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Regeneration of the central nervous system (CNS), where proliferative potency is limited, is one of the most important research themes in neuroscience and neuroengineering. Pluripotent stem cell lines have attracted broad attentions as an important model system for regenerating damaged brain. The important key for realizing regenerative medicine using pluripotent stem cells is to induce undifferentiated cell into objective cells with high efficiency and reproducibility. Although the methods to induce embryonic stem (ES) cells into specific neuronal subtype with pharmacological treatment have been proposed, however, the handling is difficult and thus the efficiency rate of objective cells is low (Barberi et al., 2003). The main reason for low differentiation rate of pluripotent stem cells is difficulty in precise control of interaction between pharmacological treatment and cell signalling. To overcome these problems, development of alternative methods for precise control of cell differentiation processes is required. During cell differentiation, both endogenous factors, such as cell-cell signal transmission, and exogenous factors, such as pharmacological application, play important roles. Micro and soft lithography-based surface modification of culture substrate enabled to control stem-cell-aggregation (EB; Embryoid body) sizes and therefore to increase cell differentiation efficiency through promotion of cell-cell signal interactions (Karp et al., 2007; Wang et al., 2009). Particularly, Park et al. reported that small-size EBs of mouse ES cells (100 ~ 200 m of diameter) tended to differentiate into ectoderm and large-size EB (500 m of diameter) into mesoderm (Park et al., 2007). These reports suggested that manipulation of EB size and shape could affect endogenous factors of stem cell EBs and thus was an important approach for precise control of differentiation processes. Another important technique for regulating cell differentiation is applying physical stimulation. It was reported that electrical or magnetic stimulation could induce cellular and molecular responses and affected the gene expressions during differentiation (Kimura et al., 1998; Piacentini et al., 2008). Particularly, Yamada et al. reported that applying electrical stimulation induced mouse ES cells efficiently into ectoderm cells (Yamada et al., 2007). These reports suggested

Toward the Precise Control of Cell Differentiation Processes by Using Micro and Soft Lithography