In vitro post-meiotic germ cell development from human embryonic stem cells.
BACKGROUND
Investigating the mechanisms of human primordial germ cell (PGC) and gamete development are important for understanding the causes of infertility and effects of environmental chemicals on reproductive development. However, there are practical and ethical difficulties associated with obtaining human tissue in early development. The aim of this study was to investigate whether human embryonic stem cell-hESC-generated germ cells could provide an in vitro model of gamete development.


METHODS
Human ESCs were differentiated as embryoid bodies (EBs) in vitro. Gene and protein marker expression profiles of EBs in different periods of culture were analysed by quantitative polymerase chain reaction (Q-PCR) and immunolocalization to monitor germ cell development. Secretion of hormones involved in germ cell maturation was measured, to detect the existence of a germ cell niche within EBs.


RESULTS
Q-PCR revealed gene expression profiles consistent with PGC formation and germ cell development. A small population of post-meiotic spermatid cells were identified using sperm-specific antibodies (Protamine 1 and 1.97). Although gene expression profiles characteristic of oocyte development and follicle-like structures were detected, a committed oocyte with extra-cellular zona pellucida was not recognized with zona pellucida-specific monoclonal antibody.


CONCLUSIONS
hESCs can form PGCs and post-meiotic spermatids in vitro, however, there remains doubt about oocyte development. Levels of steroid hormones produced by EBs were significant when compared with known values for a similar quantity of human testis, suggesting that hESC may intrinsically create a favourable hormonal niche for spermatogenesis.
Aflatoonian
B
B
Centre for Stem Cell Biology, Department of Biomedical Sciences, The University of Sheffield, Western Bank, Alfred Denny Building, Sheffield S10 2TN, UK. b.aflatoonian@shef.ac.uk
Ruban
L
L
Jones
M
M
Aflatoonian
R
R
Fazeli
A
A
Moore
H D
HD
D015415:Biomarkers; D002449:Cell Aggregation; D002453:Cell Cycle; D002454:Cell Differentiation; D002460:Cell Line; D013196:Dihydrotestosterone / Q000032:analysis; D053595:Embryonic Stem Cells; D004958:Estradiol / Q000032:analysis; D005260:Female; D005434:Flow Cytometry; D020869:Gene Expression Profiling; D018507:Gene Expression Regulation, Developmental; D005854:Germ Cells / Q000166:cytology* / Q000378:metabolism / Q000648:ultrastructure; D006801:Humans; D008297:Male; D008856:Microscopy, Fluorescence; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013091:Spermatogenesis; D013739:Testosterone / Q000032:analysis; D013997:Time Factors
D015415:Biomarkers; D012333:RNA, Messenger; D013196:Dihydrotestosterone; D013739:Testosterone; D004958:Estradiol