The Spectrum of Germline Nucleotide Variants in Gastric Cancer Patients in the Kyrgyz Republic.

Gastric cancer is a major challenge in modern oncology due to its high detection rate and prevalence. While sporadic cases make up the majority of gastric cancer, hereditary gastric cancer is caused by germline mutations in several genes linked to different syndromes. Thus, identifying hereditary forms of gastric cancer is considered crucial globally. A survey study using NGS-based analysis was conducted to determine the frequency of different types of hereditary gastric cancer in the yet-unstudied Kyrgyz population. The study cohort included 113 patients with diagnosed gastric cancer from Kyrgyzstan. The age of patients was 57.6 ± 8.9. Next-generation sequencing analysis of genomic DNA was performed using a custom Roche NimbleGen enrichment panel. The results showed that 6.2% (7/113) of the patients had pathogenic or likely pathogenic genetic variants. Additionally, 3.5% (4/113) of the patients carried heterozygous pathogenic/likely pathogenic variants in high penetrance genes, such as TP53, POLD1, RET, and BRCA2. Moreover, 2.7% (3/113) of the patients carried heterozygous mutations in genes linked to autosomal recessive conditions, specifically PALB2, FANCA, and FANCD2. We have not identified any genetic variants in hereditary GC-associated genes: CDH1, STK11, SMAD4, BMPRIA, APC, MLH1, and others. Our study included patients with sporadic features of GC. The use of recognized criteria (NCCN, Gastric Cancer, Version 2.2022) would increase the number of identified genetic variants in hereditary GC-associated genes. Further research is required to determine the clinical relevance of the genetic variants identified in the current study.

Proangiogenic Effect of 2A-Peptide Based Multicistronic Recombinant Constructs Encoding VEGF and FGF2 Growth Factors.

Coronary artery disease remains one of the primary healthcare problems due to the high cost of treatment, increased number of patients, poor clinical outcomes, and lack of effective therapy. Though pharmacological and surgical treatments positively affect symptoms and arrest the disease progression, they generally exhibit a limited effect on the disease outcome. The development of alternative therapeutic approaches towards ischemic disease treatment, especially of decompensated forms, is therefore relevant. Therapeutic angiogenesis, stimulated by various cytokines, chemokines, and growth factors, provides the possibility of restoring functional blood flow in ischemic tissues, thereby ensuring the regeneration of the damaged area. In the current study, based on the clinically approved plasmid vector pVax1, multigenic constructs were developed encoding vascular endothelial growth factor (VEGF), fibroblast growth factors (FGF2), and the DsRed fluorescent protein, integrated via picornaviruses' furin-2A peptide sequences. In vitro experiments demonstrated that genetically modified cells with engineered plasmid constructs expressed the target proteins. Overexpression of VEGF and FGF2 resulted in increased levels of the recombinant proteins. Concomitantly, these did not lead to a significant shift in the general secretory profile of modified HEK293T cells. Simultaneously, the secretome of genetically modified cells showed significant stimulating effects on the formation of capillary-like structures by HUVEC (endothelial cells) in vitro. Our results revealed that when the multicistronic multigene vectors encoding 2A peptide sequences are created, transient transgene co-expression is ensured. The results obtained indicated the mutual synergistic effects of the growth factors VEGF and FGF2 on the proliferation of endothelial cells in vitro. Thus, recombinant multicistronic multigenic constructs might serve as a promising approach for establishing safe and effective systems to treat ischemic diseases.

Psychological and Cognitive Profile of Hypertensive and Diabetic Patients.

Chronic disorders such as hypertension and diabetes mellitus are often associated with depressive and anxiety symptoms, as well as cognitive decline. Once developed, psychological support is essential for improving the quality of life. This study is aimed at identifying impaired mental health in connection with these systemic metabolic disorders. A total of 34 patients were included in this cross-sectional study: 17 hypertensive individuals with a mean age of 59 ± 10 years, and 17 diabetic patients aged 54 ± 10 years. The following psychometric tests were used: Mini-Mental State Examination (MMSE), Beck Depression Inventory (BDI), Beck Anxiety Inventory (BAI), and self-reporting questionnaire (SRQ-20). A large number of patients with high blood pressure or diabetes was associated with mental health problems (82% or 65%, respectively; p = 0.246). Affective disorder, especially moderate to severe depression, was seen mainly in diabetic patients (76%), whereas hypertensive individuals had higher prevalence of anxiety (64%). There was no cognitive impairment in this middle-aged population. This study shows a high proportion of depression and anxiety symptoms in patients with hypertension or diabetes mellitus, reinforcing the importance of psychiatric support for appropriate control of these metabolic disorders.

Non-classical animal models for studying adrenal diseases: advantages, limitations, and implications for research.

The study of adrenal disorders is a key component of scientific research, driven by the complex innervation, unique structure, and essential functions of the adrenal glands. This review explores the use of non-traditional animal models for studying congenital adrenal hyperplasia. It highlights the advantages, limitations, and relevance of these models, including domestic ferrets, dogs, guinea pigs, golden hamsters, pigs, and spiny mice. We provide a detailed analysis of the histological structure, steroidogenesis pathways, and genetic characteristics of these animal models. The morphological and functional similarities between the adrenal glands of spiny mice and humans highlight their potential as an important avenue for future research.

Peripheral blood mono-nuclear cells derived from Alzheimer's disease patients show elevated baseline levels of secreted cytokines but resist stimulation with ß-amyloid peptide.

OBJECTIVES: Among several other factors, the neuro-toxic ß-amyloid peptide (ßAP)-induced inflammatory mechanisms have also been implicated in the pathogenesis of Alzheimer's dementia (AD). Cytokines have recently emerged as prime candidates underlying this immune reaction. The purpose of this study was to evaluate the inflammatory response of peripheral blood mono-nuclear cells (PBMC) in AD. DESIGN: Cross-sectional (observational) study. SETTING: Behavioral and cognitive neurology clinic of the Universidade Federal de Minas Gerais in Belo Horizonte, Brazil. PARTICIPANTS: AD patients (n=19), healthy elderly (n=19) and young (n=14) individuals. MEASUREMENTS: Cytokine levels were assessed by enzyme-linked immuno-sorbent assay (ELISA) after exposing cells to a broad range of ßAP concentrations (10(-4)-10(-10)M) as a stimulus. AD samples were weighed against leukocytes harvested from non-demented young and elderly subjects. RESULTS: Cytokine production of PBMCs in the youth was characterized by low baseline levels when compared to cells from the older generation. In the aging population, AD cells were distinguished from the healthy elderly sub-group by an even higher basal cytokine secretion. The low resting concentration in young individuals was markedly increased after treatment with ßAP, however cells from the elderly, irrespective of their disease status, showed unchanged cytokine release following ßAP administration. Non-specific activation of PBMCs with anti-CD3/CD28 antibodies resulted in elevated interleukin (IL)-1ß concentrations in AD. CONCLUSIONS: These results demonstrate a general over-production of cytokines and resistance to ßAP in the old comparison group, with a more pronounced disruption/boosted pattern in AD. Our findings are in line with the hypothesis of "inflammaging", i.e. an enhanced inflammatory profile with normal aging and a further perturbed environment in AD. The observed cytokine profiles may serve as diagnostic biomarkers in dementia.

A direct technique for magnetic functionalization of living human cells.

Functionalized living cells are regarded as effective tools in directed cell delivery and tissue engineering. Here we report the facile functionalization of viable isolated HeLa cells with superparamagnetic cationic nanoparticles via a single-step biocompatible process. Nanoparticles are localized on the cellular membranes and do not penetrate into the cytoplasm. The magnetically responsive cells are viable and able to colonize and grow on substrates. Magnetically facilitated microorganization of functionalized cells into viable living clusters is demonstrated. We believe that the technique described here may find a number of potential applications in cell-based therapies and in development of whole-cell biosensors.

Quantitative changes in perineuronal nets in development and posttraumatic condition.

Perineuronal net (PNN) is a highly structured portion of the CNS extracellular matrix (ECM) regulating synaptic plasticity and a range of pathologic conditions including posttraumatic regeneration and epilepsy. Here we studied Wisteria floribunda agglutinin-stained histological sections to quantify the PNN size and enrichment of chondroitin sulfates in mouse brain and spinal cord. Somatosensory cortex sections were examined during the period of PNN establishment at postnatal days 14, 21 and 28. The single cell PNN size and the chondroitin sulfate intensity were quantified for all cortex layers and specifically for the cortical layer IV which has the highest density of PNN-positive neurons. We demonstrate that the chondroitin sulfate proteoglycan staining intensity is increased between P14 and P28 while the PNN size remains unchanged. We then addressed posttraumatic changes of the PNN expression in laminae 6 and 7 of cervical spinal cord following hemisection injury. We demonstrate increase of the chondroitin sulfate content at 1.6-1.8 mm rostrally from the injury site and increase of the density of PNN-bearing cells at 0.4-1.2 mm caudally from the injury site. We further demonstrate decrease of the single cell PNN area at 0.2 mm caudally from the injury site suggesting that the PNN ECM takes part in the posttraumatic tissue rearrangement in the spinal cord. Our results demonstrate new insights on the PNN structure dynamics in the developing and posttraumatic CNS.

Caroli syndrome: a clinical case with detailed histopathological analysis.

Herein we present a clinical case of the Caroli syndrome caused by the compound heterozygous mutation in the PKHD1 gene. Histopathological assessment of liver detected biliary cirrhosis, numerous dilated bile ducts of various sizes, hyperplastic cholangiocytes containing a large amount of acid mucopolysaccharides, decreased ß-tubulin expression and increased proliferation of cholangiocytes. A significant proportion of hepatic tissue was composed of giant cysts lined with a single layer of cholangiocytes, containing pus and bile in its lumen and surrounded by granulation tissue. An accumulation of neutrophils in the lumen of the bile ducts was observed, as well as an infiltration of the ducts and cysts surrounding connective tissue by CD4+ and to a lesser extent CD8+ lymphocytes. This may be caused by the expression of HLA-DR by cholangiocytes. Atrophy and desquamation of the epithelium of collecting tubules with the formation of microcysts were detected in the kidneys without a clinically significant loss of renal function. Morphopathogenetic mechanisms of the Caroli syndrome can be targets for a potential pathogenetic therapy and prevention of its manifestations and complications.

Adipose-Derived Mesenchymal Stem Cells Applied in Fibrin Glue Stimulate Peripheral Nerve Regeneration.

Mesenchymal stem cells (MSCs) hold a great promise for cell therapy. To date, they represent one of the best choices for the treatment of post-traumatic injuries of the peripheral nervous system. Although autologous can be easily transplanted in the injured area, clinical advances in this filed have been impaired by lack of preservation of graft cells into the injury area after transplantation. Indeed, cell viability is not retained after injection into the blood stream, and cells injected directly into the area of injury either are washed off or inhibit regeneration through scar formation and neuroma development. This study proposes a new way of MSCs delivery to the area of traumatic injury by using fibrin glue, which not only fixes cells at the site of application but also provides extracellular matrix support. Using a sciatic nerve injury model, MSC derived from adipose tissue embedded in fibrin glue were able to enter the nerve and migrate mainly retrogradely after transplantation. They also demonstrated a neuroprotective effect on DRG L5 sensory neurons and stimulated axon growth and myelination. Post-traumatic changes of the sensory neuron phenotype were also improved. Importantly, MSCs stimulated nerve angiogenesis and motor function recovery. Therefore, our data suggest that MSC therapy using fibrin glue is a safe and efficient method of cell transplantation in cases of sciatic nerve injury, and that this method of delivery of regeneration stimulants could be beneficial for the successful treatment of other central and peripheral nervous system conditions.

Human umbilical cord blood cells transfected with VEGF and L(1)CAM 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.

Gene Therapy Using Plasmid DNA Encoding VEGF164 and FGF2 Genes: A Novel Treatment of Naturally Occurring Tendinitis and Desmitis in Horses.

This clinical study describes the intralesional application of the plasmid DNA encoding two therapeutic species-specific growth factors: vascular endothelial growth factor (VEGF164) and fibroblast growth factor 2 (FGF2) in seven horses to restore naturally occurring injuries of the superficial digital flexor tendon (SDFT) (tendinitis) and in three horses with suspensory ligament branch desmitis. Following application all horses were able to commence a more rapid exercise program in comparison to standardized exercise programs. Clinical observation and ultrasonic imaging was used to evaluate the regeneration rate of the tendon and ligament injury recovery and to confirm the safety of this gene therapy in horses, throughout a 12 month period. Follow-up data of the horses revealed a positive outcome including significant ultrasonographic and clinical improvements in 8 out of 10 horses with SDFT and suspensory ligament branch lesions, with return to their pre-injury level of performance by 2-6 months after the completion of treatment. The ninth horse initially presenting with severe suspensory ligament branch desmopathy, showed no significant ultrasonographic improvements in the first 2 months after treatment, however, it improved clinically and became less lame. The final horse, presenting with severe tendinitis of the SDFT returned to their pre-injury level of performance, but experienced re-injury 6 months after treatment. This data is highly promising, however, further research in experimental models, with the histopathological, immunohistochemical and gene expression evaluation of the equine tendon/ligament after gene therapy application is required in order to fully understand the mechanisms of action. This treatment and the significant clinical impacts observed represents an important advancement in the field of medicine.

Gene Therapy Using Plasmid DNA Encoding Vascular Endothelial Growth Factor 164 and Fibroblast Growth Factor 2 Genes for the Treatment of Horse Tendinitis and Desmitis: Case Reports.

In this clinical study, for the first time we used the direct gene therapy to restore severe injuries of the suspensory ligament branch and superficial digital flexor tendon in horses (Equus caballus). We injected the plasmid DNA encoding two therapeutic species-specific growth factors: vascular endothelial growth factor 164 and fibroblast growth factor 2 at the site of injury in the suspensory ligament branch and tendon. Treatment effects were evaluated with the use of clinical observation and ultrasound imaging during a period of a few months. We showed that gene therapy used within a period of 2-3 months after the injury resulted in the complete recovery of functions and full restoration of the severely damaged suspensory ligament and superficial digital flexor tendon.

Cytochalasin B-induced membrane vesicles convey angiogenic activity of parental cells.

Naturally occurring extracellular vesicles (EVs) play essential roles in intracellular communication and delivery of bioactive molecules. Therefore it has been suggested that EVs could be used for delivery of therapeutics. However, to date the therapeutic application of EVs has been limited by number of factors, including limited yield and full understanding of their biological activities. To address these issues, we analyzed the morphology, molecular composition, fusion capacity and biological activity of Cytochalasin B-induced membrane vesicles (CIMVs). The size of these vesicles was comparable to that of naturally occurring EVs. In addition, we have shown that CIMVs from human SH-SY5Y cells contain elevated levels of VEGF as compared to the parental cells, and stimulate angiogenesis in vitro and in vivo.

Spatial patterns and cell surface clusters in perineuronal nets.

Perineuronal nets (PNN) ensheath GABAergic and glutamatergic synapses on neuronal cell surface in the central nervous system (CNS), have neuroprotective effect in animal models of Alzheimer disease and regulate synaptic plasticity during development and regeneration. Crucial insights were obtained recently concerning molecular composition and physiological importance of PNN but the microstructure of the network remains largely unstudied. Here we used histochemistry, fluorescent microscopy and quantitative image analysis to study the PNN structure in adult mouse and rat neurons from layers IV and VI of the somatosensory cortex. Vast majority of meshes have quadrangle, pentagon or hexagon shape with mean mesh area of 1.29µm(2) in mouse and 1.44µm(2) in rat neurons. We demonstrate two distinct patterns of chondroitin sulfate distribution within a single mesh - with uniform (nonpolar) and node-enriched (polar) distribution of the Wisteria floribunda agglutinin-positive signal. Vertices of the node-enriched pattern match better with local maxima of chondroitin sulfate density as compared to the uniform pattern. PNN is organized into clusters of meshes with distinct morphologies on the neuronal cell surface. Our findings suggest the role for the PNN microstructure in the synaptic transduction and plasticity.

Human adipose-derived stem cells stimulate neuroregeneration.

Traumatic brain injuries and degenerative neurological disorders such as Alzheimer's dementia, Parkinson's disease, amyotrophic lateral sclerosis and many others are characterized by loss of brain cells and supporting structures. Restoring microanatomy and function using stem cells is a promising therapeutic approach. Among the many various sources, adipose-derived stem cells (ADSCs) are one of the most easily harvested alternatives, they multiply rapidly, and they demonstrate low immunogenicity with an ability to differentiate into several cell types. The objective of this study was to evaluate the effect of xenotransplanted human ADSCs on post-traumatic regeneration of rat sciatic nerve. Peripheral reconstruction following complete sciatic transection and autonerve grafting was complemented by intra-operative injection of hADSCs into the proximal and distal stumps. The injury caused gliosis and apoptosis of sensory neurons in the lumbar 5 (L5) ganglia in the control rodents; however, animals treated with hADSCs demonstrated a smaller amount of cellular loss. Formation of amputation neuroma, which hinders axonal repair, was less prominent in the experimental group, and immunohistochemical analysis of myelin basic protein showed good myelination 65 days after surgery. At this point, control groups still exhibited high levels of microglia/macrophage-specific marker Iba-1 and proliferating cell nuclear antigen, the mark of an ongoing inflammation and incomplete axonal growth 2 months after the injury. This report demonstrates that hADSCs promote neuronal survival in the spinal ganglion, fuel axonal repair and stimulate the regeneration of peripheral nerves.

Transcriptional Analysis of Blood Lymphocytes and Skin Fibroblasts, Keratinocytes, and Endothelial Cells as a Potential Biomarker for Alzheimer's Disease.

Alzheimer's disease (AD) is a devastating and progressive form of dementia that is typically associated with a build-up of amyloid-ß plaques and hyperphosphorylated and misfolded tau protein in the brain. Presently, there is no single test that confirms AD; therefore, a definitive diagnosis is only made after a comprehensive medical evaluation, which includes medical history, cognitive tests, and a neurological examination and/or brain imaging. Additionally, the protracted prodromal phase of the disease makes selection of control subjects for clinical trials challenging. In this study we have utilized a gene-expression array to screen blood and skin punch biopsy (fibroblasts, keratinocytes, and endothelial cells) for transcriptional differences that may lead to a greater understanding of AD as well as identify potential biomarkers. Our analysis identified 129 differentially expressed genes from blood of dementia cases when compared to healthy individuals, and four differentially expressed punch biopsy genes between AD subjects and controls. Additionally, we identified a set of genes in both tissue compartments that showed transcriptional variation in AD but were largely stable in controls. The translational products of these variable genes are involved in the maintenance of the Golgi structure, regulation of lipid metabolism, DNA repair, and chromatin remodeling. Our analysis potentially identifies specific genes in both tissue compartments that may ultimately lead to useful biomarkers and may provide new insight into the pathophysiology of AD.

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.

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-HT(2C) in natural killer cells in Alzheimer's dementia.

Alzheimer's 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 ß-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.

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 (VEGF(165)) and human fibroblast growth factor 2 (FGF(2)) (pBud-VEGF-FGF(2)). These genetically modified hUCBCs were injected retro-orbitally into presymptomatic ALS transgenic animal models ((G)93(A) mice). Lumbar spinal cords of rodents were processed for immunofluoresent staining with antibodies against human nuclear antigen (HNA), oligodendrocyte-specific protein, S100, iba1, neuronal ß(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-FGF(2). Double staining in control animals treated with unmodified hUCBCs, however, revealed HNA+ cells expressing iba1 and CD34. Neuron-specific ß(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-FGF(2), hUCBCs transform into astrocytes (S100+). Autocrine regulation of VEGF and FGF(2) 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.