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. Single-cell RNA-Seq profiling (scRNA-Seq) was performed to determine consequences of culture expansion upon adult cardiac progenitor cells (CPCs) as well as relationships with other cell populations. Bioinformatic analyses demonstrate 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.