Table 1.
List of scRNA-seq and scATAC-seq studies in human brain organoids.
| Authors | Reference | Main focus of study | Type of organoid protocol | Single-cell technology | Number of PSC line | Stages | Biological samples | Total cells | Main analytical toolkits: visualization, clustering, batch effect, trajectory, gene network | Main cluster annotations | Brain tissue reference (human scRNA-seq/RNA-seq) | Main findings and observations related to the scRNA-seq of human organoids | ||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| P | D | E | M | L | ||||||||||||
| Camp et al. | [85] | Organoid model validation | Undirected | Fluidigm C1 | 1 iPSC, 1 ESC | X | 11 | 508 | Seurat, BackSPIN, t-SNE, Monocle | early/ventral NPC, dorsal NPC, dorsal neur., ventral neur., mesenchymal cells, RSPO + cells | 226 cells neocortex (12–13 pcw) | First study to demonstrate a similar single-cell expression profile in organoid and cortex. Yet, organoids presented less basal progenitor than apical progenitors compare with brain. | ||||
| Quadrato et al. | [145] | Organoid model validation | Undirected | Drop-Seq | 1 iPSC, 1 ESC | X | X | 31 | ##### | Seurat, t-SNE | NEC, div., Forebrain (RG, IPC, Callosal and Corticofugal neur., IN), Ast., dopa neur., retina, mesodermal prog. | Pollen et al. [93] | Organoid mimick brain neurogenesis with coherent cell subtypes progression. Presence of mesodermal cells. Bioreactors batch influences cell composition. | |||
| Birey et al. | [134] | Regional specification and interaction | Directed: cortical and subpallial + assembloids | BD Resolve/ Smart-Seq2 | 1 iPSC (?) +DLX-lineage | X | 4 | ##### | Seurat, t-SNE | hCS: RG, IPC, Glut. Neur., Choroid, hSS: GABA neur., ventral prog., Ast., OPC | Specified forebrain subregions. Assembloids reproduce regional interactions, with migration of interneurons expressing subpallial and cortical IN markers (e.g., DLXs) | |||||
| Sloan et al. | [123] | Cell-subtype specification | Directed: cortical spheroid | Smart-Seq2 | 2 iPSC | X | X | 8 | 710 | PAGODA, t-SNE, Monocle | Neur., div. glial, intermediate glial, mature Ast. | Pollen et al. [93], Darmanis et al. [77] | Described the maturation of astrocytes over ~20 months in cortical spheroids, with transcriptional and functional similarities to primary tissue. | |||
| Xiang et al. | [135] | Regional specification and interaction | Directed: cortical and MGE | 10X Chromium | 1 ESC, 1 iPSC | X | X | 8 | ##### | Seurat, t-SNE, random-forest | NPC, IPC, cortical neur., IN, Non-committed neur., Subplate, Oligo., Ast., Glia, OPC, Ependymocyte | Darmanis et al. [77] | MGE organoids generate NKX2-1+ IN lineage and oligodendrocytes. Cortical organoids generate interneurons (NKX2-1 negative). | |||
| Madhavan et al. | [125] | Cell-subtype specification | Directed: oligocortical | 10X Chromium | 1 ESC | X | 1? | ##### | CellRanger, t-SNE | Glial and neuronal clusters | Nowakowski et al. [86] | Ability to promote oligodendrocyte lineage while keeping overall cortical fate in spheroids. | ||||
| Amiri et al. | [70] | Organoid model validation | Directed: dorsal forebrain | 10X Chromium | 1 iPSC | X | X | 3 | ##### | Seurat, t-SNE, Monocle | early RG, oRG, tRG, MGE-RG, divRG, IPC, early EN, late EN, Glycolysis, Choroid, U | Nowakowski et al. [86]; Liu et al. [104] | Organoids model cortical development equivalent to 5 and 16 pcw brain tissue, reproducing radial glial and early neuronal diversity. Mapping of enhancer elements and target genes active over organoid development. | |||
| Yoon et al. | [144] | Reproducibility | Directed: cortical | BD Rhapsody system | 3 iPSC | X | 3 | ##### | BD Rhapsody, t-SNE | RG, IPC, ventral prog., Glut. Neur., GABA neur., Ast., OPC, choroid | Previous data | Reliability of cell composition over multiple IPS lines, culture conditions, and differentiation. | ||||
| Marton et al. | [124] | Cell-subtype specification | Directed: oligodendrocyte | Smart-Seq2 | 2 iPSC | X | 2 | 295 | k-mean, t-SNE, Monocle | Neurons, Astrocytes, Microglia, Endothelial, OPC, div. prog., myelinating Oligo. | Darmanis et al. [77]; Sloan et al. [123] | Induced oligodendrocytes myelinated neurons with transcriptional similarity with in vivo counterparts. Transcriptional variability of cells decreased over time. | ||||
| Pollen et al. | [151] | Evolution | Directed: telencephalic | Fluidigm C1 | 10 PSC | X | X | 32 | 1223 | NN-Louvain, CCA, t-SNE, WGCNA | RG, div (G1/S/G2/M), IPC, Reelin+ EN, Maturing EN, IN, Glycolysis, hindbrain, Choroid, retina, mesenchymal cells, outliers | Gene networks are preserved in organoids. Some lines generate off-target cells (mesenchymal, hindbrain, retina). Noted a variability in cortical cells composition, presence of glycolysis related gene modules and less interneurons than in primary samples. | ||||
| Field et al. | [150] | Evolution and lncRNA | Directed: cortical | 10X Chromium | 1 ESC | X | X | X | 4 | ##### | k-mean, t-SNE | RG, Neuroepithelium, Cajal–Retzius cells | Described the cell type-specific presence of evolutionary conserved transiently-expressed lncRNA in human organoids. | |||
| Xiang et al. | [116] | Regional specification and interaction | Directed: Thalamic | 10X Chromium | 1 ESC | X | X | 2 | ##### | Seurat, CCA, Destiny? | NPC, IPC, immature neur., mature neur., glial prog., Ast., BMP-response, Cilium, proteoglycan, UPR | Darmanis et al. [77]; Thalamus (Allen Brain Atlas) | Thalamic organoids mostly produced glutamatergic neurons and beared more similarity with cortical organoids than MGE organoids, while exhibiting specific gene expression. | |||
| Giandomenico et al. | [120] | Regional interaction | Undirected | 10X Chromium | 2 ESC | X | 6 | ##### | Seurat, t-SNE, Monocle | VZ-SVZ RG, div. prog., deep-layer neur., immature upper layer neur., mature neur., IN | Camp et al. [85] | Air–liquid interface promotes survival and axon outgrowth with intracortical and subcortical identities. | ||||
| Velasco et al. | [143] | Reproducibility | Directed: Dorsal forebrain | 10X Chromium | 4 iPSC, 1 ESC | X | X | 21 | ##### | Seurat, CCA, t-SNE, Monocle | RG, oRG, div. prog., IPC, Cajal–Retzius cells, immat./mat. projection neur. (callosal, corticofugal), div. IN prog., immat.IN, U, Ast. | Nowakowski et al. [86]; Habib et al. [179], Fan et al. [89] | Dorsally patterned forebrain organoids generate similar cell type composition with low intra- and inter-line variabilities, similarly to brain tissue datasets. | |||
| Trujillo et al. | [119] | Organoid model validation | Directed: cortical spheroid | 10X Chromium | Not explicited (?) | X | X | X | 4 | ##### | Seurat, UMAP | Prog., div. Prog., IPC, Glut. neur., GABA neur., Glia, U | Functional GABAergic interneurons develop after 6 months in cortical organoids. Glial cells become more abundant than progenitors after 3 months. | |||
| Cakir et al. | [127] | Cell-subtype specification | Directed: cortical and vascular cells | 10X Chromium | 1 ESC | X | 2 | ##### | Seurat, CCA, t-SNE, Monocle | RG, IPC, cortical neur., IN, Ast., glial prog., endothelial cells and progenitors, cilium, BMP-response, proteoglycan, EMT, UPR | Zhong et al. [88] | Induction of proto-vascularization in organoids resulted in acceleration of neuronal maturation. Endothelial-like cells expressed vasculogenesis, pericytes, collagen and cell adhesion markers. | ||||
| Kanton et al. | [152] | Evolution | Undirected | 10X Chromium | 6 IPSC, 1 ESC | X | X | X | X | 36 | ##### | Seurat, CCA, t-SNE, RSS, SPRING Diffusion map, RNA velocity | Stem cells, NPC forebrain (dorsal and ventral telencephalon, diencephalon), midbrain, hindbrain and retina, EN, IN, Ast. | Nowakowski et al. [86]; Pollen et al. [151]; Miller et al. [6]; BrainSpan; Camp et al. [85]; Mora-Bermúdez et al. [149] | Excitatory and inhibitory lineages are distinguished after 2 months, and astrocytes at 4 months. iPSC lines varied in cell composition but lineage-specific signatures remained highly correlated. Human organoids present a delayed maturation compare with chimpanzee and macaque organoids. | |
| scATAC-seq C1 Fluidigm | 9 | 521 | MACS2, Cicero, t-SNE, Diffusion map | Pluripotency, Neuroectoderm, Neuroepithelium, NPC, Neuron | Human VISTA enhancer | Differentially accessible peaks with chimpanzee with cell specific pattern are enriched for single-nucleotide evolutionary change. | ||||||||||
| Bhaduri et al. | [87] | Organoid model validation | 2 directed + 1 undirected | 10X Chromium | 3 iPSC, 1 ESC | X | X | X | X | 36 | ##### | Seurat, t-SNE, WGCNA, variance partition | RG, mixed prog./neur., EN, IN, endothelial, Ast., U | 5 cortical samples (6-22 GW), 7 regions, 189,409 cells | Stress pathway is activated in organoid models and impaired cell diversity (low IPC, oRG, and upper layer neurons) and specification compare with brain samples. Yet organoids display diverse regional signatures. | |
| Qian et al. | [122] | Organoid model validation | Directed: cortical (sliced) | SPLiT-seq (nuclei) | 1 iPSC | X | 1 | ##### | Seurat, t-SNE | RG, div, IPC, Neurons Layer I, upper layer, deep layer | Zhong et al. [88]; Zhang et al. [180] | Presence of layer specific neocortical neurons and mature astrocytes. | ||||
The information aggregated in this table focuses solely on human pluripotent stem cell (PSC) i.e., ESC- or iPSC-derived brain organoid scRNA-seq data published as of April 2020. The table highlights the historic progression of scRNA-seq usage and reflects the evolution of the technology (from low number of cells at high coverage to high number at lower coverage) and methods. All study metrics (col. F-M) were reported from the published “Method” section, main text, figures, supplementary information, or tables to the best of our comprehension, as no standard for scRNA-seq report exists. Annotations of the main cell types (col. O) have been harmonized when judged equivalent. The difference in type of clusters depends on the protocol used (col D). For instance, undirected protocol generates multiple regional identities (forebrain, midbrain, hindbrain) and mesenchymal fates and regional-directed protocols focus the annotation on relevant cell types. Difficulties and inconsistencies in annotation can partially be explained by the fact that accurate unsupervised clustering (col N) is difficult and its level of resolution is highly dependent on the question central to the study. This highlights the importance of validating annotations though some form of correlation analysis with scRNA-seq or RNA-seq from human fetal brain references (col P).
Abbreviations for stages (col G-K): P pluripotent stem cell stage, D differentiation stage (below 1 month from the beginning of the protocol), E Early (1–3 months), M Middle (3–6 months), and L Late stage organoids (>6 months).
Abbreviation for methods (col N): NN-Louvain Nearest-neighbor graph combined with Louvain clustering method (also used in Seurat), CCA canonical correlation analysis for batch effect, WGCNA weighted correlation network analysis, RSS reference similarity spectrum.
Abbreviations for cluster annotations (col O): NPC neural precursor cells, NEC neuropithelial cell, neur. neurons, div. dividing/cycling cells, RG radial glia, IPC intermediate precursor cell, IN interneuron, Ast. astrocyte, dopa dopaminergic, prog. progenitor, Glut. glutamatergic, OPC oligodendrocyte precursor cell, Choroid Choroid Plexus cells, Oligo. oligodendrocyte, U unknown/undetermined/unlabeled, EN excitatory neurons, UPR unfolded protein response related, VZ ventricular zone, SVZ subventricular zone, (im)mat. (im)mature, EMT epithelial-mesenchymal transition cells, int. intermediate.