Generation of human cerebral organoids with a structured outer subventricular zone

Abstract

Outer radial glia (oRG) emerge as cortical progenitor cells that support the development of an enlarged outer subventricular zone (oSVZ) and the expansion of the neocortex. The in vitro generation of oRG is essential to investigate the underlying mechanisms of human neocortical development and expansion. By activating the STAT3 signaling pathway using leukemia inhibitory factor (LIF), which is not expressed in guided cortical organoids, we define a cortical organoid differentiation method from human pluripotent stem cells (hPSCs) that recapitulates the expansion of a progenitor pool into the oSVZ. The oSVZ comprises progenitor cells expressing specific oRG markers such as GFAP, LIFR, and HOPX, closely matching human fetal oRG. Finally, incorporating neural crest-derived LIF-producing cortical pericytes into cortical organoids recapitulates the effects of LIF treatment. These data indicate that increasing the cellular complexity of the organoid microenvironment promotes the emergence of oRG and supports a platform to study oRG in hPSC-derived brain organoids routinely.

Keywords: CP: Neuroscience; CP: Stem cell research; LIF; cortical assembloids; hPSC; hPSC-derived brain organoids; human pluripotent stem cells; oRG; oSVZ; outer radial glia; outer subventricular zone; pericytes.

Figure 1.. LIF promotes oRG emergence in hPSC-derived guided cortical organoids

(A) Schematic outline of the cortical organoid differentiation protocol with or without LIF treatment. Y corresponds to ROCK inhibitor (Y-27632, 10 μM); LSBX corresponds to LDN193189 (100 nM), SB431542 (10 μM), and XAV939 (5 μM); LSB corresponds to LDN193189 (100 nM) and SB431542 (10 μM); for organoid media composition, see the relative section in STAR Methods. Human LIF was added at 10 ng/mL. (B) SOX2 (gray), TBR1 (red), and EOMES (yellow) staining in control and LIF-treated WA09 cortical organoids, sectioned and stained at day 60. The dotted lines highlight the regions of the ventricular zone (VZ), subventricular zone (SVZ), and outer subventricular zone (oSVZ). Nuclei are stained in blue with DAPI. Scale bars, 100 μm. (C) Rosette quantifications of WA09 control and LIF-treated cortical organoids at day 60, based on the separation of the regions as shown in (B). One-way ANOVA with Tukey’s test. Adjusted p values are shown on the graph. (D) SOX2 (gray), GFAP (red), and NESTIN (yellow) staining in day 60 control and LIF-treated WA09 brain organoids. Scale bars, 100 μm. (E) SOX2 (gray), HOPX (red), and phospho-VIMENTIN (pVIM, yellow) staining in day 60 control and LIF-treated WA09 brain organoids. Scale bars, 100 μm. (F) Quadruple staining for HOPX (cyan), GFAP (red), pVIM (yellow), and SOX2 (gray), in day 60 control and LIF-treated WA09 brain organoids. Scale bars, 50 μm. (G and H) Single-cell RNA-seq (scRNA-seq) experiments at day 60 showing plots for original identity (G) and label transfer, cell type annotation, and data integration with in vivo dataset from Bhaduri et al. (H). Two independent batches of 10 organoids each were processed and analyzed by scRNA-seq for both control and LIF conditions. (I–L) Feature plots depicting the distribution of the expression of SOX2 (I), EOMES (J), VIMENTIN (K), and HOPX (L) in control and LIF-treated cortical organoids on day 60.

Figure 2.. In vitro, LIF-induced oRG-like cells are transcriptionally similar to fetal oRG

(A) scRNA-seq Seurat clusters in control and LIF-treated organoids on day 60. The progenitor cluster 0 from control cortical organoids and the oRG cluster 2 from LIF-treated cortical organoids are highlighted. (B) Genes from analysis in (A) to mark selected populations of interest (neuronal cells, neuronal progenitors, astrocytes, and progenitor cells, including oRG) in control and LIF-treated organoids. (C) Enrichment analysis showing upregulation of STAT3 (left) and mTORC1 signaling (right) pathways in LIF-enriched cluster 2 compared to control cluster 0. (D) Gene Ontology (GO) Biological Process terms enriched in the LIF-specific cluster 2 compared to control cluster 0. (E) Migration-related genes upregulated in the LIF-specific cluster 2 compared to control cluster 0. (F) scRNA-seq signature enriched in LIF-treated organoids at day 60 (cluster 2) compared to control organoids (cluster 0) and mapped to fetal brain populations. The transcriptional profile of hPSC-derived oRG cells is enriched in fetal oRG compared to other radial glia populations and fetal astrocytes. (G) RNAscope for the LIFR, HOPX, and CDC42 transcripts, combined with a GFAP staining in control and LIF-treated WA09 cortical organoids at day 60. Nuclei are stained with DAPI and depicted either in blue. Scale bars, 50 μm.

Figure 3.. Neurogenic competence of LIF-induced oRG

(A) FACS sorting strategy to isolate SOX2⁺ HOPX⁺ progenitor cells from LIF-treated brain organoids and test their neurogenic differentiation potential in a monolayer culture. MAP2 (gray) and EdU (red) staining on dissociated monolayers from LIF-treated organoids. Nuclei are stained with DAPI in blue. Scale bar, 50 μm. (B–F) Manual patch-clamp results for the total number of recorded action potentials (APs) on dissociated monolayers from control and LIF-treated brain organoids (B). Patch-clamp results for the resting membrane potential (C), cell capacitance (D), AP amplitude (E), and firing frequency (F); Welch’s t test. (G) MAP2 (gray), SYN1 (red), and PSD95 (yellow) staining and quantifications of monolayer cultures derived upon dissociation of control and LIF-treated brain organoids. Welch’s t test. Scale bars, 25 μm. (H–J) Feature plots depicting the distribution of the expression of PAX6 (H), DCX (I), and TBR1 (J) in control and LIF-treated organoids at day 60. (K) Seurat clusters in control and LIF-treated organoids at day 60. The neuronal cluster 5 is highlighted. (L) OXPHOS-related genes enriched in cluster 5 at day 60. (M) GO Biological Process terms involved in metabolism enriched in cluster 5 and upregulated in LIF-treated organoids. (N) Violin plot showing expression levels of MEF2C in control and LIF-treated organoids at day 60 in the neuronal cell cluster 5. (O) Velocity pseudotime plots showing all neural cells undergoing maturation. (P) Ridge plots showing expression levels of neural cells that undergo maturation in LIF-treated organoids compared to control organoids at day 60. (Q–S) Calcium imaging (Q) and analysis showing quantification of Ca²⁺ intensity (Welch’s t test) (R) and frequency (Welch’s t test) (S) measured in control and LIF-treated organoids at day 60. Scale bars, 50 μm.

Figure 4.. Pericytes secrete LIF and promote oRG emergence

(A) PDGFRβ (yellow) and LIF (red) staining in human fetal choroid plexus at gestational week (GW) 8 and 11. Nuclei are stained in blue with Hoechst. Scale bars, 50 μm. (B) Schematic representation of the pericyte differentiation protocol from WA09-SOX10-GFP hPSC-derived neural crest (NC) cells. CBSB corresponds to CHIR (600 nM), BMP4 (1 ng/mL), and SB (10 μM). CSB corresponds to CHIR (1.5 μM) and SB (10 μM). On day 10, hPSC-derived NC cells were sorted based on GFP expression to derive SOX10⁺ NC cells. SOX10⁺ cells were maintained in pericyte medium to generate NC-derived pericytes. (C) Western blot data showing LIF expression in day 17 NC-derived pericytes but not in control cortical organoids (COs) at day 50 and 60 of differentiation. (D) Immunofluorescence images of day 17 NC-derived pericytes for pericyte markers PDGFRβ (yellow), NG2 (yellow), and αSMA (white), endothelial marker CD31 (white), and LIF (red). Cell nuclei are shown by DAPI. Scale bars, 10 μm. (E) ELISA assay for LIF in control NC-derived pericytes, LIF KO NC-derived pericytes, HUVEC cells, and hPSC-derived microglia. Positive and negative controls are cortical organoid media with or without LIF addition, one-way ANOVA with Tukey’s test. (F and G) Schematic outline showing the protocol to form pericyte microtissues (F) and cortical-pericyte (CP) assembloids (G). (H) Representative bright-field images of the morphological appearance of WA09 control organoids, LIF-treated organoids, and CP assembloids at day 60. Scale bars, 100 μm. (I) SOX2 (gray), TBR1 (red), and EOMES (green) staining showing rosette areas in control, LIF-treated organoids, and CP assembloids. Cell nuclei are stained by DAPI and shown in blue. Scale bars, 100 μm. (J–L) scRNA-seq experiments showing plots for original identity (J), label transfer, cell type annotation, and data integration with in vivo dataset from Bhaduri et al. (K), and Seurat clusters (L) in control organoids, LIF-treated organoids, and CP assembloids at day 65. (M) Genes selected from (J) to mark selected populations of interest (dividing cells, neuronal cell types, and neuronal progenitors, astrocytes, postmitotic neurons, and radial glia) in control organoids, LIF-treated organoids, and CP assembloids at day 65. (N) Violin plots showing expression levels of HOPX in control organoids, LIF-treated organoids, and CP assembloids at day 65. (O) SOX2 (gray), GFAP (red), and NESTIN (yellow) staining in day 60 WT CP assembloids and LIF KO CP assembloids. Cell nuclei were stained by DAPI. Scale bars, 100 μm.