Epiblast Cell Number and Primary Embryonic Stem Cell Colony Generation Are
Increased by Culture of Cleavage Stage Embryos in Insulin |
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| Authors: | Jared M CAMPBELL Michelle LANE Ivan VASSILIEV Mark B NOTTLE |
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| Institution: | 1)School of Paediatrics and Reproductive Health, Discipline of Obstetrics and Gynaecology, University of Adelaide, Adelaide, SA 5005, Australia;2)Centre for Stem Cell Research, University of Adelaide, Adelaide, SA 5005, Australia;3)Repromed, Dulwich, SA 5065, Australia |
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| 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. |
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| Keywords: | Blastocyst Embryonic stem cell Epiblast Insulin Pluripotency Preimplantation development |
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