In vitro adipose-derived stromal cell myogenic differentiation

Abstract

Adipose-derived stromal cells (ASCs) are multipotent cells obtained from adipose tissue. ASCs are able to differentiate into multiple cell lineages including adipose, cartilage, bone and muscle with the appropriate stimulus. The multipotency of ASCs has brought attention to these cells as possible therapeutic agents in regenerative medicine. The aim of this study was to optimise the methods involved in differentiating ASCs isolated from lipoaspirates into a myogenic lineage for the Institute for Cellular and Molecular Medicine (ICMM, University of Pretoria, Department of Immunology) using two previously published induction methods. ASCs, isolated from lipoaspirates, were immunophenotyped by flow cytometry, and myogenesis evaluated at the protein level using immunocytochemistry (ICC) and the transcriptomic level using reverse transcriptase quantitative real time polymerase chain reaction (RT-qPCR). ASC myogenesis was induced using either dexamethasone/ hydrocortisone (DH)- or 5-Azacytidine (5-Aza)-based induction media over 42- and 24-days respectively. Relative gene expression of myogenic targets desmin, myogenic differentiation (MyoD) and myogenin (MyoG) was determined using RT-qPCR. The presence of myogenic target proteins paired box proteins (Pax) 3/7, MyoD, MyoG and desmin was qualitatively determined by ICC and visualised using a confocal microscope. In ASCs induced to differentiate, neither MyoD nor MyoG mRNA were amplified in any condition, at any time-point. The expression of desmin was confirmed; however, there was no statistically significant change in desmin expression using either the DH or 5-Aza-based methods. The fold-increase in the expression of desmin mRNA was the highest on day twelve post-induction for the 5-Aza-based method relative to the non-induced control sample. Interestingly, when comparing the induced and non-induced samples relative to day zero, the non-induced samples showed the highest fold-increase in the expression of desmin on day six, whilst all three conditions indicated an increase in the expression on desmin on day twelve. ICC confirmed the presence of desmin from as early as day three and at every subsequent time-point, and the expression of Pax 3/7 from day six in both the DH-based induction methods. According to recent ASC myogenic modelling, where ASC differentiation is described in terms of 6 stages based on the presence of myogenic markers Pax 3/7 and desmin, ASCs in this study only achieved stage two myogenic differentiation, based on the presence of desmin and pax 3/7. Based on the data collected in this study, no conclusion could be made as to which induction medium most efficiently induced myogenesis. As the aim of this study was to optimise the differentiation of ASCs into a myogenic lineage, it could be concluded that the assays involved were optimised. However, taking into consideration limitations that were identified through the course of this study, suggestions for future experimental endeavours have been made in order to further optimise the myogenic process and accompanying assays. These include the further optimisation of primer melting temperatures, using a fluorescent signal amplification system in ICC, methods that could potentially be used to obtain quantitative data from fluorescent images and the concept of differentiating ASCs into a myogenic lineage using both chemical and mechanical stimuli under dynamic conditions.

Key words: Adipose-derived stromal cells; ASCs; myogenesis; muscle; differentiation; mesenchymal stromal cells; MSCs.