Leonardo J. Solmesky

Serum free cultured bone marrow mesenchymal stem cells as a platform to characterize the effects of specific molecules.

Human mesenchymal stem cells (hMSC) are easily isolated from the bone marrow by adherence to plastic surfaces. These cells show self-renewal capacity and multipotency. A unique feature of hMSC is their capacity to survive without serum. Under this condition hMSC neither proliferate nor differentiate but maintain their biological properties unaffected. Therefore, this should be a perfect platform to study the biological effects of defined molecules on these human stem cells. We show that hMSC treated for five days with retinoic acid (RA) in the absence of serum undergo several transcriptional changes causing an inhibition of ERK related pathways. We found that RA induces the loss of hMSC properties such as differentiation potential to either osteoblasts or adipocytes. We also found that RA inhibits cell cycle progression in the presence of proliferating signals such as epidermal growth factor (EGF) combined with basic fibroblast growth factor (bFGF). In the same manner, RA showed to cause a reduction in cell adhesion and cell migration. In contrast to these results, the addition of EGF+bFGF to serum free cultures was enough to upregulate ERK activity and induce hMSC proliferation and cell migration. Furthermore, the addition of these factors to differentiation specific media instead of serum was enough to induce either osteogenesis or adipogenesis. Altogether, our results show that hMSCs ability to survive without serum enables the identification of signaling factors and pathways that are involved in their stem cell biological characteristics without possible serum interferences.

Structural profiling and biological performance of phospholipid-hyaluronan functionalized single-walled carbon nanotubes

In spite of significant insolubility and toxicity, carbon nanotubes (CNTs) erupt into the biomedical research, and create an increasing interest in the field of nanomedicine. Single-walled CNTs (SWCNTs) are highly hydrophobic and have been shown to be toxic while systemically administrated. Thus, SWCNTs have to be functionalized to render water solubility and biocompatibility. Herein, we introduce a method for functionalizing SWCNT using phospholipids (PL) conjugated to hyaluronan (HA), a hydrophilic glycosaminoglycan, with known receptors on many types of cancer and immune cells. This functionalization allowed for CNT solubilization, endowed the particles with stealth properties evading the immune system, and reduced immune and mitochondrial toxicity both in vitro and in vivo. The CNT-PL-HA internalized into macrophages and showed low cytotoxicity. In addition, CNT-PL-HA did not induce an inflammatory response in macrophages as evidenced by the cytokine profiling and the use of image-based high-content analysis approach in contrast to non-modified CNTs. In addition, systemic administration of CNT-PL-HA into healthy C57BL/6 mice did not alter the total number of leukocytes nor increased liver enzyme release as opposed to CNTs. Taken together, these results suggest an immune protective mechanism by the PL-HA coating that could provide future therapeutic benefit.

Assessing cellular toxicities in fibroblasts upon exposure to lipid-based nanoparticles: a high content analysis approach

Lipid-based nanoparticles (LNPs) are widely used for the delivery of drugs and nucleic acids. Although most of them are considered safe, there is confusing evidence in the literature regarding their potential cellular toxicities. Moreover, little is known about the recovery process cells undergo after a cytotoxic insult. We have previously studied the systemic effects of common LNPs with different surface charge (cationic, anionic, neutral) and revealed that positively charged LNPs ((+)LNPs) activate pro-inflammatory cytokines and induce interferon response by acting as an agonist of Toll-like receptor 4 on immune cells. In this study, we focused on the response of human fibroblasts exposed to LNPs and their cellular recovery process. To this end, we used image-based high content analysis (HCA). Using this strategy, we were able to show simultaneously, in several intracellular parameters, that fibroblasts can recover from the cytotoxic effects of (+)LNPs. The use of HCA opens new avenues in understanding cellular response and nanotoxicity and may become a valuable tool for screening safe materials for drug delivery and tissue engineering.

Bone morphogenetic protein signaling is involved in human mesenchymal stem cell survival in serum-free medium.

Bone marrow human mesenchymal stem cells (hMSCs) are known to survive in serum-free media, when most normal somatic cells do not survive. We found that the endogenously-activated bone morphogenetic protein (BMP) pathway is involved in this cellular behavior. Under this culture condition, phosphorylated Smad1 (PSmad1), the transducer of this signal, is localized in the hMSC nuclei. In addition, inhibition of this pathway with noggin, a BMP antagonist, elicits a caspase-dependent hMSCs death in a concentration-dependent manner. Furthermore, exogenously added BMP4 alleviates the noggin effect, restoring cell survival, and suggesting that BMP signal is essential for hMSC survival under serum deprivation conditions. Altogether these findings demonstrate for the first time an endogenous survival pathway of hMSCs driven by a BMP signal. Such a survival mechanism might be involved in the maintenance of the hMSC population within their bone marrow niche.

Characterization of human sporadic ALS biomarkers in the familial ALS transgenic mSOD1G93A mouse model

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder of motor neurons. Although most cases of ALS are sporadic (sALS) and of unknown etiology, there are also inherited familial ALS (fALS) cases that share a phenotype similar to sALS pathological and clinical phenotype. In this study, we have identified two new potential genetic ALS biomarkers in human bone marrow mesenchymal stem cells (hMSC) obtained from sALS patients, namely the TDP-43 (TAR DNA-binding protein 43) and SLPI (secretory leukocyte protease inhibitor). Together with the previously discovered ones-CyFIP2 and RbBP9, we investigated whether these four potential ALS biomarkers may be differentially expressed in tissues obtained from mutant SOD1(G93A) transgenic mice, a model that is relevant for at least 20% of the fALS cases. Quantitative real-time PCR analysis of brain, spinal cord and muscle tissues of the mSOD1(G93A) and controls at various time points during the progression of the neurological disease showed differential expression of the four identified biomarkers in correlation with (i) the tissue type, (ii) the stage of the disease and (iii) the gender of the animals, creating thus a novel spatiotemporal molecular signature of ALS. The biomarkers detected in the fALS animal model were homologous to those that were identified in hMSC of our sALS cases. These results support the possibility of a molecular link between sALS and fALS and may indicate common pathogenetic mechanisms involved in both types of ALS. Moreover, these results may pave the path for using the mSOD1(G93A) mouse model and these biomarkers as molecular beacons to evaluate the effects of novel drugs/treatments in ALS.

A novel image-based high-throughput screening assay discovers therapeutic candidates for adult polyglucosan body disease

Glycogen storage disorders (GSDs) are caused by excessive accumulation of glycogen. Some GSDs [adult polyglucosan (PG) body disease (APBD), and Tarui and Lafora diseases] are caused by intracellular accumulation of insoluble inclusions, called PG bodies (PBs), which are chiefly composed of malconstructed glycogen. We developed an APBD patient skin fibroblast cell-based assay for PB identification, where the bodies are identified as amylase-resistant periodic acid-Schiffs-stained structures, and quantified. We screened the DIVERSet CL 10 084 compound library using this assay in high-throughput format and discovered 11 dose-dependent and 8 non-dose-dependent PB-reducing hits. Approximately 70% of the hits appear to act through reducing glycogen synthase (GS) activity, which can elongate glycogen chains and presumably promote PB generation. Some of these GS inhibiting hits were also computationally predicted to be similar to drugs interacting with the GS activator protein phosphatase 1. Our work paves the way to discovering medications for the treatment of PB-involving GSD, which are extremely severe or fatal disorders.

Personalized Drug Discovery: HCA Approach Optimized for Rare Diseases at Tel Aviv University

The Cell screening facility for personalized medicine (CSFPM) at Tel Aviv University in Israel is devoted to screening small molecules libraries for finding new drugs for rare diseases using human cell based models. The main strategy of the facility is based on smartly reducing the size of the compounds collection in similarity clusters and at the same time keeping high diversity of pharmacophores. This strategy allows parallel screening of several patient derived - cells in a personalized screening approach. The tested compounds are repositioned drugs derived from collections of phase III and FDA approved small molecules. In addition, the facility carries screenings using other chemical libraries and toxicological characterizations of nanomaterials.

Guaiacol as a drug candidate for treating adult polyglucosan body disease

Adult polyglucosan body disease (APBD) is a late-onset disease caused by intracellular accumulation of polyglucosan bodies, formed due to glycogen-branching enzyme (GBE) deficiency. To find a treatment for APBD, we screened 1,700 FDA-approved compounds in fibroblasts derived from APBD-modeling GBE1-knockin mice. Capitalizing on fluorescent periodic acid-Schiff reagent, which interacts with polyglucosans in the cell, this screen discovered that the flavoring agent guaiacol can lower polyglucosans, a result also confirmed in APBD patient fibroblasts. Biochemical assays showed that guaiacol lowers basal and glucose 6-phosphate-stimulated glycogen synthase (GYS) activity. Guaiacol also increased inactivating GYS1 phosphorylation and phosphorylation of the master activator of catabolism, AMP-dependent protein kinase. Guaiacol treatment in the APBD mouse model rescued grip strength and shorter lifespan. These treatments had no adverse effects except making the mice slightly hyperglycemic, possibly due to the reduced liver glycogen levels. In addition, treatment corrected penile prolapse in aged GBE1-knockin mice. Guaiacols curative effects can be explained by its reduction of polyglucosans in peripheral nerve, liver, and heart, despite a short half-life of up to 60 minutes in most tissues. Our results form the basis to use guaiacol as a treatment and prepare for the clinical trials in APBD.

Preparation and epitope characterization of monoclonal antibodies suitable for detection of Tomato yellow leaf curl virus

Tomato yellow leaf curl virus (TYLCV) is a begomovirus that seriously threatens tomato crops worldwide. Current immunodiagnostic methods for this pathogen employ commercially produced mAbs raised against TYLCV. However, despite the existence of these mAbs, little information regarding their characterization or strategy of production has been published. In addition, research on TYLCV would certainly benefit were more mAbs available, thus allowing efficient examination of the virus life cycle, modes of pathogenesis and possibly the development of resistant cultivars. The coat protein (CP) of TYLCV is the only known building block of the viral capsid. Therefore, in this study we used CP as an immunogen for the production of novel mAbs. We employed a strategy in which the CP was truncated at its C-terminus to avoid intra- and inter-protein interactions that could impair epitope exposure. For the same reason, we used a denaturated antigen to expose linear epitopes during the immunization. This effort yielded three mAbs: they were characterized biochemically and immunologically, and their epitopes were mapped. Possible applications of these mAbs are discussed.