Patient specific stem cell models: focus on glioblastoma and neuronal differentiation aspects of chronic granulomatous disease

Pluripotent stem cells can in vitro replicate key pathophysiological aspects of central nervous system. In the first project, we have developed a 3D co-culture system using cancer stem cells isolated from glioblastoma patients and a human stem cell-derived engineered neural tissue, which allowed us to reproduce numerous hallmarks of glioblastoma in vivo, including invasion and formation of secondary foci. Transcriptomic analysis identified that IFN response genes was induced specifically in the co-culture system that was significantly correlating with patient survival. In a second project, we have focused on the role of ROS generating NOX2 enzyme during neural differentiation. Our study demonstrates that during early stages of neurogenesis there is a regulatory role of NOX2, which is conserved from mouse to human. This suggests a contribution of redox mechanisms in the maintenance of neural stem/progenitor. In conclusion, this study exemplifies the potential of stem cells for the study of human pathophysiology

Platelet Rich Plasma Promotes Proliferation of Adipose Derived Mesenchymal Stem Cells via Activation of AKT and Smad2 Signaling Pathways

Recently, autologous platelet-rich plasma (PRP) has been proposed as a substitute for xenogenic or allogenic culture media used for in vitro cell expansion. Although PRP has been demonstrated to promote adipose-derived mesenchymal stem cell (ASC) expansion, its mechanism of action has not yet been investigated. In this study, we aimed to assess the growth factors and molecular pathways implicated in enhancement of ASC proliferation by PRP. Cell proliferation was analyzed in ASCs cultured for 10 days with 20% autologous PRP and compared to those supplemented with 10% fetal bovine serum (FBS). The secretion of PDGF-AB, FGF, TGFβ, VEGF, and MIF in the culture media was investigated. In addition, AKT, ERK, and Smad2 signalling pathway activation involved in ASC proliferation was assessed using western blot analysis. The expansion rate of cultured ASCs was 14 times greater with 20% PRP than with 10% FBS. Proliferation rate of ASCs was higher in 20% PRP-supplemented medium than in 10% FBS. PDGF-AB, FGF, TGFβ, and VEGF were present in the medium supplemented with 20% PRP up to 10 days. Macrophage migration inhibitory factor (MIF) secretion was confirmed in both media, and a higher level was seen in 20% PRP. The AKT, ERK and Smad2 signalling pathways were more activated in ASCs cultured with PRP compared to FBS. In summary, our results indicate that PRP regulates ASC proliferation through secreted proteins (PDGF-AB, FGF, TGFβ, VEGF, and MIF). Growth factor/receptor complexes activate mainly AKT and Smad2 and to a lesser extent, ERK signalling pathways

The relationship between brain tumor cell invasion of engineered neural tissues and in vivo features of glioblastoma

Glioblastoma is an aggressive brain tumor characterized by its high propensity for local invasion, formation of secondary foci within the brain, as well as areas of necrosis. This study aims to (i) provide a technical approach to reproduce features of the disease in vitro and (ii) characterize the tumor/host brain tissue interaction at the molecular level. Human engineered neural tissue (ENT) obtained from pluripotent stem cells was generated and co-cultured with human glioblastoma-initiating cells. Within two weeks, glioblastoma cells invaded the nervous tissue. This invasion displayed features of the disease in vivo: a primary tumor mass, diffuse migration of invading single cells into the nervous tissue, secondary foci, as well as peritumoral cell death. Through comparative molecular analyses, this model allowed the identification of more than 100 genes that are specifically induced and up-regulated by the nervous tissue/tumor interaction. Notably the type I interferon response, extracellular matrix-related genes were most highly represented and showed a significant correlation with patient survival. In conclusion, glioblastoma development within a nervous tissue can be engineered in vitro, providing a relevant model to study the disease and allows the identification of clinically-relevant genes induced by the tumor/host tissue interaction

Decreased neural precursor cell pool in NADPH oxidase 2-deficiency: From mouse brain to neural differentiation of patient derived iPSC

There is emerging evidence for the involvement of reactive oxygen species (ROS) in the regulation of stem cells and cellular differentiation. Absence of the ROS-generating NADPH oxidase NOX2 in chronic granulomatous disease (CGD) patients, predominantly manifests as immune deficiency, but has also been associated with decreased cognition. Here, we investigate the role of NOX enzymes in neuronal homeostasis in adult mouse brain and in neural cells derived from human induced pluripotent stem cells (iPSC). High levels of NOX2 were found in mouse adult neurogenic regions. In NOX2-deficient mice, neurogenic regions showed diminished redox modifications, as well as decrease in neuroprecursor numbers and in expression of genes involved in neural differentiation including NES, BDNF and OTX2. iPSC from healthy subjects and patients with CGD were used to study the role of NOX2 in human in vitro neuronal development. Expression of NOX2 was low in undifferentiated iPSC, upregulated upon neural induction, and disappeared during neuronal differentiation. In human neurospheres, NOX2 protein and ROS generation were polarized within the inner cell layer of rosette structures. NOX2 deficiency in CGD-iPSCs resulted in an abnormal neural induction in vitro, as revealed by a reduced expression of neuroprogenitor markers (NES, BDNF, OTX2, NRSF/REST), and a decreased generation of mature neurons. Vector-mediated NOX2 expression in NOX2-deficient iPSCs rescued neurogenesis. Taken together, our study provides novel evidence for a regulatory role of NOX2 during early stages of neurogenesis in mouse and human

Evaluation of NADPH oxidases as drug targets in a mouse model of familial amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is an incurable neurodegenerative disease characterized by progressive loss of motor neurons, gliosis, neuroinflammation and oxidative stress. The aim of this study was to evaluate the involvement of NADPH oxidases (NOX) in the oxidative damage and progression of ALS neuropathology. We examined the pattern of NOX expression in spinal cords of patients and mouse models of ALS and analyzed the impact of genetic deletion of the NOX1 and 2 isoforms as well as pharmacological NOX inhibition in the SOD1(G93A) ALS mouse model. A substantial (10-60 times) increase of NOX2 expression was detected in three etiologically different ALS mouse models while up-regulation of some other NOX isoforms was model-specific. In human spinal cord samples, high NOX2 expression was detected in microglia. In contrast to previous publications, survival of SOD1(G93A) mice was not modified upon breeding with constitutive NOX1 and NOX2 deficient mice. As genetic deficiency of a single NOX isoform is not necessarily predictive of a pharmacological intervention, we treated SOD1(G93A) mice with broad-spectrum NOX inhibitors perphenazine and thioridazine. Both compounds reached in vivo CNS concentrations compatible with NOX inhibition and thioridazine significantly decreased superoxide levels in the spinal cord of SOD1(G93A) mice in vivo. Yet, neither perphenazine nor thioridazine prolonged survival. Thioridazine, but not perphenazine, dampened the increase of microglia markers in SOD1(G93A) mice. Thioridazine induced an immediate and temporary enhancement of motor performance (rotarod) but its precise mode of action needs further investigation. Additional studies using specific NOX inhibitors will provide further evidence on the relevance of NOX as drug targets for ALS and other neurodegenerative disorders

Comprehensive metagenomic analysis of glioblastoma reveals absence of known virus despite antiviral-like type I interferon gene response

Glioblastoma is a deadly malignant brain tumor and one of the most incurable forms of cancer in need of new therapeutic targets. As some cancers are known to be caused by a virus, the discovery of viruses could open the possibility to treat, and perhaps prevent, such a disease. Although an association with viruses such as cytomegalovirus or Simian virus 40 has been strongly suggested, involvement of these and other viruses in the initiation and/or propagation of glioblastoma remains vague, controversial and warrants elucidation. To exhaustively address the association of virus and glioblastoma, we developed and validated a robust metagenomic approach to analyze patient biopsies via high-throughput sequencing, a sensitive tool for virus screening. In addition to traditional clinical diagnostics, glioblastoma biopsies were deep-sequenced and analyzed with a multistage computational pipeline to identify known or potentially discover unknown viruses. In contrast to the studies reporting the presence of viral signatures in glioblastoma, no common or recurring active viruses were detected, despite finding an antiviral-like type I interferon response in some specimens. Our findings highlight a discrete and non-specific viral signature and uncharacterized short RNA sequences in glioblastoma. This study provides new insights into glioblastoma pathogenesis and defines a general methodology that can be used for high-resolution virus screening and discovery in human cancers