Differential and Interactive Effects of Substrate Topography and Chemistry on Human Mesenchymal Stem Cell Gene Expression

Abstract

Variations in substrate chemistry and the micro-structure were shown to have a significant effect on the biology of human mesenchymal stromal cells (hMSCs). This occurs when differences in the surface properties indirectly modulate pathways within numerous signaling networks that control cell fate. To understand how the surface features affect hMSC gene expression, we performed RNA-sequencing analysis of bone marrow-derived hMSCs cultured on tissue culture-treated polystyrene (TCP) and poly(l-lactide) (PLLA) based substrates of differing topography (Fl: flat and Fs: fibrous) and chemistry (Pr: pristine and Am: aminated). Whilst 80% of gene expression remained similar for cells cultured on test substrates, the analysis of differentially expressed genes (DEGs) revealed that surface topography significantly altered gene expression more than surface chemistry. The Fl and Fs topologies introduced opposite directional alternations in gene expression when compared to TCP control. In addition, the effect of chemical treatment interacted with that of topography in a synergistic manner with the Pr samples promoting more DEGs than Am samples in all gene ontology function groups. These findings not only highlight the significance of the culture surface on regulating the overall gene expression profile but also provide novel insights into cell-material interactions that could help further design the next-generation biomaterials to facilitate hMSC applications. At the same time, further studies are required to investigate whether or not the observations noted correlate with subsequent protein expression and functionality of cells.

Effect of Substrate Topography and Chemistry on Human Mesenchymal Stem Cell Markers: A Transcriptome Study

“…3, the effect of chemistry treatments on the gene expression profile was relatively smaller than that of surface topography induced changes, and the samples cultured on flat surfaces exhibited significantly smaller changes to the control cluster than those in the fibrous samples. This feature conformed to the gene expression patterns reported for all genes (26), strongly suggesting that the marker set genuinely represented the overall gene expression. To our knowledge, this study is the first of its kind reporting the effect of substrate topography and chemistry on the expression of 177 biomarkers reported in the literature.…”

“…There was no significant change in the fibre diameter after amine functionalization, as confirmed by SEM. Besides, qualitative and quantitative analysis of cell adhesion and proliferation on various substrates was also previously reported by our group (26). Briefly, the cellular adhesion and proliferation was relatively high on control TCP substrate compared to test PLLA substrates.…”

“…Details of the material characteristics such as morphological features of Fl-PLLA and Fs-PLLA before and after amination by SEM, confirmation of amine functionalization by FITC staining and ATR-FTIR was previously reported by our group (26). The diameter of electrospun fibres of Fs-PLLA scaffold used in the study was 603±197 nm (Mean±SD).…”

“…The detailed information on surface preparation, cell culture and RNA extraction has been reported previously (26). A total of four synthesized surfaces and tissue culture plate were used for cell culture.…”

“…RNA extraction was done using the RNeasy Mini Kit and followed the supplier’s instructions (Qiagen, UK). RNA agarose gel electrophoresis and Nanodrop spectrophotometer (1000, Thermo Scientific) were used to check the quality and quantity of the extracted RNA (26). RNA was isolated at each time point; however, RNA Seq was done for day 3 sample to capture the substrate-induced early commitment of MSCs.…”