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The origin of phenotypic heterogeneity in a clonal cell population in vitro.

Stockholm D, Benchaouir R, Picot J, Rameau P, Neildez TM, Landini G, Laplace-Builhé C, Paldi A - PLoS ONE (2007)

Bottom Line: The key predictions of the two models were confronted with the results obtained experimentally using a myogenic cell line.The observations emphasize the importance of the "ecological" context and suggest that, consistently with the "extrinsic" model, local stochastic interactions between phenotypically identical cells play a key role in the initiation of phenotypic switch.Nevertheless, the "intrinsic" model also shows some other aspects of reality: The phenotypic switch is not triggered exclusively by the local environmental variations, but also depends to some extent on the phenotypic intrinsic robustness of the cells.

View Article: PubMed Central - PubMed

Affiliation: GENETHON-Centre National de la Recherche Scientifique (CNRS), UMR 8115, Evry, France.

ABSTRACT

Background: The spontaneous emergence of phenotypic heterogeneity in clonal populations of mammalian cells in vitro is a rule rather than an exception. We consider two simple, mutually non-exclusive models that explain the generation of diverse cell types in a homogeneous population. In the first model, the phenotypic switch is the consequence of extrinsic factors. Initially identical cells may become different because they encounter different local environments that induce adaptive responses. According to the second model, the phenotypic switch is intrinsic to the cells that may occur even in homogeneous environments.

Principal findings: We have investigated the "extrinsic" and the "intrinsic" mechanisms using computer simulations and experimentation. First, we simulated in silico the emergence of two cell types in a clonal cell population using a multiagent model. Both mechanisms produced stable phenotypic heterogeneity, but the distribution of the cell types was different. The "intrinsic" model predicted an even distribution of the rare phenotype cells, while in the "extrinsic" model these cells formed small clusters. The key predictions of the two models were confronted with the results obtained experimentally using a myogenic cell line.

Conclusions: The observations emphasize the importance of the "ecological" context and suggest that, consistently with the "extrinsic" model, local stochastic interactions between phenotypically identical cells play a key role in the initiation of phenotypic switch. Nevertheless, the "intrinsic" model also shows some other aspects of reality: The phenotypic switch is not triggered exclusively by the local environmental variations, but also depends to some extent on the phenotypic intrinsic robustness of the cells.

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Clonal population derived from a human primary myoblast.A: The cells were fixed and immunostained with an anti-desmin antibody. A series of 55 pictures were obtained and compiled into a single picture showing the whole population. The variation in the intensity of desmin immunostain is higher at the periphery of the growing population. The left panel shows 4 high resolution images of different parts of the population pointed by the blue arrows. B: Colour coded image of the same population as on the A panel. The colour code is based on the intensity of the pixels (red: low, green: high intensity). On the left are shown 2 high resolution images with the same color code. In the upper left, a region of interest is indicated in white with the corresponding pixel histogram shown beneath. Two additional histograms show the pixel intensities of two regions of interest: high-(green) and a low-desmin expressing cells (red). Note that the low desmin expressing cells are more frequent at the periphery of the culture.
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pone-0000394-g007: Clonal population derived from a human primary myoblast.A: The cells were fixed and immunostained with an anti-desmin antibody. A series of 55 pictures were obtained and compiled into a single picture showing the whole population. The variation in the intensity of desmin immunostain is higher at the periphery of the growing population. The left panel shows 4 high resolution images of different parts of the population pointed by the blue arrows. B: Colour coded image of the same population as on the A panel. The colour code is based on the intensity of the pixels (red: low, green: high intensity). On the left are shown 2 high resolution images with the same color code. In the upper left, a region of interest is indicated in white with the corresponding pixel histogram shown beneath. Two additional histograms show the pixel intensities of two regions of interest: high-(green) and a low-desmin expressing cells (red). Note that the low desmin expressing cells are more frequent at the periphery of the culture.

Mentions: In order to clarify whether the non-random distribution of the rare phenotype cells is observed only in the C2C12 line or whether it is a general feature, we analysed clonal populations of primary human myoblasts. These were obtained by cloning of individual cells from a primary myoblast (muscle biopsy) culture. The cultures were allowed to grow to several hundred cells, fixed and immunostained with an anti-desmin antibody (a muscle-specific intermediate filament protein and one of the earliest markers of activated muscle precursor cells). Desmin is present in all myoblasts, but its expression level depends on the degree of commitment of the cell to the myogenic differentiation path [13]. This marker is frequently used to isolate pure myoblast populations [14]. A typical isogenic population is shown on Fig 7. Although no accurate quantitative measures were performed, it is obvious that the level of the desmin protein was highly variable within the cluture. Cells with the lowest expression levels (presumed to be less engaged in differentiation) were observed preferentially on the periphery. These observations show that phenotypic heterogeneity can also emerge spontaneously in a clonal population of primary cells and the uneven distribution of the different cells is reminiscent of the results obtained using the C2C12 cell line.


The origin of phenotypic heterogeneity in a clonal cell population in vitro.

Stockholm D, Benchaouir R, Picot J, Rameau P, Neildez TM, Landini G, Laplace-Builhé C, Paldi A - PLoS ONE (2007)

Clonal population derived from a human primary myoblast.A: The cells were fixed and immunostained with an anti-desmin antibody. A series of 55 pictures were obtained and compiled into a single picture showing the whole population. The variation in the intensity of desmin immunostain is higher at the periphery of the growing population. The left panel shows 4 high resolution images of different parts of the population pointed by the blue arrows. B: Colour coded image of the same population as on the A panel. The colour code is based on the intensity of the pixels (red: low, green: high intensity). On the left are shown 2 high resolution images with the same color code. In the upper left, a region of interest is indicated in white with the corresponding pixel histogram shown beneath. Two additional histograms show the pixel intensities of two regions of interest: high-(green) and a low-desmin expressing cells (red). Note that the low desmin expressing cells are more frequent at the periphery of the culture.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC1851097&req=5

pone-0000394-g007: Clonal population derived from a human primary myoblast.A: The cells were fixed and immunostained with an anti-desmin antibody. A series of 55 pictures were obtained and compiled into a single picture showing the whole population. The variation in the intensity of desmin immunostain is higher at the periphery of the growing population. The left panel shows 4 high resolution images of different parts of the population pointed by the blue arrows. B: Colour coded image of the same population as on the A panel. The colour code is based on the intensity of the pixels (red: low, green: high intensity). On the left are shown 2 high resolution images with the same color code. In the upper left, a region of interest is indicated in white with the corresponding pixel histogram shown beneath. Two additional histograms show the pixel intensities of two regions of interest: high-(green) and a low-desmin expressing cells (red). Note that the low desmin expressing cells are more frequent at the periphery of the culture.
Mentions: In order to clarify whether the non-random distribution of the rare phenotype cells is observed only in the C2C12 line or whether it is a general feature, we analysed clonal populations of primary human myoblasts. These were obtained by cloning of individual cells from a primary myoblast (muscle biopsy) culture. The cultures were allowed to grow to several hundred cells, fixed and immunostained with an anti-desmin antibody (a muscle-specific intermediate filament protein and one of the earliest markers of activated muscle precursor cells). Desmin is present in all myoblasts, but its expression level depends on the degree of commitment of the cell to the myogenic differentiation path [13]. This marker is frequently used to isolate pure myoblast populations [14]. A typical isogenic population is shown on Fig 7. Although no accurate quantitative measures were performed, it is obvious that the level of the desmin protein was highly variable within the cluture. Cells with the lowest expression levels (presumed to be less engaged in differentiation) were observed preferentially on the periphery. These observations show that phenotypic heterogeneity can also emerge spontaneously in a clonal population of primary cells and the uneven distribution of the different cells is reminiscent of the results obtained using the C2C12 cell line.

Bottom Line: The key predictions of the two models were confronted with the results obtained experimentally using a myogenic cell line.The observations emphasize the importance of the "ecological" context and suggest that, consistently with the "extrinsic" model, local stochastic interactions between phenotypically identical cells play a key role in the initiation of phenotypic switch.Nevertheless, the "intrinsic" model also shows some other aspects of reality: The phenotypic switch is not triggered exclusively by the local environmental variations, but also depends to some extent on the phenotypic intrinsic robustness of the cells.

View Article: PubMed Central - PubMed

Affiliation: GENETHON-Centre National de la Recherche Scientifique (CNRS), UMR 8115, Evry, France.

ABSTRACT

Background: The spontaneous emergence of phenotypic heterogeneity in clonal populations of mammalian cells in vitro is a rule rather than an exception. We consider two simple, mutually non-exclusive models that explain the generation of diverse cell types in a homogeneous population. In the first model, the phenotypic switch is the consequence of extrinsic factors. Initially identical cells may become different because they encounter different local environments that induce adaptive responses. According to the second model, the phenotypic switch is intrinsic to the cells that may occur even in homogeneous environments.

Principal findings: We have investigated the "extrinsic" and the "intrinsic" mechanisms using computer simulations and experimentation. First, we simulated in silico the emergence of two cell types in a clonal cell population using a multiagent model. Both mechanisms produced stable phenotypic heterogeneity, but the distribution of the cell types was different. The "intrinsic" model predicted an even distribution of the rare phenotype cells, while in the "extrinsic" model these cells formed small clusters. The key predictions of the two models were confronted with the results obtained experimentally using a myogenic cell line.

Conclusions: The observations emphasize the importance of the "ecological" context and suggest that, consistently with the "extrinsic" model, local stochastic interactions between phenotypically identical cells play a key role in the initiation of phenotypic switch. Nevertheless, the "intrinsic" model also shows some other aspects of reality: The phenotypic switch is not triggered exclusively by the local environmental variations, but also depends to some extent on the phenotypic intrinsic robustness of the cells.

Show MeSH
Related in: MedlinePlus