Cross-site reproducibility of human cortical organoids reveals consistent cell type composition and architecture

Abstract

While guided human cortical organoid (hCO) protocols reproducibly generate cortical cell types at one site, variability in hCO phenotypes across sites using a harmonized protocol has not yet been evaluated. To determine the cross-site reproducibility of hCO differentiation, three independent research groups assayed hCOs in multiple differentiation replicates from one induced pluripotent stem cell (iPSC) line using a harmonized miniaturized spinning bioreactor protocol across 3 months. hCOs were mostly cortical progenitor and neuronal cell types in reproducible proportions that were consistently organized in cortical wall-like buds. Cross-site differences were detected in hCO size and expression of metabolism and cellular stress genes. Variability in hCO phenotypes correlated with stem cell gene expression prior to differentiation and technical factors associated with seeding, suggesting iPSC quality and treatment are important for differentiation outcomes. Cross-site reproducibility of hCO cell type proportions and organization encourages future prospective meta-analytic studies modeling neurodevelopmental disorders in hCOs.

Keywords: cortical organoids; neurodevelopment; reproducibility; scRNA-seq; stem cell state; tissue clearing.

Graphical abstract

Figure 1

Cell type characterizations from day 14 and day 84 hCOs (A) Experimental design showing critical steps for differentiation, collection time points, and assays. (B) t-SNE plot of detected cell types across all sites and time points (133,980 cells from 28 hCOs). Abbreviations are as follows: NPC, G2: neural progenitor cells, G2 phase; IP, S: intermediate progenitor, S phase; NPC, S: neural progenitor cell, S phase; IP: non-dividing intermediate progenitor; oRG: outer radial glia; RG: radial glia; NSC: neuroepithelial stem cell; Neuron: unspecified neuron; Pan CN: pan-cortical neuron; ULN, a: upper-layer cortical neuron, group a; ULN, b: upper-layer cortical neuron, group b; LLN, a: lower-layer cortical neuron, group a. LLN; b: lower-layer cortical neuron group b; PN/CR: pan-neuron and Cajal-Retzius; and ChP/MZ: choroid plexus and marginal zone. (C) t-SNE plots colored by differentiation day, inferred cell cycle phase, or gene expression of previously defined markers. (D) Heatmap of normalized gene expression for selected marker genes that are expressed in at least 5% of the cells in a cluster. (E) hCO cell type correlations to primary fetal telencephalic tissue (Bhaduri et al., 2020). Some in vivo cell type names (rows) have been simplified for plotting. Pearson’s correlation coefficients above 0.3 and surviving multiple comparison corrections of FDR <0.05 are labeled.

Figure 2

Cell type proportions across sites in hCO and primary tissue samples Cell type proportions across the 3 sites at (A) day 14 (N = 12 differentiation replicates) and (B) day 84 (N = 13 differentiation replicates). Differentiation replicates are individual hCOs from each site. Site ANOVA FDR corrected p values are indicated in the heatmap. (C) Cell type proportions from 3 separate day 56 hCOs harvested from the same differentiation replicate at UNC. (D) Cell type proportions reported by (Polioudakis et al., 2019) from 5 primary fetal tissue samples at similar developmental stages. End, endothelial; ExDp1, excitatory deep layer 1; ExDp2, excitatory deep layer 1; ExM, maturing excitatory; ExM-U, maturing excitatory upper enriched; ExN, migrating excitatory; InCGE, interneuron caudal ganglionic eminence; InMGE, interneuron medial ganglionic eminence; IP, intermediate progenitors; Mic, microglia; OPC, oligodendrocyte progenitor cell; oRG, outer radial glia; Per, pericyte; PgG2M, cycling progenitors (G2/M phase); PgS, cycling progenitors (S phase); vRG, ventricular radial glia. (E) Cell type proportions reported by Bhaduri et al. (2020). Each sample is a unique primary tissue donor ordered from earlier to later developmental stages. CS, Carnegie stage; GW,gestation week.

Figure 3

Differential gene expression across sites Counts of DEGs across sites in pseudo-bulked cell types at (A) day 14 (N = 12 differentiation replicates) and (B) day 84 (N = 13 differentiation replicates). Colors indicate the post hoc pairwise comparison with significant differences if the gene was differentially expressed across all three sites (6 genes total), differentially expressed in one site relative to the other two (“site v both”) or if the gene expression varied between two sites only. Significant GO terms for biological processes associated with DEGs in cell types at (C) day 14 and (D) day 84. (E) Gene expression differences in RSPO2 in day 14 neural progenitor cells in G2 phase and gene expression of CDH18 in day 14 NSC. (F) Gene expression differences in HSPA5 and 7SK in day 84 lower layer neuron, group b. Each point is the gene expression from one hCO. Each site collected 3–5 hCOs. Differences in gene expression across site were determined by likelihood ratio tests (LRTs). Abbreviations are as follows: reg, regulation and neg, negative.

Figure 4

Qualitative assessments of hCO differentiations (A) Representative images of ranking embedded hCOs at day 14. (B) Percent hCOs with visible budding at day 14. (C) Percent hCOs with growth into the Matrigel at day 14. (D) Representative images of ranking hCOs in the bioreactor at day 35 and day 56. (E) Percent hCOs with expected morphology at day 35. (F) Percent hCOs with expected morphology at day 56. Significance of cross-site differences was evaluated with an ANOVA implemented in a linear mixed effect logistic regression model controlling for the non-independence of multiple hCOs within the same differentiation batch using a random effect, and controlling for the ranker who evaluated the images with a fixed effect. Replicates contain between 56 and 120 hCOs. (G) Example images of burst hCOs from CHOP at day 84. (H) Percent of wells with burst hCOs across sites for all differentiation replicates. (I) Count of wells with burst hCOs by motor failure at CHOP across all differentiation replicates. (J) hCO day 56 cross-sectional area and motor failure in CHOP burst hCOs. Each point is the average area of hCOs in the same well (3–9 hCOs). Significance of motor failure and cross-sectional day 56 hCO area was evaluated in a linear mixed effect logistic regression model controlling for the other factor.

Figure 5

hCO structure across sites (A) Average cross-sectional area and growth of hCOs across each site. Cross-sectional area (top) or growth rate (bottom) shows significant differences across sites. Each point represents the average area of all hCOs from the differentiation replicate (39–120 hCO). Each site performed 3–6 differentiations. (B) Example images of tissue cleared and immunolabeled with PAX6 and NCAD day 14 hCOs from each site. (C) Total volume of day 14 hCOs across sites. Quantification of neuroepithelial bud structure at day 14 as (D) percent PAX6+ volume per hCO and (E) total NCAD+ area normalized to PAX6+ volume and TOPRO+ volume. Each point represents a single hCO. Each site imaged 9–10 hCOs. Examples images of (F) largest hCOs and (G) smallest hCOs from each site at day 84. All scale bars are 500 μm. (H) Total volume of largest and smallest hCOs across sites. (I) Representative 3D image of PAX6 and CTIP2 with annotated ventricular zone-like volumes (VZ-like) and annotated cortical plate-like volumes (CP). (J) Quantification of volume overlapped between VZ-like and CP-like annotations. Each point represents a single hCO. Each site acquired 3–6 hCOs. All scale bars are 200 μm.

Figure 6

Correlations to cell type proportions Pearson’s correlations of cell type proportions in scRNA-seq data to assay results or technical variables at (A) day 14 or (B) day 84. Nominally significant test are labeled with ^∗ and test surviving FDR correction <0.05 are labeled # (N = 7–12 differentiation replicates). (C) Correlation between primed marker ZIC2 and lower layer neuron, b proportions at day 14 (N = 11 differentiation replicates). (D) Correlation between primed marker DUSP6 and NPC, G2 phase in day 14 hCOs (N = 11 differentiation replicates).

Figure 7

Correlations of technical and biological variables to differentially expressed genes at day 14 and day 84 Proportion of DEGs across site for each cell type which correlate to technical variables or other assays at (A) day 84 and (B) day 14 (FDR <0.05). (C) Correlation between 7SK expression in day 84 lower layer neuron, b and iPSC viability at day −1 (N = 12 differentiation replicates). (D) Correlation between HSPA5 expression in day 84 lower layer neuron, b and the time iPSC were in accutase during their aggregation into 3D cultures at day −1 (N = 12 differentiation replicates). (E) Correlation between RSPO2 expression in day 14 neural progenitors in G2 phase and the iPSC passage number at day −1 (N = 11 differentiation replicates). (F) Correlation between CDH18 expression in day 14 NSCs and the percent GSX2+ cells detected by fluorescence-activated cell sorting (FACS) at day 35 (N = 6 differentiation replicates).