Abstract
Spinal cord injury (SCI), especially trauma-induced spinal cord injury, is a prevalent form of central nervous system damage. Alterations in the tissue microenvironment caused by neuronal necrosis and immune cell activation in the area of injury result in the formation of scars and cavities at the site of the SCI, which makes it difficult to repair and regenerate. Cell therapy is currently a widely researched approach for SCI repair, which involves the transplantation of neural stem cells or neural progenitor cells into the injured spinal cord region for therapeutic purposes. However, the efficacy of the therapy is poor. In other studies, human pluripotent stem cells can be directly induced into spinal cord motor neuron cells in advance, but this approach fails to replicate the developmental characteristics of the spinal cord and lacks effectiveness in SCI repair.Spinal cord tissue engineering technology based on biomaterials is a rapidly developing field in regenerative medicine. Biomaterial scaffolds have demonstrated their ability to facilitate the proliferation, differentiation, and migration of neuronal cells, thereby potentially aiding in the restoration of SCI. In our group’s previous research, a type II collagen scaffold has been designed. Type II collagen scaffolds with low immunogenicity, good biocompatibility, and suitable biomechanical intensity can be used as the substrate for developing spinal cord tissue engineering. Recently, organoids have emerged as a novel three-dimensional cell culture technology in vitro. Under some specific culture conditions, human embryonic stem cells or induced pluripotent stem cells have the ability to self-organize and differentiate into multiple cell types, forming organ-like structures with partial physiological functions. Therefore, in this thesis, one type II collagen scaffold was engineered to have a penetrating channel called the patterned type II collagen (Col II). Subsequently, two modifications were made to the type II collagen scaffolds. One is the patterned type II collagen substrate-Matrigel (Col II-M), and the other is the patterned type II collagen substrate-polydopamine (Col II-PDA). Eventually, the patterned type II collagen-based substrates will be utilized for human spinal cord organoid construction in vitro and as biomaterials for SCI repair in vivo.
In Chapter 4, patterned type II collagen scaffolds were used as substrates and embedded in Matrigel to form the interpenetrating complex, Col II-M. Initially, a three-dimensional culture method was employed to generate a specific spheroid from the human induced pluripotent stem cell (hiPSC) -derived human motor neuron progenitor cells. Subsequently, the spheroid was embedded into the patterned type II collagen substrate-Matrigel for a long-term culture to form the human spinal cord organoid in patterned type II collagen substrate-Matrigel. It is evaluated as a millimeter-scale macroscopic operable engineered artificial spinal cord. The complex structure, primarily composed of patterned type II collagen substrate-Matrigel, offered a continuous extracellular microenvironment supporting the sustained differentiation, migration, and maturation of motor neuron progenitor cells, thus establishing a three-dimensional self-organized model of developing spinal cord with multiple cell types. The spheroids embedded in Matrigel droplets were set as the control group. Immunofluorescence staining was conducted on the obtained human spinal cord organoid in patterned type II collagen substrate-Matrigel (HSCO-Col II-M), revealing the presence of early CNS marker TUJ1 and astrocyte marker GFAP within the scaffold pores formed by the patterned type II collagen substrate-Matrigel. Furthermore, more motor neuron markers were detected. Proteomic analysis indicated that the most highly expressed proteins in the spinal cord organoid were associated with motor proteins, axon guidance, adherens junction, focal adhesion, and the regulation of actin cytoskeleton, suggesting the abundance of proteins relevant to motor neurons in the spinal cord organoid. Single-cell RNA sequencing analysis identified the major cell differentiation types in the spinal cord organoid, confirming the continuous differentiation and maturation of neural progenitor cells supported by patterned type II collagen-Matrigel substrates.
Then, we attempted to verify whether the type II collagen-based substrates could support the repair of completely transected SCI in mice.
In Chapter 5, firstly, we attempted to implant the human spinal cord organoid in patterned type II collagen substrate-Matrigel into nude mice spinal cord complete transection models to verify the effectiveness of spinal cord repair. In Chapter 6, we designed novel polydopamine-coated patterned type II collagen substrates, the patterned type II collagen substrate-polydopamine, for cell-free spinal cord regeneration transplantation. The Basso Mouse Scale was used to evaluate motor performance after spinal cord injury in mice, the Col II-PDA group had the best performance in hindlimb motor recovery. In the eight-week post-surgery, the mice were euthanized, and the tissues at the site of spinal cord transection were isolated and characterized to evaluate the regenerative capacity of the patterned type II collagen-based substrates in repairing spinal cord injuries. The results demonstrated that both the human spinal cord organoid in patterned type II collagen substrate-Matrigel and the patterned type II collagen substrate-polydopamine could partially improve the microenvironment of spinal cord injury in nude mice and promote neural survival at the injury. Immunostaining for macrophage-associated antibodies CD68/CD206 staining showed that the group undergoing human spinal cord organoid in patterned type II collagen substrate-Matrigel transplantation and the group patterned type II collagen substrate-polydopamine transplantation after spinal cord injury had no significant inflammatory response and could improve spinal cord injury through immune modulation.
To summarize, we successfully generated the human spinal cord organoid in patterned type II collagen substrate-Matrigel and provided new strategies for the SCI treatment by the patterned type II collagen-based substrates.
| Date of Award | 10 Sept 2024 |
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| Original language | English |
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| Supervisor | Dongan WANG (Supervisor) |