Adenovirus-mediated gene transfer of human butyrylcholinesterase results in persistent high-level transgene expression in vivo

Human serum butyrylcholinesterase (Hu BChE) is a promising therapeutic against the toxicity of chemical warfare nerve agents, pesticide intoxication, and cocaine overdose. However, its widespread application is hampered by difficulties in large-scale production of the native protein from human plasma and/or availability as a recombinant protein suitable for use in vivo. This limitation may be resolved by in vivo delivery and expression of the Hu BChE gene. In this study, recombinant (r) adenoviruses (Ads) encoding full-length and truncated rHu BChEs were tested for in vivo expression in mice. Mice injected with these rAds intraperitoneally failed to express rHu BChE. However, a single tail vein injection of both rAds resulted in persistent high serum levels of rHu BChE in BChE knockout mice, which peaked on days 4/5 at 377+/-162U/ml for full-length rHu BChE and 574+/-143U/ml for truncated rHu BChE. These activity levels are orders of magnitude higher than 1.9U/ml of mouse BChE present in wild-type mouse serum. Thereafter, rHu BChE levels dropped rapidly and very little or no activity was detected in the serum 10 days post-virus administration. In conclusion, the present study demonstrates the potential of rAd-mediated Hu BChE gene therapy to counteract multiple lethal doses of chemical warfare nerve agent toxicity.     

Therapeutic angiogenesis by ex vivo expanded erythroid progenitor cells

Recent reports have demonstrated that erythroid progenitor cells contain and secrete various angiogenic cytokines. Here, the impact of erythroid colony-forming cell (ECFC) implantation on therapeutic angiogenesis was investigated in murine models of hindlimb ischemia. During the in vitro differentiation, vascular endothelial growth factor (VEGF) secretion by ECFCs was observed from day 3 (burst-forming unit erythroid cells) to day 10 (erythroblasts). ECFCs from day 5 to day 7 (colony-forming unit erythroid cells) showed the highest VEGF productivity, and day 6 ECFCs were used for the experiments. ECFCs contained larger amounts of VEGF and fibroblast growth factor-2 (FGF-2) than peripheral blood mononuclear cells (PBMNCs). In tubule formation assays with human umbilical vein endothelial cells, ECFCs stimulated 1.5-fold more capillary growth than PBMNCs, and this effect was suppressed by antibodies against VEGF and FGF-2. Using an immunodeficient hindlimb ischemia model and laser-Doppler imaging, we evaluated the limb salvage rate and blood perfusion after intramuscular implantation of ECFCs. ECFC implantation increased both the salvage rate (38% vs. 0%, P < 0.05) and the blood perfusion (82.8% vs. 65.6%, P < 0.01). In addition, ECFCs implantation also significantly increased capillaries with recruitment of vascular smooth muscle cells and the capillary density was 1.6-fold higher than in the control group. Continuous production of human VEGF from ECFCs in the skeletal muscle was confirmed at least 7 days after the implantation. Implantation of ECFCs promoted angiogenesis in ischemic limbs by supplying angiogenic cytokines (VEGF and FGF-2), suggesting a possible novel strategy for therapeutic angiogenesis.     

Effect of dexamethasone on moesin gene expression in rabbit bone marrow stromal cells

The influence of dexamethasone on rabbit bone marrow stromal cells differentiation was studied by screening the action of dexamethasone on gene expression. Using differential display, we observed some differential amplifications. The use of five of thirteen different primers combination allowed to identify one or more differential bands. One of them was identified as moesin gene. Real-time PCR confirmed a significant reduction of moesin gene expression following dexamethasone treatment. The decrease of expression for this protein, involved in cytoskeletal organization, could explain the effects of dexamethasone treatment on bone marrow stromal cells differentiation.     

Osteoblastic gene expression during adipogenesis in hematopoietic supporting murine bone marrow stromal cells

A growing body of data suggests that the bone marrow stroma contains a population of pluripotent cells capable of differentiating into adipocytes, osteoblasts, and lymphohematopoietic supporting cells. In this work, the murine stromal cell lines BMS2 and +/+ 2.4 have been examined as preadipocytes and adipocytes for evidence of osteoblastic gene expression. Adipocyte differentiation has been quantitated using fluorescence activated cell sorting. Within 7–10 days of adipocyte induction by treatment with glucocorticoids, indomethacin, and methylisobutylxanthine, between 40% to 50% of the cells contain lipid vacuoles and exhibit a characteristic adipocyte morphology. Based on immunocytochemistry, both the adipocytes and preadipocytes express a number of osteoblastic markers; these include alkaline phosphatase, osteopontin, collagen (I, III), bone sialoprotein II, and fibronectin. Based on biochemical assays, the level of alkaline phosphatase expression is not significantly different between preadipocyte and adipocyte cells. However, unlike rat cell lines, dexamethasone exposure causes a dose-dependent decrease in enzyme activity. The steady-state mRNA levels of the osteoblast associated genes varies during the process of adiopogenesis. The relative level of collagen I and collagen III mRNA is lower in adipocyte-induced cells when compared to the uninduced controls. Osteocalcin mRNA is detected in preadipocytes but absent in adipocytes. These data indicate that osteoblastic gene expression is detected in cells capable of undergoing adipocyte differentiation, consistent with the hypothesis that these cell lineages are interrelated. © 1993 Wiley-Liss, Inc.     

Functional small-diameter neovessels created using endothelial progenitor cells expanded ex vivo

Arterial conduits are increasingly preferred for surgical bypass because of inherent functional properties conferred by arterial endothelial cells, especially nitric oxide production in response to physiologic stimuli. Here we tested whether endothelial progenitor cells (EPCs) can replace arterial endothelial cells and promote patency in tissue-engineered small-diameter blood vessels (4 mm). We isolated EPCs from peripheral blood of sheep, expanded them ex vivo and then seeded them on decellularized porcine iliac vessels. EPC-seeded grafts remained patent for 130 days as a carotid interposition graft in sheep, whereas non-seeded grafts occluded within 15 days. The EPC-explanted grafts exhibited contractile activity and nitric-oxide-mediated vascular relaxation that were similar to native carotid arteries. These results indicate that EPCs can function similarly to arterial endothelial cells and thereby confer longer vascular-graft survival. Due to their unique properties, EPCs might have other general applications for tissue-engineered structures and in treating vascular diseases.     

Adenoviral BMP-2 gene transfer in mesenchymal stem cells: in vitro and in vivo bone formation on biodegradable polymer scaffolds

The aim of this study was to determine the feasibility of adenoviral gene transfer into primary human bone marrow osteoprogenitor cells in combination with biodegradeable scaffolds to tissue-engineer bone. Osteoprogenitors were infected with AxCAOBMP-2, a vector carrying the human BMP-2 gene. Alkaline phosphatase activity was induced in C2C12 cells following culture with conditioned media from BMP-2 expressing cells, confirming successful secretion of active BMP-2. Expression of alkaline phosphatase activity, type I collagen and mineralisation confirmed bone cell differentiation and maintenance of the osteoblast phenotype in extended culture for up to 6 weeks on PLGA porous scaffolds. In vivo implantation of adenoviral osteoprogenitor constructs on PLGA biodegradeable scaffolds, using diffusion chambers, also demonstrated bone cell differentiation and production of bone tissue. The maintenance of the osteoblast phenotype in extended culture and generation of mineralised 3-D scaffolds containing such constructs indicate the potential of such bone tissue engineering approaches in bone repair.     

In vivo and ex vivo regulation of bacterial virulence gene expression

Bacteria are remarkably adaptable organisms that are able to survive and multiply in diverse and sometimes hostile environments. Adaptability is determined by the complement of genetic information available to an organism and by the mechanisms that control gene expression. In general, gene products conferring a growth or survival advantage in a particular situation are expressed, while unnecessary or deleterious functions are not. Expression of virulence gene products that allow pathogenic bacteria to multiply on and within host cells and tissues are no exception to this rule. Being of little or no use to the bacterium except during specific stages of the infectious cycle, these accessory factors are nearly always subject to tight and coordinate regulation. As a result of recent advances, we are beginning to appreciate the complexities of the interactions between bacteria and their hosts. The ability to probe virulence gene regulation in vivo has broadened our perspectives on pathogenesis.     

Effects of age and gender on WNT gene expression in human bone marrow stromal cells

WNT signaling pathways play important roles in the behavior of human bone marrow stromal cells. Although WNT expression has been examined in human bone marrow stromal cells (hMSCs) with limited numbers of subjects or from commercial sources, there are conflicting results on WNT gene expression in hMSCs. Furthermore, the effects of age and gender on WNT expression in hMSCs are largely unknown. In this study, we evaluated RNA expression of all the WNT genes in hMSCs from 19 subjects, 12 women and 7 men, aged from 36 to 85 years. Analysis of WNT gene expression in young and old groups indicated that WNT7B and 14 were expressed significantly higher in the young group. WNT2 and WNT13 showed a trend of higher expression in young group. WNT7B, 13, and 14 were inversely correlated with age. Further analysis for gender‐specific difference indicated that WNT16 was expressed significantly higher in men than in women. WNT11 showed a trend of higher expression in hMSCs from women. For the hMSCs from women, WNT13 was inversely correlated with age and WNT4 was positively correlated with age. For the hMSCs from men, WNT7B and WNT14 were inversely correlated with age. These data indicated that most of the age‐related WNT genes belong to the canonical WNT signaling pathway. Further, there are gender‐specific differences in the expression of WNT4, 7B, 13, 14, and 16 in hMSCs. Age and gender account for many of the sample‐to‐sample variations in WNT gene expression in human marrow stromal cells. J. Cell. Biochem. 106: 337–343, 2009. © 2008 Wiley‐Liss, Inc.     

Analysis of ex vivo expanded and activated clinical-grade human NK cells after cryopreservation

Background aimsSeveral methods to expand and activate (EA) NK cells ex vivo have been developed for the treatment of relapsed or refractory cancers. Infusion of fresh NK cells is generally preferred to the infusion of cryopreserved/thawed (C/T) NK cells because of concern that cryopreservation diminishes NK cell activity. However, there has been little head-to-head comparison of the functionality of fresh versus C/T NK cell products.MethodsWe evaluated activity of fresh and C/T EA NK cells generated by interleukin (IL)-15, IL-2 and CD137L expansion.ResultsAnalysis of C/T NK cell products demonstrated decreased recovery of viable CD56⁺ cells, but the proportion of NK cells in the C/T EA NK cell product did not decrease compared with the fresh EA NK cell product. Fresh and C/T EA NK cells demonstrated increased granzyme B compared with NK cells pre-expansion, but only fresh EA NK cells showed increased NKG2D. Compared with fresh EA NK cells, cytotoxic ability of C/T EA NK cells was reduced, but C/T EA NK cells remained potently cytotoxic against tumor cells via both antibody-independent and antibody-dependent mechanisms within 4 h post-thaw. Fresh EA NK cells generated high levels of gamma interferon (IFN-γ), which was abrogated by JAK1/JAK2 inhibition with ruxolitinib, but C/T EA NK cells showed lower IFN-γ unaffected by JAK1/JAK2 inhibition.DiscussionUsage of C/T EA NK cells may be an option to provide serial “boost” NK cell infusions from a single apheresis to maximize NK cell persistence and potentially improve NK-induced responses to refractory cancer.     

Impact of hypoxia and long-term cultivation on the genomic stability and mitochondrial performance of ex vivo expanded human stem/stromal cells

Recent studies have described the occurrence of chromosomal abnormalities and mitochondrial dysfunction in human stem/stromal cells (SCs), particularly after extensive passaging in vitro and/or expansion under low oxygen tensions. To deepen this knowledge we investigated the influence of hypoxia (2% O(2)) and prolonged passaging (>P10) of human bone marrow stromal cells (BMSCs) and adipose-derived stromal cells (ASCs) on the expression of genes involved in DNA repair and cell-cycle regulation pathways, as well as on the occurrence of microsatellite instability and changes in telomere length. Our results show that hypoxic conditions induce an immediate and concerted down-regulation of genes involved in DNA repair and damage response pathways (MLH1, RAD51, BRCA1, and Ku80), concomitantly with the occurrence of microsatellite instability while maintaining telomere length. We further searched for mutations occurring in the mitochondrial genome, and monitored changes in intracellular ATP content, membrane potential and mitochondrial DNA content. Hypoxia led to a simultaneous decrease in ATP content and in the number of mitochondrial genomes, whereas the opposite effect was observed after prolonged passaging. Moreover, we show that neither hypoxia nor prolonged passaging significantly affected the integrity of the mitochondrial genome. Ultimately, we present evidence on how hypoxia selectively impacts the cellular response of BMSCs and ASCs, thus pointing for the need to optimize oxygen tension according to the cell source.     

Lentiviral and MLV based retroviral vectors for ex vivo and in vivo gene transfer

Retroviral vectors have become an important tool for gene transfer in vitro and in vivo. Classical Moloney murine leukemia virus (MLV) based retroviral vectors have been used for over 20 years to transfer genes into dividing cells. Cell lines for production of retroviral vectors have become commonly available and modifications in retroviral vector design and use of envelope proteins have made the production of high titer, helper-free, infectious virus stocks relatively easy. More recently, lentiviral vectors, another class of retroviruses, have been modified for in vitro and in vivo gene transfer. The ability of lentiviral vectors to transduce non-dividing cells has made them especially attractive for in vivo gene transfer into differentiated, non-dividing tissues. Several improvements in helper plasmids and vectors have made lentivirus a safe vector system for ex vivo and in vivo gene transfer. This review will briefly summarize the background of these vector systems and provide some common protocols available for the preparation of MLV based retroviral vectors and HIV-1 based lentiviral vectors.     

Canine bone marrow mesenchymal stromal cells with lentiviral mHCN4 gene transfer create cardiac pacemakers

Background aimsThe study objective was to test the ability of canine mesenchymal stromal cells (cMSC) transfected with the mouse hyperpolarization-activated cyclic nucleotide-gated channel 4 (mHCN4) gene to deliver a biologic pacemaker to the canine heart.Methods and ResultscMSC that were transfected by lentiviral vector with the cardiac pacemaker gene mHCN4 expressed high levels of Cs⁺ -sensitive current (26.4 ± 1.8pA/pF at –140 mV; (n = 17) and were activated in the diastolic potential range with a reversal potential of –29.7 ± 2.5 mV (n = 14), confirming that the expressed current was Funny current (I_f)-like. Next, 3 × 10⁶ cMSC transfected with either control plasmid or the mHCN4 gene construct were injected subepicardially into the canine right ventricular wall in situ. During sinus arrest, all control hearts had spontaneous atrioventricular node rhythms [rate = 21 ± 5beats per minute (b.p.m.)]. In the mHCN4 group, six of eight animals developed spontaneous ventricular rhythms of right-sided origin (rate = 45 ± 9b.p.m.; P < 0.01). Moreover, immunohistochemical analysis of the injected regions demonstrated neither apoptosis nor cellular or humoral rejection at 2 weeks.ConclusionsThese results demonstrate that genetically modified cMSC can express functional HCN4 channels in vitro and in vivo and represent a novel delivery system for pacemaker genes into the heart.     

Receptor tyrosine kinase expression in human bone marrow stromal cells

Bone marrow stromal cells (BMSCs) are a heterogeneous population of cells derived from colony-forming units-fibroblastic (CFU-Fs). These cells reside in the bone marrow cavity and are capable of differentiating into several cell phenotypes including osteoblasts, chondroblasts, hematopoiesis-supporting stromal cells, and adipocytes. However, the factors that regulate the proliferation and differentiation of the BMSC population are for the most part unknown. Since many members of the receptor tyrosine kinase (RTK) family have been shown to participate in growth control of various mesenchymal cell populations, in this study we examined the expression and function of RTKs in the BMSC population. Degenerate oligonucleotides corresponding to two conserved catalytic domains of the RTK family and RT-PCR were used initially to determine which RTKs are expressed in the human BMSC (hBMSC) system. After subcloning the amplification product generated from mRNA of a multicolony-derived hBMSC strain, PDGF receptor (β), EGF receptor, FGF receptor 1, and Axl were identified by DNA sequencing of 26 bacterial colonies. Furthermore, PDGF and EGF were found to enhance BMSC growth in a dose-dependent manner and to induce tyrosine phosphorylation of intracellular molecules, including the PDGF and EGF receptors themselves, demonstrating the functionality of these receptors. On the other hand, bFGF was found to have little effect on proliferation or tyrosine phosphorylation. Since single colony-derived hBMSC strains are known to vary from one colony to another in colony habit (growth rate and colony structure) and the ability to form bone in vivo, the expression levels of these RTKs were determined in 18 hBMSC clonal strains by semiquantitative RT-PCR and were found to vary from one clonal strain to another. While not absolutely predictive of the osteogenic capacity of individual clonal strains, on average, relatively high levels of PDGF-receptor were found in bone-forming strains, while on average, nonbone-forming strains had relatively high levels of EGF-receptor. Taken together, these results indicate that RTKs play a role in the control of hBMSC proliferation, and that the differential pattern of RTK expression may be useful in correlating the biochemical properties of individual clonal strains with their ability to produce bone in vivo. J. Cell. Physiol. 177:426–438, 1998. © 1998 Wiley-Liss, Inc.     

Regulation of platelet-derived growth factor A and B chain gene expression in bone marrow stromal cells

MBA-2, bone marrow-derived endothelial stromal cells, express platelet-derived growth factor (PDGF) A and B chain mRNAs and secrete PDGF activity that is induced by TGF-beta. Either chain of the PDGF molecule could modulate hematopoiesis and stromal cell growth. Intracellular pathways that regulate PDGF expression in the marrow microenvironment are unknown. In the present study, we examined the mechanisms that mediate PDGF A and B chain mRNA induction by TGF-beta and the role of protein kinase C (PKC) and cyclic AMP in PDGF regulation. TGF-beta was tested in parallel with PMA, an activator of phorbol ester-dependent PKC isoforms. Both PMA (10^?7M) and TGF-beta (2.5 ng/ml) increased PDGF A and B chain mRNA levels. The serine/threonine protein kinase inhibitor, H7, blocked PDGF A and B chain mRNA induction in response to TGF-beta. However, down-regulation of PKC by prolonged incubation with PMA failed to abolish TGF-beta induction of PDGF A and B chain mRNAs. These findings indicate that induction of PDGF A and B chain mRNAs can be mediated via phorbol ester-dependent PKC pathway. In contrast, H7-sensitive protein kinase(s) other than phorbol ester-sensitive protein kinase C mediate the effect of TGF-beta. Agents that increase cAMP were also tested for their effect on PDGF gene expression. TGF-beta-mediated induction of PDGF A and B chain mRNAs was markedly inhibited by cAMP. cAMP also blocked stimulation of PDGF A chain mRNA by PMA. The positive and negative signaling mechanisms involved in modulating PDGF in the microenvironment may be important for determining hematopoietic and stromal cell responses in vivo. © 1995 Wiley-Liss, Inc.     

Adenovirus-mediated BMP2 expression in human bone marrow stromal cells

Recombinant adenoviral vectors have been shown to be potential new tools for a variety of musculoskeletal defects. Much emphasis in the field of orthopedic research has been placed on developing systems for the production of bone. This study aims to determine the necessary conditions for sustained production of high levels of active bone morphogenetic protein 2 (BMP2) using a recombinant adenovirus type 5 (Ad5BMP2) capable of eliciting BMP2 synthesis upon infection and to evaluate the consequences for osteoprogenitor cells. The results indicate that high levels (144 ng/ml) of BMP2 can be produced in non-osteoprogenitor cells (A549 cell line) by this method and the resultant protein appears to be three times more biologically active than the recombinant protein. Surprisingly, similar levels of BMP2 expression could not be achieved after transduction with Ad5BMP2 of either human bone marrow stromal cells or the mouse bone marrow stromal cell line W20-17. However, human bone marrow stromal cells cultured with 1 microM dexamethasone for four days, or further stimulated to become osteoblast-like cells with 50 microg/ml ascorbic acid, produced high levels of BMP2 upon Ad5BMP2 infection as compared to the undifferentiated cells. The increased production of BMP2 in adenovirus transduced cells following exposure to 1 microM dexamethasone was reduced if the cells were not given 50 microg/ml ascorbic acid. When bone marrow stromal cells were allowed to become confluent in culture prior to differentiation, BMP2 production in response to Ad5BMP2 infection was lost entirely. Furthermore, the increase in BMP2 synthesis seen during differentiation was greatly decreased when Ad5BMP2 was administered prior to dexamethasone treatment. In short, the efficiency of adenovirus mediated expression of BMP2 in bone marrow stromal cells appears to be dependent on the differentiation state of these cells.