Abstract/Summary

Astrocytes are considered a default developmental state in mammalian 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 (astrogenesis) 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. Modifying previous methodology, we created a quick and efficient method to generate functional and heterogeneous astrocytes from mouse embryonic stem cells (mESC). 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 (ADM), 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, while image analysis revealed an elevation of GFAP from DIV 7 to DIV 28 post seeding. When astrocytes were stimulated with adenosine triphosphate (ATP), intracellular calcium concentration was elevated, as revealed by Fluo4. We hypothesise this is due to the activation of the P2X and P2Y purinoreceptor pathways, which will be probed further.