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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

Analysis of morphology and marker expression in mouse ES derived progenitor cells(A) SSEA-1 immunostaining of stage I cells counterstained by Hoechst. (B) RT–PCR analysis of cells at stage I for Nanog and Oct4 genes. Panels 5, 6 and 7 show 2% agarose gels stained with ethidium bromide for the 5th, 6th and 7th passages, respectively; (C) is a positive control with one zona pellucida free blastocyst. (C) Phase contrast micrograph of EB at Stage II. (D) Phase contrast micrograph of a sphere cultured in serum-free medium at Stage III. (E) GFAP indirect immunofluorescence of accessory cell at stage III. (F) PGP immunopositive ‘rosette’ (white arrows) at stage IV. (G) Percentage of TH+ cells in subsequent stages (I, III–V) of differentiation derived from three independent experiments; ***P<0.0001 by Friedman test and Dunn post-test. (H) Increased complexity of neurites immunostained for β-III tubulin. Scale bars = 20 μm (A, D, E, F, H), 200 μm (C).
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Figure 1: Analysis of morphology and marker expression in mouse ES derived progenitor cells(A) SSEA-1 immunostaining of stage I cells counterstained by Hoechst. (B) RT–PCR analysis of cells at stage I for Nanog and Oct4 genes. Panels 5, 6 and 7 show 2% agarose gels stained with ethidium bromide for the 5th, 6th and 7th passages, respectively; (C) is a positive control with one zona pellucida free blastocyst. (C) Phase contrast micrograph of EB at Stage II. (D) Phase contrast micrograph of a sphere cultured in serum-free medium at Stage III. (E) GFAP indirect immunofluorescence of accessory cell at stage III. (F) PGP immunopositive ‘rosette’ (white arrows) at stage IV. (G) Percentage of TH+ cells in subsequent stages (I, III–V) of differentiation derived from three independent experiments; ***P<0.0001 by Friedman test and Dunn post-test. (H) Increased complexity of neurites immunostained for β-III tubulin. Scale bars = 20 μm (A, D, E, F, H), 200 μm (C).

Mentions: Undifferentiated ES cells exhibited the expression for SSEA-1 (75%, n = 1000 cells) (Figure 1A), in correspondence with RT–PCR for Nanog and Oct-4 (Figure 1B). At stages I and II, small and sparse PGP immunoreactive cells were found, mostly located inside conglomerates. These cells did not express TH and GFAP. At this point also nestin-positive cells were present at large amounts (78%, n = 1000 cells). Cultured stage I cells proliferated and formed large and spherical floating aggregates known as EB (Figure 1C). Almost half of plated cells adhered to the bottom and consistently expressed PGP by immunocytochemistry despite being TH-negative (data not shown). The cell clusters derived from modified EB are called neurospheres and form a network that becomes more evident in subsequent stages (Figure 1D). At the end of 6 days of cultivation in this medium, many extensions of cytoplasm of individual cells are highly visible, and connecting EB are often supported by a flat monolayer of mostly GFAP-positive cells (Figure 1E).


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)

Analysis of morphology and marker expression in mouse ES derived progenitor cells(A) SSEA-1 immunostaining of stage I cells counterstained by Hoechst. (B) RT–PCR analysis of cells at stage I for Nanog and Oct4 genes. Panels 5, 6 and 7 show 2% agarose gels stained with ethidium bromide for the 5th, 6th and 7th passages, respectively; (C) is a positive control with one zona pellucida free blastocyst. (C) Phase contrast micrograph of EB at Stage II. (D) Phase contrast micrograph of a sphere cultured in serum-free medium at Stage III. (E) GFAP indirect immunofluorescence of accessory cell at stage III. (F) PGP immunopositive ‘rosette’ (white arrows) at stage IV. (G) Percentage of TH+ cells in subsequent stages (I, III–V) of differentiation derived from three independent experiments; ***P<0.0001 by Friedman test and Dunn post-test. (H) Increased complexity of neurites immunostained for β-III tubulin. Scale bars = 20 μm (A, D, E, F, H), 200 μm (C).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Analysis of morphology and marker expression in mouse ES derived progenitor cells(A) SSEA-1 immunostaining of stage I cells counterstained by Hoechst. (B) RT–PCR analysis of cells at stage I for Nanog and Oct4 genes. Panels 5, 6 and 7 show 2% agarose gels stained with ethidium bromide for the 5th, 6th and 7th passages, respectively; (C) is a positive control with one zona pellucida free blastocyst. (C) Phase contrast micrograph of EB at Stage II. (D) Phase contrast micrograph of a sphere cultured in serum-free medium at Stage III. (E) GFAP indirect immunofluorescence of accessory cell at stage III. (F) PGP immunopositive ‘rosette’ (white arrows) at stage IV. (G) Percentage of TH+ cells in subsequent stages (I, III–V) of differentiation derived from three independent experiments; ***P<0.0001 by Friedman test and Dunn post-test. (H) Increased complexity of neurites immunostained for β-III tubulin. Scale bars = 20 μm (A, D, E, F, H), 200 μm (C).
Mentions: Undifferentiated ES cells exhibited the expression for SSEA-1 (75%, n = 1000 cells) (Figure 1A), in correspondence with RT–PCR for Nanog and Oct-4 (Figure 1B). At stages I and II, small and sparse PGP immunoreactive cells were found, mostly located inside conglomerates. These cells did not express TH and GFAP. At this point also nestin-positive cells were present at large amounts (78%, n = 1000 cells). Cultured stage I cells proliferated and formed large and spherical floating aggregates known as EB (Figure 1C). Almost half of plated cells adhered to the bottom and consistently expressed PGP by immunocytochemistry despite being TH-negative (data not shown). The cell clusters derived from modified EB are called neurospheres and form a network that becomes more evident in subsequent stages (Figure 1D). At the end of 6 days of cultivation in this medium, many extensions of cytoplasm of individual cells are highly visible, and connecting EB are often supported by a flat monolayer of mostly GFAP-positive cells (Figure 1E).

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