The mammalian prefrontal cortex comprises a set of highly specialized brain areas containing billions of cells and serves as the centre of the highest-order cognitive functions, such as memory, cognitive ability, decision-making and social behaviour. Although neural circuits are formed in the late stages of human embryonic development and even after birth, diverse classes of functional cells are generated and migrate to the appropriate locations earlier in development. Dysfunction of the prefrontal cortex contributes to cognitive deficits and the majority of neurodevelopmental disorders; there is therefore a need for detailed knowledge of the development of the prefrontal cortex. However, it is still difficult to identify cell types in the developing human prefrontal cortex and to distinguish their developmental features. Here we analyse more than 2,300 single cells in the developing human prefrontal cortex from gestational weeks 8 to 26 using RNA sequencing. We identify 35 subtypes of cells in six main classes and trace the developmental trajectories of these cells. Detailed analysis of neural progenitor cells highlights new marker genes and unique developmental features of intermediate progenitor cells. We also map the timeline of neurogenesis of excitatory neurons in the prefrontal cortex and detect the presence of interneuron progenitors in early developing prefrontal cortex. Moreover, we reveal the intrinsic development-dependent signals that regulate neuron generation and circuit formation using single-cell transcriptomic data analysis. Our screening and characterization approach provides a blueprint for understanding the development of the human prefrontal cortex in the early and mid-gestational stages in order to systematically dissect the cellular basis and molecular regulation of prefrontal cortex function in humans.

The cellular complexity of human brain development has been intensively investigated, although a regional characterization of the entire human cerebral cortex based on single-cell transcriptome analysis has not been reported. Here, we performed RNA-seq on over 4,000 individual cells from 22 brain regions of human mid-gestation embryos. We identified 29 cell sub-clusters, which showed different proportions in each region and the pons showed especially high percentage of astrocytes. Embryonic neurons were not as diverse as adult neurons, although they possessed important features of their destinies in adults. Neuron development was unsynchronized in the cerebral cortex, as dorsal regions appeared to be more mature than ventral regions at this stage. Region-specific genes were comprehensively identified in each neuronal sub-cluster, and a large proportion of these genes were neural disease related. Our results present a systematic landscape of the regionalized gene expression and neuron maturation of the human cerebral cortex.

Modeling the processes of neuronal progenitor proliferation and differentiation to produce mature cortical neuron subtypes is essential for the study of human brain development and the search for potential cell therapies. We demonstrated a novel paradigm for the generation of vascularized organoids (vOrganoids) consisting of typical human cortical cell types and a vascular structure for over 200 days as a vascularized and functional brain organoid model. The observation of spontaneous excitatory postsynaptic currents (sEPSCs), spontaneous inhibitory postsynaptic currents (sIPSCs), and bidirectional electrical transmission indicated the presence of chemical and electrical synapses in vOrganoids. More importantly, singlecell RNA-sequencing analysis illustrated that vOrganoids exhibited robust neurogenesis and that cells of vOrganoids differentially expressed genes (DEGs) related to blood vessel morphogenesis. The transplantation of vOrganoids into the mouse S1 cortex resulted in the construction of functional human-mouse blood vessels in the grafts that promoted cell survival in the grafts. This vOrganoid culture method could not only serve as a model to study human cortical development and explore brain disease pathology but also provide potential prospects for new cell therapies for nervous system disorders and injury.