Xeno-Free Propagation of Spermatogonial Stem Cells from Infant Boys

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.

A Xeno-free Media for the In Vitro Expansion of Human Spermatogonial Stem Cells

“…There is currently one other reported xeno-free formulation for the expansion of SSCs, [52] however it relies on undefined human platelet lysate (hPL), which is a growth factor-rich alternative to FBS, collected from human platelets. While hPL is xeno-free, lot-to-lot variations are still a concern, as is the risk of pathogen contamination.…”

Characterization and Survival of Human Infant Testicular Cells After Direct Xenotransplantation

“…At eight weeks of age, each testis of mice was injected with 80 μg busulfan (B2635, Sigma-Aldrich) to eliminate endogenous spermatogenesis. The busulfan was dissolved in dimethyl sulfoxide (DMSO) and delivered in a volume of 20μl through two different sites ( 24 , 31 ). Xenotransplantation was performed 4-5 weeks after busulfan treatment.…”

“…Taken together, technical difficulties have hampered the translation from bench to clinic because of the lingering safety and ethical considerations. Although propagation of SSCs has been reported from infant boys under xeno-free conditions ( 24 ), the genetic and epigenetic stability of the SSCs still need to be determined before the application in clinical trials. Furthermore, the regulation for genetic and epigenetic determination also remains to be standardized.…”

Ubiquitin C‐terminal hydrolase L1 (UCHL1), a double‐edged sword in mammalian oocyte maturation and spermatogenesis

“…In detail, UCHL1 is only expressed in SSCs at 8–16 days after postnatal, and detected in both Sertoli cells and SSCs at 30 days after postnatal and adult mice. 51 Recently, some researchers have used UCHL1 as a major molecular marker for human SSCs transplantation 97 and porcine SSCs line establishment. 98 However, UCHL1 is like a double‐edged sword, if its expression level is unbalanced, it will affect the normal physiological and biological processes.…”