200 Dedifferentiated or Reborn Again? Elucidating The Chromatin Remodelling Mechanisms During Endothelial Cell Reprogramming for Cardiovascular Therapy. (3rd June 2016) - Caines, Rachel
Abstract : The vascular endothelium is central to cardiovascular homeostasis. Repair and regeneration of endothelial cells (ECs) has been an important research focus for a number of years. The recent ability to derive ECs through cell reprogramming has opened new avenues. Reprogramming somatic cells to ECs is in its infancy, but the road ahead looks very promising. A new reprogramming strategy has ruled out safety issues concerned with teratoma formation. Cells exposed to reprogramming factors for 4 days become epigenetically primed and have been defined as Partially induced Pluripotent Stem (PiPS) cells. They do not transverse pluripotency, and so do not form tumours. They have shown the ability to be differentiated into ECs by culture conditions. Efficiency of reprogramming has increased from 0.01% to 30–40% in the case of PiPS-ECs, but there is scope for improvement as the underlying mechanisms are still unclear. The role of epigenetics in reprogramming has come to the forefront recently and the ability to generate a homogenous and functional EC population will be best sought through chromatin remodelling mechanisms. A protein found to be crucial in 4-day reprogramming was SETSIP, or SET similar protein. SETSIP has high sequence homology to SET with an additional 10 amino acids at the N-terminus. SET plays roles in chromatin remodelling as a transcriptional regulator and roles in differentiation, apoptosis and cell cycle progression. The study aims to elucidate a robust and efficient protocol for the production of a homogenous and functional EC population for use in personalised cardiovascular medicine. SETSIP has been overexpressed and knocked out of early PiPS-ECs and iPS-ECs to observe the effect on EC reprogramming. Luciferase assays have been undertaken to understand the EC specific pathways regulated by SETSIP. Experiments have also been performed to establish the effect of treatment with VEGF on SETSIP expression and EC differentiation. Future work will involve the employment of CRISPR technology to create a SETSIP deficient cell line to observe the differentiation potential of the cells and phenotype of any derived ECs. SETSIP was found to translocate to the cell nuclei, and capable of regulating expression of important EC markers. The functional consequences of this were assessed in vitro and in vivo where SETSIP was found to be important for the formation of vascular tubules. Furthermore, epigenetic modulators such as CBP/p300 were identified as potential mediators of the gene regulatory effects of SETSIP in ECs. These results represent an important step forward in understanding the process of EC reprogramming for use in regenerative medicine. These findings provide knowledge of the intricate processes during EC reprogramming not only to support the scientific validity of the newly generated ECs but also to ensure the safety of bringing cellular reprogramming to the bedside of cardiovascular patients.