Yamanaka factors: The Key to Muscle Regeneration?

By Isabelle Ferenczi

A new technique to improve muscle regeneration for athletes, ageing adults and patients with muscular dystrophy.

Background

The ability to maintain young cells that can divide and replace old ones disappears in the course of development and ageing. What would it take to maintain and control this ability? Dr Shinya Yamanaka and others have been working for a long time to uncover the mechanisms that control cell division. A breakthrough came in 2006 when Yamanaka isolated four key factors controlling the transformation of mouse adult skin cells into ‘induced pluripotent stem cells’ (iPS cells or iPSCs) via the addition of a unique combination of four genes (Takahashi and Yamanaka, 2006). These factors are Oct3/4, Sox2, Klf4 and c-Myc.  Yamanaka repeated the experiment with human adult skin cells (Takahashi and Yamanaka, 2006) and found a similar result. These four factors were identified from a pool of 24 transcription factors expressed in large quantities in embryonic stem cells and were found to be sufficient to confer pluripotency (Longevity Technology, 2021). Yamanaka and John B. Gurdon received a Nobel Prize for this discovery in 2012. The research challenged the view that mature cells are confined to their specialised state.

Moreover, the discovery of iPS cells is important as the use of pluripotent embryonic stem cells involves taking tissue from embryos or umbilical cord, thus being controversial. Creating pluripotent cells from adult tissue allows one to extract, modify and engraft one’s cells in a process called autologous transplantation. Autologous transplantation has the added benefit of avoiding immunological reactions that cause the rejection of foreign tissue. iPSCs provide researchers with a plentiful reservoir of pluripotent cells which can be used for therapeutic strategies and biomedical research. Potential applications rely on their differentiation into specialised cell types and further transplantation as shown in Figure 1.

Figure 1: A diagrammatic representation of potential therapeutic applications of Yamanaka’s factors (Pereira, Marote, Salgado and Silva, 2019). The diagram shows somatic cells which have been isolated from the donor, reprogrammed into iPSCs by adding the four transcription factors (OSKM), allowed to differentiate and can then be transplanted back into the donor who now acts as a recipient. As such, the donor’s somatic cells can be differentiated for the relevant transplantation.

A 2021 study by scientists from the Gene Expression Laboratory at the Salk Institute for Biological Studies in the USA explored the effects of Yamanaka factors in a novel setting. They have examined the effects of Yamanaka factors on muscle mass.  Muscle was selected as the tissue of study because ageing is correlated with a decline in muscle mass which can result in impaired mobility and associated physical traumas.  This research is particularly important to the elderly as muscle mass decreases with age, as well as athletes whose performance greatly depends on their physical form.

The Salk study

Muscle regeneration is primarily controlled by satellite cells (SCs), also referred to as muscle stem cells or myogenic precursors. These cells are located between the basal lamina and myofibers as seen in the transverse section in Figure 2.

Figure 2: Transverse section showing the plasma membranes of a satellite cell (sp), a muscle cell (mp) and basement membrane (bm) in a skeletal muscle fibre from a rat sartorius. This section was stained with PbOH and shown at a 22,000 magnification (Mauro, 1961).

While it was previously shown that partial reprogramming via the addition of the Yamanaka factors (also referred to as OSKM) improved skeletal muscle regeneration in 12-month-old mice, it is unclear whether this was due to intrinsic or niche-specific factors. Hence, the experiment involved both Satellite cells specific or myofiber specific models, generated as described in Figure 3.

Figure 3: Representation of the two types of OSKM induction mouse models which were used. OSKM is an abbreviation of the 4 Yamanaka Factors. The two OSKM induction models used are Myofiber specific and Satellite cell-specific models. In the myofiber specific model, OSKM induction will result in Wnt4 downregulation which will cause the activation/ proliferation of SCs and hence accelerate muscle regeneration. On the other hand, the Satellite Cell specific model was found to not result in muscle regeneration.

The study found that direct Yamanaka factor expression in SCs does not improve muscle regeneration (Wang et al., 2021). On the other hand, localised expression of Yamanaka factors in myofibrils was found to induce SCs and drive muscle regeneration (Wang et al., 2021). This improvement is due to these factors regulating gene expression in myofibrils, specifically, upregulating p21 and downregulating Wnt4 (Wang et al., 2021), as shown in Figure 4. Wnt4 is secreted by myofibrils to maintain SC inactivity (Wang et al., 2021) and hence its downregulation results in the activation of more SCs. Inhibited Wnt4 transcription results in the activation of MyoD and Yap in SCs. As a result, stem cells are activated, proliferate and accelerate muscle regeneration.

Figure 4: Diagrammatic representation of Yamanaka factors regulating gene expression (adapted from Wang et al., 2021). OSKM induction activates the proteins p53 and p21 which downregulates Wnt4. The Wnt4 downregulation activates MyoD and Yap which activate SCs.

The study concluded that SC activation and proliferation are directly regulated by myofibers, however, the extracellular matrix or other cells in the SC’s niche might also take an active part in inducing these events. Moreover, it was shown that direct Yamanaka factor induction in SCs does not change the quantity of SCs or myoblasts and thus, no difference in muscle regeneration is observed. Indeed, in muscles that lacked Wnt4, N- and M-cadherins or syndecan3, there was no SC pool depletion when niche molecular interactions were interrupted and as such perhaps homeostatic mechanisms are involved. Results showed that the effect of the factors was ageing-independent as they were found to remodel the extrinsic niche rather than the intrinsic SC modulators. As intrinsic SC modulators control SC postnatal myogenesis, this is ageing-dependent. Moreover, intrinsic modulators are efficient in young SCs so improving SC quality has limited potential. Furthermore, as shown by figure 3, SC-specific OSKM induction did not change the quantity of SCs or myoblasts and hence had no difference in muscle regeneration. Examples of intrinsic SC modulators and mechanisms include non-random DNA segregation which is affected by epigenetic marks, associated proteins, cell contact, cell density, and microenvironment (Yennek, Burute, Théry and Tajbakhsh, 2014).

The study also explored whether timing affected the effect of OSKM on SC proliferation. As injury triggers SC proliferation without external influence, the effect of Yamanaka factors on SC activation and proliferation was found to be negligible. Furthermore, it was found that a cyclic Wnt4 deactivation has a similar effect on muscle regeneration compared to permanent Wnt4 deactivation. As such, temporary Wnt4 deactivation is more feasible via this mechanism, especially as myofibers are accessible for viral vectors, mRNA or nanoparticles. Therefore using this mechanism will allow the acceleration of muscle regeneration to treat diseases associated with muscle degeneration.

Conclusion

The Salk study offers novel insights into the mechanisms underlying muscle regeneration. Downregulating Wnt4 to activate MyoD and Yap in SCs points us in the direction of exciting future developments, such as improving treatments to diseases of muscle degeneration. Moreover, as myofibers are accessible to viral vectors, mRNAs and nanoparticles, novel therapies could involve the introduction of a Wnt4 deactivation system directly into myofibers. Though further research is needed before this approach can be applied not only on mice but also on humans, this could have a major positive impact on muscle regeneration, especially relevant to the elderly, athletes and also to people with muscle impairment as a result of chronic disease or injury.

Appendix

Table 1: The effect of the relevant transcription factors

References

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