Jessica M. Terry

Massively parallel digital transcriptional profiling of single cells

Characterizing the transcriptome of individual cells is fundamental to understanding complex biological systems. We describe a droplet-based system that enables 3′ mRNA counting of tens of thousands of single cells per sample. Cell encapsulation, of up to 8 samples at a time, takes place in ∼6 min, with ∼50% cell capture efficiency. To demonstrate the systems technical performance, we collected transcriptome data from ∼250k single cells across 29 samples. We validated the sensitivity of the system and its ability to detect rare populations using cell lines and synthetic RNAs. We profiled 68k peripheral blood mononuclear cells to demonstrate the systems ability to characterize large immune populations. Finally, we used sequence variation in the transcriptome data to determine host and donor chimerism at single-cell resolution from bone marrow mononuclear cells isolated from transplant patients.

Non-equivalence of Wnt and R-spondin ligands during Lgr5 + intestinal stem-cell self-renewal

The canonical Wnt/β-catenin signalling pathway governs diverse developmental, homeostatic and pathological processes. Palmitoylated Wnt ligands engage cell-surface frizzled (FZD) receptors and LRP5 and LRP6 co-receptors, enabling β-catenin nuclear translocation and TCF/LEF-dependent gene transactivation. Mutations in Wnt downstream signalling components have revealed diverse functions thought to be carried out by Wnt ligands themselves. However, redundancy between the 19 mammalian Wnt proteins and 10 FZD receptors and Wnt hydrophobicity have made it difficult to attribute these functions directly to Wnt ligands. For example, individual mutations in Wnt ligands have not revealed homeostatic phenotypes in the intestinal epithelium—an archetypal canonical, Wnt pathway-dependent, rapidly self-renewing tissue, the regeneration of which is fueled by proliferative crypt Lgr5+ intestinal stem cells (ISCs). R-spondin ligands (RSPO1–RSPO4) engage distinct LGR4–LGR6, RNF43 and ZNRF3 receptor classes, markedly potentiate canonical Wnt/β-catenin signalling, and induce intestinal organoid growth in vitro and Lgr5+ ISCs in vivo. However, the interchangeability, functional cooperation and relative contributions of Wnt versus RSPO ligands to in vivo canonical Wnt signalling and ISC biology remain unknown. Here we identify the functional roles of Wnt and RSPO ligands in the intestinal crypt stem-cell niche. We show that the default fate of Lgr5+ ISCs is to differentiate, unless both RSPO and Wnt ligands are present. However, gain-of-function studies using RSPO ligands and a new non-lipidated Wnt analogue reveal that these ligands have qualitatively distinct, non-interchangeable roles in ISCs. Wnt proteins are unable to induce Lgr5+ ISC self-renewal, but instead confer a basal competency by maintaining RSPO receptor expression that enables RSPO ligands to actively drive and specify the extent of stem-cell expansion. This functionally non-equivalent yet cooperative interaction between Wnt and RSPO ligands establishes a molecular precedent for regulation of mammalian stem cells by distinct priming and self-renewal factors, with broad implications for precise control of tissue regeneration.

Direct Detection of Biologically Significant Thiols and Disulfides with Manganese(IV) Chemiluminescence

The quantification of low-molecular mass thiols and disulfides involved in cellular redox processes is hindered by oxidation or degradation of analytes during conventional sample preparation steps (including deproteinization and derivatization). Researchers therefore seek techniques that minimize sample handling and permit direct detection of thiols and disulfides within a single chromatographic separation. We demonstrate a new HPLC procedure for these biologically important analytes that incorporates direct chemiluminescence detection with a manganese(IV) reagent. A mixture of seven thiols and disulfides (cysteine, N-acetylcysteine, homocysteine, glutathione (GSH), glutathione disulfide (GSSG), cystine, and homocystine) in their native forms were separated using a C18 column within 20 min. Detection limits for these analytes ranged from 5 × 10(-8) to 1 × 10(-7) M, and the precision for retention times and peak areas was excellent, with relative standard deviations of less than 0.3% and 2%, respectively. This approach was employed to determine two key biomarkers of oxidative stress, GSH and GSSG, in whole blood taken from 12 healthy volunteers. Samples were deproteinized, centrifuged, and diluted prior to analysis using a simple procedure that was shown to avoid significant artificial oxidation of GSH.

Autocatalytic Nature of Permanganate Oxidations Exploited for Highly Sensitive Chemiluminescence Detection

Manganese(II) salts catalyze the chemiluminescent oxidation of organic compounds with acidic potassium permanganate. The formation of insoluble manganese(IV) species from the reaction between manganese(II) and permanganate can be prevented with sodium polyphosphate, and therefore, relatively high concentrations of the catalyst can be added to the reagent before the light-producing reaction is initiated. The rapid and intense emissions from these manganese(II) catalyzed chemiluminescence reactions provide highly sensitive detection and greater compatibility with liquid chromatography.

Precision milled flow-cells for chemiluminescence detection

Novel flow-cells with integrated confluence points and reaction channels designed for efficient mixing of fast chemiluminescence systems were constructed by machining opposing sides of a polymer chip and sealing the channels with transparent epoxy-acetate films. A hole drilled through the chip provided the conduit from the confluence point on one side to the centre of the reaction zone on the other side, allowing rapid presentation of the reacting mixture to the photodetector. The effectiveness of each flow-cell was evaluated by comparing the chemiluminescence intensity using flow injection analysis methodology, and examining the distribution of light emanating from the reaction zone (captured by photography in a dark room) when the reactants were continuously merged. Although previously reported chemiluminescence detectors constructed by machining channels into polymers have almost exclusively been prepared using transparent materials, we obtained far greater emission intensities using an opaque white chip with a thin transparent seal, which minimised the loss of light through surfaces not exposed to the photomultiplier tube. Furthermore, this approach enabled the exploration of reactor designs that could not be incorporated in traditional coiled-tubing flow-cells.

Chemiluminescence detection flow cells for flow injection analysis and high-performance liquid chromatography.

AbstractWe have examined a range of new and previously described flow cells for chemiluminescence detection. The reactions of acidic potassium permanganate with morphine and amoxicillin were used as model systems representing the many fast chemiluminescence reactions between oxidising agents and organic analytes, and the preliminary partial reduction of the reagent was exploited to further increase the rates of reaction. The comparison was then extended to high-performance liquid chromatography separations of α- and β-adrenergic agonists, with permanganate chemiluminescence detection. Flow cells constructed by machining novel channel designs into white polymer materials (sealed with transparent films or plates) have enabled improvements in mixing efficiency and overall transmission of light to the photodetector. FigChemiluminescence from the reaction of morphine and permanganate in a serpentine flow cell.

Chemiluminescence detectors for liquid chromatography

In this tutorial we describe the construction of chemiluminescence detectors for high performance liquid chromatography (HPLC), comprising the components required to deliver the chemiluminescence reagent, a coiled-tubing flow cell, photomultiplier tube and detector housing, and various options for data acquisition. We also discuss two state-of-the-art commercially available chemiluminescence detectors for HPLC and other flow analysis methodology.

3D-printed and CNC milled flow-cells for chemiluminescence detection

Herein we explore modern fabrication techniques for the development of chemiluminescence detection flow-cells with features not attainable using the traditional coiled tubing approach. This includes the first 3D-printed chemiluminescence flow-cells, and a milled flow-cell designed to split the analyte stream into two separate detection zones within the same polymer chip. The flow-cells are compared to conventional detection systems using flow injection analysis (FIA) and high performance liquid chromatography (HPLC), with the fast chemiluminescence reactions of an acidic potassium permanganate reagent with morphine and a series of adrenergic phenolic amines.

Ethanol as an alternative to formaldehyde for the enhancement of manganese(IV) chemiluminescence detection

Previous applications of manganese(IV) as a chemiluminescence reagent have required the use of formaldehyde to enhance the emission intensity to analytically useful levels. However, this known human carcinogen (by inhalation) is not ideal for routine application. A wide range of alternative enhancers have been examined but to date none have been found to provide the dramatic increase in chemiluminescence intensities obtained using formaldehyde. Herein, we demonstrate that ethanol offers a simple, safe and inexpensive alternative to the use of formaldehyde for manganese(IV) chemiluminescence detection, without compromising signal intensity or sensitivity. For example, chemiluminescence signals for opiate alkaloids using 50-100% ethanol were 0.8-1.6-fold those using 2M formaldehyde. This innocuous alternative enhancer is shown to be a particularly effective for the direct detection of thiols and disulfides by manganese(IV) chemiluminescence, which we have applied to a simple HPLC procedure to determine a series of biomarkers of oxidative stress.

Abstract 150: Single cell mRNA quantification from 1000s of cells in healthy and malignant tumor samples using a high-throughput droplet-based system

Advances in single cell RNA quantification techniques have enabled comprehensive study of subpopulations of cells within a heterogeneous population. The application of single cell quantification techniques to oncology is helping to elucidate the complex variability in genetic and epigenetic interactions that occur within tumor cells and their microenvironment. However, current single cell RNA-sequencing methods are limited by their reliance on costly infrastructure and laborious experimental protocols. We developed the GemCode Platform, which combines microfluidics with molecular barcoding and custom bioinformatics software to enable 3’ mRNA counting from thousands of single cells. Here we utilized the GemCode Platform to profile primary cells from healthy donors and cancer patients. Cell lines and cancer samples were obtained from commercial sources. Single cells, reagents and a single gel bead containing barcoded oligonucleotides were encapsulated into picoliter-sized droplets using the 10X Genomics GemCode Platform. The platform achieved extremely high cell loading efficiency (> 50%), enabling the creation of libraries from precious samples. Lysis and barcoded reverse transcription of RNAs from single cells were performed inside each droplet. High quality next generation sequencing libraries were finished in a single bulk reaction. The GemCode software suite was utilized for processing, interactive analysis and visualization of single cell gene expression data. We demonstrated single cell behavior through mouse- and human cell mixing experiments with a low doublet rate of 40,000 peripheral blood mononuclear cells from healthy donors and detected all major subpopulations (i.e., B cells, CD4+ T cells, CD8+ T cells, NK cells, dendritic cells, monocytes) in similar proportions to those previously reported in the literature. Notably, the high-throughput nature of the platform enabled resolution of finer sub-structures such as CD4+ effector memory cells and CD4+ central memory cells. Experiments comparing cells isolated from patients with hematologic malignancies (such as CLL, AML and CML) with whole bone marrow from healthy donors further demonstrate the power of single cell profiling for characterizing disease-associated changes in complex tissues. We demonstrate the ability to perform high-throughput gene expression profiling of mRNAs in single cells. The high-throughput platform enables detection of rare cells in a heterogeneous tumor population. Moreover, efficient cell loading enables analysis of clinically relevant sample types with limited cell input. An integrated single cell mRNA analysis will lead to novel insights into the molecular characteristics of individual cancer cells and provide targets for therapeutic intervention. Citation Format: Grace X.y. Zheng, Tarjei Mikkelsen, Jessica Terry, Phillip Belgrader, Paul Ryvkin, Ryan Wilson, Tobias D. Wheeler, Zachary Bent, Geoff McDermott, Solongo Ziraldo, Alexander Wong, Michael Schnall-Levin, Ben Hindson. Single cell mRNA quantification from 1000s of cells in healthy and malignant tumor samples using a high-throughput droplet-based system. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 150.