Materials and biological approach to gene delivery in human embryonic stem cells

Yen, Jonathan

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https://hdl.handle.net/2142/44809 Copy

Description

Title

Materials and biological approach to gene delivery in human embryonic stem cells

Author(s)

Yen, Jonathan

Issue Date

2013-05-28T19:20:45Z

Director of Research (if dissertation) or Advisor (if thesis)

• Cheng, Jianjun
• Wang, Fei

Department of Study

Bioengineering

Discipline

Bioengineering

Degree Granting Institution

University of Illinois at Urbana-Champaign

Degree Name

M.S.

Degree Level

Thesis

Keyword(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.

Graduation Semester

2013-05

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http://hdl.handle.net/2142/44809

Copyright and License Information

Copyright 2013 Jonathan Yen

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