Investigating three-dimensional (3D) gelatin methacryloyl (GelMA) hydrogel as a potential scaffold for cell transplantation in Huntington's disease

Huntington’s disease (HD) is an inherited neurodegenerative disease that is caused by an abnormal expansion of the CAG repeats that form mutant huntingtin protein. Cell replacement therapy (CRT) offers a potential pathway to treat HD by replacing the striatal neurons lost during the disease progression with healthy ones. The emergence of cell reprogramming technology offers a powerful tool to generate an expandable cell source that can differentiate striatal-specific precursor cells. Human embryonic stem cells (hESCs) and human-induced pluripotent stem cells (hiPSCs) are used in the field as key cell sources of CRT. However, hESCs face major ethical concerns due to the origin of the cells. The use of hiPSC could overcome this, though viral vectors are associated with tumorigenesis and insertional mutation, which hinders their potential for clinical translation. Our lab has established a protocol to reprogram human-induced lateral ganglionic eminence precursor cells (hiLGEPs) directly from adult human dermal fibroblasts using SOX2/PAX6 chemically modified mRNA, which were shown to differentiate into striatal neurons in vitro and in vivo. However, the current delivery approach in liquid suspension exposes the cells to mechanical stress during the injection. Therefore, encapsulating the cells in biomaterials, such as hydrogels, could resemble their native microenvironment and thus enhance their long-term viability and differentiation. Gelatin methacryloyl (GelMA) hydrogel has been used as a cell scaffold owing to its biocompatibility, biodegradability, and the ability to promote cell adhesion. This thesis explores the potential of GelMA hydrogel as a scaffold to augment the survival and differentiation of hiLGEPs-derived MSNs for CRT. First, the study optimised the flow-focusing microfluidic systems to generate GelMA hydrogel microspheres with a diameter of 100-150 µm. However, none of these systems showed a reliable performance in producing the monodispersed microspheres as proposed. Therefore, the injectable GelMA hydrogel was used as an alternative approach. The study has demonstrated, for the first time, the capability to transplant GelMA-encapsulated hiLGEPs into the striatum of the quinolinic acid-lesioned rats. At four weeks post-transplant, cells encapsulated in GelMA gave rise to MAP2+/STEM121+ and GABA+/STEM121+ cells, with a significantly higher number of PGP9.5+/STEM121+ cells compared to those transplanted with cell media. We also demonstrated the potential of TkB small molecules, LM22A-4 and 7,8-dihydroxyflavone, in generating medium spiny neurons (MSNs). The long-term stability of LM22A-4 in cell culture media has been demonstrated for the first time. Furthermore, this research shows the first evidence that hiLGEPs encapsulated in the supramolecular GelMA hydrogel gave rise to DARPP32+ and NCAM-1+ cells and promoted neurite outgrowth in vitro following 14 days of differentiation. These findings highlight the potential of GelMA hydrogel as a biocompatible scaffold for hiLGEPs for CRT. Although further analysis is required, the current work suggested that using TrkB small molecules and the supramolecular GelMA holds the potential of improving cell viability, differentiation and integration following transplantation. Overall, this thesis contributes to the therapeutic strategies to improve the outcome of the CRT in treating HD.

Name thesis:degree_name

PhD

Level thesis:degree_level

Doctoral

Discipline thesis:degree_discipline

Pharmacology

Grantor dc:publisher

ResearchSpace@Auckland

Year dc:date.issued

2025

Author dc:creator

• Nguyen, Linh

Advisors dc:contributor.advisor

• Connor, Bronwen
• Svirskis, Darren
• McCaughey-Chapman, Amy
• Raos, Brad

Statement dc:rights

• Items in ResearchSpace are protected by copyright, with all rights reserved, unless otherwise indicated.

Licence dc:rights.uri

• https://researchspace.auckland.ac.nz/docs/uoa-docs/rights.htm

Handle dc:identifier.uri

https://hdl.handle.net/2292/76127

OAI identifier oai:identifier

oai:researchspace.auckland.ac.nz:2292/76127