3D mechanical confinement directs muscle stem cell fate and function

Background:

Most of the cells in your body are terminally differentiated, meaning they don’t have the ability to change their identity. Your skin cells won’t become brain cells, your heart cells won’t become liver cells, etc. However, there are some cells in your body with the ability to differentiate. These are called stem cells. Populations of stem cells exist in and around many organs in your body and likely have roles in the growth, development, and self-renewal of those organs, as well as their regeneration after injury. Muscle stem cells (MuSCs) maintain populations around mature muscle cells, or myofibers, and are able to differentiate into and replace existing myofibers when they are damaged. The genetic and cellular pathways involved in this process have been studied by previous research groups (Han et al. 2019, Goel et al. 2017, Collins et al. 2024). However, it is not completely understood how these processes are initiated and regulated.

Due to their unique 3D nature, it is thought that mechanical stresses play a role in MuSC regulation. However, previous studies have only studied these factors in 2D by adjusting the stiffness of the media (Gilbert et al. 2010, Silver et al. 2021, Madl et al. 2024) or compressing the cells (Tao et al. 2023). The authors of this preprint wanted to test the influence of 3D confinement and asymmetric stiffness on MuSC proliferation and differentiation.

Methods:

The authors synthesized hydrogels of 3 different stiffness levels, 5kPa, 12kPa, and 35kPa, and plated primary mouse MuSCs either onto 12kPa hydrogel monolayers or bilayers with a bottom layer of 12kPa and top layers of each stiffness. The MuSCs used were isolated from Pax7EGFP mice, or mice that had been genetically engineered so that when they expressed the Pax7 protein, which is typically expressed in skeletal muscle stem cells, it would be tagged with green fluorescent protein (Tichy et al. 2018). This fluorescence allowed the researchers to visualize the state of the cells, as they would lose it if they differentiated into a different cell type. To track this differentiation, the researchers also performed antibody staining for MyoD and MyoG, markers of MuSCs that have been stimulated to differentiate, and histone H4 lysine 16 acetylation (H4K16ac), an epigenetic marking that is associated with differentiated muscle cells. The cells were cultured over 5 days with staining performed and fluorescence measured at multiple time points.

Key Findings:

3D confinement limits MuSC proliferation:

Pax7EGFP MuSCs grown on the 2D hydrogels proliferated more rapidly than cells grown in the bilayers and reached a significantly greater relative cell count by day 5. There was no significant difference between the relative cell counts of the three bilayers, but the lowest colony density was observed in the 35kPa/12kPa bilayer.

3D confinement inhibits MuSC differentiation:

After 5 days of culture, the 3D bilayer cultures exhibited a significantly greater proportion of cells expressing high-intensity Pax7EGFP (~35% vs. ~10%) and a significantly lower proportion of cells expressing MyoD (~25 or ~50% vs. 80%) and MyoG (~5% vs. 35%). However, immunostaining did not reveal any difference in the proportion of cells expressing Pax7, indicating that the reduction in intensity is due to downregulation of expression, not termination of it. The 3D bilayer cultures also exhibited a reduced proportion of cells expressing H4K16ac, but this difference was only statistically significant in the 35kPa/12kPa bilayer (~15% vs. ~95%). Notably, there were no significant differences in the expression of any of these markers between cells grown in each bilayer, with the exceptions being cells grown in the 35kPa/12kPa bilayer exhibiting a significantly lower proportion of MyoD+ cells (~25% vs. ~50%), and cells grown in the 35kPa/12kPa bilayer exhibiting a significantly lower proportion of H4K16ac+ cells (~15% vs. ~80%).

Significance:

I am highlighting this preprint because I am interested in the mechanisms that govern stem cell specification. Most of the research I have seen on this topic has detailed paracrine or juxtacrine signaling pathways, so I have not previously heard of mechanical pressure as a regulator of cell identity. I also appreciate that this preprint demonstrates the interconnectedness of different subfields of biology. The authors used methods from bioengineering (designing a hydrogel culture system) and genetics (labeling and tracking specific genes) to answer a developmental biology question (Do environmental influences affect MuSC activation and differentiation?).

Questions for the authors:

1. In the discussion, you indicate that this model does not fully capture the complexity of the wound environment, using the absence of dynamic mechanical forces as an example. Are there any other important characteristics of the wound environment that are not included in this model that you would like to implement in future experiments?
2. Do you see this model, a 3D bilayer hydrogel, being used for medical applications, such as preserving MuSCs isolated from a patient so they can be reintroduced later? Why or why not?

References:

1. W. M. Han, M. Mohiuddin, S. E. Anderson, A. J. García, Y. C. Jang, Co-delivery of Wnt7a and muscle stem cells using synthetic bioadhesive hydrogel enhances murine muscle regeneration and cell migration during engraftment. Acta Biomaterialia 94, 243–252 (2019).
2. J. Goel, M.-K. Rieder, H.-H. Arnold, G. L. Radice, R. S. Krauss, Niche cadherins control the quiescence-to-activation transition in muscle stem cells. Cell Reports 21, 2236–2250 (2017).
3. C. Collins, J. B. Shapiro, M. M. Scheib, R. V. Musci, M. Verma, G. Kardon, Three-dimensional imaging studies in mice identify cellular dynamics of skeletal muscle regeneration. Dev Cell 59, 1457-1474.e5 (2024).
4. M. Gilbert, K. L. Havenstrite, K. E. G. Magnusson, A. Sacco, N. A. Leonardi, P. Kraft, N. K. Nguyen, S. Thrun, M. P. Lutolf, H. M. Blau, Substrate elasticity regulates skeletal muscle stem cell self-renewal in culture. Science 329, 1078–1081 (2010).
5. S. Silver, K. A. Günay, A. A. Cutler, T. O. Vogler, T. E. Brown, B. T. Pawlikowski, O. J. Bednarski, K. L. Bannister, C. J. Rogowski, A. G. Mckay, F. W. DelRio, B. B. Olwin, K. S. Anseth, Injury-mediated stiffening persistently activates muscle stem cells through YAP and TAZ mechanotransduction. Sci Adv 7, eabe4501 (2021).
6. M. Madl, Y. X. Wang, C. A. Holbrook, S. Su, X. Shi, F. J. Byfield, G. Wicki, I. A. Flaig, H. M. Blau, Hydrogel biomaterials that stiffen and soften on demand reveal that skeletal muscle stem cells harbor a mechanical memory. Proc Natl Acad Sci U S A 121, e2406787121 (2024).
7. Tao, M. I. Choudhury, D. Maity, T. Kim, S. X. Sun, C.-M. Fan, Mechanical compression creates a quiescent muscle stem cell niche. Commun Biol 6, 43 (2023).
8. D. Tichy, D. K. Sidibe, C. D. Greer, N. M. Oyster, P. Rompolas, N. A. Rosenthal, H. M. Blau, F. Mourkioti, A robust Pax7EGFP mouse that enables the visualization of dynamic behaviors of muscle stem cells. Skelet Muscle 8, 27 (2018).

 

doi: https://doi.org/10.1242/prelights.40344

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