Accumulating Transcriptome Drift Precedes Cell Aging in Human Umbilical Cord‐Derived Mesenchymal Stromal Cells Serially Cultured to Replicative Senescence

In preclinical studies, mesenchymal stromal cells (MSCs) exhibit robust potential for numerous applications. To capitalize on these benefits, cell manufacturing and delivery protocols have been scaled up to facilitate clinical trials without adequately addressing the impact of these processes on cell utility nor inevitable regulatory requirements for consistency. Growing evidence indicates that culture‐aged MSCs, expanded to the limits of replicative exhaustion to generate human doses, are not equivalent to early passage cells, and their use may underpin reportedly underwhelming or inconsistent clinical outcomes. Here, we sought to define the maximum expansion boundaries for human umbilical cord‐derived MSCs, cultured in chemically defined xeno‐ and serum‐free media, that yield consistent cell batches comparable to early passage cells. Two male and two female donor populations, recovered from cryostorage at mean population doubling level (mPDL) 10, were serially cultivated until replicative exhaustion (senescence). At each passage, growth kinetics, cell morphology, and transcriptome profiles were analyzed. All MSC populations displayed comparable growth trajectories through passage 9 (P9; mPDL 45) and variably approached senescence after P10 (mPDL 49). Transcription profiles of 14,500 human genes, generated by microarray, revealed a nonlinear evolution of culture‐adapted MSCs. Significant expression changes occurred only after P5 (mPDL 27) and accumulated rapidly after P9 (mPDL 45), preceding other cell aging metrics. We report that cryobanked umbilical cord‐derived MSCs can be reliably expanded to clinical human doses by P4 (mPDL 23), before significant transcriptome drift, and thus represent a mesenchymal cell source suited for clinical translation of cellular therapies. Stem Cells Translational Medicine 2019;8:945&958 Cryopreserved umbilical cord mesenchymal cells from four donors were serially cultured from passage (P) 2 to senescence in chemically defined media. Morphology, growth kinetics, and transcriptome profiles were evaluated at each passage. Transcriptome drift from P2 accumulates over time, although significantly differentially expressed genes (decreased, blue and increased, red) are not detected before P6. Significant drift occurs before cell aging is evident and may contribute to variable clinical outcomes for cell treatments that employ mid‐ to late‐passage cells.