Laboratory-Scale Purification of a Recombinant E-Cadherin-IgG Fc Fusion Protein That Provides a Cell Surface Matrix for Extended Culture and Efficient Subculture of Human Pluripotent Stem Cells

Protocol

Abstract

The culture of human pluripotent stem cells under defined conditions most commonly relies on the use of Matrigel as a substrate upon which the cells remain in an undifferentiated state. Matrigel is a complex mixture of extracellular matrices and growth factors derived from mouse Engelbreth–Holm–Swarm ­sarcoma cells. The complexity and lot-to-lot variation of Matrigel preparations has prompted the search for more defined substrates that can support pluripotent stem cell culture. A recombinant human E-cadherin-IgG Fc domain fusion protein (E-cad-Fc) has recently been shown to be extremely efficient in facilitating the culture of human embryonic stem (ES) cells and induced pluripotent stem (iPS) cells under completely defined conditions. This fusion protein is particularly appealing because binding requires the cellular expression of E-cadherin, which is one hallmark of pluripotent stem cells, and so the substrate is selective of an undifferentiated cell state. In addition, cells can be removed from the substrate using gentle enzyme-free dissociation buffers containing chelating reagents, which ensures maintenance of cell surface epitopes and high cell viability during subculture. Here, we provide a detailed protocol for the purification of the E-cad-Fc substrate.

Key words

Human pluripotent stem cells Feeder-free Defined culture conditions Recombinant E-cadherin surface 

Copyright information

© Humana Press 2011

Authors and Affiliations

  1. 1.Department of Cell Biology, Neurobiology and AnatomyMedical College of WisconsinMilwaukeeUSA

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