Open Access Use of Lectins to Enrich Mouse ES-Derived Retinal Progenitor Cells for the Purpose of Transplantation Therapy

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Abstract:

Using the mouse ES cell line with green fluorescent protein knocked-in at the Rx locus (Rx-KI ES cell), we previously showed that photoreceptors can be efficiently obtained in defined culture conditions by enriching Rx-positive retinal progenitor cells. We aimed to explore a protocol applicable for non-Rx-labeled stem cell lines for subsequent enrichment of retinal photoreceptor precursors for transplantation. The Rx-KI ES cell line was differentiated according to the serum-free suspension conditions with serum-free suspension/Dkk1/LeftyA/serum/activin method (SFEB/DLFA) described previously. Enrichment efficacy by negative selection was compared among 20 different lectins and the lectin combination that effectively enriched the Rx-positive cells by selecting the lectin low-binding population was determined. Subsequent differentiation efficiency to photoreceptor precursors and the contamination of Nanog or Oct3/4+ cells in the culture were evaluated between the cell cultures using negative selection with lectins and Rx positive selection. The effect of cytarabine (Ara-C) for minimizing the contamination of undifferentiated cells after the selection was also studied. The combination of the lectins, wheat germ agglutinin (WGA), and Erythrina crista-galli agglutinin (ECA) enabled us to enrich the Rx-positive population by approximately twice the original Rx percentage. The selection also minimized the percentage of Oct3/4+ cells. The lectin-selected cells produced a comparable percentage of Crx/rhodopsin-positive colonies with Rx-positive selection and were differentiated into photoreceptors. The Ara-C treatment on differentiating days 24‐26 decreased Nanog and Oct3/4 expression in subsequent cultures. Enrichment of Rx-positive cells using WGA and ECA was comparable to Rx-positive selection, and the method could be applied to achieve efficient photoreceptor differentiation from other ES or iPS cell lines in which the Rx gene is not marked.

Keywords: Embryonic stem cell; Lectin; Retinal progenitors; Rx; Selection

Document Type: Research Article

DOI: http://dx.doi.org/10.3727/096368909X476599

Publication date: January 1, 2010

More about this publication?
  • Cell Transplantation publishes original, peer-reviewed research and review articles on the subject of cell transplantation and its application to human diseases. To ensure high-quality contributions from all areas of transplantation, separate section editors and editorial boards have been established. Articles deal with a wide range of topics including physiological, medical, preclinical, tissue engineering, and device-oriented aspects of transplantation of nervous system, endocrine, growth factor-secreting, bone marrow, epithelial, endothelial, and genetically engineered cells, among others. Basic clinical studies and immunological research papers are also featured. To provide complete coverage of this revolutionary field, Cell Transplantation will report on relevant technological advances, and ethical and regulatory considerations of cell transplants. Cell Transplantation is now an Open Access journal starting with volume 18 in 2009, and therefore there will be an inexpensive publication charge, which is dependent on the number of pages, in addition to the charge for color figures. This will allow work to be disseminated to a wider audience and also entitle the corresponding author to a free PDF, as well as prepublication of an unedited version of the manuscript.
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