Seamless and non-destructive monitoring of extracellular microRNAs during cardiac differentiation from human pluripotent stem cells

Monitoring cardiac differentiation and maturation from human pluripotent stem cells (hPSCs) and detecting residual undifferentiated hPSCs are indispensable for the development of cardiac regenerative therapy. MicroRNA (miRNA) is secreted from cells into the extracellular space, and its role as a biomarker is attracting attention. Here, we performed an miRNA array analysis of supernatants during the process of cardiac differentiation and maturation from hPSCs. We demonstrated that the quantification of extracellular miR-489-3p and miR-1/133a-3p levels enabled the monitoring of mesoderm and cardiac differentiation, respectively, even in clinical-grade mass culture systems. Moreover, extracellular let-7c-5p levels showed the greatest increase with cardiac maturation during long-term culture. We also verified that residual undifferentiated hPSCs in hPSC-derived cardiomyocytes (hPSC-CMs) were detectable by measuring miR-302b-3p expression, with a detection sensitivity of 0.01%. Collectively, we demonstrate that our method of seamlessly monitoring specific miRNAs secreted into the supernatant is non-destructive and effective for the quality evaluation of hPSC-CMs. [Display omitted] •Extracellular miR-489-3p could be an indicator of mesoderm differentiation•Extracellular miR-1/133a-3p levels correlated with cardiac differentiation efficiency•Extracellular let-7c-5p levels showed a great increase with cardiac maturation•miR-302b-3p was useful for the non-destructive detection of residual hPSCs Monitoring cardiac differentiation and maturation from hPSCs and detecting residual undifferentiated hPSCs are indispensable for the development of cardiac regenerative therapy. Tohyama and colleagues demonstrate that their method of seamlessly monitoring specific miRNAs secreted into the supernatant is non-destructive and effective for the quality evaluation of target cells.