Human Induced Pluripotent Stem Cell Encapsulation Geometry Impacts Three-Dimensional Developing Human Engineered Cardiac Tissue Functionality.

Cardiac tissue engineering has been working to alleviate the immense burden of cardiovascular disease for several decades. To improve cardiac tissue homogeneity and cardiomyocyte (CM) maturation, in this study, we investigated altering initial encapsulation geometry in a three-dimensional (3D) direct cardiac differentiation platform. Traditional engineered cardiac tissue production utilizes predifferentiated CMs to produce 3D cardiac tissue and often involves various cell selection and exogenous stimulation methods to promote CM maturation. Starting tissue formation directly with human induced pluripotent stem cells (hiPSCs), rather than predifferentiated CMs, simplifies the engineered cardiac tissue formation process, making it more applicable for widespread implementation and scale-up. In this study, hiPSCs were encapsulated in poly (ethylene glycol)-fibrinogen in three tissue geometries (disc-shaped microislands, squares, and rectangles) and subjected to established cardiac differentiation protocols. Resulting 3D engineered cardiac tissues (3D-ECTs) from each geometry displayed similar CM populations (∼65%) and gene expression over time. Notably, rectangular tissues displayed less tissue heterogeneity and suggested more advanced features of maturing CMs, including myofibrillar alignment and Z-line formation. In addition, rectangular tissue showed significantly higher anisotropic contractile properties compared to square and microisland tissues (MI 0.28 ± 0.03, SQ 0.35 ± 0.05, RT 0.79 ± 0.04). This study demonstrates a straightforward method for simplifying and improving 3D-ECT production without the use of exogenous mechanical or electrical pacing and has the potential to be utilized in bioprinting and drug testing applications. Impact statement Current methods for improving cardiac maturation postdifferentiation remain tedious and complex. In this study, we examined the impact of initial encapsulation geometry on improvement of three-dimensional engineered cardiac tissue (3D-ECT) production and postdifferentiation maturation for three tissue geometries, including disc-shaped microislands, squares, and rectangles. Notably, rectangular 3D-ECTs displayed less tissue heterogeneity and more advanced features of maturing cardiomyocytes, including myofibrillar alignment, Z-line formation, and anisotropic contractile properties, compared to microisland and square tissues. This study demonstrates an initial human induced pluripotent stem cell-encapsulated rectangular tissue geometry can improve cardiac maturation, rather than implementing cell selection or tedious postdifferentiation manipulation, including exogenous mechanical and/or electrical pacing.

Assessing the use of environmental DNA (eDNA) as a tool in the detection of human DNA in water.

Environmental DNA (eDNA) is a highly sensitive and cost-effective tool that is increasingly being applied to studies of biodiversity and species detection. This non-invasive method relies on the collection of environmental samples that contain genetic material being shed into surrounding environment by the target organism/s. While forensic science has a long history of using molecular tools for collecting DNA from the environment, the detection of human DNA from environmental water samples has been limited. This study investigated the detection and degradation rates of human eDNA in water samples under controlled laboratory conditions. Using a human-specific qPCR assay targeting the ND1 region of human mitochondrial DNA, eDNA degradation over time in water spiked with human blood was assessed. Recovery of nuclear DNA was investigated by determining if routine DNA short tandem repeat (STR) profiles of the blood source could be generated. Results demonstrated that human eDNA remains detectable for up to 11 days under laboratory conditions in environmental water and up to 35 days in distilled water. Partial STR profiles could be recovered from environmental water only up to 24 h, while, in distilled water, partial profiles continued to be recovered up to 840 h. These findings demonstrate that sampling human eDNA from aquatic samples can provide reliable human DNA detection within relatively short time windows, assisting law enforcement agencies by providing information about the potential time an individual may have been present in an area or assisting in the detection and location of a body or remains in aquatic environments.

Detecting marine pests using environmental DNA and biophysical models.

The spread of marine pests is occurring at record rates due to globalisation and increasing trade. Environmental DNA (eDNA) is an emerging tool for pest surveillance, allowing for the detection of genetic material shed by organisms into the environment. However, factors influencing the spatial and temporal detection limits of eDNA in marine environments are poorly understood. In this study we use eDNA assays to assess the invasive ranges of two marine pests in south-eastern Australia, the kelp Undaria pinnatifida and the seastar Asterias amurensis. We explored the temporal and spatial detection limits of eDNA under different oceanographic conditions by combining estimates of eDNA decay with biophysical modelling. Positive eDNA detections at several new locations indicate the invasive range of both pest species is likely to be wider than currently assumed. Environmental DNA decay rates were similar for both species, with a decay rate constant of 0.035 h-1 for U. pinnatifida, and a decay rate constant of 0.041 h-1 for A. amurensis, resulting in a 57-73% decrease in eDNA concentrations in the first 24 h and decaying beyond the limits of detection after 3-4 days. Biophysical models informed by eDNA decay profiles indicate passive transport of eDNA up to a maximum of 10 to 20 km from its source, with a ~90-95% reduction in eDNA concentration within 1-3 km from the source, depending on local oceanography. These models suggest eDNA signals are likely to be highly localised, even in complex marine environments. This was confirmed with spatially replicated eDNA sampling around an established U. pinnatifida population indicating detection limits of ~750 m from the source. This study highlights the value of eDNA methods for marine pest surveillance and provides a much-needed description of the spatio-temporal detection limits of eDNA under different oceanographic conditions.

Engineered cardiac tissue microsphere production through direct differentiation of hydrogel-encapsulated human pluripotent stem cells.

Engineered cardiac tissues that can be directly produced from human induced pluripotent stem cells (hiPSCs) in scalable, suspension culture systems are needed to meet the demands of cardiac regenerative medicine. Here, we demonstrate successful production of functional cardiac tissue microspheres through direct differentiation of hydrogel encapsulated hiPSCs. To form the microspheres, hiPSCs were suspended within the photocrosslinkable biomaterial, PEG-fibrinogen (25 million cells/mL), and encapsulated at a rate of 420,000 cells/minute using a custom microfluidic system. Even at this high cell density and rapid production rate, high intra-batch and batch-to-batch reproducibility was achieved. Following microsphere formation, hiPSCs maintained high cell viability and continued to grow within and beyond the original PEG-fibrinogen matrix. These initially soft microspheres (<250 Pa) supported efficient cardiac differentiation; spontaneous contractions initiated by differentiation day 8, and the microspheres contained >75% cardiomyocytes (CMs). CMs responded appropriately to pharmacological stimuli and exhibited 1:1 capture up to 6.0 Hz when electrically paced. Over time, cells formed cell-cell junctions and aligned myofibril fibers; engineered cardiac microspheres were maintained in culture over 3 years. The capability to rapidly generate uniform cardiac microsphere tissues is critical for advancing downstream applications including biomanufacturing, multi-well plate drug screening, and injection-based regenerative therapies.

Unconventional use of ablative fractional photothermolysis in arm contouring.

Brachioplasty, in addition to several nonsurgical interventions (e.g. cryolipolysis, noninvasive radiofrequency, and intense-focused ultrasound) have been described as efficacious in the elimination of excess skin laxity from the upper arms. Recently, fractional CO2 ablation has gained attention for its ability to reduce rhytids and improve skin texture on the face, neck, and hands. In this article, we report the first successful case of fractional CO2 ablation for upper arm contouring.

The Efficacy of Surgical Excision Plus Adjuvant Multimodal Therapies in the Treatment of Keloids: A Systematic Review and Meta-Analysis.

BACKGROUND: Research evaluating the efficacy of multimodal therapy for the treatment of keloids has reported combination regimens are most effective. OBJECTIVE: To compare recurrence rates for keloids treated with surgery plus one adjuvant intervention (dual therapy) versus surgery plus 2 or more adjuvant interventions (triple therapy). MATERIALS AND METHODS: Systematic literature review and meta-analysis of combination treatment for keloids. RESULTS: After full-text review, we included 60 articles representing 5,547 keloids: 5,243 received dual therapy, 259 received triple therapy, and 45 received quadruple therapy (the latter 2 groups were combined for analysis). The difference in recurrence rates between dual (19%) and triple therapy (11.2%) was not significant (p = .343). However, the difference in recurrence rates between dual therapy using surgery and radiation (18.7%) and triple therapy using surgery, radiation, and a third intervention (7.7%) was significant (p = .002). The differences for surgery and intralesional triamcinolone (TAC) showed trends toward significance, because keloids treated with dual therapy (21.7%) had a higher recurrence rate than those treated with triple therapy comprised of surgery, TAC, and another intervention (13.7%; p = .099). CONCLUSION: Triple therapy using surgery plus radiation and/or TAC as one of the adjuvant treatment modalities may achieve the lowest recurrence rates for keloids.

Direct Production of Human Cardiac Tissues by Pluripotent Stem Cell Encapsulation in Gelatin Methacryloyl.

Direct stem cell encapsulation and cardiac differentiation within supporting biomaterial scaffolds are critical for reproducible and scalable production of the functional human tissues needed in regenerative medicine and drug-testing applications. Producing cardiac tissues directly from pluripotent stem cells rather than assembling tissues using pre-differentiated cells can eliminate multiple cell-handling steps that otherwise limit the potential for process automation and production scale-up. Here we asked whether our process for forming 3D developing human engineered cardiac tissues using poly(ethylene glycol)-fibrinogen hydrogels can be extended to widely used and printable gelatin methacryloyl (GelMA) hydrogels. We demonstrate that low-density GelMA hydrogels can be formed rapidly using visible light (<1 min) and successfully employed to encapsulate human induced pluripotent stem cells while maintaining high cell viability. Resulting constructs had an initial stiffness of approximately 220 Pa, supported tissue growth and dynamic remodeling, and facilitated high-efficiency cardiac differentiation (>70%) to produce spontaneously contracting GelMA human engineered cardiac tissues (GEhECTs). GEhECTs initiated spontaneous contractions on day 8 of differentiation, with synchronicity, frequency, and velocity of contraction increasing over time, and displayed developmentally appropriate temporal changes in cardiac gene expression. GEhECT-dissociated cardiomyocytes displayed well-defined and aligned sarcomeres spaced at 1.85 ± 0.1 µm and responded appropriately to drug treatments, including the ß-adrenergic agonist isoproterenol and antagonist propranolol, as well as to outside pacing up to 3.0 Hz. Overall results demonstrate that GelMA is a suitable biomaterial for the production of developing cardiac tissues and has the potential to be employed in scale-up production and bioprinting of GEhECTs.

Modeling the Kinetics of Serum Glial Fibrillary Acidic Protein, Ubiquitin Carboxyl-Terminal Hydrolase-L1, and S100B Concentrations in Patients with Traumatic Brain Injury.

Glial fibrillary acidic protein (GFAP), ubiquitin carboxyl-terminal hydrolase-L1 (UCH-L1), and S100B have been shown to be predictive of patients with brain injury. Kinetics of these biomarkers in injured humans have not been extensively examined. This prospective multi-center study included patients with mild-to-moderate traumatic brain injury. Blood samples obtained at enrollment and every 6 h up to 24 h post-injury were assayed for GFAP, UCH-L1, and S100B. Random effects models examined changes in the biomarkers' level over time. A total of 167 patients were enrolled; mean age was 46.0 ± 17.8, 61.1% were male, 143 (85.6%) had a Glasgow Coma Scale score of 15, and 33 (19.8%) had a positive head computed tomography (CT) scan. Baseline median biomarker concentrations for all three were higher among CT-positive patients (p < 0.0001) but GFAP was the only biomarker that significantly increased over time among CT-positive patients relative to CT-negative patients (log transformed values 0.037; 95% confidence interval 0.02, 0.05; p < 0.001), indicating a 3.7% per hour rise in GFAP concentration. There was no significant increase in either UCH-L1 or S100B in CT-positive patients (p = 0.15 and p = 0.47, respectively). GFAP concentrations increased 3.7% per hour among CT-positive patients whereas neither UCH-L1 nor S100B increased, compared with CT-negative patients. The kinetics and temporal profile of GFAP suggest it may be a more robust biomarker to detect patients with positive CT findings, particularly at later post-injury times. Further study is needed to determine if GFAP is a useful test to follow throughout a patient's clinical course.

Utility of point of care assessment of platelet reactivity (using the PFA-100®) to aid in diagnosis of stroke.

BACKGROUND: Rapid and accurate diagnosis of patients presenting with symptoms of stroke is needed to facilitate the timely delivery of proven effective treatment for patients with acute ischemic stroke (AIS). The aim of this study was to determine whether early assessment of platelet reactivity in patients presenting with symptoms of AIS was associated with a diagnosis of AIS, transient ischemic attack (TIA), or stroke mimic. METHODS: This prospective study included patients with symptoms of AIS treated at an inner-city emergency department (ED). Blood samples were obtained and assayed for platelet reactivity (quantified by closure time). Patients were grouped by discharge diagnosis into: AIS, TIA, or stroke mimic. Binary logistic regression model was used to predict the association of closure time with the final diagnosis of 1) either AIS or TIA or, 2) stroke mimic. RESULTS: Of 114 patients enrolled, 32 were diagnosed with AIS, 33 TIA, and 49 were diagnosed as a stroke mimic. There was no significant difference in closure times among patients with a diagnosis of AIS or TIA versus stroke mimic. A history of migraines and history of seizures were independently associated with lower odds of an AIS or TIA diagnosis (OR 0.31, 95% CI 0.10 to 0.94 and OR 0.08, 95% CI 0.01 to 0.88, respectively). CONCLUSION: Closure time was not found to be a clinically reliable differentiator of patients with a diagnosis of AIS, TIA, or stroke mimic in the ED.

Characterization of a peptide inhibitor of Janus kinase 2 that mimics suppressor of cytokine signaling 1 function.

Positive and negative regulation of cytokines such as IFN-gamma are key to normal homeostatic function. Negative regulation of IFN-gamma in cells occurs via proteins called suppressors of cytokine signaling (SOCS)1 and -3. SOCS-1 inhibits IFN-gamma function by binding to the autophosphorylation site of the tyrosine kinase Janus kinase (JAK)2. We have developed a short 12-mer peptide, WLVFFVIFYFFR, that binds to the autophosphorylation site of JAK2, resulting in inhibition of its autophosphorylation as well as its phosphorylation of IFN-gamma receptor subunit IFNGR-1. The JAK2 tyrosine kinase inhibitor peptide (Tkip) did not bind to or inhibit tyrosine autophosphorylation of vascular endothelial growth factor receptor or phosphorylation of a substrate peptide by the protooncogene tyrosine kinase c-src. Tkip also inhibited epidermal growth factor receptor autophosphorylation, consistent with the fact that epidermal growth factor receptor is regulated by SOCS-1 and SOCS-3, similar to JAK2. Although Tkip binds to unphosphorylated JAK2 autophosphorylation site peptide, it binds significantly better to tyrosine-1007 phosphorylated JAK2 autophosphorylation site peptide. SOCS-1 only recognizes the JAK2 site in its phosphorylated state. Thus, Tkip recognizes the JAK2 autophosphorylation site similar to SOCS-1, but not precisely the same way. Consistent with inhibition of JAK2, Tkip inhibited the ability of IFN-gamma to induce an antiviral state as well as up-regulate MHC class I molecules on cells at a concentration of approximately 10 microM. This is similar to the K(d) of SOCS-3 for the erythropoietin receptor. These data represent a proof-of-concept demonstration of a peptide mimetic of SOCS-1 that regulates JAK2 tyrosine kinase function.