hMGE cell grafting substantially reduced SRS in chronically epileptic rats (CERs)

The effect of grafting hMGE progenitors expressing the Gi-protein-coupled receptor hM4Di into the hippocampus of CERs was evaluated on SRS activity in the fourth month after grafting through continuous video-electroencephalographic (video-EEG) recordings (Fig. 1). The CERs receiving grafts were immunosuppressed with daily cyclosporine A injections (10mg/kg) starting two days before grafting and continuing until the experimental endpoint to avoid transplant rejection. Ungrafted control CERs also received the same regimen of cyclosporine injections to identify any cyclosporine-induced effects on seizures. The total numbers of SRS and stage V-SRS and the total time spent in seizure activity were measured. Compared to ungrafted CERs, grafted CERs displayed substantial reductions in the number of SRS/hour (76% reduction, p<0.0001, unpaired, two-tailed Students t test Fig. 2a), number of stage V-SRS/hour (87% reduction, p<0.0001, unpaired, two-tailed Students t test, Fig. 2b), and the total time spent in seizure activity (76% reduction, p<0.0001, unpaired, two-tailed Students t test, Fig. 2c). Thus, grafting of hPSC-derived hMGE progenitors into the hippocampus in the chronic phase of TLE significantly reduced both frequency and intensity of SRS.

Quantification in the 4th month after grafting via continuous video-EEG recordings revealed that compared to the group of CERs receiving no grafts, the group of CERs receiving hMGE cell grafts displayed greatly decreased frequencies of all SRS (a) and stage V SRS (b). The grafted animals also spent much less time in seizure activity (c). df illustrate electroencephalographic (EEG) traces during the pre-clozapine-N-oxide (CNO), CNO, and post-CNO periods. Values in bar charts are presented as meanS.E.M. ****p<0.0001 (unpaired, two-tailed Students t test).

Continuous video-EEG recordings before (days 15), during (days 68), and 2 days after (days 1114) silencing the graft-derived GABA-ergic interneurons through CNO injections evaluated the influence of graft-derived interneurons in controlling SRS activity in CERs receiving grafts. Examples of EEG traces during the pre-CNO, CNO, and post-CNO periods are illustrated (Fig. 2df). Because the action of CNO is expected to last 23h after each administration and to avoid effects associated with its accumulation due to repeated administration, we administered CNO once every 8h to activate DREADDs. Also, we employed 2 days of washout period to avoid any trace amounts of CNO interfering with the post-CNO results. Silencing of graft-derived neurons substantially escalated SRS activity in CERs compared to the extent of SRS activity before CNO administration (Fig. 3ac). Overall, one-way analysis of variance (ANOVA) with the NewmanKeuls multiple comparison tests revealed that there was a 1.49.7-fold increase in the frequency of all SRS (p<0.01, Fig. 3a), 1.26.4-fold increase in the frequency of stage V-SRS (p<0.05, Fig. 3b), and 1.36.2-fold increase in the total time spent in SRS activity (p<0.05, Fig. 3c). Then, the effect of CNO washout on SRS activity was evaluated two days after the last CNO injection. All parameters of SRS activity were restored to pre-CNO levels. One-way ANOVA with Newman-Keuls multiple comparison tests showed that compared to the CNO period, the frequencies of SRS and stage V-SRS were reduced by 5771% (p<0.01, Fig. 3a, b), and the time spent in seizure activity was reduced by 60% (p<0.05, Fig. 3c).

The bar charts ac compare all SRS and stage V SRS frequencies and times spent in SRS activity (% of recorded time) during pre-CNO, CNO, and post-CNO periods. The bar charts df compare all SRS and stage V SRS frequencies and times spent in SRS activity during the pre-CNO (days 15), CNO (days 68), and post-CNO (days 1114) periods. The bar chart g compares the average electroencephalographic (EEG) power (i.e., spectral density) recorded in interictal periods during pre-CNO, CNO, and post-CNO phases. Values in bar charts are presented as meanS.E.M. *p<0.05; **p<0.01; NS, non-significant (one-way ANOVA with NewmanKeuls multiple comparisons test).

We also evaluated the seizure parameters/day in the pre-CNO (days 15), CNO (days 68), and post-CNO periods (days 1114; Fig. 3df) using one-way ANOVA with the NewmanKeuls multiple comparison tests. In CERs receiving grafts, the total SRS and stage-V SRS/day and the time spent in SRS activity/day were lower in the pre-CNO period. There was no difference in seizure activity over five days in this phase (p>0.05). The administration of CNO enhanced the total SRS and stage-V SRS/day and the time spent in SRS/day. Furthermore, comparable seizure activity was seen over the 3-day CNO period (p>0.05; Fig. 3df). The total number of all SRS on day 7 in the CNO period was higher than all SRS on pre-CNO days 15 (p<0.05; Fig. 3d). Also, the number of stage-V SRS on day 7 in the CNO period was significantly higher than stage-V SRS recorded on pre-CNO days 1 and 3 (p<0.05; Fig. 3e). Notably, all parameters of seizures/day declined in the post-CNO period after two days of CNO washout (Fig. 3df). Also, there was no difference in seizure activity during the four-day post-CNO period (p>0.05). The total numbers of all SRS on days 1214 in the post-CNO period were significantly lower than all SRS recorded on day 7 in the CNO period (p<0.05; Fig. 3d). Additionally, all parameters of seizures were comparable between pre-CNO (days 15) and post-CNO (days 1114) periods (p>0.05; Fig. 3df), implying that the inhibitory function of graft-derived interneurons is restored after the CNO washout period.

Furthermore, we performed spectral analysis of EEG activity in interictal periods by measuring randomly chosen thirty-minute duration interictal segments devoid of noise signals (610 segments/animal, n=5/group). One-way ANOVA with the Newman-Keuls multiple comparison tests revealed that compared to the pre-CNO period, the average EEG power enhanced in the CNO period (p<0.05, Fig. 3g). However, following the CNO washout, the EEG power declined substantially (p<0.05, Fig. 3g). Also, the percentage of waves is significantly reduced in the CNO period compared to the pre-CNO period (meanS.E.M., pre-CNO period 17.53.0; CNO period, 8.71.2; p<0.05) but increased following CNO washout (111.9). Overall, in addition to enhancing the frequency and intensity of SRS, silencing graft-derived GABA-ergic interneurons through CNO injections resulted in enhanced interictal activity, which subsequently waned after the CNO washout.

Next, to examine the direct effect of CNO on SRS activity, we measured SRS activity in CERs that did not receive grafts with CNO administration. One-way ANOVA with the NewmanKeuls multiple comparison tests demonstrated that the frequencies of all SRS and stage V-SRS and the time spent in SRS activity remained comparable across pre-CNO, CNO administration, and post-CNO periods (p>0.05, Fig. 4ac). Thus, in CERs receiving hMGE cell grafts, SRS activity increased when graft-derived GABA-ergic interneuron function was blocked, implying the direct involvement of graft-derived interneurons in seizure control. Furthermore, CNO alone did not affect SRS activity, as CNO administration in CERs receiving no grafts did not change all SRS and stage V-SRS frequencies or the time spent in SRS activity.

The bar charts ac compare all SRS and stage V SRS frequencies and times spent in SRS activity (% of recorded time) during the pre-CNO, CNO, and post-CNO periods in CERs receiving no grafts. Values in bar charts are presented as meanS.E.M. NS, non-significant (one-way ANOVA with NewmanKeuls multiple comparisons test).

We employed an object location test (OLT) to examine the cognitive ability of animals to detect subtle changes in their immediate environment (Fig. 5a), a function linked to normal network activity in the hippocampus36,37. In OLT, the animals with altered hippocampal circuitry/dysfunction consistently show an inability to detect minor alterations in the environment. Naive control rats recognized the change that occurred in the position of one of the objects by exploring the object in the novel place (OINP) for significantly greater periods than the object that remained in the familiar place (OIFP, p<0.0001, unpaired, two-tailed Students t test, Fig. 5b) in trial-3 (T3). In contrast, CERs receiving no grafts showed impaired cognitive function by spending nearly equal amounts of their object exploration time with the OINP and the OIFP (p>0.05, unpaired, two-tailed Students t test, Fig. 5c). Notably, CERs receiving hMGE cell grafts behaved similarly to naive control rats by showing a greater affinity for the OINP than the OIFP (p<0.01, unpaired, two-tailed Students t test, Fig. 5d). These results suggest that grafting of hPSC-derived hMGE cells into the hippocampus could alleviate chronic epilepsy-related object location memory impairment.

a depicts the various trials involved in an object location test (OLT). The bar charts in be compare percentages of time spent with the object in the familiar place (OIFP) and the object in the novel place (OINP) in naive control rats (b), chronically epileptic rats (CERs; c), and CERs with hMGE grafts before and during the clozapine-N-oxide (CNO) treatment (d, e). The bar chart in f compares the time spent with the OINP across the four groups with ANOVA. Object location memory was impaired in CERs with no grafts and CERs with grafts when graft-derived interneurons were silenced. g shows the various trials involved in a pattern separation test (PST). The bar charts in hk compare percentages of time spent with the familiar object on pattern 2 (FO on P2) and the novel object on pattern 2 (NO on P2) in naive control rats (h), CERs (i), and CERs with hMGE grafts before and during the clozapine-N-oxide CNO treatment (j, k). The bar chart in l compares the time spent with the NO on P2 across the four groups with ANOVA. Note that pattern separation ability was impaired in CERs with no grafts. However, CERs with grafts displayed pattern separation ability even when the graft-derived interneurons were silenced with CNO. Values in bar charts are presented as meanS.E.M. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, NS, non-significant (be, hk, unpaired, two-tailed Students t test; f, l, one-way ANOVA with NewmanKeuls multiple comparisons test).

To investigate the role of graft-derived GABA-ergic interneurons in the object location memory task, we silenced the transplant-derived DREADDs expressing interneurons through CNO administration and performed the OLT with new objects. With the silencing of transplant-derived interneurons, CERs lost their ability to distinguish the OINP from the OIFP, which was evident from their exploration of OINP and OIFP for almost equal periods (p>0.05, unpaired, two-tailed Students t test, Fig. 5e). The parameters such as total object exploration times, distances traveled, or movement velocities were comparable between the pre-CNO and CNO periods (data not illustrated). Comparison of the time spent with the OINP across groups (naive, CERs, CERs + grafts in the pre-CNO and CNO periods) using one-way ANOVA with the Newman-Keuls multiple comparison tests revealed impaired object location memory in CERs with no grafts, and CERs with grafts when graft-derived interneurons were silenced (Fig. 5f). However, in the absence of CNO, CERs with grafts displayed similar object location memory as naive control rats. Thus, graft-derived GABA-ergic interneurons in CERs directly influenced the object location memory function, a hippocampus-dependent cognitive task.

The pattern separation test (PST) examines proficiency in discriminating similar experiences by storing similar representations in a non-overlapping manner and is linked to the dentate gyrus and adult hippocampal neurogenesis38,39. For this test, the movement of each rat was investigated in an open field with four successive trials (5min each), separated by 30-min intervals. The first three trials successively involved the exploration of an open field apparatus (T1), a type of identical objects placed on a floor pattern 1 (P1; T2), and the second type of identical objects placed on a floor pattern 2 (P2; T3). In T4, the animal explored objects on P2, with one of the objects from T3 replaced with an object from T2. The object from T2 is a novel object on pattern 2 (NO on P2), whereas the object retained from T3 is a familiar object on P2 (FO on P2) (Fig. 5g). Naive control animals displayed a greater propensity to explore the NO on P2 than the FO on P2 in T4 (p<0.001, unpaired, two-tailed Students t test, Fig. 5h). CERs receiving no grafts displayed a pattern separation deficit, which was evident from their lack of interest in exploring the NO on P2 for higher periods than the FO on P2 (p>0.05, unpaired, two-tailed Students t test, Fig. 5i). In contrast, CERs receiving grafts showed similar behavior as naive control animals by displaying a greater propensity to explore the NO on P2 than the FO on P2 (p<0.01, unpaired, two-tailed Students t test, Fig. 5j). Thus, grafting of hPSC-derived hMGE cells into the hippocampus alleviated chronic epilepsy-induced pattern separation dysfunction.

To determine whether graft-derived interneurons played a role in restoring the pattern separation function in CERs, we silenced the transplant-derived DREADDs expressing interneurons through CNO administration and performed the PST with new objects and floor patterns. With the silencing of transplant-derived interneurons, CERs did not lose their ability to distinguish the NO on P2 from the FO on P2, which was evident from their exploration of the NO on P2 for higher periods than FO on P2 (p<0.01, unpaired, two-tailed Students t test, Fig. 5k). Furthermore, the parameters such as total object exploration times, distances traveled, or movement velocities were comparable between the pre-CNO and CNO periods (data not illustrated). Comparison of the time spent with the NO on P2 across groups (naive, CERs, CERs + grafts in the pre-CNO and CNO periods) using one-way ANOVA with the Newman-Keuls multiple comparison tests revealed impaired pattern separation function in CERs with no grafts, but not in CERs with grafts even when graft-derived interneurons were silenced (Fig. 5l). Thus, CERs with grafts displayed similar pattern separation ability as naive control rats in the absence and presence of CNO, implying that graft-derived GABA-ergic interneurons in CERs did not directly influence the pattern separation function.

Stereological quantification of HNA+ cells per hippocampus revealed that the overall graft cell yield is >800,000 cells/hippocampus (meanS.E.M=886,26655,967, n=4). Since the graft cell yield per hippocampus was higher than the number of cells initially injected (~300,000 live cells in 3 grafts, ~100,000 cells/graft), the results implied that the grafted progenitors divided a few times after grafting as some donor cells likely die during transplantation.

To confirm DREADD expression in transplant-derived cells, we performed immunofluorescence studies on tissue sections through the hippocampus to visualize human nuclear antigen (HNA, a marker of grafted human cells) and neuron-specific nuclear protein (NeuN, a marker of neurons). Confocal microscopic analyses of HNA and mCherry (the reporter of DREADD expression) revealed that virtually all HNA+ cells in grafts expressed DREADDs (Fig. 6ac). Similar analysis of NeuN and mCherry showed that all neurons within grafts expressed DREADDs (Fig. 6df). The hESC line employed in the study was built by inserting a construct of DREADD and mCherry separated by 2A. Furthermore, the expression of DREADD and mCherry in the cell line is under the control of the universal CAG promoter, and hence mCherry is expressed stably in all cells. Earlier grafting studies have demonstrated similar results using this cell line35,40.

Note that mCherry is displayed in virtually all HNA+ graft-derived cells (ac), NeuN+ neurons (df), and GABA-ergic interneurons (jl). gi demonstrate that a vast majority (meanS.E.M, 80.81.1%) of HNA+ graft-derived cells differentiated into GABA-ergic interneurons. Scale bars: al, 20m.

Next, we determined the differentiation of graft-derived cells into NeuN+ neurons or GABA+ interneurons through HNA and NeuN, or HNA and GABA dual immunofluorescence and Z-section analysis in a confocal microscope. Such quantification demonstrated that ~85% of HNA+ expressed NeuN (meanS.E.M=85.21.2, n=6) and ~81% of HNA+ cells expressed GABA (meanS.E.M=80.81.1%, n=6). Examples of hMGE cells differentiating into GABA-ergic interneurons are illustrated (Fig. 6gi). The overall differentiation is consistent with our earlier grafting study using hiPSC-derived MGE cells as donor cells in an SE model22. Next, to confirm the expression of DREADDs in graft-derived GABA-ergic interneurons, we examined mCherry expression in these interneurons. Virtually all GABA-ergic interneurons expressed mCherry (Fig. 6jl). In addition, transplanted hMGE cells also differentiated into subclasses of GABA-ergic interneurons expressing PV or NPY, which also displayed DREADDs (Fig. 7af). These results suggest that CNO administration could block the function of graft-derived interneurons because of their robust expression of DREADDs.

Gi-protein-coupled receptor hM4Di expression (with mCherry reporter) in parvalbumin (PV) and neuropeptide Y (NPY) expressing interneurons derived from human medial ganglionic eminence progenitor cell grafts in the hippocampus of chronically epileptic rats (af), and putative synapse formation between graft-derived axons and host neurons (gp). Note that mCherry is apparent in PV and NPY+ interneurons derived from graft-derived cells (af). g, l illustrate putative synapse formation between graft-derived presynaptic boutons (green colored structures expressing human synaptophysin (hSyn) and the host postsynaptic density protein 95 (PSD95, red particles) elements on microtubule-associated protein-2 (MAP-2) positive dendrites (blue) in the host CA1 stratum radiatum (g) and the dentate gyrus molecular layer (l). h, m are magnified views of boxed regions in g, l, respectively. ik, np illustrate MAP-2, hSyn, and PSD95 elements in red, green, and blue channels. Scale bars: af, 20m; g, l, 5m; ik, np, 0.5m.

Enhanced frequency and intensity of SRS following silencing of graft-derived GABA-ergic interneurons expressing DREADDs implied connectivity between hMGE graft-derived GABA-ergic interneurons and the host neurons. To confirm this, we employed Z-section analyses in a confocal microscope of brain tissue sections through the hippocampus processed for triple immunofluorescence to localize the human-specific synaptophysin (hSyn, the presynaptic protein in graft-derived neurons), postsynaptic density protein-95 (PSD-95), and microtubule-associated protein-2 (MAP-2) in soma and dendrites of host neurons. Such analysis suggested the formation of putative synaptic contacts by graft-derived neurons on the dendrites of host CA1 pyramidal neurons in the stratum radiatum (Fig. 7g) and dentate granule cells in the molecular layer (Fig. 7l). Magnified views showing the possible contacts between the presynaptic component derived from graft-derived interneurons (h-Syn+ structures in green) and the host postsynaptic component (PSD95+ structures in blue) on the dendrites of CA1 pyramidal neurons and dentate granule cells (in red) are illustrated (Fig. 7hk, mp). In addition, hSyn+ structures were also seen on the soma of dentate granule cells. Thus, transplanted GABA-ergic interneurons appeared to have integrated synaptically with the host neurons in the dentate gyrus and the CA1 subfield. Such synaptic connectivity likely explains the control of seizures and object location memory task by transplant-derived GABA-ergic interneurons.

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