U.S. Department of Health and Human ServicesHHS National Institutes of HealthNIH National Center for Advancing Translational SciencesNCATS

Controlled astrogliogenesis enables automated, high-throughput generation of astrocytes from human pluripotent stem cells

Posted on August 27th, 2019 by claire.malley@nih.gov

Members of SCTL presented a poster at the ISSCR 2019 and Keystone Symposia 2019 conferences. The poster is entitled: Controlled astrogliogenesis enables automated, high-throughput generation of astrocytes from human pluripotent stem cells.

Authors (underlined, presenting): Vukasin M. Jovanovic, Claire Malley, Carlos A. Tristan, Pinar Ormanoglu, Christopher P. Austin, Anton Simeonov, Ilyas Singec╠ž

Download the full size poster here.

More information about ISSCR 2019 or the Keystone Symposia 2019 meetings.

Abstract

Astrocytes play important roles in normal brain development, synaptic function, neurodegenerative diseases, and various pathological conditions (e.g. opioid addiction). Derivation of human astrocytes from a scalable source such as induced pluripotent stem cells (iPSCs) is an attractive approach for disease modeling and drug discovery; however, currently available protocols are variable, inefficient, and lengthy (lasting up to several months). Here, we developed a highly efficient and controlled astrocyte differentiation protocol that overcomes the limitations of previously published methods. By identifying and simultaneously manipulating several critical pathways, we directly induced astrogliogenesis from iPSCs with over 90% efficiency in less than 30 days. These cells displayed astrocyte morphologies and expressed typical markers such as GFAP, NF-IA and S100-B. Unlike previous protocols, our approach enabled the direct transition of pluripotent cells into PAX6+ neuroepithelia and then into BLBP+ radial glial cells in only 7 days. By day 14, radial glial cells differentiated into S100B+ astroglia, thereby largely bypassing neurogenesis, followed by NF-IA expression at day 21 as demonstrated by immunocytochemistry and time-course RNA-Seq experiments. Single-cell analysis and comparison of iPSC-derived neuroepithelia to astrocytes confirmed strong enrichment of astroglial genes and absence of genes indicative of other cell types (e.g. neurons, oligodendrocytes, microglia, endothelial cells, pluripotent cells). Importantly, iPSC-derived astrocytes were functional and capable of taking up the neurotransmitter glutamate, storing glycogen intracellularly, and promoting neuronal survival and synaptic activity when co-cultured with neurons. Finally, the differentiation protocol was automated using a robotic cell culture system, which now enables standardized production of large quantities of astrocytes for high-throughput screening and other translational applications.