TY - JOUR ID - 696800 TI - Generation and Differentiation of Induced Pluripotent Stem Cells from Mononuclear Cells in An Age-Related Macular Degeneration Patient JO - Cell Journal (Yakhteh) JA - CELLJ LA - en SN - 2228-5806 AU - Wang, Tongmiao AU - Liu, Jingwen AU - Chen, Jianhua AU - Qin, Bo AD - Shenzhen Aier Eye Hospital, Shenzhen, China AD - Shenzhen Aier Eye Hospital, Shenzhen, China Y1 - 2022 PY - 2022 VL - 24 IS - 12 SP - 764 EP - 773 KW - Age-related macular degeneration KW - Differentiation KW - Induced Pluripotent Stem Cell KW - Reprogramming KW - Retinal Pigment Epithelium DO - 10.22074/cellj.2022.557559.1072 N2 - ObjectiveWe aimed to generate induced pluripotent stem cells (iPSCs)-derived retinal pigmented epithelium (RPE)cells from peripheral blood mononuclear cells (PBMCs) and age-related macular degeneration (AMD) patient to providepotential cell sources for both basic scientific research and clinical application.Materials and MethodsIn this experimental study, PBMCs were isolated from the whole blood of a 70-year-oldfemale patient with AMD and reprogrammed into iPSCs by transfection of Sendai virus that contained Yamanakafactors (OCT4, SOX2, KLF4, and c-MYC). Flow cytometry, real-time quantitative polymerase chain reaction (qPCR),karyotype analysis, embryoid body (EB) formation, and teratoma detection were performed to confirm that AMD-iPSCsexhibited full pluripotency and maintained a normal karyotype after reprogramming. AMD-iPSCs were induced intoRPE cells by stepwise induced differentiation and specific markers of RPE cells examined by immunofluorescence andflow cytometry.ResultsThe iPSC colonies started to form on three weeks post-infection. AMD-iPSCs exhibited typical morphologyincluding roundness, a large nucleus, sparse cytoplasm, and conspicuous nucleoli. QPCR data showed that AMDiPSCsexpressed pluripotency markers (endo-OCT4, endo-SOX2, NANOG and REX1). Flow cytometry indicated99.7% of generated iPSCs was TRA-1-60 positive. Methylation sequencing showed that the regions of OCT4 andNANOG promoter were demethylated in iPSCs. EBs and teratomas formation assay showed that iPSCs had strongdifferentiation potential and pluripotency. After a series of inductions with differentiation mediums, a monolayer of AMDiPSC-RPE cells was observed on day 50. The AMD-iPSC-RPEs highly expressed specific RPE markers (MITF, ZO-1,Bestrophin, and PMEL17).ConclusionA high quality iPSCs could be established from the PBMCs obtained from elderly AMD patient. The AMDiPSCdisplayed complete pluripotency, enabling for scientific study, disease modeling, pharmacological testing, andtherapeutic applications in personalized medicine. Collectively, we successfully differentiated the iPSCs into RPE withnative RPE characteristics, which might provide potential regenerative treatments for AMD patients. UR - https://www.celljournal.org/article_696800.html L1 - https://www.celljournal.org/article_696800_b276de90813035d557cefefd420737ac.pdf ER -