Targeting delivery of lipocalin 2-engineered mesenchymal stem cells to colon cancer in order to inhibit liver metastasis in nude mice

One of the major obstacles in cancer therapy is the lack of anticancer agent specificity to tumor tissues. The strategy of cell-based therapy is a promising therapeutic option for cancer treatment. The specific tumor-oriented migration of mesenchymal stem cells (MSCs) makes them a useful vehicle to deliver anticancer agents. In this study, we genetically manipulated bone marrow-derived mesenchymal stem cells with their lipocalin 2 (Lcn2) in order to inhibit liver metastasis of colon cancer in nude mice. Lcn2 was successfully overexpressed in transfected MSCs. The PCR results of SRY gene confirmed the presence of MSCs in cancer liver tissue. This study showed that Lcn2-engineered MSCs (MSC-Lcn2) not only inhibited liver metastasis of colon cancer but also downregulated the expression of vascular endothelial growth factor (VEGF) in the liver. Overall, MSCs by innate tropism toward cancer cells can deliver the therapeutic agent, Lcn2, and inhibit cancer metastasis. Hence, it could be a new modality for efficient targeted delivery of anticancer agent to liver metastasis. © 2015, International Society of Oncology and BioMarkers (ISOBM)

Neutrophil Gelatinase-Associated Lipocalin induces the expression of heme oxygenase-1 and superoxide dismutase 1, 2

Lipocalin-2 (Lcn2, NGAL) is a member of the lipocalin super family with diverse function such as the induction of apoptosis, the suppression of bacterial growth, and modulation of inflammatory response. Much interest has recently been focused on the physiological/pathological role of the lipocalin-2 that is considered to be a novel protective factor against oxidative stress. However, its precise biological roles in this protection are not fully understood. In this report we intended to test the effect of lipocalin-2 on the expression of heme oxygenase (1, 2) and superoxide dismutase (1, 2) which are two strong antioxidants. NGAL was cloned to pcDNA3.1 plasmid by using genetic engineering method. The recombinant vector was transfected to CHO and HEK293T to establish stable cell expressing NGAL and the expression of HO-1, 2 and SOD1, 2 were compared with appropriate controls byRT-PCR and western blot. On the other hand, expression of NGAL was suppressed by siRNA transfection in order to study the effect of lipocalin-2 on mentioned genes/proteins. The results showed that the expression of HO-1 and SOD 1, 2 enzymes were higher in cells expressing recombinant lipocalin-2 compared with the control cells. Although the expression of HO-1 was lower in NGAL silencing cells, the expression of SOD1 and SOD2 were higher. Our data suggest that NGAL is a potent inducer of HO-1 and somewhat SOD1 and SOD2 and it appears that part of antioxidant property of NGAL could be attributed to the induction of HO-1and SOD 1, 2. © Cell Stress Society International 2009

Development of a Cell-Based Biosensor for Residual Detergent Detection in Decellularized Scaffolds.

Ex vivo engineering of organs that uses decellularized whole organs as a scaffold with autologous stem cells is a potential alternative to traditional transplantation. However, one of the main challenges in this approach is preparing cytocompatible scaffolds. So far, high-precision and specific evaluation methods have not been developed for this purpose. Cell-based biosensors (CBBs) are promising tools to measure analytes with high sensitivity and specificity in a cost-effective and noninvasive manner. In this paper, using the NF-κB inducible promoter we developed a CBB for residual detergent detection. Proximal and core sections of the inducible promoter, containing NF-κB binding sequence, are designed and cloned upstream of the reporter gene (secreted alkaline phosphatase (SEAP)). After transfection into HEK293 cells, stable and reliable clones were selected. After confirmation of induction of this gene construct by sodium dodecyl sulfate (SDS), the stability and function of cells treated by qPCR and SEAP activity were measured. This biosensor was also used to evaluate the cytocompatibility of decellularized tissue. Results showed that the developed biosensor could detect very small amounts of SDS detergent (3.467 pM). It has the best performance 8 h after exposure to detergent, and its stability in high passage numbers was not significantly reduced. Applying this biosensor on decellularized tissues showed that SEAP activity higher than 4.36 (U/L) would lead to a viability reduction of transplanted cells below 70%. This paper presents a novel method to evaluate the cytocompatibility of decellularized tissues. The developed CBB can detect residual detergents (such as SDS) in tissues with high sensitivity and efficiency

Induction of multipotency in umbilical cord-derived mesenchymal stem cells cultivated under suspension conditions

Due to the limitations in the clinical application of embryonic stem cells (ESC) and induced pluripotent stem cells, mesenchymal stem cells (MSCs) are now much more interesting for cell-based therapy. Although MSCs have several advantages, they are not capable of differentiating to all three embryonic layers (three germ layers) without cultivation under specific induction media. Hence, improvement of MSCs for cell therapy purposes is under intensive study now. In this study, we isolated MSCs from umbilical cord tissue at the single-cell level, by treatment with trypsin, followed by cultivation under suspension conditions to form a colony. These colonies were trypsin resistant, capable of self-renewal differentiation to the three germ layers without any induction, and they were somewhat similar to ESC colonies. The cells were able to grow in both adherent and suspension culture conditions, expressed both the MSCs markers, especially CD105, and the multipotency markers, i.e., SSEA-3, and had a limited lifespan. The cells were expanded under simple culture conditions at the single-cell level and were homogenous. Further and complementary studies are required to understand how trypsin-tolerant mesenchymal stem cells are established. However, our study suggested non-embryonic resources for future cell-based therapy. © 2014 Cell Stress Society International

Positive selection of wharton�s jelly-derived CD105+ cells by MACS technique and their subsequent cultivation under suspension culture condition: A simple, versatile culturing method to enhance the multipotentiality of mesenchymal stem cells

Objective: Wharton�s jelly (WJ), an appropriate source of mesenchymal stem cells (MSCs), has been shown to have a wide array of therapeutic applications. However, the WJ-derived MSCs are very heterogeneous and have limited expression of pluripotency markers. Hence, improvement of their culture condition would promote the efficiency of WJ-MSCs. This study aims to employ a simple method of cultivation to obtain WJ-MSCs which express more pluripotency markers. Methods: CD105+ cells were separated by magnetic-associated (activated) cell sorting from umbilical cord mucous tissue. CD105+ cells were added to Methocult medium diluted in α-minimum essential medium (α-MEM) and seeded in poly(2-hydroxyethyl methacrylate) (poly-HEMA)-coated plates for suspension culture preparation. Differentiation capacity of isolated cells was evaluated in the presence of differentiation-inducing media. The expression of pluripotency markers such as Oct3/4, Nanog, and Sox2 was also analyzed by RT-PCR and western blot techniques. Moreover, immunocytochemistry was performed to detect alpha-smooth muscle actin (antigene) (α-SMA) protein. Results: WJ-MSCs grew homogeneously and formed colonies when cultured under suspension culture conditions (Non-adhesive WJ-MSCs). They maintained their growth ability in both adherent and suspension cultures for several passages. Non-adhesive WJ-MSCs expressed Oct3/4, Nanog, and Sox2 both at transcriptional and translational levels in comparison to those cultured in conventional adherent cultures. They also expressed α-SMA protein. Discussion: In this study, we isolated WJ-MSCs using a slightly modified culture condition. Our simple non-genetic method resulted in a homogeneous population of WJ-MSCs, which highly expressed pluripotency markers. Conclusion: In the future, more multipotent WJ-MSCs can be harnessed as a non-embryonic source of MSCs in MSC-based cell therapy. © W. S. Maney & Son Ltd 2015

High-level expression of functional recombinant human coagulation factor VII in insect cells

Recombinant coagulation factor VII (FVII) is used as a potential therapeutic intervention in hemophilia patients who produce antibodies against the coagulation factors. Mammalian cell lines provide low levels of expression, however, the Spodoptera frugiperda Sf9 cell line and baculovirus expression system are powerful systems for high-level expression of recombinant proteins, but due to the lack of endogenous vitamin K-dependent carboxylase, expression of functional FVII using this system is impossible. In the present study, we report a simple but versatile method to overcome the defect for high-level expression of the functional recombinant coagulation FVII in Sf9 cells. This method involves simultaneous expression of both human γ-carboxylase (hGC) and human FVII genes in the host. It may be possible to express other vitamin K-dependent coagulation factors using this method in the future. © 2010 Springer Science+Business Media B.V