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Variation in neuronal differentiation of a newly isolated mouse embryonic stem cell line: a detailed immunocytochemistry study.

Tavares RL, Cortes PA, de Azevedo CI, Cangussú SD, Camargos AF, Arantes RM - Cell Biol Int Rep (2010) (2012)

Bottom Line: The first glial cell type appears in stage III.Variations among other literature findings can be explained by the choice we made to use a newly isolated ES cell line.As colonies may behave differently during neuronal differentiation, it reinforces the necessity of studying original ES cell lines.

View Article: PubMed Central - PubMed

Affiliation: The Professor Aroldo Fernando Camargos Laboratory of Human Reproduction, The Teaching Hospital of the Federal University of Minas Gerais, Belo Horizonte, Minas Gerais, Brazil.

ABSTRACT
Neural precursor differentiation from mouse ES (embryonic stem) cells have been demonstrated using EB (embryoid body), co-culture on stromal feeder layers, and in the absence of external inducing signals. Most of available mouse ES cell original research articles have worked with only six different cell lines. Our goals were to isolate one new mouse ES lineage, and perform a detailed immunocytochemistry study during neural differentiation, making use of an EB strategy protocol following the generation of neural progenitors, glial cells and postmitotic neurons. The dynamics of differentiation of ES cell derived neuronal precursors into differentiated glia cells and neurons were followed in vitro and correlated to exposure to specific elements of feeder medium. Morphological aspects of generated cellular types, including its immunocytochemical expression of differentiation markers were studied. Immuno-positivity against β-III tubulin, PGP and TH (tyrosine hydroxylase) was observed from stage I. Approximately 80% of cells were positive for TH at stage I. The first glial cell type appears in stage III. TH, PGP or β-III tubulin-positive cells with neuronal typical morphology only being seen in stage III when TH-positive cells corresponded to approximately 12% of total cells. Variations among other literature findings can be explained by the choice we made to use a newly isolated ES cell line. As colonies may behave differently during neuronal differentiation, it reinforces the necessity of studying original ES cell lines.

No MeSH data available.


Related in: MedlinePlus

Percentage of positive cells stained by phenotypic markers of undifferentiation (SSEA-1, nestin) and cell differentiation (β-III tubulin, PGP and TH) at each stage of our protocol*Indicates statistically significant differences from stage I to the others (P<0.001).
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Figure 2: Percentage of positive cells stained by phenotypic markers of undifferentiation (SSEA-1, nestin) and cell differentiation (β-III tubulin, PGP and TH) at each stage of our protocol*Indicates statistically significant differences from stage I to the others (P<0.001).

Mentions: Stage I was positive for SSEA-1 and Oct-4. At stage III, neuroespheres presented cells strongly stained by PGP exhibiting neuronal soma and some neurites. The number of these cells was increased further at stages IV and V. GFAP and TH were expressed from stages III–V. The percentages of TH+ cells in subsequent stages (I, III–V) of differentiation derived from 3 separate experiments are illustrated in Figure 1(G). The percentage of positive cells stained by phenotypic markers of undifferentiation (SSEA-1) and cell differentiation (β-III tubulin, PGP and TH) were determined at each stage of our protocol and all results are expressed in Figure 2. All markers decreased significantly from stage I to the other stages (P<0.0001). SSEA-1 and Nestin decreased significantly from stages III–IV to IV–V (P<0.0001). PGP and tubulin immunopositive cells percentage did not vary between stages III and V (Figure 2).


Variation in neuronal differentiation of a newly isolated mouse embryonic stem cell line: a detailed immunocytochemistry study.

Tavares RL, Cortes PA, de Azevedo CI, Cangussú SD, Camargos AF, Arantes RM - Cell Biol Int Rep (2010) (2012)

Percentage of positive cells stained by phenotypic markers of undifferentiation (SSEA-1, nestin) and cell differentiation (β-III tubulin, PGP and TH) at each stage of our protocol*Indicates statistically significant differences from stage I to the others (P<0.001).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3475445&req=5

Figure 2: Percentage of positive cells stained by phenotypic markers of undifferentiation (SSEA-1, nestin) and cell differentiation (β-III tubulin, PGP and TH) at each stage of our protocol*Indicates statistically significant differences from stage I to the others (P<0.001).
Mentions: Stage I was positive for SSEA-1 and Oct-4. At stage III, neuroespheres presented cells strongly stained by PGP exhibiting neuronal soma and some neurites. The number of these cells was increased further at stages IV and V. GFAP and TH were expressed from stages III–V. The percentages of TH+ cells in subsequent stages (I, III–V) of differentiation derived from 3 separate experiments are illustrated in Figure 1(G). The percentage of positive cells stained by phenotypic markers of undifferentiation (SSEA-1) and cell differentiation (β-III tubulin, PGP and TH) were determined at each stage of our protocol and all results are expressed in Figure 2. All markers decreased significantly from stage I to the other stages (P<0.0001). SSEA-1 and Nestin decreased significantly from stages III–IV to IV–V (P<0.0001). PGP and tubulin immunopositive cells percentage did not vary between stages III and V (Figure 2).

Bottom Line: The first glial cell type appears in stage III.Variations among other literature findings can be explained by the choice we made to use a newly isolated ES cell line.As colonies may behave differently during neuronal differentiation, it reinforces the necessity of studying original ES cell lines.

View Article: PubMed Central - PubMed

Affiliation: The Professor Aroldo Fernando Camargos Laboratory of Human Reproduction, The Teaching Hospital of the Federal University of Minas Gerais, Belo Horizonte, Minas Gerais, Brazil.

ABSTRACT
Neural precursor differentiation from mouse ES (embryonic stem) cells have been demonstrated using EB (embryoid body), co-culture on stromal feeder layers, and in the absence of external inducing signals. Most of available mouse ES cell original research articles have worked with only six different cell lines. Our goals were to isolate one new mouse ES lineage, and perform a detailed immunocytochemistry study during neural differentiation, making use of an EB strategy protocol following the generation of neural progenitors, glial cells and postmitotic neurons. The dynamics of differentiation of ES cell derived neuronal precursors into differentiated glia cells and neurons were followed in vitro and correlated to exposure to specific elements of feeder medium. Morphological aspects of generated cellular types, including its immunocytochemical expression of differentiation markers were studied. Immuno-positivity against β-III tubulin, PGP and TH (tyrosine hydroxylase) was observed from stage I. Approximately 80% of cells were positive for TH at stage I. The first glial cell type appears in stage III. TH, PGP or β-III tubulin-positive cells with neuronal typical morphology only being seen in stage III when TH-positive cells corresponded to approximately 12% of total cells. Variations among other literature findings can be explained by the choice we made to use a newly isolated ES cell line. As colonies may behave differently during neuronal differentiation, it reinforces the necessity of studying original ES cell lines.

No MeSH data available.


Related in: MedlinePlus