Kyoto scientists coax human stem cells in lab to enter egg development pathway

To better understand human fertility and developmental biology and disease, researchers need reliable laboratory systems for exploring the origin and characteristics of eggs and sperm. An important step is finding a way to provide a steady and viable supply of egg and sperm precursor cells, called germ cells.

Research on cultured cells from mice has provided some practical hints. Stem cells, which can turn into any cell type, can be induced by chemicals to closely look and function like germ cells. These germ-like cells can become either eggs or sperm, depending on the presence of other cells extracted from the female or male sex glands (ovaries and testes, respectively).

Now, inspired by those findings and techniques, a team at the Advanced Study of Human Biology (ASHBi) of Kyoto University has developed an effective method of culturing human germ-like cells. Not only can the cells multiply continuously at least 20 times in four months, but they can also be triggered to develop into early-stage egg cells that survive long enough for thorough genetic analyses to be done.

Mitinori Saitou, leader of the Kyoto University group, says, “Cultured human germ cells have shown poor survivability in the past, which has limited further investigations. With our new technique, one germ-like cell keeps dividing to eventually exceed a million cells in four months. This performance is even better than that of mouse germ cell models, in which one cell becomes only 50 cells in one week.”

First, the researchers adapted mouse protocols and chemically induced human stem cells to become germ-like cells. By testing many different types and combinations of nutrients and chemical growth supplements, the researchers found an ideal culturing conditions that favored cell growth and proliferation. Gene expression profiling revealed that earlier and later generations of cells had similar patterns of gene activity.

Next, the team looked at DNA methylation, where methyl (–CH3) groups had been temporarily added to DNA to influence gene activity. Methyl groups tag the DNA to give it extra, nongenetic (epigenetic) information, in the same way that a small sticky label can be used to tag a line of text on a page in a book. In general, DNA methylation suppresses the expression of a gene, and methylation status reflects cell development. The team found that germ-like cells from different generations contained similar levels of DNA methylation, suggesting that they had reached the same developmental stage.

In contrast, past studies have reported that DNA in mouse germ-like cells gradually loses its methyl groups as the cells repeatedly multiply. That observation may be related to the reduced ability of mouse germ-like cells to keep dividing to expand their numbers, the researchers note.

Finally, human germ-like cells were grown in an ovary-like environment by co-culturing them with non-germ cells that had originated from an ovary of a mouse embryo. The presence of the mouse ovary cells triggered the germ-like cells to develop into early-stage egg cells (oogonia/gonocyte-like cells), as revealed by the increased expression of genes associated with egg development. In parallel, methylation levels dropped markedly throughout the DNA, confirming a new pattern of gene activity for cells entering the egg-making pathway. The results are consistent with epigenetic reprogramming, which is an in vivo characteristic of actual human germ cells, the researchers note.

Saitou says, “Our study has uncovered some critical species-specific differences in expansion potential and DNA methylation between human and mouse germ cell lineages. Moreover, our technique to culture germ-like cells will be useful in the future for exploring the mechanisms of human germ cell development, survival, propagation, and epigenetic reprogramming.”

Paper Information

Yusuke Murase, Yukihiro Yabuta, Hiroshi Ohta, Chika Yamashiro, Tomonori Nakamura, Takuya Yamamoto, Mitinori Saitou (2020). Long‐term expansion with germline potential of human primordial germ cell‐like cells in vitro, The EMBO Journal, DOI: https://doi.org/10.15252/embj.2020104929

Credit: Nano Pico Science