A survey of human brain transcriptome diversity at the single cell level

Abstract

The human brain is a tissue of vast complexity in terms of the cell types it comprises. Conventional approaches to classifying cell types in the human brain at single cell resolution have been limited to exploring relatively few markers and therefore have provided a limited molecular characterization of any given cell type. We used single cell RNA sequencing on 466 cells to capture the cellular complexity of the adult and fetal human brain at a whole transcriptome level. Healthy adult temporal lobe tissue was obtained during surgical procedures where otherwise normal tissue was removed to gain access to deeper hippocampal pathology in patients with medical refractory seizures. We were able to classify individual cells into all of the major neuronal, glial, and vascular cell types in the brain. We were able to divide neurons into individual communities and show that these communities preserve the categorization of interneuron subtypes that is typically observed with the use of classic interneuron markers. We then used single cell RNA sequencing on fetal human cortical neurons to identify genes that are differentially expressed between fetal and adult neurons and those genes that display an expression gradient that reflects the transition between replicating and quiescent fetal neuronal populations. Finally, we observed the expression of major histocompatibility complex type I genes in a subset of adult neurons, but not fetal neurons. The work presented here demonstrates the applicability of single cell RNA sequencing on the study of the adult human brain and constitutes a first step toward a comprehensive cellular atlas of the human brain.

Keywords: RNAseq; human brain; interneurons; neurons; single cells.

Fig. 1.

(A) Single cells colored by cluster on a 3D space. Ten clusters were identified. (B) Hierarchical clustering of all adult brain cells using a subset of cell-type–enriched genes. Biased groups are groups of cells resulting from the hierarchical clustering, whereas unbiased groups are groups of cells as defined using an unbiased approach. (C) A 3D representation of all cells using the unbiased approach. For each of the unbiased groups of cells belonging to a major cell type, the agreement with the biased groups is shown, with cells classified as members of a particular cell type by both approaches colored in green and cells differentially classified shown in red. (D) Agreement between the biased and unbiased cell-type assignments for each cell type. Number of cells is shown on the y axis.

Fig. 2.

(A) Minimum spanning tree for all neuronal cells. Colors indicate communities of cells separated using the Walktrap community finding algorithm. (B) Enrichment of genes TAC1, LHX6, CRHBP, and SOX6 in the PVALB-expressing community of interneurons. The seven neuronal communities are shown along the x axis, consistently colored as in Fig. 2A. (C) Costaining for proteins CRHBP, SOX6, and PVALB in human cortical sections. Arrows indicate costained cells for each panel of markers. Images were taken using a 20x-objective. (Scale bar, 100μm.)

Fig. 3.

(A) Expression of excitatory neuron markers in excitatory communities of neurons four and seven. (B) Expression of known inhibitory neuron markers in the interneuron communities one, two, three, five, and six. (C) Costaining for proteins PVALB and CALB2 in human cortical sections shows expression of the two proteins in distinct populations of cells. Arrows indicate PVALB-positive cells. Images were taken using a 20× objective. (Scale bar, 100 μm.) (D) Costaining for proteins VIP, PVALB, and CALB2 in human cortical sections shows VIP expression in a subset of CALB2 neurons that lack expression of PVALB. (Upper) Images were taken using a 20× objective. The white dashed box shows an area of the tissue imaged at 63×. (Scale bars, 100 μm for the 20× image and 20 μm for the 63× image.) Arrows indicate VIP/CALB2-positive cells that are negative for PVALB.

Fig. 4.

(A) PCA on adult and fetal neuronal cells for the first three principal components. (B) Boxplots show expression levels of known markers and newly identified markers for adult neurons, fetal neurons, and neuronal progenitors.

Fig. 5.

Expression of selected genes of the MHCI pathway for all fetal and adult neurons, endothelial cells, and microglia. For the SEC61 complex, the average of SEC61A1, A2, B, and G is shown.