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Novel culture conditions for the improvement of the in vitro expansion of human Spermatogonial Stem Cells. Future stem cell therapies to restore fertility in prepuberal boys enrolled in our experimental fertility preservation program with patients with cancer or genetic syndromes

M Martin-Inaraja(1,2) L Herrera(1,2) C Rodriguez(1,2) S Santos(1,2) E Solorzano(3) R Lopez-Almaraz(3) M B Prieto(4) R Matorras(4) C Eguizabal(1,2)

1:1. Cell Therapy, Stem Cells and Tissues Group, Basque Centre for Blood Transfusion and Human Tissues, Barrio Labeaga 46A, Galdakao 48960, Spain; 2:2. Biocruces Bizkaia Health Research Institute, Cruces Plaza 12, Barakaldo 48903, Spain; 3:3. Biocruces Bizkaia Health Research Institute-Cruces University Hospital, Pediatric Department, Barakaldo 48903, Spain; 4:4. Biocruces Bizkaia Health Research Institute-Cruces University Hospital-Basque Country University-IVI Bilbao, Human Reproduction Unit, Barakaldo 48903, Spain

Some of the cancer treatments and genetic syndromes, such as Klinefelter Syndrome, can cause fertility problems in adulthood in prepuberal children. Prepuberal patient do not have the option to cryopreserve the sperm so, to the date, there is no alternative to restore the fertility in the future. The aim of this experimental fertility program is to preserve testicular tissue (with Spermatogonial Stem Cells (SSCs)) to expand in vitro these stem cells for future autologous transplantation of the tissue or expanded SSCs in the adulthood. We collected and processed testicular biopsies of both adults and children (oncologic and KS). Each biopsy was divided into 3 fragments: for histological study, for clinical use and for research. We observed that adult patients, all oncologic prepuberal patients and 20% of Klinefelter Syndrome prepuberal patients express both VASA and MAGEA4 germ cell markers. Firstly, we expanded human male fetal germ cells (hPGCs) in vitro under several culture conditions. Our findings provide a 2D culture system to expand hPGCs that could be useful to study propagation to SSCs. Secondly, we pursued in vitro expansion cultures of adult SSCs using a modification of our previous culture conditions for hPGCs. We observed an increase of GPR125 + cells (3,3%) in comparison with the control culture condition (1,5%) after 28 days in culture. After these preliminary data in adult SSCs, we will use this novel culture condition for improvement of in vitro expansion of prepuberal SSCs for future cell therapy.

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