Stem Cells and Regenerative Medicine Research Group, Dow Research Institute of Biotechnology and Biomedical Sciences, Dow University of Health Sciences, Karachi, Pakistan.
Department of Pathology, Dow International Medical College, Dow University of Health Sciences, Karachi, Pakistan.
Pediatr Int. 2021 Sep;63(9):1038-1047. doi: 10.1111/ped.14655. Epub 2021 Jul 13.
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.
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.
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.
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.
最常见的肌肉营养不良症，杜氏肌营养不良症（DMD），是一种致命的 X 连锁疾病，目前尚无广泛的治疗方法。在全球范围内，涉及新的、针对突变的治疗方法和再生疗法的体外研究正在使用疾病特异性、患者特异性细胞。然而，由于人类基因组的多样性以及突变类型和频率的地理差异，这些方法可能不完全适用于巴基斯坦儿童。因此，本研究旨在从巴基斯坦 DMD 儿童的尿液中生成 DMD 诱导多能干细胞（iPSC），作为分化细胞的宝贵来源，如巴基斯坦 DMD 心肌细胞，用于未来的疾病建模、药物测试和基因治疗。
从中段尿液中分离出尿液衍生细胞（UDC），使用附加体载体系统进行分子特征分析和向 iPSC 的细胞重编程，然后对 iPSC 进行分子特征分析。
接种后 4-7 天可观察到长而纺锤形或圆形米粒状 UDC 的集落，2-3 周后进行第二次传代，细胞迅速扩增。多色流式细胞术证实了间充质干细胞标志物的表达。重编程的 iPSC 由圆形、紧密堆积的细胞组成，细胞核较大，对多能性标志物八聚体结合转录因子-4（OCT-4）、肿瘤耐药抗原 1-60（TRA-1-60）和阶段特异性胚胎 4 抗原（SSEA-4）呈阳性荧光，但对阴性多能性标志物 SSEA-1 呈阴性。据我们所知，这是首次为巴基斯坦儿童生成 DMD-iPSC。
本研究采用无整合、无饲养层、高效、可重复的重编程方法，使用 UDC。尿液是一种低成本、非侵入性、无痛、可重复的儿童和病态个体快速扩增细胞的来源，可用于获得用于药物检测和疾病建模的自体细胞，适用于 DMD 和其他衰弱性疾病。
