Abstract
Transient cell states that precede and support human myogenic lineage commitment, and the intrinsic and extrinsic signals that control them, remain poorly defined in vitro. Here, we used longitudinal single-nucleus profiling, together with a SIX1:H2B-GFP hPSC reporter for lineage tracing, resolved previously uncaptured transient intermediates and sequential waves of human myogenesis across differentiation and in vivo . We show that hPSC-directed myogenesis gives rise in parallel to paraxial mesoderm and a transient PAX8+ intermediate mesoderm population that forms a 3-dimensional pre-myogenic niche supporting the PAX3-to-PAX7 myogenic progenitor transition. LIANA+ analysis further identified a temporally restricted BMP7-BMPR1B interaction, together with laminin-linked signaling, between PAX8+ niche cells and skeletal muscle progenitors before commitment. We further show that dynamic SIX1 cofactor switching, including EYA3 activity, is required for PAX3-to-PAX7 progression, and that disruption of this program compromises multi-lineage niche integrity. Together, these findings define how transient niche populations and intrinsic regulatory networks coordinate early human myogenic lineage progression and provide a human in vitro platform to study parallel intermediate and paraxial mesoderm development during myogenesis. HIGHLIGHTS: SIX1+PAX8+ niche progenitors promote myogenic differentiation via BMP7- and laminin-dependent signaling.Loss of SIX1-EYA3 activity disrupts the pre-myogenic niche and impairs the PAX3-to-PAX7 transitionMulti-omics single-cell optimal transport resolves previously uncaptured transient intermediates and sequential waves of human myogenesisSIX1 lineage tracing identifies CREB5 as a top regulator of the PAX7+ state in human myogenesis.