Generation of pluripotent stem cells from adult human testis

Human primordial germ cells and mouse neonatal and adult germline stem cells are pluripotent and derive embryonic stem cell properties. Here we report the successful establishment of stable pluripotent human adult germline stem cells (haGSCs) derived from spermatogonial cells of adult human testis. Cellular and molecular characterization of haGSCs revealed many similarities to human embryonic stem (hES) cells and haGSCs produced teratomas after subcutaneous transplantation into immunodeficient mice. The haGSCs differentiated into various types of somatic cells of all three germ layers when grown under conditions used to induce the differentiation of hES cells. We conclude that the generation of haGSCs from testicular biopsies may provide simple and non-controversial access to individual cell-based therapy without the ethical and immunological problems associated with hES cells. Keywords: pluripotent stem cells characterisation • Spermatogonial cells (hGS) from 3 different normal patients (patient 59, 73 and 81, passage 0) were cultured for 4 days in Knockout medium, 20% Knockout-FBS, 1% L-glutamine and 4ng/ml GDNF. After enrichment with CD49f and matrix selection with collagen and laminin the cells were washed with phosphate buffered saline (PBS) and pelleted at 1000rpm/10min./4°C, lysed with RLT buffer + ß-Mercaptoethanol and stored at -80°C. • hES(H1) cells (hES) were cultured for 10 days (passage 48, 57 and 67) in Knockout medium, 20% Knockout-Serum-Replacer, 1% L-glutamine, 1% NEAA, 1% penicillin/streptomycin and 4ng/ml FGF-2, on CF1 inactivated feeder on 0,1% gelatine coated dishes. After the clusters were manually cut, the isolated clusters were washed with phosphate buffered saline (PBS) and pelleted at 1000rpm/10min./4°C and lysed with RLT buffer + ß-Mercaptoethanol and stored at -80°C. • human adult germ stem cells (haGSCs) were derived from hGS cells from 3 different normal patients after the enrichment and further cultivation in LIF supplemented medium. haGSC from patient 52, 54 and 35 (from lower and higher passages) were cultured for 10 days in basic medium (DMEM high glucose, 15% FCS (Biochrom), 1% non-essential amino acids (NEAA), 1% L-glutamine and 0.05 mM ß-mercaptoethanol (Gibco) with 103 units/ml leukemia inhibitory factor (LIF, human, Chemicon) on 0,1% gelatine coated dishes. After the clusters were manually cut, the isolated clusters were washed with phosphate buffered saline (PBS) and pelleted at 1000rpm/10min./4°C and lysed with RLT buffer + ß-Mercaptoethanol and stored at -80°C.