Multimodal Single-Cell Analysis Reveals Physiological Maturation in the Developing Human Neocortex.

In the developing human neocortex, progenitor cells generate diverse cell types prenatally. Progenitor cells and newborn neurons respond to signaling cues, including neurotransmitters. While single-cell RNA sequencing has revealed cellular diversity, physiological heterogeneity has yet to be mapped onto these developing and diverse cell types.

Establishing Cerebral Organoids as Models of Human-Specific Brain Evolution.

Direct comparisons of human and non-human primate brains can reveal molecular pathways underlying remarkable specializations of the human brain. However, chimpanzee tissue is inaccessible during neocortical neurogenesis when differences in brain size first appear. To identify human-specific features of cortical development, we leveraged recent innovations that permit generating pluripotent stem cell-derived cerebral organoids from chimpanzee.

Regulation of cell-type-specific transcriptomes by microRNA networks during human brain development.

MicroRNAs (miRNAs) regulate many cellular events during brain development by interacting with hundreds of mRNA transcripts. However, miRNAs operate nonuniformly upon the transcriptional profile with an as yet unknown logic. Shortcomings in defining miRNA-mRNA networks include limited knowledge of in vivo miRNA targets and their abundance in single cells.

Spatiotemporal gene expression trajectories reveal developmental hierarchies of the human cortex.

Systematic analyses of spatiotemporal gene expression trajectories during organogenesis have been challenging because diverse cell types at different stages of maturation and differentiation coexist in the emerging tissues. We identified discrete cell types as well as temporally and spatially restricted trajectories of radial glia maturation and neurogenesis in developing human telencephalon. These lineage-specific trajectories reveal the expression of neurogenic transcription factors in early radial glia and enriched activation of mammalian target of rapamycin signaling in outer radial glia.

Corrigendum: Fluidic Logic Used in a Systems Approach to Enable Integrated Single-Cell Functional Analysis.

[This corrects the article on p. 70 in vol. 4, PMID: 27709111.

The nature and nurture of cell heterogeneity: accounting for macrophage gene-environment interactions with single-cell RNA-Seq.

Background: Single-cell RNA-Seq can be a valuable and unbiased tool to dissect cellular heterogeneity, despite the transcriptome's limitations in describing higher functional phenotypes and protein events. Perhaps the most important shortfall with transcriptomic 'snapshots' of cell populations is that they risk being descriptive, only cataloging heterogeneity at one point in time, and without microenvironmental context. Studying the genetic ('nature') and environmental ('nurture') modifiers of heterogeneity, and how cell population dynamics unfold over time in response to these modifiers is key when studying highly plastic cells such as macrophages.

Fluidic Logic Used in a Systems Approach to Enable Integrated Single-Cell Functional Analysis.

The study of single cells has evolved over the past several years to include expression and genomic analysis of an increasing number of single cells. Several studies have demonstrated wide spread variation and heterogeneity within cell populations of similar phenotype. While the characterization of these populations will likely set the foundation for our understanding of genomic- and expression-based diversity, it will not be able to link the functional differences of a single cell to its underlying genomic structure and activity.

Molecular identity of human outer radial glia during cortical development.

Radial glia, the neural stem cells of the neocortex, are located in two niches: the ventricular zone and outer subventricular zone. Although outer subventricular zone radial glia may generate the majority of human cortical neurons, their molecular features remain elusive. By analyzing gene expression across single cells, we find that outer radial glia preferentially express genes related to extracellular matrix formation, migration, and stemness, including TNC, PTPRZ1, FAM107A, HOPX, and LIFR.

Low-coverage single-cell mRNA sequencing reveals cellular heterogeneity and activated signaling pathways in developing cerebral cortex.

Large-scale surveys of single-cell gene expression have the potential to reveal rare cell populations and lineage relationships but require efficient methods for cell capture and mRNA sequencing. Although cellular barcoding strategies allow parallel sequencing of single cells at ultra-low depths, the limitations of shallow sequencing have not been investigated directly. By capturing 301 single cells from 11 populations using microfluidics and analyzing single-cell transcriptomes across downsampled sequencing depths, we demonstrate that shallow single-cell mRNA sequencing (~50,000 reads per cell) is sufficient for unbiased cell-type classification and biomarker identification.

High-throughput microfluidic single-cell analysis pipeline for studies of signaling dynamics.

Time-dependent analysis of dynamic processes in single live cells is a revolutionary technique for the quantitative studies of signaling networks. Here we describe an experimental pipeline and associated protocol that incorporate microfluidic cell culture, precise stimulation of cells with signaling molecules or drugs, live-cell microscopy, computerized cell tracking, on-chip staining of key proteins and subsequent retrieval of cells for high-throughput gene expression analysis using microfluidic quantitative PCR (qPCR). Compared with traditional culture dish approaches, this pipeline enhances experimental precision and throughput by orders of magnitude and introduces much-desired new capabilities in cell and fluid handling, thus representing a major step forward in dynamic single-cell analysis.

Single-cell dissection of transcriptional heterogeneity in human colon tumors.

Cancer is often viewed as a caricature of normal developmental processes, but the extent to which its cellular heterogeneity truly recapitulates multilineage differentiation processes of normal tissues remains unknown. Here we implement single-cell PCR gene-expression analysis to dissect the cellular composition of primary human normal colon and colon cancer epithelia. We show that human colon cancer tissues contain distinct cell populations whose transcriptional identities mirror those of the different cellular lineages of normal colon.

Biocompatibility and reduced drug absorption of sol-gel-treated poly(dimethyl siloxane) for microfluidic cell culture applications.

Poly(dimethyl siloxane) (PDMS)-based microfluidic devices are now commonly used for a wide variety of biological experiments, including cell culture assays. However, the porous, hydrophobic polymer matrix of PDMS rapidly absorbs small hydrophobic molecules, including hormones and most small-molecule drugs. This makes it challenging to perform experiments that require such substances in PDMS microfluidic devices.

Versatile, fully automated, microfluidic cell culture system.

There is increasing demand for automated and quantitative cell culture technology, driven both by the intense activity in stem cell biology and by the emergence of systems biology. We built a fully automated cell culture screening system based on a microfluidic chip that creates arbitrary culture media formulations in 96 independent culture chambers and maintains cell viability for weeks. Individual culture conditions are customized in terms of cell seeding density, composition of culture medium, and feeding schedule, and each chamber is imaged with time-lapse microscopy.

Morphological analysis of tumor cell/endothelial cell interactions under shear flow.

In the process of hematogenous cancer metastasis, tumor cells (TCs) must shed into the blood stream, survive in the blood circulation, migrate through the vascular endothelium (extravasation) and proliferate in the target organs. However, the precise mechanisms by which TCs penetrate the endothelial cell (EC) junctions remain one of the least understood aspects of TC extravasation. This question has generally been addressed under static conditions, despite the important role of flow induced mechanical stress on the circulating cell-endothelium interactions.

Measuring cell viscoelastic properties using a force-spectrometer: influence of protein-cytoplasm interactions.

Cell adhesive and rheological properties play a very important role in cell transmigration through the endothelial barrier, in particular in the case of inflammation (leukocytes) or cancer metastasis (cancer cells). In order to characterize cell viscoelastic properties, we have designed a force spectrometer (AFM) which can stretch cells thereby allowing measurement of their rheological properties. This custom-made force spectrometer allows two different visualizations, one lateral and one from below.