Functional analysis of serially expanded human iPS cell-derived RPE cultures

Area of Science:

• Stem cell biology
• Retinal cell biology
• Tissue engineering

Background:

• Human induced pluripotent stem cells (hiPSCs) offer a renewable source for generating patient-specific cell types.
• Retinal pigment epithelium (RPE) cells are crucial for photoreceptor health and are a target for regenerative medicine.
• Serial expansion of cell cultures is often necessary for sufficient cell numbers in research and clinical applications.

Purpose of the Study:

• To investigate the impact of serial passaging on the cellular, molecular, and functional properties of hiPSC-derived RPE (hiPSC-RPE).
• To determine the optimal expansion limits for maintaining hiPSC-RPE characteristics for downstream applications.

Main Methods:

• Fibroblasts were reprogrammed to hiPSCs and differentiated into RPE cells.
• Pigmented hiPSC-RPE patches were dissected, dissociated, and serially passaged as monolayers.
• Gene and protein expression, morphology, and functional assays (tight junctions, calcium transients, phagocytosis) were performed at different passages and compared to human fetal RPE (hfRPE).

Main Results:

• hiPSC-RPE cultures achieved over 1000-fold expansion through serial passaging.
• Passages 1-3 (P1-P3) maintained RPE morphology, gene/protein expression, and key functions like tight junction formation, calcium signaling, and phagocytosis.
• Passage 4 (P4) hiPSC-RPE cells lost monolayer formation ability and exhibited altered characteristics.

Conclusions:

• Limited serial expansion (up to P3) of hiPSC-RPE monolayers preserves critical cellular and functional attributes.
• Passaging beyond senescence (P4) results in a loss of desired RPE properties.
• Controlled passaging of hiPSC-RPE is feasible for generating sufficient cells for in vitro disease modeling, drug screening, and transplantation.