Isolation of muscle satellite cells

Abstract

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

Description

[0001] Government funding

[0002] This invention was made with government support under National Institutes of Health Approval No. 5RO1AR07452605. The U.S. government holds certain rights to this invention.

[0003] Cross-reference to related applications

[0004] 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 Technology

[0005] Muscle injuries and degenerative diseases, such as Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD), represent a major challenge in modern medicine, necessitating the development of therapies for muscle regeneration. Over the past three decades, numerous clinical trials of cell therapies have been conducted in patients with DMD and BMD, involving the transplantation of primary human skeletal muscle myoblasts expanded in vitro from skeletal muscle biopsies (Skuk et al., J Neuropathol Exp Neurol These trials demonstrated feasibility, but with limited efficacy because the transplanted myoblasts showed limited regenerative potential. Since these earlier trials, PAX7+ SCs have been shown to be suitable resident stem cells in adult skeletal muscle, exhibiting tremendous regenerative capacity (Sacco et al., Nature 456, 502-506, 2008). These cells can generate a large number of myoblasts capable of rebuilding myofibrils. They can also self-renew, ensuring the long-term regenerative potential of the tissue. Therefore, SCs represent a better candidate cell for cell therapy applications compared to myoblasts transplanted in earlier DMD trials. However, there are significant limitations to the use of SCs in a clinical setting. SCs are a rare population, comprising less than 5% of the cell nuclei in skeletal muscle (Tierney and Sacco, 2006). Trends Cell Biol 26 (Montarras et al., 434-444, 2016). Therefore, generating large quantities of these cells for cell therapy applications from human muscle biopsies requires in vitro expansion. However, this method is currently not feasible because adult SCs lose their regenerative properties during in vitro culture, making them incompatible with clinical applications (Montarras et al., 434-444, 2016). Science 309, 2064-2067, 2005). An alternative approach is to generate PAX7+ SCs from human PSCs. Several protocols describing in vitro SC production have recently been reported (Caron et al., J NeuromusculDis10, 761-776, 2023). These protocols can produce 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 by mouse fetal SCs (Tierney et al., Vol. 27, Issue 1, 158-176, 2020). Trends Cell Biol 26, 434-444, 2016). However, myogenic cultures produced using these protocols that mimic dermatomyosal development are heterogeneous because they contain most natural derivatives, including fibroblasts or neurons derived from the primordial neural mesodermal precursor populations induced under these conditions (Xi et al., 26, 434-444, 2016). Cell stem cell (Vol. 27, Issue 1, 158-176, 2020). After 30-80 days in vitro, the PAX7+ SC population accounted for only 25-35% of the monocyte population (Xiet al., Vol. 27, Issue 1, 158-176, 2020). Cell stem cell (Vol. 27, Issue 1, 158-176, 2020). Therefore, the fraction of PAX7+ cell nuclei in the total population is even lower, since syncytial myofibrils are not included in this count. Thus, purification strategies, such as fluorescence-activated cell sorting (FACS) or magnetic bead-based sorting, are required to obtain a sufficiently high population of SC-rich cells for clinical trials. This purification step significantly increases the complexity of producing cell populations for cell therapy applications under GMP conditions.

[0006] Advantageously, it provides a method for isolating substantially homogeneous populations of functional SCs and SCs from any muscle culture, and provides a substantially pure population of functional SCs and SCs. Summary of the Invention

[0007] A filtering / sorting method is described to obtain significantly enriched functional SCs and populations of SCs from any muscle culture with unprecedented efficiency. The method is based on any PSC-derived myogenic culture defined by culturing / directing pluripotent cells into myofibrils or progenitor cells, which can be characterized by, for example but not limited to, their transcriptome (e.g., cultures composed of cells expressing PAX7, MYOD1, MYF5, MYOG, ACTN2, TTN). SCs adhere to the substrate of the culture; these cells are named 'INFRA cells', and they are defined by expressing the gene PAX7+ but lacking PDGFRA expression.

[0008] In one aspect, the present invention provides an INFRA cell composition comprising human muscle stem cells (SCs) expressing the transcription factor PAX7 and lacking expression of the transmembrane receptor PDGFRA, wherein at least 70% of the population of human muscle SCs is PAX7+ and PDGFRA-, and wherein the SCs have not undergone modification during isolation.

[0009] In some embodiments, human muscle SCs also express the transcription factor PAX and / or transmembrane receptors FGFR4, CD82, ITGA7, CDH15, CD56, and / or NOTCH3. In another embodiment, human muscle SCs are derived from pluripotent stem cell (PSC)-derived cultures produced by treating precursor mesodermal cells with a Wnt activator. In yet another embodiment, PSC-derived cultures are produced by treating precursor mesodermal cells with a Wnt activator and a bone morphogenetic protein (BMP) inhibitor.

[0010] In some embodiments, INFRA cells are isolated by generating a myogenic culture derived from pluripotent stem cells (PSCs) and removing the supernatant of the culture to expose an adherent monolayer of INFRA cells. In another embodiment, INFRA cells are further purified by purifying a PDGFRA-negative population using flow cytometry. In yet another embodiment, additional SCs are isolated from the isolated INFRA cells by flow cytometry using antibodies against ITGA7, CD56, NOTCH3, CD82, and / or CDH15.

[0011] Another aspect of the present invention provides a method for preparing 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 superlayer of the myogenic culture, leaving a monolayer of INFRA cells attached to the culture substrate; (c) washing the monolayer of INFRA cells with a buffer solution; and (d) mechanically dissociating the INFRA cells from the cell substrate without inducing cellular stress or modification. In some embodiments, the superlayer of the myogenic culture is removed by peeling, and in some embodiments, the buffer solution is phosphate-buffered saline (PBS).

[0012] In some embodiments, the INFRA cells comprise human muscle stem cells (SCs) that express the transcription factor PAX7 and lack expression of the transmembrane receptor PDGFRA. In another embodiment, at least 70% of the INFRA cell population is PAX7+ and PDGFRA-. In yet another embodiment, the method further includes the step of purifying the INFRA cells by flow cytometry using antibodies against ITGA7, CD56, NOTCH3, CD82, and / or CDH15.

[0013] Another aspect of the invention provides a method for treating muscle injury in a subject. The method includes contacting the subject's muscle injury with a therapeutically effective amount of INFRA cells, wherein the INFRA cells comprise human muscle stem cells (SCs) that express the transcription factor PAX7 and lack expression of the transmembrane receptor PDGFRA, wherein at least 70% of the human muscle SC population is PAX7+ and PDGFRA-, and wherein the SCs have not undergone modification during isolation. In some embodiments, the INFRA cells are administered via injection to contact the muscle injury, while in other embodiments, the muscle injury is muscular dystrophy, traumatic injury, or cachexia. Attached Figure Description

[0014] The invention can be more easily understood by referring to the following figures, in which: Figure 1A – Figure 1G Figures and images showing the new protocol design are provided: A) UMAP, showing cells from the paraxial mesoderm lineage extracted from mouse embryonic development E8.5, E9.5, E10.5, and E11.5 scRNAseq datasets (Qiu et al., Nat Genet (54, 328-341, 2022), B) Leiden clustering analysis of the corresponding cell types in the paraxial mesodermal dataset shown in A, C) Computational trajectory of the paraxial mesodermal dataset shown in A (pseudo-time from dark blue to yellow), D) Projection of the major biological pathway activities of the osteomyeloid / dermatomyosal branches identified from the prosomal mesoderm onto the dataset shown in A. E) A schematic diagram of a new protocol designed based on a bioinformatics workflow, illustrating developmental stages and showing regulated signaling pathways. F) Immunohistochemical detection of PAX7 (anti-GFP, green) and ACTN2 (anti-mKate2, red) expression in cultures of the dual-reporter gene PAX7-Venus-ACTN2-mKate2 line (n=3 / sample). G) FACS analysis of PAX7-Venus-ACTN2-mKate2 iPS reporter gene line cultures on day 31, showing the percentages of PAX7+, MYOD+, PAX7+MYOD+, and PAX7-MYOD-, shown here as mean and SD (n=5). Red indicates the new protocol, and blue indicates the reference Chal protocol.

[0015] Figure 2A – Figure 2IFigures and images showing the increased production of PAX7+ SCs in vitro are provided: A) Leiden cluster analysis of combined SnRNAseq analysis of cells differentiated with the Chal and new myogenic protocols on day 30; B) Distribution of cell nuclei in the Chal and new protocol datasets on the UMAP shown in A; C) Bar chart showing the percentage of cell nuclei in each cluster of the snRNAseq datasets of the Chal protocol (blue) and the new protocol (red); D) (Top panel) UMAP showing the cluster analysis of myogenic cell nuclei extracted from the snRNAseq dataset of day 30 cultures produced using the new protocol. Green: PAX7+ cells, Orange: Intermediate myogenic stage (MYOMX), Red: Myocytes. (Bottom panel) Expression of specific genes in each cluster; E) (Top panel): STREAM analysis showing the trajectory on a pseudo-timeline summarizing the myogenic differentiation program shown in D. (Below): Projection of gene expression of the genes shown in D onto the pseudo-time axis of the STREAM plot, F) (Above): UMAP, showing cluster analysis of myogenic cell nuclei extracted from the snRNAseq dataset of day 30 cultures generated using the Chal protocol. Green: PAX7+ cells, Orange: Intermediate myogenic stage (MYOMX), Red: Myocytes. (Below): Expression of specific genes in each cluster, G) (Above): STREAM analysis, showing the trajectory on the pseudo-time axis summarizing the myogenic differentiation program of the clusters shown in F. (Below): Projection of gene expression of the genes shown in F onto the pseudo-time axis of the STREAM plot, H) Dot plot, showing the average expression level of specific genes in each cluster of the new protocol, I) Dot plot, showing the average expression level of specific genes in each cluster of the Chal protocol.

[0016] Figure 3A – Figure 3KFigures and images showing the mechanical enrichment of human satellite cells are provided: A) Schematic diagram of the step-by-step procedure for harvesting INFRA cells from PSC-derived muscle cultures; B) (Left panel) FACS scatter plot showing FSC-A (x1000) and fluorescence intensity of PAX7-Venus cells; (Right panel) Bar chart showing the percentage of PAX7+ cells in the dissected INFRA cell population, shown here as mean and SD (n=3); C) The dissection process of harvesting INFRA cells from a 6-well plate containing a day 30 myogenic culture produced using the new protocol using forceps; D) Immunostaining showing PAX7-expressing cells in the INFRA population adhering to the culture substrate after dissection; anti-GFP antibody (green) on PAX7-Venus cells differentiated 30 days according to the new protocol; E) Differentiation of INFRA cells derived from PAX7-Venus-ACTN2-mkate2 cells after 2 weeks of culture. Immunostaining with anti-GFP (green) and anti-mkate2 antibody (red), F) (left panel) UMAP, showing Leiden cluster analysis of INFRA cells immediately after harvest by scRNAseq. (right panel) PAX7 expression shown in the same UMAP, G) dot plot showing the specific marker gene for each cluster identified in F, H) bar plot showing the percentage of cells expressing the marker gene in each cluster of INFRA cells immediately after harvest. Green: PAX7+, Gray: PAX7-, Red background: PAX7+MYOD1+, Blue background: PAX7+PDGFRA+, Red top: PAX7-MYOD1+, Blue top: PAX7-PDGFRA+, I) (left panel) UMAP, showing Leiden cluster analysis of Venus+ FACS-sorted cells from differentiated PAX7-Venus lines immediately after harvest by scRNAseq. (Right panel) PAX7 expression shown in the same UMAP, J) dot plot showing the specific marker gene for each cluster identified in F, K) bar plot showing the percentage of cells expressing the marker gene in each cluster of PAX7-Venus+ cells immediately after harvest. Green: PAX7+, Gray: PAX7-, Red background: PAX7+MYOD1+, Blue background: PAX7+PDGFRA+, Red top: PAX7-MYOD1+, Blue top: PAX7-PDGFRA+.

[0017] Figure 4A – Figure 4RFigures and images are provided demonstrating the effective colonization of mouse muscles by human PAX7+ cells and the restoration of strength production: A) Schematic diagram of the transplantation protocol, B) X-ray image of NOD mice one month after INFRA cell transplantation, overlaid with iridescent luciferase activity. Units are P / s / mm / sq (n=3), C) Tibialis anterior (TA) muscle dissected one month after transplantation. From left to right: from untreated age-matched mice (WT). TA Irradiated cells injected with cardiotoxin and transplanted with INFRA cells TA Control (CTL) irradiated and injected with cardiotoxin TA (n=3), D) One month post-transfer using standard one-factor ANOVA TA Weight comparison. From left to right: Healthy TA (WT), irradiated and injected with cardiotoxin and transplanted with INFRA cells TA Irradiated and injected with cardiotoxin TA (n=3), E) Bar graph showing the percentage of human cell nuclei counted in transverse frozen sections of TA treated 1 month post-transplantation, shown here as mean and SD. (n=4), F) 1 month post-transplantation, irradiated and cardiotoxin-injected sections transplanted with INFRA cells. TA (Left) Low-magnification images of transverse frozen sections and control (right). Hoechst (blue), anti-human laminin B2 (green), anti-human dystrophin (red). 20x tiled confocal images, G) 1 month post-transplantation, irradiated and injected with cardiotoxin and transplanted with INFRA cells. TA High-magnification images of transverse frozen sections, labeled with anti-laminin antibody (white); H) Section identical to F, labeled with anti-human laminin B2 (green); I) Section identical to F, labeled with anti-human dystrophin (red); J) Combined image of G and H; K) One month post-transplantation, irradiated sections injected with cardiotoxin and transplanted with INFRA cells. TA High-magnification image of a transverse frozen section. Hoechst is shown in blue, anti-CD31 is highlighted in green to indicate blood vessels (arrow), and anti-MYH3 is shown in red. One month post-transplantation, the patient was irradiated and injected with cardiotoxin and transplanted with INFRA cells. TA High-magnification image of a transverse frozen section. Hoechst is shown in blue, anti-PAX7 is shown in red (SC) (arrow), and anti-lamin B2 is shown in green (M). One month post-transplantation, irradiated cells were injected with cardiotoxin and transplanted with INFRA cells. TAHigh-magnification view of transverse frozen sections. Hoechst is shown in blue, anti-NG2 is shown in red indicating pericytes (arrows), and anti-lamin B2 is shown in green (N). A standard one-way ANOVA was used to compare healthy controls. TA Muscle (WT), irradiated muscle injected with cardiotoxin and transplanted with INFRA cells TA (Transplanted) and irradiated, injected with cardiotoxin TA (Comparison) TA Strength measurements were performed, shown here as mean and SD (n=3), and UMAP was observed in mice after 1 month. TA Clustering of moderately differentiated human myogenic cells (P) dot plot, showing the effect of transplantation. TA Differentially expressed genes in three human myogenic cell clusters identified in the study, Q-dot plot, showing in vitro (novel) and in vivo ( TA Differential gene expression of GO terminology glycolysis in PAX7+ cells, R) dot plot, showing in vitro (novel) and in vivo ( TA Differential gene expression of GO terminology skeletal muscle contraction in PAX7+ cells. TA : transplanted to TA Cells in vitro; new: cells produced in vitro using optimized protocols. Detailed Implementation

[0018] This invention provides a composition of INFRA cells. The composition comprises human muscle stem cells (SCs) that express the transcription factor PAX7 and lack expression of the transmembrane receptor PDGFRA, wherein at least 70% of the human muscle SC population is PAX7+ and PDGFRA-. A method for preparing the INFRA cell composition and a method for treating muscle injury using INFRA cells are also provided.

[0019] definition

[0020] 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 pertain. The terminology used in this description is for the purpose of describing particular exemplary embodiments only and is not intended to be limiting of the exemplary embodiments. As used in the specification and appended claims, the singular forms “a,” “an,” and “the” are intended to include the plural forms as well, unless the context clearly specifies otherwise. All publications, patent applications, patents, and other references mentioned herein are incorporated herein by reference in their entirety.

[0021] Where a numerical range is provided, it should be understood that, unless the context explicitly specifies otherwise, every intermediate value between the upper and lower limits of the range (to one-tenth of the lower limit unit), as well as any other specified or intermediate value within the specified range, is included in this invention. The upper and lower limits of these smaller ranges may be independently included within the smaller range and also within this invention, but are subject to any explicit exclusions within the specified range. Where a specified range includes one or two limits, the range excluding any one or both of these included limits is also included in this invention.

[0022] The “cell growth medium” defined herein is an aqueous medium containing factors and nutrients suitable for supporting the growth of pluripotent or multipotent cells (particularly human pluripotent or multipotent cells). For the maintenance of pluripotent or multipotent cells, the preferred basal medium is DMEM-F12 (Gibco, Invitrogen cell culture, USA catalog 11320-033), supplemented with 10% KOSR (Life Technologies, catalog 10828-028), non-essential amino acids (1%, Gibco, catalog 11140-050), 2 ng / ml bFGF (Invitrogen, 13256-029), and L-glutamine (1% v / v, Gibco, catalog 25030-081). In some embodiments, such as those involving staged directed differentiation, the basal medium used to support the first stage of stem cell differentiation from pluripotency into somatic cells is RPMI 1640 supplemented with bovine serum albumin (2%, Sigma). The cell culture media used are well-known in the field of cell culture and are all commercially available.

[0023] "Pluripotent" refers to a cell capable of producing each of the three embryonic cell lineages and extraembryonic cells. The pluripotency used herein is limited to the inner cell mass state and is never used to describe lineage-restricted progenitor cells formed during development. The pluripotent cell state is a natural state but can be propagated in vitro under specific conditions known to those skilled in the art. Under these culture conditions, the permanent maintenance of the stem cell or pluripotent state occurs. The defining characteristics of pluripotent cells are not limited to their functional features but can also be described by gene expression patterns, and therefore can be identified by staining patterns of markers they exhibit. Pluripotent cells, such as embryonic stem cells, may also exhibit alkaline phosphatase activity, which is commonly used for identification.

[0024] "Progenitor cell" refers to a normal cellular state at any given time that represents the direct lineage ancestor of terminally differentiated cells. By definition, progenitor cells are irreversibly destined to adopt the stated terminal fate, but can be multipotent for more than one terminal state. Therefore, they exhibit the ability to differentiate into more than one single progeny fate. The gradual loss of this ability, and thus the loss of pluripotency, as development progresses in any organism is the hallmark of differentiation and the temporally defined formation of tissues and organs.

[0025] "Stem cells" refers to cells capable of self-renewal (i.e., producing offspring with the same differentiation potential) and also capable of generating progeny cells with more limited differentiation potential. In the context of this invention, stem cells will also include more differentiated cells that have already dedifferentiated, for example, through nuclear transfer, through fusion with a more primitive stem cell, through the introduction of specific transcription factors, or through 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).

[0026] As used herein, “subject” can refer to a vertebrate or mammal. Examples of subjects include livestock, test animals, and pets, such as sheep, cattle, pigs, dogs, cats, and rodents, as well as reptiles, birds, and fish. The terms “patient” and “subject” are used interchangeably herein. Preferably, a subject is a mammal. Mammals can be humans, non-human primates, mice, rats, dogs, cats, horses, or cattle, but are not limited to these examples.

[0027] Regarding cells in a cell culture or population of cells, the term "substantially absent" means that a specific cell type is not present in the cell culture or population of cells, and 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 population of cells.

[0028] "Therapeutic effective dose" refers to the amount of cells that have been determined to produce any therapeutic response in a subject. As used herein, "therapeutic effective dose" refers to a dose sufficient to stimulate muscle growth or reduce or prevent muscle atrophy. The exact amount required will vary from subject to subject, depending on the subject's species, age and general condition, the specific therapeutic agent, and the manner and / or route of administration.

[0029] The term "treatment" is used extensively in this invention, and each such term includes, but is not limited to, the prevention, improvement, suppression, or cure of defects, dysfunctions, diseases, or other harmful processes, including those that interfere with treatment and / or are caused by treatment. In various embodiments, the symptoms of the disease or condition are reduced by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, or at least 50%.

[0030] All scientific and technical terms used in this application have their common meaning in the art, unless otherwise stated. The definitions provided herein are for ease of understanding of certain terms frequently used herein and are not intended to limit the scope of this application.

[0031] INFRA cells

[0032] In one aspect, the present invention provides an INFRA cell composition. The INFRA cell composition comprises human muscle stem cells (SCs) that express the transcription factor PAX7 and lack expression of the transmembrane receptor PDGFRA, wherein at least 70% of the human muscle SC population is PAX7+ and PDGFRA-.

[0033] In some implementations, SCs are not modified during the separation process. Existing separation methods typically stress SCs, leading to cellular modifications. Modifications caused by the separation process can include chemical and genetic modifications.

[0034] In some embodiments, the human muscle SCs of the INFRA cell composition also express a specific set of biomarkers. For example, in some embodiments, the SCs also express transcription factors PAX3 and / or PAX7 and / or transmembrane receptors FGFR4, CD82, ITGA7, CDH15, CD56 and / or NOTCH3.

[0035] In some embodiments, human muscle SCs are derived from pluripotent stem cell (PSC)-derived cultures produced by treating precursor mesodermal cells with a Wnt activator. Precursor mesodermal cells are mesenchymal cells that form bilateral bands adjacent to the notochord in early paraaxial mesoderm and are derived from neural mesodermal progenitor cells in the primitive bands or tail buds.

[0036] The Wnt gene belongs to the proto-oncogene family and encodes more than 20 cysteine-rich secretory glycoproteins that activate the Wnt signaling pathway by binding to coiled (Fz) receptors on target cells. In some embodiments, Wnt signaling pathway activators may include Wnt receptor ligands or agonists, such as coiled receptor agonists. Examples of Wnt ligands include Wnt1, Wnt2, Wnt2b, Wnt3, Wnt3a, Wnt4, Wnt5a, Wnt5b, Wnt6, Wnt7a, Wnt7b, Wnt8a, Wnt8b, Wnt9a, Wnt9b, Wnt10a, Wnt10b, Wnt11, and Wnt16. These Wnt ligands and their accession numbers are described in U.S. Patent No. 8,460,928, which is incorporated herein by reference. Any one or more of these can be used to activate Wnt signaling in TrPC. Wnt ligands are available from R&D Systems (Minnesota, USA) and PeproTech, Inc (New Jersey, USA).

[0037] In some embodiments, PSC-derived cultures are generated by treating precursor mesoderm cells with a Wnt activator and a bone morphogenetic protein (BMP) inhibitor. BMPs are secreted cytokines that interact with cells via type I and type II serine / threonine kinase receptors, and BMP inhibitors are substances that block BMP activity. BMP inhibitors include small molecule inhibitors such as dorsomorphin, proteins that bind to BMP receptors such as DAN proteins (e.g., Nb11, tendinin, tendin-like proteins, DAN, sclerosing proteins, dermal polyglycans, grimlin 1, grimlin 2, Cerberus, and Dand5), secreted modular calcium-binding proteins, and BMP-specific nanobodies. See Rosen, V., Ann NY Acad Sci., 1068: 19-25 (2006).

[0038] In some embodiments, INFRA cells are isolated by generating a myogenic culture derived from pluripotent stem cells (PSCs) and removing the top layer of the culture to expose an adherent monolayer of INFRA cells. Other methods for isolating INFRA cells are described herein.

[0039] In some embodiments, 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 be used to positively purify specific cell types using specific trait of the cells. For example, in some embodiments, additional SCs are isolated from 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 traits by selecting additional characteristics. For example, in some embodiments, flow cytometry can be used to purify PDGFRA-negative populations. See Mushahary et al., Cytometry A., 93(1): 19-31 (2018).

[0040] In some implementations, INFRA cells represent a population that has been significantly enriched. As used herein, “significantly enriched” means that, after a filtration / sorting process, at least 60%, at least 70%, or at least 80% of the culture consists of SCs.

[0041] Population of INFRA cells

[0042] Another aspect of the present invention provides a method for preparing a population of substantially pure 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 superlayer of the myogenic culture, leaving a monolayer of INFRA cells attached to the culture substrate; (c) washing the monolayer of INFRA cells with a buffer solution; and (d) mechanically dissociating the INFRA cells from the cell substrate without inducing cellular stress or modification.

[0043] The first step of the method involves generating a myogenic culture derived from pluripotent stem cells on a culture substrate. A culture substrate is a surface suitable for growing cell cultures, such as a culture dish. In some embodiments, the culture substrate may contain 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 pluripotent cells and myogenic cells.

[0044] Myogenic cells are generated from pluripotent stem cells (PSCs) by stimulating them with appropriate chemical factors. For example, transcription factors can be used to stimulate the formation of myogenic cells from pluripotent cells. In some embodiments, PSC-derived cultures are generated by treating precursor mesoderm cells with a Wnt activator, while in other embodiments, PSC cells are stimulated with a Wnt activator and a bone morphogenetic protein (BMP) inhibitor.

[0045] In the remaining steps of the invention, the PSC-derived myofibrils and the superlayer of the SC culture are mechanically removed (e.g., peeled off) and then discarded or used for other purposes. The SCs adhering to the culture substrate are now separated from the rest of the culture and remain in the wells for any purpose. Specifically, the operation begins with myogenic cultures of any size or shape (e.g., but not limited to, myofibrils and SC cultures derived from human or mouse PSCs, myofibrils and SC cultures derived from human or mouse embryonic stem cells, myofibrils and SC cultures extracted from patients, etc.). The culture is washed with an aqueous saline buffer such as PBS (phosphate-buffered saline), and then the superlayer is removed (e.g., lifted, aspirated, etc.) using a clean or sterile sharp tool (e.g., forceps, pipette tips, etc.), followed by washing with PBS. Sufficient PBS is then added to cover the SC monolayer adhering to the culture substrate, and the mixture is kept at a temperature ranging from 20 to 37 °C for several minutes (e.g., 5–15 min). Preferably, the aqueous saline buffer is free of calcium and magnesium to allow for cell detachment. Other dissociation media (e.g., acutase, trypsin, collagenase) can be used, and different temperatures and incubation times can be used accordingly. Cells are then detached from the bottom of the plate by gentle mechanical dissociation in a dissociation medium (e.g., calcium / magnesium-free PBS) by pipetting up and down (several times per culture zone, e.g., but not limited to 10 times in total).

[0046] Once INFRA cells are detached, they can be used directly for other applications or further purified using FACS cell sorting. Cells can be used directly for any application (e.g., but not limited to cell therapy, drug screening, cell culture, sequencing, etc.), and can be resuspended or not resuspended (e.g., growth medium can be added directly to the culture) in the desired volume, rotated down using a centrifuge (e.g., 300 g for 4 minutes), and / or filtered or not filtered (e.g., but not limited to a 40 μm filter). Figure 3A This document outlines the general procedures and methods for preparing INFRA cells. In summary, the steps for obtaining INFRA cells include: Step 1: Generate myogenic cultures derived from pluripotent stem cells (PSCs); Step 2: Seize the upper layer containing muscle fibers; Step 3: Peel off the top layer to expose muscle stem cells.

[0047] The INFRA cells obtained using this method may possess any of the characteristics of the INFRA cell composition described herein. In some embodiments, the INFRA cells comprise human muscle stem cells (SCs) that express the transcription factor PAX7 and lack expression of the transmembrane receptor PDGFRA. In other embodiments, at least 70% of the INFRA cell population is PAX7+ and PDGFRA-.

[0048] In some embodiments, the method further includes a step of purifying cells using flow cytometry, as described herein. For example, in some embodiments, the method includes a step of purifying INFRA cells by flow cytometry using antibodies against ITGA7, CD56, NOTCH3, CD82, and / or CDH15.

[0049] Methods for treating muscle injuries

[0050] Another aspect of the invention provides a method for treating muscle injury in a subject. The method includes contacting the subject's muscle injury with a therapeutically effective amount of INFRA cells, wherein the INFRA cells comprise human muscle stem cells (SCs) that express the transcription factor PAX7 and lack expression of the transmembrane receptor PDGFRA, wherein at least 70% of the human muscle SC population is PAX7+ and PDGFRA-. In some embodiments, the SCs are not modified during isolation.

[0051] Muscles can be classified into three types: skeletal muscle, cardiac muscle, and smooth muscle. Skeletal muscle is a type of muscular tissue that generates force and transmits that force to the bones to maintain breathing, movement, and posture. Cardiac muscle is the muscle of the heart. Smooth muscle is the muscular tissue of arteries and the intestinal walls. The methods and compositions of this invention are primarily applied to skeletal muscle, but can also have a positive effect on smooth muscle. “Skeletal muscle” is defined as muscle that interacts with bones, tendons, and joints.

[0052] In some implementations, a method is provided for treating diseases, ailments, conditions, and injuries that cause a decrease in muscle strength (also referred to herein as musculoskeletal diseases, as well as muscle dysfunction and wasting diseases). Examples of muscle injuries include muscular dystrophy, traumatic injury, and cachexia.

[0053] In some embodiments, the present invention provides a method for treating musculoskeletal disorders, including muscle dysfunction and wasting diseases or conditions, including hereditary myopathy, neuromuscular disease, muscle atrophy, drug-induced myopathy, or any disease, condition, symptom, or illness that results in decreased muscle strength. In some embodiments, the subject suffers from a muscle disease selected from the group consisting of: sarcopenia, cachexia, type II myofibril atrophy, and genetically determined muscular dystrophy, or acquired autoimmune primary muscle disease associated with impaired regenerative radial growth phase.

[0054] In some implementations, administering INFRA cells can be used to treat muscular dystrophy. Muscular dystrophy is a group of inherited or genetic diseases. It is primarily caused by a genetic defect in skeletal muscle proteins, leading to weakness, usually progressive weakness, due to loss of muscle integrity and degeneration of skeletal or voluntary muscles that control body movement. Some types of muscular dystrophy can also affect the heart and involuntary muscles.

[0055] Examples of muscular dystrophy that can be treated with INFRA cells include Duchenne muscular dystrophy, Becker muscular dystrophy, myotonic dystrophy (also known as Steinart disease), limb-girdle muscular dystrophy, inotropic diseases, myotonic dystrophy, Emory-Dreyfus muscular dystrophy, congenital muscular dystrophy (e.g., regional protein deficiency congenital muscular dystrophy, Betleme myopathy, Ulrich congenital muscular dystrophy), facioscapulohumeral muscular dystrophy, spinal muscular dystrophy, ankylosing spondylitis, distal muscular dystrophy, and oculopharyngeal muscular dystrophy.

[0056] In some implementations, INFRA cells can be used to treat muscle atrophy. Muscle atrophy is a general term used to describe conditions characterized by the consumption or loss of muscle tissue due to various diseases, conditions, other illnesses, or events. Muscle atrophy can be caused by, but is not limited to: prolonged immobility (as in the recovery period from severe burns), major joint replacement surgery, neuropathic pain, peripheral neuropathy, necrotizing vasculitis, zero-gravity environments (e.g., astronauts and spacefarers), prolonged hospitalization, degenerative diseases (e.g., amyotrophic lateral sclerosis), organ transplantation, as well as spinal cord injury, chronic hemodialysis, and stroke.

[0057] In some implementations, INFRA cells can be used to treat disuse atrophy. Disuse atrophy is a condition characterized by the wasting or loss of muscle tissue due to prolonged inactivity. Disuse atrophy can be caused by, but is not limited to: prolonged immobility (as in the recovery period from severe burns), major joint replacement surgery, neuropathic pain, zero gravity environments (e.g., astronauts and spacefarers), long-term hospitalization, anorexia, organ transplantation, as well as spinal cord injury, chronic hemodialysis, and stroke.

[0058] In some implementations, INFRA cells can be used to treat age-related muscle atrophy. Age-related muscle atrophy is a condition characterized by the consumption or loss of muscle tissue and the replacement of muscle tissue with fibrous tissue as the subject ages.

[0059] In some implementations, INFRA cells can be used to treat sarcopenia, a condition characterized by the consumption or loss of muscle tissue and the replacement of muscle tissue with fibrous tissue as the subject ages.

[0060] In some implementations, INFRA cells can be used to treat muscle wasting in cachexia. Cachexia refers to weight loss, muscle atrophy, fatigue, weakness, and significant loss of appetite in some individuals who have not actively attempted to lose weight but are suffering from chronic illness. Muscle wasting in cachexia can be caused by, but is not limited to, cancer, multiple sclerosis, tuberculosis, acquired immunodeficiency 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.

[0061] In some implementations, muscle injury is traumatic muscle injury. Traumatic muscle injury is damage to muscle tissue caused by high stress or strain or external impact. Traumatic muscle injury is usually caused by activation of the muscle during stretching, resulting in indirect, non-contact muscle injury (strain or rupture), or by direct muscle injury (contusion or tear) due to external impact. Edouard et al., Nat Rev Dis Primers., 9(1):56 (2023).

[0062] As used in this article, “contact” refers to the placement or administration of INFRA cells that enable them to interact with the injured muscle. Contact includes both direct and indirect contact, in which INFRA cells are administered or placed so that they can migrate to the site of muscle injury through natural processes. Placing INFRA cells can include placing the cells on a wound dressing, while administering the cells can include injecting the cells.

[0063] Effective therapeutic doses can be determined using various methods, such as animal studies. In some embodiments, data obtained from cell culture assays and animal studies can be used to formulate a range of doses for human use. The doses of such compounds are preferably within a range of circulating concentrations that include the ED50 and have little or no toxicity. The dose may vary within this range depending on the dosage form and route of administration used.

[0064] The therapeutically effective dose of a composition containing a population of INFRA cells can also be preliminarily estimated from cell culture assays. Doses can be formulated in vivo in animal models to achieve a significant effect on muscle damage. Alternatively, the effect of any specific dose can be monitored using appropriate bioassays.

[0065] Typically, the dosage is approximately 10 x 10 6 Cells / kg subject body weight or lower, approximately 9 x 10⁻⁶ cells / kg subject body weight or less. 6 Cells / kg or lower, approximately 8 x 10 6 Cells / kg or lower, approximately 7 x 10 6 Cells / kg or lower, approximately 6 x 10 6 Cells / kg or lower, approximately 5 x 10 6 Cells / kg or lower. In an alternative embodiment, the dosage may be approximately 0.25 x 10⁻⁶ cells / kg or lower. 6 cells / kg to approximately 5 x 10 6 Between 1 x 10 cells / kg; or more preferably about 1 x 10 6 cells / kg to approximately 5 x 10 6 Cells / kg. Therefore, in another alternative embodiment, the dosage may be approximately 0.25 x 10⁻⁶ cells / kg. 6 cells / kg, 0.5 x 10 6 cells / kg, 0.6 x 10 6 cells / kg, 0.7 x 10 6 cells / kg; 0.8 x 10 6 cells / kg; 0.9 x 10 6 1.1 x 10 cells / kg; 6 cells / kg; 1.2 x 10 6 1.3 x 10 cells / kg; 6 cells / kg; 1.4 x 10 6 cells / kg; 1.5 x 10 6 cells / kg; 1.6 x 10 6 1.7 x 10 cells / kg; 6 1.8 x 10 cells / kg;6 1.9 x 10 cells / kg; 6 1 cell / kg or 2 x 10 6 Cells / kg. In other embodiments, the dosage may 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.

[0066] Regarding the duration and frequency of treatment, skilled clinicians typically monitor subjects to determine when the treatment provides therapeutic benefit and to determine whether to increase or decrease the dose, increase or decrease the frequency of administration, discontinue treatment, resume treatment, or make other changes to the treatment regimen. Administration schedules can range from once weekly to once daily, depending on many clinical factors, such as the subject's sensitivity to the peptide. The required dose can be administered once or divided into multiple subdose, such as 2-4 subdose, administered over time intervals, such as at appropriate intervals throughout the day or other suitable schedules. Such subdose can be administered as a unit dosage form. In some implementations, administration is long-term, such as once or multiple times daily for weeks or months. Examples of administration schedules include once, twice, three times, or four or more times daily for periods of 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, or 6 months or longer.

[0067] Examples of administration methods include, but are not limited to, injection, infusion, drip, inhalation, or ingestion. "Injection" includes, but is not limited to, intravenous, intramuscular, intraarticular, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, tracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, intraspinal, intracerebrospinal, and intrasternal injection and infusion. In a preferred embodiment, the composition is administered via intravenous infusion or injection.

[0068] INFRA cells can be provided as pharmaceutical compositions containing isotonic excipients prepared under sufficiently aseptic conditions for human administration. The composition may be sterile. The formulation should be suitable for the route of administration. For general principles of pharmaceutical formulation, readers may refer to Cell Therapy: Stem Cell Transplantation, Gene Therapy, and Cellular Immunotherapy, G. Morstyn & W. Sheridan eds, Cambridge University Press, 1996; and Hematopoietic Stem Cell Therapy, ED Ball, I. Lister & P. ​​Law, Churchill Livingstone, 2000. The selection of cell excipients and any accompanying elements of the composition containing populations of INFRA cells can be tailored to the route of administration and the device used.

[0069] In some embodiments, 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 alcohol, polyethylene glycol, gelatin, sugars such as lactose, amylose or starch, dextrose, magnesium stearate, talc, silica, viscous paraffin, perfume oils, fatty acid esters, hydroxymethyl cellulose, polyvinylpyrrolidone, and combinations thereof. If desired, the pharmaceutical formulation may be mixed with excipients, such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts that affect osmotic pressure, buffers, colorants, flavoring agents, and / or aromatic substances, which do not react adversely with the active compound.

[0070] Suitable preservatives and buffers can be used in such formulations. To minimize or eliminate injection site irritation, such compositions may contain one or more nonionic surfactants with a hydrophilic-lipophilic balance (HLB) of about 12 to about 17. The amount of surfactant in such formulations is about 5% to about 15% by weight. Suitable surfactants include polyoxyethylene sorbitan fatty acid esters, such as sorbitan monooleate, and high molecular weight adducts of ethylene oxide and hydrophobic bases formed by the condensation of propylene oxide and propylene glycol. Parenteral formulations may be available in single-dose or multi-dose sealed containers, such as ampoules and vials, and may be stored under lyophilized (freeze-dried) conditions, requiring only the addition of a sterile liquid carrier for injection, such as water for injection, immediately before use. Ready-to-use injectable solutions and suspensions can be prepared from sterile powders, granules, and tablets.

[0071] Preferably, the cells are administered by injection, for example, intravenously. Pharmaceutically acceptable carriers for cell injection may include any isotonic carrier, such as, for example, physiological saline (about 0.90% w / v aqueous NaCl solution, about 300 mOsm / L NaCl solution, 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% aqueous glucose solution, or Ringer's lactate solution. In one embodiment, the pharmaceutically acceptable carrier is supplemented with human serum albumin.

[0072] One example is included to more clearly illustrate a particular embodiment of the invention. However, a wide variety of other embodiments exist within the scope of the invention and should not be limited to the specific example provided herein.

[0073] Example

[0074] Optimize the in vitro production of human satellite cells for skeletal muscle cell therapy

[0075] Here, we describe an optimized protocol that allows for the production of populations of human iPS-derived cells containing up to 75% PAX7+SCs without antibody-based sorting. This protocol significantly simplifies the production of SCs for clinical applications. We further demonstrate that these cells can be very effectively implanted into mouse muscle, generating myofibrils and SCs, and contributing to the recovery of strength in chimeric muscles.

[0076] To define this protocol, we developed an integrated workflow combining bioinformatics and in vitro cell culture. We analyzed the entire embryo from mouse embryos at stages E8.5, E9.5, E10.5, and E11.5 (staged in one segment increment) (Qiu et al., Nat Genet We began with a multi-timepoint single-cell RNA sequencing (scRNAseq) dataset (54, 328-341, 2022) because they matched the timeframe of prestomy mesoderm (PSM) differentiation into myogenic precursors. From this dataset, we extracted paraaxial mesoderm to investigate the bifurcation from PSM to osteomyeloids and dermatomyosalms (characterized by PAX1 / 9 and PAX3 expression, respectively). Figure 1A -B). We used Palantir diffusion maps and force maps (Setty et al., Nat Biotechnol37, 451-460, 2019) created a 2D representation of the subset of interest and confirmed annotations for different cell types using differentially expressed genes. We then assigned PSM nodes as trajectory roots and learned the tree structure on our data using a simple master-tree algorithm. Next, we used a pseudo-temporal algorithm (scfates) (Faure et al., Bioinformatics 39, 2023) to assign temporality within the tree branches. Figure 1C Two main recognition branches from the anterior PSM (characterized by MEOX1) are myogenic derivatives on one branch (characterized by PAX7 and TTN) and cartilage / connective tissue progenitor cells (SOX5, PAX9, and PAX1) on the other branch. Figure 1B -C). Then, for each cell, we scored the average expression of the set of genes associated with known signaling pathways described in BioPlanet (Huang et al., Front Pharmacol 10, 445, 2019). The score is the average expression of the gene set minus the average expression of the reference gene set (randomly sampled from genes contained in the dataset). The score is calculated for each signaling pathway and then processed using a machine learning algorithm (mgcv: generalized additive model) (Wood, Generalized Additive Models: An Introduction with R, Second Edition (2nd ed.) (2017) Fits to the trajectory and can be visualized over pseudo-time in our force-map spectral space ( Figure 1D Therefore, we identified that the tyrosine receptor kinase B (TRKB), bone morphogenetic protein (BMP), platelet-derived growth factor (PDGF), and transforming growth factor β (TGFB) pathways are activated in the osteophyte branch and inhibited in the dermatomyosal branch.

[0077] This knowledge was then used to establish a new myogenic differentiation protocol, in which the enrichment of SCs (similar to those derived from osteophytes) was aimed at reducing the contribution of osteophyte-derived lineages. Figure 1E We retain the initial steps required for PSM generation, which involve Wnt and FGF activation and BMP inhibition as described in our original muscle differentiation protocol (Chal) (Chal et al., Nature protocols11, 1833-1850, 2016). The Chal protocol is based on myogenic cues described in the literature and uses BMP inhibitors (LDN), hepatocyte growth factor (HGF), fibroblast growth factor (FGF-2), and insulin-like growth factor (IGF-1) from day 6 to day 8, followed by IGF-1 from day 8 to day 12, and finally IGF-1 and HGF from day 12 onwards. Based on new findings from our bioinformatics pipeline, we treated anterior PSM cells with BMP (LDN) and TGFb (SB 431542) inhibitors from day 6 to day 9 to block osteoblast differentiation. Subsequently, from day 9 to day 12, in addition to LDN and TGFb inhibition, we added platelet-derived growth factor (PDGF) (AC 710) and tyrosine receptor kinase B (TrkB) (ANA 12) inhibitors. Finally, starting from day 12, the BMP and PDGF pathways were inhibited to suppress cell differentiation into fibroblasts. Figure 1E This new protocol is serum-free because the inhibitor was added to Dulbecco modified Eagle medium (DMEM) with 15% knockout serum substitute (KSR). To track the cells that yielded osteoblast / chondrocyte fate, we engineered the SOX9-Venus human iPS reporter line. Using flow cytometry, we observed a reduction (-2.4%) in SOX9-Venus+ cells at day 9 compared to the Chal protocol. This was achieved using an iPS reporter line targeting PAX3 expression (Rao et al., Dev Cell (58, 2359-2375, 2023) We demonstrated that on day 9, this reduction in SOX9 was associated with a significant increase (+13.4%) in PAX3-GFP+ cells, which primarily correspond to dermatomyosal progenitors. To track myogenic differentiation in live cells, we designed a dual-reporter human iPS line expressing both the PAX7-Venus and α-actin mKate2 reporter genes. On day 14, we detected the first PAX7+ cells and myofibrils expressing ACTN2 ( Figure 1F In subsequent culture phases, the number of multinucleated skeletal muscle fibers and PAX7+ cells significantly increased. To quantify the myogenic cell population, we introduced mCherry fluorescent protein into the MYOD1 locus of the PAX7-Venus reporter line. On day 31, using our optimized protocol and the PAX7-MYOD1 reporter line, we observed a significant increase in Venus+ cells (+23.1%), mCherry+ cells (+9.8%), and double-positive cells (+21.1%), and a decrease in double-negative cells (-15.5%). Figure 1GTherefore, the protocol optimized using our bioinformatics workflow allows for the generation of up to 52.3% PAX7-Venus+ cells on average in the mononuclear fraction, resulting in a significant enrichment of the PAX7+ population compared to the Chal protocol.

[0078] Multiple reports have used scRNA-seq to describe cell compositions from myogenic cultures derived from human PSCs (Xi et al., Cell stem cell (Vol. 27, Issue 1, 158-176, 2020). However, these reports provide an incomplete description of cellular diversity because syncytial myofibrils cannot be encapsulated and sequenced using this technique. Therefore, we used single-nuclear RNA sequencing (snRNAseq) to generate a comprehensive characterization of myogenic cultures produced using our optimized protocol and compared it with the original Chal protocol. The analysis here allows for the capture of myofibril nuclei that could otherwise not be quantified by FACS or scRNAseq analysis. We performed nuclear extraction on cultures at day 31, followed by 10x encapsulation and sequencing. In total, we analyzed 1881 cells from the Chal protocol and 6763 cells from the new protocol. Both datasets were similarly processed to generate UMAP projections and Leiden clusters. The identity of the clusters was determined using differential gene expression. Merging and re-clustering the two datasets revealed that they contained the same cell types corresponding to PSM derivatives, including fibroblast progenitor cells (PDGFRA+), satellite cells (PAX7+), myocytes (ACTN2+), and nerve cells (MAP2+). Figure 2A -B). However, the proportions of these cell types produced by the two protocols differed. Compared to the Chal protocol (10.8%), our new protocol produced twice as many SC nuclei (25.1%). Figure 2C This was accompanied by a decrease in the proportion of all other populations (fibroblasts, myocytes, and nerve cells), thus confirming our analysis using reporter gene lines.

[0079] To better characterize skeletal muscle lineage trajectories in vitro, we extracted all myogenic nuclei (corresponding to SCs and myogenic fibers) from two datasets. We then independently re-clustered and annotated them. Figure 2D -I). For both protocols, this identified three similar clusters that were connected along developmental trajectories ( Figure 2D-G). One cluster contained PAX7-expressing SCs, which also expressed well-known markers of this lineage, including NOTCH3, FGFR4, and CD82. We also identified two additional myogenic clusters corresponding to different stages of myofibril maturation. One myoblast type cluster was rich in cells expressing MYOD1, MYOG, MYMK, MYMX, HES6, and CKB, thus corresponding to the previously described transitional state between SCs and myofibrils (Al Tanoury et al., Development Jun 26; 147(12), 2020). This cluster was linked to a myocyte cluster rich in genes encoding myofibril structural proteins, such as TTN, DMD, MYH3, and MYH8 expressed by postmitotic myofibrils. STREAM trajectory analysis revealed a generalization of satellite cell differentiation into myofibrils, accompanied by the orderly expression of characteristic markers of myogenesis. Figure 2E (G) (Chen et al., Nature communications (10, 1903, 2019). Next, we created a “satellite cell score” based on the expression of SC markers PAX7, NOTCH3, FGFR4, CD82, and CDH15, and displayed it in a matrix plot. The SC cluster from our new protocol showed the highest score among all clusters from both protocols. We also calculated the normalized enrichment scores of the signaling pathways that we are regulating in both myogenic protocols. This score was calculated using the pre-sorting function of GSEA and the Bioplanet signaling pathway gene set. Compared with the Chal protocol, the new protocol reduced the number of repressed pathways. Therefore, snRNAseq analysis confirmed that our optimized protocol increased the proportion of SCs in myogenic cultures.

[0080] To determine the developmental stage of in vitro-generated myogenic cells, we compared their gene expression profiles with snRNA-seq datasets of human embryonic limbs from week 5 post-conception (PCW) to week 9 (Zhang et al., bioRxivWe first extracted cells from the myogenic lineage (based on the original annotations) and clustered the myogenic dataset using Leiden. This analysis identified three myogenic clusters: SC (PAX7+), myoblasts (MYMK+), and myocytes (MYH3+). STREAM pseudo-time analysis identified developmental trajectories connecting these three clusters. Comparison of the three clusters identified in in vitro datasets (Chal and New) using a classifier trained on a human embryonic myogenic dataset showed that the three clusters detected in vitro were similar to their in vivo counterparts. Furthermore, the classifier indicated that in vitro-generated SCs were most closely approximating PAX7+ SCs at 8 weeks of age in human embryos; therefore, staging in vitro SCs within the embryonic / fetal transition period, as previously suggested (Xi et al., 2022). Cell stem cell , Vol. 27, Issue 1, 158-176, 2020).

[0081] Next, we developed a method to enrich functional SCs without relying on immunolabeling and cell sorting. During myogenic differentiation at days 20–30 using our original (Chal) and optimized protocols, most cultures formed a thick superlayer containing myofibrils, while most PAX7+ cells adhered to the culture plate substrate. This thick superlayer could be mechanically removed (e.g., peeled off using forceps) and then discarded, leaving a population enriched with myogenic cells (INFRA cells) adhering to the culture plate. Figure 3A Dissociation and flow cytometry analysis of this adhesive mononuclear cell fraction generated using the PAX7-Venus reporter line showed that the population produced by this strategy contained an average of 74.1% PAX7+ cells. Figure 3B Immunostaining with PAX7 antibody immediately after peeling confirmed significant enrichment of PAX7+ cells. Figure 3C -D). The exfoliated cultures, maintained in vitro for 2 weeks in DMEM / 15% KSR, differentiated into myofibrils capable of spontaneous contraction and self-renewal to generate PAX7+ cells in vitro, thus exhibiting characteristics very similar to mouse fetal SC cells. Figure 3E (Tierney et al., Cell Reports (1-13, 2016).

[0082] Next, we performed scRNA-seq on INFRA cells generated directly using our new protocol after peeling the supernatant from the culture on day 31. We will use the scRNA-seq data from 3423 INFRA cells generated using the new protocol ( Figure 3F-H) and 3135 PAX7-Venus positive cells sorted from day 31 cultures produced using the Chal protocol by FACS (-H) Figure 3I We compared the results using -K). Using similar preprocessing, followed by UMAP projection and Leiden clustering, we were able to identify clusters of the same cell types within populations of INFRA and PAX7-Venus+ cells sorted by FACS. These included SC (PAX7+), myocytes (MYOG+), and fibroblasts (PDGFRA+), which expressed similar sets of characteristic genes (…). Figure 3G -J). When we merged the two datasets and then performed Leiden clustering, the same clusters were identified. Using a classifier, we confirmed the similarity of the clusters identified in INFRA and PAX7+ sorted cells. The population of INFRA cells generated using the new protocol contained 74% PAX7+ cells, while the population of PAX7+ Venus cells generated using the Chal protocol in FACS sorting yielded 89% PAX7+ cells. Figure 3H The presence of myocytes with downregulated PAX7 (and therefore lacking PAX7 expression despite positive selection) can be explained by the stability of the YFP marker used to sort cells (Al Tanoury et al., K). Development (Jun 26;147(12), 2020). The small population of fibroblasts detected after FACS purification may represent contamination of PAX7+ cells. Alternatively, these cells may represent fibroblast progeny of PAX7+ cells that retain the fluorescent marker after downregulation of PAX7 expression, as observed in myogenic cells. Therefore, combining our new protocol with this mechanical procedure allows for the enrichment of true PAX7+ cells to near-FACS sorting levels.

[0083] Next, we established a protocol to efficiently colonize mouse skeletal muscle using in vitro-generated iPS-derived human PAX7+ SCs. To track the fate of transplanted cells in vivo, we introduced luciferase into the safe harbor AAVS of the human PAX7-Venus-ACTN2-mKateiPS reporter line. PAX7+ cells were generated 31 days after in vitro myogenic differentiation, following the Chal protocol. These cultures contained an average of 28.18% PAX7+ cells in the mononuclear fraction. Two days prior to cell injection, the legs of immunodeficient NOD mice were irradiated (18 Gray) to block the proliferation of endogenous satellite cells. To induce muscle damage and promote regeneration, we injected cardiotoxin into the tibialis anterior muscle (TA) 24 hours before transplantation. We purified PAX7-Venus+ cells from the day 31 cultures using FACS and injected one million Venus-positive SCs into the right TA, while the left TA received the same treatment but without transplanted cells. One month after transplantation, we quantified the bioluminescence emitted by the injected human cells. Luciferase signaling was observed only in the transplanted TA region, indicating that human cells were predominantly localized in their injection sites. Control TAs without transplantation showed severe atrophy, while the weight of transplanted TAs was not significantly different from healthy muscle. Transverse frozen sections of transplanted and control TA muscles labeled with a human-specific laminin B2 antibody showed that the percentage of human cell nuclei within the transplanted TA was an average of 75.5% of the total cell nuclei (n=3). Immunostaining of transverse frozen sections of hindlimb muscle with human dystrophin and laminin demonstrated successful engraftment of human cells throughout almost the entire transplanted TA. No human cells were observed in adjacent uninjected muscle, suggesting that the colonization potential of PAX7+ cells is limited to the injected muscle. Most human myofibrils expressed embryonic myosin MYH3, indicating that they retained immature characteristics. CD31+ endothelial cells were observed within the transplanted area, indicating that the newly formed muscle region was vascularized. Therefore, this protocol allows for the large-scale colonization of mouse TAs by progeny of injected human PAX7+ cells, which form myofibrils expressing human dystrophin.

[0084] Finally, we tested the transplantation capacity and functional properties of SCs generated using our optimized protocol. Using the aforementioned transplantation protocol, we directly injected 500k INFRA cells generated using our new protocol (without cell sorting) after peeling on day 30. Figure 4A One month later, bioluminescence analysis showed the presence of human cells in the injected TA, but not in the control TA. Figure 4B The size difference between transplanted and non-transplanted TAs is visible at a macroscopic level. Figure 4C The transplanted TA muscle weighed significantly more than the control group, but there was no significant difference compared to normal TA muscle. Figure 4D The percentage of human cell nuclei in transplanted TA cells averaged 62% of the total cell nuclei (n=4). Figure 4E -F). Immunostaining of transverse frozen sections of the hindlimb with human cell nuclei, human dystrophin, and laminin demonstrated the presence of human cells in almost the entire transplanted TA. Figure 4F -J). High-magnification images of TA transplanted sections show that immature human muscle fibers are parallel to the long axis of the muscle, thus exhibiting elongation as intrinsic muscle fibers ( Figure 4G These fibers express MYH3, indicating their fetal nature (-J). Figure 4K We also identified human PAX7+ cells, indicating that the injected cells can self-renew. Figure 4L We also observed CD31+ vessels in areas rich in human cells, indicating vascularization in these areas. Figure 4K To assess the function of the transplanted muscle, we performed in vivo strength tests. The force (nM) generated by the transplanted TA, normalized to body weight (mg), was significantly higher than that of the control TA, but significantly lower than that of the TA in age-matched (10-week-old) untreated NOD mice. Figure 4N In summary, these experiments demonstrate the remarkable colonization potential of in vitro generated human PAX7+ SCs, as well as their ability to generate muscle fibers and restore strength production in damaged muscles.

[0085] To examine the fate of human cells in mouse TA after transplantation, we performed snRNA-seq of the entire TA muscle one month after transplantation. A total of 3833 cell nuclei were purified and sequenced using a 10x pipeline. Based on Cellranger attribution (by mapping reads of the GRCh38 and mm10 genomes), 86% of these nuclei were identified as human-origin, while 14% were mouse-origin. Human cell nuclei were processed to generate UMAP projections and Leiden clusters. This indicated that the original injected cell population differentiated into four major cell types, including myocytes, fibroblasts, pericytes, and SCs. Using an NG2 antibody combined with a human cell nucleus antibody, we confirmed the presence of human pericytes after injection of INFRA cells. Figure 4MIn contrast, the mouse cell population was primarily composed of immune cells, including macrophages (MITF, CD14), dendritic cells (CD11C, FLT3), and vascular cells (PECAM1). Within the myogenic cluster, we identified populations expressing embryonic myosin MYH3 (91%), slow myosin MYH7 (22%), perinatal myosin MYH8 (41%), and fast myosin MYH2 (3%). Next, we extracted and clustered human myogenic cells from the dataset for more detailed analysis. Myogenic cells were divided into three clusters, exhibiting characteristics similar to the SC, myoblast, and myocyte clusters described in vivo and in vitro. Figure 4P Therefore, transplanted human PAX7SCs were able to reconstruct normal developmental myogenic trajectory in vivo in mouse TA.

[0086] To determine the identity of transplanted cells in vivo, we used a classifier to compare the clusters of human myogenic limb cells from transplanted TAs with myogenic clusters from the human limb dataset. This demonstrated excellent correspondence between the SC, myoblast, and myocyte clusters. Next, we performed a DEG analysis between myogenic cells from the novel protocol in vitro dataset and in vivo clusters from human embryonic limbs. We analyzed the GO-term enrichment in the DEG list. Interestingly, this analysis revealed a metabolic switch involving a significant increase in the expression of all glycolytic enzymes, indicating upregulated glycolytic activity of transplanted SC cells in vivo. Figure 4Q This metabolic shift has also been observed in mice, in fetal and adult activated SCs, where SCs proliferate and exhibit many similarities to transplanted cells (Paia et al., J Cell Sci 131, 2018). As expected, we also observed a significant increase in the expression levels of sarcomatin in myocyte clusters in vivo, consistent with expected cell maturation in vivo ( Figure 4R Increased expression of ECM protein-coding genes (such as laminin, fibronectin, and collagen) and the angiogenesis factor VEGFA was observed in SCs in vivo. Therefore, these data suggest that transplanted PAX7 cells undergo maturation upon transplantation into mouse TA and participate in local tissue remodeling.

[0087] Here, we leveraged a machine learning-based strategy to identify signaling cues deployed in the early stages of myogenic differentiation to optimize our original protocol. This allowed us to double the yield of human PAX7+ cells in the original in vitro protocol from 25% to ~50%. This was achieved by inhibiting the PDGF, TGFb, and BMP pathways to prevent cell differentiation into osteophytes, pathways known to play a role in mouse osteophyte development (Alkhatib et al., Curr Mol Biol Rep4,132-141, 2018). We also determined that the TRKB (NTRK2) pathway is selectively activated in osteogenic joints. This pathway has been studied primarily in the nervous system where TRKB binds to the neurotrophic factor BDNF, and has also been involved in fibroblast survival and proliferation (Glass et al., 2018). Cell 66, 405-413, 1991). The recombinant growth factor used in our original myogenic differentiation protocol was completely replaced by compounds, which should greatly facilitate the transfer to GMP conditions to optimize cell-based therapeutic protocols. We also added a simple mechanical step, resulting in an enrichment of PAX7+ SCs exceeding 70%. This level of enrichment is advantageous compared to the 70-90% myoblast purity used in clinical trials (Skuk et al., Neuromuscul Disord 17, 38-46, 2007). In this graft context, it remains to be determined whether the significant proliferation of residual fibroblasts in vivo is a positive or negative factor. Therefore, this approach circumvents the need for antibody-based purification, which significantly increases the cost and complexity of cell preparation protocols. Finally, we demonstrate that in vivo transplanted human myogenic precursor cells can effectively colonize mouse muscle and differentiate into dystrophin-positive muscle fibers. The transplanted cells regain quality and strength, supporting their application as a cell therapy for treating muscle degenerative diseases such as Duchenne muscular dystrophy.

[0088] method: Human induced pluripotent stem cell (iPSC) culture This study used NCRM1 iPSC cells (RUCDR, Rutgers University). Written informed consent was obtained from the donors at the time of sample collection. Work on the human iPS cell line was approved by the MassGeneral Brigham IRB. Cells were maintained in mTeSR1 medium (StemCell Technologies 05851) on Matrigel-coated plates (Corning 35277) and passaged every four days. In short, cultures at 90% confluence were dissociated with Accutase (Corning 25058CI), and 500,000 cells were seeded into one well of a 6-well plate containing mTeSR1 and 10 μM Y-27632 dihydrochloride (Tocris Bioscience 1254). Fresh mTeSR1 medium was added daily over the next few days. All cell lines were maintained for no more than 15 passages and were periodically tested for mycoplasma contamination.

[0089] Generation of reporter cell lines

[0090] Using the CRISPR-Cas9 system for genome editing, with previously described methods (Miao et al., Nature (21 Dec 2022, 614(7948):500-508) generated all human iPSC reporter cell lines. The following reporter cell lines were generated from previous studies: cell line #1, mKate2-ACTN2 knock-in reporter cell line (Mao et al., 21 Dec 2022, 614(7948):500-508). eLife 11, 2022); Cell line #2, Venus-NLS-T2A-PAX7 reporter cell line (Al Tanoury et al., Development Jun 26; 147(12), 2020). In this study, we generated cell lines #3-6.

[0091] To prepare a dual reporter cell line (cell line #3) for mKate2-ACTN2 and Venus-NLS-T2A-PAX7, 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 via in-fusion cloning (Takara Bio 638909), consisting of 1-kb 3' and 5' homologous arms flanking the YFP variant Venus, a nuclear localization signal (NLS), and a self-cleaving T2A peptide sequence within the pUC19 vector backbone. pGuide-it-tdTomato and the repair plasmid were delivered to cell line #1 iPS cells via nuclear transfection (Lonza VPH-5022). Twenty-four hours after nuclear transfection, cells were sorted using an S3 cell sorter (Biorad) based on TdTomato expression and seeded at a low density (500 cells per 35 mm culture dish) in mTeSR1 cells containing 10 μM Y-27632 and CloneR (Stemcell Technologies 05888) on Matrigel-coated plates. Single-cell clones were expanded, and individual colonies were screened by PCR to identify Venus-NLS-T2A cells targeting homozygous insertions immediately following the PAX7 start codon.

[0092] Other reporter cell lines were prepared using the same method. To prepare a dual reporter cell line of Venus-NLS-T2A-PAX7 and MYOD1-T2A-H2B-mCherry (cell line #4), a single guide RNA targeting the 3' end of MYOD1 was designed and cloned into the pGuide-it-tdTomato vector. The repair plasmid consisted of 1-kb 3' and 5' homologous arms flanking the T2A-H2B-mCherry sequence in the pUC19 vector backbone. These plasmids were delivered to cell line #2 iPS cells to generate cell line #4 with homozygous insertion of T2A-H2B-mCherry immediately preceding the MYOD1 stop codon. To prepare a SOX9-T2A-H2B-GFP reporter cell line (cell 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 together with the repair plasmid (Addgene 167972) to the NCRM1 iPSC cell line to generate cell line #5, which had a homozygous insertion of T2A-H2B-GFP immediately before the SOX9 stop codon. Sanger sequencing was performed in all cases to ensure in-frame localization of the reporter gene fragment and to rule out insertions or deletions at recombination sites.

[0093] We inserted constitutively expressed pCAG-luciferase into the safe harbor AAVS1 locus in cell line #3 to generate cell line #6. Briefly, we cloned the luciferase sequence into the AAVS1-pCAG vector (Addgene 80490) and co-transfected it with the pXAT2 vector (Addgene 80494) into cell line #3 iPS cells. Positive clones were selected one day after nuclear transfection by supplementing mTeSR1 with puromycin (0.5 μg / ml, Sigma-Aldrich P7255) for a total of 4 days. Single colonies were amplified and the insertion was confirmed by PCR.

[0094] Skeletal muscle fibers and satellite cells derived from human induced pluripotent stem cell optimization protocol

[0095] (Day -1) Mature iPS cell cultures (100% confluence) were dissociated in Accutase, and 200,000–240,000 cells were seeded into a Matrigel-coated 6-well plate containing 1.5 ml mTeSR1 medium and 10 μM Y-27632. Seeding density is important for further differentiation, with lower ranges being more effective. (Day 0) Cells were treated for 3 days with DMEM / F12 GlutaMAX supplemented with 1% ITS, 3 μM CHIR99021, and 0.5 μM LDN-193189 (PSM medium). (Day 3) Cells were then cultured for 3 days in DMEM / F12 GlutaMAX supplemented with 1% ITS, 3 μM CHIR99021, 0.5 μM LDN-193189, and 20 ng / ml FGF-2. (Day 6) Cells were then treated for 3 days with high-glucose DMEM supplemented with 15% KSR, 1X NEAA, 0.01 mM bME, 1% ITS, 0.5 μM LDN-193189, and 10 μM SB 431542. (Day 9) Cells were then treated for 3 days with high-glucose DMEM supplemented with 15% KSR, 1X NEAA, 0.01 mM bME, 1% ITS, 0.5 μM LDN-193189, 10 μM SB 431542, 15 nM AC 710, and 5 μM ANA 12. (Day 12) Cells were then cultured continuously for 3 days in high-glucose DMEM supplemented with 15% KSR, 1X NEAA, 0.01 mM bME, 1% ITS, 0.5 μM LDN-193189, and 10 μM SB 431542. The medium was changed every three days thereafter. The skeletal muscle fibers and satellite cells generated using this protocol can then be maintained for at least 90 days in high-glucose DMEM supplemented with 15% KSR, 1X NEAA, 0.01 mM bME, 1% ITS, 0.5 μM LDN-193189, and 15 nM AC 710.

[0096] INFRA cells

[0097] On days 20-30, myogenic cultures form a thick outer layer containing myofibrils, which can be mechanically removed and discarded. PAX7+ satellite cells adhering to the culture substrate remain attached during this stage. iPS-derived myogenic cultures are then performed in 6-well plates and washed with calcium- and magnesium-free PBS (phosphate-buffered saline). The upper layer of the culture is then removed using a clean or sterile, sharp tool, such as Dumont forceps. To harvest INFRA cells, the wells are incubated for 10 minutes at 27 °C in a sufficient amount of PBS to cover the remaining adhering cells. Cells are then detached from the bottom of the plate by gentle mechanical dissociation via pipetting up and down in PBS (several times per culture zone). After filtration (Falcon cell filter, 40 μm mesh), the cell pellet is centrifuged (300 g, 4 min), resuspended in PBS, and used for further analysis.

[0098] Flow cytometry analysis

[0099] To determine the fraction of cells expressing the fluorescent reporter gene, cultures were dissociated in 0.05% trypsin (ThermoFisher Scientific, 25200-056), type IV collagenase (ThermoFisher Scientific, 17104019), and PBS (2:2:5) and analyzed by flow cytometry using an ARIA cell sorter (BD). Cells from the parental NCRM1 line, which does not express the fluorescent protein, were used as a negative control for gating purposes. Results are expressed as the percentage of fluorescently positive cells in the sorted fraction after excluding debris and duplexes.

[0100] Immunostaining

[0101] Cells were fixed in 4% paraformaldehyde solution (PFA; Electron Microscopy Sciences 15710) at room temperature for 15 minutes, followed by washing three times with PBS. Tibialis anterior muscle was flash-frozen in isopentane and sectioned into 8–10 μm thick sections using a cryostat. Slides were post-fixed in 4% PFA before immunohistochemical treatment. Typically, samples were washed three times for 3 minutes each in Tris-buffered saline (TBS) containing 0.1% Tween, permeated in PBS containing 0.5% Triton for 10 minutes, and blocked for 1 hour at room temperature in TBS containing 0.1% Triton and 3% donkey serum. Primary antibodies were diluted in this blocking solution and incubated overnight at 4°C. After three TBST washes, cultures were incubated for 1.5 hours at 4°C with Alexa-Fluor-conjugated secondary antibody (1:500) and Hoechst33342 (1:1000). Cells were fixed in PBS and imaged after three final TBST washes and one PBS rinse. All immunostainings, except for PAX7 (see Methods below), were performed in this manner.

[0102] Antibody reference and concentration

[0103] Human cell nucleus: Laminin 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: ab13970, abcam, 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.

[0104] Antigen repair

[0105] For PAX7 immunostaining, slides were boiled in target retrieval solution (S2369, Dako) for 10 min to expose the antigen. The slides were then washed with PBS and permeated in PBS with 0.5% Triton X-100 for 15 min. The sections were incubated for 30 min in blocking solution (PBS, 2% horse serum, 2% goat serum, 2% BSA component V, 0.1% Triton X-100), followed by incubation in Mouse On Mouse blocking solution (Vector Laboratories) for 30 min. The sections were then incubated overnight at 4°C with primary antibody against Pax7 (1:100 in MOM dilution). Secondary antibody incubation was performed using a goat anti-mouse IgG1 isotype-specific antibody conjugated to AF647 (A-21240, Invitrogen, 1 / 500, 1 h at room temperature).

[0106] Microscopic examination

[0107] Wide-field fluorescence imaging was performed on a Leica DMi8 wide-field imaging system. Confocal imaging was performed on a Zeiss LSM880 confocal inverted microscope equipped with a large temperature incubation chamber and a CO2 module.

[0108] Single cell nucleus extraction

[0109] Cell culture: Remove the culture medium and add 500 μl homogenization buffer to the culture wells. Scrape the cells off the plate and add another 500 μl homogenization buffer. Then transfer the mixture to a 2 ml homogenizer on ice.

[0110] Mouse muscle tissue: post-euthanasia autopsy TA Muscles. According to the description (McLaughlin et al., STAR Protoc3, 101417, 2022), muscle was washed in PBS, transferred to 1.5 ml Eppendorf tubes, and minced with scissors in 1 ml homogenization buffer (250 mM sucrose (Sigma-Aldrich), 10 μM Tris ph 8.0 (Thermo Fisher Scientific), 25 mM KCl (Thomas Scientific), 5 mM MgCl2 (Thermo Fisher Scientific), 0.1% Triton-X 100 (Millipore Sigma), 0.5% Rnasin Plus (Promega), 1x protease inhibitor (Promega), 0.1 mM DTT (Thermo Fisher Scientific)). The mixture was then transferred to ice. Two types of homogenizers, loose and tight, were used 20 and 40 times respectively to obtain nuclear lysates. Lysates were filtered through 35 μm FACS tubes (Corning), then through a 40 μm FLOWMI filter (Scienceware), and transferred to 1.5 ml low-binding tubes (Eppendorf). The tubes were centrifuged at 1000 g at 4°C for 10 min in a swing-bar centrifuge. The precipitate was resuspended in 500 μl–1 ml (PBS + 1% BSA + RNase inhibitor + 1:1000 DAPI) and filtered again through a 40 μm FLOWMI filter. Nuclei were sorted using a 70 μm nozzle on an FACS (ARIA, BD) machine. Nucleus concentration was then manually counted, and quality control was performed by examining morphology under a fluorescence microscope.

[0111] Single-cell and nucleus preparation and sequencing

[0112] For each sample, 10,000 cell nuclei or cells were encapsulated using a 10x Chromium 3' v3.1 (Dual Index) kit (Rev E). Libraries were prepared according to the manufacturer's instructions (Rev E). Quality control was performed using a TapeStation system, and sequencing was performed at the Harvard Medical School Biopolymer Center using a NovaSeq Full 6000 SP Flowcell.

[0113] Analysis of single-cell RNA sequencing data

[0114] The transcriptome library was aligned to the human genome assembly GRCh38 (hg38) using Cell Ranger 6.1.1. The raw counting matrix was converted to anndata objects using Scanpy. Cells were filtered by count to remove outliers. Cells with excessive mitochondrial gene expression were also filtered. The raw data were normalized, logarithmically transformed, and scaled. Linear regression was performed on cell cycle genes to identify highly variable genes, and PCA and neighbor calculations were performed before the final UMAP embedding. Leiden clustering and sorting of all genes in each cluster (with p-values ​​of the Wilcoxon test corrected by Benjamini-Hochberg) allowed us to annotate the populations of cells present in the dataset. The number of cells in each cluster was plotted using Matplotlib. Dot plots were created using a signature gene list on a gene-based standard scale and plotted on the processed data using a custom matplotlib palette. Sub-clustering was created by separating the master dataset and reprocessing the data from the raw dataset. Enrichment analysis of the BioPlanet 2019 signaling pathway gene set was performed using the GSEA prerank function. Satellite cell scores are calculated by subtracting the average expression of a randomly sampled set of reference genes from the average expression of a set of genes.

[0115] Analysis of single-cell RNA sequencing and single-nuclear RNA sequencing data

[0116] Single-cell and nuclear RNA sequencing datasets were analyzed using Python 3.11, scanpy 1.9.5, and anndata 0.9.1. Datasets were analyzed individually or in merging for comparison. For datasets containing human and mouse cells, species attribution was performed using 10x CellRanger software based on the percentage of alignment with both genomes. Cells were filtered by count, gene count, and gene number based on sample quality control. Cells with high mitochondrial content were filtered, and a cell cycle score for each cell was calculated using the Regev Labs cell cycle gene list. Matrix normalization was performed using log1p, regression of total count and cell cycle phase, and scaling was applied at the end. Highly variable genes were calculated for principal component analysis, and batch correction was performed using Harmony based on samples. Neighbors were calculated, and if the analysis contained multiple datasets, neighbors were calculated based on Harmony or Bbknn for integration. Visualization was performed using UMAP, Force Mapping, and Palantir, and cell clustering was performed using Leiden. Clustering subsets were performed by using the raw data and applying preprocessing techniques again. Differential expression was assessed using the original data and Wilcoxon statistical tests with Benjamin-Hochberg correction. Dot plots were created using a signature gene list on gene-based standard scales and plotted on the processed data using a matplotlib palette. The proportion of cells expressing specific genes was calculated on the original data. The number and proportion of cells in each cluster were plotted using Matplotlib, seaborn, and prism. Trajectory and pseudo-temporal analyses were performed using stream2 and scfates with a master tree structure. A score was calculated for a specific gene set list using scanpy. This score is the average expression of the gene set minus the average expression of the reference gene set (randomly sampled from the sample gene pool). A pre-sorted GSEA analysis was performed on the specific gene set on the differentially expressed gene list using gseapy. Cell comparisons were performed using the KNeighboursClassifier classifier method from scikit-learn (0.20.3). The KNeighboursClassifier classifier was trained on a dataset with PCA subspace projections of cluster annotations using default settings and k = 20. After matching gene symbols of a subset to a highly variable gene list, cell states are predicted and projected into a defined PCA subspace. The results are plotted in a confusion matrix.

[0117] animal

[0118] Male NOD.Cg-Prkdcscid Il2rgtm1Sug / JicTac mice from Taconic Biosciences were used. The research protocol was approved by Brigham and Women's Hospital IACUC / CCM (protocol number 2016N000476). Mouse experiments were conducted in accordance with local regulations (Brigham and Women's Hospital) and national and international guidelines.

[0119] Animal anesthesia and irradiation

[0120] Animals were anesthetized by intraperitoneal injection of ketamine (100 mg / kg, Henry Schein Animal Health) + xylazine (10 mg / kg, Henry Schein Animal Health). Animals were covered with a perforated lead shield, allowing only the hind limbs of mice to be irradiated. Mice were then subjected to a single dose of gamma radiation (18 gray) from a cesium source for 20 min (Gammacell 40 Exactor).

[0121] Cardiac toxin damage

[0122] Perform this procedure to trigger the hind limbs TA The regeneration process in the muscle, 24 hours after irradiation, involves anesthetizing the animal as described above, shaving the injection site and cleaning it with 70% ethanol. Insulin is then administered percutaneously bilaterally using a specialized insulin injector (27G needle). TA Injection is performed. The solution is evenly distributed in... TA 25 μl of cardiotoxin (0.5 mg / μl) was injected into 25 injection sites in the mice. The mice were then examined for recovery after proper anesthesia and injection.

[0123] Intramuscular cell injection

[0124] Twenty-four hours after the cardiotoxin injection, the animal was anesthetized as described above, the injection site was shaved and cleaned with 70% ethanol. Cells were then injected percutaneously using a specialized insulin injector (0.5 x 10⁻⁶). 6 Up to 10 6 The total volume was 20-25 μL (depending on the experimental setup). Cell lines used for cell preparation were tested to be mycoplasma-negative and negative for standard viral contaminants (including HBV, HCV, and HIV from human cell lines). Recovery of mice after appropriate anesthesia was examined.

[0125] Bioluminescence / fluorescence screening

[0126] We used a bioluminescence readout combined with an X-ray scanner (Bruker In Vivo Xtreme imaging system) to detect luciferase-expressing human cells. We achieved a sustained maximum signal exceeding 5000 photons / second / mm in the leg region. 2 Successful transplantation was defined as a signal intensity higher than the background signal detected on the non-transplant side. Animals were anesthetized and injected intraperitoneally with D-fluorescein (150 mg / kg, IVISbrite D-fluorescein) (lower left quadrant). Images were acquired 15 minutes after D-fluorescein injection and bioluminescence imaging was performed using a 30-s exposure and 8x8 pixel binning.

[0127] In-situ strength test

[0128] Lower extremities were measured 4 weeks after cell injection. TA The force generated by the dorsiflexor muscles. Under general anesthesia as described above, the animal's... TA The ligament end is inserted and secured to a rope. Electromyography (EMG) electrodes are placed... TA Internally, the muscles were stimulated by stimulating the peroneal nerve. Using the device program (610A Dynamic Muscle Control LabBook v6, AuroraScientific, Aurora, Ontario, Canada), resting tension was adjusted until the maximum twitching force was generated by a single pulse (pulse width 0.2 ms). The maximum force was measured as the tetanic force generated under this optimized setting, normalized to the animal's body weight (mg).

[0129] Statistics and repeatability

[0130] The legend provides detailed information on the statistical analysis. All experiments were conducted three or more independent trials.

[0131] All patents, patent applications, publications, and electronically available materials cited herein are incorporated herein by reference in their entirety. The foregoing detailed descriptions and examples are provided for clarity of understanding. Unnecessary limitations should not be construed therein. The invention is not limited to the exact details shown and described, as variations apparent to those skilled in the art will be included within the scope of the invention as defined in the claims.

Claims

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

2. The composition according to claim 1, wherein, The human muscle SC also expresses transcription factors PAX3 and / or PAX7 and / or transmembrane receptors FGFR4, CD82, ITGA7, CDH15, CD56 and / or NOTCH3.

3. The composition according to claim 1, wherein, The human muscle SC is derived from a culture of pluripotent stem cells (PSCs) produced by treating precursor mesodermal cells with Wnt activator.

4. The composition according to claim 3, wherein, The PSC-derived culture was produced by treating precursor mesodermal cells with Wnt activator and bone morphogenetic protein (BMP) inhibitor.

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

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

7. The composition according to claim 6, wherein, The SCs were further isolated from the isolated INFRA cells by flow cytometry using antibodies against ITGA7, CD56, NOTCH3, CD82 and / or CDH15.

8. A method for preparing a population of substantially pure INFRA cells, comprising: (a) Producing myogenic cultures derived from pluripotent stem cells on a culture substrate; (b) Remove the upper layer of the myogenic culture, leaving a monolayer of INFRA cells attached to the bottom of the culture medium; (c) Wash the monolayer of INFRA cells with a buffer solution; and (d) Mechanically dissociate the INFRA cells from the cell substrate without causing stress or modification to the cells.

9. The method according to claim 8, wherein, The INFRA cells contain human muscle stem cells (SCs) that express the transcription factor PAX7 and lack transmembrane receptor PDGFRA expression.

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

13. The method according to claim 8, wherein, The buffer solution is phosphate-buffered saline (PBS).

14. A method for 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 comprise human muscle stem cells (SCs) that express the transcription factor PAX7 and lack transmembrane receptor PDGFRA expression, wherein at least 70% of the human muscle SC population is PAX7+ and PDGFRA-.

15. The method according to claim 14, wherein, As a result of administration via injection, the INFRA cells come into contact with the muscle injury.

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

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