Derivation of purified smooth muscle cells from mouse induced pluripotent stem (iPS) cells
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Abstract
Cardiac and vascular disease syndromes and abnormalities have long been the leading causes of death in the United States, but the cause of congenital defects remain unclear due to the lack of appropriate model systems for scientific investigation. Moreover, the predominance of cardiovascular disease has stimulated scientists to focus on developing tissue-engineered blood vessels (TEBV) for vascular reconstruction and replacement. Major challenges remain in generating large amounts of epithelial cells (EC) and vascular smooth muscle cells (VSMC) for vascular reconstruction and in reducing the immunoresponse in patients after replacement. To address both issues of disease model generation and vascular tissue engineering, we can use induced pluripotent stem (iPS) cells discovered by Shinya Yamanaka in 2006: iPS cells generated from adult tissue and organs through the forced expression of two to four transcription factors are phenotypically indistinguishable from embryonic stem (ES) cells. First, by creating iPS from cardiovascular patients, we can generate patient-specific cell lines for scientific research investigation (i.e. elucidate molecular mechanisms and potential drug targets). Second, EC and VSMC derived from patient-specific iPS cell lines can be used for vascular reconstruction with cells of the patient's own genetic background, which should overcome many of the immunological limitations that currently impede vascular replacement (i.e. provide therapeutic interventions).
The goal of this project is to study the differentiation capacity of iPS cells into smooth muscle cells (SMC). This project aims to develop a protocol that can maximize the derivation of purified smooth muscle cells from mouse induced pluripotent stem (iPS) cells through three linear developmental stages: induction of a posterior primitive- streak (PS) like population, formation of Flk1+ mesoderm, and induction of smooth muscle cells. Low dosage of Activin A and Wnt was used to differentiate iPS into a PS-like population, while the administration of BMP4 differentiates the cells to mesoderm via a posterior PS intermediate. Smooth muscle cells were induced from mesoderm by the addition of platelet-derived growth factor (PDGF-BB) and transforming growth factor b (TGF-β). The linear developmental progression from PS formation through mesoderm induction to smooth muscle cells were tracked by RT-qPCR and FACS for the expression of genes indicative of each individual stage, Flk1, and SMαA respectively. The results of this project can be used as a basis for in vitro derivation of purified mammalian smooth muscle cells from a mouse model system that can be further modified. Moreover, the differentiated SMCs can be further used in cell sheet construction as vascular patches for drug testing.
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Thesis (M.S.)--Boston University