PD39-10 A XENO-FREE CULTURE METHOD FOR THE IN VITRO EXPANSION OF HUMAN SPERMATOGONIAL STEM CELLS

Abstract

You have accessJournal of UrologyInfertility: Basic Research & Pathophysiology (PD39)1 Sep 2021PD39-10 A XENO-FREE CULTURE METHOD FOR THE IN VITRO EXPANSION OF HUMAN SPERMATOGONIAL STEM CELLS Meghan Robinson, Ryan Flannigan, and Luke Witherspoon Meghan RobinsonMeghan Robinson More articles by this author , Ryan FlanniganRyan Flannigan More articles by this author , and Luke WitherspoonLuke Witherspoon More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002049.10AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: In vitro expansion of spermatogonial stem cells (SSCs) has been established using animal-derived fetal bovine serum (FBS), however, animal components introduce the risk of contaminating with pathogens, making them unsuitable for medical use. This study set out to develop xeno-free culture conditions for the expansion of human SSCs. METHODS: SSCs were derived from human induced pluripotent stem cells (hiPSCs) and tested in various xeno-free media conditions in combination with growth factors Prostaglandin D2 (PDG2) and Insulin-Like Growth Factor 1 (IGF1). Primary SSCs then underwent 3 passages in the best condition, were cryopreserved and thawed, and then analysed for changes in gene and protein expression by real time polymerase chain reaction (RT-qPCR) and immunocytochemistry. RESULTS: 10 ng/mL PDG2 with 10 ng/mL IGF1 was found to replace FBS and BSA without loss of viability or growth. ROCK inhibitor Y-27632 was determined to be necessary for viability upon thawing. Compared to established conditions, the SSCs shared identical protein expression profiles for the SSC markers Glial Cell-Derived Neurotrophic Factor Family Receptor Alpha 1 (GFRA1), G-Protein Coupled Receptor 125 (GPR125), Thy-1 Cell Surface Antigen (CD90), and Stage-Specific Embryonic Antigen 4 (SSEA4) (Figure 1A-B). RT-qPCR analyses revealed no significant variation in gene expression of undifferentiated germ cell markers and Inhibitor Of Differentiation 4 (ID4), and Fibroblast Growth Factor Receptor 3 (FGFR3). The greatest change seen was an increase in Deleted In Azoospermia Like (DAZL), a regulator of both SSC proliferation and differentiation (23-fold) (Figure 1C). CONCLUSIONS: This study identified a combination of growth factors capable of replacing FBS and BSA components in established SSC expansion cell culture. This xeno-free defined formulation allows standardized SSC culture and removes the risk of animal pathogens. Source of Funding: VCHRI, CUASF, CIHR, UBC © 2021 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 206Issue Supplement 3September 2021Page: e671-e672 Advertisement Copyright & Permissions© 2021 by American Urological Association Education and Research, Inc.MetricsAuthor Information Meghan Robinson More articles by this author Ryan Flannigan More articles by this author Luke Witherspoon More articles by this author Expand All Advertisement Loading ...