Direct induction of meiosis from human iPSCs under defined conditions
Meiotic failure is a significant cause of infertility, but the lack of an in vitro model of human meiosis is a barrier to understanding its mechanism. Here, we establish a method to initiate meiosis directly from male or female human induced pluripotent stem cells (iPSCs). DNMT1 inhibition, retinoid signaling activation, and overexpression of regulatory factors (anti-apoptotic BCL2, and pro-meiotic HOXB5, BOLL, or MEIOC) rapidly activates meiosis over a 15-day protocol. Our protocol bypasses the primordial germ cell stage and directly generates cells expressing genes similar to meiotic oogonia, including oogonia markers, all synaptonemal complex components, and meiotic recombination machinery. DNMT1 inhibition rapidly erases DNA methylation, including at imprinting control regions and promoters of meiotic genes. Microscopy shows key aspects of meiosis, including chromosome axis formation and synapsis in live human cells. Our model of human meiosis provides opportunities for studying this critical reproductive process under chemically defined conditions in vitro. Overall design: A constitutive expression vector for the anti-apoptotic factor BCL2, as well as doxycycline-inducible expression vectors for meiosis-promoting factors (HOXB5, BOLL, and MEIOC), were integrated into three lines of human iPSCs (F2 female, F3 female, and PGP1 male) using PiggyBac transposase. 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, and incubated at 37 °C. For 6-well plates, 1.5 mL of medium was used per well. After one day, the medium was replaced with APEL2 containing 5 µM GSK3484862 and 1 µg/mL doxycycline. A 50% media change was performed every 2 days. Three days after seeding the cells, they were moved to a 34 °C incubator. GSK3484862 was withdrawn starting on day 7. Cells were harvested on day 15 and cryopreserved in CryoStor CS10 prior to sequencing. One sample was sequenced for each of the three iPSC lines.