Karl Kashofer

A Systems Biology Approach to Deciphering the Etiology of Steatosis Employing Patient-Derived Dermal Fibroblasts and iPS Cells

Non-alcoholic fatty liver disease comprises a broad spectrum of disease states ranging from simple steatosis to non-alcoholic steatohepatitis. As a result of increases in the prevalences of obesity, insulin resistance, and hyperlipidemia, the number of people with hepatic steatosis continues to increase. Differences in susceptibility to steatohepatitis and its progression to cirrhosis have been attributed to a complex interplay of genetic and external factors all addressing the intracellular network. Increase in sugar or refined carbohydrate consumption results in an increase of insulin and insulin resistance that can lead to the accumulation of fat in the liver. Here we demonstrate how a multidisciplinary approach encompassing cellular reprogramming, transcriptomics, proteomics, metabolomics, modeling, network reconstruction, and data management can be employed to unveil the mechanisms underlying the progression of steatosis. Proteomics revealed reduced AKT/mTOR signaling in fibroblasts derived from steatosis patients and further establishes that the insulin-resistant phenotype is present not only in insulin-metabolizing central organs, e.g., the liver, but is also manifested in skin fibroblasts. Transcriptome data enabled the generation of a regulatory network based on the transcription factor SREBF1, linked to a metabolic network of glycerolipid, and fatty acid biosynthesis including the downstream transcriptional targets of SREBF1 which include LIPIN1 (LPIN) and low density lipoprotein receptor. Glutathione metabolism was among the pathways enriched in steatosis patients in comparison to healthy controls. By using a model of the glutathione pathway we predict a significant increase in the flux through glutathione synthesis as both gamma-glutamylcysteine synthetase and glutathione synthetase have an increased flux. We anticipate that a larger cohort of patients and matched controls will confirm our preliminary findings presented here.

Human platelet lysate can replace fetal bovine serum for clinical-scale expansion of functional mesenchymal stromal cells.

BACKGROUND: Human multipotent mesenchymal stromal cells (MSCs) are promising candidates for a growing spectrum of regenerative and immunomodulatory cellular therapies. Translation of auspicious experimental results into clinical applications has been limited by the dependence of MSC propagation from fetal bovine serum (FBS).

Humanized large-scale expanded endothelial colony-forming cells function in vitro and in vivo

Endothelial progenitor cells are critically involved in essential biologic processes, such as vascular homeostasis, regeneration, and tumor angiogenesis. Endothelial colony-forming cells (ECFCs) are endothelial progenitor cells with robust proliferative potential. Their profound vessel-forming capacity makes them a promising tool for innovative experimental, diagnostic, and therapeutic strategies. Efficient and safe methods for their isolation and expansion are presently lacking. Based on the previously established efficacy of animal serum-free large-scale clinical-grade propagation of mesenchymal stromal cells, we hypothesized that endothelial lineage cells may also be propagated efficiently following a comparable strategy. Here we demonstrate that human ECFCs can be recovered directly from unmanipulated whole blood. A novel large-scale animal protein-free humanized expansion strategy preserves the progenitor hierarchy with sustained proliferation potential of more than 30 population doublings. By applying large-scale propagated ECFCs in various test systems, we observed vascular networks in vitro and perfused vessels in vivo. After large-scale expansion and cryopreservation phenotype, function, proliferation, and genomic stability were maintained. For the first time, proliferative, functional, and storable ECFCs propagated under humanized conditions can be explored in terms of their therapeutic applicability and risk profile.

Cognitive impairment by antibiotic-induced gut dysbiosis: Analysis of gut microbiota-brain communication

Emerging evidence indicates that disruption of the gut microbial community (dysbiosis) impairs mental health. Germ-free mice and antibiotic-induced gut dysbiosis are two approaches to establish causality in gut microbiota-brain relationships. However, both models have limitations, as germ-free mice display alterations in blood-brain barrier and brain ultrastructure and antibiotics may act directly on the brain. We hypothesized that the concerns related to antibiotic-induced gut dysbiosis can only adequately be addressed if the effect of intragastric treatment of adult mice with multiple antibiotics on (i) gut microbial community, (ii) metabolite profile in the colon, (iii) circulating metabolites, (iv) expression of neuronal signaling molecules in distinct brain areas and (v) cognitive behavior is systematically investigated. Of the antibiotics used (ampicillin, bacitracin, meropenem, neomycin, vancomycin), ampicillin had some oral bioavailability but did not enter the brain. 16S rDNA sequencing confirmed antibiotic-induced microbial community disruption, and metabolomics revealed that gut dysbiosis was associated with depletion of bacteria-derived metabolites in the colon and alterations of lipid species and converted microbe-derived molecules in the plasma. Importantly, novel object recognition, but not spatial, memory was impaired in antibiotic-treated mice. This cognitive deficit was associated with brain region-specific changes in the expression of cognition-relevant signaling molecules, notably brain-derived neurotrophic factor, N-methyl-d-aspartate receptor subunit 2B, serotonin transporter and neuropeptide Y system. We conclude that circulating metabolites and the cerebral neuropeptide Y system play an important role in the cognitive impairment and dysregulation of cerebral signaling molecules due to antibiotic-induced gut dysbiosis.

Comparative Analysis of Multidimensional, Quantitative Data

When analyzing multidimensional, quantitative data, the comparison of two or more groups of dimensions is a common task. Typical sources of such data are experiments in biology, physics or engineering, which are conducted in different configurations and use replicates to ensure statistically significant results. One common way to analyze this data is to filter it using statistical methods and then run clustering algorithms to group similar values. The clustering results can be visualized using heat maps, which show differences between groups as changes in color. However, in cases where groups of dimensions have an a priori meaning, it is not desirable to cluster all dimensions combined, since a clustering algorithm can fragment continuous blocks of records. Furthermore, identifying relevant elements in heat maps becomes more difficult as the number of dimensions increases. To aid in such situations, we have developed Matchmaker, a visualization technique that allows researchers to arbitrarily arrange and compare multiple groups of dimensions at the same time. We create separate groups of dimensions which can be clustered individually, and place them in an arrangement of heat maps reminiscent of parallel coordinates. To identify relations, we render bundled curves and ribbons between related records in different groups. We then allow interactive drill-downs using enlarged detail views of the data, which enable in-depth comparisons of clusters between groups. To reduce visual clutter, we minimize crossings between the views. This paper concludes with two case studies. The first demonstrates the value of our technique for the comparison of clustering algorithms. In the second, biologists use our system to investigate why certain strains of mice develop liver disease while others remain healthy, informally showing the efficacy of our system when analyzing multidimensional data containing distinct groups of dimensions.

A New Technology for Stabilization of Biomolecules in Tissues for Combined Histological and Molecular Analyses

For accurate diagnosis, prediction of outcome, and selection of appropriate therapies, the molecular characterization of human diseases requires analysis of a broad spectrum of altered biomolecules, in addition to morphological features, in affected tissues such as tumors. In a high-throughput screening approach, we have developed the PAXgene Tissue System as a novel tissue stabilization technology. Comprehensive characterization of this technology in stabilized and paraffin-embedded human tissues and comparison with snap-frozen tissues revealed excellent preservation of morphology and antigenicity, as well as outstanding integrity of nucleic acids (genomic DNA, miRNA, and mRNA) and phosphoproteins. Importantly, PAXgene-fixed, paraffin-embedded tissues provided RNA quantity and quality not only significantly better than that obtained with neutral buffered formalin, but also similar to that from snap-frozen tissue, which currently represents the gold standard for molecular analyses. The PAXgene tissue stabilization system thus opens new opportunities in a variety of molecular diagnostic and research applications in which the collection of snap-frozen tissue is not feasible for medical, logistic, or ethical reasons. Furthermore, this technology allows performing histopathological analyses together with molecular studies in a single sample, which markedly facilitates direct correlation of morphological disease phenotypes with alterations of nucleic acids and other biomolecules.

Gene Therapy Progress and Prospects: Stem cell plasticity

With the identification of stem cell plasticity several years ago, multiple reports raised hopes that tissue repair by stem cell transplantation could be within reach in the near future. Krause et al reported that a single purified hematopoietic stem cell not only repopulated the bone marrow of a host animal, but also integrated into unrelated tissues. Lagasse et al demonstrated that in a genetic model of liver disease, purified hematopoietic stem cells can give rise to hepatocytes and rescue fatal liver damage. More recent work by Jiang et al demonstrated that cultured cells can retain their stem cell potential. There are a number of possible mechanisms that could explain these phenomena, and recent experiments have raised controversy about which mechanism is prevalent. One possibility is transdifferentiation of a committed cell directly into another cell type as a response to environmental cues. Transdifferentiation has been shown mainly in vitro, but some in vivo data also support this mechanism. Direct transdifferentiation would clinically be limited by the number of cells that can be introduced into an organ without removal of resident cells. If bone marrow cells could on the other hand give rise to stem cells of another tissue, then they could in theory repopulate whole organs from a few starting cells. This model of dedifferentiation is consistent with recent data from animal models. Genetic analysis of cells of donor origin in vivo and in vitro has brought to light another possible mechanism. The fusion of host and donor cells can give rise to mature tissue cells without trans- or dedifferentiation. The resulting heterokaryons are able to cure a lethal genetic defect and do not seem to be prone to give rise to cancer. All these models will clinically face the problem of accessibility of healthy primary cells for transplantation. This underlines the importance of the recent identification of a population of mesenchymal stem cells (MSCs) with stem cell properties similar to embryonic stem (ES) cells. These cells can be cultured and expanded in vitro without losing their stem cell potential making them an attractive target for cell therapy. Finally, it is still not clear if stem cells for various tissues are present in peripheral blood, or bone marrow and thus can be directly purified from these sources. Identification of putative tissue stem cells would be necessary before purification strategies can be devised. In this review, we discuss the evidence for these models, and the conflicting results obtained to date.

A stress-induced early innate response causes multidrug tolerance in melanoma

Acquired drug resistance constitutes a major challenge for effective cancer therapies with melanoma being no exception. The dynamics leading to permanent resistance are poorly understood but are important to design better treatments. Here we show that drug exposure, hypoxia or nutrient starvation leads to an early innate cell response in melanoma cells resulting in multidrug resistance, termed induced drug-tolerant cells (IDTCs). Transition into the IDTC state seems to be an inherent stress reaction for survival toward unfavorable environmental conditions or drug exposure. The response comprises chromatin remodeling, activation of signaling cascades and markers implicated in cancer stemness with higher angiogenic potential and tumorigenicity. These changes are characterized by a common increase in CD271 expression concomitantly with loss of differentiation markers such as melan-A and tyrosinase, enhanced aldehyde dehydrogenase (ALDH) activity and upregulation of histone demethylases. Accordingly, IDTCs show a loss of H3K4me3, H3K27me3 and gain of H3K9me3 suggesting activation and repression of differential genes. Drug holidays at the IDTC state allow for reversion into parental cells re-sensitizing them to the drug they were primarily exposed to. However, upon continuous drug exposure IDTCs eventually transform into permanent and irreversible drug-resistant cells. Knockdown of CD271 or KDM5B decreases transition into the IDTC state substantially but does not prevent it. Targeting IDTCs would be crucial for sustainable disease management and prevention of acquired drug resistance.

High-Throughput miRNA and mRNA Sequencing of Paired Colorectal Normal, Tumor and Metastasis Tissues and Bioinformatic Modeling of miRNA-1 Therapeutic Applications

MiRNAs are discussed as diagnostic and therapeutic molecules. However, effective miRNA drug treatments with miRNAs are, so far, hampered by the complexity of the miRNA networks. To identify potential miRNA drugs in colorectal cancer, we profiled miRNA and mRNA expression in matching normal, tumor and metastasis tissues of eight patients by Illumina sequencing. We validated six miRNAs in a large tissue screen containing 16 additional tumor entities and identified miRNA-1, miRNA-129, miRNA-497 and miRNA-215 as constantly de-regulated within the majority of cancers. Of these, we investigated miRNA-1 as representative in a systems-biology simulation of cellular cancer models implemented in PyBioS and assessed the effects of depletion as well as overexpression in terms of miRNA-1 as a potential treatment option. In this system, miRNA-1 treatment reverted the disease phenotype with different effectiveness among the patients. Scoring the gene expression changes obtained through mRNA-Seq from the same patients we show that the combination of deep sequencing and systems biological modeling can help to identify patient-specific responses to miRNA treatments. We present this data as guideline for future pre-clinical assessments of new and personalized therapeutic options.

Germline variants in the SEMA4A gene predispose to familial colorectal cancer type X

Familial colorectal cancer type X (FCCTX) is characterized by clinical features of hereditary non-polyposis colorectal cancer with a yet undefined genetic background. Here we identify the SEMA4A p.Val78Met germline mutation in an Austrian kindred with FCCTX, using an integrative genomics strategy. Compared with wild-type protein, SEMA4AV78M demonstrates significantly increased MAPK/Erk and PI3K/Akt signalling as well as cell cycle progression of SEMA4A-deficient HCT-116 colorectal cancer cells. In a cohort of 53 patients with FCCTX, we depict two further SEMA4A mutations, p.Gly484Ala and p.Ser326Phe and the single-nucleotide polymorphism (SNP) p.Pro682Ser. This SNP is highly associated with the FCCTX phenotype exhibiting increased risk for colorectal cancer (OR 6.79, 95% CI 2.63 to 17.52). Our study shows previously unidentified germline variants in SEMA4A predisposing to FCCTX, which has implications for surveillance strategies of patients and their families.