Publications

Abstract Bioprinting is currently the most promising method to biofabricate complex tissues in vitro with the potential to transform the future of organ transplantation and drug discovery. Efforts to create such tissues are, however, almost exclusively based on animal-derived materials, such as gelatin methacryloyl, which have demonstrated efficacy in bioprinting of complex tissues. While these materials are already used in clinical applications, uncertainty about their safety still remains due to their animal origin. Alternatively, synthetic bioinks have been developed that match the printability of natural bioinks but lack their biological complexity, and thereby often fail to support cell growth and facilitate tissue formation. Additionally, most synthetic materials do not meet the mechanical demands of bioprint stable constructs while providing a suitable environment for cells to grow, limiting the number of available bioinks. To bridge this gap and synergize bioprinting and 3D cell culture, we developed a polyethylene glycol-based bioink system to promote the growth and spreading of cell spheroids that consist of human primary endothelial cells and fibroblasts. The 3D bioprinted centimeter-scale constructs have a high shape fidelity and accelerated softening to provide sufficient space for cells to grow. Adjusting the rate of degradability, induced by the integration of ester-functionalized crosslinkers in addition to protease cleavable crosslinkers into the hydrogel network, improves the growth of spheroids in larger printed hydrogel constructs containing an interconnected channel structure. The perfusable constructs enable extensive spheroid sprouting and the formation of a cellular network upon fusion of sprouts as initial steps toward tissue formation with the potential for clinical translation.

Protein C inhibitor (PCI) is a nonspecific, heparin-binding serpin (serine protease inhibitor) that inactivates many plasmatic and extravascular serine proteases by forming stable 1:1 complexes. Proteases inhibited by PCI include the anticoagulant activated protein C, the plasminogen activator urokinase, and the sperm protease acrosin. In humans PCI circulates as a plasma protein but is also present at high concentrations in organs of the male reproductive tract. The biological role of PCI has not been defined so far. However, the colocalization of high concentrations of PCI together with several of its target proteases in the male reproductive tract suggests a role of PCI in reproduction. We generated mice lacking PCI by homologous recombination. Here we show that PCI(-/-) mice are apparently healthy but that males of this genotype are infertile. Infertility was apparently caused by abnormal spermatogenesis due to destruction of the Sertoli cell barrier, perhaps due to unopposed proteolytic activity. The resulting sperm are malformed and are morphologically similar to abnormal sperm seen in some cases of human male infertility. This animal model might therefore be useful for analyzing the molecular bases of these human conditions.

Abstract The energy cost of neuronal activity is mainly sustained by glucose 1,2 . However, in an apparent paradox, neurons only weakly metabolize glucose through glycolysis 3,4,5,6 , a circumstance that can be accounted for by the constant degradation of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-3 (Pfkfb3) 3,7,8 , a key glycolysis-promoting enzyme. To evaluate the in vivo physiological significance of this hypo-glycolytic metabolism, here we genetically engineered mice with their neurons transformed into active glycolytic cells through Pfkfb3 expression. In vivo molecular, biochemical, and metabolic flux analyses of these neurons revealed an accumulation of anomalous mitochondria, complex I disassembly, bioenergetic deficiency and mitochondrial redox stress. Notably, glycolysis-mediated NAD + reduction impaired sirtuin-dependent autophagy. Furthermore, these mice displayed cognitive decline and a metabolic syndrome that was mimicked by confining Pfkfb3 expression to hypothalamic neurons. Neuron-specific genetic ablation of mitochondrial redox stress corrected these alterations. Thus, the weak glycolytic nature of neurons is required to sustain higher-order organismal functions.

Background: Mutations in the cardiac sodium channel, SCN5A, have been associated with one type of long-QT syndrome, with isolated cardiac conduction defects and Brugada syndrome. The sodium channelopathies exhibit marked variation in clinical phenotypes. The mechanisms underlying the phenotypical diversity, however, remain unknown. Exonic SCN5A mutations can be detected in 20% of Brugada syndrome patients. Results: An intronic mutation (c.4810+3_4810+6dupGGGT) in the SCN5A gene, located outside the consensus splice site, was detected in this study in a family with a highly variable clinical phenotype of Brugada syndrome and/or conduction disease and in a patient with Brugada syndrome. The mutation was not found in a control panel of 100 (200 alleles) ethnically matched normal control subjects. We provide in vivo and in vitro evidence that the mutation can disrupt the splice donor site, activate a cryptic splice site, and create a novel splice site. Notably, our data show that normal transcripts can be also derived from the mutant allele. Conclusions: This is the first report of an unconventional intronic splice site mutation in the SCN5A gene leading to cardiac sodium channelopathy. We speculate that its phenotypical diversity might be determined by the ratio of normal/abnormal transcripts derived from the mutant allele.