Investigating the Molecular Mechanisms Underlying the Loss of Stem Cell Potential in Human Adipose-Derived Stem Cells

Abstract

Human adipose-derived stem cells (ASCs) are of increasing interest to the field of regenerative medicine due to their multipotent nature and immunomodulatory capabilities. The majority of current therapeutics using ASCs require serial passaging of the cells to obtain a pure population of ASCs with clinically useful numbers. This is problematic as an extended time in culture correlates with a decrease in stem cell properties of ASCs such as their differentiation and proliferative potentials. There is general agreement within current literature that this loss of potential exists, however the molecular mechanisms underlying the change in potency are not yet fully characterised. This project examined microarray data of differentially expressed miRNAs and mRNAs over time in culture and revealed targets of interest for further validation and functional analysis. qPCR validation of mRNA expression highlighted KLF4 as a potential marker for stem cell potency of ASCs as it has a known role in stem cell maintenance and was significantly upregulated in freshly isolated cells compared with serially passaged cells that have lost potency. miR-378 was found to be expressed at significantly higher levels in freshly isolated (day 0) cells. The opposite was seen for miR-21, which was significantly upregulated in ASCs that had been in extended culture (day 28 cells). The effects of miR-378 and miR-21 on both adipogenic and osteogenic differentiation potential were investigated in late passage ASCs. Overexpression of miR-378 was shown to significantly increase adipogenic differentiation potential of late passage ASCs and to have the potential to do the same down the osteogenic lineage. Current clinical trials using ASCs are potentially using suboptimal cells due to isolation and expansion methods, yet have produced encouraging results. In light of miRNA targeting emerging as a viable prospect for therapeutics, manipulation of miR-378 to restore or maintain stem cell potential in serially passaged ASCs is promising for enhancing their efficacy for future clinical research.