Directing pluripotent cell differentiation using "diced RNA" in transient transfection.
Embryonic stem (ES) and embryonic carcinoma (EC) cells are pluripotent and have the capacity to differentiate into many cell types. The ability to direct their differentiation should have considerable practical applications. Here, we first report the use of diced short interfering RNAi against Oct4 in a transient approach, to direct differentiation of ES towards the trophectoderm lineage. We then apply this approach to downregulate Smad4 in mouse P19 EC cells. We have found that this leads to an increase in the levels of Pax6 (a neuroectoderm marker), reduction in the levels of Brachyury (a mesoderm marker), and a 3-fold increase in the number of betaIII tubulin-positive colonies when these cells were allowed to differentiate. This indicates a redirection of cell fate towards the neuroectoderm lineage. Thus, transient RNAi could provide a valuable tool to direct pluripotent cells along specific pathways of differentiation while circumventing permanent genetic changes.
Carpenter
Lee
L
University of Cambridge, Department of Genetics, Downing Street, Cambridge CB2 3EH, UK.
Zernicka-Goetz
Magdalena
M
D000818:Animals; D002454:Cell Differentiation; D019070:Cell Lineage; D002478:Cells, Cultured; D004268:DNA-Binding Proteins / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D004622:Embryo, Mammalian / Q000166:cytology; D005136:Eye Proteins; D005326:Fetal Proteins / Q000378:metabolism; D005786:Gene Expression Regulation; D018398:Homeodomain Proteins / Q000378:metabolism; D051379:Mice; D050814:Octamer Transcription Factor-3; D000071841:PAX6 Transcription Factor; D051761:Paired Box Transcription Factors; D039904:Pluripotent Stem Cells / Q000166:cytology* / Q000378:metabolism; D034622:RNA Interference; D012097:Repressor Proteins; D051901:Smad4 Protein; D020825:T-Box Domain Proteins / Q000378:metabolism; D015534:Trans-Activators / Q000378:metabolism; D014157:Transcription Factors / Q000037:antagonists & inhibitors / Q000235:genetics*; D014162:Transfection; D014404:Tubulin / Q000378:metabolism; D000098908:Brachyury Protein
D004268:DNA-Binding Proteins; D005136:Eye Proteins; D005326:Fetal Proteins; D018398:Homeodomain Proteins; D050814:Octamer Transcription Factor-3; D000071841:PAX6 Transcription Factor; D051761:Paired Box Transcription Factors; D012097:Repressor Proteins; D051901:Smad4 Protein; D020825:T-Box Domain Proteins; D015534:Trans-Activators; D014157:Transcription Factors; D014404:Tubulin; D000098908:Brachyury Protein; C000605732:Pax6 protein, mouse; C494174:Pou5f1 protein, mouse; C490810:Smad4 protein, mouse; C462941:beta3 tubulin, mouse