Use of lectins to enrich mouse ES-derived retinal progenitor cells for the purpose of transplantation therapy.
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.
Mandai
Michiko
M
Laboratory for Retinal Regeneration, Center for Developmental Biology, Riken Kobe Institute, Kobe, Hyogo, Japan. mmandai@cdb.riken.jp
Ikeda
Hanako
H
Jin
Zi-Bing
ZB
Iseki
Kyoko
K
Ishigami
Chie
C
Takahashi
Masayo
M
D000818:Animals; D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology; D015415:Biomarkers / Q000032:analysis / Q000378:metabolism; D002452:Cell Count / Q000379:methods; D018929:Cell Culture Techniques / Q000379:methods; D002454:Cell Differentiation / Q000187:drug effects* / Q000502:physiology; D002460:Cell Line; D002469:Cell Separation / Q000379:methods; D016895:Culture Media, Serum-Free / Q000494:pharmacology; D003561:Cytarabine / Q000494:pharmacology; D018398:Homeodomain Proteins / Q000032:analysis / Q000378:metabolism; D037102:Lectins / Q000494:pharmacology*; D051379:Mice; D000071317:Nanog Homeobox Protein; D050814:Octamer Transcription Factor-3 / Q000032:analysis / Q000378:metabolism; D020419:Photoreceptor Cells, Vertebrate / Q000166:cytology / Q000502:physiology / Q000637:transplantation*; D037121:Plant Lectins / Q000494:pharmacology; D012164:Retinal Diseases / Q000628:therapy; D033581:Stem Cell Transplantation / Q000379:methods*; D013234:Stem Cells / Q000166:cytology / Q000187:drug effects* / Q000502:physiology; D014909:Wheat Germ Agglutinins / Q000494:pharmacology
D000964:Antimetabolites, Antineoplastic; D015415:Biomarkers; D016895:Culture Media, Serum-Free; D018398:Homeodomain Proteins; D037102:Lectins; D000071317:Nanog Homeobox Protein; C475540:Nanog protein, mouse; D050814:Octamer Transcription Factor-3; D037121:Plant Lectins; C494174:Pou5f1 protein, mouse; D014909:Wheat Germ Agglutinins; C045629:erythrina lectin; D003561:Cytarabine