Project description:Organoids derived from human pluripotent stem cells recapitulate the early three-dimensional organization of human brain, but whether they establish the epigenomic and transcriptional programs essential for brain development is unknown. We compared epigenomic and gene regulatory features in cerebral organoids and human fetal brain, using genome-wide, base resolution DNA methylome and transcriptome sequencing. Transcriptomic dynamics in organoids faithfully modeled gene expression trajectories in early-to-mid human fetal brains. We found that early non-CG methylation accumulation at super-enhancers in both fetal brain and organoids marks forthcoming transcriptional repression in the fully developed brain. 74% of 35,627 demethylated regions identified during organoid differentiation overlapped with fetal brain regulatory elements. Interestingly, pericentromeric repeats showed widespread demethylation in multiple types of in vitro human neural differentiation models but not in fetal brain. Our study reveals that organoids recapitulate many epigenomic features of mid-fetal human brain and also identified novel non-CG methylation signatures of brain development.
Project description:Cerebral organoids â three-dimensional cultures of human cerebral tissue derived from pluripotent stem cells â have emerged as models of human cortical development. However, the extent to which in vitro organoid systems recapitulate neural progenitor cell proliferation and neuronal differentiation programs observed in vivo remains unclear. Here we use single-cell RNA sequencing (scRNA-seq) to dissect and compare cell composition and progenitor-to-neuron lineage relationships in human cerebral organoids and fetal neocortex. Covariation network analysis using the fetal neocortex data reveals known and novel interactions among genes central to neural progenitor proliferation and neuronal differentiation. In the organoid, we detect diverse progenitors and differentiated cell types of neuronal and mesenchymal lineages, and identify cells that derived from regions resembling the fetal neocortex. We find that these organoid cortical cells use gene expression programs remarkably similar to those of the fetal tissue in order to organize into cerebral cortex-like regions. Our comparison of in vivo and in vitro cortical single cell transcriptomes illuminates the genetic features underlying human cortical development that can be studied in organoid cultures. 734 single-cell transcriptomes from human fetal neocortex or human cerebral organoids from multiple time points were analyzed in this study. All single cell samples were processed on the microfluidic Fluidigm C1 platform and contain 92 external RNA spike-ins. Fetal neocortex data were generated at 12 weeks post conception (chip 1: 81 cells; chip 2: 83 cells) and 13 weeks post conception (62 cells). Cerebral organoid data were generated from dissociated whole organoids derived from induced pluripotent stem cell line 409B2 (iPSC 409B2) at 33 days (40 cells), 35 days (68 cells), 37 days (71 cells), 41 days (74 cells), and 65 days (80 cells) after the start of embryoid body culture. Cerebral organoid data were also generated from microdissected cortical-like regions from H9 embryonic stem cell derived organoids at 53 days (region 1, 48 cells; region 2, 48 cells) or from iPSC 409B2 organoids at 58 days (region 3, 43 cells; region 4, 36 cells).
Project description:The goal of the study was to compare gene profile between iPSC-derived cerebral organoids, human fetal brain tissue, and human adult brain tissue
Project description:To understand whether the intrinsic capacity of the fetal brain in vitro self-organizes into organoids (FeBOs) to secrete a tissue-like “matrisome” is linked to the maintenance of cell-to-cell organization and integrity, we compared the secretome of intact FeBOs and FeBO-derived neurospheres. We also performed side-by-side comparative proteomic analysis of FeBOs and human fetal brain tissue to investigate if the extracellular matrix (ECM) production in the FeBOs resembled a proper tissue-like ECM niche. Moreover, we also included proteomic analysis of unguided pluripotent stem cell (PSC)-cerebral organoids and PSC-cortical spheroids to understand how the human brain tissue ECM-niche compared not only to the FeBOs but also to PSC-derived 3D brain models.
Project description:Cerebral organoids – three-dimensional cultures of human cerebral tissue derived from pluripotent stem cells – have emerged as models of human cortical development. However, the extent to which in vitro organoid systems recapitulate neural progenitor cell proliferation and neuronal differentiation programs observed in vivo remains unclear. Here we use single-cell RNA sequencing (scRNA-seq) to dissect and compare cell composition and progenitor-to-neuron lineage relationships in human cerebral organoids and fetal neocortex. Covariation network analysis using the fetal neocortex data reveals known and novel interactions among genes central to neural progenitor proliferation and neuronal differentiation. In the organoid, we detect diverse progenitors and differentiated cell types of neuronal and mesenchymal lineages, and identify cells that derived from regions resembling the fetal neocortex. We find that these organoid cortical cells use gene expression programs remarkably similar to those of the fetal tissue in order to organize into cerebral cortex-like regions. Our comparison of in vivo and in vitro cortical single cell transcriptomes illuminates the genetic features underlying human cortical development that can be studied in organoid cultures.
Project description:Human pluripotent stem cell-derived 3D neural organoids have been shown to recapitulate the major features and cytoarchitecture of early human brain development. We used three commercially available kits: STEMdiff™ Cerebral Organoid Kit, STEMdiff™ Dorsal Forebrain Organoid Differentiation Kit, and STEMdiff™ Ventral Forebrain Organoid Differentiation Kit. These neural organoids contain unique and diverse cell types which model the early brain and are patterned with developmental cues. Here, we sought to investigate the cellular diversity of these organoids using single-cell RNA sequencing.
Project description:The human cerebral cortex possess distinct structural and functional features that are not found in the lower species traditionally used to model brain development and disease. Accordingly, considerable attention has been placed on the development of methods to direct pluripotency stem cells to form human brain-like structures termed organoids. However, many organoid differentiation protocols are inefficient and display marked variability in their ability to recapitulate the three-dimensional architecture and course of neurogenesis in the developing human brain. Here, we report optimized organoid culture methods that efficiently and reliably produce cortical and basal ganglia structures similar to those in the human fetal brain in vivo. Neurons within the organoids are functional and exhibit network-like activities. We further demonstrate the utility of the organoid system for modeling the teratogenic effects of Zika virus on the developing brain and identifying new candidate receptors and therapeutic compounds that can mitigate its desructive actions.
Project description:The human cerebral cortex possess distinct structural and functional features that are not found in the lower species traditionally used to model brain development and disease. Accordingly, considerable attention has been placed on the development of methods to direct pluripotency stem cells to form human brain-like structures termed organoids. However, many organoid differentiation protocols are inefficient and display marked variability in their ability to recapitulate the three-dimensional architecture and course of neurogenesis in the developing human brain. Here, we report optimized organoid culture methods that efficiently and reliably produce cortical and basal ganglia structures similar to those in the human fetal brain in vivo. Neurons within the organoids are functional and exhibit network-like activities. We further demonstrate the utility of the organoid system for modeling the teratogenic effects of Zika virus on the developing brain and identifying new candidate receptors and therapeutic compounds that can mitigate its desructive actions.
Project description:Brain organoids derived from human pluripotent stem cells recapitulate the course of human brain development. We applied proteomic analysis to human brainstem organoids which were developed by a novel method.