Oocyte Arrested at Metaphase II Stage were Derived from Human Pluripotent Stem Cells in vitro.

Initiation of meiosis is the most difficult aspect of inducing competent oocytes differentiation from human stem cells in vitro. Human induced pluripotent stem cells (hiPSCs) and embryonic stem cells (hESCs) were cultured with follicle fluid, cytokines and small molecule to induced oocyte-like cells (OLCs) formation through a three-step induction procedure. Expression of surface markers and differentiation potential of germ cells were analyzed in vitro by flow cytometry, gene expression, immunocytochemistry, western blotting and RNA Sequencing. To induce the differentiation of hiPSCs into OLCs, cells were firstly cultured with a primordial germ cell medium for 10 days. The cells exhibited similar morphological features to primordial germ cells (PGCs), high expressing of germ cell markers and primordial follicle development associated genes. The induced PGCs were then cultured with the primordial follicle-like cell medium for 5 days to form the induced follicle-like structures (iFLs), which retained both primordial oocytes-like cells and granulosa-like cells. In the third step, the detached iFLs were harvested and transferred to the OLC-medium for additional 10 days. The cultured cells developed cumulus-oocyte-complexes (COCs) structures and OLCs with different sizes (50-150 μm diameter) and a zona pellucida. The in vitro matured OLCs had polar bodies and were arrested at metaphase II (MII) stage. Some OLCs were self-activated and spontaneously developed into multiple-cell structures similar to preimplantation embryos, indicating that OLCs were parthenogenetically activated though in vitro fertilization potential of OLCs are yet to be proved. in vitro maturation of OLCs derived from hiPSCs provides a new means to study human germ cell formation and oogenesis.