single cell RNAseq of mouse E12.5 fetal ovarian somatic cells

A major technology for generating oocytes by in vitro gametogenesis involves generating ovary models called reconstituted ovaries (rOvaries). Widespread use of rOvaries depends on the reliable manufacturing of primordial germ cell like cells (PGCLCs) and fetal ovarian somatic cells (FOSCs). Using scRNA-seq, we identify that upon thaw, FOSCs are composed largely of Foxl2+ pre-granulosa cells, KRT19+ epithelial cells and Nr2f2+ mesenchymal cells which self-assemble together with PGCLCs into disc-like organoids containing multiple follicles embedded in NR2F2 stroma without an ovarian surface epithelium and no steroidogenic theca. The absence of an ovarian surface epithelium combined with the small size of GV-oocytes indicates that follicle production in the rOvary corresponds to first-wave folliculogenesis. Ovarian somatic cells for each corresponding lot were obtained from E12.5 mice from ten pregnant female CD1(Charles River, Strain code: 022) mice. Gonads, which included the mesonephros and ovaries, were collected into cell culture plates filled with MEF media on ice. Gonads were transferred into glass plates containing MEF media and mesonephros were removed using surgical blade and fine forceps under a SZX16 (Olympus) microscope. Ovaries were collected into a well of a 4-well plate containing MEF media and DNase I. Ovaries were washed twice by transferring them into wells containing PBS. Dissociation of ovaries was performed in 0.05% Trypsin for 10 minutes at 37C. MEF media was used to quench and tissues were dissociated by vigorous pipetting. Cells were strained using a 70uM cell strainer and then washed using MEF media. The ovarian somatic cell pellet was then resuspended in MACs Buffer and incubated with the MACs antibodies SSEA1 and CD31 at 4C for 30 minutes. Afterwards labeled cells were washed by adding MACS buffer and spinning for 5 minutes at 1,600rpm. The cell pellet was resuspended in 500uL MACS buffer and then loaded into a MS column held by a MiniMACS™ Separator magnet. Flow through was collected, including the MACS media added for washes. The number of cells collected was counted and aliquots of 300,000 cells per vial were frozen down in Cell Banker 2 at -80C then transferred to Liquid nitrogen storage tanks for long term storage.