Efficient gene delivery in differentiated human embryonic stem cells

Human embryonic stem (hES) cells are capable of differentiating into pluralistic cell types, however, spontaneous differentiation generally gives rise to a limited number of specific differentiated cell types and a large degree of cell heterogeneity. In an effort to increase the efficiency of specified hES cell differentiation, we performed a series of transient transfection of hES cells with EGFP expression vectors driven by different promoter systems, including human cellular polypeptide chain elongation factor 1 alpha (hEF1alpha), human cytomegalo-virus, and chicken beta-actin. All these promoters were found to lead reporter gene expression in undifferentiated hES cells, but very few drug-selectable transfectants were obtained and failed to maintain stable expression of the transgene with either chemical or electroporation methods. In an attempt to increase transfection efficiency and obtain stable transgene expression, differentiated hES cells expressing both mesodermal and ectodermal markers were derived using a defined medium. Differentiated hES cells were electroporated with a hEF1alpha promoter-driven EGFP or human noggin expression vector. Using RT-PCR, immunocytochemistry and fluorescence microscopy, the differentiated hES cells transfected with foreign genes were confirmed to retain stable gene and protein expression during prolonged culture. These results may provide a new tool for introducing exogenous genes readily into hES cells, thereby facilitating more directed differentiation into specific and homogenous cell populations.     

Quantitative measurement of quantum dot uptake at the cell population level using microfluidic evanescent-wave-based flow cytometry

The intracellular uptake of nanoparticles (NPs) is an important process for molecular and cellular labeling, drug/gene delivery and medical imaging. The vast majority of investigations into NP uptake have been conducted using confocal imaging that is limited to observation of a small number of cells. Such data may not yield quantitative information about the cell population due to the tiny sample size and the potential heterogeneity. Flow cytometry is the technique of choice for studying cell populations with single cell resolution. Unfortunately, classic flow cytometry detects fluorescence from whole cells and does not shed light on subcellular dynamics. In this report, we demonstrate the use of microfluidics-based total internal reflection fluorescence flow cytometry (TIRF-FC) for examining initial quantum dot (QD) entry into cells and the associated subcellular movement at the single cell level with a rate of ～200 cells s(-1). Our cytometric tool allows extraction of quantitative data from a large cell population and reveals details about the QD transport in the periphery of the cell membrane (～100 nm deep into the cytosol). Our data indicate that the fluorescence density at the membrane vicinity decreases after initial QD dosage due to the decline in the density of QDs in the evanescent field and the transport into the cytosol is very rapid.     

Characterisation of membrane surfaces: direct measurement of biological adhesion using an atomic force microscope

An atomic force microscope (AFM) in conjunction with coated colloid probe and cell probe techniques has been used to measure directly the adhesive force between both the protein bovine serum albumin (BSA) and a yeast cell at two different membranes. These were polymeric ultrafiltration membranes of similar MWCO (4000 Da) but of different materials (ES 404 and XP 117, PCI Membrane Systems, UK). The XP 117 membrane is made from a mixture of polymers chosen with the aim of achieving low fouling. The BSA was adsorbed on a 5 μm silica colloid probe formed from a tipless V-shaped AFM cantilever. The cell probe was created by immobilising a single yeast cell on such a tipless cantilever. Measurements were made in 10⁻² M NaCl solution. It was found for both protein and cell systems that the adhesive force at the ES 404 membrane was greater than that at the XP 117 membrane. The paper shows that coated colloid probe and cell probe techniques can provide useful means of directly quantifying the adhesion of biological materials to membrane surfaces.     

Single cell analysis at the nanoscale

The fundamental life processes such as signal transduction, intracellular trafficking, protein degradation, and DNA repair often occur in nanometric subcellular compartments. It is essential to conduct single cell analysis specifically at the nanoscale to fully understand the critical cellular processes while providing important medical applications. However, there are great challenges in achieving high spatial resolution in single cells for uncovering spatial heterogeneity, high sensitivity for biomolecule detections and high specificity in complicated cellular environment. In this tutorial review, we survey recent progress toward single cell analysis at the nanoscale by emphasizing how the advancement in nanotechnology has brought a plethora of nanotools to interrogate single cells with high spatiotemporal resolutions. In particular, analysis principle, nanoscale probe fabrication, high resolution cellular analysis, data collection and processing are introduced. New cell biochemistry and biology insights revealed by the unique single cell analysis methods are highlighted. The perspectives on future opportunities and unsolved challenges are also discussed.     

Microfluidic electroporation for selective release of intracellular molecules at the single-cell level

Analysis of intracellular materials at the single-cell level presents opportunities for probing the heterogeneity of a cell population. Lysis by electroporation has been gaining popularity as a rapid method for disruption of the cell membrane and release of intracellular contents. In this report, we selectively released specific intracellular molecules for interrogation at the single-cell level by tuning the parameters of electroporation. We examined the release of a small molecule, calcein (MW approximately 600), and a 72-kDa protein kinase, Syk, tagged by enhanced green fluorescent protein (EGFP) from chicken B cells during electroporation at the single-cell level. We studied the effects of the field intensity and the field duration on the release of the two molecules. We found that calcein in general was released at lower field intensities and shorter durations than did SykEGFP. By tuning the electrical parameters, we were able to deplete calcein from the cells before SykEGFP started to release. This approach potentially provides a high-throughput alternative for probing different intracellular molecules at the single-cell level compared to chemical cytometry by eliminating complete disruption of the cell membrane.     

Differentiation of individual human mesenchymal stem cells probed by FTIR microscopic imaging

Objective of this study is the novel application of Fourier transform infrared (FTIR) microscopic imaging to identify the differentiation state of individual human mesenchymal stem cells with or without osteogenic stimulation. IR spectra of several hundred single cells with lateral resolution of 5-10 microm were recorded using a FTIR imaging spectrometer coupled to a microscope with a focal plane array detector. A classification model based on linear discriminant analysis was trained to distinguish four cell types by their IR spectroscopic fingerprint. Without stimulation two cell types dominated, showing low or high levels of glycogen accumulation at the cell periphery. After stimulation, the protein composition in the cells changed and some cells started expressing calcium phosphate salts such as octacalciumphosphate, a precursor of the bone constituent hydroxyapatite. Few cells were identified which remained in their non-stimulated state. This study demonstrated for the first time that FTIR microscopic imaging can probe stem cell differentiation at the single cell level rapidly, non-destructively and with minimal preparation.     

Electron transfer reaction in a single protein molecule observed by total internal reflection fluorescence microscopy

To observe an electron transfer (ET) process in a single protein molecule, we constructed a model system, Alexa-HCytb5, in which cytochrome b5 (Cytb5) is modified with a fluorescent probe, Alexa Fluor 647 dye. In this model system, intramolecular transfer of an electron from the Alexa dye to heme in Cytb5 is supposed to oxidize the probe and quench its fluorescence, and the ET reaction at the single-molecule level can be monitored as the intermittent change in the fluorescence intensity. Alexa-HCytb5 was fixed on the glass surface, and illumination of laser light by the total internal reflection resulted in blinking of the fluorescence from the single Alexa-HCytb5 molecule in the time scale of several hundred milliseconds. Each Alexa-HCytb5 molecule is characterized by its own rate constant of the blinking, corresponding to the ET rate constant at the single-molecule level, and its variation ranges between 1 and 10 s(-1). The current system thus enables us to visualize the ET reaction in the single protein molecule, and the protein ET reaction was found to be explained by the distribution of the rate constants. On the basis of the Marcus theory, we suggest that the origin of this rate distribution is the distance change associated with the structural fluctuation in the protein molecule.     

In situ probing of cholesterol in astrocytes at the single‐cell level using laser desorption ionization mass spectrometric imaging with colloidal silver

Mass spectrometric imaging has been utilized to localize individual astrocytes and to obtain cholesterol populations at the single‐cell level in laser desorption ionization (LDI) with colloidal silver. The silver ion adduct of membrane‐bound cholesterol was monitored to detect individual cells. Good correlation between mass spectrometric and optical images at different cell densities indicates the ability to perform single‐cell studies of cholesterol abundance. The feasibility of quantification is confirmed by the agreement between the LDI‐MS ion signals and the results from a traditional enzymatic fluorometric assay. We propose that this approach could be an effective tool to study chemical populations at the cellular level. Published in 2010 by John Wiley & Sons, Ltd.     

Transport, retention and fluorescent measurement of single biological cells studied in microfluidic chips

Cellular manipulation and fluorescent measurement were performed on two types of biological cells. First, transport and retention of yeast cells were demonstrated on a glass microfluidic chip, which consists of special U-shaped microstructures. These microstructures have the openings parallel to the liquid flow and weirs perpendicular to the flow. These allow the retention of yeast cells in the U-shaped pocket and drainage of liquid over the weirs. Thereafter, the same chip was used to carry out real-time fluorescent measurement for the cellular changes in single Jurkat T cells. In this case, the Jurkat cells were localized inside the straight portion of a microchannel. Fluorescent imaging on the same, single suspension cell was carried out to study two cellular processes occurring in viable cells, (1) the intracellular conversion of fluorescein diacetate (FDA) to fluorescein; (2) the degradation of an inhibitory protein, IkappaB, as involved in the NF-kappaB signalling pathway. In the former, the increase in fluorescent intensity of single Jurkat T cells (due to fluorescein formation) was measured; whereas in the latter, the decrease in the fluorescent intensity of a single transfected Jurkat cell (due to the degradation of the IkappaB-EGFP fusion protein) was monitored. In addition, we employed a Jurkat cell expressed with IkappaB-EGFP to probe any possible action of an herbal compound, isoliquiritigenin (IQ), on the degradation of IkappaB-EGFP. These examples have demonstrated that Jurkat cells remain viable within microfluidic channels for cellular studies and that the microfluidic chip can facilitate monitoring of cellular changes of biological cells at the single cell level and in the same cell.     

Nanofluidics for sub-single cellular studies: Nascent progress,critical technologies,and future perspectives

In the field of cell studies, there is a burgeoning trend to further downscale the investigation from a single-cell level to a sub-single-cell level. Subcellular matter is the basic content in cells and correlates with cell heterogeneity. Sub-single cellular studies focus on the subcellular matter in single cells and aim to understand the details and heterogeneity of individual cells in terms of the subcellular matter or even at the single component/vesicle/molecule level. Hence, sub-single cell...     

Intracellular protein labeling with prodrug-like probes using a mutant β-lactamase tag

Intracellular protein labeling with small molecular probes that do not require a washing step for the removal of excess probe is greatly desired for real-time investigation of protein dynamics in living cells. Successful labeling of proteins on the cell membrane has been performed using mutant β-lactamase tag (BL-tag) technology. In the present study, intracellular protein labeling with novel cell membrane permeable probes based on β-lactam prodrugs is described. The prodrug-based probes quickly permeated the plasma membranes of living mammalian cells, and efficiently labeled intracellular proteins at low probe concentrations. Because these cell-permeable probes were activated only inside cells, simultaneous discriminative labeling of intracellular and cell surface BL-tag fusion proteins was attained by using cell-permeable and impermeable probes. Thus, this technology enables adequate discrimination of the location of proteins labeled with the same protein tag, in conjunction with different color probes, by dual-color fluorescence. Moreover, the combination of BL-tag technology and the prodrug-based probes enabled the labeling of target proteins without requiring a washing step, owing to the efficient entry of probes into cells and the fast covalent labeling achieved with BL-tag technology after bioactivation. This prodrug-based probe design strategy for BL-tags provides a simple experimental procedure with application to cellular studies with the additional advantage of reduced stress to living cells.     

Parallel single-cell analysis microfluidic platform

We report a PDMS microfluidic platform for parallel single-cell analysis (PaSCAl) as a powerful tool to decipher the heterogeneity found in cell populations. Cells are trapped individually in dedicated pockets, and thereafter, a number of invasive or non-invasive analysis schemes are performed. First, we report single-cell trapping in a fast (2-5 min) and reproducible manner with a single-cell capture yield of 85% using two cell lines (P3x63Ag8 and MCF-7), employing a protocol which is scalable and easily amenable to automation. Following this, a mixed population of P3x63Ag8 and MCF-7 cells is stained in situ using the nucleic acid probe (Hoechst) and a phycoerythrin-labeled monoclonal antibody directed at EpCAM present on the surface of the breast cancer cells MCF-7 and absent on the myeloma cells P3x63Ag8 to illustrate the potential of the device to analyze cell population heterogeneity. Next, cells are porated in situ using chemicals in a reversible (digitonin) or irreversible way (lithium dodecyl sulfate). This is visualized by the transportation of fluorescent dyes through the membrane (propidium iodide and calcein). Finally, an electrical protocol is developed for combined cell permeabilization and electroosmotic flow (EOF)-based extraction of the cell content. It is validated here using calcein-loaded cells and visualized through the progressive recovery of calcein in the side channels, indicating successful retrieval of individual cell content.     

A sensitive electrochemiluminescence cytosensor for quantitative evaluation of epidermal growth factor receptor expressed on cell surfaces

A sensitive electrochemiluminescence (ECL) strategy for evaluating the epidermal growth factor receptor (EGFR) expression level on cell surfaces was designed by integrating the specific recognition of EGFR expressed on MCF-7 cell surfaces with an epidermal growth factor (EGF)-funtionalized CdS quantum dots (CdSQDs)-capped magnetic bead (MB) probe. The high sensitivity of ECL probe of EGF-funtionalized CdSQD-capped-MB was used for competitive recognition with EGFR expressed on cell surfaces with recombinant EGFR protein. The changes of ECL intensity depended on both the cell number and the expression level of EGFR receptor on cell surfaces. A wide linear response to cells ranging from 80 to 4 × 10⁶ cells mL⁻¹ with a detection limit of 40 cells mL⁻¹ was obtained. The EGF-cytosensor was used to evaluate EGFR expression levels on MCF-7 cells, and the average number of EGFR receptor on single MCF-7 cells was 1.35 × 10⁵ with the relative standard deviation of 4.3%. This strategy was further used for in-situ and real-time evaluating EGFR receptor expressed on cell surfaces in response to drugs stimulation at different concentration and incubation time. The proposed method provided potential applications in the detection of receptors on cancer cells and anticancer drugs screening.     

Preparation and characterization of poly(lipid)-coated, fluorophore-doped silica nanoparticles for biolabeling and cellular imaging

The fabrication, characterization, and implementation of poly(lipid)-coated, highly luminescent silica nanoparticles as fluorescent probes for labeling of cultured cells are described. The core of the probe is a sol-gel-derived silica nanoparticle, 65-100 nm in diameter, in which up to several thousand dye molecules are encapsulated (Lian, W.; et al. Anal. Biochem. 2004, 334, 135-144). The core is coated with a membrane composed of bis-sorbylphosphatidylcholine, a synthetic polymerizable lipid that is chemically cross-linked to enhance the environmental and chemical stability of the membrane relative to a fluid lipid membrane. The poly(lipid) coating has two major functions: (i) to reduce nonspecific interactions, based on the inherently biocompatible properties of the phosphorylcholine headgroup, and (ii) to permit functionalization of the particle, by doping the coating with lipids bearing chemically reactive or bioactive headgroups. Both functions are demonstrated: (i) Nonspecific adsorption of dissolved proteins to bare silica nanoparticles and of bare nanoparticles to cultured cells is significantly reduced by application of the poly(lipid) coating. (ii) Functionalization of poly(lipid)-coated nanoparticles with a biotin-conjugated lipid creates a probe that can be used to target both dissolved protein receptors as well as receptors on the membranes of cultured cells. Measurements performed on single nanoparticles bound to planar supported lipid bilayers verify that the emission intensity of these probes is significantly greater than that of single protein molecules labeled with several fluorophores.     

Sensing lymphoma cells based on a cell-penetrating/apoptosis-inducing/electron-transfer peptide probe

To electrochemically sense lymphoma cells (U937), we fabricated a multifunctional peptide probe that consists of cell-penetrating/apoptosis-inducing/electron-transfer peptides. Electron-transfer peptides derive from cysteine residue combined with the C-terminals of four tyrosine residues (Y₄). A peptide whereby Y₄C is bound to the C-terminals of protegrin 1 (RGGRLCYCRRRFCVCVGR-NH₂) is known to be an apoptosis-inducing agent against U937 cells, and is referred to as a peptide-1 probe. An oxidation response of the peptide-1 probe has been observed due to a phenolic hydroxyl group, and this response is decreased by the uptake of the peptide probe into the cells. To improve the cell membrane permeability against U937 cells, the RGGR at the N-terminals of the peptide-1 probe was replaced by RRRR (peptide-2 probe). In contrast, RNRCKGTDVQAWY₄C (peptide-3 probe), which recognizes ovalbumin, was constructed as a control. Compared with the other probes, the change in the peak current of the peptide-2 probe was the greatest at low concentrations and occurred in a short amount of time. Therefore, the cell membrane permeability of the peptide-2 probe was increased based on the arginine residues and the apoptosis-inducing peptides. The peak current was linear and ranged from 100 to 1000 cells/ml. The relative standard deviation of 600 cells/ml was 5.0% (n = 5). Furthermore, the membrane permeability of the peptide probes was confirmed using fluorescent dye.     

Cell membranes suspended across nanoaperture arrays

We present a method for spreading large (>100 microm(2)) cell membrane fragments across nanoapertures in planar supports. Electron-beam and focused-ion-beam lithography were used to fabricate arrays of 50-600 nm diameter holes in free-standing silicon nitride (SiN) solid films 100-500 nm thick. By pressing adhering live cells onto the nanostructured SiN surface and then removing them, planar cell membrane sheets (CMSs) were transferred in a well-defined orientation onto the SiN support. We demonstrate the accessibility to both extracellular and intracellular surfaces of CMSs by targeting membrane constituents side-specifically with fluorescent markers. Our approach is of interest for studying ligand-receptor interactions using optical, electrical, and scanning probe techniques at the single-molecule level.