Accepted Manuscript

Development of a human skeletal micro muscle platform with pacing capabilities

Richard J. Mills, Benjamin L. Parker, Pauline Monnot, Elise.J Needham, Celine J.Vivien, Charles Ferguson, Robert G. Parton, David E. James, Enzo R. Porrello,James E. Hudson

PII: S0142-9612(18)30813-5

DOI: https://doi.org/10.1016/j.biomaterials.2018.11.030

Reference: JBMT 18994

To appear in: Biomaterials

Received Date: 9 March 2018

Revised Date: 28 September 2018

Accepted Date: 22 November 2018

Please cite this article as: Mills RJ, Parker BL, Monnot P, Needham EJ, Vivien CJ, Ferguson C, PartonRG, James DE, Porrello ER, Hudson JE, Development of a human skeletal micro muscle platform withpacing capabilities, Biomaterials (2018), doi: https://doi.org/10.1016/j.biomaterials.2018.11.030.

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Development of a human skeletal micro muscle platform with pacing capabilities 1 2 Richard J. Mills1,2,3, Benjamin L. Parker4, Pauline Monnot1,5, Elise J, Needham4, Celine J. 3 Vivien1,2,6, Charles Ferguson7, Robert G. Parton7,8, David E. James4, Enzo R. Porrello 1,2,6,9,*, 4 James E. Hudson1,2,3* 5 6 1School of Biomedical Sciences, The University of Queensland, St Lucia 4072, Queensland, 7 Australia. 8 2Centre for Cardiac and Vascular Biology, The University of Queensland, St Lucia 4072, 9 Queensland, Australia. 10 3 QIMR Berghofer Medical Research Institute, Brisbane 4006, Queensland, Australia. 11 4Charles Perkins Centre, School of Life and Environmental Science, The University of 12 Sydney, Sydney 2006, NSW, Australia. 13 5Laboratoire de Biologie du Développement-Institut de Biologie, CNRS, Sorbonne 14 Université, 75005 Paris, France. 15 6Murdoch Children’s Research Institute, Royal Children's Hospital, Parkville 3052, Victoria, 16 Australia. 17 7Institute for Molecular Bioscience, The University of Queensland, St Lucia 4072, 18 Queensland, Australia. 19 8Centre for Microscopy and Microanalysis, The University of Queensland, St Lucia 4072, 20 Queensland, Australia. 21 9Department of Physiology, School of Biomedical Sciences, The University of Melbourne, 22 Parkville 3010, Victoria, Australia. 23 *Co-corresponding authors. 24 25 Corresponding author contact information: 26 James E. Hudson, Ph.D. 27 QIMR Berghofer Medical Research Institute 28 Brisbane, QLD, 4006, Australia 29 Tel: +61 7 3362 0141 30 Email:james.hudson@qimrberghofer.edu.au 31 32 Enzo R. Porrello, Ph.D. 33 Murdoch Children’s Research Institute 34 The Royal Children’s Hospital 35 50 Flemington Rd, Parkville, VIC 3052, Australia 36 Tel: +61 3 9336 6140 37 Email: enzo.porrello@mcri.edu.au 38 39 Keywords: Skeletal Muscle, Bioengineering, Differentiation, Exercise, Optogenetics, 40 Metabolism 41

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ABSTRACT 1 Three dimensional engineered culture systems are powerful tools to rapidly expand our 2 knowledge of human biology and identify novel therapeutic targets for disease. 3 Bioengineered skeletal muscle has been recently shown to recapitulate many features of 4 native muscle biology. However, current skeletal muscle bioengineering approaches require 5 large numbers of cells, reagents and labour, limiting their potential for high-throughput 6 studies. Herein, we use a miniaturized 96-well micro-muscle platform to facilitate semi-7 automated tissue formation, culture and analysis of human skeletal micro muscles (hµMs). 8 Utilising an iterative screening approach we define a serum-free differentiation protocol that 9 drives rapid, directed differentiation of human myoblast to skeletal myofibres. The resulting 10 hµMs comprised organised bundles of striated and functional myofibres, which respond 11 appropriately to electrical stimulation. Additionally, we developed an optogenetic approach 12 to chronically stimulate hµM to recapitulate known features of exercise training including 13 myofibre hypertrophy and increased expression of metabolic proteins. Taken together, our 14 miniaturized approach provides a new platform to enable high-throughput studies of human 15 skeletal muscle biology and exercise physiology. 16 17

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INTRODUCTION 1 Skeletal muscle makes up ~40% of an average adult’s body mass and plays an essential role 2 in whole body locomotion and metabolism [1]. Despite its robust regenerative ability, skeletal 3 muscle function can be compromised due to a number of myopathies including 4 developmental disorders, neuromuscular diseases and muscular dystrophies [2]. Furthermore, 5 exercise induces adaptations in skeletal muscle that are regarded as some of the best 6 preventions and treatments for many chronic diseases including cancer, cardiovascular 7 disease and mental health [3, 4]. Understanding the molecular mechanisms that drive both the 8 positive adaptations of exercise and the negative effects of myopathies requires the 9 development of better model systems that recapitulate human skeletal muscle physiology and 10 pathophysiology. 11 12 Cell lines such as C2C12 mouse and L6 rat myoblasts are regularly used as in vitro 2D 13 muscle models. Whilst these cells readily proliferate, and have the capacity to differentiate 14 and fuse, these models fail to mimic cellular function, organisation, and interactions present 15 in muscle tissue and are not representative of in vivo biology [5]. In addition, signalling 16 pathways are known to be altered in a 2D versus 3D setting, which may contribute to the 17 inability of standard 2D culture models to predict potential therapeutics, with 9 out of 10 18 candidates failing from Phase I trials to the clinic [6]. Recent advances in cell biology and 19 bioengineering have led to significant progress in the development of human stem cell 20 derived three-dimensional (3D) culture systems, including mini-gut/intestinal organoids [7], 21 liver buds [8], cerebral organoids [9], kidney organoids [10], and cardiac organoids [11, 12]. 22 These 3D tissues are more representative of in vivo biology [13, 14], promote higher levels of 23 cell differentiation and tissue organisation, which recapitulate tissue-tissue interfaces and 24 mechanical microenvironments of living organs [13, 14]. This allows for the study of human 25 physiology in an organ-specific context and the development of physiologically relevant in 26 vitro models [13]. 27 28 Pioneering work by Shansky et al. [15] described the formation of functional muscle bundles 29 in vitro from rodent myoblasts. Subsequently, the Bursac lab has developed state of the art 30 tissue engineering techniques to generate functional bioengineered skeletal muscle from 31 rodent, human and pluripotent stem cell derived myoblasts [16-18]. These tissues are 32 functional, have multinucleated and striated myofibers, a resident satellite cell niche, are 33 capable of self-regeneration in vitro and respond appropriately to pharmacological agents 34 [16-18]. However, these models are expensive and require large numbers of cells, and 35 therefore miniaturization is required to unlock the potential of bioengineered skeletal muscle 36 for higher-throughput studies. 37 38 The primary goal of this study was to miniaturize the scale and automate the generation of 39 bioengineered skeletal muscle tissues in a format amenable to higher-throughput studies. We 40 recently developed a 96 well micro-muscle platform for high-throughput functional screening 41 of human cardiac organoids [11]. This platform is designed to facilitate automatic formation 42 and culture of dense muscle bundles from minimal cells and reagents, whilst also enabling 43 assessment of contractile function and marker expression analysis without any tissue 44 handling [11]. Herein, we adapt this platform to generate functional human skeletal micro 45 muscles (hµMs). Using an iterative screening approach, we define a serum-free 46 differentiation protocol that drives rapid, directed differentiation of human myoblast to 47 myofibres and subsequently develop an optogenetic approach to stimulate hµM contraction to 48 recapitulate muscle adaptations observed after exercise training. We show that these hµMs 49

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recapitulate some features of native human muscle thus providing a useful model for muscle 1 biology, biomedical research and exercise physiology. 2

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MATERIALS & METHODS 1 2 Human Myoblast Growth 3 Primary human myoblasts were purchased from GIBCO and grown as per [19]. Briefly, 4 myoblasts were grown on Matrigel (ThermoFisher Scientific) coated flasks in a media 5 consisting of a 1:1 mixture of DMEM:MCDB (ThermoFisher Scientific) supplemented with 6 20% fetal bovine serum (FBS, ThermoFisher Scientific), 1% insulin-transferrin-selenium 7 (ITS, Invitrogen), 1% penicillin-streptomycin (P/S, ThermoFisher Scientific) and 10µM of 8 SB203580 (p38 MAPK inhibitor, Stem Cell Technologies). When required, myoblasts were 9 harvested using Trypsin enzymatic digestion. Immunohistochemistry staining revealed 10 myoblasts were Pax7 positive prior to isolation for skeletal muscle bioengineering 11 (Supplementary Figure 1A). 12 13 Miniturized Culture Platform Fabrication 14 Cell-culture inserts were fabricated using standard SU-8 photolithography and PDMS 15 moulding practices, as per [11]. 16 17 Generation of Human Skeletal Micro Muscles 18 For each hµMs, 32,000 myoblasts in growth medium were mixed with collagen I gel to make 19 a 3.2 µl final solution containing 3.3 mg/ml collagen I and 22% (v/v) Matrigel. The bovine 20 acid-solubilized collagen I (Devro) was first salt balanced and pH neutralized using 10X 21 DMEM and 0.1 M NaOH, respectively, prior to mixing with Matrigel and then combined 22 with the cells. The mixture was prepared on ice and pipetted into the cell-culture inserts. The 23 mixture was then gelled at 37°C for 30-60 min prior to the addition of myoblast growth 24 medium (150 µl/ hµMs). After 2 days, cells had aggregated around the two elastomeric poles 25 and media was changed to induce myoblast differentiation. 26 27 Screening for Optimal Differentiation Conditions 28 A number of different conditions were tested for myoblast differentiation (as per Figure 1). 29 For supplements and small molecules used in the study refer to Supplementary Table S1. 30 31 DAPT and Dabrafenib (D&D) Differentiation Protocol 32 2 days after seeding, myoblasts had aggregated around the two elastomeric poles and media 33 was changed to D&D media to induce myoblast differentiation. D&D media consists of 34 MEM α (ThermoFisher Scientific) with 1% P/S (ThermoFisher Scientific), 0.5% ITS (ITS, 35 ThermoFisher Scientific) and 2% B-27 supplement (ThermoFisher Scientific), with 10 µM 36 DAPT (Stem Cell Technologies) and 1 µM Dabrafenib (Stem Cell Technologies). 2% horse 37 serum (HS) differentaition media consists of MEM α (ThermoFisher Scientific) with 1% P/S 38 (ThermoFisher Scientific), 0.5% ITS (ITS, Invitrogen), 2% B-27 supplement (ThermoFisher 39 Scientific), and 2% HS (ThermoFisher Scientific). 40 41 Modelling Muscle Physiology in Human Skeletal Micro Muscles 42 D7 D&D hµMs were treated with 0.1%DMSO or 10µM Simvastatin (Sigma). For YAP1 43 overexpression, D7 D&D hµMs were treated with an AAV6 containing a mutated version of 44 human YAP1, CMV-YAP(S127A) (Vector Biolabs) or a control AAV6-MCS (Vector 45 Biolabs) at 6 x 109 vg/ hµMs [11]. Force recordings were taken after 72 hrs of treatment. 46 47 48 49 50

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2D Differentiation of Myoblasts 1 Myoblast were seeded at 90% confluence on Matrigel coated wells in growth medium. After 2 4 hrs, media was changed to MEM α with 1% P/S, 0.5% ITS and 2% B-27 supplement, with 3 either 2% horse serum or 10 µM DAPT and 1 µM Dabrafenib. 4 5 Force Analysis of Human Skeletal Micro Muscles 6 hµMs were electrically stimulated at 1, 2, 5 and 20 Hz with 5ms square pulses with 20 mA 7 current using a Panlab/Harvard Apparatus Digital Stimulator. During stimulation, a Leica 8 DMi8 inverted high content Imager was used to capture a 5s time-lapse of each hµM 9 contracting in real time at 37°C. Pole deflection was used to approximate the force of 10 contraction as per [11]. Custom batch processing files were written in Matlab R2013a 11 (Mathworks) to convert the stacked TIFF files to AVI, track the pole movement (using 12 vision.PointTracker), produce a force-time figure, and export the batch data to an Excel 13 (Microsoft) spreadsheet. For more information regarding force analysis please refer to [11]. 14 15 Whole-mount immunostaining 16 hµMs were fixed for 60 min with 1% paraformaldehyde (Sigma) at room temperature and 17 washed 3X with PBS, after which they were incubated with primary antibodies in Blocking 18 Buffer, 5% FBS and 0.2% Triton-X-100 (Sigma) in PBS overnight at 4°C. Cells were then 19 washed in Blocking Buffer 2X for 2 h and subsequently incubated with the appropriate 20 secondary antibodies, and Hoechst33342 (1:1000), overnight at 4°C. Cells were then washed 21 in Blocking Buffer 2X for 2 h and imaged in situ or mounted on microscope slides using 22 Fluoromount-G (Southern Biotech). For a list of antibodies used in the study please refer to 23 Supplementary Table S2. 24 25 Immunostaining Analysis 26 For screening, hµMs were imaged using a Leica DMi8 high content imaging microscope for 27 in situ imaging. Custom batch processing files were written in Matlab R2013a (Mathworks) 28 to remove the background, calculate the image intensity, and export the batch data to an 29 Excel (Microsoft) spreadsheet. For higher magnification images, an Olympus IX81 confocal 30 microscope was used for slide-mounted hµM imaging. 31 32 Human RNA Samples 33 The adult human skeletal muscle sample was obtained from Clontech. The adult tissue was 34 pooled from 3 skeletal muscle samples from Asian, Caucasian male/females aged 30, 44 and 35 86. 36 37 RNA Extraction and qPCR 38 RNA was isolated using a Qiagen RNAeasy kit, as per manufacturer’s instructions and 39 DNase I treated (Roche). cDNA was reverse transcribed using Superscript III (random 40 primers, ThermoFisher Scientific) and qPCR performed using SYBR Mastermix 41 (ThermoFisher Scientific) on a Applied Biosystems Step One Plus. The 2-∆∆Ct method was 42 used to determine gene expression changes using 18S as the endogenous control. Primer 43 sequences are listed in Supplementary Table S3 and were used at 200 nM. Gene expression 44 is presented relative to adult human skeletal muscle samples (SkMuscle). 45 46 Proteomics and Data Processing 47 Single hµMs were washed 2X in PBS and snap frozen and stored at -80°C. Tissues were 48 lysed by tip-probe sonication in 1% SDS containing 100 mM Tris pH 8.0, 10 mM tris(2-49 carboxyethyl)phosphine, 40 mM 2-chloroacetamide and heated to 95°C for 5 min. Proteins 50

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were purified using a modified Single-Pot Solid-Phase-enhanced Sample Preparation (SP3) 1 strategy [20]. Briefly, Proteins were bound to Sera-Mag carboxylate coated paramagnetic 2 beads in 50% acetonitrile containing 0.8% formic acid (v/v) (ThermoFisher Scientific). The 3 beads were washed twice with 70% ethanol (v/v) and once with 100% acetonitrile. Proteins 4 were digested on the beads in 100 mM Tris pH 7.5 containing 10% 2,2,2-Trifluoroethanol 5 overnight at 37°C with 200 ng of sequencing grade LysC (Wako Chemicals) and trypsin 6 (Sigma). Beads were removed and peptides acidified to 1% trifluoroacetic acid prior to 7 purification by styrene divinyl benzene – reversed phase sulfonated solid phase extraction 8 microcolumns. Peptides were spiked with iRT peptides (Biognosys) and analysed on an 9 Easy-nLC1200 coupled to a Q-Exactive HF in positive polarity mode. Peptides were 10 separated using an in-house packed 75 µm x 50 cm pulled column (1.9 µm particle size, 11 C18AQ; Dr Maisch) with a gradient of 2 – 35% acetonitrile containing 0.1% FA over 120 12 min at 300 nl/min at 60°C. The instrument was operated in data-independent acquisition 13 (DIA) mode essentially as described previously [21]. Briefly, an MS1 scan was acquired 14 from 350 – 1650 m/z (120,000 resolution, 3e6 AGC, 50 ms injection time) followed by 20 15 MS/MS variable sized isolation windows with HCD (30,000 resolution, 3e6 AGC, 27 NCE). 16 A spectral library was created by fractionating a pooled mix of peptides from 4 separate 17 hµMs on an inhouse packed 320 µm x 25 cm column (3 µm particle size, BEH; Waters) with 18 a gradient of 2 – 40% acetonitrile containing 10 mM ammonium formate over 60 min at 6 19 µl/min using an Agilent 1260 HPLC. A total of 12 concatenated fractions were analysed 20 using the identical LC-MS/MS conditions above except the instrument was operated in data-21 dependent acquisition (DDA) mode. Briefly, an MS1 scan was acquired from 350 – 1650 m/z 22 (60,000 resolution, 3e6 AGC, 50 ms injection time) followed by 20 MS/MS with HCD (1.4 23 m/z isolation, 15,000 resolution, 1e5 AGC, 27 NCE). DDA data were processed with with 24 Andromeda in MaxQuant v1.5.8.3 [22] against the human UniProt database (January 2016) 25 using all default settings with peptide spectral matches and protein false discovery rate (FDR) 26 set to 1% . DIA data were processed with Spectronaut v11 [23] using all default settings with 27 precursor and protein FDR set to 1% and quantification performed at MS2. 28 29 Proteomic Bioinformatics 30 All proteomic data were Log2 transformed and median normalised. Differential abundance 31 was determined with two sample t-tests with FDR-based multiple hypothesis testing 32 correction. GO analysis was performed with DAVID v6.8 and gene ontology terms from 33 BP_ALL [24] and heat-maps and hierarchical clustering was performed using GENE-E 34 (Broad Institute). 35 36 SPARCLIGHT Adenovirus Generation 37 A red-shifted variant of channelrhodopsin (C1V1) [25] was linked to the fluorescence protein 38 voltage sensor Arc Light [26] via a p2A linker sequence, thus enabling membrane 39 depolarisation via optical stimulation and simultaneous recording of membrane potential via 40 changes in fluorescence. The dual reporter system (hereto referred to as SPARCLIGHT) was 41 synthesised by GeneCopeia and sub-cloned into the DUAL-CCM+ plasmid for adenovirus 42 production by Vector Biolabs (PA, USA). 43 44 Transmission electron microscopy 45 Samples were processed for electron microscopy as described previously [27]. Sections were 46 analyzed unstained in a Jeol1011 transmission electron microscope. 47 48 Optogenetic Stimulation 49

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After 7 days of differentiation, hµMs were cultured in a maintenance media consisting of 1 MEM α (ThermoFisher Scientific) with 1% P/S (ThermoFisher Scientific), 0.5% ITS (ITS, 2 ThermoFisher Scientific) and 2% B-27 supplement (ThermoFisher Scientific). hµMs were 3 treated with the SPARCLIGHT adenovirus at 500 MOI for 3 days. hµMs were subsequently 4 washed, and exposed to pulses of green light using a 96-well green LED array (Lumidox) 5 connected to a Panlab/Harvard Apparatus Digital Stimulator. hµMs were stimulated for 2 hrs 6 per day, using a 200ms light pulse every 5 seconds (5,760 total contractions). After 4 days of 7 optical stimulation, hµMs were taken for proteomics, immunohistochemistry and force 8 analysis. For force recordings, hµMs were electrically stimulated at 1, and 20 Hz with 5ms 9 square pulses with 20 mA current as described above. 10 11

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RESULTS 1 2 Iterative Screening for Optimal Serum-free Differentiation 3 Adult human satellite cells (myoblasts) were expanded as previously described [19]. Human 4 myoblasts were harvested and seeded in a Collagen I (3.3 mg/ml), Matrigel (22% (v/v)) and 5 DMEM supplemented matrix. The elliptical geometry of our micro-muscle platform is 6 designed so that the 32,000 myoblasts automatically condense around the two elastic posts 7 over 2 days (Figure 1A,B). After cell condensation, growth media was exchanged for 8 differentiation media to promote fusion and differentiation of myoblasts into myofibers 9 (Figure 1B). However, traditional horse serum (HS) based differentiation approaches failed 10 to yield substantial myoblast fusion and myofibre formation over a 5-day period (Figure 1C). 11 Therefore, an iterative screening approach was taken to identify media conditions that 12 promoted rapid, directed myofibre formation. 13 14 The micro-muscle platform facilitates screening based on marker expression by utilising 15 whole mount-immunostaining combined with high-content image analysis [11]. Based on the 16 expression of the skeletal muscle markers desmin and titin (representative of an early and late 17 stage differentiation marker, respectively), we investigated the role of a number of factors, 18 including base media, supplementation, small molecules and serum concentration in driving 19 muscle differentiation (Figure 1D). In combination with traditional 2% HS, we first 20 investigated the optimal base media and media supplements, and found it was advantageous 21 to use MEM α, supplemented with 0.5% insulin transferrin selenium (ITS) and 2% B-27 22 (Figure 1E,F). Using small molecules, we subsequently investigated the function of a 23 number of highly conserved cell signalling pathways involved in myoblast proliferation and 24 differentiation, including the Wnt [28], Notch [29] and Raf pathways [30]. CHIR99021, a 25 potent inhibitor of GSK3 and activator of Wnt signalling, was detrimental to myoblast 26 differentiation, reducing desmin and titin expression by ~60% compared to control conditions 27 (Figure 1G), whilst IWR-1, a Wnt inhibitor, had little effect on differentiation. Interestingly, 28 both DAPT, a γ-secretase inhibitor that blocks the Notch pathway, and Dabrafenib, a potent 29 Raf inhibitor and key regulator of cell cycle, led to increased expression of titin (Figure 30 1G,H). These molecules were subsequently investigated and tested in a factorial array 31 between 1 and 10µM. All combinations using both molecules led to a dramatic increase in the 32 fusion and formation of myofibres (Figure 1H, I and Supplementary Figure 1A), with a 2-33 fold increase and 4-fold increase in desmin and titin expression, respectively, using 10µM 34 DAPT and 1µM Dabrafenib (Figure 1H,I). This combination of molecules was further tested 35 and found to be most effective over a 7-day differentiation period (Figure 1J). Furthermore, 36 removal of horse serum was not detrimental to differentiation and indeed increased the 37 relative expression of titin compared to media containing 5% horse serum (Figure 1K). 38 DAPT and Dabrafenib (D&D) could also be used serum-free in 2D culture to generate 39 myotubes (Supplementary Figure 1B). Thus, by performing iterative screening, in which 40 the optimal condition was used for ensuing screens, we identified a rapid, directed, serum-41 free protocol to generate hµMs from human myoblasts. This protocol was termed ‘D&D’, 42 which we subsequently phenotypically characterised. 43 44

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1 Figure 1: Iterative screening in a micro-muscle culture platform identifies serum-free muscle 2 differentiation protocol. 3

A) Schematic representation of cell culture insert containing an eliptical seeding well with two elastomeric 4 poles. Each well of a 96 well-plate contains an insert. 5

B) Automatic tissue formation within the micro-muscle platform. Myoblasts are seeded into the eliptical 6 well in combination with matrix and allowed to gel at 37°C. Cells subsequently condense around the 7 two elastomeric poles. 8

C) Myoblasts failed to sufficently fuse and differentiate following treatment with traditional differentiation 9 media of 2% HS. Whole mount immunostaining for titin (green), desmin (red) and DNA (blue). Scale 10 bar- 500µm. 11

D) Culture schematic of screening strategy to identify conditions that promote myotube formation. 12 E) MEM α is the optimal base media for induction of titin and desmin in the presence of 2% HS. Titin and 13

desmin relative intensity heat map after 5 days of differentiation (relative to LG-DMEM). n=3, from 1 14 experiment. 15

F) Screening identifies optimal media supplementation of 0.5% ITS and 2% B-27 for expression of titin 16 and desmin in the presence of 2% HS. Titin and desmin relative intensity heat map after 5 days of 17

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differentiation (relative to MEM α). n=4-6, from 2 experiments. I- 0.5% ITS, A- 200µM Ascorbate, B- 1 2% B-27 supplement. 2

G) Distinct inhibition of the Notch and Raf pathways resulted in increased expression of titin. Titin and 3 desmin relative intensity heat map after 5 days of differentiation (relative to DM). n=3-9, from 3 4 experiments. DM- control condition using MEM α with 0.5% ITS, 2% B-27 and 2% HS. 5

H) Wholemount immunostaining of desmin, titin, and DNA after 5 day treatment with DAPT, dabrafenib 6 or in combination. Scale bar represents- 500µm. 7

I) Combination of DAPT and dabrafenib led to a dramatic upregulation of titin and desmin expression. 8 Desmin (left) and titin (Right) relative intensity heat map after 5 days of differentiation in response to 9 DAPT and Dabrafenib at 0, 1 or 10 µM (relative to 0 µM DAPT and dabrafenib). n=3-7, from 2 10 experiments. Base media consisted of MEM α with 0.5% ITS, 2% B-27 and 2% HS. 11

J) 7 day treatment with 10 µM DAPT and 1 µM dabrafenib is optimal for desmin and titin expression. 12 Titin and desmin relative intensity heat map after 3, 5, and 7 days of differentiation (relative to 5 day 13 differentiation). n=3-4, from 1 experiment. Differentiation media consisted of MEM α with 0.5% ITS, 14 2% B-27, 2% HS, 10 µM DAPT and 1 µM Dabrafenib. 15

K) Differentiation protocol can be performed serum-free and does not impact the expression of titin or 16 desmin. Titin and desmin relative intensity heat map using 5%, 2% or 0% HS over 7 days of 17 differentiation (relative to 5 % HS), n=3-4, from 1 experiment. Differentiation media consisted of 18 MEM α with 0.5% ITS, 2% B-27, 10 µM DAPT and 1 µM dabrafenib. 19

*P < 0.05, **P < 0.01, ***P<0.001, ****P < 0.0001, using one-way ANOVA with Dunnet’s post-test in 20 comparison to ‘relative’ condition. 21

22 Structure, Function and Gene Expression Analysis of Human Skeletal Micro Muscles 23 After defining a rapid, directed differentiation approach, we characterised the structure, 24 function and gene expression of hµMs generated via standard 2% HS and D&D optimized 25 differentiation (Figure 2A). Consistent with Figure 1, D&D hµMs had more abundant 26 (Figure 2B) and well developed myofibres (Supplementary Video 1). D&D hµM myofibres 27 had defined titin and desmin striations throughout the tissue (Figure 2C), located within a 28 laminin and collagen IV rich-matrix (Supplementary Figure 2A,B). Transmission electron 29 microscopy confirmed D&D huMs have aligned sarcomeres with alternating A-bands and I-30 bands, electron-dense Z-lines and distinct M-lines and H-zones (Figure 2D). Together, these 31 data indicate a mature sarcomeric structure in D&D huMs. Additionally, we also identified 32 junctional structures between sarcoplasmic reticulum and T-tubules, including triads, 33 reflective of native skeletal muscle fibers (Figure 2D). 34 35 Functional properties were assessed by tracking of elastomeric poles during electrical 36 stimulation of the hµMs. The micro-muscle platform allows contractile force to be assessed 37 in situ, without any tissue handling [11]. Consistent with other bioengineered skeletal muscle 38 systems [16-18], hµMs displayed twitch and tetanic contractions and a positive force 39 frequency relationship in respose to electrical stimulation (Figure 2E and Supplementary 40 Video 2-4). Active force (Figure 2F) and specific force (Figure 2G) were consistently 41 higher in hµMs differentiated using D&D compared to 2%HS. D&D hµMs had an average 42 specific force of 2.0± 0.7 mN.mm-2 and 4.9 ±0.8 mN.mm-2 for twitch and tetanus, 43 respectively (Figure 2G). Specific tetanic forces generated within our system are similar to 44 other reported tissue engineered skeletal muscle [16-18]. However, this is more reflective of 45 fetal human muscle function, and an order of magnitude lower than specific forces reported 46 for adult human muscle [31, 32]. Additionally, the tetanus-to-twitch ratio was higher in D&D 47 (≈2.4) compared to 2% HS hµM (≈1.2) (Figure 2H), closer, but not equivalent, to values 48 reported for adult human muscle (≈2-4 fold lower) [31]. 49 50

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Our optimised D&D protocol was capable of generating >90% viable, force generating hµMs 1 (Supplementary Figure 2C), important for functional screening approches in academia and 2 industry. Consistent with differentiation of myoblasts into functional myotubes there was a 3 rapid decrease in the myoblast marker PAX7 (Figure 2I), increase in the myotube markers 4 MYH2 and MYH3 (Figure 2J,K), and increase in the calcium handling genes SERCA1 and 5 RYR1 (Figure 2L,M). Both MYH3 and SERCA1 were higher in D&D compared to 2% HS 6 hµM, while PAX7 was similar between the two conditions. Taken together, this indicates that 7 both conditions are capable of inducing differentiation of skeletal myoblasts into myotubes, 8 but our optimised D&D protocol leads to more rapid differentiation and better functional 9 outcomes.10

11 Figure 2: Comparison of D&D differentiation versus 2% HS in human skeletal micro muscles. 12

A) Schematic of the protocol for the generation of hµM using 2% HS or D&D. 13

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B) Whole-tissue images of hµM stained with desmin (red), titin (green), and DNA (Blue). Scale bars- 500 1 µm. 2

C) High magnification imaging of D&D hµMs reveal the presence of multinucleated myotubes with 3 defined titin striations throughout the tissue. Desmin (red), titin (green), and DNA (Blue). Scale bar = 4 20 µm. Inset- close-up image of striated titin. 5

D) Transmission electron micrographs of D&D hµMs. T – T-Tubule, SR- sarcoplasmic reticulum, S- 6 Sarcomere, I- I band, Z- Z line, A- A band, M- M line, H- H zone. Scale Bar- 1µm. 7

E) Representative force trace curves of D&D hµMs in response to electrical stimulation under twitch 8 conditions (2 Hz) or tetanus (20 Hz). 9

F) Active force production is higher in D&D versus 2% HS hµMs under twitch and tetanus. n=7, from 2 10 experiments. 11

G) Specific force production is higher in D&D versus 2% HS hµMs under twitch and tetanus. n=7, from 2 12 experiments. 13

H) The tetanic to twitch force production ratio is higher in D&D versus 2% HS. n=7, from 2 experiments 14 I) PAX7, a marker of muscle stem cells, is rapidly downregulated during differentiation in both 2% HS or 15

D&D hµMs by day 5 using qPCR. n = 4-6. 16 J) MYH2, a marker of differentiated skeletal muscle, is rapidly upregulated during differentiation in both 17

2% HS or D&D hµMs by day 5 using qPCR. n = 4-6. 18 K) MYH3, a marker of differentiated skeletal muscle, is rapidly upregulated during differentiation in both 19

2% HS or D&D hµMs and is higher in D&D hµMs by day 5 using qPCR. n = 4-6. 20 L) The calcium handling gene RYR1 is rapidly upregulated during differentiation in both 2% HS or D&D 21

hµMs and is higher in D&D hµMs by day 5 using qPCR. n = 4-6. 22 M) The calcium handling gene ATP1A1 aka SERCA1 is rapidly upregulated during differentiation in D&D 23

hµMs by day 5 using qPCR. n = 4-6. 24 Data is presented as mean ± s.e.m. * P < 0.05, ** P< 0.01, *** P < 0.001 using one-way ANOVA with Tukey’s 25 post test (I-M) or t-test (F-H). 26 27 Proteomic Analysis of Human Skeletal Micro Muscles 28 Proteomics on single hµMs was used to profile the development of hµMs under D&D 29 differentiation conditions. This facilitated quantification of over 4000 proteins per hµMs at 30 day 0, 3, 5 and 7 of differentiation. Fetal myosin heavy chain isoforms rapidly increased 31 during differentiation including MYH3 and MYH8, and were similar in abundance at day 3 32 versus day 7 (Figure 3A). However, many adult myosin heavy chain isoforms were not 33 strongly regulated at any of the time-points (MYH1, MYH2 and MYH7) (Figure 3A). Other 34 sarcomeric proteins such as TTN and calcium handling genes (CASQ2, CASQ1, ATP2A1 35 aka SERCA1, ATP2A2 aka SERCA2 and RYR1) were rapidly increased during hµM 36 differentiation (Figure 3A). Together these results are consistent with the development of 37 functional hµMs as presented in Figure 1 and Figure 2, and the rapid, directed 38 differentiation process using D&D. 39 40 Analysis of protein abundance at day 7 versus day 0 revealed that over 1,500 proteins were 41 significantly up- or down-regulated during D&D induced differentiation (Figure 3B). Some 42 of these proteins were progressively increased over the 7 day protocol and a principal 43 component analysis revealed that differentiation proceeded in an ordered fashion with the 44 hµMs clustering at each time-point (Figure 3C). Hierarchical clustering of significantly 45 regulated proteins supported this developmental progression with samples from each time-46 point clustering distinctly (Figure 3D). As supported by the analysis of critical skeletal 47 muscle genes in Figure 3A, the most differentially clustered samples were from day 0 to day 48 3 indicating rapid differentiation of hµMs with D&D. This is highlighted by gene ontology 49 (GO) analysis of the “up-regulated early” cluster being enriched for proteins involved in 50

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skeletal muscle development and the “down-regulated early” cluster being enriched for 1 proteins involved in extracellular matrix organisation, which is a feature of stromal cells prior 2 to differentiation (Figure 3D). There was a distinct cluster of proteins “up-regulated late” 3 which were enriched in proteins involved in metabolism and the production of energy 4 (Figure 3D). Together this indicates that D&D induces a rapid differentiation into a skeletal 5 muscle phenotype and metabolic processes gradually mature over time. 6 7

8 Figure 3: Proteomic analysis of human skeletal micro muscle development reveals a rapid differentiation 9 response using D&D. 10

A) Rapid increase in the abundance of critical skeletal muscle sarcomeric and calcium handling genes 11 during hµM development, n = 4 per time point.Data is presented as mean ± s.e.m. and * P < 0.05, * P < 12 0.01, *** P < 0.001, **** P < 0.0001 using one-way ANOVA compared to day 0. 13

B) Volcano plot of significantly regulated genes comparing day 7 to day 0 hµMs reveals over 1,500 14 proteins are regulated. 15

C) Principal component analysis showing hµM development is consistent between tissues and follows a 16 developmental program. 17

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D) Hierarchically clustered heat map of significantly regulated genes during hµM development. Gene 1 ontologies (biological processes) are shown together with their p-value for each gene cluster. 2

Human Skeletal Micro Muscles are Representative of Immature Human Muscle 3 Protein expression was compared between adult human skeletal muscle tissue (hSkM tissue) 4 and D7 hµMs derived via D&D differentiation. Although there was a significant relationship 5 between protein abundance in hµM compared to hSkM tissue (Figure 4A), the Pearson 6 correlation co-efficient was 0.57 indicating that there were also some differences. These 7 differences include higher expression of fetal myosin heavy chain isoforms, lower expression 8 of adult myosin heavy chain isoforms and lower expression of some calcium handling genes 9 (Figure 4B), all of which are consistent with the ‘fetal-like’ contractile function observed in 10 our system. Taken together this indicates hµMs are more reflective of fetal human skeletal 11 muscle. GO analysis of the top 50 proteins with higher abundance in hSkM tissue compared 12 to hµMs revealed that most biological processes were enriched for metabolism and energy 13 production (Figure 4C). Together this indicates that even though D&D induces rapid 14 differentiation in hµMs and induction of metabolic processes by day 7, the sarcomeric 15 proteins are still fetal and the expression of the metabolic proteins still lower than hSkM 16 tissue. 17

18 Figure 4: Proteomic comparison of day 7 hµµµµMs to adult human skeletal muscle reveals immaturity. 19

A) Protein abundance of day 7 hµM (x-axis) versus adult human skeletal muscle (hSKM Tissue, y-axis) 20 reveals that they are significantly correlated. 21

B) Abundance of adult skeletal muscle sarcomeric and calcium handling proteins is lower and abundance 22 of fetal skeletal muscle proteins is higher in 7 day hµMs versus adult human skeletal muscle. 23

C) Gene ontologies (biological processes) of top 50 differentially expressed proteins with higher 24 abundance in adult human skeletal muscle (hSkM Tissue) versus day 7 hµMs. Gene ontologies relate to 25 metabolic processes. Number of genes in each process are shown in the bars. 26

Muscle Physiology can be Modelled using Human Skeletal Micro Muscles 27 The potential to model muscle physiology was subsequently evaluated within D&D hµMs. 28 To evaluate whether hµMs display appropriate responses to pharmacological agents with 29 known muscle toxicity, D7 hµMs were treated with 10 µM Simvastatin. A well known side-30 effect of statin usage is sigificant myopathic weakness and rhabdomyolysis . After 72 hrs of 31 treatment, hµMs displayed reduced twitch and tetanus active force (Supplementary Figure 32 3A). To further evaluate the ability of hµMs to model skeletal muscle physiology, we also 33 treated hµMs with constitutively active human Yes-associated protein 1 (YAP1), a known 34 regulator of muscle mass and function in mice [34, 35]. Over-expression of constitutively 35 active human Yes-associated protein 1 (YAP1) increased tetanus active force in hµMs 36 (Supplementary Figure 3B). Collectively, these findings demonstrate that hµMs respond 37 appropriately to known pharmacological agents and signalling effectors and can be utilised as 38 a model system to study human muscle physiology. 39 40

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Optogenetic Stimulation Recapitulates some Adaptations of Exercise 1 Functional and proteomic analysis of hµMs revealed that they were more reflective of a fetal 2 muscle phenotype. As the main differences between native skeletal muscle tissue and hµMs 3 were related to metabolism and energy production (Figure 4), we hypothesized that 4 contractile stimulation may promote maturation. Furthermore, hµM contractile stimulation 5 may recapitulate an exercise-training regime, thus allowing the in vitro analysis of exercise 6 adaptations in an isolated human system. 7 8 In order to stimulate the hµMs, we developed an optogentic approach to enable chronic 9 contraction of hµMs in vitro (Figure 5A and Supplementary Figure 4A). This was chosen 10 over electrical stimulation regimes, as electrical stimulation can cause significant myofibre 11 damage and cell death [36], which was observed in our miniaturized format (Supplementary 12 Figure 4B). For these experiments, the red-shifted channelrhodopsin, for optogenetic 13 stimulation [25], and ArcLight, as a fluorescent voltage sensor [26], were delivered to hµMs 14 using an adenovirus under the control of a CMV promoter. We termed this dual stimulation-15 reporter system SPARCLIGHT. hµMs were treated with the SPARCLIGHT adenovirus to 16 allow for (1) control over hµM contraction and (2) provide an optical approach to visualise 17 cell depolarisation (Figure 5B). To stimulate contraction, hµMs were exposed to a single 18 200ms pulse of light every 5 seconds (0.2Hz), for 2 hrs a day. This caused a single tetanic 19 contraction after each light pulse (Supplementary Video 5 and Figure 5C). After 4 days of 20 stimulation (STIM), a total of 5760 contractions, there was no significant change in active 21 force or the tetanus-to-twitch ratio compared to time-matched, unstimulated control tissues 22 (Figure 5D,E). However, there was an increase in tetanus to twitch ratios compared to week 23 1 tissues (Figure 2E), which approached levels similar to adult skeletal muscle [31], 24 indicative that further culture time leads to improvements in function (Figure 5E). Chronic 25 stimulation increased hµM projected area (Figure 5F), myotube fibre diameter (Figure 5G) 26 and titin intensity (Figure 5H,I), consistent with changes observed in native muscle during 27 resistance exercise [37]. Proteomic analysis revealed that 204 proteins were differentially 28 expressed and that changes were associated with mitochondrial biogenesis and organisation, 29 generation of energy and cellular respiration (Figure 5J), indicative of a progression towards 30 a more metabolically active muscle similar to that derived in vivo (Figure 4C). Additionally, 31 we observed increased protein expression of MYH7B, a slow-twitch myosin, and decreased 32 expression of MYH2, a fast-twitch myosin, indicative of a switch in fibre type observed after 33 endurance exercise [37] (Supplementary Figure 4C). Together this suggests that (1) 34 optogenetic stimulation can be used to mature bioengineered skeletal muscle and (2) our 35 model is capable of recapitulating some in vivo features of the physiological adaption to 36 exercise in vitro. 37 38

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1 Figure 5: Optogenetic Stimulation can induce some aspects of maturation and recapitulate some features 2 of exercise. 3

A) Culture schematic for generating and optogenetically stimulating hµMs. 4 B) SPARCLIGHT construct schematic to enable optgenetic stimulation of hµMs. 5 C) A single tetanic contraction is induced by a 200ms light pulses every 5s (STIM). 6 D) Active force including both twitch and tetanic are not altered by the STIM. n=10-12, 2 experiments. 7 E) Tetanus-to-twitch ratio increase with additional culture time, but not with STIM. n=7-12, from 2 8

experiments 9 F) Projected tissue area increases with STIM. n=12, 3 experiments. 10 G) Fibre diameter increases with STIM. n=12, 3 experiments. 11 H) Titin intensity increases with STIM. n=12, 3 experiments. 12 I) Representative images of CTRL and STIM hµMs. (Left) Whole-mount immunostaining of titin (green) 13

and DNA (blue). Scale bar- 500µm. (Right) Higher magnification confocal imaging of hµMs. Scale 14 Bar- 20 µm. 15

J) Gene ontologies (biological processes) of regulated proteins with higher abundance in STIM versus 16 CTRL. Most gene ontologies relate to metabolic processes. Number of genes in each process are shown 17 in the bars. 18

Data is presented as mean ± s.e.m. and * P < 0.05, * P < 0.01, *** P < 0.001, **** P < 0.0001 using one-19 way ANOVA with Dunnett’s multiple comparison test (E) or students T-test (F-H). 20

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DISCUSSION 1 Exercise training induces adaptations that have enormous health benefits [3, 4]. Identification 2 of the underlying molecular mechanisms of these adaptations may lead to new therapeutic 3 targets for a range of different diseases [38]. However, exercise produces a complex, 4 cascading set of responses within skeletal muscle and also elicits body-wide physiological 5 adaptations in other organ systems making it extremely complicated to dissect underlying 6 molecular mechanisms in vivo. In vitro culture models that recapitulate human skeletal 7 muscle physiology are needed to rapidly expand our knowledge of disease mechanisms and 8 exercise adaptations. As traditional 2D models have well known limitations [6, 13, 14], 3D 9 models could provide more predictive assays of human physiology for biological discovery 10 and drug development [17]. Recent studies suggest that organoid systems are more reflective 11 of in vivo biology compared with 2D assays [6, 13, 14]. For example, it was recently shown 12 that intestinal organoids can be used to successfully predict the outcome of stage I/II clinical 13 trials in cancer patients [39]. 14 15 In order to facilitate rapid biological discovery, 3D physiological systems need to be 16 amenable to high-throughput, large-scale screening. This requires a miniaturised platform 17 that is relatively inexpensive and requires minimal labour. We recently published a micro-18 tissue screening platform for the culture of human cardiac organoids [11]. We have now 19 adapted this platform to skeletal muscle, allowing automated tissue formation, culture and 20 analysis of hµMs for the first time. Furthermore, this approach enabled us to reduce the size, 21 reagents and cost of hµMs by a factor of ≈25 in comparison to state-of-the-art skeletal muscle 22 bioengineering approaches [16-18]. 23 24 In our platform, traditional skeletal muscle differentiation protocols using horse serum [16, 25 17], failed to yield any substantial myofibres and only developed low active contractile 26 forces. We therefore sought to optimise a differentiation protocol to drive rapid, directed, 27 differentiation of myoblasts. Through iterative screening (≈30 iterations), we identified a 28 serum-free protocol based on Notch and Raf signalling inhibition, that enables rapid 29 production of functional hµM within 7 days. DAPT is a potent inhibitor of the γ-secretase 30 complex, which inhibits the Notch signalling pathway. Notch signalling is known to play a 31 critical role in development and regeneration of skeletal muscle, with Notch activation 32 driving self-renewal of undifferentiated PAX7 myoblasts [40] and inhibiting myogenic 33 differentiation [29]. Dabrafenib is a B-Raf specific inhibitor. B-Raf is known to activate 34 PAX3 and is required for muscle precursor cell migration and proliferation [41]. 35 Furthermore, activated Raf has been shown to prevent L6 rat myoblast differentiation [30]. 36 Our optimised differentiation protocol targets these two key signalling pathways to enhance 37 myoblast differentiation and drive rapid formation of myofibres. 38 39 Functional and proteomic analysis revealed that our hµMs resembled fetal skeletal muscle. 40 This was not unexpected as our protocol was assessed over a short 7-day period. Although, 41 the arrangement of muscle fibres within our hµMs are not completely unidirectional, forces 42 obtained are comparable with the forces produced in other bioengineered formats that contain 43 unidirectional, aligned myofibres [17, 18, 33, 42]. Therefore, it is likely the high expression 44 of fetal myosin heavy chain isoforms and fetal-like maturity of the muscle fibres that leads to 45 ‘fetal’ like contractile function. An increase in culture time has been previously shown to 46 promote maturation of human bioengineered skeletal muscle, however, even over a period of 47 4 weeks, adult-like function, structure and myogenic capacity were not achieved [17, 18]. By 48 optogenetically stimulating hµM contraction, we were able to promote some features of 49 increased maturation; for example, myotube hypertrophy and enhanced metabolism. Key 50

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upstream drivers of skeletal muscle maturation are poorly defined and require further 1 investigation to enhance maturation of engineered skeletal muscle. Muscle innervation [43], 2 metabolic substrates [11], support cell populations (e.g. fibroblasts, endothelial cell, 3 macrophages), different synthetic or natural hydrogels (e.g. GelMA [44])and different 4 exercise intensity and regimes (e.g. endurance or resistance) [37], all warrant further 5 investigation and may be key to generate an adult-like phenotype. High-throughput functional 6 screening approaches have the capacity to rapidly facilitate the identification of key drivers of 7 maturation in order to successfully model diseases such as sarcopenia, metabolic disorders 8 and motor neuron disease. 9 10 3D organoid technologies, in combination with high throughput screening platforms, have the 11 ability to rapidly expand our knowledge of human biology and could lead to the development 12 of novel therapeutics. However, large-scale generation of skeletal muscle tissues are currently 13 limited by an insufficient supply of human myoblasts. To this end, recent research into 14 myoblast expansion protocols [19] and the development of human pluripotent stem cell 15 myoblast differentiation protocols [2, 18] will be critical in facilitating large scale hµM 16 research. Our study provides proof-of-concept evidence that high-throughput studies can be 17 performed in bioengineered skeletal muscle, thus enabling future high-throughput 18 investigations of human muscle physiology, pathophysiology and exercise adaptation. 19

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DATA AVAILABILITY 1 Proteomic data-sets generated in this manuscript will be uploaded to PRIDE and made 2 publicly available. 3 4 ACKNOWLEDGEMENTS 5 We used the Australian National Fabrication Facility Queensland Node for the fabrication of 6 the micro-muscle platform molds. The authors acknowledge the use of the Australian 7 Microscopy & Microanalysis Research Facility at the Center for Microscopy and 8 Microanalysis at The University of Queensland. B.L.P., D.E.J., E.R.P., R.G.P. and J.E.H. are 9 supported by Fellowships and Project Grants from the National Health and Medical Research 10 Council, the National Heart Foundation, Stem Cells Australia, The University of Queensland 11 or QIMR Berghofer Medical Research Institute. The Murdoch Children’s Research Institute 12 is supported by the Victorian Government's Operational Infrastructure Support Program. The 13 contents of the published material are solely the responsibility of the authors and do not 14 reflect the views of the funding bodies. 15 16

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