Zhao Zhenqiang, Ma Yanlin, Chen Zhibin, Liu Qian, Li Qi, Kong Deyan, Yuan Kunxiong, Hu Lan, Wang Tan, Chen Xiaowu, Peng Yanan, Jiang Weimin, Yu Yanhong, Liu Xinfeng
Department of Neurology, Jinling Hospital, Southern Medical UniversityNanjing, China; Department of Neurology, First Affiliated Hospital, Hainan Medical UniversityHaikou, China.
Department of Obstetrics and Gynecology, Nanfang Hospital, Southern Medical UniversityGuangzhou, China; Hainan Provincial Key Laboratory for Human Reproductive Medicine and Genetic Research, Hainan Reproductive Medical Center, First Affiliated Hospital, Hainan Medical UniversityHaikou, China.
Front Cell Neurosci. 2016 Dec 20;10:291. doi: 10.3389/fncel.2016.00291. eCollection 2016.
Mouse embryonic fibroblasts (MEFs) and human foreskin fibroblasts (HFFs) are used for the culture of human embryonic stem cells (hESCs). MEFs and HFFs differed in their capacity to support the proliferation and pluripotency of hESCs and could affect cardiac differentiation potential of hESCs. The aim of this study was to evaluate the effect of MEFs and HFFs feeders on dopaminergic differentiation of hESCs lines. To minimize the impact of culture condition variation, two hESCs lines were cultured on mixed feeder cells (MFCs, MEFs: HFFs = 1:1) and HFFs feeder, respectively, and then were differentiated into dopaminergic (DA) neurons under the identical protocol. Dopaminergic differentiation was evaluated by immunocytochemistry, quantitative fluorescent real-time PCR, transmission and scanning electron microscopy, and patch clamp. Our results demonstrated that these hESCs-derived neurons were genuine and functional DA neurons. However, compared to hESCs line on MFCs feeder, hESCs line on HFFs feeder had a higher proportion of tyrosine hydroxylase (TH) positive cells and expressed higher levels of FOXA2, PITX3, NURR1, and TH genes. In addition, the values of threshold intensity and threshold membrane potential of DA neurons from hESCs line on HFFs feeder were lower than those of DA neurons from hESCs line on the MFCs feeder. In conclusion, HFFs feeder not only facilitated the differentiation of hESCs cells into dopaminergic neurons, but also induced hESCs-derived DA neurons to express higher electrophysiological excitability. Therefore, feeder cells could affect not only dopaminergic differentiation potential of different hESCs lines, but also electrophysiological properties of hESCs-derived DA neurons.
小鼠胚胎成纤维细胞（MEFs）和人包皮成纤维细胞（HFFs）被用于人类胚胎干细胞（hESCs）的培养。MEFs和HFFs在支持hESCs增殖和多能性的能力方面存在差异，并且会影响hESCs的心脏分化潜能。本研究的目的是评估MEFs和HFFs饲养层对hESCs系多巴胺能分化的影响。为了尽量减少培养条件变化的影响，分别将两条hESCs系培养在混合饲养细胞（MFCs，MEFs:HFFs = 1:1）和HFFs饲养层上，然后按照相同方案将其分化为多巴胺能（DA）神经元。通过免疫细胞化学、定量荧光实时PCR、透射和扫描电子显微镜以及膜片钳技术评估多巴胺能分化情况。我们的结果表明，这些源自hESCs的神经元是真正的功能性DA神经元。然而，与培养在MFCs饲养层上的hESCs系相比，培养在HFFs饲养层上的hESCs系酪氨酸羟化酶（TH）阳性细胞比例更高，并且FOXA2、PITX3、NURR1和TH基因的表达水平更高。此外，培养在HFFs饲养层上的hESCs系所分化出的DA神经元的阈值强度和阈值膜电位值低于培养在MFCs饲养层上的hESCs系所分化出的DA神经元。总之，HFFs饲养层不仅促进了hESCs细胞向多巴胺能神经元的分化，还诱导源自hESCs的DA神经元表现出更高的电生理兴奋性。因此，饲养层细胞不仅会影响不同hESCs系的多巴胺能分化潜能，还会影响源自hESCs的DA神经元的电生理特性。
