Optogenetic and chemogenetic approaches for modeling neurological disorders in vivo.

Animal models of human neurological disorders provide valuable experimental tools which enable us to study various aspects of disorder pathogeneses, ranging from structural abnormalities and disrupted metabolism and signaling to motor and mental deficits, and allow us to test novel therapies in preclinical studies. To be valid, these animal models should recapitulate complex pathological features at the molecular, cellular, tissue, and behavioral levels as closely as possible to those observed in human subjects. Pathological states resembling known human neurological disorders can be induced in animal species by toxins, genetic factors, lesioning, or exposure to extreme conditions. In recent years, novel animal models recapitulating neuropathologies in humans have been introduced. These animal models are based on synthetic biology approaches: opto- and chemogenetics. In this paper, we review recent opto- and chemogenetics-based animal models of human neurological disorders. These models allow for the creation of pathological states by disrupting specific processes at the cellular level. The artificial pathological states mimic a range of human neurological disorders, such as aging-related dementia, Alzheimer's and Parkinson's diseases, amyotrophic lateral sclerosis, epilepsy, and ataxias. Opto- and chemogenetics provide new opportunities unavailable with other animal models of human neurological disorders. These techniques enable researchers to induce neuropathological states varying in severity and ranging from acute to chronic. We also discuss future directions for the development and application of synthetic biology approaches for modeling neurological disorders.

Hydrogen peroxide is not generated intracellularly in human neural spheroids during ischemia-reperfusion.

Reactive oxygen species (ROS) are considered a primary source of damage during ischemic stroke. However, the precise timing of ROS production (during hypoxia or reperfusion) remains unclear. Cellular 3D spheroids are often proposed as an optimal alternative to both 2D cell cultures and animal models in modeling disease conditions. Here we report live imaging of hydrogen peroxide dynamics during the acute phase of hypoxia and reperfusion in human iPSC-derived neural spheroids, stably expressing fluorescent biosensor HyPer7. Contrary to previous reports, we did not observe a hydrogen peroxide production burst neither during hypoxia nor in course of reperfusion. Our data suggest either lack of oxidative stress during ischemia-reperfusion in spheroids or existence of different mechanisms of oxidative damage.

Soil organic carbon stock retrieval from Sentinel-2A using a hybrid approach.

Digital soil maps find application in numerous fields, making their accuracy a crucial factor. Mapping soil properties in homogeneous landscapes where the soil surface is concealed, as in forests, presents a complex challenge. In this study, we evaluated the spatial distribution of soil organic carbon stocks (SOCstock) under forest vegetation using three methods: regression kriging (RK), random forest (RF), and RF combined with ordinary kriging of residuals (RFOK) in combination with Sentinel-2A satellite data. We also compared their accuracies and identified key influencing factors. We determined that SOCstock ranged from 0.6 to 10.9 kg/m2 with an average value of 4.9 kg/m2. Among the modelling approaches, we found that the RFOK exhibited the highest accuracy (RMSE = 1.58 kg/m2, NSE = 0.33), while the RK demonstrated a lack of spatial correlation of residuals, rendering this method inapplicable. An analysis of variable importance revealed that the SWIR B12 band of the Sentinel-2A satellite contributed the most to RFOK predictions. We concluded that the RFOK hybrid approach outperformed the others, potentially serving as a foundation for digital soil mapping under similar environmental conditions. Therefore, it is essential to consider spatial correlations when mapping soil properties in ecosystems that are inaccessible for capturing the spectral response of the soil surface.

Metabolic Fate of Human Immunoactive Sterols in Mycobacterium tuberculosis.

Mycobacterium tuberculosis (Mtb) infection is among top ten causes of death worldwide, and the number of drug-resistant strains is increasing. The direct interception of human immune signaling molecules by Mtb remains elusive, limiting drug discovery. Oxysterols and secosteroids regulate both innate and adaptive immune responses. Here we report a functional, structural, and bioinformatics study of Mtb enzymes initiating cholesterol catabolism and demonstrated their interrelation with human immunity. We show that these enzymes metabolize human immune oxysterol messengers. Rv2266 - the most potent among them - can also metabolize vitamin D3 (VD3) derivatives. High-resolution structures show common patterns of sterols binding and reveal a site for oxidative attack during catalysis. Finally, we designed a compound that binds and inhibits three studied proteins. The compound shows activity against Mtb H37Rv residing in macrophages. Our findings contribute to molecular understanding of suppression of immunity and suggest that Mtb has its own transformation system resembling the human phase I drug-metabolizing system.

Cell Sheet Comprised of Mesenchymal Stromal Cells Overexpressing Stem Cell Factor Promotes Epicardium Activation and Heart Function Improvement in a Rat Model of Myocardium Infarction.

Cell therapy of the post-infarcted myocardium is still far from clinical use. Poor survival of transplanted cells, insufficient regeneration, and replacement of the damaged tissue limit the potential of currently available cell-based techniques. In this study, we generated a multilayered construct from adipose-derived mesenchymal stromal cells (MSCs) modified to secrete stem cell factor, SCF. In a rat model of myocardium infarction, we show that transplantation of SCF producing cell sheet induced activation of the epicardium and promoted the accumulation of c-kit positive cells in ischemic muscle. Morphometry showed the reduction of infarct size (16%) and a left ventricle expansion index (0.12) in the treatment group compared to controls (24-28%; 0.17-0.32). The ratio of viable myocardium was more than 1.5-fold higher, reaching 49% compared to the control (28%) or unmodified cell sheet group (30%). Finally, by day 30 after myocardium infarction, SCF-producing cell sheet transplantation increased left ventricle ejection fraction from 37% in the control sham-operated group to 53%. Our results suggest that, combining the genetic modification of MSCs and their assembly into a multilayered construct, we can provide prolonged pleiotropic effects to the damaged heart, induce endogenous regenerative processes, and improve cardiac function.

Transplantation of Adipose Stromal Cell Sheet Producing Hepatocyte Growth Factor Induces Pleiotropic Effect in Ischemic Skeletal Muscle.

Cell therapy remains a promising approach for the treatment of cardiovascular diseases. In this regard, the contemporary trend is the development of methods to overcome low cell viability and enhance their regenerative potential. In the present study, we evaluated the therapeutic potential of gene-modified adipose-derived stromal cells (ADSC) that overexpress hepatocyte growth factor (HGF) in a mice hind limb ischemia model. Angiogenic and neuroprotective effects were assessed following ADSC transplantation in suspension or in the form of cell sheet. We found superior blood flow restoration, tissue vascularization and innervation, and fibrosis reduction after transplantation of HGF-producing ADSC sheet compared to other groups. We suggest that the observed effects are determined by pleiotropic effects of HGF, along with the multifactorial paracrine action of ADSC which remain viable and functionally active within the engineered cell construct. Thus, we demonstrated the high therapeutic potential of the utilized approach for skeletal muscle recovery after ischemic damage associated with complex tissue degenerative effects.

Enhanced angiogenesis in ischemic skeletal muscle after transplantation of cell sheets from baculovirus-transduced adipose-derived stromal cells expressing VEGF165.

INTRODUCTION: Cell therapy using adipose-derived stromal cells (ADSC) is an intensively developing approach to promote angiogenesis and regeneration. Administration technique is crucial and among others minimal constructs - cell sheets (CS) have certain advantages. Delivery of CS allows transplantation of cells along with matrix proteins to facilitate engraftment. Cells' therapeutic potential can be also increased by expression of proangiogenic factors by viral transduction. In this work we report on therapeutic efficacy of CS from mouse ADSC transduced to express human vascular endothelial growth factor 165 a/a isoform (VEGF165), which showed potency to restore perfusion and protect tissue in a model of limb ischemia. METHODS: Mouse ADSC (mADSC) isolated from C57 male mice were expanded for CS formation (10(6)cells per CS). Constructs were transduced to express human VEGF165 by baculoviral (BV) system. CS were transplanted subcutaneously to mice with surgically induced limb ischemia and followed by laser Doppler perfusion measurements. At endpoint animals were sacrificed and skeletal muscle was evaluated for necrosis and vessel density; CS with underlying muscle was stained for apoptosis, proliferation, monocytes and blood vessels. RESULTS: Using BV system and sodium butyrate treatment we expressed human VEGF165 in mADSC (production of VEGF165 reached ≈ 25-27 ng/ml/10(5) cells) and optimized conditions to ensure cells' viability after transduction. Implantation of mock-transduced CS resulted in significant improvement of limb perfusion, increased capillary density and necrosis reduction at 2 weeks post-surgery compared to untreated animals. Additional improvement of blood flow and angiogenesis was observed after transplantation of VEGF165-expressing CS indicating enhanced therapeutic potential of genetically modified constructs. Moreover, we found delivery of mADSC as CS to be superior to equivalent dose of suspended cells in terms of perfusion and angiogenesis. Histology analysis of extracted CS detected limited proliferation and approximately 10 % prevalence of apoptosis in transplanted mADSC. Significant vascularization of CS and infiltration by monocytes were found in both - BV-transduced and control CS indicating graft and host interaction after transplantation. CONCLUSIONS: Delivery of ADSC by subcutaneous transplantation of CS is effective for stimulation of angiogenesis and tissue protection in limb ischemia with a potential for efficacy improvement by BV transduction to express VEGF165.

Transplantation of modified human adipose derived stromal cells expressing VEGF165 results in more efficient angiogenic response in ischemic skeletal muscle.

BACKGROUND: Modified cell-based angiogenic therapy has become a promising novel strategy for ischemic heart and limb diseases. Most studies focused on myoblast, endothelial cell progenitors or bone marrow mesenchymal stromal cells transplantation. Yet adipose-derived stromal cells (in contrast to bone marrow) are abundantly available and can be easily harvested during surgery or liposuction. Due to high paracrine activity and availability ADSCs appear to be a preferable cell type for cardiovascular therapy. Still neither genetic modification of human ADSC nor in vivo therapeutic potential of modified ADSC have been thoroughly studied. Presented work is sought to evaluate angiogenic efficacy of modified ADSCs transplantation to ischemic tissue. MATERIALS AND METHODS: Human ADSCs were transduced using recombinant adeno-associated virus (rAAV) serotype 2 encoding human VEGF165. The influence of genetic modification on functional properties of ADSCs and their angiogenic potential in animal models were studied. RESULTS: We obtained AAV-modified ADSC with substantially increased secretion of VEGF (VEGF-ADSCs). Transduced ADSCs retained their adipogenic and osteogenic differentiation capacities and adhesion properties. The level of angiopoetin-1 mRNA was significantly increased in VEGF-ADSC compared to unmodified cells yet expression of FGF-2, HGF and urokinase did not change. Using matrigel implant model in mice it was shown that VEGF-ADSC substantially stimulated implant vascularization with paralleling increase of capillaries and arterioles. In murine hind limb ischemia test we found significant reperfusion and revascularization after intramuscular transplantation of VEGF-ADSC compared to controls with no evidence of angioma formation. CONCLUSIONS: Transplantation of AAV-VEGF- gene modified hADSC resulted in stronger therapeutic effects in the ischemic skeletal muscle and may be a promising clinical treatment for therapeutic angiogenesis.

Combined transfer of human VEGF165 and HGF genes renders potent angiogenic effect in ischemic skeletal muscle.

Increased interest in development of combined gene therapy emerges from results of recent clinical trials that indicate good safety yet unexpected low efficacy of "single-gene" administration. Multiple studies showed that vascular endothelial growth factor 165 aminoacid form (VEGF165) and hepatocyte growth factor (HGF) can be used for induction of angiogenesis in ischemic myocardium and skeletal muscle. Gene transfer system composed of a novel cytomegalovirus-based (CMV) plasmid vector and codon-optimized human VEGF165 and HGF genes combined with intramuscular low-voltage electroporation was developed and tested in vitro and in vivo. Studies in HEK293T cell culture, murine skeletal muscle explants and ELISA of tissue homogenates showed efficacy of constructed plasmids. Functional activity of angiogenic proteins secreted by HEK293T after transfection by induction of tube formation in human umbilical vein endothelial cell (HUVEC) culture. HUVEC cells were used for in vitro experiments to assay the putative signaling pathways to be responsible for combined administration effect one of which could be the ERK1/2 pathway. In vivo tests of VEGF165 and HGF genes co-transfer were conceived in mouse model of hind limb ischemia. Intramuscular administration of plasmid encoding either VEGF165 or HGF gene resulted in increased perfusion compared to empty vector administration. Mice injected with a mixture of two plasmids (VEGF165+HGF) showed significant increase in perfusion compared to single plasmid injection. These findings were supported by increased CD31+ capillary and SMA+ vessel density in animals that received combined VEGF165 and HGF gene therapy compared to single gene therapy. Results of the study suggest that co-transfer of VEGF and HGF genes renders a robust angiogenic effect in ischemic skeletal muscle and may present interest as a potential therapeutic combination for treatment of ischemic disorders.