Physiological Analysis on Toti-potential Differentiation of ascidian cleavage-arrested half-embryonic cells as an embryonic stem cell model

海鞘是早期脊椎动物发育的雏形，在其发育过程中，两细胞胚胎（2C细胞）的一侧（海鞘蝌蚪的所有细胞类型都来源于此）应该具有多重命运。将两细胞胚胎中的2C细胞与任何一对前神经外胚层细胞（2C-aa三胞胎）、推定脊索神经细胞（2C-aa三胞胎）、推定后表皮细胞（2C-bb三胞胎）或推定大肌细胞（2C-bb三胞胎）进行接触培养，制备卵裂阻滞细胞三胞胎。通过观察细胞类型特异性电流，在电生理学上鉴定了三胞胎中所有三个细胞的分化。在这些2C三胞胎中，当两细胞胚胎的受精时间比8细胞胚胎的受精时间晚3小时时，来自两细胞胚胎的2C细胞发育成任何前神经元细胞、后神经元细胞或肌肉细胞，这取决于接触细胞对的细胞类型。然而，当2细胞胚胎发育时间早于8细胞胚胎时，2C细胞主要发育为表皮细胞型。当它们同时发育时，2C细胞被诱导分化为上述三种类型中的任何一种，或发育为表皮细胞的概率几乎相等。综上所述，通过选择接触细胞对的类型作为诱导剂，以及2C细胞与接触细胞对的发育时间差，控制了海鞘2C细胞的分化，揭示了蝌蚪幼虫的主要细胞类型。从单纯性干细胞分化的简单模型出发，讨论了海鞘2C细胞与脊椎动物胚胎干细胞的相似性。

During early ascidian development, which is a prototype of early vertebrate development, one side of the two-cell embryo (2C cell), from which all cell types in the ascidian tadpoles are derived, should have multiple fates. The cleavage-arrested cell triplet was prepared from a 2C cell of the two-cell embryo and any pair of anterior neuroectoderm cells (2C-aa triplet), of presumptive notochordal neural cells (2C-AA triplet), of presumptive posterior epidermal cells (2C-bb triplet), or of presumptive major muscle cells (2C-BB triplet) derived from the 8-cell embryo, and cultured in contact. Differentiation of all three cells in the triplet was identified electrophysiologically by observing cell type-specific currents. In these 2C triplets, when the batch of two-cell embryos were fertilized and developed 3 hours later than that of 8-cell embryos for the cell pairs, the 2C cell from two-cell embryos developed into any of anterior neuronal, posterior neuronal, or muscle cells, depending upon the cell types of contacting cell pairs. However, when the two-cell embryos developed at earlier time than the 8-cell embryos, from which the cell pairs were derived, the 2C cells developed mostly into epidermal cell type. When they developed at the same time, the 2C cells were induced to differentiate into any of the above three types, or developed as the epidermal cell, with almost equal probability. In conclusion, the differentiation of the cleavage-arrested ascidian 2C cell was controlled to reveal the major cell types in the tadpole larva by selecting the types of contacting cell pairs as the inducers and the developmental time difference between the 2C cell and the contacting pair. The similarity of the cleavage-arrested ascidian 2C cell to the vertebrate embryonic stem cell was discussed in respect to the simple model for toti-potent stem cell differentiation.