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USE OF ENDOTHELIAL-SPECIFIC PROMOTERS TO IDENTIFY AND SELECT DIFFERENTIATING STEM CELLS

Kim, Saejeong

Abstract Details

2009, Doctor of Philosophy, Case Western Reserve University, Biomedical Engineering.
Development of efficient ways to identify and isolate EPCs derived from stem cell sources is in high demand because ECs can be used in many tissue engineering applications and cell therapy. We have chosen to examine endothelial selection strategies using mouse ESCs because of their unlimited proliferation potential. Mouse ESCs were genetically transformed with GFP or a drug resistance gene (PuroR) under the control of different endothelial-lineage specific promoters (Flk1, PECAM, Tie1, and VE-Cadherin). These cells were then differentiated in the presence of VEGF followed by continuous selection with the drug resistance. The differentiated cells were then analyzed for their GFP expression or drug resistance under endothelial promoters. Using GFP as a reporter, we examined differentiation profiles of ESCs selected using three different endothelial promoters (Flk1, PECAM, Tie1) that correspond to endothelial proteins expressed at different time points (early, middle, and late) in ESC differentiation. All three promoters yielded cells with EC-specific protein expression and DiI-Ac-LDL uptake when sorted for GFP+ population; however, Flk1- driven GFP+ cells yielded both SMCs and ECs whereas Tie1- driven GFP+ cells yielded mostly ECs. Both Flk1 and PECAM promoters had significant background GFP expression at an undifferentiated state, making the elimination of undifferentiated cells difficult. Use of GFP:PuroR fusion protein under the endothelial promoters enabled continuous selection of ECs. PuroR -selected cells under all of the EC-promoters were also positive for capillary-like network formation in Matrigel. These cells also showed low, but detectable levels of PGI2 secretion which increased with orbital shear stress exposure. tPA secretion by PuroR-selected cells was comparable to EOMAs, however these cells lacked alignment with direction of flow upon shear stress exposure. The selected ESC-derived ECs were then used to examine angiogenesis participation in in vivo 3D tumor model. ESC-derived ECs were injected into a tail vein of mouse bearing B16 melanoma, then recruitment of endothelial cells by tumor vasculature were assessed by presence of GFP expressing cells or RT-PCR. Tracking of survival and function of the ESC-derived ECs in vivo with Matrigel plug implantation was explored with firefly luciferase, red fluorescent protein, and truncated thymidine kinase under Tie1 promoter.
Horst von Recum, PhD (Advisor)
Roger Marchant, PhD (Committee Member)
Linda Graham, MD (Committee Member)
Melissa Knothe Tate, PhD (Committee Member)
Kathleen Molyneaux, PhD (Committee Member)
230 p.

Recommended Citations

Citations

  • Kim, S. (2009). USE OF ENDOTHELIAL-SPECIFIC PROMOTERS TO IDENTIFY AND SELECT DIFFERENTIATING STEM CELLS [Doctoral dissertation, Case Western Reserve University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=case1237483364

    APA Style (7th edition)

  • Kim, Saejeong. USE OF ENDOTHELIAL-SPECIFIC PROMOTERS TO IDENTIFY AND SELECT DIFFERENTIATING STEM CELLS. 2009. Case Western Reserve University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=case1237483364.

    MLA Style (8th edition)

  • Kim, Saejeong. "USE OF ENDOTHELIAL-SPECIFIC PROMOTERS TO IDENTIFY AND SELECT DIFFERENTIATING STEM CELLS." Doctoral dissertation, Case Western Reserve University, 2009. http://rave.ohiolink.edu/etdc/view?acc_num=case1237483364

    Chicago Manual of Style (17th edition)