Akil, Putri Nur Hidayah Al-Zikri Mohamad
(2021)
The molecular, cellular and functional changes in the treatment of rat spinal cord injury with neuro-progenitor cells derived from bone marrow mesenchymal stem cells.
PhD thesis, Universiti Sains Malaysia.
Abstract
Recently, there has been growing interest in the differentiation of mesenchymal stem cells (MSCs) into neural lineages under the specific influence of paracrine factors. To unravel the roles and effects of paracrine factors, we have differentiated BM-MSCs into neural progenitor-like cells (NPCs) using insulin-like growth factor 1 (IGF-1) and standard paracrine factors; epidermal growth factor (EGF) and basic fibroblast growth factor (BFGF). The differentiated NPCs were evaluated for functional recovery in the spinal cord. Various strategies have managed to improve functional recovery after spinal cord injury (SCI) induction but an optimal strategy has not been developed yet. Thus, this study aims to graft Bone Marrow Mesenchymal Stem Cells (BM-MSCs)-derived neural progenitor-like cells (NPCs) after a spinal cord hemisection injury and transplantation into Sprague Dawley rats to assess their effects on functional recovery, histology, gene expression, and proteomic study. BM-MSCs were differentiated into NPCs in three groups; Group A (without growth factor), Group B (EGF + BFGF), and Group C (EGF + BFGF + IGF-1). The BM-MSCs-derived NPCs (neurospheres-like cells) formed were analyzed for cell proliferation and apoptosis using MTS and Annexin V assay, respectively. In in vivo study, rats SCI were divided into three groups; Group 1 (injured + vehicle-collagen), Group 2 (injured + vehicle-collagen + NPCs [bFGF & EGF]-treated) and Group 3 (injured + vehicle-collagen + NPCs [bFGF, EGF & IGF-1]-treated). In order to evaluate the treatment efficacy and functional improvement on T10 rat SCI, behavioural studies on locomotor and sensory function were conducted. The behavioural outcomes in in vivo study is the most important factor for evaluating the treatment efficacy and the improvement of functional recovery. The locomotor function was tested by utilizing the Modified Open-field gait assessment using Basso, Beatie, and Bresnahan (BBB) rating scale. Motor coordination and balance were determined using a wooden beam test. Meanwhile, the sensory function was determined by Electronic Von Frey test. The investigation was followed by histology, gene expression and proteomic evaluation after the SCI investigation to help elucidate possible mechanisms leading to the observed functional recovery. Isolation of BM-MSCs showed positive expression on Vimentin, Nestin, Fibronectin, CD90, and CD44 markers whereas negative expression was recorded for CD11b marker. Among the groups treated with growth factors, differentiation of MSCs-derived NPCs treated with IGF-1 showed the highest cell proliferation and the lowest inhibited apoptosis. IGF-1 has a crucial role in the differentiation of BM-MSCs into neural lineage by enhancing the proliferation and reducing the apoptosis in the NPCs. In the in vivo study, the open field test using BBB rating scale results exhibited that the mean scores overtime was higher in Group 3 as compared to Group 1 and 2. The difference between Group 3 and the control group was statistically significant at p<0.05. Meanwhile, there was a positive response in the Von Frey test for Group 3 as compared to the control group. H&E results after the behavioural study indicated that Group 1 exhibited a clear trend towards injury with vacuolation of cells within the white matter as well as areas of necrosis. Group 2 and 3 had very limited residual lesions and the profile of tissue damage showed considerable improvements. However, there was a cyst formation observed in Group 2. IHC staining results demonstrated that all groups expressed all antibodies including GFAP (for astrocytes), APC (for oligodendrocyte), SOX2 (for stemness), and MBP (for myelination) antibodies except Nestin (for neural precursor cell) antibody, 4 weeks after transplantation. The results at gene expression level using representative genes involved were upregulated in neural development or differentiation (Beta III Tubulin, SOX2, MAP2, Nestin, GFAP, Olig2, and MBP genes) and angiogenesis (Ang-1 and Ang-2). In the protein level, bands were evident for immature neuronal markers such as Beta III-tubulin and SOX2 antibodies; and mature neuronal markers such as MAP2 antibody after SCI recovery, except for Nestin. Besides, bands were also present for astrocyte marker (GFAP), oligodendrocyte marker (APC), and myelin marker (MBP). As a conclusion, BM-MSCs-derived NPCs transplantation on spinal cord hemisection injury of Sprague Dawley rats demonstrated positive effects on functional recovery including histology, gene expression, and proteomic levels. Therefore, this information will be beneficial for improving cell-based therapy in regenerative medicine.
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