1) Differentiation of forebrain cortical neurons and astrocytes from hiPSC-derived primitive neural stem cells:
We differentiated neural stem cells (NSCs) from hiPSCs. For neuronal differentiation, NSCs were plated in neuronal differentiation medium. At 14 days in vitro, cells exhibited neuronal morphology and were positive for the neuron-specific cytoskeletal marker β-III-tubulin and forebrain marker FOXG1. For the differentiation of astrocytes, dissociated NSCs were plated in an astrocyte differentiation medium and immunocytochemical analysis at passage 7 confirmed the expression of the astrocyte marker S100 β in cells maintained in astrocyte differentiation medium (Figure 1).
2) In vitro differentiated hiPSC-derived cortical neurons exhibit fundamental electrophysiological properties.
The functional assessment of the differentiated neurons were performed using patch-clamp recordings from individual neurons. The recordings demonstrated the presence of fast activating and inactivating inward currents and slow and sustained outward currents resembling voltage-gated Na+ (Nav) and K+ (Kv) channels. Whole-cell voltage clamp recordings revealed spontaneous postsynaptic currents (sPSCs) at 21-28 days in vitro differentiation (Figure 2).
3. Expression of P2X7Rs in hiPSC-derived neurons and astrocytes
To investigate if P2X7Rs are expressed in hiPSC-derived neurons, immunohistochemistry was performed on neurons and astrocytes. In double labelling experiments using the neuronal marker β-III tubulin and P2X7-specific affinity purified antibody, expression of P2X7Rs was evident by a punctate staining pattern. Likewise, to assess the expression of P2X7Rs on hiPSC-derived astrocytes, double immunostaining was performed on astrocytes at 4 weeks in vitro using antibodies against the astrocyte marker GFAP and P2X7 (Figure 3).
4. BzATP evokes AFC-5128-sensitive Ca2+ transients in hiPSC-derived neurons
To confirm the results obtained using immunocytochemistry, we next sought to assess whether P2X7Rs also respond to the application of P2X7-stimulating agonists. During appication of 300 μM BzATP, hiPSc-derived neurons responded to application by a discernable increase in fluorescence. In contrast, when the cells were pre-incubated with the P2X7R antagonist AFC-5128 (30 nM) pulse ejection of 300 μM BzATP and 30 nM AFC-5128 the change in [Ca2+]i was significantly reduced. Taken together, these results show that the BzATP-gated change in [Ca2+]i in hiPSC-derived neurons is mediated via the P2X7R (Figure 4).
5. BzATP evokes AFC-5128-sensitive Ca2+ transients in iPSC-derived astrocytes
Similar to neurons, we then assessed the functional expression of P2X7Rs in hiPSC-derived astrocytes by monitoring changes in [Ca2+]i upon BzATP application. To assess the functional expression of P2X7Rs, hiPSC-derived astrocytes grown on coverslips were loaded with 2 μM Cal-520 AM. Pulse ejection of BzATP (300 μM) for 5 s evoked an increase in [Ca2+]i in of hiPSC-derived astrocytes.
In support of the role for the P2X7R in mediating this response, when astrocytes were pre-incubated with the P2X7R antagonist AFC-5128, co-application of BzATP with AFC-5128 (30 nM) significantly reduced the [Ca2+]i response.
Plans for exploitation and dissemination of results: To explore the potential of the research findings, and the use of in vitro seizure model using human iPSC-derived neuron and glia as preclinical cellular models neurological drug discovery industry will be further exploited with support from the RCSI Office of Research and Innovation and Enterprise Ireland –funded activities. The market survey/feasibility study is already underway with funding from Enterprise Ireland to explore such a possibility. If a potential market is identified in this feasibility study, efforts to establish an RCSI spin-off focused on a platform for drug screening and toxicity testing using human brain-relevant cellular models of disease will be initiated.
The research findings have been submitted for publication and is under revision in an open access journal (bioRxiv doi: 10.1101/2021.03.28.437391
https://biorxiv.org/cgi/content/short/2021.03.28.437391v1(si apre in una nuova finestra))
.
