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Egg whites may be perfect for a health-conscious breakfast, but egg yolks turned out to be the key ingredient for cultivating bird embryonic stem cells (ESCs) in the lab. Using a growing medium of egg yolk along with a few other key factors, a USC Stem Cell-led team of scientists has succeeded in deriving and maintaining authentic ESCs from chickens and seven other bird species.

These bird ESCs hold tremendous promise for applications ranging from studying embryonic development to producing lab-grown poultry to reviving endangered or even extinct birds.

The study in Nature Biotechnology.

"A true embryonic stem cell has two key qualities: it is self-renewing and can multiply to produce more stem cells; and it is pluripotent, meaning it can differentiate into all cell types of the embryo—both in a culture dish and after being reintroduced into an embryo," said corresponding author Qi-Long Ying, Ph.D., a professor of stem cell biology and regenerative medicine at the Keck School of Medicine of USC.

"In this study, we successfully derived and maintained true self-renewing and pluripotent ESCs from chicken, quail, pheasant, turkey, duck, goose, peafowl, and ostrich."

First author Xi Chen, now at Caltech, spent a decade advancing this research, first during his Ph.D. and postdoctoral training in the Ying Lab at USC, and later while continuing the work in Carlos Lois's lab at Caltech. The project complements previous research from Ying's lab, which independently derived the first true ESCs from rats and published the findings in Cell in 2008, in parallel with a separate report from Austin Smith's lab at the University of Cambridge.

In the current study, Chen and his collaborators first optimized conditions to support ESCs from chickens, the most well-studied avian species.

Starting with cells from the early embryonic stage known as the blastoderm, they added two chemicals known to encourage ESC formation by inhibiting signals that sometimes promote stem cell differentiation.

The first chemical, IWR-1, inhibits cell signals related to the proteins Wnt and β-catenin, and the second chemical, Gö6983, inhibits cell signals from the protein kinase C family. When exposed to these two chemicals, the chicken cells exhibited genetic markers of stem cell pluripotency, but still could not be maintained in the lab long-term.

The scientists then made a game-changing observation: when the blastoderm cells were transferred from their eggs along with larger amounts of yolk, they tended to self-renew better in the lab. The scientists began to suspect that the missing ingredient in their ESC-promoting cocktail was a natural component of egg yolk.

A careful study of the yolk revealed that it does indeed contain a protein, ovotransferrin, that completed their cocktail and enabled them to derive and maintain ESCs from several chicken breeds.

Surprisingly, the three-ingredient cocktail that worked for chicken ESCs didn't show the same success in promoting ESCs from other avian species. Different species required variations of the cocktail.

For example, pheasant, duck, and turkey ESCs needed a fourth ingredient, the chemical SB431542, to block differentiation into beating heart muscle cells and enable maintenance of stem cell identity. Quail, geese, and peafowl ESCs required all four of these components plus a fifth ingredient, the pluripotency-related signaling protein LIF derived from chickens.

Meanwhile, ostrich ESCs failed in this five-ingredient cocktail, but successfully maintained their stem cell identity when the scientists removed the chicken LIF and reverted to the four-ingredient version.

When exposed to the five-ingredient cocktail rather than the original three-ingredient cocktail, chicken ESCs could self-renew over a long period of time while maintaining a high degree of pluripotency.

Under these optimized conditions, chicken ESCs could form the three primary cell layers of the early embryo that ultimately give rise to all tissues and organs in the body—the hallmark of true pluripotency. They could also differentiate into reproductive germ cells such as sperm, as well as non-reproductive or "somatic" cells that make up other tissues in the body—another sign of pluripotency.

Chicken ESCs also passed the gold standard test for pluripotency: the ability to make a "chimera," an animal composed of a mosaic of cells from two genetically distinct individuals. When chicken ESCs were introduced into a developing chicken embryo, the resulting animal developed from a mosaic of cells from both sources.

As a clear demonstration, when the developing embryo was albino, chicken ESCs could contribute pigmented feathers to the chimera.

Chicken ESCs could also be genome edited using CRISPR and other tools, expanding their usefulness for applications in both research and biotechnology.

"Compared to naïve ESCs derived from mice, chicken ESCs are a little less stem cell-like but are not primed to commit to specific cell lineages," said Chen. "This type of ESC sits on the developmental continuum between naïve and primed."

ESCs from the other avian species also passed key tests of pluripotency. Quail, goose, and ostrich ESCs displayed classic pluripotency-associated genetic markers. Quail ESCs could differentiate into all three early embryonic layers, while goose ESCs could contribute to germ cells. And quail and goose ESCs could both contribute to chimeras.

Importantly, the lab of co-author Professor Guojun Sheng at Kumamoto University in Japan has independently derived chimera-competent chicken and peafowl ESCs using the methods described in this study—demonstrating that the approach is robust and reproducible.

"By demonstrating that we can derive and maintain authentic ESCs from several avian species, this research opens up so many possibilities," said Ying.

"As we continue to expand our ability to derive avian ESCs, we can imagine engineering healthier chickens for the poultry industry, incubating therapeutic proteins inside of eggs for pharmaceutical development, or reviving endangered or extinct species to support conservation and biodiversity efforts. There are so many exciting applications of this technology, and this study is only the beginning."

Ying, Chen, Zheng Guo, Xinyi Tong and Xugeng Liu are inventors on patent WO2023158627A3, titled "Methods for derivation and propagation of avian pluripotent stem cells and applications thereof" that arose from this work.