Improved Cell Survival and Cytoprotection Enhance Reproducibility and Proper Differentiation of Cerebral Organoids Derived from Human Pluripotent Stem Cells Seungmi Ryu, Claire Malley, Pei-Hsuan Chu, Ben Ernest, Carlos Tristan, Vukasin Jovanovic, Tao Deng, Anton Simeonov, Ilyas Singeç National Center for Advancing Translational Sciences (NCATS), Stem Cell Translation Laboratory (SCTL), NIH, Rockville, MD 20850

Figure 3. Single-cell analysis A CEPT Y-27632 0 1 2 3 B Introduction Results Reelin Cajal-Retzius (scRNA-seq) of two-months 1 2 3 4 5 6 7 Cell average Calretinin cells expression SATB2 Upper layer old cerebral organoids A B C CUX1 neuron generated with either Y-27632 Self-organizing organoid models from human pluripotent stem cells (hPSCs) recapitulate some Day 1 Day 5 60 CTIP2 Lower layer aspects of brain development and function. However, current protocols are hampered by Y-27632 C+E CEPT 2020 4040 Y-27632 C+E CEPT Cluster 7 TBR1 neuron or CEPT. (A) Average 1515 3030 expression of established cell Cluster 6 TBR2 Subventricular uncontrolled cell death, considerable organoid-to-organoid heterogeneity, and lack of 1010 2020 30 PAX6 Zone

9000 type-specific markers (Trujillo et 55 1010 Cluster 3 Cluster 4 Ventricular standardization. We found that ROCK inhibitor Y-27632, the most widely used reagent to improve SOX2 Zone al., 2019) projected on UMAP 00 00 - Glo(a.u.) CellTiter

6000 0 Cluster 5 cell survival during embryoid body (EB) formation, is deficient in preventing cellular stress and tSNE_2 Radial glia plots showing higher neuronal 600 700700 6009000 6000 4500 3000 1500 9000 6000 4500 3000 1500 Cluster 1 marker expression after CEPT apoptosis, leading to nonoptimal organoid formation. Here, we used a newly developed small 500 600600 SOX2 PAX6 EOMES TBR1 BCL11B 500 Projection Maximum 4500 -30 3 treatment. (B) Schematic of molecule cocktail termed “CEPT” (Chroman 1, Emricasan, Polyamines, Trans-ISRIB according to 400400 500500 Cluster 2 2 cortical layer markers in the 300300 400400 1 3000

Chen et al., 2019, bioRxiv), which greatly enhanced cell survival and cell quality during EB Starting cell number -50 -25 0 25 50 0 developing cortex and box plots tSNE_1 Neural progenitor formation. The data demonstrated that improved cell survival during the early stages of EB 9000 6000 4500 3000 1500 9000 6000 4500 3000 1500 CUX1 SATB2 CALB2 RELN (zero-value removed) showing section, - section, Average diameter(µm) Average 1500 formation has long-lasting consequences affecting not only total cell numbers and organoid size but Starting cell number Z 3 relative expression of genes in Y- 50 µm 50 µm depth 2 Expression Level Expression 27632 vs. CEPT-generated also enhanced neuronal differentiation of organoids cultured up to 60 days. Single-cell RNA Y-27632 C+E CEPT 1 sequencing from two-month old organoids revealed that CEPT-generated organoids vs. Y-27632 0 organoids. treatment contained higher expression levels of neuronal markers representative of different cortical D Y-27632 C+E CEPT E Cortical neuron Lower cortex Upper cortex Y-27632 CEPT **** layers. Moreover, transcriptomic analysis of CEPT-generated organoids indicated significantly 60 ******** eIF2A CDH1 H9 **** **** Y-27632 CEPT 40 **** ******** **** Figure 4. Gene expressions A higher correlation to datasets from the developing human brain. Importantly, CEPT-generated **** p-eIF2A TJP1 LiPSC- LiPSC- profile of CEPT-organoids 2 organoids resulted in experimental reproducibility based on RNA sequencing analysis across 20 GM23279 GM25256 GR1.1 GM23279 GM25256 GR1.1 R

GR1.1 1.0 LiPSC - γH2AX Paxillin show stronger correlation with Replicate 1 2 3 4 5 6 1 2 3 4 5 6 7 1 2 3 4 5 1 2 3 4 5 6 1 2 3 4 5 6 1 2 3 4 5 6 7 0.9 1 individual organoids. In summary, our study identified uncontrolled cell death at the onset of 1

- Glo (a.u.) CellTiter 0.8 2 0 developing human cortex and 2 3 organoid formation as a critical quality control determinant and demonstrated that using the newly p-CHK2 3 0.7 4 less variation across individual 4 0.6 5 5

GM23279 0.5 GM23279 GM23279 6 developed CEPT cocktail dramatically improved morphogenesis, neuronal differentiation, and GAPDH organoids. (A) Pearson 6 1 1 2 overall reproducibility of cerebral organoids. correlogram based on mRNA 2 3 Y-27632 C+E CEPT 3 4 4 GM25256 transcriptomes of individual 5 5 GM25256 6 6 Figure 2. CEPT improves cell viability, generates larger EBs, and mitigates cellular stress. (A) Morphology of single organoids showing higher GM25256 1 7 2 1 - EBs generated by varying cell numbers and treatment with one (Y-27632), two (Chroman 1 + Emricasan) or four factors correlation across individual - 3 2 4 Materials and Methods CEPT-generated organoids. (B) 3 5 (CEPT) for 24 h. (B) Quantitative analyses of EB viability (CellTiter-Glo assay) and EB size (Celigo Imaging Cytometer) 4 GR1.1 LiPSC GR1.1 LiPSC 6 Silhouette plot of whole mRNA 5 from varying starting cell numbers. (C) Confocal images (z-scan) visualize EBs containing live and dead cells after a 5- 7 CEPT Cocktail: day culture period. (D) Cell survival in EBs generated by using various hPSC lines confirming reproducibility of findings. transcriptomes from individual A B organoids indicate more Chroman 1 (E) Western blot analysis showing lower expression of cellular stress markers (left panel) and higher expression of cell B Silhouette value C Y-27632 CEPT Emricasan membrane-associated proteins (right panel) in CEPT-generated EBs compared to Y-27632. homogeneous profiles in CEPT- -0.3 0 0.5 1 Polyamines 0.95 correlation

generated organoids with Spearman Trans-ISRIB comparable average silhouette 0.9 A C 0.85 Day 30 Day 60 DCX TBR1 Overlay - 27632 Figure 3. Maturation and functionality of values relative to Y-27632. (C) Y 0.8 cerebral organoids generated by CEPT. Heatmap of Spearman correlation 0.75 100 μm (A) H&E staining of representative coefficients of differentially 0.7

- 27632 TBR2 SATB2 Overlay organoids. (B) Immunostaining (day 60) for Y expressed genes in CEPT vs. Y- 600 μm 300 µm 300 μm mature neurons (MAP2) and progenitors 27632 organoids with human BrainSpansamples Cerebral cortex

CEPT Hippo.-Amygdala (SOX2), shows properly organized neural brain samples at 8-9 pcw (data Ventral forebrain CTIP2 SATB2 Overlay tube-like structures in CEPT-generated source: Allen BrainSpan GM23279 Thalamus GM25256 Cerebellum CEPT organoid. (C) Appropriate positioning of transcriptome). LiPSC-GR1.1 neurons in deep (green) and superficial TBR2 TBR1 Overlay (red) cortical layers in CEPT-treated organoids. (D) Ca2+ oscillation traces B DAPI MAP2 SOX2 Overlay indicate spontaneous electrophysiological Conclusion activity in CEPT-generated organoid 0.30 We utilized a new cytoprotective small molecule cocktail named “CEPT” that dramatically improved 0.25

- 27632 D (black, DMSO vehicle control). Inhibition of

Y cell viability and demonstrated how controlling cell survival and cell quality affects morphogenesis, Figure 1. High-throughput screening for development of the small molecule cocktail CEPT and strategy for 200 μm 0.20 GABAA receptors (red) led to an increased 0.15 differentiation, and reproducibility of hPSC-derived cerebral organoids. Optimized reproducibility of cerebral organoid formation. (A) Quantitative high-throughput screening was performed to identify a four-factor small 0.10 magnitude of each surge. ∆F/F molecule combination that is superior to the widely used ROCK inhibitor Y-27632 for improving cell survival of hPSCs 0.05 Bicuculline organoids by CEPT will allow more rigorous studies of neurodevelopment and disease modeling in 0.00

(Chen et al. (2019) bioRxiv). (B) Schematic of cerebral organoid protocol to generate EBs by using the CEPT cocktail CEPT 50 100150200250300350400450500550600

-0.050.025 vitro. Lastly, organoid reproducibility will leverage drug screening and drug testing under more versus Y-27632. Note that CEPT and Y-27632 were only applied for the first 24 h. -0.1025 sec Untreated controlled and predictable conditions. Funding Source: NIH Common Fund; NCATS Intramural Research