SIX1+PAX3+ identify a progenitor for myogenic lineage commitment from hPSCs

Area of Science:

• Developmental biology
• Stem cell biology
• Molecular genetics

Background:

• Skeletal muscle progenitor cells (SMPCs) derived from human pluripotent stem cells (hPSCs) are challenging to identify early.
• Defining early transcriptional checkpoints is crucial for efficient hPSC differentiation into skeletal muscle.

Purpose of the Study:

• To identify early markers for myogenic commitment in hPSC differentiation.
• To investigate the role of SIX1 in skeletal muscle development.
• To establish methods for improving hPSC-derived skeletal muscle generation.

Main Methods:

• Analysis of myogenic factors in human embryos and hPSC differentiations.
• CRISPR interference (dCas9-KRAB) to inhibit SIX1 expression in hPSCs.
• RNA sequencing (RNA-seq) to compare SIX1 expression across developmental stages.
• Optimization of differentiation conditions (seeding density, metabolic monitoring, CHIR99021 concentration).

Main Results:

• SIX1+PAX3+ co-expression was identified as a critical indicator of myogenesis.
• Early SIX1 inhibition reduced PAX3 expression, PAX7+ SMPCs, and subsequent myotube formation.
• Optimized differentiation conditions improved the emergence of SIX1+PAX3+ precursors and co-emergence of non-myogenic lineages.
• SIX1 expression is conserved across human development, but its co-factor expression is developmentally regulated.

Conclusions:

• SIX1 plays a crucial role in early myogenic commitment during hPSC differentiation.
• SIX1+PAX3+ co-expression serves as a reliable marker for skeletal muscle development.
• Optimized protocols enhance the efficiency of generating skeletal muscle from hPSCs, providing a valuable resource.