Heart disease remains the leading cause of death worldwide. By understanding the regulating networks during cardiac development we can exploit those networks to manipulate adult cells into cardiac progenitors and provide an alternative for repairing diseased hearts. Mesp1 is considered to have critical roles during cardiac development but the molecular mechanisms need to be further studied. The roles of ETS transcription factors have been primarily limited to hematopoietic differentiation and cancer progression. The ETS transcription factors are known to have proliferating roles and were hypothesized to also be involved in cardiac differentiation and may potentially be used for cell reprogramming.
The first part of this study characterizes the expression pattern of Mesp1 protein in early mouse embryo from E6.5 to E9.5 and provides a full expression profile in differentiating embryoid bodies in vitro from the undifferentiated stage to Day10. Our work showed Mesp1 expresses in the posterior region of E6.5 embryo then starts migrating through the primitive streak toward anterior mesoderm and endoderm in E7.5. A Mesp1 linage tracing ES cell line was established, and it allowed us to trace the Mesp1 derived cell population. The lineage tracing system confirmed Mesp1 expressing cells give rise to a major part of the heart and also contributes to some endodermal derived organs such as the pancreas. The direct DNA binding targets of Mesp1 were determined using a Mesp1 specific antibody to perform ChIP of bound DNA that could then be used in next generation sequencing. The resulting sequence data included cardiac genes such as Gata4, Hand2, and Myocd. Endoderm correlated genes Foxa2, Pitx2, and Gata6 were also shown to be Mesp1 targets. The targeted genes were validated as transcriptional targets using an ES cell line with inducible Mesp1 followed by qPCR of target gene transcript levels with and without Mesp1 expression. Secondly, the complete gene expression profiles of over 20 ETS transcription factors were generated. By comparing the ETS factor expression patterns and identifying which showed cardiac gene activation, ETV4 and ETS2 were chosen for further study of their roles in cardiac differentiation. ETS2 was used in combination with Mesp1 to reprogram Normal Human Dermal Fibroblast into cardiac progenitors. The reprogrammed cells were then characterized for gene expression patterns, surface marker, and structural protein presentation. This work provides thorough insights into the roles of Mesp1 and ETS transcription factors during germ layer development and led to the development of a method to reprogram adult cells into cardiac progenitors that could be applied for clinical use in the future
