Urine-derived stem cells are a novel approach for transcriptomic studies in Duchenne Muscular Dystrophy

Background: Nowadays, urine derived stem cells (USCs) are considered as a new approach for modelling a wide variety of human diseases. In fact, these cell lines have many advantages in terms of the easy isolation, low costs and the absence of ethical issues. Aims: The goals of this study included examining the gene expression profile of both native and myogenically transformed (MyoD) USCs derived from DMD patients and healthy individuals. In addition to that, we exploited the new technology Celector ® (StemSel Ltd.), in order to distinguish both control and DMD USCs through their segregation and consequently discovering the cell-subpopulations. Methods: In the current study, USCs were obtained from urine samples collected from six healthy donors and three DMD patients, having different deletions. Myogenic transformation, using the adenovirus type 5 (Ad5) was applied on USCs, derived from three healthy controls and one DMD patient, bearing the exon 45 deletion. Afterwards, in order to evaluate the gene expression profile in both native and myogenically transformed USCs, we performed RNA sequencing analysis using illumina Hiseq4000. Furthermore, USCs obtained from both healthy individuals and DMD patients, with recognized mutations, were subjected to the segregation, through the use of the Celector ® (StemSel Ltd); a new apparatus to distinguish cells, according to their physical features, such as shape, density, dimension etc. Following this, a flow cytometry was performed in order to test the mesenchymal stem cell markers. Then, the DMD transcript of the cells was profiled, using FluiDMD; a TaqMan gene expression assay. Results: This study demonstrated that subsequently to induced myogenesis, many pathways correlated to DMD were modulated, examples of that are: muscle function and inflammation. Furthermore, a clear heterogeneity between, healthy and DMD USCs, regarding the cell-type constitution was spotted through the use of RNA seq. We also characterized USCs and consequently unveiled the presence of 3 three distinct subpopulations with particular physical and expression features using the new cell sorting 5 technique Celector®. Consequently, we assumed that USCs are extremely versatile and heterogeneous. Conclusion: USCs are a very useful, in vitro cell model, to profile transcriptomics in DMD pathology and, thus, facilitate functional studies aiming at addressing drug efficacy.