BackgroundMesenchymal stromal cells (MSCs) hold promise for cell-based therapies due to their ability to stimulate tissue repair and modulate immune responses. Umbilical cord-derived MSCs from Wharton jelly (WJ) offer advantages such as low immunogenicity and potent immune modulatory effects. However, ensuring consistent quality and safety throughout their manufacturing process remains critical. RNA sequencing (RNA-seq) emerges as a crucial tool for assessing genetic stability and expression dynamics in cell-based therapeutic products. MethodsWe examined the secretome and transcriptome of WJ-MSC signatures throughout Good Manufacturing Practice (GMP) production, focusing on the performance of total RNA or Massive Analysis of cDNA Ends (MACE) sequencing. ResultsThrough extensive transcriptomic analysis, we demonstrated consistent stability of WJ-MSC expression signatures across different manufacturing stages. Notably, MACE-seq showed improved identification of key expression patterns related to senescence and immunomodulation. ConclusionsThese findings highlight the potential of MACE-seq as a quality assessment tool for WJ-MSC-based therapies, ensuring their efficacy and safety in clinical applications. Importantly, MACE-seq demonstrated its value in characterizing WJ-MSC-derived products, offering insights that traditional assays cannot provide.