Neural Induction from ES Cells Portrays Default Commitment but Instructive Maturation

The neural induction has remained a debatable issue pertaining to whether it is a mere default process or it involves precise instructive cues. We have chosen the embryonic stem (ES) cell model to address this issue. In a devised monoculture strategy, the cell�Ccell interaction availed through optimum cell plating density could define the niche for the attainment of efficient in vitro neurogenesis from the ES cells. The medium plating density was found ideal in generating optimum number of progenitors and also yielded about 80% mature neurons in a serum free culture set up barring any exogenous inducers. We could also demarcate and quantify the neural stem cells/progenitors among the heterogeneous cell population of differentiating ES cells using nestin intron II driven EGFP expression as a tool. The one week post-plating was determined to be the critical time window for optimum neural progenitor generation from ES cells that helped us further in purifying these cells and in demonstrating their proliferation and multipotent differentiation potential. Seeding cells at varying densities, we could decipher an interesting paradoxical scenario that interlinked both commitment and maturation with the initial plating density having a vital influence on neuronal maturation but not specification and the secretory factors were apparently playing a key role during this process. Thus it was comprehended that, the neural specification was a default process independent of exogenous factors and cellular interaction. Conversely, a defined number of cells at the specification stage itself seemed critical to provide an auto-/paracrine means of signaling threshold for the maturation process to materialize.

神经诱导（neural induction）始终是一个颇具争议的科学问题：其究竟仅为一种默认发生的过程，还是依赖于精准的指导性信号提示？本研究选用胚胎干细胞（embryonic stem, ES）模型来探究这一争议问题。在本研究设计的单培养策略中，通过优化细胞铺板密度所构建的细胞间相互作用微环境，可实现胚胎干细胞高效的体外神经发生过程。研究发现，适宜的铺板密度可生成数量最优的祖细胞，且在无需添加任何外源性诱导剂的无血清培养体系中，可获得约80%的成熟神经元。本研究还可利用巢蛋白内含子II（nestin intron II）驱动的增强型绿色荧光蛋白（enhanced green fluorescent protein, EGFP）表达作为标记工具，在分化中的胚胎干细胞异质性细胞群中区分并定量神经干细胞/祖细胞。研究确定，铺板后的第7天为胚胎干细胞生成最优神经祖细胞的关键时间窗，这一发现可为后续的细胞纯化工作，以及验证这些细胞的增殖能力与多向分化潜能提供支持。通过调整细胞接种密度，本研究揭示了一个有趣的悖论现象：细胞定型与成熟过程均与初始铺板密度相关，初始铺板密度对神经元成熟具有显著影响，但对神经特化过程并无作用；而分泌因子在此过程中似乎发挥了关键调控作用。据此本研究推断，神经特化是一种不依赖外源性因子与细胞间相互作用的默认过程；与之相反，处于特化阶段的特定细胞数量，似乎是为成熟过程提供足够自分泌/旁分泌信号阈值的关键条件，以此保障成熟过程顺利启动。