Bone marrow-derived mesenchymal stem cells (MSC) have been shown to provide neuroprotection after transplantation into the injured nervous system. The present thesis investigates whether adult human and rat MSC differentiated along a Schwann cell lineage could increase their expression of neurotrophic factors and promote regeneration after transplantation into the injured peripheral nerve and spinal cord. Human and rat mesenchymal stem cells (hMSC and rMSC) expressed characteristic stem cell surface markers, mRNA transcripts for different neurotrophic factors and demonstrated multi-lineage differentiation potential. Following treatment with a cocktail of growth factors, the hMSC and rMSC expressed typical Schwann cells markers at both the transcriptional and translational level and significantly increased production of brain-derived neurotrophic factor (BDNF) and vascular endothelial growth factor (VEGF). Age and time in culture are of relevance for clinical settings and growth-promoting effects of hMSC from young donors (16-18 years) and old donors (67-75 years) were compared. Undifferentiated hMSC from both young and old donors increased total neurite length of cultured dorsal root ganglion (DRG) neurons. Differentiation of hMSC from the young donors, but not the eldery donors, further enhanced the neurite outgrowth. Undifferentiated hMSC were cultured for eleven weeks in order to examine the effect of in vitro expansion time on neurite outgrowth. hMSC from the young donors maintained their proliferation rate and their ability to enhance neurite outgrowth from DRG neurons. Using a sciatic nerve injury model, a 10mm gap was bridged with either an empty tubular fibrin glue conduit, or conduits containing hMSC, with and without cyclosporine treatment. Cells were labeled with PKH26 prior to transplantation. At 3 weeks after injury the conduits with cells and immunosuppression increased regeneration compared with an empty conduit. PKH26 labeled human cells survived in the rat model and the inflammatory reaction could be suppressed by cyclosporine. After cervical C4 hemisection, BrdU/GFP-labeled rMSC were injected into the lateral funiculus rostral and caudal to the spinal cord lesion site. Spinal cords were analyzed 2-8 weeks after transplantation. Transplanted MSC remained at the injection sites and in the trauma zone for several weeks and were often associated with numerous neurofilament-positive axons. Transplanted rMSC induced up-regulation of vascular endothelial growth factor in spinal cord tissue rostral to the injury site, but did not affect expression of brain-derived neurotrophic factor. Although rMSC provided neuroprotection for rubrospinal neurons and significantly attenuated astroglial and microglial reaction, cell transplantation caused aberrant sprouting of calcitonin gene-related peptide immunostained sensory axons in the dorsal horn. In summary these results demonstrate that both rat and human MSC can be differentiated towards the glial cell lineage, and show functional characteristics similar to Schwann cells. hMSC from the young donors represent a more favorable source for neurotransplantation since they maintain proliferation rate and preserve their growth-promoting effects in long-term cultures. The data also suggest that differentiated MSC increase expression of neurotrophic factors and support regeneration after peripheral nerve and spinal cord injury.

Bone is an important organ influenced by mechanical load, hormones, nutrition and disease. During bone remodelling, osteoclasts resorb bone and osteoblasts form new bone. Osteoblasts are derived from mesenchymal stem cells (MSCs) such as adipose-derived stromal cells (ASCs). The mitogen-activated protein kinase (MAPK) pathway has been shown to interfere with osteoblast differentiation from an early stage. Runt related transcription factor 2 (RUNX2) exerts an effect downstream from p38 MAPK. RUNX2 phosphorylation by p38 MAPK may increase osteoblast differentiation markers such as alkaline phosphatase (ALP), osteoprotegerin (OPG) and receptor activator of nuclear factor kB ligand (RANKL). Palmitoleic acid (PLA), an omega-7 monounsaturated fatty acid (MUFA), promotes anti-osteoclastogenic effects, however, the effects of PLA on osteoblasts has not been reported. Osteoporosis is a condition which has debilitating effects in the elderly. Unsaturated fatty acids (UFA) have been studied for their beneficial effects on human health for a number of years. Polyunsaturated fatty acids (PUFA) have been studied as a potential therapeutic agent to prevent and assist in managing the condition. Few studies have been conducted on the effects of MUFA on bone therefore this study aimed to investigate the effects of PLA on osteoblast differentiation using ASCs and MG-63 osteosarcoma cells as an osteoblast model. ASCs and MG-63 osteosarcoma cell lines were exposed to PLA (20-100 μM) in osteogenic media (OM). The effects of PLA on cell viability was evaluated on undifferentiated cells. Thereafter, cells were exposed to PLA for 7, 14 or 21 days. Subsequently ALP activity, calcium mineralisation, gene expression, protein expression and adipogenesis were assessed. In this study, PLA had no significant effects on cell viability in undifferentiated cells. Furthermore, PLA had no significant effects on ALP activity, calcium mineralisation or phosphorylated extracellular signal-regulated kinase (pERK)/extracellular signal-regulated kinase (ERK) expression in differentiating cells, however, ALP activity increased at 7 day in ASCs and 21 days in MG-63 cells. Alizarin Red S staining increased at 21 days in both cell lines with a significant increase in the ASCs, however, calcium nodules were not visible. In the ASCs, PLA significantly increased the gene expression of ALP at 7 and 14 days compared to control (p<0.01 and p<0.05) while RANKL was significantly decreased at 7 days compared to the control (p<0.05). In the MG-63 cells, RUNX2 and OCN were significantly reduced at 7 days compared to control (p<0.05) and ALP, RUNX2, Osx and RANKL were significantly reduced at 14 days compared to control (p<0.001 and p<0.05). In the ASCs, lipid accumulation was not present after 21 days while in MG-63 cells, there was a significant increase in lipid accumulation at a high concentration of PLA after 21 days compared to control (p<0.05). This is the first study to explore the effects of PLA on osteoblast formation using ASCs and MG-63 osteosarcoma cells. Results suggest that PLA exerted changes in the ASCs and MG-63 cells during osteoblast differentiation, however, these changes were not significant. To conclude, PLA showed some significant effects on osteoblast-specific gene expression, however, most of the osteoblast-specific gene expression was downregulated, particularly in the MG-63 cells, after PLA treatment. / Dissertation (MSc)--University of Pretoria, 2020. / Physiology / MSc / Unrestricted

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第18131号 / 医博第3851号 / 新制||医||1001(附属図書館) / 30989 / 京都大学大学院医学研究科医学専攻 / (主査)教授 千葉 勉, 教授 坂井 義治, 教授 羽賀 博典 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Introduction It is well known that resident adipose stem/stromal cells (ASCs) are a heterogeneous population of multipotent cells characterized by (a) their ability to adhere to plastic; (b) immunophenotypic expression of certain cell surface markers, while lacking others; and (c) the capacity to differentiate into cells of mesodermal origin including osteocytes, chondrocytes and adipocytes. Adipose derived stromal cells offer great therapeutic potential in multiple medical fields, including, orthopedics, cardiology, oncology and degenerative diseases, to name a few. Combining different disciplines of medicine and engineering, organ and tissue repair can be achieved through tissue engineering and regenerative medicine. Adipose derived stromal cells (ASCs) can be utilized as biological vehicles for vector-based gene delivery systems, since they home to sites of inflammation and infection in vivo. In order to reach the long-term aim of clinical translation of cell-based therapy, preclinical safety and efficacy need to be shown in animal models. This has motivated the development of standardized isolation, characterization and differentiation operating procedures as well as an in vivo tracking system for ASCs and lentiviral vector transduction for a vector-based gene delivery system. Methodology Human ASCs were isolated from lipoaspirate, expanded in culture, immunophenotyped using flow cytometery and induced to differentiate into adipogenic, osteogenic and chondrogenic lineages. Tri-lineage differentiation was confirmed by microscopy. The ASCs were then transduced with green fluorescent protein (GFP)-expressing lentiviral vectors in vitro. The effect of the GFP lentiviral vector on ASCs was investigated by studying ASC immunophenotypic expression of surface markers as well as their capacity to differentiate into osteocytes, chondrocytes and adipocytes. Results The isolated and expanded cell population, from harvested lipoaspirate adhered to recommended ASC identity criteria. The heterogeneity of ASCs was confirmed by the presence of sub-populations. Transduction efficiency in ASC cultures of approximately 80% was observed after introducing a total of 300 μl of concentrated lentiviral vector suspension per 4.8 x 104 cells. No immunophenotypic differences were observed between GFP positive and GFP negative cultures. Flow cytometric analysis revealed a progressive increase in GFP expression following in vitro expansion of transduced ASCs. Both non-transduced and transduced cultures successfully differentiated into osteocytes, chondrocytes and adipocytes. Conclusion The isolated and expanded cell population conformed to the recommended characterization criteria. Heterogeneity was demonstrated with the identification of immunophenotypic sub-populations and semi-quantification of adipogenesis was performed. ASCs were efficiently transduced using the GFP lentiviral vectors produced in our facility. In addition, transduced ASCs maintained adherence to plastic, ASC immunophenotype and were able to differentiate successfully into cells of the three lineages of mesodermal origin. This optimized GFP-ASC transduction technique offers a feasible tracking system as well as a vector-based gene delivery system for future preclinical studies. / Dissertation (MSc)--University of Pretoria, 2015. / tm2015 / Immunology / MSc / Unrestricted