Hindawi Publishing Corporation Journal of Biomedicine and Biotechnology Volume 2012, Article ID 291038, 15 pages doi:10.1155/2012/291038 Research Article Potential Stemness of Frozen-Thawed Testicular Biopsies without Sperm in Infertile Men Included into the In Vitro Fertilization Programme Martin Stimpfel, 1 Thomas Skutella, 2 Mikael Kubista, 3, 4 Elvira Malicev, 5 Sabine Conrad, 6 and Irma Virant-Klun 1 1 Reproductive Unit, Department of Obstetrics and Gynaecology, University Medical Centre Ljubljana, 1000 Ljubljana, Slovenia 2 Institute for Anatomy and Cell Biology, School of Medicine, University of Heidelberg, 69120 Heidelberg, Germany 3 TATAA Biocenter, 41103 G¨ oteborg, Sweden 4 Laboratory of Gene Expression, Institute of Biotechnology, Academy of Sciences of the Czech Republic, 14220 Prague, Czech Republic 5 Blood Transfusion Centre of Slovenia, 1000 Ljubljana, Slovenia 6 Institute for Anatomy, School of Medicine, University of T¨ ubingen, 72074 T¨ ubingen, Germany Correspondence should be addressed to Irma Virant-Klun, irma.virant@kclj.si Received 31 May 2011; Revised 28 October 2011; Accepted 30 October 2011 Academic Editor: Ji Wu Copyright © 2012 Martin Stimpfel et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. We describe the potential stemness of a small amount of frozen-thawed testicular tissue without sperm obtained by biopsy from six patients undergoing assisted reproductive treatment. The patients were diagnosed with Sertoli Cell-Only Syndrome alone or combined with maturation arrest. Trying to provide the natural stem cell niche for cultured stem cells, all isolated cells from enzymatically degraded biopsies where cultured together in different culture media and the presence of putative mesenchymal and putative pluripotent ES-like stem cells was indicated using different methods. High throughput real-time quantitative PCR followed by multivariate analysis revealed the formation of distinct cell clusters reflecting high degree of similarity and some of these cell clusters expressed the genes characteristic for pluripotent stem cells. In the presence of the follicular fluid, prepared as serum, putative testicular stem cells showed a certain degree of plasticity, and spontaneously differentiated into adipose-like and neuronal-like cells. Additionally, using differentiation protocols putative testicular stem cells were differentiated into neuronal- and pancreatic-like cells. This study shows that in assisted reproduction programmes, testicular tissue with no sperm might be an important source of stem cells, although it is discarded in daily medical practice; this requires further research. 1. Introduction Stem cells derived from adult human tissues are of great sci- entific interest to provide potential individual cell-based therapy without ethical and immunological problems asso- ciated with human embryonic stem cells. Testicular tissue retrieved in the assisted reproduction programme might be an important source of stem cells. In infertile men with azoo- spermia (no sperm in the ejaculate), a diagnostic testicular biopsy is usually performed to find sperm and to cryopre- serve it until use for in vitro fertilization. In a certain number of these patients, there is no sperm in the testicular tissue due to Sertoli Cell-Only Syndrome (SCOS) or maturation arrest (MA) of germ cells at different stages of development. In these patients, fresh or frozen-thawed testicular tissue is thrown away in daily medical practice, but could be used for the personalized cell therapy in the future. The majority of studies on testicular stem cells have been performed with the whole animal or human testicles or large biopsies [1–14], but in a clinical practice only small testicular biopsies of infertile men are available. Most of the earlier studies concerning testicular stem cells were performed in the mouse model, mostly to study sper- matogonial stem cells and their in vitro reprogramming [1– 10]. This work was followed by studies on human testicular tissue [11–14], and the results have shown that germinal stem