Developing methods for co-culture of human testicular cells and pluripotent stem cells

Abstract

Human Sertoli and Leydig cells are of great value for research community as tools for studying testicular physiology and effects of environmental pollutants. However, traditional cell culture conditions containing serum pose limitations for evaluation of the role of active molecules on cell functions. Therefore, our first objective was to establish serum-free culture conditions to maintain somatic testicular cell lines. The ability to perform live cell microscopy to model testicular physiology is of great importance. Thus, our second objective was to develop methods for genetic manipulation of human somatic testicular cells and establish cell lines that express fluorescent markers. Our long term goal is to use human pluripotent stem cells (hPSCs) to generate testicular somatic and germ cells. Our final objective was to co-culture our genetically modified testicular cell lines with hPSCs in 3D microenvironment. Using de-identified donor testes we established cell lines, which were used for testing serum-free culture conditions and genetic modification. Characterized human embryonic stem cell (hESC) line was used for comparison in genetic manipulations and in co-culture with testicular cells. De-identified adult donor testes were mechanically dissociated and plated into serum-containing medium. Suspension cells were removed, and adherent cells were further propagated to establish cell lines, which were further characterized for testicular somatic cell marker expression by immunocytochemistry and RT-PCR. For comparative studies, cells were grown in serum-containing medium or under serum-free conditions for consecutive passages and evaluated for increase in cell number. Fluorescent markers were introduced into testicular cells and hESCs using lipofection. Transfected cells were further selected for antibiotic resistance and subcloned. Genetic modification was confirmed by genotyping, and transgene expression was assessed using live cell imaging. In co-culture studies, fluorescent hESCs were first induced to differentiate in embryoid bodies. Formed embryoid bodies and fluorescent testicular cells were embedded into extracellular matrix for stationary culture and transferred for culture in spin bioreactors. Cell co-cultures were assessed by live imaging. Our study has shown, that established cell lines are represented by cells expressing Sertoli cell markers with a minor contribution of other testicular cell types. We have shown that cells can be maintained under serum-free conditions without the loss of proliferative activity. Established cell lines can be genetically modified to express a marker of interest. Importantly, we have demonstrated that testicular cells and hESCs can be co-cultured for prolonged time in a 3D microenvironment. Our research extends possible applications of human testis-derived somatic cells for further studies of human male reproductive biology and shows that these cells can be successfully adapted for PSC research.