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。本研究旨在探讨在匹罗卡品诱导的TLE小鼠成年神经系统中，gaba能中间神经元移植到海马齿状回突触整合的调节机制。成体齿状回gaba能间神经元移植抑制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通过将神经元移植到出生在癫痫大脑环境中的颗粒细胞（GCs）上形成抑制性突触。为了验证这一假设，我们将比较三种来源的gaba能前体移植对癫痫发作的抑制和抑制性突触的形成：1)胎鼠大脑的内侧神经节突起（MGE）， 2)小鼠胚胎干细胞（mESCs）， 3)小鼠诱导多能干细胞（mIPS细胞）。鉴定胚胎mge来源的gaba能中间神经元移植到TLE小鼠齿状回的功能和突触伙伴。这一目的验证了胎儿gaba能中间神经元移植抑制癫痫发作的假设，这是由于在癫痫大脑环境中出生的GCs上形成了抑制性突触。目的1.1：观察胚胎基底前脑细胞移植对TLE小鼠癫痫发作的脑电图抑制作用。目的1.2：通过膜片钳电生理记录胚胎间神经元移植的TLE小鼠海马切片，测量逆转录病毒标记的GCs群体的突触后抑制电流。目的1.3：比较胚胎gaba能中间神经元移植到逆转录病毒标记的GCs后，TLE小鼠轴突生长和抑制性突触形成的程度。目的2：鉴定小鼠多能干细胞（mPSC）来源的gaba能中间神经元移植到TLE小鼠齿状回的功能和突触伴侣。本研究的目的是验证mpsc衍生的gaba能前体移植会抑制癫痫发作的假设，这是由于在癫痫大脑环境中出生的GCs上形成了抑制性突触。目的2.1：从具有Nkx2.1 BAC报告基因构建的mESCs中获得mge样基底前脑细胞。目的2.2：从具有Nkx2.1 BAC报告基因构建的miPS细胞中获得mge样基底前脑细胞。目的2.3：观察移植mpsc源性mge样基底前脑细胞对小鼠癫痫发作的脑电图抑制作用。目的2.4：通过膜片钳电生理记录移植mpsc来源的mge样基底前脑细胞的小鼠海马切片，测量逆转录病毒标记的GCs群体的突触后抑制电流。

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.