Induction of meiosis from human pluripotent stem cells [Bisulfite-seq]

An in vitro model of human meiosis would accelerate research into this important reproductive process and development of therapies for infertility. We have developed a method to induce meiosis starting from male or female human pluripotent stem cells. We demonstrate that DNMT1 inhibition, retinoid signaling activation, and overexpression of regulatory factors (anti-apoptotic BCL2, and pro-meiotic HOXB5, BOLL, or MEIOC) rapidly activates meiosis, with leptonema beginning at 6 days, zygonema at 9 days, and pachynema at 12 days. Immunofluorescence microscopy shows key aspects of meiosis, including chromosome synapsis and sex body formation. The meiotic cells express genes similar to meiotic oogonia in vivo, including all synaptonemal complex components and machinery for meiotic recombination. These findings establish an accessible system for inducing human meiosis in vitro. Plasmids for constitutive expression of BCL2 and doxycycline-inducible expression of HOXB5, BOLL, and MEIOC were integrated into F2 D4TDZG, F3 D4TT2G, and PGP1 D4TR8G reporter hiPSCs. The iPSCs were seeded at 50,000 cells/cm2 on Matrigel-coated plates in mTeSR1 supplemented with 5 µM GSK3484862, 1 µg/mL doxycycline, and 10 µM Y-27632. For 6-well plates, 1.5 mL of medium was used per well. After one day, the media was replaced with APEL2 containing 5 µM GSK3484862 and 1 µg/mL doxycycline. A 50% media change was performed every 2 days, and GSK3484862 was withdrawn starting on day 7. Cells were harvested at each day from day 0 (hiPSC) to day 15. For the pilot experiment, methyl-seq was performed on cells that had been fixed using the Parse Biosciences scRNAseq fixation kit. For the follow-up experiment, methyl-seq was performed on unfixed cells, some of which were sorted based on DDX4-tdTomato reporter expression. Library preparation was performed using the NEB Enzymatic Methyl-Seq kit. For the pilot experiment, sequencing was performed using a MiSeq Micro kit. For the follow-up experiment, sequencing was performed on two lanes of a Novaseq X Plus 25B PE150 flowcell.