serotonin receptor modulators, histamine receptor modulators, adenosine receptor agonists, ionophores, ion channel modulators, gamma-secretase modulators, corticosteroids, and any combination thereof. Examples of proliferation enhancers, but not limited to, those described in International Patent Application PCT / US2017 / 016099 (which is incorporated herein by reference). The compounds may induce, enhance, or increase satellite cell proliferation by at least 5%, 10%, 20%, 30%, 40%, 50%, 50%, 70%, 80%, 90%, 1x, 1.1x, 1.5x, 2x, 3x, 4x, 5x, 10x, 50x, 100x or more compared to untreated satellite cells.

[0110] Composition containing satellite cells Some embodiments of the present invention relate to a cell composition (e.g., a cell culture or cell population) comprising satellite cells, wherein the satellite cells are derived from at least one myoblast. In some embodiments, the cell composition comprises myoblasts. In some embodiments, the cell composition comprises differentiated skeletal muscle cells. In some embodiments, the cell composition comprises proliferative myofiocytes.

[0111] According to certain embodiments, the chemically induced satellite cells are mammalian cells, and in preferred embodiments, such satellite cells are human satellite cells. In some embodiments, myoblasts are derived from germinal muscle cells. In other embodiments, myoblasts are isolated from the skeletal muscle of the subject.

[0112] Other embodiments of the present invention relate to compositions (e.g., isolated cell populations or cell cultures) comprising satellite cells produced by the methods disclosed herein. In such embodiments, the satellite cells constitute less than about 90%, less than about 85%, less than about 80%, less than about 75%, less than about 70%, less than about 65%, less than about 60%, less than about 55%, less than about 50%, less than about 45%, less than about 40%, less than about 35%, less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 12%, less than about 10%, less than about 8%, less than about 6%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than 1% of the total cells in the satellite cell population. In some embodiments, the composition includes a population of satellite cells that constitute more than about 90% of the total cells in the cell population, for example, at least 95%, at least 96%, at least 97%, at least 98%, at least about 99%, or at least 100% of the total cells in the cell population are satellite cells.

[0113] Compositions and kits Kits for carrying out the methods disclosed herein and for producing satellite cells disclosed herein are described herein. Kits for treating muscle injury are also described herein. In one embodiment, the kit comprises at least one myoblast and at least one culture medium as described herein, and optionally the kit may further include instructions for converting at least one myoblast into a population of satellite cells using the methods described herein. In some embodiments, the kit comprises at least two culture media. In some embodiments, the kit comprises at least three culture media. In some embodiments, the kit comprises at least one supplemental agent. In some embodiments, the kit comprises at least two supplemental agents. In some embodiments, the kit comprises at least three supplemental agents. In some embodiments, the kit comprises a culture medium and / or supplemental agent for dedifferentiation of myoblast into satellite cells. In some embodiments, the kit comprises, for example, any combination of a culture medium and / or supplemental agent for dedifferentiation of myoblast into satellite cells.

[0114] In some embodiments, the kit includes at least one myoblast medium. In some embodiments, the kit includes at least one spin medium. In some embodiments, the kit includes at least one differentiation medium. In some embodiments, the kit includes myoblast medium, spin medium, and differentiation medium. In some embodiments, the kit includes compounds selected from the group consisting of kinase inhibitors, G protein-coupled receptor (GPCR) modulators, histone deacetylase (HDAC) modulators, Hedgehog signaling pathway modulators, neuropeptides, dopamine receptor modulators, serotonin receptor modulators, histamine receptor modulators, ionophores, ion channel modulators, gamma-secretase modulators, and any combination thereof.

[0115] In some embodiments, the compounds in the kit may be supplied in waterproof or airtight containers that are substantially free of other components of the kit. The compounds may be supplied in more than one container (for example, it may be supplied in a container having sufficient reagents for a predetermined number of reactions for inducing myoblasts into satellite cells, e.g., one, two, three or more separate reactions). The culture medium and / or auxiliaries may be supplied in any form, e.g., liquid, dry, or lyophilized form. The compounds described herein (e.g., culture medium or drug) are preferably substantially pure and / or sterile. If the compounds described herein are supplied in solution, the solution is preferably an aqueous solution, and preferably a sterile aqueous solution. If the compounds described herein are supplied in dry form, reconstitution is generally by the addition of a suitable solvent. The solvent, e.g., sterile water or buffer, may be supplied in the kit as needed.

[0116] In some embodiments, the kit may further include materials as necessary. These materials may be explanatory materials, instructional materials, marketing materials, and other materials relating to the methods and / or the use of the compounds described herein in the methods described herein.

[0117] The materials in a kit are not limited to instructions or helpful information. In one embodiment, the materials may include information about the manufacture of the compound, its molecular weight, concentration, expiration date, batch or place of manufacture, etc. In one embodiment, the materials concern methods of administering the compound. In addition, the materials in a kit are not limited to their form. Often, materials, such as instructions, are provided in printed form, such as printed documents, diagrams and / or photographs, such as labels or printed sheets. However, materials may also be provided in other forms, such as Braille, computer-readable materials, video recordings or audio recordings. In another embodiment, the materials in a kit are contact information, such as a physical address, email address, website or telephone number, so that the user of the kit can obtain important information about the compounds described herein and / or their use in the manner described herein. Naturally, the materials may be provided in any combination of forms.

[0118] In one embodiment, the document may include instructions for administering the compounds described herein (e.g., culture media and / or auxiliaries) in a manner appropriate for carrying out the methods described herein, e.g., appropriate doses, forms of administration or methods of administration (e.g., doses, forms of administration or methods of administration described herein) (to cells in vitro or in vivo). In another embodiment, the document may include instructions for administering the compounds described herein to a suitable subject, e.g., a human, e.g., a human having or at risk of having one of the disorders described herein, or to cells in vitro.

[0119] In addition to the compounds described herein, the kit composition may include other components, such as solvents or buffers, stabilizers, preservatives, flavoring agents (e.g., bitter antagonists or sweeteners), fragrances or cosmetic ingredients, and / or further agents for treating the conditions or disorders described herein. Alternatively, the other components may be present in a different composition or container than those included in the kit, but different from the compounds described herein. In such embodiments, the kit may include instructions for mixing the compounds described herein with the other components, or for using the compounds described herein together with other components, such as instructions for combining the two agents before administration.

[0120] A kit may include one or more containers for a composition containing at least one culture medium and / or auxiliary agent as described herein. In some embodiments, the kit includes separate containers (e.g., two separate containers for two drugs), dividers, or compartments for the composition and the material. For example, the composition may be contained in a bottle, vial, or syringe, and the material may be contained in a plastic sleeve or packet. In other embodiments, the separate elements of the kit are contained within a single, undivided container. For example, the composition is contained in a bottle, vial, or syringe, with the material attached in the form of a label. In some embodiments, the kit includes a plurality of individual containers (e.g., packs), each containing one or more unit dose forms (e.g., dose forms as described herein) of the compounds described herein. For example, the kit includes a plurality of syringes, ampoules, foil packets, or blister packs, each containing a single unit dose of the compound described herein. The containers of the kit may be airtight, waterproof (e.g., impermeable to changes in moisture or evaporation), and / or light-shielding.

[0121] The kit includes, as needed, devices suitable for administering the composition, such as syringes, inhalers, pipettes, forceps, measuring spoons, drip injectors (e.g., eye droppers), swabs (e.g., cotton swabs or wooden swabs), or any such delivery devices. In preferred embodiments, the device is a medical implantable device, packaged for surgical insertion, for example.

[0122] The kit may also include markers for satellite cells, for example, components for detecting the markers described herein, for example, reagents for detecting satellite cells. Alternatively, in some embodiments, the kit may also include reagents for detecting negative markers for satellite cells for the purpose of negative selection of satellite cells, or for identifying cells that do not express these negative markers (e.g., satellite cells). The reagent may be, for example, an antibody against the marker, or a primer for an RT-PCR or PCR reaction, for example, a semi-quantitative or quantitative RT-PCR or PCR reaction. Such markers may be used to assess whether iPS cells have been produced. If the detection reagent is an antibody, it may be supplied as a dry preparation, for example, a lyophilized preparation or a solution. The antibody or other detection reagent may be linked to a label for use in detection, for example, an radiolabel, a fluorescent (e.g., GFP) label or a colorimetric label. If the detection reagent is a primer, it may be supplied as a dry preparation, for example, a lyophilized preparation or a solution.

[0123] The kit may include satellite cells, for example, satellite cells derived from the same type of myoblast, for use as a positive cell type control.

[0124] Methods for administering cells In one embodiment, the cells described herein, for example, a population of satellite cells, are transplantable, and for example, a population of satellite cells can be administered to a subject. In some embodiments, the subject administered with the population of satellite cells is the same subject from which myoblasts used to dedifferentiate into satellite cells (for example, for autologous cell therapy) were obtained. In some embodiments, the subject is a different subject. In some embodiments, the subject is suffering from muscle injury or is a normal subject. For example, the cells for transplantation (e.g., a composition containing a population of satellite cells) may be in a form suitable for transplantation.

[0125] The method may further include administering the cells to a subject in need, for example, a mammalian subject, for example, a human subject. The source of the cells may be a mammal, preferably a human. The source or recipient of the cells may also be a non-human subject, for example, an animal model. The term “mammal” includes organisms including mice, rats, cattle, sheep, pigs, rabbits, goats, horses, monkeys, dogs, cats, and preferably humans. Similarly, transplantable cells may be obtained from any of these organisms, for example, a non-human transgenic organism. In one embodiment, the transplantable cells are genetically engineered, for example, the cells contain exogenous genes or are genetically engineered to inactivate or alter endogenous genes.

[0126] Compositions containing a population of satellite cells may be administered to subjects using implantable devices. Implantable devices and related technologies are known in the art and are useful as delivery systems, with continuous-release or timed-release delivery of the compounds or compositions described herein being preferred. In addition, delivery systems using implantable devices are useful for targeting specific points of delivery of compounds or compositions (e.g., local sites or organs). Negrin et al., Biomaterials, 22(6):563 (2001). Timed-release technologies with alternative delivery methods may also be used in the present invention. For example, timed-release formulations based on polymer technology, sustained-release technology, and encapsulation technology (e.g., polymers, liposomes) may also be used for the delivery of the compounds and compositions described herein.

[0127] Pharmaceutical composition For administration to a subject, a population of cells produced by the methods disclosed herein, for example, a population of satellite cells (produced by contacting at least one myoblast with at least one culture medium (e.g., myoblast medium, spin medium, and / or differentiation medium as described herein)), may be administered to the subject, for example, in a pharmaceutically acceptable composition. These pharmaceutically acceptable compositions comprise a therapeutically effective amount of the above-mentioned population of satellite cells formulated with one or more pharmaceutically acceptable carriers (additives) and / or diluents.

[0128] As described in detail below, the pharmaceutical compositions of the present invention may be specifically formulated for administration in solid or liquid form, including those adapted for: (1) oral administration, e.g., liquid drugs (aqueous or non-aqueous solutions or suspensions), lozenges, sugar-coated tablets, capsules, pills, tablets (e.g., those intended for absorption in the cheek, sublingually, and systemically), boluses, powders, granules, pastes for application to the tongue; (2) parenteral administration, e.g., as sterile solutions or suspensions or as sustained-release formulations, e.g., by subcutaneous, intramuscular, intravenous, or epidural injection; (3) topical application, e.g., creams, ointments, or controlled-release patches or sprays applied to the skin; (4) vaginal or rectal, e.g., pessaries, creams, or foams; (5) sublingual; (6) ocular; (7) transdermal; (8) transmucosal; or (9) transnasal. In addition, the compounds may be implanted in the patient or injected using a drug delivery system. For example, see Urquhart, et al., Ann. Rev. Pharmacol. Toxicol. 24:199-236 (1984); Lewis, ed. “Controlled Release of Pesticides and Pharmaceuticals” (Plenum Press, New York, 1981); U.S. Patent No. 3,773,919; and U.S. Patent No. 35,3270,960.

[0129] As used herein, the term “pharmaceutically acceptable” means a compound, material, composition and / or dosage form that is appropriate for use in contact with human and animal tissues within the bounds of sound medical judgment and in proportion to a reasonable benefit / risk ratio, without excessive toxicity, irritation, allergic response or other problem or complication.

[0130] As used herein, the term “pharmaceutically acceptable carrier” means a pharmaceutically acceptable material, composition, or vehicle involved in transporting or delivering the compound of interest from one organ or part of the body to another, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, magnesium talc, calcium stearate or zinc stearate or stearic acid) or solvent encapsulation material. Each carrier should be “acceptable” in the sense that it is compatible with the other components of the formulation and is not harmful to the patient. Some examples of materials that can function as pharmaceutically acceptable carriers include: (1) sugars, e.g., lactose, glucose, and sucrose; (2) starches, e.g., corn starch and potato starch; (3) cellulose and its derivatives, e.g., sodium carboxymethylcellulose, methylcellulose, ethylcellulose, microcrystalline cellulose, and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricants, e.g., magnesium stearate, sodium lauryl sulfate, and talc; (8) excipients, e.g., cocoa butter and suppository waxes; (9) oils, e.g., peanut oil, cottonseed oil, safflower oil. (10) Glycols, e.g., propylene glycol; (11) Polyols, e.g., glycerin, sorbitol, mannitol, and polyethylene glycol (PEG); (12) Esters, e.g., ethyl oleate and ethyl laurylate; (13) Agar; (14) Buffers, e.g., magnesium hydroxide and aluminum hydroxide; (15) Alginic acid; (16) Water free of pyrogens; (17) Isotonic saline; (18) Ringer's solution; (19) Ethyl alcohol; (20) pH buffered solution; (21) Polyesters, polycarbonates, and / or polyacid anhydrides; (22) Bulking agents, e.g., polypeptides and amino acids; (23) Serum components, e.g., serum albumin, HDL, and LDL; (22) C2-C 12Examples include alcohols, such as ethanol; and (23) other non-toxic, suitable substances used in pharmaceutical formulations. Wetting agents, colorants, release agents, coating agents, sweeteners, flavoring agents, fragrances, preservatives, and antioxidants may also be present in the formulations. The terms used herein, such as “excipient,” “carrier,” and “pharmaceutically acceptable carrier,” are interchangeable.

[0131] Where used herein in relation to cell populations, the term “therapeutic dose” means an amount of relevant cells in a cell population, e.g., satellite cells, or a composition comprising the satellite cells of the present invention, that is effective in producing some desired therapeutic effect in at least a subpopulation of cells in an animal, with a reasonable benefit / risk ratio applicable to any medical treatment. For example, a population of satellite cells sufficient to produce a statistically significant measurable change in at least one symptom of muscle injury, e.g., muscle repair or muscle regeneration, is administered to a subject. Determining the therapeutic dose is well within the capabilities of those skilled in the art. Generally, the therapeutic dose may vary depending on the subject’s medical history, age, condition, sex, and the severity and type of the medical condition in the subject, as well as the administration of other pharmaceutically active agents.

[0132] As used herein, the term “repair” refers to the process by which damage to muscle tissue is mitigated or completely eliminated. In some embodiments, at least one symptom of muscle tissue damage is mitigated by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, or at least 50%.

[0133] "Treatment," "prevention," or "improvement" of a disease or disorder means delaying or preventing the onset of such disease or disorder, progression, worsening or exacerbation, reversal of progression or severity of a condition associated with such disease or disorder, mitigation, improvement, inhibition, slowing or cessation. In one embodiment, the symptoms of the disease or disorder are mitigated by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, or at least 50%.

[0134] As used herein, the term “administer” refers to the placement of a composition in a subject by a method or route that results in at least partial localization of the composition at a desired site to produce the desired effect. Suitable routes of administration for the methods of the present invention include both local and systemic administration. Generally, local administration results in the delivery of many administered satellite cells (or growth-enhancing agent-treated satellite cells) to a specific site compared to systemic administration of the subject, whereas systemic administration essentially results in the delivery of satellites to the entire subject. One method of local administration is by intramuscular injection.

[0135] In the context of administering compound-treated cells, the term “administer” also includes the transplantation of such cells into a subject. As used herein, the term “transplantation” refers to the process of implanting or transferring at least one cell into a subject. The term “transplantation” includes, for example, autotransplantation (taking cells from one location in a patient and moving them to the same or another location in the same patient), allotransplantation (transplantation between members of the same species), and xenotransplantation (transplantation between members of different species). Those skilled in the art will be well aware of methods for implanting or transplanting cells for muscle repair and muscle regeneration that are suitable for the present invention. See, for example, U.S. Patent No. 7,592,174 and U.S. Patent Application Publication No. 2005 / 0249731 (both incorporated herein by reference).

[0136] Satellite cells or compositions containing them may be administered by any suitable route known in the Art, for example, by oral or parenteral routes, such as intravenous, intramuscular, subcutaneous, transdermal, respiratory (aerosol), lung, nasal, rectal, and topical (e.g., cheek and sublingual) administration.

[0137] Exemplary modes of administration include, but are not limited to, injection, infusion, intravenous infusion, inhalation, or oral ingestion. "Injection" includes, but is not limited to, intravenous, intramuscular, intraarterial, intrathecal, intraventricular, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, intraspinal, intracerebrospinal, and intrasternal injections and infusions. In preferred embodiments, the composition is administered by intravenous infusion or injection.

[0138] As used herein, “subject” means human or animal. Typically, animals are vertebrates, e.g., primates, rodents, domesticated or game animals. Examples of primates include chimpanzees, crab-eating macaques, spider monkeys, and macaques, e.g., rhesus macaques. Examples of rodents include mice, rats, woodchucks, ferrets, rabbits, and hamsters. Examples of domesticated and game animals include cattle, horses, pigs, deer, bison, buffalo, feline species, e.g., domestic cats, canine species, e.g., dogs, foxes, wolves, birds, e.g., chickens, emus, ostriches, and fish, e.g., trout, catfish, and salmon. A patient or subject can be any subset of the foregoing, e.g., all of the above, but excluding one or more groups or species, e.g., humans, primates, or rodents. In certain embodiments of the aspects described herein, the subject is a mammal, e.g., a primate, e.g., a human. The terms “patient” and “subject” are used interchangeably herein. A subject may be male or female.

[0139] Preferably, the subject is a mammal. The mammal may be, but is not limited to, humans, non-human primates, mice, rats, dogs, cats, horses, or cattle. Non-human mammals can be advantageously used as subjects corresponding to animal models of autoimmune diseases or inflammation-related disorders. In addition, the methods and compositions described herein may be used to treat livestock and / or pets.

[0140] Subjects may be individuals who have been previously diagnosed with, or are identified as having, a disorder characterized by muscle injury or muscle atrophy / wasting. Subjects may be individuals who have been previously diagnosed with, or are identified as having, a muscle degenerative disorder.

[0141] In some embodiments of the aspects described herein, the method further includes diagnosing and / or selecting a subject for muscle injury or muscle atrophy / waste before treating the subject for muscle repair or muscle regeneration.

[0142] In some embodiments, damaged muscle tissue is due to sarcopenia. As used herein, the term “sarcopenia” refers to the loss of muscle mass and function that inevitably occurs with aging. Sarcopenia is a cause of decreased physical activity levels and can lead to increased body fat and further muscle loss. The loss of muscle mass is due to a negative net balance between muscle protein synthesis and muscle protein breakdown. The pathogenesis of this loss of skeletal muscle mass and function is not considered clear. Decreased physical activity levels, loss of motor units secondary to changes in the central nervous system, and insufficient protein uptake are all thought to be involved.

[0143] In some embodiments, the damaged muscle tissue results from a physical injury or trauma. The damaged muscle may be skeletal muscle. In some embodiments, the subject has or is otherwise affected by a muscle injury, wound, trauma or disease.

[0144] In some embodiments, the disease resulting in damaged muscle tissue is myopathy. Myopathy may be congenital or acquired, but is not limited to these. Exemplary myopathy may include, but is not limited to, dystrophy, myotonia (neuromytonia), congenital myopathy (e.g., nemaline myopathy, multi / minicore myopathy, central nucleus myopathy (or myotyl myopathy)), mitochondrial myopathy, familial periodic paralysis, inflammatory myopathy, metabolic myopathy (e.g., gluconocyte storage disorders and lipid storage disorders), dermatomyositis, polymyositis, inclusion body myositis, myositis ossificans, rhabdomyolysis, and myoglobinuria.

[0145] In this embodiment and some other embodiments described herein, myopathy is a dystrophy selected from the group consisting of muscular dystrophy, Duchenne muscular dystrophy, Becker muscular dystrophy, reflex sympathetic dystrophy, retinal dystrophy, conal dystrophy, myotonic dystrophy, corneal dystrophy, and any combination thereof.

[0146] Congenital myopathy is a term sometimes applied to hundreds of different neuromuscular disorders that may be present at birth, but it is reserved for a group of rare hereditary primary muscle disorders that typically cause hypotonia and weakness at birth or in the neonatal period, and in some cases, delayed motor development in childhood. Patients suffer weakness ranging from mild (onset in late childhood and inability to walk throughout adulthood) to severe (respitatory insufficiency and death within the first year of life).

[0147] The most common types of congenital myopathy are nemaline myopathy, myotulotubular myopathy, central nucleus myopathy, congenital fibrous type disequilibrium, and multicore myopathy. They are primarily distinguished by their histological features, symptoms, and prognosis. Diagnosis is indicated by characteristic clinical findings and confirmed by muscle biopsy.

[0148] Specific examples of myopathy include, but are not limited to, those described in International Patent Application PCT / US2017 / 016099 (which is incorporated herein by reference).

[0149] The satellite cell compositions described herein may be co-administered to a subject in combination with the growth enhancers and / or pharmaceutically active agents described herein. Examples of pharmaceutically active compounds include, but are not limited to, Harrison's Principles of Internal Medicine, 13 th Edition,Eds.TRHarrison et al.,McGraw-Hill NY,NY;Physicians'Desk Reference,50 th Edition,1997,Oradell New Jersey,Medical Economics Co.;Pharmacological Basis of Therapeutics,8 th Examples include those found in Edition, Goodman and Gilman, 1990; United States Pharmacopeia, The National Formulary, USP XII NF XVII, 1990; the latest edition of Goodman and Oilman's The Pharmacological Basis of Therapeutics; and the latest edition of The Merck Index (all of which are incorporated herein by reference in their entirety).

[0150] In some embodiments of the embodiments described herein, the pharmaceutically active agent is a growth factor. Exemplary growth factors include, but are not limited to, basic epidermal growth factor (bEGF), fibroblast growth factor (FGF), FGF-1, FGF-2 (bFGF), FGF-4, thymosin, platelet-derived growth factor (PDGF), epidermal growth factor (EGF), transforming growth factor (TGF), TGF-alpha, TGF-beta, cartilage-inducing factor-A and -B, osteoid-inducing factor, osteogenin, bone morphogenetic proteins and other bone growth factors, collagen growth factor, heparin-binding growth factor-1 or -2, and their bioactive derivatives.

[0151] Satellite cell compositions, growth enhancers, and / or pharmaceutically active agents may be administered to a subject (simultaneously or at different times) in the same or different pharmaceutically active compositions. When administered at different times, the satellite cell composition, growth enhancer, and / or pharmaceutically active agent may be administered within 5 minutes, 10 minutes, 20 minutes, 60 minutes, 2 hours, 3 hours, 4 hours, 8 hours, 12 hours, or 24 hours of the other administration. When the satellite cell composition, growth enhancer, and / or pharmaceutically active agent are administered in different pharmaceutically active compositions, the route of administration may differ.

[0152] The toxicity and therapeutic efficacy of a composition containing a population of satellite cells can be determined, for example, by standard pharmaceutical procedures in cell culture or experimental animals to determine the LD50 (lethal dose for 50% of the population) and ED50 (therapeutably effective dose for 50% of the population). Compositions containing a population of satellite cells exhibiting a large therapeutic index are preferred.

[0153] The amount of composition containing a population of satellite cells can be tested using multiple well-established animal models.

[0154] In some embodiments, data obtained from cell culture assays and animal studies may be used to formulate a range of doses for use in humans. Doses of such compounds are preferably within the range of circulating concentrations containing little to no toxicity or a non-toxic ED50. Doses may vary within this range depending on the dosage form used and the route of administration employed.

[0155] The therapeutically effective dose of a composition containing a population of satellite cells can be initially estimated from a cell culture assay. Alternatively, the effect of any particular dose can be monitored by an appropriate bioassay.

[0156] Regarding the duration and frequency of treatment, it is typical for a skilled clinician to monitor the subject to determine when the treatment is providing therapeutic benefit and to decide whether to increase or decrease the dosage, increase or decrease the frequency of administration, discontinue the treatment, resume the treatment, or make other changes to the treatment regimen. The administration schedule can vary from once a week to daily, depending on numerous clinical factors. The desired dosage may be administered at once or divided into partial doses, e.g., 2 to 4 partial doses, and administered over a period of time, e.g., at appropriate intervals throughout the day or at other appropriate schedules. Such partial doses may be administered as unit doses. In some embodiments, the administration is once daily or more over a long period, e.g., several weeks or months. Examples of administration schedules include daily, twice daily, three times daily, four times daily, or more frequently for one week, two weeks, three weeks, four weeks, one month, two months, three months, four months, five months, or six months or more.

[0157] In another aspect of the present invention, the method provides the use of an isolated population of satellite cells disclosed herein. In one embodiment of the present invention, an isolated population of satellite cells disclosed herein may be used to produce a pharmaceutical composition for use in transplantation into a subject requiring treatment, for example, a subject having or at risk of developing a muscle disease or muscle disorder (e.g., muscle degenerative disorder). Examples include subjects having muscle atrophy or muscle injury. In one embodiment, the isolated population of satellite cells may be genetically modified. In another embodiment, the subject may have or be at risk of having muscle injury or muscle atrophy. In some embodiments, an isolated population of satellite cells, such as those disclosed herein, may be autologous and / or homogeneous. In some embodiments, the subject is a mammal, and in other embodiments, the mammal is a human.

[0158] One embodiment of the present invention relates to a method for treating a muscle injury or muscle disease in a subject, comprising administering an effective amount of a composition containing a population of satellite cells disclosed herein to a subject having a muscle injury or muscle disease. Another embodiment relates to a method for treating a muscle degenerative disorder in a subject, comprising administering an effective amount of a composition containing a population of satellite cells disclosed herein to a subject having a muscle degenerative disorder. In a further embodiment, the present invention provides a method for treating a muscle disease, comprising administering a composition containing a population of satellite cells disclosed herein to a subject having a muscle disease or being at increased risk of developing it.

[0159] In some embodiments, the satellite cell populations disclosed herein may be administered with any physiologically acceptable excipient, and the satellite cells may find suitable sites for replication, proliferation, and / or engraftment. In some embodiments, the satellite cell populations disclosed herein may be introduced by injection, catheter, etc. In some embodiments, the satellite cell populations disclosed herein may be frozen at liquid nitrogen temperature, stored for extended periods, and available upon thawing. When frozen, the satellite cell populations would typically be stored in 10% DMSO, 50% FCS, 40% RPMI1640 medium. Upon thawing, the cells may be expanded and proliferated by the use of growth factors and / or feeder cells associated with the culture of satellite cells disclosed herein.

[0160] In some embodiments, the satellite cell populations disclosed herein may be supplied in the form of a pharmaceutical composition containing an isotonic excipient prepared under sufficiently sterile conditions for administration to humans. For general principles of medical formulations, readers refer to *Cell Therapy: Stem Cell Transplantation, Gene Therapy, and Cellular Immunotherapy*, by G. Morstyn & W. Sheridan eds, Cambridge University Press, 1996; and *Hematopoietic Stem Cell Therapy*, EDBall, J. Lister & P. ​​Law, Churchill Livingstone, 2000. The selection of cell excipients and any accompanying elements in the composition containing the satellite cell populations disclosed herein will be adopted based on the route and device used for administration. In some embodiments, the composition containing the satellite cell population may also contain, or be accompanied by, one or more other components that facilitate the engraftment or functional recruitment of the satellite cells. Suitable components include matrix proteins that support or promote the adhesion of satellite cells or complementary cell types. In another embodiment, the composition may include an absorbable or biodegradable matrix scaffold.

[0161] Gene therapy can be used to modify cells, either by replacing gene products, promoting tissue regeneration, treating diseases, or improving cell viability after implantation in a subject (i.e., preventing rejection).

[0162] In some embodiments, populations of satellite cells may be administered and prescribed based on standards of medical quality control, taking into account the clinical condition of the individual patient, the site and method of administration, the administration schedule, the patient's age, sex, weight, and other factors known to healthcare professionals. Thus, the pharmaceutically effective dose for the purposes of this specification is determined by such considerations, as is known in the art. The dose must be effective in achieving improvement, e.g., improved survival rate, faster recovery, or improvement or elimination of symptoms and other indicators, as selected as an appropriate measure by those skilled in the art. Populations of satellite cells may be administered to subjects in the following locations: clinics, hospitals, emergency departments, wards, intensive care units, operating rooms, catheterization suites, and radiologic suites.

[0163] In other embodiments, a population of satellite cells is stored for later implantation / injection. The population of satellite cells may be divided into one or more aliquots or units so that a portion of the population is retained for later application and a portion is applied immediately to the subject. Medium to long-term storage of all or part of the cells in a cell bank is also within the scope of the present invention, as disclosed in U.S. Patent Application Publication 2003 / 0054331 and International Publication 03 / 024215 (which are incorporated in whole by reference). At the end of processing, the enriched cells may be filled into a delivery device, such as a syringe, for placement into the recipient by any means known to those skilled in the art.

[0164] In some embodiments, a population of satellite cells may be applied alone or in combination with other cells, tissues, tissue fragments, growth factors, such as VEGF and other known angiogenic or angiogenic growth factors, biologically active or inactive compounds, absorbable plastic scaffolds, or other additives intended to enhance the delivery, efficacy, tolerance, or function of the population. In some embodiments, a population of satellite cells may be modified by DNA insertion or arrangement in cell culture to alter, enhance, or add to the function of the cells in order to achieve structural or therapeutic purposes. For example, gene transfer techniques for stem cells are known to those skilled in the art, as disclosed in (Morizono et al., 2003; Mosca et al., 2000), and include viral transfection techniques, and more specifically, adeno-associated virus gene transfer techniques disclosed in (Walther and Stein, 2000) and (Athanasopoulos et al., 2000). Non-viral techniques may also be implemented, as disclosed in (Murarnatsu et al., 1998).

[0165] In other embodiments, in some cases, a population of satellite cells may be combined with a gene encoding a pro-angiogenic growth factor. Genes encoding anti-apoptotic factors or drugs may also be applied. The addition of genes (or combinations of genes) may be by any technique known in the art, for example, adenovirus transduction, “gene guns,” liposome-mediated transduction, and retrovirus or lentivirus-mediated transduction, plasmid adeno-associated viruses. Cells may be embedded with carrier material having a gene delivery vehicle capable of releasing and / or presenting genes to the cells over time so that transduction may continue or be initiated. In particular, if cells and / or tissues containing cells are administered to a patient other than the patient from whom the cells and / or tissues were obtained, one or more immunosuppressants may be administered to the patient receiving the cells and / or tissues to reduce, and preferably prevent, graft rejection. As used herein, the term “immunosuppressant or immunosuppressant” is intended to include pharmaceuticals that inhibit or interfere with normal immune function. Examples of immunosuppressants suitable for the methods disclosed herein include agents that inhibit the simultaneous stimulation pathway of T cells / B cells, such as agents that interfere with T-cell and B-cell coupling via the CTLA4 and B7 pathways, as disclosed in U.S. Patent Application Publication 2002 / 0182211 (which is incorporated herein by reference). In one embodiment, the immunosuppressant is cyclosporine A. Other examples include mycophenolate mofetil, rapamycin, and anti-thymocyte globulin. In one embodiment, the immunosuppressant is administered together with at least one other therapeutic agent. The immunosuppressant is administered in a formulation adapted to the route of administration and in a dose sufficient to achieve the desired therapeutic effect. In another embodiment, the immunosuppressant is administered transiently for a sufficient period of time to induce tolerance to the satellite cells of the present invention.

[0166] Pharmaceutical compositions containing an effective population of satellite cells are also intended by the present invention. These compositions contain an effective number of satellite cells, as may be combined with pharmaceutically acceptable carriers, additives, or excipients as needed. In certain indications, systemic administration of the satellite cell population to the subject may be preferred, while in other indications, direct administration to or near the site of the affected and / or damaged tissue may be preferred.

[0167] In some embodiments, the satellite cell population may be packaged in a suitable container, along with written instructions, if necessary, regarding the desired purpose, such as the reconstitution or thawing (if frozen) of the satellite cell population before administration to a subject. [Examples]

[0168] Example 1 - Myoblasts cultured in a 3D spin environment self-assemble into skeletal muscle organoids composed of differentiated skeletal muscle and proliferative myogenic populations. WT(C57bl / 6), MyoDCre ROSATdtomato, Pax7 nGFP Myoblasts isolated from Pax7Cre ROSATdtomato self-assemble into 3D spheres when cultured under 3D spin conditions (Figure 1A). To determine the differentiation state of cells present within skeletal muscle organoids, we collected spheres at days 10, 20, and 30 of growth under 3D conditions. At each time point, the collected spheres were frozen in OCT:sucrose and sectioned to 10 μm. Sections were stained with Pax7(DSHB) (Seale et al., 2000) to identify myoblasts or satellite cells, and with an antibody recognizing myosin heavy chain (MyHC, MF20-DSHB) (a marker of terminal differentiation of skeletal muscle) (Bader et al., 1982). Skeletal muscle organoids at each of the three time points were identified as Pax7 + It is composed of a combination of MyHC+ cells and Pax7 + The proportion of cells decreases over time.

[0169] To determine whether these findings apply to human cells, the inventors grew WT human skeletal myoblasts (Life Technologies #A12555) in myoblast medium containing a higher concentration of bFGF (10 ng / ml). The human myoblasts were seeded in 96-well spheroid microplates (Corning-4515) for 4 days, then transferred to 10 cm low-adhesion plates maintained on an orbital shaker. After 10 days of culture, human skeletal muscle organoids were formed (Figure 1B). Frozen sections of the human skeletal muscle organoids, stained with Pax7 and MyHC, showed a majority of cells to be MyHC-positive. Interestingly, since human myoblasts do not express the transcription factor Pax7 under normal culture conditions, it is important to note the presence of Pax7-positive human cells within the skeletal muscle organoids (Figure 1C). The inventors' data suggest that, upon seeding in spheroid plates, human myoblasts can self-assemble into 3D spheres. After sphere formation, Pax7 + The emergence of human myoblasts suggests that dedifferentiation or reprogramming occurred, causing myoblasts to revert to a state similar to that of satellite cells.

[0170] Next, the inventors performed RNAseq analysis to identify the transcriptional fingerprint of satellite cell state (Figure 2A). In satellite cells (Pax7, Myf5), multiple core myogenic genes are highly expressed, but the master transcription factor of skeletal muscle MyoD is preferentially expressed in myoblasts and not present in satellite cells. These myogenic markers, in combination with a panel of quiescence-related genes (Spry1, Nm1, Nfia, Fos, Dusp1) and members of the Notch signaling pathway (Notch1, 2, 3, HeyL, Hey2, Hes), function as a transcriptional fingerprint to identify satellite cells. To determine whether Pax7-positive cells present in skeletal muscle organoids share a transcriptional profile similar to that of satellite cells, the inventors isolated total RNA from nGFP cells purified from skeletal muscle organoids cultured for 30 days by fluorescence-activated cell sorting (FACS) (Figure 2B). After qPCR analysis, the inventors determined an increase in Pax7 (about 4.5-fold) and Myf5 (about 18-fold), along with a significant decrease in MyoD (about 1 / 50). Furthermore, nGFP + In cells derived from skeletal muscle organoids on day 30, markers of the Notch signaling pathway, Notch1 (about 2.5-fold), Notch2 (about 4-fold), Notch3 (about 35-fold), HeyL (about 580-fold), Hey2 (about 37-fold), and Hes1 (about 11-fold), were increased. In the nGFP + cell population derived from skeletal muscle organoids on day 30, activation quiescence-related genes or markers, including Nm1 (about 5-fold), Nfia (5-fold), Fos (about 480-fold), Spry1 (about 34-fold), and Dusp1 (about 20-fold), were enhanced (Figure 2B). This data suggests that nGFP+ cells isolated from skeletal muscle organoids have a transcriptional profile similar to that of satellite cells.

[0171] To address the mechanism by which skeletal muscle organoids enable myofibrils to revert to a satellite cell state, we investigated the environmental differences between standard 2D and 3D cultures. Adult stem cells have the ability to divide asymmetrically, thus generating a precursor population while simultaneously self-renewing and maintaining the stem cell pool (Kuang et al., 2007). Factors within the stem cell niche are crucial for this to occur. We evaluated our RNA-seq datasets for extracellular molecules that could explain these differences in the niches presented to myoblasts in 2D and 3D cultures. After analysis, a core group of proteoglycans emerged that was enriched in satellite cells compared to myoblasts (Figure 3A). After 3D culture, nGFP+ cells were highly enriched for these proteoglycan markers (Figure 3B). To further confirm the presence of these proteoglycans at the protein level, the inventors sectioned and stained skeletal muscle organoids cultured in spin medium for 20 days or in spin medium for 10 days followed by 10 days in serum-free differentiation medium (Figure 3C). After differentiation in serum-free medium, the inventors observed an increase in the presence of Bgn and TgfbrIII (Figure 3D). The inventors' data suggest that 3D culture more accurately resembles the satellite cell niche in vivo, thus generating cells similar to satellite cells in transcriptional profiles and secreting proteoglycans important for establishing the satellite cell niche.

[0172] material and method Culture medium Myoblast medium - Ham F10 (Wisent), 20% heat-inactivated fetal bovine serum, 5 ng / ml bFGF (Millipore), 1 × non-essential amino acids (Life Technologies), 1 × Glutamax (Life Technologies).

[0173] Spin medium - DMEM:F12 (Life Technologies), 20% heat-inactivated fetal bovine serum, 10 ng / ml bFGF (Millipore), 1 × non-essential amino acids (Life Technologies), 1 × Glutamax (Life Technologies).

[0174] Differentiation medium - DMEM (Life Technologies), 1 x non-essential amino acids (Life Technologies), 1 x Glutamax (Life Technologies).

[0175] cell line Methods for generating satellite cells were developed using multiple mouse strains and human strains (including Bl / 6, MyoDiCre, Pax7nGFP, Pax7CreROSATdt, and Pax7CreROSALuc), as well as human strains from Gibco and Cook.

[0176] Myoblast proliferation and expansion Myoblasts from multiple mouse strains were isolated from hindlimb skeletal muscle and cultured on collagen-coated dishes in myoblast culture medium. The myoblast cell lines were subcultured using trypsin (Life Technologies). The myoblast cell lines were expanded on 10 cm culture dishes to obtain 10 plates. Subsequently, the cells were subcultured and seeded onto 10 15 cm plates, which were cultured until they reached approximately 80 confluences before being seeded into spinner flasks.

[0177] 3D sphere formation conditions Myoblasts are seeded at a density of 1 million cells / ml into a 125 ml spinner flask (Corning). The flask rotation speed is set to 55 rpm. The culture is supplied every two days by removing half of the culture medium and replacing it with fresh medium.

[0178] Differentiation conditions Myoblasts in spin culture (spin medium) formed myospheres or skeletal muscle organoids composed of differentiated and proliferating cells. After culturing under serum-free conditions (differentiation medium), proliferating myofibrioforms became quiescent and returned to a satellite cell state. Skeletal muscle organoids were grown in spin medium for 20 days. Differentiation was carried out for 10 days.

[0179] [ka]

Claims

1. A composition containing satellite cells that do not exist in nature, wherein: a. The aforementioned naturally occurring satellite cells have the ability to rearrange the satellite cell niche; b. The aforementioned naturally occurring satellite cells induce muscle regeneration after transplantation; c. The naturally occurring satellite cells express two or more genes selected from the group consisting of Nr2f1, Dmp1, Wisp2 / Ccn5, Ecm1, Mgp, Igfbp5, Itgb5, Sdc2, and Bgn, wherein the expression of these two or more genes is upregulated to more than twice the level of expression by myoblasts, and these two or more genes are not expressed in endogenous satellite cells, and d. The aforementioned satellite cells, which do not exist in nature, are generated through the dedifferentiation of myoblasts isolated from organisms. composition.

2. The composition according to claim 1, wherein the naturally occurring satellite cells express at least one quiescence-related gene selected from the group consisting of Spry1, Nm1, Nfia, Fos, and Dusp1.

3. The composition according to claim 1, wherein the naturally occurring satellite cells express at least one Notch signaling pathway gene selected from the group consisting of Notch1, Notch2, Notch3, HeyL, Hey2, and Hes.

4. The composition according to claim 1, wherein the naturally occurring satellite cells express Myf5 and / or Pax7.

5. The composition according to claim 1, wherein the satellite cells, which do not exist naturally, do not express MyoD.

6. The composition according to claim 1, wherein the naturally occurring satellite cells exhibit a response to muscle injury.

7. The composition according to claim 1, wherein the satellite cells, which do not exist in nature, can be expanded and proliferated in culture.

8. The composition according to claim 1, wherein the satellite cells that do not exist in nature are human.

9. The composition according to claim 1, wherein the satellite cells, which do not exist in nature, are not genetically modified.

10. The composition according to claim 1, wherein the satellite cells, which do not exist in nature, are genetically modified.

11. A microcapsule containing an isolated population of naturally occurring satellite cells as described in any one of claims 1 to 10, which is encapsulated within the microcapsule.

12. The composition according to any one of claims 1 to 10, characterized in that it is administered to a subject requiring administration.

13. The composition according to claim 12, characterized in that the population of satellite cells that do not exist in nature is encapsulated in microcapsules and administered to the subject.

14. The composition according to claim 12, wherein the subject has a muscle degeneration disorder or has an increased risk of developing a muscle degeneration disorder.

15. The composition according to claim 12, wherein the subject has a muscle injury or has an increased risk of developing a muscle injury.

16. The composition according to claim 12, wherein the subject has sarcopenia or has an increased risk of developing sarcopenia.

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