Differentiation and Functional Incorporation of Embryonic Stem Cell-Derived GABAergic Interneurons in the Dentate Gyrus of Mice with Temporal Lobe Epilepsy

mRNA expression profiles for Sox1-GFP ESNPs. A, The expression of transcription factor mRNA increases from day 2 to day14, except for Oct 4, a transcription factor expressed by pluripotent stem cells, which decreases during this period. B, RT-qPCR analyses of MAP2, Dlx2, GAD2, and Pax6 mRNA expression at different stages of differentiation were compared with expression profiles of pluripotent ES cells on day 2. Map2 mRNA increased by 143-fold by day 14, Pax6 mRNA increased 53-fold, and Dlx2 mRNA increased 103-fold. Strikingly, GAD2 mRNA increased 540-fold. Significance values were calculated by ANOVA; *p < 0.1 between days 2 and 14; **p < 0.05 and a significant difference comparing day 2 with day 14; ^#p < 0.1, significant difference comparing day 11 with day 14; ^##p < 0.05, significant difference in expression between days 11 and 14. White, Day 2; black, day 9; light gray, day 11; dark gray, day 14.

FISH analyses show that transplanted ESNPs in the dentate gyrus differentiate into GABAergic interneurons. A–J, Approximately 90% of the transplanted cells differentiated into neurons, ascertained by immunofluorescent staining for NeuN (87.4 ± 2.6% NeuN⁺ cells; n = 6). B–J, Magnified views of boxed regions in A show NeuN-expressing transplanted cells. K–O, GAD1/2 mRNA expression was detected in nearly half of the transplanted cells (48.9 ± 3.0%; n = 6), as assessed by FISH. Arrowheads indicate RFP⁺ cells expressing GAD1/2 mRNA. Scale bars: A, 50 μm; B–J, L–O, 20 μm; K, 200 μm.

ESNPs differentiate into neurochemically distinct interneuron types after transplantation into the hilus of the dentate gyrus. A–D, Confocal images of RFP⁺ ESNP-derived neurons in the dentate gyrus. Approximately 5% of transplanted cells express the calcium-binding protein parvalbumin (4.6 ± 0.7%; n = 4; arrowheads). E–H, Approximately 8% express calbindin indicated by arrowheads (7.5 ± 0.7%; n = 5). Rare cells coexpress calbindin and ctip2 (arrows), suggesting that some ESNPs differentiate into excitatory, glutamatergic neurons. I–K, Nearly 10% (9.3 ± 0.4%, n = 4) of the cells in hilar transplants expressed calretinin (arrows). L, Quantification of the neurochemical phenotypes ascertained for ESNP-derived neurons. Scale bars: A–K, 20 μm.

Transplanted cells displace mossy fibers in the hilus but fail to suppress MFS in the inner molecular layer. Dual immunohistochemical staining for transplanted ESNP-derived neurons in RFP (red) and mossy fibers expressing ZnT-3 (green) was used to evaluate MFS in the hilus and iml of the dentate gyrus in TLE mice. A, MFS is not observed in the granule cell layer or iml in normal mice without prior seizure experience, B, TLE mice show moderate MFS by 6 weeks after SE after control stereotaxic injections of cell-culture medium devoid of ESNPs. C, More than 3 months after SE, much more extensive MFS in the iml is typical (five of eight TLE cases showed extensive MFS in iml). In the hilus, however, mossy fibers rarely overlapped with ESNP-derived neuronal arbors, and the mossy fiber projections were often displaced in the vicinity of the graft cores (n = 8). D, H, Typically, RFP⁺ axons from ESNP transplants invaded the iml and overlapped extensively with mossy fibers (n = 5 of 8). E–G, I–K, L, M–O, Only three of eight mice with transplants exhibited reduced MFS (L), despite extensive innervation of the iml by the transplanted neurons in all cases (E–G, I–K, M–O). hi, Hilus; gcl, granule cell layer. E–G, I–K, M–O are magnified views of boxed regions in D, H, and L, respectively. Arrowheads in E–G, I–K, M–O represent the outer limits of ZnT₃ staining. Scale bars: A–C, 50 μm; D, H, L, 200 μm; E–G, I–K, M–O, 100 μm.

Locations of ESNP-derived neurons that were electrophysiologically characterized. Whole-cell current- and voltage-clamp recordings were performed 55–81 d after transplantation. A–C, Transplanted cells were selected in the red fluorescent channel, and the recordings were made under DIC optics. The arrows point to an RFP⁺ neuron in contact with a patch pipette. C, Overlay of the red fluorescent view and DIC view after obtaining a gigaohm seal of the RFP⁺ neuron. D, Diagram showing locations of the neurons that were recorded and filled with biocytin. Transplanted neurons were located in the dentate gyrus, with one exception in the stratum oriens of CA1 (cell 19). The morphologies of 15 neurons were examined after biocytin staining. Scale bars: A–C, 50 μm.

The majority of neurons displayed electrophysiological characteristics of endogenous hippocampal GABAergic interneurons. Top, Neurons are classified based on the presence (types I and II) or absence of I_h current (types III, IV, and V). Types I and II are distinguished by AP drop and AP amplitude. Types III and IV are distinguished by the maximum number of APs in response to depolarizing currents. The type V category contains one cell with a large AP half-width recorded from the granule cell layer. A, Type I cells exhibit I_h currents (arrow) and prominent AP drops. B, Type II cells exhibit I_h currents (arrow) and significantly smaller APs compared with other types. This type also exhibits trains of fast APs. C, Type III neurons resemble type I cells, but without I_h currents. D, Type IV neurons exhibit small AP drops and fast-spiking APs without I_h currents. E, Type V neurons are found in the granule cell layer of the dentate gyrus and resemble granule neurons based on AP half-width and small fast afterhyperpolarizations. F, A GIN neuron recorded from the hilus exhibits firing patterns similar to those from ESNP-derived transplants. Calibration is as indicated.

Electrophysiology of transplanted neurons into the host brain circuitry. A, The top trace shows a 2 s sample from a voltage-clamp recording at −70 mV demonstrating spontaneous EPSPs in a transplanted neuron. In the bottom traces, for comparison, spontaneous EPSPs were recorded from an endogenous somatostatin-expressing interneuron in a GIN mouse. B, Enlarged views of the individual EPSCs from neurons in A are presented in the highlighted region. Calibration is as indicated.