Lingwen Zeng

A Mesenchymal-to-Epithelial Transition Initiates and Is Required for the Nuclear Reprogramming of Mouse Fibroblasts

Epithelial-to-mesenchymal transition (EMT) is a developmental process important for cell fate determination. Fibroblasts, a product of EMT, can be reset into induced pluripotent stem cells (iPSCs) via exogenous transcription factors but the underlying mechanism is unclear. Here we show that the generation of iPSCs from mouse fibroblasts requires a mesenchymal-to-epithelial transition (MET) orchestrated by suppressing pro-EMT signals from the culture medium and activating an epithelial program inside the cells. At the transcriptional level, Sox2/Oct4 suppress the EMT mediator Snail, c-Myc downregulates TGF-beta1 and TGF-beta receptor 2, and Klf4 induces epithelial genes including E-cadherin. Blocking MET impairs the reprogramming of fibroblasts whereas preventing EMT in epithelial cells cultured with serum can produce iPSCs without Klf4 and c-Myc. Our work not only establishes MET as a key cellular mechanism toward induced pluripotency, but also demonstrates iPSC generation as a cooperative process between the defined factors and the extracellular milieu. PAPERCLIP:

Vitamin C Enhances the Generation of Mouse and Human Induced Pluripotent Stem Cells

Somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs) by defined factors. However, the low efficiency and slow kinetics of the reprogramming process have hampered progress with this technology. Here we report that a natural compound, vitamin C (Vc), enhances iPSC generation from both mouse and human somatic cells. Vc acts at least in part by alleviating cell senescence, a recently identified roadblock for reprogramming. In addition, Vc accelerates gene expression changes and promotes the transition of pre-iPSC colonies to a fully reprogrammed state. Our results therefore highlight a straightforward method for improving the speed and efficiency of iPSC generation and provide additional insights into the mechanistic basis of the reprogramming process.

The Histone Demethylases Jhdm1a/1b Enhance Somatic Cell Reprogramming in a Vitamin-C-Dependent Manner

Reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) resets the epigenome to an embryonic-like state. Vitamin C enhances the reprogramming process, but the underlying mechanisms are unclear. Here we show that the histone demethylases Jhdm1a/1b are key effectors of somatic cell reprogramming downstream of vitamin C. We first observed that vitamin C induces H3K36me2/3 demethylation in mouse embryonic fibroblasts in culture and during reprogramming. We then identified Jhdm1a/1b, two known vitamin-C-dependent H3K36 demethylases, as potent regulators of reprogramming through gain- and loss-of-function approaches. Furthermore, we found that Jhdm1b accelerates cell cycle progression and suppresses cell senescence during reprogramming by repressing the Ink4/Arf locus. Jhdm1b also cooperates with Oct4 to activate the microRNA cluster 302/367, an integral component of the pluripotency machinery. Our results therefore reveal a role for H3K36me2/3 in cell fate determination and establish a link between histone demethylases and vitamin-C-induced reprogramming.

Aptamer-nanoparticle strip biosensor for sensitive detection of cancer cells.

We report an aptamer-nanoparticle strip biosensor (ANSB) for the rapid, specific, sensitive, and low-cost detection of circulating cancer cells. Known for their high specificity and affinity, aptamers were first selected from live cells by the cell-SELEX (systematic evolution of ligands by exponential enrichment) process. When next combined with the unique optical properties of gold nanoparticles (Au-NPs), ANSBs were prepared on a lateral flow device. Ramos cells were used as a model target cell to demonstrate proof of principle. Under optimal conditions, the ANSB was capable of detecting a minimum of 4000 Ramos cells without instrumentation (visual judgment) and 800 Ramos cells with a portable strip reader within 15 min. Importantly, ANSB has successfully detected Ramos cells in human blood, thus providing a rapid, sensitive, and low-cost quantitative tool for the detection of circulating cancer cells. ANSB therefore shows great promise for in-field and point-of-care cancer diagnosis and therapy.

Disposable Nucleic Acid Biosensors Based on Gold Nanoparticle Probes and Lateral Flow Strip

In this article, we describe a disposable nucleic acid biosensor (DNAB) for low-cost and sensitive detection of nucleic acid samples in 15 min. Combining the unique optical properties of gold nanoparticles (Au-NP) and the high efficiency of chromatographic separation, sandwich-type DNA hybridization reactions were realized on the lateral flow strips, which avoid multiple incubation, separation, and washing steps in the conventional nucleic acid biosensors. The captured Au-NP probes on the test zone and control zone of the biosensor produced the characteristic red bands, enabling visual detection of nucleic acid samples without instrumentation. The quantitative detection was performed by reading the intensities of the produced red bands with a portable strip reader. The parameters (e.g., the concentration of reporter probe, the size of Au-NP, the amount of Au-NP-DNA probe, lateral flow membranes, and the concentration of running buffer) that govern the sensitivity and reproducibility of the sensor were optimized. The response of the optimized device is highly linear over the range of 1-100 nM target DNA, and the limit of detection is estimated to be 0.5 nM in association with a 15 min assay time. The sensitivity of the biosensor was further enhanced by using horseradish peroxidase (HRP)-Au-NP dual labels which ensure a quite low detection limit of 50 pM. The DNAB has been applied for the detection of human genomic DNA directly with a detection limit of 2.5 microg/mL (1.25 fM) by adopting well-designed DNA probes. The new nucleic acid biosensor thus provides a rapid, sensitive, low cost, and quantitative tool for the detection of nucleic acid samples. It shows great promise for in-field and point-of-care diagnosis of genetic diseases and detection of infectious agents or warning against biowarfare agents.

BMPs functionally replace Klf4 and support efficient reprogramming of mouse fibroblasts by Oct4 alone

Generation of induced pluripotent stem cells by defined factors has become a useful model to investigate the mechanism of reprogramming and cell fate determination. However, the precise mechanism of factor-based reprogramming remains unclear. Here, we show that Klf4 mainly acts at the initial phase of reprogramming to initiate mesenchymal-to-epithelial transition and can be functionally replaced by bone morphogenetic proteins (BMPs). BMPs boosted the efficiency of Oct4/Sox2-mediated reprogramming of mouse embryonic fibroblasts (MEFs) to ∼1%. BMPs also promoted single-factor Oct4-based reprogramming of MEFs and tail tibial fibroblasts. Our studies clarify the contribution of Klf4 in reprogramming and establish Oct4 as a singular setter of pluripotency in differentiated cells.

Lateral flow biosensor for DNA extraction-free detection of Salmonella based on aptamer mediated strand displacement amplification.

Convenient and sensitive point-of-care rapid diagnostic tests for food-borne pathogens have been a long-felt need of clinicians. Traditional approaches such as culture-based methods have good sensitivity and specificity, but they tend to be tedious and time-consuming. Herein we present a simple and sensitive aptamer based biosensor for rapid detection of Salmonella enteritidis (S. enteritidis). One of the aptamers specific for the outmembrane of S. enteritidis was used for magnetic bead enrichments. Another aptamer against S. enteritidis was used as a reporter for this pathogen, which was amplified by isothermal strand displacement amplification (SDA) and further detected by a lateral flow biosensor. As low as 10(1) colony forming unit (CFU) of S. enteritidis was detected in this study. Without DNA extraction, the reduced handling and simpler equipment requirement render this assay a simple and rapid alternative to conventional methods.

A sensitive lateral flow biosensor for Escherichia coli O157:H7 detection based on aptamer mediated strand displacement amplification.

Foodborne diseases caused by pathogens are one of the major problems in food safety. Convenient and sensitive point-of-care rapid diagnostic tests for food-borne pathogens have been a long-felt need of clinicians. Commonly used methods for pathogen detection rely on conventional culture-based tests, antibody-based assays and polymerase chain reaction (PCR)-based techniques. These methods are costly, laborious and time-consuming. Herein, we present a simple and sensitive aptamer based biosensor for rapid detection of Escherichia coli O157:H7 (E. coli O157:H7). In this assay, two different aptamers specific for the outmembrane of E. coli O157:H7 were used. One of the aptamers was used for magnetic bead enrichment, and the other was used as a signal reporter for this pathogen, which was amplified by isothermal strand displacement amplification (SDA) and further detected by a lateral flow biosensor. Only the captured aptamers on cell membrane were amplified, limitations of conventional DNA amplification based method such as false-positive can be largely reduced. The generated signals (red bands on the test zone of a lateral flow strip) can be unambiguously read out by the naked eye. As low as 10 colony forming units (CFU) of E. coli O157:H7 were detected in this study. Without DNA extraction, the reduced handling and simpler equipment requirement render this assay a simple and rapid alternative to conventional methods.

Colorimetric detection of copper(II) ion using click chemistry and hemin/G-quadruplex horseradish peroxidase-mimicking DNAzyme.

G-quadruplex-forming sequence can be formed through a copper(I) ion (Cu(+))-catalyzed click chemistry between azide- and alkyne-modified short G-rich sequences in aqueous solution, eliminating immobilization and washing steps of conventional assays. The source for Cu(+) was generated from the reduction of Cu(2+) with the reductant of sodium ascorbate. In the presence of hemin and K(+), the self-assembly of hemin/G-quadruplex structure has the activity of horseradish peroxidase (HRP), which can catalyze its colorless substrate tetrazmethyl benzidine (TMB) into a colored product. Hence, the concentration of Cu(2+) can be evaluated visually for qualitative analysis according to the color change of the solution, and the optical density (OD) value of the resulting solution at 450 nm was also recorded using a microplate reader for quantitative analysis.

Molecular beacon-functionalized gold nanoparticles as probes in dry-reagent strip biosensor for DNA analysis

The highly specific molecule recognition properties of molecular beacons (MB) are combined with the unique optical properties of gold nanoparticles (Au-NPs) for the development of a dry-reagent strip-type nucleic acid biosensor (DSNAB) that enables sensitive and low-cost detection of nucleic acid samples within 15 min.