Regenerative therapeutic approaches for myocardial diseases often involve delivery of stem cells expanded ex vivo. Prior studies indicate that cell culture conditions affect functional and phenotypic characteristics, but relationship(s) of cultured cells derived from freshly isolated populations and the heterogeneity of the cultured population remain poorly defined. Functional and phenotypic characteristics of ex vivo expanded cells will determine outcomes of interventional treatment for disease, necessitating characterization of the impact that ex vivo expansion has upon isolated stem cell populations. Researchers at San Diego State University performed Single-cell RNA-Seq profiling (scRNA-Seq) to determine consequences of culture expansion upon adult cardiac progenitor cells (CPCs) as well as relationships with other cell populations. Bioinformatic analyses demonstrated that identity marker genes expressed in freshly isolated cells become undetectable in cultured CPCs while low level expression emerges for thousands of other genes. Transcriptional profile of CPCs exhibited greater degree of similarity throughout the cultured population relative to freshly isolated cells. Findings were validated by comparative analyses using scRNA-Seq datasets of various cell types generated by multiple scRNA-Seq technology. Increased transcriptome diversity and decreased population heterogeneity in the cultured cell population may help account for reported outcomes associated with experimental and clinical use of CPCs for treatment of myocardial injury.
Transcriptomic differences between freshly isolated and cultured cells revealed by scRNA-Seq
(a,b) Workflow of scRNA-Seq of CPCs using 10X Genomics (a) or Smart-Seq2 technology (b). Datasets for Smart-Seq2 analyses were derived from open access transcriptome repositories for both fresh or cultured samples (scRNA-Seq datasets, blue cylinder; see supplemental table for detail information) or samples prepared in-house from cultured CPCs (represented as dish). (c,d) t-SNE plots show freshly isolated cells cluster separately from cultured cells using 10X Genomics dataset (1,615 fresh cells and 850 cultured cells; c) or Smart-seq. 2 dataset (576 fresh cells and 550 cultured cells; d). Arrow indicates cultured CPCs in (b,d). CPC, cardiac progenitor cell; BM.MSC, bone marrow-derived mesenchymal stromal cell; MEF, mouse embryonic fibroblast; ESC, embryonic stem cell; iN, induced neuronal cell; HL-1, atrial cardiomyocyte cell line; Fusion, Fusion cell of BM-MSC and HL-1; AV macrophage, atrioventricular node macrophage; E9, E11.5, E14.5, embryo 9, 11.5, 14.5 days, respectively; P7, postnatal 7 days; HSC, hematopoietic stem cell; Two-cell, embryonic cells at two-cell stage; Four-cell, embryonic cells at four-cell stage.