Elucidating the cellular architecture of the human cerebral cortex is central to understanding our cognitive abilities and susceptibility to disease. Here we applied single nucleus RNA-sequencing to perform a comprehensive analysis of cell types in the middle temporal gyrus of human cortex. We identified a highly diverse set of excitatory and inhibitory neuronal types that are mostly sparse, with excitatory types being less layer-restricted than expected. Comparison to similar mouse cortex single cell RNA-sequencing datasets revealed a surprisingly well-conserved cellular architecture that enables matching of homologous types and predictions of human cell type properties. Despite this general conservation, we also find extensive differences between homologous human and mouse cell types, including dramatic alterations in proportions, laminar distributions, gene expression, and morphology. These species-specific features emphasize the importance of directly studying human brain.

The human neocortex is created from diverse intermixed progenitors in the prenatal germinal zones. These progenitors have been difficult to characterize since progenitors—particularly radial glia (RG)—are rare, and are defined by a combination of intracellular markers, position and morphology. To circumvent these problems we developed a method called FRISCR for transcriptome profiling of individual fixed, stained and sorted cells. After validation of FRISCR using human embryonic stem cells, we profiled primary human RG that constitute only 1% of the mid-gestation cortex. These RG could be classified into ventricular zone-enriched RG (vRG) that express ANXA1 and CRYAB, and outer subventricular zone-localized RG (oRG) that express HOPX. Our study identifies the first markers and molecular profiles of vRG and oRG cells, and provides an essential step for understanding molecular networks driving the lineage of human neocortical progenitors. Furthermore, FRISCR allows targeted single-cell transcriptomic profiling of tissues that lack live-cell markers.

The neocortex is disproportionately expanded in human compared with mouse1,2, both in its total volume relative to subcortical structures and in the proportion occupied by supragranular layers composed of neurons that selectively make connections within the neocortex and with other telencephalic structures. Single-cell transcriptomic analyses of human and mouse neocortex show an increased diversity of glutamatergic neuron types in supragranular layers in human neocortex and pronounced gradients as a function of cortical depth3. Here, to probe the functional and anatomical correlates of this transcriptomic diversity, we developed a robust platform combining patch clamp recording, biocytin staining and single-cell RNA-sequencing (Patch-seq) to examine neurosurgically resected human tissues. We demonstrate a strong correspondence between morphological, physiological and transcriptomic phenotypes of five human glutamatergic supragranular neuron types. These were enriched in but not restricted to layers, with one type varying continuously in all phenotypes across layers 2 and 3. The deep portion of layer 3 contained highly distinctive cell types, two of which express a neurofilament protein that labels long-range projection neurons in primates that are selectively depleted in Alzheimer’s disease4,5. Together, these results demonstrate the explanatory power of transcriptomic cell-type classification, provide a structural underpinning for increased complexity of cortical function in humans, and implicate discrete transcriptomic neuron types as selectively vulnerable in disease.