Abstract P347: Transcriptional Features Of Biological Age Maintained In Cultured Cardiac Interstitial Cells

Introduction: Ex vivo expansion of cells is necessary in regenerative medicine to generate large populations for therapeutic use. Adaptation to culture conditions prompt an increase in transcriptome diversity and decreased population heterogeneity in cKit+ cardiac interstitial cells (cCICs). The “transcriptional memory” influenced by cellular origin remains unexplored and is likely to differ between neonatal versus senescent input cells undergoing culture expansion. Approach: cCICs isolated from neonatal and adult cardiac tissue (Left Ventricular Assist Device; LVAD). Single cell libraries from in vitro expanded cells were prepared following five passages as previously demonstrated by our group to promote transcriptional homogeneity. “Transcriptional memory” was surveyed via bioinformatic analysis including unsupervised clustering, differential expression analysis, gene ontology and pathway analysis. Transitional states were assessed through pseudotime analysis. Results: Cell cycle imprint associated with biological age after culture was observed in Neonatal cCICs via upregulation of G2M genes. LVAD derived cCICs retained a widespread senescent profile, in particular high expression of interleukins and elements of the senescence associated secretory phenotype (SASP). Nuclear pore complex TPR and UBC9 and translation initiation factors, displayed age-associated downregulation of elements in the RNA transport and processing pathway. Pathway and co-expression analysis of fibroblast markers Ddr2, Tcf21, Vimentin, Periostin and Collagen deposition markers indicated a primed fibrotic phenotype in senescent cells. A small subset of cCICs exist in a transcriptional continuum between “youthful” phenotype and the damaged microenvironment of adult tissue in which they were embedded. Conclusion: The influence of age, pathology and the cellular stress associated to the in vivo tissue microenvironment persist after culture adaptation, influencing targets of 1) cell cycle, 2) senescence associated secretory phenotype (SASP), 3) RNA transport, and 4) ECM-receptor/fibrosis. The connate transcriptional phenotypes offer fundamental biological insight and highlights cellular input as a consideration in culture expansion and adoptive transfer protocols.