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细胞分离培养主要由神经上皮干细胞（NESCs）组成。在不同条件下进行体外培养，并在原代培养7天后进行分析。当它们作为组织培养时，在无血清和含血清培养基中，几乎所有细胞都分化为神经丝（NF）阳性神经元，延伸出长神经突。星形胶质细胞未表达胶质纤维酸性蛋白（GFAP）。纳米化学标志物评价中，谷氨酸阳性细胞较多，γ-氨基丁酸（GABA）和酪氨酸羟化酶（TH）阳性细胞较少。另一方面，当它们被分离培养时，原代细胞在含有成纤维细胞生长因子-2 （FGF-2）的培养基中培养7天后产生神经球。FGF-2反应性神经球被巢蛋白染色呈阳性，并能维持。在分化条件下，原代神经球细胞全部分化为NF阳性神经元，未分化为GFAP阳性细胞，分化神经球中谷氨酸阳性细胞较多，GABA和TH阳性细胞较少，与组织培养相同。提示FGF-2给药后大鼠中脑神经板细胞可保持神经元分化趋势至1周。我们还分析了移植到正常成年大鼠脑中的中脑神经板和FGF-2应答神经球的免疫组化生物学性质。移植数周后，在宿主脑内观察到存活的移植物，移植物中可见许多hu阳性神经元细胞和少量gfap阳性细胞，特别是在FGF-2应答的移植物中。移植2周后，未见gfap阳性细胞，但有hui阳性神经元细胞和少量巢蛋白阳性细胞。这些结果表明，NESCs在成人大脑环境中表现出向神经元分化的倾向。将E17-18猪胚胎的中脑神经管组织移植物立即植入帕金森模型大鼠的睾丸，观察数周，仅在移植后给予免疫抑制剂1周。猪源移植物在宿主大鼠纹状体中有许多nf阳性和th阳性细胞。电镜研究显示，移植物中供体来源的未成熟神经元或th阳性供体细胞均有传出和传入突触形成。除髓的轴突，猪特异性NF抗体阳性和阴性，在移植物中混合在一起。移植后8周，迷你猪源性NESCs和大鼠源性NESCs在帕金森大鼠纹状体中均存活。在安非他明诱导的旋转实验中，移植大鼠NESCs的大鼠帕金森行为缺陷完全恢复。另一方面，迷你猪NESCs和大鼠NESCs移植后，安非他明诱导大鼠旋转的平均次数占移植前的百分比直到移植后4周才发生变化，但在移植后8周下降到30%，移植后2周有许多TH阳性的供体来源细胞用于免疫组织学评估。因此，NESCs是一种有吸引力的候选供体材料，可用于脑内神经回路重建移植

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