Journal: Cellular and Molecular Life Sciences

Article Title: Transcription factor-based modulation of neural stem cell differentiation using direct protein transduction

doi: 10.1007/s00018-010-0347-1

Figure Lengend Snippet: Nkx2.2 protein transduction promotes oligodendrocyte differentiation in mouse ESC-derived NSC. Mouse ESC-derived NSC ( a ), largely negative for endogenous Nkx2.2, were induced to differentiate by growth factor withdrawal in the presence of T3 and ascorbic acid (AA) and incubated with recombinant Nkx2.2 protein (5 µg/ml b ). c – e After 5 days transduced populations showed a twofold increase of cells immunopositive for the oligodendrocyte-specific O4 antigen (Nkx 2.2: 13.8 ± 1.8%, vehicle control: 7.4 ± 0.6%; mean ± SD). *** P < 0.001 (Student’s t test). f Concentration-dependent effect of Nkx2.2-NTH transduction on mouse ESC-derived NSC. A twofold increase in the number of O4-positive oligodendrocytes ( blue curve ) compared to vehicle-treated control cells ( red dashed line ) could be detected already with 5 μg/ml protein. Higher concentrations had no further effect on oligodendroglial differentiation (mean ± SD). g Quantitative RT–PCR analysis of neural marker genes in differentiated cultures of Nkx2.2-NTH-transduced NSC relative to control populations. Nkx2.2 transduction resulted in an increase in transcripts for early ( sox10, ng2 ) and advanced ( cnp, plp, mbp ) oligodendroglial differentiation, whereas astrocytic markers ( gfap, s100ß ) were slightly decreased (mean ± SEM). DAPI was used for nuclear counterstaining ( blue ). Scale bars a , c , d 50 µm

Article Snippet: After washing in PBS, cells were blocked with 5% normal goat serum in PBS and incubated overnight in 1% normal goat serum in PBS with the following primary antibodies: Olig2 (rabbit IgG; 1:3,000; Chemicon, Hofheim, Germany), Nkx2.2 (mouse IgG; 1:1,000; Thomas M. Jessell, Columbia University, New York), Sox10 (mouse IgG; 1:1,500; Michael Wegner, University of Erlangen-Nürnberg, Germany), Sox9 (rabbit IgG; 1:300; Chemicon), O4 (mouse IgM; 1:100; Chemicon), GFAP (rabbit IgG; 1:1,000; DAKO, Hamburg, Germany), NG2 (rabbit IgG; 1:75; Chemicon), bIII-tubulin (rabbit IgG; 1:1000; Covance, Denver, USA) and GFP (rabbit IgG; 1:500; Acris Antibodies GmbH, Hiddenhausen, Germany).

Techniques: Transduction, Derivative Assay, Incubation, Recombinant, Concentration Assay, Quantitative RT-PCR, Marker

Journal: Cellular and Molecular Life Sciences

Article Title: Transcription factor-based modulation of neural stem cell differentiation using direct protein transduction

doi: 10.1007/s00018-010-0347-1

Figure Lengend Snippet: Nkx2.2 protein transduction promotes oligodendrocyte maturation in mouse ESC-derived NSC. a , b Immunofluorescence analysis of the O4 antigen reveals differences in morphological maturation between Nkx2.2-transduced and control oligodendrocytes. c Quantification of oligodendrocyte maturation. Oligodendrocytes with three or more branches were classified as mature. Nkx2.2-transduced NSC generated more oligodendrocytes with a mature, tertiary ramified phenotype compared to control cells. d Oligodendrocytes generated from Nkx2.2-transduced NSC formed myelin sheath-like membrane protrusions ( arrows ). e , f Immunostaining for the early oligodendroglial marker NG2 and O4 confirmed the differences in the developmental stages of oligodendrocytes in Nkx2.2-transduced and control populations. g Quantitative analysis of cells co-expressing NG2 and O4. The majority of NG2-positive oligodendrocytes in the control population represented pre-oligodendrocytes, which could not be stained for the oligodendrocyte marker O4. Shown are mean values ± SD. *** P < 0.001 (Student’s t test). DAPI was used for nuclear counterstaining ( blue ). Scale bars a , b , e , f 25 μm, d 10 μm

Article Snippet: After washing in PBS, cells were blocked with 5% normal goat serum in PBS and incubated overnight in 1% normal goat serum in PBS with the following primary antibodies: Olig2 (rabbit IgG; 1:3,000; Chemicon, Hofheim, Germany), Nkx2.2 (mouse IgG; 1:1,000; Thomas M. Jessell, Columbia University, New York), Sox10 (mouse IgG; 1:1,500; Michael Wegner, University of Erlangen-Nürnberg, Germany), Sox9 (rabbit IgG; 1:300; Chemicon), O4 (mouse IgM; 1:100; Chemicon), GFAP (rabbit IgG; 1:1,000; DAKO, Hamburg, Germany), NG2 (rabbit IgG; 1:75; Chemicon), bIII-tubulin (rabbit IgG; 1:1000; Covance, Denver, USA) and GFP (rabbit IgG; 1:500; Acris Antibodies GmbH, Hiddenhausen, Germany).

Techniques: Transduction, Derivative Assay, Immunofluorescence, Generated, Immunostaining, Marker, Expressing, Staining

Journal: Cellular and Molecular Life Sciences

Article Title: Transcription factor-based modulation of neural stem cell differentiation using direct protein transduction

doi: 10.1007/s00018-010-0347-1

Figure Lengend Snippet: Effects of Nkx2.2 protein transduction on oligodendrocyte differentiation and maturation are comparable to lentiviral-mediated gene transfer. a NSC derived from ESC were transduced with EGFP- and Nkx2.2/EGFP-expressing lentiviruses (pPGK-IRES2-EGFP, pPGK-Nkx2.2-IRES2-EGFP). Transgene expression in EGFP- ( b ) and Nkx2.2/EGFP-transduced cells ( c ) was confirmed by immunofluorescence analysis using anti-EGFP and anti-Nkx2.2 antibodies. d Infected cells were selected with blasticidine (Bsd) for 6–10 days before they were differentiated. Following a 4-day growth factor-mediated differentiation in the presence of T3 and AA, cultures of Nkx2.2-overexpressing cells contained more oligodendrocytes positive for the O4 antigen (EGFP control: 6.4 ± 1.2%; Nkx2.2: 12.2 ± 2.1%; mean ± SD, *** P < 0.001, Student’s t test). Oligodendrocytes in cultures stably overexpressing Nkx2.2 also adopted a more ramified mature phenotype compared to EGFP-expressing control cells ( f , g ). NG2 immunostaining revealed the generation of oligodendrocytes with complex ramified processes in Nkx2.2-transduced cultures ( f 2 ), whereas control populations predominantly exhibited NG2-positive pre-oligodendrocytes ( g 2 ). DAPI was used for nuclear counterstaining ( blue ). Scale bars b , c 50 µm, f 1 , g 1 50 µm, f 2 , g 2 25 µm

Article Snippet: After washing in PBS, cells were blocked with 5% normal goat serum in PBS and incubated overnight in 1% normal goat serum in PBS with the following primary antibodies: Olig2 (rabbit IgG; 1:3,000; Chemicon, Hofheim, Germany), Nkx2.2 (mouse IgG; 1:1,000; Thomas M. Jessell, Columbia University, New York), Sox10 (mouse IgG; 1:1,500; Michael Wegner, University of Erlangen-Nürnberg, Germany), Sox9 (rabbit IgG; 1:300; Chemicon), O4 (mouse IgM; 1:100; Chemicon), GFAP (rabbit IgG; 1:1,000; DAKO, Hamburg, Germany), NG2 (rabbit IgG; 1:75; Chemicon), bIII-tubulin (rabbit IgG; 1:1000; Covance, Denver, USA) and GFP (rabbit IgG; 1:500; Acris Antibodies GmbH, Hiddenhausen, Germany).

Techniques: Transduction, Derivative Assay, Expressing, Immunofluorescence, Infection, Stable Transfection, Immunostaining