Mesenchymal stem cells—adult stem cells that are constantly renewing our bone, cartilage, and muscle—are thought to hold tremendous promise for the treatment of disease based partly on their ability to churn out a variety of different healing factors. Numerous clinical trials are investigating these cells as therapies for an array of conditions ranging from diabetes to spinal cord injury.
About a decade ago, researchers discovered that the surfaces on which these mysterious cells were cultured guided them toward specific fates. Cells maintained on a hard surface tend to turn into bone cells, whereas those grown on a soft surface turn into softer cell types, such as fat cells (1).
Now they are one step closer: Reporting in the journal eLife, the Wang and colleagues explain how pushing and pulling the outsides of individual mesenchymal stem cells can affect the dynamics of calcium release within (2).
“What is really nice about this paper is that it is shedding light into some of the completely unknown mechanisms of mechanical regulation of cell behavior,” said Gordana Vunjak-Novakovic, professor of biomedical engineering and medicine at Columbia University in New York, who was not involved with the work.
That cells respond to physical forces in our bodies, such as blood flow or exercise, is not new or surprising. Exploring the mechanical aspects of biology is important for a variety of diseases such as atherosclerosis, asthma, and arthritis. Research is now beginning to give physical triggers the same due as chemical stimuli.
“There’s a huge interest in this,” Vunjak-Novakovic said. “The difficulty is that we know very little about the actual mechanisms of what happens between physical action at the cell membrane and changes in nucleus.”
Bead, meet cell
To answer that question requires single-cell manipulation. Wang has refined a combination of techniques — developing and harnessing Förster resonance energy transfer (FRET)-based biosensors in cells, optical tweezing, and RNA interference — for more than a decade.
In 2014, Wang’s group showed that deforming individual mesenchymal stem cells by pulling the gel substrate on which they were growing triggered calcium oscillations within the cells (3).In the new study, Wang’s group employed a FRET-based calcium biosensor and its variants that are targeted for specific organelles within the cell, in particular the endoplasmic reticulum (ER). The ER is responsible for the cell’s characteristic calcium fluctuations that are, in turn, important for a host of functions, including differentiation.
The scientists attached sticky nanoparticle beads (10 m in diameter) to the outside of mesenchymal stem cells and used a laser to pull the beads, applying minute amounts of mechanical force that Wang likens to a “cell massage.”
Cells bathed in culture media lacking calcium lit up in response to the “massage,” indicating that calcium was being released by the ER. Surprisingly, blocking a stretch-activated calcium channel in the outer membrane prevented calcium release from the organelle.
How exactly a pull on the outer membrane triggered calcium release from the ER was unclear. The team used additional biosensors to distinguish between two possible mechanisms: either the pulling was activating biochemical signaling cascades that resulted in ER calcium release, or the pulling was directly mechanically altering the channels on the ER.
The group found that the latter was true. In particular, the ER’s sensing capability depended on the cytoskeleton as well as contractility of actomyosin. “This was very exciting to us,” Wang said.
The touchy toolbox
Roger Kamm, a professor of biological and mechanical engineering at MIT who was not involved with the research, said, “the thinking before has been, when you apply a force to the outside of the cell, what you’re mainly stressing is the [calcium-sensitive channels in the] outer lipid bilayer. What this study shows is that the process is really more complex.”
The fact that the force is transmitted through the cytoskeleton to elicit calcium release from the ER is something that people had postulated for some time, but not shown convincingly until now, Kamm added.
The techniques Wang’s group used enabled a satisfying look into the dynamics of the process—and it is a dynamic process, Vunjak-Novakovic said: “You see things as they happen.”
Integrating the techniques Wang uses is challenging, and a big reason why not many others do what he does. He often fields questions from scientists who want to learn. Wang is adamant that what his group does is within everyone’s reach, if they are motivated. He is planning a hands-on workshop in late summer that will target graduate students and postdocs in Southern California. If it goes well, he plans to expand.
In addition to learning how these mechanical forces affect other cell types, Wang’s group is interested in knowing how the calcium signals affect epigenetics and genomic regulation—and ultimately, the correct development of tissue and organs.
To do this, the team is developing different biosensors that will allow them to visualize histone modifications and other epigenetic marks. The modification of histones, the protein spools on which DNA winds, can affect the packaging of DNA and the resulting patterns of gene expression.
Single-cell manipulation will be particularly crucial for circumventing the noise that plagues epigenetics studies in cell populations. “If you are looking at the same cell, you have much better chance to tease out or extract information with precision,” Wang said.
Also on the horizon are Wang’s ambitious goals of translating that knowledge not only into a comprehensive map of a cell’s interior (inspired by Google Earth) but also into specific controls over the cells. Meanwhile, “I enjoy watching how the cells behave whenever we perturb them. We really have a lot of fun,” he said.
1. Engler AJ, Sen S, Sweeney HL, Discher DE. Matrix elasticity directs stem cell lineage specification. Cell. 2006;126:677-89.
2. Kim TJ, Joo C, Seong J, Vafabakhsh R, Botvinick EL, Berns MW, Palmer AE, Wang N, Ha T, Jakobsson E, Sun J, Wang Y. Distinct mechanisms regulating mechanical force-induced Ca²⁺ signals at the plasma membrane and the ER in human MSCs. Elife. 2015 Feb 10;4:e04876.
3. Kim TJ, Sun J, Lu S, Qi YX, Wang Y. Prolonged mechanical stretch initiates intracellular calcium oscillations in human mesenchymal stem cells. PLoS One. 2014 Oct 20;9(10):e109378.