Julie Audet

Current techniques for single-cell lysis

Owing to the small quantities of analytes and small volumes involved in single-cell analysis techniques, manipulation strategies must be chosen carefully. The lysis of single cells for downstream chemical analysis in capillaries and lab-on-a-chip devices can be achieved by optical, acoustic, mechanical, electrical or chemical means, each having their respective strengths and weaknesses. Selection of the most appropriate lysis method will depend on the particulars of the downstream cell lysate processing. Ultrafast lysis techniques such as the use of highly focused laser pulses or pulses of high voltage are suitable for applications requiring high temporal resolution. Other factors, such as whether the cells are adherent or in suspension and whether the proteins to be collected are desired to be native or denatured, will determine the suitability of detergent-based lysis methods. Therefore, careful selection of the proper lysis technique is essential for gathering accurate data from single cells.

Stem cell bioengineering for regenerative medicine.

Stem cells can be used to treat a variety of diseases and several recent studies in animal models demonstrate the potential of bioengineering strategies targeting adult and embryonic stem cells. In order to obtain the desired cells for transplantation, stem cell bioengineering approaches entail the manipulation of environmental signals influencing cell survival, proliferation, self-renewal and differentiation. In that regard, multivariate analytical approaches have been used with success to optimise different stem cell culture processes. The genetic or molecular enhancement of stem cells is also a powerful means to control their proliferation or differentiation or to correct genetic defects in recipients. In the future, systems-level approaches have the potential to revolutionise the field of stem cell bioengineering by improving our understanding of regulatory networks controlling cellular behaviour. This advance in basic biology will be instrumental for the implementation of many stem cell-based regenerative therapies at the clinical level, as treatment accessibility will depend on the development of robust technologies to produce sufficient cell numbers.

Single Amino Acid Resolution of Proteolytic Fragments Generated in Individual Cells

The complex nature of enzyme regulation mandates that enzyme activity profiles be measured in the context of the intact cell. Single‐cell capillary electrophoresis (CE) coupled with laser‐induced fluorescence is a powerful approach for quantitation and separation of analytes present in small samples and single live cells; however, it does not allow for the definitive identification of the reaction products. On the other hand, mass spectrometry (MS) is able to identify analytes but still lacks the requisite sensitivity for most single‐cell analysis applications. Thus, it follows that by determining the relative amounts of reaction products generated in single cells using CE and by producing larger quantities of these products using bulk cell populations to identify them using MS, it is possible to determine enzyme activity profiles in single cells. In this study, the applicability of this approach was demonstrated by examining the intracellular fate of a protease substrate derived from the β‐amyloid precursor protein (β‐APP). In single live TF‐1 cells, three distinct fragments were generated from the β‐APP peptide, which differed by a single uncharged amino acid. The CE measurements indicated that the proteolytic fragment profiles (i.e., the relative amounts of each fragment) were consistent from cell to cell but that they were different from those obtained in cell lysates. Furthermore, measurements obtained at the single cell level made it possible to observe a modest but statistically significant negative correlation between the total amount of β‐APP peptide loaded in cells and the fraction of peptide that remained intact. This study demonstrates how single‐cell CE, MS, and peptide substrates can be combined to identify and measure enzyme activities in single live cells.

Sampling Efficiency of a Single-Cell Capillary Electrophoresis System

Capillary electrophoresis (CE) combined with a laser‐induced fluorescence (LIF) detection scheme is a powerful approach for single‐cell analysis. For measurements requiring a high temporal resolution, CE–LIF is often combined with cell lysis systems based on pulsed lasers. Although extremely rapid, laser lysis has raised some concerns about the efficiency at which the cell contents are sampled. We have assembled a single‐cell CE‐LIF mounted on the stage of a microscope. This system was coupled with a nanosecond pulsed laser for cell lysis. We have analyzed green fluorescent protein (GFP) expressed in single mammalian cells and developed a novel approach to estimate the cell sampling efficiency (SE) based on the use of fluorescent calibration microspheres and flow cytometry. A significant advantage of this method is that it does not require any knowledge or assumption regarding the cell volume. We have evaluated the SE for different laser pulse energies (from 2 to 9 μJ) and two different pulse focal positions in the xy plane (0–10 μm from the center of the cell). We found the maximum SE at the lowest energy (2 μJ), with the pulse focused directly on the cell. We have demonstrated the utility of a novel method to measure the SE of a single‐cell CE system. The measurements presented in this study indicate that rapid cell lysis with nanosecond lasers requires careful optimization of pulse parameters for maximum sampling of the cell contents.

Measurement of generation-dependent proliferation rates and death rates during mouse erythroid progenitor cell differentiation

Herein, we describe an experimental and computational approach to perform quantitative carboxyfluorescein diacetate succinimidyl ester (CFSE) cell‐division tracking in cultures of primary colony‐forming unit‐erythroid (CFU‐E) cells, a hematopoietic progenitor cell type, which is an important target for the treatment of blood disorders and for the manufacture of red blood cells. CFSE labeling of CFU‐Es isolated from mouse fetal livers was performed to examine the effects of stem cell factor (SCF) and erythropoietin (EPO) in culture. We used a dynamic model of proliferation based on the Smith‐Martin representation of the cell cycle to extract proliferation rates and death rates from CFSE time‐series. However, we found that to accurately represent the cell population dynamics in differentiation cultures of CFU‐Es, it was necessary to develop a model with generation‐specific rate parameters. The generation‐specific rates of proliferation and death were extracted for six generations (G0–G5) and they revealed that, although SCF alone or EPO alone supported similar total cell outputs in culture, stimulation with EPO resulted in significantly higher proliferation rates from G2 to G5 and higher death rates in G2, G3, and G5 compared with SCF. In addition, proliferation rates tended to increase from G1 to G5 in cultures supplemented with EPO and EPO + SCF, while they remained lower and more constant across generations with SCF. The results are consistent with the notion that SCF promotes CFU‐E self‐renewal while EPO promotes CFU‐E differentiation in culture.

Adventures in time and space: Nonlinearity and complexity of cytokine effects on stem cell fate decisions

Cytokines are central factors in the control of stem cell fate decisions and, as such, they are invaluable to those interested in the manipulation of stem and progenitor cells for clinical or research purposes. In their in vivo niches or in optimized cultures, stem cells are exposed to multiple cytokines, matrix proteins and other cell types that provide individual and combinatorial signals that influence their self-renewal, proliferation and differentiation. Although the individual effects of cytokines are well-characterized in terms of increases or decreases in stem cell expansion or in the production of specific cell lineages, their interactions are often overlooked. Factorial design experiments in association with multiple linear regression is a powerful multivariate approach to derive response-surface models and to obtain a quantitative understanding of cytokine dose and interactions effects. On the other hand, cytokine interactions detected in stem cell processes can be difficult to interpret due to the fact that the cell populations examined are often heterogeneous, that cytokines can exhibit pleiotropy and redundancy and that they can also be endogenously produced. This perspective piece presents a list of possible biological mechanisms that can give rise to positive and negative two-way factor interactions in the context of in vivo and in vitro stem cell-based processes. These interpretations are based on insights provided by recent studies examining intra- and extra-cellular signaling pathways in adult and embryonic stem cells. Cytokine interactions have been classified according to four main types of molecular and cellular mechanisms: (i) interactions due to co-signaling; (ii) interactions due to sequential actions; (iii) interactions due to high-dose saturation and inhibition; and (iv) interactions due to intercellular signaling networks. For each mechanism, possible patterns of regression coefficients corresponding to the cytokine main effects, quadratic effects and two-way interactions effects are provided. Finally, directions for future mechanistic studies are presented.

Identification of enzyme-converted peptide products from single cells using capillary electrophoresis and liquid chromatography-mass spectrometry.

Single-cell analysis using chemical methods, otherwise known as chemical cytometry, promises to provide significant leaps in understanding signaling processes which result in cellular behavior. Sensitive methods for chemical cytometry such as capillary electrophoresis can detect and quantify multiple targets; however, conclusive identification of detected analytes is required for useful data to be obtained. Here, we demonstrate a method for determining the identity of enzyme-converted peptide products from single cells using a combination of capillary electrophoresis and liquid chromatography-mass spectrometry (LC-MS).

Single-Cell Approaches to Dissect Cellular Signaling Networks

Progress in understanding signal transduction, especially in rare and heterogeneous stem cell populations, is dependent on advances in single-cell assays. Newly developed techniques based on flow cytometry, capillary electrophoresis and live-cell imaging have enabled researchers to study kinase activity at the single-cell level. Since kinase activation is central to the regulation of virtually all cellular functions, single-cell kinase assays promise to help elucidating the molecular mechanisms controlling stem cell fate decisions.

Measurement of cell-penetrating peptide-mediated transduction of adult hematopoietic stem cells.

The ability of cell-penetrating peptides (CPPs) to cross cell membranes and transport cargo into cells makes them an attractive tool for the molecular engineering of stem cells. Even though the exact mechanism of transduction remains unclear, their potential has been demonstrated for diverse applications, including hematopoietic stem cell expansion and the generation of islets cells from embryonic stem cells. Several parameters can affect the intracellular delivery of CPP-based constructs. Those include the type of cells targeted, the type of CPP used, and the properties of the cargo. For this reason, it is important to have a means to quantitatively assess the transduction efficiency of specific constructs in the cell type of interest in order to select the best vector for a specific application. In this chapter, we describe a method to measure the uptake of HIV transactivator of transcription (TAT) and the homeobox protein Antennapedia (Antp) constructs in primary hematopoietic progenitor cells and hematopoietic cell lines. This method is useful to compare, select, and optimize different strategies to deliver CPP-based constructs into a given cell type.