Image informatics strategies for deciphering neuronal network connectivity

Brain function relies on an intricate network of highly dynamic neuronal connections that rewires dramatically under the impulse of various external cues and pathological conditions. Among the neuronal structures that show morphologi- cal plasticity are neurites, synapses, dendritic spines and even nuclei. This structural remodelling is directly connected with functional changes such as intercellular com- munication and the associated calcium-bursting behaviour. In vitro cultured neu- ronal networks are valuable models for studying these morpho-functional changes. Owing to the automation and standardisation of both image acquisition and image analysis, it has become possible to extract statistically relevant readout from such networks. Here, we focus on the current state-of-the-art in image informatics that enables quantitative microscopic interrogation of neuronal networks. We describe the major correlates of neuronal connectivity and present workflows for analysing them. Finally, we provide an outlook on the challenges that remain to be addressed, and discuss how imaging algorithms can be extended beyond in vitro imaging studies

Prospective isolation of human bone marrow stromal cell subsets: a comparative study between Stro-1-, CD146- and CD105-enriched populations

Stro-1 has proved an efficacious marker for enrichment of skeletal stem and progenitor cells although isolated populations remain heterogeneous, exhibiting variable colony-forming efficiency and osteogenic differentiation potential. The emerging findings that skeletal stem cells originate from adventitial reticular cells have brought two further markers to the fore including CD146 and CD105 (both primarily endothelial and perivascular). This study has compared CD146-, CD105- and Stro-1 (individual and in combination)-enriched human bone marrow stromal cell subsets and assessed whether these endothelial/perivascular markers offer further selection over conventional Stro-1. Fluorescent cell sorting quantification showed that CD146 and CD105 both targeted smaller (2.22% ± 0.59% and 6.94% ± 1.34%, respectively) and potentially different human bone marrow stromal cell fractions compared to Stro-1 (16.29% ± 0.78%). CD146+, but not CD105+, cells exhibited similar alkaline phosphatase-positive colony-forming efficiency in vitro and collagen/proteoglycan deposition in vivo to Stro-1+ cells. Molecular analysis of a number of select osteogenic and potential osteo-predictive genes including ALP, CADM1, CLEC3B, DCN, LOXL4, OPN, POSTN and SATB2 showed Stro-1+ and CD146+ populations possessed similar expression profiles. A discrete human bone marrow stromal cell fraction (2.04% ± 0.41%) exhibited positive immuno-labelling for both Stro-1 and CD146. The data presented here show that CD146+ populations are comparable but not superior to Stro-1+ populations. However, this study demonstrates the critical need for new candidate markers with which to isolate homogeneous skeletal stem cell populations or skeletal stem cell populations which exhibit homogeneous in vitro/in vivo characteristics, for implementation within tissue engineering and regenerative medicine strategies

Zika virus impairs the development of blood vessels in a mouse model of congenital infection

Zika virus (ZIKV) is associated with brain development abnormalities such as primary microcephaly, a severe reduction in brain growth. Here we demonstrated in vivo the impact of congenital ZIKV infection in blood vessel development, a crucial step in organogenesis. ZIKV was injected intravenously in the pregnant type 2 interferon (IFN)-deficient mouse at embryonic day (E) 12.5. The embryos were collected at E15.5 and postnatal day (P)2. Immunohistochemistry for cortical progenitors and neuronal markers at E15.5 showed the reduction of both populations as a result of ZIKV infection. Using confocal 3D imaging, we found that ZIKV infected brain sections displayed a reduction in the vasculature density and vessel branching compared to mocks at E15.5; altogether, cortical vessels presented a comparatively immature pattern in the infected tissue. These impaired vascular patterns were also apparent in the placenta and retina. Moreover, proteomic analysis has shown that angiogenesis proteins are deregulated in the infected brains compared to controls. At P2, the cortical size and brain weight were reduced in comparison to mock-infected animals. In sum, our results indicate that ZIKV impairs angiogenesis in addition to neurogenesis during development. The vasculature defects represent a limitation for general brain growth but also could regulate neurogenesis directly

3D Microfluidic model for evaluating immunotherapy efficacy by tracking dendritic cell behaviour toward tumor cells

Immunotherapy efficacy relies on the crosstalk within the tumor microenvironment between cancer and dendritic cells (DCs) resulting in the induction of a potent and effective antitumor response. DCs have the specific role of recognizing cancer cells, taking up tumor antigens (Ags) and then migrating to lymph nodes for Ag (cross)-presentation to naïve T cells. Interferon-α-conditioned DCs (IFN-DCs) exhibit marked phagocytic activity and the special ability of inducing Ag-specific T-cell response. Here, we have developed a novel microfluidic platform recreating tightly interconnected cancer and immune systems with specific 3D environmental properties, for tracking human DC behaviour toward tumor cells. By combining our microfluidic platform with advanced microscopy and a revised cell tracking analysis algorithm, it was possible to evaluate the guided efficient motion of IFN-DCs toward drug-treated cancer cells and the succeeding phagocytosis events. Overall, this platform allowed the dissection of IFN-DC-cancer cell interactions within 3D tumor spaces, with the discovery of major underlying factors such as CXCR4 involvement and underscored its potential as an innovative tool to assess the efficacy of immunotherapeutic approaches

Improving treatment of glioblastoma: new insights in targeting cancer stem cells effectively

Glioblastoma is the most common primary malignant brain tumour in the adult population. Despite multimodality treatment with surgery, radiotherapy and chemotherapy, outcomes are very poor, with less than 15% of patients alive after two years. Increasing evidence suggests that glioblastoma stem cells (GSCs) are likely to play an important role in the biology of this disease and are involved in treatment resistance and tumour recurrence following standard therapy. My thesis aims to address two main aspects of this research area: 1) optimization of methods to evaluate treatment responses of GSCs and their differentiated counterparts (non-GSCs), with a particular focus on a tissue culture model that resembles more closely the tumoral niche; 2) characterization of cell division and centrosome cycle of GSCs, investigating possible differences between these cells and non-GSCs, that would allow the identification of targets for new therapeutic strategies against glioblastomas. In the first part of my project, I optimized a clonogenic survival assay, to compare sensitivity of GSCs and non-GSCs to various treatments, and I developed the use of a 3-dimentional tissue culture system, that allows analysis of features and radiation responses of these two subpopulations in the presence of specific microenvironmental factors from the tumoral niche. In the second part, I show that GSCs display mitotic spindle abnormalities more frequently than non-GSCs and that they have distinctive features with regards to the centrosome cycle. I also demonstrate that GSCs are more sensitive than non-GSCs to subtle changes in Aurora kinase A activity, which result in a rapid increase in polyploidy and subsequently in senescence, with a consistent reduction in clonogenic survival. Based on these findings, I propose that kinases involved in the centrosome cycle need to be explored as a novel strategy to target GSCs effectively and improve outcomes of glioblastoma patients

Lymphatic endothelium stimulates melanoma metastasis and invasion via MMP14-dependent Notch3 and b1-integrin activation

Lymphatic invasion and lymph node metastasis correlate with poor clinical outcome in melanoma. However, the mechanisms of lymphatic dissemination in distant metastasis remain incompletely understood. We show here that exposure of expansively growing human WM852 melanoma cells, but not singly invasive Bowes cells, to lymphatic endothelial cells (LEC) in 3D co-culture facilitates melanoma distant organ metastasis in mice. To dissect the underlying molecular mechanisms, we established LEC co-cultures with different melanoma cells originating from primary tumors or metastases. Notably, the expansively growing metastatic melanoma cells adopted an invasively sprouting phenotype in 3D matrix that was dependent on MMP14, Notch3 and β1-integrin. Unexpectedly, MMP14 was necessary for LEC-induced Notch3 induction and coincident β1-integrin activation. Moreover, MMP14 and Notch3 were required for LEC-mediated metastasis of zebrafish xenografts. This study uncovers a unique mechanism whereby LEC contact promotes melanoma metastasis by inducing a reversible switch from 3D growth to invasively sprouting cell phenotype

Cell-based gene therapy for mending infarcted hearts

The goal of this study was to analyse the efficiency of a combinatorial cell/growth factor therapy to improve function of infarcted murine hearts. The Insulin-like Growth Factor-1 (IGF-1) isoform, IGF-1Ea, has been shown to reduce scar formation and decrease cell death after MI. The present study utilized P19Cl6-derived, IGF-1Ea over-expressing cardiomyocytes to achieve its goal. The P19Cl6 cells were stably transduced with IGF-1Ea using a lentiviral vector and investigated first in vitro for their feasibility for in vivo cell therapy. The engineered pluripotent cells over-expressing IGF-1Ea survived better to hypoxia-induced injury than the control cells. The cells maintained their pluripotency and efficient differentiation capacity towards ventricular cardiomyocyte lineage, generating large quantities of cardiomyocytes optimal for the transplantation study. The generated cardiomyocytes were functionally active and exhibited a mature phenotype. Transplantation of the cardiomyocytes into allogeneic wild type murine infarcted hearts conferred a tendency for maintenance of function at short-term time point. At long-term however, this effect was lost, returning to the level of the control infarcted hearts. Cell tracing assessment revealed engraftment of both IGF-1Ea- and empty-cells, although the cells failed to couple with the recipient tissue. Scar size and capillary density analyses revealed no significant difference between the cells transplanted compared to the saline treated hearts, corroborating with the long-term functional data. Interestingly, the IGF- 1Ea-cell transplanted hearts expressed significantly higher amount of VEGFa compared to the controls, albeit no change in capillary density. Further investigation revealed that the enhanced VEGFa expression in IGF-1Ea-cells transplanted hearts was associated with reduced hypertrophy, marked by reduced cell cross-sectional area at the border-zone, aSK and bMHC expression compared to the control hearts. Nonetheless, modulation of hypertrophic response and transplantation of IGF-1Ea-cells were not able to confer lasting functional preservation, possibly due to lack of sufficient engraftment and coupling of the transplanted cells

Molecular effect of an OPTN common variant associated to Paget's disease of bone

Paget's disease of bone (PDB) is a chronic bone disorder and although genetic factors appear to play an important role in its pathogenesis, to date PDB causing mutations were identified only in the Sequestosome 1 (SQSTM1) gene at the PDB3 locus. PDB6 locus, also previously linked to PDB, contains several candidate genes for metabolic bone diseases. We focused our analysis in the most significantly associated variant with PDB, within the Optineurin (OPTN) gene, i.e. the common variant rs1561570. Although it was previously shown to be strongly associated with PDB in several populations, its contribution to PDB pathogenesis remains unclear. In this study we have shown that rs1561570 may contribute to PDB since its Tallele results in the loss of a methylation site in patients' DNA, leading to higher levels of OPTN gene expression and a corresponding increase in protein levels in patients' osteoclasts. This increase in OPTN expression leads to higher levels of NF-KB translocation into the nucleus and increasing expression of its target genes, which may contribute to the overactivity of osteoclasts observed in PDB. We also reported a tendency for a more severe clinical phenotype in the presence of a haplotype containing the rs1561570 T allele, which appear to be re-enforced with the presence of the SQSTM1/P392L mutation. In conclusion, our work provides novel insight towards understanding the functional effects of this variant, located in OPTN intron 7, and its implication in the contribution to PDB pathogenesis.national funds from Foundation for Science and Technology (FCT) [UID/Multi/04326/2013]; Canadian Institutes for Health Research, Canada [MOP130457]; CHU de Quebec Foundation; Canadian Foundation for Innovation; Fonds de recherche du Quebec-sante; Laval University; CHU de Quebec-Universite Laval Research Centre; FCT [SFRH/BD/77227/2011, SFRH/BPD/111898/2015]; Fonds de recherche Quebec-Sante (FRQ-S), Quebec, Canad