Open AccessExpression of GFP Under the Control of the RNA Helicase VASA Permits Fluorescence‐Activated Cell Sorting Isolation of Human Primordial Germ Cells
Author(s) -
Tilgner Katarzyna,
Atkinson Stuart P.,
Yung Sun,
Golebiewska Anna,
Stojkovic Miodrag,
Moreno Ruben,
Lako Majlinda,
Armstrong Lyle
Publication year - 2010
Publication title -
stem cells
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.159
H-Index - 229
eISSN - 1549-4918
pISSN - 1066-5099
DOI - 10.1002/stem.263
Subject(s) - biology , cell sorting , embryonic stem cell , reprogramming , microbiology and biotechnology , germ cell , population , rna helicase a , stem cell , cellular differentiation , reporter gene , cell , genetics , rna , gene expression , gene , helicase , demography , sociology
Abstract The isolation of significant numbers of human primordial germ cells at several developmental stages is important for investigations of the mechanisms by which they are able to undergo epigenetic reprogramming. Only small numbers of these cells can be obtained from embryos of appropriate developmental stages, so the differentiation of human embryonic stem cells is essential to obtain sufficient numbers of primordial germ cells to permit epigenetic examination. Despite progress in the enrichment of human primordial germ cells using fluorescence‐activated cell sorting (FACS), there is still no definitive marker of the germ cell phenotype. Expression of the widely conserved RNA helicase VASA is restricted to germline cells, but in contrast to species such as Mus musculus in which reporter constructs expressing green fluorescent protein (GFP) under the control of a Vasa promoter have been developed, such reporter systems are lacking in human in vitro models. We report here the generation and characterization of human embryonic stem cell lines stably carrying a VASA ‐pEGFP‐1 reporter construct that expresses GFP in a population of differentiating human embryonic stem cells that show expression of characteristic markers of primordial germ cells. This population shows a different pattern of chromatin modifications to those obtained by FACS enrichment of Stage Specific Antigen one expressing cells in our previous publication. S TEM C ELLS 2010;28:84–92