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Human fetal brain-derived neural stem/progenitor cells grafted into the adult epileptic brain restrain seizures in rat models of temporal lobe epilepsy.

Lee H, Yun S, Kim IS, Lee IS, Shin JE, Park SC, Kim WJ, Park KI - PLoS ONE (2014)

Bottom Line: However, NSPC grafting neither improved spatial learning or memory function in pilocarpine-treated animals.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.

View Article: PubMed Central - PubMed

Affiliation: Brain Korea 21 Plus Project for Medical Science, Yonsei University College of Medicine, Seoul, Korea.

ABSTRACT
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. In vitro, 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.

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Multipotent human NSPCs and differentiation into GABAergic neurons in culture.(A–C) Under differentiation conditions, differentiation of fractions of NSPCs into TUJ1+ neurons visualized by Texas Red (A), PDGFR-α+ oligodendrocyte progenitors identified by fluorescein (B), and GFAP+ astrocytes imaged using fluorescein (C) could be observed. Nuclei were counterstained with DAPI. (D–F) Many NSPC-derived differentiated neurons were co-labeled with TUJ1 (red) and GABA (green) with small bipolar processes. (G, H) Fractions of NSPC-derived cells also expressed CALB2 (calbindin2, green) and GAD2 (GAD65, green). Scale bar, 50 µm. (I) Western blot analysis shows that GAD1 and GAD2 were expressed highly in NSPCs under differentiation conditions. (J) The bar chart illustrates percentages of NSPCs that exhibit differentiation into TUJ1+ neurons, PDGFR-α+ oligodendrocyte progenitors, GFAP+ astrocytes, GABA+ neurons, CALB2+ neurons, and GAD2+ neurons. Quantification of the data presented in (J); mean ± SEM (n = 3).
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pone-0104092-g001: Multipotent human NSPCs and differentiation into GABAergic neurons in culture.(A–C) Under differentiation conditions, differentiation of fractions of NSPCs into TUJ1+ neurons visualized by Texas Red (A), PDGFR-α+ oligodendrocyte progenitors identified by fluorescein (B), and GFAP+ astrocytes imaged using fluorescein (C) could be observed. Nuclei were counterstained with DAPI. (D–F) Many NSPC-derived differentiated neurons were co-labeled with TUJ1 (red) and GABA (green) with small bipolar processes. (G, H) Fractions of NSPC-derived cells also expressed CALB2 (calbindin2, green) and GAD2 (GAD65, green). Scale bar, 50 µm. (I) Western blot analysis shows that GAD1 and GAD2 were expressed highly in NSPCs under differentiation conditions. (J) The bar chart illustrates percentages of NSPCs that exhibit differentiation into TUJ1+ neurons, PDGFR-α+ oligodendrocyte progenitors, GFAP+ astrocytes, GABA+ neurons, CALB2+ neurons, and GAD2+ neurons. Quantification of the data presented in (J); mean ± SEM (n = 3).

Mentions: huNSPCs have the ability to differentiate into all three neural cell types in vitro: neurons, oligodendrocytes, and astrocytes (Fig. 1A–C). At 7 days after plating of neurosphere-derived single cells under differentiation conditions, ∼61% of NSPCs had differentiated into TUJ1+ neurons, ∼2% into PDGFR-α+ oligodendrocyte progenitors, and ∼5% into GFAP+ astrocytes (Fig. 1J). To investigate whether NSPCs could differentiate into GABAergic interneurons, we examined the expression of GABAergic neuronal markers in NSPCs using Western blots and immunocytochemistry (Fig. 1D-J). Histological analysis showed that ∼26% of NSPC-derived differentiated cells expressed GABA (Fig. 1E, J), and ∼37% of NSPC-derived TUJ1+ neurons expressed GABA with small bipolar processes (Fig. 1D–F, J). Additionally, ∼30% of cells expressed interneuron subtype marker CALB2, also known as calretinin [26] (Fig. 1G, J), and ∼11% of cells expressed GABA-synthesizing enzyme, GAD2 (Fig. 1H, J). Western blot showed that protein levels of GAD1 and GAD2, the two isoforms of the GABA-synthesizing enzyme, were elevated markedly in cells under differentiation (Diff) conditions, versus proliferation (Prol) conditions (Fig. 1I).


Human fetal brain-derived neural stem/progenitor cells grafted into the adult epileptic brain restrain seizures in rat models of temporal lobe epilepsy.

Lee H, Yun S, Kim IS, Lee IS, Shin JE, Park SC, Kim WJ, Park KI - PLoS ONE (2014)

Multipotent human NSPCs and differentiation into GABAergic neurons in culture.(A–C) Under differentiation conditions, differentiation of fractions of NSPCs into TUJ1+ neurons visualized by Texas Red (A), PDGFR-α+ oligodendrocyte progenitors identified by fluorescein (B), and GFAP+ astrocytes imaged using fluorescein (C) could be observed. Nuclei were counterstained with DAPI. (D–F) Many NSPC-derived differentiated neurons were co-labeled with TUJ1 (red) and GABA (green) with small bipolar processes. (G, H) Fractions of NSPC-derived cells also expressed CALB2 (calbindin2, green) and GAD2 (GAD65, green). Scale bar, 50 µm. (I) Western blot analysis shows that GAD1 and GAD2 were expressed highly in NSPCs under differentiation conditions. (J) The bar chart illustrates percentages of NSPCs that exhibit differentiation into TUJ1+ neurons, PDGFR-α+ oligodendrocyte progenitors, GFAP+ astrocytes, GABA+ neurons, CALB2+ neurons, and GAD2+ neurons. Quantification of the data presented in (J); mean ± SEM (n = 3).
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pone-0104092-g001: Multipotent human NSPCs and differentiation into GABAergic neurons in culture.(A–C) Under differentiation conditions, differentiation of fractions of NSPCs into TUJ1+ neurons visualized by Texas Red (A), PDGFR-α+ oligodendrocyte progenitors identified by fluorescein (B), and GFAP+ astrocytes imaged using fluorescein (C) could be observed. Nuclei were counterstained with DAPI. (D–F) Many NSPC-derived differentiated neurons were co-labeled with TUJ1 (red) and GABA (green) with small bipolar processes. (G, H) Fractions of NSPC-derived cells also expressed CALB2 (calbindin2, green) and GAD2 (GAD65, green). Scale bar, 50 µm. (I) Western blot analysis shows that GAD1 and GAD2 were expressed highly in NSPCs under differentiation conditions. (J) The bar chart illustrates percentages of NSPCs that exhibit differentiation into TUJ1+ neurons, PDGFR-α+ oligodendrocyte progenitors, GFAP+ astrocytes, GABA+ neurons, CALB2+ neurons, and GAD2+ neurons. Quantification of the data presented in (J); mean ± SEM (n = 3).
Mentions: huNSPCs have the ability to differentiate into all three neural cell types in vitro: neurons, oligodendrocytes, and astrocytes (Fig. 1A–C). At 7 days after plating of neurosphere-derived single cells under differentiation conditions, ∼61% of NSPCs had differentiated into TUJ1+ neurons, ∼2% into PDGFR-α+ oligodendrocyte progenitors, and ∼5% into GFAP+ astrocytes (Fig. 1J). To investigate whether NSPCs could differentiate into GABAergic interneurons, we examined the expression of GABAergic neuronal markers in NSPCs using Western blots and immunocytochemistry (Fig. 1D-J). Histological analysis showed that ∼26% of NSPC-derived differentiated cells expressed GABA (Fig. 1E, J), and ∼37% of NSPC-derived TUJ1+ neurons expressed GABA with small bipolar processes (Fig. 1D–F, J). Additionally, ∼30% of cells expressed interneuron subtype marker CALB2, also known as calretinin [26] (Fig. 1G, J), and ∼11% of cells expressed GABA-synthesizing enzyme, GAD2 (Fig. 1H, J). Western blot showed that protein levels of GAD1 and GAD2, the two isoforms of the GABA-synthesizing enzyme, were elevated markedly in cells under differentiation (Diff) conditions, versus proliferation (Prol) conditions (Fig. 1I).

Bottom Line: However, NSPC grafting neither improved spatial learning or memory function in pilocarpine-treated animals.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.

View Article: PubMed Central - PubMed

Affiliation: Brain Korea 21 Plus Project for Medical Science, Yonsei University College of Medicine, Seoul, Korea.

ABSTRACT
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. In vitro, 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.

Show MeSH
Related in: MedlinePlus