How do young cells decide what they want to be when they grow up? The biological process of cellular differentiation is a critical moment in the development of a tissue, during which less specialized cells become more specialized and undergo changes in size and shape, as well as in membrane charge and metabolic activity.
Cellular differentiation is largely controlled by changes in gene regulation patterns - that is, cells begin expressing different genes - which is controlled by numerous cell signaling pathways. But researchers are uncovering external, nano-scale methods for predicting and possibly changing cell fate.
In a recent publication in the journal Biomaterials, Hopkins researchers Gregory Christopherson, Hongjung Song and Hai-Quan Mao collaboratively showed the influence of the fiber diameter of electrospun substrates on neural stem/progenitor cell (NSC) differentiation and proliferation. NSCs can turn into almost any cell in the nervous system, depending on the conditions they are exposed to.
By imposing different biochemical and topological conditions on the cells by changing the electrospun substrate, they found that NSC differentiation and proliferation could be guided and predicted.
The substrate that the team used was laminin-coated electrospun Polyethersulfone (PES) in fiber meshes, with average fiber diameters of 283 to 1,452 nanometers. This is about the size of a chromosome.
It is important to use a protein like laminin to adhere to the substrate and "disguise" it before it is exposed to cells, because cells prefer to attach to proteins and don't stick to polymers. Additionally, the study necessitated the use of three-dimensional (3D) fibers, as opposed to a flat substrate.
"We chose laminin because it is the main adhesion protein used for neural stem cell culture. Laminin is a key component of the naturally occuring basement membrane and is the most efficient molecule for mediating NSC adhesion for in vitro culture, so [it] was an obvious choice," Christopherson said.
Electrospinning is a straightforward method for making nanoscale fibrous substrates. It involves the use of an electrical charge for drawing fine fibers from a liquid. When a high voltage is applied to the liquid, it becomes charged and stretches due to the balance between electrostatic repulsion and surface tension.
"The PES system is a very stable and easily tailorable polymer system for electrospinning and is non-degradable, which is desirable for in vitro culture (obviously not suitable for implantation or in vivo study).
There are many types of fibrous membranes in the body,?so using laminin-coated electrospun fibers was our best attempt at creating an ex vivo system for studying the effect of topographical cues in rat neural stem cell culture," Christopherson said.
The rat NSCs that the researchers used for cell culture on the fibers met different fates depending on the types of fibers they used, both in cell morphology (how the cells are shaped) and in fate-specification. In typical lab conditions (which are 2D), the NSCs would form a mix of neural cells with various functions.
In this case, when the fiber diameter was small (about 273 nm), cells preferentially formed oligodendrocytes, a variety of neuroglia or neural support cells. However, on fiber meshes with larger diameters (749 or 1,452 nm), cells usually differentiated into neurons. Christopherson attributed this phenomenon to the nature of cell interaction with the substrate.
"The cell interaction with the substrate is much different for each fiber size, which is also different from a 2D surface. ?Cells align longitudinally along single fibers if the diameter is large enough, whereas they stretch thin among several fibers if they are of a smaller diameter. The mechanisms dictating how this directs the cell differentiation still need to be worked out but appear to result from the contact guidance offered by the underlying fiber matrix," he said.
The team ran into minor problems working with the electrospun fiber meshes during the research, which are tougher to work with than petri dishes or flasks unless one has had practice. Christopherson attributed hindrances mainly to the optical properties of the fibers.
"They are not optically transparent, so it's more difficult to monitor the cell's development through a light microscope.?It can also be difficult to run certain assays because of the fiber network impeding or scattering light normally used for quantification, either limiting some things we can do or causing us to be more creative," he said.
Clearly, this work is both intellectually intriguing and useful for clinical applications. Although much more work needs to be done to understand the cell signaling pathways and cellular mechanisms to understand why fiber diameter influences cell fate, from the point of the cell, the work can be used in development of studies focusing on human beings.
"We need to start unravelling the mechanisms that control why the cell fate is different on a fiber versus 2D substrate, as well as test further combinations of biochemical and topographical cues, some of which are currently underway; it's great that we see a difference, but what is actually occuring in the cell reorganization or internal signaling to induce it? That information can help our understanding of cell differentiation, as well as possible ways to enhance or further control the cell behavior," Christopherson said.
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