Patent Application
20190256821
The present described inventions relate, inter alia, to methods and compositions that provide for improved production and efficacy of cells for use, e.g. in cell-based therapies.
Cell-based therapies provide for exciting possibilities for treatment of various diseases. However, the use of cellular therapy methods is often hindered by inefficient production techniques that can compromise efficacy.
For example, satellite cells are the major effector cell responsible for eliciting muscle regeneration and have potential for use in cell-based treatment of neuromuscular dystrophy diseases. Such treatment requires injection of expanded satellite cells that engraft and incorporate into skeletal muscle fibers. Preparation of these cells is cumbersome as, among others, in vitro mimicking of the satellite cell “niche”—including extracellular matrix (ECM) adhesion proteins which influence satellite cell activity—is often required. Extended passaging of muscle satellite cells using traditional methods and ECMs results in the progressive loss of engraftment potential. Traditional methods lead to poor cellular survival and only minimal integration of injected cells into skeletal muscle fibers.
There is a need for improved compositions and methods for the handling and production of cell-based therapies.
Accordingly, in general, methods and compositions which improve the production and utility of cell-based therapies are disclosed herein.
In one aspect, the present invention relates to compositions and methods for producing a progenitor cell, such as a muscle progenitor cell (satellite cell) in which cells are cultured in the presence of one or more laminin α5 proteins, such as laminin 521. In various embodiments, the present compositions and methods produce cells which have a differentiation and engraftment potential that is suitable for use as a cell-based therapy in humans or in drug discovery. In some embodiments, the present compositions and methods comprising one or more laminin α5, such as laminin 521, provide improved cell differentiation and engraftment potential as compared to cells not cultured in laminin α5, such as when cultured in any one or more of laminin 211, laminin 111, fibronectin, gelatin, collagen, hydrogel and matrigel (sometimes referred to as MG) i.e. a gelatinous protein mixture secreted by Engelbreth-Holm-Swarm mouse sarcoma cells, Corning Life Sciences). In some embodiments, the present compositions and methods allow for large-scale expansion and/or long term (e.g. multiple passage in vitro) culture of cells as compared to cells not cultured in laminin α5, such as when cultured in any one or more of laminin 211, laminin 111, fibronectin, gelatin, collagen, hydrogel, and matrigel.
In some embodiments, the present compositions and methods provide increased cellular proliferation during expansion of the cells, for example, in the early stages of expansion as compared to cells not cultured in laminin α5, such as when cultured in any one or more of laminin 211, laminin 111, fibronectin, gelatin, collagen, hydrogel, and matrigel.
In other embodiments, the present compositions and methods provide improved fusion, including following multiple cell passages as compared to cells not cultured in laminin α5, such as when cultured in any one or more of laminin 211, laminin 111, fibronectin, gelatin, collagen, hydrogel, and matrigel.
In some embodiments, the present compositions and methods provide improved fusion and therefore an increase in multinucleated myotubes as compared to cells not cultured in laminin a5, such as when cultured in any one or more of laminin 211, laminin 111, fibronectin, gelatin, collagen, hydrogel, and matrigel. In further embodiments, the present compositions and methods provide increased functional muscle fibers, e.g. when provided to a subject.
In various embodiments, the present compositions and methods provide improved differentiation and diminished spontaneous differentiation as compared to cells not cultured in laminin α5, such as when cultured in any one or more of laminin 211, laminin 111, fibronectin, gelatin, collagen, hydrogel, and matrigel. For example, the present compositions and methods provide for cells that express and/or up-regulate myosin heavy chain (MHC) or comparable differentiation-specific markers, such as, for example, alpha-actinin and troponin-T.
In some aspects, the present compositions and methods provide a cell, e.g. a progenitor cell, such as a muscle progenitor cell (satellite cell), culture on one or more laminin α5, such as laminin 521, which allows for expansion of satellite cells in vitro while maintaining their ability to be used for cell-based therapy applications. For example, the present invention provides methods of treatment of various neuromuscular diseases or disorders in which muscle progenitor cells (satellite cells) are prepared as described herein and implanted into a patient. Such methods find use in the treatment of a variety of neuromuscular diseases or disorders, such as, for example, muscular dystrophies.
The present invention is based, in part, on the surprising discovery that culture of muscle progenitor cells (satellite cells), including long-term culture (e.g. with multiple passages), with a laminin α5, such as laminin 521, is useful to maintain differentiation and engraftment potential of the cells, e.g. for therapeutic and drug discovery purposes.
Methods involving conventional molecular biology techniques are described herein. Such techniques are generally known in the art and are described in detail in methodology treatises, such as Current Protocols in Molecular Biology, ed. Ausubel et al., Greene Publishing and Wiley-Interscience, New York, 1992 (with periodic updates). Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention pertains. Commonly understood definitions of molecular biology terms can be found in, for example, Rieger et al., Glossary of Genetics: Classical and Molecular, 5th Ed., Springer-Verlag: New York, 1991, and Lewin, Genes V, Oxford University Press: New York, 1994. The definitions provided herein are to facilitate understanding of certain terms used frequently herein and are not meant to limit the scope of the present invention.
As used herein, the term “progenitor cell” refers to primary cells or cell lines that are committed to differentiate into a specific type of cell or to form a specific type of tissue (e.g., a muscle progenitor cell).
As used herein, the term “muscle progenitor cell” (used herein interchangeably with “satellite cell”) refers to progenitor cells that differentiate into muscle cells. For example, in mice, the muscle progenitor is integrin alpha 7 positive, and in human, the muscle progenitor is CD56 positive. In some embodiments, the human and mouse muscle progenitor is pax7 positive and/or myoD positive. In some embodiments, the muscle progenitor cell is derived from muscle tissue, and is not a pericyte or a mesoangioblast.
As used herein, the term “laminin α5” refers to extracellular matrix molecules or active fragments thereof encoded by the LAMAS gene (e.g., homo sapiens laminin subunit alpha 5 (LAMAS) mRNA, NCBI Ref. Seq. NM_05560.4, 11445 bp). In some embodiments, the laminin α5 protein is complexed with a laminin β and γ chain. In some embodiments, the laminin β and γ chain can be selected from laminin subunit 1, 2, or 3. In certain embodiments, the laminin α5 may be recombinant or non-recombinant. Examples of laminin α5 include, but are not limited to, laminin 521 (α5β2γ1 chain composition), laminin 511 (α5β1γ1 chain composition), laminin 522 (α5β2γ2 chain composition), laminin 523 (α5β2γ3 chain composition), and active fragments thereof. See also, e.g., 51. Spenlé et al., Cell Adh. Migr. 2013; 7(1):90-100, Macdonald et al., J Struct. Biol. 2010; 170(2):398-405, and Siler et al., Br. J. Haematol. 2002; 119:212-220.
As used herein, the terms “cultured” or “cell cultured” refers to growing cells outside of their natural environment. A “culture” refers to the cells and the structure holding them.
As used herein, the terms “passage” or “passaged” or “subculturing” refers to the process of transferring some cells from a previous culture to a new culture. In some embodiments, one or more passages are conducted. For example, in some embodiments, greater than about 5, 6, 7, 8, 9, 10, 15, or 25 passages are conducted without loss of beneficial properties.
As used herein, the term “fusion capacity” refers to the time it takes to obtain cells with more than one nucleus, and the extent of fusion as a function of the number of myotubes per total number of nuclei and number of nuclei per myotube. “Improved fusion capacity” results in an increase in multinucleated myotubes.
As used herein, “spontaneous differentiation” refers to a cell differentiating without induction.
Cell Production
In one aspect, the present invention relates to compositions and methods for producing a progenitor cell, such as a muscle progenitor cell (satellite cell), in which cells are cultured in the presence laminin α5. For example, in some embodiments, progenitor cells are cultured in a cell medium as known in the art and laminin α5 as the ECM substrate.
The progenitor cells may be primary cells or cell lines. Further, the methods of the invention can be used in vivo, ex vivo, or in vitro. For example, the primary cell can be autologous (derived from and provided to the same subject) or allogenic (derived from and provided to a different subject).
In various embodiments, the present compositions and methods provide cells which have a differentiation and engraftment potential that is suitable for use as a cell-based therapy or in neuromuscular drug discovery.
In some embodiments, the present compositions and methods comprising laminin α5, such as laminin 521, provide improved cell differentiation and engraftment potential as compared to cells not cultured in laminin α5, such as compared to compositions and methods comprising one or more of laminin 211, laminin 111, fibronectin, gelatin, collagen, hydrogel, and matrigel.
In some embodiments, the present compositions and methods provide increased cellular proliferation during expansion of the cells, for example, in the early stages of expansion. In some embodiments, the present compositions and methods provide improved fusion, including following multiple cell passages. In some embodiments, the present compositions and methods provide improved fusion and therefore an increase in multinucleated myotubes. In further embodiments, the present compositions and methods provide increased functional muscle fibers, e.g. when provided to a subject. In further embodiments, the present compositions and methods provide improved differentiation and diminished spontaneous differentiation. In some embodiments, the present compositions and methods provide for cells that up-regulate myosin heavy chain (MHC) or comparable differentiation-specific markers. In some embodiments, any of the features described in this paragraph are increased, decreased or otherwise improved as compared to cells not cultured in laminin α5, such as compared to compositions and methods comprising one or more of laminin 211, laminin 111, fibronectin, gelatin, collagen, hydrogel, and matrigel.
In some embodiments, the present compositions and methods allow progenitor cells to maintain their stem cell and differentiation potential after long term culture.
In some embodiments, the present compositions and methods allow for long term growth without loss of beneficial properties (e.g. for use as a cell-based therapy). For example, in some embodiments, the present compositions and methods support greater than about 5, or 6, or 7, or 8, or 9, or 10, or 15, or 25 passages without loss of beneficial properties (e.g. for use as a cell-based therapy). By way of further example, in some embodiments, the present compositions and methods support greater than about 2500-, or 3000-, or 3500-, or 4000-, or 4500-, or 5000-, or 5500-, or 6000-, or 6500-, or 7000-, or 7500-, or 8000-, or 8500-, or 9000-, or 9500-, or 10,000-, or 11,000-, or 12,000-, or 13,000-, or 14,000-, or 15,000-, or 16,000-, or 17,000-, or 18,000-, or 19,000-, or 20,000-, or 25,000-, or 30,000-, or 35,000-, or 40,000-, or 45,000-, or 50,000-, or 55,000-, or 60,000-, or 65,000- or 70,000-, or 75,000-, or 80,000-, or 85,000-, or 90,000-, or 95,000-, or 100,000-, or 105,000-fold expansion of cells without loss of beneficial properties (e.g. for use as a cell-based therapy).
In various embodiments, the laminin α5 is one or more of laminin 521, laminin 511, laminin 522, laminin 523, or active fragment thereof. In various embodiments, the laminin α5 is recombinant. In various embodiments, the laminin α5 is laminin 521 or active fragment thereof.
In various embodiments, and without wishing to be bound by theory, the laminin α5, e.g. laminin 521, interacts with one or more of with six integrin binding sites (α3β1 (twice), αVβ3, α6β1, α6β4, α7β1). This is distinguishable from the four binding sites in laminin 111 and MG (α1β1, α2β1, α6β1, α7β1).
Combination Culture Agents
In various embodiments, in addition to laminin α5, cells can also optionally be contacted with compounds that promote cell adhesion, proliferation, differentiation, and/or maintenance, including but not limited to any of the collagens, other laminin types, fibronectin, integrins, glycoproteins, proteoglycans, heparan sulfate proteoglycan, glycosaminoglycans, entactin, nidogen, and peptide fragments thereof.
In various embodiments, the present cells are produced by contacting the cells with laminin α5 (e.g., without limitation, laminin 521) and one or more additional ECM agents. Such additional ECM agents include one or more laminin other than laminin α5 (e.g. one or more of laminin 111, laminin 211, laminin 121, laminin 221, laminin 332/laminin 3a32, laminin 3b32, laminin 311/laminin 3a11, laminin 321/laminin-3a21, laminin-411, laminin-421, laminin-213, and laminin-423), fibronectin (e.g. type I or II), gelatin, collagen (e.g. one or more of collagen type I, III, IV, V, and VI), and matrigel.
In various embodiments, the present methods allow for broad substrate transfer compatibility. For example, in various embodiments, the present methods allow for cells to be expanded and/or maintained on a substrate comprising the agent comprising laminin α5 (e.g. laminin 521) and transferred to another of the substrate, e.g. laminin other than laminin α5 (e.g. one or more of laminin 111, laminin 211, laminin 121, laminin 221, laminin 332/laminin 3a32, laminin 3b32, laminin 311/laminin 3a11, laminin 321/laminin-3a21, laminin-411, laminin-421, laminin-213, and laminin-423), a fibronectin (e.g. type I or II), gelatin, a collagen (e.g. one or more of collagen type I, III, IV, V, and VI), hydrogel, or matrigel, without substantial loss of differentiation capacity.
In various embodiments, the present methods allow for expansion and/or maintenance on fibronectin and differentiation when moved to a substrate comprising the one or more laminin α5 (e.g. laminin 521).
Coatings
In various embodiments, the present methods provide for coating on the surface of a cell growth substrate.
In some embodiments, the one or more laminin α5 (e.g., without limitation, laminin 521) is used to coat the surface of a substrate to promote cell adhesion to the substrate, and to stimulate cell proliferation, differentiation, and/or maintenance. The substrate used herein may be any desired substrate. For laboratory use, the substrate may be glass or plastic or other cells. For use in vivo, the substrate may be any biologically compatible material capable of supporting cell growth. Illustrative suitable substrate materials include shaped articles made of or coated with such materials as collagen, regenerated collagen, polyglycolic acid, polygalactose, polylactic acid or derivatives thereof; biocompatible metals such as titanium and stainless steel; ceramic materials including prosthetic material such as hydroxylapatite; synthetic polymers including polyesters and nylons; polystyrene; polyacrylates; polytetrafluoroethylene, and virtually any other material to which biological molecules can readily adhere.
In various embodiments, the invention provides for coating cell culture plastic or glass with human laminin 521. In some embodiments, the laminin 521 is coated at a coating concentration of about 10 ug/ml to about 20 ug/ml (e.g. about 10 ug/ml, about 11 ug/ml, about 12 ug/ml, about 13 ug/ml, about 14 ug/ml, about 15 ug/ml, about 16 ug/ml, about 17 ug/ml, about 18 ug/ml, about 19 ug/ml, about 20 ug/ml, or about 10-20 ug/ml, or about 10-17.5 ug/ml, or about 10-15 ug/ml, or about 10-12.5 ug/ml. In various embodiments, coating is conducted for about 2 hours at 37° C. or overnight at 4° C. Afterwards, laminin 521 coating solution is decanted and replaced with satellite cell growth media, e.g. DMEM/F12.
In various embodiments, the coating is in the presence of calcium and/or magnesium.
Methods of Treatment
In one aspect, the present invention relates to a method of treating a neuromuscular disease or disorder by administering muscle progenitor cells (satellite cells) of the present invention to a subject. In various embodiments, the one or more laminin α5 (e.g., without limitation, laminin 521) is used to prepare an effective amount of the progenitor cell for use as a cell-based therapy for a neuromuscular disease or disorder.
In some embodiments, a method for treating or preventing a neuromuscular disease or disorder is provided comprising preparing a progenitor cell as described above, e.g., by culturing a progenitor cell in the presence of laminin α5 and administering an effective amount of the cultured progenitor cell to a subject in need thereof.
In some embodiments, the neuromuscular disease or disorder is an injury (e.g. muscular injury) and the present cells are useful for repair.
In some embodiments, the neuromuscular disease or disorder is a myopathy.
In some embodiments, the neuromuscular disease or disorder includes muscular dystrophies (e.g. myotonic dystrophy (Steinert disease), Duchenne muscular dystrophy, Becker muscular dystrophy, limb-girdle muscular dystrophy, facioscapulohumeral muscular dystrophy, congenital muscular dystrophy, oculopharyngeal muscular dystrophy, distal muscular dystrophy, Emery-Dreifuss muscular dystrophy), motor neuron diseases (e.g. amyotrophic lateral sclerosis (ALS), Infantile progressive spinal muscular atrophy (type 1, Werdnig-Hoffmann disease), intermediate spinal muscular atrophy (Type 2), juvenile spinal muscular atrophy (Type 3, Kugelberg-Welander disease), adult spinal muscular atrophy (Type 4), spinal-bulbar muscular atrophy (Kennedy disease)), inflammatory Myopathies (e.g. polymyositis dermatomyositis, inclusion-body myositis), diseases of neuromuscular junction (e.g. myasthenia gravis, Lambert-Eaton (myasthenic) syndrome, congenital myasthenic syndromes), diseases of peripheral nerve (e.g. Charcot-Marie-Tooth disease, Friedreich's ataxia, Dejerine-Sottas disease), metabolic diseases of muscle (e.g. phosphorylase deficiency (McArdle disease) acid maltase deficiency (Pompe disease) phosphofructokinase deficiency (Tarui disease) debrancher enzyme deficiency (Cori or Forbes disease) mitochondrial myopathy, carnitine deficiency, carnitine palmityl transferase deficiency, phosphoglycerate kinase deficiency, phosphoglycerate mutase deficiency, lactate dehydrogenase deficiency, myoadenylate deaminase deficiency), myopathies due to endocrine abnormalities (e.g. hyperthyroid myopathy, hypothyroid myopathy), and other myopathies (e.g. myotonia congenita paramyotonia congenita central core disease nemaline myopathy myotubular myopathy periodic paralysis).
In some embodiments, the neuromuscular disease or disorder is a muscular dystrophy or related myopathy, e.g. Becker muscular dystrophy (OMIM 300376, the entire contents of which are hereby incorporated by reference), congenital muscular dystrophy, Duchenne muscular dystrophy (OMIM 310200, the entire contents of which are hereby incorporated by reference and including Steinert's Disease and DM2), distal muscular dystrophy (OMIM 254130, the entire contents of which are hereby incorporated by reference), Emery-Dreifuss muscular dystrophy OMIM 310300 and 181350, the entire contents of which are hereby incorporated by reference), facioscapulohumeral muscular dystrophy (OMIM 158900, the entire contents of which are hereby incorporated by reference), limb-girdle muscular dystrophy, myotonic muscular dystrophy (OMIM160900 and 602668, the entire contents of which are hereby incorporated by reference), and oculopharyngeal muscular dystrophy (OMIM 164300, the entire contents of which are hereby incorporated by reference)).
Muscular dystrophy refers to a group of diseases that cause weakness and progressive degeneration of skeletal muscles. There are different forms of muscular dystrophy which differ in their mode of inheritance, age of onset, severity and pattern of muscles affected. The most well-known muscular dystrophies are Duchenne, Becker, limb girdle, congenital, facioscapulohumeral, myotonic, oculopharyngeal, distal, Miyoshi myopathy and Emery-Dreifuss but there are more than 100 myopathies with similarities to muscular dystrophy, all of which are included within the scope of the present invention. The term “myopathy” refers to a muscular disease in which the skeletal muscle fibers do not function for any one of many reasons, resulting in muscular weakness.
Duchenne muscular dystrophy has been often considered as a model muscular dystrophy. Duchenne muscular dystrophy results from mutations in the gene coding for the protein dystrophin, which localizes at the inner face of the sarcolemma. Dystrophin associates with a large complex of membrane proteins, called the dystrophin glycoprotein complex, important for cell membrane integrity. Without the dystrophin complex to tether the actin cytoskeleton inside the muscle cell to the extracellular matrix, forces generated by the muscle fiber result in tears of sarcolemma leading to muscle damage. The mdx mice strain is the most widely used animal model for Duchenne muscular dystrophy, having a nonsense mutation in exon 23 which eliminates dystrophin expression. Human patients with Duchenne muscular dystrophy and mdx mice suffer progressive skeletal muscle degeneration.
Muscle degeneration is a common feature of muscular dystrophy patients. Skeletal fiber loss is initially compensated by proliferation and fusion with preexisting fibers of satellite cells, resulting in an increase in muscle size. After repetitive cycles of muscle degeneration and regeneration, the dystrophic muscle damage can, however, ultimately not be repaired anymore and the dystrophic fibers become gradually replaced, initially by fibrotic infiltrates and subsequently by fat tissue. In fact, muscles of Duchenne muscular dystrophy patients or mdx mice, as well as other muscular dystrophy patients, present high fibrosis. The whole degenerative process leads to loss of normal muscle function.
Thus, in some embodiments, the present improved cells and cell productions allow for improved treatments of muscular dystrophies. The muscular dystrophy to be treated is selected from Duchenne muscular dystrophy, Becker's muscular dystrophy, limb girdlemuscular dystrophy, congenital muscular dystrophy, facioscapulohumeral muscular dystrophy, myotonic muscular dystrophy, oculopharyngeal muscular dystrophy, distal muscular dystrophy, and Emery-Dreifuss muscular dystrophy. In a particular embodiment, the treatment provided is for Duchenne muscular dystrophy.
In various embodiments, the present methods provide a reduction or alleviation of one or more of muscle pain, muscle weakness, muscle stiffness, difficulty in walking, myotonia, fatigue, scoliosis, axonal peripheral neuropathy, cardiomyopathy, cardiac arrhythmia, mental retardation, hypersomnia, sleep apnea, iridescent posterior subcapsular cataracts, insulin insensitivity, type II diabetes mellitus, premature balding, testicular failure, infantile hypotonia, and respiratory deficits.
In some embodiments, the muscle progenitor cells that are cultured are autologous (derived from and provided to the same subject) or allogenic (derived from and provided to a different subject).
In various embodiments, the present treatment methods further comprise an additional therapeutic agent which is selected based on the disease state for which the cell-based therapy is being used.
In various embodiments, the present cells are used to treat a neuromuscular disease or disorder (e.g. a muscular dystrophy, e.g. Duchenne muscular dystrophy) in combination with an additional therapeutic agent. Illustrative additional therapeutic agents include corticosteroids such as prednisone, deflazacort and VBP15. Further illustrative additional therapeutic agents include ataluren (TRANSLARNA, PTC Therapeutics), PTC 124, eteplirsen (Sarepta), SRP-4045 (Sarepta), SRP-4052 (Sarepta), SRP-4053 (Sarepta), tamoxifen, idebenone, PB1046, vamorolone, TAS-205, NS-065/NCNP-01, Rimeporide, DS-5141b, Drisapersen, FG-3019, Deflazacort, Sustanon (testosterone), BMS-986089, HT-100, CAP-1002, and CAT-1004. Further still illustrative additional therapeutic agents include supplements, such as coenzyme Q10, carnitine, amino acids (e.g. glutamine, arginine), anti-inflammatories/anti-oxidants (e.g. fish or krill oil, vitamin E, green-tea extract), and others.
In various embodiments, routes of administration include, for example: intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, oral, sublingual, intranasal, intracerebral, intravaginal, transdermal, rectally, by inhalation, or topically, particularly to the ears, nose, eyes, or skin. In some embodiments, the administering is effected orally or by parenteral injection. In various embodiments, the cells of the present invention can be administered by intravenous infusion or bolus injection. In various embodiments, the cells of the present invention can be administered by infusion or engraftment. The mode of administration can be left to the discretion of the practitioner, and depends in-part upon the site of the medical condition. In most instances, administration results in the release of any agent described herein into the bloodstream.
Dosage forms suitable for parenteral administration (e.g. intravenous, intramuscular, intraperitoneal, subcutaneous and intra-articular injection and infusion) include, for example, solutions, suspensions, dispersions, emulsions, and the like. They may also be manufactured in the form of sterile solid compositions (e.g. lyophilized composition), which can be dissolved or suspended in sterile injectable medium immediately before use. They may contain, for example, suspending or dispersing agents known in the art.
Further, the cells described herein can take the form of solutions, suspensions, emulsion, drops, tablets, pills, pellets, capsules, capsules containing liquids, powders, sustained-release formulations, suppositories, emulsions, aerosols, sprays, suspensions, or any other form suitable for therapeutic use. In one embodiment, the composition is in the form of a capsule (see, e.g., U.S. Pat. No. 5,698,155). Other examples of suitable pharmaceutical excipients are described in Remington's Pharmaceutical Sciences 1447-1676 (Alfonso R. Gennaro eds., 19th ed. 1995), incorporated herein by reference.
In some embodiments, the subject and/or animal is a human. In some embodiments, the human is a pediatric human. In other embodiments, the human is an adult human. In other embodiments, the human is a geriatric human. In other embodiments, the human may be referred to as a patient.
In certain embodiments, the human has an age in a range of from about 0 months to about 6 months old, from about 6 to about 12 months old, from about 6 to about 18 months old, from about 18 to about 36 months old, from about 1 to about 5 years old, from about 5 to about 10 years old, from about 10 to about 15 years old, from about 15 to about 20 years old, from about 20 to about 25 years old, from about 25 to about 30 years old, from about 30 to about 35 years old, from about 35 to about 40 years old, from about 40 to about 45 years old, from about 45 to about 50 years old, from about 50 to about 55 years old, from about 55 to about 60 years old, from about 60 to about 65 years old, from about 65 to about 70 years old, from about 70 to about 75 years old, from about 75 to about 80 years old, from about 80 to about 85 years old, from about 85 to about 90 years old, from about 90 to about 95 years old or from about 95 to about 100 years old.
In other embodiments, the subject is a non-human animal, and therefore the invention pertains to veterinary use. In a specific embodiment, the non-human animal is a household pet. In another specific embodiment, the non-human animal is a livestock animal.
Drug Discovery
In various embodiments, the present invention provides for methods of drug discovery with the muscle progenitor cells (satellite cells) of the present invention. In various embodiments, the present methods provide sufficient scale-up of skeletal muscle precursors into hundreds of millions or billions of cells for high throughput drug screening. In order to perform drug discovery in relevant models derived from stem cells isolated from patients, it is critical that the cells maintain the expansion and differentiation potential of the primary cell type. Given that target cells for assays addressing the majority of neuromuscular disease are multinucleated and differentiated myotubes and not transit amplifying myogenic cells, it is critical that culture conditions support the ability of the expanded primary cells to differentiate effectively into myotubes. Accordingly, the present invention allows for such drug discovery methods.
Cells made according to the methods and compositions described here may be used to screen for factors (such as solvents, small molecule drugs, peptides, polynucleotides, and the like) or environmental conditions (such as culture conditions or manipulations) that affect the characteristics of differentiated cells.
In some applications, the present cells are used to screen factors that promote maturation, or promote proliferation and maintenance of such cells in long-term culture. For example, candidate maturation factors or growth factors are tested by adding them to progenitor cells or differentiated cells in different wells, and then determining any phenotypic change that results, according to desirable criteria for further culture and use of the cells.
Furthermore, gene expression profiling of the present cell may be used to identify receptors, transcription factors, and signaling molecules that are unique or highly expressed in these cells. Specific ligands, small molecule inhibitors or activators for the receptors, transcription factors and signaling molecules may be used to modulate differentiation and properties of progenitor cell lines and differentiated cells.
Particular screening applications relate to the testing of pharmaceutical compounds in drug research. For instance, the methodologies in In vitro Methods in Pharmaceutical Research, Academic Press, 1997 (Eds. Castell and Gómez-Lechó) and U.S. Pat. No. 5,030,015, the entire contents of which are incorporated by reference in their entireties, provide various drug discovery methods which are applicable to the present invention.
In various embodiments, assessment of the activity of candidate pharmaceutical compounds generally involves combining the cells with the candidate compound, determining any change in the morphology, marker phenotype, or metabolic activity of the cells that is attributable to the compound (compared with untreated cells or cells treated with an inert compound), and then correlating the effect of the compound with the observed change.
The screening may be done, for example, either because the compound is designed to have a pharmacological effect on certain cell types, or because a compound designed to have effects elsewhere may have unintended side effects. Two or more drugs can be tested in combination (by combining with the cells either simultaneously or sequentially), to detect possible drug-drug interaction effects. In some applications, compounds are screened initially for potential toxicity (see, e.g., In vitro Methods in Pharmaceutical Research, Academic Press, 1997 (Eds. Castell and Gómez-Lechó), pp. 375-410, the entire contents of which incorporated by reference in its entirety). Cytotoxicity can be determined in the first instance by the effect on cell viability, survival, morphology, and expression or release of certain, markers, receptors or enzymes. Effects of a drug on chromosomal DNA can be determined by measuring DNA synthesis or repair. [3H]thymidine or BrdU incorporation, especially at unscheduled times in the cell cycle, or above the level required for cell replication, is consistent with a drug effect. Unwanted effects can also include unusual rates of sister chromatid exchange, as determined by metaphase spread.
In various embodiments, the present cells are useful in drug discovery efforts for agents that are beneficial for muscle health, e.g. those which may be used to treat or prevent one or more neuromuscular disease or disorder.
Kits
The invention provides kits that can simplify the cell production or treatment methods described herein. An illustrative kit of the invention comprises any composition, including produced cells, described herein in unit dosage form. The kit may comprise progenitor cells in a medium suitable for culturing and laminin α5. The kit can further comprise a label or printed instructions instructing the use of any agent described herein. The kit may also include a lid speculum, topical anesthetic, and a cleaning agent for the administration location. The kit can also further comprise one or more additional agent described herein. In one embodiment, the kit comprises a container containing an effective amount of a composition of the invention and an effective amount of another composition, such those described herein.
In one embodiment, the kit can comprise articles for cell culture, such as a cell growth substrate that is optionally pre-coated with agents described herein and culture media.
This invention is further illustrated by the following non-limiting examples.
Materials and Methods
Isolation and Culture of Murine Satellite Cells
Primary murine satellite cells were isolated from tibialis anterior and quadriceps muscles from 12 week old DBA/2J male mice. Dissected muscles were minced with scalpel blades and digested in DMEM/F12 (Life Technologies, 1:1 mixture) containing 2% collagenase II (Worthington Biochemicals) and 1.2 U/ml dispase (Worthington Biochemicals) with 2.5 mM CaCl2. Digestions were incubated at 37° C. for 1 hour with trituration and mixing every 15 minutes. Cells were filtered through 100 μM and 40 μM cell strainers (BD). Cells were pelleted by centrifugation for 5 min at 300 g. Cells were resuspended in FACS staining buffer (DMEM/F12/0.5% BSA/25 mM HEPES) and distributed in 200 μl aliquots into staining tubes. Cells were blocked using anti-CD16/CD32 antibody (Ebioscience) at 1:100 dilution for 10 min on ice. Cells were stained with the following antibodies on ice for 30 min: CD31-FITC (1:50, Ebioscience, 390) CD45-FITC (1:50, Ebioscience, 30-F11), PDGFRα-BV421 (1:40, BD, APAS), Scal-BV605 (1:100, BD, D7), and Integrin α7 (1:400, Ablab, R2F2). Cells were washed twice with sort buffer (HBSS/0.5% BSA/25mM HEPES) including centrifugation for 5 min at 300 g. Compensation controls were prepared using Ultracomp beads (Ebioscience). Single only bead controls were stained in 100 μl with 2 μl of each antibody for 15 min at room temperature. Beads were washed once with sort buffer and resuspended in sort buffer. Compensation was calculated using single stained and unstained bead controls with FACS DIVA compensation wizard. Gating was determined by using fluorescence minus one plus isotype controls. Dead cells were gated out using propidium iodide (Life Technologies).
ECM Coating and Culture
Laminins including laminin 111, laminin 211, laminin 332, laminin 411, laminin 421, laminin 511, and laminin 521 are human recombinant isoforms obtained from Biolamina. Laminins were diluted at a concentration of 10 ug/ml in HBSS with calcium and magnesium, and coated overnight at 4° C. Fibronectin was from human placenta (Corning #354008), and coated at 10 μg/ml in distilled water for 1 hour at room temperature. Growth factor reduced MATRIGEL (Corning) was diluted 1:5 with DMEM/F12 media and thinly coated by covering plastic, removing excess, and drying matrigel for 20 minutes at 37° C.
For initial characterization, cells were plated at a density of 2,000 cells per well in 96 well format in DMEM/F12/20% FBS/Primocin (Live Technologies/ Invivogen) with 10 ng/ml mouse FGF-2 (R&D). Media was replaced after 5 days and refreshed every 3 days afterwards. To induce differentiation at day 8, media was switched to Differentiation Media (DM), DMEM/F12/5% HI-HS/Primocin, and maintained until day 11.
For long term growth, cells were plated at a density of 10,000 cells per well in 6 well format. Cells were grown in growth media as previously described and refreshed every 3-4 days with growth media and 10 ng/ml FGF-2. Cells were split using Accutase and maintained on the same substrate for 6-8 passages. To assay differentiation, cells were split using Accutase and seeded in 96 well format at a density of 4,000 cells per well. Cells were grown in GM for 5 days, and then switched to DM for an additional 5 days.
For ECM substitution experiments, cells were thawed, expanded and passaged twice before analysis. At second passage, cells were transferred to a 96 well plate containing five of the ECM substrates (laminin 111, laminin 211, laminin 521, FN, and MG). Cells were grown and differentiated similarly to the previously mentioned long term growth procedure.
Immunocytochemistry and Imaging
Immunostaining was performed in black Corning 96 well plates. For myosin heavy chain (MHC) staining, cells were fixed using Cytoperm/Cytofix for 15 min at room temperature. Cells were rinsed twice and then subsequently blocked using 10% HI-HS/0.1% Triton for 1 hour at room temperature. Cells were stained with MHC-Alexa488 antibody at 1:100 overnight at 4° C. Cells were rinsed 4 times with PBS and stained with Hoechst to identify nuclei. Images were acquired using a 10× objective on a Cellomics ArrayScan. Analysis was performed using the Cellomics HCS Studio Version 6.5 software analyzing MHC positive cells containing 2 or more nuclei. Software algorithm used was the “myotube formation” package using dynamic thresholding, 3 sigma or isodata, for myotube identification.
For Pax7/MyoD staining cells were fixed using foxp3/ki67 nuclear fixation buffer (Ebioscience) for 15 min at room temperature. Cells were rinsed twice and blocked with Block Aid (Life Technologies) for 1 hour at room temperature. Pax7 (1:50, R&D) and MyoD (1:50, 5F11, Millipore) were coincubated overnight at 4° C. in Block Aid. Cells were rinsed 3× and secondary antibodies (donkey anti-mouse Alexa488, donkey anti-rat Alexa647; 1:200) were incubated for 1 hour at room temperature. Cells were rinsed 4× and stained with Hoechst for nuclei identification. Images were acquired using a 20× objective on a Cellomics ArrayScan. Analysis was performed using the nuclear colocalization algorithm (Cellomics HCS Studio 6.5) analyzing proportion of Pax7 or MyoD positive nuclei.
Integrin FACS Analysis
Passage 8 mouse satellite cells were split using Accutase, collected and centrifuged at 300 g for 5 min, and resuspended in FACS staining buffer. Cells were blocked with FC block (BD biosciences) at 1:50 for 10 min on ice. Afterwards cells were stained with the following PE conjugated antibodies: integrin alphal (BD 562115) at 1:40, integrin alpha2 (Ebioscience 12-5971-81) at 1:40, integrin alpha3 (R&D FAP2787P) at 1:10, integrin alpha4 (Ebioscience 12-0492-81) at 1:20, integrin alpha5 (BD 553930) at 1:40, integrin alpha6 (Ebioscience 12-0495-81) at 1:200, integrin alpha7 (Ablab) at 1:200, integrin alphaV (Ebioscience 12-0512-82) at 1:50, integrin beta1 (Ebioscience 12-0291-81) at 1:20, integrin beta2 (Ebioscience 12-0181-81) at 1:20, integrin beta3 (Ebioscience 12-0611-81) at 1:40, integrin beta4 (R&D FAB4054P) at 1:20, and integrin beta5 (Ebioscience 12-0497-41) at 1:20. Cells were stained for 30 min on ice followed by two washes in FACS stain buffer. Cells were resuspended in 300 μl of FACS buffer and analyzed on the FACS Aria II. Gating was set according to negative unstained and isotype control Rat IgG2a K-PE (Ebioscience 12-4321-81).
Human Myogenic Cell Isolation
Post-mortem non-diseased skeletal muscle gracillus tissue was obtained through Asterand. Muscle was trimmed of fat and connective tissue. Tissue was minced for approximately 10 minutes. Tissue was digested using Collagenase II (Worthington Biochemicals) and Dispase (Worthington Biochemicals), for approximately 75 minutes at 3° C. Digestions were performed in gentleMACS™ Dissociators. Tissue was pulsed every 15 minutes. Following digestion, cells were strained through 100 μM, 70 μM, and 30 μM cell strainers (Miltenyi), respectively. Cells were resuspended in approximately 200 μl of MACS stain buffer (Miltenyi). Cells are stained for 1 hour on ice with the following antibodies: CD11b-FITC, Miltenyi Biotec, Catalog Number:130-081-201, CD31-FITC, Miltenyi Biotec, Catalog Number; 130-092-654, CD45-FITC, Miltenyi Biotec, Catalog Number: 130-080-202, CD34-APC, BD Biosciences, Catalog Number: 560940, CD56-PE, Miltenyi Biotec, Catalog Number; 130-090-755. Afterwards cells were rinsed twice and subsequently incubated with anti-FITC microbeads (Miltenyi Biotec, 130-048-701) for 30 min on ice followed by two washes. Afterwards, cells were passed through a Miltenyi magnetic depletion column. The column binds magnetically labelled FITC+ cells (CD31, CD45, CD11b) while allowing FITC− cells to flow through. Cells moved passively through the column into a collection tube. Afterwards cells were centrifuged, resuspended in FACS buffer, and FACS sorted (FACS ARIA II) for CD56+, CD34−, CD45−, CD31−, CD11b− cells. Myogenic cells were grown in growth media DMEM/F12 (Gibco) supplemented with 20% FBS (Gibco)/Primocin and 10 ng/ml human FGF2 (R&D). For differentiation of human cells, cells were seeded at a density of 16,000 cells per well in 96 well format. After 3 days, half of the media was replaced with differentiation media consisting of DMEM/F12 supplemented with 5% HS-HI (Gibco) and Primocin. Afterwards, half of the media was replaced every other day until day 11 when cells were fixed with Ctyoperm/Cytofix (BD).
Statistics
Statistics for multiple comparisons were conducted using one-way ANOVA with Bonferroni correction. Significance is annotated as less than 0.05(*), less than 0.01(**), less than 0.001 (. ***), and less than 0.0001 (****). All comparisons were conducted using laminin 521 as control. Significance for myotube nuclei distribution was determined using linear regression. Statistical calculations were conducted using Graphpad Prism 6.
Short-Term and Long-Term Myogenic Cell Analysis
To compare the activity of freshly isolated mouse satellite cells, Integrinα7+/PDGFRα−/Sca1−/CD31−/CD45− cells were FACS sorted (
While satellite cells typically have strong differentiation potential when freshly isolated, no study has evaluated a variety of ECMs to expand satellite cells to maintain their stem cell and differentiation potential after long term culture. Therefore laminin 111, FN, and MG due to their common usage in the literature were selected, as well as laminin 211 and laminin 521 due to their expression in vivo. Additionally, laminin 521 was selected over laminin 511 due to the observed performance benefit in the short term study in
ECM Exchange
To determine the utility of using laminin 521 for cell types already expanded on substrates other than laminin 521, a substrate substitution experiment was performed evaluating previously isolated primary mouse satellite cells on each of the other substrates in the study. Cells expanded on laminin 521 show robust differentiation when transferred to any of the substrates tested here including laminin 111, laminin 211, FN, and MG (
Marker Analysis
Due to the large differences observed in myogenic cell performance a set of control experiments to rule out the presence of contaminating non-myogenic cells in the primary mouse satellite cell cultures was performed. FACS staining revealed 99% of cells stained positive for integrin α7 (
Integrin Profiling
Integrin receptor signaling plays many critical roles during myogenesis. Since laminins, FN, and components of MG, activate many of their functions via integrin receptors, it was hypothesized, without wishing to be bound by theory, that the observed differences in long term culture may be caused by shifts in integrin expression on different ECMs. Previously expanded mouse cells were assayed at passage 8 in growth conditions on each ECM (laminin 111, laminin 211, laminin 521, FN, and MG) by FACS staining using integrin α1-7, integrin αV, and integrin β1-5 antibodies (
Human Satellite Cell Culture and Expansion
In an effort to determine if the result obtained with the mouse model translates to humans, a similar evaluation was undertaken with freshly isolated human muscle cells. Human satellite cells were isolated from the gracilis muscle obtained from a post-mortem patient lacking diagnosed skeletal muscle disease. CD56+/CD31−/CD45−/CD11b− satellite cells were isolated using a dual MACS/FACS approach (
Since the mouse studies revealed unexpected expansion and differentiation effects on long term culture of satellite cells on specific substrates, we next performed a similar study on the newly generated human satellite cells. Human satellite cells expanded at a faster rate on laminin 521 compared to other substrates following 5 passages (
In addition to the mouse study presented here, an analysis of freshly isolated mdx/BL10 cells was performed, and a very similar pattern with laminin 521 outperforming laminin 111, laminin 211, FN, and MG in differentiation was observed (
Engraftment
Muscle stem cells were isolated from pax7 reporter mice expressing firefly luciferase (Pax7 Rydl satellite cells, Yfp/luci/DTR). Isolation was performed using enzymatic digestion and fluorescent activated cell sorting using muscle stem cell specific antibody integrin α7. Cells were passaged on each substrate independently on laminin 111, laminin 521, and growth factor-reduced matrigel (MG). Laminin coatings were prepared by coating cell culture ware with 10 μg/ml laminin in HBSS containing calcium and magnesium overnight at 4° C. MG was prepared by diluting 1:5 in serum-free media and thin coating plastic ware followed by drying at 37° C. for 30 min. Initial seeding was performed in 6-well plate format. Cells were expanded and split when cells reached approximately 60-75% confluency. Cells were split using Accutase enzymatic solution. Cells were frozen at passage 6. Cells were thawed and passaged two additional times on the same corresponding substrates as their initial expansion. To prepare the skeletal muscle for engraftment experiments, limb muscles (tibialis anterior, gastrocnemius, and quadriceps) of recipient mice were injected with 75 μl of 10 μM cardiotoxin 3 days prior to cellular injection. Cardiotoxin injection induces muscle degeneration and subsequent muscle regeneration. On day 3 following cardiotoxin injection, cells were lifted with Accutase, counted, and resuspended in 0.5% BSA DMEM/F12 with 25 mM HEPES/Phenol Red-free media (Gibco). Mice were anesthetized using Isoflurane gas. 100,000 cells were injected intramuscularly into the left gastrocnemius and quadriceps, and 50,000 cells were injected into the left tibialis anterior. Volume for tibialis anterior is 50 μl, gastrocnemius and quadriceps received 100 μl each.
The first engraftment study followed 18 mice (6 per substrate group) for 28 days. At Day 1 post-injection, mice were imaged in a LagoX live animal imager. Mice were injected via intraperitoneal route with 200 μl of Rediject Luciferin-D to visualize luciferase (
The second engraftment study followed 30 mice (10 per substrate group) for 49 days (2 mice in the MG group passed before day 49) using the same methods described above. At Day 1 post-injection, mice were imaged in a LagoX live animal imager. Mice were injected via intraperitoneal route with 200 μl of Rediject Luciferin-D to visualize luciferase (
Long-Term Passage
Satellite cells previously cultured for 14 passages on laminin 521 were passed one additional time to a total of 15 passages and differentiated in differentiation media (DMEM/F12+5% HI-HS+Primocin). Cells were seeded at density of 10,000 cells per well and media was changed 50% every two days. After 10 days in differentiation conditions, cells were fixed and analyzed for MHC expression to quantify myotubes. Cells were fixed using 4% PFA for 15 minutes, two washes in PBS, staining with Myosin Heavy Chain antibody at 1:100 dilution (Ebioscience) overnight at 4° C., followed by 4 washes in PBS, staining with Donkey-anti-mouse (1:400 dilution) Rhodamine Red labeled for 1 hour at room temperature, 4 washes in PBS, stain with Hoecsht 1:10,000 dilution for 5 min, wash out with PBS once. Imaging was conducted with Arrayscan VTi (
Other than in the examples herein, or unless otherwise expressly specified, all of the numerical ranges, amounts, values and percentages, such as those for amounts of materials, elemental contents, times and temperatures of reaction, ratios of amounts, and others, in the following portion of the specification and attached claims may be read as if prefaced by the word “about” even though the term “about” may not expressly appear with the value, amount, or range. Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.
Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains error necessarily resulting from the standard deviation found in its underlying respective testing measurements. Furthermore, when numerical ranges are set forth herein, these ranges are inclusive of the recited range end points (e.g., end points may be used). When percentages by weight are used herein, the numerical values reported are relative to the total weight.
Also, it should be understood that any numerical range recited herein is intended to include all sub-ranges subsumed therein. For example, a range of “1 to 10” is intended to include all sub-ranges between (and including) the recited minimum value of 1 and the recited maximum value of 10, that is, having a minimum value equal to or greater than 1 and a maximum value of equal to or less than 10. The terms “one,” “a,” or “an” as used herein are intended to include “at least one” or “one or more,” unless otherwise indicated.
While this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.
Any patent, publication, or other disclosure material, in whole or in part, that is said to be incorporated by reference herein is incorporated herein only to the extent that the incorporated material does not conflict with existing definitions, statements, or other disclosure material set forth in this disclosure. As such, and to the extent necessary, the disclosure as explicitly set forth herein supersedes any conflicting material incorporated herein by reference. Any material, or portion thereof, that is said to be incorporated by reference herein, but which conflicts with existing definitions, statements, or other disclosure material set forth herein will only be incorporated to the extent that no conflict arises between that incorporated material and the existing disclosure material.
For instance, the following references are incorporated by reference:
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| Number | Date | Country | Kind |
|---|---|---|---|
| 17305745.6 | Jun 2017 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2017/044170 | 7/27/2017 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62367812 | Jul 2016 | US |
