Cells grown in culture are not alone: They are constantly communicating with one another by sending signals through their culture media that are picked up and transmitted by other cells in the media. When thousands of cells are cultured together in a dish, there are hundreds of thousands of these signals present every minute, all competing to be heard.
Scientists trying to direct cells to do useful things — like causing stem cells to turn into neurons or heart cells — typically try to overcome these signals by adding their own exogenous factors. These exogenous factors are often added at saturating concentrations, blanketing the cells with a particular growth factor or cytokine to activate specific pathways to produce a desired outcome, such as controlling stem cell differentiation. However, the constant din of cell communications is still present, causing alternate and perhaps opposing pathways to be stimulated.
This unstoppable secretion by cells in culture makes it difficult to determine the exact “recipe” of exogenous factors needed to elicit a specific phenotype, particularly in fast-growing cells like embryonic stem cells. MIT researchers Laralynne Przybyla, a graduate student in biology, and Joel Voldman, associate professor of electrical engineering and computer science, report in a paper published this week in Proceedings of the National Academy of Sciences how they were able to silence this din by using a microfluidic device to culture embryonic stem cells under continuous liquid flow (known as perfusion) such that factors secreted by the cells were removed before they could be transmitted to other cells. They used this device to investigate the influence of these factors on stem cells.
Mouse embryonic stem cells (mESCs) can be maintained as stem cells indefinitely in culture, a characteristic known as self-renewal. One recipe used by scientists to maintain mESC self-renewal involves addition of the signaling proteins known as leukemia inhibitory factor (LIF) and bone morphogenetic protein 4 (BMP4). It has been previously thought that LIF and BMP4 together were sufficient to maintain mESC self-renewal, an assumption that was difficult to challenge given the problem of cell-secreted background noise.
However, using their microfluidic perfusion device, Przybyla and Voldman found that mESCs actually exit their self-renewing state when the culture liquid is perfused in the presence of LIF and BMP4. “This shows that these two factors are not in fact sufficient in isolation, and that a previously unrecognized cell-secreted signal is required to maintain embryonic stem cells as such,” notes Przybyla, who was first author on the study.
She and Voldman additionally found that the cells entered a more primed stem cell state corresponding to a more advanced embryonic developmental stage, and that proteins that normally remodel the fibrous scaffolding that cells secrete, known as the extracellular matrix, were partially responsible for the change.
“By showing that microfluidic perfusion can be used to study not only the necessity but also the sufficiency of specific factors to elicit a desired phenotype, this work has the potential for broader applications such as determination of cancer cell-secreted signals that are required for tumor growth or metastasis,” Voldman says. The results also directly inform stem cell biology, because understanding self-renewal is essential as stem cell research advances toward clinical and biotechnological applications.