WO2020028957A1

WO2020028957A1 - Skeletal muscle cell maturation

Info

Publication number: WO2020028957A1
Application number: PCT/AU2019/050837
Authority: WO
Inventors: James Hudson; Richard Mills; Enzo PORRELLO
Original assignee: Queensland Institute of Medical Research QIMR
Current assignee: QIMR Berghofer Medical Research Institute
Priority date: 2018-08-09
Filing date: 2019-08-09
Publication date: 2020-02-13
Anticipated expiration: 2021-02-09

Abstract

Provided herein is a skeletal muscle cell differentiation medium comprising a base medium and at least one of a Notch inhibitor and a Raf inhibitor. Also described herein is a skeletal muscle cell culture vessel comprising one or more wells that each comprise opposed poles that extend substantially perpendicularly from a basal surface of the well. A method of producing skeletal muscle cells by contacting one or more skeletal muscle progenitor cells with the skeletal muscle cell differentiation medium and optionally in the skeletal muscle cell culture vessel to induce or promote differentiation of one or a plurality of skeletal muscle cells from the progenitor cell is also provided.

Description

TITLE

SKELETAL MUSCLE CELL MATURATION TECHNICAL FIELD

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.

BACKGROUND

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.

Cell lines such as C2C12 mouse and L6 rat myoblasts are regularly used as in vitro 2D muscle models. Whilst these cells readily proliferate, and have the capacity to differentiate and fuse, these models fail to mimic cellular function, organisation, and interactions present in muscle tissue and are not representative of in vivo biology [5] In addition, signalling pathways are known to be altered in a 2D versus 3D setting, which may contribute to the inability of standard 2D culture models to predict potential therapeutics, with 9 out of 10 candidates failing from Phase I trials to the clinic [6] Recent advances in cell biology and bioengineering have led to significant progress in the development of human stem cell derived three-dimensional (3D) culture systems, including mini-gut/intestinal organoids [7], liver buds [8], cerebral organoids [9], kidney organoids [10], and cardiac organoids [11, 12] These 3D tissues are more representative of in vivo biology [13, 14], promote higher levels of cell differentiation and tissue organisation, which recapitulate tissue-tissue interfaces and mechanical microenvironments of living organs [13, 14] This allows for the study of human physiology in an organ-specific context and the development of physiologically relevant in vitro models [13] Pioneering work by Shansky et al. [15] described the formation of functional muscle bundles in vitro from rodent myoblasts. Subsequently, the Bursae lab has developed state of the art tissue engineering techniques to generate functional bioengineered skeletal muscle from rodent, human and pluripotent stem cell derived myoblasts [16-18] These tissues are functional, have multinucleated and striated myofibers, a resident satellite cell niche, are capable of self-regeneration in vitro and respond appropriately to pharmacological agents [16-18] However, these models are expensive and require large numbers of cells, therefore miniaturization is required to unlock the potential of bioengineered skeletal muscle for higher-throughput studies.

SUMMARY

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. In a particular broad form, 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.

In an embodiment, the Notch inhibitor is DAPT.

In an embodiment, the Raf inhibitor is or comprises a BRAF inhibitor, such as Dabrafenib.

Suitably, the differentiation medium is serum free.

In one embodiment, 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.

In one embodiment, 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:

(i) the skeletal muscle cell differentiation medium of the first aspect; and (ii) the culture vessel of the second aspect.

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.

In one embodiment, 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.

In particular embodiments, 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.

In one embodiment, 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.

In a particular embodiment, the optogenetic actuator molecule is channelrhodopsin. In a particular embodiment, 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.

In an embodiment, 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.

In an embodiment, 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.

Suitably, the stimulus is, or recapitulates or mimics, exercise.

In one particular embodiment, the stimulus is light, which facilitates optogenetic analysis of the skeletal muscle cell, tissue or organoid. In another particular embodiment, 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.

In one embodiment, the modulator at least partly inhibits or suppresses skeletal muscle cell differentiation.

In another embodiment, 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.

In particular embodiments, the method determines, assesses or monitors the therapeutic efficacy, safety or toxicity of the one or more molecules.

In an embodiment, 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.

Suitably, 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.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1: Iterative screening in a micro-muscle culture platform identifies serum- free muscle differentiation protocol.

A) Schematic representation of cell culture insert containing an eliptical seeding well with two elastomeric poles. Each well of a 96 well-plate contains an insert. B) Automatic tissue formation within the micro-muscle platform. Myoblasts are seeded into the eliptical well in combination with matrix and allowed to gel at 37°C. Cells subsequently condense around the two elastomeric poles.

C) Myoblasts failed to sufficiently fuse and differentiate following treatment with traditional differentiation media of 2% HS. Whole mount immunostaining for titin

(green), desmin (red) and DNA (blue). Scale bar- 500pm.

D) Culture schematic of screening strategy to identify conditions that promote myotube formation.

E) MEM a is the optimal base media for induction of titin and desmin in the presence of 2% HS. Titin and desmin relative intensity heat map after 5 days of differentiation (relative to LG-DMEM). n=3, from 1 experiment.

F) Screening identifies optimal media supplementation of 0.5% ITS and 2% B-27 for expression of titin and desmin in the presence of 2% HS. Titin and desmin relative intensity heat map after 5 days of differentiation (relative to MEM a). n=4-6, from 2 experiments. I- 0.5% ITS, A- 200pM Ascorbate, B- 2% B-27 supplement.

G) Distinct inhibition of the Notch and Raf pathways resulted in increased expression of titin. Titin and desmin relative intensity heat map after 5 days of differentiation (relative to DM). n=3-9, from 3 experiments. DM- control condition using MEM a with 0.5% ITS, 2% B-27 and 2% HS.

H) Wholemount immunostaining of desmin, titin, and DNA after 5 day treatment with DAPT, dabrafenib or in combination. Scale bar represents- 500pm.

I) Combination of DAPT and dabrafenib led to a dramatic upregulation of titin and desmin expression. Desmin (left) and titin (Right) relative intensity heat map after 5 days of differentiation in response to DAPT and Dabrafenib at 0, 1 or 10 pM

(relative to 0 pM DAPT and dabrafenib). n=3-7, from 2 experiments. Base media consisted of MEM a with 0.5% ITS, 2% B-27 and 2% HS.

J) 7 day treatment with 10 pM DAPT and 1 pM dabrafenib is optimal for desmin and titin expression. Titin and desmin relative intensity heat map after 3, 5, and 7 days of differentiation (relative to 5 day differentiation). n=3-4, from 1 experiment. Differentiation media consisted of MEM a with 0.5% ITS, 2% B-27, 2% HS, 10 pM DAPT and 1 pM Dabrafenib.

K) Differentiation protocol can be performed serum-free and does not impact the expression of titin or desmin. Titin and desmin relative intensity heat map using 5%, 2% or 0% HS over 7 days of differentiation (relative to 5 % HS), n=3-4, from 1 experiment. Differentiation media consisted of MEM a with 0.5% ITS, 2% B- 27, 10 mM DAPT and 1 mM dabrafenib.

* < 0.05, ** < 0.0l, *** <0.00l, ****p < 0.0001, using one-way ANOVA with

Dunnet’s post-test in comparison to‘relative’ condition.

Figure 2: Comparison of D&D differentiation versus 2% HS in human skeletal micro muscles.

A) Schematic of the protocol for the generation of hpM using 2% HS or D&D.

B) Whole-tissue images of hpM stained with desmin (red), titin (green), and DNA (Blue). Scale bars- 500 pm.

C) High magnification imaging of D&D hpMs reveal the presence of multinucleated myotubes with defined titin striations throughout the tissue. Desmin (red), titin (green), and DNA (Blue). Scale bar = 20 pm. Inset- close-up image of striated titin.

D) Transmission electron micrographs of D&D hpMs. T - T-Tubule, SR- sarcoplasmic reticulum, S- Sarcomere, I- 1 band, Z- Z line, A- A band, M- M line, H- H zone. Scale Bar- lpm.

E) Representative force trace curves of D&D hpMs in response to electrical stimulation under twitch conditions (2 Hz) or tetanus (20 Hz).

F) Active force production is higher in D&D versus 2% HS hpMs under twitch and tetanus. n=7, from 2 experiments.

G) Specific force production is higher in D&D versus 2% HS hpMs under twitch and tetanus. . n=7, from 2 experiments.

H) The tetanic to twitch force production ratio is higher in D&D versus 2% HS. n=7, from 2 experiments

I) PAX7 , a marker of muscle stem cells, is rapidly downregulated during differentiation in both 2% HS or D&D hpMs by day 5 using qPCR. n = 4-6.

J) 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. K) 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.

L) The calcium handling gene RYR1 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.

M) The calcium handling gene ATP1A1 aka SERCA1 is rapidly upregulated during differentiation in D&D hpMs by day 5 using qPCR. n = 4-6.

Data is presented as mean ± s.e.m. * P < 0.05, ** P< 0.01, *** P < 0.001 using one-way ANOVA with Tukey’s post test (I-M) or t-test (E-G).

Figure 3: Proteomic analysis of human skeletal micro muscle development reveals a rapid differentiation response using D&D.

A) Rapid increase in the abundance of critical skeletal muscle sarcomeric and calcium handling genes during hpM development. Data are presented as mean ± s.e.m. and * P < 0.05, * P < 0.01, *** P < 0.001, **** p < 0.0001 using one-way ANOVA compared to day 0.

B) Volcano plot of significantly regulated genes comparing day 7 to day 0 hpMs reveals over 1,500 proteins are regulated.

C) Principal component analysis showing hpM development is consistent between tissues and follows a developmental program.

D) Hierarchically clustered heat map of significantly regulated genes during hpM development. Gene ontologies (biological processes) are shown together with their p-value for each gene cluster. Figure 4: Proteomic comparison of day 7 hpMs to adult human skeletal muscle reveals immaturity.

A) Protein abundance of day 7 hpM (x-axis) versus adult human skeletal muscle (hSKM Tissue, y-axis) reveals that they are significantly correlated.

B) Abundance of adult skeletal muscle sarcomeric and calcium handling proteins is lower and abundance of fetal skeletal muscle proteins is higher in 7 day hpMs versus adult human skeletal muscle. C) Gene ontologies (biological processes) of top 50 differentially expressed proteins with higher abundance in adult human skeletal muscle (hSkM Tissue) versus day 7 hpMs. Gene ontologies relate to metabolic processes. Number of genes in each process are shown in the bars. Figure 5: Optogenetic Stimulation can induce some aspects of maturation and recapitulate some features of exercise.

A) Culture schematic for generating and optogenetically stimulating hpMs.

B) SPARCLIGHT construct schematic to enable optgenetic stimulation of hpMs.

C) A single tetanic contraction is induced by a 200ms light pulses every 5s (STIM). D) Active force including both twitch and tetanic are not altered by the STIM. h=10-

12, 2 experiments.

E) Tetanus-to-twitch ratio increase with additional culture time, but not with STEM. n=7-l2, from 2 experiments

F) Projected tissue area increases with STEM. n=l2, 3 experiments.

G) Fibre diameter increases with STEM. n=l2, 3 experiments.

H) Titin intensity increases with STEM. n=l2, 3 experiments.

I) Representative images of CTRL and STEM hpMs. (Left) Whole-mount immunostaining of titin (green) and DNA (blue). Scale bar- 500p (Right) Higher magnification confocal imaging of hpMs. Scale Bar- 20 pm.

J) Gene ontologies (biological processes) of regulated proteins with higher abundance in STEM versus CTRL. Most gene ontologies relate to metabolic processes. Number of genes in each process are shown in the bars.

K) Data is presented as mean ± s.e.m. and * P < 0.05, * P < 0.01, *** P < 0.001, **** P < 0.0001 using one-way ANOVA with Dunnett’s multiple comparison test (E) or students T-test (F-H).

Figure 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 7: High magnification imaging. High magnification imaging of D&D hpMs reveal myotubes are surrounded by a laminin (A) and collagen IV rich matrix (B). Scale bar = 20 gm. C) Conditions for the generation of viable, force producing hpMs was >90% using D&D.

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.

Figure 9: A) Active force is reduced in D&D hpMs treated with 10mM Simvastatin for 72hrs. n= 6-8 from 1 experiment, E) Active force is increased in D&D hpMs overexpressing constitutively active YAP1 for 72 hrs. n= 6-7 from 1 experiment. Data is presented as mean ± s.e.m. * P < 0.05, ** P< 0.01, using a t-test.

DETAILED DESCRIPTION

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.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, preferred methods and materials are described.

As used herein, except where the context requires otherwise, the term“ comprise” and variations of the term, such as“ comprising’ “ comprises” and“ comprised are not intended to exclude further additives, components, integers or steps.

It will be appreciated that the 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. For example,“a” cell includes one cell, one or more cells and a plurality of cells.

As used herein, 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. By way of example only,“about 100 mM” may be 90-110 mM, 95-105 mM or 98-102 mM.

For the purposes of this invention, by“ isolated’ is meant material that has been removed from its natural state or otherwise been subjected to human manipulation. Isolated material (e.g, cells) may be substantially or essentially free from components that normally accompany it in its natural state, or may be manipulated so as to be in an artificial state together with components that normally accompany it in its natural state.

By“ 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.

In certain aspects, 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.

Suitably, 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. Suitably, the present method is at least partly performed using the skeletal muscle cell differentiation system described hereinafter.

In certain embodiments, 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. In this regard, 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. Typically, progenitor cells are unipotent or oligopotent and are capable of at least limited self-renewal. Suitably, 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. In some embodiments, 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. By way of example only, induced myogenic progenitor cells (iMPCs) may be produced via transient overexpression of Pax7 in paraxial mesoderm cells differentiated from hPSCs (Rao et al ., 2018, Nat. Comm. 9 126).

The terms “ differentiate”, “ differentiating’’ and “ differentiated 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. As will be well understood in the art, 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. In this context, by“ 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. Suitably, protein markers are detected by an antibody or antibody fragment (which may be polyclonal or monoclonal) that binds the protein marker. Suitably, 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. Particular non-limiting examples of 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.

It will be appreciated that in the context of myoblast culture and maturation media, reference will be made to“ base media” or“ basal media" . 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.

Non-limiting examples of media and media components are provided in Table 1.

In some embodiments, the media may further comprise serum, or alternatively may be serum free or substantially serum free.

As generally used herein, 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.

In this context“ 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.

Initially, progenitor cells such as myoblasts are grown in a culture medium comprising a base medium in the presence of one or more extracellular matrix (ECM) components. As broadly used herein,“ ECM” refers to a matrix or web of molecules located outside or external to cells that regulate cell-cell communication, cell signalling, cell adhesion, spacing, location and/or orientation, although without limitation thereto. 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. In some embodiments, the ECM may be present in the form of Matrigel™. Typically, the culture medium comprises serum.

Suitably, the culture medium further comprises a mitogen-activated protein kinase (MAPK) inhibitor such as a p38 MAPK inhibitor. 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.

In“3D” cultures, typically the harvested myoblasts are cultured in a culture medium comprising a base medium and a gelling agent. Suitably, the gelling agent comprises one or more ECM agents such as Matrigel™ and collagen I. In a particular embodiment pertaining to 3D cultures, the myoblasts are cultured in a plate comprising opposed poles. The concentration of Matrigel™ may be about 5-50% (v/v) (e.g., 5, 6, 7,

8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,

32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 % v/v and any range therein), particularly at about 10-40 % (v/v), more particularly about 15-30 % (v/v) or about 20-25 % (v/v) or advantageously about 22 % (v/v) of the gelling agent. Suitably, 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 (whether in 2D or 3D cultures) are cultured in a maturation medium to induce myoblast differentiation. Suitably, 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,

100 mM and any range therein), particularly 2-80, 3-70, 4-60, 5-50, 6-40, 7-30, 8-20. 9- 12 or about 10 mM. It is contemplated that 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. In an embodiment, 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 (A-Raf, B-Raf and C-Raf) 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. It is contemplated that the Raf inhibitor may be any as are known in the art and may inhibit one or more of the Raf proteins. In an embodiment, the Raf inhibitor is a BRAF inhibitor, such as Dabrafenib. Non-limiting examples of other BRAF inhibitors include sorafenib, vemurafenib, LGX818, PLX4720, GDC 0879, and SB 590885.

In particular embodiments, the differentiation medium may include one or more Notch inhibitors and/or Raf inhibitors. For example, the differentiation medium may include 1, 2, 3, 4, 5, or more Notch inhibitors and/or Raf inhibitors, such as those hereinbefore described.

In certain embodiments, 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. Suitably, the myoblasts may be cultured in the differentiation medium for sufficient time to differentiate into skeletal muscle cells. Typically, 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.

Suitably, 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.

Typically, the well, or at least the upper perimeter of the well, is substantially oval in shape. Suitably, the well comprises opposed poles spaced apart along a long axis of the well. Suitably, 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. In a particular form, 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.

In certain embodiments, 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:

(a) the skeletal muscle cell maturation medium disclosed herein; and

(b) the skeletal muscle cell culture vessel described herein.

In particular embodiments, 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.

In one embodiment, the modulator at least partly enhances or promotes myoblast differentiation.

In another embodiment, the modulator at least partly inhibits or suppresses myoblast differentiation.

It will be appreciated that 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.

As described previously, the skeletal muscle cell maturation medium, culture vessel, system and method may be suitable for producing skeletal muscle cell suspensions, monolayers or“2D cultures”.

In other embodiments, 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. For example, 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. Also contemplated are“bioprinted” 3D tissue structures.

By way of example only, an organ printing machines and processes have been developed which use a hydrogel scaffold to place mammalian cells in a desired orientation to recreate human mammalian organs. It will also be appreciated that the 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. For example, 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. These disorders are exemplified by the various muscular dystrophies ( e.g ., Duchenne's dystrophy, Becker's dystrophy, LG dystrophy, FSH dystrophy, and myotonic dystrophy) in which skeletal muscle performance is severely impaired due to muscle wasting and weakness in spite of largely normal pathways for muscle ATP resynthesis.

In this context, it will also be appreciated that the skeletal muscle cells and organoids described herein may be suitable for disease modelling. By way of example, 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. In a further embodiment, 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.

In other embodiments, 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).

Accordingly, 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.

In particular embodiments of 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. By way of example, 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. Accordingly, 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.

In some embodiments the skeletal muscle cells, tissues and/or organoids may be obtained from progenitor cells of an individual having one or more particular genetic defects. By way of example, 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. In one embodiment, 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. In another embodiment, 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.

In an embodiment, 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. However, 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. In particular embodiments, 3D organoid models, such as those described herein, could provide more predictive assays of human physiology for biological discovery and drug development.

In one particular embodiment, the stimulus is a light stimulus which mimics or recapitulates the effect of physical exertion or exercise. According to this embodiment, the skeletal muscle cell, tissue or organoid is genetically engineered to express an optogenetic actuator molecule which is suitably a light-responsive protein. Non-limiting examples of optogenetic actuator molecules include channelrhodopsin, halorhodopsin and achaerhodopsin, although without limitation thereto. Suitably, 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.

In this context, it will be appreciated that“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). Suitably, the genetic construct comprises a constitutive or regulatable promoter that is operable in the cell to facilitate expression of the exogenous protein(s). A more detailed explanation of genetic engineering methods may be found, for example, in Chapter 9 of CURRENT PROTOCOLS IN MOLECULAR BIOLOGY Eds. Ausubel et al. (John Wiley & Sons Inc NY, 1995-2017).

According to a particular embodiment, 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.

In some embodiments, 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. Suitably, the genes and encoded proteins may be associated with mitochondrial biogenesis and organisation, generation of energy and/or cellular respiration.

As will be described in more detail in the Examples, 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.

In another particular embodiment, 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. In some embodiments, the bioreactor may comprise the vessel of the third aspect comprising skeletal muscle cells in one or more of the vessel wells Typically, 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. Suitably, as hereinbefore described in the context of optogenetic stimulation, electrical stimulation essentially recapitulates or mimics exercise, physical training and/or other physical activities or exertions of skeletal muscle. By way of example only, non-limiting examples of 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. In the context of skeletal muscle and tissue engineering, it will be appreciated that 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.

Accordingly, terms such as “individuaF , “ patienf and “ subject’ are used interchangeably herein to refer to a human or non-human mammal.

So that the invention may be readily understood and put into practical effect, reference is made to the following non-limiting Examples.

EXAMPLES

INTRODUCTION

Herein, we describe the generation of functional human skeletal micro muscles (hpMs). Using an iterative screening approach, we define a serum-free differentiation protocol that drives rapid, directed differentiation of human myoblast to myofibres and subsequently develop an optogenetic approach to stimulate hpM contraction to recapitulate muscle adaptations observed after exercise training. We show that these hpMs recapitulate some features of native human muscle thus providing a useful model for muscle biology, biomedical research and exercise physiology.

MATERIALS & METHODS

Human Myoblast Growth

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. Miniaturized Culture Platform Fabrication

Cell-culture inserts were fabricated using standard SU-8 photolithography and PDMS moulding practices, as per [11]

Generation of Human Skeletal Micro Muscles

For each hpMs, 32,000 myoblasts in growth medium were mixed with collagen I gel to make a 3.2 pl final solution containing 3.3 mg/ml collagen I and 22% (v/v) Matrigel. The bovine acid-solubilized collagen I (Devro) was first salt balanced and pH neutralized using 10X DMEM and 0.1 M NaOH, respectively, prior to mixing with Matrigel and then combined with cells. The mixture was prepared on ice and pipetted into the cell-culture inserts. The mixture was then gelled at 37°C for 30 min prior to the addition of myoblast growth medium (150 mΐ/ hpMs). After 2 days, cells had aggregated around the two elastomeric poles and media was changed to induce myoblast differentiation.

Screening for Optimal Differentiation Conditions

A number of different conditions were tested for myoblast differentiation (as per Figure 1). For supplements and small molecules used in the study refer to Table 1.

DAPT and Dabrafenib (D&D) Differentiation Protocol 2 days after seeding, myoblasts had aggregated around the two elastomeric poles and media was changed to D&D media to induce myoblast differentiation. 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). 2% horse serum (HS) differentaition media consists of MEM a (ThermoFisher Scientific) with 1% P/S (ThermoFisher Scientific), 0.5% ITS (ITS, Invitrogen), 2% B-27 supplement (ThermoFisher Scientific), and 2% HS (ThermoFisher Scientific).

2D Differentiation of Myoblasts

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.

Force Analysis of Human Skeletal Micro Muscles

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. During stimulation, 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. For more information regarding force analysis please refer to [11]

Whole-mount immunostaining

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.

Immunostaining Analysis

For screening, 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.

Human RNA Samples

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.

RNA Extraction and qPCR

RNA was isolated using a Qiagen RNAeasy kit, as per manufacturer’s instructions and DNase I treated (Roche). cDNA was reverse transcribed using Superscript III (random primers, ThermoFisher Scientific) and qPCR performed using SYBR Mastermix (ThermoFisher Scientific) on a Applied Biosystems Step One Plus. The 2-AACt method was used to determine gene expression changes using 18S as the endogenous control. Primer sequences are listed in Table 3 and were used at 200 nM. Gene expression is presented relative to adult human skeletal muscle samples (SkMuscle). Proteomics and Data Processing

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. A total of 12 concatenated fractions were analysed using the identical LC-MS/MS conditions above except the instrument was operated in data-dependent acquisition (DDA) mode. Briefly, an 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.

Proteomic Bioinformatics

All proteomic data were Log2 transformed and median normalised. Differential abundance was determined with two sample t-tests with FDR-based multiple hypothesis testing correction. GO analysis was performed with DAVID v6.8 and gene ontology terms from BP ALL [24] and heat-maps and hierarchical clustering was performed using GENE-E (Broad Institute).

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) was synthesised by GeneCopeia and sub-cloned into the DUAL-CCM⁺ plasmid for adenovirus production by Vector Biolabs (PA, USA).

Transmission electron microscopy

Samples were processed for electron microscopy as described previously [27] Sections were analyzed unstained in a JeollOl 1 transmission electron microscope.

Optogenetic Stimulation

After 7 days of differentiation, 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.

Modelling Muscle Physiology in Human Skeletal Micro Muscles

D7 D&D hpMs were treated with 0. l%DMSO or 1 OmM Simvastatin (Sigma). For YAP1 overexpression, 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 10⁹ vg/Hco [11] Force recording were taken after 72 hrs of treatment.

RESULTS

Iterative Screening for Optimal Serum-free Differentiation Adult human satellite cells (myoblasts) were expanded as previously described [19] Human myoblasts were harvested and seeded in a Collagen I (3.3 mg/ml), Matrigel (22% (v/v)) and DMEM supplemented matrix. The elliptical geometry of our micro-muscle platform is designed so that the 32,000 myoblasts automatically condense around the two elastic posts over 2 days (Figure 1A,B). After cell condensation, growth media was exchanged for differentiation media to promote fusion and differentiation of myoblasts into myofibers (Figure IB). However, traditional horse serum (HS) based differentiation approaches failed to yield substantial myoblast fusion and myofibre formation over a 5- day period (Figure 1C). Therefore, an iterative screening approach was taken to identify media conditions that promoted rapid, directed myofibre formation.

The micro-muscle platform facilitates screening based on marker expression by utilising whole mount-immunostaining combined with high-content image analysis [11] Based on the expression of the skeletal muscle markers desmin and titin (representative of an early and late stage differentiation marker, respectively), we investigated the role of a number of factors, including base media, supplementation, small molecules and serum concentration in driving muscle differentiation (Figure 1D). In combination with traditional 2% HS, we first investigated the optimal base media and media supplements, and found it was advantageous to use MEM a, supplemented with 0.5% insulin transferrin selenium (ITS) and 2% B-27 (Figure 1E,F). ETsing small molecules, we subsequently investigated the function of a number of highly conserved cell signalling pathways involved in myoblast proliferation and differentiation, including the Wnt [28], Notch [29] and Raf pathways [30] CHIR99021, a potent inhibitor of GSK3 and activator of Wnt signalling, was detrimental to myoblast differentiation, reducing desmin and titin expression by -60% compared to control conditions (Figure 1G), whilst IWR-l, a Wnt inhibitor, had little effect on differentiation. Interestingly, both DAPT, a g-secretase inhibitor that blocks the Notch pathway, and Dabrafenib, a potent Raf inhibitor and key regulator of cell cycle, led to increased expression of titin (Figure 1G,H). These molecules were subsequently investigated and tested in a factorial array between 1 and IOmM. All combinations using both molecules led to a dramatic increase in the fusion and formation of myofibres (Figure 1H,I and Figure 6A), with a 2-fold increase and 4-fold increase in desmin and titin expression, respectively, using 10mM DAPT and ImM Dabrafenib (Figure 1H,I). This combination of molecules was further tested and found to be most effective over a 7-day differentiation period (Figure 1J). Furthermore, removal of horse serum was not detrimental to differentiation and indeed increased the relative expression of titin compared to media containing 5% horse serum (Figure IK). DAPT and Dabrafenib (D&D) could also be used serum-free in 2D culture to generate myotubes (Figure 6B). Thus, by performing iterative screening, in which the optimal condition was used for ensuing screens, we identified a rapid, directed, serum-free protocol to generate hpMs from human myoblasts. This protocol was termed‘D&D’, which we subsequently phenotypically characterised.

Structure, Function and Gene Expression Analysis of Human Skeletal Micro Muscles

After defining a rapid, directed differentiation approach, we characterised the structure, function and gene expression of hpMs generated via standard 2% HS and D&D optimized differentiaiton (Figure 2A). Consistent with Figure 1, D&D hpMs had more abundant (Figure 2B) and well developed myofibres. D&D hpM myofibres had defined titin and desmin striations throughout the tissue (Figure 2C), located within a laminin and collagen IV rich-matrix. Transmission electron microscopy confirmed D&D huMs have aligned sarcomeres with alternating A-bands and I-bands, electron-dense Z-lines and distinct M-lines and H-zones located at the center of A-bands (Figure 2D). Together, these data indicate a mature sarcomeric structure in D&D huMs. Additionally, we also identified junctional structures between sarcoplasmic reticulum and T-tubules, including triads, reflective of native skeletal muscle fibers (Figure 2D).

Functional properties were assessed by tracking of elastomeric poles during electrical stimulation of the hpMs. The micro-muscle platform allows contractile force to be assessed in situ , without any tissue handling [11] Consistent with other bioengineered skeletal muscle systems [16-18], hpMs displayed twitch and tetanic contractions and a positive force frequency relationship in respose to electrical stimulation (Figure 2E). Active force (Figure 2F) and specific force (Figure 2G) were consistently higher in hpMs differentiated using D&D compared to 2%HS. 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] Furthermore, 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]

Our optimised D&D protocol was capable of generating >90% viable, force generating hpMs (Figure 7C), important for functional screening approaches in academia and industry. Consistent with differentiation of myoblasts into functional myotubes there was a rapid decrease in the myoblast marker PAX7 (Figure 21), increase in the myotube markers MYH2 and MYH3 (Figure 2J,K), and increase in the calcium handling genes SERCA1 and RYR1 (Figure 2L,M). Both MYH3 and SERCA1 were higher in D&D compared to 2% HS hpM, while PAX7 was similar between the two conditions. Taken together, this indicates that both conditions are capable of inducing differentiation of skeletal myoblasts into myotubes, but our optimised D&D protocol leads to more rapid differentiation and better functional outcomes. Proteomic Analysis of Human Skeletal Micro Muscles

Proteomics on single hpMs was used to profile the development of hpMs under D&D differentiation conditions. This facilitated quantification of over 4000 proteins per hpMs at day 0, 3, 5 and 7 of differentiation. Fetal myosin heavy chain isoforms rapidly increased during differentiation including MYH3 and MYH8, and were similar in abundance at day 3 versus day 7 (Figure 3A). However, many adult myosin heavy chain isoforms were not strongly regulated at any of the time-points (MYH1, MYH2 and MYH7) (Figure 3A). Other sarcomeric proteins such as TTN and calcium handling genes (CASQ2, CASQ1, ATP2A1 aka SERCA1, ATP2A2 aka SERCA2 and RYR1) were rapidly increased during hpM differentiation (Figure 3A). Together these results are consistent with the development of functional hpMs as presented in Figure 1 and Figure 2, and the rapid, directed differentiation process using D&D.

Analysis of protein abundance at day 7 versus day 0 revealed that over 1,500 proteins were significantly regulated during D&D induced differentiation (Figure 3B). Some of these proteins were progressively induced over the 7 day protocol and a principal component analysis revealed that differentiation proceeded in an ordered fashion with the hpMs clustering at each time-point (Figure 3C). Hierarchical clustering of significantly regulated proteins supported this developmental progression with samples from each time-point clustering distinctly (Figure 3D). As supported by the analysis of critical skeletal muscle genes in Figure 3A, the most differentially clustered samples were from day 0 to day 3 indicating rapid differentiation of hpMs with D&D. This is highlighted by gene ontology (GO) analysis of the“up-regulated early” cluster being enriched for proteins involved in skeletal muscle development and the“down-regulated early” cluster being enriched for proteins involved in extracellular matrix organisation, which is a property of stromal cells prior to differentiation (Figure 3D). There was a distinct cluster of proteins“up-regulated late” which were enriched in proteins involved in metabolism and the production of energy (Figure 3D). Together this indicates that D&D induces a rapid differentiation into a skeletal muscle phenotype and metabolic processes gradually mature over time.

Human Skeletal Micro Muscles are Representative of Immature Human Muscle

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. GO analysis of the top 50 proteins with higher abundance in hSkM tissue compared to hpMs revealed that most biological processes were enriched for metabolism and energy production (Figure 4C). Together this indicates that even though D&D induces rapid differentiation in hpMs and induction of metabolic processes by day 7, the sarcomeric proteins are still fetal and the expression of the metabolic proteins still inferior to hSkM tissue.

Optogenetic Stimulation Recapitulates some Adaptations of Exercise

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.

In order to stimulate the hpMs, we developed an optogentic approach to enable chronic contraction of hpMs in vitro (Figure 5A and 8A). This was chosen over electrical stimulation regimes, as electrical stimulation can cause significant myofibre damage and cell death [35], which was observed in our miniaturized format (Figure 8B). For these experiments, the red-shifted channelrhodopsin, for optogenetic stimulation [25], and ArcLight, as a fluorescent voltage sensor [26], were delivered to hpMs using an adenovirus under the control of a CMV promoter. We termed this dual stimulation- reporter system SP ARCLIGHT. 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). 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). However, there was an increase in 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).

Chronic stimulation increased hpM projected area (Figure 5F), myotube fibre diameter (Figure 5G) and titin intensity (Figure 5H,I), consistent with changes observed in native muscle during resistance exercise [36] Proteomic analysis revealed that 204 proteins were differentially expressed and that changes were associated with mitochondrial biogenesis and organisation, generation of energy and cellular respiration (Figure 5 J), indicative of a progression towards a more metabolically active muscle similar to that derived in vivo (Figure 4C). Additionally, we observed increased protein expression of MYH7B, a slow-twitch myosin, and decreased expression of MYH2, a fast-twitch myosin, indicative of a switch in fibre type observed after endurance exercise [36] (Figure 8C). Together this suggests that (1) optogenetic stimulation can be used to mature bioengineered skeletal muscle and (2) our model is capable of recapitulating some in vivo features of the physiological adaption to exercise in vitro. Muscle Physiology can be Modelled using Human Skeletal Micro Muscles

The potential to model muscle physiology was subsequently evaluated within D&D hpMs. D7 hpMs were treated with 10 mM Simvastatin. A side-effect of statin usage is sigificant myopathic weakness and rhabdomyolysis [17] After 72 hrs of treatment hpMs displayed reduced twitch and tetanus active force (Figure 9A). Conversely, hpMs over expressing constitutively active human Yes-associated protein 1 (YAP1), a key regulator of muscle mass and function [33, 34], had increased tetanus active force (Figure 9B). Collectively, hpMs respond appropriately to known biological processes and can be utilised as a tool to study human muscle physiology.

DISCUSSION

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] However, 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. As traditional 2D models have well known limitations [6, 13, 14], 3D models could provide more predictive assays of human physiology for biological discovery and drug development [17] Recent studies suggest that organoid systems are more reflective of in vivo biology compared with 2D assays [6, 13, 14] For example, it was recently shown that intestinal organoids can be used to successfully predict the outcome of stage Eli clinical trials in cancer patients [36]

In order to facilitate rapid biological discovery, 3D physiological systems need to be amenable to high-throughput, large-scale screening. This requires a miniaturised platform that is relatively inexpensive and requires minimal labour. We recently published a micro tissue screening platform for the culture of human cardiac organoids, termed the Heart- Dyno [11] We have now adapted this platform to skeletal muscle, allowing automated tissue formation, culture and analysis of hpMs for the first time. Furthermore, this approach enabled us to reduce the size, reagents and cost of hpMs by a factor of ~25 in comparison to state-of-the-art skeletal muscle bioengineering approaches [16-18] In our platform, traditional skeletal muscle differentiation protocols using horse serum [16, 17], failed to yield any substantial myofibres and only developed low active contractile forces. We therefore sought to optimise a differentiation protocol to drive rapid, directed, differentiation of myoblasts. Through iterative screening (~30 iterations), we identified a serum-free protocol based on Notch and Raf signalling inhibition, that enables rapid production of functional hpM within 7 days. DAPT is a potent inhibitor of the g-secretase complex, which inhibits the Notch signalling pathway. 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] Furthermore, 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.

Functional and proteomic analysis revealed that our hpMs resembled immature skeletal muscle. This was not unexpected as our protocol was assessed over a short 7-day period. An increase in culture time has been previously shown to promote maturation of human bioengineered skeletal muscle, however, even over a period of 4 weeks, adult-like function, structure and myogenic capacity were not achieved [17, 18] By optogenetically stimulating hpM contraction, we were able to promote some features of increased maturation; for example, myotube hypertrophy and enhanced metabolism. Key upstream drivers of skeletal muscle maturation are poorly defined and require further investigation to enhance maturation of engineered skeletal muscle. Muscle innervation [39], metabolic substrates [11], stromal cell populations (e.g. fibroblasts, endothelial cell, macrophages), and different exercise intensity and regimes (e.g. endurance or resistance) [34], all warrant further investigation and may be key to generate an adult-like phenotype. 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. However, large-scale generation of skeletal muscle tissues are currently limited by an insufficient supply of human myoblasts. To this end, recent research into myoblast expansion protocols [19] and the development of human pluripotent stem cell myoblast differentiation protocols [2, 18] will be critical in facilitating large scale hpM research. Our study provides proof-of-concept evidence that high-throughput studies can be performed in bioengineered skeletal muscle, thus enabling future high-throughput investigations of human muscle physiology, pathophysiology and exerci se adaptati on .

Throughout the specification the aim has been to describe the preferred embodiments of the invention without limiting the invention to any one embodiment or specific collection of features. It will therefore be appreciated by those of skill in the art that, in light of the instant disclosure, various modifications and changes can be made in the particular embodiments exemplified without departing from the scope of the present invention.

All computer programs, algorithms, patent and scientific literature referred to herein is incorporated herein by reference.

REFERENCES

1. Frontera, W.R. and J. Ochala, Skeletal Muscle: A Brief Review of Structure and Function. Calcified Tissue International, 2015. 96(3): p. 183-195.

. Chal, J. and O. Pourquie, Making muscle: skeletal myogenesis in vivo and in vitro.

Development, 2017. 144(12): p. 2104.

3. Pedersen, B.K. and B. Saltin, Evidence for prescribing exercise as therapy in chronic disease. Scandinavian Journal of Medicine & Science in Sports, 2006. 16(Sl): p. 3-63.

. Pedersen, B.K. and B. Saltin, Exercise as medicine evidence for prescribing exercise as therapy in 26 different chronic diseases. Scandinavian Journal of

Medicine & Science in Sports, 2015. 25: p. 1-72.

5. Langhans, S.A., Three-Dimensional in Vitro Cell Culture Models in Drug Discovery and Drug Repositioning. Frontiers in Pharmacology, 2018. 9(6).

6. Horvath, P., N. Aulner, M. Bickle, A.M. Davies, E.D. Nery, D. Ebner, M.C.

Montoya, P. Ostling, V. Pietiainen, L.S. Price, S.L. Shorte, G. Turcatti, C. von

Schantz, and N.O. Carragher, Screening out irrelevant cell-based models of disease. Nature Reviews Drug Discovery, 2016. 15: p. 751.

7. Sato, T., R.G. Vries, H.J. Snippert, M. van de Wetering, N. Barker, D.E. Stange, J.H. van Es, A. Abo, P. Kujala, P.J. Peters, and H. Clevers, Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche. Nature, 2009.

459(7244): p. 262-265.

8. Takebe, T., K. Sekine, M. Enomura, H. Koike, M. Kimura, T. Ogaeri, R.-R.

Zhang, Y. ETeno, Y.-W. Zheng, N. Koike, S. Aoyama, Y. Adachi, and H. Taniguchi, Vascularized and functional human liver from an iPSC-derived organ bud transplant. Nature, 2013. 499(7459): p. 481-484.

9. Lancaster, M.A., M. Renner, C.-A. Martin, D. Wenzel, L.S. Bicknell, M.E.

Hurles, T. Homfray, J.M. Penninger, A.P. Jackson, and J.A. Knoblich, Cerebral organoids model human brain development and microcephaly. Nature, 2013. 501(7467): p. 373-379.

10. Takasato, M., P.X. Er, M. Becroft, J.M. Vanslambrouck, E.G. Stanley, A.G.

Elefanty, and M.H. Little, Directing human embryonic stem cell differentiation towards a renal lineage generates a self-organizing kidney. Nat Cell Biol, 2014.

16(1): p. 118-126.

11. Mills, R. J., D.M. Titmarsh, X. Koenig, B.L. Parker, J.G. Ryall, G. A. Quaife-Ryan, H.K. Voges, M.P. Hodson, C. Ferguson, L. Drowley, A.T. Plowright, E.J.

Needham, Q.-D. Wang, P. Gregorevic, M. Xin, W.G. Thomas, R.G. Parton, L.K. Nielsen, B.S. Launikonis, D.E. James, D.A. Elliott, E.R. Porrello, and J.E. Hudson, Functional screening in human cardiac organoids reveals a metabolic mechanism for cardiomyocyte cell cycle arrest. Proceedings of the National Academy of Sciences, 2017. 114(40): p. E8372.

12. Voges, H.K., R.J. Mills, D.A. Elliott, R.G. Parton, E.R. Porrello, and J.E. Hudson, Development of a human cardiac organoid injury model reveals innate regenerative potential. Development, 2017. 144(6): p. 1118.

13. Ranga, A., N. Gjorevski, and M.P. Lutolf, Drug discovery through stem cell- based organoid models. Advanced Drug Delivery Reviews, 2014(0).

14. Huh, D., G.A. Hamilton, and D.E. Ingber, From 3D cell culture to organs-on- chips. Trends in Cell Biology, 2011. 21(12): p. 745-754.

15. Shansky, J., J. Chromiak, M. Del Tatto, and H. Vandenburgh, A simplified method for tissue engineering skeletal muscle organoids in vitro. In Vitro Cellular & Developmental Biology - Animal, 1997. 33(9): p. 659-661. 16. Juhas, M., G.C. Engelmayr, A.N. Fontanella, G.M. Palmer, and N. Bursae, Biomimetic engineered muscle with capacity for vascular integration and functional maturation in vivo. Proceedings of the National Academy of Sciences,

2014. 111(15): p. 5508.

17. Madden, L., M. Juhas, W.E. Kraus, G.A. Truskey, and N. Bursae, Bioengineered human myohundles mimic clinical responses of skeletal muscle to drugs. eLife,

2015. 4: p. e04885.

18. Rao, L., Y. Qian, A. Khodabukus, T. Ribar, and N. Bursae, Engineering human pluripotent stem cells into a functional skeletal muscle tissue. Nature Communications, 2018. 9(1): p. 126.

19. Charville, Gregory W., Tom H. Cheung, B. Yoo, Pauline J. Santos, Gordon K.

Lee, Joseph B. Shrager, and Thomas A. Rando, ExVivo Expansion and InVivo Self-Renewal of Human Muscle Stem Cells. Stem Cell Reports. 5(4): p. 621-632.

20. Fenyo, D., Q. Wang, J.A. DeGrasse, J.C. Padovan, M. Cadene, and B.T. Chait, MALDI sample preparation: the ultra thin layer method. J Vis Exp, 2007(3): p.

192.

21. Bruderer, R., O.M. Bernhardt, T. Gandhi, Y. Xuan, J. Sondermann, M. Schmidt,

D. Gomez-Varela, and L. Reiter, Optimization of Experimental Parameters in Data-Independent Mass Spectrometry Significantly Increases Depth and Reproducibility of Results. Mol Cell Proteomics, 2017. 16(12): p. 2296-2309.

22. Cox, J. and M. Mann, MaxQuant enables high peptide identification rates, individualized p.p.b. -range mass accuracies and proteome-wide protein quantification. Nat Biotechnol, 2008. 26(12): p. 1367-72.

23. Bruderer, R., O.M. Bernhardt, T. Gandhi, S.M. Miladinovic, L.Y. Cheng, S.

Messner, T. Ehrenberger, V. Zanotelli, Y. Butscheid, C. Escher, O. Vitek, O.

Rinner, and L. Reiter, Extending the limits of quantitative proteome profiling with data-independent acquisition and application to acetaminophen-treated three- dimensional liver microtissues. Mol Cell Proteomics, 2015. 14(5): p. 1400-10.

24. Huang da, W., B.T. Sherman, and R.A. Lempicki, Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat Protoc,

2009. 4(1): p. 44-57.

25. Lin, J.Y., P.M. Knutsen, A. Muller, D. Kleinfeld, and R.Y. Tsien, ReaChR: A red- shifted variant of channelrhodopsin enables deep transcranial optogenetic excitation. Nature neuroscience, 2013. 16(10): p. 1499-1508.

26. Jin, L., Z. Han, J. Platisa, J.R.A. Wooltorton, L.B. Cohen, and V.A. Pieribone,

Single action potentials and subthreshold electrical events imaged in neurons with a novel fluorescent protein voltage probe. Neuron, 2012. 75(5): p. 779-785.

27. Takasato, M., P.X. Er, H.S. Chiu, B. Maier, G.J. Baillie, C. Ferguson, R.G. Parton,

E. J. Wolvetang, M. S. Roost, S.M. Chuva de Sousa Lopes, and M.H. Little, Kidney organoids from human iPS cells contain multiple lineages and model human nephrogenesis. Nature, 2015. 526: p. 564.

28. Cistemas, P., J.P. Henriquez, E. Brandan, and N.C. Inestrosa, Wnt Signaling in Skeletal Muscle Dynamics: Myogenesis, Neuromuscular Synapse and Fibrosis. Molecular Neurobiology, 2014. 49(1): p. 574-589.

29. Luo, D., V.M. Renault, and T.A. Rando, The regulation of Notch signaling in muscle stem cell activation and postnatal myogenesis. Seminars in Cell & Developmental Biology, 2005. 16(4): p. 612-622.

30. Winter, B. and H.H. Arnold, Activated raf kinase inhibits muscle cell differentiation through a MEF 2 -dependent mechanism. Journal of Cell Science, 2000. 113(23): p. 4211. 31. Cheng, C.S., B.N.J. Davis, L. Madden, N. Bursae, and G.A. Truskey, Physiology and Metabolism of Tissue Engineered Skeletal Muscle. Experimental biology and medicine (Maywood, N.J.), 2014. 239(9): p. 1203-1214.

32. Racca Alice, W., E. Beck Anita, S. Rao Vijay, V. Flint Galina, D. Lundy Scott, E. Bom Donald, J. Bamshad Michael, and M. Regnier, Contractility and kinetics of human fetal and human adult skeletal muscle. The Journal ofPhysiology, 2013. 591(12): p. 3049-3061.

33. Hirt, M.N., J. Boeddinghaus, A. Mitchell, S. Schaaf, C. Bomchen, C. Miiller, H.

Schulz, N. Hubner, J. Stenzig, A. Stoehr, C. Neuber, A. Eder, P.K. Luther, A. Hansen, and T. Eschenhagen, Functional improvement and maturation of rat and human engineered heart tissue by chronic electrical stimulation. Journal of Molecular and Cellular Cardiology, 2014. 74: p. 151-161.

34. Ruas, J.L., J.P. White, R.R. Rao, S. Kleiner, K.T. Brannan, B.C. Harrison, N.P.

Greene, J. Wu, J.L. Estall, B.A. Irving, I.R. Lanza, K.A. Rasbach, M. Okutsu, K.S. Nair, Z. Yan, L.A. Leinwand, and B.M. Spiegelman, A PGC-la isoform induced by resistance training regulates skeletal muscle hypertrophy. Cell, 2012. 151(6): p. 1319-1331.

35. Fan, W. and R.M. Evans, Exercise Mimetics: Impact on Health and Performance.

Cell Metabolism, 2017. 25(2): p. 242-247.

36. Vlachogiannis, G., S. Hedayat, A. Vatsiou, Y. Jamin, J. Fernandez-Mateos, K.

Khan, A. Lampis, K. Eason, I. Huntingford, R. Burke, M. Rata, D.-M. Koh, N. Tunariu, D. Collins, S. Hulkki-Wilson, C. Ragulan, I. Spiteri, S.Y. Moorcraft, I. Chau, S. Rao, D. Watkins, N. Fotiadis, M. Bali, M. Darvish-Damavandi, H. Lote, Z. Eltahir, E.C. Smyth, R. Begum, P.A. Clarke, J.C. Hahne, M. Dowsett, J. de Bono, P. Workman, A. Sadanandam, M. Fassan, O.J. Sansom, S. Eccles, N.

Starling, C. Braconi, A. Sottoriva, S.P. Robinson, D. Cunningham, and N. Valeri, Patient-derived organoids model treatment response of metastatic gastrointestinal cancers. Science, 2018. 359(6378): p. 920.

37. Wen, Y., P. Bi, W. Liu, A. Asakura, C. Keller, and S. Kuang, Constitutive Notch Activation Upregulates Pax7 and Promotes the Self -Renewal of Skeletal Muscle

Satellite Cells. Molecular and Cellular Biology, 2012. 32(12): p. 2300-2311.

38. Shin, J., S. Watanabe, S. Hoelper, M. Kriiger, S. Kostin, J. Poling, T. Kubin, and T. Braun, BRAF activates PAX3 to control muscle precursor cell migration during forelimb muscle development. eLife, 2016. 5: p. el 8351.

39. Mills, R., H. Taylor-Weiner, J.C. Correia, L.Z. Agudelo, I. Allodi, C. Kolonelou,

V. Martinez-Redondo, D.M.S. Ferreira, S. Nichterwitz, L.H. Comley, V. Lundin, E. Hedlund, J.L. Ruas, and A.I. Teixeira, Neurturin is a PGC-lal -controlled myokine that promotes motor neuron recruitment and neuromuscular junction formation. Molecular Metabolism, 2018. 7: pl2-22. Table 1: Media supplements, and small molecules used in this study.

Table 2: Antibodies used in this study.

Table 3: qPCR primers used in this study.

Claims

1. A skeletal muscle cell differentiation medium comprising a base medium and at least one of a Notch inhibitor and a Raf inhibitor.

2. The skeletal muscle cell differentiation medium of Claim 1, which is serum free.

3. The skeletal muscle cell differentiation medium of Claim 1 or Claim 2, wherein the Raf inhibitor is or comprises a BRAF inhibitor.

4. The skeletal cell differentiation medium of Claim 3, wherein the BRAF inhibitor is or comprises Dabrafenib.

5. The skeletal muscle differentiation medium of any one of the preceding claims, wherein the Notch inhibitor is or comprises DAPT.

6. The skeletal muscle cell differentiation medium of any one of the preceding claims, which comprises a gelling agent.

7. 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.

8. The skeletal muscle cell culture vessel of Claim 7, wherein displacement of the opposed poles caused by muscle bundles in the well facilitates contractile force measurements.

9. A skeletal muscle cell culture system comprising:

(i) the skeletal muscle cell differentiation medium of any one of Claims 1-6; and

(ii) the skeletal muscle cell culture vessel of Claim 7 or Claim 8.

10. A method of producing skeletal muscle cells, organoid or tissue said method including the step of contacting one or more skeletal muscle progenitor cells with the skeletal muscle cell differentiation medium of any one of Claims 1-6 for sufficient time and under suitable conditions to induce or promote differentiation of one or a plurality of skeletal muscle cells from the progenitor cell.

11. The method of Claim 10, which is at least partly performed using the skeletal muscle cell differentiation system of Claim 9.

12. The method of Claim 10 or Claim 11, wherein the one or more skeletal progenitor cells is or comprises a myoblast.

13. A skeletal muscle cell, organoid or tissue produced by the method of any one of Claims 10-12.

14. The skeletal muscle cell, tissue or organoid of Claim 13 which is engineered to express an optogenetic actuator molecule; and a protein that emits light in response to detecting changes in plasma membrane voltage.

15. The skeletal muscle cell, tissue or organoid of Claim 14, wherein the optogenetic actuator molecule is channelrhodopsin.

16. The skeletal muscle cell, tissue or organoid of Claim 14 or Claim 15, wherein the protein that emits light in response to detecting changes in plasma membrane voltage is ArcLight.

17. A method of determining, assessing or monitoring the effect of a stimulus upon a skeletal muscle cell, tissue or organoid of any one of Claims 13 to 16, 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.

18. The method of Claim 17, wherein the stimulus is, recapitulates or mimics, exercise.

19. The method of Claim 18, wherein the stimulus is a light stimulus which facilitates optogenetic analysis of the skeletal muscle cell, tissue or organoid.

20. The method of Claim 18, wherein the stimulus is an electrical stimulus.

21. The method of any one of Claims 17 to 20, wherein determining, assessing or monitoring the effect of the stimulus upon the skeletal muscle cell, tissue or organoid is at least partly performed using the cell culture vessel of Claim 7 or Claim 8.

22. A method of identifying one or more molecules that modulate skeletal muscle cell differentiation, said method including the step of contacting one or more skeletal muscle progenitor cells with one or more candidate molecules in the culture medium of any one of Claims 1-6 or the culture system of Claim 9, whereby modification of the differentiation 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.

23. The method of Claim 22, wherein the method is at least partly performed using the cell culture vessel of Claim 7 or Claim 8.

24. A method of determining, assessing or monitoring the effect of one or more molecules upon a skeletal muscle cell, tissue or organoid according to any one of Claims 13 to 16, said method including the steps of contacting the skeletal muscle cell, tissue or organoid and determining, assessing or monitoring the effect of the one or more molecules upon the skeletal muscle cell, organoid or tissue.

25. The method of Claim 24, wherein determining, assessing or monitoring the effect of one or molecules upon the skeletal muscle cell, tissue or organoid is at least partly performed using the cell culture vessel of Claim 7 or Claim 8.