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  • Küçük Resim
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    Propagation of spermatogonial stem cell-like cells from infant boys
    (Frontiers Media, 2019) Dong, Lihua; Kristensen, Stine Gry; Hildorf, Simone; Gül, Murat; Clasen-Linde, Erik; Fedder, Jens; Hoffmann, Eva R.; Cortes, Dina; Thorup, Jorgen; Andersen, Claus Yding
    Background: Gonadotoxic treatment of malignant diseases as well as some nonmalignant 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) worldwide. 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 Methods: A 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. Results: Proliferation 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. Conclusion: This study has demonstrated that SSCs from infant boys possess the capacity for in vitro proliferation and advance a fertility preservation strategy for prepubertal boys who may otherwise lose their fertility.
  • Küçük Resim
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    Xeno-free propagation of spermatogonial stem cells from infant boys
    (MDPI AG, 2019) Dong, Lihua; Gül, Murat; Hildorf, Simone; Susanne Elisabeth; Kristensen, Stine Gry; Hoffmann, Eva R.; Cortes, Dina; Thorup, Jorgen Mogens; Andersen, Claus Yding
    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 (GFR?-1) F? 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, GFR?1 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.