Expression of GFP under the control of the RNA helicase VASA permits fluorescence-activated cell sorting isolation of human primordial germ cells.
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.
Tilgner
Katarzyna
K
North East Stem Cell Institute, Newcastle University, International Centre for Life, NE1 3BZ, United Kingdom.
Atkinson
Stuart P
SP
Yung
Sun
S
Golebiewska
Anna
A
Stojkovic
Miodrag
M
Moreno
Ruben
R
Lako
Majlinda
M
Armstrong
Lyle
L
D002454:Cell Differentiation / Q000235:genetics; D002460:Cell Line; D002469:Cell Separation / Q000379:methods*; D042002:Chromatin Assembly and Disassembly; D053487:DEAD-box RNA Helicases / Q000235:genetics*; D053595:Embryonic Stem Cells / Q000378:metabolism*; D005260:Female; D005434:Flow Cytometry; D020869:Gene Expression Profiling / Q000379:methods; D018507:Gene Expression Regulation, Developmental; D005854:Germ Cells / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000096:biosynthesis* / Q000235:genetics; D006801:Humans; D008540:Meiosis; D018613:Microscopy, Confocal; D020411:Oligonucleotide Array Sequence Analysis; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013997:Time Factors; D014162:Transfection
D011994:Recombinant Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; C498288:DDX4 protein, human; D053487:DEAD-box RNA Helicases