Abstract/Summary

Astrocytes are considered a default developmental state in mammalian nervous system differentiation. Even though astrocytes are involved in the maturation of the nervous system, information regarding their own development is sparse. Evidence has shown that astrocyte development (astrocytogenesis) commences after neurogenesis, leading to a heterogeneous population of astrocytes within the CNS. However, generating astrocytes in vitro has proven to be more challenging than previously thought, where complex use of morphogens, long time culture incubation (>180 days), and reduced focus on subtype astrocyte generation prevents full understanding of astrocytes. By modifying previous methodologies, here we describe a quick and efficient method to generate functional and heterogeneous astrocytes from mouse embryonic stem cells (mESC) which contributes towards ever growing need towards standardising protocols for in vitro astrocytogenesis. Under the cell suspension protocol, mESCs formed embryoid bodies (EBs), which were then inducted into a neural lineage using retinoic acid (RA) (DIV 3). At DIV 6 EBs were seeded onto laminin coated glass coverslips, in astrocyte differentiation media, containing heparin and N2. Cells morphologically resembling astrocytes were observed migrating from attached EBs, two days post seeding. Migrating cells stained positive for astrocyte markers GFAP, ALDH1L1 and S100β. When astrocytes were stimulated with adenosine triphosphate (ATP), intracellular calcium concentration was elevated, as revealed by Fluo4. Analysis of P2X and P2Y purinoreceptor pathways, revealed significant contributions of each pathways and a functional diversity of the generated astrocytes. Furthermore, these generated astrocytes displayed potential to be used as a tool for future studies via successful removal from substrate, and rescuing damaged neuron rich network. In summary, we describe here the generation of functional astrocytes derived from mESCs, which display heterogeneity in form and function that is commonly observed in astrocytes in vivo.