SUMMARY The development of the nervous system involves a coordinated succession of events including the migration of GABAergic neurons from ventral to dorsal forebrain and their integration into cortical circuits. However, these interregional interactions have not yet been modelled with human cells. Here, we generate from human pluripotent cells three-dimensional spheroids resembling either the dorsal or ventral forebrain and containing cortical glutamatergic or GABAergic neurons. These subdomain-specific forebrain spheroids can be assembled to recapitulate the saltatory migration of interneurons similar to migration in fetal forebrain. Using this system, we find that in Timothy syndrome– a neurodevelopmental disorder that is caused by mutations in the CaV1.2 calcium channel, interneurons display abnormal migratory saltations. We also show that after migration, interneurons functionally integrate with glutamatergic neurons to form a microphysiological system. We anticipate that this approach will be useful for studying development and disease, and for deriving spheroids that resemble other brain regions to assemble circuits in vitro.

The differentiation of pluripotent stem cells in three-dimensional cultures can recapitulate key aspects of brain development, but protocols are prone to variable results. Here we differentiated multiple human pluripotent stem cell lines for over 100 d using our previously developed approach to generate brain-region-specific organoids called cortical spheroids and, using several assays, found that spheroid generation was highly reliable and consistent. We anticipate the use of this approach for large-scale differentiation experiments and disease modeling.

We present a technically simple approach for gene expression cytometry combining next-generation sequencing with stochastic barcoding of single cells. A combinatorial library of beads bearing cell- and molecular-barcoding capture probes is used to uniquely label transcripts and reconstruct the digital gene expression profile of thousands of individual cells in a single experiment without the need for robotics or automation. We applied the technology to dissect the human hematopoietic system and to characterize heterogeneous response to in vitro stimulation. High sensitivity is demonstrated by detection of low-abundance transcripts and rare cells. Under current implementation, the technique can analyze a few thousand cells simultaneously and can readily scale to 10,000s or 100,000s of cells.

Significance Circulating cell-free RNA in the blood provides a potential window into the health, phenotype, and developmental programs of a variety of human organs. We used high-throughput methods of RNA analysis such as microarrays and next-generation sequencing to characterize the global landscape of circulating RNA in human subjects. By focusing on tissue-specific genes, we were able to identify the relative contributions of these tissues to circulating RNA and monitor changes during tissue development and neurodegenerative disease states.

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