小鼠前庭支持细胞诱导人羊水干细胞定向分化为功能性神经元的初步研究

Mouse vestibular supporting cells induce directional differentiation of human amniotic fluid stem cells into functional neurons:a pilot study

目的探讨通过支持接触共培养模式,小鼠前庭支持细胞体外诱导人羊水干细胞向功能性神经元分化的可行性。方法分离培养人来源的羊水干细胞。从新生C57BL/6J小鼠获取内耳前庭组织,经酶解消化、细胞培养,选取其空心球体,制备单细胞饲养层。将标记慢病毒载体介导产生绿色荧光蛋白nGFP的羊水干细胞种植在饲养层表面,形成支持接触共培养,期间不添加任何外源性神经生长因子及神经元信号诱导因子。检测分化后的羊水干细胞中神经元标记物β微管蛋白(β-Tubulin,Tuj1)、突触后致密蛋白PSD95的表达;标记功能性突触小泡技术(FM1-43穿透)检测分化后细胞是否具有神经元离子通道。同时观察羊水干细胞单独分化、小鼠前庭支持细胞单独分化、Transwell共培养体系中羊水干细胞和饲养层细胞神经元的分化情况。结果单细胞饲养层表达内耳支持细胞特异性标记物细胞周期蛋白激酶抑制因子P27kip1。nGFP标记过的羊水干细胞接种于饲养层后,nGFP表达阳性的部位有(52.0±3.0)%的细胞表达Tuj1,并具有典型的神经元形态特征,突起长度平均(110.7±6.2)μm。饲养层细胞单独分化,仅有(1.1±0.6)%的细胞表达Tuj1阳性且形态典型的神经元;羊水干细胞单独分化,(92.0±1.0)%的细胞表达神经元标记物Tuj1,但无典型的神经元形态特征,突起长度平均(16.0±4.1)μm。将羊水干细胞与饲养层在Transwell中共培养,虽然(92.0±1.0)%的羊水干细胞表达Tuj1,但仍无典型的神经元形态特征,突起长度平均(17.0±4.5)μm,饲养层仅有(1.2±0.9)%的细胞Tuj1表达阳性且具有典型的神经元形态。结论通过支持接触共培养模式,由小鼠前庭来源的内耳支持细胞制备的饲养层,可成功将人羊水干细胞定向诱导分化为功能性神经元。

Objective To investigate the feasibility of inducing human amniotic fluid stem cells into functional neurons by supporting contact co-culture depended on feeder layer from mouse vestibular supporting cells.Methods Human amniotic fluid stem cells were isolated to culture.The vestibular tissues were obtained from the newborn C57BL/6J mouse by enzymatic digestion and cell culture,the hollow spheres were selected to prepare a monolayer feeder layer.The nGFP-labeled amniotic fluid stem cells were planted on the surface of the feeder layer to form the supporting contact co-culture without adding any exogenous nerve growth factor and neuronal signal inducing factor,and detected the expression of Tuj1 and PSD95,and investigated whether there were ion channels in neurons by FM1-43.Human amniotic fluid stem cells and mouse vestibular supporting cells,which were differentiated separately,and Transwell coculture was used as the control group.Results The feeder layer expressed the special marker P27kip1of the inner ear supporting cell.The nGFP-labeled amniotic fluid stem cells were inoculated on the feeder layer,and(52.0±3.0)%of the nGFP cells expressed Tuj1,which had typical neurons morphological characteristics[protrusion length(110.7±6.2)μm];the feeder layer cells were differentiated separately,of which(1.1±0.6)%expressed Tuj1 in the control group;the amniotic fluid stem cells were differentiated independently without typical neuron morphological features[protrusion length(16±4.1)μm],of which(92.0±1.0)%expressed Tuj1.The amniotic fluid stem cells and the feeder layer were co-cultured in Transwell:although(92.0±1.0)%of amniotic fluid stem cells had the expression of Tuj1,which had no typical neurons morphological feature[protrusion length(17±4.5)μm],only(1.2±0.9)%of Tuj1 were observed in the feeder layer.Conclusion By supporting contact co-culture,the feeder layer from the vestibular supporting cells can successfully differentiate human amniotic fluid stem cells into neurons.