TY - DATA T1 - Initial germ cell to somatic cell ratio impacts the efficiency of SSC expansion in vitro PY - 2017/12/01 AU - Itai Gat AU - Leila Maghen AU - Melissa Filice AU - Shlomit Kenigsberg AU - Brandon Wyse AU - Khaled Zohni AU - Peter Saraz AU - Andrée Gauthier Fisher AU - Clifford Librach UR - https://tandf.figshare.com/articles/dataset/Initial_germ_cell_to_somatic_cell_ratio_impacts_the_efficiency_of_SSC_expansion_i_in_vitro_i_/5660473 DO - 10.6084/m9.figshare.5660473.v1 L4 - https://ndownloader.figshare.com/files/9874429 L4 - https://ndownloader.figshare.com/files/9874432 KW - Differential plating KW - fertility preservation KW - GFRα1 KW - gonadotoxic treatments KW - prebubertal boys KW - spermatogonial stem cells N2 - Spermatogonial Stem Cell (SSC) expansion in vitro remains a major challenge in efforts to preserve fertility among pubertal cancer survivor boys. The current study focused on innovative approaches to optimize SSC expansion. Six- to eight-week-old CD-1 murine testicular samples were harvested by mechanical and enzymatic digestion. Cell suspensions were incubated for differential plating (DP). After DP, we established two experiments comparing single vs. repetitive DP (S-DP and R-DP, respectively) until passage 2 (P2) completion. Each experiment included a set of cultures consisting of 5 floating-to-attached cell ratios (5, 10, 15, 20, and 25) and control cultures containing floating cells only. We found similar cell and colony count drops during P0 in both S- and R-DP. During P2, counts increased in S-DP in middle ratios (10, 15, and especially 20) relative to low and high ratios (5 and 25, respectively). Counts dropped extensively in R-DP after passage 2. The superiority of intermediate ratios was demonstrated by enrichment of GFRα1 by qPCR. The optimal ratio of 20 in S-DP contained significantly increased proportions of GFRα1-positive cells (25.8±5.8%) as measured by flow cytometry compared to after DP (1.9±0.7%, p<0.0001), as well as positive immunostaining for GFRα1 and UTF1, with rare Sox9-positive cells. This is the first report of the impact of initial floating-to-attached cell ratios on SSC proliferation in vitro. Abbreviations: SSC: spermatogonial stem cells; DP: differential plating; NOA: non-obstructive azoospermia; MACS: magnetic-activated cells sorting; FACS: fluorescence-activated cells sorting ER -