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Derivation of Functional Endothelial Cells from Human Vascular Smooth Muscle Cells through Reprogramming

Student thesis: Doctoral ThesisDoctor of Philosophy

Endothelial damage and dysfunction are implicated in cardiovascular pathological changes and the development of vascular diseases. In view of the fact that the spontaneous endothelial cell (EC) regeneration is a slow and insufficient process, it would be of great significance to explore alternative cell sources capable of generating functional ECs to repair damaged endothelium. Indeed, recent achievements of cell reprogramming to convert somatic cells directly to other cell types provide new powerful approaches to study endothelial regeneration. Endothelial damage is often followed by smooth muscle cell (SMC) proliferation and accumulation. If we could arbitrarily convert SMC into EC, a novel cell regeneration strategy may be achieved. The aim of the present study is to test the hypothesis whether functional ECs could be derived from human vascular SMCs through reprogramming.

Based on a combined protocol of reprogramming and differentiation, human vascular SMCs were first reprogrammed for 4 days to achieve a partially converted state expressing vascular progenitor marker CD34, by introducing four transcription factors OCT4, SOX2, KLF4 and c-MYC. Partially reprogrammed SMCs were then subjected to defined media and culture conditions to induce the differentiation towards an endothelial lineage. The differentiated cells expressed EC markers at the mRNA and protein level. Next, CD34-positive cells were selected from the heterogeneous population of vascular progenitors and further maintained in endothelial-promoting conditions. The CD34-positive cells were able to give rise to a more homogenous endothelial population with a repertoire of endothelial characteristics. These SMC-converted ECs displayed typical endothelial functional properties including Nitric Oxide (NO) production, acetylated-low density lipoprotein (LDL) uptake and angiogenic ability in vitro and in vivo. More importantly, these human SMC-derived ECs showed therapeutic angiogenic capacity that improved blood flow recovery when applied in a murine hindlimb ischaemia model as well as the ability to assemble into endothelium-like layer in tissue engineered vascular graft. Furthermore, the mechanisms involved in SMC to EC conversion were explored. Comprehensive analyses indicated that mesenchymal-to- epithelial transition was required to initiate the conversion from SMCs into vascular progenitors. In addition, components of the Notch signalling pathway, hairy and enhancer of split 5 (HES5) and Jagged1 (JAG1), regulated the differentiation of vascular progenitors towards an endothelial lineage.

Together, we provide the first evidence of the conversion of human SMC towards functional endothelial lineage through mechanisms involving mesenchymal-to-epithelial transition and the Notch signalling pathway.

Original languageEnglish
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Award date2016

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