Abstract:

Adipose-derived stem cell (ASC) is a valuable source of cell therapy, and we aimed to explore the regenerative capabilities of ASC in scaffold-free three-dimensional (3D) culture conditions. We manipulated spheroid formation of human ASCs by culturing them on fabricated biomaterial films, and we also stimulated extracellular matrix (ECM) secretion of ASCs and fabricated cell sheets by treatment with ascorbate 2-phosphate. Enhanced expression of stemness markers Sox-2, Oct-4 and Nanog was noted in ASCs within cell spheroids and sheets. However, previous reports have found decreased expression of vascular endothelial growth factor (VEGF) in ASC sheets.
RNA-sequencing analysis revealed that upregulation of angiogenesis-related genes was found only in ASC spheroids. Hence, we further integrated ASC spheroids into ASC sheets to enhance the angiogenic capability of cell sheets. The stimulating effect of spheroid formation on ASCs toward endothelial lineage was demonstrated by enhanced CD31 expression, which maintained after ASC spheroids were seeded on cell sheets. Relative to ASC sheets, enhanced expression of VEGF and hepatocyte growth factor was also noted in ASC spheroid-sheets, and conditioned medium of ASC spheroid-sheets significantly enhanced tube formation of endothelial cells in vitro. Moreover, chick embryo chorioallantoic membrane assay showed a significantly higher capillary density with more branch points after applying ASC spheroid-sheets, and immunohistochemistry also revealed a significantly higher ratio of CD31-positive area.
In the spheroid-sheet construct, ASC spheroids can augment the pro-angiogenesis capability of ASC sheets without the use of exogenous biomaterial or genetic manipulation. The strategy of this composite system holds promise as an advance in 3D culture technique of ASCs for future application in angiogenesis and regeneration therapies.

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