Andrey P. Kiyasov

Human umbilical cord blood cells transfected with VEGF and L1CAM do not differentiate into neurons but transform into vascular endothelial cells and secrete neuro-trophic factors to support neuro-genesis—a novel approach in stem cell therapy

Genetically modified mono-nuclear cell fraction from human umbilical cord blood (HUCB) expressing human vascular endothelial growth factor (VEGF) and mouse neural L(1) cell adhesion molecule (L(1)CAM) were used for gene-stem cell therapy of transgenic (G)93(A) mice adopted as an animal amyotrophic lateral sclerosis (ALS) model. We generated non-viral plasmid constructs, expressing human VEGF(165) (pcDNA-VEGF) and mouse neural L(1) cell adhesion molecule (pcDNA-mL(1)CAM). Mono-nuclear fraction of HUCB cells were transiently transfected by electro-poration with a mixture of expression plasmids (pcDNA-VEGF+pcDNA-mL(1)CAM). Sixteen transgenic female and male mice were randomly assigned to three groups: (1) transplantation of genetically modified HUCB cells expressing L(1) and VEGF (n=6), (2) transplantation of un-transfected HUCB cells (n=5), and (3) control group (n=5). In first two experimental groups 1x10(6) cells were injected retro-orbitally in pre-symptomatic 22-25-week-old (G)93(A) mice. Our results demonstrate that HUCB cells successfully grafted into nervous tissue of ALS mice and survived for over 3 months. Therefore, genetically modified HUCB cells migrate in the spinal cord parenchyma, proliferate, but instead of transforming into nerve cells, they differentiate into endothelial cells forming new blood vessels. We propose that: (A) expression of mouse neural L(1)CAM is responsible for increased homing and subsequent proliferation of transplanted cells at the site of neuro-degeneration, (B) expression of human VEGF directs HUCB cell differentiation into endothelial cells, and (C) neuro-protective effect may stem from the delivery of various neuro-trophic factors from newly formed blood vessels.

Coronary artery bypass surgery provokes alzheimer's disease-like changes in the cerebrospinal fluid

Several biomarkers are used in confirming the diagnosis of cognitive disorders. This study evaluates whether the level of these markers after heart surgery correlates with the development of cognitive dysfunction, which is a frequent complication of cardiac interventions. Concentrations of amyloid-β peptide, tau, and S100β in the cerebro-spinal fluid were assessed, as well as cognitive functions were evaluated before and after coronary artery bypass grafting, utilizing immuno-assays and psychometric tests, respectively. A drastic rise in the level of S100β was observed one week after the surgery, a mark of a severe generalized cerebral injury. The level of amyloid-β peptide significantly decreased, whereas the concentration of tau markedly increased six months postoperatively. Gradual cognitive decline was also present. These findings clearly demonstrate post-surgical cognitive impairment associated with changes in biomarkers similar to that seen in Alzheimers disease, suggesting a unifying pathognomic factor between the two disorders. A holistic approach to coronary heart disease and Alzheimers type dementia is proposed.

Genetically modified human umbilical cord blood cells expressing vascular endothelial growth factor and fibroblast growth factor 2 differentiate into glial cells after transplantation into amyotrophic lateral sclerosis transgenic mice

Current therapy of a number of neuropsychiatric maladies has only symptomatic modality. Effective treatment of these neuro-degenerative diseases, including amyotrophic lateral sclerosis (ALS), may benefit from combined gene/stem-cell approaches. In this report, mononuclear fraction of human umbilical cord blood cells (hUCBCs) were transfected by electroporation with dual plasmid constructs, simultaneously expressing vascular endothelial growth factor 165 (VEGF165) and human fibroblast growth factor 2 (FGF2) (pBud-VEGF-FGF2). These genetically modified hUCBCs were injected retro-orbitally into presymptomatic ALS transgenic animal models (G93A mice). Lumbar spinal cords of rodents were processed for immunofluoresent staining with antibodies against human nuclear antigen (HNA), oligodendrocyte-specific protein, S100, iba1, neuronal β 3-tubulin and CD34. Co-localization of HNA and S100 was found in the spinal cord of mice after transplantation of genetically modified hUCBCs over-expressing VEGF-FGF2. Double staining in control animals treated with unmodified hUCBCs, however, revealed HNA+ cells expressing iba1 and CD34. Neuron-specific β 3-tubulin or oligodendrocyte-specific protein were not expressed in hUCBCs in either control or experimental mice. These results demonstrate that genetically naïve hUCBCs may differentiate into endothelial (CD34+) and microglial (iba1+) cells; however when over-expressing VEGF-FGF2, hUCBCs transform into astrocytes (S100+). Autocrine regulation of VEGF and FGF2 on hUCBCs, signal molecules from dying motor neurons in spinal cord, as well as self-differentiating potential may provide a unique microenvironment for the transformation of hUCBCs into astrocytes that eventually serve as a source of growth factors to enhance the survive potential of surrounding cells in the diseased regions.

Human Tooth Germ Stem Cells Preserve Neuro-Protective Effects after Long-Term Cryo-Preservation

The use of mesenchymal stem cells (MSCs) has been shown to be promising in chronic disorders such as diabetes, Alzheimers dementia, Parkinsons disease, spinal cord injury and brain ischemia. Recent studies revealed that human tooth germs (hTG) contain MSCs which can be easily isolated, expanded and cryo-preserved. In this report, we isolated human tooth germ stem cells (hTGSCs) with MSC characteristics from third molar tooth germs, cryo-preserved them at -80( degrees )C for 6 months, and evaluated for their surface antigens, expression of pluri-potency associated genes, differentiation capacity, karyotype, and proliferation rate. These characteristics were compared to their non-frozen counterparts. In addition, neuro-protective effects of cryo-preserved cells on neuro-blastoma SH-SY5Y cells were also assessed after exposure to stress conditions induced by hydrogen-peroxide (oxidative stress) and paclitaxel (microtubule stabilizing mitotic inhibitor). After long term cryo-preservation hTGSCs expressed surface antigens CD29, CD73, CD90, CD105, and CD166, but not CD34, CD45 or CD133, which was typical for non-frozen hTGSCs. Cryo-preserved hTGSCs were able to differentiate into osteo-, adipo- and neuro-genic cells. They also showed normal karyotype after high number of population doublings and unchanged proliferation rate. On the other hand, cryo-preserved cells demonstrated a tendency for lower level of pluri-potency associated gene expression (nanog, oct4, sox2, klf4, c-myc) than non-frozen hTGSCs. hTGSCs conditioned media increased survival of SH-SY5Y cells exposed to oxidative stress or paclitaxel. These findings confirm that hTGSCs preserve their major characteristics and exert neuro-protection after long-term cryo-preservation, suggesting that hTGSCs, harvested from young individuals and stored for possible use later as they grow old, might be employed in cellular therapy of age-related degenerative disorders.

Symptomatic Improvement, Increased Life-Span and Sustained Cell Homing in Amyotrophic Lateral Sclerosis After Transplantation of Human Umbilical Cord Blood Cells Genetically Modified with Adeno-Viral Vectors Expressing a Neuro-Protective Factor and a Neural Cell Adhesion Molecule

Amyotrophic lateral sclerosis (ALS) is an incurable, chronic, fatal neuro-degenerative disease characterized by progressive loss of moto-neurons and paralysis of skeletal muscles. Reactivating dysfunctional areas is under earnest investigation utilizing various approaches. Here we present an innovative gene-cell construct aimed at reviving inert structure and function. Human umbilical cord blood cells (hUCBCs) transduced with adeno-viral vectors encoding human VEGF, GDNF and/or NCAM genes were transplanted into transgenic ALS mice models. Significant improvement in behavioral performance (open-field and grip-strength tests), as well as increased life-span was observed in rodents treated with NCAM-VEGF or NCAM-GDNF co-transfected cells. Active trans-gene expression was found in the spinal cord of ALS mice 10 weeks after delivering genetically modified hUCBCs, and cells were detectable even 5 months following transplantation. Our gene-cell therapy model yielded prominent symptomatic control and prolonged life-time in ALS. Incredible survivability of xeno-transpanted cells was also observed without any immune-suppression. These results suggest that engineered hUCBCs may offer effective gene-cell therapy in ALS.

Disease-specific expression of the serotonin-receptor 5-HT2C in natural killer cells in Alzheimer's dementia

Alzheimers dementia (AD) is a degenerative brain disorder characterized mainly by cholinergic failure, but other neuro-transmitters are also deficient especially at late stages of the disease. Misfolded β-amyloid peptide has been identified as a causative agent, however inflammatory changes also play a pivotal role. Even though the most prominent pathology is seen in the cognitive functions, specific abnormalities of the central nervous system (CNS) are also reflected in the periphery, particularly in the immune responses of the body. The aim of this study was to characterize the dopaminergic and serotonergic systems in AD, which are also markedly disrupted along with the hallmark acetyl-choline dysfunction. Peripheral blood mono-nuclear cells (PBMCs) from demented patients were judged against comparison groups including individuals with late-onset depression (LOD), as well as non-demented and non-depressed subjects. Cellular sub-populations were evaluated by mono-clonal antibodies against various cell surface receptors: CD4/CD8 (T-lymphocytes), CD19 (B-lymphocytes), CD14 (monocytes), and CD56 (natural-killer (NK)-cells). The expressions of dopamine D(3) and D(4), as well as serotonin 5-HT(1A), 5-HT(2A), 5-HT(2B) and 5-HT(2C) were also assessed. There were no significant differences among the study groups with respect to the frequency of the cellular sub-types, however a unique profound increase in 5-HT(2C) receptor exclusively in NK-cells was observed in AD. The disease-specific expression of 5-HT(2C), as well as the NK-cell cyto-toxicity, has been linked with cognitive derangement in dementia. These changes not only corroborate the existence of bi-directional communication between the immune system and the CNS, but also elucidate the role of inflammatory activity in AD pathology, and may serve as potential biomarkers for less invasive and early diagnostic purposes as well.

Interaction and self-organization of human mesenchymal stem cells and neuro-blastoma SH-SY5Y cells under co-culture conditions: A novel system for modeling cancer cell micro-environment

The common drawback of many in vitro cell culture systems is the absence of appropriate micro-environment, which is formed by the combination of factors such as cell-cell contacts, extracellular matrix and paracrine regulation. Micro-environmental factors in a tumor tissue can influence physiological status of the cancer cells and their susceptibility to anticancer therapies. Interaction of cancer cells with their micro-environment and regional stem cells, therefore, is of particular interest. Development of in vitro systems which allow more accurate modeling of complex relations occurring in real tumor environments can increase efficiency of preclinical assays for screening anticancer drugs. The aim of this work was to study interactions between human mesenchymal stem cells (MSCs) and neuro-blastoma cancer SH-SY5Y cells under co-culture conditions on different coated surfaces to determine the effect of co-existence of cancer and stem cells on each cellular population under various stress conditions. We developed an efficient in vitro system for studying individual cancer and stem cell populations during co-culture using differential live fluorescent membrane labeling, and demonstrated self-organization of cancer and stem cells during co-culture on various coated surfaces. Our findings support the evidence that cancer and stem cell interactions play important roles in cellular behavior of cancer cells. These properties can be used in different fields of cancer research, tissue engineering and biotechnology.

Improved cognitive, affective and anxiety measures in patients with chronic systemic disorders following structured physical activity

Mental illnesses are frequent co-morbid conditions in chronic systemic diseases. High incidences of depression, anxiety and cognitive impairment complicate cardiovascular and metabolic disorders such as hypertension and diabetes mellitus. Lifestyle changes including regular exercise have been advocated to reduce blood pressure and improve glycaemic control. The purpose of this project was to evaluate the effect of physical training on the most prevalent corollary psychiatric problems in patients with chronic organic ailments. This longitudinal study assessed the mental health of hypertensive (age: 57 ± 8 years) and/or diabetic (age: 53 ± 8 years) patients using mini-mental state examination, Beck’s depression inventory, Beck’s anxiety inventory and self-reporting questionnaire-20 before and after a 3-month supervised resistance and aerobic exercise programme comprising structured physical activity three times a week. Clinically relevant improvement was observed in the Beck’s depression inventory and Beck’s anxiety inventory scores following the 12-week training (61%, p = 0.001, and 53%, p = 0.02, respectively). Even though statistically not significant (p = 0.398), the cognitive performance of this relatively young patient population also benefited from the programme. These results demonstrate positive effects of active lifestyle on non-psychotic mental disorders in patients with chronic systemic diseases, recommending exercise as an alternative treatment option.

Evaluation of Post-Surgical Cognitive Function and Protein Fingerprints in the Cerebro-Spinal Fluid Utilizing Surface-Enhanced Laser Desorption/Ionization Time-of-Flight Mass-Spectrometry (SELDI-TOF MS) After Coronary Artery Bypass Grafting: Review of Proteomic Analytic Tools and Introducing a New Syndrome

Cognitive dysfunction following surgery is a common complication, which increases the incidence of other co-morbid conditions, hospital and health-care costs. The reported rate of the occurrence of post-operative cognitive decline varies with different studies, depending on population profile, type of surgery, definition of cognitive disorder and detection methods, design of study, etc. It remains unclear whether these psychiatric signs and symptoms are direct results of the effects of surgery or general anesthesia. Nonetheless they are more frequent after cardiac surgery and are likely to be multi-factorial, but the patho-mechanisms are not yet fully characterized. This communication provides a synopsis of proteomics tools and delineates novel SELDI-TOF results to evaluate biomarkers in this regard. Presented for the first time is a classification of the clinically relevant forms of post-operative cognitive decline with the advent of a novel subclass.

Changes in the Count of Pancreatic b- and a-Cells and Blood Glucose Level in Rats with Alloxan-Induced Diabetes

We estimated the count of pancreatic alpha- and beta-cells and blood glucose level at various stages of alloxan-induced diabetes in rats. Alloxan decreased the count of insulin-producing beta-cells, but increased the number of glucagon-secreting alpha-cells in the pancreas (week 1 of diabetes). These changes were accompanied by hyperglycemia. The decrease in blood glucose level in diabetic rats was associated with an increase in beta-cell count against the background of high density of pancreatic alpha-cells.