UC San Francisco researchers have, for the first time, developed a light-based method to precisely control embryonic stem cell differentiation, converting cells into neurons in response to a defined external cue.
This optogenetic approach also uncovered an internal timing mechanism inside stem cells that filters out fleeting molecular fluctuations yet allows a swift, decisive switch into a mature cell state when an appropriate, sustained signal is detected, the team reports in a study published online August 26 in Cell Systems.
“We’ve identified a fundamental way cells decide whether to ignore a developmental cue or to act on it,” said co-senior author Matthew Thomson, PhD, of UCSF’s Department of Cellular and Molecular Pharmacology and the Center for Systems and Synthetic Biology.
During embryonic development, pluripotent stem cells undergo tightly regulated transitions from an undifferentiated state into specialized cell types that form organs and tissues. Researchers have cataloged many molecular signals that instruct stem cells when to become neurons, liver cells, muscle, and other tissues at precise times. Controlling these decisions in the lab has major potential for regenerative medicine, but reliably directing large numbers of stem cells to adopt a specific fate has proven challenging.
Part of the difficulty stems from the fact that genes encoding developmental cues often flicker on and off in undifferentiated stem cells. How cells ignore such noisy fluctuations while responding rapidly to true developmental signals has been unclear.
“These cells receive many overlapping inputs,” said lead author Cameron Sokolik, a research assistant in the Thomson laboratory at the time of the study. “The key question is how a cell determines the right moment to differentiate.”
To probe how embryonic stem cells distinguish meaningful cues from noise, the authors engineered mouse embryonic stem cells so that a pulse of blue light would activate an endogenous neural differentiation gene, Brn2. Using controlled light pulses allowed precise tuning of Brn2 dose and duration so the researchers could observe how cells respond to different signaling patterns.
The experiments showed a clear threshold behavior: when the Brn2 signal was sufficiently strong and sustained, stem cells rapidly initiated neuronal differentiation. By contrast, brief or weak Brn2 pulses were ignored and produced no change in cell fate.
“The cells are sensitive to the duration of the signal,” Thomson said. “That was an important and unexpected finding.”
To investigate how cells filter transient Brn2 activity yet respond to persistent stimulation, co-senior author Stanley Qi, PhD, and co-author Yanxia Liu, PhD, both now at Stanford University, used CRISPR-Cas9 to fuse a fluorescent tag to Nanog, a key transcription factor that maintains pluripotency and acts as a brake on differentiation. This Nanog-GFP reporter allowed the researchers to track the internal state of cells as they processed Brn2 inputs.
The results revealed that Nanog functions as an internal timer. Activation of Brn2 disrupts a positive feedback loop that stabilizes the undifferentiated state, causing Nanog protein levels to fall. Because Nanog decays with an intrinsic half-life of around four hours, cells can tolerate brief Brn2 fluctuations: Nanog rebounds if the input stops. But if Brn2 remains active long enough for Nanog to fall below a critical level, the cell crosses a threshold and differentiation proceeds rapidly. “Once Nanog is depleted and the signal persists, it’s like a buzzer goes off,” Thomson said. “The conversion to neurons then happens quickly and irreversibly.”
These findings indicate that the architecture and dynamics of the pluripotency network—specifically positive feedback combined with the degradation rate of a key regulator—enable stem cells to classify inputs as either noise or authentic developmental signals.
Thomson and colleagues suggest that similar timing mechanisms may be a general feature of how stem cells make fate decisions across many tissue types. “It’s difficult for a biological system to be both tolerant and fast: to ignore minor perturbations but respond precisely when a real signal appears,” Thomson said. “This internal timing mechanism achieves both goals.”
Thomson is supported by UCSF’s Sandler and Systems Biology fellowship programs, initiatives that recruit early-career scientists to pursue ambitious, high-risk research.
Looking ahead, Thomson envisions applying his lab’s light-inducible differentiation tools to study 3-D tissue formation. He imagines using patterned light to instruct undifferentiated cells in a culture to form organized structures—blood vessels, nerves, liver tissue—that could one day be assembled into transplantable tissue or organs. But he cautions that success requires understanding the cell’s own information-processing rules. “The cell is not a passive puppet; it actively interprets signals. To control cell fate precisely, we must map the mechanisms by which cells process those signals,” he said.
David A. Sivak, PhD, now at Simon Fraser University, is listed as a senior author. Additional co-authors from UCSF include David Bauer, PhD; Jade McPherson, PhD; Michael Broeker, PhD; and Graham Heimberg, PhD.
Funding: This work was supported by the UCSF Center for Systems and Synthetic Biology, the National Institute of General Medical Sciences, the NIH Office of the Director, the National Cancer Institute, and the National Institute of Dental & Craniofacial Research (grants NIGMS P50 GM081879, NIH DP5 OD012194, NIH DP5 OD017887).
Source: Nicholas Weiler – UCSF
Image Source: Image credit to UCSF
Original Research: Abstract for “Transcription Factor Competition Allows Embryonic Stem Cells to Distinguish Authentic Signals from Noise” in Cell Systems, published online April 26, 2015 (doi:10.1016/j.cels.2015.08.001).
Abstract
Transcription Factor Competition Allows Embryonic Stem Cells to Distinguish Authentic Signals from Noise
Embryonic stem cells must reliably differentiate in variable environments where gene expression fluctuates stochastically. Using optogenetics, the authors controlled an endogenous neural differentiation cue (Brn2) with light and tracked differentiation with a Nanog-GFP reporter. Pulsed Brn2 inputs revealed that the pluripotency network filters out inputs below specific magnitude or duration thresholds, but triggers rapid differentiation when both thresholds are exceeded. These filtering properties arise from the network’s positive feedback and the intrinsic half-life of Nanog, which sets the duration threshold. The data indicate that the dynamic behavior of positive feedback networks helps stem cells classify inputs as signal or noise.
“Transcription Factor Competition Allows Embryonic Stem Cells to Distinguish Authentic Signals from Noise” by Cameron Sokolik, Yanxia Liu, David Bauer, Jade McPherson, Michael Broeker, Graham Heimberg, Lei S. Qi, David A. Sivak, and Matt Thomson in Cell Systems. Published online April 26, 2015. doi:10.1016/j.cels.2015.08.001