Journal: bioRxiv

Article Title: Tracking long-term functional connectivity maps in human stem-cell-derived neuronal networks by holographic-optogenetic stimulation

doi: 10.1101/2021.05.11.443589

Figure Lengend Snippet: (A) Experimental scheme of iNGN-astrocyte co-culture preparation (left) and experimental procedure (right) over time. Functional recordings, microscopy imaging, full-field and holographic stimulations were studied on different days post induction (dpi). (B) Schematic of the so-called Banker culture, a neuron-glia co-culture system within the MEA device. (C) Representative microscopy images of iNGN cells. Upper panel, iNGN cells grown on Matrigel-coated well plates at 3 dpi. Lower panel, PDL-laminin-coated MEA chambers at 15 dpi. ChR2-EYFP-labeled cells are visualized by live cell microscopy. Scale bars, 100 µm. mTeSR1: standardized medium for the feeder-independent maintenance of hiPSCs. BrainPhys: serum-free neurophysiological basal medium for improved neuronal function. LV-ChR2-EYFP: lentiviral particles delivering ChR2-EYFP to iNGN cells. Dox: doxycycline. Ara-C: cytosine arabinoside to remove undifferentiated cells.

Article Snippet: Then, complete BrainPhys™ (BrainPhys™ Neuronal Medium (Stemcell Technologies) + 1% P/S + NeuroCult™ SM1 Neuronal Supplement (Stemcell Technologies) + N2 Supplement-A + 20 ng/ml recombinant human BDNF (Peprotech) + 20 ng/ml recombinant human GDNF (Peprotech) + 200 nM ascorbic acid (Sigma)) was added to the MEA rings, covering neuronal and astrocyte cultures ( ).

Techniques: Co-Culture Assay, Functional Assay, Microscopy, Imaging, Labeling

Journal: bioRxiv

Article Title: MAPT expression is regulated by long-range interactions with cis -regulatory elements

doi: 10.1101/2023.03.07.531520

Figure Lengend Snippet: A) Loop plot of HiCCUPs nominated loops to the MAPT promoter. Top panel: Nominated loops by dataset. Top: HiC loops to the MAPT promoter from iCell GlutaNeuron cultures. Middle: Capture-C loops to the MAPT promoter from iCell GlutaNeuron cultures. Bottom: Capture-C loops to the MAPT promoter from iCell GABANeuron cultures. Bottom panel: Top: Track of ENCODE annotated candidate CREs. Middle: ATAC-seq peaks from KOLF2.1J-hNGN2 differentiated neurons. Bottom: H3K27ac and CTCF ChIP-seq peaks from Day 14 BrainPhys differentiated neurons. B) Proportion of HiC global loop calls by ENCODE annotation. C) Proportion of Capture-C MAPT promoter loops by ENCODE annotation for both iCell GlutaNeurons and GABANeurons.

Article Snippet: On day 3 neurites were present, and cells were renewed with Cortical Neuron Culture Media: BrainPhys neuronal medium (StemCell Technologies), 50X SM1 supplement (StemCell Technologies), 10 μ g/mL BDNF (StemCell Technologies), 10 μ g/mL NT-3 (StemCell Technologies), and 1 mg/mL Mouse Laminin (Gibco).

Techniques: Capture-C, ChIP-sequencing

Journal: bioRxiv

Article Title: MAPT expression is regulated by long-range interactions with cis -regulatory elements

doi: 10.1101/2023.03.07.531520

Figure Lengend Snippet: A) Heatmap showing expression of MAPT and cell type marker genes in NPCs, Day 14 BrainPhys differentiated neurons (BrainPhys Neurons), KOLF2.1J-hNGN2 Neurons (NGN2 Neuron), and iCell GlutaNeurons. B) Differential Capture-C directional p-value track. Top panel: Comparison of NPCs and Day 14 BrainPhys Neurons. Middle panel: Comparison of iCell GlutaNeurons (Excitatory) and iCell GABANeurons (Inhibitory). C) Zoom in of chr17: 45,225,000-45,320,000. Top panels: Chromatin accessibility across cell types generated by snATAC-seq in DLPFC nuclei. Bottom panels: Differential capture-C comparing BrainPhys differentiated neurons and NPCs with the neuron-specific region highlighted in gray.

Article Snippet: On day 3 neurites were present, and cells were renewed with Cortical Neuron Culture Media: BrainPhys neuronal medium (StemCell Technologies), 50X SM1 supplement (StemCell Technologies), 10 μ g/mL BDNF (StemCell Technologies), 10 μ g/mL NT-3 (StemCell Technologies), and 1 mg/mL Mouse Laminin (Gibco).

Techniques: Expressing, Marker, Capture-C, Generated

Journal: bioRxiv

Article Title: MAPT expression is regulated by long-range interactions with cis -regulatory elements

doi: 10.1101/2023.03.07.531520

Figure Lengend Snippet: A) Boxplots showing elements tested in luciferase assays in iCell GlutaNeurons (*p<0.05, ANOVA with Fisher’s LSD). B) Boxplots showing elements tested in luciferase assays in KOLF2.1J-hNGN2 neurons (*p<0.05, ANOVA with Fisher’s LSD). C) RT-qPCR of MAPT expression. Barplots show regions tested in Day 14 BrainPhys differentiated neurons by CRISPRi experiments (*p<0,05, ANOVA with Fisher’s LSD).

Article Snippet: On day 3 neurites were present, and cells were renewed with Cortical Neuron Culture Media: BrainPhys neuronal medium (StemCell Technologies), 50X SM1 supplement (StemCell Technologies), 10 μ g/mL BDNF (StemCell Technologies), 10 μ g/mL NT-3 (StemCell Technologies), and 1 mg/mL Mouse Laminin (Gibco).

Techniques: Luciferase, Quantitative RT-PCR, Expressing

Journal: bioRxiv

Article Title: MAPT expression is regulated by long-range interactions with cis -regulatory elements

doi: 10.1101/2023.03.07.531520

Figure Lengend Snippet: A) Boxplots showing statistically significant (*p<0.05, ANOVA with Fisher’s LSD) elements tested in luciferase assays in iCell GlutaNeurons. B) Boxplots showing statistically significant (*p<0.05, ANOVA with Fisher’s LSD) elements tested in luciferase assays in KOLF2.1J-hNGN2 neurons. C) RT-qPCR of MAPT expression. Barplots show statistically significant regions tested in Day 14 BrainPhys differentiated neurons by CRISPRi experiments. Red asterisk indicates regions significant by 3’ mRNA-seq and RT-qPCR. Gray asterisk indicates regions significant in either RT-qPCR or 3’ mRNA-seq. (*p<0.05, ANOVA with Fisher’s LSD). D) Heatmap showing differential expression of MAPT and all genes expressed (cpm > 30) within 1 Mb of MAPT TSS. Left y-axis indicates the distance of the midpoint of the target region tested in CRISPRi experiments from the MAPT promoter (chr17:45894000). textbfE) Browser view of significant nominated regions. Green: iCell GlutaNeuron luciferase assay. Blue: KOLF2.1J-hNGN2 neuron luciferase assay. Teal: CRISPRi. Red: significant in luciferase and CRISPRi.

Article Snippet: On day 3 neurites were present, and cells were renewed with Cortical Neuron Culture Media: BrainPhys neuronal medium (StemCell Technologies), 50X SM1 supplement (StemCell Technologies), 10 μ g/mL BDNF (StemCell Technologies), 10 μ g/mL NT-3 (StemCell Technologies), and 1 mg/mL Mouse Laminin (Gibco).

Techniques: Luciferase, Quantitative RT-PCR, Expressing

Journal: bioRxiv

Article Title: MAPT expression is regulated by long-range interactions with cis -regulatory elements

doi: 10.1101/2023.03.07.531520

Figure Lengend Snippet: Zoom in on regions validated by qPCR and 3’ mRNA-seq ( A) −652,338, B) −464,677 I&-461,949, C) −44,905, D) 48,416). Top panel: H3K27ac signal from Day14 BrainPhys Neurons. Middle panel: Normalized Accessibility from KOLF2.1J-hNGN2 differentiated Neurons. Bottom panel: Tracks of design region, luciferase region (Green: tested in iCell Gluta Neurons; Blue: tested in KOLF2.1J-hNGN2 neurons), gRNA position, and nominated regions (DLPFC Links: Blue: control-specific link; gray: common link; Red: AD-specific link).

Article Snippet: On day 3 neurites were present, and cells were renewed with Cortical Neuron Culture Media: BrainPhys neuronal medium (StemCell Technologies), 50X SM1 supplement (StemCell Technologies), 10 μ g/mL BDNF (StemCell Technologies), 10 μ g/mL NT-3 (StemCell Technologies), and 1 mg/mL Mouse Laminin (Gibco).

Techniques: Luciferase