Abstract
Purpose :
Age-related macular degeneration (AMD) is thought to be caused by atrophy of retinal pigmented epithelial (RPE) cells leading to photoreceptor degeneration, and in advanced disease stages, neovascularization in the choroid. We have developed 3D models of AMD that include cell types found in both choroid and RPE including endothelial cells, pericytes, fibroblasts, RPE, and monocytes. With the goal to make an AMD patient-specific disease model, the purpose of this work is to make a fully isogenic tissue. Here, we aim to derive monocytes from iPSCs and compare them to primary monocytes to advance the development of a fully isogenic 3D AMD model.
Methods :
iPSC derived monocytes were generated in vitro by treating attached embryoid bodies with m-CSF and IL-3. Monocytes were repeatedly harvested every 3-4 days over two weeks and were compared to primary monocytes via flow cytometry analysis. Monocytes were stained with CD45 and CD14 to indicate true monocyte phenotype, CD11b for overall macrophages, CD86/CD206 for polarized macrophage subtypes. Monocytes were differentiated into polarized macrophages via solutions with gm-CSF for M1-like or m-CSF for M2-like. Macrophages were analyzed with the same panel of surface markers.
Results :
iPSC derived monocytes showed compatible surface marker between experiments, harvests, and cell lines demonstrating reproducibility. Batches of monocytes were then compared to primary monocytes. Primary sources showed >95% CD45 and CD14 co-expression while iPSC monocytes showed CD14 expression at an average 82% from 4 harvests and 85% from 4 different iPSC lines. CD11b expression was observed at 0-4% in both populations indicating monocytes did not undergo spontaneous macrophage differentiation. When differentiated into activated macrophages, both primary and iPSC cells demonstrated M1 or M2 appropriate morphologies with CD86/CD206 protein expression.
Conclusions :
Our protocol to generate monocytes from iPSCs confirmed consistency across multiple differentiation batches by surface protein expression. This characterization also shows that our monocytes do not spontaneously differentiate into macrophages. Primary monocytes and iPSC derived monocytes showed similar marker expression, indicating the potential to replace their primary counterpart in our 3D-printed iPSC based AMD model.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.