Developing a fully human cell culture system with IPSC-derived neurons and astrocytes that supports pre- and postsynaptic maturation and function for translatable research

Abstract

With the prospect of better translation compared to mouse-derived systems, hIPSC-derived neural networks are a promising model system for overcoming the translational gap between basic research and cognitive drug development for brain disorders. To not only target neurodevelopmental disorders but also neurodegenerative diseases, a fully human neural network capable of synapse strengthening and weakening that is supported by human astrocytes and microglia is required. Human IPSC-derived neurons have been shown to express presynaptic markers and show activity in multielectrode array recordings in many reports. But convincing postsynaptic immunostainings are rare, as are functional assays investigating pre- and postsynaptic function and maturation. In our hIPSC-derived NGN2-iNeurons co-cultured with mouse astrocytes, we see presynaptic markers sparsely distributed on day 22 that increase in number until day 65 in culture, while the post-synaptic markers PSD95 and NMDAR-1 appeared and colocalized with presynaptic terminals later, at 45 or 71 days in vitro, respectively. These iNeurons show spontaneous and evoked calcium responses, as well as FM1-43 dye release kinetics with exponential decay, indicating mature synaptic vesicle recycling, when in co-culture with mouse astrocytes. To replace mouse with human astrocytes we created and characterized a stable, doxycycline-inducible NFIB+SOX9 human IPSC line, which matures to functional astrocytes (iAstros) that can reliably support iNeurons in culture. Immunostains confirmed that our iAstros express the typical astrocyte markers S100B and GFAP in a subpopulation. With this work’s progress toward a standardized fully human IPSC-derived neural network, including glial cells, with mature neurons capable of synapse strengthening and weakening, we hope to contribute to advancing disease-related translational research.