UP-156 A xeno-free defined culture method for the in vitro expansion of human spermatogonial stem cells
Thursday June 27, 2019 from
TBD
Presenter

Meghan A Robinson, Canada

Research Assistant

Department of Urology

Vancouver Prostate Centre, University of British Columbia

Abstract

A xeno-free defined culture method for the in vitro expansion of human spermatogonial stem cells

Meghan Robinson1, Ryan Flannigan1,3,4, Luke Witherspoon1,2,3.

1Urology, Vancouver Prostate Centre, Vancouver, BC, Canada; 2Urology, Ottawa Hospital, Ottawa, ON, Canada; 3Urology, University of British Columbia, Vancouver, BC, Canada; 4Urology, Weill Cornell Medicine, New York, NY, United States

Vancouver Coastal Health Research Institute (VCHRI). Canadian Urological Association Scholarship Foundation (CUASF). Canadian Institute of Health Research (CIHR). University of British Columbia Department of Urological Sciences.

Introduction: 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). RT-qPCR analyses revealed no significant variation in gene expression of undifferentiated germ cell markers Undifferentiated Embryonic Cell Transcription Factor 1 (UTF1), Inhibitor Of Differentiation 4 (ID4), and Fibroblast Growth Factor Receptor 3 (FGFR3). An increase in Deleted In Azoospermia Like (DAZL), a regulator of both SSC proliferation and differentiation, was observed (13-fold) (Figure 1B).

Conclusion: 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.


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