{"id":{"repo_id":"uiuc","oai_identifier":"oai:www.ideals.illinois.edu:2142/78586"},"canonical_url":"https://search.dev.ndltd.org/etd/uiuc/oai:www.ideals.illinois.edu:2142/78586","repository":{"repo_id":"uiuc","name":"University of Illinois - Urbana-Champaign","base_url":"https://www.ideals.illinois.edu/oai-pmh"},"display":{"title":"Materials and biological approach to gene delivery in human embryonic stem cells","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.","abstract_html":"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.","abstract_has_math":false,"creators":["Yen, Jonathan"],"institution":"University of Illinois at Urbana-Champaign","degree_name":"Ph.D.","degree_level":"Dissertation","degree_discipline":"Bioengineering","degree_department":null,"school":null,"contributors":["Cheng, Jianjun","Wang, Ning","Underhill, Gregory","Ma, Jian","Kilian, Kristopher A."],"advisors":[],"committee_chairs":[],"committee_members":[],"year":2015,"date_issued":"2015-07-22T22:32:52Z","date_published":"2015-07-22T22:32:52Z","updated_at":"2026-07-22T22:26:12Z","subjects":["endosomal escape","hyaluronic acid","Fibroblasts","Stem Cells","Human Pluripotent Stem Cells","polypeptides","Human Embryonic Stem Cells","Gene Delivery"],"languages":[],"rights":["Copyright by Jonathan Yen 2015"],"rights_urls":[],"identifier_entries":[]},"links":{"outbound_url":"http://hdl.handle.net/2142/78586","outbound_label":"Handle","outbound_source":"dc:identifier"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor","label":"Contributor","values":["Cheng, Jianjun","Wang, Ning","Underhill, Gregory","Ma, Jian","Kilian, Kristopher A."]},{"key":"dc:creator","label":"Author","values":["Yen, Jonathan"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date","label":"Dc Date","values":["2015-07-22T22:32:52Z","2017-07-23T09:15:24Z","2015-05","2015-03-02","2015-5"]},{"key":"dc:type","label":"Dc Type","values":["text"]},{"key":"thesis:degree_discipline","label":"Discipline","values":["Bioengineering"]},{"key":"thesis:degree_level","label":"Degree Level","values":["Dissertation"]},{"key":"thesis:degree_name","label":"Degree Name","values":["Ph.D."]},{"key":"thesis:institution_name","label":"Thesis Institution Name","values":["University of Illinois at Urbana-Champaign"]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["endosomal escape","hyaluronic acid","Fibroblasts","Stem Cells","Human Pluripotent Stem Cells","polypeptides","Human Embryonic Stem Cells","Gene Delivery"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:rights","label":"Dc Rights","values":["Copyright by Jonathan Yen 2015"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier","label":"Identifier","values":["http://hdl.handle.net/2142/78586"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description","label":"Description","values":["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.","Submission published under a 24 month embargo labeled 'U of I only', the embargo will last until 2017-05-01","The student, Jonathan Yen, accepted the attached license on 2015-02-09 at 12:06.","The student, Jonathan Yen, submitted this Dissertation for approval on 2015-02-09 at 12:16.","This Dissertation was approved for publication on 2015-03-02 at 09:28.","DSpace SAF Submission Ingestion Package generated from Vireo submission #7713 on 2015-07-22 at 14:16:45","Made available in DSpace on 2015-07-22T22:32:52Z (GMT). No. of bitstreams: 3 Yen_Jonathan12.pdf: 2593954 bytes, checksum: 3378f696ae7f4889e9ed8e3ce3c5ed9c (MD5) Dissertation-Jonathan Yen- 2015 Submit.docx: 30673352 bytes, checksum: 738b1a1f40828d9baf8e5080cf2f391a (MD5) license.txt: 4059 bytes, checksum: 7828f24dbb12123a1fe19aff97014234 (MD5) Previous issue date: 2015-03-02","Embargo set by: Seth Robbins for item 79827 Lift date: 2017-07-22T22:34:16Z Reason: Author requested U of Illinois access only (OA after 2yrs) in Vireo ETD system","U of I Only Restriction Lifted for Item 79827 on 2017-07-23T09:15:24Z."]},{"key":"dc:format","label":"Dc Format","values":["application/pdf"]},{"key":"dc:title","label":"Title","values":["Materials and biological approach to gene delivery in human embryonic stem cells"]}]}],"canonical_facts":{"dc:contributor":["Cheng, Jianjun","Wang, Ning","Underhill, Gregory","Ma, Jian","Kilian, Kristopher A."],"dc:creator":["Yen, Jonathan"],"dc:date":["2015-07-22T22:32:52Z","2017-07-23T09:15:24Z","2015-05","2015-03-02","2015-5"],"dc:description":["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.","Submission published under a 24 month embargo labeled 'U of I only', the embargo will last until 2017-05-01","The student, Jonathan Yen, accepted the attached license on 2015-02-09 at 12:06.","The student, Jonathan Yen, submitted this Dissertation for approval on 2015-02-09 at 12:16.","This Dissertation was approved for publication on 2015-03-02 at 09:28.","DSpace SAF Submission Ingestion Package generated from Vireo submission #7713 on 2015-07-22 at 14:16:45","Made available in DSpace on 2015-07-22T22:32:52Z (GMT). No. of bitstreams: 3 Yen_Jonathan12.pdf: 2593954 bytes, checksum: 3378f696ae7f4889e9ed8e3ce3c5ed9c (MD5) Dissertation-Jonathan Yen- 2015 Submit.docx: 30673352 bytes, checksum: 738b1a1f40828d9baf8e5080cf2f391a (MD5) license.txt: 4059 bytes, checksum: 7828f24dbb12123a1fe19aff97014234 (MD5) Previous issue date: 2015-03-02","Embargo set by: Seth Robbins for item 79827 Lift date: 2017-07-22T22:34:16Z Reason: Author requested U of Illinois access only (OA after 2yrs) in Vireo ETD system","U of I Only Restriction Lifted for Item 79827 on 2017-07-23T09:15:24Z."],"dc:format":["application/pdf"],"dc:identifier":["http://hdl.handle.net/2142/78586"],"dc:rights":["Copyright by Jonathan Yen 2015"],"dc:subject":["endosomal escape","hyaluronic acid","Fibroblasts","Stem Cells","Human Pluripotent Stem Cells","polypeptides","Human Embryonic Stem Cells","Gene Delivery"],"dc:title":["Materials and biological approach to gene delivery in human embryonic stem cells"],"dc:type":["text"],"thesis:degree_discipline":["Bioengineering"],"thesis:degree_level":["Dissertation"],"thesis:degree_name":["Ph.D."],"thesis:institution_name":["University of Illinois at Urbana-Champaign"]},"updated_at":"2026-07-22T22:26:12Z"}