Substrate-dependent gene regulation of self-assembled human MSC spheroids on chitosan membranes

Background: Three-dimensional (3D) multicellular spheroids of mesenchymal stem cells (MSCs) are generally regarded to have beneficial properties over MSCs in monolayer. Recent literatures have documented that MSCs can self-assemble into 3D spheroids with a greater capacity for differentiation into various cell types when grown on chitosan (CS), a biopolymer. The genomic modulation occurring in these MSC spheroids is thus of essential importance for understanding their uniqueness and therapeutic potentials. In this study, 3D spheroids self-assembled from human umbilical cord MSCs grown on CS membranes were analyzed by mRNA as well as microRNA microarrays, which helped identify the critical signaling events that may alter the cellular functions during the spheroid forming process. Results: Genes screened from mRNA and microRNA cross-correlation analyses were further confirmed with the quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) analysis. Results revealed the regulation of a significant number of calcium-associated genes, which suggested the crucial role of calcium signaling in CS-derived MSC spheroids. In addition, many genes associated with the multilineage differentiation capacities and those associated with the antiinflammatory and antitumor properties of MSCs were upregulated. The genetic modulation was significantly more remarkable and endured longer for MSC spheroids derived on CS substrates compared to those derived on a non-adherent (polyvinyl alcohol) substrate. Conclusions: Based on the study, the culture substrates used to prepare 3D MSC spheroids may predefine their properties through cell-substrate interaction.