KIT ligand and bone morphogenetic protein signaling enhances human embryonic stem cell to germ-like cell differentiation.
BACKGROUND
Signaling mechanisms involved in early human germ cell development are largely unknown and believed to be similar to mouse germ cell development; however, there may be species specific differences. KIT ligand (KITL) and Bone morphogenetic protein 4 (BMP4) are necessary in mouse germ cell development and may play an important role in human germ cell development.


METHODS
KITL signaling studies were conducted by differentiating human embryonic stem cells (hESCs) on KITL wild-type, hetero- or homozygous knockout feeders for 10 days, and the effects of BMP signaling was determined by differentiation in the presence of BMP4 or its antagonist, Noggin. The formation of germ-like cells was ascertained by immunocytochemistry, flow cytometry and quantitative RT-PCR for germ cell markers.


RESULTS
The loss of KITL in enrichment and differentiation cultures resulted in significant down-regulation of germ cell genes and a 70.5% decrease in germ-like (DDX4+ POU5F1+) cells, indicating that KITL is involved in human germ cell development. Moreover, endogenous BMP signaling caused germ-like (DDX4+ POU5F1+) cell differentiation, and the inhibition of this pathway caused a significant decrease in germ cell gene expression and in the number of DDX4+ POU5F1+ cells. Further, we demonstrated that eliminating feeders but maintaining their secreted extracellular matrix is sufficient to sustain the increased numbers of DDX4+ POU5F1+ cells in culture. However, this resulted in decreased germ cell gene expression.


CONCLUSIONS
From these studies, we establish that KITL and BMP4 germ cell signaling affects in vitro formation of hESC derived germ-like cells and we suggest that they may play an important role in normal human germ cell development.
West
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FD
Department of Animal and Dairy Science, University of Georgia, Athens, GA 30602, USA. sstice@uga.edu
Roche-Rios
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MI
Abraham
S
S
Rao
R R
RR
Natrajan
M S
MS
Bacanamwo
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M
Stice
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SL
D000818:Animals; D055415:Bone Morphogenetic Protein 4 / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D002352:Carrier Proteins / Q000494:pharmacology; D002454:Cell Differentiation; D018920:Coculture Techniques; D053487:DEAD-box RNA Helicases / Q000378:metabolism; D019175:DNA Methylation; D053595:Embryonic Stem Cells / Q000166:cytology / Q000187:drug effects*; D015894:Genome, Human; D005854:Germ Cells / Q000166:cytology* / Q000254:growth & development / Q000378:metabolism; D006801:Humans; D051379:Mice; D050814:Octamer Transcription Factor-3 / Q000378:metabolism; D015398:Signal Transduction; D019089:Stem Cell Factor / Q000494:pharmacology*; D000099176:Noggin Protein
D055415:Bone Morphogenetic Protein 4; D002352:Carrier Proteins; D053487:DEAD-box RNA Helicases; D050814:Octamer Transcription Factor-3; D019089:Stem Cell Factor; D000099176:Noggin Protein; C525809:BMP4 protein, human; C494173:POU5F1 protein, human; C498288:DDX4 protein, human