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Abstract Molecular profiles of mesenchymal stem cells (MSCs) are needed to standardize the composition and effectiveness of MSC therapeutics. This study employs RNA sequencing to identify genes to be used in concert with CD264 as a molecular profile of aging MSCs at a clinically relevant culture passage. CD264⁻and CD264⁺populations were isolated by fluorescence-activated cell sorting from passage 4 MSC cultures. CD264⁺MSCs exhibited an aging phenotype relative to their CD264⁻counterpart. Donor-matched CD264^−/+mRNA samples from 5 donors were subjected to pair-ended, next-generation sequencing. An independent set of 5 donor MSCs was used to validate differential expression of select genes with quantitative reverse transcription PCR. Pairwise differential expression analysis identified 2,322 downregulated genes and 2,695 upregulated genes in CD264⁺MSCs relative to donor-matched CD264⁻MSCs with a Benjamini–Hochberg adjustedp-value (BHp_adj) < 0.1. Nearly 25% of these genes were unique to CD264^−/+MSCs and not differentially expressed at a significance level of BHp_adj < 0.1 in previous RNA sequencing studies of early- vs. late-passage MSCs. Least Absolute Shrinkage and Selection Operator regression identified microtubule-associated protein 1A (MAP1A) and pituitary tumor-transforming gene 1 (PTTG1) as predictive genes of CD264⁺MSCs. CombinedMAP1AandPTTG1expression correctly classified CD264 status of MSC samples with an accuracy of 100%. Differential expression and predictive ability ofMAP1AandPTTG1compared favorably with that of existing senescence markers expressed in early passage CD264^−/+MSCs. This study provides the first linkage ofMAP1Ato CD264, aging and senescence. Our findings have application as quality metrics to standardize the composition of MSC therapies and as molecular targets to slow/reverse cellular aging. 

Cell-to-cell variation in the regenerative potential of mesenchymal stromal cells (MSCs) impedes the translation of MSC therapies into clinical practice. Cellular heterogeneity is ubiquitous across MSC cultures from different species and tissues. This review highlights advances to elucidate molecular profiles that identify cell subsets with specific regenerative properties in heterogeneous MSC cultures. Cell surface markers and global signatures are presented for proliferation and differentiation potential, as well as immunomodulation and trophic properties. Key knowledge gaps are discussed as potential areas of future research. Molecular profiles of MSC heterogeneity have the potential to enable unprecedented control over the regenerative potential of MSC therapies through the discovery of new molecular targets and as quality attributes to develop robust and reproducible biomanufacturing processes. These advances would have a positive impact on the nascent field of MSC therapeutics by accelerating the development of therapies with more consistent and effective treatment outcomes.