Study Of Ciliary Neurotrophic Factor Via ERK1/2 Pathway During Myogenic Differentiation Of Adult Human Myoblasts In Vitro

Myoblasts are a derivative of the somite and exhibit a remarkable capacity for self-renewal. They have been long considered a distinct myogenic lineage responsible for postnatal growth, repair, and maintenance of skeletal muscle. The myoblasts used as autologous"seed cells"and its potential therapeutic implication has prompted new investigations into finding the novel regulators of the proliferation and differentiation of myoblasts in vitro. Ciliary neurotrophic factor (CNTF) has been well known to play a tropic, survival and protective role in a variety of neuronal cell types. Furthermore, it is also able to regulate the proliferation and differentiation of these neurons. Recently, its trophic effects on muscular tissues in vivo have been documented. However, because CNTF was primarily characterized by its ability to sustain the survival of motoneurons in vivo and in vitro, the documented experiment results in vivo may be related to a possible effect of the CNTF on the neuron activity and/or directly on the myoblasts.In the present study, we used a modified procedure to rapidly isolate and harvest more myoblasts derived from skeletal muscle of adult human, and then cloned the myoblasts by limiting dilution to obtain clones of individual myoblasts. These clones could divide for more than 25 passages with an average division time of 23.6±4.8h, and preserve their normal phenotypes. The myoblasts in these clones are homogeneous by morphological criteria and immolunocytochemistry identification. Thus, it is an excellent culture system to study the effect of exogenous factors on myoblasts in vitro. All the experiments described here were performed on these clones of individual myoblasts.In order to determine whether CNTF may be involved in regulating the myoblast differentiation, we directly added recombinant human CNTF to the subconfluent myoblasts cultured in the low serum culture medium, and observed the effect of CNTF on myoblast differentiation in vitro and the role of p42/p44-MAPK pathway in the mechanism. Our results demonstrate the exogenous CNTF (≤10ng/mL) can inhibit myogenic differentiation of myoblasts and the inhibition is dose-dependent and reversible. The CNTF-induced undifferentiated myoblasts are similar to the defined"reserve cells"at morphological and molecular level. Furthermore, PD098059, an inhibitor of p42/p44-MAPK pathway, can rescue the CNTF-induced inhibition of myoblast differentiation. Here, we for the first time proved that the recombinant human CNTF could inhibit the myogenic differentiation of myoblasts by increases"reserve cells"through p42/p44-MAPK pathway, and suggested that the recombinant human CNTF may be a novel negative regulator of myogenic differentiation of myoblasts of skeletal muscle of adult human in vitro. The fact that recombinant human CNTF could function to increase the pool of"reserve cells"during terminal differentiation of the myoblasts has important implications in autologous therapy of muscular injury and dystrophy.Collectedly, in the present study, we used a modified procedure to rapidly isolate and harvest more myoblasts derived from skeletal muscle of adult human, and provide an excellent culture system of clones from the individual myoblast to study the effect of exogenous factors on myoblasts in vitro. The most important dedications in the study are for the first time to demonstrate that the lower concentration of recombinant human CNTF could be a novel negative regulator of the myoblast differentiation of skeletal muscle of adult human in vitro and function to increase the pool of"reserve cells"; For the first time to reveal that the recombinant human CNTF through ERK1/2 pathway during myogenic differentiation of adult human myoblasts in vitro. These findings have important implications in further investigation of the mechanism of myoblast differentiation and autologous therapy of degenerative diseases, such as Parkinson's disease.