Human Mesenchymal Stem Cells in vivo: The Cytoskeleton

Since their description in the 1970s1, mesenchymal stem cells (MSC) have been at the center of very active research with the challenge of using them as therapy in organ and tissue repair. For that purpose, it has been essential to develop culture protocols that would ensure their growth and, eventually, their differentiation into specific tissues2–8.

This living sample of human MSC from bone marrow kindly provided by PromoCell GmbH, was pre-coated with human fibronectin and cultured with low-serum cell growth medium.

Once incubated, the sample was observed under the 3D Cell Explorer. Nanolive imaging is a method of choice for stem cell imaging, as its technology avoids intense light exposure or labelling-induced perturbations, which limit the usage of imaging methods like fluorescence microscopy.

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Human mesenchymal stem cells growing on fibronectin

This blogpost and our previous mitosis in human mesenchymal stem cells (MSCs) blog post feature the same cell type. However, the MSCs presented here grow on fibronectin, which resulted in a difference in shape (Figure 1). Fibronectin is a glycoprotein from the extracellular matrix that enhances attachment and spreading of cells cultured in vivo and in vitro9.

The use of fibronectin as substrate influences the cytoskeletal structures of the growing cells, leading to the formation of stress fibers in this case.

Stress fibers appear in most cell types, but most commonly on those of mesenchymal origin10 (Figure 2). Actin can be found in the cytosol as a monomer (G-actin) or as a polymer (F-actin). Stress fibers are bundles of highly compacted filamentous actin resulting from actin polymerization, that are associated with several cytoskeletal proteins like myosin II and α-actinin. They are connected to the underlying matrix -fibronectin in our case-, at one or both ends in structures known as focal adhesions1.

Cytoskeleton - With and without fibronectin

Figure 1: Difference in shape between human mesenchymal stem cells cultured without (left) and with (right) fibronectin.

Similarly to the distinguishable metaphase plate on our mitosis blog post, the high concentration of proteins in stress fibers leads to a detectable refractive index that allows us to visualize the structure under the microscope (Figure 2).

Stress fibers play a role in stem cell differentiation into specific tissues11.

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Figure 2. Characterization of stress fibers in human mesenchymal stem cells. Right: schema from Burridge, K., & Guilluy, C. (2016). Focal adhesions, stress fibers and mechanical tension. Experimental cell research, 343(1), 14–20. Right: Image of hMSC obtained with the 3D Cell Explorer. Stress fibers are highlighted

  1. Dexter, T. M., Allen, T. D. & Lajtha, L. G. Conditions controlling the proliferation of haemopoietic stem cells in vitro. J. Cell. Physiol. 91, 335–344 (1977).
  2. Wolfe, M., Pochampally, R., Swaney, W. & Reger, R. L. Isolation and Culture of Bone Marrow-Derived Human Multipotent Stromal Cells (hMSCs). in Mesenchymal Stem Cells 3–25 (Humana Press, 2008). doi:10.1007/978-1-60327-169-1_1
  3. Pittenger, M. F. Mesenchymal Stem Cells from Adult Bone Marrow. in Mesenchymal Stem Cells 27–44 (Humana Press, 2008). doi:10.1007/978-1-60327-169-1_2
  4. Reger, R. L., Tucker, A. H. & Wolfe, M. R. Differentiation and Characterization of Human MSCs. in Mesenchymal Stem Cells 93–107 (Humana Press, 2008). doi:10.1007/978-1-60327-169-1_7
  5. Zigova, T., Sanberg, P. R. & Sanchez-Ramos, J. R. Neural stem cells : methods and protocols. (Humana Press, 2002).
  6. Fraser, J. K., Zhu, M., Wulur, I. & Alfonso, Z. Adipose-Derived Stem Cells. in Mesenchymal Stem Cells 59–67 (Humana Press, 2008). doi:10.1007/978-1-60327-169-1_4
  7. Mesenchymal Stem Cells. (Humana Press, 2008). doi:10.1007/978-1-60327-169-1
  8. Mesenchymal Stem Cell Culture | PromoCell. Available at: https://www.promocell.com/portfolio/mesenchymal-stem-cell-culture/#application-notes. (Accessed: 20th May 2019)
  9. Birdwell, C. R., Gospodarowicz, D. & Nicolson, G. L. Identification, localization, and role of fibronectin in cultured bovine endothelial cells. Proc. Natl. Acad. Sci. 75, 3273–3277 (1978).
  10. Burridge, K. & Guilluy, C. Focal adhesions, stress fibers and mechanical tension. Exp. Cell Res. 343, 14–20 (2016).
  11. Steward, A. J. & Kelly, D. J. Mechanical regulation of mesenchymal stem cell differentiation. J. Anat. 227, 717–31 (2015).

 

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