Yang Jenn-Rong, Liao Chia-Hsin, Pang Cheng-Yoong, Huang Lynn Ling-Huei, Lin Yu-Ting, Chen Yi-Ling, Shiue Yow-Ling, Chen Lih-Ren
Division of Physiology, Livestock Research Institute, Council of Agriculture, Executive Yuan, Tainan, Taiwan, ROC.
Cell Reprogram. 2010 Aug;12(4):447-61. doi: 10.1089/cell.2009.0078.
This study was conducted to direct porcine embryonic stem (pES) cells differentiating into neural lineages and to investigate therapeutic potential of GFP-expressing pES (pES/GFP(+)) in the rat model of Parkinson's disease (PD). Directed differentiation of pES into neural lineages was induced by suspension culture in medium containing RA, SHH, and FGF combinations without going through embryoid body formation. A high yield of nestin-expressing neural precursors was found in all treatments on day 2 after the 12-day induction. On day 6 after replating, more than 86.2 and 83.4% of the differentiated cells stained positively for NFL and MAP2, respectively. The expression of TH, ChAT, and GABA specific markers were also observed in these NFL-positive neural cells. The undifferentiated pES/GFP(+) cells and their neuronal differentiation derivatives were transplanted into the Sprague-Dawley (SD) rat's brain, and their survival and development was determined by using live animal fluorescence optical imaging system every 15 days. The results showed that fluorescent signals from the injection site of SD rats' brain could be detected through the experimental period of 3 months. The level of fluorescent signal detected in the treatment group was twofold that of the control group. The results of behavior analysis showed that PD rats exhibited stably decreased asymmetric rotations after transplantation with pES/GFP(+)-derived D18 neuronal progenitors. The dopaminergic differentiation of grafted cells in the brain was further confirmed by immunohistochemical staining with anti-TH, anti-DA, and anti-DAT antibodies. These results suggested that the differentiation approach we developed would direct pES cells to differentiate into neural lineages and benefit the development of novel therapeutics involving stem cell transplantation.
本研究旨在引导猪胚胎干细胞（pES）分化为神经谱系，并研究表达绿色荧光蛋白的pES（pES/GFP(+)）在帕金森病（PD）大鼠模型中的治疗潜力。通过在含有视黄酸（RA）、音猬因子（SHH）和碱性成纤维细胞生长因子（FGF）组合的培养基中进行悬浮培养，诱导pES定向分化为神经谱系，而无需经过胚状体形成阶段。在12天诱导后的第2天，所有处理组均发现了高产量的表达巢蛋白的神经前体细胞。在重新接种后的第6天，分别有超过86.2%和83.4%的分化细胞对神经丝轻链蛋白（NFL）和微管相关蛋白2（MAP2）染色呈阳性。在这些NFL阳性神经细胞中也观察到了酪氨酸羟化酶（TH）、胆碱乙酰转移酶（ChAT）和γ-氨基丁酸（GABA）特异性标志物的表达。将未分化的pES/GFP(+)细胞及其神经元分化衍生物移植到Sprague-Dawley（SD）大鼠脑中，每隔15天使用活体动物荧光光学成像系统确定它们的存活和发育情况。结果显示，在3个月的实验期内均可检测到SD大鼠脑注射部位的荧光信号。治疗组检测到的荧光信号水平是对照组的两倍。行为分析结果表明，用pES/GFP(+)衍生的D18神经元祖细胞移植后，PD大鼠的不对称旋转稳定减少。通过用抗TH、抗多巴胺（DA）和抗多巴胺转运体（DAT）抗体进行免疫组织化学染色，进一步证实了移植细胞在脑中的多巴胺能分化。这些结果表明，我们开发的分化方法可引导pES细胞分化为神经谱系，并有利于涉及干细胞移植的新型治疗方法的开发。
