Human Fetal Brain-Derived Neural Stem/Progenitor Cells Grafted into the Adult Epileptic Brain Restrain Seizures in Rat Models of Temporal Lobe Epilepsy

<div><p>Cell transplantation has been suggested as an alternative therapy for temporal lobe epilepsy (TLE) because this can suppress spontaneous recurrent seizures in animal models. To evaluate the therapeutic potential of human neural stem/progenitor cells (huNSPCs) for treating TLE, we transplanted huNSPCs, derived from an aborted fetal telencephalon at 13 weeks of gestation and expanded in culture as neurospheres over a long time period, into the epileptic hippocampus of fully kindled and pilocarpine-treated adult rats exhibiting TLE. <i>In vitro</i>, huNSPCs not only produced all three central nervous system neural cell types, but also differentiated into ganglionic eminences-derived γ-aminobutyric acid (GABA)-ergic interneurons and released GABA in response to the depolarization induced by a high K⁺ medium. NSPC grafting reduced behavioral seizure duration, afterdischarge duration on electroencephalograms, and seizure stage in the kindling model, as well as the frequency and the duration of spontaneous recurrent motor seizures in pilocarpine-induced animals. However, NSPC grafting neither improved spatial learning or memory function in pilocarpine-treated animals. Following transplantation, grafted cells showed extensive migration around the injection site, robust engraftment, and long-term survival, along with differentiation into β-tubulin III⁺ neurons (∼34%), APC-CC1⁺ oligodendrocytes (∼28%), and GFAP⁺ astrocytes (∼8%). Furthermore, among donor-derived cells, ∼24% produced GABA. Additionally, to explain the effect of seizure suppression after NSPC grafting, we examined the anticonvulsant glial cell-derived neurotrophic factor (GDNF) levels in host hippocampal astrocytes and mossy fiber sprouting into the supragranular layer of the dentate gyrus in the epileptic brain. Grafted cells restored the expression of GDNF in host astrocytes but did not reverse the mossy fiber sprouting, eliminating the latter as potential mechanism. These results suggest that human fetal brain-derived NSPCs possess some therapeutic effect for TLE treatments although further studies to both increase the yield of NSPC grafts-derived functionally integrated GABAergic neurons and improve cognitive deficits are still needed.</p></div

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hNSCs express diverse trophic factors. (A) In vitro proliferating and differentiated hNSCs expressed BDNF, NTF3, NTF4, NGF, VEGF, FGF2, and GDNF. (B) Western blotting analysis showed that hNSCs secreted higher levels of BDNF, NTF3, NTF4, NGF, and VEGF into the culture medium than human foreskin fibroblasts secrete. (TIFF 127 kb

Differentiation of human NSPCs into GABAergic neurons in the hippocampus of kindled rats.

<p>(A–J) BrdU⁺ grafted cells (green, A, C, D, F, G, I, J), were co-labeled with GABA (red, B, C, E, F, H–J), in the molecular and granular layer of the dentate gyrus, hilus of the hippocampus (A–C), and the radiatum layer of the CA3 region (D–J) of rats. (G–I) A large fraction of BrdU⁺ grafted cells co-expressed GABA, as viewed under the dual-filter microscope. (J) Orthogonal view from confocal <i>z</i>-series visualized co-expression of BrdU (green) and GABA (red) in a grafted cell. (K–N) Orthogonal images show that hNP or hNuc⁺ grafted cells (red) co-expressed GABA (green) in different layers of the CA3: the stratum lucidum (K), stratum pyramidale (L and M), and stratum oriens (N). (O–Q) A BrdU⁺ grafted cells (green) were also co-labeled with calbindin2 (CALB2, red) in the stratum oriens around the CA3 area. Scale bar, 200 µm (A), 100 µm (D), 50 µm (G), 10 µm (J), 20 µm (K), 10 µm (O).</p

Differentiation of human NSPCs following transplantation into the hippocampus of kindled rats.

<p>(A–F) BrdU⁺ grafted cells (green) were co-stained with TUJ1 (red, arrowheads in B, C, E, F) in the CA3 region of the hippocampus (A–C) and fimbria (D–F) in rats. (G–J) hNP⁺ grafted cells (red, arrowheads in G, I, J) were co-localized with TUJ1 (green, arrowheads in H–J) in the hilus of the hippocampus. (J) Orthogonal view from confocal <i>z</i>-series showed that hNP (red) in nuclei and TUJ1 (green) in cytoplasm were expressed in the same cell. (K, M) Under the dual-filter microscope, BrdU⁺ grafted cells co-expressed APC-CC1 in the fimbria (arrows in K), and hNuc⁺ grafted cells co-expressed GFAP in the CA3 region (an arrow in M). Scale bar; 200 µm (A), 20 µm (D), 10 µm (G, J), 20 µm (K).</p

Effect of human NSPC grafting on aberrant mossy fiber sprouting in pilocarpine-treated animals.

<p>(A–F) Timm staining exhibits mossy fiber staining from age-matched intact control (A, D), vehicle-injected pilocarpine-treated (B, E) and huNSPCs-transplanted pilocarpine-treated rats (C, F) 3 months following transplantation. D–F are high-magnification views of areas indicated by arrows in A–C, respectively. Arrowheads in D–F indicate the supragranular region of the dentate gyrus. Pilocarpine-treated rats reveal mossy fiber sprouting with an increased density of Timm granules in the supragranular region (B, C, E, F), whereas control rats did not (A, D). Scale bar, 500 µm (C), 100 µm (F). (G) NSPC-transplanted and vehicle injected rats had higher Timm scores than the control group (*<i>P</i><0.05). Error bars, SEM. No significant difference regarding Timm score was found between NSPC- and vehicle-injected groups.</p

Transcript expression profiling of human NSPCs.

<p>(A–F) Expression patterns of genes associated with development and function of GABAergic neurons were analyzed in NSPCs under proliferation (Prol) and differentiation conditions (Diff) using quantitative RT-PCR. The expression levels of each mRNA expression were normalized to levels of GAPDH. Panels show: telencephalic (A), ventral telencephalic GABAergic neuronal lineage (B), medial ganglionic eminence (MGE) (C), caudal ganglionic eminence (CGE) (D), GABAergic neuron (E), and interneuron (IN) subtype markers (F). The expression levels of makers were significantly elevated under Diff condition compared to under Prol condition except <i>OLIG2</i>. Data represented as mean ± SEM (n = 3).</p

Engraftment and distribution of human NSPCs into the hippocampus of kindled rats.

<p>(A–E) Serial sections (320 µm apart) throughout the hippocampus were used to determine the location of BrdU⁺ grafted cells, visualized with fluorescein at 4 weeks following transplantation. A–E panels are representative images of serial coronal sections of the hippocampus which are ordered from anterior to posterior. Grafted cells had migrated apart from the injection site (arrowhead in C) and dispersed throughout the adjacent hippocampal subfields. Many grafted cells were predominantly placed in the radiatum layer (Rad) of the CA3 region, molecular (ML) and granular cell layer (GCL) of the dentate gyrus (DG), and hilus (HI) of the hippocampus. Dotted lines denote the boundary between HI and GCL of the DG. In panel C, the boxed regions of the Rad (1), and ML, GCL and HI (2) are magnified in panel C-1 and C-2, respectively. Scale bar, 500 µm (A–E) and 100 µm (C-1, C-2). (F) The bar chart illustrates the distribution of grafted cells versus rostrocaudal distances to injection site at 4 and 8 weeks after transplantation. The number of grafted cells/distance of serial sections was expressed as a percentage of the total number of surviving cells. Values are represented as mean ± SEM.</p

Additional file 8: Table S1. of Human neural stem cells alleviate Alzheimer-like pathology in a mouse model

List of primary antibodies used in immunohistochemistry (IHC) and western blot (WB). (DOCX 28 kb

HPLC analysis for GABA in human NSPCs.

<p>(A) huNSPCs contain GABA under both proliferation (Prol) and differentiation (Diff) conditions in culture. Note that the total intracellular GABA content of NSPCs was significantly higher under Diff conditions than under Prol conditions. (B, C) NSPCs under Diff conditions were incubated in basal (4 mM KCl) or high K⁺ (53 mM KCl) medium, and intracellular GABA content (B) and GABA release into the medium (C) were quantified. * Significantly different from that under Prol conditions at <i>P</i><0.05; † significantly different from that in the basal medium at <i>P</i><0.05; error bars indicate ±SEM.</p

Effect of human NSPC grafting on hippocampal-kindled seizures in rats.

<p>The mean values of afterdischarge duration (ADD) in electroencephalograms (A), behavioral seizure duration (B), and seizure stage (C) between vehicle-injected and NSPC-transplanted groups were compared before (pre-Tx) and after NSPC grafting (post-Tx). Error bars indicate ±SEM. huNSPC grafting significantly reduced all three seizure parameters—ADD, behavioral seizure duration, and seizure stage—although this seizure-suppressing effect was not permanent. * Significantly different from the vehicle-injected group at <i>P</i><0.05; ** significantly different from the vehicle-injected group at <i>P</i><0.01.</p