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  1. Evaluation Summary:

    This manuscript describes pioneering work providing detailed description of iPS-derived muscle fiber differentiation in culture. It demonstrates that muscle fibers show self-organising capacities in vitro and form bundles with identified attachment points; this self-organisation generates internal tension within myofibers. Overall, this study suggests that tension drives sarcomerogenesis in multi fibrillar vertebrate muscles and will be of interest to researchers in the muscle field and also biophysicists interested in collective cell behaviour.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)

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  2. Reviewer #1 (Public Review):

    Starting with iPSCs the authors build a 2D cell culture system and observe the gradual process of muscle differentiation in order to view myofiber formation in real time. Authors perform a detailed study looking at the time course of muscle development both in terms of expression of markers in real time as well as transcriptomic analysis. During their descriptive studies the authors note general rules of myofiber formation, myotube differentiation follows the emergence of stable attachment of foci as well as a seemingly coordinated strong induction of sarcomeric genes. These rules are consistent with a uniform cue for myofiber formation which they hypothesize to be tension. They test this hypothesis using laser microsurgery and explore the hypothesis that force-resistant integrin-based fiber-fiber attachments stabilise the myofibers and serve as a mechanism to explain their other observations.

    Overall this is an important pioneering study. It stands on it's own but opens up an exciting avenue of research. The ability to view the entire process of myofiber and sarcomere formation in real time allows tissue engineering approaches to be used including varying the matrix (they currently use minimal culture conditions), varying the mechanical environment (using stretchable tissue media), and performing knockout screens to understand the process better. Moreover, since the authors start with iPSC this technique could be used to generate muscle cultures from patients to provide clinical insight.

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  3. Reviewer #2 (Public Review):

    The article from Q Mao et al describes that muscle cells derived from iPSC differentiate into myofibers bundles that attach to each other at their ends in an integrin-dependent manner. To do so, the cells progressively arrange themselves together to form domains of alignment. The authors also observe that formation of sarcomeres creates tension forces which are in turn needed for building additional sarcomeres, ending in the stabilization of the myofibrils. Those results bring light into the collective behavior effect of muscle cells on their maturation.

    The study will interest both researchers in the muscle field but also biophysicists interested in collective cell behavior.

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  4. Reviewer #3 (Public Review):

    The manuscript by Mao et al., applies previously described protocol of in vitro muscle differentiation from iPS-derived human myogenic progenitor cells to test whether tension is required for the formation of sarcomeric pattern in multi fibrillar context of human muscle.

    Authors elegantly describe and document, step-by-step, in vitro muscle differentiation from myotubes to mature myofibers organized in bundles. They apply quantitative and bioinformatics-assisted methods that clearly demonstrate that this process is biphasic with the formation of self-organising bundles of myofibers whose number and area increase rapidly until day 7 and then remain stable until day 15. Interestingly, this correlates with the transcriptional burst of several genes encoding sarcomeric components and the appearance of more and more regular striated patterns of titin, actin and MyHC along the myofibers. Strikingly, bundles of myofibers tend to cluster their ends in a way to form foci in which myofibers from adjacent bundles attach to each other. Authors suggest that this organisation and attachment between myofibers allows generation of tension, which increases during differentiation and is necessary for the formation of sarcomeres. This view is supported by an increased level of expression of actin, integrin, myosin and N-term titin in mature myofibers but also by micro-lesion experiments that allow measuring tension. Overall this is well executed and documented with innovative methods to study uncovering capacities of myofibers to self-organise in vitro and providing new standards for analysing iPS-derived muscle differentiation.

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