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A differential response to newt regeneration extract by C2C12 and primary mammalian muscle cells.

Kawesa S, Vanstone J, Tsilfidis C - Skelet Muscle (2015)

Bottom Line: We isolated extract from early newt forelimb regenerates and assessed its effects on differentiation of proliferating primary and C2C12 myoblasts.We have confirmed the results obtained in C2C12 cells and expanded these studies to also examine the effects of newt regeneration extracts on primary muscle cells.However, unlike C2C12 cells, primary muscle cells do not re-enter the cell cycle in response to treatment with newt extracts.

View Article: PubMed Central - PubMed

Affiliation: Ottawa Hospital Research Institute, Vision Research/Regenerative Medicine Program, 501 Smyth Road, Box 307, Ottawa, Ontario K1H 8L6 Canada ; Department of Cellular and Molecular Medicine, University of Ottawa, 451 Smyth Road, Ottawa, Ontario K1H 8M5 Canada.

ABSTRACT

Background: Dedifferentiation, a process whereby differentiated cells lose their specialized characteristics and revert to a less differentiated state, plays a key role in the regeneration process in urodele amphibians such as the red spotted newt, Notophthalmus viridescens. Dedifferentiation of fully mature tissues is generally absent in mammalian cells. Previous studies have shown that mouse C2C12 multinucleated myotubes treated with extract derived from regenerating newt forelimbs can re-enter the cell cycle, fragment into mononucleated cells, and proliferate. However, this response has been difficult to replicate.

Methods: We isolated extract from early newt forelimb regenerates and assessed its effects on differentiation of proliferating primary and C2C12 myoblasts. We also treated fully differentiated primary and C2C12 myotube cultures with extract and assessed cell cycle re-entry and myotube fragmentation.

Results: We have confirmed the results obtained in C2C12 cells and expanded these studies to also examine the effects of newt regeneration extracts on primary muscle cells. Newt extract can block differentiation of both C2C12 and primary myoblasts. Once differentiation is induced, treatment with newt extract causes cell cycle re-entry and fragmentation of C2C12 myotubes. Downregulation of p21 and muscle-specific markers is also induced. Primary myotubes also fragment in response to extract treatment, and the fragmented cells remain viable for long periods of time in culture. However, unlike C2C12 cells, primary muscle cells do not re-enter the cell cycle in response to treatment with newt extracts.

Conclusions: Dedifferentiation of fully mature muscle occurs during regeneration in the newt forelimb to contribute cells to the regeneration process. Our study shows that extracts derived from regenerating newt forelimbs can induce dedifferentiation, cell cycle re-entry, and fragmentation of mouse C2C12 cells but can only induce fragmentation in primary muscle cells.

No MeSH data available.


Related in: MedlinePlus

Inhibition of muscle differentiation in C2C12 cells treated with newt extract. a C2C12 myoblasts treated with 0.3 mg/ml of newt extract in either differentiation medium (DM) or growth medium (GM) showed impaired differentiation and reduced numbers of MHC-positive cells. b The percentage of MHC-positive cells was calculated by adding all MHC-positive cells (myotubes and myocytes) and dividing by the total number of cells. The percentage of MHC-positive cells was reduced in extract-treated cells in both DM and GM. Scale bar = 200 μm. *p < 0.0001
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Fig1: Inhibition of muscle differentiation in C2C12 cells treated with newt extract. a C2C12 myoblasts treated with 0.3 mg/ml of newt extract in either differentiation medium (DM) or growth medium (GM) showed impaired differentiation and reduced numbers of MHC-positive cells. b The percentage of MHC-positive cells was calculated by adding all MHC-positive cells (myotubes and myocytes) and dividing by the total number of cells. The percentage of MHC-positive cells was reduced in extract-treated cells in both DM and GM. Scale bar = 200 μm. *p < 0.0001

Mentions: Myogenic differentiation is typically studied in vitro by the fusion of myoblasts into multinucleated myotubes. To examine the effect of newt extract on myoblast differentiation, the newt extract at a concentration 0.3 mg/ml was added to subconfluent C2C12 myoblasts, and when these cultures reached confluence, they were transferred to differentiation medium (DM) or GM for 4 days; then the cells were analyzed for their ability to undergo myogenic differentiation. When confluent myoblasts were cultured without newt extract in both DM and GM for 4 days, the cells underwent differentiation to form multinucleated myotubes (Fig. 1a). Cells grown in differentiation medium formed larger and more numerous myotubes, as might be expected, but even in growth medium, confluent myoblasts exited the cell cycle and formed myosin heavy chain (MHC)-positive myotubes. In contrast, the newt extract-treated cells formed fewer multinucleated myotubes than the control (Fig. 1a) and they were much smaller in size. A significant decrease in the expression of myosin heavy chain (MHC) was observed in the extract-treated culture compared to the control in GM and DM (Fig. 1b). This suggests that newt extract has effects on myoblast differentiation and on myoblast fusion into multinucleated C2C12 myotubes.Fig. 1


A differential response to newt regeneration extract by C2C12 and primary mammalian muscle cells.

Kawesa S, Vanstone J, Tsilfidis C - Skelet Muscle (2015)

Inhibition of muscle differentiation in C2C12 cells treated with newt extract. a C2C12 myoblasts treated with 0.3 mg/ml of newt extract in either differentiation medium (DM) or growth medium (GM) showed impaired differentiation and reduced numbers of MHC-positive cells. b The percentage of MHC-positive cells was calculated by adding all MHC-positive cells (myotubes and myocytes) and dividing by the total number of cells. The percentage of MHC-positive cells was reduced in extract-treated cells in both DM and GM. Scale bar = 200 μm. *p < 0.0001
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4471912&req=5

Fig1: Inhibition of muscle differentiation in C2C12 cells treated with newt extract. a C2C12 myoblasts treated with 0.3 mg/ml of newt extract in either differentiation medium (DM) or growth medium (GM) showed impaired differentiation and reduced numbers of MHC-positive cells. b The percentage of MHC-positive cells was calculated by adding all MHC-positive cells (myotubes and myocytes) and dividing by the total number of cells. The percentage of MHC-positive cells was reduced in extract-treated cells in both DM and GM. Scale bar = 200 μm. *p < 0.0001
Mentions: Myogenic differentiation is typically studied in vitro by the fusion of myoblasts into multinucleated myotubes. To examine the effect of newt extract on myoblast differentiation, the newt extract at a concentration 0.3 mg/ml was added to subconfluent C2C12 myoblasts, and when these cultures reached confluence, they were transferred to differentiation medium (DM) or GM for 4 days; then the cells were analyzed for their ability to undergo myogenic differentiation. When confluent myoblasts were cultured without newt extract in both DM and GM for 4 days, the cells underwent differentiation to form multinucleated myotubes (Fig. 1a). Cells grown in differentiation medium formed larger and more numerous myotubes, as might be expected, but even in growth medium, confluent myoblasts exited the cell cycle and formed myosin heavy chain (MHC)-positive myotubes. In contrast, the newt extract-treated cells formed fewer multinucleated myotubes than the control (Fig. 1a) and they were much smaller in size. A significant decrease in the expression of myosin heavy chain (MHC) was observed in the extract-treated culture compared to the control in GM and DM (Fig. 1b). This suggests that newt extract has effects on myoblast differentiation and on myoblast fusion into multinucleated C2C12 myotubes.Fig. 1

Bottom Line: We isolated extract from early newt forelimb regenerates and assessed its effects on differentiation of proliferating primary and C2C12 myoblasts.We have confirmed the results obtained in C2C12 cells and expanded these studies to also examine the effects of newt regeneration extracts on primary muscle cells.However, unlike C2C12 cells, primary muscle cells do not re-enter the cell cycle in response to treatment with newt extracts.

View Article: PubMed Central - PubMed

Affiliation: Ottawa Hospital Research Institute, Vision Research/Regenerative Medicine Program, 501 Smyth Road, Box 307, Ottawa, Ontario K1H 8L6 Canada ; Department of Cellular and Molecular Medicine, University of Ottawa, 451 Smyth Road, Ottawa, Ontario K1H 8M5 Canada.

ABSTRACT

Background: Dedifferentiation, a process whereby differentiated cells lose their specialized characteristics and revert to a less differentiated state, plays a key role in the regeneration process in urodele amphibians such as the red spotted newt, Notophthalmus viridescens. Dedifferentiation of fully mature tissues is generally absent in mammalian cells. Previous studies have shown that mouse C2C12 multinucleated myotubes treated with extract derived from regenerating newt forelimbs can re-enter the cell cycle, fragment into mononucleated cells, and proliferate. However, this response has been difficult to replicate.

Methods: We isolated extract from early newt forelimb regenerates and assessed its effects on differentiation of proliferating primary and C2C12 myoblasts. We also treated fully differentiated primary and C2C12 myotube cultures with extract and assessed cell cycle re-entry and myotube fragmentation.

Results: We have confirmed the results obtained in C2C12 cells and expanded these studies to also examine the effects of newt regeneration extracts on primary muscle cells. Newt extract can block differentiation of both C2C12 and primary myoblasts. Once differentiation is induced, treatment with newt extract causes cell cycle re-entry and fragmentation of C2C12 myotubes. Downregulation of p21 and muscle-specific markers is also induced. Primary myotubes also fragment in response to extract treatment, and the fragmented cells remain viable for long periods of time in culture. However, unlike C2C12 cells, primary muscle cells do not re-enter the cell cycle in response to treatment with newt extracts.

Conclusions: Dedifferentiation of fully mature muscle occurs during regeneration in the newt forelimb to contribute cells to the regeneration process. Our study shows that extracts derived from regenerating newt forelimbs can induce dedifferentiation, cell cycle re-entry, and fragmentation of mouse C2C12 cells but can only induce fragmentation in primary muscle cells.

No MeSH data available.


Related in: MedlinePlus