Generation and Proteome Profiling of PBMC-Originated, iPSC-Derived Corneal Endothelial Cells

Investigative Ophthalmology & Visual Science
Muhammad AliS Amer Riazuddin

Abstract

Corneal endothelial cells (CECs) are critical in maintaining clarity of the cornea. This study was initiated to develop peripheral blood mononuclear cell (PBMC)-originated, induced pluripotent stem cell (iPSC)-derived CECs. We isolated PBMCs and programmed the mononuclear cells to generate iPSCs, which were differentiated to CECs through the neural crest cells (NCCs). The morphology of differentiating iPSCs was examined at regular intervals by phase contrast microscopy. In parallel, the expression of pluripotent and corneal endothelium (CE)-associated markers was investigated by quantitative real-time PCR (qRT-PCR). The molecular architecture of the iPSC-derived CECs and human corneal endothelium (hCE) was examined by mass spectrometry-based proteome sequencing. The PBMC-originated, iPSC-derived CECs were tightly adherent, exhibiting a hexagonal-like shape, one of the cardinal characteristics of CECs. The CE-associated markers expressed at significantly higher levels in iPSC-derived CECs at days 13, 20, and 30 compared with their respective levels in iPSCs. It is of importance that only residual expression levels of pluripotency markers were detected in iPSC-derived CECs. Cryopreservation of iPSC-derived CECs did not affect the...Continue Reading

References

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Citations

Dec 8, 2019·Scientific Reports·Muhammad AliS Amer Riazuddin
Jan 26, 2021·Experimental Eye Research·Pierre Antoine FayeYohan Benayoun

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Datasets Mentioned

BETA
PXD009142

Methods Mentioned

BETA
dissection
flow cytometry
PCR
proteome sequencing
density gradient centrifugation
phase

Software Mentioned

Capture
Andromeda
Visual Annotation Display ( VLAD
MaxQuant
VLAD
Perseus
Slidebook
Adobe Photoshop

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