Cell replacement therapies, which utilize renewable stem cell sources, hold tremendous potential to treat a wide range of degenerative diseases. Although many studies have established techniques to successfully differentiate stem cells into different mature cell lineages, their practicality is limited by the lack of control during the differentiation process and low yields of differentiated cells. In order to address these issues, we have previously established a murine embryonic stem cell alginate-poly-L-lysine microencapsulation differentiation system [1]. We demonstrated that ES cell differentiation could be mediated by cell-cell aggregation in the encapsulation microenvironment. We have demonstrated that both cell aggregation and hepatocyte functions, such as urea and albumin secretions, as well as increased expression of cytokeratin 18 and cyp4507a, occur concomitantly with surface E-cadherin expression [2]. In the present studies, we assessed the feasibility of inducing neuronal lineage differentiation in the alginate microenvironment by incorporating soluble inducers, such as retinoic acid, into the permeable microcapsule system. We demonstrated decreased cell aggregation and enhanced neuronal lineage differentiation with the expression of various neuronal specific markers, including neurofilament, A2B5, O1 and glial fibrillary acidic protein (GFAP). In addition, we demonstrated that, by blocking the cell aggregation using anti-E-cadherin antibody, encapsulated cells increased neuronal marker expression at a later stage of the encapsulation, even in the absence of retinoic acid. In conjunction with the mechanical and physical characterizations of the alginate crosslinking network, we show that 2.2% alginate concentration is most conducive to neuronal differentiation from embryonic stem cells in the presence of retinoic acid.

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