Individual brain organoids reproducibly form cell diversity of the human cerebral cortex

Abstract

Experimental models of the human brain are needed for basic understanding of its development and disease 1. Human brain organoids hold unprecedented promise for this purpose; however, they are plagued by high organoid-to-organoid variability 2,3. This has raised doubts as to whether developmental processes of the human brain can occur outside the context of embryogenesis with a degree of reproducibility comparable to the endogenous tissue. Here, we show that an organoid model of the dorsal forebrain can achieve reproducible generation of a rich diversity of cell types appropriate for the human cerebral cortex. Using single-cell RNA sequencing of 166,242 cells isolated from 21 individual organoids, we find that 95% of the organoids generate a virtually indistinguishable compendium of cell types, through the same developmental trajectories, and with organoid-to-organoid variability comparable to that of individual endogenous brains. Furthermore, organoids derived from different stem cell lines show consistent reproducibility in the cell types produced. The data demonstrate that reproducible development of complex central nervous system cellular diversity does not require the context of the embryo, and that establishment of terminal Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:

A critical look: Challenges in differentiating human pluripotent stem cells into desired cell types and organoids

“…For instance, hPSC-derived brain organoids often harbor neurons radially organized around ventricular zone-like progenitor regions (Kadoshima et al, 2013;Lancaster et al, 2013;Pasca et al, 2015). However, these ventricular zone regions have been reported to disperse after several weeks (Velasco et al, 2019) (Figure 6c). Moreover, the positions of TBR1 + / SATB2 + deep and BRN2 + middle cortical layer neurons appears to be reversed in hPSC-derived brain organoids (Qian et al, 2016) (Figure 6c).…”

Robust Production of Uniform Human Cerebral Organoids from Pluripotent Stem Cells

“…Several protocols have been developed to generate cerebral organoids including both directed and self-directed (Kadoshima, Sakaguchi et al, 2013, Velasco et al, 2019, Yoon et al, 2019 and here we selected a previously established method of selfpatterned organoids (Lancaster & Knoblich, 2014a, Lancaster et al, 2013 as a starting point. Our work to standardize production revealed that optimization of the early steps of organoid generation including the embryoid body formation and neural induction phases were key in ensuring the production of similar COs.…”

“…To establish a method to produce highly similar brain organoids (Fig. 1a), we explored modifications to a previously established protocol for generating selfpatterned whole-brain organoids (Lancaster & Knoblich, 2014a, Lancaster et al, 2013, which yields organoids with variable morphology and cell type composition (Quadrato, Nguyen et al, 2017, Velasco, Kedaigle et al, 2019, Yoon, Elahi et al, 2019. We primarily used female H9 human Embryonic Stem Cells (hESCs), and also validated results in a male hESC model (H1; see below).…”

Next‐generation disease modeling with direct conversion: a new path to old neurons

“…While marker expression and electrophysiological characteristics often correlate with cell identity, they are considered to be downstream of epigenetic and proteomic changes and likely follow earlier pathogenic events in neurological diseases. This concept was later extended toward new insights into cellular diversity, once also in vivo single-cell data of the developing human brain became available [130][131][132][133]. Conversely, gene expression changes related to synapse formation, voltage-gated potassium channels, or mitochondrial oxidative phosphorylation critically define mature neuronal identity [122], and transcriptomes of single human iPSCderived neurons can clearly predict neuronal functionality [123].…”

“…Camp et al [129] compared single-cell transcriptome data derived from iPSC cerebral organoids to in vivo data and found that the cells recapitulate gene expression trajectories that correspond specifically to human fetal neocortical development. This concept was later extended toward new insights into cellular diversity, once also in vivo single-cell data of the developing human brain became available [130][131][132][133]. In the iN field, Vadodaria et al [35] used a transcriptome comparison to confirm serotonergic iN identity, as they found a high similarity of serotonergic iNs to the transcriptomes of the human raphe nucleus.…”