Journal: bioRxiv

Article Title: Directed Differentiation of Human Pluripotent Stem Cells into Radial Glia and Astrocytes Bypasses Neurogenesis

doi: 10.1101/2021.08.23.457423

Figure Lengend Snippet: a , Enhanced neuronal activity of glutamatergic cells upon co-culture with hPSC-astrocytes. Multi-electrode array (MEA) analysis was performed at day 8 of co-culture show. b-d . Quantification of spikes (b), active electrodes (c) and reduced coefficient of variation between recorded wells (d) in co-culture as compared to glutamatergic neurons only (n=6 wells per group). e , Immunoreactivity for synaptic marker synapsin-1 (SYN1) expressed by motor neurons co-cultured with astrocytes for 21 days versus neuron-only cultures. f , MEA recording (1 out of 48 electrodes represented) to visualize sporadic spikes in-between bursts in neuronal mono-culture versus higher synchrony in co-cultures. g , MEA analysis shows reduced inter burst interval when motor neurons are co-cultured with hPSC-astrocytes (unpaired t-test, p=0.18, nMN = 11; nMN + Astro = 6 wells). h , Video-microscopy and quantification of RFP + neurite length over the course of 12 days in neuron-only versus neuron-astrocyte co-cultures. i3-neurons were co-cultured with astrocytes in a ratio of 1 astrocyte per 3 neurons (1:3). Images obtained with IncuCyte S3 live imaging system (each dot represents a well of a 24-well plate; and for each dot 4 images were taken and analyzed). Neurite length is normalized to cell body clusters. Note the clear separation of co-cultures from day 2 onward. i , Representative images of RFP + i3-neurons and image mask of neurites and cell bodies (NeuroTrack software). j . Uptake of glutamate by iPSC-astrocytes is comparable to controls (primary mouse astrocytes and human iCell astrocytes (FUJIFILM CDI). k , Secretion of human complement C3 by hPSC-astrocytes into the culture medium after stimulation with inflammatory cytokines (Interleukin-1b, TNF-alpha and C1q) for 24 h. l , Calcium transients in iPSC-astrocyte cultures after stimulation with KCL, ATP and L-glutamate. m . Periodic acid-Schiff stain of intracellular glycogen in hPSC-astrocytes and controls (iCell Astro from FUJIFILM CDI). Scale bar, 100 µm ( e, h, m ).

Article Snippet: Primary antibodies used for immunocytochemistry are as follows: mouse anti-PAX6 1:200 (561462, BD Biosciences), rabbit anti-FABP7 (BLBP) 1:200 (ABN14, EMD Millipore), rabbit anti-CD44 1:400 (ab157107, Abcam), rat anti-CD44 1:100 (A25528), mouse anti-VIMENTIN 1:500 (M0725, DAKO), rabbit anti-NFIA 1:250 (NBP-1-81406, Novus), rabbit anti-S100-beta 1:100 (ab52642, Abcam), mouse anti-TUJ1 1:1000 (801201, BioLegend), rabbit anti-GFAP 1:1000 (Z0334, Dako), rabbit anti-synapsin-1 1:500 (106-011, Synaptic Systems), rabbit anti-ASPM 1:100 (NB-100-227, Novus), rabbit anti-FAT1 1:100 (HPA023882 Millipore Sigma) and mouse anti-Pan-Neuronal Marker (PNM) 1:200 (MAB2300, EMD Millipore).

Techniques: Activity Assay, Co-Culture Assay, Marker, Cell Culture, Microscopy, Imaging, Software, Staining

Journal: STAR Protocols

Article Title: Protocol for generating human cerebral organoids from two-dimensional cultures of pluripotent stem cells bypassing embryoid body aggregation

doi: 10.1016/j.xpro.2025.103678

Figure Lengend Snippet: Characterization of the main cell types in the hCOs at maturation (A) hCOs immunostained for cell cycle marker Ki67 (green) and neural precursor marker Sox2 (green), different neuronal markers (DCX, βIIITubulin and MAP2) (red) and synaptic marker (SYN1) (green). Cell nuclei were identified by Höechst 33258 staining (blue). VZs are marked with white dashed lines. Scale bar = 100 μm. (B) Whole-mount immunohistochemistry of hCOs stained for Sox2 (red) and MAP2 (green). Cell nuclei were identified by Höechst 33258 staining (blue). VZs are marked with white dashed lines. Scale bar = 100 μm. hCOs, human cerebral organoids.

Article Snippet: Rabbit polyclonal anti-Synapsin 1 (Syn1) (1:200) , Merck (Millipore) , Cat#AB1543; RRID: AB_2200400.

Techniques: Marker, Staining, Immunohistochemistry

Journal: STAR Protocols

Article Title: Protocol for generating human cerebral organoids from two-dimensional cultures of pluripotent stem cells bypassing embryoid body aggregation

doi: 10.1016/j.xpro.2025.103678

Figure Lengend Snippet:

Article Snippet: Rabbit polyclonal anti-Synapsin 1 (Syn1) (1:200) , Merck (Millipore) , Cat#AB1543; RRID: AB_2200400.

Techniques: Recombinant, Knock-Out, Software