Propagation of human spermatogonial stem cells in vitro.
BACKGROUND
Young boys treated with high-dose chemotherapy are often confronted with infertility once they reach adulthood. Cryopreserving testicular tissue before chemotherapy and autotransplantation of spermatogonial stem cells at a later stage could theoretically allow for restoration of fertility.


OBJECTIVE
To establish in vitro propagation of human spermatogonial stem cells from small testicular biopsies to obtain an adequate number of cells for successful transplantation.


METHODS
Study performed from April 2007 to July 2009 using testis material donated by 6 adult men who underwent orchidectomy as part of prostate cancer treatment. Testicular cells were isolated and cultured in supplemented StemPro medium; germline stem cell clusters that arose were subcultured on human placental laminin-coated dishes in the same medium. Presence of spermatogonia was determined by reverse transcriptase polymerase chain reaction and immunofluorescence for spermatogonial markers. To test for the presence of functional spermatogonial stem cells in culture, xenotransplantation to testes of immunodeficient mice was performed, and migrated human spermatogonial stem cells after transplantation were detected by COT-1 fluorescence in situ hybridization. The number of colonized spermatogonial stem cells transplanted at early and later points during culture were counted to determine propagation.


METHODS
Propagation of spermatogonial stem cells over time.


RESULTS
Testicular cells could be cultured and propagated up to 15 weeks. Germline stem cell clusters arose in the testicular cell cultures from all 6 men and could be subcultured and propagated up to 28 weeks. Expression of spermatogonial markers on both the RNA and protein level was maintained throughout the entire culture period. In 4 of 6 men, xenotransplantation to mice demonstrated the presence of functional spermatogonial stem cells, even after prolonged in vitro culture. Spermatogonial stem cell numbers increased 53-fold within 19 days in the testicular cell culture and increased 18,450-fold within 64 days in the germline stem cell subculture.


CONCLUSIONS
Long-term culture and propagation of human spermatogonial stem cells in vitro is achievable.
Sadri-Ardekani
Hooman
H
Center for Reproductive Medicine, Department of Obstetrics and Gynaecology, University of Amsterdam, Amsterdam, The Netherlands.
Mizrak
Sefika C
SC
van Daalen
Saskia K M
SK
Korver
Cindy M
CM
Roepers-Gajadien
Hermien L
HL
Koruji
Morteza
M
Hovingh
Suzanne
S
de Reijke
Theo M
TM
de la Rosette
Jean J M C H
JJ
van der Veen
Fulco
F
de Rooij
Dirk G
DG
Repping
Sjoerd
S
van Pelt
Ans M M
AM
D048868:Adaptor Proteins, Signal Transducing; D000328:Adult; D000818:Animals; D018929:Cell Culture Techniques; D002478:Cells, Cultured; D015925:Cryopreservation; D003470:Culture Media; D005455:Fluorescent Antibody Technique; D015870:Gene Expression; D006801:Humans; D007150:Immunohistochemistry; D017404:In Situ Hybridization, Fluorescence; D039503:Integrin alpha6 / Q000235:genetics; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics; D051741:Kruppel-Like Transcription Factors / Q000235:genetics; D008297:Male; D008565:Membrane Proteins / Q000235:genetics; D051379:Mice; D000075722:Promyelocytic Leukemia Zinc Finger Protein; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013093:Spermatogonia / Q000166:cytology* / Q000502:physiology / Q000637:transplantation*; D033581:Stem Cell Transplantation; D013234:Stem Cells / Q000166:cytology* / Q000502:physiology; D013737:Testis / Q000166:cytology; D014183:Transplantation, Heterologous
D048868:Adaptor Proteins, Signal Transducing; D003470:Culture Media; C108257:ITGB1BP1 protein, human; D039503:Integrin alpha6; D047908:Intracellular Signaling Peptides and Proteins; D051741:Kruppel-Like Transcription Factors; D008565:Membrane Proteins; D000075722:Promyelocytic Leukemia Zinc Finger Protein; C092037:ZBTB16 protein, human