Abstract
Autologous transplantation of spermatogonial stem cells is a promising new avenue to restore fertility in infertile recipients. Expansion of the initial spermatogonial stem cell pool through cell culturing is a necessary step to obtain enough cells for effective repopulation of the testis after transplantation. Since in vitro propagation can lead to (epi-)genetic mutations and possibly malignant transformation of the starting cell population, we set out to investigate genome-wide DNA methylation status in uncultured and cultured primary testicular ITGA6+ sorted cells and compare them with germ cell tumor samples of the seminoma subtype. Seminomas displayed a severely global hypomethylated profile, including loss of genomic imprinting, which we did not detect in cultured primary testicular ITGA6+ cells. Differential methylation analysis revealed altered regulation of gamete formation and meiotic processes in cultured primary testicular ITGA6+ cells but not in seminomas. The pivotal POU5F1 marker was hypomethylated in seminomas but not in uncultured or cultured primary testicular ITGA6+ cells, which is reflected in the POU5F1 mRNA expression levels. Lastly, seminomas displayed a number of characteristic copy number variations that were not detectable in primary testicular ITGA6+ cells, either before or after culture. Together, the data show a distinct DNA methylation patterns in cultured primary testicular ITGA6+ cells that does not resemble the pattern found in seminomas, but also highlight the need for more sensitive methods to fully exclude the presence of malignant cells after culture and to further study the epigenetic events that take place during in vitro culture.
Highlights
In vitro propagation of cryopreserved spermatogonial stem cells (SSCs) followed by autologous transplantation of cultured SSCs (SSCT) into the testes is viewed as a promising new technique to treat male survivors of childhood cancer for sub- or infertility [1,2,3,4,5]
We have previously demonstrated that our culture method, either with or without selection of integrin alpha-6 (ITGA6)+ cells, yields a population of cells that have the ability to migrate to the basal membrane of the seminiferous tubules after xenotransplantation to mouse recipients, as evidenced by positive immunofluorescent staining of COT-1 DNA sequences in the transplanted tissues [40, 43, 54]
Human SSCs cannot differentiate in the mouse testis microenvironment due to the large phylogenetic difference between mice and humans [17], these findings form the golden standard to evaluate the presence of spermatogonial stem cells in a transplanted population of cells and confirm that the human ITGA6+ testicular population contains SSCs
Summary
In vitro propagation of cryopreserved spermatogonial stem cells (SSCs) followed by autologous transplantation of cultured SSCs (SSCT) into the testes is viewed as a promising new technique to treat male survivors of childhood cancer for sub- or infertility [1,2,3,4,5]. The robustness of spermatogenic rescue following SSCT has been demonstrated for various species including mice, cattle and primates [6,7,8,9,10] and SSCT treated animals are capable of producing offspring which appears healthy [11,12,13,14,15] and fertile, at least in rodents [12, 14,15,16]. Since the proportion of true SSCs will be limited in a biopsy from a human prepubertal small testis, in vitro propagation of the initial SSC pool is a necessary step in the SSCT protocol
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