Fig. 2

Differentiation of hypoimmunogenic hPSCs into electrophysiologically mature neuronal networks. A Schematic of the protocol and stages for neural differentiation of hPSCs. B Representative neural tube-like rosette structures formed by PAX6⁺ neuroepithelial cells. Scale bar, 100 μm. C FACS quantification showing the percentage of PAX6⁺ cells on day 12 neural differentiation of WT and hypoimmunogenic hPSCs. Data are presented as mean ± SEM. n.s., no significance, n = 3, Student’s t test. D Bright-field images showing neuron morphology derived from WT and hypoimmunogenic hPSCs. Scale bar, 25 μm. E Representative immunofluorescence images of TUJ1, SYN1 and MAP2 in WT and hypoimmunogenic hPSCs-derived neurons. Scale bars, left, 25 µm; middle, 5 µm; right, 25 µm. F Quantification of the percentage of TUJ1⁺ cells in E. Data are presented as mean ± SEM. n.s., no significance; n = 3, Student’s t test. G MLR analysis showing relative proliferation of PBMCs after coculturing with neurons differentiated from WT, B2M^null, B2M^mHLAG, and B2M^m/sHLAG hPSCs. PBMCs cultured only were used as a negative control (NC). Data are presented as mean ± SEM. **p < 0.01, n = 3, Student’s t test. H Representative traces from neurons derived from hypoimmunogenic and WT hPSCs firing repetitive APs during depolarizing constant-current injections. Current step is shown in the bottom panel. I Violin plots of frequency–current (F-I) among firing neurons (current injection = 0 and 40 pA). Data are presented as mean ± SEM. ** p < 0.01, n.s., no significance, n (WT) = 8, n (B2M^null) = 5, n (B2M^mHLAG) = 11, n (B2M^m/sHLAG) = 6, Student’s t test. J, K Resting membrane potentials (J) and peak amplitudes (K) of neurons derived from hypoimmunogenic and WT hPSCs. Parameters were calculated from the first evoked spike. Data are presented as mean ± SEM. For resting membrane potentials, n (WT) = 9, n (B2M^null) = 5, n (B2M^mHLAG) = 7, n (B2M^m/sHLAG) = 6. For peak amplitudes, n (WT) = 9, n (B2M^null) = 5, n (B2M^mHLAG) = 9, n (B2M^m/sHLAG) = 6. n.s., no significance, Student’s t test. L Left, representative voltage-clamp recording from neurons derived from hypoimmunogenic and WT hPSCs with spontaneous synaptic input (Vm = −70 mV). Right, zoom-in of the region in left marked by the red five-pointed star, containing one postsynaptic event. M, N Amplitudes (M) and frequencies (N) of spontaneous postsynaptic currents of neurons derived from hypoimmunogenic and WT hPSCs. Data are represented as mean ± SEM. n.s., no significance, n (WT) = 10, n (B2M^null) = 9, n (B2M^mHLAG) = 16, n (B2M^m/sHLAG) = 8, Student’s t test