title: Epiblast cell number and primary embryonic stem cell colony generation are increased by culture of cleavage stage embryos in insulin.
abstract: Human embryos for hESC derivation are often donated at the cleavage stage and of reduced quality. Poor quality embryos have lower efficiency for hESC derivation. However, cleavage stage mouse embryos develop into higher quality expanded blastocysts if they are cultured with insulin, suggesting that this approach could be used to improve hESC derivation from poor quality cleavage stage embryos. The present study used a mouse model to examine this approach. In particular we examined the effect of insulin on the number of epiblast cells in blastocysts on days 4, 5 and 6 using Oct4 and Nanog co-expression. Second we examined the effect of insulin on the frequency with which outgrowths can be derived from these. Finally, we tested whether prior culture in the presence of insulin results in blastocysts with increased capacity to generate ESC colonies. Culture of cleavage stage embryos with insulin increased the number of Oct4 and Nanog positive cells in blastocysts at all time points examined. Prior culture with insulin had no effect on outgrowths generated from blastocysts plated on days 4 or 5. However, insulin treatment of blastocysts plated on day 6 resulted in increased numbers of outgrowths with larger epiblasts compared with controls. 13% of insulin treated day 6 blastocysts produced primary ESC colonies compared with 6% of controls. In conclusion, treatment with insulin can improve epiblast cell number in mice leading to an increase with which primary ESC colonies can be generated and may improve hESC isolation from reduced quality embryos donated at the cleavage stage.
authors.affiliation: School of Paediatrics and Reproductive Health, Discipline of Obstetrics and Gynaecology, University of Adelaide, Adelaide, SA 5005, Australia.
authors.forename: Jared M
authors.initials: JM
authors.lastname: Campbell
authors.forename: Michelle
authors.initials: M
authors.lastname: Lane
authors.forename: Ivan
authors.initials: I
authors.lastname: Vassiliev
authors.forename: Mark B
authors.initials: MB
authors.lastname: Nottle
mesh_terms: D000818:Animals; D001755:Blastocyst / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D002452:Cell Count; D046149:Embryo Culture Techniques / Q000379:methods*; D053595:Embryonic Stem Cells / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D005855:Germ Layers / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D018398:Homeodomain Proteins / Q000378:metabolism; D007004:Hypoglycemic Agents / Q000494:pharmacology*; D007328:Insulin / Q000494:pharmacology*; D051379:Mice; D000071317:Nanog Homeobox Protein; D050814:Octamer Transcription Factor-3 / Q000378:metabolism
chemical_list: D018398:Homeodomain Proteins; D007004:Hypoglycemic Agents; D007328:Insulin; D000071317:Nanog Homeobox Protein; C475540:Nanog protein, mouse; D050814:Octamer Transcription Factor-3; C494174:Pou5f1 protein, mouse