Isolation of muscle satellite cells

Abstract

A composition of INFRA cells is described. The composition includes human muscle stem cells (SCs) expressing the transcription factor PAX7 and lacking the expression of the transmembrane receptor PDGFRA, wherein at least 70% of the population of human muscle SCs are PAX7+ and PDGFRA-. Methods of preparing the INFRA cell compositions, and methods of using the INFRA cells to treat muscle injury are also described.

Description

GOVERNMENT FUNDING

[0001] The present invention was made with government support under Grant No. 5RO1AR07452605, awarded by the National Institutes of Health. The US government has certain rights in this invention. CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Patent Application Serial No. 63 / 621,660, filed January 17, 2024, which is incorporated herein by reference. BACKGROUND

[0003] Muscle injuries and degenerative diseases such as Duchenne Muscular Dystrophy (DMD) and Becker Muscular Dystrophy (BMD) represent significant challenges in modern medicine, necessitating the development of therapies for muscle regeneration. In the past thirty years, several cell therapy clinical trials involving transplantation of primary human skeletal myoblasts amplified in vitro from skeletal muscle biopsies have been carried out in DMD and BMD patients (Skuk et al., J Neuropathol Exp Neurol 65, 371-386, 2006). These trials demonstrated feasibility but poor efficacy as the grafted myoblasts showed limited regeneration potential. Since these early trials, PAX7+ SCs have been shown to act as resident stem cells in adult skeletal muscle, demonstrating tremendous regenerative power (Sacco et al., Nature 456, 502-506, 2008). These cells can generate large amounts of myoblasts which can reconstruct myofibers. They can also self-renew, ensuring the long-term regeneration potential of the tissue. Therefore, compared to the myoblasts grafted in the early DMD trials, SCs represent a much better candidate for cell therapy applications. There are however significant limitations for the use of SCs in a clinical setting. SCs is a rare population, constituting less than 5% of the nuclei in skeletal muscle (Tierney and Sacco, Trends Cell Biol 26, 434-444, 2016). Thus, producing large amounts of these cells from human muscle biopsies for cell therapy applications would require in vitro amplification. However, this approach is currently not viable as adult SCs lose their regenerative properties when cultured in vitro, making them incompatible with clinical applications (Montarras et al., Science 309, 2064-2067, 2005). An alternative approach is to produce PAX7+ SCs from human PSCs. Several protocols describing SCs production in vitro have been recently reported (Caron et al., J Neuromuscui Dis 10, 761776, 2023). These protocols can generate immature PAX7+ SCs exhibiting embryonic / fetal characteristics (Xi et al., Cell stem cell, Vol. 27, Issue 1, 158-176, 2020). Importantly, these immature human SCs appear to retain their self-renewal properties in vitro as demonstrated for mouse fetal SCs (Tierney et al., Trends Cell Biol 26, 434-444, 2016). However, the myogenic cultures produced with these protocols which mimic dermomyotome development are heterogenous as they contain most natural derivatives including fibroblasts or neural cells derived from the founding neuro-mesodermal precursor population induced in these conditions (Xi et al., Cell stem cell, Vol. 27, Issue 1, 158-176, 2020). The PAX7+ SC population represents a fraction of up to 25-35% of the mononucleated population after 30-80 days in vitro (Xi et al., Cell stem cell, Vol. 27, Issue 1, 158-176, 2020). Therefore, the fraction of PAX7+ nuclei in the total population is even lower as the syncytial myofibers are excluded from this count. Consequently, purification strategies such as Fluorescence-activated Cell Sorting (FACS) or magnetic beads-based sorting would need to be applied to obtain a cell population sufficiently enriched in SCs for clinical trials. Such purification steps significantly complicate the production of cell populations in GMP conditions for cell therapy applications.

[0004] It would be advantageous to provide a method of isolating a substantially homogeneous population of functional SCs and SCs from any muscle culture, and to provide a substantially pure population of functional SCs and SCs. SUMMARY OF THE INVENTION

[0005] A filtration / sorting method to obtain a significantly enriched population of functional SCs and SCs from any muscle culture with unprecedented levels of efficiency is described. The method is based on any PSC-derived myogenic culture defined by the culture / directed growth of pluripotent cells into muscle fibers or progenitors that can be characterized for example but not limited to by their transcriptome (e.g., cultures composed of cells expressing PAX7, MYODI, MYF5, MYOG, ACTN2, TTN). The SCs are adherent to the substrate of the culture, and we named these cells 'INFRA cells’ and they are defined by expressing the gene PAX7+ but lacking expression of PDGFRA.

[0006] In one aspect, the present invention provides a composition of INFRA cells, comprising human muscle stem cells (SCs) expressing the transcription factor PAX7 and lacking the expression of the transmembrane receptor PDGFRA, wherein at least 70% of the population of human muscle SCs are PAX7+ and PDGFRA-, and wherein the SCs have not undergone modification in the isolation process.

[0007] In some embodiments, the human muscle SCs further express the transcription factors PAX and / or the transmembrane receptors FGFR4, CD82, ITGA7, CDH15, CD56 and / or NOTCH3. In further embodiments, the human muscle SCs are derived from pluripotent stem cell (PSC)-derived cultures generated by treating presomitic mesoderm cells with a Wnt activator. In yet further embodiments, the PSC-derived cultures are generated by treating presomitic mesoderm cells with a Wnt activator and a bone morphogenic protein (BMP) inhibitor.

[0008] In some embodiments, the INFRA cells are isolated by generating a myogenic culture derived from pluripotent stem cells (PSCs) and removing the upper layer of the culture to expose an adherent monolayer of INFRA cells. In further embodiments, the INFRA cells are further purified by flow cytometry to purify the PDGFRA negative population. In yet further embodiments, the SCs are further isolated from the isolated INFRA cells by flow cytometry using antibodies against ITGA7, CD56, NOTCH3, CD82 and / or CDH15.

[0009] Another aspect of the invention provides a method of creating a substantially pure population of INFRA cells comprising: (a) generating a myogenic culture derived from pluripotent stem cells on a culture substrate; (b) removing the upper layer of the myogenic culture, leaving behind a monolayer of INFRA cells attached to the culture substrate; (c) washing the monolayer of INFRA cells with a buffered solution; and (d) mechanically dissociating the INFRA cells from the cell substrate without causing stress or modification of the cells. In some embodiments, the upper layer of myogenic culture is removed by peeling, and in some embodiments the buffered solution is phosphate buffered saline (PBS).

[0010] In some embodiments, the INFRA cells comprise human muscle stem cells (SCs) expressing the transcription factor PAX7 and lacking the expression of the transmembrane receptor PDGFRA. In further embodiments, at least 70% of the population of INFRA cells are PAX7+ and PDGFRA-. In yet further embodiments, the method further comprises the step of purifying the INFRA cells by flow cytometry using antibodies against ITGA7, CD56, NOTCH3, CD82 and / or CDH15.

[0011] Another aspect of the invention provides a method of treating muscle injury in a subject. The method includes contacting the muscle injury of the subject with a therapeutically effective amount of INFRA cells, wherein the INFRA cells comprising human muscle stem cells (SCs) expressing the transcription factor PAX7 and lacking the expression of the transmembrane receptor PDGFRA, wherein at least 70% of the population of human muscle SCs are PAX7+ and PDGFRA-, and wherein the SCs have not undergone modification in the isolation process. In some embodiments, the INFRA cells contact the muscle injury as a result of administration by injection, while in additional embodiments the muscle injury is muscular dystrophy, traumatic injury, or cachexia. BRIEF DESCRIPTION OF THE FIGURES

[0012] The present invention may be more readily understood by reference to the following figures, wherein:

[0013] Figs. 1A - 1G provide graphs and images showing new protocol design: A) UMAP showing cells of the paraxial mesoderm lineage extracted from E8.5, E9.5, E10.5, El 1.5 scRNAseq datasets from mouse embryonic development (Qiu et al., Nat Genet 54, 328-341, 2022), B) Leiden clustering analysis showing the corresponding cell types of the paraxial mesoderm dataset shown in A, C) Computed trajectory of the paraxial mesoderm dataset shown in A (pseudotime going from dark blue to yellow), D) Projection of the activity of the main biological pathways identified in sclerotome / dermomyotome branching from the presomitic mesoderm on the dataset shown in A. Brown maximal and blue minimal activation of the pathways, E) Schematic of the new protocol designed based on the bioinformatics pipeline, illustrating developmental stages and showing the signaling pathways modulated, F) Expression of PAX7 (Anti-GFP in green) and ACTN2 (anti-mKate2 in red) detected by immunohistochemistry in cultures of the double reporter PAX7-Venus-ACTN2-mKate2 line (n=3 / sample), G) FACS analysis at day 31 of PAX7-Venus-ACTN2-mKate2 iPS reporter line cultures showing the percentages of PAX7+, MY0D+, PAX7+MY0D+ and PAX7-MY0D-, shown here with mean and SD (n=5). New protocol in red and the reference Chai protocol in blue.

[0014] Figs. 2A - 21 provide graphs and images showing increased production of PAX7+ SCs in vitro: A) Leiden clustering analysis of the combined SnRNAseq analyses of day 30 cells differentiated with the Chai and New myogenic protocol, B) Distribution of the nuclei of the Chai and New protocol datasets on the UMAP shown in A, C) Bar plot showing the percentage of nuclei in each cluster in the snRNAseq datasets of the Chai protocol (blue) and New protocol (red), D) (Top) UMAP showing clustering analysis of the myogenic nuclei extracted from the snRNAseq dataset of day 30 cultures generated with the New protocol. Green: PAX7+ cells, orange: intermediate myogenic stage (MYOMX), red: myocytes. (Bottom) Expression of specific genes for each cluster, E) (Top): STREAM analysis showing the trajectory on a pseudotime axis recapitulating the myogenic differentiation program of the clusters shown in D. (Bottom): Projection of gene expression of the genes shown in D on the pseudotime axis of the STREAM graph, F) (Top) UMAP showing clustering analysis of the myogenic nuclei extracted from the snRNAseq dataset of day 30 cultures generated with the Chai protocol. Green: PAX7+ cells, orange: intermediate myogenic stage (MYOMK), red: myocytes. (Bottom) Expression of specific genes for each cluster, G) (Top): STREAM analysis showing the trajectory on a pseudotime axis recapitulating the myogenic differentiation program of the clusters shown in F. (Bottom): Projection of gene expression of the genes shown in F on the pseudotime axis of the STREAM graph, H) Dot plot showing the mean expression level of specific genes in each cluster of the New protocol, I) Dot plot showing the mean expression level of specific genes in each cluster of the Chai protocol.

[0015] Figs. 3 A - 3K provide graphs and images showing mechanical enrichment of human Satellite cells: A) Schematic of the step-by-step procedure to harvest INFRA cells from a PSC-derived muscle culture, B) (Left) FACS scatter plot showing the FSC-A (xlOOO) and the fluorescence intensity of PAX7-Venus cells. (Right) Bar plot showing the percentage of PAX7+ cells in the INFRA cell population post-peeling, shown here with mean and SD (n=3), C) Peeling process using a forceps to harvest INFRA cells from a 6-well plate containing day 30 myogenic cultures generated using the New protocol, D) Immunostaining showing PAX7-expressing cells in the INFRA population adherent to culture substrate post-peeling. Anti-GFP antibody (in green) on PAX7-Venus cells differentiated for 30 days according to the New protocol, E) Differentiation of INFRA cells generated from the PAX7-Venus-ACTN2-mkate2 cells after 2 weeks in culture. Immunostaining using an anti-GFP (green) and anti-mkate2 antibody (red), F) (Left) UMAP showing a Leiden clustering analysis of INFRA cells analyzed by scRNAseq immediately post harvesting. (Right) PAX7 expression shown in the same UMAP, G) Dot plot showing specific marker genes for each cluster identified in F, H) Bar plots showing percentages of cells expressing marker genes for each cluster in INFRA cells immediately post harvesting. Green: PAX7+, gray: PAX7-, red bottom: PAX7+MY0D1+, blue bottom: PAX7+PDGFRA+, red top: PAX7-MY0D1+, blue top: PAX7-PDGFRA+, I) (Left) UMAP showing a Leiden clustering analysis of Venus+ FACS-sorted cells from the differentiated PAX7-Venus line analyzed by scRNAseq immediately post harvesting. (Right) PAX7 expression shown in the same UMAP, J) Dot plot showing specific marker genes for each cluster identified in F, K) Bar plots showing percentages of cells expressing marker genes for each cluster in PAX7-Venus+ cells immediately post harvesting. Green: PAX7+, gray: PAX7-, red bottom: PAX7+MY0D1+, blue bottom: PAX7+PDGFRA+, red top: PAX7-MY0D1+, blue top: PAX7-PDGFRA+.

[0016] Figs. 4A - 4R provide graphs and images showing Human PAX7+ cells efficiently colonize mouse muscles and can restore force production: A) Schematic of the grafting protocol, B) X-ray of NOD mice one month after the graft of INFRA cells overlayed with luciferase activity in rainbow colors. Unit is P / sec / mm / sq (n=3), C) Dissected Tibialis Anterior (TA) muscle one month post graft. From left to right: TA from untreated age matched mouse (WT), irradiated TA injected with cardiotoxin and grafted with INFRA cells, control (CTL) irradiated TA injected with cardiotoxin (n=3), D) Comparison of TA weights 1 month post graft using ordinary one-way ANOVA. From left to right: healthy TA (WT), TA irradiated and injected with cardiotoxin grafted with INFRA cells, TA irradiated and injected with cardiotoxin (n=3), E) Bar plot showing percentage of human nuclei counted in transversal cryosections of treated TA, 1 month post graft, shown here with mean and SD (n=4), F) Low magnification of a transverse cryosection of the TA irradiated and injected with cardiotoxin grafted with INFRA cells (left), and control (right) 1 month post-graft. Hoechst (blue), anti-human Lamin B2 (green), anti-human dystrophin (red). 20x tiled confocal images, G) Higher magnification of a transverse cryosection of the TA irradiated and injected with cardiotoxin grafted with INFRA cells 1 month post-graft, labeled with an anti-laminin antibody (white), H) Same section as F labeled with an anti-human Lamin B2 (green), I) Same section as F labeled with an anti-human dystrophin (red), J) Merged image of the G and H panels, K) Higher magnification of a transverse cryosection of the TA irradiated and injected with cardiotoxin grafted with INFRA cells 1 month post-graft. Hoechst in blue, anti-CD31 to highlight blood vessels (arrowhead) in green, and anti-MYH3 in red, L) Higher magnification of a transverse cryosection of the TA irradiated and injected with cardiotoxin grafted with INFRA cells 1 month post-graft. Hoechst in blue, anti-PAX7 to show SCs (arrowheads) in red, and anti-LaminB2 in green, M) Higher magnification of a transverse cryosection of the TA irradiated and injected with cardiotoxin grafted with INFRA cells 1 month post-graft. Hoechst in blue, anti-NG2 to identify pericytes (arrowhead) in red, and anti-LaminB2 in green, N) Force measurements using ordinary oneway ANOVA of the healthy control TA muscle (WT), TA irradiated and injected with cardio toxin and grafted with INFRA cells (grafted) and TA irradiated and injected with cardiotoxin (control) TA, shown here with mean and SD (n=3), O) UMAP showing the human myogenic clusters of cells differentiated in the mouse TA after 1 month, P) Dot plot showing genes differentially expressed in the three clusters of human myogenic cells identified in the grafted TA, Q) Dot plot showing differential expression of genes of the GO term glycolysis in PAX7+ cells in vitro (New) and in vivo (TA), R) Dot plot showing differential expression of genes of the GO term Striated muscle contraction in PAX7+ cells in vitro (New) and in vivo (TA). TA: cells grafted in the TA; New: cells generated in vitro with the optimized protocol. DETAILED DESCRIPTION OF THE INVENTION

[0017] The present invention provides a composition of INFRA cells. The composition includes human muscle stem cells (SCs) expressing the transcription factor PAX7 and lacking the expression of the transmembrane receptor PDGFRA, wherein at least 70% of the population of human muscle SCs are PAX7+ and PDGFRA-. Methods of preparing the INFRA cell compositions, and methods of using the INFRA cells to treat muscle injury are also provided. Definitions

[0018] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which these exemplary embodiments belong. The terminology used in the description herein is for describing particular exemplary embodiments only and is not intended to be limiting of the exemplary embodiments. As used in the specification and the appended claims, the singular forms “a,” “an,” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety.

[0019] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0020] A “cell growth medium,” as defined herein, is an aqueous media containing the factors and nutrients suitable for supporting the growth of multipotent or pluripotent cells, in particular human multipotent or pluripotent cells. For maintenance of multipotent or pluripotent cells, the preferred basic medium is DMEM-F12 (Gibco, Invitrogen cell culture, USA Cat. No. 11320-033) supplemented with 10% KOSR (Life Technologies, Catalog Number 10828-028), non-essential amino acids (1%, Gibco, Catalog Number, 11140-050), 2 ng / ml bFGF (Invitrogen, 13256-029), and L-Glutamine (1% v / v, Gibco, Catalog Number, 25030-081). In some embodiments, such as those pertaining to stage-wise directed differentiation, the basic medium in supporting the first stage of stem cells differentiating into somatic cells from pluripotency is RPMI1640 supplemented with Bovine Serum Albumin (2%, Sigma). The cell culture media applied are well known in the art of cell cultures and all are commercially available.

[0021] "Pluripotent" is meant that the cell can give rise to each of the three embryonic cell lineages as well as extraembryonic cells. Pluripotency as used herein is limited to the inner cell mass state, and is not used at any point to describe lineage restricted progenitors that form during development. The pluripotent cell state is a natural one, but can be propagated in-vitro under specific conditions known to those skilled in the art. In such culture conditions, perpetual maintenance of the stem cell, or pluripotent state, occurs. The defining qualities of the pluripotent cells are not limited to their functional characteristic, but can also be described through gene expression patterns and consequently the identification of pluripotent cells can be accomplished through staining patterns for markers they exhibit. Pluripotent cells, such as embryonic stem cells, can also exhibit alkaline phosphatase activity which is often used in the identification of pluripotent cells.

[0022] “Progenitor cell” refers to a normal cellular state, at any point, which represented a direct lineage ancestor for the terminally differentiated cell. By definition progenitors are not irreversibly destined to adopt the said terminal fate, but may be multipotent for more than one terminal state. As such, they display competence for differentiation into more than a single descendant fate. As development proceeds in any organism, the gradual loss of competency, and thus loss of multipotency, is the hall mark of differentiation and the temporally defined formation of tissues and organs.

[0023] “Stem cell” means a cell that can undergo self-renewal (i.e., progeny with the same differentiation potential) and also produce progeny cells that are more restricted in differentiation potential. Within the context of the invention, a stem cell would also encompass a more differentiated cell that has de-differentiated, for example, by nuclear transfer, by fusion with a more primitive stem cell, by introduction of specific transcription factors, or by culture under specific conditions. See, for example, Wilmut et al., Nature, 385:810-813 (1997); Ying et al., Nature, 416:545-548 (2002); Guan et al., Nature, 440:1199-1203 (2006); Takahashi et al., Cell, 126:663-676 (2006); Okita et al., Nature, 448:313-317 (2007); and Takahashi et al., Cell, 131:861-872 (2007).

[0024] A "subject", as used therein, can be a vertebrate or a mammal. Examples of subjects include livestock, test animals, and pets, such as ovine, bovine, porcine, canine, feline and murine mammals, as well as reptiles, birds and fish. The terms, "patient" and "subject" are used interchangeably herein. Preferably, the subject is a mammal. The mammal can be a human, non-human primate, mouse, rat, dog, cat, horse, or cow, but are not limited to these examples.

[0025] With respect to cells in cell cultures or in cell populations, the term "substantially free of" means that the specified cell type of which the cell culture or cell population is free, is present in an amount of less than about 10%, less than about 9%, less than about 8%, less than about 7%, less than about 6%, less than about 5%, less than about 4%, less than about 3%, less than about 2% or less than about 1% of the total number of cells present in the cell culture or cell population.

[0026] A “therapeutically effective amount” refers to the amount of cells determined to produce any therapeutic response in a subject. A “therapeutically effective amount,” as used herein, refers to a sufficient amount of agent to stimulate muscle growth or decrease or prevent muscle atrophy. The exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the particular therapeutic agent, its mode and / or route of administration, and the like.

[0027] “Treat,” “treating,” or “treatment” are used broadly in relation to the invention and each such term encompasses, among others, preventing, ameliorating, inhibiting, or curing a deficiency, dysfunction, disease, or other deleterious process, including those that interfere with and / or result from a therapy. In various embodiments, the symptoms of a disease or disorder are alleviated by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, or at least 50%.

[0028] All scientific and technical terms used in the present application have meanings commonly used in the art unless otherwise specified. The definitions provided herein are to facilitate understanding of certain terms used frequently herein and are not meant to limit the scope of the present application. INFRA Cells

[0029] In one aspect, the present invention provides a composition of INFRA cells. The INFRA cell composition includes human muscle stem cells (SCs) expressing the transcription factor PAX7 and lacking the expression of the transmembrane receptor PDGFRA, wherein at least 70% of the population of human muscle SCs are PAX7+ and PDGFRA-.

[0030] In some embodiments, the SCs have not undergone modification in the isolation process. Prior art methods of isolation typically stress the SCs, resulting in modification of the cells. Modification caused by the isolation process can include chemical modification and genetic modification.

[0031] In some embodiments, the human muscle SCs of the INFRA cell composition further express a specific set of biomarkers. For example, in some embodiments, the SCs further express the transcription factors PAX3 and / or PAX7 and / or the transmembrane receptors FGFR4, CD82, ITGA7, CDH15, CD56 and / or NOTCH3.

[0032] In some embodiments, the human muscle SCs are derived from pluripotent stem cell (PSC)-derived cultures generated by treating presomitic mesoderm cells with a Wnt activator. Presomitic mesoderm cells are mesenchymal cells that form bilateral streaks next to the notochord in the early paraxial mesoderm, and are derived from the primitive streak or neuromesodermal progenitors in the tail bud.

[0033] The Wnt genes belong to a family of proto-oncogenes and encode over 20 cysteine-rich secreted glycoproteins that activate the Wnt signaling pathway by binding to Frizzled (Fz) receptors found on target cells. In some embodiments, the Wnt signaling pathway activation agent can include a Wnt receptor ligand or agonist, such as a Frizzled receptor agonist. Examples of Wnt ligands include Wntl, Wnt2, Wnt2b, Wnt3, Wnt3a, Wnt4, Wnt5a, Wnt5b, Wnt6, Wnt7a, Wnt7b, Wnt8a, Wnt8b, Wnt9a, Wnt9b, WntlOa, WntlOb, Wntll and Wntl6. Such Wnt ligands, as well as their accession numbers, are described in U.S. Patent No. U.S. 8,460,928, which is herein incorporated by reference. Any one or more of these may be employed to activate Wnt signaling in the TrPCs. Wnt ligands may be obtained from R&D Systems (Minnesota, USA) and from PeproTech, Inc (New lersey, USA).

[0034] In some embodiments, the PSC-derived cultures are generated by treating presomitic mesoderm cells with a Wnt activator and a bone morphogenic protein (BMP) inhibitor. BMPs are secreted cytokines that interact with cells through type I and type II serine / threonine kinase receptors, while BMP inhibitors are substances that block the activity of BMPs. BMP inhibitors include small molecule inhibitors such as dorsomorphin, proteins that bind to BMP receptors such as DAN proteins (e.g., Nbll, chordin, chordin-like proteins, DAN, sclerostin, decorin, gremlin 1, gremlin2, cerberus, and Dand5), secreted modular calcium binding proteins, and nanobodies that are specific for BMP. See Rosen, V., Ann N Y Acad Sci., 1068:19-25 (2006).

[0035] In some embodiments, the INFRA cells are isolated by generating a myogenic culture derived from pluripotent stem cells (PSCs) and removing the upper layer of the culture to expose an adherent monolayer of INFRA cells. Methods of isolating INFRA cells are further described herein.

[0036] In some embodiments, the INFRA cells are further purified by flow cytometry. Flow cytometry is a laser-based method that uses fluorescently labeled antibodies to detect surface antigens and intracellular molecules and uses the light scattering properties of cells to determine their size and complexity. Flow cytometry can use specific traits of the cells to positively purify specific types of cells. For example, in some embodiments, the SCs are further isolated from the isolated INFRA cells by flow cytometry using antibodies against ITGA7, CD56, NOTCH3, CD82 and / or CDH15. In other embodiments, flow cytometry can be used to isolate cells lacking certain features by selecting for other features. For example, in some embodiments, flow cytometry can be used to purify the PDGFRA negative population. See Mushahary et al., Cytometry A., 93(1):19-31 (2018).

[0037] In some embodiments, the INFRA cells represent a population that has been significantly enriched. “Significantly enriched,” as used herein, means that at least 60%, at least 70%, or at least 80% of the culture consists of SCs after the filtration / sorting procedure. Creating a Population of INFRA cells

[0038] Another aspect of the invention provides a method of creating a substantially pure population of INFRA cells. The method includes the steps of (a) generating a myogenic culture derived from pluripotent stem cells on a culture substrate; (b) removing the upper layer of the myogenic culture, leaving behind a monolayer of INFRA cells attached to the culture substrate; (c) washing the monolayer of INFRA cells with a buffered solution; and (d) mechanically dissociating the INFRA cells from the cell substrate without causing stress or modification of the cells.

[0039] The first step of the method includes generating a myogenic culture derived from pluripotent cells on a culture substrate. The culture substrate is a suitable surface for growing the cell culture, such as a culture dish. In some embodiments, the culture substrate can include a layer of inactivated feeder cells such as mouse embryonic fibroblasts to provide a supportive environment for cell growth and adhesion. The cell culture should also include a growth medium that supports the growth of both pluripotent cells and myogenic cells.

[0040] Myogenic cells are generated from pluripotent stem cells (PSCs) by stimulating them with the appropriate chemical factors. For example, transcription factors can be used to stimulate formation of myogenic cells from pluripotent cells. In some embodiments, the PSC-derived cultures are generated by treating presomitic mesoderm cells with a Wnt activator, while in further embodiments the PSC cells are stimulated using a Wnt activator and a bone morphogenic protein (BMP) inhibitor. [0041 ] In the remaining steps of the invention, the upper layer of the PSC-derived muscle fibers and SCs cultures is removed mechanically (e.g., peeled) and then discarded or used for other purposes. The SCs, being adherent to the substrate of the culture, are now isolated from the rest of the culture and remain in the well and can be used for any purposes. In detail, the manipulation starts with a myogenic culture of any size or shape (e.g., but not limited to muscle fibers and SCs cultures derived from human or mouse PSCs, muscle fibers and SCs cultures derived from human or mouse embryonic stem cells, muscle fibers and SCs cultures extracted from patients, etc...). The culture is washed using an aqueous saline buffer such as PBS (phosphate buffered saline), then using a clean or sterile and sharp tool (e.g., tweezers, pipette tips, etc...), the upper layer of the culture is removed (e.g., lifted, aspirated, etc...) from the culture which is washed again using PBS. Then, enough PBS to cover the monolayer of SCs adherent to the culture substrate is added for a few minutes (e.g., 5-15 min) at a temperature ranging from 20 to 37 °C. Preferably the aqueous saline buffer does not contain calcium and magnesium to allow the cells to detach). Other dissociation media could be used (e.g. accutase, trypsin, collagenase...) and other temperatures and time of incubation can be used accordingly. Cells are then detached from the bottom of the plate by pipetting up and down (a few strokes per culture area e.g., but not limited to 10 strokes total) in the dissociation media (e.g. PBS without calcium / magnesium) to perform gentle mechanical dissociation.

[0042] Once the INFRA cells have been detached, they may be used directly for other applications, or they may be further purified using FACS cell sorting. The cells can be used directly for any applications (e.g. but not limited to cell therapy, drug screening, cell culture, sequencing, etc...) with or without resuspension (e.g. growth media could directly be added to the culture) in the desired volume by spinning them down using a centrifuge (e.g. 300 g for 4 minutes) and / or with or without filtration (e.g. but not limited to strainers of 40 qm). A scheme recapitulating the overall procedure and method preparing INFRA cells is shown in Figure 3A. To summarize, the steps of obtaining the INFRA cells include:

[0043] Step 1: Generate a myogenic culture derived from pluripotent stem cells (PSC);

[0044] Step 2: Seize the upper layer containing the muscle fibers;

[0045] Step 3: Peel the upper layer to uncover the muscle stem cells.

[0046] The INFRA cells obtaining using this method can have any of the characteristics described herein for INFRA cell compositions. In some embodiments, the INFRA cells comprise human muscle stem cells (SCs) expressing the transcription factor PAX7 and lacking the expression of the transmembrane receptor PDGFRA. In other embodiments, at least 70% of the population of INFRA cells are PAX7+ and PDGFRA-

[0047] In some embodiments, the method also includes the step of purifying the cells using flow cytometry, as described herein. For example, in some embodiments, the method comprises the step of purifying the INFRA cells by flow cytometry using antibodies against ITGA7, CD56, NOTCH3, CD82 and / or CDH15. Methods of Treating Muscle Injury

[0048] Another aspect of the invention provides a method of treating muscle injury in a subject. The method includes contacting the muscle injury of the subject with a therapeutically effective amount of INFRA cells, wherein the INFRA cells comprising human muscle stem cells (SCs) expressing the transcription factor PAX7 and lacking the expression of the transmembrane receptor PDGFRA, wherein at least 70% of the population of human muscle SCs are PAX7+ and PDGFRA-. In some embodiments, the SCs have not undergone modification in the isolation process.

[0049] Muscle can be divided into three types: skeletal muscle, cardiac muscle, and smooth muscle. Skeletal muscle is muscle tissue capable of generating force and transferring that force to the skeleton to enable breathing, movement, and posture maintenance. Cardiac muscle is muscle of the heart. Smooth muscle is muscle tissue of the arterial and bowel walls. The methods and compositions of the present invention apply primarily to skeletal muscle and, but may additionally positively affect smooth muscles. "Skeletal muscle" and "skeletal muscles" are defined as muscles with interactions with bones, tendons, and joints.

[0050] In some embodiments, a method of treatment of illnesses, diseases, disorders, and injuries that cause a decrease in muscle strength (also referred to herein as musculoskeletal diseases, and as muscle dysfunction and muscle-wasting diseases). Examples of muscle injury include muscular dystrophy, traumatic injury, and cachexia.

[0051] In some embodiments, the invention provides methods for the treatment of musculoskeletal diseases, including muscle dysfunction and muscle-wasting diseases or disorders, including hereditary myopathy, neuromuscular disease, muscular atrophy, drug-induced myopathy, or an illness, disease, disorder or condition that causes a decrease in muscle strength. In some embodiments, the subject has a muscle disease selected from the group consisting of sarcopenia, cachexia, type II muscle fiber atrophy and genetically determined muscular dystrophies or acquired autoimmune primary muscle disorders associated with impaired radial growth phase of regeneration.

[0052] In some embodiments, administration of INFRA cells can be used to treat muscular dystrophies. Muscular dystrophies are family of hereditary or genetic diseases. Genetic defects in genes mainly in striated muscle proteins cause weakness, usually progressive weakness, through a loss of muscle integrity and degeneration of the skeletal or voluntary muscles that control physical movement. Some muscular dystrophies also affect heart and involuntary muscles.

[0053] Examples of muscular dystrophies that can be treated by administration of INFRA cells include Duchenne muscular dystrophy, Becker muscular dystrophy, myotonic muscular dystrophy (also known as Steinert's disease), limb-girdle muscular dystrophy, sarcoglycanopathies, myotonic dystrophy, Emery-Dreifuss muscular dystrophy, congenital muscular dystrophy (e.g., Merosin-deficient congenital muscular dystrophy, Bethlem myopathy, Ullrich congenital muscular dystrophy), fascioscapulohumeral muscular dystrophy, spinal muscular dystrophy, rigid spine muscular dystrophy, distal muscular dystrophy, and oculopharyngeal muscular dystrophy.

[0054] In some embodiments, INFRA cells can be used to treat muscular atrophy. Muscular atrophy is a general term used to describe a condition marked by the wasting or loss muscle tissue resulting from a variety of diseases, disorders, other conditions, or events. Muscle atrophies can be the result of, but are not limited to, protracted immobilization resulting from recovery from severe burns, major joint replacement surgery, neuropathic pain, peripheral neuropathy, necrotizing vasculitis, zero gravity environment (e.g., astronauts and cosmonauts), extended hospitalization, degenerative disease (e.g., amyotrophic lateral sclerosis) and organ transplant as well as spinal cord injury, chronic hemodialysis, and stroke.

[0055] In some embodiments, INFRA cells can be used to treat disuse muscular atrophy. Disuse muscular atrophy is a condition marked by the wasting or loss muscle tissue resulting from long periods of inactivity. Disuse muscular atrophy can be result of, but are not limited to, protracted immobilization resulting from recovery from severe burns, major joint replacement surgery, neuropathic pain, zero gravity environment (e.g., astronauts and cosmonauts), extended hospitalization, anorexia, and organ transplant as well as spinal cord injury, chronic hemodialysis, and stroke.

[0056] In some embodiments, INFRA cells can be used to treat age-related muscular atrophy. Age-related muscular atrophy is a condition marked by the wasting or loss muscle tissue and the replacement of muscle tissue with fibrosis tissue as the subject ages.

[0057] In some embodiments, INFRA cells can be used to treat sarcopenia. Sarcopenia is a condition marked by the wasting or loss muscle tissue and the replacement of muscle tissue with fibrosis tissue as the subject ages.

[0058] In some embodiments, INFRA cells can be used to treat the muscle wasting in cachexia. Cachexia is loss of weight, muscle atrophy, fatigue, weakness and significant loss of appetite in someone who is not actively trying to lose weight, but rather as the result of chronic disease. The muscle wasting component of cachexia can be result of, but are not limited to, cancer, multiple sclerosis, tuberculosis, acquired immune deficiency syndrome, human immunodeficiency virus, malnutrition, Parkinson's disease, emphysema, heart failure, motor neuron disease, cystic fibrosis, dementia, sarcopenia, chronic obstructive pulmonary disease, kidney disease, and kidney failure.

[0059] In some embodiments, the muscle injury is a traumatic muscle injury. Traumatic muscle injury is damage to muscle tissue caused by high stress or strain, or by an external impact. Traumatic muscle injury is often due to muscle activation while the muscle is lengthening, resulting in indirect and non-contact muscle injuries (strains or ruptures), or from external impact, resulting in direct muscle injuries (contusion or laceration). Edouard et al., Nat Rev Dis Primers., 9(1):56 (2023).

[0060] Contacting, as used herein, refers to placing or administering the INFRA cells so that they can interact with the injured muscle. Contacting includes both direct contact, and indirect contact in which the INFRA cells are administered or placed such that they will move through natural processes to the site of muscle injury. Placing the INFRA cells can include putting the cells on a wound dressing, while administering the cells can include injection of the cells.

[0061] Therapeutically effective dosages can be determined using various methods, such as animal studies. In some embodiments, data obtained from the cell culture assays and in animal studies can be used in formulating a range of dosage for use in humans. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.

[0062] The therapeutically effective dose of a composition comprising a population of INFRA cells can also be estimated initially from cell culture assays. A dose may be formulated in animal models in vivo to achieve a significant effect on muscle injury. Alternatively, the effects of any particular dosage can be monitored by a suitable bioassay.

[0063] Typically said dose is about 10 x 106 cells / kg of subject weight or lower, is about 9 x 106 cells / kg or lower, is about 8 x 106 cells / kg or lower, is about 7 x 106 cells / kg or lower, is about 6 x 106 cells / kg or lower, is about 5 x 106 cells / kg or lower. In an alternative embodiment said dose may be between about 0.25 x 106 cells / kg to about 5 x 106 cells / kg; or more preferably about 1 x 106 cells / kg to about 5 x 106 cells / kg. Accordingly in further alternative embodiments the dose may be about 0.25 x 106 cells / kg, 0.5 x 106 cells / kg, 0.6 x 106 cells / kg, 0.7 x 106 cells / kg; 0.8 x 106 cells / kg; 0.9 x 106 cells / kg; 1.1 x 106 cells / kg; 1.2 x 106 cells / kg; 1.3 x 106 cells / kg; 1.4 x 106 cells / kg; 1.5 x 106 cells / kg; 1.6 x 106 cells / kg; 1.7 x 106 cells / kg; 1.8 x 106 cells / kg; 1.9 x 106 cells / kg or 2 x 106 cells / kg. The dose may, in other embodiments, be between 0.1 and 1 million cells / kg; or between 1 and 2 million cells / kg; or between 2 and 3 million cells / kg; or between 3 and 4 million cells / kg; or between 4 and 5 million cells / kg; or between 5 and 6 million cells / kg; or between 6 and 7 million cells / kg; or between 7 and 8 million cells / kg; or between 8 and 9 million cells / kg; or between 9 and 10 million cells / kg.

[0064] With respect to duration and frequency of treatment, it is typical for skilled clinicians to monitor subjects in order to determine when the treatment is providing therapeutic benefit, and to determine whether to increase or decrease dosage, increase or decrease administration frequency, discontinue treatment, resume treatment or make other alteration to treatment regimen. The dosing schedule can vary from once a week to daily depending on a number of clinical factors, such as the subject's sensitivity to the polypeptides. The desired dose can be administered at one time or divided into subdoses, e.g., 2-4 subdoses and administered over a period of time, e.g., at appropriate intervals through the day or other appropriate schedule. Such sub-doses can be administered as unit dosage forms. In some embodiments, administration is chronic, e.g., one or more doses daily over a period of weeks or months. Examples of dosing schedules are administration daily, twice daily, three times daily or four or more times daily over a period of 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, or 6 months or more.

[0065] Exemplary modes of administration include, but are not limited to, injection, infusion, instillation, inhalation, or ingestion. "Injection" includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intraventricular, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, sub capsular, subarachnoid, intraspinal, intracerebro spinal, and intrasternal injection and infusion. In preferred embodiments, the compositions are administered by intravenous infusion or injection.

[0066] INFRA cells can be supplied in the form of a pharmaceutical composition, comprising an isotonic excipient prepared under sufficiently sterile conditions for human administration. The composition can be sterile. The formulation should suit the mode of administration. For general principles in medicinal formulation, the reader is referred 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, E. D. Ball, I. Lister & P. Law, Churchill Livingstone, 2000. Choice of the cellular excipient and any accompanying elements of the composition comprising a population of INFRA cells will be adapted in accordance with the route and device used for administration.

[0067] In some embodiments, the INFRA cells are administered together with a pharmaceutically acceptable carrier. Suitable pharmaceutically acceptable carriers include but are not limited to water, salt solutions (e.g., NaCl), saline, buffered saline, alcohols, glycerol, ethanol, gum arabic, vegetable oils, benzyl alcohols, polyethylene glycols, gelatin, carbohydrates such as lactose, amylose or starch, dextrose, magnesium stearate, talc, silicic acid, viscous paraffin, perfume oil, fatty acid esters, hydroxymethylcellulose, polyvinyl pyrolidone, etc., as well as combinations thereof. The pharmaceutical preparations can, if desired, be mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring, flavoring and / or aromatic substances and the like that do not deleteriously react with the active compounds.

[0068] Suitable preservatives and buffers can be used in such formulations. In order to minimize or eliminate irritation at the site of injection, such compositions may contain one or more nonionic surfactants having a hydrophile-lipophile balance (HLB) of from about 12 to about 17. The quantity of surfactant in such formulations ranges from about 5% to about 15% by weight. Suitable surfactants include polyethylene sorbitan fatty acid esters, such as sorbitan monooleate and the high molecular weight adducts of ethylene oxide with a hydrophobic base, formed by the condensation of propylene oxide with propylene glycol. The parenteral formulations can be presented in unit-dose or multi-dose sealed containers, such as ampoules and vials, and can be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example, water, for injections, immediately prior to use. Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules, and tablets.

[0069] Preferably, the cells are administered by injection, e.g., intravenously. The pharmaceutically acceptable carrier for the cells for injection may include any isotonic carrier such as, for example, normal saline (about 0.90% w / v of NaCl in water, about 300 mOsm / L NaCl in water, or about 9.0 g NaCl per liter of water), NORMOSOL R electrolyte solution (Abbott, Chicago, Ill.), PLASMA-LYTE A (Baxter, Deerfield, Ill.), about 5% dextrose in water, or Ringer's lactate. In an embodiment, the pharmaceutically acceptable carrier is supplemented with human serum albumen.

[0070] An example has been included to more clearly describe a particular embodiment of the invention. However, there are a wide variety of other embodiments within the scope of the present invention, which should not be limited to the particular example provided herein. EXAMPLE Optimization of in vitro production of human satellite cells for skeletal muscle cell therapy

[0071] Here, we describe an optimized protocol allowing the production of human iPS-derived cell populations containing up to 75% of PAX7+SC without antibody-based sorting. This protocol could considerably simplify the production of SCs for clinical applications. We further show that these cells can engraft very efficiently in mouse muscles, generating myofibers and SCs and contributing to the restoration of force in the chimeric muscles.

[0072] To define such a protocol, we developed an integrated pipeline that combines bioinformatics with in vitro cell culture. We started with the analysis of a multi-time points single cell RNA sequencing (scRNAseq) dataset of mouse embryos from stages E8.5, E9.5, E10.5 and El 1.5 (whole embryos staged in one-somite increments (Qiu et al., Nat Genet 54, 328-341, 2022) as they match the time frame of Presomitic Mesoderm (PSM) differentiation into myogenic precursors. We extracted the paraxial mesoderm from this dataset to study the bifurcation from PSM to sclerotome and dermomyotome characterized by the expression of PAX1 / 9 and PAX3 respectively (Fig 1A-B). We used Palantir diffusion maps and force atlas (Setty et al., Nat Biotechnol 37,451-460,2019) to create a 2D representation of our final subset of interest and confirmed the annotation of the different cell types using differentially expressed genes. Then, we assigned the PSM node as the trajectory root and used a simple principal tree algorithm to learn a tree structure on our data. We next used a pseudotime algorithm (scfates) (Faure et al., Bioinformatics 39, 2023) to assign temporality within the tree branches (Fig IC). The two main identified branches coming from the anterior PSM (characterized by ME0X1) are the myogenic derivatives on one branch (characterized by PAX7 and TTN) and the cartilage / connective tissue progenitors (SOX5, PAX9 and PAX1) on the other branch (Fig 1B-C). Then, for each cell, we scored the average expression of sets of genes related to known signaling pathways described in BioPlanet (Huang et al., Front Pharmacol 10,445,2019). The score is the average expression of the set of genes subtracted with the average expression of the reference set of genes (being randomly sampled from the genes contained within the dataset). This score is calculated for every signaling pathway, then fitted to the trajectory using a machine learning algorithm (mgcv : a generalized additive model) (Wood, Generalized Additive Models: An Introduction with R, Second Edition (2nd ed.), 2017) and can be visualized in our force atlas space over pseudotime (Fig ID). As a result, we identified the Tyrosine receptor kinase B (TRKB), Bone Morphogenetic Protein (BMP), Platelet-derived growth factor (PDGF) and Transforming growth factor beta (TGFB) pathways as being activated in the sclerotome branch and inhibited in the dermomyotome branch.

[0073] This knowledge was then used to establish a new myogenic differentiation protocol in which we aimed to enrich SCs by reducing the contribution of sclerotome-derived lineages (Fig IE). We kept the initial steps necessary for PSM generation, which involve Wnt and FGF activation and BMP inhibition described in our original muscle differentiation protocol (Chai) (Chai eta / ., Nature protocols 11, 1833-1850, 2016). The Chai protocol was based on myogenic cues described in the literature and used BMP inhibition (LDN), Hepatocyte growth factor (HGF), Fibroblast growth factor (FGF-2) and Insulin-Like Growth Factor (IGF-1) from day 6 to day 8, then IGF-1 from day 8 to day 12, finally IGF-1 and HGF starting from day 12. Based on the new findings from our bioinformatics pipeline, we treated the anterior PSM cells with BMP (LDN) and TGFb (SB 431542) inhibitors from day 6 to day 9 to block sclerotome differentiation. Next, from day 9 to day 12, we added Platelet-Derived Growth Factor (PDGF) (AC 710) and Tyrosine receptor kinase B (TrkB) (ANA 12) inhibitors in addition to the LDN and TGFb inhibition. Finally, we inhibited BMP and PDGF pathways starting from day 12 to inhibit the differentiation of cells into fibroblasts (Fig IE). This new protocol is serum-free, as inhibitors were added to Dulbecco's Modified Eagle Medium (DMEM) with 15% Knock-Out Serum Replacement (KSR). To track cells acquiring the sclerotome / chondrocyte fate, we engineered a SOX9-Venus human iPS reporter line. Using flow cytometry, we observed a decrease of the SOX9-Venus+ cells (-2.4%) at day 9 compared to the Chai protocol. Using an iPS reporter line for PAX3 expression (Rao et al., Dev Cell 58, 2359-2375, 2023), we showed that this SOX9 decrease correlates with a significant increase (+13.4%) in the PAX3-GFP+ cells which mostly correspond to dermomyotome precursors at day 9. To follow the myogenic differentiation in live cells, we engineered a double reporter human iPS line expressing a PAX7-Venus and an alpha actinin mKate2 reporter. At day 14, we detected the first PAX7+ cells and myofibers expressing ACTN2 (Fig IF). The number of multinucleated striated myofibers and PAX7+ cells dramatically increased during the subsequent phase of the culture. To quantify the myogenic cell populations, we introduced a mCherry fluorescent protein in the MYODI locus in the PAX7-Venus reporter line. At day 31, with our optimized protocol, using the PAX7-MY0D1 reporter line, we observed a significant increase in Venus+ cells (+23.1%), mCherry+ cells (+9.8%), and double-positive cells (+21.1%) together with a decrease of double-negative cells (-15.5%) (Fig 1G). Thus, the protocol optimized with our bioinformatics pipeline allows the generation of up to 52.3% of PAX7-Venus+ cells on average in the mononucleated fraction, resulting in a significant enrichment in the PAX7+ population compared to the Chai protocol. L0074J Several reports have used scRNAseq to describe the cell composition of myogenic cultures generated from human PSCs (Xi et al.. Cell stem cell, Vol. 27, Issue 1, 158-176, 2020). However, these reports provide an incomplete description of the cellular diversity, as the syncytial myofibers cannot be encapsulated and sequenced with this technology. We therefore used single nuclei RNA sequencing (snRNAseq) to generate a full characterization of the myogenic cultures generated with our optimized protocol in comparison to the original Chai protocol. Such an analysis allows capturing myofibers nuclei that are otherwise not quantified by FACS or scRNAseq analyses. We performed nuclei extraction from day 31 cultures followed by lOx encapsulation and sequencing. In total, we analyzed 1881 cells from the Chai protocol and 6763 cells from the new protocol. Both datasets were similarly processed to generate UMAP projections and Leiden clustering. The identity of the clusters was established using differential gene expression. Merging and reclustering the two datasets show that they contain the same cell types corresponding to PSM derivatives including fibroblastic progenitors (PDGFRA+), satellite cells (PAX7+), and myocytes (ACTN2+) as well as neural cells (MAP2+) (Fig 2 A-B). However, the two protocols yield different proportions of these cell types. Our new protocol yielded twice as many SC nuclei (25.1%) compared to the Chai protocol (10.8%) (Fig 2C). This was accompanied with a lower proportion of all other populations (fibroblastic, myocytes and neural) thus confirming our analyses with the reporter lines.

[0075] To better characterize the skeletal muscle lineage trajectory in vitro, we extracted all the myogenic nuclei (corresponding to SCs and myogenic 1'ibers) from the two datasets. We then re-clustered and annotated them independently (Fig 2D-I). For both protocols, this identified three similar clusters which are connected along a developmental trajectory (Fig 2D-G). One cluster contains the PAX7-expressing SCs which also express well-known markers of this lineage including NOTCH3, FGFR4, and CD82. We also identified two additional myogenic clusters corresponding to distinct stages of myofibers maturation. One myoblasttype cluster is enriched in cells expressing MYODI, MYOG, MYMK, MYMX, HES6 and CKB thus corresponding to a previously described transitional state between SCs and myofibers (Al Tanoury et al., Development. Jun 26; 147(12), 2020). This cluster was connected to a myocyte cluster enriched in genes coding for myofiber structural proteins such as TTN, DMD, MYH3 and MYH8 expressed by post-mitotic myofibers. STREAM trajectory analysis shows the recapitulation of satellite cells differentiation into muscle fibers with the sequential expression of characteristic markers of myogenesis (Fig 2E, G) (Chen et al., Nature communications 10, 1903, 2019). Next, we created a ‘satellite cell score’ based on expression of the SC markers PAX7, NOTCH3, FGFR4, CD82, and CDH15 and displayed it in a matrix plot. The SC cluster from our new protocol exhibits the highest score of all clusters from both protocols. We also calculated a normalized enrichment score for the signaling pathways that we are modulating in the two myogenic protocols. This score was computed using the pre-rank function of GSEA and Bioplanet signaling pathways gene sets. The pathways inhibited in the new protocol were all decreased compared to the Chai protocol. Thus, snRNAseq analysis confirms that our optimized protocol increases the proportion of SCs in the myogenic cultures.

[0076] To identify the developmental stage of the myogenic cells generated in vitro, we compared their gene expression profiles to a snRNAseq dataset of human embryonic limbs ranging from post-conception week (PCW) 5 to 9 (Zhang el al., bioRxiv, 2022). We first extracted cells of the myogenic lineage (based on the original annotations) and performed clustering of this myogenic dataset using Leiden. This analysis identified three myogenic clusters, including SCs (PAX7+), myoblasts (MYMK+) and myocytes (MYH3+). STREAM pseudotime analysis identified the developmental trajectory linking these three clusters. Comparison of the three clusters identified in the in vitro datasets (Chai and New) using a classifier trained against the human embryo myogenic dataset indicated that the three clusters detected in vitro are similar to their in vivo counterpart. Further, the classifier indicated that the SCs generated in vitro are closest to the 8 weeks PAX7+SCs in the human embryo, thus, staging the in vitro SC at the embryonic / fetal transition, as previously suggested (Xi et al., Cell stem cell, Vol. 27, Issue 1, 158-176, 2020).

[0077] We next developed a method to enrich for functional SCs without resorting to immunolabeling and cell sorting. At day 20-30 of myogenic differentiation with our original (Chai) as well as our optimized protocol, most of the culture forms a thick upper layer containing the myofibers, whereas most of the PAX7+ cells adhere to the culture plate substrate. The thick upper layer can be removed mechanically (e.g., peeled using forceps) and then discarded, leaving a population enriched in myogenic cells adherent to the culture dish (INFRA cells) (Fig 3A). Dissociation and analysis by flow cytometry of this adherent mononuclear cell fraction generated using the PAX7-Venus reporter line demonstrated that this strategy yields a population containing 74.1% PAX7+ cells on average (Fig 3B). Immunostaining with a PAX7 antibody directly post-peeling confirms the remarkable enrichment in PAX7+ cells (Fig 3C-D). Peeled cultures maintained for 2 weeks in vitro in DMEM / 15% KSR differentiate into myofibers able to spontaneously contract in vitro and selfrenew to generate PAX7+ cells, thus demonstrating properties very similar to that of mouse fetal SC cells (Fig 3E) (Tierney et al., Cell Reports, 1-13, 2016).

[0078] We next performed scRNAseq of the INFRA cells generated with our new protocol directly after peeling the top layer in day 31 cultures. We compared the scRNAseq data of 3423 INFRA cells generated with the new protocol (Fig 3F-H) to 3135 PAX7-Venus positive cells sorted by FACS from 31-day cultures generated with the Chai protocol (Fig 3I-K). Using a similar preprocessing followed by UMAP projection and Leiden clustering, we were able to identify clusters of the same cell types in both INFRA and PAX7-Venus+ cell populations sorted by FACS. These include SCs (PAX7+), myocytes (MY0G+) and fibroblastic cells (PDGFRA+) which express similar sets of characteristic genes (Fig 3G-J). The same clusters were identified when we merged the two datasets followed by Leiden clustering. Using a classifier, we confirmed the similarity of the clusters identified in INFRA and in PAX7+ sorted cells. The INFRA cell population generated with the new protocol contained 74% PAX7+ cells, while 89% PAX7+ cells were obtained following FACS-sorting of the PAX7+Venus cells generated with the Chai protocol (Fig 3H, K). The presence of myocytes that have downregulated PAX7 (and therefore lack PAX7 expression despite their positive selection) can be explained by the stability of the YFP marker used for sorting the cells (Al Tanoury et al., Development. Jun 26;147(12), 2020). The minor fibroblast population detected upon FACS purification could represent a contamination of the PAX7+ cells. Alternatively, these cells could represent fibroblastic descendants of the PAX7+ cells that retained the fluorescent marker after downregulation of PAX7 expression as observed for the myogenic cells. Therefore, combining our new protocol with this mechanical procedure allows enriching in true PAX7+ cells to levels close to FACS sorting.

[0079] We next established a protocol to efficiently colonize mouse skeletal muscles with iPS-derived human PAX7+ SCs produced in vitro. To track the fate of grafted cells in vivo, we introduced a luciferase in the safe harbor A A VS in the human PAX7-Venus-ACTN2-mKate iPS reporter line. PAX7+ cells were produced after 31 days of myogenic differentiation in vitro following the Chai protocol. These cultures contained on average 28.18% of PAX7+ cells in the mononucleated fraction. The legs of immunocompromised NOD mice were irradiated (18 Gray) 2 days before cell injection to block proliferation of endogenous satellite cells. To induce muscle injury and promote regeneration, we injected cardiotoxin in the Tibialis Anterior (TA) muscle 24 hours before the graft. We purified PAX7-Venus+ cells by FACS from day 31 cultures and injected one million Venus-positive SCs in the right TA while the left TA received the same treatment but no grafted cells. After 1 month of engraftment, we quantified the bioluminescence emitted by the injected human cells. The luciferase signal was exclusively observed in the grafted TA region suggesting that the human cells remained largely localized in the area where they were injected. The control non grafted TA was severely atrophic, whereas the grafted TA weight was not significatively different from that of the healthy muscle. Transverse cryosections of grafted and control TA muscles labeled with a human-specific laminB2 antibody indicated that the percentage of human nuclei inside the grafted TA averaged 75.5% of the total number of nuclei (n=3). Immunostainings of transverse hindlimb muscle cryosections for human dystrophin and laminin, demonstrated successful engraftment of human cells in the quasi-totality of the grafted TA. No human cells were observed in adjacent muscles which were not injected thus suggesting that the colonization potential of the PAX7+ cells is restricted to the injected muscle. Most human myofibers express embryonic myosin MYH3, suggesting that they retain an immature character. CD31+ endothelial cells were observed within the grafted territory indicating that the newly formed muscle territory is vascularized. Therefore, this protocol allows massive colonization of the mouse TA by descendants of injected human PAX7+ cells which form myofibers expressing human dystrophin.

[0080] Finally, we tested the engraftment capacity and the functional properties of the SC produced with our optimized protocol. We injected 500k INFRA cells produced with our new protocol directly after peeling at day 30 (without cell sorting), using the grafting protocol described above (Fig 4A). After 1-month, bioluminescence analysis demonstrated the presence of human cells in the injected but not in the control TA (Fig 4B). The difference in size between grafted and ungrafted TAs was visible at the macroscopic level (Fig 4C). The weight of the grafted TA was significatively higher compared to control and not significatively different from the normal TA muscle (Fig 4D). The percentage of human nuclei in grafted TAs averaged 62% of the total number of nuclei (n=4; Fig 4E-F). Immunostainings of transverse hindlimb cryosections for human nuclei, human dystrophin and laminin demonstrates the presence of human cells in the quasi-totality of the grafted TA (Fig 4F-J). Higher magnification of sections of the grafted TA shows that the immature human myofibers are parallel to the long axis of the muscle thus elongating as the endogenous myofibers (Fig 4G-J). These fibers expressed MYH3, indicating their fetal nature (Fig 4K). We also identified human PAX7+ cells, suggesting that the injected cells can self-renew (Fig 4L). We also observed CD31+ blood vessels in the areas rich in human cells indicating vascularization of these territories (Fig 4K). To assess the functionality of the grafted muscles, we performed force tests in vivo. The force (nM) normalized by body weight (mg) developed by the grafted TA was significantly higher than the control TA but significantly lower than the TA of same age (10-week-old) untreated NOD mice (Fig 4N). Together, these experiments demonstrate the striking colonization potential of human PAX7+ SCs produced in vitro and their ability to generate myofibers and rescue force production in damaged muscles.

[0081] To examine the fate of the human cells in the mouse TA post-engraftment, we performed snRNA-seq of a whole TA muscle one month after grafting. A total of 3833 nuclei were purified and sequenced using the lOx pipeline. 86% of these nuclei were identified as human while 14% were of mouse origin based on the cellranger attribution (by mapping the reads of both GRCh38 and mm 10 genomes). Human nuclei were processed to generate UMAP projections and Leiden clustering. This showed that the original injected cell population differentiated into four major cell types including myocytes, fibroblasts, pericytes and SCs. Using an NG2 antibody combined with human nuclei antibody, we confirmed the presence of human pericytes post-injection of INFRA cells (Figure 4M). In contrast, the mouse cell population was mostly composed of immune cells including macrophages (MITF, CD 14) and Dendritic Cells (CD11C, FLT3) as well as Vascular Cells (PECAM1). In the myogenic clusters, we identified populations expressing the embryonic myosin MYH3 (91%), the slow myosin MYH7 (22%), the perinatal myosin MYH8 (41%) and the fast myosin MYH2 (3%). We next extracted and clustered the human myogenic cells from the dataset to perform more detailed analyses. The myogenic cells segregated into three clusters exhibiting similar characteristics to the SC, myoblasts and myocytes clusters described in vivo and in vitro (Figure 4P). Therefore, the grafted human PAX7 SC are able to reconstitute a normal developmental myogenic trajectory in vivo in the mouse TA.

[0082] To establish the identity of the grafted cells in vivo, we used a classifier to compare clusters of human myogenic limb cells from the grafted TA to the myogenic clusters of the human limb dataset. This demonstrated an excellent correspondence between the SC, myoblasts and myocytes clusters. We next performed DEG analysis between myogenic cells from the New protocol in vitro dataset and in vivo clusters from the human embryonic limb. We analyzed GO-term enrichment in the lists of DEGs. Interestingly, this analysis revealed a metabolic switch involving a strong increase in the expression of all glycolytic enzymes suggesting an upregulation of the glycolytic activity in grafted SC cells in vivo (Fig 4Q). In mouse, such a metabolic switch is also observed in fetal and adult activated SCs, which proliferate and exhibit many similarities with the grafted cells (Pala etal., J Cell Sci 131, 2018). As expected, we also observed a strong increase in expression levels of sarcomeric proteins for the myocyte cluster in vivo, consistent with the expected maturation of cells in vivo (Fig 4R). An increase in expression of ECM protein-coding genes such as laminin, fibronectin, collagens as well as the angiogenic factor VEGFA was observed in SC in vivo. These data therefore argue that the grafted PAX7 cells undergo maturation when grafted in the mouse TA and participate to the local tissue remodeling.

[0083] Here, we leveraged a machine learning-based strategy to identify signaling cues deployed during early stages of myogenic differentiation to optimize our original protocol. This allowed us to approximately double the yield of our original protocol from 25 to ~50 % human PAX7+ cells in vitro. This was achieved by preventing the cells to differentiate towards sclerotome via inhibition of PDGF, TGFb, and BMP pathways which are well-known to play a role in sclerotome development in mouse (Alkhatib et al., Curr Mol Biol Rep 4, 132-141, 2018). We also identified the TRKB (NTRK2) pathway as being selectively activated in the sclerotome. This pathway, which has been mostly studied in the nervous system where TRKB binds the neurotrophin BDNF, has also been implicated in fibroblast survival and proliferation (Glass et al., Cell 66, 405-413, 1991). The recombinant growth factors used in our original myogenic differentiation protocol were entirely substituted by chemical compounds, which should considerably favor the transfer to GMP conditions for the optimization of cell-based therapy protocols. We also added a simple mechanical step which results in an enrichment of more than 70% in PAX7+ SCs. This enrichment level compares favorably with the 70-90% myoblast purity which was used in clinical trials (Skuk et al., Neuromuscul Disord 17, 38-46, 2007). Whether the remaining fraction of fibroblasts which appear to proliferate significantly in vivo is a positive or negative factor in the context of such grafts remains to be established. Therefore, this method may circumvent the need for antibody-based purification which significantly increase the cost and the complexity of the cell preparation protocol. Finally, we show that the human myogenic precursors cells grafted in vivo can efficiently colonize mouse muscles and differentiate in dystrophin-positive muscle fibers. The grafted cells can restore mass and force, supporting their use for cell therapy applications for muscle degenerative diseases such as Duchenne Muscular Dystrophy. Methods: Human induced pluripotent stem cell (iPSC) culture

[0084] NCRM1 iPSC cells (RUCDR, Rutgers University) were used throughout this study. Written informed consent from the donor was obtained by Rutgers University at the time of sample collection. Work on human iPS cell lines was approved by Mass General Brigham IRB. Cells were maintained in Matrigel-coated plates (Coming 35277) in mTeSRl medium (StemCell Technologies 05851) and passed every four days. Briefly, cultures of 90% confluency were dissociated with Accutase (Corning 25058CI) and 500,000 cells were seeded into one well of a 6-well plate with mTeSRl and 10 pM Y-27632 dihydrochloride (Tocris Biosciencel254). Fresh mTeSRl medium was supplemented daily in the following days. All cell lines were maintained no longer than 15 passages and were regularly tested for mycoplasma contamination. Generation of reporter cell lines

[0085] The CRISPR-Cas9 system for genome editing was used to generate all human iPSC reporter lines with a previously described approach (Miao et al.. Nature, 21 Dec 2022, 614(7948):500-508). The following reporter lines were generated from previous studies: Line#l, mKate2-ACTN2 knock-in reporter line (Mao et al., eLife 11, 2022); Line#2, Venus-NLS-T2A-PAX7 reporter line (Al Tanoury et al., Development. Jun 26; 147(12), 2020). We generated Line#3-6 in this study.

[0086] To make the double reporter line of mKate2-ACTN2 and Venus-NLS-T2A-PAX7 (Line#3), a single-guide RNA targeting the 5' end of PAX7 was designed using an online tool and cloned into the pGuide-it-tdTomato vector (Takara 632604). We also generated a repair plasmid consisting of 1-kb 3' and 5' homology arms flanking a YFP variant Venus, a nuclear localization signal (NLS), and a self-cleaving T2A peptide sequence in a pUC19 vector backbone by In-Fusion cloning (Takara Bio 638909). Both the pGuideit-tdTomato and repair plasmids were delivered to Line#l iPS cells by nucleofection (Lonza VPH-5022). 24 hours after nucleofection, cells were sorted by TdTomato expression using an S3 cell sorter (Biorad) and seeded at low density (500 cells per 35mm dish) in Matrigel coated plates in mTeSRl with lOpM Y-27632 and CloneR (Stemcell Technologies 05888). Single cells were expanded clonally, and individual colonies were screened by PCR for targeted homozygous insertion of Venus-NLS-T2A immediately after the start codon of PAX7.

[0087] An identical approach was used to make additional reporter cell lines. To make the double reporter line of Venus-NLS-T2A-PAX7 and MYODl-T2A-H2B-mCherry (Line#4), a single-guide RNA targeting the 3' end of MYODI was designed and cloned into the pGuide-it-tdTomato vector. The repair plasmid consisted of 1-kb 3' and 5' homology arms flanking a T2A-H2B-mCherry sequence in a pUC19 vector backbone. These plasmids were delivered to Line#2 iPS cells to generate Line#4 with homozygous insertion of T2A-H2B-mCherry immediately before the stop codon of MYODI. To make the SOX9-T2A-H2B-GFP reporter line (Line #5), a single-guide RNA targeting the 3' end of SOX9 was designed and cloned into the pGuide-it-tdTomato vector. It was delivered to NCRM1 iPSC line, together with the repair plasmid (Addgene 167972), to generate Line#5 with homozygous insertion of T2A-H2B-GFP immediately before the stop codon of SOX9. Sanger sequencing was performed in all cases to ensure in-frame positioning of the reporter fragment and to rule out the presence of indels at recombination site.

[0088] We inserted the constitutively expressed pCAG-luciferase in the safe harbor AAVS1 locus of Line#3 to generate Line#6. In brief, we cloned the luciferase sequence into the AAVSl-pCAG vector (Addgene 80490) and co-transfected it along with the pXAT2 vector (Addgene 80494) into Line#3 iPS cells. One day after nucleofection, we selected positive clones by supplementing mTeSRl with puromycin (0.5 pg / ml, Sigma-Aldrich P7255) for a total of 4 days. Single colonies were expanded and insertions were confirmed by PCR. Skeletal Muscle Fibers and Satellite cells derived from human induced Pluripotent Stem Cells optimized protocol

[0089] (Day -1) Mature iPS cell cultures (100% confluency) are dissociated in Accutase and 200,000-240,000 cells are seeded into one Matrigel-coated well of a 6-well plate with 1.5 ml mTeSRl medium and 10 pM Y-27632. The seeding number is important for further differentiation, with a lower range working best. (Day 0) Cells are treated with DMEM / F12 GlutaMAX supplemented with 1 % ITS, 3 pM CHIR99021 and 0.5 pM LDN-193189 (PSM medium) for 3 days. (Day 3) Cells are then cultured in DMEM / F12 GlutaMAX supplemented with 1 % ITS, 3 pM CHIR99021, 0.5 pM LDN-193189 and 20 ng / ml FGF-2 for 3 days. (Day 6) Cells are then treated with DMEM high glucose supplemented with 15% KSR, IX NEAA, 0.01 mM bME, 1 % ITS, 0.5 pM LDN-193189, 10 pM SB 431542 for 3 days. (Day 9) Cells are then treated with DMEM high glucose supplemented with 15% KSR, IX NEAA, 0.01 mM bME, 1 % ITS, 0.5 pM LDN-193189, 10 pM SB 431542, 15 nM AC 710, 5 pM ANA 12 for 3 days. (Day 12) Cells were then cultured in DMEM high glucose supplemented with 15% KSR, IX NEAA, 0.01 mM bME, 1 % ITS, 0.5 pM LDN-193189, 10 pM SB 431542, for 3 consecutive days. Culture medium was then changed every three days. Skeletal Muscle fibers and satellite cells, generated with this protocol, can then be maintained for at least 90 days in DMEM high glucose supplemented with 15% KSR, IX NEAA, 0.01 mM bME, 1 % ITS, 0.5 pM LDN-193189, 15 nM AC 710. INFRA cells

[0090] Day 20-30 myogenic cultures form a thick superficial layer containing the myofibers which can be removed mechanically and then discarded. The PAX7+ satellite cells which at this stage are adherent to the culture substrate, remain attached. iPS derived myogenic cultures were performed in 6 well plates and then washed with calcium and magnesium-free PBS (phosphate buffered saline). Then using a clean or sterile sharp tool, such as Dumont forceps, the upper layer of the culture was removed. To harvest INFRA cells, wells were incubated in enough PBS to cover the remaining adherent cell monolayer for 10 minutes at 27 °C. Cells were then detached from the bottom of the plate by pipetting up and down (a few strokes per culture area) in PBS to perform gentle mechanical dissociation. Cells were then spun down (300 g for 4 minutes) after filtration (Falcon cell strainer, mesh of 40 pm), suspended in PBS and used for further analysis. Flow cytometry analysis

[0091] To determine the fraction of cells expressing a fluorescent reporter, cultures were dissociated in Trypsin 0.05% (Thermo Fisher Scientific, 25200-056), Collagenase Type IV (Thermo Fisher Scientific, 17104019) and PBS (2:2:5) and analyzed by flow cytometry using an ARIA cell sorter (BD). Cells from parental NCRM1 line, which do not express the fluorescent protein, were used as a negative control for gating purposes. Results were presented as the percentage of fluorescent positive cells in the sorted fraction after exclusion of debris and doublets. Immunostaining

[0092] Cells were fixed in a 4% paraformaldehyde solution (PFA; Electron Microscopy Sciences 15710) for 15 minutes at room temperature, then washed 3 times with PBS. Tibialis Anterior muscles were flash frozen in isopentane and 8-10 pm thick sections were cut using a cryostat. Slides were post-fixed in 4% PFA before processing for immunohistochemistry. Typically, samples were washed 3 times for 3 min each in Tris buffered saline (TBS) with 0.1% Tween (TBST), permeabilized in PBS with 0.5% Triton for lOmin, and blocked for Ih at room temperature in TBS with 0.1% Triton and 3% Donkey serum. Primary antibodies were diluted in this blocking solution and incubated overnight at 4 °C. Following 3 TBST washes, cultures were incubated with Alexa-Fluor-conjugated secondary antibodies (1:500), and Hoechst33342 (1:1000) for 1.5h at 4°C. Three final TBST washes and a PBS rinse were performed, and cells were mounted in PBS and imaged. All immunostainings were done accordingly except for PAX7 (see method below). Antibodies references and concentration

[0093] Human nuclei: Lamin B2 (D8P3U) Rabbit mAb #12255, Cell Signaling Technology, 1 / 500. Human dystrophin: MANDYS106(2C6), DSHB, 1 / 100. MYH3 : BF-G6, DSHB, 1 / 100. PAX3 : Pax3-c, DSHB, 1 / 500. SOX9 : AB5535, Sigma-Aldrich, 1 / 500. GFP : abl3970, abeam, 1 / 500. mKate2 : TagRFP Polyclonal Antibody (R10367), Invitrogen, 1 / 500. Laminin : Polyclonal Chicken (LS-C96142-10000, LSBio, 1 / 500. CD31 : CD31 / PECAM1 Rabbit mAb (A19014), ABclonal, 1 / 500. Pax7 : pax7-c, DSHB, 1 / 100. Antigen Retrieval

[0094] For PAX7 immunostaining, slides were boiled for 10 min with target retrieval solution (S2369, Dako) to unmask antigen. Slides were then washed with PBS and permeabilized with 0.5% Triton X-100 in PBS for 15 min. Sections were incubated in blocking solution (2% horse serum, 2% goat serum, 2% BSA fraction V, 0.1% Triton X-100 in PBS) for 30 min, followed by Mouse On Mouse blocking solution (Vector Laboratories) incubation for 30 min. Sections were then incubated with primary antibody against Pax7 (1:100 in M.O.M diluent) overnight at 4°C. Secondary incubation was done using Goat anti-mouse IgGl isotype-specific antibody coupled to AF647 (A-21240, Invitrogen, 1 / 500, Ih at RT). Microscopy

[0095] Wide-field fluorescence imaging was performed on a Leica DMi8 Widefield imaging system. Confocal imaging was performed on a Zeiss LSM880 confocal inverted microscope fitted with a large temperature incubation chamber and a CO2 module. Single nuclei extraction

[0096] Cell cultures: medium was removed and 500 pl homogenization buffer were added to the culture well, cells were scrapped from the plate, and an additional 500 pl homogenization buffer was added before transferring the mix into a 2 ml douncer on ice.

[0097] Mouse muscle tissues: TA muscle was dissected post-euthanasia. The muscle was washed in PBS and transferred to a 1.5ml Eppendorf tube and chopped using scissors in a 1ml homogenization buffer solution (250 mM sucrose (Sigma-Aldrich), 10 pM Tris ph 8.0 (Thermo Fisher Scientific), 25 mM KCL (Thomas Scientific), 5 mM MgCh (Thermo Fisher Scientific), 0.1% Triton-X 100 (Millipore Sigma), 0.5% Rnasin Plus (Promega), lx protease inhibitor (Promega), 0.1 mM DTT (Thermo Fisher Scientific)) before transferring the mix on ice as described (McLaughlin et al., STAR Protoc 3, 101417, 2022). Two types of douncers were used: loose and tight and used sequentially for 20 and 40 strokes respectively to obtain a nuclei lysate. The lysate was filtered through a 35 pm-mesh FACS tube (Corning), followed by a filtration on a 40pm-mesh FLOWMI filter (Scienceware) and transferred into a 1.5 ml low binding tube (Eppendorf). The tubes were spinned in a swinging-bucket centrifuge at 1000g for 10 min at 4 °C. The pellet was resuspended in 500pl-lml (PBS + 1%BSA + RNAse inhibitor + 1:1000 DAPI) before being filtered again through the 40pm-mesh FLOWMI filter. The nuclei were sorted through a FACS (ARIA, BD) machine with a 70 pm nozzle. Nuclei concentration was then manually counted, and quality control was performed by checking the shape under a fluorescence microscope. Single cell and nuclei preparation and Sequencing

[0098] For every sample, 10,000 nuclei or cells were encapsulated using lOx Chromium 3’ v3.1 (Dual Index) kits (Rev E). Libraries were prepared according to the manufacturer instructions (Rev E). Quality control was done using TapeStation System and sequencing was done using a NovaSeq Full 6000 SP Flowcell at the Biopolymer core from Harvard Medical School. Analysis of Single-cell RNA sequencing data

[0099] Transcriptome libraries were aligned to the Homo sapiens genome assembly GRCh38 (hg38) using cell ranger 6.1.1. Raw count matrices were converted to anndata objects using Scanpy. Cells were filtered by counts to remove outliers. Cells with excessive mitochondrial gene expression were also filtered. Raw data were normalized, log-transformed and scaled. Cell cycle genes were regressed linearly, highly variables genes were identified, PCA was performed along with neighbors’ computation before final UMAP embedding. Leiden clustering and ranking of all genes per cluster (Wilcoxon test p-value corrected by Benjamini-Hochberg) allowed us to annotate the cell populations present in the datasets. Matplotlib was used to plot the number of cells in every cluster. Dot plots were made with a list of signature genes with a gene-based standard scale and plotted using a custom matplotlib color palette on processed data. Subclusters were made by isolation of the main dataset and re-processing of the data starting from the raws. Enrichment analysis was done using GSEA prerank function on BioPlanet 2019 signaling pathway gene sets. Satellite cell score was calculated using the average expression of a set of genes subtracted with the average expression of a reference set of genes being randomly sampled. Analysis of single-cell RNA-sequencing and single-nuclei RNA-sequencing data

[00100] Single cell and nuclei RNA sequencing datasets analysis was made using Python 3.11, scanpy 1.9.5 and anndata 0.9.1. Datasets were analyzed individually or merged to be compared. For datasets that contain both human and mouse cells, the species was attributed by lOx cellranger software based on the percent of alignment to the two genomes. Cells were filtered by number of counts, number of genes and genes were filtered by number of cells based on sample quality controls. Cells with high mitochondrial contents were filtered and cell cycle score was calculated for each cell using Regev lab cell cycle gene list. Matrices were log Ip and normalized, regressed for total number of counts and cell cycle phase, and finally scaled. Highly variable genes were computed for principal component analysis and Harmony was used for batch correction based on the samples. Neighbors were calculated and if the analysis contains multiple datasets: neighbors are based on Harmony or Bbknn computations for integration purposes. UMAP, Force atlas and Palantir were used for visualization and Leiden for cell clustering. Cluster subsets were done by using raw data and applying preprocessing techniques again. Differential expression was done using raw data and Wilcoxon statistical tests with Benjamin-Hochberg corrections. Dot plots were made with a list of signature genes with a gene-based standard scale and plotted using a matplotlib color palette on processed data. Proportions of cells that express a specific gene were calculated on the raw data. Matplotlib, seaborn and prism were used to plot the number and proportions of cells in every cluster. Trajectory and pseudotime analysis were performed using stream2 and scfates with a principal tree structure. Scores were calculated using scanpy for specific gene set lists. The score is the average expression of the set of genes subtracted with the average expression of the reference set of genes (randomly sampled from the gene pool of the sample). Prerank GSEA analysis was done using gseapy with specific gene sets on differentially expressed gene lists. Cells comparison was done using the KNeighboursClassifier classifier methods from scikit-learn (0.20.3). Classifiers were trained on cluster annotated PCA subspace-projected datasets with default settings and k = 20 for KNeighboursClassifier. Cell states were predicted after subsetting matching gene symbols for the highly variable gene list, and projecting into the defined PCA subspace. The result was plotted in a confusion matrix. Animals

[00101] NOD.Cg-Prkdcscid I12rgtmlSug / JicTac male mouse from Taconic Biosciences were used. The study protocol was approved by Brigham and Women’s Hospital IACUC / CCM (protocol number 2016N000476). Experiments on mice were done according to local regulations (Brigham and Women’s Hospital), in agreement with national and international guidelines. Animal anesthesia and irradiation

[00102] Animals were anesthetized with an intraperitoneal injection of ketamine (100 mg / kg, Henry Schein Animal Health) + xylazine (10 mg / kg, Henry Schein Animal Health). They were covered with a lead shielding block with aperture allowing only mouse hind limbs to be irradiated. Mice were then gamma-irradiated (18 gray) in a single dose using a cesium-source for 20 min (Gammacell 40 Exactor). Cardio toxin injury

[00103] This procedure was performed to trigger the regeneration process in the hind limb TA muscle Twenty-four hours after irradiation, animals were anesthetized as above, the injection site was shaved and cleaned with 70% Ethanol. Injections were performed percutaneously in both TA with a dedicated insulin syringe (27G needle). Twenty-five pl of cardiotoxin (0.5 mg / pl) were injected over 25 injection sites evenly distributed in the TA. Mice were then checked for proper post-anesthesia and injection recovery. Intra-muscular cell injections

[00104] Twenty-four hours after cardiotoxin injection, animals were anesthetized as above, the injection sites were shaved and cleaned with 70% Ethanol. Injections of cells (0.5 106 to 106 depending on experimental setting) were performed percutaneously with a dedicated insulin syringe, in a total volume of 20-25pL as above. Cell lines used for cell preparations were tested negative for mycoplasma and standard viral contaminants (including HBV, HCV and HIV for human cell lines). Mice were checked for proper post-anesthesia recovery. Bioluminescent / Fluorescent screening

[00105] We used a bioluminescence reader combined with an X-ray scanner (Bruker In Vivo Xtreme imaging system) to detect luciferase-expressing human cells. We defined successful engraftment by a persistence of a maximum signal on the leg area of more than 5000 photons / seconds / millimiter2, which is above the background signal detected on non-grafted side. Animals were anesthetized and injected received D-Luciferin (150 mg / kg, IVISbrite D-Luciferin) IP (lower left quadrant). Images were captured 15 minutes after D-Luciferin injection, bioluminescence imaging was performed using an exposure of 30 s and a binning of 8x8 pixels. In situ force test

[00106] Force produced by the dorsiflexor muscle of the lower limb TAs was measured 4 weeks post-cell injection. Under general anesthesia as above, the animal’s TA ligament terminal insertion was secured to a string. The muscle was stimulated by placing the Electromyography (EMG) electrodes inside the TA to stimulate the fibular nerve. Using the device program (610A Dynamic Muscle Control LabBook v6, Aurora Scientific, Aurora, Ontario, Canada), the resting tension was adjusted until maximum twitch force was produced by a single pulse with the pulse width of 0.2 ms. The maximal force was measured as the tetanic force produced at this optimized setting, normalized to the body weight of the animal (mg). Statistics and reproducibility

[00107] Details of statistical analyses are indicated in the figure legends. Three or more independent experiments were performed for all experiments.

[00108] The complete disclosure of all patents, patent applications, and publications, and electronically available material cited herein are incorporated by reference. The foregoing detailed description and examples have been given for clarity of understanding only. No unnecessary limitations are to be understood there from. The invention is not limited to the exact details shown and described, for variations obvious to one skilled in the art will be included within the invention defined by the claims.

Claims

1. A composition of INFRA cells, comprising human muscle stem cells (SCs) expressing the transcription factor PAX7 and lacking the expression of the transmembrane receptor PDGFRA, wherein at least 70% of the population of human muscle SCs are PAX7+ and PDGFRA-.

2. The composition of claim 1 wherein the human muscle SCs further express the transcription factors PAX3 and / or PAX7 and / or the transmembrane receptors FGFR4, CD82, ITGA7, CDH15, CD56 and / or N0TCH3.

3. The composition of claim 1, wherein the human muscle SCs are derived from pluripotent stem cell (PSC)-derived cultures generated by treating presomitic mesoderm cells with a Wnt activator.

4. The composition of claim 3, wherein the PSC-derived cultures are generated by treating presomitic mesoderm cells with a Wnt activator and a bone morphogenic protein (BMP) inhibitor.

5. The composition of claim 1, wherein the INFRA cells are isolated by generating a myogenic culture derived from pluripotent stem cells (PSCs) and removing the upper layer of the culture to expose an adherent monolayer of INFRA cells.

6. The composition of claim 5, wherein the INFRA cells are further purified by flow cytometry to purify the PDGFRA negative population.

7. The composition of claim 6 wherein the SCs are further isolated from the isolated INFRA cells by flow cytometry using antibodies against ITGA7, CD56, N0TCH3, CD82 and / or CDH15.

8. A method of creating a substantially pure population of INFRA cells comprising:(a) generating a myogenic culture derived from pluripotent stem cells on a culture substrate;(b) removing the upper layer of the myogenic culture, leaving behind a monolayer of INFRA cells attached to the culture substrate;(c) washing the monolayer of INFRA cells with a buffered solution; and(d) mechanically dissociating the INFRA cells from the cell substrate without causing stress or modification of the cells.

9. The method of claim 8, wherein the INFRA cells comprise human muscle stem cells (SCs) expressing the transcription factor PAX7 and lacking the expression of the transmembrane receptor PDGFRA10. The method of claim 8, wherein at least 70% of the population of INFRA cells are PAX7+ and PDGFRA-11. The method of claim 8, further comprising the step of purifying the INFRA cells by flow cytometry using antibodies against ITGA7, CD56, NOTCH3, CD82 and / or CDH15.

12. The method of claim 8, wherein the upper layer of myogenic culture is removed by peeling.

13. The method of claim 8, wherein the buffered solution is phosphate buffered saline (PBS).

14. A method of treating muscle injury in a subject, comprising contacting the muscle injury of the subject with a therapeutically effective amount of INFRA cells, wherein the INFRA cells comprising human muscle stem cells (SCs) expressing the transcription factor PAX7 and lacking the expression of the transmembrane receptor PDGFRA, wherein at least 70% of the population of human muscle SCs are PAX7+ and PDGFRA-.

15. The method of claim 14, wherein the INFRA cells contact the muscle injury as a result of administration by injection.

16. The method of claim 14, wherein the muscle injury is muscular dystrophy, traumatic injury, or cachexia.

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