4000/人胚胎干细胞源TH 阳性细胞电压门控性通道的初步研究

目的：检测人胚胎干细胞源TH阳性细胞的神经元性电生理特性。方法：采用我们实验室改良后的“无血清四步法经拟胚体培养体系”的方法，体外诱导人胚胎干细胞源性TH阳性细胞，在对其细胞核型及特异性标志物进行检测的基础上，运用全细胞膜片钳记录的方法，检测其细胞膜上电压门控性离子通道的电生理特性。结果：分化前后细胞核型保持正常；诱导得到的细胞形态一致，大多数细胞（>90%）表达多巴胺能神经元的标志β-tubulion和TH，并仍表达神经前体细胞的标志nestin；膜片钳检测显示诱导分化的TH阳性细胞具有神经元性电压门控钠、钾离子通道。结论：人胚胎干细胞经体外定向诱导分化为TH阳性细胞后具有一定的DA能神经元特性，特别是神经元性电生理特性。

Differentiation of Human Embryonic Stem Cells into Functional TH-positive Cells by Using Modified Four-step Method in Vitro

Human embryonic stem cells (hESCs) are excellent candidates as a source of functional differentiated cells for the replacement therapy of degenerative disease, such as Parkinson's disease (PD), which is characterized by progressive and selective loss of dopamine (DA) neurons in the midbrain substantia nigra. In order to make them safety and utility, we must detect their characters before transplantation. In the present study, we efficiently generated tyrosine hydroxylase (TH)-positive cells in vitro from our hESCs cells by using our modified four-step method including embryoid body (EB) formation. The characteristics of these human embryonic stem (hES)-derived cells were confirmed by immunohistochemical staining, patch clamp recording and karyotype analysis. The results showed that at the end of differentiation schedule, more than 90% of the differentiated cells were positive for both the DA neuronal marker TH and the neuronal marker TuJ1. In addition, some of the hES-derived cells were also positive for nestin, a common neural progenitor marker. Cell karyotype analysis and electrophysiological study further demonstrated that these hES-derived cells not only had a normal karyotype but also showed general neural potassium and sodium currents. These data suggest that the hES-derived cells are likely to be differentiated into the mature functional TH-positive DA neurons, which are characterized by stable normal karyotype and general electrophysiological functional properties, by using modified four-step method in vitro.