Astrocyte Transcriptomics in a Three-Dimensional Tissue-Engineered Rostral Migratory Stream

The glial tube is a longitudinal structure predominantly composed of densely bundled, aligned astrocytes that projects from subventricular zone (SVZ) to olfactory bulb. Neural precursor cells (NPCs) generated in the SVZ migrate through this glial tube - referred to as the rostral migratory stream (RMS) - to replace olfactory bulb interneurons in the mammalian brain. RMS astrocytes have distinct morphological and functional characteristics facilitating their unique purpose as an endogenous living scaffold directing NPC migration and maturation. However, the transcriptomic factors underlying these unique structure-function attributes versus standard stellate astrocytes have not been examined. We previously developed biofabrication techniques to create the first tissue-engineered rostral migratory stream (TE-RMS) that replicates key features of the glial tube in vivo. We have shown that TE-RMS astrocytes exhibit elongated nuclei, longitudinally aligned intermediate filaments, and enrichment of key functional proteins - cytoarchitectural and surface features characteristic of native RMS astrocytes. In the current study, we performed RNAseq on TE-RMS astrocytes in comparison to planar astrocyte cultures to identify gene expression patterns that may underlie their profound morphological and functional differences. Remarkably, we found 4008 differentially expressed genes in TE-RMS astrocytes, with 2076 downregulated (e.g. LOC690251, ccn5) and 1932 upregulated (e.g. lrrc45, cntn1) compared to planar astrocytes. Moreover, there were 256 downregulated and 91 upregulated genes with >3-fold change. We also conducted analyses of gene sets related to cytoskeleton and nuclear structure, revealing greatest enrichment of actin-related components. Overall, the TE-RMS offers a platform to study interplay between transcriptomic and cytoarchitectural dynamics in a unique astrocyte population. ### Competing Interest Statement DKC is a co-founder of two University of Pennsylvania spin-out companies concentrating in applications of neuroregenerative medicine: Innervace, Inc and Axonova Medical, Inc. There are two patent applications related to the methods, composition, and use of microtissue engineered glial networks: U.S. Patent App. 15/534,934 titled "Methods of promoting nervous system regeneration" (DKC) and U.S. Provisional Patent App 63/197,007 titled "Tissue-engineered rostral migratory stream for neuronal replacement" (DKC, EMP, JO'D).