Improved Methods of Expanding Skeletal Muscle Progenitors in Culture.
There is general agreement within the muscle research community that methods which allow the in vitro expansion of skeletal muscle progenitor cells (MuSCs, also known as satellite cells) without sacrificing their future myogenic potential, will benefit applications in basic research, drug discovery, and cell therapy. Two recent publications discussed below, present contrasting approaches to achieving this objective.
Judson et al. [(2018) Cell Stem Cell 22 177-190] used small molecule inhibition of the Setd7 lysine methyl-transferase to prevent MuSCs obtained from mouse or human skeletal muscle from differentiating, in vitro, into myoblasts; the authors had earlier found that Setd7 expression was upregulated as myogenesis proceeded and that genetic deletion of Setd7 or its inhibition using the small molecule PFI-2 impeded adult skeletal muscle differentiation. In this way, they sought to address the problem that although myoblasts themselves can proliferate in culture for some time before fusing into myotubes, their myogenic potential both in vitro or after transplantation, diminishes rapidly. Strikingly, PFI-2 inhibition facilitated greater expansion of MuSCs in vitro and once differentiated, these resulting myoblasts displayed much improved engraftment potential after transplantation into the tiberialus anterior (TA) muscles of mice.
Using a radically different approach, Rao et al. [(2018) Nature Communications DOI: 10.1038/s41467-017-02636-4, PMID: 29317646] achieved MuSC expansion by forced expression of Pax7 in paraxial mesoderm cells prepared from pluripotent stem cells (PSC; human embryonic or induced pluripotent stem cells). Pax7 expression (along with a co-expressed reporter GFP gene) was controlled by doxycycline added to the culture medium. Over a 30 day period, 300,000 PSCs could be differentiated into 200 million MuSCs using two different media and an intervening FACS sorting step. These cells were then capable of highly efficient monolayer (2-D) differentiation into functional myotubes complete with some resident Pax7 cells. However, these myotubes were not able to maintain maturity for long periods in culture. This situation was rectified when a 3-D culture system was implemented, comprising embedding expanding cell populations in fibrin gel before inducing final differentiation. The resulting 3-D skeletal muscle (ISKM) bundles exhibited calcium transients and improved contractile function and strength over 2-D muscle, and were able to successfully engraft into muscle (TA) and dorsal skin sites in mice. Overall, although functionally the ISKM bundles were significantly inferior to those made with human primary myoblasts, this work marks a major step forward. A very striking and encouraging observation was that the muscle gene expression in the ISKM bundles changed during a 4 week culture period from embryonic/fetal like towards an adult pattern. This transformation was not seen in the 2-D cultures, and represents an important departure from the embryonic/fetal-like expression profiles mostly seen in cells and tissues made from PSCs.
Both papers record notable advances in the quest to make large quantities of functional muscle in vitro from expanded cell populations. It will be interesting to see how the PFI-2 expanded human MuSCs made from primary human myoblasts measure up to the PSC-derived MuSCs. Furthermore, one wonders whether the PFI-2 method could satisfactorily substitute for the use of Pax7 and thereby provide a simpler, generic and arguably more clinically relevant way of prolonging MuSC expansion. Genea Biocells is encouraged by these developments although at the present time, it finds the myoblasts and myotubes it generates using its proprietary media, to provide excellent assays for its drug discovery activities.
Alan Colman Feb 5, 2018