Fluorescent hiPSC-derived MYH6-mScarlet cardiomyocytes for real-time tracking, imaging, and cardiotoxicity assays.

Human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) hold great potential in the cardiovascular field for human disease modeling, drug development, and regenerative medicine. However, multiple hurdles still exist for the effective utilization of hiPSC-CMs as a human-based experimental platform that can be an alternative to the current animal models. To further expand their potential as a research tool and bridge the translational gap, we have generated a cardiac-specific hiPSC reporter line that differentiates into fluorescent CMs using CRISPR-Cas9 genome editing technology. The CMs illuminated with the mScarlet fluorescence enable their non-invasive continuous tracking and functional cellular phenotyping, offering a real-time 2D/3D imaging platform. Utilizing the reporter CMs, we developed an imaging-based cardiotoxicity screening system that can monitor distinct drug-induced structural toxicity and CM viability in real time. The reporter fluorescence enabled visualization of sarcomeric disarray and displayed a drug dose-dependent decrease in its fluorescence. The study also has demonstrated the reporter CMs as a biomaterial cytocompatibility analysis tool that can monitor dynamic cell behavior and maturity of hiPSC-CMs cultured in various biomaterial scaffolds. This versatile cardiac imaging tool that enables real time tracking and high-resolution imaging of CMs has significant potential in disease modeling, drug screening, and toxicology testing.

Shiga-like toxin binds with high avidity to multivalent O-linked blood group P1 determinants on mucin-type fusion proteins.

The binding of Shiga-like toxin 1 (Stx1) and Shiga-like toxin 2 (Stx2) to a mucin-like fusion protein, P-selectin glycoprotein ligand-1/mouse IgG2b (PSGL-1/mIgG2b), carrying multiple copies of the blood group P1 determinant on O-glycans was investigated with western blot and the biosensor Biacore. Chinese hamster ovary K-1 (CHO-K1) cells were stably transfected with linearized plasmids encoding the PSGL-1/mIgG2b fusion protein, the pigeon α1,4-galactosyltransferase (α4Gal-T) and the core 2 ß1,6-N-acetylglucosaminyltransferase (C2GnT-I). Western blot analyses of purified PSGL-1/mIgG2b and liquid chromatography-mass spectrometry (LC-MS) of released O-glycans confirmed the presence of the P1 determinant. Western blot analysis indicated strong binding of Stx1, but not Stx2, to PSGL-1/mIgG2b. In a Biacore assay, Stx1 and Stx2 were immobilized on a dextran chip and the binding of purified PSGL-1/mIgG2b and a P(k)-albumin neoglycoprotein was analyzed. Stx1 and Stx2 bound with high avidity to both PSGL-1/mIgG2b and P(k)-albumin, while the Stx1 binding was the strongest. In summary, we have shown that the pigeon α4Gal-T can be aberrantly expressed in CHO cells together with the core 2 enzyme to generate multiple, O-linked P1 determinants on a simultaneously expressed mucin-type fusion protein. P1-decorated PSGL-1/mIgG2b bound with high avidity to both Stx1 and Stx2, and as such constitutes a potential therapeutic inhibitor of these toxins.

Mechanically Biomimetic Gelatin-Gellan Gum Hydrogels for 3D Culture of Beating Human Cardiomyocytes.

To promote the transition of cell cultures from 2D to 3D, hydrogels are needed to biomimic the extracellular matrix (ECM). One potential material for this purpose is gellan gum (GG), a biocompatible and mechanically tunable hydrogel. However, GG alone does not provide attachment sites for cells to thrive in 3D. One option for biofunctionalization is the introduction of gelatin, a derivative of the abundant ECM protein collagen. Unfortunately, gelatin lacks cross-linking moieties, making the production of self-standing hydrogels difficult under physiological conditions. Here, we explore the functionalization of GG with gelatin at biologically relevant concentrations using semiorthogonal, cytocompatible, and facile chemistry based on hydrazone reaction. These hydrogels exhibit mechanical behavior, especially elasticity, which resembles the cardiac tissue. The use of optical projection tomography for 3D cell microscopy demonstrates good cytocompatibility and elongation of human fibroblasts (WI-38). In addition, human-induced pluripotent stem cell-derived cardiomyocytes attach to the hydrogels and recover their spontaneous beating in 24 h culture. Beating is studied using in-house-built phase contrast video analysis software, and it is comparable with the beating of control cardiomyocytes under regular culture conditions. These hydrogels provide a promising platform to transition cardiac tissue engineering and disease modeling from 2D to 3D.

The haloarchaeal MCM proteins: bioinformatic analysis and targeted mutagenesis of the ß7-ß8 and ß9-ß10 hairpin loops and conserved zinc binding domain cysteines.

The hexameric MCM complex is the catalytic core of the replicative helicase in eukaryotic and archaeal cells. Here we describe the first in vivo analysis of archaeal MCM protein structure and function relationships using the genetically tractable haloarchaeon Haloferax volcanii as a model system. Hfx. volcanii encodes a single MCM protein that is part of the previously identified core group of haloarchaeal MCM proteins. Three structural features of the N-terminal domain of the Hfx. volcanii MCM protein were targeted for mutagenesis: the ß7-ß8 and ß9-ß10 ß-hairpin loops and putative zinc binding domain. Five strains carrying single point mutations in the ß7-ß8 ß-hairpin loop were constructed, none of which displayed impaired cell growth under normal conditions or when treated with the DNA damaging agent mitomycin C. However, short sequence deletions within the ß7-ß8 ß-hairpin were not tolerated and neither was replacement of the highly conserved residue glutamate 187 with alanine. Six strains carrying paired alanine substitutions within the ß9-ß10 ß-hairpin loop were constructed, leading to the conclusion that no individual amino acid within that hairpin loop is absolutely required for MCM function, although one of the mutant strains displays greatly enhanced sensitivity to mitomycin C. Deletions of two or four amino acids from the ß9-ß10 ß-hairpin were tolerated but mutants carrying larger deletions were inviable. Similarly, it was not possible to construct mutants in which any of the conserved zinc binding cysteines was replaced with alanine, underlining the likely importance of zinc binding for MCM function. The results of these studies demonstrate the feasibility of using Hfx. volcanii as a model system for reverse genetic analysis of archaeal MCM protein function and provide important confirmation of the in vivo importance of conserved structural features identified by previous bioinformatic, biochemical and structural studies.