Stem Cell Transplantation for Epilepsy

描述（由申请人提供）：我们的长期目标是推进严重颞叶癫痫（TLE）的细胞治疗。拟开展的工作研究了匹罗卡品诱导的TLE小鼠成年神经系统中移植到海马齿状回的GABA能中间神经元的突触整合调节机制。GABA能中间神经元移植到成年齿状回抑制TLE小鼠颞叶癫痫发作。我们的工作假设是，癫痫发作抑制的机制是由移植的神经元形成抑制性突触到颗粒细胞（GC）出生在癫痫脑环境。为了验证这一假设，我们将比较来自三个来源的GABA能前体移植物的癫痫抑制和抑制性突触形成：1）胎鼠脑的内侧神经节隆起（MGE），2）小鼠胚胎干细胞（mESC），和3）小鼠诱导的多能干细胞目的1：鉴定移植到TLE小鼠的齿状回中的胎儿MGE衍生的GABA能中间神经元的功能和突触配偶体。该目的检验胎儿GABA能中间神经元移植由于在出生于癫痫脑环境的GC上形成抑制性突触而抑制癫痫发作的假设。目的1.1：通过EEG评价胚胎基底前脑细胞移植对TLE小鼠癫痫发作的抑制作用。目标1.2：通过膜片钳电生理学记录，测量植入胎儿中间神经元的TLE小鼠海马切片中逆转录病毒标记的GC群体中的抑制性突触后电流。目标1.3：比较TLE小鼠中胎儿GABA能中间神经元移植到逆转录病毒标记的GC上的轴突生长和抑制性突触形成的程度。目标二：鉴定移植到TLE小鼠齿状回的小鼠多能干细胞（mPSC）衍生的GABA能中间神经元的功能和突触伴侣。该目的测试了mPSC衍生的GABA能前体移植物将由于在出生于癫痫脑环境中的GC上形成抑制性突触而抑制癫痫发作的假设。目的2.1：用Nkx2.1 BAC报告基因构建体从mESC体外衍生MGE样基底前脑细胞。目的2.2：用Nkx2.1 BAC报告基因构建体从miPS细胞体外衍生MGE样基底前脑细胞。目的2.3：通过EEG评价移植mPSC衍生的MGE样基底前脑细胞的小鼠的癫痫发作抑制。目标2.4：通过膜片钳电生理学记录，测量移植mPSC衍生的MGE样基底前脑细胞的小鼠海马切片中逆转录病毒标记的GC群体中的抑制性突触后电流。

DESCRIPTION (provided by applicant): Our long-term goal is to advance cell-based treatments for severe temporal lobe epilepsy (TLE). The proposed work examines the mechanisms regulating synaptic integration of transplanted GABAergic interneurons transplanted into the dentate gyrus of the hippocampus in the adult nervous system of mice with pilocarpine-induced TLE. GABAergic interneuron grafts made into the adult dentate gyrus suppress temporal lobe seizures in TLE mice. Our working hypothesis is that the mechanism for seizure suppression is formation of inhibitory synapses by the transplanted neurons onto granule cells (GCs) born into the epileptic brain environment. To test this hypothesis we will compare seizure suppression and inhibitory synapse formation by transplants of GABAergic precursors derived from three sources: 1) the medial ganglionic eminence (MGE) of fetal mouse brain, 2) mouse embryonic stem cells (mESCs), and 3) mouse induced pluripotent stem cells (mIPS cells) in the following two Specific Aims: Aim 1: Identify the functionality and synaptic partners of fetal MGE-derived GABAergic interneurons transplanted into the dentate gyrus of TLE mice. This aim tests the hypothesis that fetal GABAergic interneuron transplants suppress seizures due to formation of inhibitory synapses onto GCs born into the epileptic brain environment. Aim 1.1: Evaluate seizure suppression by EEG in TLE mice with transplants of fetal basal forebrain cells. Aim 1.2: Measure inhibitory post-synaptic currents in retrovirally-labeled populations of GCs by patch-clamp electrophysiological recordings in hippocampal slices from TLE mice with fetal interneuron transplants. Aim 1.3: Compare the extent of axon outgrowth and inhibitory synapse formation by the fetal GABAergic interneuron transplants onto retrovirally-labeled GCs in TLE mice. Aim 2: Identify the functionality and synaptic partners of mouse pluripotent stem cell (mPSC)-derived GABAergic interneurons transplanted into the dentate gyrus of TLE mice. This aim tests the hypothesis that mPSC-derived GABAergic precursor transplants will suppress seizures due to formation of inhibitory synapses onto GCs born into the epileptic brain environment. Aim 2.1: Derive MGE-like basal forebrain cells in vitro from mESCs with an Nkx2.1 BAC reporter construct. Aim 2.2: Derive MGE-like basal forebrain cells in vitro from miPS cells with an Nkx2.1 BAC reporter construct. Aim 2.3: Evaluate seizure suppression by EEG in mice with transplants of mPSC-derived MGE-like basal forebrain cells. Aim 2.4: Measure inhibitory post-synaptic currents in retrovirally-labeled populations of GCs by patch-clamp electrophysiological recordings in hippocampal slices from mice with transplants of mPSC-derived MGE-like basal forebrain cells.