During embryonic development, stem cells not only differentiate, they start to organize themselves. Rather than merely clumping, they form a distinctly layered structure. Actually, differentiation and self-organization occur together, with stem cells in different embryonic germ layers taking on distinctive roles.

Replicating this process in the lab has proven to be difficult. Researchers tried flooding stem cells with chemical signals. However, simple application of growth factors produced multiple fates without inducing consistent spatial order. Researchers also tried controlling colony geometry. They noted a shift in the proportion of cells adopting different fates as the colony size was changed, but they did not observe spatial organization.

Now researchers at The Rockefeller University report that they have developed an approach that essentially combines chemistry and geometry. Their emphasis, however, is on geometry. In a paper that appeared June 29 in Nature Methods, they wrote: “We show that geometric confinement is sufficient to trigger self-organized patterning in hESCs.”

The research team, led by Ali Brivanlou, Robert and Harriet Heilbrunn Professor and head of the Laboratory of Stem Cell Biology and Molecular Embryology at The Rockefeller University, confined human embryonic stem cells originally derived at Rockefeller to tiny circular patterns on glass plates that had been chemically treated to form “micropatterns” that prevent the colonies from expanding outside a specific radius. When the researchers introduced chemical signals spurring the cells to begin gastrulation, they found the colonies that were geometrically confined in this way proceeded to form endoderm, mesoderm, and ectoderm and began to organize themselves just as they would have under natural conditions. Cells that were not confined did not.

By monitoring specific molecular pathways the human cells use to communicate with one another to form patterns during gastrulation—something that was not previously possible because of the lack of a suitable laboratory model—the researchers also learned how specific inhibitory signals generated in response to the initial chemical cues function to prevent the cells within a colony from all following the same developmental path.

Additional details appeared in the Nature Methods article (“A method to recapitulate early embryonic spatial patterning in human embryonic stem cells”). “We provide a proof of principle for this approach by determining the spatial patterning phenotypes of knocking down gene products with siRNA,” they noted. Also, they provided hints about future work: “The advent of clustered, regularly interspaced, short palindromic repeats (CRISPR) technology will allow for the same assay to be performed with complete gene knockouts.”

“At the fundamental level, what we have developed is a new model to explore how human embryonic stem cells first differentiate into separate populations with a very reproducible spatial order just as in an embryo,” said Aryeh Warmflash, a postdoc at Rockefeller who contributed to the research. “We can now follow individual cells in real time in order to find out what makes them specialize, and we can begin to ask questions about the underlying genetics of this process.”

The research also has direct implications for biologists working to create “pure” populations of specific cells, or engineered tissues consisting of multiple cell types, for use in medical treatments. “These cells have a powerful intrinsic tendency to form patterns as they develop,” Dr. Warmflash added. “Varying the geometry of the colonies may turn out to be an important tool that can be used to guide stem cells to form specific cell types or tissues.”

“Understanding what happens in this moment, when individual members of this mass of embryonic stem cells begin to specialize for the very first time and organize themselves into layers, will be a key to harnessing the promise of regenerative medicine,” Dr. Brivanlou concluded. “It brings us closer to the possibility of replacement organs grown in petri dishes and wounds that can be swiftly healed.”

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