Our studies explored the small molecule pharmacological approach to de-differentiation and reprogramming that does not involve over-expression of exogenous genes, which is the currently searched for method in the field of cell reprogramming .The use of a broad pharmacological inhibitor of tyrosine phosphatases simultaneously with the inhibition of apoptosis was, in our hands, sufficient to induce actual reprogramming of terminally differentiated post-mitotic multinucleated skeletal muscle cells into their progenitors. The use of small molecule inhibitors for reprogramming studies has high translational significance. The apoptosis inhibitors used in our studies is reversible treatment, has short half life and is not present in cells when expanded in vitro for transplantation experiments. Hence the reprogrammed myogenic progenitor cells transplanted with the aim to alleviate myopathic conditions will not be resistant to apoptosis and consequentially will not pose risk of cancers. In this work the irreversible cell-fate lineage marking of myotubes was based on the fact that terminally differentiated skeletal muscle cells are normally produced by the fusion of myoblasts. Thus, Lox-YFP Rosa 26 nuclei and Cre containing nuclei at some point coexisted in a multinucleated cell in order to produce YFP+ myotubes. The Cre-Lox myotubes labeling method efficiently distinguishes reserve cells from multinucleated myotubes. Our data shows that 4 day old cultures of YFP+ primary myotubes express typical muscle differentiation markers such as myogenin, eMyHC, express high levels of cdk inhibitor p21 and do not incorporate BrdU which strongly suggests that these YFP marked cells are indeed terminally differentiated (). The observation that dividing YFP+ mononucleated myogenic progeny were obtained from YFP+ myotubes unambiguously establishes the reprogramming step toward a less differentiated precursor cell. Importantly, such genetic labeling for the first time demonstrates de-differentiation of mature 4-day old multinucleated primary myotubes into proliferating fusion-competent myoblasts that expand in vitro and repair muscle in vivo.
The calculation of reprogramming efficiency from terminally differentiated myotubes to the muscle progenitor cells is complicated by the fact that heterogenous Cre and Lox YFP myoblasts form YFP+ myotubes where varying number of both myonuclei co-exist in same multinucleated myotube. Further, only Lox YFP myonuclei and not Ad-Cre MB nuclei co-existing in YFP+
myotubes will give rise to YFP+ mono-nucleated cells when labeled myotube de-differentiates. Moreover, the de-differentiation of myotubes that are produced by syngeneic fusion events were not accounted for in our YFP labeling method. Hence, we estimated efficiency by two different methods. By method 1 total number of YFP+ mononucleated cells were divided by the total number of YFP+ myotubes before inhibitor treatment. Based on this method, efficiency was estimated ~12-13% in the presence of inhibitor mix as compared to BpV alone which was around ~ 1.18% (Figure S2d
). By method 2, the total number of YFP+ mononucleated cells was divided by an estimated number of Lox YFP myonuclei present in all labeled YFP+ myotubes before inhibitor treatment (see methods section).This method gave an estimate of ~ 5% in presence of inhibitor mix and ~0.4% in presence of BpV alone (Figure S2d
and methods section). For the above reasons, we feel these calculations give very conservative estimates of de-differentiation. No matter, the method of quantification, BpV alone gave poor reprogramming efficiency and apoptotic inhibitor was needed to augment the de-differentiation likely by facilitating the survival of those myotubes which undergo reprogramming in the presence of phosphatase inhibitor.
Recent reports have shown that cells expressing higher levels of anti proliferative genes and those involved in senescence are indeed difficult to reprogram (Li et al., 2009
; Utikal et al., 2009
). Since myotubes are post-mitotically arrested cells which express high levels of CDK inhibitors, these may have low reprogramming efficiency. Studies have indicated that experimental down regulation of CDK inhibitors in post-mitotic myotubes results in accumulation of DNA damage, hinders cell cycle reentry and cause DNA fragmentation and apoptosis (Pajalunga et al., 2010
) It has been shown that dividing cells robustly repair DNA damage (Nouspikel and Hanawalt, 2002
) and since our de-differentiated cells are cultured in mitogen-high growth medium (following the BpV and apoptosis inhibitor treatments) and are actively dividing, any DNA damage accumulated in myotubes is likely to be repaired during the process of DNA replication. Notably, our results definitively demonstrate that reprogrammed myotubes give rise to functional muscle progenitor cells which form new muscle in vitro and in vivo, hence no irreversible DNA damage or mutations occurred to compromise the myogenic properties of the de-differentiated cells. Recent studies has also shown that inhibition of Rb and p16/p19 can induce cell cycle entry in post mitotic myocytes (Pajcini et al., 2010
). Our work conducted on 4 day old mature myotubes not only downregulated CDK inhibitors and muscle differentiation markers but also decreased gene expression of chromatin remodelers that maintain differentiated state. The role of chromatin organization in establishment and maintenance of cell fates has been well defined. It has also been shown to play a role in commitment of myoblast to terminally differentiated myotubes as different signaling pathways have been shown to modulate chromatin signaling in muscle progenitor cells upon differentiation (Caretti et al., 2004
; de la Serna et al., 2001
; McKinsey et al., 2002
; Palacios and Puri, 2006
; Saccone and Puri). Recent work has also demonstrated the stage specific role of Ezh2 in muscle regeneration where Ezh2 occupies the Pax7 regulatory sequences in differentiated state (Palacios et al., 2010
). This suggests that in spite of downregulation of Ezh2 upon differentiation as reported earlier (Caretti et al., 2004
), a certain level of Ezh2 along with other polycomb members would be required to maintain muscle progenitor genes in repressed state. Our PCR arrays on Cre-Lox myotubes after inhibitor mix treatment demonstrate significant down regulation of Ezh2 and other polycomb members as compared to untreated myotubes suggesting the creation of sensitized background in myotubes that may force them to re-express muscle progenitor genes and enter cell cycle. The conversion of cell fates due to enhanced cell fate plasticity in polycomb compromised backgrounds has been documented in other model systems such as flies (Lee et al., 2005
) and worms (Yuzyuk et al., 2009
). In this regard, our work highlights the fact that key players in maintaining and establishing terminally differentiated state need to be erased transiently at epigenetic level in order to switch a differentiated state to a less differentiated state within the same cell lineage.
Since we used a generic tyrosine phosphatase and apoptosis inhibitor which is expected to modulate signaling in many biochemical pathways, the down-regulation of specific chromatin remodeling factors predisposed primary myotubes to respond to the mitogens of GM thereby changing their cell fate to that of proliferating myogenic precursor cells. Interestingly, while upon addition of inhibitor mix there were profound changes in the morphology of many myotubes, only a subset of these myotubes de-differentiated into proliferating mono-nucleated precursor cells. Since there is temporal progression towards the degree of terminal differentiation in myotubes there might be a specific time window when myotubes would be more responsive to the treatment and capable of undergoing cell fate reversal. Future work would determine the exact role of chromatin remodeling factors in acquiring muscle progenitor cell fate from multinucleated post-mitotic differentiated state. As there is temporal progression of gene expression upon myotube differentiation, it would be interesting to study in the future whether the inhibitor mix is sufficient to induce de-differentiation in mature myofibres formed in vivo or indeed other factors are required to yield regenerative cells.
Use of pharmacological inhibitors to modulate different signaling pathways without gene over expression is therapeutically relevant in coaxing differentiated cells to yield regenerative cells. Our data highlights the combinatorial use of tyrosine phosphatase and apoptosis inhibitors for primary myotube de-differentiation to yield myogenic proliferating cells which aided in muscle regeneration both in vitro and in vivo in SCID mice. The novel myotube labeling technique developed for this study served as a powerful tool to clearly show the origin of reprogrammed cells and to sort them away from reactivated reserve myoblasts. Importantly, small molecule inhibitors induced changes in myotubes at epigenetic level and facilitated them to enter proliferative state in mitogen rich medium. All together, the novel labeling technique employed the use of small molecule inhibitors advance the ongoing research in regenerative medicine and would enable unique clinical strategies for enhancing tissue regeneration.