IMPROVING CELL SURVIVAL TO CONTROL CEREBRAL FORMATION FROM HUMAN PLURIPOTENT STEM CELLS Seungmi Ryu, Yu Chen, Pei-Hsuan Chu, Claire Malley, Carlos Tristan, Christopher P. Austin, Anton Simeonov, Ilyas Singeç National Center for Advancing Translational Sciences (NCATS), Translation Laboratory (SCTL), NIH, Rockville, MD 20850

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Introduction Day 30 Day 60

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Generation of in vitro organoid models from pluripotent stem cell holds great promise for disease modeling, -

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aspects of the physiologically complex, multicellular, and layered architecture of the . However,

C+E C+E significant variability and experimental challenges exist in organoid formation protocols. One such C+E challenge is the large amount of cell death when pluripotent cells are dissociated and aggregated into 3D structures. Currently, the ROCK inhibitor Y-27632 is the most widely used reagent to improve cell survival.

Nevertheless, poor cell survival and emergence of debris are evident even after treatment with Y-27632 CEPT

suggesting that uncontrolled cell stress introduces an inherent systematic shortcoming in currently used CEPT CEPT protocols. Here, we used a novel small molecule cocktail, recently developed at NCATS, that greatly enhances cell survival during cerebral organoid formation. Improved cell survival at the onset of sphere Fig. 3: Gross and histology images of cerebral organoids at day 60. formation generated larger, healthier and better controlled organoids. Ongoing molecular and functional (A) Digital image of organoids at day 60 cultured in ultra-low attachment plates (6-well) on an orbital shaker. Each well contained experiments are aimed at demonstrating that optimal cell survival enhances morphogenesis, differentiation, equal numbers of 14 organoids. (B) H&E staining of representative organoids (day 30 and 60) derived from iPSCs. (C) and reproducibility of cerebral organoids. Immunostaining of sectioned organoids at day 60 for neural progenitors (SOX2) and (TUJ1). Note the larger amounts of SOX2+ and TUJ1+ cells in C+E and CEPT versus Y-27632. Scale bar, 300 µm.

Materials and Methods A B DAPI FOXG1 Overlay DAPI DAPI DAPIOverlay DAPI Fig. 4: Immunohistochemistry for brain region- and -specific

27632 markers. (A) Representative images - Y illustrating more numerous cells C+E A B Y-27632 expressing the forebrain marker 12,744 MAP2 TBR2 FOXG1Overlay CTIP2 FOXG1 after CEPT exposure as hESC compounds CEPT compared to other treatment groups

Small molecule C+E Media: STEMdiffTM Cerebral Organoid Kit from STEMCELL Technologies at day 36. (B) Staining for MAP2 addition Optimized formulation based on previous literatures (Lancaster MA et al. (2013) Nature; Lancaster MA &Knoblich JA. (2014) Nat. Protoc.) (marker for mature neurons), TBR2 Resuspend Embed in (Intermediate Progenitor Neuron), (EB) formation EBs Matrigel® Overlay OverlayISL1 OverlayOverlay Overlay FOXG1 (forebrain marker), and

24hr after, change Change media every 3 days, CTIP2 (deep-layer neurons), of

0 media every 2 days 5 7 10 Culture on orbital shaker 40+ CEPT CellTiter-Glo CEPT-treated organoids at day 60. Feeder free, EB Formation Induction Expansion Maturation Scale bar, 300 µm. mTesRTM1 Medium Medium Medium Medium

neuron projection development (GO:0031175) A central neuron development (GO:0021954) Establishment of CEPT cocktail based on establishment of synaptic vesicle localization (GO:0097480) B neuroepithelial cell differentiation (GO:0060563) quantitative high-throughput (qHTS) combinatorial screening glial cell differentiation (GO:0010001) nervous system development (GO:0007399) Y-27632 NEUROD6 DCX neuron development (GO:0048666) ❖ Fully automated screening of 15,333 compounds in 1536-well format -> 29 hits CEPT regulation of neuron differentiation (GO:0045664) ❖ Combinatorial matrix screening of the 29 compounds revealed synergistic roles of C+E compound axon guidance (GO:0007411) EB Formation Induction Expansion Organoid Maturation noradrenergic neuron differentiation (GO:0003357) ❖ Additional screening of 7,599 compounds at ultra-low cell density (10 cells/well) axonogenesis (GO:0007409) neural tube development (GO:0021915) ❖ Discovery of a small-molecule cocktail “CEPT” that dramatically improves hPSC survival autonomic nervous system development (GO:0048483) central nervous system neuron differentiation (GO:0021953) glial cell development (GO:0021782) generation of neurons (GO:0048699) Fig. 1: High-throughput screening for development of cell survival cocktail and strategy for cerebral organoid formation. receptor localization to synapse (GO:0097120) positive regulation of axon extension (GO:0045773) Combined score neuron migration (GO:0001764) -log10(Value) (A) Quantitative HTS was performed to identify a small molecule combination that is superior to the widely used ROCK inhibitor Y-27632 regulation of neuron projection development (GO:0010975) for improving cell survival of human pluripotent stem cells (hPSCs). (B) Cerebral organoids were generated from hPSCs using a 0 5 10 15 commercially available kit adapted based on previous literatures (Lancaster MA et al. (2013) Nature; Lancaster MA &Knoblich JA. (2014) TBR1 FOXG1 MAP2 C Y-27632 CEPT Nat. Protoc.). In comparison to Y-27632, novel drug combinations C+E or CEPT produced larger, healthier, and more reproducible kidney (bulk tissue) neuronal epithelium blood dendritic cells fetal brain cortex organoids. dendritic cell midbrain midbrain prefrontal cortex CD34+ cell kidney (bulk tissue) neuronal epithelium motor neuron Results fetal brain cortex pancreatic islet human cord blood blood dendritic cells Combined score Combined score myoblast fetal brain -log10(Value) -log10(Value) A B C bone marrow (bulk tissue) neuronal progenitor cells Day 1 (24 h after EB formation) Day 5 (end of EB culture phase) 0 20 40 60 80 0 30 60 90 120 150 Y-27632 C+E CEPT Day 1 Day 5 Bright Live Dead Bright Live Dead Fig. 5: Single-cell analysis (scRNA-seq) of cerebral organoid at day 72 generated with either Y-27632 or CEPT.

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Seeded cell number Seeded cell number CEPT 1. We utilized a small molecule cocktail named “CEPT” that dramatically improved cell viability and demonstrated how optimization of cell survival affects morphogenesis, differentiation, and reproducibility of hPSC-derived cerebral organoids. Fig. 2: Improved cell viability and superior morphology of EBs generated with CEPT cocktail. 2. CEPT treatment for 24 h during EB formation resulted in larger and more consistent brain organoids. (A) Morphology of single EB in 96-well ultra-low attachment plates with various starting cell number 24 h after compound treatment. (B) 3. Optimization and reproducibility of CEPT-treated organoids will allow for the improved study of neurodevelopment and Quantitative analyses of single EB measuring viability and size. Cell viability was measured by using the CellTiter-Glo assay (Promega) relevant disease phenotypes in vitro. Also, organoid reproducibility will leverage drug screening and drug testing under and EB diameter was analyzed using high-content imaging (Celigo). (C) Visualization of live and dead cells within single EBs. Cells were more controlled and predictable conditions. stained with a Live/Dead assay kit (Thermo Fisher). Scale bar, 200 µm. Funding Source: NIH Common Fund; NCATS Intramural Research