Hui Qi

Role of Injured Pancreatic Extract Promotes Bone Marrow-Derived Mesenchymal Stem Cells Efficiently Differentiate into Insulin-Producing Cells

Mesenchymal stem cells (MSCs) can be successfully induced to differentiate into insulin-producing cells （IPCs) by a variety of small molecules and cytokines in vitro. However, problems remain, such as low transdifferentiation efficiency and poor maturity of trans-differentiated cells. The damaged pancreatic cells secreted a large amount of soluble proteins, which were able to promote pancreative islet regeneration and MSCs differentiation. In this study, we utilized the rat injured pancreatic tissue extract to modulate rat bone marrow-derived MSCs differentiation into IPCs by the traditional two-step induction. Our results showed that injured pancreatic tissue extract could effectively promote the trans-differentiation efficiency and maturity of IPCs by the traditional induction. Moreover, IPCs were able to release more insulin in a glucose-dependent manner and ameliorate better the diabetic conditions of streptozotocin (STZ)-treated rats. Our study provides a new strategy to induce an efficient and directional differentiation of MSCs into IPCs.

A rapid and highly sensitive protocol for the detection of Escherichia coli O157:H7 based on immunochromatography assay combined with the enrichment technique of immunomagnetic nanoparticles

Background Escherichia coli O157:H7 (E. coli O157:H7) is an important pathogenic bacterium that threatens human health. A rapid, simple, highly sensitive, and specific method for the detection of E. coli O157:H7 is necessary. Methods In the present study, immunomagnetic nanoparticles (IMPs) were prepared with nanopure iron as the core, coated with E. coli O157:H7 polyclonal antibodies. These IMPs were used in combination with immunochromatographic assay (ICA) and used to establish highly sensitive and rapid kits (IMPs+ICA) to detect E. coli O157:H7. The kits were then used to detect E. coli O157:H7 in 150 food samples and were compared with conventional ICA to evaluate their efficacy. Results The average diameter of IMPs was 56 nm and the amount of adsorbed antibodies was 106.0 μg/mg. The sensitivity of ICA and IMPs+ICA was 105 colony-forming units/mL and 103 CFUs/mL, respectively, for purified E. coli O157:H7 solution. The sensitivity of IMPs+ICA was increased by two orders, and its specificity was similar to ICA. Conclusion The kits have the potential to offer important social and economic benefits in the screening, monitoring, and control of food safety.

Role of histone deacetylase inhibitors in the aging of human umbilical cord mesenchymal stem cells

Mesenchymal stem cells (MSCs) are self‐renewing cells that exhibit differentiation capacity and immune regulation ability. These versatile cells have a wide range of potential applications. However, the spontaneous differentiation and aging of MSCs during long‐term culturing restrict the amount of cells available for therapies and tissue engineering. Thus, maintaining the biological characteristics of MSCs during long‐term culturing is crucial. Chromatic modification via epigenetic regulatory mechanisms (e.g., histone acetylation, deacetylation, and methylation) is crucial in stem cell pluripotency. We investigated the effects of largazole or trichostatin A (TSA), a novel histone deacetylase inhibitor (HDACi), against human umbilical cord (hUC)‐MSCs aging. Results show that low concentrations of largazole or TSA can significantly improve hUC‐MSCs proliferation and delay hUC‐MSCs aging. Largazole can better improve MSCs proliferation than TSA. HDAC is modulate histone H3 acetylation and methylation in the telomerase reverse‐transcriptase, octamer‐binding transcription factor 4, Nanog, C‐X‐C chemokine receptor 4, alkaline phosphatase, and osteopontin genes. HDACis can promote hUC‐MSCs proliferation and suppress hUC‐MSCs spontaneous osteogenic differentiation. HDACis can affect histone H3 lysine 9 or 14 acetylation and histone H3 lysine 4 dimethylation, thus increasing the mRNA expression of pluripotent and proliferative genes and suppressing the spontaneous differentiation of hUC‐MSCs. J. Cell. Biochem. 114: 2231–2239, 2013.

Immunogenicity of allogeneic mesenchymal stem cells transplanted via different routes in diabetic rats

Due to their hypoimmunogenicity and unique immunosuppressive properties, mesenchymal stem cells (MSCs) are considered one of the most promising adult stem cell types for cell therapy. Although many studies have shown that MSCs exert therapeutic effects on several acute and subacute conditions, their long-term effects are not confirmed in chronic diseases. Immunogenicity is a major limitation for cell replacement therapy, and it is not well understood in vivo. We evaluated the immunogenicity of allogeneic MSCs in vivo by transplanting MSCs into normal and diabetic rats via the tail vein or pancreas and found that MSCs exhibited low immunogenicity in normal recipients and even exerted some immunosuppressive effects in diabetic rats during the initial phase. However, during the later stage in the pancreas group, MSCs expressed insulin and MHC II, eliciting a strong immune response in the pancreas. Simultaneously, the peripheral blood mononuclear cells in the recipients in the pancreas group were activated, and alloantibodies developed in vivo. Conversely, in the tail vein group, MSCs remained immunoprivileged and displayed immunosuppressive effects in vivo. These data indicate that different transplanting routes and microenvironments can lead to divergent immunogenicity of MSCs.Cellular & Molecular Immunology advance online publication, 22 September 2014; doi:10.1038/cmi.2014.70

Alpha-1-antitrypsin for the improvement of autoimmunity and allograft rejection in beta cell transplantation

Islet transplantation offers hope for patients with type 1 diabetes, which is an autoimmune disease. However, islet transplant recipients must overcome two obstacles in both allograft rejection and autoimmune reaction. Alpha-1-antitrypsin (a1-proteinase inhibitor, AAT) possesses anti-inflammatory properties, reduces cytokine-mediated islet damage, and induces specific immune tolerance. In this study, an insulinoma cell line, NIT-1, was transfected with human AAT (hAAT), named NIT-hAAT, and was transplanted to the left renal subcapsular spaces of 7-week-old female non-obese diabetic (NOD) mice (n=22). Cyclophosphamide(CY) was administered to synchronize and accelerate the development of diabetes. Thus, the immunosuppressive and cytoprotective activity of hAAT in β-cell transplantation was investigated. NIT-hAAT has immunomodulatory properties, which delay the onset of autoimmune diabetes, reduce diabetes incidence, inhibit insulitis and β-cell apoptosis, and dampen transplant site inflammation. We propose that NIT-hAAT has a dual function by improving islet autoimmunity and protecting transplanted β-cells from allograft rejection. However, the low expression of hAAT in vivo results in the inability of NIT-hAAT to induce long-term specific immune tolerance and to completely block allograft rejection.

Detection of circulating tumor cells in breast cancer with a refined immunomagnetic nanoparticle enriched assay and nested-RT-PCR

UNLABELLED Early detection of circulation tumor cells (CTCs) in breast cancer patients has great clinical relevance. Currently, immunomagnetic microparticles enriched assays require Fe3O4 inner cores, making it difficult to improve sensitivity. In this study, we prepared magnetic nanoparticles with carbon-coated pure iron (Fe@C) acted as the core, Conjugating with EpCAM monoclonal antibody for immunomagnetic nanoparticles(IMPs). IMPs were used in conjunction with immunocytochemistry (ICC) to develop a refined immunomagnetic nanoparticles enriched assay (IMPEA) for detection of circulating tumor cells (CTCs) in breast cancer patients. Compared with nested RT-PCR, this method achieved the same sensitivity, but with a significantly reduced false-positive rate. This method will help find hidden micrometastases, establish clinical stage, and guide individual treatment post-surgery, suggesting potentially significant value in the clinic. FROM THE CLINICAL EDITOR This team of investigators prepared magnetic nanoparticles with carbon-coated pure iron as core and conjugated them with EpCAM monoclonal antibody to form immunomagnetic nanoparticles for circulating tumor cell (CTC) detection. Compared with nested RT-PCR, this method achieved the same sensitivity, but with a significantly reduced false-positive rate, paving the way to the development of a tool that enables enhanced detection of micrometastases and post-surgical treatment monitoring.

Detection of micrometastases in peripheral blood of non-small cell lung cancer with a refined immunomagnetic nanoparticle enrichment assay

Fe3O4 particles are currently used as the core of immunomagnetic microspheres in the immunomagnetic enrichment assay of circulating tumor cells (CTCs). It is difficult to further improve the sensitivity of CTC detection or to improve tumor cell-type identification and characterization. In the present study, we prepared immunomagnetic nanoparticles with nanopure iron as the core, coated with anti-cytokeratin 7/8 (CK7/8) monoclonal antibody. These immunomagnetic nanoparticles (IMPs) were used in conjunction with immunocytochemistry (ICC) to establish a refined immunomagnetic nanoparticle enrichment assay for CTC detection in non-small cell lung cancer (NSCLC). The assay was compared with nested reverse transcription polymerase chain reaction (RT-PCR) to detect CK19 mRNA and lung specific X protein (LUNX) mRNA. Human lung adenocarcinoma cell line A549 was used for sensitivity and specificity evaluation. Peripheral blood samples were collected from each group for CTC detection. The average diameter of the immunomagnetic nanoparticles was 51 nm, and the amount of adsorbed antibodies was 111.2 μg/mg. We could detect down to one tumor cell in 5 × 107 peripheral blood mononuclear cells. The sensitivity was consistent with that of nested RT-PCR; however, the false positive rate was significantly reduced. The modified assay combined with ICC did not differ from nested RT-PCR in sensitivity, but it had significantly increased specificity. This approach could, therefore, contribute to identification of micrometastases, re-defining clinical staging, and guiding individual postoperative treatments. The technique shows considerable potential clinical value and further clinical trials are warranted.

The immunoregulation effect of alpha 1-antitrypsin prolong β-cell survival after transplantation.

Islet transplantation has considerable potential as a cure for diabetes. However, the difficulties that arise from inflammation and the immunological rejection of transplants must be addressed for islet transplantation to be successful. Alpha 1-antitrypsin (AAT) inhibits the damage on β cells caused by inflammatory reactions and promotes β-cell survival and proliferation. This protein also induces specific immune tolerance to transplanted β cells. However, whether the expression of AAT in β cells themselves could eliminate or decrease immunological rejection of transplants is not clear. Therefore, we established a β cell line (NIT-hAAT) that stably expresses human AAT. Interestingly, in a cytotoxic T lymphocyte (CTL)-killing assay, we found that hAAT reduced apoptosis and inflammatory cytokine production in NIT-1 cells and regulated the Th1/Th2 cytokine balance in vitro. In vivo transplantation of NIT-hAAT cells into mice with diabetes showed hAAT inhibited immunological rejection for a short period of time and increased the survival of transplanted β cells. This study demonstrated that hAAT generated remarkable immunoprotective and immunoregulation effects in a model of β cell islet transplantation for diabetes model.

Reversible immortalization of Nestin-positive precursor cells from pancreas and differentiation into insulin-secreting cells

Pancreatic stem cells or progenitor cells posses the ability of directed differentiation into pancreatic β cells. However, these cells usually have limited proliferative capacity and finite lifespan in vitro. In the present study, Nestin-positive progenitor cells (NPPCs) from mouse pancreas that expressed the pancreatic stem cells or progenitor cell marker Nestin were isolated to obtain a sufficient number of differentiated pancreatic β cells. Tet-on system for SV40 large T-antigen expression in NPPCs was used to achieve reversible immortalization. The reversible immortal Nestin-positive progenitor cells (RINPPCs) can undergo at least 80 population doublings without senescence in vitro while maintaining their biological and genetic characteristics. RINPPCs can be efficiently induced to differentiate into insulin-producing cells that contain a combination of glucagon-like peptide-1 (GLP-1) and sodium butyrate. The results of the present study can be used to explore transplantation therapy of type I diabetes mellitus.

A novel sensitive immunoassay by nucleic acid barcode dot and its application in the detection of prostate-specific antigen.

Abstract Background: The sensitivity and selectivity of traditional methods limits ultramicro detection of proteins. Bio-barcode amplification detection methods based on nanotechnology enables ultramicro detection of protein. However, bio-barcode amplification detection depends on the oligonucleotides being fixed on a glass chip. It also requires specialized equipment, which limits its application. We introduce a nano-nucleic acid barcode dot detection technology to determine ultramicro concentrations of protein. The method is simple, quick and accurate. Methods: Magnetic probe (IgG-M) and dual-labeled gold nanoparticle bio-probe (IgG-Au-DNA) were prepared. Protein was captured using a sandwich assay technique and magnetic separation was used. The DNA barcode was released with dithiothreitol (DTT) and detected directly without the requirement for polymerase chain reaction (PCR). Serum prostate-specific antigen (PSA) from 135 patients was detected with this method and compared with enzyme linked immunosorbent assay (ELISA) and radioimmunoassay (RIA). Results: Each IgG-Au-DNA could be covered with 138±47 oligonucleotides and 11±3 antibodies. The IgG-M could bind 118 μg of antibody per mg. The sensitivity of nano-nucleic acid barcode dot detection technology might allow detection of 1 fg/mL. There were no significant differences in serum PSA from 135 patients when comparing the three methods (compared with ELISA, r=0.950; and with RIA, r=0.967). Conclusions: The nucleic acid barcode dot method does not require special equipment or complex procedures, but its detection limit is 2–3 orders of magnitude lower than ELISA. Clin Chem Lab Med 2010;48:279–83.