Purification and characterization of human neural stem and progenitor cells

Abstract

The human brain undergoes rapid development at mid-gestation from a pool of neural stem and progenitor cells (NSPCs) that give rise to the neurons, oligodendrocytes, and astrocytes of the mature brain. Functional study of these cell types has been hampered by a lack of precise purification methods. We describe a method for prospectively isolating ten distinct NSPC types from the developing human brain using cell-surface markers. CD24^-THY1^-/lo cells were enriched for radial glia, which robustly engrafted and differentiated into all three neural lineages in the mouse brain. THY1^hi cells marked unipotent oligodendrocyte precursors committed to an oligodendroglial fate, and CD24⁺THY1^-/lo cells marked committed excitatory and inhibitory neuronal lineages. Notably, we identify and functionally characterize a transcriptomically distinct THY1^hiEGFR^hiPDGFRA^- bipotent glial progenitor cell (GPC), which is lineage-restricted to astrocytes and oligodendrocytes, but not to neurons. Our study provides a framework for the functional study of distinct cell types in human neurodevelopment.

Keywords: fluorescence-activated cell sorting; glial progenitor cells; index sorting; intermediate progenitor cells; neural stem cells; neurodevelopment; neuronal maturation; oligodendrocyte progenitor cells; radial glia; single-cell transcriptomics.

Figure 1.. Prospective isolation of NSPCs from the developing human brain

(A) Tissue processing and experimental workflow for isolation and characterization of NSPCs via transcriptomic and functional methods. (B) Gating scheme for isolation of distinct NSPC populations using FACS, based on varying cell-surface expression of THY1 (CD90), CD24, EGFR, PDGFRA, and CXCR4. Events are pre-gated on live single cells and negative for non-neural lineage markers PECAM1 (CD31), CD34, PTPRC (CD45), ENG (CD105), and GYPA (CD235a). (C) Single cell RNA sequencing of index-sorted NSPCs using Smart-seq3. Plot showing PAGAembedded Leiden clusters with annotated cell type identities based on expressed transcripts of known genes. (D) Index-sort data was used to map the sequenced single cells to their original immunophenotype with respect to cell-surface CD24 and THY1 expression levels.

Figure 2.. CD24⁻THY1^−/lo expression identifies NSCs

(A) Gating scheme for CD24⁻THY1^−/lo NSC compartment. (B) Index-sort data was used to map the transcriptomically-identified vRG (red), oRG (orange), and AC (brown) clusters to their original immunophenotype. (C) Plot showing the quantification of neurosphere initiation frequency of each CD24⁻THY1^−/lo subset based on in vitro limiting dilution assays. n=5–11 donors per population, mean ± standard deviation (S.D.) (D) Right: immunofluorescence (IF) images of CD24⁻THY1^−/lo subsets showing DCX (green) and GFAP (red) expression after 5 days of in vitro differentiation post-sort. Scale bar 50 μm. Left: bar-graph showing quantification of percent DCX⁺ and GFAP⁺ cells in images similar those shown in right panel. Error bars show standard deviation. (E–G) Confocal IF images of mouse brains engrafted with CD24⁻THY1^−/lo human NSCs. 40 μm thick sections were stained with anti-human GFAP (E) or human cytoplasmic antigen-specific STEM121 antibody (F, G), in addition to species cross-reactive antibodies against SOX2, OLIG2, or NeuN and MAP2 (E, F, G, respectively). Imaged regions: medulla (Med), third ventricle (3V), hypothalamus (HY), olfactory bulb (OB), cerebellum (CB), corpus callosum (cc), optic chiasm (och), and subventricular zone (SVZ). Scale bar 25 μm. (H) Visualization of CD24⁻THY1^−/loEGFR⁻ (top) and CD24⁻THY1^−/loEGFR^hi (middle) NSCs engrafted into the mouse brain. Each dot represents an engrafted GFAP⁺ (orange) or an OLIG2⁺ (green) human cell. Bottom: bar graph showing quantification of orange GFAP⁺ and green OLIG2⁺ cells per mm³ in brain sections similar to those shown in the upper two panels. n=4 quantified brains per population; mean ± S.D.

Figure 3.. THY1^hi expression identifies OPCs

(A) Gating scheme for THY1^hi OPC compartment. (B) Index-sort data was used to map the transcriptomically-identified pre-OPC (light green), OPC (green), and OL (dark green) clusters to their original immunophenotype. (C) (left) Quantification of processes on EGFR⁺PDGFRA⁺ and EGFR⁻PDGFRA⁺ cells in fetal human cerebral cortex (18 gestational weeks, GW18). (right) Confocal IF images of GW18 fetal human cerebral cortex stained for EGFR (green), PDGFRA (red), and DAPI (blue). Empty arrowheads to EGFR⁺PDGFRA⁺ cells; solid arrowheads point to EGFR⁻PDGFRA⁺ cells. Scale bar 50 μm. (D) Plot showing the quantification of neurosphere initiation frequency of each THY1^hi subset based on limiting dilution assays. n=4–7 donors per population, mean ± S.D. (E) IF images of the bulk-sorted THY1^hi subsets that were cultured in the absence of growth factors for 4 days, and subsequently stained with anti-O4 (green) antibody and DAPI (blue) marking the nuclei, along with quantification of percent O4⁺ cells in images similar to those in the left panel. Mean ± S.D. Scale bar 50 μm. (F) Visualization of THY1^hi subsets engrafted into the mouse brain. Each dot represents an engrafted GFAP⁺ (orange) or OLIG2⁺ (green) human cell. (G) Confocal IF images of mouse brains engrafted with THY1^hi human OPCs. 40 μm thick sections were stained with human cytoplasmic antigen specific STEM121 antibody (green) and OLIG2 (red). Imaged regions: corpus callosum (cc), cerebellum (CB), dentate gyrus (DG), and midbrain (MB). Scale bar 25 μm.

Figure 4.. Identification of a bipotent glial progenitor

(A) mRNA expression matrix showing astrocyte and oligodendrocyte marker genes in transcriptomic cell clusters. (B) PAGA pseudotime analysis of expressed transcripts along the maturation trajectory from ventricular and outer radial glia (vRG, oRG) to glial progenitor cells (GPC) to oligodendrocyte precursor cells (OPC) to oligodendrocytes (OL). (C) Confocal immunofluorescence (IF) images of fetal human brain sections (18 gestational weeks; GW18) from the cortex. 14 μm thick sections were stained with antibodies against GFAP (green) and OLIG2 (red). Scale bar 50 μm. (D) Confocal IF images of GW18 fetal human cerebral cortex, stained with antibodies against GFAP (green), OLIG2 (red), ETV4 (cyan, top), and HOPX (cyan, bottom). Scale bar 10 μm. (E) Anatomical distribution of GFAP⁺OLIG2⁺ cells in GW18 fetal human cortex across the ventricular/subventricular zone (VZ/SVZ), outer subventricular zone (OSVZ), intermediate zone/subplate (IZ/SP), and cortical plate (CP). (F) Index-sort data was used to map the transcriptomically-identified glial progenitors to their original immunophenotype. Glial progenitors were found to be enriched in the THY1^hiEGFR^hiPDGFRA⁻ gate. (G) Experimental strategy for clonal differentiation assay. (H) IF images of cells after differentiation stained with DAPI (blue) and antibodies against GFAP (red), O4 (green), and DCX (cyan). Clonal neurospheres were derived from single THY1^hiEGFR^hiPDGFRA⁻ cells, the cells from clonal neurospheres were dissociated then subjected to differentiation conditions. Scale bar 50 μm. (I) Quantification of lineage output of clonal neurospheres derived from THY1^hiEGFR^hiPDGFRA⁻, THY1^hiEGFR^midPDGFRA⁻, THY1^hiEGFR⁺PDGFRA⁺, or CD24−THY1^−/lo cells. Each column represents a distinct clonal neurosphere. Differentiated cells were classified based on their expression of GFAP, DCX, or OLIG2. (J) Confocal IF images of mouse brains engrafted with THY1^hiEGFR^hiPDGFRA⁻ putative glial progenitors. 40 μm thick sections were stained with antibodies against human GFAP (left, green), SOX2 (left, red), human cytoplasmic antigen (right, green) and OLIG2 (right, red). Imaged regions: medulla (Med) and midbrain (MB). Scale bar 50 μm. (K) Visualization of THY1^hiEGFR^hiPDGFRA⁻ cells engrafted into the mouse brain. Each dot represents an engrafted GFAP⁺ (orange) or OLIG2⁺ (green) human cell.

Figure 5.. CD24⁺THY1^−/lo expression identifies neuron precursors

(A) Gating scheme for CD24⁺THY1^−/lo neuron compartment. (B) Index-sort data was used to map the transcriptomically-identified early ExN (light blue), late ExN (dark blue), and InN (purple) clusters to their original immunophenotype. (C) PAGA pseudotime analysis of expressed transcripts along the neuronal maturation trajectory. (D) Top: IF images of cultured CD24⁺THY1^−/loCXCR4⁻ cells stained with anti-DCX (red) and anti-MAP2 (green) antibodies. Bottom: bar-graph showing the quantification of percent DCX⁺, MAP2⁺, GFAP⁺, and OLIG2⁺ cells in images similar to those shown in the top panel. Scale bar 50 μm. (E) IF images of cultured CD24⁻THY1^−/loCXCR4⁻ cells stained with anti-SYN1 (red) and anti-MAP2 (green) antibodies. Scale bar 50 μm.

Figure 6.. NSPC surface markers are conserved across diverse brain regions

(A) Dissected regions of fetal human brain. (B) Sorting strategy for neural stem and progenitor cell types. Each gate has been assigned a letter from a-i. Stacked bar graphs show the composition of transcriptomically-defined cell types purified from each gate, for each brain region. (a) CD24⁻THY1^−/lo, (b) CD24⁺THY1^−/lo, (c) THY1^hi, (d) CD24⁺THY1^−/loCXCR4⁻, (e) CD24⁺THY1^−/loCXCR4⁺, (f) THY1^hiEGFR^hiPDGFRA⁻, (g) THY1^hiEGFR⁺PDGFRA⁺, (h) THY1^hiEGFR⁻PDGFRA⁺, (i) THY1^hiEGFR⁻PDGFRA⁻.

Figure 7.. Prospective isolation strategy for human fetal NSPCs

Graphical summary of NSPC types and their corresponding surface marker expression identified in this study.