Abstract
BackgroundGonadotoxic treatment of malignant diseases as well as some non-malignant conditions such as cryptorchidism in young boys may result in infertility and failure to father children later in life. As a fertility preserving strategy, several centers collect testicular biopsies to cryopreserve spermatogonial stem cells (SSCs) world-wide. One of the most promising therapeutic strategies is to transplant SSCs back into the seminiferous tubules to initiate endogenous spermatogenesis. However, to obtain sufficient numbers of SSC to warrant transplantation, in vitro propagation of cells is needed together with proper validation of their stem cell identity.Materials and MethodsA minute amount of testicular biopsies (between 5 mg and 10 mg) were processed by mechanical and enzymatic digestion. SSCs were enriched by differential plating method in StemPro-34 medium supplemented with several growth factors. SSC-like cell clusters (SSCLCs) were passaged five times. SSCLCs were identified by immunohistochemical and immunofluorescence staining, using protein expression patterns in testis biopsies as reference. Quantitative polymerase chain reaction analysis of SSC markers LIN-28 homolog A (LIN28A), G antigen 1 (GAGE1), promyelocytic leukemia zinc finger protein (PLZF), integrin alpha 6 (ITGA6), ubiquitin carboxy-terminal hydrolase L1 (UCHL1) and integrin beta 1 (ITGB1) were also used to validate the SSC-like cell identity.ResultsProliferation of SSCLCs was achieved. The presence of SSCs in SSCLCs was confirmed by positive immunostaining of LIN28, UCHL1 and quantitative polymerase chain reaction for LIN28A, UCHL1, PLZF, ITGA6, and ITGB1, respectively.ConclusionThis study has demonstrated that SSCs from infant boys possess the capacity for in vitro proliferation and advance a fertility preservation strategy for pre-pubertal boys who may otherwise lose their fertility.
Highlights
Cryptorchidism occurs with a frequency of around 3% in fullterm pregnancies and in 30% of boys born prematurely
Lin-28 homolog A (LIN28) and Ubiquitin carboxy-terminal hydrolase L1 (UCHL1) were expressed in gonocytes and spermatogonia, including spermatogonia with rarefaction-zone in their nuclei (Figures 2A–F)
Further quantification of LIN28 positive cells and A dark (Ad) spermatogonia demonstrated that LIN28 was expressed around 60% of Ad spermatogonia and 5% of LIN28 positive germ cells were Ad spermatogonia (Figures 2G,H)
Summary
Cryptorchidism occurs with a frequency of around 3% in fullterm pregnancies and in 30% of boys born prematurely. Around one third of boys with cryptorchidism, especially bilateral cryptorchidism, suffer from infertility in adult life despite the presence of spermatogonial stem cells (SSCs) in the majority of testes biopsies (Hadziselimovic and Herzog, 2001). Another group of young boys in need of fertility preservation is those suffering from childhood cancer. Prior to chemotherapeutic treatment this group of patients may benefit from having a biopsy excised and frozen from which SSCs can be isolated and, in theory, auto-transplanted after remission Due to this potential, reproductive centers worldwide currently freeze testis biopsies to preserve SSCs and the germ cell niche. To obtain sufficient numbers of SSC to warrant transplantation, in vitro propagation of cells is needed together with proper validation of their stem cell identity
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