A combined approach facilitates the reliable detection of human spermatogonia in vitro

STUDY QUESTION Does a combined approach allow for the unequivocal detection of human germ cells and particularly of spermatogonia in vitro? SUMMARY ANSWER Based on our findings, we conclude that an approach comprising: (i) the detailed characterization of patients and tissue samples prior to the sel...

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Published in:Human reproduction (Oxford) 2013-11, Vol.28 (11), p.3012-3025
Main Authors: Kossack, N., Terwort, N., Wistuba, J., Ehmcke, J., Schlatt, S., Schöler, H., Kliesch, S., Gromoll, J.
Format: Article
Language:English
Subjects:
Antigens, Neoplasm - genetics
Antigens, Neoplasm - metabolism
Biopsy
Cell Culture Techniques
DEAD-box RNA Helicases - genetics
DEAD-box RNA Helicases - metabolism
Gene Expression Profiling
Genetic Markers
Humans
Immunohistochemistry
Male
Neoplasm Proteins - genetics
Neoplasm Proteins - metabolism
Nuclear Proteins - genetics
Nuclear Proteins - metabolism
Real-Time Polymerase Chain Reaction
Receptor, Fibroblast Growth Factor, Type 3 - genetics
Receptor, Fibroblast Growth Factor, Type 3 - metabolism
Spermatogonia - cytology
Spermatogonia - metabolism
Testis - cytology
Testis - pathology
Trans-Activators - genetics
Trans-Activators - metabolism
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Summary:STUDY QUESTION Does a combined approach allow for the unequivocal detection of human germ cells and particularly of spermatogonia in vitro? SUMMARY ANSWER Based on our findings, we conclude that an approach comprising: (i) the detailed characterization of patients and tissue samples prior to the selection of biopsies, (ii) the use of unambiguous markers for the characterization of cultures and (iii) the use of biopsies lacking the germ cell population as a negative control is the prerequisite for the establishment of human germ cell cultures. WHAT IS KNOWN ALREADY The use of non-specific marker genes and the failure to assess the presence of testicular somatic cell types in germ cell cultures may have led to a misinterpretation of results and the erroneous description of germ cells in previous studies. STUDY DESIGN, SIZE, DURATION Testicular biopsies were selected from a pool of 264 consecutively obtained biopsies. Based on the histological diagnosis, biopsies with distinct histological phenotypes were selected (n = 35) to analyze the expression of germ cell and somatic cell markers. For germ cell culture experiments, gonadotrophin levels and clinical data were used as selection criteria resulting in the following two groups: (i) biopsies with qualitatively intact spermatogenesis (n = 4) and (ii) biopsies from Klinefelter syndrome Klinefelter patients lacking the germ cell population (n = 3). PARTICIPANTS/MATERIALS, SETTING, METHODS Quantitative real-time PCR analyses were performed to evaluate the specificity of 18 selected germ cell and 3 somatic marker genes. Cell specificity of individual markers was subsequently validated using immunohistochemistry. Finally, testicular cell cultures were established and were analyzed after 10 days for the expression of germ cell- (UTF1, FGFR3, MAGE A4, DDX4) and somatic cell-specific markers (SMA, VIM, LHCGR) at the RNA and the protein levels. MAIN RESULTS AND THE ROLE OF CHANCE Interestingly, only 9 out of 18 marker genes reflected the presence of germ cells and cell specificity could be validated using immunohistochemistry. Furthermore, VIM, SMA and LHCGR were found to reflect the presence of testicular somatic cells at the RNA and the protein levels. Using this validated marker panel and biopsies lacking the germ cell population (n = 3) as a negative control, we demonstrated that germ cell cultures containing spermatogonia can be established from biopsies with normal spermatogenesis (n = 4) and that these cultures
ISSN:0268-1161
1460-2350
DOI:10.1093/humrep/det336