Zehravi Mehwish, Wahid Mohsin, Ashraf Junaid
Mehwish Zehravi, Dow Research Institute of Biotechnology and Biomedical Sciences, Dow University of Health Sciences, Karachi, Pakistan.
Mohsin Wahid, Dow Research Institute of Biotechnology & Biomedical Sciences and Department of Pathology, Dow International Medical College, Dow University of Health Sciences, Karachi, Pakistan.
Pak J Med Sci. 2021 Mar-Apr;37(2):432-438. doi: 10.12669/pjms.37.2.3388.
To derive Duchenne muscular dystrophy patient specific induced pluripotent stem cells (iPSCs) from CD3T cells of DMD patients using episomal reprogramming and characterization of these DMD-iPSCs using immunofluorescence to confirm their pluripotent state.
DMD patients were selected based upon their clinical history and examination. Peripheral blood mononuclear cells were isolated from peripheral blood of DMD patients (n=3) by density gradient centrifugation and were used to generate DMD patient specific T cells (DMD-T cells) using rhIL-2, plate bound anti CD3 antibody and T cell specific media along with specific culture conditions that promote T cell expansion. CD3 T cells were characterized by flow cytometry and reprogrammed using episomal plasmid to generate DMD-iPSCs. These DMD-iPSCs were characterized using immunofluorescence. The study was carried out at Dow Research Institute of Biotechnology and Biomedical Sciences during August 2017- July 2018 for a period of approximately 12 months.
The peripheral blood mononuclear cells (PBMNC) derived T cells appeared as suspended cell clumps morphologically were positive for the expression of CD3 and negative for CD31. The DMD patient specific iPSCs appeared as round, compact and flat colonies with well-defined edges morphologically and were positive for the expression of pluripotency markers OCT4, SSEA-4 and TRA-1-81 on immunofluorescence.
CD3 T cell derived DMD-iPSCs were obtained under feeder free and xeno-free culture conditions using episomal reprogramming.
利用游离型重编程从杜氏肌营养不良症（DMD）患者的CD3⁺T细胞中获得患者特异性诱导多能干细胞（iPSC），并通过免疫荧光对这些DMD-iPSC进行鉴定，以确认其多能状态。
根据临床病史和检查结果选择DMD患者。通过密度梯度离心从DMD患者（n = 3）的外周血中分离外周血单个核细胞，并使用重组人白细胞介素-2、板结合抗CD3抗体和T细胞特异性培养基以及促进T细胞扩增的特定培养条件，用于生成DMD患者特异性T细胞（DMD-T细胞）。通过流式细胞术对CD3⁺T细胞进行鉴定，并使用游离型质粒进行重编程以生成DMD-iPSC。使用免疫荧光对这些DMD-iPSC进行鉴定。该研究于2017年8月至2018年7月在陶氏生物技术与生物医学科学研究所进行，为期约12个月。
外周血单个核细胞（PBMNC）来源的T细胞在形态上表现为悬浮的细胞团块，CD3表达呈阳性，CD31表达呈阴性。DMD患者特异性iPSC在形态上表现为圆形、紧密且扁平的集落，边缘清晰，免疫荧光检测多能性标志物OCT4、SSEA-4和TRA-1-81的表达呈阳性。
使用游离型重编程在无饲养层和无外源因子的培养条件下获得了CD3⁺T细胞来源的DMD-iPSC。
