Synchrotron Infrared Microspectroscopy for Stem Cell Research.

Stem cells have shown great potential functions for tissue regeneration and repair because of their unlimited self-renewal and differentiation. Stem cells reside in their niches, making them a hotspot for the development and diagnosis of diseases. Complex interactions between niches and stem cells create the balance between differentiation, self-renewal, maturation, and proliferation. However, the multi-facet applications of stem cells have been challenged since the complicated responses of stem cells to biological processes were explored along with the limitations of current systems or methods. Emerging evidence highlights that synchrotron infrared microspectroscopy, known as synchrotron radiation-based Fourier transform infrared microspectroscopy, has been investigated as a potentially attractive technology with its non-invasive and non-biological probes in stem cell research. With their unique vibration bands, the quantitative mapping of the content and distribution of biomolecules can be detected and characterized in cells or tissues. In this review, we focus on the potential applications of synchrotron infrared microspectroscopy for investigating the differentiation and fate determination of stem cells.

Neferine Protects Endothelial Glycocalyx via Mitochondrial ROS in Lipopolysaccharide-Induced Acute Respiratory Distress Syndrome.

Damage to the endothelial glycocalyx is a critical factor in increased pulmonary vascular permeability, which is the basic pathological feature of acute respiratory distress syndrome (ARDS). Neferine (Nef), a bisbenzylisoquinoline alkaloid isolated from green seed embryos of Nelumbo nucifera Gaertn, has extensive pharmacological activity. In this study, we showed that Nef reduced lung-capillary permeability, down-regulated the production of cytokines (IL-1ß, IL-6, TNF-α, and IL-10) and inhibited the activation of the NF-κB signaling pathway in mice with lipopolysaccharide (LPS)-induced ARDS. Further analysis indicated that Nef provided protection against endothelial glycocalyx degradation in LPS-induced ARDS mice (in vivo) and in LPS-stimulated human umbilical vein endothelial cells (in vitro). The glycocalyx-protective effect of Nef may be initiated by suppressing the production of mitochondrial ROS (mtROS) and decreasing oxidative damage. Nef was also found to promote glycocalyx restoration by accelerating the removal of mtROS in endothelial cells in LPS-induced ARDS. These results suggested the potential of Nef as a therapeutic agent for ARDS associated with Gram-negative bacterial infections and elucidated the mechanisms underlying the protection and restoration of the endothelial glycocalyx.

Glycerol facilitates the disaggregation of recombinant adeno-associated virus serotype 2 on mica surface.

Preparation of distributed virus on a solid substrate is a prerequisite for investigation of the properties and individualism of virus, while many previous studies showed that virus has a tendency to aggregate on solid substrates. In this communication, we report a novel approach by which well-separated recombinant adeno-associated virus serotype 2 (rAAV2) could be prepared on bare mica surface. The key technique in this approach is the addition of less than 3% (v/v) glycerol into the virus solution and subsequently deposition onto mica surface for the sample preparation. The possible mechanisms are also briefly discussed.

Purification of the recombinant hepatitis B virus core antigen (rHBcAg) produced in the yeast Saccharomyces cerevisiae and comparative observation of its particles by transmission electron microscopy (TEM) and atomic force microscopy (AFM).

Hepatitis B virus core antigen (HBcAg) gene (C gene) was expressed in Saccharomyces cerevisiae and the products (rHBcAg or core particles) were purified from a crude lysate of the yeast by three steps: Sephrose CL-4B chromatography, Sucrose step-gradient ultracentrifugation and CsCl-isopycnic ultracentrifugation. It has been observed that HBcAg was synthesized in yeast cells as a particle consisting of polypeptides with a molecular weight of 21.5 kDa (p21.5). Results of ELISA test and density analysis of CsCl-isopycnic ultracentrifugation indicated that the purified products (rHBcAg particles) with HBcAg antigenicity mainly located at the densities of 1.27 and 1.40 g ml(-1), respectively. Observation and analysis of the purified rHBcAg products by TEM indicated that rHBcAg peptides could mainly self-assemble into two size classes of core particles. The larger particles were approximately 30.1 nm and the smaller were approximately 21.5 nm in mean diameter. Further observation and analysis of the same rHBcAg (core) particles by AFM also indicated that rHBcAg (core) particles were similar to the native HBcAg (core) particles from infected human hepatocytes and mainly composed of two size classes of partides core. The larger particles were approximately 31.3 nm and the smaller were approximately 22.5 nm in mean diameter which was similar to the results obtained by TEM. All results from both TEM and AFM suggested that core particles (capsids) produced in S. cerevisiae possessed dimorphism.

Nanomanipulation of extended single-DNA molecules on modified mica surfaces using the atomic force microscopy.

The technique of nanometer scale manipulation is very important in constructing nano-structures and nano-devices. By using atomic force microscope, three kinds of controllable manipulation on single-DNA molecules were introduced in the paper. DNA molecules deposited and extended on modified mica surface were first imaged by tapping mode, then cutting, bending, and pushing were respectively performed on single-DNA molecules. The results of the manipulation depend on the interaction between tip and DNA as well as between substrate and DNA.

TGF-ß1 induces the formation of vascular-like structures in embryoid bodies derived from human embryonic stem cells.

Human embryonic stem cells (ESCs) can differentiate into endothelial cells in response to stimuli from extracellular cytokines. Transforming growth factor (TGF)-ß1 signaling is involved in stem cell renewal and vascular development. Previously, human ESCs were isolated from inner cell mass and a stable ESC line was developed. In the present study, the effects of extracellular TGF-ß1 were investigated on human ESC-derived embryoid bodies (EB) in suspension. The structures of the EBs were analyzed with light and electron microscopy, while the cellular composition of the EBs was examined via the expression levels of specific markers. Vascular-like tubular structures and cardiomyocyte-like beating cells were observed in the EBs at day 3 and 8, respectively. The frequencies of vascular-like structures and beating cells in the TGF-ß1 treated group were significantly higher compared with the control group (84.31 vs. 12.77%; P<0.001; 37.25 vs. 8.51%; P<0.001, respectively). Electron microscopy revealed the presence of lumens and gap junctions in the sections of the tubular structures. Semiquantitative polymerase chain reaction revealed elevated expression levels of CD31 and fetal liver kinase-1 in EBs cultured with TGF-ß1. In addition, extensive staining of von Willebrand factor was observed in the vascular-like structures of TGF-ß1-treated EBs. Therefore, the results of the present study may aid the understanding of the underlying mechanisms of human ESC differentiation and improve the methods of propagating specific cell types for the clinical therapy of cardiovascular diseases.

Improved cryopreservation of human embryonic stem cells with trehalose.

Human embryonic stem (ES) cells have been established either from fresh or frozen embryos. The recovery rates of undifferentiated human ES cells after cryopreservation with conventional slow-rate freezing and rapid-thawing methods are relatively low. The purpose of this study was to improve cryopreservation efficiency by modifying conventional methods with addition of trehalose. Immature oocytes donated from patients undergoing IVF treatment were utilized to generate blastocysts. One human ES cell line (named hES1) was established and characterized in detail. The hES1 cells expressed regular human ES cell markers, including stage-specific embryonic antigens SSEA-3, SSEA-4, tumour rejection antigens TRA-1-60, TRA-1-81 and octamer-binding transcription factor Oct-4 with high levels of alkaline phosphatase and telomerase activities. Cells could be differentiated to form teratomas in vivo. With slow-rate freezing and rapid-thawing methods modified by adding trehalose, the recovery rate of undifferentiated hES1 cells has been greatly improved from 15 to 48%. Cells retained pluripotency with normal karyotype after thawing. The results indicated that the use of trehalose is efficient and convenient for cryopreservation of human ES cells.

[Biological characterization of immortalized porcine chondrocytes by introduction of telomerase].

The aim of this project was to acquire a clonal immortalized porcine chondrocyte cell line and to determine its biological characterization. Porcine auricle chondrocytes were infected with a retroviral vector pBABE-hTERT followed by selection with 2 micrograms/ml puromycin. RT-PCR and PCR-ELISA performed on separated colonies showed a cell line (TL1) with high and stable ectopic expression of hTERT. The growth curve, HE staining, flow cytometry, and tumorigenicity were analyzed to define the biological characterization of the immortalized chondrocytes. TL1 cells showed an increase in cellular viability and the reduction in apoptosis. But there was no malignant transformation and tumor development in nude mice. The changes of chondrocyte marker expression in this cell line revealed that it remained unstable phenotype. So high ectopic expression of hTERT is able to extend the life span of chondrocytes, but it unable to keep their differentiated phenotype during serial monolayer culture in vitro.

[Construction and expression of retroviral vector containing bone morphogenetic protein-7].

AIM: To achieve stable and long-term expression of BMP-7 gene in bone marrow stem cells (BMSCs). METHODS: Retrovirus expression vector containing BMP-7 gene was constructed and transfected into packaging cells PT67. After puromycin selection and cells cloning, cell clones producing high level of recombinant virus were obtained. The viruses were used to infect directly BMSCs and the expression of BMP-7 gene in BMSCs was analyzed by immunohistochemical staining. RESULTS: Retrovirus vector containing BMP-7 gene was successfully reconstructed and BMP-7 was expressed in the BMSCs.The transfection rates was about 30%-40%. CONCLUSION: The construction of recombinant retrovirus vector containing BMP-7 gene can provide a reliable tool for the formation of bone or cartilage seed cells in the research of tissue engineering.

Positioning isolation and biochemical analysis of single DNA molecules based on nanomanipulation and single-molecule PCR.

Recently, the isolation and biochemical analysis of DNA at the single-molecule level has been recognized as very important for genetic research and clinical analysis. A unique technique for the positioning, dissection, and isolation of single DNA molecules using atomic force microscopy (AFM) has been demonstrated. Full-length genome DNA molecules were first deposited and stretched by a modified "molecular combing" technique onto a 3-aminopropyl triethoxysilane-coated mica substrate. A single DNA fragment was dissected from one of those genome DNA strands with the AFM tip at the desired position, and then isolated (or picked up) after a special operation called "kneading". All the operations including imaging, dissection, and isolation could be carried out with one tip. The isolated DNA fragment on the AFM tip could be successfully amplified by single-molecule PCR.