Telomerase Activity and Myogenesis Ability as an Indicator of Cultured Turkey Satellite Cell Ability for In Vitro Meat Production

Skeletal muscles are generated through proliferation, differentiation and fusion of embryonic myoblasts. In adulthood, muscle growth and regeneration is facilitated by highly proliferative satellite cells, detectable in skeletal muscles of chicken, turkey, pig, cattle and lamb [1]. These cells can be characterized by the expression of markers specific for their lineage, such as ALDH1 and ALDH2 [2]. Satellite cells are located between basal lamina and sarcolemma of the mature muscle fiber, possessing the ability to proliferate and differentiate into myotubes or myofibers [3]. Satellite cell telomere length is a factor of particular significance in muscle regeneration. Telomeres are composed of DNA and multiple protein components. Located at the end of eukaryotic chromosomes, they protect chromosomes end against enzymatic degradation to maintain chromosome stability [4]. They are dynamic structures, with their length able to greatly vary due to environmental conditions [5]. In vertebrates, telomeres consist of repeated sequences of TTAGGG synthesized by the telomerase enzyme [6]. Telomerase activity is present in germ line cells, stem cells, cancer cells, and immortal cell lines, but has not yet been detected in normal somatic cells [7,8]. Its activity can be measured through the analysis of expression of its genes, TERT1 and TERT2, as well as its proven regulators, such as Dyskerin [9]. The absence of telomerase activity in cells allows shortening of telomeres during each cell division, as DNA polymerase is not able to replicate the very ends of the DNA strand [10]. Incompletely replicated ends of chromosomal DNA are inherited by daughter cells and progressively shorten the ends of chromosomes. Through this mechanism, telomerase regulates cell division before senescence [11,12,13]. Furthermore, telomerase activity seems to be an important factor in production of continuous cell lines. Although the absence of telomerase activity has been demonstrated in somatic adult cells in vertebrates, turkey satellite cells appear to be an exception of that rule [14]. While satellite cells are partly mitotically active in early postnatal skeletal muscles [15], their population shrinks during aging as their proliferation decreases [16], becoming quiescent in mature muscles [17]. However, even such dormant populations of myosatellites have the potential to become mitotically active during muscle repair following injuries [18]. Therefore, their proliferative capacity is partly maintained even during growth and aging. While telomerase activity has not been well studied in myoblasts and satellite cells, it was observed to be relatively high in immortalized mouse C2C12 muscle cell progenitor line [19]. Hence, this study aims at examining the activity of telomerase related genes during an attempted longterm (1 year) suspension culture, as well as compare them to its short-term (3 months) and plastic adherent culture counterparts. The ability to maintain such culture, as well as the maintenance of telomerase related gene expression could suggest that while the muscle satellite cells might lose a significant portion of their stemness due to the ex vivo conditions, they can still be successfully propagated for a relatively long periods of time in suspension cultures, which could bear significance for applications such as in vitro meat production.

Telomerase activity is a very important factor in continuous cell line production, as it prevents cell senescence and death. While most somatic cells do not exhibit telomerase activity, it has been reported in some tissues of certain animals. Satellite cells are muscle stem cells which show robust telomerase activity throughout adult life. However, their immediate primary myoblast progeny dramatically downregulates telomerase activity [3]. Despite that, it has been previously shown that turkey breast muscle satellite cells exhibit a certain extent of telomerase activity [14]. In this research, satellite cells were isolated and cultured as adherent primary myoblasts and suspended primary myoblasts to evaluate their ability to preserve their telomerase properties after a long-term culture. Results of this research indicated that there is telomerase activity in cultured turkey myoblasts, as telomerase bands were detected in those cells while rat myoblasts didn’t show any bands (Fig. 1). Furthermore, differentiation of myoblasts into myotubes was performed and telomerase activity was observed in myotubes (Fig. 2), which may be due to the presence of a population of non-differentiated cells, or remains of the previous high telomerase activity that could disappear after more passages. In the mature muscle fibers, there are quiescent satellite cells located in sarcolemma which can be induced to proliferate after injury [17]. Telomerase detection in differentiated myotubes is probably caused by the presence of quiescent satellite cells.

Furthermore, suspension cultures are being used increasingly in the biotechnology as they have the ability to be easily scaled up to produce therapeutic proteins or edible biomass [22]. In this research, to scale up myoblast cell culture, myoblasts were transferred placed in a suspension culture flasks and were cultured for a long-term (a year). After the culture period, telomerase activity was detected in the cells. As figure 3 shows, the telomerase bands were more similar to positive control after long-term suspension culture. Therefore, culture of myoblast cells in suspension had no adverse effect on telomerase activity, with long-term culture probably inducing more telomerase activity. As can be seen in Figure 4, the expression of genes related to telomerase activity and myogenesis in myotubes versus myoblast is indicating down regulation of telomerase related genes including TERT1, TERT2 and Dyskerin. It has been demonstrated that in somatic cells the expression level of TERT decreases after differentiation [12], with overexpression of this gene able to cause significant raise in telomerase activity [23]. Dyskerin is a nuclear protein that binds to the H/ACA box of telomerase RNA and forms ribonucleoprotein (RNP), with its dysfunction causing telomerase shortening [24]. In turn, figure 5 shows TERT1, TERT2, and Dyskerin expression in suspended cells compared to adherent cells. While upregulation of all those genes is seen, TERT1 and TERT2 expression was significantly higher than Dyskerin. In a research on immortalized human mammary epithelial cells, despite increased Dyskerin, overexpression of hTERT was observed. However, there was no correlation between hTERT expression or telomerase activity and Dyskerin expression level., A significant upregulation of TETR1 was detected compared to other genes in cells that has been cultured in suspension for long-term, compared to short-term (Fig. 6) indicating that long-term culture of myoblasts in suspension probably induces higher expression of TERT1 and telomerase activity.

Figure 5

Figure 6

Expression level of myogenic genes in myotubes has been different than in myoblasts. As it is shown in figure 4, MyoD, MRF4 and Myogenin were upregulated and Myf5 and PAX7 were downregulated in myotubes compared to myoblasts. Myogenic regulatory factors including Myf5, myogenin and MyoD are members of the helix-loop-helix family of transcription factors that control differentiation of skeletal muscle cells during prenatal and postnatal development [25]. As differentiation of myoblasts into myotubes occurs, the MyoD locus relocates to the lumen of the nucleus where the factors that promote MyoD expression are located [26]. In the next step, MyoD induces myogenic expression, which causes downregulation of Myf5 [27]. Paired box7 (Pax7) is one of the important factors in the regulation of differentiation and specification [28]. It has been shown that Pax7 inhibits differentiation of myoblasts by inhibiting Myod, resulting in myogenin activation [29,30].

All of the investigated genes related to myogenesis has been upregulated in suspended versus adherent cells. MyoD, Myf5 and Pax7 upregulation was significantly lower than that of Mrf4 and myogenin. Higher expression of myogenin probably indicates that the presence of MyoD in cells increases myogenin expression. While significant upregulation of myogenin indicates differentiation of myoblasts, significant upregulation of TERT1 in long-term versus short-term cultured myoblasts indicates high proliferation capacity of the myoblast cells in suspension. The data indicates that there should be both undifferentiated and differentiated myoblasts present in the suspension cell culture. It has been shown that ALDH1 and ALDH2 expression is a good marker of human myoblasts cells [31]. Presence of ALDH1 and ALDH2 in differentiated and undifferentiated cells indicates the presence of myoblasts cells in all culture systems that we were evaluated.

To our knowledge, this the first report on the establishment of a year-long myoblast cell culture in suspension, with the results indicating that such form of culture is possible and viable. However, improvement of cell culture conditions, such as the addition of growth factors to cell culture media to prevent differentiation of myoblasts, may help to obtain a better efficiency of large-scale in vitro cell production. In vitro meat manufacturing is a prospective and widely investigated potential source of human nutrition [32]. Hence, as the establishment of suspended muscle cell lines is a promising method for successful culture of those cells in large scale for meat production purposes, it could become a significant contributor to the development of this novel industry.