Fibroblasts impair muscle stem cell self-renewal via excessive fibronectin deposition in viscoelastic hydrogel co-cultures

What I like about the preprint

This preprint attracted me because it explores the important role of the local microenvironment in regulating stem cells during ageing, rather than focusing solely on stem cell age. The authors use an elegant co-culture system and, in their preprint, present tricky experiments clearly. The repeated use of immunofluorescence to assess different markers makes the experimental logic easy to follow. The results presented here raise interesting questions and opens avenues for future research (see below), while also having potential therapeutic relevance given the use of fibronectin to improve wound healing in clinical trials.

Background

Following injury, our tissues need to clear away damaged cells and rebuild tissue to maintain function and protect the rest of the body. Macrophages play an important role in clearing damaged tissue and cellular debris, as well as coordinating the inflammatory and repair responses following an injury (Wang and Zhou, 2022). To rebuild the tissue, a healthy population of stem cells is needed to proliferate and differentiate and thereby replenish local cell populations, as well as produce chemical signals to promote repair processes (Jiang et al., 2024 and Lau et al., 2009).

It is well established that stem-cell ageing contributes to a decline in stem-cell function (Brunet et al., 2023 and Rando et al., 2025), which can impair efficient wound healing with age (Rajendren et al., 2021). Both intrinsic and extrinsic factors affect stem cell regulation (Dumont et al., 2015), including the effects of the extracellular environment on stem cell ageing. Transplanting an aged stem cell into a young niche does not fully restore its function (Bernet et al., 2014), although this can reverse nearly half of the age-associated transcriptional changes (Lazure et al., 2023).

In this preprint, the authors find that fibronectin deposition after injury transiently increases in young mice but is sustained in aged mice. They link this to the young or aged fibroblast population where they observe similar trends in their activity. Finally, they show that these changes in fibroblasts and fibronectin deposition impacts renewal and quiescence of the stem cell population in myofibres cultured in vitro.

Key findings

Injury induces transient changes in stem cell proliferation and fibroblast-mediated fibronectin deposition.

Chang and colleagues used injections of Barium chloride to chemically induce muscle injury through degeneration and necrosis in the Tibialis anterior of mice, a more controlled method than creating a cut or tear. They then performed immunofluorescence staining for different markers, imaging at four and seven days after injury, in young and old mice, as well as uninjured controls (Figure 1 and 2 from Chang et al., 2026).

Using Pax7 to label stem cells, they observed that the number of stem cells in young mice transiently increased at four days after injury and decreased towards the baseline at seven. In the same muscles, fibronectin coverage also followed this pattern. However, in older mice, the increase in stem cell number and fibronectin coverage was not transient and remained high at seven days (Figure 1 from Chang et al., 2026).

By analysing single-cell and single-nucleus RNA-seq data from another study (Kurland et al., 2023), they determined that fibroblasts are the most likely source of the fibronectin changes. They investigated the number and activation of fibroblasts by labelling platelet-derived growth factor receptor ⍺ (PDGFR⍺) and ⍺-smooth muscle actin (⍺SMA) respectively. Both markers showed the same pattern as seen with fibronectin deposition: a transient increase at four days in young mice, but a sustained increase in older mice (Figure 2 from Chang et al., 2026).

Activation of fibroblasts and fibronectin deposition are intrinsic to the age of the fibroblast, and not the surrounding environment.

The team next looked at the extent to which fibronectin deposition is determined by the age of the fibroblast vs the age of the surrounding microenvironment. To do this, they began by culturing fibroblasts derived from young and aged mice on gelatine-coated tissue culture plates. They observed that the aged fibroblasts had more ⍺SMA labelling, indicating higher activity, and greater fibronectin deposition.

Next, they used a previously developed hydrogel formulation to co-encapsulate and culture myofibres in vitro, without spontaneous activation and differentiation of stem cells (Chang et al., 2025). With this system, they were able to co-culture young or aged myofibres, with fibroblasts from young vs aged mice. They observed that fibroblast activation (⍺SMA) and fibronectin deposition are hardly impacted by the age of the myofibre, whereas the age of the fibroblasts showed a large change with both measures increasing with the aged fibroblasts (Figure 5 from Chang et al., 2026).

Fibroblast age regulates stem cell quiescence and proliferation in a fibronectin-dependent manner.

Finally, the team investigated how the age of fibroblasts can affect stem cell maintenance. They again used the in vitro co-culture method of young and old myofibres with young or old fibroblasts, but this time they labelled Pax7 to identify stem cells, and MyoD to identify stem cell activation. Additionally, they included a pulse of EdU labelling two hours before fixation to quantify proliferation (Figure 6 and 7 from Chang et al., 2026).

Comparing young and old myofibres, without addition of fibroblasts, there was a similar proportion of nuclei positive for Pax7 (Figure 6 from Chang et al., 2026), however in the young myofibres, the proportion of these Pax7 positive nuclei that were also positive for EdU and MyoD was much higher than in the aged myofibres (Figure 6 from Chang et al., 2026). Co-culturing with young fibroblasts did little to affect the EdU and MyoD labelling of Pax7 cells in the aged myofibres, however in the young myofibres it reduced both markers (Figure 6 from Chang et al., 2026). This suggests that young fibroblasts can promote a more quiescent stem cell population.

Adding aged fibroblasts had the opposite effect, with the young myofibres having little difference in EdU and MyoD labelling of Pax7-positive cells when cultured alone or with aged fibroblasts, however a large increase in both markers was seen for aged myofibres co-cultured with aged fibroblasts (Figure 6 from Chang et al., 2026). Therefore, aged fibroblasts can promote proliferation and differentiation.

To determine if fibronectin deposition is an important factor in the regulation of stem cells by fibroblasts, the team used siRNA targeted to Fn1, reducing fibronectin production in fibroblasts derived from young and aged mice. They showed that knocking down fibronectin in the aged fibroblasts largely attenuated their effect on stem cells, with less proliferation or differentiation (measured by EdU and MyoD labelling) compared to aged fibroblasts “knocked down” with a scrambled control siRNA (Figure 7 from Chang et al., 2026).

Future directions and questions for the authors

1. How do you predict fibronectin is cleared between day four and seven in young mice?
2. As a non-chemist, can you explain more about how you designed the hydrogel? Why did you choose hyaluronic acid?
3. Comparing the young and old myofibres cultured in vitro without fibroblasts, there is a large increase in the number of Pax7 positive cells that are also MyoD and EdU positive in young myofibres compared to old (Figure 6 of Chang et al., (2026)).
   • Why is this reduced by adding young fibroblasts?
   • Are young fibroblasts contributing some other unidentified factor?
4. What are the implications for short-term injury recovery? Less fibronectin promotes stem cell renewal, which is beneficial in the long term, but does it delay or prevent short-term activation and recovery?
5. Ageing has also been linked to a reduction in fibronectin in the stem cell niche, leading to reduced stem cell adhesion and impaired tissue regeneration (Lukjanenko et al., 2016). How do you reconcile this with your own data showing fibronectin deposition increased with age?
6. Could the findings presented as part of this work also extend to cardiac and smooth muscle?

References

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• Kurland, Jesse V., Alicia A. Cutler, Jacob T. Stanley, et al. ‘Aging Disrupts Gene Expression Timing during Muscle Regeneration’. Stem Cell Reports 18, no. 6 (2023): 1325–39. https://doi.org/10.1016/j.stemcr.2023.05.005.
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Tags: extracellular matrix, fibroblasts, fibronectin, muscles, niche, regeneration, stem cells

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