The basic research for the establishment of the donor for the neural transplantation with the miniature swine neuroepitherial stem cells.

胚胎10.5天（E10.5）Wister大鼠中脑神经板的组织和细胞分离培养，由妻子神经上皮干细胞（neuroepitheralstemcells，NESCs）组成。在各种条件下体外培养，并在原代培养后7天进行分析。当它们作为组织培养时，几乎所有的细胞在无血清和含血清的培养基中分化成神经丝（NF）阳性神经元，延伸长的神经突。而星形胶质细胞不表达胶质纤维酸性蛋白（GFAP）。神经化学标记物的评价，有许多谷氨酸阳性细胞，少数γ-氨基丁酸（GABA）和酪氨酸羟化酶（TH）阳性细胞。另一方面，当将它们接种在解离培养物中时，在含有成纤维细胞生长因子-2（FGF-2）的培养基中原代细胞培养7天后产生神经球。成纤维细胞生长因子2反应性神经球被巢蛋白阳性染色，并能维持。在微分条件下， ...更多信息 原代神经球细胞均分化为NF阳性神经元，而未分化为GFAP阳性细胞，分化后的神经球内有较多的谷氨酸阳性细胞，少数GABA和TH阳性细胞，与组织培养相同。结果表明，大鼠中脑神经板细胞经FGF-2处理后可保持神经元分化的趋势，并对移植到正常成年大鼠脑内的中脑神经板细胞和FGF-2反应性神经球的生物学特性进行了免疫组化分析。移植后数周内移植物在宿主脑内存活，可见大量Hu阳性神经细胞和少量GFAP阳性细胞，尤其是FGF-2反应性移植物。移植后2周可见少量nestin阳性细胞和Hu阳性神经元，但未见GFAP阳性细胞。将E17-18猪胚胎的中脑神经管组织移植物立即植入帕金森病模型大鼠纹状体内，观察数周，仅在移植后1周给予免疫抑制剂。猪源性移植物在宿主大鼠纹状体中有许多NF阳性和TH阳性细胞。电子显微镜研究显示，在供体来源的未成熟神经元或TH阳性供体细胞的移植物中传出和传入突触的形成。移植后8周，小型猪源性神经干细胞和大鼠源性神经干细胞均存活于帕金森病大鼠纹状体内。大鼠神经干细胞移植组大鼠在苯丙胺诱导的旋转实验中表现出完全恢复帕金森病的行为缺陷。另一方面，用移植前数量的百分比表示的小型猪NESC移植大鼠的安非他明诱导旋转的平均数量直到移植后4周才发生变化，但在小型猪NESC和大鼠NESC的移植物中，在移植后8周时下降到30%，许多TH阳性供体-因此，NESC是一种有吸引力的候选者，可作为脑内移植的供体材料来源。神经回路重建中的移植

The tissue and cell dissociation culture of the embryonic day 10.5 (E10.5) Wister rats mesencephalic neural plate, consisted wife neuroepitheral stem cells (NESCs). were cultured in vitro under various conditions and were analyzed 7 days after the primary culture. When they were cultured as a tissue, almost all cells differentiated into neurofilament (NF) positive neuron extending long neurites in a serum-free and serum containing medium. But, there were no astrocyte expressed glial fibrially acidic protein (GFAP). For the evaluation of the naerochemical marker, many glutamate positive cells, few γ-aminoburyric acid (GABA) and tyrosine bydroxylase (TH) positive cells were there. On the other hand, when they were plated in a dissociation culture, neurosphere generated 7 days after the primary cell culture in the medium containing fibroblast growth factor-2 (FGF-2). The FGF-2 responsive neurosphere were positively stained by nestin and able to maintain. Under the condition of differentia … More tion all cells of the primary neurosphere differentiated into NF positive neuron, not into GFAP positive cell, in the differentiated neurosphere, many glutamate positive cells few GABA and TH positive cells were there as same as the tissue culture. These indicated that the rat mesencephalic neural plate cells can keep the neuronal differentiation tendency until one week by FGF-2 administration.We also analyzed immunohisochemically the biological nature of the mesensephalic neural plate and FGF-2 responsive neurospheres transplanted into the normal adult rat brain. The viable graft in the host brain was observed for several weeks after transplantation and many Hu-positive neuronal cells and a few GFAP-positive cells were seen in the graft Especially in the FGF-2 responsive graft marry. Hu-positive neuronal cells and a few nestin-positive cells were observed, but no GFAP-positive cells could be identified 2 weeks after transplantation. These results suggested that NESCs demonstrated a propensity to differentiate into neurons in the adult brain environment.The mesencephalic neural tube tissue graft from E17-18 swine embryos implanted immediately into the strianum of the Parkinsonian model rat were observed for several weeks with 1-week administration of the immunosuppressants after transplantation only. The swine derived grafts had many NF-positive and TH-positive cells in the host rat striatum. An electron microscopic study revealed both efferent and afferent synaptic formations in the donor-derived immature neurons or TH-positive donor cells in the grafts. Myelimated axons, bath positive and negative for swine-specific NF antibody, were mingled together in the graft.Both mini-swine derived NESCs and rat derived NESCs survived in Parkinsonian rat striatum at 8 weeks after transplantation. The rat NESCs grafted rat showed complete recovery from Parkinsonian behavioral defects in amphetamine-induced rotation test. On the other hand, the mean number of amphetamine-induced rotations made by the mini-swine NESCs grafted rats expressed as a percentage of the number before grafting had not changed until 4 weeks after grafting but decreased to 30% at 8 weeks after grafted in the grafts of both mini-swine NESCs and rat NESCs, many TH positive donor-derived cells were there 2 weeks after transplantation for the immunohistolgical evaluation.NESCs are therefore an attractive candidate as a source of donor material for intracerebral grafting in neural circuitry reconstruction Less