The transcriptomic signature of age and sex is not conserved in human primary myotubes.

Principal component analysis showed that tissue type (biopsy vs. myotube) was the dominant source of transcriptomic variation, separating samples along PC1.

• Biopsies were collected from the vastus lateralis of 10 young males (18-30), 10 young females (18-30), and 10 older males (60-75).
• PCA clearly separated muscle tissue samples from their corresponding differentiated HPMC lines along the first principal component.
• Age and sex groupings were not distinguishable within the HPMC samples in PCA space, unlike in the tissue samples.

Differential gene expression between young males and young females in muscle biopsies was largely not conserved in their corresponding HPMC lines.

• A large number of genes were differentially expressed by sex in muscle tissue biopsies.
• The overlap between differentially expressed genes in vivo and in vitro was minimal, indicating near-complete loss of sex-specific transcriptomic signatures in culture.
• The exception was several Y-chromosome encoded genes, which remained differentially expressed in male vs. female HPMC lines.

Differential gene expression between young males and older males in muscle biopsies was largely not conserved in their corresponding HPMC lines.

• Age-associated gene expression differences identified in tissue biopsies (young males aged 18-30 vs. older males aged 60-75) were not retained in differentiated HPMCs.
• Pathway enrichment analysis confirmed that age-related biological pathways active in vivo were not reflected in the in vitro transcriptome.
• This indicates a near-complete lack of retention of age-specific transcriptomic signatures in culture.

Y-chromosome encoded genes were the notable exception, retaining differential expression between male and female HPMC lines.

• Several genes encoded on the Y-chromosome remained differentially expressed between male and female HPMCs in vitro.
• This finding was consistent with the retention of the donors' genome in culture regardless of the cellular milieu.
• All other sex-specific gene expression differences were lost in the in vitro environment.

Single-cell deconvolution analysis indicated that the diversity of resident cell populations in muscle tissue contributes to the transcriptomic differences between biopsies and HPMCs.

• Muscle tissue biopsies contained a diverse mix of cell types including satellite cells, immune cells, endothelial cells, and other non-myogenic cells.
• Differentiated HPMC lines are predominantly composed of myogenic cells, lacking this cellular diversity.
• The distinct cellular composition of tissue versus culture was identified as a major contributor to the transcriptomic divergence.

The absence of sex- and age-dependent circulating factors in the in vitro cellular milieu is proposed as a contributing factor to the loss of in vivo transcriptomic signatures.

• HPMCs retain their genome in culture, but many endogenous circulating factors are not present in the in vitro environment at physiological concentrations.
• Sex hormones and other age-related circulating factors that influence gene expression in vivo are absent from standard cell culture conditions.
• This loss of circulating cues likely contributes to the failure to maintain sex- and age-specific gene expression patterns in cultured myotubes.

The study calls for caution when using differentiated HPMCs as an experimental model of human muscle sex or age.

• The near-complete lack of retention of in vivo transcriptomic signatures for both sex and age was observed across all 30 donor-matched biopsy and HPMC pairs.
• Pathway enrichment analysis confirmed that biologically relevant sex- and age-associated pathways identified in tissue were not recapitulated in HPMCs.
• The authors conclude that differentiated HPMCs are not valid models of age and sex-specificity in human muscle research without additional experimental modifications.