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    Cell Therapy of Male Infertility: Characterization of Human Prepubertal Germ Stem Cells


    Anne-Sophie Gille

     

     

    Thursday october 28 2021 at 2 pm

    Rosalind Franklin room

    Institut Cochin, 22 rue Méchain, Paris 75014

    and by videoconference


    Supervisor: Jean-Philippe Wolf

    Team: From Gametes to Birth: Genomics, Epigenetics and Physiopathology of Reproduction

    Department: Developpement, Reproduction, Cancer (DRC)

    SUMMARY

    Germ stem cells (GSC) found in the prepubertal testis are the basis of spermatogenesis in adults. Chemotherapy can severely damage the GSC pool, resulting in the steriity. Preservation of fertility in children is done by cryopreservation of immature testicular tissue (ITT). Restoration of fertility from ITT have been successfully performed in many animals models. Its feasibility and its safety remain to be demonstrated in humans. My thesis work consisted of 3 parts. The aims were to contribute to the characterization of human GSC in prepubertal ITT (GermCell-A01824-47) (parts 1 and 2), and to take part in the study of the effects of hypoxia, a component of the testicular niche, on GSC, in order to develop culture conditions that support their in vitro expansion (part 3).

    1) Evaluation of the pool of prepubertal GSC in preserved human ITT

    A reduction in the spermatogonia pool was observed in our cohort of 30 sickle cell patients (4.2-15.0 years), and among 75 leukemia patients (0.9-13.7 years). In sickle cell patients, this depletion has been attributed to the disease, and not to hydroxyurea. In leukemia boys, it is all the more reduced the more patients have been exposed to cyclophosphamide, with a notable decrease when the cumulative doses of this alkylating agent exceed 4 g / m².

    2) Development of an in vivo model of prepubertal GSC

    ITT fragments from 17 patients (2.8-15.6 years), were used to develop a human ITT xenograft model in immunodeficient mouse testes, as a model for the study of human GSC in vivo and as a preclinical tissue autograft strategy. The functionality of the testicular niche, capable of supporting the maturation of GSC and their long-term self-renewal, ie 8 months after xenografting, was observed. Continuous administration of FSH / LH gonadotropins did not show any effect on the maintenance of GSC, nor on tissue maturation and meiosis initiation.

    3) Study of the effects of hypoxia, a key parameter in the control of the fate of the GSC in the testicular niche

    The ability of murine GSC to form colonies and their proliferation were reduced when they were exposed to extremely low O2 content (1% and 0.1%) in comparison with culture at atmospheric content (21% O2). In our study, the initial GSC cell density is a critical parameter for the effects of moderate hypoxia (3.5% O2), on cell growth and on self-renewal of GSC in vitro. An increase in cell density under hypoxic conditions could modulate the response of GSC, also influenced by the culture system with murine embryonic fibroblasts.

     

    KEYWORDS : germ stem cells, fertility preservation, prepubertal, spermatogenesis, spermatogonia, infertility