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WO2020028957A1 - Maturation de cellules de muscle squelettique - Google Patents

Maturation de cellules de muscle squelettique Download PDF

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Publication number
WO2020028957A1
WO2020028957A1 PCT/AU2019/050837 AU2019050837W WO2020028957A1 WO 2020028957 A1 WO2020028957 A1 WO 2020028957A1 AU 2019050837 W AU2019050837 W AU 2019050837W WO 2020028957 A1 WO2020028957 A1 WO 2020028957A1
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WIPO (PCT)
Prior art keywords
skeletal muscle
muscle cell
tissue
organoid
differentiation
Prior art date
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Ceased
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PCT/AU2019/050837
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English (en)
Inventor
James Hudson
Richard Mills
Enzo PORRELLO
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QIMR Berghofer Medical Research Institute
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Queensland Institute of Medical Research QIMR
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Priority claimed from AU2018902910A external-priority patent/AU2018902910A0/en
Application filed by Queensland Institute of Medical Research QIMR filed Critical Queensland Institute of Medical Research QIMR
Publication of WO2020028957A1 publication Critical patent/WO2020028957A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
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Definitions

  • THIS INVENTION relates to skeletal muscle. More particularly, this invention relates to a culture medium, system and method that promotes skeletal muscle cell differentiation and/or maturation in vitro.
  • Skeletal muscle makes up -40% of an average adult’s body mass and plays an essential role in whole body locomotion and metabolism [1] Despite its robust regenerative ability, skeletal muscle function can be compromised due to a number of myopathies including developmental disorders, neuromuscular diseases and muscular dystrophies [2] Furthermore, exercise induces adaptations in skeletal muscle that are regarded as some of the best preventions and treatments for many chronic diseases including cancer, cardiovascular disease and mental health [3, 4] Understanding the molecular mechanisms that drive both the positive adaptations of exercise and the negative effects of myopathies requires the development of better model systems that recapitulate human skeletal muscle physiology and pathophysiology.
  • the invention is broadly directed to a medium having defined constituents that promote or enhance skeletal muscle differentiation and/or maturation.
  • the invention is also broadly directed to a culture system that facilitates the formation of skeletal muscle cells and/or organoids.
  • the invention may facilitate determining the effects of drugs and/or other molecules, exercise and/or other actions or stimuli on skeletal muscle, skeletal muscle tissue and/or organ engineering such as for medical, veterinary and/or food technology applications.
  • a first aspect of the invention provides a skeletal muscle cell differentiation medium comprising a base medium and at least one of a Notch inhibitor and a Raf inhibitor.
  • the Notch inhibitor is DAPT.
  • the Raf inhibitor is or comprises a BRAF inhibitor, such as Dabrafenib.
  • the differentiation medium is serum free.
  • the medium further comprises a gelling agent.
  • a second aspect of the invention provides a skeletal muscle cell culture vessel comprising one or a plurality of wells that each comprise opposed poles that extend substantially perpendicularly from a basal surface of the well.
  • displacement of the opposed poles caused by muscle bundles in the well facilitates contractile force measurements.
  • a third aspect of the invention provides a skeletal muscle cell culture system comprising:
  • a fourth aspect of the invention provides a method of culturing or producing skeletal muscle cells, a skeletal muscle organoid or skeletal muscle tissue, said method including the step of contacting one or more skeletal muscle progenitor cells with the skeletal muscle cell differentiation medium of the first aspect for sufficient time and under suitable conditions to induce or promote differentiation of one or a plurality of skeletal muscle cells from the progenitor cells.
  • the method is at least partly performed using the cell culture vessel of the second aspect and/or the skeletal muscle cell differentiation system of the third aspect.
  • the one or more skeletal progenitor cells is or comprises a myoblast.
  • a fifth aspect provides a skeletal muscle cell, a skeletal muscle tissue or a skeletal muscle organoid produced by the method of the aforementioned aspect.
  • the skeletal muscle cell, tissue or organoid of this aspect is engineered to express an optogenetic actuator molecule which is a light-responsive protein; and a protein that emits light in response to detecting changes in plasma membrane voltage.
  • the optogenetic actuator molecule is channelrhodopsin.
  • the protein that emits light in response to detecting changes in plasma membrane voltage is ArcLight.
  • a sixth aspect of the invention provides a method of determining, assessing or monitoring the effect of a stimulus upon a skeletal muscle cell, tissue or organoid of the fifth aspect, said method including the steps of exposing the skeletal muscle cell, tissue or organoid to the stimulus and determining, assessing or monitoring the effect of the stimulus upon the skeletal muscle cell, tissue or organoid.
  • the skeletal muscle cell, tissue or organoid is produced using the medium of the first aspect or according to the method of the fourth aspect.
  • determining, assessing or monitoring the effect of a stimulus upon the skeletal muscle cell, tissue or organoid is performed using the culture vessel of the second aspect.
  • the stimulus is, or recapitulates or mimics, exercise.
  • the stimulus is light, which facilitates optogenetic analysis of the skeletal muscle cell, tissue or organoid.
  • the stimulus is an electrical stimulus.
  • a seventh aspect of the invention provides a method of identifying one or more molecules that modulate skeletal muscle cell differentiation in the medium of the first aspect, said method including contacting one or more skeletal muscle progenitor cells with one or more candidate molecules, whereby modification of the maturation of one or a plurality of the skeletal muscle progenitor cells indicates that the candidate molecule is a modulator of skeletal muscle progenitor cell differentiation.
  • the modulator at least partly inhibits or suppresses skeletal muscle cell differentiation.
  • the modulator at least partly enhances or promotes skeletal muscle cell differentiation.
  • An eighth aspect of the invention provides a method of determining, assessing or monitoring the effect of one or more molecules upon a skeletal muscle cell, tissue or organoid, said method including the steps of contacting the skeletal muscle cell, tissue or organoid produced according to the method of the third aspect to the one or more molecules and determining, assessing or monitoring the effect of the one or more molecules upon the skeletal muscle cell, tissue or organoid.
  • the method determines, assesses or monitors the therapeutic efficacy, safety or toxicity of the one or more molecules.
  • the skeletal muscle cell, tissue or organoid is produced using the differentiation medium of the first aspect or according to the method of the third aspect.
  • the method of the sixth, seventh and eighth aspects is at least partly performed using the cell culture vessel of the second aspect and/or the skeletal muscle cell differentiation system of the third aspect.
  • Figure 1 Iterative screening in a micro-muscle culture platform identifies serum- free muscle differentiation protocol.
  • Base media consisted of MEM a with 0.5% ITS, 2% B-27 and 2% HS.
  • Figure 2 Comparison of D&D differentiation versus 2% HS in human skeletal micro muscles.
  • MYH2 a marker of differentiated skeletal muscle, is rapidly upregulated during differentiation in both 2% HS or D&D hpMs by day 5 using qPCR.
  • n 4-6.
  • MYH3 a marker of differentiated skeletal muscle, is rapidly upregulated during differentiation in both 2% HS or D&D hpMs and is higher in D&D hpMs by day 5 using qPCR.
  • n 4-6.
  • Figure 3 Proteomic analysis of human skeletal micro muscle development reveals a rapid differentiation response using D&D.
  • FIG. 6 A) Whole-mount immunostaining. Whole-mount immunostaining of titin (green), desmin (red) and DNA (blue) in hpMs treated with DAPT and Dabrafenib at combinations of 1 pM and 10 pM. Scale bar- 500 pm.
  • B 2D differentiation of human myotubes using 2%HS and D&D protocols, immunostaining displays F-Actin (green) and DNA (blue). Scale bar- 200 pm.
  • Figure 8 A) LED array stimulator, upright (left) & placed on top of 96 well screening platform (right). B) Whole-mount immunostaining of hpMs after chronic electrical stimulation reveals significant myotube damage and cell death. Whole-mount immunostaining of titin (green), desmin (red) and DNA (blue). Scale bar- 500 pm. C) Protein expression changes of skeletal muscle sarcomeric and calcium handling proteins of stimulated hpMs compared to control hpMs. Data is presented as mean ⁇ s.e.m., * P ⁇ 0.05 using student t-test.
  • the present invention has arisen from work that aimed to identify a suitable differentiation medium for differentiation of myoblasts into skeletal muscle cells and organoids comprising skeletal muscle cells.
  • the present invention is therefore directed to a method, culture medium and/or system for facilitating differentiation of skeletal muscle tissue, such as in the form of 3D organoids.
  • the skeletal muscle cells and organoids may be useful for assessing the effects of exercise upon skeletal muscle and/or determining the effects of various compounds, drugs and other molecules upon skeletal muscle.
  • the invention may also provide skeletal muscle tissue and/or organ engineering such as for medical, veterinary and/or food technology applications. These include organ replacement or repair and the production of edible meat having enhanced skeletal muscle quality, although without limitation thereto.
  • indefinite articles“a” and“an” are not to be read as singular indefinite articles or as otherwise excluding more than one or more than a single subject to which the indefinite article refers.
  • “a” cell includes one cell, one or more cells and a plurality of cells.
  • the term“about” qualifies a stated value to encompass a range of values above or below the stated value. Suitably, in this context the range may be 2, 5 or 10% above or below the stated value.
  • “about 100 mM” may be 90-110 mM, 95-105 mM or 98-102 mM.
  • isolated material that has been removed from its natural state or otherwise been subjected to human manipulation.
  • Isolated material e.g, cells
  • enriched or purified is meant having a higher incidence, representation or frequency in a particular state (e.g an enriched or purified state) compared to a previous state prior to enrichment or purification.
  • the invention is broadly directed to a cell culture medium, vessel, system and/or method suitable for differentiating skeletal muscle cells from progenitor cells such as myoblasts.
  • An aspect of the invention provides a differentiation medium for skeletal muscle comprising a base medium and at least one of a Notch inhibitor and a Raf inhibitor.
  • the differentiation medium is serum free or substantially serum free.
  • Yet another aspect of the invention provides a method of culturing or producing skeletal muscle cells, said method including the step of contacting one or more skeletal muscle progenitor cells with the skeletal muscle cell differentiation medium of the first mentioned aspect for sufficient time and under suitable conditions to induce or promote differentiation of one or a plurality of skeletal muscle cells from the one or more skeletal muscle progenitor cells.
  • the present method is at least partly performed using the skeletal muscle cell differentiation system described hereinafter.
  • the method further includes the step of exposing the skeletal muscle cells to a stimulus, such as an optogenetic or electrical stimulus, that recapitulates or mimics muscular contraction or exercise.
  • a stimulus such as an optogenetic or electrical stimulus
  • the method of the present aspect may further include the step of engineering the skeletal muscle cells and/or the skeletal muscle progenitor cells to express an optogenetic actuator molecule, such as a light-responsive protein; and a protein that emits light in response to detecting changes in plasma membrane voltage.
  • A“ progenitor celF is a cell which is capable of differentiating along one or a plurality of developmental pathways, with or without self-renewal.
  • progenitor cells are unipotent or oligopotent and are capable of at least limited self-renewal.
  • the progenitor cell is a“ myoblast” , which is a progenitor cell that can differentiate through myogenesis to give rise to a muscle cell, such as a skeletal muscle myocyte, myotube, micro-muscle and/or myofibre.
  • the progenitor cell may be a primary cell obtainable or derivable from a mammal or may be a cell line such as mouse C2C12 cells or rat L6 myoblasts.
  • the myoblasts may be differentiated, derived or otherwise obtained from progenitor cells, such as embryonic stem cells (ES), pluripotent stem cells (PSCs) or from genetically-reprogrammed cells, such as induced pluripotent stem cells (iPSCs), although without limitation thereto.
  • progenitor cells such as embryonic stem cells (ES), pluripotent stem cells (PSCs) or from genetically-reprogrammed cells, such as induced pluripotent stem cells (iPSCs), although without limitation thereto.
  • progenitor cells such as embryonic stem cells (ES), pluripotent stem cells (PSCs) or from genetically-reprogrammed cells, such as induced pluripotent stem cells (iPSCs), although without limitation thereto.
  • iMPCs induced myogenic progenitor cells
  • iMPCs may be produced via transient overexpression of Pax7 in paraxial mesoderm cells differentiated from hPSCs (Rao et al
  • differentiate relate to progression or maturation of a cell from an earlier or initial stage of a developmental pathway to a later or more mature stage of the developmental pathway. It will be appreciated that in this context“ differentiated' does not mean or imply that the cell is fully differentiated and has lost pluropotentiality or capacity to further progress along the developmental pathway or along other developmental pathways. Differentiation may, or may not, be accompanied by cell division. In some embodiments, differentiation may further include, or be followed by, “ maturation”, which includes progression or development of skeletal muscle cells to a more mature phenotype, genotype and/or function.
  • the stage or state of differentiation of a cell may be characterized by the expression and/or non-expression of one of a plurality of markers.
  • markers is meant nucleic acids or proteins that are encoded by the genome of a cell, cell population, lineage, compartment or subset, whose expression or pattern of expression changes throughout development.
  • Nucleic acid marker expression may be detected or measured by any technique known in the art including nucleic acid sequence amplification (e.g . polymerase chain reaction) and nucleic acid hybridization (e.g. microarrays, Northern hybridization, in situ hybridization), although without limitation thereto.
  • Protein marker expression may be detected or measured by any technique known in the art including flow cytometry, immunohistochemistry, immunofluorescence, immunoblotting, protein arrays, and protein profiling (e.g 2D gel electrophoresis), although without limitation thereto.
  • protein markers are detected by an antibody or antibody fragment (which may be polyclonal or monoclonal) that binds the protein marker.
  • the antibody is labelled, such as with a radioactive label, a fluorophore (e.g Alexa dyes), digoxogenin or an enzyme (e.g alkaline phosphatase, horseradish peroxidase), although without limitation thereto.
  • markers useful for marker detection according to the invention include titin, desmin, the myoblast marker PAX7, myotube markers MYH2 and MYH3 and calcium handling genes SERCA1 and RYR1.
  • Markers may alternatively be metabolic enzymes or“metabolites” that are the product of metabolic processes accompanying cellular changes as a result of differentiation or development. Non-limiting examples of such markers are provided in Tables 2 and 3.
  • Base media may be, or include any medium such as aMEM, DMEM, MCDB, Iscove’s medium (IMDM) or RPMI1640 or combinations of these.
  • Media may further comprise a supplement such as, but not limited to, B27 and one or more other components such as insulin-transferrin-selenium (ITS) and antibiotics, such as penicillin-streptomycin.
  • ITS insulin-transferrin-selenium
  • antibiotics such as penicillin-streptomycin.
  • Non-limiting examples of media and media components are provided in Table 1.
  • the media may further comprise serum, or alternatively may be serum free or substantially serum free.
  • the term“serum” refers to a substantially cell-free proteinacious blood fraction obtained or obtainable from an animal (e.g fetal bovine serum, horse serum) and may include purified or recombinant synthetic serum components such as albumin.
  • a particular embodiment of the differentiation medium disclosed herein is serum free or substantially serum free.
  • serum free' or“ substantially serum free”, in a serum free medium, means a complete absence of serum, a level of serum substantially below that which is used for optimal myoblast culture conditions, or no more than about 0.5%, 0.2%, 0.1% or 0.05% (v/v) serum.
  • a particular feature of the differentiation culture medium disclosed herein is the selection of components or constituents that optimize the production of myocytes and/or formation of skeletal muscle organoids.
  • ECM extracellular matrix
  • ECM extracellular matrix
  • the molecular components of ECM may include proteoglycans, heparan sulphate, chondroitin sulphate, keratin, collagens (e.g types I-XIV), elastins, laminin and fibronectin, although without limitation thereto.
  • the ECM may be present in the form of MatrigelTM.
  • the culture medium comprises serum.
  • the culture medium further comprises a mitogen-activated protein kinase (MAPK) inhibitor such as a p38 MAPK inhibitor.
  • MAPK mitogen-activated protein kinase
  • a non-limiting example of a MAPK inhibitor is SB203580.
  • Other non-limiting examples of p38 MAPK inhibitors include SB239063, SB202190 and VX-702.
  • Initially cultured myoblasts are subsequently harvested and subjected to monolayer, suspension, two-dimensional (“2D”) or three-dimensional (“3D”) culture.
  • the harvested myoblasts are cultured in a culture medium comprising a base medium and a gelling agent.
  • the gelling agent comprises one or more ECM agents such as MatrigelTM and collagen I.
  • the myoblasts are cultured in a plate comprising opposed poles.
  • the concentration of MatrigelTM may be about 5-50% (v/v) (e.g., 5, 6, 7,
  • collagen is also present at a concentration of about 1-5 mg/mL (e.g., 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5 mg/mL and any range therein), particularly about 2-4 mg/mL or more particularly about 3.3 mg/mL of the gelling agent.
  • the myoblasts are cultured in a maturation medium to induce myoblast differentiation.
  • the maturation medium comprises a base medium and at least one of a Notch inhibitor and a Raf inhibitor.
  • the Notch inhibitor may be present at a concentration in the range about 1 -100 mM (e.g., 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95,
  • the Notch inhibitor may be any as are known in the art, such as g-secretase inhibitors, a-secretase inhibitors, small-molecule blockers, endosomal acidification inhibitors, soluble decoys, blocking peptides and blocking antibodies.
  • the Notch inhibitor is DAPT.
  • Non-limiting examples of other Notch inhibitors include RO4929097, MK-0752, SAHM1, FLI 06, DBZ, OMP- 21M18 antibody, EGFL7, SAHM1 and PF0384014.
  • the Raf proteins are a family of three serine-threonine kinases and a component of the RAS-RAF-MAPK signalling pathway.
  • the Raf inhibitor may be present at a concentration in the range about 0.1-10 mM (e.g., 0.1, 0.25, 0.5, 0.75, 1, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10 mM and any range therein), particularly about 0.2-8, 0.3-7, 0.4-6, 0.5-5, 0.6-4, 0.7-3, 0.8-2, 0.9- 1.2 or about 1 mM.
  • the Raf inhibitor may be any as are known in the art and may inhibit one or more of the Raf proteins.
  • the Raf inhibitor is a BRAF inhibitor, such as Dabrafenib.
  • BRAF inhibitors include sorafenib, vemurafenib, LGX818, PLX4720, GDC 0879, and SB 590885.
  • the differentiation medium may include one or more Notch inhibitors and/or Raf inhibitors.
  • the differentiation medium may include 1, 2, 3, 4, 5, or more Notch inhibitors and/or Raf inhibitors, such as those hereinbefore described.
  • the differentiation medium may include a Notch inhibitor alone or in isolation (i.e., with no Raf inhibitor), a Raf inhibitor alone or in isolation (i.e., with no Notch inhibitor) or both of a Notch inhibitor and a Raf inhibitor.
  • the myoblasts may be cultured in the differentiation medium for sufficient time to differentiate into skeletal muscle cells.
  • the culture period is about 2-14 days ( e.g ., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 days and any range therein), particularly about 5-10 days or optimally about 7 days.
  • a further aspect of the invention resides in a skeletal muscle cell culture vessel comprising one or a plurality of wells that each comprise opposed poles that extend substantially perpendicularly from a basal surface of the well.
  • the well and opposed poles of the culture vessel are dimensioned, shaped and oriented to maximize the formation of 3D organoids comprising dense skeletal muscle bundles that engage and surround the opposed poles. Displacement of the opposed poles caused by the muscle bundles facilitates contractile force measurements, as described in more detail in the Examples.
  • the well or at least the upper perimeter of the well, is substantially oval in shape.
  • the well comprises opposed poles spaced apart along a long axis of the well.
  • the poles are spaced symmetrically along the long axis.
  • the poles may be substantially perpendicular to a base of the well, projecting no further than the upper perimeter or boundary of the well.
  • Particular, non-limiting dimensions of the well include: a 3 mm long axis and a 2 mm short axis.
  • the opposed poles may be block-shaped having a square or rectangular cross-section. In a particular form, the opposed poles may be rectangular in cross-section, the rectangle having dimensions of 0.2mm by 0.5 mm.
  • the opposed poles are symmetrically spaced about 1.0 mm from the centres of the poles along the long axis.
  • the culture vessel may comprise a plurality of wells disclosed herein, such as in 24, 48, 96, 384 or other multi-well formats known in the art.
  • the culture vessel of the present aspect is adapted to receive or be electrically coupled to a bioreactor, stimulator or other device or apparatus capable of delivering an electrical stimulus to one or more skeletal muscle cells in the culture vessel and measuring or monitoring the response of the skeletal muscle cells.
  • Another aspect of the invention provides a skeletal muscle cell differentiation system comprising:
  • the present system may further comprise a bioreactor or other apparatus capable of delivering an electrical stimulus to one or more skeletal muscle cells in the culture vessel and measuring or monitoring the response of the skeletal muscle cells.
  • a related aspect of the invention provides a method of identifying one or more molecules that modulate myoblast differentiation, said method including contacting one or more myoblasts in the maturation medium or cell culture system disclosed herein with one or more candidate molecules, whereby modification of the maturation of one or a plurality of the myoblasts indicates that the candidate molecule is a modulator of myoblast differentiation.
  • the modulator at least partly enhances or promotes myoblast differentiation.
  • the modulator at least partly inhibits or suppresses myoblast differentiation.
  • this aspect of the invention provides a method or system for identifying, assaying or screening candidate molecules that may modulate myoblast maturation.
  • Candidate molecules may be present in combinatorial libraries, natural product libraries, synthetic chemical libraries, phage display libraries, lead compound libraries and any other libraries or collections of molecules suitable for screening, as are known in the art.
  • a further aspect of the invention provides one or more skeletal muscle cells, or skeletal muscle tissues or organoids comprising same, produced by the method disclosed herein.
  • the skeletal muscle cell maturation medium, culture vessel, system and method may be suitable for producing skeletal muscle cell suspensions, monolayers or“2D cultures”.
  • the skeletal muscle cell maturation medium, system and method may be suitable for producing skeletal muscle tissue in three dimensional (3D) structures, such as“organoids”.
  • Organoids may be used for producing engineered or artificial skeletal muscle tissue.
  • skeletal muscle organoids may be incorporated within a scaffold, such as decellularised human skeletal muscle tissue, polyester fleece or biodegradable polymer scaffold, to thereby produce a 3D tissue structure.
  • a scaffold such as decellularised human skeletal muscle tissue, polyester fleece or biodegradable polymer scaffold, to thereby produce a 3D tissue structure.
  • Also contemplated are“bioprinted” 3D tissue structures.
  • skeletal muscle cells, tissues and organoids described herein may provide potential sources of purified, differentiated cells and tissues for muscular therapy, such as for the treatment of a skeletal muscle-associated disease, disorder or condition.
  • Disorders of skeletal muscle may be categorized in distinct groups. One group consists of primary disorders of muscle energy metabolism, including defects in muscle carbohydrate and lipid metabolism, disorders of mitochondrial electron transport, and abnormalities of purine nucleotide metabolism affecting the capacity for ATP resynthesis.
  • oxidative phosphorylation is the dominant quantitative source of energy for ATP resynthesis under most exercise conditions. Consequently, disordered oxidative metabolism (i.e., in patients with defects in the availability or utilization of oxidizable substrate, such as those with phosphorylase or PFK deficiency or those with defects in mitochondrial electron transport) may lead to severely impaired skeletal muscle performance, intolerance to sustained exercise and premature fatigability.
  • Another group of disorders includes patients with decreased muscle mass due to muscle necrosis, atrophy, and replacement of muscle by fat and connective tissue.
  • muscular dystrophies e.g ., Duchenne's dystrophy, Becker's dystrophy, LG dystrophy, FSH dystrophy, and myotonic dystrophy
  • skeletal muscle performance is severely impaired due to muscle wasting and weakness in spite of largely normal pathways for muscle ATP resynthesis.
  • the skeletal muscle cells and organoids described herein may be suitable for disease modelling.
  • the effect of genetic defects upon skeletal muscle function may be investigated, such as by determining the contractile properties of skeletal muscle organoids comprising skeletal muscle cells having the genetic defect.
  • the efficacy of drugs or other molecules in treating or correcting the genetic defect may be assessed in respect of skeletal muscle cells and/or organoids described herein.
  • skeletal muscle cells and/or organoids described herein may be used in applications such as patient specific disease modelling and biology, such as modelling, investigating or predicting the effects of modulating skeletal muscle gene expression (e.g., gene“knock out”,“knock-down” or over-expression).
  • a particular aspect of the invention provides a method of determining, assessing or monitoring the effect of one or more molecules upon skeletal muscle cells, tissues and/or organoids described herein, said method including the steps of contacting the skeletal muscle cells, tissues and/or organoids described herein or produced according to the method disclosed herein with the one or more molecules and determining assessing or monitoring the effect of the one or more molecules upon the skeletal muscle cells and/or organoids.
  • the effect may be, or relate to, therapeutic efficacy in treating diseases, conditions or disorders of skeletal muscle, drug dosage determination, toxicity and/or safety (e.g ., assessing side-effects of a drug, such as rhabdomyolysis) and contractile properties of the skeletal muscle cell, tissue or organoid, although without limitation thereto.
  • the one or more molecules may be known or pre-existing drugs or may be present in combinatorial libraries, natural product libraries, synthetic chemical libraries, phage display libraries, lead compound libraries and any other libraries or collections of molecules suitable for use in the present method.
  • skeletal muscle cells, tissues and/or organoids described herein may be useful for toxicity screening or for in vitro drug safety testing.
  • some drugs used for therapeutic interventions can cause unexpected toxicity in muscle tissue, often leading to significant morbidity and disability.
  • Myotoxic drugs can cause myopathies through a variety of mechanisms by directly affecting muscle organelles such as mitochondria, lysosomes, and myofibrillar proteins; altering muscle antigens and generating an immunologic or inflammatory reaction; or by disturbing the electrolyte or nutritional balance, which can subsequently impact muscle function.
  • Muscle tissue can be particularly susceptible to drug-related injury because of its mass, high blood flow, and mitochondrial energy metabolism.
  • drugs may be screened against skeletal muscle cells, tissues and/or organoids described herein to determine general toxicity or to determine if skeletal muscle cells, tissues and/or organoids produced from a particular individual display sensitivity, or not, to potentially toxic drugs or other molecules or compounds.
  • the skeletal muscle cells, tissues and/or organoids may be obtained from progenitor cells of an individual having one or more particular genetic defects.
  • the invention contemplates a“genetic background test” where a candidate drug or other molecule could be tested against skeletal muscle cells, tissues and/or organoids disclosed herein having different genetic backgrounds to determine whether there are differential drug efficacies and/or side effects that correlate with a particular genetic background. This may enable selection of appropriate drug therapies for patients with a particular genetic background.
  • Further embodiments of the invention may relate to the production of edible skeletal muscle, such as in the form of edible animal muscle.
  • the invention disclosed herein may provide a medium, method or system suitable for the production of high-quality skeletal muscle tissue of non-human origin (e.g ., porcine, ovine, bovine) that is suitable for human consumption.
  • the invention disclosed herein may provide a medium, method or system suitable for the production of high-volume, low-cost skeletal muscle tissue of non-human origin that is suitable for use in animal feeds, fertilizers or as a low-cost, high-volume muscle protein source for other applications.
  • a further aspect of the invention provides a method of determining, assessing or monitoring the effect of a stimulus upon a skeletal muscle cell, engineered tissue or organoid, said method including the steps of exposing the skeletal muscle cell, tissue or organoid to the stimulus and determining, assessing or monitoring the effect of the stimulus upon the skeletal muscle cell, tissue or organoid.
  • the stimulus recapitulates or mimics exercise, physical training and/or other physical activities or exertions of skeletal muscle.
  • Exercise training induces adaptations that have health benefits for an individual. Identification of the underlying molecular mechanisms of these adaptations may lead to new therapeutic targets for a range of different diseases.
  • exercise produces a complex, cascading set of responses within skeletal muscle and also elicits body-wide physiological adaptations in other organ systems making it extremely complicated to dissect underlying molecular mechanisms in vivo.
  • In vitro culture models, such as those described herein that may recapitulate human skeletal muscle physiology are needed to rapidly expand our knowledge of disease mechanisms and exercise adaptations.
  • 3D organoid models such as those described herein, could provide more predictive assays of human physiology for biological discovery and drug development.
  • the stimulus is a light stimulus which mimics or recapitulates the effect of physical exertion or exercise.
  • the skeletal muscle cell, tissue or organoid is genetically engineered to express an optogenetic actuator molecule which is suitably a light-responsive protein.
  • optogenetic actuator molecules include channelrhodopsin, halorhodopsin and achaerhodopsin, although without limitation thereto.
  • the skeletal muscle cell, tissue or organoid is also genetically engineered to express a protein that emits light in response to detecting changes in plasma membrane voltage.
  • Non-limiting examples include an ArcLight protein, a Bongwoori protein and variants and derivatives thereof.
  • “genetically engineered” means subjected to recombinant DNA technology to thereby express one or more exogenous proteins, such as the optogenetic actuator molecule and/or the protein that emits light in response to detecting changes in plasma membrane voltage.
  • Such methods typically include the delivery to a cell of one or more genetic constructs comprising one or more nucleic acids encoding the exogenous protein(s).
  • the genetic construct comprises a constitutive or regulatable promoter that is operable in the cell to facilitate expression of the exogenous protein(s).
  • a skeletal muscle cell, tissue or organoid expressing the light responsive protein and the protein that emits light in response to detecting changes in plasma membrane voltage is repeatedly exposed to light of a defined wavelength, to thereby stimulate skeletal muscle cell contraction wherein plasma membrane depolarization may be visually monitored.
  • skeletal muscle cells, tissues or organoids subjected to optogenetic stimulation may be subjected to further analysis to identify modulation of genes or encoded protein expression in response to light stimulation.
  • the genes and encoded proteins may be associated with mitochondrial biogenesis and organisation, generation of energy and/or cellular respiration.
  • this optogenetic system induces further maturation of relatively immature or fetal-like micro-muscle into more mature skeletal muscle, which may mimic or recapitulate the effect of exercise.
  • the stimulus is an electrical stimulus which mimics or recapitulates the effect of physical exertion or exercise.
  • Electrical stimulation may be applied to skeletal muscle cells, such by way of a bioreactor or other apparatus capable of delivering an electrical stimulus to the skeletal muscle cells and measuring or monitoring the response of the skeletal muscle cells.
  • the bioreactor may comprise the vessel of the third aspect comprising skeletal muscle cells in one or more of the vessel wells
  • the bioreactor includes an electrical source and electrodes that deliver a controlled electrical stimulus to the skeletal muscle cells in the vessel.
  • the electrodes may comprise steel, copper, platinum, silver, gold or other electrically-conductive metals or materials such as carbon, silicon or lithium, or combinations of these, although without limitation thereto.
  • electrical stimulation essentially recapitulates or mimics exercise, physical training and/or other physical activities or exertions of skeletal muscle.
  • apparatus for electrical stimulation and monitoring of muscle cells that may be adapted for use according to the present invention include those described in Hirt et al ., 2014, Molecular and Cellular Cardiology 74 151 and Stoehr et al., 2014, Am. J. Heart Circ. Physiol.306 H1353.
  • the invention disclosed herein may be generally useful in human and other mammalian species inclusive of livestock such as cattle, sheep and pigs, performance animals such as racehorses, camels and greyhounds and domestic pets such as dogs and cats, although without limitation thereto.
  • livestock such as cattle, sheep and pigs
  • performance animals such as racehorses, camels and greyhounds
  • domestic pets such as dogs and cats
  • the invention contemplates xenogeneic transfer or transplantation of tissue, organ and organoid production from one non-human mammalian species to another, or from a non-human mammalian species to a human.
  • Non-limiting examples include xenogeneic transfer or transplantation from pigs to humans or non-human primates to humans.
  • hpMs functional human skeletal micro muscles
  • Human myoblasts were purchased from GIBCO and grown as per [19] Briefly, myoblasts were grown on Matrigel (ThermoFisher Scientific) coated flasks in a media consisting of a 1 : 1 mixture of DMEM:MCDB (ThermoFisher Scientific) supplemented with 20% fetal bovine serum (FBS, ThermoFisher Scientific), 1% insulin-transferrin- selenium (ITS, Invitrogen), 1% penicillin-streptomycin (P/S, ThermoFisher Scientific) and 10mM of SB203580 (p38 MAPK inhibitor, Stem Cell Technologies). When required, myoblasts were harvested using Trypsin enzymatic digestion.
  • D&D media consists of MEM a (ThermoFisher Scientific) with 1% P/S (ThermoFisher Scientific), 0.5% ITS (ITS, ThermoFisher Scientific) and 2% B-27 supplement (ThermoFisher Scientific), with 10 mM DAPT (Stem Cell Technologies) and 1 mM Dabrafenib (Stem Cell Technologies).
  • HS horse serum
  • Myoblast were seeded at 90% confluence on Matrigel coated wells in growth medium. After 4 hrs, media was changed to MEM a with 1% P/S, 0.5% ITS and 2% B-27 supplement, with either 2% horse serum or 10 pM DAPT and 1 pM Dabrafenib.
  • hpMs were electrically stimulated at 1, 2, 5 and 20 Hz with 5ms square pulses with 20 mA current using a Panlab/Harvard Apparatus Digital Stimulator.
  • a Leica DMi8 inverted high content Imager was used to capture a 5s time-lapse of each hpM contracting in real time at 37°C.
  • Pole deflection was used to approximate the force of contraction as per [11]
  • Custom batch processing files were written in Matlab R20l3a (Mathworks) to convert the stacked TIFF files to AVI, track the pole movement (using vision. PointTracker), determine the contractile parameters, produce a force-time figure, and export the batch data to an Excel (Microsoft) spreadsheet.
  • Matlab R20l3a Mathworks
  • hpMs were fixed for 60 min with 1% paraformaldehyde (Sigma) at room temperature and washed 3X with PBS, after which they were incubated with primary antibodies in Blocking Buffer, 5% FBS and 0.2% Triton-X-lOO (Sigma) in PBS overnight at 4°C. Cells were then washed in Blocking Buffer 2X for 2 h and subsequently incubated with the appropriate secondary antibodies, and Hoechst33342 (1 : 1000), overnight at 4°C. Cells were then washed in Blocking Buffer 2X for 2 h and imaged in situ or mounted on microscope slides using Fluoromount-G (Southern Biotech). For a list of antibodies used in the study please refer to Table 2.
  • hpMs were imaged using a Leica DMi8 high content imaging microscope for in situ imaging. Custom batch processing files were written in Matlab R20l3a (Mathworks) to remove the background, calculate the image intensity, and export the batch data to an Excel (Microsoft) spreadsheet. For higher magnification images, an Olympus 1X81 confocal microscope was used for slide-mounted hpM imaging.
  • the adult human skeletal muscle sample was obtained from Clontech.
  • the adult tissue was pooled from 3 skeletal muscle samples from Asian, Caucasian male/females aged 30, 44 and 86.
  • Single hpMs were washed 2X in PBS and snap frozen and stored at -80°C. Tissues were lysed by tip-probe sonication in 1% SDS containing 100 mM Tris pH 8.0, 10 mM tris( 2- carboxyethyl)phosphine, 40 mM 2-chloroacetamide and heated to 95°C for 5 min. Proteins were purified using a modified Single-Pot Solid-Phase-enhanced Sample Preparation (SP3) strategy [20] Briefly, Proteins were bound to Sera-Mag carboxylate coated paramagnetic beads in 50% acetonitrile containing 0.8% formic acid (v/v) (ThermoFisher Scientific).
  • the beads were washed twice with 70% ethanol (v/v) and once with 100% acetonitrile. Proteins were digested on the beads in 100 mM Tris pH 7.5 containing 10% 2,2,2-Trifluoroethanol overnight at 37°C with 200 ng of sequencing grade LysC (Wako Chemicals) and trypsin (Sigma). Beads were removed and peptides acidified to 1% trifluoroacetic acid prior to purification by styrene divinyl benzene - reversed phase sulfonated solid phase extraction microcolumns.
  • Peptides were spiked with iRT peptides (Biognosys) and analysed on an Easy-nLCl200 coupled to a Q- Exactive HF in positive polarity mode. Peptides were separated using an in-house packed 75 pm x 50 cm pulled column (1.9 pm particle size, C18AQ; Dr Maisch) with a gradient of 2 - 35% acetonitrile containing 0.1% FA over 120 min at 300 nl/min at 60°C.
  • the instrument was operated in data-independent acquisition (DIA) mode essentially as described previously [21] Briefly, an MS1 scan was acquired from 350 - 1650 m/z (120,000 resolution, 3e6 AGC, 50 ms injection time) followed by 20 MS/MS variable sized isolation windows with HCD (30,000 resolution, 3e6 AGC, 27 NCE).
  • a spectral library was created by fractionating a pooled mix of peptides from 10 separate hpMs on an inhouse packed 320 pm x 25 cm column (3 pm particle size, BEH; Waters) with a gradient of 2 - 40% acetonitrile containing 10 mM ammonium formate over 60 min at 6 pl/min using an Agilent 1260 HPLC.
  • DDA data-dependent acquisition
  • MS1 scan was acquired from 350 - 1650 m/z (60,000 resolution, 3e6 AGC, 50 ms injection time) followed by 20 MS/MS with HCD (1.4 m/z isolation, 15,000 resolution, le5 AGC, 27 NCE).
  • DDA data were processed with Andromeda in MaxQuant vl.5.8.3 [22] against the human UniProt database (January 2016) using all default settings with peptide spectral matches and protein false discovery rate (FDR) set to 1% .
  • DIA data were processed with Spectronaut vl l [23] using all default settings with precursor and protein FDR set to 1% and quantification performed at MS2.
  • SPARCLIGHT Adenovirus Generation A red-shifted variant of channelrhodopsin (C1V1) [25] was linked to the fluorescence protein voltage sensor Arc Light [26] via a p2A linker sequence, thus enabling membrane depolarisation via optical stimulation and simultaneous recording of membrane potential via changes in fluorescence.
  • the dual reporter system hereto referred to as SPARCLIGHT
  • SPARCLIGHT was synthesised by GeneCopeia and sub-cloned into the DUAL-CCM + plasmid for adenovirus production by Vector Biolabs (PA, USA).
  • hpMs were cultured in a maintenance media consisting of MEM a (ThermoFisher Scientific) with 1% P/S (ThermoFisher Scientific), 0.5% ITS (ITS, ThermoFisher Scientific) and 2% B-27 supplement (ThermoFisher Scientific).
  • hpMs were treated with the SPARCLIGHT adenovirus at 500 MOI for 3 days.
  • hpMs were subsequently washed, and exposed to pulses of green light using a 96-well green LED array (Lumidox) connected to a Panlab/Harvard Apparatus Digital Stimulator.
  • hpMs were stimulated for 2 hrs per day, using a 200ms light pulse every 5 seconds (5,760 total contractions). After 4 days of optical stimulation, hpMs were taken for proteomics, immunohistochemistry and force analysis. For force recordings, hpMs were electrically stimulated at 1, and 20 Hz with 5ms square pulses with 20 mA current as described above.
  • D7 D&D hpMs were treated with 0. l%DMSO or 1 OmM Simvastatin (Sigma).
  • D7 D&D hpMs were treated with an AAV6 containing a mutated version of human YAP1, CMV-YAP(Sl27A) (Vector Biolabs) or a control AAV6-MCS (Vector Biolabs) at 6 x 10 9 vg/Hco [11] Force recording were taken after 72 hrs of treatment.
  • the micro-muscle platform facilitates screening based on marker expression by utilising whole mount-immunostaining combined with high-content image analysis [11]
  • base media including base media, supplementation, small molecules and serum concentration in driving muscle differentiation
  • Figure 1E,F insulin transferrin selenium
  • DAPT and Dabrafenib could also be used serum-free in 2D culture to generate myotubes ( Figure 6B).
  • This protocol was termed‘D&D’, which we subsequently phenotypically characterised.
  • D&D hpMs had an average specific force of 2.0 ⁇ 0.7 mN.mm 2 and 4.9 ⁇ 0.8 mN.mm 2 for twitch and tetanus, respectively (Figure 2G).
  • Specific tetanic forces generated within our system are an order of magnitude lower than that reported for adult human muscle, and are more reflective of fetal human muscle [31, 32]
  • the tetanus-to-twitch ratio was higher in D&D (-2.4) compared to 2% HS hpM (-1.2) ( Figure 2H), closer, but not equivalent, to values reported for adult human muscle (-2-4 fold lower) [31]
  • Protein expression was compared between adult human skeletal muscle tissue (hSkM tissue) and D7 hpMs derived via D&D differentiation. Although there was a significant relationship between protein abundance in hpM compared to hSkM tissue ( Figure 4A), the Pearson correlation co-efficient was 0.57 indicating that there were also some differences. These differences include higher expression of fetal myosin heavy chain isoforms, lower expression of adult myosin heavy chain isoforms and lower expression of some calcium handling genes (Figure 4B). Taken together with contractile function, hpMs are more reflective of fetal human skeletal muscle.
  • Optogenetic Stimulation Recapitulates some Adaptations of Exercise
  • hpMs Functional and proteomic analysis of hpMs revealed that they were more reflective of a fetal muscle phenotype. As the main differences between native skeletal muscle tissue and hpMs were related to metabolism and energy production (Figure 4), we hypothesized that contractile stimulation may promote maturation. Furthermore, hpM contractile stimulation may recapitulate an exercise-training regime, thus allowing the in vitro analysis of exercise adaptations in an isolated human system.
  • hpMs were treated with the SP ARCLIGHT adenovirus to allow for (1) control over hpM contraction and (2) provide an optical approach to visualise cell depolarisation (Figure 5B).
  • To stimulate contraction hpMs were exposed to a single 200ms pulse of light every 5 seconds (0.2Hz), for 2 hrs a day. This caused a single tetanic contraction after each light pulse ( Figure 5C).
  • STIM After 4 days of stimulation (STIM), a total of 5760 contractions, there was no significant change in active force or the tetanus-to-twitch ratio compared to time-matched, unstimulated control tissues (Figure 5D,E).
  • tetanus to twitch ratios compared to week 1 tissues ( Figure 2E), which approached levels similar to adult skeletal muscle [31], indicative that further culture time leads to improvements in function (Figure 5E).
  • Exercise training induces adaptations that have enormous health benefits [3, 4] Identification of the underlying molecular mechanisms of these adaptations may lead to new therapeutic targets for a range of different diseases [35]
  • exercise produces a complex, cascading set of responses within skeletal muscle and also elicits body-wide physiological adaptations in other organ systems making it extremely complicated to dissect underlying molecular mechanisms in vivo.
  • In vitro culture models that recapitulate human skeletal muscle physiology are needed to rapidly expand our knowledge of disease mechanisms and exercise adaptations.
  • Notch signalling is known to play a critical role in development and regeneration of skeletal muscle, with Notch activation driving self-renewal of undifferentiated PAX7 myoblasts [37] and inhibiting myogenic differentiation [29]
  • Dabrafenib is a B-Raf specific inhibitor.
  • B-Raf is known to activate PAX3 and is required for muscle precursor cell migration and proliferation [38]
  • activated Raf has been shown to prevent L6 rat myoblast differentiation [30]
  • Our optimised differentiation protocol targets these two key signalling pathways to enhance myoblast differentiation and drive rapid formation of myofibres.
  • fibroblasts endothelial cell, macrophages
  • exercise intensity and regimes e.g. endurance or resistance
  • High- throughput functional screening approaches have the capacity to rapidly facilitate the identification of key drivers of maturation in order to successfully model diseases such as sarcopenia, metabolic disorders and motor neuron disease.
  • 3D organoid technologies, in combination with high throughput screening platforms, have the ability to rapidly expand our knowledge of human biology and could lead to the development of novel therapeutics.
  • large-scale generation of skeletal muscle tissues are currently limited by an insufficient supply of human myoblasts.
  • Kidney organoids from human iPS cells contain multiple lineages and model human nephrogenesis. Nature, 2015. 526: p. 564.

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Abstract

La présente invention concerne un milieu de différenciation de cellules de muscle squelettique comprenant un milieu de base et au moins l'un d'un inhibiteur de la voie de signalisation Notch et d'un inhibiteur de Raf. L'invention concerne également un récipient de culture de cellules de muscle squelettique comprenant un ou plusieurs puits qui comprennent chacun des pôles opposés qui s'étendent sensiblement perpendiculairement à partir d'une surface de base du puits. L'invention concerne également un procédé de production de cellules de muscle squelettique par mise en contact d'une ou de plusieurs cellules progénitrices de muscle squelettique avec le milieu de différenciation de cellules de muscle squelettique et éventuellement dans le récipient de culture de cellules de muscle squelettique pour induire ou stimuler la différenciation d'une ou de plusieurs cellules de muscle squelettique à partir de la cellule progénitrice.
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US20230183647A1 (en) * 2020-05-19 2023-06-15 Association Francaise Contre Les Myopathies (Afm) Method for generating functional skeletal muscle fibers innervated by motoneurons
CN117589740A (zh) * 2024-01-04 2024-02-23 南京市产品质量监督检验院(南京市质量发展与先进技术应用研究院) 一种高通量快速检测和评估化合物骨骼肌毒性的方法
WO2025101068A1 (fr) * 2023-11-06 2025-05-15 Erasmus University Medical Center Rotterdam Modèles de maladie modifiés par des tissus 3d et leurs utilisations

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20230183647A1 (en) * 2020-05-19 2023-06-15 Association Francaise Contre Les Myopathies (Afm) Method for generating functional skeletal muscle fibers innervated by motoneurons
WO2022114955A1 (fr) * 2020-11-25 2022-06-02 Mosa Meat B.V. Milieu sans sérum pour la différenciation d'une cellule progénitrice
US20240010984A1 (en) * 2020-11-25 2024-01-11 Mosa Meat B.V. Serum-free medium for differentiation of a progenitor cell
WO2025101068A1 (fr) * 2023-11-06 2025-05-15 Erasmus University Medical Center Rotterdam Modèles de maladie modifiés par des tissus 3d et leurs utilisations
CN117589740A (zh) * 2024-01-04 2024-02-23 南京市产品质量监督检验院(南京市质量发展与先进技术应用研究院) 一种高通量快速检测和评估化合物骨骼肌毒性的方法

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