Directed Differentiation of Embryonic Stem Cells Into Cardiomyocytes by Bacterial Injection of Defined Transcription Factors

Forced expression of defined transcriptional factors has been well documented as an effective method for cellular reprogramming or directed differentiation. However, transgene expression is not amenable for therapeutic application due to potential insertional mutagenesis. Here, we have developed a bacterial type III secretion system (T3SS)-based protein delivery tool and shown its application in directing pluripotent stem cell differentiation by a controlled delivery of transcription factors relevant to early heart development. By fusing to an N-terminal secretion sequence for T3SSdependent injection, three transcriptional factors, namely Gata4, Mef2c, and Tbx5 (abbreviated as GMT), were translocated into murine embryonic stem cells (ESCs), where the proteins are effectively targeted to the nucleus with an average intracellular half-life of 5.5 hours. Exogenous GMT protein injection activated the cardiac program, and multiple rounds of GMT protein delivery significantly improved the efficiency of ESC differentiation into cardiomyocytes. Combination of T3SS-mediated GMT delivery and Activin A treatment showed an additive effect, resulting in on average 60% of the ESCs differentiated into cardiomyocytes. ESC derived cardiomyocytes displayed spontaneous rhythmic contractile movement as well as normal hormonal responses. This work serves as a foundation for the bacterial delivery of multiple transcription factors to direct cell fate without jeopardizing genomic integrity.