Influence of basement membrane components on the in vitro functionality of hESC-RPE

Abstract

Purpose : Transplantation of human pluripotent stem cell (hPSC) derived retinal pigment epithelium (RPE) to treat diseases such as age-related macular degeneration is currently under way. For cell replacement therapy, RPE of uniform quality and consistent level of maturation is required with potential implications to functional cell integration and survival after transplantation. We have previously shown, that RPE basement membrane (BM) proteins, type IV collagen, laminin 521, and nidogen-1, affect the maturation of human embryonic stem cell (hESC) derived RPE. In the present study, we investigated the role of BM proteins to the functionality of the hESC-RPE focusing on the highly important Ca²⁺ signaling.

Methods : Differentiated hESC-RPE cells were seeded (2 x 10⁵ cells/cm²) on 1 µm polyethylene terephthalate (PET) culture inserts coated with collagen IV or laminin 521 alone, or in combination with and without nidogen-1. Cells were cultured from nine to twelve weeks in serum-free culture conditions after which live-cell Ca²⁺ imaging experiments and immunolabeling of RPE specific markers and Ca²⁺ channels were performed. For Ca²⁺ imaging, Fluo-4-AM was used as an indicator, and both spontaneous Ca²⁺ activity and responses to 2-minute exposures of 100 µM adenosine triphosphate (ATP) were recorded.

Results : Collagen IV coating alone resulted in a morphologically poor quality epithelium compared to the combination of collagen IV and laminin 521 with and without nidogen-1. Regarding functionality, BM protein coatings had a clear effect on both spontaneous and ATP induced intracellular Ca²⁺ activity in hESC-RPE. Preliminary results indicated that the use of laminin alone reduces the spontaneous Ca²⁺ activity, and especially the ability of cells to respond to ATP stimuli. The addition of nidogen-1 to the combination of collagen IV and laminin 521 seemed to increase the intracellular Ca²⁺ activity.

Conclusions : Collagen IV and laminin 521 are commonly used to coat cell culture surfaces for hESC-RPE. However, these proteins are typically used only individually. Our results indicate that it would be advantageous to combine these BM proteins and use extracellular matrix linker molecules such as nidogen to obtain cells with improved functionality.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.