In resting muscle, SCs are imprisoned in a reversible quiescent phase and they have capacity to quickly reactivate in response to injury [ 45 ]. The molecular systems involved in G0 exit have recently been the subject of intense research [ 15 , 46 – 48 ]. However , SCs are difficult to maintain with this quiescent state in vitro and the molecular analysis associated with quiescent SCs on the materials used as culture substrates (tissue culture polystyrene, plastic coated with proteins) is usually technically challenging due to the spontaneous shift toward commitment. Hence, most of the recent studies of quiescence in SCs are usually performed either directly in vivo [ 15 , 46 ], within situ with SCs on the fiber [ 47 ] or ex vivo but a few hours after isolation [ 49 ], already with a potential and variable shift towards an activated state. These conditions greatly limit the quantity of available cells and the full characterization of the studied tissues. Furthermore, it is difficult to make direct comparisons between these types of studies due to the unknown phenotypes of SCs under these types of various conditions. A key to control SC behavior may be in order to modulate the biophysical properties of the in-vitro microenvironment because are known to influence cell fate [ 20 ].
Using a biopolymeric platform for the in-vitro culture and study of adherent quiescent SCs, we all show that human SCs are sensitive to the crosslinking level of the biopolymeric films, as reported previously with regard to mouse skeletal myoblasts [ 31 ]. While SCs within culture spread on plastic or stiff substrates, these people remain round and exhibit blebs on EDC10 movies. These round-blebbing cells are reminiscent of the SC form reported previously in vivo during migration [ 50 ]. This mechanism is indeed poorly studied ex vivo because of the high SC cell spreading on plastic culture substrates and due to the difficulty of imaging SCs on a single dietary fiber at high resolution. Our results suggest that SCs cultured former mate vivo on soft EDC10 biopolymeric film maintained the phenotype close to their native state in vivo through physical signals.
The repair of the quiescent state from day 1 to time 10 for SCs cultured on EDC10 films has been shown by several concomitant findings: sustained Pax7 expression without appearance of MyoG; absence of proliferation; confinement of more than 95% associated with SCs in the G1/G0 phase; the absence of cyclin D1 expression, which suggests that the cells are in the G0 stage; and cell reactivation after replating on plastic. Prior studies showed that SCs can also enter a much deeper state of quiescence, called dormancy [ 6 ]. Right here, we showed the possibility to reactivate SCs by replating them on plastic, exploiting the reversible process of getting out of and entering the cell cycle. Importantly, we demonstrated that the proportion of quiescent cells in the population boosts significantly after preculture on plastic and replating upon EDC10 films, either by upregulation of Pax7 or selection of the preexisting Pax7 + population. These outcomes also indicate that amplification on plastic is possible just before culture on EDC10 films to recover a larger population associated with quiescent cells.
Interestingly, immediate culture of SCs on EDC10 films resulted in circular morphology associated with low fibronectin and collagen I release, while SCs cultured on plastic secreted significantly more collagen I. However , longer-term culture on EDC10 films led to some cell spreading, which was associated with the secretion of ECM proteins. We hypothesize that fibronectin secreted by SCs in GM hides the mechanical cues, allowing the pv cells to spread independently of the underlying substrate. It is likely that the particular biochemical signal of the fibronectin layer overrides the mechanised signal from EDC10 films. This result sheds a few light on the role of the underlying material substrate within the secretion of ECM proteins by SCs: both the mechanised and the biochemical microenvironment of SCs can influence their particular morphology and protein secretion, as well as their fate. Fibronectin expressed by SCs is known to modulate their expansion inside their niche by potentiating Wnt7a signaling and transiently redesigning their environment [ 21 ]. In addition , the activation associated with specific cell receptors (e. g., integrins) by manufactured materials is a means to control cell adhesion, proliferation plus differentiation [ 21 , 27 ], and muscle stem cells had been also shown to remodel their own ECM [ 28 ].
Mechanical signals have been shown to be since important as biomechanical signals [ 51 ]. Mechanical attributes are often characterized by the stiffness (also called rigidity) of the material, which has been shown to be a crucial parameter in stem cellular fate determination [ 20 ] and SC self-renewal [ 13 ]. Plastic traditionally used for cell culture is an extremely stiff material (~3 GPa). In contrast, the elasticity associated with muscle was found to be ~12 kPa [ 29 ], the elasticity of undifferentiated C2C12 myoblasts ~11 kPa and the elasticity of isolated myofibers 45 kPa [ 52 ]. Thus, there are approximately five orders of degree of difference between muscle stiffness and the traditional cellular culture substrate.
Our answers are in agreement with a previous study showing that there has been an optimal stiffness of PEG hydrogels (~12 kPa) to foster SC self-renewal in culture and continual Pax7 appearance [ 13 ]. These PEG hydrogel substrates allowed the particular maintenance of Pax7 expression in 32% of the daughter cells, which is beneath the frequency reached on soft biopolymeric films.
Recently, Schroder and coworkers suggested a new culture model by culturing SCs in a semi-solid suspension medium of methyl cellulose [ 53 ]. Oddly enough, the authors suggested that the absence of cell adhesion towards the material substrate triggered the cells to enter G0 police arrest. This is reminiscent of our findings of a low adhesive/round condition of SCs cultured on EDC10 films. Because their own study was done over 4 days [ 53 ], it would be interesting to further evaluate the effective capacity of SCs to remain quiescent over longer periods of time. Although very interesting, this particular culture model does not provide morphological information for SCs, because the cells need to be cytospun for microscopic observation [ 53 ]. Another study used a cocktail of substances (drugs, proteins, growth factors and other biomolecules) combined with a good engineered 3D microscaffold to mimic a muscle dietary fiber [ 48 ]. In this artificial niche with a well-defined cellular culture medium, cells can remain quiescent. However , the consequence of all the biochemical signals composing the cocktail of bioactive molecules would need to be tightly controlled in the long term.
These strategies provide information on the native condition of SCs but none of them featured an adherent lifestyle platform, such as the biopolymeric films, for the ex-vivo culture associated with quiescent SCs to facilitate translation studies. According to Negroni et al. [ 7 ], the ideal cell candidate just for cell therapy should fit the following requirements: high number, simple isolation and amplification, high myogenic potential, specific homing capacities [ 54 ] and genetically modifiable. Benefit regeneration potential demonstrated by quiescent SCs in their market implanted in vivo implies that, for ex-vivo culture plus retransplantation, a high amplification rate is not needed to obtain vast amounts of injected cells. In view of the regenerative potential of SCs, as few as seven SCs associated with one transplanted myofiber may generate over 100 new myofibers, because these cells can expand in vivo and self-renew relatively efficiently [ 54 ]. Thus, quiescent SCs are among the most promising applicants for muscle cell therapy [ 55 ].
SCs cultured on EDC10 appear to meet several requirements for cell therapy because they are quiescent, could be preamplified by culture on plastic and are transduced simply by lentivirus. Notably, in our experimental conditions, the precultured cellular material on EDC10 films exhibited the highest transduction capacity. The particular biopolymeric EDC10 film may thus be further investigated in the context of cell therapy: the autologous affected person cells may be first preamplified on plastic, possibly reprogrammed genetically, before being replated on EDC10 films to assist them recover their quiescent state, prior to transplantation. At the same time, transduction may be done after replating on EDC10 movies and prior to transplantation.