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Title:Materials and biological approach to gene delivery in human embryonic stem cells
Author(s):Yen, Jonathan
Advisor(s):Cheng, Jianjun; Wang, Fei
Department / Program:Bioengineering
Discipline:Bioengineering
Degree Granting Institution:University of Illinois at Urbana-Champaign
Degree:M.S.
Genre:Thesis
Subject(s):Stem Cells
gene delivery
human embryonic stem cells
pluripotent stem cells
peptides
helical peptides
fibroblasts
small molecule
Abstract:Gene delivery is an important tool to study and manipulate human pluripotent stem cells for regenerative medicine purposes. Yet current methods of transient gene delivery are still highly inefficient. Using materials and biologically based concepts, we aim to develop new methods and protocols to enhance the efficiency of gene delivery to be useful. For the materials aspect, diblock copolymers consisting of poly(ethylene glycol)-block-poly(γ-4-(((2-(piperidin-1-yl)ethyl)amino)methyl)benzyl-L-glutamate) (PEG-b-PVBLG-8) were synthesized and evaluated for their ability to mediate gene delivery in hard-to-transfect cells like IMR-90 human fetal lung fibroblasts and human embryonic stem cells (hESCs). The PEG-b-PVBLG-8 contained a membrane-disruptive, cationic, helical polypeptide block (PVBLG-8) for complexing with DNA and a hydrophilic PEG block to improve the biocompatibility of the gene delivery vehicle. PEG-b-PVBLG-8 diblock polymers with a high degree of polymerization have a greater transfection efficiency and lower toxicity in IMR-90 cells than the commercial reagent Lipofectamine 2000. The usefulness of PEG-b-PVBLG-8 was further demonstrated via the successful transfection of hESCs without a measured loss in cell pluripotency markers. From the biological aspect, a small molecule that selectively inhibits the Rho-associated kinase inhibitor (Y-27632) was discovered that transiently alters the hESC morphology to induce spreading and reduced membrane tension. These morphological changes allowed the increase of plasmid transfection, siRNA transfection and nanoparticle uptake to increase substantially. Treating the cells with Y-27632 and passaging them as single cells, we were able to obtain a transfection efficiency of ~90% in hESCs. Cells were also able to recover after treatment back to normal pluripotent stem cell morphology and express important pluripotency markers.
Issue Date:2013-05-28
URI:http://hdl.handle.net/2142/44809
Rights Information:Copyright 2013 Jonathan Yen
Date Available in IDEALS:2013-05-28
2015-05-28
Date Deposited:2013-05


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