MP66-18 EVIDENCE OF SPERMATOGENIC DIFFERENTIATION OF HUMAN EMBRYONIC STEM CELLS IN AN IN VITRO HOLLOW FIBER MODEL

Abstract

You have accessJournal of UrologyInfertility: Basic Research, Physiology & Pathophysiology1 Apr 2014MP66-18 EVIDENCE OF SPERMATOGENIC DIFFERENTIATION OF HUMAN EMBRYONIC STEM CELLS IN AN IN VITRO HOLLOW FIBER MODEL Meenakshi Gaur, Connie John, Cyril Ramathal, Renee Reijo Pera, and Paul Turek Meenakshi GaurMeenakshi Gaur More articles by this author , Connie JohnConnie John More articles by this author , Cyril RamathalCyril Ramathal More articles by this author , Renee Reijo PeraRenee Reijo Pera More articles by this author , and Paul TurekPaul Turek More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2014.02.2065AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Replicating human spermatogenesis in vitro has applications in reproductive toxicology of drugs and chemicals and, in the long-term, could involve the treatment of male infertility. Although mouse embryonic stems cells have been successfully differentiated to functional sperm in vitro, the full sequence of human spermatogenesis has not been similarly reproduced. By employing a sterile, hollow fiber model, we sought to differentiate human embryonic stem cells along the spermatogenic sequence and report our success to date. METHODS A 0.5 mm pore size membrane hollow fiber (HF) bioreactor unit was connected to a Cellmax Artificial Capillary Oxygenator Module (Spectrum Laboratories, Inc.). Human Sertoli cells (20 x 106) derived from cadaveric tissue and H1 human embryonic stem cells (hESs) transfected to express mcherry, DAZ, DAZL, and Boule (7 x 106) were introduced to the HF. Inner and outer chambers were filled with complete medium and the assembly incubated at 37°C in 5% CO2. Medium samples were collected from the inside and outside HF chambers and tested for glucose levels. Cells within the bioreactor were then forcibly eluted and evaluated histologically after parafilm embedding and also assessed for viability, immunofluorescence, and gene expression. RESULTS The sterile HF bioreactor containing viable Sertoli cells was maintained for 42 days. At that time, viable hESs were also observed in the bioreactor (see Figure, left panel, green stain). Gene expression studies showed active DAZL (see Figure, right panel, red stain) and VASA expression in cultured HF cells indicating the active differentiation along the germ cell pathway within the HF bioreactor. CONCLUSIONS It is possible to promote the viability and partial germ line differentiation of hESs in vitro in a sterile hollow fiber model that mimics the architecture of the natural seminiferous tubule. Further research is needed to create culture conditions that allow for mature sperm production and increased efficiency of spermatogenesis. © 2014FiguresReferencesRelatedDetails Volume 191Issue 4SApril 2014Page: e747 Advertisement Copyright & Permissions© 2014MetricsAuthor Information Meenakshi Gaur More articles by this author Connie John More articles by this author Cyril Ramathal More articles by this author Renee Reijo Pera More articles by this author Paul Turek More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...