The recent advent of methods for high-throughput single-cell molecular profiling has catalyzed a growing sense in the scientific community that the time is ripe to complete the 150-year-old effort to identify all cell types in the human body. The Human Cell Atlas Project is an international collaborative effort that aims to define all human cell types in terms of distinctive molecular profiles (such as gene expression profiles) and to connect this information with classical cellular descriptions (such as location and morphology). An open comprehensive reference map of the molecular state of cells in healthy human tissues would propel the systematic study of physiological states, developmental trajectories, regulatory circuitry and interactions of cells, and also provide a framework for understanding cellular dysregulation in human disease. Here we describe the idea, its potential utility, early proofs-of-concept, and some design considerations for the Human Cell Atlas, including a commitment to open data, code, and community.

The Human Cell Atlas

The unabated progress in next-generation sequencing technologies is fostering a wave of new genomics, epigenomics, transcriptomics and proteomics technologies. These sequencing-based technologies are increasingly being targeted to individual cells, which will allow many new and longstanding questions to be addressed. For example, single-cell genomics will help to uncover cell lineage relationships; single-cell transcriptomics will supplant the coarse notion of marker-based cell types; and single-cell epigenomics and proteomics will allow the functional states of individual cells to be analysed. These technologies will become integrated within a decade or so, enabling high-throughput, multi-dimensional analyses of individual cells that will produce detailed knowledge of the cell lineage trees of higher organisms, including humans. Such studies will have important implications for both basic biological research and medicine.

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Single-cell sequencing-based technologies will revolutionize whole-organism science

Single-cell genome sequencing can provide detailed insights into the composition of single genomes that are not readily apparent when studying bulk cell populations. This Review discusses the considerable technical challenges of amplifying and interrogating genomes from single cells, emerging innovative solutions and various applications in microbiology and human disease, in particular in cancer.

Single-cell genome sequencing: current state of the science

The functional importance of gene enhancers in regulated gene expression is well established. In addition to widespread transcription of long non-coding RNAs (lncRNAs) in mammalian cells, bidirectional ncRNAs are transcribed on enhancers, and are thus referred to as enhancer RNAs (eRNAs). However, it has remained unclear whether these eRNAs are functional or merely a reflection of enhancer activation. Here we report that in human breast cancer cells 17β-oestradiol (E2)-bound oestrogen receptor α (ER-α) causes a global increase in eRNA transcription on enhancers adjacent to E2-upregulated coding genes. These induced eRNAs, as functional transcripts, seem to exert important roles for the observed ligand-dependent induction of target coding genes, increasing the strength of specific enhancer-promoter looping initiated by ER-α binding. Cohesin, present on many ER-α-regulated enhancers even before ligand treatment, apparently contributes to E2-dependent gene activation, at least in part by stabilizing E2/ER-α/eRNA-induced enhancer-promoter looping. Our data indicate that eRNAs are likely to have important functions in many regulated programs of gene transcription.

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Functional roles of enhancer RNAs for oestrogen-dependent transcriptional activation

Microfluidics consist of microfabricated structures for liquid handling, with cross-sections in the 1–500 μm range, and small volume capacity (fL-nL) Capillary tubes connected with fittings,1 although utilizing small volumes, are not considered microfluidics for the purposes of this paper since they are not microfabricated Likewise, millifluidic systems, made by conventional machining tools, are excluded due to their larger feature sizes (>500 μm)

Though micromachined systems for gas chromatography were introduced in the 1970’s,2 the field of microfluidics did not gain much traction until the 1990’s3 Silicon and glass were the original materials used, but then the focus shifted to include polymer substrates, and in particular, polydimethylsiloxane (PDMS) Since then the field has grown to encompass a wide variety of materials and applications The successful demonstration of electrophoresis and electroosmotic pumping in a microfluidic device provided a nonmechanical method for both fluid control and separation4 Laser induced fluorescence (LIF) enabled sensitive detection of fluorophores or fluorescently labeled molecules The expanded availability of low-cost printing allowed for cheaper and quicker mask fabrication for use in soft lithography5 Commercial microfluidic systems are now available from Abbott, Agilent, Caliper, Dolomite, Micralyne, Microfluidic Chip Shop, Micrux Technologies and Waters, as a few prominent examples For a more thorough description of the history of microfluidics, we refer the reader to a number of comprehensive, specialized reviews,3, 6–11 as well as a more general 2006 review12

The field of microfluidics offers many advantages compared to carrying out processes through bulk solution chemistry, the first of which relates to a lesson taught to every first-year chemistry student Simply stated, diffusion is slow! Thus, the smaller the distance required for interaction, the faster it will be Smaller channel dimensions also lead to smaller sample volumes (fL-nL), which can reduce the amount of sample or reagents required for testing and analysis Reduced dimensions can also lead to portable devices to enable on-site testing (provided the associated hardware is similarly portable) Finally, integration of multiple processes (like labeling, purification, separation and detection) in a microfluidic device can be highly enabling for many applications

Microelectromechanical systems (MEMS) contain integrated electrical and mechanical parts that create a sensor or system Applications of MEMS are ubiquitous, including automobiles, phones, video games and medical and biological sensors13 Micro-total analysis systems, also known as labs-on-a-chip, are the chemical analogue of MEMS, as integrated microfluidic devices that are capable of automating multiple processes relevant to laboratory sciences For example, a typical lab-on-a-chip system might selectively purify a complex mixture (through filtering, antibody capture, etc), then separate target components and detect them

Microfluidic devices consist of a core of common components Areas defined by empty space, such as reservoirs (wells), chambers and microchannels, are central to microfluidic systems Positive features, created by areas of solid material, add increased functionality to a chip and can consist of membranes, monoliths, pneumatic controls, beams and pillars Given the ubiquitous nature of negative components, and microchannels in particular, we focus here on a few of their properties Microfluidic channels have small overall volumes, laminar flow and a large surface-to-volume ratio Dimensions of a typical separation channel in microchip electrophoresis (μCE) are: 50 μm width, 15 μm height and 5 cm length for a volume of 375 nL Flow in these devices is normally nonturbulent due to low Reynolds numbers For example, water flowing at 20°C in the above channel at 1 μL/min (222 cm/s) results in a Reynolds number of ~05, where <2000 is laminar flow Since flow is nonturbulent, mixing is normally diffusion-limited Small channel sizes also have a high surface-to-volume ratio, leading to different characteristics from what are commonly found in bulk volumes The material surface can be used to manipulate fluid movement (such as by electroosmotic flow, EOF) and surface interactions For a solution in contact with a charged surface, a double layer of charge is created as oppositely charged ions are attracted to the surface charges This electrical double layer consists of an inner rigid or Stern Layer and an outer diffuse layer An electrostatic potential known as the zeta potential is formed, with the magnitude of the potential decreasing as distance from the surface increases The electrical double layer is the basis for EOF, wherein an applied voltage causes the loosely bound diffuse layer to move towards an electrode, dragging the bulk solution along Charges on the exposed surface also exert a greater influence on the fluid in a channel as its size decreases Larger surface-to-volume ratios are more prone to nonspecific adsorption and surface fouling In particular, non-charged and hydrophobic microdevice surfaces can cause proteins in solution to denature and stick

We focus our review on advances in microfluidic systems since 2008 In doing this, we occasionally must cover foundational work in microfluidics that is considerably less recent We do not focus on chemical synthesis applications of microfluidics although it is an expanding area, nor do we delve into lithography, device fabrication or production costs Our specific emphasis herein is on four areas within microfluidics: properties and applications of commonly used materials, basic functions, integration, and selected applications For each of these four topics we provide a concluding section on opportunities for future development, and at the end of this review, we offer general conclusions and prospective for future work in the field Due to the considerable scope of the field of microfluidics, we limit our discussion to selected examples from each area, but cite in-depth reviews for the reader to turn to for further information about specific topics We also refer the reader to recent comprehensive reviews on advances in lab-on-a-chip systems by Arora et al10 and Kovarik et al14

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Advances in microfluidic materials, functions, integration, and applications.

A long-sought milestone in microfluidics research has been the development of integrated technology for scalable analysis of transcription in single cells Here we present a fully integrated microfluidic device capable of performing high-precision RT-qPCR measurements of gene expression from hundreds of single cells per run Our device executes all steps of single-cell processing, including cell capture, cell lysis, reverse transcription, and quantitative PCR In addition to higher throughput and reduced cost, we show that nanoliter volume processing reduced measurement noise, increased sensitivity, and provided single nucleotide specificity We apply this technology to 3,300 single-cell measurements of (i) miRNA expression in K562 cells, (ii) coregulation of a miRNA and one of its target transcripts during differentiation in embryonic stem cells, and (iii) single nucleotide variant detection in primary lobular breast cancer cells The core functionality established here provides the foundation from which a variety of on-chip single-cell transcription analyses will be developed

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Mani Hamidi

Papers

High-throughput microfluidic single-cell RT-qPCR