title: Isolation of primordial germ cells from differentiating human embryonic stem cells.
abstract: Of all the cell types that can be obtained from the differentiation of embryonic stem cells, primordial germ cells are arguably the most fascinating, as they represent the in vitro completion of the reproductive cycle of the organism from which the embryonic stem cell line was derived. It is also possible to obtain these cells from embryos at an appropriate stage of development, but this process yields only small numbers that are not applicable to investigations of their epigenetic architecture. A considerable body of data has been generated from the differentiation of mouse embryonic stem cells to this cell type, but despite the demonstration of their presence in human embryoid bodies, there has been little progress toward methods of producing human primordial germ cells in useful numbers. We present here a robust protocol to differentiate two human embryonic stem cell lines (H9 and hES-NCL1) that maximizes the numbers of primordial germ cells that may be obtained using a simple fluorescence-activated cell sorting strategy for their isolation. These primordial germ cells demonstrate high-level expression of the germ cell-specific VASA gene and show removal of parental imprints and chromatin modification changes that support their primordial germ cell identity.
authors.affiliation: North East Institute for Stem Cell Research, University of Newcastle upon Tyne, Newcastle upon Tyne, United Kingdom.
authors.forename: Katarzyna
authors.initials: K
authors.lastname: Tilgner
authors.forename: Stuart P
authors.initials: SP
authors.lastname: Atkinson
authors.forename: Anna
authors.initials: A
authors.lastname: Golebiewska
authors.forename: Miodrag
authors.initials: M
authors.lastname: Stojkovic
authors.forename: Majlinda
authors.initials: M
authors.lastname: Lako
authors.forename: Lyle
authors.initials: L
authors.lastname: Armstrong
mesh_terms: D000818:Animals; D018929:Cell Culture Techniques / Q000379:methods*; D002454:Cell Differentiation; D002460:Cell Line; D002469:Cell Separation / Q000379:methods*; D002843:Chromatin / Q000378:metabolism; D018899:CpG Islands; D044127:Epigenesis, Genetic; D005434:Flow Cytometry; D005854:Germ Cells / Q000166:cytology* / Q000378:metabolism; D006801:Humans; D051379:Mice; D018613:Microscopy, Confocal / Q000379:methods; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection
chemical_list: D002843:Chromatin