Rapid specification of human pluripotent stem cells to functional astrocytes

Astrocytes are essential for the formation and maintenance of neural networks through metabolic support, facilitation of synaptic function, and optimization of electrophysiological activity. However, a major technical challenge for investigating astrocyte function and disease-related pathophysiology has been the limited ability to obtain functional human astrocytes. Here we present a novel method to efficiently differentiate human pluripotent stem cell (hPSC)-derived neural progenitors to functional astrocytes in 28 days using a culture medium containing leukemia inhibitory factor (LIF) and bone morphogenetic protein 4 (BMP4). This approach yields highly pure populations of astrocytes expressing canonical astrocyte markers, which we confirmed by immunofluorescence, flow cytometry and RNA sequencing. Human PSC-derived astrocytes efficiently buffer glutamate and robustly support neural network activity. Co-cultures of hPSC-derived astrocytes and neurons on multi-electrode arrays generated robust network activity within 2 days and synchronous network bursts after 6 days. Whole cell patch-clamp recordings revealed an increased frequency of postsynaptic currents in human hPSC-derived neurons co-cultured with hPSC-derived versus primary rodent astrocytes, consistent with a corresponding increase in synapse density. Furthermore, hPSC-derived astrocytes retained their hominid morphology when transplanted into a mouse brain. In conclusion, we present a novel protocol to obtain functional astrocytes from human pluripotent stem cells, providing a platform for investigating human astrocyte function and neuronal-glial interactions.