unprocessed confocal images  (Mendeley Ltd)

Journal: The Journal of neuroscience : the official journal of the Society for Neuroscience

Article Title: BMP4 sufficiency to induce choroid plexus epithelial fate from embryonic stem cell-derived neuroepithelial progenitors

doi: 10.1523/JNEUROSCI.3227-12.2012

Figure Lengend Snippet: (A) Schematic of neural differentiation using the SFEBq method (Eiraku et al., 2008). (B) Cells/well optimization for neural induction (RT-qPCR, normalized to starting mESCs). 2000 and 6000 cells/well (green arrows) yield optimal gene expression profiles for the M1 and M2 mESC lines, respectively. (C) Temporal analysis of 2000-cell (M1) or 6000-cell (M2) aggregates (RT-qPCR, normalized to mESCs). Plateaus for the four NEC markers are seen in M1 aggregates, whereas they peak, then decrease in M2 aggregates. Green arrows designate the time point at which most CPEC differentiations were initiated. (D–F′) Nestin, Pax6, and Sox1 ICC of 7-day M1 aggregates and 5-day M2 aggregates reveal efficient neural induction. Scale bars: 50 um.

Article Snippet: For blinded scoring of Sox1 and Nestin immunostains, matched unprocessed confocal images (Zeiss LSM 510) were scored as follows: 2+, 1+ and 0 corresponded to >50%, <50%, and undetectable Sox1 positivity, respectively; 3+, 2+, 1+, and 0 corresponded to >50%, 10–50%, <10%, and undetectable Nestin positivity, respectively.

Techniques: Quantitative RT-PCR, Gene Expression

Journal: The Journal of neuroscience : the official journal of the Society for Neuroscience

Article Title: BMP4 sufficiency to induce choroid plexus epithelial fate from embryonic stem cell-derived neuroepithelial progenitors

doi: 10.1523/JNEUROSCI.3227-12.2012

Figure Lengend Snippet: (A) SFEBq aggregates treated with BMP4 for 7 DIV (M1) or 5 DIV (M2) (RT-qPCR normalized to no BMP4 controls). 5-day M1 and M2 aggregates show much stronger CPEC gene induction than 7-, 9-, or 11-day aggregates. (B–E) RC2 ICC of sectioned M1 and M2 aggregates. 5-day aggregrates exhibit low RC2 expression levels characteristic of NECs, while strong RC2 expression typical of RG is present throughout 7-day aggregates. (F–G) Sox1 and Nestin ICC of sectioned M2 aggregates. 5-day aggregates exhibit the strong Sox1 and weak Nestin patterns characteristic of NECs, whereas 7-day aggregates display the opposite RG-like pattern. (H) Scoring of Sox1/Nestin staining patterns in M2 aggregates. Scale bars: 100 um.

Article Snippet: For blinded scoring of Sox1 and Nestin immunostains, matched unprocessed confocal images (Zeiss LSM 510) were scored as follows: 2+, 1+ and 0 corresponded to >50%, <50%, and undetectable Sox1 positivity, respectively; 3+, 2+, 1+, and 0 corresponded to >50%, 10–50%, <10%, and undetectable Nestin positivity, respectively.

Techniques: Quantitative RT-PCR, Expressing, Staining

Journal: The Journal of neuroscience : the official journal of the Society for Neuroscience

Article Title: BMP4 sufficiency to induce choroid plexus epithelial fate from embryonic stem cell-derived neuroepithelial progenitors

doi: 10.1523/JNEUROSCI.3227-12.2012

Figure Lengend Snippet: (A) Schematic of the differentiation method. The hESCs form multiple and less uniform SFEBq aggregates than mESCs. (B) Temporal analysis of neural induction in hSFEBq aggregates (9000 hESCs/well, RT-qPCR normalized to hESCs). Dramatic increases in forebrain (LHX2, FOXG1) and generic neural progenitor gene expression (SOX1, NESTIN) occur by 6 DIV and are stably maintained thereafter. (C) ICC of 9000 cells/well hSFEBq aggregates at 21 DIV for PAX6 and NESTIN demonstrate efficient neural induction. (D) Temporal- and BMP4 concentration-dependent induction of CPEC genes in hSFEBq aggregates (9000 cells/well, RT-qPCR normalized to no BMP4 controls). 18-day aggregates show marked BMP4-mediated upregulation of TTR and LMX1A, while little to no induction is seen in 9-, 12-, or 15-day aggregates. TTR and LMX1A baselines also increased with aggregation time (~64X for TTR, 4X for LMX1A), but these baseline changes were much less than the BMP4-mediated changes in 18-day aggregates (>1000X for TTR, >100X for LMX1A). (E) CPEC and neural progenitor markers, 18-day aggregates (RT-qPCR normalized to no BMP4 controls). BMP4 induces CPEC markers TTR, LMX1A, and OTX2 at the expense of neural progenitor markers FOXG1 and NESTIN. Scale bars: 50 um.

Article Snippet: For blinded scoring of Sox1 and Nestin immunostains, matched unprocessed confocal images (Zeiss LSM 510) were scored as follows: 2+, 1+ and 0 corresponded to >50%, <50%, and undetectable Sox1 positivity, respectively; 3+, 2+, 1+, and 0 corresponded to >50%, 10–50%, <10%, and undetectable Nestin positivity, respectively.

Techniques: Quantitative RT-PCR, Gene Expression, Stable Transfection, Concentration Assay