Compositions and methods for muscle regeneration using prostaglandin e2

Abstract

To provide effective treatment methods and compositions for regenerating or rejuvenating damaged, impaired, dysfunctional and / or atrophied muscle in a subject in need thereof.SOLUTION: There are provided herein, compositions, methods and kits for proliferating muscle cells by exposing the muscle cells to a prostaglandin E2 (PGE2) compound or a compound that activates PGE2 signaling. Also there are provided methods for regenerating muscles in a subject suffering from muscular atrophy, muscular dystrophy and / or muscular injury by administering a PGE2 compound alone or in combination with isolated muscle cells. The PGE2 compound in combination with the isolated muscle cells can be administered prophylactically to prevent a muscle disease or a disease condition of the muscles.SELECTED DRAWING: None

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 62 / 303,979, filed March 4, 2016, and U.S. Provisional Patent Application No. 62 / 348,116, filed June 9, 2016, the disclosures of which are incorporated by reference in their entireties for all purposes.

[0002] STATEMENT REGARDING RIGHTS TO INVETIONS MADE UNDER FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT This invention was made with Government support under Grant No. AG020961 awarded by the National Institutes of Health. The Government has certain rights in this invention. [Background technology]

[0003] 2. Background of the Invention In skeletal muscle, ageing leads to progressive regeneration failure and loss of muscle mass, strength, and function. Decreasing muscle function is a major public health problem that often leads to severely reduced mobility and poor quality of life in the ever-growing elderly population. A major determinant of muscle function decline is the impaired ability of skeletal muscle stem cells (MuSCs) to regenerate muscle after acute injury or damage during the aging process. There is also a need to enhance muscle regeneration in muscles that have been damaged, injured, and / or atrophied due to, for example, immobilization or disuse after surgery, cachexia in cancer and HIV, muscular dystrophies, acute injury, and aging. Resident MuSCs are rare but essential to maintain and repair muscles, e.g., skeletal, smooth, and cardiac muscles, during adulthood. With age, the number of functional stem cells decreases, thus increasing the need to increase the number and function of MuSCs.

[0004] Prostaglandin E2 (PGE2), also known as dinoprostone, is used in various clinical situations, including inducing labor in women and enhancing hematopoietic stem cell transplantation. PGE2 can be used as an anticoagulant and antithrombotic agent. The role of PGE2 as a lipid mediator that can resolve inflammation is also well known. Nonsteroidal anti-inflammatory drugs (NSAIDs), COX-1 and / or COX-2 inhibitors, suppress inflammation by inhibiting prostanoids mainly via PGE2 biosynthesis.

[0005] PGE2 is synthesized from arachidonic acid by the enzymes cyclooxygenase (COX) and prostaglandin E synthase. PGE2 levels are physiologically regulated by the PGE2 degrading enzyme 15-hydroxyprostaglandin dehydrogenase (15-PGDH). 15-PGDH catalyzes the inactivation of PGE2 15-OH to a 15-keto group.

[0006] There remains a need in the art for effective treatments to regenerate or rejuvenate damaged, impaired, dysfunctional, and / or atrophied muscles in subjects in need thereof. The present invention fulfills this need and provides advantages as well. Summary of the Invention

[0007] BRIEF SUMMARY OF THE INVETION In one aspect of the present invention, a method for promoting the proliferation of isolated muscle stem cell population is provided herein.The method comprises culturing isolated muscle cell population with a compound selected from the group consisting of prostaglandin E2 (PGE2), PGE2 prodrug, PGE2 receptor agonist, compound that attenuates PGE2 catabolism, compound that negates PGE2 inhibition, its derivative, its analog, and combinations thereof.

[0008] In a second aspect of the present invention, provided herein is a composition comprising an isolated muscle cell population and a compound selected from the group consisting of prostaglandin E2 (PGE2), a PGE2 prodrug, a PGE2 receptor agonist, a compound that attenuates PGE2 catabolism, a compound that negates PGE2 inhibition, derivatives thereof, analogs thereof, and combinations thereof.

[0009] In a third aspect of the present invention, provided herein is a kit comprising a composition comprising an isolated muscle cell population and a compound selected from the group consisting of prostaglandin E2 (PGE2), a PGE2 prodrug, a PGE2 receptor agonist, a compound that attenuates PGE2 catabolism, a compound that negates PGE2 inhibition, derivatives thereof, analogs thereof, and combinations thereof, and instructions for use.

[0010] In a fourth aspect, a method is provided herein for regenerating muscle cell mass in a subject with a condition or disease involving muscle damage, injury or atrophy.The method comprises administering to the subject a therapeutically effective amount of a compound selected from the group consisting of prostaglandin E2 (PGE2), PGE2 prodrugs, PGE2 receptor agonists, compounds that attenuate PGE2 catabolism, compounds that negate PGE2 inhibition, derivatives thereof, analogs thereof, and combinations thereof, and a pharma- ceutically acceptable carrier, so as to increase muscle cell mass and / or enhance muscle function in the subject.

[0011] In a fifth aspect, provided herein is a method for preventing or treating a condition or disease that involves muscle damage, injury or atrophy in a subject in need thereof.The method includes administering to the subject (i) a therapeutically effective amount of a compound selected from the group consisting of prostaglandin E2 (PGE2), PGE2 prodrugs, PGE2 receptor agonists, compounds that attenuate PGE2 catabolism, compounds that negate PGE2 inhibition, its derivatives, its analogs, and combinations thereof, and a pharma- ceutically acceptable carrier, and (ii) an isolated muscle cell group, to prevent or treat a condition or disease that involves muscle damage, injury or atrophy.

[0012] Other objects, features and advantages of the present invention will become apparent to those skilled in the art from the following detailed description and drawings. [Brief description of the drawings]

[0013] [Figure 1-1]Figures 1A-1H show that transient PGE2 treatment promotes the proliferation of juvenile MuSCs in vitro. Figure 1A: PGE2 levels after injury (notexin, NTX) of young tibialis anterior (TA) muscles; control is uninjured contralateral TA examined by ELISA; (n=4 mice per time point). Figure 1B: Expression of PGE2 synthesis enzymes (Ptges2 and Ptges) by MuSCs after notexin injury by RT-qPCR, (n=3 mice per time point). Figure 1C: Increase in MuSC numbers after 24 h treatment with vehicle (-) or PGE2 (10 ng / ml) followed by culture on hydrogels up to day 7 (short-term treatment); (n=12 mice, 4 independent experiments). Figure 1D: Increase in MuSC numbers after 24 h treatment with vehicle (-) or PGE2 (10 ng / ml) in the absence or presence of EP4 antagonist (ONO-AE3-208, 1 μM); (n=9 mice, examined in 3 independent experiments). Figure 1E-1G: Proliferation of EP4-deficient MuSCs. EP4f / f (deficient) MuSCs were transduced with a lentiviral vector encoding GFP / luciferase and treated with a lentiviral vector encoding Cre (+Cre) or not (-Cre; empty vector) to delete the EP4 allele. MuSCs were subsequently treated with vehicle (-) or PGE2 (10 ng / ml) for 24 h and cultured on hydrogels for 3 days. Figure 1E: Scheme representing the analysis of EP4-deficient MuSCs. Figure 1F: Number of EP4-deficient MuSCs; (n=6 mice, examined in 2 independent experiments). Figure 1G: Representative images. Scale bar = 40 μm; GFP, green; mCherry, red. Figure 1H: MuSC numbers after culture on hydrogels in charcoal-stripped medium treated with vehicle (-) or PGE2 (10 ng / ml) every 2 days for 7 days; (n = 3 mice, 3 technical replicates). *P < 0.05, **P < 0.001, ***P < 0.0005, ****P < 0.0001. ANOVA test with Bonferroni correction for multiple comparisons (Figures 1A, 1B, 1D, and 1F); paired t test (Figure 1C); Mann-Whitney test (Figure 1H). Mean + standard error of the mean. ns, not significant. [Figure 1-2] See description of Figure 1-1. [Figure 2-1]Figures 2A-2J show the unusual response of aged MuSCs to PGE2. Figure 2A: PGE2 levels after injury (notexin, NTX) of aged TA; control is uninjured contralateral TA examined by ELISA; (n=4 mice per time point). Figure 2B: PGE2 levels in TA of uninjured young (n=7 mice) and old (n=5 mice) mice examined by ELISA. Figure 2C: Scheme showing PGE2 catabolism to the inactive PGE metabolite 13,14-dihydro-15-ketoPGE2 (PGEM) via the degradative enzyme 15-PGDH. Figure 2D: Levels of PGEM quantified by mass spectrometry; (n=4 mice per age group). Figure 2E: Expression of PGE2 degradative enzyme 15-PGDH (Hpgd); (n=3 mice, 2 technical replicates). Figure 2F: Increase in the number of senescent MuSCs after short-term treatment for 24 h with vehicle (-), PGE2 (10 ng / ml), or the 15-PGDH inhibitor SW033291 (1 μM; SW), examined on day 7; (n = 15 mice, 5 independent experiments). Figure 2G: Number of senescent MuSCs after culture on hydrogels in charcoal-stripped medium treated with vehicle (-) or PGE2 (10 ng / ml) every 2 days for 7 days; (n = 3 mice, 3 technical replicates). Figure 2H: Scheme representing the effect of PGE2 on MuSCs. PGE2 acts via the EP4 receptor / cyclic AMP (cAMP) signaling pathway to promote proliferation. In senescent MuSCs, PGE2 catabolism to the inactive form, PGEM, is mediated by 15-PGDH following intracellular transport by PGT (prostaglandin transporter). Figure 2I: Trajectories from clones of senescent MuSCs tracked by time-lapse microscopy in microwells for 48 hours for control (left) and after short-term treatment with PGE2 (right). Trajectories of the original cells and their respective newly born progeny are represented by different colors. Figure 2J: Changes in viable cell number (number) of senescent MuSCs in clones tracked by time-lapse microscopy for control (left, clones n=32) and after short-term treatment with PGE2 (right, clones n=45).The percentage of live cells of each generation (G1-G6) at all time points is shown as cell number normalized to the starting population of 100 single MuSCs. The percentage increase in the number of live cells was 4.0% (control) and 5.4% (PGE2-treated) (upper panel). Change in the number of dead cells (number) of senescent MuSCs in control (left) and after short-term treatment with PGE2 (right) clones followed by time-lapse microscopy. The percentage of dead cells of each generation (G1-G6) at all time points is shown as cell number normalized to the starting population of 100 single MuSCs. The percentage increase in the number of dead cells was 1.0% (control) and 0.1% (PGE2-treated) (lower panel). *P<0.05, **P<0.001, ***P<0.0005. ANOVA test with Bonferroni correction for multiple comparisons (Figures 2A and 2F); Mann-Whitney test (Figures 2B, 2D, 2E, and 2G). Means ± standard error of the mean. ns, not significant. [Figure 2-2] See description of Figure 2-1. [Figure 2-3] See description of Figure 2-1. [Figure 3-1]Figures 3A-3D show that short-term PGE2 treatment promotes MuSC engraftment and regeneration in vivo. Figure 3A: Transplantation of GFP / luc-labeled juvenile MuSCs (250 cells) isolated and cultured from transgenic mice after short-term treatment with vehicle (-) or PGE2 as described in Figure 1C. Transplantation scheme (top). Non-invasive bioluminescence imaging (BLI) signal measured as radiance for each TA; (n=5 mice per condition) (bottom). Figure 3B: Transplantation of GFP / luc-labeled EP4f / f MuSCs (1,000 cells) treated with Cre (+Cre) or without Cre (-Cre; empty vector) to delete the EP4 allele during culture. For BLI, EP4f / f MuSCs were transduced with a lentiviral vector encoding GFP / luciferase. Transplantation scheme (top). BLI signal after transplantation (n=5 mice per condition) (bottom). Figure 3C: Transplantation of freshly sorted GFP / luc-labeled young MuSCs (250 cells) co-injected with vehicle (-) or dmPGE2. Transplantation scheme (top). BLI signal after transplantation (n=4 and n=5 mice for vehicle and dmPGE2 treatment, respectively). Figure 3D: Transplantation of GFP / luc-labeled senescent MuSCs (250 cells) co-injected with vehicle (-) or dmPGE2; (n=3 mice per condition) (bottom). For BLI, senescent MuSCs were transduced with a lentiviral vector encoding GFP / luciferase. Transplantation scheme (top). BLI signal after transplantation expressed as mean radiance (ps-1 cm-2 sr-1). Representative BLI images for each condition. Scale bar = 5 mm (Figures 3A-3D). Data are representative of two independent experiments. *P<0.05, **P<0.001, and ***P<0.0005. Significant differences in endpoints by ANOVA test and Fisher's test for group comparison. Mean + standard error of the mean. [Figure 3-2] See description of Figure 3-1. [Figure 4-1]Figures 4A-4P show that intramuscular injection of PGE2 alone promotes MuSC expansion, improves regeneration, and increases force. Young: (Figures 4A-4D) TA muscles of young mice were injected with vehicle (-) or dmPGE2 at 48 hours after cardiotoxin (CTX) injury; (n=3 mice per condition). Figure 4A: Scheme of experimental procedure (top). Representative TA cross sections (bottom) stained for nuclei (DAPI; blue), laminin (green), and PAX7 (red) at 14 days after cardiotoxin injury. Arrowheads indicate PAX7+ MuSCs. Scale bar=40 μm. Figure 4B: Increase in endogenous MuSCs based on immunofluorescence of PAX7-expressing satellite cells per 100 fibers in TA cross sections of young mice. Figure 4C: Myofiber cross-sectional area (CSA) of vehicle (-, open white bars) and dmPGE2-treated (solid blue bars) young TAs quantified using the Baxter algorithm for myofiber analysis. Figure 4D: Distribution of small (<1,000 μm2 CSA) and large (>1,000 μm2 CSA) myofibers. (Figures 4E-4G) Increase in endogenous MuSCs examined based on Pax7-luciferase. Pax7CreERT2;Rosa26-LSL-Luc mice were treated intraperitoneally with tamoxifen (TAM) and TAs were subjected to cardiotoxin (CTX) injury and 3 days later injected with vehicle (-) or dmPGE2 and monitored based on BLI; (n=3 mice per condition). Figure 4E: Scheme of the experimental procedure. Figure 4F: BLI (n=3 mice per condition). Figure 4G: Representative BLI images. Scale bar = 5 mm. Aged: (Fig. 4H-4K) TAs from aged mice were treated in vivo with vehicle (-) or dmPGE2 treatment (n = 3 mice per condition) 48 hours after cardiotoxin (CTX) injury. Fig. 4H: Scheme of experimental procedure (top). Representative TA cross-sections (bottom) stained for nuclei (DAPI; blue), laminin (green), and PAX7 (red) 14 days after cardiotoxin injury. Arrowheads indicate PAX7+ muscle stem cells. Scale bar = 40 μm. Fig. 4I: Increase in endogenous MuSCs as in Fig. 4B for aged mice. Fig. 4J: Myofiber cross-sectional area (CSA) as in Fig. 4C for aged TA. Fig. 4K: Distribution of CSA as in Fig. 4D for aged TA.(Figure 4L-4P) Increased force in aged mice, measured in vivo as muscle contraction force after downhill treadmill running. Mice were subjected to 20° downhill treadmill running for 2 consecutive weeks and force was examined at week 5. During the first week, the medial gastrocnemius (GA) and lateral gastrocnemius (GA) of aged mice were injected with either vehicle (-) or dmPGE2. Biological replicates were n=10 or 8 for vehicle (-) or dmPGE2 treatment, respectively, and technical replicates were 5 each. Figure 4L: Experimental scheme. Representative twitch force (Figure 4M) and tetanic force (Figure 4N). Muscle twitch force per unit (Figure 4O) and muscle tetanic force per unit (Figure 4P) were calculated by normalizing force to physiological cross-sectional area (PCSA). Paired t-test (Figures 4B, 4D, 4I, and 4K); ANOVA test for group comparison and Fisher's test for significant differences in endpoints (Figure 4F); Mann-Whitney test (Figures 4O and 4P). *P<0.05, **P<0.001, and ****P<0.0001. Means + standard error of the mean. [Figure 4-2] See description of Figure 4-1. [Figure 4-3] See description of Figure 4-1. [Figure 5-1]Figures 5A-5K show that PGE2 promotes MuSC expansion. Figure 5A: PGE2 levels 3 days after cold injury in hindlimb anterior tibial (TA) muscles of young mice compared to contralateral uninjured controls examined by ELISA; (n=4 mice per condition and time point). Figure 5B: Representative images of dividing muscle stem cells (MuSCs) labeled with EdU (red) for 1 hour and stained for myogenin (green) after treatment with PGE2 (10 ng / ml) for 24 hours (days 0-1) or vehicle (-). Scale bars correspond to 40 μm. Figure 5C: Proportion of dividing MuSCs labeled with EdU as in (b); (n=6 mice, 3 technical replicates in 2 independent experiments). FIG. 5D: Increased proliferation measured by metabolic viability assay VisionBlue after treatment with vehicle (-) or the indicated doses of PGE2 (1-200 ng / ml); (n=6 mice, 3 technical replicates in 2 independent experiments). FIG. 5E: Expression of prostaglandin receptors (Ptger1-4) by MuSCs after treatment with vehicle (-) or PGE2 for 24 h; (n=3 mice, 2 technical replicates). FIG. 5F: Increased cAMP levels in MuSCs after treatment with PGE2 for 1 h compared to the untreated group (-); (n=6 mice, 3 technical replicates examined in 2 independent experiments). FIG. 5G-5H: Expression of Pax7 (FIG. 5G) and myogenin (FIG. 5H) by MuSCs after treatment with vehicle (-) or PGE2 for 24 h; (n=3 mice, 2 technical replicates). Figures 5I-5J: EP4f / f MuSCs were transduced with a lentiviral vector encoding GFP / luciferase and treated with a lentiviral vector encoding Cre (+Cre) or not (-Cre; empty vector) to delete the EP4 allele. Bar graphs show the percentage of +Cre MuSCs (Figure 5I) and GFP / Luc+ MuSCs (Figure 5J). Figure 5K: Representative images of MuSCs in hydrogel cultures after 7 days in myoblast medium containing vehicle (-) or charcoal-stripped fetal bovine supplemented with PGE2 (10 ng / ml) every 2 days. Scale bars correspond to 40 μm. *P<0.05, **P<0.001, ***P<0.0005.Paired t-test (Figures 5A, 5E, 5G, and 5H); Mann-Whitney test (Figure 5C). Means + standard error of the mean. ns, not significant. [Figure 5-2] See description of Figure 5-1. [Figure 6A] Figures 6A-6C show mass spectrometry analysis of young and aged muscles to detect prostaglandins and PGE2 metabolites. Figure 6A: Chemical structures, formulas, exact masses, and molecular weights of the analyzed prostaglandins (PGE2, PGF2α, and PGD2) and PGE2 metabolites (15-keto PGE2 and 13,14-dihydro-15-keto PGE2). Both composite standards were spiked with internal standards PGF2α-D9 and PGE2-D9. [Figure 6B] Figures 6A-6C show mass spectrometry analysis of young and aged muscles to detect prostaglandins and PGE2 metabolites. Figure 6B: Calibration curves for liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS / MS) analysis were generated by diluting the stock solutions to final concentrations of 0.1 ng / ml to 500 ng / ml. The equations and correlation coefficients of the calibration curves are shown for each standard. [Figure 6C] Figures 6A-6C show mass spectrometric analysis of young and aged muscles to detect prostaglandins and PGE2 metabolites. Figure 6C: Representative chromatogram. Separate peaks indicate excellent chromatographic resolution for the analyzed prostaglandins and their metabolites. cps, counts per second. [Figure 7-1]Figures 7A-7G show that proliferation and cell viability of senescent MuSCs are increased in response to PGE2 treatment. Figures 7A-7C: mRNA levels measured by qRT-PCR were normalized to Gapdh for young and senescent MuSCs (n=3 mice, 2 technical replicates). Figure 7A: Prostaglandin transporter (PGT) encoded by Slco2a1 gene. Figure 7B: PGE2 synthesis enzymes Ptges and Ptges2. Figure 7C: EP1-4 receptors encoded by genes Ptger1-4. Figure 7D: Pax7 mRNA levels in MuSCs after 24 h treatment with vehicle (-) or PGE2 treatment; (n=3 mice, 2 technical replicates). Figure 7E: Senescent MuSC single clones followed by time-lapse microscopy after short-term treatment with vehicle (-, top) and PGE2 (bottom). For each clone, the resulting number of live (open bars) and dead (black bars) cells after 48 h of time-lapse tracking is shown. Figure 7F: Proliferation curves of tracked live senescent MuSCs assessed by time-lapse microscopy for 48 h of vehicle (-) or transient PGE2 treatment. Figure 7G: Flow cytometric analysis of apoptotic Annexin V+ in senescent MuSCs analyzed 7 days after growth on hydrogels after 24 h of treatment with vehicle (-) or PGE2; (n=9 mice, 3 independent experiments). Mann-Whitney test (Figures 7A-7D) and paired t-test at α=0.05 (Figure 7G). Means + standard error of the mean. ns, not significant. [Figure 7-2] See description of Figure 7-1. [Figure 8] Figures 8A-8B show the Baxter algorithm for myofiber analysis of muscle cross-sectional area. Figure 8A: Representative cross-sectional images of tibialis anterior muscle fibers from young mice treated in vivo with vehicle (-) or PGE2 at 48 hours after cardiotoxin (CTX) injury. Images show laminin (green) and DAPI (blue) staining. Figure 8B: Segmented images corresponding to Figure 8A analyzed by the Baxter algorithm for myofiber analysis to measure (bottom) the cross-sectional area (CSA) of myofibers at 14 days after injury. Scale bar corresponds to 40 µm. [Figure 9-1] Figures 9A-9G show that deletion of the PGE2 receptor EP4 in MuSCs reduces skeletal muscle regeneration and force after injury. Tibialis anterior (TA) muscles of tamoxifen-treated Pax7-specific EP4 conditional knockout mice (Pax7CreERT2; EP4fl / fl) were examined at days 6 (Figures 9C-9D), 21 (Figures 9B and 9E), and 14 (Figures 9F and 9G) after notexin injury; (n=3 mice per condition). Figure 9A: Experimental scheme. Figure 9B: Expression of Ptger4 (EP4 receptor) in sorted MuSCs (α7+ CD34+ lin-) derived from control or EP4 KO mice after injury. Figure 9C: Representative TA cross-sections. DAPI, blue; embryonic myosin heavy chain (eMyHC), red. Scale bar=40 μm. Figure 9D: Proportion of eMyHC+ fibers. Figure 9E: Muscle fiber cross-sectional area (CSA) of control and Pax7-specific EP4 knockout TAs. Figure 9F: Muscle twitch force and (Figure 9G) muscle tetanic force 14 days after notexin injury. Significant differences in each category by Mann-Whitney test (Figures 9B, 9C, 9F, and 9G); ANOVA test for group comparison and Fisher's test (Figure 9E). *P<0.05, ***P<0.0005, and ****P<0.0001. Means + standard error of the mean. [Figure 9-2] See description of Figure 9-1. [Figure 10]Figures 10A-10C show that blocking endogenous PGE2 signaling in muscle at early time points of regeneration reduces regeneration and force. Endogenous MuSCs were examined in Pax7CreERT2;Rosa26-LSL-Luc mice treated with tamoxifen (TAM) based on non-invasive bioluminescence imaging (BLI) after injection of vehicle (-) or NSAID (indomethacin) following cardiotoxin injury to the tibialis anterior (TA); (n=3 mice per condition). Figure 10A: Experimental scheme. Figure 10B: BLI (n=3 mice per condition). Figure 10C: Muscle twitch force 14 days after notexin injury (n=8 vehicle-treated, n=10 NSAID-treated). ANOVA test for group comparison and significant differences in endpoints by Fisher's test (Figure 10B). Mann-Whitney test (Figure 10C). *P<0.05, **P<0.001, ***P<0.0005, and ****P<0.0001. Mean + standard error of the mean. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] Detailed Description of the Invention I. Introduction The present invention is based in part on the discovery that prostaglandin E2 (PGE2) can improve muscle cell proliferation and function. PGE2 can be used alone or in combination with isolated muscle cells to repair muscle damage caused by injury, atrophy, or disease. Indeed, muscle cells treated with PGE2 show enhanced muscle regeneration and improved muscle function when administered. Thus, novel therapeutic methods, compositions, and kits are provided herein for promoting muscle regeneration and rejuvenation of damaged, injured, or atrophied muscles.

[0015] II. Overview The practice of the present invention employs conventional techniques in the field of molecular biology. Essential reference texts that disclose the general methods useful in the present invention include Sambrook and Russell, Molecular Cloning, A Laboratory Manual (3rd ed. 2001); Kriegler, Gene Transfer and Expression: A Laboratory Manual (1990); and Current Protocols in Molecular Biology (Ausubel et al., eds., 1994).

[0016] For nucleic acids, sizes are expressed in either kilobases (kb), base pairs (bp), or nucleotides (nt). Sizes of single-stranded DNA and / or RNA can be expressed in nucleotides. These are estimates derived from agarose or acrylamide gel electrophoresis, sequenced nucleic acids, or published DNA sequences. For proteins, sizes are expressed in kilodaltons (kDa) or number of amino acid residues. Protein sizes are estimated from gel electrophoresis, sequenced proteins, derived amino acid sequences, or published protein sequences.

[0017] Non-commercially available oligonucleotides can be chemically synthesized, for example, using an automated synthesizer as described in Van Devanter et. al., Nucleic Acids Res. 12:6159-6168 (1984) according to the solid-phase phosphoramidite triester method first described by Beaucage and Caruthers, Tetrahedron Lett. 22: 1859-1862 (1981). Purification of oligonucleotides is performed using any art-recognized strategy, for example, native acrylamide gel electrophoresis or anion-exchange high performance liquid chromatography (HPLC) as described in Pearson and Reanier, J. Chrom. 255:137-149 (1983).

[0018] III Definition As used herein, the following terms have the meanings ascribed to them unless specified otherwise.

[0019] As used herein, the terms "a," "an," or "the" include aspects including one member as well as aspects including multiple members. For example, the singular forms "a," "an," and "the" include multiple referents unless the context clearly dictates otherwise. Thus, for example, reference to "a cell" includes a plurality of such cells, reference to "the agent" includes reference to one or more agents known to those of skill in the art, and so forth.

[0020] The term "prostaglandin E2" or "PGE2" refers to a prostaglandin that can be synthesized from arachidonic acid by cyclooxygenase (COX) enzymes and terminal prostaglandin E synthase (PGES). PGE2 plays a role in several biological functions, including vasodilation, inflammation, and regulation of the sleep-wake cycle.

[0021] The term "prostaglandin E2 receptor agonist" or "PGE2 receptor agonist" refers to a chemical compound, small molecule, polypeptide, biological product, etc., that is capable of binding to and activating any PGE2 receptor, thereby stimulating the PGE2 signaling pathway.

[0022] The term "compound that attenuates PGE2 catabolism" refers to a chemical compound, small molecule, polypeptide, biological product, etc. that is capable of reducing or decreasing the breakdown of PGE2.

[0023] The term "compound that negates PGE2 inhibition" refers to a chemical compound, small molecule, polypeptide, biological product, etc., that can interfere with or impede inhibitors of the synthesis, activity, secretion, and function of PGE2.

[0024] The term "derivative" in the context of a compound includes, but is not limited to, amide, ether, ester, amino, carboxyl, acetyl, and / or alcohol derivatives of a given compound.

[0025] The term "embryonic stem cell-derived muscle cells" or "ESC-derived muscle cells" refers to muscle cells that are derived from or differentiated from embryonic stem cells.

[0026] The term "induced pluripotent stem cell-derived muscle cells" or "iPSC-derived muscle cells" refers to muscle cells derived from or differentiated from induced pluripotent stem cells.

[0027] The term "isolated" in the context of cells refers to a single cell of interest or a group of cells of interest that is at least partially isolated and / or purified from other cell types or other cellular materials that naturally coexist with the cell in the tissue from which it is derived (e.g., muscle tissue). A group of muscle cells is "isolated" if it is at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, and in some cases at least about 99% free of cells that are not muscle cells. Purity can be measured by any suitable method, for example, by fluorescence-activated cell sorting.

[0028] The term "autologous" refers to any material (eg, cells) derived from an individual that is to be subsequently reintroduced into that same individual.

[0029] The term "allogeneic" refers to any material (e.g., cells) derived from a different animal of the same species as the individual to whom the material is introduced. Two or more individuals are said to be allogeneic to one another if the genes at one or more loci are not identical. In some aspects, allogeneic material derived from individuals of the same species may be sufficiently genetically distinct to interact antigenically.

[0030] The term "treating" or "treatment" means any one of the following: ameliorating one or more symptoms of the disease; preventing the onset of such symptoms before they occur; slowing down or completely preventing the progression of the disease (which may be manifested by a longer period between recurrent episodes, slowing down or preventing the worsening of symptoms, etc.); promoting the onset of a period of remission; slowing down the irreversible damage caused during the progression-chronic phase of the disease (both primary and secondary stages); delaying the onset of said progression phase; or any combination thereof.

[0031] The terms "administer", "administering" or "administration" refer to methods that can be used to allow delivery of agents or compositions, such as the compounds and cells described herein, to a desired biological site of action. These methods include, but are not limited to, parenteral administration (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular, intraarterial, intravascular, intracardiac, intrathecal, intranasal, intradermal, and intravitreal), transmucosal injection, oral administration, administration as a suppository, and topical administration. Those skilled in the art will know other methods for administering a therapeutically effective amount of the compounds and / or cells described herein to prevent or alleviate one or more symptoms associated with a disease or condition.

[0032] The term "therapeutically effective amount" or "therapeutically effective dose" or "effective amount" refers to an amount of a compound, therapeutic agent (e.g., cells), and / or pharmaceutical agent that is sufficient to produce a beneficial or desired clinical effect. The therapeutically effective amount or therapeutically effective dose may be based on factors specific to each patient, including, but not limited to, the patient's age, size, type or extent of disease, stage of disease, route of administration of regenerative cells, type or extent of adjunctive therapy used, ongoing disease process, and type of treatment desired (e.g., active or conventional treatment). Therapeutically effective amounts of pharmaceutical compounds or compositions described herein can be estimated initially from cell culture and animal models. For example, IC measured by cell culture methods. 50 The IC values ​​can serve as a starting point in animal models and are consistent with IC values ​​measured in animal models. 50 The values ​​can be used to find a therapeutically effective dose in humans.

[0033] The term "pharmaceutically acceptable carrier" means a carrier or diluent that does not cause significant irritation to an organism and does not destroy the biological activity and properties of the compound with which it is administered.

[0034] The terms "subject," "individual," and "patient" are used interchangeably herein to refer to a vertebrate, preferably a mammal, more preferably a human. Mammals include, but are not limited to, mice, rats, monkeys, humans, farm animals, sport animals, and pets.

[0035] The term "short-term exposure" in the context of administration of a compound refers to a temporary or brief application of a compound to a subject, e.g., a human subject or cell. In some embodiments, short-term exposure includes a single administration of a compound during a treatment period or chronically.

[0036] The term "intermittent exposure" in the context of administration of a compound means repeated application of a compound to a subject, eg, a human subject or cell, wherein a desired period of time elapses between each application.

[0037] The term "short-term treatment regimen" in the context of administering a compound refers to the temporary or short-term application of a compound to a subject, e.g., a human subject, or the repeated application of a compound to a subject, e.g., a human subject, with a desired period of time (e.g., one day) between each application.In some embodiments, a short-term treatment regimen includes short-term exposure of a compound to a subject (e.g., a single administration) during the treatment period or during a long period.In other embodiments, a short-term treatment regimen includes intermittent exposure of a compound to a subject (e.g., repeated administration), with a desired period of time between each exposure.

[0038] The term "continuous exposure" in the context of administration of a compound means chronic repeated application of a compound to a subject, eg, a human subject or cell, over an extended period of time.

[0039] The term "chronic treatment regimen" in the context of administration of a compound refers to the chronic repeated application of a compound to a subject, e.g., a human subject, over an extended period of time such that the amount or level of the compound remains substantially constant over a selected period of time. In some embodiments, a chronic treatment regimen involves continuous exposure of a compound to a subject over an extended period of time.

[0040] IV. DETAILED DESCRIPTION OF EMBODIMENTS In one aspect, a method is provided herein for promoting the proliferation, expansion, and / or transplantation of isolated muscle cells by culturing the isolated muscle cells with a compound selected from the group consisting of prostaglandin E2 (PGE2), PGE2 prodrugs, PGE2 receptor agonists, compounds that attenuate PGE2 catabolism, compounds that negate PGE2 inhibition, derivatives thereof, analogs thereof, and combinations thereof.In some embodiments, the isolated muscle cells are substantially purified or purified (e.g., separated from non-muscle cells or other cells that are not of interest).In some examples, the isolated muscle cells include skeletal muscle cells, smooth muscle cells, cardiomyocytes, embryonic stem cell-derived muscle cells, induced pluripotent stem cell-derived muscle cells, dedifferentiated muscle cells, or combinations thereof.In certain embodiments, the isolated muscle cells include muscle stem cells, satellite cells, myocytes, myoblasts, myotubes, myofibers, or combinations thereof.

[0041] The isolated muscle cells can be obtained from a subject. In other embodiments, the isolated muscle cells are derived from a cell line, such as a primary cell line. In some instances, the subject has a condition or disease involving muscle damage, injury, or atrophy. The condition or disease involving muscle damage, injury, or atrophy may be selected from the group consisting of acute muscle injury, tear, or trauma, soft tissue injury of the hand, Duchenne muscular dystrophy (DMD), Becker muscular dystrophy, limb-girdle muscular dystrophy, amyotrophic lateral sclerosis (ALS), distal muscular dystrophy (DD), hereditary myopathy, myotonic muscular dystrophy (MDD), mitochondrial myopathy, myotubular myopathy (MM), myasthenia gravis (MG), congestive heart failure, periodic paralysis, polymyositis, rhabdomyolysis, dermatomyositis, cancer cachexia, AIDS cachexia, cardiac cachexia, stress-induced urinary incontinence, and sarcopenia.

[0042] In some embodiments, the PGE2 derivative comprises 16,16-dimethyl prostaglandin E2. In other embodiments, the compound that attenuates PGE2 catabolism comprises a compound, neutralizing peptide, or neutralizing antibody that inactivates or interferes with 15-hydroxyprostaglandin dehydrogenase (15-PGDH) or inactivates or interferes with the prostaglandin transporter (PTG or SLCO2A1) that transports PGE2 into the cell for catabolism by 15-PGDH.

[0043] In some embodiments, culturing the isolated muscle cells with a compound comprises exposing the isolated muscle cells to the compound short-term, intermittently, or continuously. The compound may be exposed to the isolated cells once for a short period of time. In other cases, the compound may be exposed to the isolated cells multiple times such that time elapses between each exposure. In yet other cases, the compound may be exposed to the isolated cells continuously such that the level of the compound in direct contact with the cells does not fall below a preselected amount.

[0044] In certain embodiments, the present application provides a method for promoting muscle cell transplantation in subject.The method includes culturing or contacting isolated muscle cell groups with an effective amount of a compound selected from the group consisting of prostaglandin E2 (PGE2), PGE2 prodrug, PGE2 receptor agonist, compound that attenuates PGE2 catabolism, compound that negates PGE2 inhibition, its derivative, its analog, and combinations thereof, to promote muscle cell transplantation in subject, and administering the cultured or contacted muscle cell to subject.

[0045] In another aspect, provided herein is a composition comprising an isolated muscle cell population and a compound selected from the group consisting of prostaglandin E2 (PGE2), PGE2 prodrugs, PGE2 receptor agonists, compounds that attenuate PGE2 catabolism, compounds that negate PGE2 inhibition, derivatives thereof, analogs thereof, and combinations thereof.In some embodiments, the isolated muscle cell population comprises skeletal muscle cells, smooth muscle cells, cardiomyocytes, embryonic stem cell-derived muscle cells, induced pluripotent stem cell-derived muscle cells, dedifferentiated muscle cells, or combinations thereof.In some examples, the isolated muscle cell population comprises muscle stem cells, satellite cells, myocytes, myoblasts, myotubes, myofibers, or combinations thereof.The composition may also comprise a pharmaceutically acceptable carrier.

[0046] In yet another aspect, provided herein is a kit comprising any of the compositions disclosed herein and instructions.

[0047] In another aspect, the present specification provides a method for regenerating muscle cell mass in a subject with a condition or disease that involves muscle damage, injury or atrophy.The method includes administering to the subject a therapeutically effective amount of a compound selected from the group consisting of prostaglandin E2 (PGE2), PGE2 prodrug, PGE2 receptor agonist, compound that attenuates PGE2 catabolism, compound that negates PGE2 inhibition, its derivative, its analog, and combinations thereof, and a pharma- ceutically acceptable carrier, so as to increase muscle cell mass in the subject and / or enhance muscle function in the subject.

[0048] In a related aspect, a method is provided herein for promoting the proliferation and / or increase of muscle cell population in a subject having a condition or disease involving muscle damage, injury, or atrophy.The method comprises administering to the subject a therapeutically effective amount of a compound selected from the group consisting of prostaglandin E2 (PGE2), a PGE2 prodrug, a PGE2 receptor agonist, a compound that attenuates PGE2 catabolism, a compound that negates PGE2 inhibition, its derivatives, its analogs, and combinations thereof, and a pharma- ceutically acceptable carrier to increase muscle cell population in the subject and / or enhance muscle function in the subject.

[0049] In some embodiments, the muscle cells include endogenous muscle cells. In other embodiments, the muscle cells include isolated muscle cells that have been administered (e.g., injected or implanted) to the subject. In yet other embodiments, the muscle cells include both endogenous muscle cells and isolated muscle cells that have been administered to the subject.

[0050] In some embodiments, the condition or disease involving muscle damage, injury, or atrophy is selected from the group consisting of acute muscle injury or trauma, soft tissue injury of the hand, Duchenne muscular dystrophy (DMD), Becker muscular dystrophy, limb-girdle muscular dystrophy, amyotrophic lateral sclerosis (ALS), distal muscular dystrophy (DD), hereditary myopathy, myotonic muscular dystrophy (MDD), mitochondrial myopathy, myotubular myopathy (MM), myasthenia gravis (MG), congestive heart failure, periodic paralysis, polymyositis, rhabdomyolysis, dermatomyositis, cancer cachexia, AIDS cachexia, cardiac cachexia, stress-induced urinary incontinence, and sarcopenia.

[0051] In some embodiments, the muscle cells include skeletal muscle cells, smooth muscle cells, cardiomyocytes, embryonic stem cell-derived muscle cells, induced pluripotent stem cell-derived muscle cells, dedifferentiated muscle cells, or combinations thereof. In some cases, the muscle cells include muscle stem cells, satellite cells, myocytes, myoblasts, myotubes, myofibers, or combinations thereof.

[0052] In some embodiments, the PGE2 derivative comprises 16,16-dimethyl prostaglandin E2.

[0053] In some embodiments, compounds that attenuate PGE2 catabolism include compounds, neutralizing peptides, or neutralizing antibodies that inactivate or interfere with 15-hydroxyprostaglandin dehydrogenase (15-PGDH) or inactivate or interfere with prostaglandin transporters (PTG or SLCO2A1).

[0054] In some embodiments, administering the compound comprises oral, intraperitoneal, intramuscular, intraarterial, intradermal, subcutaneous, intravenous, or intracardiac administration. In some cases, the compound is administered according to a short-term treatment regimen. In some examples, the short-term treatment regimen comprises short-term exposure (e.g., single administration) of the compound to the subject. In other examples, the short-term treatment regimen comprises intermittent exposure (e.g., repeated administration) of the compound to the subject. As a non-limiting example, the short-term PGE2 treatment regimen may comprise a series of intermittent (e.g., daily) PGE2 administrations over a desired period (e.g., over a period of 2, 3, 4, 5, 6, or 7 days).

[0055] In other embodiments, the administering step further comprises administering isolated muscle cells to the subject. The isolated muscle cells may be autologous to the subject. The isolated muscle cells may be allogeneic to the subject. In some examples, the isolated muscle cells are substantially purified or have been purified. In other examples, the isolated muscle cells are cultured with the compound prior to administration to the subject. Culturing the isolated muscle cells with the compound may include exposing the isolated muscle cells to the compound acutely, intermittently, or continuously. Administering the isolated muscle cells may include injecting or implanting the cells into the subject. The isolated muscle cells and the compound may be administered to the subject simultaneously. Alternatively, the isolated muscle cells and the compound may be administered to the subject sequentially.

[0056] In another aspect, provided herein is a method for preventing or treating the condition or disease that involves muscle damage, injury or atrophy in a subject that requires it.The method includes: (i) administering to the subject a therapeutically effective amount of a compound selected from the group consisting of prostaglandin E2 (PGE2), PGE2 prodrug, PGE2 receptor agonist, compound that attenuates PGE2 catabolism, compound that negates PGE2 inhibition, its derivative, its analog, and combinations thereof, and a pharma- ceutically acceptable carrier, and (ii) administering to the subject a group of isolated muscle cells, to prevent or treat the condition or disease that involves muscle damage, injury, or atrophy.

[0057] In a related aspect, provided herein is a method for promoting the proliferation and / or augmentation of muscle cells in a subject having a condition or disease involving muscle damage, injury, or atrophy by administering to the subject (i) a therapeutically effective amount of a compound selected from the group consisting of prostaglandin E2 (PGE2), a PGE2 prodrug, a PGE2 receptor agonist, a compound that attenuates PGE2 catabolism, a compound that negates PGE2 inhibition, its derivatives, its analogs, and combinations thereof, and a pharma- ceutically acceptable carrier, and (ii) an isolated muscle cell population. In some embodiments, the muscle cell population comprises endogenous muscle cells. In other embodiments, the muscle cell population comprises isolated muscle cells administered (e.g., injected or implanted) to the subject. In yet another embodiment, the muscle cell population comprises both endogenous muscle cells and isolated muscle cells administered to the subject. In certain embodiments, a therapeutically effective amount of a compound includes an amount that is sufficient to increase endogenous muscle cells in a subject and / or to increase isolated muscle cells administered to a subject and / or to enhance muscle function in a subject.

[0058] In some embodiments, the PGE2 derivative comprises 16,16-dimethyl prostaglandin E2. In some examples, the compound that attenuates PGE2 catabolism comprises a compound, a neutralizing peptide, or a neutralizing antibody that inactivates or interferes with 15-hydroxyprostaglandin dehydrogenase (15-PGDH) or inactivates or interferes with prostaglandin transporter (PTG or SLCO2A1).

[0059] In some embodiments, the muscle cells include skeletal muscle cells, smooth muscle cells, cardiomyocytes, embryonic stem cell-derived muscle cells, induced pluripotent stem cell-derived muscle cells, dedifferentiated muscle cells, or combinations thereof. In some cases, the muscle cells include muscle stem cells, satellite cells, myocytes, myoblasts, myotubes, myofibers, or combinations thereof. The isolated muscle cells may be substantially purified or purified.

[0060] In some embodiments, the isolated muscle cells are cultured with a compound prior to administration to the subject. In some cases, culturing the isolated muscle cells with a compound comprises acutely, intermittently, or continuously exposing the isolated muscle cells to the compound.

[0061] In some embodiments, the isolated muscle cells are autologous to the subject. In other embodiments, the isolated muscle cells are allogeneic to the subject.

[0062] The administration of the compound may be oral, intraperitoneal, intramuscular, intraarterial, intradermal, subcutaneous, intravenous, or intracardiac. In some cases, the compound is administered according to a short-term treatment regimen. In some examples, the short-term treatment regimen includes short-term exposure (e.g., single administration) of the compound to the subject. In other examples, the short-term treatment regimen includes intermittent exposure (e.g., repeated administration) of the compound to the subject. As a non-limiting example, the short-term PGE2 treatment regimen may include a series of intermittent (e.g., daily) administrations of PGE2 over a desired period (e.g., over a period of 2, 3, 4, 5, 6, or 7 days). The administration of the isolated muscle cells may include injecting or transplanting the cells into the subject. The compound and the isolated muscle cells may be administered to the subject simultaneously. Optionally, the compound and the isolated muscle cells may be administered to the subject sequentially.

[0063] In some embodiments, the subject is suspected to have or is at risk of developing a condition or disease that involves muscle damage, injury or atrophy.In some cases, the condition or disease that involves muscle damage, injury or atrophy is selected from the group consisting of acute muscle injury or trauma, soft tissue injury of the hand, Duchenne muscular dystrophy (DMD), Becker muscular dystrophy, limb-girdle muscular dystrophy, amyotrophic lateral sclerosis (ALS), distal muscular dystrophy (DD), hereditary myopathy, myotonic muscular dystrophy (MDD), mitochondrial myopathy, myotubular myopathy (MM), myasthenia gravis (MG), congestive heart failure, periodic paralysis, polymyositis, rhabdomyolysis, dermatomyositis, cancer cachexia, AIDS cachexia, cardiac cachexia, stress-induced urinary incontinence, and sarcopenia.

[0064] A. Methods for promoting muscle cell proliferation or transplantation Provided herein are in vitro or ex vivo methods for promoting or facilitating the proliferation and / or engraftment of isolated muscle cells.These methods include culturing or contacting isolated muscle cells with prostaglandin E2 (PGE2), PGE2 prodrugs, PGE2 receptor agonists, compounds that attenuate PGE2 catabolism, compounds that negate PGE2 inhibition, derivatives thereof, analogs thereof, or combinations thereof.The compounds may be added to any medium used to maintain or grow cells.

[0065] The compound may be any small molecule, prodrug, biological product, etc. that can mimic, activate, or promote PGE2 signaling. In some cases, the compound is PGE2 (i.e., dinoprostone), synthetic PGE2 derivative (e.g., 16,16-dimethyl prostaglandin E2; dmPGE2), synthetic PGE2 analog, synthetic PGE2 mutant, or muscle-specific PGE2 mutant. In other cases, the compound is a PGE2 prodrug, e.g., a PGE2 prodrug that can be metabolized to a pharmacologically active PGE2 drug when exposed to muscle cells or in close proximity to muscle cells. In yet another case, the compound may be an agonist of any one of the PGE2 receptors, including PGE2 receptor 1, PGE2 receptor 2, PGE2 receptor 3, and PGE2 receptor 4. The agonist can specifically bind to or activate one or more PGE2 receptors. In some cases, the compound may be a compound that attenuates, interferes with, inhibits, or reduces PGE2 catabolism, such as a compound that inactivates or interferes with an enzyme that breaks down or metabolizes PGE2, e.g., 15-hydroxyprostaglandin dehydrogenase (15-PGDH), or a neutralizing (blocking) antibody. In other cases, the compound interferes with, interferes with, or counteracts the inhibition of PGE2 and / or PGE2 synthesis, activity, and / or secretion.

[0066] In some embodiments, the compounds described herein can initiate proliferation of muscle cells, including quiescent muscle cells. The isolated muscle cell population can be a pure or substantially pure population of muscle cells, such that at least about 90% of the muscle cells are a single type of muscle cell. In other embodiments, the population is a mixture of muscle cells, such that less than about 90% of the cells are one type of cell. In some cases, the muscle cells include skeletal muscle cells, smooth muscle cells, and / or cardiomyocytes taken from a subject. In other cases, the muscle cells arise from or are differentiated from embryonic stem cells, such as human embryonic stem cells, or induced pluripotent stem cells, such as human induced pluripotent stem cells. In still other cases, the muscle cells are dedifferentiated muscle cells. In some embodiments, the isolated muscle cell population includes muscle stem cells, satellite cells, myocytes, myoblasts, myotubes, myofibers, or combinations thereof. For example, the isolated muscle cells can be a pure or substantially pure population of muscle stem cells. Alternatively, the isolated muscle cells can be a pure or substantially pure population of satellite cells. In other cases, the isolated muscle cells may be a heterogeneous mixture of muscle stem cells, satellite cells, myocytes, myoblasts, myotubes, myofibers, or any combination thereof. Thus, the mixture may include muscle stem cells and satellite cells, and optionally myocytes.

[0067] In some embodiments, muscle cells or induced pluripotent stem cells are derived from a subject with a condition or disease that involves muscle damage, injury, or atrophy.In some embodiments, the condition or disease that involves muscle damage, injury, or atrophy is acute muscle injury, tear, or trauma, hand soft tissue injury, Duchenne muscular dystrophy (DMD), Becker muscular dystrophy, limb-girdle muscular dystrophy, amyotrophic lateral sclerosis (ALS), distal muscular dystrophy (DD), hereditary myopathy, myotonic muscular dystrophy (MDD), mitochondrial myopathy, myotubular myopathy (MM), myasthenia gravis (MG), congestive heart failure, periodic paralysis, polymyositis, rhabdomyolysis, dermatomyositis, cancer cachexia, AIDS cachexia, cardiac cachexia, stress-induced urinary incontinence, sarcopenia, or any combination thereof.

[0068] In certain embodiments, ex vivo methods are provided for promoting muscle cell engraftment in a subject. These methods include culturing or contacting isolated muscle cells with an effective amount of a compound selected from the group consisting of prostaglandin E2 (PGE2), PGE2 prodrugs, PGE2 receptor agonists, compounds that attenuate PGE2 catabolism, compounds that negate PGE2 inhibition, derivatives thereof, analogs thereof, and combinations thereof, to promote muscle cell engraftment in the subject. These methods also include administering the cultured or contacted muscle cells to the subject. In some cases, the isolated muscle cells are autologous to the subject. In other cases, the isolated muscle cells are allogeneic to the subject. In some embodiments, the subject is a human. In some embodiments, the subject has a condition or disease involving muscle damage, injury, or atrophy. In some embodiments, these methods further comprise administering to the subject a therapeutically effective amount of a compound selected from the group consisting of prostaglandin E2 (PGE2), PGE2 prodrugs, PGE2 receptor agonists, compounds that attenuate PGE2 catabolism, compounds that negate PGE2 inhibition, derivatives thereof, analogs thereof, and combinations thereof, and a pharma- ceutically acceptable carrier. The compound may be administered to the subject before, simultaneously, and / or after the cultured or contacted muscle cells are administered to the subject. In some cases, the isolated muscle cell population is cultured or contacted with the very compound that is administered to the subject. In other cases, the isolated muscle cell population is cultured or contacted with a compound that is different from the compound that is administered to the subject.

[0069] Muscle cells include, but are not limited to, the pectoral complex, latissimus dorsi, teres major and subscapularis, brachioradialis, biceps, brachialis, pronator quadratus, pronator teres, flexor carpi radialis, flexor carpi ulnaris, flexor digitorum superficialis, flexor digitorum profundus, flexor pollicis brevis, opponens hallucis, adductor pollicis, flexor pollicis brevis, iliopsoas, psoas, rectus abdominis, rectus femoris, gluteus maximus, gluteus medius, medial hamstrings, gastrocnemius, lateral hamstrings, and lateral hamstrings. The muscle may be derived from any muscle of the body, including the hamstrings, quadriceps mechanism, adductor longus, adductor brevis, adductor magnus, medial gastrocnemius, lateral gastrocnemius, soleus, tibialis posterior, tibialis anterior, flexor digitorum longus, flexor brevis, flexor pollicis longus, extensor pollicis longus, hand muscles, arm muscles, foot muscles, lower leg muscles, chest muscles, stomach muscles, back muscles, gluteal muscles, shoulder muscles, as well as muscles of the head and neck.

[0070] In some embodiments, muscle cells are obtained from a particular muscle, expanded according to the methods disclosed herein, and then transplanted back into the same muscle or into another muscle. In some cases, the source of muscle cells and the transplantation site are the same muscle of a subject. In other cases, the source of muscle cells and the transplantation site are different muscles of a subject. In other cases, the source of muscle cells and the transplantation site are the same type of muscle from different subjects. In yet other cases, the source of muscle cells and the transplantation site are different types of muscle from different subjects.

[0071] The compounds disclosed herein may be cultured with isolated muscle cells short-term, intermittently, or continuously.In some embodiments, the compound is exposed to cells in a single dose for a certain duration.In other embodiments, the compound is exposed to cells at least twice or more times, with a certain period between each dose, for example, one day, two days, one week, or more.In some embodiments, the compound is exposed to cells chronically or continuously for a certain duration, for example, without changing the compound concentration or the effect on cells.

[0072] B. Methods for regenerating damaged muscle cells in a subject The methods provided herein can be used to regenerate or rejuvenate muscle in subjects, such as human subjects.Muscle regeneration includes forming new muscle fibers from muscle stem cells, satellite cells, muscle progenitor cells, and any combination thereof.These methods are also useful for enhancing or enhancing muscle repair and / or maintenance.

[0073] The PGE2 compound of the present invention can be administered to a subject who is experiencing muscle degeneration or atrophy.Muscle atrophy can include a decrease in muscle mass and / or muscle strength.This can affect any muscle of the subject.In some cases, the subject who is in need of the compositions, methods and kits provided herein shows or experiences muscle loss, for example, due to age, inactivity, injury, disease, and any combination thereof.

[0074] In some embodiments, the compound can activate muscle cell proliferation, differentiation, and / or muscle cell fusion. In some cases, muscle tissue is regenerated. In other cases, muscle function (e.g., muscle mass, muscle strength, and / or muscle contraction) is restored or enhanced. In some cases, muscle weakness and muscle atrophy are ameliorated.

[0075] Injured muscles include, but are not limited to, the pectoralis complex, latissimus dorsi, teres major and subscapularis, brachioradialis, biceps, brachialis, pronator quadratus, pronator teres, flexor carpi radialis, flexor carpi ulnaris, flexor digitorum superficialis, flexor digitorum profundus, flexor pollicis brevis, opponens hallucis, adductor pollicis, flexor pollicis brevis, iliopsoas, psoas, rectus abdominis, rectus femoris, gluteus maximus, gluteus medius, medial hamstrings, gastrocnemius, and lateral hamstrings. The muscle may be any muscle of the body, including: quadriceps mechanism, adductor longus, adductor brevis, adductor magnus, medial gastrocnemius, lateral gastrocnemius, soleus, tibialis posterior, tibialis anterior, flexor digitorum longus, flexor brevis, flexor pollicis longus, extensor pollicis longus, hand muscles, arm muscles, foot muscles, lower leg muscles, chest muscles, stomach muscles, back muscles, gluteal muscles, shoulder muscles, head and neck muscles, facial muscles, as well as eye-pharyngeal muscles, and the like.

[0076] Subjects in need of muscle regeneration include those with musculoskeletal injuries (e.g., fractures, strains, sprains, acute injuries, and overuse injuries), post-traumatic injuries to the limbs or face, athletic injuries, post-fracture injuries in the elderly, soft tissue injuries of the hand, muscle atrophy (e.g., loss of muscle mass), Duchenne muscular dystrophy (DMD), Becker muscular dystrophy, Fukuyama congenital muscular dystrophy (FCMD), limb-girdle muscular dystrophy (LGMD), congenital muscular dystrophies, facioscapulohumeral muscular dystrophy (FHMD), myotonic dystrophy, and the like. Patients may have muscular dystrophy, oculopharyngeal dystrophy, distal muscular dystrophy, Emery-Dreifuss muscular dystrophy, congenital myotonia, myotonic dystrophy, other muscular dystrophies, muscle wasting diseases such as cachexia due to cancer, end-stage renal disease (ESRD), acquired immune deficiency syndrome (AIDS), or chronic obstructive pulmonary disease (COPD), muscle weakness after surgery, muscle weakness after trauma, sarcopenia, inactivity (e.g., muscle disuse or immobility), urethral sphincter insufficiency, urethral sphincter insufficiency, and neuromuscular diseases.

[0077] Non-limiting examples of neuromuscular diseases include acid maltase deficiency, amyotrophic lateral sclerosis, Andersen-Tawil syndrome, Becker muscular dystrophy, Becker congenital myotonia, Bethlem myopathy, spinal-bulbar muscular atrophy, carnitine deficiency, carnitine palmityltransferase deficiency, central core disease, centronuclear myopathy, Charcot-Marie-Tooth disease, congenital muscular dystrophies, congenital myasthenic syndromes, congenital myotonic dystrophy, Cori's disease, Debranchier's enzyme deficiency, Dejerine-Sottas disease, skin Myositis, Distal Muscular Dystrophy, Duchenne Muscular Dystrophy, Myotonic Dystrophy, Emery-Dreifuss Muscular Dystrophy, Endocrine Myopathy, Eulenberg Disease, Facioscapulohumeral Muscular Dystrophy, Distal Tibial Myopathy, Friedreich's Ataxia, Fukuyama Congenital Muscular Dystrophy, Glycogen Storage Disease Type 10, Glycogen Storage Disease Type 11, Glycogen Storage Disease Type 2, Glycogen Storage Disease Type 3, Glycogen Storage Disease Type 5, Glycogen Storage Disease Type 7, Glycogen Storage Disease Type 9, Gowers-Lein Distal Myopathy, Hereditary Inclusion Body Myositis, Hyperthyroid Myopathy, Hypothyroid Myopathy myasthenia, inclusion body myositis, hereditary myopathy, integrin deficiency congenital muscular dystrophy, spinobulbar muscular atrophy, spinal muscular atrophy, lactate dehydrogenase deficiency, Lambert-Eaton myasthenic syndrome, McArdle disease, merosin deficiency congenital muscular dystrophy, muscle metabolic disease, mitochondrial myopathy, Miyoshi distal myopathy, motor neuron disease, muscle-eye-brain disease, myasthenia gravis, myoadenylate deaminase deficiency, myofibrillar myopathy, muscle phosphorylase deficiency, congenital myotonia, myotonic muscular dystrophy Myotubular myopathy, nemaline myopathy, Nonaka distal myopathy, oculopharyngeal muscular dystrophy, paramyotonia congenita, Pearson syndrome, periodic paralysis, phosphofructokinase deficiency, phosphoglycerate kinase deficiency, phosphoglycerate mutase deficiency, phosphorylase deficiency, polymyositis, Pompe disease, progressive external ophthalmoplegia, spinal muscular atrophy, Ullrich congenital muscular dystrophy, Welander distal myopathy, and ZASP-related myopathy.

[0078] Muscle atrophy (e.g., muscle wasting) may be caused by or associated with, for example, normal aging (e.g., sarcopenia), genetic abnormalities (e.g., mutations or single nucleotide polymorphisms), inadequate nutrients, poor circulation, reduced hormonal support, disuse of muscles due to lack of exercise (e.g., bed rest, immobilization of a limb in a cast, etc.), aging, damage to the nerves that innervate muscles, poliomyelitis, amyotrophic lateral sclerosis (ALS or Lou Gehrig's disease), heart failure, liver disease, diabetes, obesity, metabolic syndrome, demyelinating diseases, demyelinating diseases (e.g., multiple sclerosis, Charcot-Marie-Tooth disease, Pelizaeus-Merzbacher disease, encephalomyelitis, neuromyelitis optica, adrenoleukodystrophy, and Guillain-Barré syndrome), denervation, fatigue, exercise-induced muscle fatigue, weakness, neuromuscular disease, debilitation, and chronic pain.

[0079] In some aspects, provided herein is a method for regenerating muscle in a subject in need thereof by administering to the subject a therapeutically effective amount of a compound selected from the group consisting of prostaglandin E2 (PGE2), PGE2 prodrugs, PGE2 receptor agonists, compounds that attenuate PGE2 catabolism, compounds that negate PGE2 inhibition, derivatives thereof, analogs thereof, and combinations thereof, and a pharma- ceutically acceptable carrier to increase muscle cell population and / or enhance muscle function in the subject. The muscle cell population of the subject may include skeletal muscle cells, smooth muscle cells, cardiomyocytes, embryonic stem cell-derived muscle cells, induced pluripotent stem cell-derived muscle cells, dedifferentiated muscle cells, or any combination thereof. Furthermore, the muscle cells of the subject may be muscle stem cells, satellite cells, myocytes, myoblasts, myotubes, muscle fibers, or any combination thereof. The compound may be administered to the subject by oral, intraperitoneal, intramuscular, intra-arterial, intradermal, subcutaneous, intravenous, or intracardiac administration. In some cases, the compound is administered directly to the dysfunctional, injured, damaged, and / or atrophied muscle. The compound may be administered according to a short-term treatment regimen (e.g., single dose or intermittent dosing) or a long-term treatment regimen (e.g., continuous dosing).

[0080] In some embodiments, the subject is also administered a population of isolated (or isolated and purified) muscle cells that are either autologous or allogeneic to the subject. These cells may be isolated and / or purified by any method known to those of skill in the art. These cells may be an allogeneic or heterogeneous population of muscle cells.

[0081] In some embodiments, cells are stimulated to proliferate by culturing cells with PGE2 compound before administering them to a subject.Cells can be exposed to compound short-term, intermittently, or continuously during in vitro culturing.In some cases, muscle cell population increases by at least about 1%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 200%, at least about 500%, at least about 1000% or more after culturing with PGE2 compound.

[0082] In order to regenerate or repair muscle in a subject, the compound of the present invention and isolated muscle cells are administered to the subject simultaneously.In some embodiments, the compound and cultured muscle cells are administered to the subject simultaneously.In other embodiments, the compound and isolated muscle cells are administered to the subject sequentially.In yet another embodiment, the compound and cultured muscle cells are administered to the subject sequentially.

[0083] Using the methods described herein, the number of muscle fibers can be increased by at least about 1%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 200%, at least about 500%, at least about 1000%, or more. In some embodiments, these methods can increase the proliferation of damaged, injured, atrophied, or degenerated muscles.

[0084] C. Methods for preventing or treating conditions or diseases affecting muscles The method provided herein can be used to prevent or treat conditions or diseases involving muscle damage, injury, or atrophy in subjects in need thereof.The method can provide preventive treatment to subjects who are likely to suffer muscle damage, injury, or atrophy.In some embodiments, the subject may have a condition or disease that may cause secondary symptoms that affect muscle.In other embodiments, the subject has undergone surgical or therapeutic intervention to treat muscle condition or disease, and the method disclosed herein is used to prevent or suppress recurrence or relapse.In some embodiments, the subject has any one of the conditions or diseases described herein that affect muscle.

[0085] As used herein, the term "treatment" or "treating" includes administration of an appropriate form of compound and / or cells prior to the onset of disease symptoms and / or after clinical or other manifestation of a condition or disease to lessen disease severity, halt disease progression, or eliminate the disease. The term "prevention of" a disease or "preventing" a disease preferably includes prolonging or delaying the onset of symptoms of a condition or disease in a subject predisposed to the condition or disease.

[0086] The method includes administering to a subject (i) a therapeutically effective amount of a compound selected from the group consisting of prostaglandin E2 (PGE2), a PGE2 prodrug, a PGE2 receptor agonist, a compound that attenuates PGE2 catabolism, a compound that negates PGE2 inhibition, derivatives thereof, analogs thereof, and combinations thereof, and a pharma- ceutically acceptable carrier, and (ii) an isolated population of muscle cells to prevent or treat a condition or disease involving muscle damage, injury, or atrophy. The muscle cells may be autologous or allogeneic to the subject.

[0087] The compound may be administered orally, intraperitoneally, intramuscularly, intraarterially, intradermally, subcutaneously, intravenously, or by intracardiac injection. The compound may be administered according to a short-term treatment regimen (e.g., single dose or intermittent dosing) or a long-term treatment regimen (e.g., continuous dosing). Isolated muscle cells may be administered by injection or transplantation. In some embodiments, the compound and cells are administered together or simultaneously. In other embodiments, the compound and cells are administered sequentially. In some cases, the compound is administered before the cells. In other cases, the cells are administered before the compound.

[0088] The isolated muscle cells may be substantially purified or purified before being injected or transplanted into a subject. These cells may also be expanded or stimulated to grow in culture before administration. As described herein, isolated muscle cells, including skeletal muscle cells, smooth muscle cells, cardiomyocytes, embryonic stem cell-derived muscle cells, induced pluripotent stem cell-derived muscle cells, dedifferentiated muscle cells, muscle stem cells, satellite cells, myoblasts, myocytes, myotubes, myofibers, and any combination thereof, may be cultured with the compounds of the present invention. By exposing the cells to the compounds short-term, intermittent, or continuous, the muscle cells are expanded and increased in number. The expanded cells may be transplanted into a subject experiencing muscle damage, injury, or atrophy.

[0089] D Prostaglandin E2 (PGE2) compounds In some embodiments, the compound of the present invention is selected from the group consisting of PGE2, PGE2 prodrugs, PGE2 receptor agonists, compounds that attenuate PGE2 catabolism, compounds that negate PGE2 inhibition, derivatives thereof, analogs thereof, and combinations thereof.In some cases, the compound that attenuates PGE2 catabolism can be a compound, neutralizing peptide, or neutralizing antibody that inactivates or interferes with 15-hydroxyprostaglandin dehydrogenase (15-PGDH) or inactivates or interferes with prostaglandin transporter that transports PGE2 into cells for catabolism by 15-PGDH.Prostaglandin transporter is also known as 2310021C19Rik, MATR1, Matrin F / Q, OATP2A1, PGT, PHOAR2, SLC21A2, solute transporter organic anion transporter family member 2A1, and SLCO2A1.

[0090] PGE2 receptor agonist may be a small molecule compound, an activating antibody that specifically binds to PGE2 receptor, and the like.In some embodiments, the compound is a PGE2 derivative or a PGE2 analog.In some embodiments, the compound is a PGE2 prodrug.PGE2 prodrug can be metabolized to pharmacologically active PGE2 drug, for example, at the site of administration or muscle regeneration, or when the prodrug is exposed to muscle cells.

[0091] In certain embodiments, the compound is a PGE2 derivative or PGE2 analog that includes one or more modifications to PGE2 that enhance PGE2 stability, activity, resistance to degradation, transport into muscle cells (e.g., to facilitate cellular uptake), and / or retention in muscle cells (e.g., to reduce secretion from muscle cells following uptake).

[0092] Non-limiting examples of PGE2 derivatives and analogs include 2,2-difluoro-16-phenoxy-PGE2 compounds, 2-decarboxy-2-hydroxymethyl-16-fluoro-PGE2 compounds, 2-decarboxy-2-hydroxymethyl-11-deoxy-PGE2 compounds, 19(R)-hydroxy PGE2, 16,16-dimethyl PGE2, 16,16-dimethyl PGE2, These include p-(p-acetamidobenzamido)phenyl ester, 11-deoxy-16,16-dimethyl PGE2, 9-deoxy-9-methylene-16,16-dimethyl PGE2, 9-deoxy-9-methylene PGE2, butaprost, sulprostone, enprostil, PGE2 serinolamide, PGE2 methyl ester, 16-phenyltetranor PGE2, 5-trans-PGE2, 15(S)-15-methyl PGE2, and 15(R)-15-methyl PGE2. Other PGE2 derivatives and analogs are described, for example, in U.S. Patent No. 5,409,911.

[0093] Other non-limiting examples of PGE2 derivatives and analogs include hydantoin derivatives of PGE2, i.e., more stable PGE2 analogs described by Zhao et al. (Bioorganic & Medicinal Chemistry Letters, 17:6572-5 (2007)) in which the hydroxycyclopentanone ring is replaced with a heterocycle and the unsaturated α-alkenyl chain is replaced with a phenethyl chain, PGE2 analogs described by Ungrin et al. (Mol. Pharmacol., 59: 1446-56 (2001)), 13-dehydro derivatives of PGE2 described by Tanami et al. (Bioorg. Med. Chem. Lett., 8: 1507-10 (1998)), and substituted cyclopentanes described in U.S. Pat. Nos. 8,546,603 and 8,158,676.

[0094] In some embodiments, the compound is an agonist of PGE2 receptor, such as EP1 receptor, EP2 receptor, EP3 receptor, and EP4 receptor.Non-limiting examples of PGE2 receptor agonists include ONO-DI-004, ONO-AE1-259, ONO-AE-248, ONO-AE1-329, ONO-4819CD (Ono Pharmaceutical Co., Japan), L-902688 (Cayman Chemical), CAY10598 (Cayman Chemical), and CP-533536 (Pfizer). Other PGE2 receptor agonists are described, for example, in U.S. Patent Nos. 6,410,591, 6,610,719, 6,747,037, 7,696,235, 7,662,839, 7,652,063, 7622,475, and 7,608,637.

[0095] E. Isolated muscle cells The muscle (myogenic) cells of the present invention include, but are not limited to, muscle stem cells, skeletal muscle stem cells, smooth muscle stem cells, cardiac muscle stem cells, muscle satellite cells, myogenic progenitor cells, myoblasts, myocytes, myoblasts, myotubes, postmitotic myotubes, multinucleated myofibers, and postmitotic myofibers. In some embodiments, the isolated muscle cells include muscle stem cells. In other embodiments, the isolated muscle cells include muscle satellite cells. The muscle cells may be derived from stem cells, such as bone marrow-derived stem cells, or from pluripotent stem cells, such as embryonic stem cells or induced pluripotent stem cells. In some embodiments, the isolated muscle cells include dedifferentiated muscle cells. In other embodiments, the muscle cells are genetically modified to correct genetic mutations associated with disease in some cases.

[0096] Satellite cells are small mononuclear precursor cells that can exist inside muscle tissue. These cells can be induced to proliferate and differentiate into muscle cells, and in some cases, to fuse into muscle fibers. During muscle damage or injury, quiescent satellite cells (e.g., satellite cells that are not currently differentiated or undergoing cell division) and muscle stem cells can be activated to proliferate and / or migrate out of the muscle stem cell niche. Satellite cells and muscle stem cells can also differentiate into myocytes, myoblasts, or other muscle cell types.

[0097] Methods and protocols for generating muscle cells from embryonic stem cells are described, for example, in Hwang et al., PLoS One, 2013, 8(8):e72023; and Darabi et al., Cell Stem Cell, 2012, 10(5):610-9. Methods and protocols for generating muscle cells from induced pluripotent stem cells are described, for example, in Darabi et al., Cell Stem Cell, 2012, 10(5):610-9; Tan et al., PLoS One, 2011; and Mizuno et al., FASEB J., 2010, 24(7):2245-2253.

[0098] In some embodiments, muscle cells are obtained by biopsy from muscle, such as mature or grown muscle, for example, quadriceps, gluteus maximus, biceps, triceps, or any muscle from an individual.Muscle can be skeletal muscle, smooth muscle, or cardiac muscle.Detailed description of the method of isolating smooth muscle stem cells can be found, for example, in U.S. Patent No. 8,747,838 and U.S. Patent Application Publication No. 20070224167.The method of isolating muscle cells of interest, such as muscle stem cells or satellite cells, from muscle tissue is described in detail, for example, in Blanco-Bose et al., Exp. Cell Res., 2001, 26592:212-220.

[0099] Methods for purifying muscle cells of interest, such as muscle stem cells, muscle satellite cells, myocytes, myoblasts, myotubes, and / or muscle fibers, include selecting, isolating, or enriching cells that have a specific cell surface marker or a specific polypeptide expressed on the cell surface of the muscle cells of interest. Useful cell surface markers are described, for example, in Fukada et al., Front. Physiol., 2013, 4:317. Cell sorting methods such as flow cytometry, e.g., fluorescence-activated cell sorting (FACS); magnetic bead cell separation, e.g., magnetic-activated cell sorting (MACS), and other antibody-based cell sorting methods, can be performed to isolate or separate the muscle cells of interest from other cell types.

[0100] The isolated muscle cell population of interest can be expanded or multiplied using conventional culture-based methods. Methods for culturing muscle cells are described, for example, in U.S. Patent No. 5,324,656. In some cases, cells are cultured on the surface of a gel, such as a scaffold or hydrogel.

[0101] F. Method of Administration The compound of the present invention may be administered locally to or near the site of injury in a subject, or systemically. In some embodiments, the compound may be administered, for example, intraperitoneally, intramuscularly, intraarterially, orally, intravenously, intracranially, intrathecally, intraspinally, intralesionally, intranasally, subcutaneously, intracerebroventricularly, locally, and / or by inhalation. The compound may be administered simultaneously or sequentially with the muscle cells of interest. When the compound is administered simultaneously with the cells, both the compound and the cells may be administered in the same composition. When administered separately, the compound may be provided in a pharma- ceutically acceptable carrier. In some cases, the compound is administered before or after the administration of the cells.

[0102] In some embodiments, the compound is administered according to a short-term treatment regimen. In some cases, the compound is administered to the subject once. In other cases, the compound is administered at one time point and then administered again at a second time point. In still other cases, the compound is administered to the subject repeatedly (e.g., once or twice daily) as intermittent administration over a short period of time (e.g., 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, or more than 1 month). In some cases, the period between compound administrations is about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, or more than 1 month. In other embodiments, the compound is administered continuously or chronically according to a long-term treatment regimen for a desired period of time. For example, the compound can be administered so that the amount or level of the compound is substantially constant over a selected period of time.

[0103] Administration of isolated muscle cells to a subject can be accomplished by methods commonly used in the art. In some embodiments, administration is by implantation or injection, such as intramuscular injection. The number of cells introduced takes into account factors such as sex, age, weight, type of disease or disorder, stage of disorder, ratio of desired cells in the cell population (e.g., purity of the cell population), and the number of cells required to produce the desired result. Typically, when cells are administered for therapeutic purposes, they are given in a pharmacologically effective dose. A "pharmacologically effective amount" or "pharmacologically effective dose" is an amount sufficient to produce a desired physiological effect or to achieve a desired result, particularly for the treatment of a condition or disease, including reducing or eliminating one or more symptoms or manifestations of the condition or disease. A pharmacologically effective dose also applies to therapeutic compounds used in combination with cells as described herein.

[0104] Cells can be administered in one injection or by continuous injection for a predetermined period of time sufficient to produce a therapeutic effect.When treatment involves continuous injection, different muscle cell groups can be injected.A pharmaceutically acceptable carrier, as described further below, can be used to inject cells into a subject.Typically, these include, for example, buffered saline (e.g., phosphate buffered saline) or plain basal cell culture medium, or media known in the art.

[0105] Any number of muscles of the body may be directly injected with the compound and / or cells of the present invention.Muscles include, for example, biceps muscle; triceps muscle; brachioradialis muscle; brachialis muscle (brachialis anterior); superficial compartment wrist flexor; deltoid muscle; biceps femoris muscle, gracilis muscle, semitendinosus and semimembranosus muscles of hamstrings; rectus femoris muscle, vastus lateralis, vastus medialis, and vastus intermedius muscles of quadriceps; gastrocnemius muscle (lateral and medial), tibialis anterior, and soleus muscle of calf; pectoralis major and pectoralis minor muscles of chest; latissimus dorsi muscle of upper back; rhomboid muscle (large and small); trapezius muscle that spans neck, shoulder, and back; rectus abdominis muscle of abdomen; and gluteus maximus, gluteus medius, and gluteus minimus muscles of buttocks.

[0106] G. Pharmaceutical Compositions Pharmaceutical compositions comprising the compounds and cells of the present invention may include a pharma- ceutically acceptable carrier. In certain aspects, the pharma- ceutically acceptable carrier will be determined, in part, based on the particular composition to be administered, as well as the particular method used to administer the composition. Thus, there are a wide variety of suitable formulations of the pharmaceutical compositions of the present invention (see, e.g., REMINGTON, et al., J. Clin. Immunol. 1999, 143:131-132, 1999). ' S PHARMACEUTICAL SCIENCES, 18TH ED., Mack Publishing Co., Eastern, PA (1990)).

[0107] As used herein, " pharmaceutically acceptable carrier " includes any of the pharmaceutically acceptable standard carriers known to those skilled in the art when formulating pharmaceutical compositions. Thus, cells or compounds that exist alone, for example, as pharmaceutically acceptable salts or conjugates, can be prepared as formulations using pharmaceutically acceptable diluents; for example, saline, phosphate buffered saline (PBS), aqueous ethanol, or solutions of glucose, mannitol, dextran, propylene glycol, oils (for example, vegetable oils, animal oils, synthetic oils, etc.), microcrystalline cellulose, carboxymethylcellulose, hydroxypropylmethylcellulose, magnesium stearate, calcium phosphate, gelatin, or polysorbate 80, or as solid formulations using suitable excipients.

[0108] Pharmaceutical compositions often further comprise one or more buffers (e.g., neutral buffered saline or phosphate buffered saline), carbohydrates (e.g., glucose, mannose, sucrose, or dextran), mannitol, proteins, polypeptides, or amino acids such as glycine, antioxidants (e.g., ascorbic acid, sodium metabisulfite, butylated hydroxytoluene, butylated hydroxyanisole, and the like), bacteriostatic agents, chelating agents such as EDTA or glutathione, solutes to render the formulation isotonic, hypotonic, or weakly hypertonic with the blood of the recipient, suspending agents, thickening agents, preservatives, flavoring agents, sweetening agents, and coloring compounds as required.

[0109] The pharmaceutical composition of the present invention is administered in a manner compatible with the dosage formulation and in an amount that is therapeutically effective.The amount administered depends on various factors, including, for example, the age, weight, physical activity, and diet of the individual, the pathology or disease being treated, and the stage or severity of the pathology or disease.In certain embodiments, the amount of dose can also be determined based on the existence, nature, and extent of any adverse side effects that accompany the administration of each therapeutic agent in each individual.

[0110] It will be understood, however, that the specific dose level and frequency of dosing for any particular patient may vary and will depend upon a variety of factors, including the activity of the particular compound used, the metabolic stability and length of action of that compound, age, body weight, genetics, general health, sex, diet, mode and time of administration, rate of excretion, drug combination, the severity of the particular condition, and the recipient receiving the therapy.

[0111] In certain embodiments, the dose of the compound may be in the form of a solid, semi-solid, lyophilized powder, or liquid dosage form, such as a tablet, pill, pellet, capsule, powder, solution, suspension, emulsion, suppository, retention enema, cream, ointment, lotion, gel, aerosol, or foam, preferably in a unit dosage form suitable for easy administration of precise dosage amounts.

[0112] As used herein, the term "unit dosage form" refers to a physically discrete unit suitable as a unitary dosage for humans and other mammals, each unit containing a predetermined amount of therapeutic substance calculated to produce a desired onset, tolerability, and / or therapeutic effect in association with suitable pharmaceutical excipients (e.g., ampule).In addition, more concentrated dosage forms may be prepared, from which more dilute unit dosage forms may then be made.Thus, more concentrated dosage forms contain substantially more therapeutic compound, for example, at least 1x, 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, 10x, or more than 10x the amount.

[0113] Methods for preparing such dosage forms are known to those skilled in the art (see, e.g., REMINGTON'S PHARMACEUTICAL SCIENCES, supra). Typically, these dosage forms contain conventional pharmaceutical carriers or excipients, and may further contain other medicinal substances, carriers, adjuvants, diluents, tissue permeation enhancers, solubilizers, and the like. Appropriate excipients can be tailored to the particular dosage form and route of administration by methods well known in the art (see, e.g., REMINGTON'S PHARMACEUTICAL SCIENCES, supra).

[0114] Examples of suitable excipients include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum acacia, calcium phosphate, alginate, gum tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, saline, syrup, methylcellulose, ethylcellulose, hydroxypropylmethylcellulose, and polyacrylic acid, such as carbopol, e.g., carbopol 941, carbopol 980, carbopol 981, etc. The dosage form may further include lubricants such as talc, magnesium stearate, and mineral oil; wetting agents; emulsifying agents; suspending agents; preservatives such as methyl, ethyl, and propyl hydroxybenzoates (i.e., parabens); pH adjusting agents, such as inorganic and organic acids and bases; sweetening agents; and flavoring agents. Dosage forms may also include biodegradable polymer beads, dextran, and cyclodextrin inclusion complexes.

[0115] For oral administration, the therapeutically effective dose can be in the form of tablets, capsules, emulsions, suspensions, liquids, syrups, sprays, lozenges, powders, and sustained-release preparations.Suitable excipients for oral administration include pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, gelatin, sucrose, magnesium carbonate, etc.

[0116] The therapeutically effective dose may also be provided in lyophilized form. Such dosage forms may contain a buffer, such as bicarbonate, for reconstitution before administration, or a buffer may be included in the lyophilized dosage form for reconstitution, such as with water. The lyophilized dosage form may further contain a suitable vasoconstrictor, such as epinephrine. The lyophilized dosage form may be provided in a syringe, optionally packaged in combination with a buffer solution for reconstitution, so that the reconstituted dosage form can be administered immediately to an individual.

[0117] H Kit Another embodiment of the compositions described herein is a kit comprising an isolated muscle cell population and a compound selected from the group consisting of prostaglandin E2 (PGE2), a PGE2 prodrug, a PGE2 receptor agonist, a compound that attenuates PGE2 catabolism (e.g., a 15-hydroxyprostaglandin dehydrogenase (15-PGDH) inhibitor or a prostaglandin transporter (PTG or SLCO2A1) inhibitor), a compound that negates PGE2 inhibition, its derivatives, its analogs, and combinations thereof. Typically, the kit includes containers that can be formed from a variety of materials, such as glass or plastic, and may include, for example, bottles, vials, syringes, and test tubes. Typically, a label is attached to the kit, and the label includes any written or recorded material, which may be in electronic or computer readable form, that provides instructions or other information for using the contents of the kit. EXAMPLES

[0118] V. Working Example The following examples are offered to illustrate, but not to limit, the claimed invention.

[0119] Example 1: Short-term prostaglandin E2 delivery enhances skeletal muscle regeneration and strength in aged mice. This example shows that PGE2 signaling is required for muscle stem cell function during regeneration.

[0120] Elderly people suffer from progressive skeletal muscle weakness and failure to regenerate, which reduces mobility and quality of life 1、2 Adult muscle stem cells (MuSCs), which reside in a niche within muscle tissue and are ready to respond to injury and repair skeletal muscles throughout life, are crucial for muscle regeneration. 3~8 During aging, the proportion of functional MuSCs is significantly decreased, slowing muscle regeneration. 9~13To date, no therapeutics targeting MuSCs to counter this decline in regeneration have been used clinically. Herein, we identify prostaglandin E2 (PGE2), a natural immunomodulator, as a potent regulator of MuSC function essential for muscle regeneration. We find that the PGE2 receptor, EP4, is essential for MuSC proliferation in vitro and for in vivo transplantation in mice. The PGE2 pathway is dysregulated in MuSCs from aged mice due to a defect in a cell-intrinsic molecule, namely an increase in the prostaglandin degrading enzyme (15-PGDH) that inactivates PGE2. This defect is overcome by transient short-term exposure of MuSCs to 16,16-dimethyl PGE2 (dmPGE2), a stable degradation-resistant PGE2, simultaneously with MuSC transplantation into injured muscle. Remarkably, a single intramuscular injection of dmPGE2 alone was sufficient to accelerate regeneration, as evidenced by an early increase in endogenous MuSC numbers and muscle fiber size after injury. Furthermore, the ability of aged mice to generate muscle force was enhanced in response to exercise-induced regeneration and a short-term dmPGE2 treatment regimen. Our findings reveal a novel therapeutic indication for PGE2 as a potent inducer of muscle regeneration and strength.

[0121] To counteract this waning muscle regenerative potential, we sought therapeutics that target MuSCs, also known as satellite cells, a population of stem cells dedicated to muscle regeneration. 3~8 Transient inflammatory and fibroadipogenic responses play a crucial role in muscle regeneration. 14~17 Therefore, we sought to identify injury-induced inflammatory regulators that could counteract the age-related decline in MuSC function. Analysis of our transcriptome database revealed that PGE2, a natural and potent lipid mediator during acute inflammation, 18We found that the Ptger4 receptor for PGE2 was expressed at high levels on the surface of freshly isolated MuSCs. In muscle tissue lysates, we observed a rapid increase in PGE2 levels 3 days after injury of young (2-4 months) mouse muscles by standard injury with notexin injection or cold injury (Figures 1A and 5A) and a concomitant upregulation of its synthetic enzymes, Ptges and Ptges2 (Figure 1B). This early and transient time window is consistent with the well-documented kinetics of MuSC expansion and inflammatory cytokine accumulation after injury. 8、15、16 To determine whether PGE2 treatment improved MuSC behavior, we FACS-purified MuSCs from hindlimb muscles of young mice (2–4 months old). 6 , and seeded them on hydrogels with a stiffness of 12 kPa to maintain stem cell function. 19 We found that PGE2 (10 ng / ml) increased cell division as assessed by EDU incorporation (Figures 1B-1D) and that acute 1-day exposure to PGE2 increased MuSC numbers six-fold after 1 week compared to controls (Figure 1C).

[0122] PGE2 is known to transmit signals via four G protein-coupled receptors (Ptger1-4; EP1-4). 18、20 However, the expression of these receptors in MuSCs has not been described so far. Analysis of the transcript levels of the different receptors (Ptger1-4) revealed that only Ptger1 and Ptger4 were upregulated after treatment of MuSCs with PGE2 (Figure 5E). Intracellular cAMP was increased in PGE2-stimulated MuSCs, suggesting that Ptger1 and Ptger4 are upregulated after treatment of MuSCs with PGE2. 18、20These results confirmed that PGE2 signals through EP4 to promote proliferation and transcriptional state of stem cells (Fig. 5F-5H). In the presence of EP4 antagonist ONO-AE3-208, PGE2-induced proliferation was slowed (Fig. 1D). However, the specificity of PGE2 for EP4 was most clearly demonstrated in MuSCs lacking the receptor after cre-mediated conditional cleavage (Fig. 1E-1G and Fig. 5I-5J). Indeed, even in the presence of growth factor-rich medium, these EP4-deficient MuSCs were unable to proliferate. Finally, we showed that MuSCs growth-arrested by exposure to medium containing charcoal-stripped serum were able to grow in vitro and in vivo. 21 We found that the addition of PGE2 induced division (Figure 1H and Figure 5K), thus highlighting that PGE2 / EP4 is both necessary and sufficient for MuSC proliferation.

[0123] We sought to determine whether the previously reported defects in muscle regeneration of aged MuSCs could be ameliorated by PGE2. 9~13In contrast to young mouse muscles (2-4 months), injury of aged muscles (18-20 months) with notexin did not increase PGE2 synthesis. On the contrary, steady-state PGE2 levels in aged muscles remained unchanged after injury (Figure 2A) and were significantly higher than in young tibialis anterior (TA) muscles (Figure 2B). We hypothesized that PGE2 in aged muscles may be dysfunctional due to defective catabolism. Indeed, when we analyzed the PGE2 present in young and aged TA muscle tissues by mass spectrometry, we found that the relative amount of the inactive form, 13,14-dihydro-15-keto PGE2 (PGEM), was significantly increased in aged muscle tissue (Figures 2C-2D and 6A-6C). This was found to be due to a concomitant seven-fold increase in the levels of mRNA encoding the PGE2 degrading enzyme (15-PGDH), the first step in the conversion of PGE2 to its inactive form (Figure 2E). In contrast, the relative levels of prostaglandin transporters (PGTs), PGE2 synthase, and EP4 receptors did not differ between young and senescent MuSCs (Figures 7A-7C). Furthermore, when senescent MuSCs were exposed to a brief daily application of PGE2 or an inhibitor of 15-PGDH (SW033291), 22 In culture, the effect of 15-PGDH was suppressed, and a characteristic increase in proliferation and maintenance of Pax7 expression were observed (Fig. 2F and Fig. 7D). Similar to young MuSCs, senescent MuSCs were unable to proliferate in medium composed of charcoal-stripped serum, but were rescued by the addition of PGE2 alone (Fig. 2G). We speculated that the PGE2 pathway was dysregulated in senescent MuSCs due to a defect in a cell-intrinsic molecule, i.e., increased 15-PGDH, that could be overcome in culture by short-term exposure to PGE2 or SW (Fig. 2H).

[0124] Senescent MuSCs are heterogeneous 10Thus, we sought to clarify the effects of PGE2 at the single cell level. Clonal analysis can reveal differences that are hidden in the analysis of the population as a whole. We therefore performed long-term time-lapse microscopy in hydrogel "microwells" containing single senescent MuSCs and untreated control MuSCs that were transiently exposed to PGE2 for 1 day. Data were collected over a 48-h period and then analyzed using our previously described Baxter algorithm for cell tracking and lineage restoration. 10、19、23 We observed a marked increase in cumulative cell number in response to PGE2 over six generations in the most robust clones (Figs. 2I-2J). The number of cells per clone after PGE2 treatment was significantly increased by a marked increase in proliferation (Figs. 2I-2J and 7E-7F) accompanied by a strong decrease in cell death (Figs. 2J and 7E-7G). These synergistic effects led to the observed increase in the number of senescent MuSCs in response to PGE2.

[0125] To test whether transient treatment of young MuSCs with PGE2 enhances regeneration, we transplanted PGE2-treated cultured MuSCs into injured hindlimb muscles of mice. To quantitatively monitor the time-course pattern of regeneration in vivo, we utilized a sensitive and quantitative bioluminescence imaging (BLI) assay that we previously developed to monitor MuSC function after transplantation. 6、10、19MuSCs were isolated from young transgenic mice (2–4 months) expressing GFP and luciferase (GFP / Luc mice), exposed to short-term 1-day PGE2 treatment, harvested, and transplanted on day 7. Equal numbers of dmPGE2-treated and control MuSCs (250 cells) were transplanted into injured hind limbs of young (2–4 months) NOD-SCID mice. After short-term treatment with PGE2, the regenerative capacity of young MuSCs, as assessed by BLI, was an order of magnitude greater (Figure 3A). In contrast, after transplantation of four-fold greater numbers of cultured MuSCs lacking the EP4 receptor due to conditional ablation (Figure 3B), the initially detected BLI signal gradually declined to levels below the threshold for significance (Figure 3B).

[0126] Furthermore, a mouse model of muscle stem cell-specific deletion of EP4 (Pax7 CreERT2 ;EP4 fl / fl When notexin injury was performed in mice (Figures 9A-9B), muscle regeneration was attenuated, as observed by an elevated number of embryonic myosin heavy chain (eMHC)-positive fibers (Figures 9C-9D). This was assessed at the end of the regeneration time point (day 21), when Pax7 CreERT2 ;EP4 fl / f This was accompanied by a decrease in the cross-sectional area of ​​mouse fibers in the group (Figure 9E). A significant decrease in force output (tetanus) was also detected 14 days after injury (Figure 9F-9G). Thus, PGE2 signaling via the EP4 receptor is required for MuSC regeneration in vivo.

[0127] To test whether direct injection of PGE2 without culture could be effective in promoting regeneration in vivo, we co-injected PGE2 with freshly isolated MuSCs. In all subsequent in vivo injection experiments, we used a modified, more stable form of PGE2, 16,16-dimethyl PGE2 (dmPGE2). 24 We hypothesized that delivery of modified 15-PGDH-resistant dmPGE2 may be particularly important for senescent MuSC experiments, since 15-PGDH is significantly increased in senescent MuSCs (Figure 2E). 24Using dmPGE2, we observed significantly enhanced engraftment of young and senescent MuSCs compared to controls, further increasing in response to notexin insult, a rigorous test of well-recognized stem cell function (Figures 3C-3D). Thus, delivery of dmPGE2 together with MuSC populations is sufficient to enhance regeneration.

[0128] We hypothesized that muscle regeneration could be promoted by delivery of PGE2 alone. To test this, we injured the muscles of young mice using cardiotoxin, and 3 days later, we injected a large amount of dmPGE2 into the hindlimb muscles of young mice. We observed an increase (60±15%) in endogenous PAX7-expressing MuSCs in the typical satellite cell niche beneath the basement membrane and above the muscle fibers at 14 days after injury (Figures 4A-4B), whereas dmPGE2 had no effect in the absence of injury. Furthermore, at this early time point, the distribution of muscle fibers was shifted to larger sizes, as assessed by cross-sectional area using the Baxter algorithm for muscle fiber analysis, suggesting that regeneration was accelerated by PGE2 (Figures 4C-4D and Figures 8A-8B). Furthermore, we demonstrated that in a transgenic mouse model Pax7 CreERT2 Rosa26-LSL-Luc was used to track the response of endogenous MuSCs to injury and dmPGE2 based on luciferase expression (Figure 4E).BLI data were consistent with the histological data (Figures 4F-4G).

[0129] We investigated the effect of injection of the nonsteroidal anti-inflammatory drug (NSAID) indomethacin and a COX2 inhibitor that reduces PGE2 synthesis on muscle regeneration. CreERT2When indomethacin was injected into the hindlimb muscles of the Rosa26-LSL-Luc mouse model, we observed a marked decrease in luciferase activity, suggesting impaired muscle stem cell activation and regeneration (Fig. 10A-10B). Injection of indomethacin into cardiotoxin-injured muscles also resulted in a marked decrease in twitch force compared to controls, assessed 14 days after injury (Fig. 10C). In aged mice, we also observed a substantial increase in the number of endogenous MuSCs (24±2%) (Fig. 4H-4I) and a concomitant increase in muscle fiber size (Fig. 4J-4K) 14 days after injury after a single injection of dmPGE2. Thus, exposure to dmPGE2 alone has a strong impact on the magnitude and time course of endogenous repair.

[0130] As a final test, we determined whether dmPGE2-enhanced regeneration could increase muscle strength after natural injury induced by downhill treadmill running. In this scenario, injury was induced by running on a downhill treadmill for 10 minutes each day with a 20° downhill gradient. 25 During the first week, treated aged mice ran for 5 consecutive days and received daily injections of dmPGE2 after exercise. During the second week, treated aged mice ran for 5 consecutive days but received no additional treatment (Figure 4L). Comparison of unit twitch and tetanic forces for dmPGE2-treated and dmPGE2-untreated gastrocnemius mouse muscles (GA) revealed that both were significantly increased (Figure 4M-4P). Thus, a significant increase in aged muscle strength can be achieved by short-term exposure to dmPGE2 concomitant with exercise-induced injury.

[0131] We have discovered a new indication for PGE2 in skeletal muscle regeneration. Previous studies on the effects of PGE2 on skeletal muscle have shown that PGE2 alters proliferation, fusion, proteolysis, and differentiation of myoblasts in tissue culture. 26~30Therefore, these studies differ from ours because myoblasts are progenitor cells that have lost stem cell function. Satellite cells (MuSCs) are crucial for development and regeneration. 3~8、31 , and their numbers increase with running or other very strenuous exercise in young and old mice and humans. 15、32~36 It has been reported that nonsteroidal anti-inflammatory drugs attenuate exercise-induced increases in MuSCs. 15、32~36 Our data suggest that inflammation characterizes effective muscle regeneration. 15 We provide novel evidence that the beneficial effect of early and transient enhancement of PGDH is in part contributed by PGE2 and its receptor EP4, which are essential and sufficient for MuSC proliferation and engraftment. Delivery of SW033291, a 15-PGDH inhibitor, has recently been shown to enhance regeneration in hematopoietic, hepatic, and colonic tissues. 22 Of note, PGE2 and its analogs have been used to induce labor, e.g. 37 , and to promote hematopoietic stem cell transplantation. 38 It has been safely used in human patients for decades, paving the way for its clinical use in repairing muscle after injury. In summary, our findings show that a short-term PGE2 treatment regimen is sufficient to rapidly and steadily enhance regeneration of exercise-induced damage and overcome age-related limitations resulting in increased force.

[0132] References TIFF2024116386000001.tif102146TIFF2024116386000002.tif213147TIFF2024116386000003.tif138146

[0133] method mouse We performed all experiments and protocols in accordance with Stanford University's school guidelines and the Administrative Committee for Laboratory Animal Care (APLAC). We obtained wild-type aged C57BL / 6 (18-20 months) mice from the National Institute on Aging (NIA) for the study of aging muscle and wild-type young C57BL / 6 mice from the Jackson laboratory. Double transgenic GFP / luc mice were generated as previously described. 1 Briefly, mice expressing a firefly luciferase (luc) transgene under the control of the ubiquitous Actb promoter were maintained in the FVB strain. Mice expressing a green fluorescent protein (GFP) transgene under the control of the ubiquitous UBC promoter were maintained in the C57BL / 6 strain. We used cells derived from GFP / luc for allogeneic transplantation experiments into NOD-SCID (Jackson lab) recipient mice. EP4 flox / flox (EP4 f / f ) Mice were kindly provided by K. Andreasson (Stanford University). 2 Pax7 obtained from the Jackson laboratory CreERT2 Mouse (Stock number 017763) 3 and Rosa26-LSL-Luc (stock no. 005125) obtained from the Jackson laboratory. 4 Double transgenic Pax7 CreERT2 ;Rosa26-LSL-Luc. We confirmed these genotypes by a suitable PCR-based strategy. All mice derived from the transgenic lines were young. For all lines, the young mice were 2-4 months old and the old mice were 18-20 months old. All mice used in these studies were female.

[0134] Muscle stem cell isolation We isolated and enriched muscle stem cells as previously described. 1、5、6Briefly, mild collagenase digestion and mincing with a MACs dissociator allowed dissociation of numerous single fibers, followed by dispase digestion to liberate mononuclear cells from their niches. Cells representing hematopoietic lineages and non-myocytes were then depleted from the cell mixture using a magnetic bead column (Miltenyi) (Figure 1). - / CD11b - / CD31 - ). The remaining cell mixture was then subjected to FACS analysis to select MuSCs that co-expressed CD34 and α7-integrin markers. We generated and analyzed flow cytometry scatter plots using FlowJo v10.0. For each sort, we pooled together MuSCs (approximately 5000 each) from at least three unrelated female donor mice.

[0135] muscle stem cell transplant The inventors performed the following procedure as previously described: 1、5、6 We directly transplanted 250 MuSCs (Figures 3a, 3c, and 3d) or 1,000 MuSCs (Figure 3b) into the tibialis anterior (TA) muscle of recipient mice immediately after FACS isolation or after harvesting from cell culture. For studies of young MuSCs, we transplanted cells derived from GFP / luc mice (2–4 months old) into irradiated NOD-SCID mice in the hind limb. For studies of aged MuSCs, we transplanted cells from GFP / luc mice (2–4 months old) into irradiated NOD-SCID mice in the hind limb as previously described. 5We transplanted cells derived from aged C57BL / 6 mice (18-20 months, NIH) that had been transduced with luc-IRES-GFP lentivirus (GFP / luc virus) for 24 hours on the second day of culture prior to transplantation (see section Muscle Stem Cell Culture, Treatment, and Lentiviral Infection below for details). Prior to transplanting muscle stem cells, we anesthetized NOD-SCID recipient mice by intraperitoneal injection of ketamine (2.4 mg per mouse). We then irradiated the hind limbs once with a dose of 18 Gy while the rest of the body was shielded in a lead jig. We performed transplantation within 2 days of irradiation. Cultured cells were treated as described (vehicle or PGE2 treated 10 ng / ml), harvested from hydrogel cultures by incubation with 0.5% trypsin in PBS for 2 min at 37°C, and counted using a hemocytometer. We resuspended the cells in 0.1% gelatin / PBS at the desired cell concentration and then transplanted them (250 MuSCs per TA) by intramuscularly injecting a volume of 10 μl into the TA muscle. For new MuSC transplants, we co-injected the sorted cells with 13 nmol of 16,16-dimethyl prostaglandin E2 (dmPGE2) (Tocris, Cat. No. 4027) or vehicle control (PBS). We compared cells derived from different conditions by transplanting into the TA muscle of the opposite lower limb of the same mice. One month after transplantation, we added 10 μl of notexin (10 μg / ml -1 MuSCs were then injected into the recipient muscle to injure it and activate the MuSCs in vivo. Eight weeks after transplantation, the mice were euthanized and the TAs were harvested for analysis.

[0136] Bioluminescence imaging The inventors performed the following procedure as previously described: 1、5、6Bioluminescence imaging (BLI) was performed using a Xenogen-100 system. Briefly, we anesthetized mice by isofluorane inhalation and administered 120 μL of D-luciferin (0.1 mmol / kg 200 mM NaCl) by intraperitoneal injection. -1 , reconstituted in PBS, Caliper LifeSciences) was administered. We acquired BLI 5 min after luciferin injection using an exposure time of 60 s at F-stop = 1.0. Digital images were recorded and analyzed using Living Image software (Caliper LifeSciences). We analyzed images using consistent regions of interest (ROIs) placed throughout each hind paw to calculate the bioluminescence signal. We determined the luminescence signal (units: radians (ps -1 cm -2 sr -1 )) to 10 4 This radiance threshold of 10 4 is approximately equal to the previously reported total flux threshold in units of p / s. This BLI threshold corresponds to the histological detection of one or more GFP+ myofibers. 1、5、6 We performed BLI imaging every week after transplantation.

[0137] muscle injury We administered 10 μl of notexin (10 μg / ml -1 An injury model involving intramuscular injection of cryotoxin (10 μM; Latoxan) or cardiotoxin (10 μM; Latoxan) was used. For cold injury, an incision was made in the skin overlying the TA muscle and a copper probe cooled in liquid nitrogen was applied to the TA muscle for three 10-second intervals, allowing the muscle to thaw between each cryoprobe application. When required, 48 hours after injury, the TA muscle was injected with either 16,16-dimethyl prostaglandin E2 (dmPGE2) (13 nmol, Tocris, Cat. No. 4027) or vehicle control (PBS). The contralateral TA was used as an internal control. We harvested tissue for analysis 14 days after injury.

[0138] Pax7 CreERT2 For the Rosa26-LSL-Luc mouse experiment, we treated the mice with five consecutive daily intraperitoneal injections of tamoxifen to activate luciferase expression under the control of the Pax7 promoter. One week after the last tamoxifen injection, the mice were subjected to an intramuscular injection of 10 μl of cardiotoxin (10 μM; Latoxan), which we considered as day 0 of the assay. Three days later, either 13 nmol of dmPGE2 (13 nmol) or vehicle control (PBS) was injected into the TA muscle. The contralateral TA was used as an internal control. Bioluminescence was examined on days 3, 7, 10, and 14 after injury.

[0139] Histological examination of tissues The inventors performed the following procedure as previously described: 5、6Recipient TA muscle tissue was harvested and prepared for histological examination. We incubated cross sections with primary anti-laminin (Miliipore, clone A5, catalog number 05-206, 1:200) and anti-PAX7 (Santa Craz Biotechnology, catalog number sc-81648, 1:50) antibodies, followed by AlexaFluor secondary antibody (Jackson ImmunoResearch Laboratories, 1:200). We counterstained nuclei with DAPI (Invitrogen). We acquired images using an AxioPlan2 epifluorescence microscope (Carl Zeiss Microimaging) with a Plan NeoFluar 10x / 0.30NA objective or a Plan NeoFluar 20x / 0.75NA objective (Carl Zeiss) and an ORCA-ER digital camera (Hamamatsu Photonics) managed by SlideBook (3i) software. The images were cropped by Adobe Photoshop with a consistent contrast adjustment for all images from the same experiment. Composite images were created using Adobe Illustrator. We analyzed the number of PAX7 positive cells using MetaMorph image analysis software (Molecular Devices) and analyzed fiber area using the Baxter algorithm for myofiber analysis, which identifies fibers, segments fibers in the image, and analyzes the area of ​​each fiber. For PAX7 quantification, we examined serial sections of the TA spanning at least 2 mm depth. For fiber area, at least 10 fields of laminin-stained myofiber cross-sections containing more than 400 myofibers were captured for each mouse. Data analysis was blinded. The investigators performing image acquisition and scoring were unaware of the treatment conditions given to the sample groups being analyzed.

[0140] Hydrogel fabrication The inventors performed the following procedure as previously described: 6Polyethylene glycol (PEG) hydrogels were made from a synthesized PEG precursor. Briefly, we fabricated the hydrogels by using a published formulation to achieve a 1 mm thick hydrogel with a stiffness of 12 kPa (Young's modulus), which is the optimal condition for culturing MuSCs and maintaining stem cell fate in culture. 6 We fabricated 12 kPa hydrogel microwell arrays for clonal growth experiments as previously described. 6 We cut and attached all hydrogels to cover the surface area of ​​a 12-well or 24-well culture plate.

[0141] Muscle stem cell culture, treatment, and lentiviral infection After isolation, we cultured the cells in DMEM / F10 (50:50), 15% FBS, 2.5 ng -1 MuSCs were resuspended in myogenic culture medium containing 100 / ml fibroblast growth factor-2 (FGF-2, also known as bFGF), and 1% penicillin-streptomycin. 2MuSC suspensions were seeded at a density of 500 cells per well. We maintained cell cultures at 37°C in 5% CO2 and changed the medium daily. To study PGE2 treatment, 15-PGDH inhibitor treatment, and EP4 receptor antagonist treatment, we added 1-200 ng / ml prostaglandin E2 (Cayman Chemical) (10 ng / ml was the standard concentration used unless specified in the figure legends), and / or 1 μM EP4 antagonist (ONO-AE3-208, Cayman Chemical), or 1 μM 15-PGDH inhibitor (SW033291, Cayman Chemical) to MuSCs cultured in collagen-coated dishes for the first 24 hours. We then trypsinized the cells and reseeded the cells on the hydrogels for further culture for 6 days. All treatments were compared to a solvent (DMSO) vehicle control. For the depleted serum assay, we used DMEM / F10 (50:50), 15% charcoal stripped FBS (Gibco, Cat. No. 12676011), 2.5 ng / ml -1 Isolated MuSCs were resuspended in medium containing bFGF and 1% penicillin-streptomycin. Where figures are annotated, we further supplemented serum-free cell culture medium with 1.5 μg / ml insulin (Sigma, I0516) and 0.25 μM dexamethasone (Sigma, D8893). For these experiments, MuSCs were cultured on hydrogels and vehicle (DMSO) or 10 ng / ml PGE2 (Cayman Chemical) was added to the cultures at each medium change (every 2 days). Proliferation (see below) was examined after 7 days. Unless otherwise noted, we performed all MuSC culture assays and transplantation after 1 week of culture. When studying the transplantation of senescent MuSCs, we performed the same procedure as previously described. 3 MuSCs were infected with a lentivirus encoding elongation factor 1a promoter-driven luc-IRES-GFP (GFP / luc virus) for 24 h in culture. f / fWhen studying MuSCs, we isolated MuSCs as described above (Muscle stem cell isolation), infected all cells with GFP / luc virus, and co-infected a portion of them with lentivirus encoding pLM-CMV-R-Cre (mCherry / Cre virus) for 24 h in culture. pLM-CMV-R-Cre was a kind gift from Michel Sadelain (Addgene plasmid no. 27546). 7 We used senescent MuSCs (250 cells) or EP4 f / f MuSCs (1,000 cells) were transplanted into young (2-4 months) 18gy irradiated TAs of NOD-SCID recipient mice. For in vitro proliferation assays, EP4 f / f MuSCs were seeded onto hydrogels and treated with vehicle (DMSO) or 10 ng / ml PGE2 for 24 h and proliferation was examined 3 days later. At 48 h post-infection, cells were examined for GFP and / or mCherry expression using an inverted fluorescence microscope (Carl Zeiss Microimaging). MuSCs were freshly isolated from mice by FACS and kept in culture for periods up to 1 week and therefore were not assessed for mycoplasma contamination.

[0142] Proliferation Assay To examine proliferation, we used three different assays (hemocytometer, VisionBlue, and EdU). For each, we plated 1 cm2 cells with a surface area on flat hydrogels (hemocytometer and VisionBlue) or collagen-coated plates (EdU assay). 2MuSCs were seeded at a density of 500 cells per well. For cell counting with a hemocytometer, we harvested cells at the indicated time points by incubation with 0.5% trypsin in PBS at 37°C for 5 min and quantified them at least three times using a hemocytometer. In addition, we used the VisionBlue Rapid Cell Viability Fluorescence Assay Kit (BioVision, Cat. No. K303) as a readout of cell proliferation during culture. Briefly, we incubated MuSCs with 10% VisionBlue in medium for 3 h and measured the fluorescence intensity at Ex=530-570 nm, Em=590-620 nm using a fluorescence plate reader (Infinite M1000 PRO, Tecan). We examined proliferation using the Click-iT EdU Alexa Fluor 555 Imaging Kit (Life Technologies). Briefly, we incubated live cells with EdU (20 μM) for 1 h before fixation and stained the nuclei with anti-myogenin (Santa Cruz, catalog number sc576, 1:250) according to the manufacturer's guidelines to examine differentiation. We counterstained the nuclei with DAPI (Invitrogen). We acquired images using an AxioPlan2 epifluorescence microscope (Carl Zeiss Microimaging) with a Plan NeoFluar 10x / 0.30NA objective or a Plan NeoFluar 20x / 0.75NA objective (Carl Zeiss) and an ORCA-ER digital camera (Hamamatsu Photonics) managed by SlideBook (3i) software. We quantified EdU-positive cells using MetaMorph image analysis software (Molecular Devices). Data analysis was blinded, and the investigator performing the cell scoring was unaware of the treatment conditions given to the sample groups being analyzed.

[0143] Proliferation and fate analysis of clonal muscle stem cells The inventors performed the following procedure as previously described: 5、6We investigated the proliferation of clonal muscle stem cells by time-lapse microscopy. Briefly, we treated isolated senescent MuSCs with PGE2 (Cayman Chemical) or vehicle (DMSO) for 24 h. After 5 days of growth on the hydrogel, the cells were plated in 600 μm diameter hydrogel microwells with a surface area of ​​1 cm. 2 Single cells were reseeded at a density of 500 cells per well. For time-lapse microscopy, we monitored cell growth in the above wells containing single cells starting 12 hours after seeding (day 0) until day 2, recording images every 3 minutes at 10x magnification using a PALM / AxioObserver Z1 system (Carl Zeiss MicroImaging) equipped with a custom environmental chamber and a motorized stage. We changed the medium every other day during the acquisition time interval. We analyzed the time-lapse image series using the Baxter algorithm for cell tracking and lineage reconstruction to identify and track single cells and generate lineage trees. 5、6、8~10 .

[0144] Live and dead cells were differentiated in a series of time-lapse photographs based on the phase contrast boundary and the maintenance or reduction of motility, respectively. We found that the rates of proliferation (division) and death in the two conditions changed over time. Therefore, we estimated the rates for the first 24-hour period and the second 24-hour period separately. These values ​​were: 6 The growth rates in the two periods were estimated using the formula described in and listed in Table 1. 24 and p 48 and the corresponding death rate d 24 and d 48 As an example, the growth rate in the treatment condition for the second 24 hour period is 5.38% / hr. Table 1 (below) shows that the growth and death rates in the two conditions are similar for the first period, and that the difference in cell numbers at the end of the experiment is due to differences in both division and death rates during the second period. The modeled cell numbers for these two periods are given by the following equation: TIFF2024116386000004.tif11128 where c0 is the starting cell number. The model curve is plotted together with the actual cell number in Figure 7F.

[0145] (Table 1) Estimated growth rate and death rate per hour TIFF2024116386000005.tif28142

[0146] Data analysis was blinded: the investigators performing the image acquisition and scoring were unaware of the treatment conditions given to the sample groups being analyzed.

[0147] Quantitative RT-PCR We isolated RNA from MuSCs using RNeasy micro kit (Qiagen). For muscle samples, we flash-frozen the tissue in liquid nitrogen, homogenized the tissue using a mortar and pestle, followed by grinding with a syringe and needle, and then isolated RNA using Trizol (Invitrogen). We reverse-transcribed cDNA from total mRNA from each sample using SensiFAST™ cDNA synthesis kit (Bioline). We subjected the cDNA to RT-PCR using SYBR Green PCR master mix (Applied Biosystems) or TaqMan Assays (Applied Biosystems) in an ABI 7900HT real-time PCR system (Applied Biosystems). We cycled the samples at 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. To quantify relative transcript levels, we compared treated and untreated samples using 2-ΔΔCt and expressed the results relative to Gapdh. For SYBR Green qRT-PCR, we used the following primer sequences: TIFF2024116386000006.tif84151TaqMan assays (Applied Biosystems) were used with the TaqMan Universal PCR Master Mix Reagent Kit (Applied Biosystems) according to the manufacturer's instructions to quantify Pax7, Myogenin, Slco2a1 (PGT), Ptger3, and Ptger4 in the samples. Transcript levels were expressed relative to Gapdh levels. For SYBR Green qPCR, Gapdh qPCR was used to normalize input cDNA samples. For Taqman qPCR, multiplex qPCR allowed target signals (FAM) to be individually normalized based on the internal Gapdh signal (VIC).

[0148] PGE2 ELISA Muscles were harvested, rinsed in ice-cold PBS containing indomethacin (5.6 μg / ml) and flash frozen in liquid nitrogen. Frozen samples were pulverized in liquid nitrogen. The powder was transferred to an Eppendorf tube containing 500 μl of lysis buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 4 mM CaCl, 1.5% Triton X-100, protease inhibitors, and micrococcal nuclease) and then homogenized using a tissue homogenizer. PGE2 levels in the supernatants were measured using a PGE2 ELISA kit (R&D Systems, Cat. No. KGE004B) and expressed relative to total protein measured by BCA assay (BioRad) and expressed as ng of PGE2. Each sample was examined in duplicate in each of two independent experiments.

[0149] cAMP Activity Assay MuSCs were treated with DMSO (vehicle) or PGE2 (10 ng / ml) for 1 h, and cyclic AMP levels were measured according to the cAMP-Glo ​​assay protocol optimized by the manufacturer (Promega). Each sample was tested in triplicate and in two independent experiments.

[0150] Flow cytometry We examined Annexin V as a readout of apoptosis in MuSCs after initial short-term (24 h) treatment with vehicle (DMSO) or PGE2 (10 ng / ml) and cultured on hydrogels for 7 days. We used a FITC Annexin V Apoptosis Detection Kit (Biolegend, Cat. No. 640914) according to the manufacturer's protocol. We analyzed cells for Annexin V by a FACS LSR II cytometer using FACSDiva software (BD Biosciences) in a shared FACS facility purchased with an NIH S10 shared equipment grant (S10RR027431-01).

[0151] mass spectrometry Analyte: Prostaglandin standards—PGF2α; PGE2; PGD2: 15-keto PGE2; 13,14-dihydro 15-keto PGE2; PGE2-D4; and PGF2α-D9—were all purchased from Cayman Chemical. For the PGE2-D4 internal standard, positions 3 and 4 were labeled with a total of four deuterium atoms. For PGF2α-D9, positions 17, 18, 19, and 20 were labeled with a total of nine deuterium atoms.

[0152] Creating a calibration curve: Analyte stock solutions (5 mg / mL) were prepared in DMSO. These stock solutions were serially diluted with acetonitrile / water (1:1 by volume) to obtain a series of standard working solutions, which were used to generate calibration curves. Calibration curves were generated by adding 10 uL of each working solution to 200 μL of homogenization buffer (1:1 acetone / water by volume; 0.005% BHT to prevent oxidation), followed by the addition of 10 uL of internal standard solution (3000 ng / mL, PGF2α-D9 and PGE2-D4, respectively). Calibration curves were generated fresh with each sample set. Calibration curve ranges: for PGE2 and 13,14-dihydro15-ketoPGE2, 0.05 ng / mL to 500 ng / mL; for PGD2 and PGF2α, 0.1 ng / mL to 500 ng / mL; and for 15-ketoPGE2, 0.025 ng / mL to 500 ng / mL.

[0153] Extraction Procedure: The extraction procedure was as described by Prasain et al. 11 The method was modified from that of previous studies and involved acetone protein precipitation followed by a two-step liquid-liquid extraction; the latter step enhances the sensitivity of LC-MS / MS. Butylated hydroxytoluene (BHT) and evaporation under nitrogen (N2) gas were used to prevent oxidation.

[0154] Solid tissues were harvested, weighed, and flash frozen using liquid nitrogen. Muscle tissues were mixed with homogenization beads and 200 μL of homogenization buffer in a polypropylene tube and processed in a FastPrep 24 homogenizer (MP Biomedicals) at a speed of 6 m / s for 40 seconds. After homogenization, 10 μl of internal standard solution (3000 ng / mL) was added to the tissue homogenate, followed by sonication and shaking for 10 minutes. The samples were centrifuged and the supernatant was transferred to a clean Eppendorf tube. 200 μL of hexane was added to the sample, followed by shaking for 15 minutes and then centrifugation. The samples were frozen at -80°C for 40 minutes. The hexane layer was poured off from the frozen lower aqueous layer and discarded. After thawing, 25 μL of 1N formic acid was added to the lower aqueous layer and the samples were vortexed. For the second extraction, 200 μL of chloroform was added to the aqueous phase. The samples were shaken for 15 min to ensure complete extraction. Centrifugation was performed to separate the layers. The lower chloroform layer was transferred to a new Eppendorf tube and evaporated to dryness under nitrogen at 40° C. The dried residue was reconstituted in 100 μl of acetonitrile / 10 mM ammonium acetate (volume ratio 2:8) and analyzed by LC-MS / MS.

[0155] LC-MS / MS: Chromatographic separation is essential since many prostaglandins are positional isomers with the same mass and similar fragmentation patterns. Two SRM transitions—one quantifier and one qualifier—were carefully selected for each analyte. Characteristic qualifier ion intensity ratios and retention times were essential to confirm the target analytes. All analyses were performed on an LC-20AD XRThe analysis was carried out by negative electrospray LC-MS / MS using a Prominence liquid chromatograph and 8030 triple quadrupole mass spectrometer (Shimadzu). HPLC conditions: An Acquity UPLC BEH C18 2.1x100mm, 1.7um particle size column was run at 50°C with a flow rate of 0.25mL / min. The mobile phase consisted of A: 0.1% acetic acid in water and B: 0.1% acetic acid in acetonitrile. Elution profile: initial hold at 35% B for 5 min, followed by a gradient from 35% to 40% in 3 min, then 40% to 95% in 3 min; total run time was 14 min. Injection volume was 20μL. Using these HPLC conditions, we achieved baseline separation of the analytes of interest.

[0156] Selected reaction monitoring (SRM) was used for quantification. Mass transitions were as follows: PGD2: m / z 351.10 → m / z 315.15 (Quantifier) ​​and m / z 351.10 → m / z 233.05 (Qualifier); PGE2: m / z 351.10 → m / z 271.25 (Quantifier) ​​and m / z 351.10 → m / z 315.20 (Qualifier); PGF2α: m / z 353.10 → m / z 309.20 (Quantifier) ​​and m / z 353.10 → m / z 19 3.20 (Qualifier); 15-keto-PGE2: m / z 349.30 → m / z 331.20 (Quantifier) ​​and m / z 349.30 → m / z 113.00 (Qualifier); 13,14-dihydro 15-keto PGE2: m / z 351.20 → m / z 333.30 (Quantifier) ​​and m / z 351.20 → m / z 113.05 (Qualifier); PGE2-D4; m / z 355.40 → m / z 275.20; and PGF2α-D9: m / z 362.20 → m / z 318.30. Retention times were 20-30 ms.

[0157] Quantitative analysis was performed using LabSolutions LCMS (Shimadzu). The internal standard method was used for quantification: PGE2-D4 was used as the internal standard for the quantification of PGE2, 15-ketoPGE2, and 13,14-dihydro15-ketoPGE2. PGF2α-D9 was the internal standard for the quantification of PGD2 and PGF2α. 1 / X 2 Calibration curves were linear (R>0.99) over the concentration range with a weighting factor of (X is the concentration). Back-calculated standard concentrations were ±15% from the nominal values ​​and ±20% at the lower limit of quantification (LLOQ).

[0158] In vivo muscle force measurements Aged mice (18 months) were subjected to downhill treadmill running for 2 consecutive weeks. During the first week, the mice ran daily for 5 days and rested on the 6th and 7th days. Two hours after each treadmill run during the first week, each mouse was injected with one dose of either PBS (vehicle control) or 13 nM dmPGE2 (experimental group) into each (lateral and medial) gastrocnemius (GA) muscle of both legs. During the second week, the mice were subjected to treadmill running only for 5 days. Treadmill running was performed using an Exer3 / 6 (Columbus Instruments). The mice ran on a 20° downhill treadmill for 10 min, starting at a speed of 7 m / min. After 3 min, the speed was increased by 1 m / min to a final speed of 14 m / min. A 10-min running time was chosen because exhaustion, defined as the inability of the animal to remain on the treadmill despite electrical stimulation, was observed at a median of 12 min in an independent control group of aged mice. Force measurements were performed in GA muscles at week 5, based on a previously published protocol. 5Briefly, for each mouse, an incision was made to expose the GA. We cut the calcaneus with the intact Achilles tendon and attached the tendon-bone complex to a 300C-LR force transducer (Aurora Scientific) using a fine metal hook. The muscle and tendon were kept moist by periodic wetting with saline (0.9% sodium chloride) solution. The lower limbs were immobilized below the knee using metal clamps to avoid compromising the blood supply to the leg. Throughout the force measurement procedure, the mice received an inhalation anesthetic (2% isofluorane) and body temperature was maintained by a heat lamp. For both measurements, we used 0.1 ms pulses at the prescribed supramaximal stimulation voltage. The GA muscle was stimulated via the proximal sciatic nerve using a bipolar electrical stimulation cuff delivering a constant current of 2 mA (square wave width 0.1 ms). For twitch force measurements, the GA muscle was stimulated with a single 0.1 ms pulse, and for tetanic force measurements, the GA muscle was stimulated with a train of 150 Hz, 0.3 s pulses. We performed five twitch force measurements followed by five tetanic force measurements for each muscle, with n=5 mice per group and 2-3 min recovery between measurements. Data were collected using a PCI-6251 acquisition card (National Instruments) and analyzed in Matlab. We calculated per unit force values ​​by standardizing force measurements based on the physiological cross-sectional area (PCSA) of the muscle, which was similar in the control and PGE2-treated experimental groups (Table 2). PCSA (units mm 2 (measured at) was calculated according to the following formula 12 : PCSA(mm 2 )=[Mass(g)×Cosθ]÷[p(g / mm 3 ) × fiber length (mm)] In the above formula, θ is the fiber pennation angle and p is the muscle density (0.001056 g / mm 3 ).

[0159] statistical analysis We performed cell culture experiments in at least three independent experiments, with three biological replicates for each experiment. Typically, we performed MuSC transplantation experiments in at least two independent experiments, with a total of at least three to five transplants per condition. We used paired t-tests for the experiments, with control samples coming from the same experiment in vitro or from the contralateral limb muscle in vivo. A nonparametric Mann-Whitney test was used to determine significant differences between the untreated group (-) and the treated groups (PGE or dmPGE2) with α = 0.05. ANOVA or multiple t-tests were performed for multiple comparisons, with significance levels determined using Bonferroni correction or Fisher's test as indicated in the figure legends. Data are presented as mean ± standard error of the mean, unless otherwise stated.

[0160] Method References TIFF2024116386000007.tif71146TIFF2024116386000008.tif71146

[0161] (Table 2) Physiological cross-sectional area (PCSA) of aged gastrocnemius muscle 5 weeks after exercise TIFF2024116386000009.tif130146

[0162] Example 2: Increased muscle strength after prostaglandin E2 (PGE2) injection This example shows that gastrocnemius muscle twitch force per unit is increased in aged mice injected with PGE2. Aged mice (18 months old) were subjected to treadmill running until exhaustion for 10 days daily. Treadmill running was performed using Exer3 / 6 (Columbus Instruments). Mice ran on a 20° downhill treadmill starting at a speed of 10 meters / min. After 3 minutes, the speed was increased by 1 meter / min to a final speed of 20 meters / min. Exhaustion was defined as the inability of the animals to remain on the treadmill despite electrical stimulation. Two hours after each treadmill run, both gastrocnemius muscles of each mouse were injected with either PBS (control group) or 3 nM PGE2 (experimental group). Four weeks after the last treadmill run, force measurements were performed using a 300C-LR force transducer (Aurora Scientific) with a single 0.1 ms pulse at the prescribed supramaximal stimulation voltage.

[0163] Representative raw force traces of a single gastrocnemius muscle are provided in Figures 4M-4N. Muscle forces and synchronous pulses were recorded through a PCI-6251 acquisition card (National Instruments) and analyzed using Matlab. Figures 4O-4P show per-unit muscle force values, calculated by normalizing force measurements using the physiological cross-sectional area of ​​the muscle. Per-unit twitch force values ​​(kN / m 2 ) are presented with box plots showing minimum, maximum and median values. Five replicate measurements were taken from each muscle. N=4 for control group and n=5 for PGE2-injected group. ** denotes statistically significant value of p<0.005 by two-tailed Mann-Whitney test.

[0164] VI. EXEMPLARY EMBODIMENTS Exemplary embodiments provided in accordance with the presently disclosed subject matter include, but are not limited to, the following embodiments and the claims. 1. Culturing an isolated population of muscle cells with a compound selected from the group consisting of prostaglandin E2 (PGE2), a PGE2 prodrug, a PGE2 receptor agonist, a compound that attenuates PGE2 catabolism, a compound that negates PGE2 inhibition, derivatives thereof, analogs thereof, and combinations thereof. 23. A method for promoting proliferation of an isolated muscle cell population, comprising: 2. The method of embodiment 1, wherein the isolated muscle cells are purified. 3. The method of embodiment 1 or 2, wherein the isolated muscle cells comprise skeletal muscle cells, smooth muscle cells, cardiac muscle cells, embryonic stem cell-derived muscle cells, induced pluripotent stem cell-derived muscle cells, dedifferentiated muscle cells, or a combination thereof. 4. The method of any one of aspects 1-3, wherein the isolated muscle cell population comprises muscle stem cells, satellite cells, myocytes, myoblasts, myotubes, myofibers, or a combination thereof. 5. The method of any one of aspects 1 to 4, wherein the isolated muscle cells are obtained from a subject. 6. The method of embodiment 5, wherein the subject has a condition or disease involving muscle damage, injury, or atrophy. 7. The method of embodiment 6, wherein the condition or disease involving muscle damage, injury, or atrophy is selected from the group consisting of acute muscle injury or trauma, soft tissue injury of the hand, Duchenne muscular dystrophy (DMD), Becker muscular dystrophy, limb-girdle muscular dystrophy, amyotrophic lateral sclerosis (ALS), distal muscular dystrophy (DD), hereditary myopathy, myotonic muscular dystrophy (MDD), mitochondrial myopathy, myotubular myopathy (MM), myasthenia gravis (MG), congestive heart failure, periodic paralysis, polymyositis, rhabdomyolysis, dermatomyositis, cancer cachexia, AIDS cachexia, cardiac cachexia, stress-induced urinary incontinence, and sarcopenia. 8. The method of any one of aspects 1-7, wherein the PGE2 derivative comprises 16,16-dimethyl prostaglandin E2. 9. Compounds that attenuate PGE2 catabolism Compounds, neutralizing peptides, or neutralizing antibodies that inactivate or interfere with 15-hydroxyprostaglandin dehydrogenase (15-PGDH) or inactivate or interfere with prostaglandin transporters (PTG or SLCO2A1) The method according to any one of aspects 1 to 7, comprising: 10. The method of any one of aspects 1-9, wherein the step of culturing the isolated muscle cells with a compound comprises exposing the isolated muscle cells to the compound acutely, intermittently, or continuously. 11. Culturing the isolated muscle cell population with an effective amount of a compound selected from the group consisting of prostaglandin E2 (PGE2), a PGE2 prodrug, a PGE2 receptor agonist, a compound that attenuates PGE2 catabolism, a compound that negates PGE2 inhibition, derivatives thereof, analogs thereof, and combinations thereof to promote muscle cell engraftment in the subject; and Administering the cultured muscle cells to the subject. 23. A method for promoting muscle cell engraftment in a subject, comprising: 12. The method of embodiment 11, wherein the isolated muscle cells are purified. 13. The method of embodiment 11 or 12, wherein the isolated muscle cells comprise skeletal muscle cells, smooth muscle cells, cardiac muscle cells, embryonic stem cell-derived muscle cells, induced pluripotent stem cell-derived muscle cells, dedifferentiated muscle cells, or a combination thereof. 14. The method of any one of aspects 11-13, wherein the isolated muscle cell population comprises muscle stem cells, satellite cells, myocytes, myoblasts, myotubes, myofibers, or a combination thereof. 15. The method of any one of aspects 11 to 14, wherein the isolated muscle cells are autologous to the subject. 16. The method of any one of aspects 11 to 14, wherein the isolated muscle cells are allogeneic to the subject. 17. The method of any one of aspects 11 to 16, wherein the subject has a condition or disease involving muscle damage, injury, or atrophy. 18. The method of embodiment 17, wherein the condition or disease involving muscle damage, injury, or atrophy is selected from the group consisting of acute muscle injury or trauma, soft tissue injury of the hand, Duchenne muscular dystrophy (DMD), Becker muscular dystrophy, limb-girdle muscular dystrophy, amyotrophic lateral sclerosis (ALS), distal muscular dystrophy (DD), hereditary myopathy, myotonic muscular dystrophy (MDD), mitochondrial myopathy, myotubular myopathy (MM), myasthenia gravis (MG), congestive heart failure, periodic paralysis, polymyositis, rhabdomyolysis, dermatomyositis, cancer cachexia, AIDS cachexia, cardiac cachexia, stress-induced urinary incontinence, and sarcopenia. 19. The method of any one of aspects 11 to 18, wherein the PGE2 derivative comprises 16,16-dimethyl prostaglandin E2. 20. Compounds that attenuate PGE2 catabolism Compounds, neutralizing peptides, or neutralizing antibodies that inactivate or interfere with 15-hydroxyprostaglandin dehydrogenase (15-PGDH) or inactivate or interfere with prostaglandin transporters (PTG or SLCO2A1) 19. The method according to any one of aspects 11 to 18, comprising: 21. The method of any one of aspects 11-20, wherein the step of culturing the isolated muscle cell population with the compound comprises exposing the isolated muscle cell population to the compound on a short-term, intermittent, or continuous basis. 22. A composition comprising an isolated group of muscle cells and a compound selected from the group consisting of prostaglandin E2 (PGE2), a PGE2 prodrug, a PGE2 receptor agonist, a compound that attenuates PGE2 catabolism, a compound that negates PGE2 inhibition, derivatives thereof, analogs thereof, and combinations thereof. 23. The composition of embodiment 22, wherein the isolated muscle cells comprise skeletal muscle cells, smooth muscle cells, cardiac muscle cells, embryonic stem cell-derived muscle cells, induced pluripotent stem cell-derived muscle cells, dedifferentiated muscle cells, or a combination thereof. 24. The composition of embodiment 22 or 23, wherein the isolated muscle cells comprise muscle stem cells, satellite cells, myocytes, myoblasts, myotubes, muscle fibers, or a combination thereof. 25. The composition of any one of aspects 22 to 24, further comprising a pharma- ceutically acceptable carrier. 26. A kit comprising the composition of any one of aspects 22 to 25 and instructions for use. 27. A method for regenerating muscle cells in a subject having a condition or disease involving muscle damage, injury, or atrophy, comprising: administering to the subject a therapeutically effective amount of a compound selected from the group consisting of prostaglandin E2 (PGE2), a PGE2 prodrug, a PGE2 receptor agonist, a compound that attenuates PGE2 catabolism, a compound that negates PGE2 inhibition, derivatives thereof, analogs thereof, and combinations thereof, and a pharma- ceutically acceptable carrier to increase muscle cell population and / or enhance muscle function in the subject. A method comprising: 28. The method of embodiment 27, wherein the condition or disease involving muscle damage, injury, or atrophy is selected from the group consisting of acute muscle injury or trauma, soft tissue injury of the hand, Duchenne muscular dystrophy (DMD), Becker muscular dystrophy, limb-girdle muscular dystrophy, amyotrophic lateral sclerosis (ALS), distal muscular dystrophy (DD), hereditary myopathy, myotonic muscular dystrophy (MDD), mitochondrial myopathy, myotubular myopathy (MM), myasthenia gravis (MG), congestive heart failure, periodic paralysis, polymyositis, rhabdomyolysis, dermatomyositis, cancer cachexia, AIDS cachexia, cardiac cachexia, stress-induced urinary incontinence, and sarcopenia. 29. The method of embodiment 27 or 28, wherein the muscle cells comprise skeletal muscle cells, smooth muscle cells, cardiac muscle cells, embryonic stem cell-derived muscle cells, induced pluripotent stem cell-derived muscle cells, dedifferentiated muscle cells, or a combination thereof. 30. The method of any one of aspects 27-29, wherein the muscle cell population comprises muscle stem cells, satellite cells, myocytes, myoblasts, myotubes, muscle fibers, or a combination thereof. 31. The method of any one of aspects 27 to 30, wherein the PGE2 derivative comprises 16,16-dimethyl prostaglandin E2. 32. A compound that attenuates PGE2 catabolism is Compounds, neutralizing peptides, or neutralizing antibodies that inactivate or interfere with 15-hydroxyprostaglandin dehydrogenase (15-PGDH) or inactivate or interfere with prostaglandin transporters (PTG or SLCO2A1) The method according to any one of aspects 27 to 30, comprising: 33. The method of any one of aspects 27-32, wherein the step of administering the compound comprises oral, intraperitoneal, intramuscular, intraarterial, intradermal, subcutaneous, intravenous, or intracardiac administration. 34. The method of any one of aspects 27-33, wherein the compound is administered according to a short-term treatment regimen. 35. The method of any one of aspects 27-34, wherein the administering step further comprises administering to the subject an isolated muscle cell population. 36. The method of embodiment 35, wherein the isolated muscle cells are autologous to the subject. 37. The method of embodiment 35, wherein the isolated muscle cells are allogeneic to the subject. 38. The method of any one of aspects 35 to 37, wherein the isolated muscle cell population is purified. 39. The method of any one of aspects 35-38, wherein the isolated muscle cells are cultured with the compound prior to administration to the subject. 40. The method of embodiment 39, wherein culturing the isolated muscle cell population with a compound comprises exposing the isolated muscle cell population to the compound on a short-term, intermittent, or continuous basis. 41. The method of any one of aspects 35 to 40, wherein administering the isolated muscle cells comprises injecting or transplanting the cells into the subject. 42. The method of any one of aspects 35-41, wherein the isolated muscle cells and the compound are administered to the subject simultaneously. 43. The method of any one of aspects 35-41, wherein the isolated muscle cells and the compound are administered sequentially to the subject. 44. Administering to a subject (i) a therapeutically effective amount of a compound selected from the group consisting of prostaglandin E2 (PGE2), a PGE2 prodrug, a PGE2 receptor agonist, a compound that attenuates PGE2 catabolism, a compound that negates PGE2 inhibition, derivatives thereof, analogs thereof, and combinations thereof, and a pharma- ceutically acceptable carrier, and (ii) a population of isolated muscle cells, to prevent or treat a condition or disease involving muscle damage, injury, or atrophy. 23. A method for preventing or treating a condition or disease involving muscle damage, injury, or atrophy in a subject in need thereof, comprising: 45. The method of embodiment 44, wherein the PGE2 derivative comprises 16,16-dimethyl prostaglandin E2. 46. ​​A compound that attenuates PGE2 catabolism is Compounds, neutralizing peptides, or neutralizing antibodies that inactivate or interfere with 15-hydroxyprostaglandin dehydrogenase (15-PGDH) or inactivate or interfere with prostaglandin transporters (PTG or SLCO2A1) 45. The method of embodiment 44, comprising: 47. The method of any one of aspects 44-46, wherein the isolated muscle cells comprise skeletal muscle cells, smooth muscle cells, cardiac muscle cells, embryonic stem cell-derived muscle cells, induced pluripotent stem cell-derived muscle cells, dedifferentiated muscle cells, or a combination thereof. 48. The method of any one of aspects 44-47, wherein the isolated muscle cell population comprises muscle stem cells, satellite cells, myoblasts, myocytes, myotubes, muscle fibers, or a combination thereof. 49. The method of any one of aspects 44 to 48, wherein the isolated muscle cell population is purified. 50. The method of any one of aspects 44-49, wherein the isolated muscle cells are cultured with the compound prior to administration to the subject. 51. The method of embodiment 50, wherein culturing the isolated muscle cell population with a compound comprises exposing the isolated muscle cell population to the compound acutely, intermittently, or continuously. 52. The method of any one of aspects 44-51, wherein the isolated muscle cells are autologous to the subject. 53. The method of any one of aspects 44-51, wherein the isolated muscle cells are allogeneic to the subject. 54. The method of any one of aspects 44-53, wherein the step of administering the compound comprises oral, intraperitoneal, intramuscular, intraarterial, intradermal, subcutaneous, intravenous, or intracardiac administration. 55. The method of any one of aspects 44-54, wherein the compound is administered according to a short-term treatment regimen. 56. The method of any one of aspects 44 to 55, wherein administering the isolated muscle cells comprises injecting or transplanting the cells into the subject. 57. The method of any one of aspects 44-56, wherein the compound and the isolated muscle cells are administered to the subject simultaneously. 58. The method of any one of aspects 44-56, wherein the compound and the isolated muscle cells are administered sequentially to the subject. 59. The method of any one of aspects 44 to 58, wherein the subject is suspected of having, or is at risk of developing, a condition or disease involving muscle damage, injury, or wasting. 60. The method of any one of aspects 44 to 59, wherein the condition or disease involving muscle damage, injury, or atrophy is selected from the group consisting of acute muscle injury or trauma, soft tissue injury of the hand, Duchenne muscular dystrophy (DMD), Becker muscular dystrophy, limb-girdle muscular dystrophy, amyotrophic lateral sclerosis (ALS), distal muscular dystrophy (DD), hereditary myopathy, myotonic muscular dystrophy (MDD), mitochondrial myopathy, myotubular myopathy (MM), myasthenia gravis (MG), congestive heart failure, periodic paralysis, polymyositis, rhabdomyolysis, dermatomyositis, cancer cachexia, AIDS cachexia, cardiac cachexia, stress-induced urinary incontinence, and sarcopenia.

[0165] Although the foregoing invention has been described in some detail by way of illustration and example for clarity of understanding, those skilled in the art will appreciate that certain changes and modifications may be practiced within the scope of the appended claims. Further, each reference provided herein is incorporated by reference in its entirety to the same extent as if each reference was individually incorporated by reference.

[0166] Unofficial sequence listing TIFF2024116386000010.tif156128

[0167] Sequence information SEQUENCE LISTING <110> The Board of Trustees of the Leland Stanford Junior University <120> COMPOSITIONS AND METHODS FOR MUSCLE REGENERATION USING PROSTAGLANDIN E2 <150> US 62 / 303,979 <151> 2016-03-04 <150> US 62 / 348,116 <151> 2016-06-09 <160> 12 <170> PatentIn version 3.5 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Gapdh forward primer <400> 1 ttcaccacca tggagaaggc 20 <210> 2 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Gapdh reverse primer <400> 2 cccttttggc tccaccct 18 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Hpgd forward primer <400> 3 tccagtgtga tgtggctgac 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Hpgd reverse primer <400> 4 attgttcacg cctgcattgt 20 <210> 5 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Ptges forward primer <400> 5 gctgtcatca caggccaga 19 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Ptges reverse primer <400> 6 ctccacatct gggtcactcc 20 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Ptges2 forward primer <400> 7 ctcctacagg aaagtgccca 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Ptges2 reverse primer <400> 8 accaggtagg tcttgagggc 20 <210> 9 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Ptger1 forward primer <400> 9 gtggtgtcgt gcatctgct 19 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Ptger1 reverse primer <400> 10 ccgctgcagg gagttagagt 20 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Ptger2 forward primer <400> 11 accttcgcca tatgctcctt 20 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Ptger2 reverse primer <400> 12 ggaccggtgg cctaagtatg 20

Claims

1. culturing the isolated muscle cells with a compound selected from the group consisting of prostaglandin E2 (PGE2), a PGE2 prodrug, a PGE2 receptor agonist, a compound that attenuates PGE2 catabolism, a compound that negates PGE2 inhibition, derivatives thereof, analogs thereof, and combinations thereof.

23. A method for promoting proliferation of an isolated muscle cell population, comprising:

2. 2. The method of claim 1, wherein the isolated muscle cells are purified.

3. 2. The method of claim 1, wherein the isolated muscle cells comprise skeletal muscle cells, smooth muscle cells, cardiac muscle cells, embryonic stem cell derived muscle cells, induced pluripotent stem cell derived muscle cells, dedifferentiated muscle cells, or a combination thereof.

4. 2. The method of claim 1, wherein the isolated muscle cells comprise muscle stem cells, satellite cells, myocytes, myoblasts, myotubes, myofibers, or a combination thereof.

5. 2. The method of claim 1, wherein the isolated muscle cells are obtained from a subject.

6. The method of claim 5, wherein the subject has a condition or disease involving muscle damage, injury, or atrophy.

7. 7. The method of claim 6, wherein the condition or disease involving muscle damage, injury, or atrophy is selected from the group consisting of acute muscle injury or trauma, soft tissue injury of the hand, Duchenne muscular dystrophy (DMD), Becker muscular dystrophy, limb-girdle muscular dystrophy, amyotrophic lateral sclerosis (ALS), distal muscular dystrophy (DD), hereditary myopathy, myotonic muscular dystrophy (MDD), mitochondrial myopathy, myotubular myopathy (MM), myasthenia gravis (MG), congestive heart failure, periodic paralysis, polymyositis, rhabdomyolysis, dermatomyositis, cancer cachexia, AIDS cachexia, cardiac cachexia, stress-induced urinary incontinence, and sarcopenia.

8. 2. The method of claim 1, wherein the PGE2 derivative comprises 16,16-dimethyl prostaglandin E2.

9. Compounds that attenuate PGE2 catabolism Compounds, neutralizing peptides, or neutralizing antibodies that inactivate or interfere with 15-hydroxyprostaglandin dehydrogenase (15-PGDH) or inactivate or interfere with prostaglandin transporters (PTG or SLCO2A1) 2. The method of claim 1, comprising:

10. 2. The method of claim 1, wherein the step of culturing the isolated muscle cells with the compound comprises exposing the isolated muscle cells to the compound on a short-term, intermittent or continuous basis.

11. Culturing the isolated muscle cells with an effective amount of a compound selected from the group consisting of prostaglandin E2 (PGE2), a PGE2 prodrug, a PGE2 receptor agonist, a compound that attenuates PGE2 catabolism, a compound that negates PGE2 inhibition, derivatives thereof, analogs thereof, and combinations thereof to promote muscle cell engraftment in the subject; and Administering the cultured muscle cells to the subject.

23. A method for promoting muscle cell engraftment in a subject, comprising:

12. 12. The method of claim 11, wherein the isolated muscle cells are purified.

13. 12. The method of claim 11, wherein the isolated muscle cells comprise skeletal muscle cells, smooth muscle cells, cardiac muscle cells, embryonic stem cell derived muscle cells, induced pluripotent stem cell derived muscle cells, dedifferentiated muscle cells, or a combination thereof.

14. 12. The method of claim 11, wherein the isolated muscle cells comprise muscle stem cells, satellite cells, myocytes, myoblasts, myotubes, myofibers, or a combination thereof.

15. 12. The method of claim 11, wherein the isolated muscle cells are autologous to the subject.

16. 12. The method of claim 11, wherein the isolated muscle cells are allogeneic to the subject.

17. 12. The method of claim 11, wherein the subject has a condition or disease involving muscle damage, injury, or atrophy.

18. 18. The method of claim 17, wherein the condition or disease involving muscle damage, injury, or atrophy is selected from the group consisting of acute muscle injury or trauma, soft tissue injury of the hand, Duchenne muscular dystrophy (DMD), Becker muscular dystrophy, limb-girdle muscular dystrophy, amyotrophic lateral sclerosis (ALS), distal muscular dystrophy (DD), hereditary myopathy, myotonic muscular dystrophy (MDD), mitochondrial myopathy, myotubular myopathy (MM), myasthenia gravis (MG), congestive heart failure, periodic paralysis, polymyositis, rhabdomyolysis, dermatomyositis, cancer cachexia, AIDS cachexia, cardiac cachexia, stress-induced urinary incontinence, and sarcopenia.

19. 12. The method of claim 11, wherein the PGE2 derivative comprises 16,16-dimethyl prostaglandin E2.

20. Compounds that attenuate PGE2 catabolism Compounds, neutralizing peptides, or neutralizing antibodies that inactivate or interfere with 15-hydroxyprostaglandin dehydrogenase (15-PGDH) or inactivate or interfere with prostaglandin transporters (PTG or SLCO2A1) 12. The method of claim 11, comprising:

21. 12. The method of claim 11, wherein culturing the isolated muscle cells with the compound comprises acutely, intermittently, or continuously exposing the isolated muscle cells to the compound.

22. A composition comprising an isolated group of muscle cells and a compound selected from the group consisting of prostaglandin E2 (PGE2), a PGE2 prodrug, a PGE2 receptor agonist, a compound that attenuates PGE2 catabolism, a compound that negates PGE2 inhibition, derivatives thereof, analogs thereof, and combinations thereof.

23. 23. The composition of claim 22, wherein the isolated muscle cells comprise skeletal muscle cells, smooth muscle cells, cardiac muscle cells, embryonic stem cell derived muscle cells, induced pluripotent stem cell derived muscle cells, dedifferentiated muscle cells, or a combination thereof.

24. 23. The composition of claim 22, wherein the isolated muscle cells comprise muscle stem cells, satellite cells, myocytes, myoblasts, myotubes, muscle fibers, or a combination thereof.

25. 23. The composition of claim 22, further comprising a pharma- ceutically acceptable carrier.

26. 23. A kit comprising the composition of claim 22 and instructions.

27. 1. A method for regenerating muscle cells in a subject having a condition or disease involving muscle damage, injury, or atrophy, comprising: administering to the subject a therapeutically effective amount of a compound selected from the group consisting of prostaglandin E2 (PGE2), a PGE2 prodrug, a PGE2 receptor agonist, a compound that attenuates PGE2 catabolism, a compound that negates PGE2 inhibition, derivatives thereof, analogs thereof, and combinations thereof, and a pharma- ceutically acceptable carrier to increase muscle cell population and / or enhance muscle function in the subject. A method comprising:

28. 28. The method of claim 27, wherein the condition or disease involving muscle damage, injury, or atrophy is selected from the group consisting of acute muscle injury or trauma, soft tissue injury of the hand, Duchenne muscular dystrophy (DMD), Becker muscular dystrophy, limb-girdle muscular dystrophy, amyotrophic lateral sclerosis (ALS), distal muscular dystrophy (DD), hereditary myopathy, myotonic muscular dystrophy (MDD), mitochondrial myopathy, myotubular myopathy (MM), myasthenia gravis (MG), congestive heart failure, periodic paralysis, polymyositis, rhabdomyolysis, dermatomyositis, cancer cachexia, AIDS cachexia, cardiac cachexia, stress-induced urinary incontinence, and sarcopenia.

29. 28. The method of claim 27, wherein the muscle cells comprise skeletal muscle cells, smooth muscle cells, cardiac muscle cells, embryonic stem cell derived muscle cells, induced pluripotent stem cell derived muscle cells, dedifferentiated muscle cells, or a combination thereof.

30. 28. The method of claim 27, wherein the muscle cells comprise muscle stem cells, satellite cells, myocytes, myoblasts, myotubes, muscle fibers, or a combination thereof.

31. 28. The method of claim 27, wherein the PGE2 derivative comprises 16,16-dimethyl prostaglandin E2.

32. Compounds that attenuate PGE2 catabolism Compounds, neutralizing peptides, or neutralizing antibodies that inactivate or interfere with 15-hydroxyprostaglandin dehydrogenase (15-PGDH) or inactivate or interfere with prostaglandin transporters (PTG or SLCO2A1) 28. The method of claim 27, comprising:

33. 28. The method of claim 27, wherein administering the compound comprises oral, intraperitoneal, intramuscular, intraarterial, intradermal, subcutaneous, intravenous, or intracardiac administration.

34. 28. The method of claim 27, wherein the compound is administered according to a short-term treatment regimen.

35. 30. The method of claim 27, wherein the administering step further comprises administering to the subject a population of isolated muscle cells.

36. 36. The method of claim 35, wherein the isolated muscle cells are autologous to the subject.

37. 36. The method of claim 35, wherein the isolated muscle cells are allogeneic to the subject.

38. 36. The method of claim 35, wherein the isolated muscle cells are purified.

39. 36. The method of claim 35, wherein the isolated muscle cells are cultured with the compound prior to administration to the subject.

40. 40. The method of claim 39, wherein culturing the isolated muscle cells with the compound comprises exposing the isolated muscle cells to the compound on a short-term, intermittent or continuous basis.

41. 36. The method of claim 35, wherein administering the isolated muscle cells comprises injecting or transplanting the cells into the subject.

42. 36. The method of claim 35, wherein the isolated muscle cells and the compound are administered to the subject simultaneously.

43. 36. The method of claim 35, wherein the isolated muscle cells and the compound are administered sequentially to the subject.

44. (i) administering to a subject a therapeutically effective amount of a compound selected from the group consisting of prostaglandin E2 (PGE2), a PGE2 prodrug, a PGE2 receptor agonist, a compound that attenuates PGE2 catabolism, a compound that negates PGE2 inhibition, derivatives thereof, analogs thereof, and combinations thereof, and a pharma- ceutically acceptable carrier to prevent or treat a condition or disease involving muscle damage, injury, or atrophy, and (ii) an isolated population of muscle cells.

23. A method for preventing or treating a condition or disease involving muscle damage, injury, or atrophy in a subject in need thereof, comprising:

45. 45. The method of claim 44, wherein the PGE2 derivative comprises 16,16-dimethyl prostaglandin E2.

46. Compounds that attenuate PGE2 catabolism Compounds, neutralizing peptides, or neutralizing antibodies that inactivate or interfere with 15-hydroxyprostaglandin dehydrogenase (15-PGDH) or inactivate or interfere with prostaglandin transporters (PTG or SLCO2A1) 45. The method of claim 44, comprising:

47. 45. The method of claim 44, wherein the isolated muscle cells comprise skeletal muscle cells, smooth muscle cells, cardiac muscle cells, embryonic stem cell derived muscle cells, induced pluripotent stem cell derived muscle cells, dedifferentiated muscle cells, or a combination thereof.

48. 45. The method of claim 44, wherein the isolated muscle cells comprise muscle stem cells, satellite cells, myoblasts, myocytes, myotubes, muscle fibers, or a combination thereof.

49. 45. The method of claim 44, wherein the isolated muscle cells are purified.

50. 45. The method of claim 44, wherein the isolated muscle cells are cultured with the compound prior to administration to the subject.

51. 51. The method of claim 50, wherein culturing the isolated muscle cells with the compound comprises exposing the isolated muscle cells to the compound on a short-term, intermittent, or continuous basis.

52. 45. The method of claim 44, wherein the isolated muscle cells are autologous to the subject.

53. 45. The method of claim 44, wherein the isolated muscle cells are allogeneic to the subject.

54. 45. The method of claim 44, wherein administering the compound comprises oral, intraperitoneal, intramuscular, intraarterial, intradermal, subcutaneous, intravenous, or intracardiac administration.

55. 45. The method of claim 44, wherein the compound is administered according to a short-term treatment regimen.

56. 45. The method of claim 44, wherein administering the isolated muscle cells comprises injecting or transplanting the cells into the subject.

57. 45. The method of claim 44, wherein the compound and the isolated muscle cells are administered to the subject simultaneously.

58. 45. The method of claim 44, wherein the compound and the isolated muscle cells are administered sequentially to the subject.

59. 45. The method of claim 44, wherein the subject is suspected of having or is at risk of developing a condition or disease involving muscle damage, injury or wasting.

60. 45. The method of claim 44, wherein the condition or disease involving muscle damage, injury, or atrophy is selected from the group consisting of acute muscle injury or trauma, soft tissue injury of the hand, Duchenne muscular dystrophy (DMD), Becker muscular dystrophy, limb-girdle muscular dystrophy, amyotrophic lateral sclerosis (ALS), distal muscular dystrophy (DD), hereditary myopathy, myotonic muscular dystrophy (MDD), mitochondrial myopathy, myotubular myopathy (MM), myasthenia gravis (MG), congestive heart failure, periodic paralysis, polymyositis, rhabdomyolysis, dermatomyositis, cancer cachexia, AIDS cachexia, cardiac cachexia, stress-induced urinary incontinence, and sarcopenia.

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