Patent Description:
Prostaglandin E2 (PGE2), also known as dinoprostone, has been employed in various clinical settings including to induce labor in women and to augment hematopoietic stem cell transplantation. PGE2 can be used as an anticoagulant and antithrombotic agent. PGE2's role as a lipid mediator that can resolve inflammation is also well known. Nonsteroidal anti-inflammatory drugs (NSAIDs), inhibitors of COX-<NUM> and/or COX-<NUM>, suppress inflammation by inhibiting prostanoids, mainly via PGE2 biosynthesis.

PGE2 is synthesized from arachidonic acid by a cyclooxygenase (COX) and prostaglandin E synthase enzymes. Levels of PGE2 are physiologically regulated by the PGE2 degrading enzyme, <NUM>- hydroxyprostaglandin dehydrogenase (<NUM>-PGDH). <NUM>-PGDH catalyzes the inactivating conversion of the PGE2 <NUM>-OH to a <NUM>-keto group.

There remains a need in the art for effective treatments for regenerating or rejuvenating damaged, impaired, dysfunctional, and/or atrophied muscle in a subject in need thereof. The present invention satisfies this need and provides advantages as well.

<CIT> discloses methods and compositions that induce, increase and/or enhance differentiation of endogenous stem cells and progenitor cells in a subject to replenish injured or damaged cardiomyocytes. <CIT> discloses pretreating myoblast cultures with growth or trophic factors before transplantation to treat a myopathy. <NPL> describes adult stem cells for use in therapeutic purposes to treat animal models of chronic muscle degeneration. Otis et al.

(<NPL> suggests that the COX2 pathway is critical for myoblast proliferation in response to stretching. <NPL> discloses clinical applications and roles in myogenic differentiation of PGE2.

The invention provides a composition comprising a therapeutically effective amount of a compound, neutralizing peptide, or neutralizing antibody that inactivates or blocks <NUM>-hydroxyprostaglandin dehydrogenase (<NUM>-PGDH) for use in regenerating a population of muscle cells and treating muscle damage, muscle injury, or muscle atrophy in a subject in need thereof.

Other objects, features, and advantages of the present invention will be apparent to one of skill in the art from the following detailed description and figures.

The present invention is based, in part, on the discovery that prostaglandin E2 (PGE2) can improve muscle cell proliferation and function. As such, provided herein is a compound, neutralizing peptide, or neutralizing antibody that inactivates or blocks <NUM>-PGDH for use in regenerating a population of muscle cells and treating muscle damage, muscle injury, or muscle atrophy in a subject in need thereof.

Practicing this invention utilizes routine techniques in the field of molecular biology. Basic texts disclosing the general methods of use in this invention include <NPL>); <NPL>); and <NPL>)).

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

Oligonucleotides that are not commercially available can be chemically synthesized, e.g., according to the solid phase phosphoramidite triester method first described by <NPL>), using an automated synthesizer, as described in <NPL>). Purification of oligonucleotides is performed using any art-recognized strategy, e.g., native acrylamide gel electrophoresis or anion-exchange high performance liquid chromatography (HPLC) as described in <NPL>).

As used herein, the following terms have the meanings ascribed to them unless specified otherwise.

The terms "a," "an," or "the" as used herein not only include aspects with one member, but also include aspects with more than one member. For instance, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a cell" includes a plurality of such cells and reference to "the agent" includes reference to one or more agents known to those skilled in the art, and so forth.

The term "prostaglandin E2" or "PGE2" refers to prostaglandin that can be synthesized via arachidonic acid via cyclooxygenase (COX) enzymes and terminal prostaglandin E synthases (PGES). PGE2 plays a role in a number of biological functions including vasodilation, inflammation, and modulation of sleep/wake cycles.

The term "prostaglandin E2 receptor agonist" or "PGE2 receptor agonist" refers to a chemical compound, small molecule, polypeptide, biological product, etc. that can bind to and activate any PGE2 receptor, thereby stimulating the PGE2 signaling pathway.

The term "compound that attenuates PGE2 catabolism" refers to a chemical compound, small molecule, polypeptide, biological product, etc. that can reduce or decrease the breakdown of PGE2. In the context of the invention, a "compound that attenuates PGE2 catabolism" refers to a compound, neutralizing peptide, or neutralizing antibody that inactivates or blocks <NUM>-hydroxyprostaglandin dehydrogenase (<NUM>-PGDH).

The term "compound that neutralizes PGE2 inhibition" refers to a chemical compound, small molecule, polypeptide, biological product, etc. that can block or impede an inhibitor of PGE2 synthesis, activity, secretion, function, and the like.

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.

The term "embryonic stem cell-derived muscle cell" or "ESC-derived muscle cell" refers to a muscle cell that is derived from or differentiated from an embryonic stem cell.

The term "induced pluripotent stem cell-derived muscle cell" or "iPSC-derived muscle cell" refers to a muscle cell that is derived from or differentiated from an induced pluripotent stem cell.

The term "isolated," in the context of cells, refers to a single cell of interest or a population of cells of interest, at least partially isolated and/or purified from other cell types or other cellular material with which it naturally occurs in the tissue of origin (e.g., muscle tissue). A population of muscle cells is "isolated" when it is at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>% and, in certain cases, at least about <NUM>% free of cells that are not muscle cells. Purity can be measured by any appropriate method, for example, by fluorescence-activated cell sorting.

The term "autologous" refers to any material (e.g., a cell) derived from the same individual to whom it is later to be re-introduced into the individual.

The term "allogeneic" refers to any material (e.g., a cell) 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 when the genes at one or more loci are not identical. In some aspects, allogeneic material from individuals of the same species may be sufficiently unlike genetically to interact antigenically.

The term "treating" or "treatment" refers to any one of the following: ameliorating one or more symptoms of disease; preventing the manifestation of such symptoms before they occur; slowing down or completely preventing the progression of the disease (as may be evident by longer periods between reoccurrence episodes, slowing down or prevention of the deterioration of symptoms, etc.); enhancing the onset of a remission period; slowing down the irreversible damage caused in the progressive-chronic stage of the disease (both in the primary and secondary stages); delaying the onset of said progressive stage; or any combination thereof.

The term "administer," "administering," or "administration" refers to the methods that may be used to enable delivery of agents or compositions such as the compounds and cells described herein to a desired site of biological action. These methods include, but are not limited to, parenteral administration (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular, intra-arterial, intravascular, intracardiac, intrathecal, intranasal, intradermal, intravitreal, and the like), transmucosal injection, oral administration, administration as a suppository, and topical administration. One skilled in the art will know of additional methods for administering a therapeutically effective amount of the compounds and/or cells described herein for preventing or relieving one or more symptoms associated with a disease or condition.

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 drug that is sufficient to bring about a beneficial or desired clinical effect. A therapeutically effective amount or dose may be based on factors individual to each patient, including, but not limited to, the patient's age, size, type or extent of disease, stage of the disease, route of administration of the regenerative cells, the type or extent of supplemental therapy used, ongoing disease process and type of treatment desired (e.g., aggressive vs. conventional treatment). Therapeutically effective amounts of a pharmaceutical compound or compositions, as described herein, can be estimated initially from cell culture and animal models. For example, IC<NUM> values determined in cell culture methods can serve as a starting point in animal models, while IC<NUM> values determined in animal models can be used to find a therapeutically effective dose in humans.

The term "pharmaceutically acceptable carrier" refers to refers to a carrier or a diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered compound.

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, murines, rats, simians, humans, farm animals, sport animals, and pets.

The term "acute exposure," in the context of administration of a compound, refers to a temporary or brief application of a compound to a subject, e.g., human subject, or cells. In some embodiments, an acute exposure includes a single administration of a compound over the course of treatment or over an extended period of time.

The term "intermittent exposure," in the context of administration of a compound, refers to a repeated application of a compound to a subject, e.g., human subject, or cells, wherein a desired period of time lapses between applications.

The term "acute regimen," in the context of administration of a compound, refers to a temporary or brief application of a compound to a subject, e.g., human subject, or to a repeated application of a compound to a subject, e.g., human subject, wherein a desired period of time (e.g., <NUM> day) lapses between applications. In some embodiments, an acute regimen includes an acute exposure (e.g., a single dose) of a compound to a subject over the course of treatment or over an extended period of time. In other embodiments, an acute regimen includes intermittent exposure (e.g., repeated doses) of a compound to a subject in which a desired period of time lapses between each exposure.

The term "continuous exposure," in the context of administration of a compound, refers to a repeated, chronic application of a compound to a subject, e.g., human subject, or cells, over an extended period of time.

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

The subject has a condition or disease associated with muscle damage, injury, or atrophy. The condition or disease associated with muscle damage, injury, or atrophy can be selected from the group consisting of acute muscle injury, tear, or trauma, soft tissue hand injury, Duchenne muscular dystrophy (DMD), Becker muscular dystrophy, limb girdle muscular dystrophy, amyotrophic lateral sclerosis (ALS), distal muscular dystrophy (DD), inherited myopathies, myotonic muscular dystrophy (MDD), mitochondrial myopathies, 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.

In the invention, the compound that attenuates PGE2 catabolism comprises a compound, neutralizing peptide, or neutralizing antibody that inactivates or blocks <NUM>-hydroxyprostaglandin dehydrogenase (<NUM>-PGDH).

The invention provides a composition comprising a therapeutically effective amount of a compound, neutralizing peptide, or neutralizing antibody that inactivates or blocks <NUM>-hydroxyprostaglandin dehydrogenase (<NUM>-PGDH) for use in a method of regenerating a population of muscle cells and treating muscle damage, muscle injury, or muscle atrophy in a subject in need thereof. The method may include administering to the subject a therapeutically effective amount of the compound and a pharmaceutically acceptable carrier to increase the population of muscle cells in the subject and/or to enhance muscle function in the subject.

In a related aspect, the invention stimulates the proliferation and/or expansion of a population of muscle cells in a subject having a condition or disease associated with muscle damage, injury, or atrophy. The method may include administering to the subject a therapeutically effective amount of the compound and a pharmaceutically acceptable carrier to increase the population of muscle cells in the subject and/or to enhance muscle function in the subject.

In some embodiments, the population of muscle cells comprises an endogenous population of muscle cells. In other embodiments, the population of muscle cells comprises a population of isolated muscle cells that has been administered (e.g., injected or transplanted) to the subject. In yet other embodiments, the population of muscle cells comprises both an endogenous population of muscle cells and a population of isolated muscle cells that has been administered to the subject.

In some embodiments, the condition or disease associated with muscle damage, injury, or atrophy is selected from the group consisting of acute muscle injury or trauma, soft tissue hand injury, Duchenne muscular dystrophy (DMD), Becker muscular dystrophy, limb girdle muscular dystrophy, amyotrophic lateral sclerosis (ALS), distal muscular dystrophy (DD), inherited myopathies, myotonic muscular dystrophy (MDD), mitochondrial myopathies, 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.

In some embodiments, the population of muscle cells comprises 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. In some cases, the population of muscle cells comprises muscle stem cells, satellite cells, myocytes, myoblasts, myotubes, myofibers, or a combination thereof.

In some embodiments, the step of administering the compound comprises oral, intraperitoneal, intramuscular, intra-arterial, intradermal, subcutaneous, intravenous, or intracardiac administration. In some cases, the compound is administered in accordance with an acute regimen. In certain instances, the acute regimen comprises acute exposure (e.g., a single dose) of the compound to the subject. In other instances, the acute regimen comprises intermittent exposure (e.g., repeated doses) of the compound to the subject. As a non-limiting example, an acute PGE2 regimen can comprise a series of intermittent (e.g., daily) doses of PGE2 over a desired period of time (e.g., over the course of <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, or <NUM> days).

In other embodiments, the step of administering further comprises administering a population of isolated muscle cells to the subject. The population of isolated muscle cells can be autologous to the subject. The population of isolated muscle cells can be allogeneic to the subject. In some instances, the population of isolated muscle cells is substantially purified or purified. In other instances, the population of isolated muscle cells is cultured with the compound prior to being administered to the subject. The step of culturing the population of isolated muscle cells with the compound can include acute, intermittent, or continuous exposure of the population of isolated muscle cells to the compound. Administering the population of isolated muscle cells can comprise injecting or transplanting the cells into the subject. The population of isolated muscle cells and the compound can be administered to the subject concomitantly. Alternatively, the population of isolated muscle cells and the compound can be administered to the subject sequentially.

The invention provides a composition comprising a therapeutically effective amount of a compound, neutralizing peptide, or neutralizing antibody that inactivates or blocks <NUM>-hydroxyprostaglandin dehydrogenase (<NUM>-PGDH) for use in a method of regenerating a population of muscle cells and treating muscle damage, muscle injury, or muscle atrophy in a subject in need thereof. The method may include administering to the subject (i) a therapeutically effective amount of the compound and a pharmaceutically acceptable carrier, and (ii) a population of isolated muscle cells, to prevent or treat the condition or disease associated with muscle damage, injury, or atrophy.

In a related aspect, the invention stimulates the proliferation and/or expansion of a population of muscle cells in a subject having a condition or disease associated with muscle damage, injury, or atrophy by administering to the subject (i) a therapeutically effective amount of the compound and a pharmaceutically acceptable carrier, and (ii) a population of isolated muscle cells. In some embodiments, the population of muscle cells comprises an endogenous population of muscle cells. In other embodiments, the population of muscle cells comprises the population of isolated muscle cells that has been administered (e.g., injected or transplanted) to the subject. In yet other embodiments, the population of muscle cells comprises both an endogenous population of muscle cells and the population of isolated muscle cells that has been administered to the subject. In certain embodiments, the therapeutically effective amount of the compound comprises an amount that is sufficient to increase the population of endogenous muscle cells in the subject and/or to increase the population of isolated muscle cells that has been administered to the subject and/or to enhance muscle function in the subject.

In some embodiments, the population of muscle cells comprises 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. In some cases, the population of muscle cells comprises muscle stem cells, satellite cells, myocytes, myoblasts, myotubes, myofibers, or a combination thereof. The population of isolated muscle cells can be substantially purified or purified.

In some embodiments, the population of isolated muscle cells is cultured with the compound prior to being administered to the subject. In some cases, culturing the population of isolated muscle cells with the compound comprises acute, intermittent, or continuous exposure of the population of isolated muscle cells to the compound.

In some embodiments, the population of isolated muscle cells is autologous to the subject. In other embodiments, the population of isolated muscle cells is allogeneic to the subject.

Administration of the compound can be oral, intraperitoneal, intramuscular, intra-arterial, intradermal, subcutaneous, intravenous, or intracardiac administration. In some cases, the compound is administered in accordance with an acute regimen. In certain instances, the acute regimen comprises acute exposure (e.g., a single dose) of the compound to the subject. In other instances, the acute regimen comprises intermittent exposure (e.g., repeated doses) of the compound to the subject. As a non-limiting example, an acute PGE2 regimen can comprise a series of intermittent (e.g., daily) doses of PGE2 over a desired period of time (e.g., over the course of <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, or <NUM> days). Administration of the population of isolated muscle cells can include injecting or transplanting the cells into the subject. The compound and the population of isolated muscle cells can be administered to the subject concomitantly. Optionally, the compound and the population of isolated muscle cells can be administered to the subject sequentially.

In some embodiments, the subject is suspected of having or at risk for developing the condition or disease associated with muscle damage, injury, or atrophy. In some cases, the condition or disease associated with muscle damage, injury or atrophy is selected from the group consisting of acute muscle injury or trauma, soft tissue hand injury, Duchenne muscular dystrophy (DMD), Becker muscular dystrophy, limb girdle muscular dystrophy, amyotrophic lateral sclerosis (ALS), distal muscular dystrophy (DD), inherited myopathies, myotonic muscular dystrophy (MDD), mitochondrial myopathies, 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.

The compositions provided herein can be used to regenerate or rejuvenate muscle in a subject, such as a human subject. Regeneration of muscle includes forming new muscle fibers from muscle stem cells, satellite cells, muscle progenitor cells, and any combination thereof. The compositions are also useful for enhancing or augment muscle repair and/or maintenance.

The PGE2 compounds of the present invention can be administered to a subject experiencing muscle degeneration or atrophy. Muscle atrophy can include loss of muscle mass and/or strength. It can affect any muscle of a subject. In some cases, the subject in need of the compositions provided herein is exhibiting or experiencing muscle loss due to, e.g., age, inactivity, injury, disease, and any combination thereof.

In some embodiments, compounds can activate muscle cell proliferation, differentiation, and/or fusion of muscle cells. In some cases, the 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 atrophy are ameliorated.

The damaged muscle can be any muscle of the body, including but not limited to, musculi 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 pollicis, adductor pollicis, flexor pollicis brevis, iliopsoas, psoas, rectus abdominis, rectus femoris, gluteus maximus, gluteus medius, medial hamstrings, gastrocnemius, lateral hamstring, quadriceps mechanism, adductor longus, adductor brevis, adductor magnus, gastrocnemius medial, gastrocnemius lateral, soleus, tibialis posterior, tibialis anterior, flexor digitorum longus, flexor digitorum brevis, flexor hallucis longus, extensor hallucis longus, hand muscles, arm muscles, foot muscles, leg muscles, chest muscles, stomach muscles, back muscles, buttock muscles, shoulder muscles, head and neck muscles, facial muscles, oculopharyngeal muscles, and the like.

Subjects in need of muscle regeneration may have musculoskeletal injuries (e.g., fractures, strains, sprains, acute injuries, overuse injuries, and the like), post-trauma damages to limbs or face, athletic injuries, post-fractures in the aged, soft tissue hand injuries, 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 dystrophy, facioscapulohumeral muscular dystrophy (FHMD), myotonic muscular dystrophy, oculopharyngeal muscular dystrophy, distal muscular dystrophy, Emery-Dreifuss muscular dystrophy, myotonia congenita, myotonic dystrophy, other muscular dystrophies, muscle wasting disease, such as cachexia due to cancer, end stage renal disease (ESRD), acquired immune deficiency syndrome (AIDS), or chronic obstructive pulmonary disease (COPD), post-surgical muscle weakness, post-traumatic muscle weakness, sarcopenia, inactivity (e.g., muscle disuse or immobility), urethral sphincter deficiency, urethral sphincter deficiency, neuromuscular disease, and the like.

Non-limiting examples of neuromuscular diseases include, but are not limited to, acid maltase deficiency, amyotrophic lateral sclerosis, Andersen-Tawil syndrome, Becker muscular dystrophy, Becker myotonia congenita, Bethlem myopathy, bulbospinal muscular atrophy, carnitine deficiency, carnitine palmityl transferase deficiency, central core disease, centronuclear myopathy, Charcot-Marie-Tooth disease, congenital muscular dystrophy, congenital myasthenic syndromes, congenital myotonic dystrophy, Cori disease, Debrancher enzyme deficiency, Dejerine-Sottas disease, dermatomyositis, distal muscular dystrophy, Duchenne muscular dystrophy, dystrophia myotonica, Emery-Dreifuss muscular dystrophy, endocrine myopathies, Eulenberg disease, facioscapulohumeral muscular dystrophy, tibial distal myopathy, Friedreich's ataxia, Fukuyuma congenital muscular dystrophy, glycogenosis type <NUM>, glycogenosis type <NUM>, glycogenosis type <NUM>, glycogenosis type <NUM>, glycogenosis type <NUM>, glycogenosis type <NUM>, glycogenosis type <NUM>, Gowers-Laing distal myopathy, hereditary inclusion-body myositis, hyperthyroid myopathy, hypothyroid myopathy, inclusion-body myositis, inherited myopathies, integrin-deficient congenital muscular dystrophy, spinal-bulbar muscular atrophy, spinal muscular atrophy, lactate dehydrogenase deficiency, Lambert-Eaton myasthenic syndrome, McArdel disease, merosin-deficient congenital muscular dystrophy, metabolic diseases of muscle, mitochondrial myopathy, Miyoshi distal myopathy, motor neuron disease, muscle-eye-brain disease, myasthenia gravis, myoadenylate deaminase deficiency, myofibrillar myopathy, myophosphorylase deficiency, myotonia congenital, myotonic muscular dystrophy, myotubular myopathy, nemaline myopathy, Nonaka distal myopathy, oculopharyngeal muscular dystrophy, paramyotonia congenital, 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, ZASP-related myopathy, and the like.

Muscle atrophy (e.g., muscle wasting) can be caused by or associated with, for example, normal aging (e.g., sarcopenia), genetic abnormalities (e.g., mutations or single nucleotide polymorphisms), poor nourishment, poor circulation, loss of hormonal support, disuse of the muscle due to lack of exercise (e.g., bedrest, immobilization of a limb in a cast, etc.), aging, damage to the nerve innervating the muscle, poliomyelitis, amyotrophic lateral sclerosis (ALS or Lou Gehrig's disease), heart failure, liver disease, diabetes, obesity, metabolic syndrome, demyelinating diseases (e.g., multiple sclerosis, Charcot-Marie-Tooth disease, Pelizaeus-Merzbacher disease, encephalomyelitis, neuromyelitis optica, adrenoleukodystrophy, and Guillian-Barre syndrome), denervation, fatigue, exercise-induced muscle fatigue, frailty, neuromuscular disease, weakness, chronic pain, and the like.

In some aspects, provided herein is regeneration of muscle in a subject in need thereof by administering to the subject a therapeutically effective amount of a compound, neutralizing peptide, or neutralizing antibody that inactivates or blocks <NUM>-hydroxyprostaglandin dehydrogenase (<NUM>-PGDH) and a pharmaceutically acceptable carrier, to increase the population of muscle cells and/or to enhance muscle function in the subject. The population of muscle cells in the subject can include 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 any combinations thereof. Additionally, the muscle cells in the subject can be muscle stem cells, satellite cells, myocytes, myoblasts, myotubes, myofibers, or any combination thereof. The compound can 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 can be administered in accordance with an acute regimen (e.g., single or intermittent dosing) or a chronic regimen (e.g., continuous dosing).

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. The cells can be isolated and/or purified by any method known to those of skill in the art. The cells can be a homogenous or heterogeneous population of muscle cells.

In some embodiments, the cells are stimulated to proliferate by culturing the cells with the PGE2 compound prior to administering them to the subject. The cells can be acutely, intermittently or continuously exposed to the compound during in vitro culturing. In some cases, the population of muscle cells increases by at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, or more after culturing with the PGE2 compound.

To regenerate or repair muscle in the subject, the compound and the isolated muscle cells are administered to the subject concomitantly. In some embodiments, the compound and the cultured muscle cells are administered to the subject concomitantly. In other embodiments, the compound and the isolated muscle cells are administered to the subject sequentially. In yet other embodiments, the compound and the cultured muscle cells are administered to the subject sequentially.

The compositions described herein can be used to increase the number of muscle fibers by at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, or more. In some embodiments, the compositions can increase the growth of a damaged, injured, atrophied, or degenerated muscle.

The compositions provided herein can be used in methods to prevent or treat a condition or disease associated with muscle damage, injury, or atrophy in a subject in need thereof. The method can provide prophylactic treatment to a subject who is likely to experience muscle damage, injury or atrophy. In some embodiments, the subject can have a condition or disease with possible secondary symptoms that affect muscle. In other embodiments, the subject has undergone a surgical or therapeutic intervention to treat the muscle condition or disease, and the method disclosed here is used to prevent or inhibit recurrence or relapse. In some embodiments, the subject has any one of the conditions or diseases described herein that affects muscle.

As used herein, the term "treatment" or "treating" encompasses administration of compounds and/or cells in an appropriate form prior to the onset of disease symptoms and/or after clinical manifestations, or other manifestations of the condition or disease to reduce disease severity, halt disease progression, or eliminate the disease. The term "prevention of" or "preventing" a disease includes prolonging or delaying the onset of symptoms of the condition or disease, preferably in a subject with increased susceptibility to the condition or disease.

The method may include administering to the subject (i) a therapeutically effective amount of a compound, neutralizing peptide, or neutralizing antibody that inactivates or blocks <NUM>-hydroxyprostaglandin dehydrogenase (<NUM>-PGDH) and a pharmaceutically acceptable carrier, and (ii) a population of isolated muscle cells, to prevent or treat the condition or disease associated with muscle damage, injury, or atrophy. The muscle cells can be autologous or allogeneic to the subject.

The compound can be administered orally, intraperitoneally, intramuscularly, intra-arterially, intradermally, subcutaneously, intravenously, or by intracardiac injection. The compound can be administered in accordance with an acute regimen (e.g., single or intermittent dosing) or a chronic regimen (e.g., continuous dosing). The isolated muscle cells can be administered by injection or transplantation. In some embodiments, the compound and the cells are administered together or concomitantly. In other embodiments, the compound and the cells are administered sequentially. In some cases, the compound is administered before the cells. In other cases, the cells are administered before the compound.

The isolated muscle cells can be substantially purified or purified prior to injection or transplantation into the subject. The cells can also be expanded or stimulated to proliferate in culture prior to administration. As described herein, isolated muscle cells including 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, muscle stem cells, satellite cells, myoblasts, myocytes, myotubes, myofibers, and any combination thereof can be cultured with the compounds of the present inventio. By exposing the compound to the cells acutely, intermittently or continuously, the muscle cells proliferate and increase in number. The expanded cells can be transplanted into a subject experiencing muscle damage, injury or atrophy.

The composition comprises a compound, neutralizing peptide, or neutralizing antibody that inactivates or blocks <NUM>-hydroxyprostaglandin dehydrogenase (<NUM>-PGDH)).

Muscle (myogenic) cells 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 precursor cells, myogenic cells, myocytes, myoblasts, myotubes, postmitotic myotubes, multinucleated myofibers, and postmitotic muscle fibers. In some embodiments, the isolated muscle cells encompass muscle stem cells. In other embodiments, the isolated muscle cells include muscle satellite cells. The muscle cells can be derived from a stem cell such as a bone marrow-derived stem cell, or a pluripotent stem cell such as an embryonic stem cell or an induced pluripotent stem cell. In some embodiments, the isolated muscle cells include dedifferentiated muscle cells. In other embodiments, the muscle cells have been genetically modified to, in some cases, correct disease-associated gene mutations.

Satellite cells are small mononuclear progenitor cells that can reside within muscle tissue. These cells can be induced to proliferate and differentiate into muscle cells, and in some instances, fuse to muscle fibers. During muscle damage or injury, quiescent satellite cells (e.g., satellite cells that are not differentiating or undergoing cell division at present) and muscle stem cells can be activated to proliferate, and/or migrate out of the muscle stem cell niche. The satellite cells and muscle stem cells can also differentiate into myocytes, myoblasts, or other muscle cell types.

Methods and protocols for generating muscle cells from embryonic stem cells are described, e.g., in <NPL>; and <NPL>. Methods and protocols for generating muscle cells from induced pluripotent stem cells are described, e.g., in <NPL>; <NPL>; and <NPL>.

In some embodiments, muscle cells are obtained by biopsy from a muscle such as a mature or adult muscle, e.g., quadriceps, gluteus maximus, bicep, tricep, or any muscle from an individual. The muscle can be a skeletal muscle, smooth muscle, or cardiac muscle. Detailed descriptions of methods of isolating smooth muscle stem cells can be found, e.g., in <CIT>, and <CIT>. Methods of isolating muscle cells of interest such as muscle stem cells or satellite cells from muscle tissue are described in detail, for example, in <NPL>.

Methods for purifying a population of muscle cells of interest, e.g., muscle stem cells, muscle satellite cells, myocytes, myoblasts, myotubes, and/or myofibers include selecting, isolating or enriching for a cell having a specific cell surface marker or a specific polypeptide that is expressed on the cell surface of the muscle cell of interest. Useful cell surface markers are described in, e.g., <NPL>. 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.

The isolated population of muscle cells of interest can be expanded or multiplied using conventional culture-based methods. Methods for culture muscle cells are found in, e.g., <CIT>. In some cases, the cells are cultured on a scaffold or gel such as a hydrogel.

The compounds used in the present invention can be administered locally at or near a site of injury in the subject or systemically. In some embodiments, the compounds can be administered, for example, intraperitoneally, intramuscularly, intra-arterially, orally, intravenously, intracranially, intrathecally, intraspinally, intralesionally, intranasally, subcutaneously, intracerebroventricularly, topically, 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 cells can be administered in the same composition. When administered separately, the compound can be provided in a pharmaceutically acceptable carrier. In some embodiments, the compound is administered before or after the administration of the cells.

In some embodiments, the compound is administered in accordance with an acute regimen. In certain instances, the compound is administered to the subject once. In other instances, the compound is administered at one time point, and administered again at a second time point. In yet other instances, the compound is administered to the subject repeatedly (e.g., once or twice daily) as intermittent doses over a short period of time (e.g., <NUM> days, <NUM> days, <NUM> days, <NUM> days, <NUM> days, a week, <NUM> weeks, <NUM> weeks, <NUM> weeks, a month, or more). In some cases, the time between compound administrations is about <NUM> day, <NUM> days, <NUM> days, <NUM> days, <NUM> days, <NUM> days, a week, <NUM> weeks, <NUM> weeks, <NUM> weeks, a month, or more. In other embodiments, the compound is administered continuously or chronically in accordance with a chronic regimen over a desired period of time. For instance, the compound can be administered such that the amount or level of the compound is substantially constant over a selected time period.

Administration of the isolated muscle cells into a subject can be accomplished by methods generally used in the art. In some embodiments, administration is by transplantation or injection such as intramuscular injection. The number of cells introduced will take into consideration factors such as sex, age, weight, the types of disease or disorder, stage of the disorder, the percentage of the desired cells in the cell population (e.g., purity of cell population), and the cell number needed to produce the desired result. Generally, for administering the cells for therapeutic purposes, the cells are given at a pharmacologically effective dose. By "pharmacologically effective amount" or "pharmacologically effective dose" is an amount sufficient to produce the desired physiological effect or amount capable of achieving the desired result, particularly for treating the condition or disease, including reducing or eliminating one or more symptoms or manifestations of the condition or disease. Pharmacologically effective doses will also apply to therapeutic compounds used in combination with the cells, as described herein.

Cells can be administered in one injection, or through successive injections over a defined time period sufficient to generate a therapeutic effect. Different populations of muscle cells may be injected when treatment involves successive injections. A pharmaceutically acceptable carrier, as further described below, may be used for injection of the cells into the subject. These will typically comprise, for example, buffered saline (e.g., phosphate buffered saline) or unsupplemented basal cell culture medium, or medium as known in the art.

Any number of muscles of the body may be directly injected with the compound and/or cells of the present invention, such as, for example, the biceps muscle; the triceps muscle; the brachioradialus muscle; the brachialis muscle (brachialis anticus); the superficial compartment wrist flexors; the deltoid muscle; the biceps femoris, the gracilis, the semitendinosus and the semimembranosus muscles of the hamstrings; the rectus femoris, vastus lateralis, vastus medialis and vastus intermedius muscles of the quadriceps; the gastrocnemius (lateral and medial), tibialis anterior, and the soleus muscles of the calves; the pectoralis major and the pectoralis minor muscles of the chest; the latissimus dorsi muscle of the upper back; the rhomboids (major and minor); the trapezius muscles that span the neck, shoulders and back; the rectus abdominis muscles of the abdomen; and the gluteus maximus, gluteus medius and gluteus minimus muscles of the buttocks.

The pharmaceutical compositions used in the present invention may comprise a pharmaceutically acceptable carrier. In certain aspects, pharmaceutically acceptable carriers are determined in part by the particular composition being administered, as well as by the particular method used to administer the composition. Accordingly, there is a wide variety of suitable formulations of pharmaceutical compositions of the present invention (see, e.g., <NPL>)).

As used herein, "pharmaceutically acceptable carrier" comprises any of standard pharmaceutically accepted carriers known to those of ordinary skill in the art in formulating pharmaceutical compositions. Thus, the cells or compounds, by themselves, such as being present as pharmaceutically acceptable salts, or as conjugates, may be prepared as formulations in pharmaceutically acceptable diluents; for example, saline, phosphate buffer saline (PBS), aqueous ethanol, or solutions of glucose, mannitol, dextran, propylene glycol, oils (e.g., vegetable oils, animal oils, synthetic oils, etc.), microcrystalline cellulose, carboxymethyl cellulose, hydroxylpropyl methyl cellulose, magnesium stearate, calcium phosphate, gelatin, polysorbate <NUM> or the like, or as solid formulations in appropriate excipients.

The pharmaceutical compositions will often further comprise one or more buffers (e.g., neutral buffered saline or phosphate buffered saline), carbohydrates (e.g., glucose, mannose, sucrose or dextrans), mannitol, proteins, polypeptides or amino acids such as glycine, antioxidants (e.g., ascorbic acid, sodium metabisulfite, butylated hydroxytoluene, butylated hydroxyanisole, etc.), bacteriostats, chelating agents such as EDTA or glutathione, solutes that render the formulation isotonic, hypotonic or weakly hypertonic with the blood of a recipient, suspending agents, thickening agents, preservatives, flavoring agents, sweetening agents, and coloring compounds as appropriate.

The pharmaceutical compositions of the invention are administered in a manner compatible with the dosage formulation, and in such amount as will be therapeutically effective. The quantity to be administered depends on a variety of factors including, e.g., the age, body weight, physical activity, and diet of the individual, the condition or disease to be treated, and the stage or severity of the condition or disease. In certain embodiments, the size of the dose may also be determined by the existence, nature, and extent of any adverse side effects that accompany the administration of a therapeutic agent(s) in a particular individual.

It will be understood, however, that the specific dose level and frequency of dosage for any particular patient may be varied and will depend upon a variety of factors including the activity of the specific compound employed, the metabolic stability and length of action of that compound, the age, body weight, hereditary characteristics, general health, sex, diet, mode and time of administration, rate of excretion, drug combination, the severity of the particular condition, and the host undergoing therapy.

In certain embodiments, the dose of the compound may take the form of solid, semisolid, lyophilized powder, or liquid dosage forms, such as, for example, tablets, pills, pellets, capsules, powders, solutions, suspensions, emulsions, suppositories, retention enemas, creams, ointments, lotions, gels, aerosols, foams, or the like, preferably in unit dosage forms suitable for simple administration of precise dosages.

As used herein, the term "unit dosage form" refers to physically discrete units suitable as unitary dosages for humans and other mammals, each unit containing a predetermined quantity of a therapeutic agent calculated to produce the desired onset, tolerability, and/or therapeutic effects, in association with a suitable pharmaceutical excipient (e.g., an ampoule). In addition, more concentrated dosage forms may be prepared, from which the more dilute unit dosage forms may then be produced. The more concentrated dosage forms thus will contain substantially more than, e.g., at least <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, or more times the amount of the therapeutic compound.

Methods for preparing such dosage forms are known to those skilled in the art (see, e.g., REMINGTON'S PHARMACEUTICAL SCIENCES, supra). The dosage forms typically include a conventional pharmaceutical carrier or excipient and may additionally include other medicinal agents, 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).

Examples of suitable excipients include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate, alginates, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, saline, syrup, methylcellulose, ethylcellulose, hydroxypropylmethylcellulose, and polyacrylic acids such as Carbopols, e.g., Carbopol <NUM>, Carbopol <NUM>, Carbopol <NUM>, etc. The dosage forms can additionally include lubricating agents such as talc, magnesium stearate, and mineral oil; wetting agents; emulsifying agents; suspending agents; preserving agents such as methyl-, ethyl-, and propyl-hydroxy-benzoates (i.e., the parabens); pH adjusting agents such as inorganic and organic acids and bases; sweetening agents; and flavoring agents. The dosage forms may also comprise biodegradable polymer beads, dextran, and cyclodextrin inclusion complexes.

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

The therapeutically effective dose can also be provided in a lyophilized form. Such dosage forms may include a buffer, e.g., bicarbonate, for reconstitution prior to administration, or the buffer may be included in the lyophilized dosage form for reconstitution with, e.g., water. The lyophilized dosage form may further comprise a suitable vasoconstrictor, e.g., epinephrine. The lyophilized dosage form can be provided in a syringe, optionally packaged in combination with the buffer for reconstitution, such that the reconstituted dosage form can be immediately administered to an individual.

The following examples are offered to illustrate, but not to limit, the claimed invention. Only the sections involving the use of an inhibitor of <NUM>-PGDH fall within the invention.

This example illustrates that PGE2 signaling is required for muscle stem cell function during regeneration.

The elderly suffer from progressive skeletal muscle wasting and regenerative failure that decreases mobility and quality of life<NUM>,<NUM>. Crucial to muscle regeneration are adult muscle stem cells (MuSCs) that reside in niches in muscle tissues, poised to respond to damage and repair skeletal muscles throughout life<NUM>-<NUM>. During aging, the proportion of functional MuSCs markedly decreases, hindering muscle regeneration<NUM>-<NUM>. To date, no therapeutic agents are in clinical use that target MuSCs to combat this regenerative decline. Here, we identify a natural immunomodulator, prostaglandin E2 (PGE2), as a potent regulator of MuSC function essential to muscle regeneration. We found that the PGE2 receptor, EP4, is essential for MuSC proliferation in vitro and engraftment in vivo in mice. In MuSCs of aged mice, the PGE2 pathway is dysregulated due to a cell intrinsic molecular defect, elevated prostaglandin degrading enzyme (<NUM>-PGDH) that renders PGE2 inactive. This defect is overcome by transient acute exposure of MuSCs to a stable degradation-resistant PGE2, <NUM>,<NUM>-dimethyl PGE2 (dmPGE2), concomitant with MuSC transplantation into injured muscles. Notably, a single intramuscular injection of dmPGE2 alone suffices to accelerate regeneration, evident by an early increase in endogenous MuSC numbers and myofiber sizes following injury. Furthermore, aged mouse muscle force generating capacity was increased in response to exercise-induced regeneration and an acute dmPGE2 treatment regimen. Our findings reveal a novel therapeutic indication for PGE2 as a potent inducer of muscle regeneration and strength.

To counter the decline in muscle regenerative potential we sought therapeutic agents that target MuSCs, also known as satellite cells, a stem cell population dedicated to muscle regeneration<NUM>-<NUM>. Since a transient inflammatory and fibroadipogenic response plays a crucial role in muscle regeneration<NUM>-<NUM>, we sought to identify inflammatory modulators induced by injury that could overcome the age-related decline in MuSC function. An analysis of our transcriptome database revealed that the Ptger4 receptor for PGE2, a natural and potent lipid mediator during acute inflammation<NUM>, was expressed at high levels on freshly isolated MuSCs. In muscle tissue lysates, we detected a surge in levels of PGE2 three days after injury to young (<NUM>-<NUM> mo) mouse muscles by standard injury paradigms entailing notexin injection or cryoinjury (<FIG> and <FIG>), and a concomitant upregulation of its synthesizing enzymes, Ptges and Ptges2 (<FIG>). This early and transient time window coincides with the well-documented kinetics of MuSC expansion and inflammatory cytokine accumulation post injury<NUM>,<NUM>,<NUM>. To determine if PGE2 treatment enhanced MuSC behavior, we FACS-purified MuSCs from hindlimb muscles from young mice (<NUM>-<NUM> mo)<NUM> and plated them on hydrogels of <NUM> kpa stiffness to maintain stem cell function<NUM>. We found that PGE2 (10ng/ml) increased cell division assayed by EDU incorporation (<FIG>) and that an acute <NUM>-day exposure to PGE2 induced a <NUM>-fold increase in the number of MuSCs relative to controls one week later (<FIG>).

PGE2 is known to signal through four G-protein coupled receptors (Ptger<NUM>-<NUM>; EP1-<NUM>)<NUM>,<NUM>, but the expression of these receptors in MuSCs has not previously been described. An analysis of the transcript levels of the different receptors (Ptger<NUM>-<NUM>) revealed that the only receptors upregulated after PGE2 treatment of MuSCs are Ptger1 and Ptger4 (<FIG>). PGE2 stimulated MuSCs had elevated intracellular cAMP<NUM>,<NUM> confirming that PGE2 signals through EP4 to promote proliferation and a stem cell transcriptional state (<FIG>). In the presence of an EP4 antagonist, ONO-AE3-<NUM>, proliferation induced by PGE2 was blunted (<FIG>). However, the specificity of PGE2 for EP4 was most clearly shown in MuSCs lacking the receptor following cre-mediated conditional ablation (<FIG> and <FIG>). Indeed, even in the presence of growth factor-rich media, these EP4-null MuSCs failed to proliferate. Finally, we found that MuSCs growth arrested by exposure to medium with charcoal stripped serum<NUM>, divided upon addition of PGE2 (<FIG> and <FIG>). Thus, PGE2/EP4 stands out as necessary and sufficient for MuSC proliferation.

We sought to determine if PGE2 could ameliorate the muscle regenerative defects previously reported for aged MuSCs<NUM>-<NUM>. By contrast with young mouse muscles (<NUM>-<NUM> mo), notexin damage to aged muscles (<NUM>-<NUM> mo) did not lead to an increase in PGE2 synthesis. Instead, steady state PGE2 levels in aged muscle remained unchanged post injury (<FIG>) and were significantly higher than in young limb tibialis anterior (TA) muscles (<FIG>). We hypothesized that the PGE2 in aged muscle might be dysfunctional due to a catabolic defect. 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, <NUM>,<NUM>-dihydro-<NUM>-keto PGE2 (PGEM), was significantly increased in the aged (<FIG> and <FIG>). This proved to be due to a concomitant <NUM>-fold increase in levels of mRNA encoding the PGE2 degrading enzyme (<NUM>-PGDH), the initial step in the conversion of PGE2 to its inactive form (<FIG>). In contrast, the relative levels of the prostaglandin transporter (PGT), PGE2 synthesizing enzymes, and EP4 receptor did not differ between young and aged MuSCs (<FIG>). Additionally, when aged MuSCs were exposed to a <NUM>-day pulse of PGE2 or to an inhibitor of <NUM>-PGDH (SW033291)<NUM>, the effects of <NUM>-PGDH were overcome and the characteristic increase in proliferation and maintenance of Pax7 expression was observed (<FIG> and <FIG>). Like young, aged MuSCs failed to proliferate in medium comprised of charcoal stripped serum, but were rescued by addition of PGE2 alone (<FIG>). We surmised that in aged MuSCs the PGE2 pathway is dysregulated due to a cell intrinsic molecular defect, elevated <NUM>-PGDH that can be surmounted in culture by acute exposure to PGE2 or SW (<FIG>).

Since aged MuSCs are heterogeneous<NUM>, we sought to determine the effect of PGE2 at the single cell level. Clonal analysis can reveal differences that are masked by analysis of the population as a whole. Accordingly, we performed long-term time-lapse microscopy in hydrogel 'microwells' of single aged MuSCs transiently exposed to PGE2 for <NUM> day and untreated control MuSCs. Data were collected over a <NUM> time period and then analyzed using our previously described Baxter Algorithms for Cell Tracking and Lineage Reconstruction<NUM>,<NUM>,<NUM>. We observed a remarkable increase in cumulative cell numbers in response to PGE2, spanning <NUM> generations for the most robust clones (<FIG>). The numbers of cells per clone following PGE2 treatment were significantly augmented due to a marked increase in proliferation (<FIG> and <FIG>) that was accompanied by a profound reduction in cell death (<FIG> and <FIG>-<NUM>). These synergistic effects led to the observed increases in aged MuSC numbers in response to PGE2.

To test whether transient treatment of young MuSCs with PGE2 augments regeneration, we transplanted cultured PGE2 treated MuSCs into injured hindlimb muscles of mice. To monitor the dynamics of regeneration over time in a quantitative manner in vivo, we capitalized on a sensitive and quantitative bioluminescence imaging (BLI) assay we previously developed for monitoring MuSC function post-transplantation<NUM>,<NUM>,<NUM>. MuSCs were isolated from young transgenic mice (<NUM>-<NUM> mo) expressing GFP and luciferase (GFP/Luc mice), exposed to an acute <NUM>-day PGE2 treatment, harvested and transplanted on day <NUM>. Equivalent numbers of dmPGE2 treated and control MuSCs (<NUM> cells) were transplanted into injured hindlimbs of young (<NUM>-<NUM> mo) NOD-SCID mice. Following acute treatment with PGE2, young MuSC regenerative capacity was enhanced by an order of magnitude when assessed by BLI (<FIG>). In contrast, following transplantation of <NUM>-fold greater numbers of cultured MuSCs that lacked the EP4 receptor due to conditional ablation (<FIG>), the BLI signal that was initially detected progressively declined to levels below the threshold of significance (<FIG>).

Furthermore, when notexin injury was performed in the mouse model of muscle stem cell specific deletion of EP4 (Pax7CreERT2;EP4fl/fl) (<FIG>), muscle regeneration was impaired as observed by the elevated number of embryonic myosin heavy chain (eMHC) positive fibers (<FIG>). This was accompanied by the reduction in cross-sectional area of the mouse fibers in the Pax7CreR12; EP4fl/f group, assessed at the end of the regeneration time point (day <NUM>) (<FIG>). A significant reduction in force output (tetanus) was also detected at day <NUM> post-injury (<FIG>). Thus, PGE2 signaling via the EP4 receptor is required for MuSC regeneration in vivo.

To test if direct injection of PGE2 without culture could be effective in promoting regeneration in vivo, we coinjected PGE2 together with freshly isolated MuSCs. For all subsequent in vivo injection experiments, we used a modified, more stable form of PGE2, <NUM>,<NUM>-dimethyl PGE2 (dmPGE2)<NUM>. We hypothesized that for the aged MuSC experiments, the delivery of the modified <NUM>-PGDH-resistant dmPGE2 was particularly important, as <NUM>-PGDH is significantly elevated in aged MuSCs (<FIG>)<NUM>. Using dmPGE2, we observed significantly enhanced engraftment of young and aged MuSCs relative to controls that was further increased in response to notexin injury, a well-accepted stringent test of stem cell function (<FIG>). Thus, the delivery of dmPGE2 together with MuSC cell populations suffices to augment regeneration.

We postulated that delivery of PGE2 alone could stimulate muscle regeneration. To test this, muscles of young mice were injured with cardiotoxin and three days later a bolus of dmPGE2 was injected into the hindlimb muscles of young mice. We observed an increase (<NUM> ± <NUM>%) in endogenous PAX7-expressing MuSCs in the classic satellite cell niche beneath the basal lamina and atop myofibers fourteen days post injury (<FIG>), whereas dmPGE2 had no effect in the absence of injury. Further, at this early time point, the distribution of myofibers shifted toward larger sizes, assessed as cross-sectional area using the Baxter Algorithms for Myofiber Analysis, suggesting that regeneration is accelerated by PGE2 (<FIG> and <FIG>). In addition, we tracked the response to injury and dmPGE2 of endogenous MuSCs by luciferase expression using a transgenic mouse model, Pax7creERT2;Rosa26-LSL-Luc (<FIG>). The BLI data were in agreement with the histological data (<FIG>).

We tested the effects of injecting indomethacin, a nonsteroidal anti-inflammatory drug (NSAID) and an inhibitor of COX2 which reduces PGE2 synthesis, on muscle regeneration. Upon indomethacin injection into the hindlimb muscles of the same Pax7creERT2;Rosa26-LSL-Luc mouse model three days post-cardiotoxin injury, we observed a significant decrease in luciferase activity indicative of an impairment in muscle stem cell activation and regeneration (<FIG>). Injection of indomethacin into cardiotoxin-injured muscles also led to a significant loss in Twitch force as compared to the control group assessed at day <NUM> post-injury (<FIG>). In aged mice, we also detected a substantial increase (<NUM> ± <NUM>%) in the number of endogenous MuSCs (<FIG>), and a concomitant increase in myofiber sizes (<FIG>) fourteen days post-injury after a single dmPGE2 injection. Thus, exposure solely to dmPGE2 impacts the magnitude and time course of the endogenous repair.

As the ultimate test, we determined if dmPGE2 enhanced regeneration could lead to increased muscle strength after a natural injury induced by downhill treadmill-running. In this scenario, damage was caused by a daily <NUM> run on a downhill treadmill <NUM> degree decline<NUM>. During week one, aged mice in the treatment group ran for <NUM> days in succession and were injected daily with dmPGE2 after exercise. During week two, aged mice in the treatment group ran for <NUM> consecutive days but received no additional treatment (<FIG>). The specific twitch and tetanic force were compared for dmPGE2 treated and untreated gastrocnemius mouse muscles (GA) and both were significantly increased (<FIG>). Thus, an acute exposure to dmPGE2 concurrent with exercise-induced injury can confer a significant increase in aged muscle strength.

We have discovered a new indication for PGE2 in skeletal muscle regeneration. Prior studies of PGE2 effects on skeletal muscle have shown that it alters the proliferation, fusion, protein degradation, and differentiation of myoblasts in tissue culture<NUM>-<NUM>. Thus, these studies differ from ours as myoblasts are progenitors that have lost stem cell function. Satellite cells (MuSCs) are crucial to development and regeneration<NUM>-<NUM>,<NUM> and their numbers are increased by running or other high intensity exercise in young and aged mice and humans<NUM>,<NUM>-<NUM>. Nonsteroidal anti-inflammatory agents have been reported to attenuate the exercise-induced increase in MuSCs<NUM>,<NUM>-<NUM>. Our data provide novel evidence that the beneficial effects of the early transient wave of inflammation that characterizes efficacious muscle regeneration<NUM> is due in part to PGE2 and its receptor EP4, which are essential and sufficient for MuSC proliferation and engraftment. For hematopoietic, liver, and colon tissues, delivery of the inhibitor of <NUM>-PGDH, SW033291, was recently shown to enhance regeneration<NUM>. Notably, PGE2 and its analogues have safely been used in human patients for decades, for instance to induce labor<NUM> and to promote hematopoietic stem cell transplantation<NUM> paving the way for its clinical use in restoring muscles post-injury. In summary, our findings show that an acute PGE2 regimen suffices to rapidly and robustly enhance regeneration of exercise-induced damage and overcome age-associated limitations leading to increased strength.

We performed all experiments and protocols in compliance with the institutional guidelines of Stanford University and Administrative Panel on Laboratory Animal Care (APLAC). We obtained wild-type aged C57BL/<NUM> (<NUM>-<NUM> mo) mice from the US National Institute on Aging (NIA) for aged muscle studies and young wild-type C57BL/<NUM> mice from Jackson Laboratory. Double-transgenic GFP/luc mice were generated as described previously<NUM>. Briefly, mice expressing a firefly luciferase (luc) transgene under the regulation of the ubiquitous Actb promoter were maintained in the FVB strain. Mice expressing a green fluorescent protein (GFP) transgene under the regulation of the ubiquitous UBC promoter were maintained in the C57BL/<NUM> strain. We used cells from GFP/luc for allogenic transplantation experiments into NOD-SCID (Jackson Laboratory) recipient mice. EP4fiox/flox (EP4f/f) mice were a kind gift from K. Andreasson (Stanford University)<NUM>. Double-transgenic Pax7CreERT2;Rosa26-LSL-Luc were generated by crossing Pax7CreERT2 mice obtained from Jackson Laboratory (Stock # <NUM>)<NUM> and Rosa26-LSL-Luc obtained from Jackson Laboratory (Stock # <NUM>)<NUM>. We validated these genotypes by appropriate PCR-based strategies. All mice from transgenic strains were of young age. Young mice were <NUM>-<NUM> mo. of age and aged mice were <NUM>-<NUM> mo of age for all strains. All mice used in these studies were females.

We isolated and enriched muscle stem cells as previously described<NUM>,<NUM>,<NUM>. Briefly, a gentle collagenase digestion and mincing by the MACs Dissociator enabled numerous single fibers to be dissociated, followed by dispase digestion to release mononucleated cells from their niches. Subsequently, the cell mixture was depleted for hematopoietic lineage expressing and non-muscle cells (CD45-/CD11b-/CD31-) using a magnetic bead column (Miltenyi). The remaining cell mixture was then subjected to FACS analysis to sort for MuSCs co-expressing CD34 and α7-integrin markers. We generated and analyzed flow cytometry scatter plots using FlowJo v10. For each sort, we pooled together MuSCs (~<NUM>,<NUM> each) from at least three independent donor female mice.

We transplanted <NUM> MuSCs (<FIG>, <FIG>) or <NUM>,<NUM> MuSCs (<FIG>) immediately following FACS isolation or after collection from cell culture directly into the tibialis anterior (TA) muscles of recipient mice as previously described<NUM>,<NUM>,<NUM>. For young MuSC studies, we transplanted cells from GFP/luc mice (<NUM>-<NUM> mo of age) into hindlimb-irradiated NOD-SCID mice. For aged MuSCs studies, we transplanted cells from aged C57BL/<NUM> mice (<NUM>-<NUM> mo, NIH) that were transduced with a luc-IRES-GFP lentivirus (GFP/luc virus) on day <NUM> of culture for a period of <NUM> hr before transplantation, as previously described<NUM> (see below "Muscle stem cell culture, treatment and lentiviral infection" section for details). Prior to transplantation of muscle stem cells, we anesthetized NOD-SCID recipient mice with ketamine (<NUM> per mouse) by intraperitoneal injection. We then irradiated hindlimbs with a single <NUM> Gy dose, with the rest of the body shielded in a lead jig. We performed transplantations within <NUM> d of irradiation. Cultured cells were treated as indicated (vehicle or PGE2 treated 10ng/ml) and collected from hydrogel cultures by incubation with <NUM>% trypsin in PBS for <NUM> at <NUM> and counted using a hemocytometer. We resuspended cells at desired cell concentrations in <NUM>% gelatin/PBS and then transplanted them (<NUM> MuSCs per TA) by intramuscular injection into the TA muscles in a <NUM>µl volume. For fresh MuSCs transplantation, we coinjected sorted cells with <NUM> nmol of <NUM>,<NUM>-Dimethyl Prostaglandin E2 (dmPGE2) (Tocris, catalog # <NUM>) or vehicle control (PBS). We compared cells from different conditions by transplantation into the TA muscles of contralateral legs in the same mice. One month after transplant, we injected <NUM>µl of notexin (<NUM>µg ml-<NUM>; Latoxan, France) to injure recipient muscles and to activate MuSCs in vivo. Eight weeks after transplantation, mice were euthanized and the TAs were collected for analysis.

We performed bioluminescence imaging (BLI) using a Xenogen-<NUM> system, as previously described<NUM>,<NUM>,<NUM>. Briefly, we anesthetized mice using isofluorane inhalation and administered <NUM>µL D-luciferin (<NUM> mmol kg-<NUM>, reconstituted in PBS; Caliper LifeSciences) by intraperitoneal injection. We acquired BLI using a <NUM> exposure at F-stop=<NUM> at <NUM> minutes after luciferin injection. Digital images were recorded and analyzed using Living Image software (Caliper LifeSciences). We analyzed images with a consistent region-of-interest (ROI) placed over each hindlimb to calculate a bioluminescence signal. We calculated a bioluminescence signal in radiance (p s-<NUM> cm-<NUM> sr-<NUM>) value of <NUM><NUM> to define an engraftment threshold. This radiance threshold of <NUM><NUM> is approximately equivalent to the total flux threshold in p/s reported previously. This BLI threshold corresponds to the histological detection of one or more GFP+ myofibers<NUM>,<NUM>,<NUM>. We performed BLI imaging every week after transplantation.

We used an injury model entailing intramuscular injection of <NUM>µl of notexin (<NUM>µg ml-<NUM>; Latoxan) or cardiotoxin (<NUM>; Latoxan) into the TA muscle. For cryoinjury, an incision was made in the skin overlying the TA muscle and a copper probe, chilled in liquid nitrogen, was applied to the TA muscle for three <NUM> intervals, allowing the muscle to thaw between each application of the cryoprobe. When indicated, <NUM> hr after injury either <NUM>,<NUM>-Dimethyl Prostaglandin E2 (dmPGE2) (<NUM> nmol, Tocris, catalog # <NUM>) or vehicle control (PBS) was injected into the TA muscle. The contralateral TA was used as an internal control. We collected tissues <NUM> days post-injury for analysis.

For Pax7CreERT2; Rosa26-LSL-Luc mice experiments, we treated mice with five consecutive daily intraperitoneal injections of tamoxifen to activate luciferase expression under the control of the Pax7 promoter. A week after the last tamoxifen injection, mice were subjected to intramuscular injection of <NUM>µl of cardiotoxin (<NUM>; Latoxan), which we designated as day <NUM> of the assay. Three days later either <NUM> nmol dmPGE2 (<NUM> nmol) or vehicle control (PBS) was injected into the TA muscle. The contralateral TA was used as an internal control. Bioluminescence was assayed at days <NUM>, <NUM>, <NUM> and <NUM> post-injury.

We collected and prepared recipient TA muscle tissues for histology as previously described<NUM>,<NUM>. We incubated transverse sections with anti-LAMININ (Millipore, clone A5, catalog # <NUM>-<NUM>, <NUM>:<NUM>), and anti-PAX7 (Santa Cruz Biotechnology, catalog # sc-<NUM>, <NUM>:<NUM>) primary antibodies and then with AlexaFluor secondary Antibodies (Jackson ImmunoResearch Laboratories, <NUM>:<NUM>). We counterstained nuclei with DAPI (Invitrogen). We acquired images with an AxioPlan2 epifluorescent microscope (Carl Zeiss Microimaging) with Plan NeoFluar <NUM>×/<NUM>. 30NA or <NUM>×/<NUM>. 75NA objectives (Carl Zeiss) and an ORCA-ER digital camera (Hamamatsu Photonics) controlled by the SlideBook (3i) software. The images were cropped using Adobe Photoshop with consistent contrast adjustments across all images from the same experiment. The image composites were generated using Adobe Illustrator. We analyzed the number of PAX7 positive cells using the MetaMorph Image Analysis software (Molecular Devices), and the fiber area using the Baxter Algorithms for Myofiber Analysis that identified the fibers and segmented the fibers in the image to analyze the area of each fiber. For PAX7 quantification we examined serial sections spanning a depth of at least <NUM> of the TA. For fiber area at least <NUM> fields of LAMININ-stained myofiber cross-sections encompassing over <NUM> myofibers were captured for each mouse as above. Data analyses were blinded. The researchers performing the imaging acquisition and scoring were unaware of treatment condition given to sample groups analyzed.

We fabricated polyethylene glycol (PEG) hydrogels from PEG precursors, synthesized as described previously<NUM>. Briefly, we produced hydrogels by using the published formulation to achieve <NUM>-kPa (Young's modulus) stiffness hydrogels in <NUM> thickness which is the optimal condition for culturing MuSCs and maintaining stem cell fate in culture<NUM>. We fabricated hydrogel microwell arrays of <NUM>-kPa for clonal proliferation experiments, as described previously<NUM>. We cut and adhered all hydrogels to cover the surface area of <NUM>-well or <NUM>-well culture plates.

Following isolation, we resuspended MuSCs in myogenic cell culture medium containing DMEM/F10 (<NUM>:<NUM>), <NUM>% FBS, <NUM> ng ml-<NUM> fibroblast growth factor-<NUM> (FGF-<NUM> also known as bFGF) and <NUM>% penicillin-streptomycin. We seeded MuSC suspensions at a density of <NUM> cells per cm<NUM> surface area. We maintained cell cultures at <NUM> in <NUM>% CO<NUM> and changed medium daily. For PGE2, <NUM>-PGDH inhibitor and EP4 receptor antagonist treatment studies, we added <NUM>-<NUM> ng/ml Prostaglandin E2 (Cayman Chemical) (unless specified in the figure legends, <NUM> ng/ml was the standard concentration used), and/or <NUM> EP4 antagonist (ONO-AE3-<NUM>, Cayman Chemical), or <NUM> <NUM>-PGDH inhibitor (SW033291, Cayman Chemical) to the MuSCs cultured on collagen coated dishes for the first <NUM>. The cells were then trypsinized and cells reseeded onto hydrogels for an additional <NUM> days of culture. All treatments were compared to their solvent (DMSO) vehicle control. For stripped serum assays, we resuspended isolated MuSCs in medium containing DMEM/F10 (<NUM>:<NUM>), <NUM>% charcoal stripped FBS (Gibco, cat # <NUM>), <NUM> ng ml-<NUM> bFGF and <NUM>% penicillin-streptomycin. When noted in the figure, we additionally added <NUM>. 5µg/ml insulin (Sigma, I0516) and <NUM> dexamethasone (Sigma, D8893) to stripped serum cell medium. For these experiments MuSCs were cultured on hydrogels and vehicle (DMSO) or <NUM> ng/ml PGE2 (Cayman Chemical) was added to the cultures with every media change (every two days). Proliferation (see below) was assayed <NUM> days later. We performed all MuSC culture assays and transplantations after <NUM> week of culture unless noted otherwise. For aged MuSCs transplant studies, we infected MuSCs with lentivirus encoding elongation factor-1α promoter-driven luc-IRES-GFP (GFP/luc virus) for <NUM> in culture as described previously<NUM>. For EP4f/f MuSCs studies, we isolated MuSCs as described above (Muscle stem cell isolation), and infected all cells with the GFP/luc virus and a subset of them was coinfected with a lentivirus encoding pLM-CMV-R-Cre (mCherry/Cre virus) for <NUM> in culture. pLM-CMV-R-Cre was a gift from Michel Sadelain (Addgene plasmid # <NUM>)<NUM>. We transplanted aged MuSC (<NUM> cells) or EP4f/f MuSCs (<NUM>,<NUM> cells) into young (<NUM>-<NUM> mo) <NUM>-gy irradiated TAs of NOD-SCID recipient mice. For in vitro proliferation assays, EP4f/f MuSCs were plated on hydrogels post-infection and treated for <NUM> hr with vehicle (DMSO) or <NUM> ng/ml PGE2, and proliferation was assayed <NUM> days later. Cells were assayed for GFP and/or mCherry expression <NUM> post-infection using an inverted fluorescence microscope (Carl Zeiss Microimaging). MuSCs are freshly isolated from the mice by FACS and put in culture for a maximum time period of one week, therefore mycoplasma contamination is not assessed.

To assay proliferation, we used three different assays (hemocytometer, VisionBlue, and EdU). For each, we seeded MuSCs on flat hydrogels (hemocytometer and VisionBlue) or collagen-coated plates (EdU assay) at a density of <NUM> cells per cm<NUM> surface area. For hemocytometer cell number count, we collected cells at indicated timepoints by incubation with <NUM>% trypsin in PBS for <NUM> at <NUM> and quantified them using a hemocytometer at least <NUM> times. Additionally, we used the VisionBlue Quick Cell Viability Fluorometric Assay Kit (BioVision, catalog # K303) as a readout for cell growth in culture. Briefly, we incubated MuSCs with <NUM>% VisionBlue in culture medium for <NUM>, and measured fluorescence intensity on a fluorescence plate reader (Infinite M1000 PRO, Tecan) at Ex= <NUM>-<NUM>, Em=<NUM>-<NUM>. We assayed proliferation using the Click-iT EdU Alexa Fluor <NUM> Imaging kit (Life Technologies). Briefly, we incubated live cells with EdU (<NUM>) for 1hr prior to fixation, and stained nuclei according to the manufacturer's guidelines together with anti-MYOGENIN (Santa Cruz, catalog # sc576, <NUM>:<NUM>) to assay differentiation. We counterstained nuclei with DAPI (Invitrogen). We acquired images with an AxioPlan2 epifluorescent microscope (Carl Zeiss Microimaging) with Plan NeoFluar <NUM>×/<NUM>. 30NA or <NUM>×/<NUM>. 75NA objectives (Carl Zeiss) and an ORCA-ER digital camera (Hamamatsu Photonics) controlled by SlideBook (3i) software. We quantified EdU positive cells using the MetaMorph Image Analysis software (Molecular Devices). Data analyses were blinded, where researchers performing cell scoring were unaware of the treatment condition given to sample groups analyzed.

We assayed clonal muscle stem cell proliferation by time-lapse microscopy as previously described<NUM>,<NUM>. Briefly, we treated isolated aged MuSCs with PGE2 (Cayman Chemical) or vehicle (DMSO) for 24hr. After five days of growth on hydrogels, cells were reseeded at a density of <NUM> cells per cm<NUM> surface area in hydrogel microwells with <NUM> diameter. For time-lapse microscopy we monitored cell proliferation for those wells with single cells beginning <NUM> hr (day <NUM>) to two days after seeding and recorded images every <NUM> at <NUM>× magnification using a PALM/AxioObserver Z1 system (Carl Zeiss MicroImaging) with a custom environmental control chamber and motorized stage. We changed medium every other day in between the acquisition time intervals. We analyzed time-lapse image sequences using the Baxter Algorithms for Cell Tracking and Lineage Reconstruction to identify and track single cells and generate lineage trees<NUM>,<NUM>,<NUM>-<NUM>.

Viable and dead cells were distinguished in time-lapse sequences based on phase-contrast boundary and motility maintenance or loss, respectively. We found that the rates of proliferation (division) and death in the two conditions varied over time, Therefore, we estimated the rates for the first and the second <NUM> hour intervals separately. The values were estimated using the equations described in <NUM>, and found in Table <NUM>. We denote the proliferation rates in the two intervals p<NUM> and p<NUM> and the corresponding death rates d<NUM> and d<NUM>. As an example, the proliferation rate in the treated condition during the second <NUM> hour interval is <NUM>% per hour. Table <NUM> (below) shows that the rates of proliferation and death in the two conditions are similar in the first time interval, and that the difference in cell numbers at the end of the experiment is due to differences in both the division rates and the death rates during the second time interval. The modeled cell counts in the two time intervals are given by <MAT> where c<NUM> is the number of cells at the onset. The modeled curves are plotted together with the actual cell counts in <FIG>.

The data analysis was blinded. The researchers performing the imaging acquisition and scoring were unaware of the treatment condition given to sample groups analyzed.

We isolated RNA from MuSCs using the RNeasy Micro Kit (Qiagen). For muscle samples, we snap froze the tissue in liquid nitrogen, homogenized the tissues using a mortar and pestle, followed by syringe and needle trituration, and then isolated RNA using Trizol (Invitrogen). We reverse-transcribed cDNA from total mRNA from each sample using the SensiFAST™ cDNA Synthesis Kit (Bioline). We subjected 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 samples at <NUM> for <NUM> and then <NUM> cycles at <NUM> for <NUM> and <NUM> for <NUM>. To quantify relative transcript levels, we used <NUM>-ΔΔCt to compare treated and untreated samples and expressed the results relative to Gapdh. For SYBR Green qRT-PCR, we used the following primer sequences: Gapdh, forward <NUM>'-TTCACCACCATGGAGAAGGC-<NUM>', reverse <NUM>'-CCCTTTTGGCTCCACCCT-<NUM>'; Hpgd, forward <NUM>'- TCCAGTGTGATGTGGCTGAC -<NUM>', reverse <NUM>'-ATTGTTCACGCCTGCATTGT-<NUM>'; Ptges, forward <NUM>'-GCTGTCATCACAGGCCAGA-<NUM>', reverse <NUM>'-CTCCACATCTGGGTCACTCC-<NUM>'; Ptges2, forward <NUM>'-CTCCTACAGGAAAGTGCCCA-<NUM>', reverse <NUM>'-ACCAGGTAGGTCTTGAGGGC -<NUM>'; Ptger1, forward <NUM>' GTGGTGTCGTGCATCTGCT-<NUM>', reverse, <NUM>' CCGCTGCAGGGAGTTAGAGT-<NUM>', and Ptger2, forward <NUM>'-ACCTTCGCCATATGCTCCTT-<NUM>', reverse <NUM>'-GGACCGGTGGCCTAAGTATG-<NUM>'. TaqMan Assays (Applied Biosystems) were used to quantify Pax7, Myogenin, Slco2a1 (PGT), Ptger3 and Ptger4 in samples according to the manufacturer instructions with the TaqMan Universal PCR Master Mix reagent kit (Applied Biosystems). 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 enabled target signals (FAM) to be normalized individually by their internal Gapdh signals (VIC).

Muscle was harvested, rinsed in ice-cold PBS containing indomethacin (<NUM>µg/ml), and snap frozen in liquid nitrogen. Frozen samples were pulverized in liquid nitrogen. The powder was transferred to an Eppendorf tube with <NUM>µl of lysate buffer (<NUM> Tris-HCl pH <NUM>, <NUM> NaCl, <NUM> CaCl, <NUM>% Triton X-<NUM>, protease inhibitors and micrococcal nuclease), and then homogenized using a tissue homogenizer. The PGE2 level of the supernatant was measured using a PGE2 ELISA Kit (R&D Systems, catalog # KGE004B) and expressed relative to total protein measured by BCA assay (BioRad) and expressed as ng of PGE2. Each sample was assayed in duplicate and in each of two independent experiments.

MuSCs were treated with DMSO (vehicle) or PGE2 (<NUM> ng/ml) for <NUM> and cyclic AMP levels measured according to the cAMP-Glo Assay protocol optimized by the manufacturer (Promega). Each sample was assayed in triplicate and in two independent experiments.

We assayed Annexin V as a readout of apoptosis for MuSCs after <NUM> days in culture on hydrogels, after an initial acute (<NUM> hr) treatment of vehicle (DMSO) or PGE2 (<NUM> ng/ml). We used the FITC Annexin V Apoptosis Detection Kit (Biolegend, cat # <NUM>) according to the protocol of the manufacturer. We analyzed the cells for Annexin V on a FACS LSR II cytometer using FACSDiva software (BD Biosciences) in the Shared FACS Facility, purchased using an NIH S10 Shared Instrument Grant (S10RR027431-<NUM>).

All prostaglandin standards - PGF2α; PGE2; PGD2; <NUM>-keto PGE2; <NUM>,<NUM>-dihydro <NUM>-keto PGE2; PGE2-D4; and PGF2α-D9 - were purchased from Cayman Chemical. For the PGE2-D4 internal standard, positions <NUM> and <NUM> were labeled with a total of four deuterium atoms. For PGF2α-D9, positions <NUM>, <NUM>, <NUM> and <NUM> were labeled with a total of nine deuterium atoms.

Analyte stock solutions (<NUM>/mL) were prepared in DMSO. These stock solutions were serially diluted with acetonitrile/water (<NUM>:<NUM> v/v) to obtain a series of standard working solutions, which were used to generate the calibration curve. Calibration curves were prepared by spiking <NUM> uL of each standard working solution into <NUM>µL of homogenization buffer (acetone/water <NUM>:<NUM> v/v; <NUM>% BHT to prevent oxidation) followed by addition of <NUM> uL internal standard solution (<NUM> ng/mL each PGF2α-D9 and PGE2-D4). A calibration curve was prepared fresh with each set of samples. Calibration curve ranges: for PGE2 and <NUM>,<NUM>-dihydro <NUM>-keto PGE2, from <NUM> ng/mL to <NUM> ng/mL; for PGD2 and PGF2α, from <NUM> ng/mL to <NUM> ng/mL; and for <NUM>-keto PGE2, from <NUM> ng/mL to <NUM> ng/mL.

The extraction procedure was modified from that of Prasain et al. <NUM> and included acetone protein precipitation followed by <NUM>-step liquid-liquid extraction; the latter step enhances LC-MS/MS sensitivity. Butylated hydroxytoluene (BHT) and evaporation under nitrogen (N2) gas were used to prevent oxidation.

Solid tissues were harvested, weighed, and snap-frozen with liquid nitrogen. Muscle tissue was combined with homogenization beads and <NUM>µL homogenization buffer in a polypropylene tube and processed in a FastPrep <NUM> homogenizer (MP Biomedicals) for <NUM> seconds at a speed of <NUM>/s. After homogenization, <NUM>µL internal standard solution (<NUM> ng/mL) was added to tissue homogenate followed by sonication and shaking for <NUM> minutes. Samples were centrifuged and the supernatant was transferred to a clean eppendorf tube. <NUM>µL hexane was added to the sample, followed by shaking for <NUM> minutes, then centrifugation. Samples were frozen at -<NUM> for <NUM> minutes. The hexane layer was poured off from the frozen lower aqueous layer, and discarded. After thawing, 25µL of 1N formic acid was added to the bottom aqueous layer, and the samples were vortexed. For the second extraction, <NUM>µL chloroform was added to the aqueous phase. Samples were shaken for <NUM> minutes to ensure full 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 <NUM>. The dry residue was reconstituted in <NUM>µL acetonitrile/<NUM> ammonium acetate (<NUM>:<NUM> v/v) and analyzed by LC-MS/MS.

Since many prostaglandins are positional isomers with identical masses and have similar fragmentation patterns, chromatographic separation is critical. Two SRM transitions - one quantifier and one qualifier - were carefully selected for each analyte. Distinctive qualifier ion intensity ratios and retention times were essential to authenticate the target analytes. All analyses were carried out by negative electrospray LC-MS/MS using an LC-20ADXR prominence liquid chromatograph and <NUM> triple quadrupole mass spectrometer (Shimadzu). HPLC conditions: Acquity UPLC BEH C18 <NUM>. 1x100 mm, <NUM> particle size column was operated at <NUM> with a flow rate of <NUM>/min. Mobile phases consisted of A: <NUM>% acetic acid in water and B: <NUM>% acetic acid in acetonitrile. Elution profile: initial hold at <NUM>% B for <NUM> minutes, followed by a gradient of <NUM>%-<NUM>% in <NUM> minutes, then <NUM>%-<NUM>% in <NUM> minutes; total run time was <NUM> minutes. Injection volume was <NUM> uL. Using these HPLC conditions, we achieved baseline separation of the analytes of interest.

Selected reaction monitoring (SRM) was used for quantification. The mass transitions were as follows: PGD2: m/z <NUM> → m/z <NUM> (quantifier) and m/z <NUM> → m/z <NUM> (qualifier); PGE2: m/z <NUM> → m/z <NUM> (quantifier) and m/z <NUM> → m/z <NUM> (qualifier); PGF2α: m/z <NUM> → m/z <NUM> (quantifier) and m/z <NUM> → m/z <NUM> (qualifier); <NUM> keto-PGE2: m/z <NUM> → m/z <NUM> (quantifier) and m/z <NUM> → m/z <NUM> (qualifier); <NUM>, <NUM>-dihydro <NUM>-keto PGE2: m/z <NUM> → m/z <NUM> (quantifier) and m/z <NUM> → m/z <NUM> (qualifier); PGE2-D4: m/z <NUM> → m/z <NUM>; and PGF2α-D9: m/z <NUM> → m/z <NUM>. Dwell time was <NUM>-<NUM>.

Quantitative analysis was done using LabSolutions LCMS (Shimadzu). An internal standard method was used for quantification: PGE2-D4 was used as an internal standard for quantification of PGE2, <NUM>-keto PGE2, and <NUM>, <NUM>-dihydro <NUM>-keto PGE2. PGF2α-D9 was the internal standard for quantification of PGD2 and PGF2α. Calibration curves were linear (R><NUM>) over the concentration range using a weighting factor of <NUM>/X<NUM> where X is the concentration. The back-calculated standard concentrations were ±<NUM>% from nominal values, and ±<NUM>% at the lower limit of quantitation (LLOQ).

Aged mice (<NUM> mo. ) were subjected to downhill treadmill run for <NUM> consecutive weeks. During week <NUM>, mice ran daily for <NUM> days and rested on days <NUM> and <NUM>. Two hours after each treadmill run during week <NUM>, each (lateral and medial) gastrocnemius (GA) muscle from both legs of each mouse was injected with a dose of either PBS (vehicle control) or <NUM> dmPGE2 (experimental group). During week <NUM>, mice were subjected to <NUM> days treadmill run only. The treadmill run was performed using the Exer3/<NUM> (Columbus Instruments). Mice ran for <NUM> minutes on the treadmill at <NUM> degrees downhill, starting at a speed of <NUM> meters/min. After <NUM>, the speed was increased by <NUM> meter/min to a final speed of <NUM> meter/min. <NUM> minutes run time was chosen, as exhaustion defined as the inability of the animal to remain on the treadmill despite electrical prodding, was observed at a median of <NUM> minute in an independent control aged mouse group. Force measurements were on the GA muscles at week <NUM> based on a protocol published previously<NUM>. Briefly, for each mouse, an incision was made to expose the GA. We severed the calcaneus bone with intact achilles tendon and attached the tendon-bone complex to a 300C-LR force transducer (Aurora Scientific) with a thin metal hook. The muscles and tendons were kept moist by periodic wetting with saline (<NUM>% sodium chloride) solution. The lower limb was immobilized below the knee by a metal clamp without compromising the blood supply to the leg. The mouse was under inhaled anesthetic (<NUM>% isofluorane) during the entire force measuring procedure and body temperature was maintained by a heat lamp. In all measurements, we used <NUM>-ms pulses at a predetermined supramaximal stimulation voltage. The GA muscles were stimulated via the proximal sciatic nerve using a bipolar electrical stimulation cuff delivering a constant current of <NUM> mA (square pulse width <NUM>). GA muscles were stimulated with a single <NUM>-ms pulse for twitch force measurements, and a train of <NUM> for <NUM> pulses for tetanic force measurements. We performed five twitch and then five tetanic measurements on each muscle, with <NUM>-<NUM> recovery between each measurement with n=<NUM> mice per group. Data were collected with a PCI-<NUM> acquisition card (National Instruments) and analyzed in Matlab. We calculated specific force values by normalizing the force measurements by the muscle physiological cross-sectional areas (PCSAs), which were similar between the control and the experimental PGE2 treated group (Table <NUM>). PCSA (measured in mm<NUM>) was calculated according to the following equation<NUM>: PCSA (mm<NUM>) = [mass (g) × Cos θ] = [ρ(g/mm<NUM>) × fiber length (mm)], where θ is pennation angle of the fiber and ρ is muscle density (<NUM>/mm<NUM>).

We performed cell culture experiments in at least three independent experiments where three biological replicates were pooled in each. In general, we performed MuSC transplant experiments in at least two independent experiments, with at least <NUM>-<NUM> total transplants per condition. We used a paired t-test for experiments where control samples were from the same experiment in vitro or from contralateral limb muscles in vivo. A non-parametric Mann-Whitney test was used to determine the significance difference between untreated (-) vs treated (PGE or dmPGE2) groups using α=<NUM>. ANOVA or multiple t-test was performed for multiple comparisons with significance level determined using Bonferroni correction or with Fisher's test as indicated in the figure legends. Unless otherwise described, data are shown as the mean ± s.

This example shows an increase in specific twitch force of gastrocnemius muscles in aged mice injected with PGE2. The aged mice (<NUM> months old) were subject to treadmill run to exhaustion daily for <NUM> days. The treadmill run was performed using the Exer3/<NUM> (Columbus Instruments). Mice ran on the treadmill at <NUM> degrees downhill, starting at a speed of <NUM> meters/min. After <NUM>, the speed was increased <NUM> meter/min to a final speed of <NUM> meters/min. Exhaustion was defined as the inability of the animal to remain on the treadmill despite electrical prodding. <NUM> after each treadmill run, both gastrocnemius muscles of each mouse were injected with either PBS (control group) or <NUM> PGE2 (experimental group). The force measurement was performed <NUM> weeks after the last treadmill run using a 300C-LR force transducer (Aurora Scientific) with a single <NUM> pulse at predetermined supramaximal stimulation intensity.

Claim 1:
A composition comprising a therapeutically effective amount of a compound, neutralizing peptide, or neutralizing antibody that inactivates or blocks <NUM>-hydroxyprostaglandin dehydrogenase (<NUM>-PGDH) for use in regenerating a population of muscle cells and treating muscle damage, muscle injury, or muscle atrophy in a subject in need thereof.