| Summary: | 博士 === 國立臺灣大學 === 醫學工程學研究所 === 100 === Because of their abilities of rapid proliferation and differentiation into multiple cell types, mesenchymal stem cells are regarded to have great potentials for application in regenerative medicine. The adipose-derived stem cells (ASCs) have been shown to share similar characteristics of mesenchymal stem cells isolated from bone marrow, and they can be harvested in large amount by liposuction. However, the stem cell characteristics of ASC, indicated by the expression of pluripotency markers, quickly decreased during in vitro culture. This problem has limited the application of ASCs in cell therapy or tissue engineering. Previous reports have suggested that culture as three-dimensional spheroids can increase therapeutic potentials of mesenchymal stem cells. Therefore, we aimed to manipulate the spheroid formation of human ASCs by culturing them on chitosan films. With the increasing seeding density of ASCs on chitosan films, we found that ASC spheroids formed faster and exhibited a larger diameter. Live/dead assay further showed that ASCs within the spheroid were largely viable. Importantly, significant upregulation of stemness marker genes Sox-2, Oct-4 and Nanog was noted in cells within ASC spheroids comparing to monolayer culture. We also used western blot and immunofluorescence to confirm the enhanced expression of pluripotency markers in ASC spheroids. Secondly, we demonstrated a higher regenerative power of ASCs after spheroid formation by examining proliferation activity and colony-forming capability. Finally, we investigated the differentiation potentials of ASC spheroids when cultured in appropriate induction media. Not only differentiation capability toward adipogenic and osteogenic lineages (mesoderm) was maintained, but neurogenic (ectoderm) and hepatogenic (endoderm) differentiation of ASC spheroids was even enhanced after spheroid formation.
To facilitate ASC transfer, we further designed a composite membrane made of chitosan/gelatin (C/G) for ASC culture and subsequent application to injured tissues. Increasing chitosan content within the C/G blends enhanced the sample’s mechanical properties, including tensile strength and elongation-at-break ratio. Although ASC spheroids developed shortly after seeding on pure chitosan films, increasing gelatin proportion in the C/G blends promoted cell adhesion onto the membranes. However, gradual gelatin release from the C/G blend films, leading to enriched chitosan content in the blends, encouraged ASC detachment and spheroid formation. We found that a blend made of 75% chitosan and 25% gelatin was most efficient in modulating ASCs for cell transfer. Therefore, C/G films in combination with ASCs have potentials of clinical application, such as treating a cutaneous wound.
Healing of difficult cutaneous wounds is a challenging clinical problem, and current treatment modalities have their limitations. ASC can contribute to cutaneous wound healing through differentiation and paracrine effects. We observed that after spheroid formation of human ASCs on chitosan films, their stemness markers Sox-2, Oct-4 and Nanog were still maintained with spheroid dissociation and further monolayer culture. These spheroid-derived ASCs also expressed significantly more angiogenic growth factors, including hepatocyte growth factor (HGF) and vascular endothelial growth factor (VEGF), comparing to monolayer ASCs. The upregulation of CXCR4 in the spheroid-derived ASCs is associated with enhanced proliferation, reduced apoptosis and increased expression of matrix metalloproteinases. The interaction between stromal-derived factor-1 (SDF-1) and CXCR4 plays an important role in the homing of mesenchymal stem cells to the site of injury. In our animal study, we observed significantly more cellular engraftment of spheroid-derived ASCs in the cutaneous wound tissue with accelerated wound healing comparing to monolayer-cultured ASCs. The mechanism could be attributed to a higher cellular retention rate, enhanced differentiation and angiogenesis. Therefore, biomaterial modulation of ASCs for spheroid formation can provide important therapeutic potentials for tissue regeneration.
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