Abstract
Spermatogonial stem cell (SSC) transplantation therapy is a promising strategy to renew spermatogenesis for prepubertal boys whose fertility is compromised. However, propagation of SSCs is required due to a limited number of SSCs in cryopreserved testicular tissue. This propagation must be done under xeno-free conditions for clinical application. SSCs were propagated from infant testicular tissue (7 mg and 10 mg) from two boys under xeno-free conditions using human platelet lysate and nutrient source. We verified SSC-like cell clusters (SSCLCs) by quantitative real-time polymerase chain reaction (PCR) and immune-reaction assay using the SSC markers undifferentiated embryonic cell transcription factor 1 (UTF1), ubiquitin carboxyl-terminal hydrolase isozyme L1 (UCHL1), GDNF receptor alpha-1 (GFRα-1) Fα and promyelocytic leukaemia zinc finger protein (PLZF). The functionality of the propagated SSCs was investigated by pre-labelling using green fluorescent Cell Linker PKH67 and xeno-transplantation of the SSCLCs into busulfan-treated, therefore sterile, immunodeficient mice. SSC-like cell clusters (SSCLCs) appeared after 2 weeks in primary passage. The SSCLCs were SSC-like as the UTF1, UCHL1, GFRα1 and PLZF were all positive. After 2.5 months’ culture period, a total of 13 million cells from one sample were harvested for xenotransplantation. Labelled human propagated SSCs were identified and verified in mouse seminiferous tubules at 3–6 weeks, confirming that the transplanted cells contain SSCLCs. The present xeno-free clinical culture protocol allows propagation of SSCs from infant boys.
Highlights
Advances in cancer detection and therapy have greatly improved survival rates for childhood cancer patients and nowadays the vast majority will become long-term survivors
We investigated a clinically acceptable human substitution, which is already used in a clinical setting, by replacing fetal bovine serum (FBS) with 2% human platelet lysate (hPL) and replacing bovine serum albumin (BSA) with human serum albumin
We found undifferentiated embryonic cell transcription factor 1 (UTF1) only localized in the nucleus of spermatogonial stem cells (SSCs)-like cell clusters (SSCLCs) and very few UTF1 positive cells showed proliferation marker, indicating the most cell became quiesced in SSCLCs while only a few cells are proliferative active
Summary
Advances in cancer detection and therapy have greatly improved survival rates for childhood cancer patients and nowadays the vast majority will become long-term survivors. 1 in 530 young adults between the ages of 20 and 39 years is a childhood cancer survivor [1]. To restore fertility in childhood cancer survivors, options for fertility preservation prior to gonadotoxic cancer therapies are needed [3]. Cryopreservation of spermatozoa is not an option for prepubertal boys as they have no spermatogenesis yet [4]. Cryopreservation of immature testicular biopsy (ITT) for storage of spermatogonial stem cells (SSCs) is a preventive strategy for prepubertal boys facing gonadotoxic cancer therapy which has been available for more than a decade in different reproductive centres worldwide [5,6]. About 17% of azoospermia men have a history of cryptorchidism [8]
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