Reliability of human cortical organoid generation

Abstract

The differentiation of pluripotent stem cells in three-dimensional cultures can recapitulate key aspects of brain development, but protocols are prone to variable results. Here we differentiated multiple human pluripotent stem cell lines for over 100 d using our previously developed approach to generate brain-region-specific organoids called cortical spheroids and, using several assays, found that spheroid generation was highly reliable and consistent. We anticipate the use of this approach for large-scale differentiation experiments and disease modeling.

Figure 1.. Success of differentiation and transcriptional reliability of human cortical spheroids.

a, Scheme illustrating the derivation of hCS-FF from hPSCs and the assays used. b, Representative images of neural spheroids at day 0, 6 and 14 of differentiation. c, Circularity (4p × area/perimeter2) of day 6 neural spheroids derived from 4 hiPSC lines. A value of 1.0 indicates a perfect circle. d, Gene expression of FOXG1, PAX6, NKX2.1 relative to GAPDH in hCS-FF at day 25 of Differentiation (n = 12 hiPSC lines from 11 subjects). Mean ± s.e.m. are shown. e, Percentage of successful differentiations up to 100 days for 12 hiPSC lines (n= 85 experiments; number per line indicated inside bars). f, Principal component analysis of hCS-FF and hCS-MEF at 4 stages of in vitro differentiation. Differentiation of the same line are indicated by a gray line (days 25, 50, 75, 100: n = 22, 25, 25, 22 hCS-FF and 3, 5, 8, 4 hCS-MEF samples, respectively). g, Spearman’s correlation of samples obtained from different individuals (between individuals) or from multiple differentiations of the same hiPSC lines (within individual); two-sided Wilcoxon–Mann–Whitney test, P< 0.03. Day 25, 50, 75, 100: n = 202, 269, 281, 206 samples (between individual) and 33, 41, 47, 31 samples (within individuals), respectively. Middle hinge corresponds to median, and lower and upper hinges correspond to first and third quartiles. RNA-seq data in f and g were obtained from n = 6 hiPSC lines derived from 6 individuals and differentiated in multiple independent differentiation experiments each.

Figure 2.. Single-cell characterization of human cortical spheroids.

a,b, Single-cell profiling of hCS-MEF (n = 7,340 cells) and hSS-MEF n = 4,771 cells) (a) versus hCS-FF derived from 3 different individuals (hCS-FF-1, n = 4,649 cells; hCS-FF-2, n = 4,389 cells; hCS-FF-3, n = 3,088 cells) (b) at day 105 of differentiation. The correlation (log10 transformed mean of molecules per cell per gene) between total hCS-MEF and each of the hCS-MEF cultures or hSS-MEF is indicated. c,d, Clustering (c) and proportions (d) of all single cells across conditions (glutamatergic neuron cluster 1, n = 11,367 cells; intermediate progenitor cluster 2, n = 1,018 cells; radial glia cluster 3, n = 4,217 cells; astroglia cluster 4, n = 2,036 cells; ventral progenitor cluster 5, n = 1,915 cells; GABAergic neuron cluster 6, n = 2,520 cells; OPC cluster 7, n = 194 cells; choroid plexus cluster 8, n = 170 cells; 800 cells not assigned to a cluster). e, Representative cryosection at day 150 of differentiation stained for deep (CTIP2) and superficial (SATB2) neuronal markers and the glial marker GFAP. f,g, Proportion of cells expressing layer-specific cortical markers (TBR1, CTIP2, SATB2) at day 75 (f) and days 135ȓ150 (g) of differentiation from 4–5 hiPSC lines derived from 4–5 individuals (two-way ANOVA, F2,21 = 5.44, P = 0.01 for interaction; mean ± s.e.m. are shown; sample size indicated on each column). h, Developmental time course for GFAP⁺ cell generation, quantified in dissociated hCSs (n = 3–5 hiPSC lines per time point from 7 hiPSC lines derived from 6 individuals); ANOVA F3,11 = 21.89, P < 0.0001; mean ± s.e.m.).