Generation of high purity NRSCs with no manual selection of rosettes.

(A) Schematic representation of protocol for the generation of highly pure populations of NRSCs with no manual selection. (B) Size distribution of differentiating clusters measured on DIV 6 before seeding in 2D. Box represents minimum and maximum values, line represents mean, N=5. (C) Representative brightfield images captured on DIV (days in vitro) 0, 1, 3, 6, 8 and 10 in 4x magnification and 40x magnification. (D) Representative immunofluorescence images of DIV10 NRSCs positive for the common NSC markers NES and SOX2. (E) Higher magnification of a neural rosette. (F) NRSCs were also positive for the forebrain markers FOXG1 and OTX2. The formation of neural rosettes is visualized by the redistribution of TJP1 into the lumen of the rosettes. Scalebar = 100 µm.

NRSCs maintain stable marker expression through extended passaging.

(A) Representative immunofluorescence images showing cells positive for NSC markers NES and SOX2 at passages 2, 4, 6, 8, 10 and 12. TJP1 is used to highlight the lumen of neural rosettes. All images were taken in 20X magnification, scalebar = 50 µm. The different passages were imaged at different timepoints. (B) Representative immunofluorescence images showed NRSC positive for regional forebrain markers FOXG1 and OTX2 and NSC marker PAX6. All images were taken in 20X magnification, scalebar = 50 µm. The different passages were imaged at different timepoints. (C) Flow cytometric analysis of NSCs at passages 2, 4, 6, 8, 10 and 12. The cells showed stable marker expression through passaging of NSC markers SOX2, PAX6, neuroectoderm marker SOX1 and forebrain marker OTX2. Red dashed lines delineate the gating regions corresponding to the displayed percentages. FOM, Fluorescence Minus One.

scRNAseq reveals the presence of mainly forebrain NRSCs in cultures.

(A) Percentage of NRSCs expressing classic NSC stem cell markers at passage 2 and 8. Passage 2 shown in orange and passage 8 in blue. (B) Percentage of NRSCs expressing markers of regional identity in the developing brain. NRSCs show high and consistent expression of forebrain regional markers FOXG1, TLE4, SOX5, OTX2, with no midbrain, hindbrain, spinal cord or vNT (ventral neural tube) contaminants in both passage 2 and 8. Passage 2 shown in orange and passage 8 in blue. (C) UMAP of passage 2 and 8 NRSCs integrated, showing no major differences in the cell-types present. Passage 2 shown in yellow and passage 8 in blue. (D) UMAP of NRSC cultures, revealed large cluster of proliferating NRSC and minor populations of slightly more mature cells. Annotation is based on known markers highly expressed within each cluster. UMAP shows integrated data from passage 2 and 8. Percentage of cells in each cluster is shown as bar plot. (E) Dotplot visualizing the expression levels of NSC-, neural progenitor-, neuronal- and glial progenitor markers in the different clusters.

Differentiation of NRSCs into neurons.

(A) Schematic representation of protocol for generating neurons from NRSCs. (B) Representative brightfield images of generated neurons captured on day 0 and 8 in 20x magnification and 40x magnification. (C) UMAP based on scRNA-seq of NRSC-derived neurons reveal 2 trajectories of differentiating neuronal populations. Annotation is based on known markers expressed within each cluster. scRNAseq performed after 8 days of differentiation, with day 0 corresponding to the NRSC stage. The percentage of cells in each cluster is shown color coded in the plot. (D) UMAP visualizing the expression levels of NSC markers; SOX2 and NES, neural progenitor marker; DCX, pan-neuronal marker; ELAVL4, glutamatergic neuron marker; SLC17A6, and GABAergic neuron marker; GAD1. (E) UMAP visualizing the cell-cycle phase of the neurons. (F) Representative immunofluorescence images of NRSC-derived neurons on day 14 (with NRSC corresponding to day 0). They are positive for pan-neuronal markers TUJ1, ELAVL3/4, NeuN and MAP2. Images are taken in 20X magnification, scalebar = 50 µm. (G) Raster plot of spontaneous spiking by NRSC-derived neurons on HD-MEA chip at day 110 (with NRSC stage corresponding to day 0). Recording time = 60 seconds. Each dot corresponds to a spike. (H) Axon tracking reveal placement and axon morphology of NRSC-derived neurons on HD-MEA chip at day 110 (with NRSC stage corresponding to day 0). Higher magnification is shown to the right. Color-coding indicates recorded µV. Red lines show reconstructed axon morphology. Scalebar = 100 µm.

Differentiation of NRSCs into astrocytes.

(A) Schematic representation of protocol for generating astrocytes from our NRSCs. (B) Representative immunofluorescence images of Astrocytes positive for TNC on day 20 and GFAP and S100β on day 50 of differentiation. Day 0 corresponds to the NRSC stage. Images are taken in 20X magnification, scalebar = 50 µm. (C) Higher magnified representative image of Astrocyte markers GFAP and S100β on day 55 of differentiation.

Differentiation of NRSCs into oligodendrocytes.

(A) Schematic representation of protocol for generating oligodendrocytes from our NRSCs. (B) Representative immunofluorescence images of oligodendrocytes markers OLIG1, OLIG2, SOX10, O4, GALC, CNP. Images from day 0 and 62 are taken in 20X magnification, scalebar = 50 µm. Images from day 80 are taken in 40x magnification, scalebar = 20 µm.

NRSCs and neurons maintain high viability after cryopreservation.

(A) Viability of NRSCs after thawing and the 1st and 2nd passage after thawing. N=3. (B) Viability of NRSC-derived neurons after thawing. Shown as mean +/- SD, N=5.

Separate NRSC-line maintain stable marker expression through extended passaging.

(A) Representative immunofluorescence images show cells positive for NSC markers NES and SOX2 in at passage 2, 4, 6, 8, 10 and 12. TJP1 is used to highlight the lumen of neural rosettes. All images are taken in 20X magnification, scalebar = 50 µm. The different passages are imaged at different timepoints. (B) Representative immunofluorescence images show NRSC positive for regional forebrain markers FOXG1 and OTX2. And NSC marker PAX6. All images are taken in 20X magnification, scalebar = 50 µm. The different passages are imaged at different timepoints

Generation of NRSCs from alternative cell line.

(A) Representative brightfield images captured on DIV 1, 3, 6, 8 and 10 in 20x magnification. (B) Representative immunofluorescence images of DIV 10 NRSCs positive for common NSC markers NES and SOX2. The formation of neural rosettes is visualized by the redistribution of TJP1 into the lumen of the rosettes. Scalebar = 100 µm. (C) Size distribution of differentiating clusters measured on DIV 6 before seeding in 2D. Box represents minimum and maximum values, line is mean, N=5. (D) Representative brightfield image captured on DIV 17 in 40x magnification, revealing the formation of rosettes.

scRNA-seq analyses indicate high cell purity of NRSCs.

(A) UMAP visualization based on scRNA-seq of NRSCs show the expression levels of NSC markers; NES, SOX2, PAX6, OTX2, HES5, MSI1, TTYH1, PRDM16, MKI67, VIM, PCNA and GLI3 (B) UMAP visualization based on scRNA-seq of NRSCs show the expression levels of glutamatergic neural progenitor markers; NEUROD1, NEUROG1, TBR1, EOMES and SLC17A6. (C) UMAP visualization based on scRNA-seq of NRSCs show the expression levels of GABAergic neural progenitor markers; ASCL1, NKX2-1, DLX2, GAD1 and GAD2. (D) UMAP visualization based on scRNA-seq of NRSCs show the expression levels of glial progenitor markers; GLIS3, NKX2-1, NR2F1, NR2F2 and WLS.

scRNA-seq analyses reveal neuronal differentiation capacity of NRSCs.

(A) UMAP visualization based on scRNA-seq of NRSC-derived neurons show the expression levels of NSC marker; NES and proliferation markers: MKI67 and PCNA. (B) UMAP visualization based on scRNA-seq of NRSC-derived neurons show the expression levels of pan-neuronal markers; STMN2 and ELAVL4 and GABAergic neural progenitor markers; DLX2, GAD1, GAD2 and SLC32A1. (C) UMAP visualization based on scRNA-seq of NRSC-derived neurons show the expression levels of pan-neuronal markers; STMN2 and ELAVL4 and glutamatergic neural progenitor markers; RELN, ONECUT1 and SLC17A6.

Early hypothalamic identity of NRSC-derived neurons.

(A) UMAP visualization based on scRNA-seq of NRSC-derived neurons show the expression levels of glutamatergic early hypothalamic markers; ONECUT2, LHX1/2/5/9, KLF7, FOXP2, MEIS1 and SIX3. (B) UMAP visualization based on scRNA-seq of NRSC-derived neurons show the expression levels of GABAergic early hypothalamic markers; DLX6-AS1, LHX1/2 and ARX.

Morphological development during glial differentiations.

(A) Representative brightfield images of astrocyte differentiation captured on DIV 3, 9, 17, 22, 30, 37, 47 and 50 in 20x magnification and 40x magnification. DIV 0 corresponds to NRSC-stage. (B) Representative brightfield images of oligodendrocyte differentiation captured on DIV 7, 14, 21, 49, 62, and 80 in 20x magnification and 40x magnification. DIV 0 corresponds to NRSC-stage

hESC line characterization.

(A) Representative brightfield images of NOVOe001-A hESC line at passage 8 showing clear hESC morphology with large cell-bodies, dark nucleus and spiky edges. Taken in 4X, 10X, 20X and 40X magnification. (B) Representative immunofluorescence images of NOVOe001-A hESC line at passage 7 showing positive for pluripotency markers NANOG, OCT3/4 and SOX2. Scalebar = 50 µm. (C) Flow cytometry histrograms at passage 8 showing positive expression for pluripotency markers NANOG, OCT3/4, SOX2 and TRA-1-60.