A new technique for studying stem cell development leads to a new understanding of germ cells

Nov. 29, 2023

By Christopher Vaughan

In mathematics, physics, and engineering, it’s common to take a difficult, complex problem in three dimensions and simplify it by modelling it in two dimensions. Institute researchers have done something similar in order to study stem cell development and differentiation by creating a two-dimensional, monolayer model of cell development to precisely study what is normally a hard-to-study, three-dimensional process.

“Our system has a lot of advantages,” said institute member and associate professor of obstetrics and gynecology Vittorio Sebastiano, PhD. “We are able to answer a lot of longstanding questions about human cell development.”

A common way to study cell development and differentiation is to study embryoid bodies: a cluster of pluripotent cells that specialize, or differentiate, as the cells grow and divide. Researchers can study the direct interactions between cells in the embryoid body as it grows, but they have trouble studying how the cells are affected by molecular signals that diffuse through the embryoid body, Sebastiano said. 

“There are molecular signals that diffuse out from individual cells and it’s hard to know other cells in the embryoid body experience the concentration and timing of those signals,” Sebastiano said. “The way cells react to various local concentrations of signal can be very different.”

Sebastiano and his colleagues therefore went about creating an embryoid body in two dimensions, laying the cells out flat on media so that they knew precisely the concentrations and timing of various molecular signals that each cell was exposed to. 

“All the cells growing on the monolayer are homogenously exposed to whatever is in the culture media (e.g., signaling factors) and we can systematically interrogate their impact on cell differentiation and development ,” Sebastiano said. 

The system is a boon for discovering important new cell signaling pathways, Sebastiano says. “With a three-dimensional embryoid body, you have to already know what pathways are important,” he said. “With the monolayer approach, you can guess what signals you think will be important and then test that hypothesis.”

Sebastiano teamed up with assistant professor of developmental biology Kyle Loh, PhD and Siebel Investigator Lay Teng Ang, PhD to apply to system to a specific scientific problem. 

Primordial germ cells (PGCs) exist early in embryonic development and are later transformed into eggs and sperm. Being able to generate PGCs from human pluripotent stem cells would help our understanding of human development and might provide future treatments for infertility. But scientist have been stuck trying to understand how to generate PGCs because the 3-dimensional environment in which they develop is complex.

Sebastiano, Loh and Ang used the simplified monolayer approach to study the exact steps that were required to transition cells from pluripotent stem cells to PGCs. 

“We discovered that at a specific time in the development of the early embryo, the WNT signal needs to be active for 12 hours and then inactivated in order for PGCs to develop,” Loh said. If the WNT signal stays active for more than 12 hours, the cells go on to form the primitive streak.”

Scientists also knew that embryonic stem cells and PGCs had a very similar profile of pluripotency gene expression but were uncertain if, in the process of becoming PGCs, those pluripotency genes needed to be turned off and then turned back on. “By studying the process using the monolayer system, we now know that these gene stay on and don’t shut down temporarily,” Loh and Sebastiano said.

Knowing exactly what genes are active and when they are during the developmental process will be important for regenerative medicine, Loh said. “If you want to grow organs, you need to have a precise protocol for reproducing these conditions in the lab.”

Ultimately, Sebastiano says, the work is a first step that could lead to a new understanding of how to generate youthful patient-specific ovarian tissues, which could impact women’s health and longevity, a critical goal and focus of the Sebastiano Lab.

The work was published in the journal Nature Communications.