Human Embryonic Stem Cells Handbook
Considerable advances have taken place since the initial isolation and characterization of human embryonic stem (HES) cells; however, significant challenges remain before their potential for restoration and regeneration processes in patients can be realiz
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1. Introduction Human embryonic and induced pluripotent stem cells (hESCs/ iPSCs) have enormous potential as a source of cells for cell replacement therapies and as a model for early human development and drug discovery. An increasing number of hES and iPS cell lines have been derived worldwide. Although there have been considerable efforts to characterize some pluripotent stem cell lines, for many lines, the effectiveness of neural differentiation is unknown. It is critical to establish methods to compare the reliability and variability of each individually derived cell line as to neural differentiation potential and to identify the efficient lines for research and therapeutic use. Kursad Turksen (ed.), Human Embryonic Stem Cells Handbook, Methods in Molecular Biology, vol. 873, DOI 10.1007/978-1-61779-794-1_16, © Springer Science+Business Media, LLC 2012
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Methods for neural differentiation of hESCs have been extensively studied (1–10). But direct comparisons of neural differentiation between independently derived pluripotent stem cell lines under common culture conditions are not well documented (11–13). Our side-by-side comparison confirms the general finding that hESC lines share the properties of selfrenewal, expression of “stemness,” and pluripotency markers, and the ability to differentiate, but many distinctions remain among cell lines (11). These include the ability to maintain an undifferentiated state, to self-renew, and to differentiate. In addition to inherited variations in the sex, stage, quality, and genetic background of embryos used for hESC line derivation, these different qualities may be associated with considerable diversity in derivation methods and changes acquired during passaging that may influence the differentiation capacity of cell lines. To this end, it is important to set up standards shared by multiple laboratories for routine analysis of neural differentiation testing under common culture conditions. However, the information regarding common culture conditions permitting neural differentiation comparison is lacking. It is also challenging to determine reliable readouts to quantify the differentiation effectiveness of cell lines. In the present article, hES and iPS cell lines were maintained routinely on mouse embryonic fibroblast (MEF) feeder layers or on Matrigel using enzymatic passaging. We assessed the neural differentiation potential of both hESC and iPSC lines via embryoid body (EB) formation. We established a dynamic process to generate robust Pax6+ neural rosettes (neuroectodermal cells) and to further differentiate them into nestin+ neural progenitors (NPs) in a defined neural inducing (N2) medium. The differentiation effectiveness was analyzed by a simple measurement of the distance from the edge of the EB sphere to the far edge of the rim of nestin-positive neurospheres. As a sample, we evaluate and compare the efficiency of neural differentiation under standardized conditions from three hESC lines, TE03 (NIH Registry), TE06 (NIH Registry), and B
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