Fig 2: Long-term (6 months) engraftment and neuronal and glial differentiation of hiPSC-NPCs after grafting into striata of the immunodeficient rat. (A) IF analysis of the coronal brain section showed a consistent presence of hNUMA+ grafts in the targeted striatal region. Staining with human-specific GFAP shows the homogenous distribution of grafted NPC-derived astrocytes throughout the graft. (B) Intense human-specific NSE expression (green area) with well morphologically defined human astrocytes (red signal). (C) Areas occupied with mature grafted human neurons (hNSE) show intense human-specific synaptophysin (hSYN) expression. (D) hSYN expression is primarily present in areas with a high density of grafted neuron-derived human axons (HO14+), (yellow arrows). (E) Expression of mature neuronal marker NeuN in hNUMA+ grafted neurons. (F) A high density of VGAT punctate-like expression in areas of hSYN immunoreactivity. hSYN+ terminals show co-localization with VGAT immunoreactivity and are opposed to post-synaptic gephyrin immunoreactivity (yellow arrows). (G) Presence of vimentin+ glial progenitors at the periphery of hNUMA+ graft. (H) Expression of Olig2 (oligodendrocyte marker) immunoreactivity in hNUMA+ grafted cells. (I) Hematoxylin-eosin-stained section with normally appearing graft. No hyper-cellularity (indicative of tumor formation) can be identified (I-1, I-2). (J) Quantitative analysis of neuronal and glial marker(s) expression. Scale bars: 20 µm (A–H), 10 µm (A–H; inserts). *P < 0.05; **P < 0.01, all data are presented as mean ± SEM, n = 3, two-tailed t test. DCX: doublecortin; GFAP: glial fibrillary acidic protein; hiPSCs-NPC: human induced pluripotent stem cells–derived neural precursors; hNUMA: human-specific nuclear marker; IF: immunofluorescence; NeuN: DNA-binding neuron-specific protein; SEM: standard error of the mean; VGAT: vesicular GABA transporter.

Fig 3: Induced hiPSC-NPCs generate functional neurons in vitro. Immunofluorescence staining of 2 month–induced NPCs show expression of neuronal markers including (A) NeuN/MAP2, (B) MAP2/GABA, and, (C) HO14/DCX. (D) Expression of astrocyte marker (GFAP). (E, F) Co-localization of Syn/GAD65 and Syn/VGLUT1-3 in synaptic terminals. (G) Expression of Olig2 and Vimentin in immature glial cells. (H) Ki67 (mitotic marker) is seen in a small subpopulation of cells. (I) FISH shows the presence of VGLUT2 and VGAT transcripts in a separate population of induced neurons. (J) Quantification of NeuN, GFAP, Olig2, and mitotically active Ki67+ cells. (K) Presence of spontaneous intracellular calcium oscillation in induced neurons at 4 months after differentiation. Scale bars: 50 µm (I), 10 µm (A, B, C, D, E, F, I′, I″), 5 µm (G, H). DCX: doublecortin; GFAP: glial fibrillary acidic protein; hiPSCs-NPC: human-induced pluripotent stem cells–derived neural precursors; NeuN: DNA-binding neuron-specific protein; VGAT: vesicular GABA transporter.