Journal:

Article Title: Assessment of Stromal-Derived Inducing Activity in the Generation of Dopaminergic Neurons from Human Embryonic Stem Cells

doi: 10.1634/stemcells.2008-0039

Figure Lengend Snippet: Time course of differentiation of hESC in co-culture with PA6 stromal feeder cells. Phase contrast images of hESC in co-culture with PA6 cells for 3, 8, 10, and 12 days (A–D). (A) Two colonies after 3 days of co-culture, illustrating the development of undefined borders and more elongated cells in the periphery of the colonies. (B) Demonstration of rosette-like structures at day 8 in the center of a colony (arrows). (C, D) Colonies after 10 and 12 days of differentiation containing cells with process-bearing neuronal morphology. (E–L) Immunocytochemical marker expression analysis of hESC after 8 days and (M–P) 12 days of co-culture. (E) Sox-1, (F) Nestin, (G) NCAM, (H), PSA-NCAM, (I) Oct3/4, (J) SSEA-4, (K) β-III-tubulin, (L) TH, (M) TH and Oct3/4, (N) SSEA-4, (O) β-III-tubulin and (P) TH. Scale bars=200 µm.

Article Snippet: The following primary antibodies were used: mouse anti-Oct 3/4(1:50; Santa Cruz, CA; http://www.scbt.com ), goat anti-Oct3/4 (1:100; Santa Cruz), rabbit anti-TH (1:1000; Pel-Freez, Rogers, AK; http://www.pelfreez-bio.com ), rabbit anti–β III-tubulin (1:2000; Promega, Madison, WI; www.promega.com ), and rabbit anti-γ-aminobutyric acid (GABA) (1:2000; Sigma), mouse anti-NCAM (clone NCAM-0B11, 1:50; Sigma), mouse anti-nestin (1:50; R & D Systems) rabbit anti-GFAP (1:2000; Dako, Carpinteria, CA; http://www.dakousa.com ) and rabbit anti-MAP-2 (1:1000), rabbit anti-dopamine beta hydroxylase (DBH) (1:200), mouse anti-SSEA-4 (1:200) mouse anti-PSA-NCAM (clone 2-2B, 1:200) and rabbit anti-Sox-1 (1:500; all from Chemicon).

Techniques: Co-Culture Assay, Marker, Expressing

Journal:

Article Title: Assessment of Stromal-Derived Inducing Activity in the Generation of Dopaminergic Neurons from Human Embryonic Stem Cells

doi: 10.1634/stemcells.2008-0039

Figure Lengend Snippet: Characterization of cellular phenotypes present in differentiated cultures 18 days after removal of cells from the PA6 cell feeder layer. Immunocytochemistry for neuron subtype-specific markers after 18 additional days of differentiation in the presence of SHH, FGF8 and GDNF. (A, B) Co-expression of MAP2 and TH. 80 ± 11 percent of MAP2+ cells were positive for TH. (C) Expression of GABA was seen in a small number of cells. (D) Illustration of the large number of TH+ cells in a culture. (E) Bipolar morphology of TH+ neurons. (F) Expression of TH was seen in 34 ± 6 percent of the total cells. This frame shows an area in a typical colony. (G–I) Expression of S-100β and GFAP in astrocytes and glial cells in cultures co-stained for the neuronal marker β-III-tubulin. Scale bars=200 µm.

Article Snippet: The following primary antibodies were used: mouse anti-Oct 3/4(1:50; Santa Cruz, CA; http://www.scbt.com ), goat anti-Oct3/4 (1:100; Santa Cruz), rabbit anti-TH (1:1000; Pel-Freez, Rogers, AK; http://www.pelfreez-bio.com ), rabbit anti–β III-tubulin (1:2000; Promega, Madison, WI; www.promega.com ), and rabbit anti-γ-aminobutyric acid (GABA) (1:2000; Sigma), mouse anti-NCAM (clone NCAM-0B11, 1:50; Sigma), mouse anti-nestin (1:50; R & D Systems) rabbit anti-GFAP (1:2000; Dako, Carpinteria, CA; http://www.dakousa.com ) and rabbit anti-MAP-2 (1:1000), rabbit anti-dopamine beta hydroxylase (DBH) (1:200), mouse anti-SSEA-4 (1:200) mouse anti-PSA-NCAM (clone 2-2B, 1:200) and rabbit anti-Sox-1 (1:500; all from Chemicon).

Techniques: Immunocytochemistry, Expressing, Staining, Marker

Journal:

Article Title: Assessment of Stromal-Derived Inducing Activity in the Generation of Dopaminergic Neurons from Human Embryonic Stem Cells

doi: 10.1634/stemcells.2008-0039

Figure Lengend Snippet: Comparison of expression of Oct3/4, β-III-tubulin and TH by immunocytochemistry between cells differentiated on irradiated, fixed, mitomycin-c treated or untreated PA6 cells after 12 days of co-culture. (A) Few cells expressing β-III-tubulin or TH were observed following differentiation on irradiated PA6 cells. (B) Expression of β-III-tubulin was unchanged in cells differentiated on fixed PA6 cells, as compared to untreated cells, while TH expression was greatly reduced. (C) Mitomycin-c treatment of PA6 cells had a relatively small effect on differentiation, as measured by either β-III-tubulin or TH expression. (D) β-III-tubulin and TH expression in cells differentiated on untreated PA6 cells. (E) Quantitative analysis of expression of Oct3/4, β-III-tubulin and TH in hESC differentiated on untreated, mitomycin-c treated, fixed and irradiated PA6 cells. Data represent 60 colonies from three independent experiments. The overall effect of treatment was statistically significant for Oct3/4 (P<0.0005), β-III-tubulin (P<0.0001), and TH (P<0.0001) expressing colonies. *=P<0.05, **=P<0.01, and ***=P<0.001 as compared to the untreated condition. In addition, the mitomycin-c and the irradiation conditions were significantly different from each other for Oct3/4 and β-III-tubulin (P<0.0001) and TH (P<0.01). Scale bars=200 µm.

Article Snippet: The following primary antibodies were used: mouse anti-Oct 3/4(1:50; Santa Cruz, CA; http://www.scbt.com ), goat anti-Oct3/4 (1:100; Santa Cruz), rabbit anti-TH (1:1000; Pel-Freez, Rogers, AK; http://www.pelfreez-bio.com ), rabbit anti–β III-tubulin (1:2000; Promega, Madison, WI; www.promega.com ), and rabbit anti-γ-aminobutyric acid (GABA) (1:2000; Sigma), mouse anti-NCAM (clone NCAM-0B11, 1:50; Sigma), mouse anti-nestin (1:50; R & D Systems) rabbit anti-GFAP (1:2000; Dako, Carpinteria, CA; http://www.dakousa.com ) and rabbit anti-MAP-2 (1:1000), rabbit anti-dopamine beta hydroxylase (DBH) (1:200), mouse anti-SSEA-4 (1:200) mouse anti-PSA-NCAM (clone 2-2B, 1:200) and rabbit anti-Sox-1 (1:500; all from Chemicon).

Techniques: Expressing, Immunocytochemistry, Irradiation, Co-Culture Assay

Journal:

Article Title: Assessment of Stromal-Derived Inducing Activity in the Generation of Dopaminergic Neurons from Human Embryonic Stem Cells

doi: 10.1634/stemcells.2008-0039

Figure Lengend Snippet: Differentiation of progenitor cells isolated from co-culture with inactivated PA6 cells. Expression of MAP2 and TH 18 days after isolation from (A) irradiated (B) fixed (C) mitomycin-c and (D) untreated PA6 cells. (A) Cells that were grown on irradiated PA6 cells gave rise to the lowest number of MAP2+ and TH+ cells. (B, C) Cells isolated from fixed PA6 or mitomycin-c treated cells behaved similarly, resulting in a significantly decreased number of MAP2+ and TH+ cells. (D) Untreated PA6 cells were most efficient in generating MAP2+ and TH+ cells. (E) Comparison of expression of β-III-tubulin, GFAP, MAP2 and TH. The overall effect of treatment was statistically significant for β-III-tubulin (P<0.0001), GFAP (P<0.0005), MAP2 (P<0.0001), and TH (P<0.0001) expressing cells (F) MAP2+ cells expressing TH following differentiation for 18 days after isolation from untreated, mitomycin-c treated, fixed and irradiated PA6 cells. The overall effect of treatment was statistically significant for MAP2+ cells expressing TH (P =0.041). *=P<0.05, **=P<0.01, and ***=P<0.001 as compared to the untreated condition. In addition, the mitomycin-c and the irradiation conditions were significantly different from each other for MAP2 (P<0.05). Scale bars=200 µm.

Article Snippet: The following primary antibodies were used: mouse anti-Oct 3/4(1:50; Santa Cruz, CA; http://www.scbt.com ), goat anti-Oct3/4 (1:100; Santa Cruz), rabbit anti-TH (1:1000; Pel-Freez, Rogers, AK; http://www.pelfreez-bio.com ), rabbit anti–β III-tubulin (1:2000; Promega, Madison, WI; www.promega.com ), and rabbit anti-γ-aminobutyric acid (GABA) (1:2000; Sigma), mouse anti-NCAM (clone NCAM-0B11, 1:50; Sigma), mouse anti-nestin (1:50; R & D Systems) rabbit anti-GFAP (1:2000; Dako, Carpinteria, CA; http://www.dakousa.com ) and rabbit anti-MAP-2 (1:1000), rabbit anti-dopamine beta hydroxylase (DBH) (1:200), mouse anti-SSEA-4 (1:200) mouse anti-PSA-NCAM (clone 2-2B, 1:200) and rabbit anti-Sox-1 (1:500; all from Chemicon).

Techniques: Isolation, Co-Culture Assay, Expressing, Irradiation

Journal:

Article Title: Assessment of Stromal-Derived Inducing Activity in the Generation of Dopaminergic Neurons from Human Embryonic Stem Cells

doi: 10.1634/stemcells.2008-0039

Figure Lengend Snippet: Influence of PA6 cell-secreted factors on survival and neural differentiation of hESC. Colonies are shown 48 hrs after culturing hESC in the (A) presence and (B) absence of PA6 cell conditioned media (CM). The majority of hESC in unconditioned media (UCM) did not attach to the gelatin-coated cell culture dishes, while CM promoted cell-to-substrate attachment and enhanced subsequent survival of hESC. (C, D) β-III-tubulin expression after 12 days of differentiation in the presence (C) and absence (D) of CM. Differentiation of hESC 48 hr after sub-culturing in (E) CM and (F) UCM. (G) Expression of MAP2 and TH and (H) GFAP and TH after 18 additional days of differentiation in CM. No surviving cells were found in cultures containing UCM at this stage. (I) Quantitative analysis of expression of Oct3/4, β-III-tubulin and TH in colonies differentiated in CM or UCM. The effect of treatment was statistically significant for Oct3/4 (P=0.0039), but not for β-III-tubulin or TH. Scale bars=200 µm.

Article Snippet: The following primary antibodies were used: mouse anti-Oct 3/4(1:50; Santa Cruz, CA; http://www.scbt.com ), goat anti-Oct3/4 (1:100; Santa Cruz), rabbit anti-TH (1:1000; Pel-Freez, Rogers, AK; http://www.pelfreez-bio.com ), rabbit anti–β III-tubulin (1:2000; Promega, Madison, WI; www.promega.com ), and rabbit anti-γ-aminobutyric acid (GABA) (1:2000; Sigma), mouse anti-NCAM (clone NCAM-0B11, 1:50; Sigma), mouse anti-nestin (1:50; R & D Systems) rabbit anti-GFAP (1:2000; Dako, Carpinteria, CA; http://www.dakousa.com ) and rabbit anti-MAP-2 (1:1000), rabbit anti-dopamine beta hydroxylase (DBH) (1:200), mouse anti-SSEA-4 (1:200) mouse anti-PSA-NCAM (clone 2-2B, 1:200) and rabbit anti-Sox-1 (1:500; all from Chemicon).

Techniques: Cell Culture, Expressing

Journal:

Article Title: Assessment of Stromal-Derived Inducing Activity in the Generation of Dopaminergic Neurons from Human Embryonic Stem Cells

doi: 10.1634/stemcells.2008-0039

Figure Lengend Snippet: Combinatorial effect of PA6 cell surface activity and secreted factors. Expression of β-III-tubulin and TH was examined after 18 days of co-culture of hESC with fixed PA6 cells under the influence of (A) CM, (B) UCM, (C) CM plus heparin and (D) UCM plus heparin. Heparin dependence of secreted factors involved in dopaminergic differentiation is indicated by the high number of TH expressing cells seen in the CM, plus heparin condition, shown in (C). (E, F) Phase contrast images of differentiated hESC colonies cultured in CM and UCM, respectively, in the presence of heparin, showing neural rosette-like structures in the core of colonies. Images shown in G and H illustrate random differentiation of hESC cultured in CM and UCM, respectively, in the absence of heparin. (I) Quantitative analysis of expression of β-III-tubulin, TH and appearance of neural rosette-like structures in colonies cultured in CM or UCM in the presence or absence of heparin. The overall effect of treatment was statistically significant for β-III-tubulin- and TH- (P<0.0001) expressing colonies, and for rosette-containing colonies (P<0.0003). *=P<0.05, **=P<0.01, and ***=P<0.001 as compared to the UCM condition. In addition, the UCM+Hep and CM+Hep conditions were significantly different from each other for β-III-tubulin and TH (P<0.0001), and for rosette-containing colonies (P<0.01). Scale bars=200 µm.

Article Snippet: The following primary antibodies were used: mouse anti-Oct 3/4(1:50; Santa Cruz, CA; http://www.scbt.com ), goat anti-Oct3/4 (1:100; Santa Cruz), rabbit anti-TH (1:1000; Pel-Freez, Rogers, AK; http://www.pelfreez-bio.com ), rabbit anti–β III-tubulin (1:2000; Promega, Madison, WI; www.promega.com ), and rabbit anti-γ-aminobutyric acid (GABA) (1:2000; Sigma), mouse anti-NCAM (clone NCAM-0B11, 1:50; Sigma), mouse anti-nestin (1:50; R & D Systems) rabbit anti-GFAP (1:2000; Dako, Carpinteria, CA; http://www.dakousa.com ) and rabbit anti-MAP-2 (1:1000), rabbit anti-dopamine beta hydroxylase (DBH) (1:200), mouse anti-SSEA-4 (1:200) mouse anti-PSA-NCAM (clone 2-2B, 1:200) and rabbit anti-Sox-1 (1:500; all from Chemicon).

Techniques: Activity Assay, Expressing, Co-Culture Assay, Cell Culture

Journal: STAR Protocols

Article Title: Protocol for culturing neurospheres from progenitor cells in the dentate gyrus of aged mouse hippocampus

doi: 10.1016/j.xpro.2025.103692

Figure Lengend Snippet:

Article Snippet: Polyclonal rabbit IgG anti-β-Tubulin III/Tuj1 (dilution 1:500) , GenScript , Cat#A01627; RRID: AB_2622164.

Techniques: Recombinant, Microscopy, Electron Microscopy, Sterility, Cell Culture, Software