Oocytes Generated from Chemically Reprogrammed Mouse Granulosa Cells Produce Healthy Pups

Researchers at Nankai University in China have succeeded in chemical reprogramming adult mouse granulosa cells into genomically stable, functional mouse oocytes that were able to produce healthy offspring after fertilization. “This is the first time we turned granulosa cells (GCs) into oocytes, it is a crucial and interesting work in developmental and reproductive biology,” said Lin Liu, PhD, of the College of Life Sciences at Nankai University. “But implementing this research to humans from mice still has a long way to go. I think it has more prospect in preserving fertility and endocrine function, than in treating infertility.” Liu and colleagues report their achievement in Cell Reports, in a paper titled, “Functional Oocytes Derived from Granulosa Cells.”

Ovarian follicles are the basic functional unit of the ovary and consist of an oocyte, which is the immature egg, and supporting granulosa cells, which are crucial to the development of the follicles. The number of oocytes is determined at birth, and their declining number and quality as females age, or as a result of chemotherapy, lead to infertility, the authors explained. The ability to generate oocytes could thus hold “great potential in preserving and restoring fertility.” As Liu commented, “The thing about in vitro fertilization is that they only use the oocyte for the procedure. After the egg retrieval, the granulosa cells in the follicle are discarded. It got us thinking, what if we can utilize these granulosa cells? Since every egg has thousands of granulosa cells surrounding it, if we can induce them into pluripotent cells and turn those cells into oocytes, aren’t we killing two birds with one stone?”

Encouragingly, previous studies have shown that granulosa cells do possess plasticity that shows stem cell-like properties. It’s also possible to reprogram granulosa cells into induced pluripotent stem cells (iPSC) using just two transcription factors, and granulosa cells have been used successfully to clone animals, the authors noted. “These factors prompted us to explore the potential of GCs in deriving oocytes via reprogramming and differentiation using a simple method without cell transfection.

Their approach exploited a chemical approach to reprogram the granulosa cells into pluripotent stem using only small molecules. This approach generates what are termed chemically induced pluripotent stem cells (CiPSCs), and has been carried out successfully in several laboratories. “This approach avoids genetic manipulation, cell transfection, and ectopic expression of transcription factors for iPSCs and avoids embryo destruction for ESC derivation,” the team noted. “Hence, CiPSCs provide an alternative source with greater potential in stem cell-based therapy.”

What wasn’t known was whether the adult female cells would be able to generate germline-competent PSCs (gPSCs) using this solely chemical approach, and whether CiPSCs could be directed to differentiate into germ cells and functional oocytes. “We undertook experiments to test whether GCs isolated from adult mouse ovaries can be induced to gPSCs using a purely chemical approach and whether these gPSCs can be further induced to form oocytes with high genomic stability that produce fertile mice.”

Granulosa cells tend to undergo cell death and differentiation once removed from the follicles. Liu and the team, which included PhD students Chenglei Tian and Haifeng Fu, developed a chemical cocktail that included a Rock inhibitor (Rocki) and crotonic acid, for creating CiPSCs from granulosa cells. The Rock inhibitor was introduced to prevent cell death and promote proliferation. “By testing various compounds, we found that treatment with a Rocki effectively enhanced the attachment of GCs to the culture dish, prevented apoptosis, and promoted proliferation, factors that are important for obtaining a sufficient number of primitive GCs for reprogramming,” the team noted. They then demonstrated that in combination with other important small chemicals, crotonic acid could promote the induction of granulosa cells into germline-competent pluripotent stem cells that exhibited pluripotency similar to that of embryonic stem cells.

“It’s a surprising result,” says Liu. “The competency of induced pluripotent germline is usually lower than embryonic stem cells. Germline competency is crucial for germline cells to transfer genetic information to the next generation. With the co-formulation of Rock inhibitor and crotonic acid, it’s not only more efficient, but the quality also increased.”

Another cocktail of Rock inhibitor and vitamin C was then introduced to the germline-competent pluripotent stem cells to improve follicle development and induce meiosis. The primordial-germ-cell-like cells (PGCLCs) differentiated from gPSCs had elongated telomeres, and the resulting germ cells and oocytes exhibited high genomic stability. Importantly, these oocytes successfully produced offspring that also showed normal fertility. “Importantly, gPSCs induced by a purely chemical approach maintain genome integrity,” the scientists stated. “Germ cells and oocytes rejuvenated from GCs without genetic modifications and transfection maintain high genomic stability, and longer telomeres and the offspring show normal fertility … the derived gPSCs show high germline competence and genome … Using GCs to produce oocytes via chemical reprogramming not only has potential for treating infertility but also may have implications in preserving fertility and endocrine function.”