Introduction: Heart disease is the leading cause of death worldwide. Stem cell therapeutics have the potential to benefit patients with heart disease by providing a means to regenerate damaged tissue. Stem cells expressing islet-1 differentiate into all major cell types of the heart and represent a promising candidate cell type which can be cloned and expanded for cardiovascular repair. Interestingly, cardiovascular stem cells (CSCs) isolated from neonates have a unique transcriptome that facilitates regeneration shortly after birth. We isolated human neonatal Islet-1+ CSC clones and showed that these cells express Prostaglandin synthases (PTGSs), Prostaglandin E2 (PGE2) Receptor 4 (EP4), YAP1, and Agrin. These factors promote cardiac repair via their respective anti-fibrotic, angiogenic, pro proliferative, and immunomodulatory functions. The objective of the current study is to determine whether these transcripts are induced in vivo when neonatal islet-1+ CSCs are administered for cardiac repair and to test the hypothesis that PGE2 can induce YAP1 and Agrin through EP4 in neonatal Islet-1+ CSCs.

Methods: A myocardial infarction was introduced in four 1-2 year old Western sheep via ligation of the left anterior descending coronary artery. Sheep were injected 3-4 weeks later with 10 million allogeneic neonatal islet-1+ CSCs in the peri-infarct area. Functional recovery and significant retention of implanted CSCs was shown 2 months later. Hearts were extracted for analysis of the transcriptomic changes identified in the non-infarcted region, the regenerative zone, and fibrotic zone of the infarcted, stem cell treated hearts. RNA was extracted from the three regions and RTqPCR was conducted to identify PTGSs, EP4, YAP1 and Agrin. Validation of PGE2-mediated activation of neonatal Islet-1+ CSCs was assessed in vitro via addition of 1uM PGE2 to cloned human neonatal Islet-1+ CSCs. EP4 was blocked in PGE2 treated cells using a competitive antagonist L-161,982. Protein and RNA was collected, and the proliferation rate was determined via WST assay. Transcripts expressed in neonatal Islet-1+ CSC clones were identified by RNAseq.

Results: RTqPCR analysis of transcripts elevated in the cardiac repair zone demonstrated that YAP1, PTGS1, EP4, and Agrin were elevated between 1.5-10-fold. In vitro treatment of neonatal Islet-1+ CSC clones with PGE2 followed by RTqPCR showed that PGE2 induced YAP1, EP4, and Agrin by 1.5-6 fold. EP4 antagonism blocked this induction. RNAseq analysis showed that clones with high expression of PGE2 receptors and PTGSs expressed higher levels of stemness and proliferation markers. 1uM PGE2 treatment induced stemness markers and increased the rate of proliferation ~1.4 fold.

Conclusion: This study demonstrated that PGE2 can induce YAP1 and Agrin through EP4 in neonatal Islet-1+ CSCs. This novel pathway identifies a mechanistic basis by which neonatal CSCs respond to PGE2 and exhibit a therapeutic advantage in stem cell based cardiac repair.