Generation and Differentiation of Induced Pluripotent Stem Cells from Mononuclear Cells in An Age-Related Macular Degeneration Patient
Authors: Tongmiao Wang; Jingwen Liu; Jianhua Chen; Bo Qin
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We 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 provide
potential cell sources for both basic scientific research and clinical application.
Materials and Methods
In this experimental study, PBMCs were isolated from the whole blood of a 70-year-old
female patient with AMD and reprogrammed into iPSCs by transfection of Sendai virus that contained Yamanaka
factors (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-iPSCs
exhibited full pluripotency and maintained a normal karyotype after reprogramming. AMD-iPSCs were induced into
RPE cells by stepwise induced differentiation and specific markers of RPE cells examined by immunofluorescence and
The iPSC colonies started to form on three weeks post-infection. AMD-iPSCs exhibited typical morphology
including roundness, a large nucleus, sparse cytoplasm, and conspicuous nucleoli. QPCR data showed that AMDiPSCs
expressed pluripotency markers (endo-OCT4, endo-SOX2, NANOG and REX1). Flow cytometry indicated
99.7% of generated iPSCs was TRA-1-60 positive. Methylation sequencing showed that the regions of OCT4 and
NANOG promoter were demethylated in iPSCs. EBs and teratomas formation assay showed that iPSCs had strong
differentiation 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).
A high quality iPSCs could be established from the PBMCs obtained from elderly AMD patient. The AMDiPSC
displayed complete pluripotency, enabling for scientific study, disease modeling, pharmacological testing, and
therapeutic applications in personalized medicine. Collectively, we successfully differentiated the iPSCs into RPE with
native RPE characteristics, which might provide potential regenerative treatments for AMD patients.