P.0261 Investigation of cdh13’s role in neurodevelopmental disorders using isogenic induced pluripotent stem cell (ipsc) lines and different cdh13 snp variants

Abstract

s man IL-6 or vehicle for either 3 or 24 hours, RNA and media samples were collected. Protein samples for immunoblot-ting were collected at 15, 30, 60 and 180 minutes after IL-6 or vehicle stimulation. For secretome analysis, media samples were processed with the Bio-Techne Proteome Profiler Kit. Confirming a microglial-like phenotype, qPCR revealed increased expression of microglia signature genes MERTK (p = 0.0012 1way ANOVA) and P2RY12 (p < 0.0001; 1way-ANOVA) with longer differentiation from MGL-progenitors to mature MGLs. Receptor transcripts required for IL-6 signalling (p = 0.0009; IL6ST, p = 0.0046; 1way-ANOVA) also increased with MGL differentiation. ICC confirmed > 95% of MGL-progenitors and mature MGLs expressed both PU.1 and TMEM119 proteins. Moreover, soma size (p = 8.275x10-5) and arborization (p = 0.0022) area increased during differentiation from MGL-progenitors to mature MGLs. Exposure to IL-6 resulted in time-dependent increases in Y705-STAT-3 phos-phorylation in mature MGLs peaking at 15-30mins after IL-6 stimulation confirming activation. MGLs responded to IL-6 by increasing IL-6 expression itself (Treatment p = 0.0196; 2way-ANOVA) and the IL-6 induced secretome consisted of an efflux of specific chemokines, including MIF, MIP-1A/1B, CCL1/2, Serpin-E1 and GROa, with maximal effects observed 24 hours post-exposure. Collectively, these data confirm we can recapitulate a human mature microglia-like cell in monoculture in line with prior data [5]. These cells express the necessary receptor machinery for IL-6 signalling and IL-6 exposure induces both STAT-3 phosphorylation and increased secretion of IL-6 and pro-inflammatory chemokines. These data provide a foundation for future studies in vitro using co-cultures of hiPSC-derived MGLs with NPCs using patient derived material.