Differentiation of stem cells upon deprivation of exogenous FGF2: a general approach to study spontaneous differentiation of hESCs in vitro.
Establishing a model for in vitro differentiation of human embryonic stem cells (hESCs) towards the germ cell lineage could be used to identify molecular mechanisms behind germ cell differentiation that may help in understanding human infertility. Here, we evaluate whether a lack of exogenous fibroblast growth factor 2 (FGF2) is supporting spontaneous differentiation of hESCs cultured on human foreskin fibroblast (hFF) monolayers towards germ cell lineage. Additionally to depriving the hESCs of exogenous FGF2, cells were stimulated with all-trans retinoic acid (ATRA). To get a more comprehensive impression on effects of removal of FGF2 and stimulation with ATRA, we combined the results of three cell lines for each experimental setting. When combining gene expression profiles of three cell lines for 96 genes, only 6 genes showed a significant up-regulation in all cell lines, when no FGF2 was added to the media for 12 weeks. None of these genes are related to the germ lineage, whereas genes for neuronal cells (PAX6 and NR6A1) and endothelial cells (FLT-1 and PTF1A) were up-regulated. To induce and support the differentiation towards the germ lineage we stimulated hESCs with different concentrations of ATRA for 7 and 14 days. We observed no significant difference in gene expression on RNA level when combining all cell lines. Whereas, the overall outcome was negative, one of these cell lines demonstrated an up-regulation of DDX4 on RNA and protein level after 7 days of ATRA stimulation. In summary, our data showed that the lack of exogenous FGF2 results in up-regulation of genes crucial for neuronal and endothelial cell differentiation of hESCs, but not in the up-regulation of genes related to germ cell differentiation when cultured on hFFs. Additionally, we demonstrated that ATRA supplementation did not result in a general specific direction of hESCs towards the germ lineage.
Laboratory of Reproductive Biology, Scientific Unit, Horsens Hospital, Denmark. ros.kjartansdottir@ki.se
Kristín Rós
KR
Kjartansdóttir
Anette
A
Gabrielsen
Ahmed
A
Reda
Olle
O
Söder
Rosita
R
Bergström-Tengzelius
Claus Yding
CY
Andersen
Outi
O
Hovatta
Jan-Bernd
JB
Stukenborg
Jens
J
Fedder
D002454:Cell Differentiation / Q000187:drug effects*; D053487:DEAD-box RNA Helicases / Q000235:genetics; D053595:Embryonic Stem Cells / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D016222:Fibroblast Growth Factor 2 / Q000494:pharmacology*; D015870:Gene Expression / Q000187:drug effects; D018398:Homeodomain Proteins / Q000235:genetics; D006801:Humans; D066298:In Vitro Techniques; D000071317:Nanog Homeobox Protein; D050814:Octamer Transcription Factor-3 / Q000235:genetics; D016601:RNA-Binding Proteins / Q000235:genetics; D014212:Tretinoin / Q000494:pharmacology
C101358:DAZL protein, human; D018398:Homeodomain Proteins; C486780:NANOG protein, human; D000071317:Nanog Homeobox Protein; D050814:Octamer Transcription Factor-3; C494173:POU5F1 protein, human; D016601:RNA-Binding Proteins; D016222:Fibroblast Growth Factor 2; D014212:Tretinoin; C498288:DDX4 protein, human; D053487:DEAD-box RNA Helicases