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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. 

Abstract Preclinical animal studies are essential to the development of safe and effective stem cell therapies. Bioluminescence imaging (BLI) is a powerful tool in animal studies that enables the real‐time longitudinal monitoring of stem cells in vivo to elucidate their regenerative properties. This review describes the application of BLI in preclinical stem cell research to address critical challenges in producing successful stem cell therapeutics. These challenges include stem cell survival, proliferation, homing, stress response, and differentiation. The applications presented here utilize bioluminescence to investigate a variety of stem and progenitor cells in several different in vivo models of disease and implantation. An overview of luciferase reporters is provided, along with the advantages and disadvantages of BLI. Additionally, BLI is compared to other preclinical imaging modalities and potential future applications of this technology are discussed in emerging areas of stem cell research. 

Abstract In vivo mesenchymal stem cell (MSC) survival is relevant to therapeutic applications requiring engraftment and potentially to nonengraftment applications as well. MSCs are a mixture of progenitors at different stages of cellular aging, but the contribution of this heterogeneity to the survival of MSC implants is unknown. Here, we employ a biomarker of cellular aging, the decoy TRAIL receptor CD264, to compare the survival kinetics of two cell populations in human bone marrow MSC (hBM‐MSC) cultures. Sorted CD264⁺hBM‐MSCs from two age‐matched donors have elevated β‐galactosidase activity, decreased differentiation potential and form in vitro colonies inefficiently relative to CD264⁻hBM‐MSCs. Counterintuitive to their aging phenotype, CD264⁺hBM‐MSCs exhibited comparable survival to matched CD264⁻hBM‐MSCs from the same culture during in vitro colony formation and in vivo when implanted ectopically in immunodeficient NIH III mice. In vitro and in vivo survival of these two cell populations were independent of colony‐forming efficiency. These findings have ramifications for the preparation of hBM‐MSC therapies given the prevalence of aging CD264⁺cells in hBM‐MSC cultures and the popularity of colony‐forming efficiency as a quality control metric in preclinical and clinical studies with MSCs. 

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.