Effects of inhibition of gap junctional intercellular communication on neural differentiation of mouse embryonic stem cells in vitro

1.小鼠胚胎干细胞体外神经分化体系的建立我们建立了小鼠胚胎干细胞神经分化的体外体系。未分化的ES细胞悬浮培养8d，后半期用全反式维甲酸处理。然后，将细胞转移到贴壁培养。90%以上的类胚体内可见突起。我们用胰酶处理来分散类胚体，并开发了一种早期神经细胞的分散细胞培养系统。在这个系统中，神经元标记阳性的细胞在培养一天后形成小集落。体外ES细胞神经分化过程中缝隙连接细胞间通讯的变化我们用神经生物素偶联微量注射示踪剂测定了细胞间缝隙连接通讯。在贴壁培养的第1天，显微注射神经生物素可以扩散到所有表达A2B5的集落细胞中，但当集落周围没有A2B5表达时，染料不会扩散到扁平细胞中。在贴壁培养3天后，将神经生物素注射到集落中的单个细胞中，仅限于集落的一小部分。这些结果表明，在神经分化过程中，神经细胞内缝隙连接通讯的区间化程度迅速降低。显性负性Cx43表达载体的制备我们制备了显性负性Cx43绿色荧光蛋白(GFP)表达载体，并将其导入野生型Cx43细胞中。该载体对野生型Cx43.4表现出显性负效应。Cx43缺失ES细胞的体外分化我们发现，Cx43-/-ES细胞在体外分化为自发收缩的心肌细胞，与野生型细胞相似，但在体外分化的所有阶段都表现出极少量的荧光黄染料偶联。

1. Establishment of an in vitro system for neural differentiation using mouse embryonic stem (ES) cells in vitroWe established an in vitro system for neural differentiation using mouse ES cells. Undifferentiated ES cells were cultured in suspension for 8 days and treated with all-trans retinoic acid during the latter half of the period. Thereafter, cell were transferred to adhesion culture. Neurites were observed in more than 90% of embryoid bodies. We dispersed embryoid bodies by treatment with trypsin and developed a dispersed cell culture system for early neuronal cells. In this system, cells positive for neuronal markers formed small colonies after one day of culture.2. Changes in gap junctional intercellular communication during neural differentiation of ES cells in vitroWe measured gap junctional intercellular communication by microinjection tracer coupling with neurobiotin. Microinjected neurobiotin was found to spread into all cells constituting colonies that expressed A2B5 at day 1 of adhesion culture, but the dye did not spread into flat cells when there was no A2B5 expression around the colony. After day 3 of adhesion culture, neurobiotin injected into a single cell in the colony was restricted to small portions of the colony. These results suggest that compartmentalization of gap junctional communication within neural cells decreases rapidly during the process of neural differentiation.3. Preparation of a dominant-negative Cx43 expression vectorWe prepared a dominant-negative Cx43-green fluorescent protein (GFP) expression vector and transfected into a cell line expressing wild-type Cx43. This vector showed dominant negative effects on wild-type Cx43.4. In vitro differentiation of ES cells deficient in Cx43We found that Cx43 -/- ES cells differentiated into spontaneously contracting cardiomyocytes in vitro similar to the wild-type cells although they showed a very small extent of Lucifer Yellow dye coupling at all stages of in vitro differentiation.