Hum. Reprod. Advance Access published online on October 19, 2009
Human Reproduction, doi:10.1093/humrep/dep338
KIT ligand and bone morphogenetic protein signaling enhances human embryonic stem cell to germ-like cell differentiation
1 Department of Animal and Dairy Science, University of Georgia, Athens, GA 30602, USA 2 Regenerative Bioscience Center, University of Georgia, Rhodes ADS Center, 425 River Rd., Athens, GA 30602, USA 3 Department of Genetics, University of Georgia, Athens, GA 30602, USA 4 Department of Chemical and Life Science Engineering, Virginia Commonwealth University, Richmond, VA 23284, USA 5 Cardiovascular Research Institute, Morehouse School of Medicine, Atlanta, GA 30310, USA
6 Correspondence address. Tel: +1-706-583-0071; Fax: +1-706-542-7925; E-mail: sstice{at}uga.edu
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.
Key words: stem cells/germ cells/cell signaling/cell culture
Submitted on March 22, 2009; resubmitted on August 29, 2009; accepted on September 2, 2009.