Cardiosphere-derived cells (CDCs) are in clinical development as a regenerative cell product which can be expanded ex vivo from patient cardiac biopsies. Cardiosphere-derived cells are clonogenic, exhibit multilineage differentiation, and exert functional benefits in preclinical models of heart failure. The origin of CDCs remains unclear: are these cells endogenous to the heart, or do they arise from cells that populate the heart via blood-borne seeding? METHODS AND RESULTS: Right ventricular endomyocardial biopsies were obtained from cardiac transplant recipients (n = 10, age 57 +/- 15 years), and CDCs expanded from each biopsy. Donor-recipient mismatches were used to probe the origin of CDCs in three complementary ways. First, DNA analysis of short-tandem nucleotide repeats (STRs) was performed on genomic DNA from donor and recipient, then compared with the STR pattern of CDCs. Second, in two cases where the donor was male and the recipient female, CDCs were examined for the presence of X and Y chromosomes by fluorescence in situ hybridization. Finally, in two cases, quantitative PCR (qPCR) was performed for individual-specific polymorphisms of a major histocompatability locus to quantify the contribution of recipient cells to CDCs. In no case was recipient DNA detectable in the CDCs by STR analysis. In the two cases in which a female patient had received a male heart, all CDCs examined had an X and Y chromosome, similarly indicating exclusively donor origin. Likewise, qPCR on CDCs did not detect any recipient DNA. CONCLUSION: Cardiosphere-derived cells are of endogenous cardiac origin, with no detectable contribution from extra-cardiac seeding.