Induced pluripotent stem cells from urine of Duchenne muscular dystrophy patients

Abstract

Background The most common muscular dystrophy, Duchenne muscular dystrophy (DMD), is a lethal, X‐linked disorder with no widespread cure. Worldwide, in vitro studies involving new, mutation‐specific cures and regenerative therapies are employing disease‐specific patient‐specific cells. However, these may not be completely relevant for Pakistani children because of the human genome diversities and geographic variation in mutation type and frequency. Therefore, this study aimed to generate DMD induced pluripotent stem cells (iPSCs) from the urine of Pakistani children with DMD, to serve as a precious source of differentiated cells, such as Pakistani DMD‐cardiomyocytes, for future disease‐modelling, drug testing, and gene therapy. Methods Urine‐derived cells (UDCs) isolated from mid‐stream urine underwent molecular characterization and cellular reprogramming towards iPSCs using the episomal vector system followed by molecular profiling of the iPSCs. Results Colonies of elongated and spindle‐shaped or rounded rice‐grain like UDCs were spotted 4–7 days after plating and expanded rapidly with a second passage at 2–3 weeks. Multicolor flow cytometry confirmed the expression of mesenchymal stem‐cell markers. The reprogramed iPSCs consisted of colonies of round, tightly‐packed cells with large nuclei that were positively fluorescent for the pluripotency markers octamer binding transcription factor‐4 (OCT‐4), tumour resistance antigen 1–60 (TRA‐1‐60), and stage specific embryonic 4 antigen (SSEA‐4), but not for the negative pluripotency marker SSEA‐1. To the best of our knowledge, this was the first time DMD‐iPSCs have been generated for Pakistani children. Conclusion This integration‐free, feeder‐free, efficient, and reproducible reprogramming method employed UDCs. Urine is a low‐cost, non‐invasive, painless, and repeatable source of rapidly expandable cells from children and morbid individuals for obtaining autologous cells for drug‐assays and disease‐modelling, suitable for DMD and other debilitating diseases.