Astrocytes are important players in neuronal maturation and activity [52, 53]. Maturation of precursors into functional neurons is therefore regularly facilitated by co-culturing with astrocytes [54]. To identify neuronal-specific changes, removing astrocytes at the analysis stages is however often desired. While we expect that astrocyte-conditioned media will not be sufficient to support neuronal maturation of low-density culture of purely neurons, we have not explored these culture settings. In the direct and indirect contact cultures used for this study, we did not observe astrocyte loss. Furthermore, we were able to generate pure hPSC-derived neuronal networks when neuronal precursors were co-cultured with rat astrocytes in the indirect contact mode. This was confirmed by mass spectrometric analysis, which revealed no presence of human glial proteins after removal of the astrocytes. When we cultured neurons in complete absence of astrocytes we noticed generation of glial cells from neuronal precursor cells. This indicates that neuronal precursors that are maturing towards functional pure neuronal networks need presence of astrocytes.
