A Defined and Scalable Peptide-Based Platform for the Generation of Human Pluripotent Stem Cell-Derived Astrocytes.

Abstract

Astrocytes comprise the most abundant cell type in the central nervous system (CNS) and play critical roles in maintaining neural tissue homeostasis. In addition, astrocyte dysfunction and death has been implicated in numerous neurological disorders such as multiple sclerosis, Alzheimer’s disease, amyotrophic lateral sclerosis (ALS), and Parkinson’s disease (PD). As such, there is much interest in using human pluripotent stem cell (hPSC)-derived astrocytes for drug screening, disease modeling, and regenerative medicine applications. However, current protocols for generation of astrocytes from hPSCs are limited by the use of undefined xenogeneic components and two-dimensional (2D) culture surfaces, which limits their downstream applications where large-quantities of cells generated under defined conditions are required. Here, we report the use of a completely synthetic, peptide-based substrate that allows for the differentiation of highly pure populations of astrocytes from several independent hPSC lines, including those derived from patients with neurodegenerative disease. This substrate, which we demonstrate is compatible with both conventional 2D culture formats and scalable microcarrier (MC)-based technologies, leads to the generation of cells that express high levels of canonical astrocytic markers as well as display properties characteristic of functionally mature cells including production of apolipoprotein E (ApoE), responsiveness to inflammatory stimuli, ability to take up amyloid-β (Aβ), and appearance of robust calcium transients. Finally, we show that these astrocytes can be cryopreserved without any loss of functionality. In the future, we anticipate that these methods will enable the development of bioprocesses for the production of hPSC-derived astrocytes needed for biomedical research and clinical applications.