Manipulating Water Content in Stem Cells Influences Differentiation Fate

(A) Development of stem cells on hydrogel, a soft substrate, to pre-bone cells after the removal of water. (B) Development of stem cells on glass, a hard substrate, to pre-fat cells after the addition of water. [Harvard T.H. Chan School of Public Health researchers]

Scientists in the U.S. have shown that intentionally altering the amount of water in cells to change their volume impacts directly on cell stiffness, which can influence the direction of stem cell differentiation, a finding that could have implications for the future use of stem cells in a therapeutic context. The studies, led by Ming Guo, Ph.D., at the Massachusetts Institute of Technology (MIT), and David Weitz, Ph.D., at Harvard University, demonstrated that removing water from mouse mesenchymal stem cells (MSCs) caused the cells to condense, which changed cell stiffness and prompted the cells to differentiate into one cell type, while adding water to the cells caused them to swell and differentiate into an alternate cell type, irrespective of microenvironmental cues.

The findings could help scientists better understand celluar function, potentially in relation to disease, by providing “an entry point to think about the impact of physical properties and water content in cells,” professor Guo told GEN. “These results provide insights into the mechanisms that stem cells control their fate and cooperately regulate development of embryos and organs.” Professor Guo is the d'Arbeloff Assistant Professor at MIT’s Department of Mechanical Engineering. 

The researchers describe their studies in the Proceedings of the National Academy of Sciences (PNAS),  in a paper which is entitled, “Cell Volume Change through Water Efflux Impacts Cell Stiffness and Stem Cell Fate.”

Cell volume is a highly regulated property that impacts many cellular functions. “Volume has been considered as a well controlled quantity of cells,” professor Guo explained to GEN.  The volume of cells changes over the course of the cell’s life cycle and increases as the cell plasma membrane grows and the amount of protein, DNA, and other intracellular material increases. Cells can also, however, undergo rapid changes in size and density within minutes by absorbing or releasing water.

The latest work by the teams at Harvard and MIT originally set out to understand the effects of volume on cellular properties and function. As professor Guo further pointed out to GEN, the team want to test 1) if cellular water content changes upon extracellular enviornmental cues, and 2) if this change affects cell mechanics and stem cell fate. “We started by asking why cells change their stiffness responding to a change of the stiffness of their substrate … The stiffness of cells has been suggested to affect a variety of cell behaviors including stem cell fate … From a material science perspective, the material volume fraction typically affects the apparent mechanical property. Therefore, we wondered if the water amount in cells also varies responding to different environmental cues, thus changing the mechanical property of the cell.” 

The team's results highlighted a direct relationship between cell volume and cell stiffness, which also links to stem cell differentiation fate. Surprisingly, “we find that cell volume is a universal predictor of cell stiffness. For a given cell types, their stiffness strongly correlates with the volume.” professor Guo added.

The researchers demonstrated that as a cell spreads out on a substrate, its volume decreases and its stiffness increases. Their studies showed that when cultured on stiffer substrates, cells reduce their volume by removing water, which leads to an increase in macromolecular density. “Cells reduce their volume and increase their molecular crowding due to an accompanying water efflux,” they write. Conversely, restricting the area on which cells can spread leads to increased cell volumes.

To test whether cell volume and cell stiffness might also impact on stem cell differentiation fate, the team first placed normal mouse MSCs in a hardened hydrogel substrate that simulated the rigidness of bone cells. The cells were provided with a growth medium that supported stem cell differentiation into either bone or fat cells. In this environment, the cells tended to develop into pre-bone cells, as expected given the cues from their microenvironment.

However, when the culture conditions were changed to hypotonic, the cells took in water and swelled, and they tended to differentiate into pre-fat cells, even though they were still on the stiff substrate. Similarly, mouse MSCs grown on a soft hydrogel substrate tended to differentiate into pre-fat cells, but when water was removed from the cells so that their volume decreased, they demonstrated tendencies to differentiate into pre-bone cell types. “The results indicate that we can influence stem-cell differentiation either toward osteogenic or adipogenic fates by changing their volume,” the authors state.

The results so far indicated that in the absence of strong chemical triggers, physical properties such as substrate stiffness or external osmotic pressure affected stem-cell differentiation. In a final set of experiments, the researchers investigated whether chemical cues that biased differentiation would also trigger changes in cell volume. They grew the mouse MSCs on a soft hydrogel, which would normally bias the cells toward fat cell differentiation. However, they also added supplements to the media that pushed the cells toward differentiating into bone cells. In the process of undergoing this osteogenic differentiation, the cells also decreased in volume, even before the differentiation process began. Conversely MSCs cultured on stiff substrates increased in volume when they were chemically induced to undergo adipogenesis. The researchers note that these results suggest that cell volume and stem cell differentiation are strongly correlated.

“The findings from this study add a fascinating new tool to our understanding and utilization of stem cell biology for regenerative medicine,” commented co-author Praveen Arany, DDS, Ph.D., assistant professor in the department of oral biology in the University at Buffalo School of Dental Medicine. And as professor Guo noted to GEN, the results provide “insights into the mechanisms that stem cells use to control their fate, and cooperatively regulat development of embryos and organs.” In a clinical context, and when considering the potential future use of stem cells and development of stem cell therapies, the results suggest that “physical perterbations, such as osmotic pressure can be considerated as a new physical approach, especially in a dynamic osmotic enviornment such as the intestine.”

“We find that both cortical and cytoplasmic cell stiffness scale with volume for numerous perturbations, including varying substrate stiffness, cell spread area, and external osmotic pressure,” the authors conclude. “These observations reveal a surprising, previously unidentified, relationship between cell stiffness and cell volume that strongly influences cell biology.”

“The surprising thing about these experiments is the observation that volume seems to be related to so much about the cell,” added Prof. Weitz, who is Mallinckrodt Professor of Physics and of Applied Physics in the John A. Paulson School of Engineering and Applied Sciences at Harvard University, and also a core faculty member of the Wyss Institute for Biologically Inspired Engineering and director of the Materials Research Science and Engineering Center at Harvard. “It seems to dictate the cell stiffness as well as the cell fate….These observations may also have implications in external means of monitoring cell fate, which may be important for future biotech applications.”

Article updated to include comment from the authors, September 28 2017