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

Yen, Jonathan

Permalink

https://hdl.handle.net/2142/78586 Copy

Description

Title

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

Author(s)

Yen, Jonathan

Issue Date

2015-03-02

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

Cheng, Jianjun

Doctoral Committee Chair(s)

Cheng, Jianjun

Committee Member(s)

• Wang, Ning
• Underhill, Gregory
• Ma, Jian
• Kilian, Kristopher A.

Department of Study

Bioengineering

Discipline

Bioengineering

Degree Granting Institution

University of Illinois at Urbana-Champaign

Degree Name

Ph.D.

Degree Level

Dissertation

Keyword(s)

• endosomal escape
• hyaluronic acid
• Fibroblasts
• Stem Cells
• Human Pluripotent Stem Cells
• polypeptides
• Human Embryonic Stem Cells
• Gene Delivery

Abstract

Gene delivery is an important tool used in the study and manipulation of human pluripotent stem cells for regenerative medicine purposes. However current methods of transient gene delivery are still highly inefficient. Using materials and biologically based concepts, I aim to develop new methods and protocols to enhance the efficiency of gene delivery. 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, such as 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. This system proved to be inefficient for hESCs, thus I designed a system that uses the combination of a cell specific and materials approach. Plasmid DNA was condensed with PVBLG-8 to form nanocomplexes, which were further coated with hyaluronic acid. PVBLG-8 has proven to be an effective gene delivery material in certain cell lines, due to its membrane disruptive properties. Yet in more sensitive cell lines, like hESCs, it proves to be toxic and thus ineffective. Hyaluronic acid not only shields the positive charges from the helical peptides, but also acts as a targeting moiety for cell surface receptor CD44, which binds and facilitates the internalization of hyaluronan for degradation. Despite the negative charged surface, the gene transfection of the cells increased by 1.5 fold with reduced toxicity. I demonstrated that the increased transfection efficiency is due to the CD44 mediated targeting delivery of DNA by HA coating nanocomplex. In addition, this nanocomplex system can be further activated through the endosomal specific degradation of HA by hyaluronidase to expose PVBLG-8. From the biological aspect, a small molecule that selectively inhibits the Rho-associated kinase inhibitor (Y-27632) was discovered to transiently alter 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. Cells were also able to recover after treatment back to normal pluripotent stem cell morphology and express important pluripotency markers. These new methods expands the field of gene delivery in human pluripotent stem cells, which can be further applied to other biomedical applications.

Graduation Semester

2015-5

Type of Resource

text

Permalink

http://hdl.handle.net/2142/78586

Copyright and License Information

Copyright by Jonathan Yen 2015

Owning Collections