Abstract
The Israeli Society of Gene & Cell Therapy 2010 Oral Presentations*
The Department of Molecular Microbiology & Biotechnology, Tel Aviv University , Tel Aviv, 69978, Israel E-mail: adibarzel@gmail.com
The advance of gene therapy has been hampered by the recurrence of adverse side effects attributed to the viral vectors being used. Carcinogenicity and immunogenicity can be markedly reduced if the transgene is specifically targeted to correct the defective allele at the endogenous locus or to complement for the defect by being inserted at an intergenic “safe harbor” thus avoiding oncogene activation.
The induction of gene targeting at a locus of choice can be facilitated by the introduction of a site-specific DNA break. Indeed, Zinc-finger nucleases and engineered homing endonucleases have both been shown capable of inducing transgene integration at desired loci. However, enzyme engineering is costly, time consuming, and laborious. Furthermore, the target recognition of a given enzyme can be manipulated only to an extent and any shift may come with the cost of reduced specificity. We therefore set forth to explore whether the natural diversity of homing endonucleases together with the natural plasticity in target recognition may allow us to find native homing endonucleases that can be used for cleaving desired human loci in order to induce therapeutic gene targeting. We devised the HomeBase application, a computational assignment of homing endonucleases to the targeting of specific loci, and an experimental methodology for its validation. Our database currently holds almost a thousand different enzymes, many of which have never before been annotated. Novel insights allow us to predict the positions along each enzyme's target that are crucial for recognition and cleavage. This allows us to detect potential targets in the human genome that would otherwise be ignored. We validated the capacity of selected homing endonucleases to cleave and induce gene targeting at their predicted targets using bacterial and yeast systems. We were further able to show that some of our enzymes can cleave their predicted targets in the live human cell with efficiency and specificity comparable to that of the I-SceI golden standard and with no cytotoxicity.
We now move forward to assess the gene targeting potential of our enzymes in different human tissue cultures, primary cells and induced pluripotent stem cells.
Department of Human Molecular Genetics and Biochemistry, Sackler Faculty of Medicine, Tel Aviv University , Ramat Aviv, Tel-Aviv, 69978, Israel E-mail: berneman_dana@hotmail.com
The present study unravels fundamental key events in the reprogramming pancreas in liver which will allow us to increase the therapeutic merit of using adult cells as a source of pancreatic tissue for treating diabetes.
Reprogramming into Pancreatic Beta Cells Based on Developmental Cues: Converting Alpha into Beta Cells
The loss of Arx and/or Pax4 gene activity leads to a shift in the fate of the different islet cell subtypes in the mouse endocrine pancreas, this without affecting the total islet cell numbers. Recently, we conditionally and ectopically forced the expression of the Pax4 gene using the cre-lox system and demonstrated that Pax4 misexpression is sufficient to induce endocrine precursor cells, as well as mature α-cells, to adopt a β-cell phenotype. The ensuing glucagon deficiency was found to induce a compensatory and continuous glucagon+ cell neogenesis requiring the re-expression of the proendocrine gene Ngn3. Interestingly, these newly formed α-like cells fail to counter the hypoglucagonemia as they subsequently acquire a β-cell identity upon Pax4 misexpression. Notably, this cycle of neogenesis and redifferentiation was found sufficient to restore a functional β-cell mass and cure chemically-induced diabetes in vivo.
Identification and Prospective Isolation of Novel Human Renal Stem/Progenitor Populations from Normal and Cancerous Kidney Tissue
The precursor of the adult mammalian kidney is called the metanephros and it appears at 5 weeks of human gestation, equivalent to embryonic day 11 in mice and day 12 in rats. At this stage, the organ consists of ureteric bud epithelium which becomes enveloped by renal mesenchyme/blastema: these tissues form collecting ducts and nephron tubules, respectively. In the developing human kidney, fresh stem cells are induced into the nephrogenic pathway to form nephrons until 34 weeks of gestation.
Others and we have shown that two pathologic situations strongly recapitulate this developmental program by activating specific transcription factors that mark the early renal progenitor population: (i) renal regeneration following episodes of acute injury, suggestive of the presence of adult kidney stem cells (ii) renal tumorigenesis in the form of a Wilms' tumor, a common pediatric kidney cancer, believed to arise from multipotent embryonic renal precursors of the renal mesenchyme/blastema. Thus, the renal progenitor pool is likely to be at the heart of all of these processes.
Recent molecular advances have much contributed to our understanding of the cell lineages in the developing kidney. Nevertheless, in contrast with other organs, such as the hematopoietic system, in which the identification of surface markers enabled purification of these cells, the lack of such in the kidney has hampered progress in identifying and isolating stem cells. Microarray experiments, cell selection strategies, clonal analysis and various in vivo assays of human cells derived from normal and cancerous kidney tissue have now afforded insights into relevant shared stem cell markers and accordingly to human renal stem/progenitor populations.
This may enable the potential application of renal stem cells in kidney repair and the treatment of kidney cancers.
Ex Vivo Expansion of Adult Human Pancreatic Islet Beta Cells
Expansion of adult beta cells from the limited number of islet donors is an attractive prospect for development of abundant cells for cell therapy of diabetes. However, while evidence supports the replicative capacity of rodent beta cells in vivo, attempts at expanding both rodent and human beta cells in tissue culture result in loss of beta-cell phenotype. Our recent lineage-tracing studies support the ability of human beta cells to survive and significantly replicate in vitro. Beta-cell delamination out of the normal epithelial structure, a process that results in epithelial-mesenchymal transition and dedifferentiation, is required for significant in vitro proliferation. We hypothesized that the dedifferentiated cells (termed beta-cell derived, BCD) maintain sufficient epigenetic memory to represent good candidates for generation of insulin-producing cells. Thus, ways must be found for inducing redifferentiation of the expanded cells ex vivo, or restoring their function upon transplantation. Elucidation of the signaling pathways altered during beta-cell adaptation to growth in culture may provide clues for cell redifferentiation. We found that human beta-cell dedifferentiation and entrance into the cell cycle in vitro correlated with activation of the NOTCH pathway and downregulation of the cell cycle inhibitor p57.
Inhibition of the NOTCH downstream target HES1 using short hairpin RNA reduced beta-cell dedifferentiation and replication, suggesting a potential target for inducing cell redifferentiation following expansion in culture for use in cell replacement therapy of diabetes.
Lab. Exp Surgery, Hadassah University Hospital, Jerusalem 91120, Israel E-mail: anat.globerman@gmail.com
Patients suffering from kidney failure frequently require the formation of an arterio-venous fistula (AVF) in which an arm vein is connected to an artery resulting in arterialization of the vein to allow adequate blood flow into an external “artificial kidney”. However, neo-intimal hyperplasia (NIH) frequently ensues, causing stenosis and subsequent occlusion of the vein leading to significant morbidity. The molecular and cellular events taking place in the vein causing NIH may serve as ideal targets for molecular-based therapies. Therapeutic gene delivery into the vascular system is seriously impeded by problems related to the low efficacy and toxicity of targeted viral vector delivery.
To explore the feasibility of a clinically applicable, plasmid-based vascular gene delivery system, we have established a rat AVF model in which typical NIH develops. Intraluminal injection of plasmid DNAs into the AVF resulted in robust and long-term reporter gene expression exclusively observed throughout the venous limb including the site of NIH formation. Plasmid transfection was confined to endothelial cells and myofibroblasts that migrate inwards from the adventitia to participate in NIH formation. There were no signs of detrimental tissue reaction to plasmid delivery contrasting with the severe vein inflammatory response observed following adenovirus infection.
Preliminary results with over-expressing the biologically active KLF4 demonstrated a moderate reduction in the size of the NIH lesion. These results pave the way for intravascular plasmid delivery and may offer a clinically relevant therapeutic modality to ameliorate NIH and other vein graft pathologies.
AAV Vectors for Gene Therapy of the Brain in Lysosomal Storage Diseases
The rationale for therapeutic approaches in MPSs is based on the observation that delivery of the missing enzyme reverses phenotypic abnormalities in cells genetically deficient for a lysosomal enzyme. Phenotypic correction was initially described in cultures of MPSI patient fibroblasts and then confirmed in MPSI patients who benefited from enzyme replacement therapy. It relies on the internalisation of extracellular enzyme by deficient cells. Enzyme internalisation is made possible through binding to the mannose-6-phosphate receptors. However, due to the presence of the Blood-Brain Barrier (BBB), none of the therapeutic enzymes used in enzyme replacement therapies are able to reach the CNS and modify the course of neurodegeneration. This limitation can be circumvented by gene therapy directed to the brain, in which sources of enzyme are created by surgical injection of a viral vector directly into the central nervous system.
The assumption that enzyme delivery will allow reversion of the diseased phenotype was fully confirmed by studies performed in MPSVII mice, MPSI mice, MPSIII mice and more recently in MPSI and MPSIIIB dogs. Delivery of the missing enzyme to the entire brain through gene therapy was associated with improvement of biochemical and histological markers of the disease and improved animal behaviour. These studies provided evidence that enzyme delivery to the CNS reversed HS oligosaccharide accumulation and was sufficient to prevent neuropathology and alleviate clinical manifestations, suggesting that enzyme deficiency leading to the accumulation of abnormal heparan sulfate fragments is the primary cause of neuropathology.
Dog studies also provided evidence that stereotaxic injections of AAV vectors coding for a lysosomal enzyme in the brain was safe in a total of 28 injected dogs. They also demonstrated the absolute necessity of combining gene therapy with immunosuppression, whereas immune response against the therapeutic gene product was not observed in previous mouse studies, though it certainly needs to be considered in similar therapies for other diseases.
Departments of Pediatrics and Genetics, Stanford University , Stanford CA 94305, USA E-mail: markay@stanford.edu
Type 1 diabetes, for which there is no current cure, results from the autoimmune destruction of pancreatic β-cells. Traditional β-islet transplantation is severely hampered by the low availability of viable human islet cells and possibility for rejection. Currently, there are a number of alternative strategies being pursued as therapies, including the expansion of β-cells from pluripotent stem cells and the induced pancreatic endocrine cell reprogramming of somatic cells. This work focuses on the latter approach, using an adenoviral (Adv)/adeno-associated virus (AAV) based therapy in a streptozotocin-induced diabetes mouse model. We want to elucidate the factors required for reprogramming and establish if the reprogrammed cells are different enough from endogenous β-cells to avoid autoimmune elimination. We have used an approach originally described by Ferber et al., and others including our own lab to express various pancreatic transcription factors (PTF), including Neurogenin-3 (Ngn-3), for delivery into the liver or pancreas to successfully treat STZ-induced diabetes in mice. However, current approaches require the use of recombinant Adv vectors because of the corequirement for a component of the adenoviral capsid. Since AAV or plasmid-Ngn3 vectors only correct diabetes with codelivery of an adenoviral particle, we hypothesize that the adenoviral corequirement involves a secondary signal induced by the adenoviral capsid or an adenovirus-induced alteration in rAAV/plasmid tropism. To characterize the nature of a signaling response, we attempted to determine if liver resident Kupffer cells contributed to such response by eliminating them using GdCl3. Although loss of Kupffer cells could be demonstrated by microscopy of sectioned tissue, we found no enhancement in Adv-Ngn3 induced correction of diabetes. However, Kupffer depleted mice treated with pNgn3 + irrelevant Adv showed a slightly enhanced correction of diabetes. This suggests that a signaling cascade, if required, was not dependent on liver macrophages. Gene profiling approaches in various mouse strains are being used to further evaluate the role of adenoviral signaling in the process.
To evaluate the possibility that adenoviral tropism plays a critical role in the process, we have utilized a number of transgenic mouse-insulin promoter-Luc and -GFP mouse strains and viral vectors encoding for several fluorescent proteins to not only track vector biodistribution in live animals (healthy and diseased) but to identify the cell types most amenable to reprogramming. Using complex 3D reconstruction imaging, we have found several sites of interest in and outside the liver where reprogramming appears to be concentrated. Vector tropism studies are ongoing. A central issue that needs to be addressed in using cellular reprogramming to treat diabetes is that the new therapeutic cells must be different enough from normal β-islets that they escape autoimmune destruction by the host. Following Adv-Ngn3 treatment, over time, mice become resistant to secondary STZ-induced diabetes, suggesting that the reprogrammed cells while functional are not identical to endogenous β-cells.
These studies are being repeated in new human-relevant autoimmune models of murine diabetes to provide insight if reprogrammed cells are likely to persist in vivo in type 1 diabetes patients. Together, our approaches might provide the framework for a bona-fide clinical therapy for Type 1 Diabetes.
Dynamic Interactions Between the Nervous and Immune Systems with the Microenvironment Regulate Normal and Leukemic Human Stem Cells
Functional, preclinical models for normal and leukemic human stem cells using immune deficient NOD/SCID mice as recipients revealed that their repopulation is dependent on SDF-1/CXCR4 interactions. Homing, retention, release and stem cell mobilization are tightly regulated processes, which involve bone turnover and interplay between cytokines, chemokines, adhesion molecules and proteolytic enzymes. The roles of CD44, MT1-MMP and RECK in stem cell migration will be discussed.
Most blood forming stem cells are retained in the bone marrow (BM), anchored to specialized niches via adhesion interactions, which prevent their motility and proliferation. However, low levels of motile progenitors migrate in the circulation as part of homeostasis. These low levels are dramatically amplified during alarm situations in response to stress signals due to injury, bleeding and infections, as part of host defense and repair mechanisms. These stress signals are mimicked by repeated G-CSF stimulation in order to mobilize stem and progenitor cells to the circulation in order to harvest them for clinical transplantation protocols.
Stem cell adhesion interactions with the stromal niche supporting cells need to be dynamic in order to allow the undifferentiated cells to proliferate, differentiate and migrate. Osteoclasts have a dual role in host defense: bone remodeling and regulation of stem cells by freeing them from their inhibitory anchorage to stromal cells. Thus, osteoclast/osteoblast interactions also regulate BM leukocyte production on demand. Both, osteoclasts, osteoblasts and stem and progenitor cells functionally express receptors for neurotransmitters. Immature human CD34+ cells dynamically express dopamine and epinephrine receptors and inflammatory, myeloid cytokines such as G-CSF and GM-CSF increase catecholaminergic receptor expression in order to facilitate leukocyte production and trafficking. This upregulation activates the progenitor cells in response to stimulation by the neurotransmitters and induces their motility and proliferation via Wnt signaling. Thus, regulation of leukocyte production and trafficking by stem cells in the BM reservoir is dynamic and involves mutual, reciprocal interactions between the nervous and immune systems with the stromal microenvironment throughout the body.
Both normal and leukemic human stem and progenitor cells functionally express neurotransmitter receptors, which are involved in the regulation of their motility and proliferation. In conclusion, normal and leukemic human stem cells are directly and indirectly regulated by dynamic interactions of the nervous and immune systems with the microenvironment.
Engineering Cell Platforms for Cardiovascular Regeneration
A major challenge in cardiac tissue engineering is finding a biomaterial that can mimic the architecture of the heart tissue, integrate with it, maintain its mechanical properties and promote remodeling. Another major limitation for the successful engineering of cardiac tissue is the restricted ability to vascularize and supply adequate amount of nutrients to thick tissue, thus affecting tissue function and cellular remodeling.
Our lab focuses on decellularized thick (>100 μm) porcine cardiac extracellular matrix (pcECM) as a scaffold for cardiac engineering. The suggested thick pcECM is unique, as it has its own natural and inherent infrastructure of coronary arteries and veins, which can be utilized for the decellularization and cleaning processes, as well as for nutrient and oxygen transport to the constructed tissue. Using such scaffold, reduces the need to neither engineer artificial blood vessels nor add factors, which stimulate blood vessels infiltration to the scaffold. Moreover, the pcECM preserves the optimal structure of native extracellular skeleton, which is essential for the appropriate loading of myocytes or other possible cells, and provides them with optimal conditions and stimuli for growth. This precise and complex structure is also essential to convert the work generated by the myocytes into global contraction. We therefore hypothesize that such infrastructure, when infused with EC's, may promote cellular survival, proliferation and differentiation in this newly developed cardiac-like construct. Furthermore, a relatively simple anastomosis, of the graft to the patient-vasculature, has the potential to increase survival and functionality post transplantation.
The proposed system could also serve as a model for basic in vitro research, to study the effects of different parameters and treatments on the behavior of myocardium like constructs and the basic cell-scaffold interaction in a myocardial ECM milieu. Thus, a newly dynamic perfusion bioreactor is designed, to include controlled mechanical and electrical preconditioning, which will be synchronized with the biochemical and pharmacological stimuli.
Ella Institute of Melanoma, Sheba Medical Center , Tel Hashomer, Ramat Gan 52621, Israel E-mail: markel@post.tau.ac.il
Delineating the Molecular Mechanisms Regulating Chromatin Protein Dynamics in Embryonic Stem Cells
Embryonic stem cells (ESCs) are characterized by unique epigenetic features including decondensed chromatin, hyperdynamic association of proteins with chromatin and permissive transcriptional program. Here we investigate the mechanisms that regulate chromatin plasticity in ESCs. Using epigenetic drugs and mutant ESCs lacking various chromatin binding proteins, we find that DNA methylation and nucleosome repeat length have little or no effect on the dynamics of chromatin-binding proteins in ESCs. In contrast, histone acetylation enhances chromatin dynamics specifically in euchromatin, while histone H3 lysine 9 (H3K9) methylation and lamin A expression restrict chromatin dynamics exclusively in heterochromatin. Altered chromatin dynamics was associated with perturbed ESC differentiation.
Together, these data delineate the mechanisms responsible for chromatin plasticity in ESCs, and indicate that the epigenetic state of the genome modulates chromatin plasticity and the differentiation potential of ESCs.
Differentiated Mesenchymal Stem Cells Rescue Sciatic Nerve Injury in Rats
Sciatic nerve injury is common and may cause neurological deficits, particularly muscle weakness. Previous studies have shown that administration of neurotrophic factors (NTFs), naturally occurring proteins that support the development and survival of neurons, preserved and protected the damaged motor neuron in the injured sciatic nerve. We have been successful in converting bone marrow-derived mesenchymal stem cells into astrocyte-like cells that produce and secrete NTFs such as BDNF and GDNF (NTF+ cells). These NTF+ cells demonstrate typical astrocyte morphology, expressed characteristic astrocyte markers such as glial fibrillary acidic protein (GFAP) and glutamine synthetase and secreted high levels of NTFs. Such cells may be utilized to improve sustained NTFs delivery to damaged nerve tissues. We have already shown that these cells and their conditioned media can protect neurons in culture and in animal models of neurodegenerative diseases, e.g., Parkinson's and Huntington's diseases.
In the current study we examined whether NTF+ cells are capable of rescuing motor neurons in a unilateral rat sciatic nerve injury model, where the right hind limb sciatic nerve was crushed using a clamp. One day later, rats were inoculated with 5 × 106 rat NTF+ cells, MSCs or PBS into the site of the lesion. Five days later, in rats injected with the NTF+ cells, motor function measured by rotarod was markedly preserved (73% vs. control in the NTF+ cells treated rats as compared to 41% in the PBS groups, p < 0.01). Electromyography (EMG) study indicated that compound muscle action potential and its duration was preserved in the hind limb treated with NTF+ cells. Moreover, NTF+ cells significantly inhibited the degeneration of the neuromuscular junctions (99% vs. 72% in the PBS groups, p < 0.01).
In conclusion, our findings suggest that an autologous stem cell-based therapeutic approach can alleviate signs of sciatic nerve injury and probably other neurological disorders.
Department of Pediatrics and Pediatric Stem Cell Research Institute, Sheba Medical Center , Tel Hashomer, Ramat-Gan, Israel E-mail: naomsh@gmail.com
During development renal stem cells reside in the nephrogenic blastema of the growing fetal kidney. Wilms' tumor (WT), the most common malignant renal tumor of childhood is suggested to arise from malignant transformation of abnormally persistent renal stem cells which retain proliferative potential but also undergo partial differentiation to form the heterogeneous mature cell populations seen in this tumor. We have previously suggested NCAM1 as a marker for WT stem/progenitor cell based on in vitro assays which demonstrated this cell fraction as highly clonogenic and proliferative, overexpressing the WT “stemness” genes and topoisomerase2A (TOP2A), a poor prognostic marker for WT.
Complicating WT in vivo cellular analysis is the inability of fresh or cultured WT cells to form tumors in immmunodeficient mice. Here, we have established WT xenografts (Xn) that are characteristically highly proliferating tumors that can be rapidly propagated and sustained by individual cells and are enriched for early renal lineage markers (OSR1/SIX2), poor WT prognostic markers (TOP2A, N-MYC and CRABP2), the surface marker NCAM1 and depleted for CD133. We utilized this WT reservoir to isolate tumor-initiating cells that recapitulate the entire tumor morphology in immunodeficient mice. Accordingly, sorting experiments of Xn-derived cells and injection to mice at serial dilutions, revealed that the tumor-initiating capacity is solely contained within the NCAM1+ cell population (as few as 500 cells), which gives rise to NCAM− tubular cells and can further induce tumors when transplanted into secondary and tertiary recipients, indicative of self-renewal and differentiation capacities.
We go on to show that within the NCAM1 population CD44 and especially aldeheyde dehydrogenase activity but not PSA-NCAM enrich for the tumor-initiating cells. Importantly, the expression of nephric patterning, stemness, and poor prognostic genes in these cell fractions was intimately linked with their tumor forming ability.
In summary, this study definitely identifies a renal stem cell as the cellular origin of WT and affords a novel in vivo system for vast preclinical drug studies on WT.
hESC – From Bench to Patient
Human embryonic stem cells (hESCs) may serve as a renewable source of neural and retinal cells for basic research, the discovery of drugs and for transplantation therapy in degenerative disorders of the CNS and retina.
Here we show that hESCs may be induced to differentiate, under defined culture conditions, into highly enriched cultures of neural precursors (NPs). The NPs may be further directed to differentiate into more specialized neural and retinal cells.
We will review the roadmap towards the clinical development of these hESC-derived progeny for cell therapy.
We will demonstrate data supporting the curative potential of the hESC-derived cells in transplantation therapy of neural and retinal degenerative disorders. The beneficial therapeutic effect of transplanted hESC-derived progeny may be mediated by multiple mechanisms. We will show evidence for potential mechanisms mediating the beneficial effects of transplanted cells.
International Center for Cell Therapy & Cancer Immunotherapy (CTCI), Tel Aviv, Israel E-mail: slavin@CTCIcenter.com
Central nervous system autoimmune diseases affecting the central or peripheral nervous system, and neurodegenerative diseases are characterized by loss of specific cells or functions of neural cells, which result in potentially irreversible loss of neurological functions. Currently, therapeutic options for treating and restoring neurological functions are limited and therefore alternative novel approaches are required.
MSCs are mesoderm-derived cells that typically reside in adult bone marrow and adipose tissue at very low concentrations. In addition to their ability to differentiate to fat, bone and cartilage, MSCs have been also reported to transdifferentiate into cells with various neural phenotypes. We studied the role of MSCs in experimental autoimmune encephalitis (EAE), and found that treatment of mice with bone marrow-derived MSCs resulted in significant suppression of anti-self reactivity and improved disease manifestations. We have also demonstrated that in addition to down regulation of autoimmunity, neural differentiation of these cells may occur in vivo. These observations suggested that treatment with MSCs may be used for immune regulation of anti-self reactivity and for neuroprotective functions.
Our preliminary results of a phase I/II open clinical trial to evaluate the feasibility and safety of intrathecal and intravenous administration of autologous bone marrow-derived MSCs in 13 patients with severe MS failing conventional modalities and 14 with ALS included in this trial suggested that treatment with MSCs was feasible, safe, and potentially effective. No major side effect were developed or reported during a follow-up period of nearly 4 years. The mean EDSS score improved to 6.0 and 5.7 at 3 and 6 months respectively, and the mean ALSFRS remained stable during the 6 first months of observation (22.1–22.6). Based on the preliminary study, 60 additional patients with MS were treated at our center with MSCs, some with very significant objective improvement of disease manifestations. Objective benefits were also documented in the 2 children with autism. More recently, we have discovered novel approaches for inducing long-term neural differentiation of MSCs. With the expression of specific neuronal markers by differentiating MSCs, we could demonstrate the expression of voltage-dependent sodium channels and that of neurotrophic factors in both bone marrow and adipose-derived MSCs.
These results further emphasize potential advantages and importance of using MSCs for cell-based therapy in neurodegenerative and neuroinflammatory disorders.
Reprogramming Liver Cells into Insulin-Producing Cells by Pancreatic Transcription Factors
Effective delivery of pancreatic transcription factors (PTF) to reprogram liver cells into insulin-producing cells (IPCs) is a promising avenue for beta-cell replacement therapy. Using mice with streptozotocin (Stz)-induced diabetes, this study compares advantages and pitfalls of (a) adeno-associated virus (AAV)-mediated Pdx1-VP16(PV)/Ngn3 gene transfer and (b) direct delivery of recombinant Pdx1 protein (rPDX1) via the latter's built-in protein transduction domain (PTD).
To determine whether liver or pancreatic IPCs were responsible for restoring normoglycemia, we assessed by blood glucose levels, IPGTT, effects of near-total pancreatectomy, liver in situ perfusion, and measurement of tissue/serum insulin levels. Hyperglycemic diabetic mice were given AAV-GFP, AAV-PV, AAV-Ngn3, or AAV-PV/AAV-Ngn3 (108 viral particles/mouse; via portal vein injection). With AAV- PV, ∼10-15% of liver cells expressed Pdx1 protein, whereas AAV-Ngn3 had no effect on the blood glucose levels in diabetic mice. Significantly, AAV-PV alone, or when combined AAV-PV/Ngn3, persistently reduced blood glucose levels, the latter acting synergistically. Upon restored normoglycemia, near-total (∼80-90%) pancreatectomy had no significant effect on euglycemia, and IPGTT yielded nearly normal glucose-response curves. These findings indicate that AAV-PV/AAV-Ngn3-reprogrammed, liver-derived IPCs are glucose-responsive and mainly responsible for restoring normoglycemia in diabetic mice. In direct protein therapy studies, Stz-induced diabetic mice were treated daily with rPDX1 to observe the effects of restoring normoglycemia, liver cell reprogramming, and islet cell regeneration. Restoration of euglycemia in rPDX1-treated diabetic mice was evident by improved IPGTT, glucose-stimulated insulin release, as well as increased insulin content in liver and pancreas.
The underlying mechanism of rPDX1-mediated restoration of normoglycemia in Stz/diabetic mice includes reprogramming liver cells into IPCs, as well as promoting islet cell regeneration. Therefore, PTD-based PTF protein therapy may offer a safe way to treat patients with diabetes.
The Israeli Society of Gene & Cell Therapy 2010 Poster Presentations*
Department of Pediatrics and Pediatric Stem Cell Research Institute, Sheba Medical Center, Tel Hashomer, Israel and Sackler Faculty of Medicine, Tel Aviv University , Tel Aviv, Israel E-mail: ella_buzh@yahoo.com
Human adult kidney (hAK) stem/progenitor cells are ideal targets for gene therapy, cell transplantation, and tissue engineering. Nevertheless, their identity remains elusive. We have therefore analyzed cells from dissociated human kidney tissue obtained from nephrectomy specimens for progenitor potential based on clonogenic and sphere-forming assays concomitant with analysis of expression of molecular (early renal lineage—six2, pax2, wt1, sall1; polycomb group—ezh2, bmi1) and surface markers (NCAM1) of embryonic renal stem/progenitor cells.
With the obstacle of a low cell number retrieved from nephrectomy specimens and enzymatic digestion, we initially established optimal growth conditions for cultured hAK cells. Following low seeding density, hAK cells were expanded in serum containing (SCM) and defined serum free media (SFM). Limiting-dilution assays disclosed highest clonogenic potential of hAK cells in conditioned media obtained from hFK cultures (hFKCM) leading to clonogenic expansion (self-renewal) of a number of clones. In addition, we succeeded in growing hAK cells, in low-attachment conditions, sphere-like structures, which expressed significantly higher levels of the renal ‘stemness’ gene set compared to the hAK adherent culture. Having identified these growth characteristics, subpopulations of cultured hAK cells were then isolated by cell sorting to determine a fraction possessing progenitor potential. Cultured hAK cells immunoselected according to the NCAM1 marker, demonstrated overexpression of the early renal lineage and polycomb group genes, the pluripotency marker oct4 as well as elevated vimentin and reduced E-cadherin. Remarkably, the NCAM+ subpopulation was shown to be highly clonogenic and slow cycling in comparison to NCAM− fraction. Moreover, NCAM+ cells had enhanced well-defined sphere formation ability.
These findings allow a better understanding of the clonal nature and growth characteristics of cultured hAK cells and indicate that hAK contains cell types with enhanced stem/progenitor potential. Further functional testing in in vivo models are needed in order to evaluate their therapeutic potential.
Faculty of Biotechnology and Food Engineering , Technion, Haifa 32000, Israel E-mail: debbie@tx.technion.ac.il
Engineered Pseudotyped Lentivirus Mediate Specific Gene Targeting via a Single Chain Variable Fragment Displayed on Their Surface
Current viral-mediated gene delivery methods suffer from poor to non-existing targeting capacities when administered systemically. Although envelope glycoproteins derived from vesicular stomatitis virus glycoprotein (VSV-G), murine leukemia virus, and gibbon ape leukemia virus, are widely used to pseudotype retroviral vectors, they lack the cell specificity, which limits their current use as gene therapy regimens for only an ex vivo protocol of pre-purified cells. We have established a unique lentiviral-mediated antibody screening platform, where a 108 Single Chain Variable Fragment (scFv) functional library is displayed on the surface of HIV recombinant viral particles and human cells. scFv pseudotyped lentiviral particles can function as highly specific targeted gene delivery vehicles via their scFv moiety, maintaining their highly infectious properties.
Safety checkpoints and removal of most of the HIV genome allow convenient and safe use of lentivectors for gene therapy, minimizing a host-immune response against the virus. Both HIV-1- and MLV-based retroviral vectors can be pseudotyped with the envelope of the Alphavirus Sindbis Virus without reduction of viral titer. Importantly, the Sindbis virus encodes two transmembrane envelope proteins—E1 responsible for pH-dependent fusion and E2 responsible for receptor binding. Thus, disrupting the E2 region and replacing this binding moiety with an antibody generate viral particles that can specifically bind to cells. Such a mutant Sindbis virus envelope—m168—exhibits enhanced selectivity for targeting and reduced levels of nonspecific infectivity and was used to pseudotype lentiviral vectors.
In this study, we specifically target cells which display on their surface two distinct S1 spike glycoproteins from SARS Corona Virus (SARS CoV)—GDO3 and TOR2. To this aim, lentiviruses were pseudotyped with m168 sindbis envelope, where the E2 binding domain is mutated, while the E1 fusion motif remains functional. Specific scFv that target the S1 of SARS CoV were displayed on the surface of pseudotyped lentiviral particles and mediated specific attachment to target cells.
Overall, development of methods capable of engineering lentiviral vectors to transduce specific cells could substantially change the current practice of gene therapy and greatly expand its use for disease treatment, alleviating the concern of an off-targeting effect.
Institute of Pulmonology, Hadassah-Hebrew University Medical Center , Jerusalem, 91120, Israel E-mail: fridlender@hadassah.org.il
An immunosuppressive environment exists within tumors, induced by cancer and immune cells. 5,6-Dimethylxanthenone-4 acetic acid (DMXAA) is a small flavanoid-like compound, previously shown to have a strong anti-tumor activity in murine models of cancer, and currently being tested in phase 3 trials in humans. We have shown that part of the effect of DMXAA as monotherapy is by activating tumor-associated macrophages. We therefore hypothesized that DMXAA would be a useful adjunct to immuno-gene therapy (immunoRx) in lung cancer.
We evaluated the impact of DMXAA in three immunoRx models of non-small cell lung cancer (NSCLC): two aimed at an expressed HPV-E7 antigen, using an adenovirus or a Listeria vector, and one using an adenovirus expressing Interferon-alpha.
DMXAA markedly augmented the effect of immunoRx on large (>200 mm3) established NSCLC flank tumors in the three models and two cell lines tested, resulting in cures in 30–40% of mice. There was no significant difference in the subtypes of systemic (Splenic) immunocytes between the two groups. In contrast, DMXAA increased the intratumoral influx of neutrophils, macrophages, and CD8+ T-cells compared to the immunoRx group. Furthermore, adding DMXAA to immunoRx decreased the percentage of intratumoral M2 (CD206+) macrophages by 2-fold, while increasing the percentage of M1 macrophages by 2-fold. Although the percentage of CD8+ T-cells was increased in tumors with the combined treatment, there was no difference in their activation or E7 antigen-specificity. DMXAA up-regulated mRNA levels for a host of proinflammatory and chemoattractant cytokines including TNF-alpha, IFN-gamma, and CCL2.
We conclude that macrophage activation and differentiation using DMXAA significantly augments the efficacy of immunoRx in established murine NSCLC tumor models by increasing the traffic of immunocytes into tumors, activating and differentiating macrophages and creating an immunostimulatory microenvironment.
Our data suggests that combining agents like DMXAA with vaccines should be considered in future immunoRx trials.
Ultrasound Gene Delivery to Mesenchymal Stem Cells for Tumor Therapy
The Hadassah Human Embryonic Stem Cell Research Center, The Goldyne Savad Institute of Gene Therapy & Department of Gynecology, Hadassah-Hebrew University Medical Center , Jerusalem, 91120, Israel E-mail: mariai@hadassah.org.il
Dysfunction and loss of retinal pigment epithelium (RPE) leads to degeneration of photoreceptors in Age-Related Macular Degeneration and subtypes of Retinitis Pigmentosa. Human embryonic stem cells (hESCs) may serve as an unlimited source of RPE cells for transplantation in these blinding conditions.
Here we show the directed differentiation of hESC clusters towards an RPE fate under defined culture conditions. We demonstrate that nicotinamide promotes the differentiation of hESCs to neural, and subsequently, to an RPE fate. The enhanced expression of RPE markers is observed concomitant with the appearance of pigmented areas in more than 70% of the clusters. Following plating on laminin, the pigmented cells assume a polygonal RPE-like shape and express markers of RPE. Furthermore, the hESC-derived pigmented cells have phagocytic capability, like authentic RPE cells.
In the presence of nicotinamide, activin A, the factor from the TGFβ superfamily which presumably patterns RPE development during embryogenesis, significantly enhances the differentiation towards RPE-like cells.
Following intraocular subretinal transplantation, the hESC-derived pigmented cells integrate within the host RPE layer and express markers of mature RPE. Significant photoreceptor rescue is evident in proximity to the grafted cells. Electroretinographic recordings demonstrate improved retinal function in transplanted eyes as compared to control eyes.
In conclusion, the present study demonstrates that nicotinamide and activin A can promote, under defined culture conditions, the differentiation of hESCs towards an RPE fate. The hESC-derived pigmented cells exhibit the morphology, function, and marker expression of authentic RPE, and rescue retinal structure and function after transplantation to an animal model of retinal degeneration. These results are an important step towards the future use of hESCs for transplantation therapy and replenishment of RPE in blinding diseases.
Mesenchymal Stem Cells Promote the Proliferation and Maturation of Neural Stem Cells in the Subventricular Zone
The discovery of adult neurogenesis has opened new possibilities for therapy of neurodegenerative disorders. Neurogenesis has been demonstrated at the subventricular zone (SVZ) of the lateral ventricles and the subgranular zone (SGZ) of the hippocampus. Throughout life, SVZ-derived cells migrate to the olfactory bulb where they differentiate into mature neurons. Previous studies showed that enriched environment induced therapeutic effects in animal models of neurodegeneration and effected endogenous neurogenesis.
Since the current study aimed to enhance the endogenous neurogenesis, we employed this form of housing. In agreement with previous reports, our results show that enriched environment augments the neurogenesis in the SGZ, but not in the SVZ. However, MSCs transplantation into the SVZ of mice housed in the enriched environment stimulated the proliferation and the maturation of endogenous neural progenitors in the SVZ. Histological analysis confirmed the survival of a significant amount of cells at least 3 weeks after the transplantation and showed that the transplanted cells express BDNF, a neurotrophic factor, which may effect the differentiation of SVZ residents.
To our knowledge, this is the first study to show that MSCs stimulate proliferation and differentiation of neural stem cells in the SVZ, independent of brain induced lesion. These results may promote the development of therapeutic modalities for treatment of neurodegenerative diseases.
Department of Neurology, Laboratory of Neuroimmunology and Agnes Ginges Center for Neurogenetics and Multiple Sclerosis Center, Hadassah-Hebrew University Hospital , Ein-Kerem, Jerusalem 91120, Israel E-mail: ibrahimk@ekmd.huji.ac.il
Mesenchymal stromal cells (MSC) have shown neuroprotective and immunomodulatory effects. A phase I/II clinical trial was designed to evaluate the feasibility and safety of intrathecal and intravenous administration of autologous MSCs in patients with multiple sclerosis (MS) and amyotrophic lateral sclerosis (ALS).
Fifteen MS (mean EDSS = 6.7 ± 1.05) and 19 ALS patients (mean ALSFRS = 20.26 ± 8.56) were enrolled. Following culture, a mean number of 63.2 ± 2.5 × 106 MSCs was injected intrathecally (n = 35) and intravenously (n = 14). In 9 cases, MSCs were magnetically labeled with the superparamagnetic iron oxide, Feridex®. In 21 patients there were injection-related side effects consisting of transient fever and headache. No major side effects were reported during a follow up period of up to 25 months. The mean ALSFRS remained stable during the 6 first months of observation, whereas the mean EDSS score improved from 6.7 ± 1.05 to 5.9 ± 1.6. MR imaging visualized the MSCs in the occipital horns of the ventricles, and indicated a possible migration of Feridex®-labeled cells in the meninges, the subarachnoid space and the spinal cord. Immunological analysis, revealed an increase in the proportion of CD4+CD25+ regulatory T-cells, and a decrease in the proliferative responses of lymphocytes, the IFNγ and IL-17 production and of the expression of CD40, CD83, CD86, and HLA-DR on myeloid dendritic cells, at 24 hrs following MSC transplantation.
Our findings suggest that intrathecal and intravenous injection of MSCs in patients with MS and ALS, is a clinically feasible and relatively safe procedure. Controlled studies are needed to evaluate the long term safety and the potential clinical efficacy of MSC treatment.
Department of Virology and Developmental Genetics and the National Institute of Biotechnology in the Negev, Ben-Gurion University , Beer-Sheva 84105, Israel E-mail: kuzmina@bgu.ac.il
The Positive Elongation Factor b is a hetrodimer, composed of CDK9 and cyclin T. Cyclin T1 is an essential cellular cofactor for the transcription of the human immunodeficiency virus type 1 (HIV-1). Cyclin T1 (CycT1) associates with a viral protein, Tat, at the transactivation response RNA element (TAR), located at 5′ of all viral transcripts. This represents a critical step for the stimulation of transcriptional elongation. Therefore, CycT1 may serve as a potential target for the development of anti-HIV therapies.
To generate a dominant-negative mutant cyclin T1, random mutations were introduced in the N-terminus region of cyclin T1. Protein mutants were tested for their ability to mediate dominant negative (DN) effects and inhibit Tat transactivation in human cells. One mutant—CycT U7 with the strongest DN effect—was isolated. Upon transient over-expression of Tat and U7 cyclin T1 in human cells, efficient inhibition of Tat transactivation was detected. Western Blot analysis revealed that expression levels of Tat were much lower than in control cells that expressed wild-type cyclin T1 and Tat. Interestingly, expression levels of U7 cyclin T1 were also down-regulated relatively to WT cyclin T1. Expression levels of both proteins were restored by adding proteosome inhibitors, suggesting that binding of the DN cyclin T1 to TAT results in rapid degradation of both proteins by proteosomal pathway.
In this study, we aimed to investigate the effects of cyclin T1 U7 in inhibition of HIV replication in vivo. At first, stable cells lines that overexpress the U7 dominant negative mutant Cyclin T1 were generated and tested for inhibition of HIV replication. Stable cell lines demonstrated strong inhibition of TAT transactivation. Currently, cyclin T1-U7 mediated inhibition of HIV replication is tested in T cells which stably express cyclin T1-U7 and infected with wild-type HIV. These results suggest that CycT1-U7 could potentially be used as a tool for antiviral therapy.
Tumor Immunotherapy Using Chimeric Receptor Redirected Allogeneic T Cells Across MHC Barriers
We present a strategy for adoptive immunotherapy using fully mismatched allogeneic chimeric receptor redirected T cells (allo-‘T-bodies’) in the absence of Graft vs. Host disease (GvHD). The strategy hinges on creating a therapeutic time-window using mild preconditioning which on the one hand allows the allogeneic T-bodies enough time to effectively attack the tumor, but on the other hand, does not ablate the Host vs. Graft response (HvG) ensuring the eventual rejection of the allogeneic T cells, thus preventing GvHD.
We found that transfer of fully mismatched allogeneic T cells expressing a HER2/neu specific chimeric receptor following mild preconditioning roughly tripled the median survival of mice bearing pulmonary Renca-HER2 metastases with around 50% of mice surviving long term (>200 days) in the absence of GvHD mortality. We show that allo-T-bodies provide comparable benefit to syngeneic T-bodies, and are far superior to nonspecific allogeneic T cells, demonstrating that the response is indeed tumor-specific and not merely allo-specific.
Taken together these data provide proof-of-principle for safe and effective allogeneic adoptive therapy allowing for the use of ‘universal effector cells’ as a standardized treatment for cancer.
Department of Pediatrics and Pediatric Stem Cell Research Institute, Sheba Medical Center, Tel Hashomer, Ramat Gan and Sackler Faculty of Medicine, Tel Aviv University , Tel Aviv, 52621, Israel E-mail: salima2@walla.co.il
In the human fetal kidney (HFK), self-renewing stem cells residing in the metanephric mesenchyme (MM)/blastema are induced to form all cell types of the nephron till 34th week of gestation. Definition of useful markers is crucial for the identification of HFK stem cells. Because Wilms' tumor, a pediatric renal cancer, initiates from retention of renal stem cells, we hypothesized that surface antigens previously up-regulated in microarrays of both HFK and blastema-enriched stem-like Wilms' tumor xenografts (NCAM, ACVRIIB, DLK1/PREF, GPR39, FZD7, FZD2, NTRK2) are likely to be relevant markers. Comprehensive profiling of these putative and of additional stem cell markers (CD34, CD133, c-Kit, CD90, CD105, CD24) in mid-gestation HFK was performed using immunostaining and FACS in conjunction with EpCAM, an epithelial surface marker that is absent from the MM and increases along nephron differentiation and hence can be separated into negative, dim or bright fractions.
No marker was specifically localized to the MM. Nevertheless, FZD7 and NTRK2 were preferentially localized to the MM and emerging tubules (<10% of HFK cells) and were mostly present within the EpCAMneg and EpCAMdim fractions, indicating putative stem/progenitor markers. In contrast, single markers such as CD24 and CD133 as well as double-positive CD24+CD133+ cells comprise >50% of HFK cells and predominantly co-express EpCAMbright , indicating they are mostly markers of differentiation. Furthermore, localization of NCAM exclusively in the MM and in its nephron progenitor derivatives but also in stroma and the expression pattern of significantly elevated renal stem/progenitor genes Six2, Wt1, Cited1, and Sall1 in NCAM+EpCAM− and to a lesser extent in NCAM+EpCAM+ fractions confirmed regional identity of cells and assisted us in pinpointing the presence of subpopulations that are putative MM-derived progenitor cells (NCAM+EpCAM+FZD7+), MM stem cells (NCAM+EpCAM−FZD7+) or both (NCAM+FZD7+).
These results and concepts provide a framework for developing cell selection strategies for human renal cell-based therapies.
Frankel Laboratory, Center for Stem Cell Research, Schneider Children's Medical Center of Israel , Petah Tikva 49202, Israel E-mail: mizrahikeren@hotmail.com
Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a member of the TNF superfamily that participates in homeostatic regulation of cell death and activation. Since this ligand is selectively toxic to malignant cells, it is questioned whether it affects the function of hematopoietic progenitors. In this study we assessed the impact of TRAIL on umbilical cord blood (UCB) cells, as a source of progenitors with increasing use in the transplant setting. Among the five known receptors, DR4 (TRAIL-R1) and DR5 (TRAIL-R2) transduce apoptotic signals in human cells. DR4 is preferentially expressed in fresh mononuclear (MNC) cells (18% versus 2% DR5), lineage-negative (lin−) (DR4 = 11% and DR5 = 3%) and in CD34+ cells (DR4 = 11% and DR5 = 8%). A lethal concentration of 1.5 μg/ml TRAIL did not induce significant apoptosis during 72 hours of incubation in the MNC (33%), lin− (26%) and CD34+ (24%) subsets. DR5+ cells cycled faster than DR4+ cells (2-fold) irrespective of the presence of the cognate ligand. Supplementation of TRAIL suppressed CFU-GM activity in bulk UCB cells at low concentrations (0.25–0.5 μg/ml) and induced this activity at higher concentrations (>1 μg/ml). In contrast, dose-dependent activation was observed in lin− progenitors. Two functional assays were performed: a) to assess toxicity to committed progenitors, UCB cells were incubated with TRAIL. The activity of viable cells isolated by ficoll was markedly enriched by TRAIL preincubation, suggesting selective induction of apoptosis in nonprogenitor cells. b) SCID repopulating activity was unaffected by UCB preincubation for 24 and 72 hours with TRAIL prior to transplantation, consistent with survival of hematopoietic progenitors.
In summary, TRAIL is not toxic to UCB derived hematopoietic progenitors and exerts a trophic effect by increasing recruitment and responsiveness of progenitors to clonogenic activity, which might accelerate engraftment of UCB grafts.
Department of Pediatrics and Pediatric Stem Cell Research Institute, Sheba Medical Center, Tel Hashomer, Ramat Gan, Israel and Sackler Faculty of Medicine, Tel Aviv University , Tel Aviv, 52621, Israel E-mail: dorit.omer@gmail.com
The transcription factor Six2 defines committed stem cells in the metanephric mesenchyme that are capable of self-renewing and of differentiating towards different types of nephron epithelia. Nevertheless, as nephrogenesis commences Six2 along with other key renal lineage markers (Osr1, Sall1, Pax2, Wt1) are transcriptionally silenced in part via epigenetic inhibition. We therefore hypothesized that re-induction of genes characteristic of renal progenitors can be achieved in human somatic kidney cells by chromatin-modifying agents (VPA and TSA/AzaC).
Here, using quantitative RT-PCR and FACS analysis, we show in VPA-treated primary cultures of human adult kidney significant re-induction of Six2, Osr1, Sall1, the pluripotency genes Oct4, Nanog and Klf4 mRNA concomitant with the embryonic renal progenitor surface markers, NCAM and PSA-NCAM. Interestingly; E-cadherin transcript levels were mostly elevated while Vimentin remained unchanged in treated cells and thus cells constantly exposed to VPA retained Six2/Osr1 induction upon culture passage but did not undergo epithelial-mesenchymal transition. Robust Six2 mRNA re-expression was confirmed at the protein level by western blot and was associated with epigenetic changes of the histones at multiple sites of the Six2 promoter leading to gene activation; significantly increased acetylation of histones H4 and methylation of lysine 4 on H3. Furthermore, because VPA also activated the canonical Wnt pathway and induced strong Wnt4 expression, potentially limiting Six2, we could demonstrate the synergistic effects of Wnt antagonists on Six2 and also Osr1 re-induction.
Thus, epigenetic alterations of chromatin and Wnt pathway modulation can reactivate dormant renal-progenitor genes in terminally differentiated human kidney cells. We now aim to evaluate these chemically ‘reprogrammed’ cells in murine models of renal failure.
Lentiviral Delivery of Anti-PDEs shRNA into Activated Microglia Cells to Prevent the Secondary Damage After Spinal Cord Injury
The spinal cord trauma provokes activation of immune cells resident—microglia, and systemic phagocytes, causing neurodegenerative processes of the spinal cord tissue as a secondary damage in the injured area. The activated immune cells of the injured CNS, exacerbated by the initial insult, secrete cytotoxic neurotoxins and cytokines, and free oxygen species, and a wide range of inflammatory enzymes. Prevention of phagocyte activation or inhibition of pro-inflammatory molecules produced by activated phagocytes, provide reduction in the neural tissue loss. Several intended objectives have been identified to protect neurons from secondary damage after CNS injury. Phosphodiesterase 4 (PDE4) is the major cAMP-metabolizing enzyme found in inflammatory and microglial cells. PDE4 inhibitors have proven to be potential anti-inflammatory drugs. Moreover, inhibition of PDE4 provides cAMP elevation that supports spinal axon sparing and growth. In this study, RNAi technology was applied using lentiviral vector pLL3.7 to deliver shRNA into target cells.
The cloning site was enriched with unique BamHI downstream of HpaI, so for cloning, a vector linearized with BamHI-XhoI restriction enzymes was used. The loop of shRNA was designed with a unique SphI for insert detection. PDE4 shRNAs, designed for A, B, D isoforms, are capable of working with any of the three—human, rats and mice. Four sequences of 19 nt have been chosen at the beginning, middle and end of mRNAs. TNF-alpha-activated microglial cells (EOC-2) were transfected using a unique protocol, elaborated in our laboratory. An empty vector was used for transfection control. No cell death caused by manipulations was observed. Changes in cell-morphology and culture-morphology were observed in experimental plates, but not in control. At four time points, cells were collected for RT-PCR and immune blot-hybridization. The highest, up to 80% reduction, was observed with RNAi sequences taken mostly at the beginning of mRNA for PDE4A (5′-GCAGAATGAAGTGGAGATC-3′), Pde4B (5′-GTCCTTGGAGTTGTATCGG-3′), and PDE4D (5′-GACCATTACCATGCTGACG-3′).
Mannosylated Block Copolymer Micelles for Targeting Genes into Antigen-Presenting Cells
Antigen-presenting cells (APCs) have unique properties in initiating primary immune responses and thus can be an excellent target for DNA vaccination. Polyion complex micelles made of poly(ethylene glycol)-block-poly(ethylene imine) (PEG-b-PEI) conjugates are known to self assemble in aqueous media with DNA into a spherical polymeric micelles with a core-shell architecture, which reduces interactions with blood components. However, the PEG shielding lowers the transfection efficiency. The attachment of a specific ligand may increase cellular uptake via receptor-mediated endocytosis. The mannose receptor (ManR) involved in the uptake of particulate and soluble antigenic products of bacterial origin represents therefore a promising receptor for targeting APCs having ManR.
We assembled a di-block copolymer composed of a cationic (PEI) and hydrophilic (PEG) segments installed with Mannose (Man) targeting ligand, in a mono- and trivalency (designated as Man-PEG-b-PEI and Man3-PEG-b-PEI, respectively). Complex formation with DNA was determined by ethidium bromide assay in a range of polymer/plasmid ratios. The surface charge of the Mannosylated-PEG-PEI complexes was found to be lower than that of the linear PEI. The block copolymer micelles were further tested in vitro for their ability to enhance the transfection efficiency in THP-1, murine dendritic DC2.4 and HeLa cells. Man3-PEG-PEI demonstrated a 3-4 fold greater transfection efficiency relative to Man-PEG-PEI in both ManR positive dendritic cell lines. Furthermore, the mannosylated block copolymers demonstrated reduced transfection efficiency in a free Man competition assay, had low cytotoxicity when compared with PEI, and showed low transfection efficiency in HeLa cells.
These results suggest that the mannosylated block copolymer micelles are an effective gene delivery system for APCs.
Laboratory of Neurosciences, Human Molecular Genetics and Biochemistry Department, Sackler Faculty of Medicine, Tel Aviv University , Tel Aviv, Israel E-mail: ofersa@post.tau.ac.il
Huntington's disease (HD) is a hereditary disorder characterized by involuntary movements, emotional instability and dementia. Polymorphic region containing cytosine-adenine-guanine (CAG) repeats on the huntingtin gene (htt) is expanded in HD patients (over 37 repeats). To date, there is no effective treatment for this fatal disease. Recent controlled clinical trials using fetal neuronal grafts failed to induce a clinical benefit, suggesting that a better choice of cells is warranted.
We developed a medium-based differentiation protocol that induces bone marrow-derived mesenchymal stem cells into astrocyte-like cells. These cells express astrocytic markers such as GFAP and glutamine synthase, produce and secrete large amounts of neurotrophic factors (NTF) such as BDNF and GDNF, and are capable of glutamate uptake. We previously found that these induced cells can migrate within the CNS toward an excitotoxic lesion induced by striatal quinolinic acid (QA) injection, an animal model of HD. We found that transplanting induced NTF secreting cells, but not undifferentiated MSCs, decreased apomorphine-induced rotations by over 80%. One of the potential advantages of MSCs is the possibility of autotransplantation. Comparison of MSCs obtained from HD patients and healthy donors showed similar expression of CD markers and capacity to differentiate into osteocytes, chondrocytes, and adipocytes.
Ongoing research is trying to determine the potential of HD-derived MSCs to differentiate into astrocyte-like cells in vitro, and to test their neuroprotective capabilities in vivo.
Laboratory of Neurosciences, Human Molecular Genetics and Biochemistry Department, Sackler Faculty of Medicine, Tel Aviv University , Tel Aviv, Israel E-mail: ofersa@post.tau.ac.il
Stem cell-based treatments are a promising new frontier in the field of neurodegenerative diseases and particularly PD. We have been studying the potential of utilizing the patients' own bone marrow-derived stem cells for an improved mode of sustained delivery of neuroprotective factors in order to preserve the surviving dopaminergic neurons.
A novel protocol has been developed to induce bone marrow-derived mesenchymal stem cells into neurotrophic factors secreting cells (NTF+ cells). These cells express astrocytic markers and secrete high amounts of NTFs such as BDNF and GDNF (5 and 0.5 ng/106 cells, respectively). Transplantation of human-derived NTF-cells in a unilateral 6-hydroxydopamine induced striatal lesion, attenuated amphetamine-induced rotations by a mean of 45%, restored the number of dopaminergic terminals, and attenuated the dopamine depletions in the lesioned hemisphere (72% vs. 21% in controls). MRI study and corresponding histological assessments revealed that a significant percentage of the engrafted cells migrated toward the lesion.
To examine the possibility of autotransplantation, we used syngeneic mouse-derived NTF+ cells in the MPTP mouse model for PD. Mouse NTF+ cells were transplanted unilaterally into the striatum, and following transplantation, systemic MPTP treatment was administered for 5 days. Sixteen days later, the number of net contralateral amphetamine-induced rotations from 0.02 ± 0.67 per minute in the PBS treated group to 2.37 ± 0.83 in the NTF+ cells-treated group indicating a higher level of released dopamine in the treated hemisphere. Thus, using both 6-OHDA and MPTP models, we demonstrated that the bone marrow-derived NTF+ cells may have a therapeutic potential for a safe cell based therapy in Parkinson's disease.
The Krieger Eye Research Laboratory, FMRC, Rabin Medical Center , Petah Tikva, 49100, Israel E-mail: tamilla1@walla.co.il
Bone marrow cells have been shown to home to sites of injury and participate in the process of remodeling after injury. The smallest population of nucleated bone marrow cells has been shown to differentiate into a range of tissues, in addition to long-term hematopoietic reconstituting potential. The retina, under physiological conditions, continues to develop 3 weeks post partum, to complete neurogenesis and angiogenesis. To determine whether these cells can contribute to the retina in the absence of injury and inflammation, we conceived that the neonatal eye would be the most favorable environment for such incorporation and differentiation. Therefore, we seek to determine the participation of this subset of candidate multipotent cells to retinal modeling after birth.
Adult bone marrow cells were isolated by counterflow centrifugal elutriation that separates a subset accounting for 8-14% of the bone marrow cells at a flow rate of 25 ml/min (Fr25). To obtain a hematopoietic lineage-negative fraction (Fr25lin−), immunomagnetic depletion was performed for Ter-119, GR-1, Mac-1, CD5 and B220. Intraocular injection of Fr25lin− to neonate mice aged 4-6 days was performed under anesthesia.
Donor cells that constitutively express GFP were detected in the neonate eyes one month following transplantation of Fr25lin− cells. The cells that incorporated into the developing retinae were mainly located in the RGC layer and showed neuronal markers.
Neonates survived intraocular injection of putative stem cells from GFP+ adult bone marrow donors. The eyes maintained their normal anatomy. A few cells, which were incorporated into the retina, as detected 1 month following transplantation, demonstrated neuronal and glial differentiation. The survival and differentiation of the GFP+ cells for longer than 6 months is currently under investigation.
Department of Orthopedic Surgery, Hadassah-Hebrew University Medical Center , Jerusalem, Israel E-mail: jes@hadassah.org.il
Distal third tibial fractures have a high rate of nonunion. Mesenchymal stem cells (MSC) have been shown to be able to form bone tissue in vitro and in vivo. In this study, MSC were harvested from patients with distal tibia fractures, immune-isolated, and injected into the fracture in order to improve fracture healing.
Twenty-four patients underwent surgery for extra-articular distal tibia fracture (between 6/2009 and 02/2010). After surgery, the patients signed an informed consent form. Patients eligible for the study, had to be without any bone-modifying condition. Patients were randomized to intervention and control groups.
One month after the fracture fixation, the iliac crest was prepped, 50 ml of bone marrow were aspirated and 100 ml of blood was collected. MSCs were isolated with a CliniMACS machine. Blood was centrifuged and the platelet reach plasma (PRP) was collected. In the OR, the fracture was washed and draped. Under fluoroscopic guidance, a mix of CD105+ cells (MSC), PRP and demineralized bone matrix (DBM) was injected into the fracture site. Small fractions were grown in vitro and implanted subcutaneously in nude mice.
MSC numbers averaged at 105 to 106 cells (range: 6–300 ×106). FACS analysis (cytometry) of the MSC showed a purity of 60-80%. PRP had 1050 platelets per ml (range 400–1500). There were no complications or infections. In vitro cultures grew colony forming units (CFUs) at a rate of 5%. Mice developed bone. Average time to union was 130 days in the control group, and 71 days in the intervention group ( p < 0.015). One patient in the control group developed delayed union.
The combination of MSC, PRP, and DBM has proven to be an effective treatment, hastening fracture healing, creating bone both in mice and humans. In conclusion, we clearly show the feasibility of augmenting fracture healing by cell-based therapy.
Studying the Differentiation of Pancreatic Beta-Cells Using High-Throughput Proteomic Approaches
Human embryonic stem-cells (hESCs) are a powerful tool for analyzing pancreatic β-cell development and may generate new opportunities for diabetes therapy. While some reports have described the ability of hESC-derived endoderm precursors to produce insulin in vivo, any future clinical use would likely require identification and generation of precursor populations with higher fidelity. As part of the ongoing efforts to elucidate the early stages of pancreatic differentiation, our goal is to identify and study stage-specific pancreatic precursors using two complementary proteomic platforms. Identification of stage-specific precursors is performed by a high-throughput screen with large arrays of antibodies against cell surface markers. These arrays allow rapid identification of cell surface markers which distinguish different populations of cells that emerge during differentiation. To further screen for conditions promoting stepwise differentiation of hESC-derived pancreatic precursors, we use arrays of signaling microenvironments which provide parallel exposure of multipotent cells to diverse combinations of signaling molecules (e.g., extracellular matrix components, morphogens and other signaling proteins). Following a period of cell culture on the arrayed microenvironments the differentiation outcome is analyzed using lineage markers and automated microscopy.
Together, these methods may provide a more detailed characterization of stage-specific pancreatic precursors and improve the efficiency of the differentiation process. This may help achieve the goal of generating sufficient numbers of insulin producing β-cells or clinically pure populations of precursor cells for cell replacement therapy. (Supported by a network grant from the Juvenile Diabetes Research Foundation.)
Dana Farber Cancer Institute , Boston, MA 02115, USA
The potential of an anti HIV-Tat intrabody to promote survival of CD4+ cells and decrease levels of chimeric SIV/HIV infection was evaluated in rhesus macaques. Rhesus CD4+ cells from two animals were enriched after leukapheresis, stimulated and transduced with GaLV-pseudotyped retroviral vector expressing either an anti-HIV-1 Tat or a control single-chain intrabody.
With the optimized stimulation and transduction protocols, ex vivo CD4+ gene marking was highly efficient, averaging almost 4 proviral copies/cell in the large-scale experiments. Upon re-infusion of the engineered CD4+ cells into each animal, relatively stable levels of transduced PBMCs (peak of 0.6% and 6.8% of PBMCs) were found in vivo. Peripheral tissues were also efficiently marked, as 0.2% or 2.8% of the lymph node cells contained the vector at day seven post-infusion. One week following cell infusion, animals were challenged with the chimeric virus SHIV 89.6p. Viremia peaked at 2 weeks and was followed by a generalized loss of CD4+ cells and progressive decline in the frequency of genetically modified CD4+ T cells. However, CD4+ T cells expressing the therapeutic anti-Tat intrabody exhibited survival rates between 20–30 × higher than CD4+ cells expressing the control vector after only 1 week of SHIV challenge. In one animal, this survival benefit of anti-Tat transduced cells was associated with detectable levels of intrabody expression in the plasma and a reduction in SHIV viral load. After six months, transduced cells were still detectable in secondary lymphoid tissues (LN, spleen, gut).
Overall, these results indicate that a retroviral-mediated intrabody delivery targeting HIV-Tat provided significant levels of gene marking, in both PBMCs and peripheral tissues, and partial protection of transduced cells in vivo.
Laboratory of Neurovirology, Hadassah University Hospital , PO Box 12000, Jerusalem 91120, Israel E-mail: yaelts@ekmd.huji.ac.il
The aim of the study is to understand the mechanism of neurotropic viral infection and spread in the CNS. Neurotropic viruses, such as herpes simplex virus type I (HSV-1) and Rabies, infect the central nervous system and may cause severe diseases. In order to study neurotropic virus tropism and spread in the brain, a unique ex vivo system of organ culture was developed in our lab. The ex vivo system enables preservation of the tissue structures, including the variety of cell types and the extracellular matrix. HSV-1 may spread in the brain by retrograde axonal transport, extracellular re-infection or cell to cell transport.
In this study the infection and spread of HSV-1 vector, expressing the GFP protein, was evaluated after injection of the virus directly to the ventricle zone of the brain. The infected native tissues were observed daily under a fluorescent microscope. After a day, infection was seen as a focal site and subsequently the virus spread along distinct anatomical structures. Neutralizing antibodies failed to inhibit viral spread, suggesting that the mechanism of spread is not mediated by extracellular re-infection. Inhibition of replication using acyclovir slowed down viral spread but did not abolish it altogether.
Lentiviral based vectors have a great potential in gene therapy of neurological diseases. We, therefore, compared the infection pattern of lentivirus pseudotyped by VSV-G and rabies virus envelope proteins (RV-G) to the pattern of HSV-1 infection. While VSV-G pseudotype tropism had a striking resemblance to HSV1, the RV-G pseudotype infection was more diffused.
Understanding the mechanism of viral infection and spread will enable the development of more effective treatments than those available today, and may also enhance gene therapy related research, using vectors based on HSV-1 and lentiviruses.
Platelet–Type 12-Lipoxygenase Knockdown Using Gutless AAV Vectors Can Induce Apoptosis in Human Vascular Smooth Muscle Cells In Vitro
Abnormal accumulation of vascular smooth muscle cells (VSMCs) within the arterial intima, resulting in intima-media thickening, appears a common step in the pathogenesis of atherosclerosis driven by hypertension, hyperlipidemia or hyperglycemia. Such growth in VSMC population results from alteration in the equilibrium between cell growth and apoptosis and may negatively affect vascular function and structure. We have previously identified a unique “long form” of platelet type 12-Lipoxygenase (12-Lo) which appears necessary for human VSMC survival. We are therefore developing a gene therapy paradigm to curb VSMC growth and possibly remove pathogenic VSMC from the human vascular system.
In the current study we compare different AAV serotypes (2, 4, 5 and 12) for their ability to infect primary human VSMC in vitro. We found that AAV2 succeeded to infect 65–80% of cultured HSMC by 6 days post-infection, while the other 3 AAV serotypes did not infect more than 25% of the cells. Then 12-Lo knockdown AAV2 vectors were prepared and infected into cultured primary human VSMC in vitro and their ability to eradicate these smooth muscle cells was assessed at different viral to cell concentrations in the presence and absence of the 12-Lo product 12-Hydroxyeicosatetraenoic acid (12-HETE). Indeed, these optimal AAV2 12-Lo vectors can effectively kill human VSMC over a relatively short period of time reaching a maximum of approximately 70% at 10 days post-infection at a m.o.i of 4,000. The addition of 12-HETE to cultures prevented the viral induced cell death, thus indicating that endogenous cell deprivation of 12HETE was the cause of cell death induced by 12-Lo knockdown.
Once these vectors are optimized for expression exclusively in the pathogenic human VSMC subtype, an effective new tool may have application for the future treatment of human vascular disease.
Placental Mesenchymal Stem Cells Differentiated into Neurotrophic Factor Producing Cells Protect Neuronal Cells from Hypoxia and Oxidative Stress
Numerous studies have shown that mesenchymal stem cells (MSCs) from adult and fetal tissues have the potential to differentiate into various cell types and used for clinical applications. Multiple sources for MSC were described, including bone marrow, peripheral blood, umbilical cord blood and amniotic fluid. Placental tissue was also suggested as an abundant, ethically acceptable, less immunogenic and easily accessible source of MSC. Therefore, we aimed to test the possibility that differentiated placental MSCs (PL-MSC) will be beneficial for neurons after induce hypoxia.
First, we have shown that isolated cells from three human placenta showed typical MSC membrane markers (positive for CD105, CD90, CD73, CD29, and negative for CD14, CD34, and CD45). These cells were also capable to differentiate into the typical mesoderm lineage osteoblasts and adipocytes. Then, we induce astrocyte differentiation using a novel protocol that has been developed in our laboratory. In our previous studies, using this protocol on bone marrow MSC, we demonstrated high neuroprotective capacity in animal models of neurodegenerative diseases. In the current study we show that the differentiated PL-MSC express astrocyte marker (GFAP) and higher levels of neurotrophic factors such as BDNF and GDNF. Moreover, the conditioned media from the differentiated PL-MSC markedly protected mouse primary neuronal cultures, neuroblastoma (SH-SY5Y), and motoneuron (NSC-34) cell lines against induced hypoxia (1% O2 for 48 hours), and increased doses of H2O2 and 6-OHDA.
In conclusion, our data indicate for a new strategy for stem cell-based therapy in hypoxia and ischemia, using placental derived mesenchymal stem cells, following neural differentiation.
Derivation and Use of the Hematopoietic Human Herpesvirus 6 (HHV-6) Amplicon Vectors for Gene Therapy and Vaccination
The present talk deals with the use of Human Herpesvirus-6A (HHV-6A) amplicon vectors for gene therapy and vaccination. HHV-6A employs the CD46 receptor, allowing entry into T and B-lymphocytes, NK, monocytes, and dendritic cells. The virus does not cause any known acute disease.
Amplicon-type vectors first developed in our laboratory are packaged 150 kb concatemeric genomes, composed of multiple reiterations of cloned units, each containing the cis-acting DNA replication origin, the packaging signals as well as the selected transgene(s). Transgene(s) can be very large (e.g., up to 18 kb). They are expressed efficiently due to sequence reiterations. Amplicon vectors of 3 general types are currently employed worldwide: (i) composite vectors, containing mixtures of helper viruses and packaged concatemeric reiterated amplicons; (ii) packaged concatemeric amplicons free of helper viruses; (iii) chimeric vectors of the HSV amplicon and added vector from different viruses.
The present talk concerns three topics: (i) We describe composite amplicon-6 vectors with (a) oncolytic properties. In addition to GFP marker the vector carries the HSV thymidine kinase capable of inducing lymphocyte death in response to gancyclovir treatment; (b) vaccination vectors expressing membrane-associated or secreted proteins, e.g., HSV-1 glycoprotein D (gD) and HIV gp160. (ii) To produce the defective helper required for packaging of amplicons we cloned the 160 kb HHV-6A genome into Bacterial Artificial Chromosomes (BACs). The composite BAC could be stably retained in cells under puromycin selection and it expressed early and late genes in vitro. Current studies are directed towards derivation of hematopoietic helper-free amplicon vectors. (iii) We succeeded to induce differentiation of peripheral blood monocytes collected from healthy donors into the immature DCs as well as mature DC by incubation with the relevant cytokines.
Current studies are ongoing to test viral gene expression in these engineered DCs towards their potential use for autologous vaccination.
Goldyne Savad Institute of Gene Therapy, Hadassah Hebrew University Hospital , Jerusalem, 91120, Israel E-mail: evezeira@hadassah.org.il
Gene delivery to the retina was suggested as a therapeutic approach for several retinal pathologies among them retinal degenerations such as retinitis pigmentosa (RP), intraocular tumors, and macular degeneration (AMD). Currently, gene delivery to the retina is usually performed using viral vectors. This approach does not allow targeting of confined retinal area and, in addition, it exposes the subject to the risks associated with viral vector use.
We aim to develop a novel gene delivery: the Laser Beam Gene therapy (LBGT) mediated nonviral gene delivery model, which will facilitate efficient and long-term gene delivery for the treatments of such disorders without use of viral vectors. We also aim to assess the efficacy of LBGT gene delivery with naked DNA plasmid expressing the MERTK protein in the RCS rat, in order to demonstrate the potential of LBGT-mediated nonviral gene delivery model approaches for the treatment of retinal degenerations caused by defects specific to the RPE and/or photoreceptors of the retina of the eye. The Royal College of Surgeons (RCS) rat provides a model of recessive RP. Here the defect is due to a defect in Mertk, a gene that is expressed in the RPE and encodes a receptor tyrosine kinase that is thought to be involved in the recognition and binding of outer segment debris. The gene defect results in the inability of the RPE to phagocytose the shed outer segments from photoreceptor cells. The resulting accumulation of debris between the RPE and the neuroretina leads to progressive loss of photoreceptor cells.
An infrared femtosecond laser will be applied to the retina under direct ophthalmoscopic control through an operating microscope following a subretinal injection of plasmid. The technique will first be applied in Albinos rats (Sabre) using plasmid carrying lac-Z and GFP or RFP reporter genes. Following establishment of feasibility along with optimal laser set up and plasmid concentration, the technique will be applied to the RCS rat model, using a vector encoding the rat Merkt reporter gene. The tip of a 1 cm capillary is inserted tangentially through the sclera of the rat eye, causing a self-sealing wound tunnel. The capillary tip was brought into focus in the subretinal space and approximately 3 to 200 μg into 1 μl of plasmid suspension is injected to produce a bullous retinal detachment in the superior and inferior hemispheres around the injection sites.
In a preliminary experiment using laser beam gene transduction (LBGT), an injection of 3 μg of pCAG/EGFP, pCAG/LacZ resulted in expression on day one. Further experiment injecting 200 μg/1 μl of plasmid into RCS rats resulted in nice expression on days 1 and 3. Our next experiments will be to inject a plasmid encoding the rat Merkt vector, in order to demonstrate that subretinal injection of LBGT-mediated plasmid expressing the rat Mertk gene can significantly prolong photoreceptor cell survival in the RCS rat, and decrease the amount of debris in the subretinal space, suggesting that RPE function is restored.
LBGT-mediated naked DNA is a simple and reproducible method for in vivo eye gene expression and gene therapy approaches for RP patients with mutations in Mertk with the potential of being utilized in the clinical setting.
Department of Hematology and Bone Marrow Transplantation, Tel-Aviv Sourasky Medical Center , Tel Aviv, 642392, Israel E-mail: eti.zigman@gmail.com
Mesenchymal Progenitor Cells (MPCs) are non-hematopoietic stromal cells that can be found in many organs and are capable of differentiating into mesenchymal tissues such as bone, cartilage, skeletal muscle and adipocytes. Due to their multilineage differentiation, easy access and lack of immune response, MPCs have various potential therapeutic applications such as tissue regeneration.
The aim of our study was to develop a safe and clinically applicable method for the isolation, preservation, and thawing of adipocyte derived MPCs for reconstructive applications.
For that purpose, MPCs were isolated from fat tissue after liposuction procedure using collagenase digestion. The isolated cells were used to establish long-term culture either immediately following the harvest, or after cryopreservation and thawing. The in vitro cultures were established in DMEM containing 10% FCS for three weeks. The adherent cells were trypsinized and analyzed by flow-cytometry. Both freshly isolated and cryopreserved/thawed cultured cells showed a typical membrane phenotype characteristic for MPCs with median expression of CD90+ (94%), CD73+ (93%), CD105+ (97%), CD45− and CD34−.
We conducted a clinical trial in order to assess the safety and the potential use of fresh or thawed adipose tissue derived autologous MPCs for minor aesthetic applications such as wrinkle filling. Twenty patients (19 F, 1 M, median age 47 ± 17) were recruited for study, 10 received single injection of fresh MPCs and 10 received a second injection of thawed cells. No side effects were reported and after a follow-up of 10 months stable improvement was evident in most of the treated patients.
Our results confirm previous observations and demonstrate that adult MPCs can be easily isolated from fat tissue and potentially used as a safe source of autologous cells for soft tissue filling. Moreover, our data clearly show that these cells can be cryopreserved for further use while maintaining their potential implementation in reconstructive surgery.
Cardiomyocyte Differentiation of Human Induced Pluripotent Stem Cells
ISGCT 2010 Abstract Author Index*
Alper R, P 6, P 26
Arbel G, P 28
Ashkenasi N, Or 16
Askenasy N, P 12, P 18
Avaraham-Lubin BR, P 18
Bakimer-Kleiner R, Or 16
Banin E, P 6, P 26
Barhum Y, P 7, P 16, P 17, P 24
Barzel A, Or 1
Barzilay R, P 24
Bednarski C, Or 1
Ben-Hur H, P 11
Ben-Hur T, P 8
Ben-Shushan E, P 6
Ben-Zur T, P 7, P 24
Berman-Zaken Y, P 6
Berneman D, Or 2
Besser M, Or 11
Bobis-Wozowicz S, Or 1
Borenstein R, P 25
Braun E, P 22
Braun SE, P 21
Brodie C, Or 16
Bromberg Z, P 26
Bulte J, P 8
Buzhor E, P 1, P 11, P 13
Caspi O, P 28
Cathomen T, Or 1
Cazacu S, Or 16
Chaimov D, P 2
Chaouat M, Or 6
Cohen MA, P 6
Cohen Y, P 16, P 17
Collombat P, Or 3
Daskal A, P 19
David A, P 15
Dekel B, Or 4, Or 14, P 1, P 11, P 13
Dotan Z, P 1
Dwyer M, P 21
Eshel R, P 27
Eshhar Z, P 10
Even-Ram S, P 6
Fassler M, P 3
Ferber S, Or 2, P 2
Finnins S, Or 16
Fisher-Soval Y, P 24
Frenkel N, P 25
Fridlender ZG, P 4
Fridman E, Or 14
Fujinaga K, P 9
Galun E, Or 1, Or 6, P 26
Gazit SA, P 19
Gepstein A, P 28
Gepstein L, P 28
Gesuntheid B, Or 16
Giladi H, Or 1
Globerman AS, Or 6
Goldenberg-Cohen N, P 18
Gomori JM, P 8
Gophna U, Or 1
Gowda-Kurkalli B, Or 16, P 8
Grompe M, Or 8
Gropp M, P 6
Gur E, P 27
Halperin R, P 11
Harari-Steinberg O, Or 14, P 1, P 11, P 13
Heard JM, Or 7
Hemo I, P 6, P 26
Huber I, P 28
Johnson RP, P 21
Kan I, P 7
Kaplan O, P 12
Kapoor V, P 4
Karageorgiou C, Or 16, P 8
Karman H, P 23
Karussis D, P 8
Kassis I, P 8
Kay MA, Or 8
Keshet E, Or 6
Khaner H, P 6
Khoury A, P 19
Knoll E, P 23
Kronfeld N, Or 16
Kupiec M, Or 1
Kuzmina A, P 9
Lazary R, Or 1
Lee H-K, Or 16
Lev N, P 24
Levi C, P 26
Levin L, P 26
Lewis A, P 26
Liebergall M, P 19
Limor R, P 23
Manevitch Z, P 26
Many A, P 23
Marasco WA, P 21
Marcus A, P 10
Markel G, Or 11
Matzrafi L, P 6
Meilik B, P 27
Meivar-Levy I, Or 2, P 2
Melamed E, Or 13, P 7, P 16, P 17, P 24
Meshorer E, Or 12
Metsuyanim S, P 1, P 11, P 13
Mizrahi K, P 12
Mor Y, Or 14
Moseri A, P 25
Moshieff R, P 19
Murakami A, P 21
Naparstek E, P 27
Offen D, Or 13, P 7, P 16, P 17, P 24
Omer D, P 1, P 11, P 13
Orenstein A, Or 11
Ortenberg R, Or 11
Panet A, P 22
Pe'eri M, Or 1
Petrou P, P 8
Phillips N, Or 8
Pleniceanu O, P 1
Pode-Shakked N, Or 14, P 1, P 11, P 13
Privman E, Or 1
Pupko T, Or 1
Puzis LE, P 14
Raviv L, P 15
Rechavi G, P 6
Reubinoff BE, Or 15, P 6
Rivkin L, Or 1
Rom-Gross E, Or 14
Sadikov T, P 18
Schachter J, Or 11
Schneider D, P 11
Schroeder J, P 19
Seidman R, Or 11
Senior G, P 27
Shachar E, Or 1
Sharivkin R, P 20
Shemesh N, P 16, P 17
Shlomai Z, Or 6
Slavin S, Or 16, P 8
Smith Y, P 6
Soen Y, P 20
Stein J, P 12
Steiner I, P 22, P 24
Stern N, P 23
Sun J, P 4
Treves A, Or 11
Tsalenchuck Y, P 22
Tsvetkov P, Or 14
Walker MD, P 20
Weil Y, P 19
Weisinger G, P 23
Wiser O, P 6
Wong F Eng, P 21
Yang L-J, Or 17
Yaniv I, P 12
Yust-Katz S, P 24
Zeigerman H, P 25
Zeira E, Or 6, P 26
Zhu Q, P 21
Zigman-Hoffman E, P 27
Zilberman Y, P 19
Zundelevich A, P 13
Zwi L, P 28
Footnotes
*
The names of Presentors are underlined throughout.
*
Oral (Or) abstracts are found on pages 640–646; Poster (P) abstracts on pages 647–658.