  Citation: Ruan, T.; Harney, D.; Koay, Y.C.; Loo, L.; Larance, M.; Caron, L. Anabolic Factors and Myokines Improve Differentiation of Human Embryonic Stem Cell Derived Skeletal Muscle Cells. Cells 2022, 11, 963. https://doi.org/10.3390/ cells11060963 Academic Editor: Valentina Di Felice Received: 7 February 2022 Accepted: 9 March 2022 Published: 11 March 2022 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- iations. Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). cells Article Anabolic Factors and Myokines Improve Differentiation of Human Embryonic Stem Cell Derived Skeletal Muscle Cells Travis Ruan 1 , Dylan Harney 2 , Yen Chin Koay 3 , Lipin Loo 1 , Mark Larance 2 and Leslie Caron 1,4, * 1 Dr. John and Anne Chong Lab for Functional Genomics, Charles Perkins Centre, School of Life and Environmental Sciences, The University of Sydney, Sydney, NSW 2006, Australia; trua9904@uni.sydney.edu.au (T.R.); lipin.loo@sydney.edu.au (L.L.) 2 Larance Laboratory, Charles Perkins Centre, School of Life and Environmental Sciences, The University of Sydney, Sydney, NSW 2006, Australia; dylan.harney@sydney.edu.au (D.H.); mark.larance@sydney.edu.au (M.L.) 3 Cardiometabolic Disease Group, Heart Research Institute, Charles Perkins Centre, School of Life and Environmental Sciences, The University of Sydney, Sydney, NSW 2006, Australia; yen.koay@hri.org.au 4 MMG, Marseille Medical Genetics, Aix Marseille Univ, INSERM U1251, 13005 Marseille, France * Correspondence: leslie.caron@univ-amu.fr Abstract: Skeletal muscle weakness is linked to many adverse health outcomes. Current research to identify new drugs has often been inconclusive due to lack of adequate cellular models. We previously developed a scalable monolayer system to differentiate human embryonic stem cells (hESCs) into mature skeletal muscle cells (SkMCs) within 26 days without cell sorting or genetic manipulation. Here, building on our previous work, we show that differentiation and fusion of myotubes can be further enhanced using the anabolic factors testosterone (T) and follistatin (F) in combination with a cocktail of myokines (C). Importantly, combined TFC treatment significantly enhanced both the hESC-SkMC fusion index and the expression levels of various skeletal muscle markers, including the motor protein myosin heavy chain (MyHC). Transcriptomic and proteomic analysis revealed oxidative phosphorylation as the most up-regulated pathway, and a significantly higher level of ATP and increased mitochondrial mass were also observed in TFC-treated hESC-SkMCs, suggesting enhanced energy metabolism is coupled with improved muscle differentiation. This cellular model will be a powerful tool for studying in vitro myogenesis and for drug discovery pertaining to further enhancing muscle development or treating muscle diseases. Keywords: skeletal muscle; myotubes; myokines; human embryonic stem cell; myosin heavy chain 1. Introduction Skeletal muscle is the most abundant tissue in the human body, making up around 40% of total body weight. Skeletal muscle is essential for movement and metabolic health, and diseases of muscle function can arise due to genetic mutations; metabolic or neuromuscular dysfunctions; or natural aging [1,2]. Skeletal muscle disorders are linked to many adverse health outcomes, such as impaired mobility, falls, fractures, frailty, diminished quality of life and premature death [3]; and research to identify new drugs has often been inconclusive due to lack of adequate skeletal muscle models. Due to inter-species differences, animal models and rodent cell lines (e.g., C2C12 myoblasts, L6) do not accurately reflect all aspects of human muscle development [4,5]. Primary myoblasts obtained from patients’ biopsies have often been used for research but are limited in number, phenotypically diverse and have poor expandability, restricting their utility. Human pluripotent stem cells (hPSCs), on the other hand, offer a major advantage for studying human skeletal muscle development. Their capacity to proliferate indefinitely and differentiate into most cell types of the human body make them an excellent and renewable source of human skeletal muscle cells (SkMCs). In recent years, human Cells 2022, 11, 963. https://doi.org/10.3390/cells11060963 https://www.mdpi.com/journal/cells