RNA sequencing identifies MAP1A and PTTG1 as predictive genes of aging CD264+ human mesenchymal stem cells at early passage
Molecular profiles of mesenchymal stem cells (MSCs) are critically 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. 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. CD264+ cells accounted for 15% to 60% of the cells in MSC cultures and exhibited an aging phenotype. 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 adjusted p-value (BH padj) < 0.1. Nearly 25% of these genes were unique to CD264-/+MSCs and not differentially expressed at a significance level of BH padj < 0.1 in previous RNA sequencing studies of early- vs. late-passage MSCs. Kyoto Encylopedia of Genes and Genomes pathways for DNA replication, cell cycle, spliceosome, RNA transport and ribosome biogenesis were downregulated in CD264+ MSCs and extracellular matrix-receptor interactions was upregulated (BH padj < 0.1). Pathway results were confirmed by Database for Annotation, Visualization, and Integrated Discovery gene set enrichment analysis. Least Absolute Shrinkage and Selection Operator regression was performed on the most significant genes and pathways and identified microtubule-associated protein 1A (MAP1A) and pituitary tumor-transforming gene 1 (PTTG1) as signature genes of CD264+ MSCs. For both the sequencing and validation sets, the combination of MAP1A and PTTG1 expression clustered MSC samples into distinct groups that had the correct CD264 classification with an accuracy of 100% (p = 0.01). This study provides the first linkage of MAP1A upregulation to aging, CD264, and stem cells, and confirms previous reports of PTTG1 downregulation in aging MSCs. Our findings have application as quality metrics to standardize the composition of MSC therapies and as molecular targets to slow/reverse cellular aging. Overall design: The molecular profile of aging mesenchymal stem cells (MSCs) was investigated using subconfluent cultures of human bone marrow MSCs at passage 4 from 5 donors. Fluorescence-activated cell sorting was used to isolate passage 4 MSCs based on their expression of CD264, a cell surface marker of aging. PolyA RNA samples were isolated from donor-matched CD264- and CD264+ MSCs from 5 donors. Pair-ended RNA sequencing and pairwise differential expression analysis of the donor-matched CD264-/+ MSCs samples identified predictive genes of the aging CD264+ phenotype. Predictive genes were validated with donor-matched CD264-/+ MSCs samples from an independent set of 5 donors.