An Intermediate Mesoderm Premyogenic Niche Supports Early Human Myogenic Lineage Progression

. 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.