Studies show resulting mononucleate cells can be expanded in vitro and contribute to muscle repair in vivo.

Terminally differentiated muscle myotubes can be triggered to convert back into the actively replicating mononucleate muscle progenitor cell state by treating them with the tyrosine phosphatase inhibitor BpV and an apoptosis inhibitor Q-VD, researchers claim. The duo of investigators from the University of California, Berkeley claim that the technique doesn’t require any genetic manipulation or introduction of exogenous genes.

The studies by Preeti Paliwal, Ph.D., and Irina M. Conboy, Ph.D., showed that lineage-marked progenitor cells derived from terminally differentiated myotubes are not only capable of fusing with other mycotes and reforming myotubes but can contribute to muscle regeneration in vitro and vivo, providing new avenues of research for muscle regenerative cell therapies.

The investigators report their work in Chemistry & Biology in a paper titled “Inhibitors of Tyrosine Phosphatases and Apoptosis Reprogram Lineage-Marked Differentiated Muscle to Myogenic Progenitor Cells.”

Reprogramming myotubes back into myogenic precursor cells is particularly challenging because myogenic proliferating cells not only undergo postmitotic arrest, but they also physically fuse with each other to form multinucleated myotubes during their terminal differentiation, Drs. Paliwal and Conboy report. Indeed, once these cells terminally differentiate, they are incapable of re-entering into mitosis even when switched to serum-rich medium.

Myotube formation involves a raft of events including changes in cytoskeletal assembly, sequential expression of differentiation-specific genes, modulation of signaling pathways, and up-regulation of tyrosine phosphatases.

The team thus reasoned that global transient inactivation of tyrosine phosphatases might reset signaling in myotubes, making them receptive to mitogens present in growth medium conditions, and trigger them to re-enter the cell cycle and regress toward a less-differentiated state. Moreover, they suppositioned, the addition of an apoptosis inhibitor to a tyrosine phosphatase inhibitor might then help the survival of those myotubes that do break from mitotic arrest and as a result undergo massive restructuring of the cell cytoskeleton.

They carried out their subsequent muscle reprogramming studies in differentiated lineage-marked primary myotubes generated by the physiological fusion of Rosa26-Lox-YFP myoblasts with Cre-expressing myoblasts. Using this procedure, formation of multinucleated myotubes results in the recombination of YFP locus and expression of YFP. Effectively, the technique lineage-marks terminally differentiated myotubes following physiological myoblast fusion.

When they treated their myotube cultures with both BpV and Q-VD (the inhibitor mix) for two days, about 12–13% of the YFP+ myotubes exhibited altered morphology and cleaved into small cells. This was then followed by the appearance of YFP+ mononucleate progeny of the dedifferentiated myotubes.

Conversely, treating the myotubes with BpV alone resulted in cell death. “These results demonstrate that the inhibition of apoptosis is a critical requirement for the dedifferentiation of multinucleated primary myotubes,” the researchers note.

To further assess the properties of reprogrammed cells, mononucleated YFP+ progeny from the dedifferentiated myotubes were FACS-sorted and expanded in culture. The cells positively immunostained for Ki67 (a proliferation marker) and BrdU (confirming that the cells have re-entered the cell cycle). The majority of YFP+ mononucleated cells also expressed high levels of the myogenic markers Pax7 and MyoD1.

Importantly, when switched from a growth medium to a differentiation medium, the YFP+ mononucleated cells were found to retain their myogenic potential as they underwent rapid physiological fusion de novo into myotubes that expressed typical muscle differentiation markers eMyHC, myogenin, and p21.

“The changes in marker gene expression were also validated at the transcriptional level by qRT-PCR, which clearly showed the up-regulation of eMyHC, p21, and myogenin and down-regulation of Pax7 and MyoD mRNA levels upon differentiation of YFP+ mononucleated cells,” the researchers state.

The ultimate test was to see whether the reprogrammed cells could contribute to in vivo muscle regeneration under physiological conditions. To this end the UCB team expanded dividing dedifferentiated YFP+ cells for about 1–2 weeks and then injected them into the cardiotoxin-injured tibialis anterior (TA) muscles of immunodeficient NOD-SCID mice.

After two weeks, they found that YFP+ reprogrammed cells had readily fused with regenerating myofibers and contributed to muscle repair. “These results establish that postmitotic myotubes can dedifferentiate into functional, proliferating myogenic precursor cells that regenerate muscle tissue after an injury,” they remark.

At the gene-expression level, over 60% of the myonuclei in YFP+ myotubes treated with the inhibitor mix down-regulated myogenin (a muscle marker expressed on onset of differentiation) in comparison with untreated mytubes. Levels of p21, a negative regulator of mitosis that plays an important role in cell-cycle arrest, were found to be attenuated in approximately 25% of the myonuclei present in YFP+ myotubes. Down-regulation of the terminal muscle differentiation marker eMyHC along with p21, p15, p16, and myogenin were also confirmed in inhibitor mix-treated Ad-Cre-Lox-YFP+ myotubes by Western blotting and qRT-PCR analysis. “These results demonstrate that the myogenic cell fate is reversed at the genetic level in multinucleated myotubes, before they split into single dividing cells.”

Prior studies have also shown that myotube differentiation is accompanied by widespread chromatin remodeling, and genes necessary for differentiation are activated while those for proliferation are repressed. To analyze these changes during inhibitor mix treatment, PCR arrays for chromatin enzymes and chromatin remodeling factors were performed on untreated and inhibitor mix-treated Cre-Lox YFP myotube cultures. The results indicated that, as expected, chromatin factors and enzymes dedicated to the maintenance of a differentiated state in primary myotubes were downregulated, enabling the cells to respond to the growth factors in serum and dedifferentiate into proliferating progenitor cells.

“Use of pharmacological inhibitors to modulate different signaling pathways without gene overexpression is therapeutically relevant in coaxing differentiated cells to yield regenerative cells,” the authors conclude. And importantly, the apoptosis inhibitors used represent a reversible treatment, have a short half life, and are only needed for the initial dedifferentiation step, not for the expansion of derived cells in vitro. “Hence, the reprogrammed myogenic progenitor cells transplanted with the aim to alleviate myopathic conditions will not be resistant to apoptosis and, consequently, will not pose the risk of cancer.”

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