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摘要
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/Dkkl/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.
- 出版日期2010
- 单位RIKEN
