Human skeletal muscle organoids model fetal myogenesis and sustain uncommitted PAX7 myogenic progenitors

Reviewer #1 (Public Review):

The authors aimed to establish a cell culture system to investigate muscle tissue development and homeostasis. They successfully developed a complex 3D cell model and conducted a comprehensive molecular and functional characterization. This approach represents a critical initial step towards using human cells, rather than animals, to study muscular disorders in vitro. Although the current protocol is time-consuming and the fetal cell model may not be mature enough to study adult-onset diseases, it nonetheless provides a valuable foundation for future disease modelling studies using isogenic iPSC lines or patient-derived cells with specific mutations. The manuscript does not explore whether or how this stem cell model can advance our understanding of muscular diseases, which would be an exciting avenue for future research. Overall, the detailed protocol presented in this paper will be useful for informing future studies and provides an important resource to the stem cells community. The inclusion of data on disease modelling using isogenic iPSC lines or patient-derived cells would further enhance the manuscript's impact.

Reviewer #2 (Public Review):

This paper illustrates that PSCs can model myogenesis in vitro by mimicking the in vivo development of the somite and dermomyotome. The advantages of this 3D system include (1) better structural distinctions, (2) the persistence of progenitors, and (3) the spatial distribution (e.g. migration, confinement) of progenitors. The finding is important with the implication in disease modeling. Indeed the authors tried DMD model although it suffered the lack of deeper characterization.

The differentiation protocol is based on a current understanding of myogenesis and compelling. They characterized the organoids in depth (e.g. many time points and immunofluorescence). The evidence is solid, and can be improved more by rigorous analyses and descriptions as described below.

Major comments:

1. Consistency between different cell lines.
I see the authors used a few different PSC lines. Since organoid efficiency differ between lines, it is important to note the consistency between lines.

2. Heterogeneity among each organoid
Let's say authors get 10 organoids in one well. Are they similar to each other? Does each organoid possess similar composition of cells? To determine the heterogeneity, the authors could try either FACS or multiple sectioning of each organoid.

3. Consistency of Ach current between organoids.
Related to comment 2, are the currents consistent between each organoid? How many organoids were recorded in the figures? Also, please comment if the current differ between young and aged organoids.

4. Communication between neural cells and muscle?
The authors did scRNAseq, but have not gone deep analysis. I would recommend doing Receptor-ligand mapping and address if neural cells and muscle are interacting.

5. More characterization of DMD organoids.
One of the key applications of muscle organoids is disease model. They have generated DMD muscle organoids, but rarely characterized except for currents. I recommend conducting immunofluorescence of DMA organoids to confirm structure change. Very intriguing to see scRNAseq of DMD organoids and align with disease etiology.

6. More characterization of engraft.
Authors could measure the size of myotube between mice and human. Does PAX7+ Sattelite cell exist in engraft? To exclude cell fusion events make up the observation, I recommend to engraft in GFP+ immunodeficient mice. Could the authors comment how long engraft survive.