Exercising unprecedented delicacy, researchers probed the regulatory circuits that govern the transitions between stem cells’ pluripotent states. The researchers uncovered far more variation in pluripotent stem cells than was previously appreciated. In addition, they learned that there are many small fluctuations in the state of a stem cell’s pluripotency that can influence which developmental path it will follow.

The researchers, led by James Collins, Ph.D., at the Wyss Institute and George Daley, M.D., Ph.D., at Boston Children’s Hospital, presented their findings December 3 in Nature, in an article entitled, “Deconstructing transcriptional heterogeneity in pluripotent stem cells.”

“We set out to characterize transcriptional heterogeneity in mouse [pluripotent stem cells] by single-cell expression profiling under different chemical and genetic perturbations,” wrote the authors. “Signaling factors and developmental regulators show highly variable expression, with expression states for some variable genes heritable through multiple cell divisions.”

Researchers explored the developmental landscape of pluripotent stem cells by perturbing them with variants such as different chemicals, culture environments, and genetic knockouts. Then, they analyzed the individual genetic makeup of each cell to observe microfluctuations in each stem cell’s state of pluripotency. They discovered many small nuances in the way stem cells are influenced by internal, chemical and environmental cues, revealing a complex “decision making” circuit of developmental regulators.

“Expression variability and population heterogeneity can be influenced by perturbation of signaling pathways and chromatin regulators,” the authors continued. “Notably, either removal of mature microRNAs or pharmacological blockage of signaling pathways drives [pluripotent stem cells] into a low-noise ground state characterized by a reconfigured pluripotency network, enhanced self-renewal and a distinct chromatin state, an effect mediated by opposing microRNA families acting on the Myc/Lin28/let-7 axis.”

Looking at the findings, the researchers now believe there is a “code” that relates patterns of dynamic behavior in stem cell regulatory circuits to the developmental path a cell ends up taking. By leveraging that code, they hope to dial in precisely to specific individual cell states and to use them for a variety of purposes, such as creating certain cell types that a patient's body may be unable to produce on its own.

“Stem cell colonies contain much variability between individual cells. This has been considered somewhat problematic for developing predictive approaches in stem cell engineering,” said Dr. Collins. “Now, we have discovered that what was previously considered problematic variability could actually be beneficial to our ability to precisely control stem cells.”

In the Nature article, the authors elaborated on how their single-cell approach helped them survey stem cells’ complex transcriptional landscapes: “We found that different classes of genes manifest high or low expression variability in PSCs, with housekeeping and metabolic gene sets showing consistent expression across individual cells, while genes involved in signaling pathways and development were considerably more variable. Moreover, expression states of variable regulatory factors were coupled together, implying the presence of a regulated biological network.”

The authors concluded by noting that transcriptional heterogeneity is increasingly being recognized as a key component of many biological processes. The added that “it will be of interest to map stable and flexible regulatory nodes in networks governing other progenitor and differentiated cell types to discern common principles underlying network architecture and gene expression variability.”

“The ability to understand and program stem cells throughout changing states of pluripotency is a critical necessity for the success of regenerative medicine,” explained Wyss Institute founding director Donald Ingber, M.D., Ph.D., who is affiliated with Harvard Medical School, Boston Children’s Hospital, and the Harvard School of Engineering and Applied Sciences. “By making stem cell engineering more predictive, we hope to leverage the versatility of controllable pluripotent stem cells to address a wide range of diseases and injuries.”

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