Cardiomyocyte generation from somatic sources — current status and future directions

Transdifferentiation of one cell type to another has garnered significant research efforts in recent years. As cardiomyocyte loss following myocardial infarction becomes debilitating for cardiac patients, the option of an autologous source of cardiomyocytes not derived from multi/pluripotent stem cell sources is an attractive option. Such direct programming has been clearly realized with the use of transcription factors, microRNAs and more recently small molecule delivery to enhance epigenetic modifications, all albeit with low efficiencies in vitro. In this review, we aim to present a brief overview of the current in vitro and in vivo transdifferentiation strategies in the generation of cardiomyocytes from somatic sources. The interdisciplinary fields of tissue, cell, material and regenerative engineering offer many opportunities to synergistically achieve directly programmed cardiac tissue in vitro and enhance transdifferentiation in vivo. This review aims to present a concise outlook on this topic with these fields in mind

Preserved bioactivity and tunable release of a SDF1-GPVI bi-specific protein using photo-crosslinked PEGda hydrogels

AbstractChemokine-induced stem cell recruitment is a promising strategy for post myocardial infarction treatment. Injection of stromal cell-derived factor 1 (SDF1) has been shown to attract bone marrow-derived progenitor cells (BMPCs) from the blood that have the potential to differentiate into cardiovascular cells, which support angiogenesis, enabling the improvement of myocardial function. SDF1-GPVI bi-specific protein contains a glycoprotein VI (GPVI)-domain that serves as an anchor for collagen type I (Col I) and III, which are exposed in the wall of injured vasculature. In this study, we generated a cytocompatible hydrogel via photo-crosslinking of poly(ethylene glycol) diacrylate that serves as a reservoir for SDF1-GPVI. Controlled and sustained release of SDF1-GPVI was demonstrated over a period of 7 days. Release features were modifiable depending on the degree of the crosslinking density. Functionality of the GPVI-domain was investigated using a GPVI-binding ELISA to Col I. Activity of the SDF1-domain was tested for its CXCR4 binding potential. Preserved functionality of SDF1-GPVI bi-specific protein after photo-crosslinking and controllable release was successfully demonstrated in vitro supporting the implementation of this drug delivery system as a powerful tool for therapeutic protein delivery in the treatment of cardiovascular ischemic disease

On the reproducibility of extrusion-based bioprinting: round robin study on standardization in the field

The outcome of three-dimensional (3D) bioprinting heavily depends, amongst others, on the interaction between the developed bioink, the printing process, and the printing equipment. However, if this interplay is ensured, bioprinting promises unmatched possibilities in the health care area. To pave the way for comparing newly developed biomaterials, clinical studies, and medical applications (i.e. printed organs, patient-specific tissues), there is a great need for standardization of manufacturing methods in order to enable technology transfers. Despite the importance of such standardization, there is currently a tremendous lack of empirical data that examines the reproducibility and robustness of production in more than one location at a time. In this work, we present data derived from a round robin test for extrusion-based 3D printing performance comprising 12 different academic laboratories throughout Germany and analyze the respective prints using automated image analysis (IA) in three independent academic groups. The fabrication of objects from polymer solutions was standardized as much as currently possible to allow studying the comparability of results from different laboratories. This study has led to the conclusion that current standardization conditions still leave room for the intervention of operators due to missing automation of the equipment. This affects significantly the reproducibility and comparability of bioprinting experiments in multiple laboratories. Nevertheless, automated IA proved to be a suitable methodology for quality assurance as three independently developed workflows achieved similar results. Moreover, the extracted data describing geometric features showed how the function of printers affects the quality of the printed object. A significant step toward standardization of the process was made as an infrastructure for distribution of material and methods, as well as for data transfer and storage was successfully established

On the reproducibility of extrusion-based bioprinting: round robin study on standardization in the field

The outcome of three-dimensional (3D) bioprinting heavily depends, amongst others, on the interaction between the developed bioink, the printing process, and the printing equipment. However, if this interplay is ensured, bioprinting promises unmatched possibilities in the health care area. To pave the way for comparing newly developed biomaterials, clinical studies, and medical applications (i.e. printed organs, patient-specific tissues), there is a great need for standardization of manufacturing methods in order to enable technology transfers. Despite the importance of such standardization, there is currently a tremendous lack of empirical data that examines the reproducibility and robustness of production in more than one location at a time. In this work, we present data derived from a round robin test for extrusion-based 3D printing performance comprising 12 different academic laboratories throughout Germany and analyze the respective prints using automated image analysis (IA) in three independent academic groups. The fabrication of objects from polymer solutions was standardized as much as currently possible to allow studying the comparability of results from different laboratories. This study has led to the conclusion that current standardization conditions still leave room for the intervention of operators due to missing automation of the equipment. This affects significantly the reproducibility and comparability of bioprinting experiments in multiple laboratories. Nevertheless, automated IA proved to be a suitable methodology for quality assurance as three independently developed workflows achieved similar results. Moreover, the extracted data describing geometric features showed how the function of printers affects the quality of the printed object. A significant step toward standardization of the process was made as an infrastructure for distribution of material and methods, as well as for data transfer and storage was successfully established

Inhibition of SLPI ameliorates disease activity in experimental autoimmune encephalomyelitis

<p>Abstract</p> <p>Background</p> <p>The secretory leukocyte protease inhibitor (SLPI) exerts wide ranging effects on inflammatory pathways and is upregulated in EAE but the biological role of SLPI in EAE, an animal model of multiple sclerosis is unknown</p> <p>Methods</p> <p>To investigate the pathophysiological effects of SLPI within EAE, we induced SLPI-neutralizing antibodies in mice and rats to determine the clinical severity of the disease. In addition we studied the effects of SLPI on the anti-inflammatory cytokine TGF-β.</p> <p>Results</p> <p>The induction of SLPI neutralizing antibodies resulted in a milder disease course in mouse and rat EAE. SLPI neutralization was associated with increased serum levels of TGF-β and increased numbers of FoxP3+ CD4+ T cells in lymph nodes. <it>In vitro</it>, the addition of SLPI significantly decreased the number of functional FoxP3+ CD25^hiCD4+ regulatory T cells in cultures of naive human CD4+ T cells. Adding recombinant TGF-β to SLPI-treated human T cell cultures neutralized SLPI's inhibitory effect on regulatory T cell differentiation.</p> <p>Conclusion</p> <p>In EAE, SLPI exerts potent pro-inflammatory actions by modulation of T-cell activity and its neutralization may be beneficial for the disease.</p

Generation of Functional Neutrophils from a Mouse Model of X-Linked Chronic Granulomatous Disorder Using Induced Pluripotent Stem Cells

Murine models of human genetic disorders provide a valuable tool for investigating the scope for application of induced pluripotent stem cells (iPSC). Here we present a proof-of-concept study to demonstrate generation of iPSC from a mouse model of X-linked chronic granulomatous disease (X-CGD), and their successful differentiation into haematopoietic progenitors of the myeloid lineage. We further demonstrate that additive gene transfer using lentiviral vectors encoding gp91(phox) is capable of restoring NADPH-oxidase activity in mature neutrophils derived from X-CGD iPSC. In the longer term, correction of iPSC from human patients with CGD has therapeutic potential not only through generation of transplantable haematopoietic stem cells, but also through production of large numbers of autologous functional neutrophils

Circulating Pneumolysin Is a Potent Inducer of Cardiac Injury during Pneumococcal Infection

Streptococcus pneumoniae accounts for more deaths worldwide than any other single pathogen through diverse disease manifestations including pneumonia, sepsis and meningitis. Life-threatening acute cardiac complications are more common in pneumococcal infection compared to other bacterial infections. Distinctively, these arise despite effective antibiotic therapy. Here, we describe a novel mechanism of myocardial injury, which is triggered and sustained by circulating pneumolysin (PLY). Using a mouse model of invasive pneumococcal disease (IPD), we demonstrate that wild type PLY-expressing pneumococci but not PLY-deficient mutants induced elevation of circulating cardiac troponins (cTns), well-recognized biomarkers of cardiac injury. Furthermore, elevated cTn levels linearly correlated with pneumococcal blood counts (r=0.688, p=0.001) and levels were significantly higher in non-surviving than in surviving mice. These cTn levels were significantly reduced by administration of PLY-sequestering liposomes. Intravenous injection of purified PLY, but not a non-pore forming mutant (PdB), induced substantial increase in cardiac troponins to suggest that the pore-forming activity of circulating PLY is essential for myocardial injury in vivo. Purified PLY and PLY-expressing pneumococci also caused myocardial inflammatory changes but apoptosis was not detected. Exposure of cultured cardiomyocytes to PLY-expressing pneumococci caused dose-dependent cardiomyocyte contractile dysfunction and death, which was exacerbated by further PLY release following antibiotic treatment. We found that high PLY doses induced extensive cardiomyocyte lysis, but more interestingly, sub-lytic PLY concentrations triggered profound calcium influx and overload with subsequent membrane depolarization and progressive reduction in intracellular calcium transient amplitude, a key determinant of contractile force. This was coupled to activation of signalling pathways commonly associated with cardiac dysfunction in clinical and experimental sepsis and ultimately resulted in depressed cardiomyocyte contractile performance along with rhythm disturbance. Our study proposes a detailed molecular mechanism of pneumococcal toxin-induced cardiac injury and highlights the major translational potential of targeting circulating PLY to protect against cardiac complications during pneumococcal infections

Induction of CD4+CD25+FOXP3+ Regulatory T Cells during Human Hookworm Infection Modulates Antigen-Mediated Lymphocyte Proliferation

Hookworm infection is considered one of the most important poverty-promoting neglected tropical diseases, infecting 576 to 740 million people worldwide, especially in the tropics and subtropics. These blood-feeding nematodes have a remarkable ability to downmodulate the host immune response, protecting themselves from elimination and minimizing severe host pathology. While several mechanisms may be involved in the immunomodulation by parasitic infection, experimental evidences have pointed toward the possible involvement of regulatory T cells (Tregs) in downregulating effector T-cell responses upon chronic infection. However, the role of Tregs cells in human hookworm infection is still poorly understood and has not been addressed yet. In the current study we observed an augmentation of circulating CD4+CD25+FOXP3+ regulatory T cells in hookworm-infected individuals compared with healthy non-infected donors. We have also demonstrated that infected individuals present higher levels of circulating Treg cells expressing CTLA-4, GITR, IL-10, TGF-β and IL-17. Moreover, we showed that hookworm crude antigen stimulation reduces the number of CD4+CD25+FOXP3+ T regulatory cells co-expressing IL-17 in infected individuals. Finally, PBMCs from infected individuals pulsed with excreted/secreted products or hookworm crude antigens presented an impaired cellular proliferation, which was partially augmented by the depletion of Treg cells. Our results suggest that Treg cells may play an important role in hookworm-induced immunosuppression, contributing to the longevity of hookworm survival in infected people