Engineering of human brain organoids with a functional vascular-like system

Çakır, Barış; Xiang, Yangfei; Tanaka, Yoshiaki; Kural, Mehmet H.; Parent, Maxime; Kang, Young-Jin; Chapeton, Kayley; Patterson, Benjamin; Yuan, Yifan; He, Chuan; Raredon, Micha Sam Brickman; Dengelegi, Jake; Kim, Kun-Yong; Sun, Pingnan; Zhong, Ming; Lee, Sang-Ho; Patra, Prabir; Hyder, Fahmeed; Niklason, Laura E.; Lee, Sang-Hun; Yoon, Young-sup; Park, In‐Hyun

doi:10.1038/s41592-019-0586-5

Cited by 632 publications

(545 citation statements)

References 39 publications

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“…The 656 most advanced in vitro human cerebral organoid differentiation protocols currently available 657 can only recapitulate relatively early embryonic developmental stages (reviewed Kwiecinski and Lancaster, 2019)). Whether further development of human cerebral organoids 659 through in vitro vascularisation(Cakir et al, 2019) or transplantation into the mouse(Mansour 660 et al, 2018) can overcome this developmental obstacle and accelerate human neuron 661 maturation to experimentally tractable time scales is currently unknown. To our knowledge, 662 the mouse data presented here is the first report of mammalian neurons derived in vitro from 663 pluripotent cells that harbor mCH at levels similar to those present in neurons in vivo, and 664 opens the door to further, targeted investigations of the regulatory pathways and 665 environmental factors involved.…”

mentioning

confidence: 99%

Conserved and divergent features of DNA methylation in embryonic stem cell-derived neurons

Martin

Poppe²,

Olova

et al. 2020

Preprint

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27DNA methylation functions in genome regulation and is implicated in neuronal maturation. 28Early post-natal accumulation of atypical non-CG methylation (mCH) occurs in neurons of 29 mice and humans, but its precise function remains unknown. Here we investigate mCH 30 deposition in neurons derived from mouse ES-cells in vitro and in cultured primary mouse 31 neurons. We find that both acquire comparable levels of mCH over a similar period as in vivo. 32In vitro mCH deposition occurs concurrently with a transient increase in Dnmt3a expression, 33 is preceded by expression of the post-mitotic neuronal marker Rbfox3 (NeuN) and is enriched 34 at the nuclear lamina. Despite these similarities, whole genome bisulfite sequencing reveals 35 that mCH patterning in mESC-derived neurons partially differs from in vivo. mESC-derived 36 neurons therefore represent a valuable model system for analyzing the mechanisms and 37

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mentioning

confidence: 99%

Conserved and divergent features of DNA methylation in embryonic stem cell-derived neurons

Martin

Poppe²,

Olova

et al. 2020

Preprint

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“…To address this limitation, GBOs could be co-cultured with immune and/or endothelial cells to reconstitute the microenvironment. This has already been demonstrated for many cerebral organoid models [35][36][37][38] , and thus should be feasible in GBOs.…”

Section: Limitations Of Glioblastoma Organoid Culturementioning

confidence: 68%

“…This incubation step substantially increases cell viability. 36. Aspirate GBO freezing medium and resuspend GBO pieces in 2 mL fresh GBO freezing medium.…”

Section: Cryopreservation Of Glioblastoma Organoids (Timing 2-3 H)mentioning

confidence: 99%

Generation and biobanking of patient-derived glioblastoma organoids and their application in CAR-T cell testing

Jacob

Ming

Song

2020

Preprint

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Short Summary: Detailed procedures for generating and biobanking glioblastoma organoids from resected patient tumor tissue and testing CAR-T cell efficacy by co-culture. Additional procedures for tissue processing, immunohistology, and detecting hypoxia gradients and actively proliferating cells. AbstractGlioblastoma tumors exhibit extensive inter-and intra-tumoral heterogeneity, which has contributed to the poor outcomes of numerous clinical trials and continues to complicate the development of effective therapeutic strategies. Current in vitro models do not preserve the cellular and mutational diversity of parent tumors and often require a lengthy generation time with variable efficiency. Here, we describe detailed procedures for generating glioblastoma organoids (GBOs) from surgically resected patient tumor tissue using a chemically defined medium without cell dissociation. By preserving cell-cell interactions and minimizing clonal selection, GBOs maintain the cellular heterogeneity of parent tumors. We include methods for passaging and cryopreserving GBOs for continued use, biobanking, and long-term recovery. We further describe procedures for investigating patient-specific responses to immunotherapies by co-culturing GBOs with chimeric antigen receptor (CAR) T cells. This protocol takes approximately 2-4 weeks to generate GBOs and 5-7 days to perform CAR-T cell co-culture. Competence with human cell culture, tissue processing, and immunohistology is required for optimal results.An ideal in vitro model for glioblastoma would maintain cellular diversity, preserve native cell-cell interactions, recapitulate defining histological characteristics, and maintain the transcriptomes and mutation profiles of parent tumors. To be useful for personalized medicine approaches, the model would need a quick generation time with high fidelity for success and be scalable to test potential therapeutics. This would require maintaining patient-derived glioblastoma tissue in culture with minimal perturbation. Towards this goal, we opted to directly culture micro-dissected tumor pieces without cell dissociation to preserve cell-cell interactions and minimize clonal selection. We start from resected patient tumor tissue that can be sampled from different tumor foci or subdivided into regional samples to analyze intra-tumoral heterogeneity. Through testing of many medium formulations, we learned that tumor pieces did not require basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), serum, or added extracellular matrix such as Matrigel to survive, form organoids, and proliferate at rates similar to parent tumors 11 . We optimized a chemically defined medium that contains basic nutrients and components needed to support glial cell health with few exogenous factors to minimize clonal selection induced by growth factors and decrease potential treatment confounders. This resulted in an optimized version of our medium used to maintain human brain organoid cultures 12,13 . We culture tumor pieces on an orbital shaker to inc...

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“…endothelial, pericyte, and epithelial), reducing the cost to produce and mature multicellular tissues. A similar approach was used recently via ETV2expressing cells to vascularize cortical organoids (Cakir et al, 2019). Future work can take advantage of assembled layered genetic circuits (Kiani et al, 2014;Nissim et al, 2014) embedded in hiPSCs that can be activated based on cell state, decreasing the need for delivery of genetic cargo during differentiation in organoids.…”

Section: Discussionmentioning

confidence: 98%

Synthetic Maturation of Multilineage Human Liver Organoids via Genetically Guided Engineering

Velazquez

LeGraw

Moghadam

et al. 2020

Preprint

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Graphical AbstractHIGHLIGHTS  In vitro tissue maturation via genetically encoded molecular programs  Computational analysis to identify maturation transcription factors in liver organoids  Promoting vascularization of organoids via genetically encoded molecular programs  Single cell analysis of parenchymal and non-parenchymal cells  Modeling of native liver functions and in vivo therapeutic potential SUMMARY Pluripotent stem cell (PSC)-derived organoids are emerging as novel human-based microphysiological models but display immature phenotypes with limited subsets of endothelial or stromal cells. Here we demonstrate that in vitro manipulation of gene regulatory networks (GRNs) in PSC-derived liver organoids selected either through computational analysis or targeted tissue design can advance tissue maturation in vitro. Through an unbiased comparison with the genetic signature of mature livers, we identify downregulated GRNs in fetal liver organoids compared to adult livers. We demonstrate that overexpression of PROX1 and ATF5, together with targeted CRISPR-based transcriptional activation of endogenous CYP3A4, drives maturation in vitro. Single cell analyses reveal hepatobiliary-, endothelial-, and stellate-like cell populations. The engineered organoids demonstrate enhanced vasculogenesis, capture native liver characteristics (e.g. FXR signaling, CYP3A4 activity), and exhibit therapeutic potential in mice. Collectively, our approach provides a genetically guided framework for engineering developmentally advanced multilineage tissues from hiPSCs.

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Engineering of human brain organoids with a functional vascular-like system

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Cited by 632 publications

References 39 publications

Conserved and divergent features of DNA methylation in embryonic stem cell-derived neurons

Conserved and divergent features of DNA methylation in embryonic stem cell-derived neurons

Generation and biobanking of patient-derived glioblastoma organoids and their application in CAR-T cell testing

Synthetic Maturation of Multilineage Human Liver Organoids via Genetically Guided Engineering

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