Stem Cell Mechanotransduction

This lecture had a lot more to do with general mechanobiology than with stem cells in particular, but oh well. For an introduction to mechanobiology, see here.

Heart cell differentiation

As mentioned here, stiffness clearly has an impact on the differentiation of cardiac cells (they turned into bone when transplanted into a stiffer region). In vitro, the type of hydrogel (synthetic ECM) used can also impact differentiation. Two different types of hydrogels: polyacrylamide (a static stiffness hydrogel) and hyaluronic acid (a dynamic stiffness hydrogel that becomes stiffer with time) were trialled. Differentiation was better on the hyaluronic acid gel.

Traction force

As also mentioned here, cells can exert force on the ECM by pulling on it. This is called traction force. As mentioned in that previous post, there is a chain of proteins connecting the ECM to the nucleus: ECM - integrin - focal adhesion complex (talin and vinculin) - actin/myosin - nucleus. This chain of proteins affects mechotransduction (like a "signalling pathway" for mechanical forces) which may affect traction force. Stimuli that affect traction force include shape, size and stiffness.

Myogenic differentiation

As another example of how stiffness can affect differentiation, adipose stem cells (ASCs) were cultured on a 10kPa hydrogel (10kPa is the approximate stiffness of muscle tissue). This was found to improve the differentiation of ASCs into myocytes. When certain aspects of the mechanotransduction pathway (like integrins) were "turned off" by using silencing RNA (siRNA), differentiation was markedly reduced.

Muscle-like stiffness also promoted the fusion of ASCs into myotubes. Don't get excited though: in a study done, only around 2% of ASCs actually fused into myotubes, but that's still a big improvement over previous trials that only showed 0.2% fusion. Fused myotubes remained fused when cultured on stiff tissues, but unfused cells were more likely to become bone when cultured on stiff tissues.

Durotaxis

As mentioned here, durotaxis is the phenomenon in which cells migrate from softer to stiffer tissue. This can be tested by using gradient hydrogels. One type of gradient is a linear gradient, in which the hydrogel gradually changes from soft to stiff along the length of the gel. Another type of gradient is the step gradient which strips of soft hydrogel alternate with strips of stiff hydrogel. An example of a gel that uses a step gradient is called "zebraxis." Cells cultured on zebraxis hydrogel have a tendency to congregate in the stiffer sections.

Stiffness vs. Pore Size

Stiffness, to some extent, depends on the amount of "stuff" in the ECM. In softer ECMs (and softer hydrogels), there are larger gaps between the "stuff," resulting in larger pore sizes. Conversely, in stiffer ECMs, there are smaller pore sizes. When proteins like collagen tether to hydrogels, the distance between tethering points also depends on the pore size: larger pores (and softer ECM) means a larger distance between tethering points, and smaller pores (and stiffer ECM) means a shorter distance between tethering points. It has been hypothesised that the length of the "tether" may be sensed by cells, rather than the actual stiffness of the ECM (but this is likely incorrect, as I'm about to explain.)

One way to test this in vitro is by varying the composition of the hydrogel. Polyacrylamide hydrogels are made up of two main components: acrylamide and bis-acrylamide. Bis-acrylamide acts as a cross-linker for acrylamide. When the proportions of acrylamide and bis-acrylamide are changed without changing the overall amount of molecules in the hydrogel, you can change the pore size without changing stiffness, allowing you to test the effect of pore size independent of stiffness. By testing this, it has been found that stiffness affects mechanotransduction independent of pore size.

Lamin-A

Lamin-A is a nuclear protein that scales with stiffness, i.e. there is more of it when tissues are stiffer. Interestingly enough, it may be involved in a feedback mechanism where stiff tissues maintain Lamin-A and the stiffness of the nucleus. Lamin-A, like other proteins, is synthesised in the cytoplasm. When the cell is under stress, it can be translocated to the nucleus and help in the assembly of nuclear lamins, but when the cell is more relaxed, it remains in the cytoplasm (or already assembled nuclear lamins may be transported back to the cytoplasm), where they can be degraded by proteases. (I think I'll need to double-check that I've got this part right, though. I was pretty tired during the lecture, and the slides aren't really helping.)

Other mechanosensors

Some other mechanosensors present in the cell include YAP/TAZ, talin and vinculin. YAP/TAZ are proteins that can localise in either the nucleus or the cytoplasm. When localised in the cytoplasm, differentiation into adipocytes, growth arrest and maybe even apoptosis can occur. When localised in the nucleus, proliferation and/or osteogenic differentiation may occur.

Talin and vinculin, as mentioned earlier, are components of the focal adhesion complex. When pulled, they can undergo conformational changes. When vinculin is bound to talin, it can reveal a cryptic binding site for MAPK (MAP kinase). (A "cryptic binding site" is basically a binding site on a protein that is normally hidden due to the conformation of the protein.)

Mechanomemory

One phenomenon that has been observed is that of "mechanomemory." Basically, if you culture cells on a very stiff surface such as glass before moving them to a softer hydrogel, they won't respond as well as cells that were on the soft hydrogel from the get-go. It's been thought that this is because the cells have "mechanomemory" of the stiffer hydrogel.