Dedifferentiation of adult human myoblasts induced by ciliary neurotrophic factor in vitro

Abstract

Ciliary neurotrophic factor (CNTF) is primarily known for its important cellular effects within the nervous system. However, recent studies indicate that its receptor can be highly expressed in denervated skeletal muscle. Here, we investigated the direct effect of CNTF on skeletal myoblasts of adult human. Surprisingly, we found that CNTF induced the myogenic lineage-committed myoblasts at a clonal level to dedifferentiate into multipotent progenitor cells--they not only could proliferate for over 20 passages with the expression absence of myogenic specific factors Myf5 and MyoD, but they were also capable of differentiating into new phenotypes, mainly neurons, glial cells, smooth muscle cells, and adipocytes. These "progenitor cells" retained their myogenic memory and were capable of redifferentiating into myotubes. Furthermore, CNTF could activate the p44/p42 MAPK and down-regulate the expression of myogenic regulatory factors (MRFs). Finally, PD98059, a specific inhibitor of p44/p42 MAPK pathway, was able to abolish the effects of CNTF on both myoblast fate and MRF expression. Our results demonstrate the myogenic lineage-committed human myoblasts can dedifferentiate at a clonal level and CNTF is a novel regulator of skeletal myoblast dedifferentiation via p44/p42 MAPK pathway.

Figure 1.

Myogenic lineage-committed clones of individual myoblasts. (A) Phase micrographs of clonal myoblasts (MBs), NIH3T3 (3T3), and mesenchymal stem cells (MSCs) cultured in myoblast GM (MB GM) for 24 h or in myoblast DM (MB DM) for 72 h. Immunocytochemistry assay of these cells cultured in the GM or DM by using the anti-MyoD (MyoD) and anti-Myosin (Myosin) antibodies. (B) Immunocytochemistry assay of clonal myoblasts (MBs) and its positive control (Con) NSC and HSC by using anti-Nestin, anti-CD34, and anti-CD45 antibodies. This data presented are representative of five clones with similar results. (The myoblasts are randomly selected from 3 to 25 passage clones of the individual Desmin⁺ myoblasts.)

Figure 2.

Exogenous CNTF inhibits myoblast differentiation and induces a novel cell population in vitro. (a and b) Phase micrographs of myoblasts treated with (CNTF) or without (Control) 30 ng/ml CNTF in DM for 72 h. The data presented are representative of the experiments that are treated with 30–50 ng/ml CNTF with similar results. (c) The differentiation extent of myoblasts in a and b is expressed as fusion index indicating a percentage of the number of nuclei in the fused cells among the total number of nuclei in five randomly chosen fields at 200× magnification. Cells containing more than three nuclei are regarded as fused cells. (d) The number of spherical cells is expressed as a percentage of the number of nuclei in the spherical cells among the total number of nuclei in five randomly chosen fields at 200× magnification. Nestin⁺ cells are regarded as spherical cells in a and b. (e) Western blot analysis by using the anti-Myf5, -MyoD, and -myogenin antibodies. Extracts are prepared from the cells as described in a and b, and 100 μg of each extract is separated by SDS-PAGE and transferred to nitrocellulose. The data presented are representative of three separate experiments with similar results. (f) The intensities of the protein bands in e are quantified by densitometry with ScionImage software. *p < 0.05.

Figure 3.

Dedifferentiated myoblast clones. (A) Phase micrographs of individual MBSC isolated by limiting dilution (a). Individual MBSC clone proliferated in 48-well uncoated tissue-culture plates for 5 wk in suspension (b). The clone is further expanded in 25-cm² uncoated tissue-culture flasks to grow as spheres in suspension over the course of 8 wk (c). (B) Western blot analysis using anti-Myf5, -MyoD, and -myogenin antibodies. Extracts were prepared from MBPCs, from the positive control myotubes (MT) of the myoblasts cultured in DM for 7 d, and from the negative control NIH3T3 (3T3). One hundred micrograms of each extract was separated by SDS-PAGE and transferred to nitrocellulose. The data presented are representative of three separate experiments with similar results. (C) The intensities of the protein bands in B are quantified by densitometry with ScionImage software. (D) Semiquantitative RT-PCR analysis of Myf5 and MyoD expression in MBPCs. Total RNA was obtained from MBPCs*, from the negative control NIH3T3 (3T3), and from the positive control myotubes (MT) of the myoblasts cultured in DM for 7 d. The data presented are representative of three separate experiments with similar results. PCR products were checked by 1% agarose-TBE-ethidium bromide gels after 35 cycles. M, 100-bp molecular size marker. (The MBPCs were randomly selected from 3 to 20 passage clones of the individual MBPCs.)

Figure 4.

Molecular characterization of MBPCs. (A) Immunocytochemistry assay of MBPCs, and its control (Con) NSCs (NSC), NIH3T3 (3T3), myoblasts (MB) and HSCs (HSC) by using the anti-Vimentin, -Nestin, -Myf5, -M-cadherin, -CD34 and -CD45 antibodies. Cells are counterstained with Hoechst 33342 (blue) to show nuclei. (B) Phase micrographs of MBPCs in GM0 (a) and in NSC growth medium (b). The adipogenic potency of MBPCs (c) and NSCs (d) under the same conditions of adipogenic differentiation to MBPCs, and their cytochemistry stain by Oil Red O in cultures of c (e) and d (f).

Figure 5.

MBPCs show signs of multipotency. Immunocytochemistry assay of MBPCs, and its control myoblasts (MBs) by using (A) the specific makers of neurons: anti-NF160, TH, NSE, and chAT antibodies; (B) the specific glial makers: anti-CNPase and -GFAP antibodies and (C) the anti-SMA and -Myosin antibodies. Cells were counterstained with Hoechst 33342 (blue) to show nuclei. In addition, (C) MBPCs and MBs are stained by the lipophilic dyes, Oil Red O. (D) The percentage of cells producing a given marker protein in A–C is expressed as positive cell number indicating a percentage of the number of nuclei in the cells stained a given marker among the total number of nuclei in five randomly chosen fields at 200× magnification.

Figure 6.

p44/p42 MAPK-mediated CNTF effects. (A) Western blot assays using anti-phospho (pho-MAPK) or anti-nonphospho (MAPK)-p44/p42 MAPK antibodies. Extracts are prepared from the cells after a treatment without (Con) or with 30 ng/ml CNTF in the absence (30) or presence (30+PD) of 20 μM PD98059 at the time indicated. The graph presented is representative of three separate experiments with similar results. (B) Phase micrographs of myoblasts treated without (Con) or with 30 ng/ml CNTF in the absence (30) or presence (30+PD) of 20 μM PD98059 in DM for 72 h. (C) The differentiation extent of myoblasts in B is expressed as fusion index indicating a percentage of the number of nuclei in the fused cells among the total number of nuclei in five randomly chosen fields at 200× magnification. Cells containing more than three nuclei were regarded as fused cells. (D) The number of spherical cells is expressed as a percentage of the number of nuclei in the spherical cells among the total number of nuclei in five randomly chosen fields at 200× magnification. Nestin⁺ cells are regarded as spherical cells in B. (E) Western blot analysis by using the anti-Myf5, -MyoD, and -myogenin antibodies. Extracts were prepared from the cells as described in B, and 100 μg of each extract was separated by SDS-PAGE and transferred to nitrocellulose. The data presented are representative of three separate experiments with similar results. (F) The intensities of the protein bands in E are quantified by densitometry with ScionImage software. *p < 0.05.