Lab News

Impact of pulse duration on cardiac electroporation: nanosecond pulses enhance cardiomyocyte selectivity and promote a Raman-detected shift towards apoptotic cell death

Aims: Pulsed field ablation (PFA), a cardiac ablation technique using microsecond pulsed electric fields (µsPEF), is widely used in clinical settings, while nanosecond pulsed electric fields (nsPEF) have recently entered clinical trials. Selective ablation of cardiomyocytes over endothelial cells is critical to prevent adverse remodelling, arrhythmias, and thrombosis, yet comparative data on nsPEF vs. µsPEF remain limited. This study investigates the cytotoxic effects and cell death mechanisms induced by nsPEF and µsPEF in cardiac and endothelial cells. Methods and results: Human cardiomyocytes and endothelial cells were exposed to varying electric field intensities with nsPEF and µsPEF using custom-built automated setup to assess permeabilization and cell death. Raman spectroscopy evaluated biochemical changes in cardiomyocytes following electroporation. Ex vivo epicardial ablation was performed on murine hearts using customized electrodes. Maximal cardiomyocyte death occurred 24 h after both pulse types in vitro. Ex vivo, both pulse types produced visible myocardial lesions as early as 1 h post-exposure, with lesion size progressively increasing up to 4 h. Microsecond pulsed electric fields induced significantly greater endothelial damage (ED50: 1.18 kV/cm) than damage to cardiomyocytes (ED50: 1.28 kV/cm), whereas nsPEF affected both cell types equally (ED50: 7.27 kV/cm vs. 7.24 kV/cm). Raman spectroscopy analysis of exposed cells indicated that µsPEF predominantly triggered necrotic or unregulated cell death, while nsPEF exposure was associated with regulated, apoptotic cell death. Conclusion: Pulse duration critically determines electroporation selectivity and downstream death pathways. Nanosecond pulsed electric fields favoured regulated cell death and cardiomyocyte selectivity, highlighting its potential to improve the safety and durability of PFA.

ECM Proteins Nidogen-1 and Decorin Restore Functionality of Human Islets of Langerhans upon Hypoxic Conditions

Summary: Transplantation of donor islets of Langerhans is a potential therapeutic approach for patients with diabetes mellitus; however, its success is limited by islet death and dysfunction during the initial hypoxic conditions at the transplantation site. This highlights the need to support the donor islets in the days post-transplantation until the site is vascularized. It was previously demonstrated that the extracellular matrix (ECM) proteins nidogen-1 (NID1) and decorin (DCN) improve the functionality and survival of the β-cell line, EndoC-βH3, and the viability of human islets post-isolation. To advance the use of these ECM proteins toward a clinical application and elucidate the mechanisms of action in primary islets, the study assesses the effects of ECM proteins NID1 and DCN on isolated human donor islets cultured in normoxic and hypoxic conditions. NID1- and DCN-treatment restore β-cell functionality of human donor islets in a hypoxic environment through upregulation of genes involved in glycolytic pathways and reducing DNA fragmentation in hypoxic conditions comparable to normoxic control islets. The results demonstrate that the utilization of NID1 or DCN with islets of Langerhans may have the potential to overcome the hypoxia-induced cell death observed post-transplantation and improve transplant outcomes.

MYCT1 controls environmental sensing in human haematopoietic stem cells

Summary: The processes that govern human haematopoietic stem cell (HSC) self-renewal and engraftment are poorly understood and challenging to recapitulate in culture to reliably expand functional HSCs1–3. Here we identify MYC target 1 (MYCT1; also known as MTLC) as a crucial human HSC regulator that moderates endocytosis and environmental sensing in HSCs. MYCT1 is selectively expressed in undifferentiated human haematopoietic stem and progenitor cells (HSPCs) and endothelial cells but becomes markedly downregulated during HSC culture. Lentivirus-mediated knockdown of MYCT1 prevented human fetal liver and cord blood (CB) HSPC expansion and engraftment. By contrast, restoring MYCT1 expression improved the expansion and engraftment of cultured CB HSPCs. Single-cell RNA sequencing of human CB HSPCs in which MYCT1 was knocked down or overexpressed revealed that MYCT1 governs important regulatory programmes and cellular properties essential for HSC stemness, such as ETS factor expression and low mitochondrial activity. MYCT1 is localized in the endosomal membrane in HSPCs and interacts with vesicle trafficking regulators and signalling machinery. MYCT1 loss in HSPCs led to excessive endocytosis and hyperactive signalling responses, whereas restoring MYCT1 expression balanced culture-induced endocytosis and dysregulated signalling. Moreover, sorting cultured CB HSPCs on the basis of lowest endocytosis rate identified HSPCs with preserved MYCT1 expression and MYCT1-regulated HSC stemness programmes. Our work identifies MYCT1-moderated endocytosis and environmental sensing as essential regulatory mechanisms required to preserve human HSC stemness. Our data also pinpoint silencing of MYCT1 as a cell-culture-induced vulnerability that compromises human HSC expansion.

Translating genomic tools to Raman spectroscopy analysis enables high-dimensional tissue characterization on molecular resolution

Summary: Spatial transcriptomics of histological sections have revolutionized research in life sciences and enabled unprecedented insights into genetic processes involved in tissue reorganization. However, in contrast to genomic analysis, the actual biomolecular composition of the sample has fallen behind, leaving a gap of potentially highly valuable information. Raman microspectroscopy provides untargeted spatiomolecular information at high resolution, capable of filling this gap. In this study we demonstrate spatially resolved Raman "spectromics" to reveal homogeneity, heterogeneity and dynamics of cell matrix on molecular levels by repurposing state-of-the-art bioinformatic analysis tools commonly used for transcriptomic analyses. By exploring sections of murine myocardial infarction and cardiac hypertrophy, we identify myocardial subclusters when spatially approaching the pathology, and define the surrounding metabolic and cellular (immune-) landscape. Our innovative, label-free, non-invasive "spectromics" approach could therefore open perspectives for a profound characterization of histological samples, while additionally allowing the combination with consecutive downstream analyses of the very same specimen.

Nidogen-1 mitigates ischemia and promotes tissue survival and regeneration

Summary: Ischemia impacts multiple organ systems and is the major cause of morbidity and mortality in the developed world. Ischemia disrupts tissue homeostasis, driving cell death, and damages tissue structure integrity. Strategies to heal organs, like the infarcted heart, or to replace cells, as done in pancreatic islet beta-cell transplantations, are often hindered by ischemic conditions. Here, it is discovered that the basement membrane glycoprotein nidogen‐1 attenuates the apoptotic effect of hypoxia in cardiomyocytes and pancreatic beta-cells via the αvβ3 integrin and beneficially modulates immune responses in vitro. It is shown that nidogen-1 significantly increases heart function and angiogenesis, while reducing fibrosis, in a mouse postmyocardial infarction model. These results demonstrate the protective and regenerative potential of nidogen-1 in ischemic conditions.

Macrophage retrival from 3D biomaterials: A detailed comparison of common dissociation methods

Summary: The immune system is widely recognized as a crucial determinant in implant biocompatibility and tissue integration. In order to assess macrophage response to biomaterials, commonly used analysis techniques require the removal of cells from the material. This process inherently has a negative impact on the cells, but few studies have comprehensively compared different dissociation methods in terms of impact on cell yield, viability, phenotype and function. Cell scraping, EDTA at 4 °C, EDTA at 37 °C, trypsin, Accutase and the PromoCell macrophage detachment solution were compared in terms of cell yield and viability. The effect of Accutase on cell surface antigen conservancy and cell functionality was investigated. Lastly, effect of Accutase detachment of macrophages from electrospun biomaterials was analyzed. The efficiency of common cell detachment protocols for human monocyte-derived macrophages (MDMs) varies significantly between enzymatic and non-enzymatic approaches. In terms of surface marker detection, we showed that enzymatic detachment not only selectively cleaves the M2 markers CD206 and CD163, but we also identified that this effect is variable across donors. Furthermore, we observed that the process of cell detachment impairs macrophage endocytic ability. Lastly, we tested the efficiency of enzymatic cell detachment on electrospun 3D matrices designed for tissue engineering.

Fluorescence lifetime metabolic mapping of hypoxia-induced damage in pancreatic pseudo-islets

Summary: Pancreatic islet isolation from donor pancreases is an essential step for the transplantation of insulin-secreting beta-cells as a therapy to treat type 1 diabetes mellitus. This process however damages islet basement membranes, which can lead to islet dysfunction or death. Posttransplantation, islets are further stressed by a hypoxic environment and immune reactions that cause poor engraftment and graft failure. The current standards to assess islet quality before transplantation are destructive procedures, performed on a small islet population that does not reflect the heterogeneity of large isolated islet batches. In this study, we incorporated fluorescence lifetime imaging microscopy (FLIM) into a pancreas-on-chip system to establish a protocol to noninvasively assess the viability and functionality of pancreatic beta-cells in a three-dimensional in vitro model (= pseudoislets). We demonstrate how (pre) hypoxic beta-cell composed pseudoislets can be discriminated from healthy functional pseudo‐islets according to their FLIM-based metabolic profiles. The use of FLIM during the pretransplantation pancreatic islet selection process has the potential to improve the outcome of beta-cell islet transplantation.

Summary: The use of biomaterials and biomaterial functionalization is a promising approach to support pancreatic islet viability posttransplantation in an effort to reduce insulin dependence for patients afflicted with diabetes mellitus type 1. Extracellular matrix (ECM) proteins are known to impact numerous reparative functions in the body. Assessing how endogenously expressed pancreatic ECM proteins are affected by posttransplant-like hypoxic conditions may provide significant insights toward the development of tissue-engineered therapeutic strategies to positively influence β-cell survival, proliferation, and functionality. Here, we investigated the expression of three relevant groups of pancreatic ECM proteins in human native tissue, including basement membrane (BM) proteins (collagen type 4 [COL4], laminins [LAM]), proteoglycans (decorin [DCN], nidogen-1 [NID1]), and fibril-forming proteins (fibronectin [FN], collagen type 1 [COL1]). In an in vitro hypoxia model, we identified that ECM proteins were differently affected by hypoxic conditions, contributing to an overall loss of β-cell functionality. The use of a COL1 hydrogel as carrier material demonstrated a protective effect on β-cells mitigating the effect of hypoxia on proteoglycans as well as fibril-forming protein expression, supporting β-cell functionality in hypoxia. We further showed that providing endothelial cells (ECs) into the COL1 hydrogel improves β-cell response as well as the expression of relevant BM proteins. Our data show that β-cells benefit from a microenvironment composed of structure-providing COL1 with the incorporation of ECs to withstand the harsh conditions of hypoxia. Such hydrogels support β-cell survival and can serve as an initial source of ECM proteins to allow cell engraftment while preserving cell functionality posttransplantation.

Summary: Appropriate mechanical properties and fast endothelialization of synthetic grafts are key to ensure long-term functionality of implants. We used a newly developed biostable polyurethane elastomer (TPCU) to engineer electrospun vascular scaffolds with promising mechanical properties (E-modulus: 4.8 ± 0.6 MPa, burst pressure: 3326 ± 78 mmHg), which were biofunctionalized with fibronectin (FN) and decorin (DCN). Neither uncoated nor biofunctionalized TPCU scaffolds induced major adverse immune responses except for minor signs of polymorph nuclear cell activation. The in vivo endothelial progenitor cell homing potential of the biofunctionalized scaffolds was simulated in vitro by attracting endothelial colony-forming cells (ECFCs). Although DCN coating did attract ECFCs in combination with FN (FN + DCN), DCN-coated TPCU scaffolds showed a cell-repellent effect in the absence of FN. In a tissue-engineering approach, the electrospun and biofunctionalized tubular grafts were cultured with primary-isolated vascular endothelial cells in a custom-made bioreactor under dynamic conditions with the aim to engineer an advanced therapy medicinal product. Both FN and FN + DCN functionalization supported the formation of a confluent and functional endothelial layer.

Summary: Electrospinning is an attractive method to generate drug releasing systems. In this work, we encapsulated the cell death-inducing drug Diclofenac (DCF) in an electrospun poly-L-lactide (PLA) scaffold. The scaffold offers a system for a sustained and controlled delivery of the cytotoxic DCF over time making it clinically favourable by achieving a prolonged therapeutic effect. We exposed human dermal fibroblasts (HDFs) to the drug-eluting scaffold and employed multiphoton microscopy and fluorescence lifetime imaging microscopy. These methods were suitable for non-invasive and marker-independent assessment of the cytotoxic effects. Released DCF induced changes in cell morphology and glycolytic activity. Furthermore, we showed that drug release can be influenced by adding dimethyl sulfoxide as a co-solvent for electrospinning. Interestingly, without affecting the drug diffusion mechanism, the resulting PLA scaffolds showed altered fibre morphology and enhanced initial DCF burst release. The here described model could represent an interesting way to control the diffusion of encapsulated bio-active molecules and test them using a marker-independent, non-invasive approach.

Summary: Cryomedium toxicity is a major safety concern when transplanting cryopreserved organs. Therefore, thorough removal of potentially toxic cryoprotective agents (CPAs) is required before transplantation. CPAs such as dimethyl-sulfoxide (DMSO), propylene glycol (PG), and formamide (FMD), routinely employed in ice-free cryopreservation (IFC), have advantages in long-term preservation of tissue structures compared with conventional cryopreservation employing lower CPA concentrations. This study evaluated the impact of potential residual CPAs on human cardiac valves. Raman microspectroscopy and Raman imaging were established as nondestructive marker-independent techniques for in situ quantitative assessment of CPA residues in IFC valve tissues. In detail, IFC valve leaflets and supernatants of the washing solutions were analyzed to determine the washing efficiency. A calibration model was developed according to the CPA’s characteristic Raman signals to quantify DMSO, PG and FMD concentrations in the supernatants. Single point Raman measurements were performed on the intact tissues to analyze penetration properties. In addition, Raman imaging was utilized to visualize potential CPA residues. Our data showed that washing decreased the CPA concentration in the final washing solution by 99%, and no residues could be detected in the washed tissues, validating the multistep CPA removal protocol routinely used for IFC valves. Raman analysis of unwashed tissues showed different permeation characteristics depending on each CPA and their concentration. Our results demonstrate a great potential of Raman microspectroscopy and Raman imaging as marker-independent in situ tissue quality control tools with the ability to assess the presence and concentration of different chemical agents or drugs in preimplantation tissues.

The Central Ethics Committee for Stem Cell Research (Zentrale Ethik-Kommission für Stammzellenforschung, ZES) was established in 2002 as part of the German Stem Cell Act. The committee is an independent and interdisciplinary expert body whose activities are governed by this law, the act ensuring the protection of embryos in conjunction with the import and use of human embryonic stem cells (Stem Cell Act – StZG) dated 28 June 2002 and by the regulation concerning the Central Ethics Committee for Stem Cell Research and the competent authority pursuant to the Stem Cell Act (ZES Regulation – ZESV) dated 18 July 2002. The commission, which performs its duties on a voluntary basis, comprises 18 members and deputy members appointed by the Federal Government for a term of three years. The members represent the fields of biology, medicine and philosophical, medical and theological ethics. The committee’s remit is to review and assess applications for the import and use of human embryonic stem cells (hESCs). For every application submitted, the committee issues an opinion which is then sent to the Robert Koch Institute (RKI), the competent authority under the Stem Cell Act. Text from here

The award was given to Nian at a conference in Berlin for her excellent scientific achievement.

Prof. Dr. Katja Schenke-Layland took over the management of the Natural and Medical Institute (NMI) in Reutlingen on April 1, 2018. She succeeds Prof. Dr. med. Hugo Hämmerle, who has headed the institute since 2008. The new woman at the head of the NMI is an old face in the institute's networks: since 2011, Schenke-Layland has been a professor of medical technology and regenerative medicine at the Eberhard Karls University of Tübingen and is involved in numerous biomedical research projects with the NMI. During her career at the Fraunhofer Institute for Interfacial Engineering and Biotechnology IGB in Stuttgart, where she has been working since January 2016 together with Prof. Dr. med. Christian Oehr temporarily headed the institute. As head of the NMI, the 41-year-old biologist now brings her expertise in research and management together in Reutlingen, while at the same time continuing her professorship. "I have always felt the need to promote a socially relevant, innovation-oriented research with the aim of application in medical technology and regenerative or personalized medicine, which represent important future fields for the economy of Baden-Württemberg," says Schenke-Layland.

Summary: The extracellular matrix (ECM) is extensively remodeled in tumor tissues. Overproduction of collagens, pathological collagen crosslinking and alignment of fibers are major processes that ultimately result in an increased tissue stiffness. Although it is known that glycosaminoglycans (GAGs) play an important role in tumor signaling, their contribution to the biomechanical properties of tumor ECM is unknown. In this study, ECM structures of human colon carcinoma and normal (control) colon tissues were histologically identified. Using atomic force microscopy (AFM) nanoindentation, we show that the collagen-rich regions within the ECM of colon carcinoma tissues were significantly stiffer than the submucosal collagen-rich layer of control tissues. Screening of these regions with Raman microspectroscopy revealed significantly different molecular fingerprints for collagen fibers in colon carcinoma tissues compared to control tissues. We further showed an increased alignment of collagen fibers and elevated levels of GAG immuno-reactivity within the collagen network of colon carcinoma tissues. GAGs such as heparan sulfate and chondroitin sulfate were detected in significantly elevated levels in collagen fibers of carcinoma tissues. Moreover, immunodetection of the collagen-associated proteoglycan decorin was significantly decreased in carcinomas tissues of individual patients when compared with the corresponding control tissues. Overall a strong patient-to-patient variability was evident in the ECM composition, structure and biomechanics of individual colon carcinoma tissues. Although, biomechanical characteristics of tumor ECM were not directly influenced by GAG content, GAGs might play an important role during the mechanical and structural remodeling of pathological tumor ECM. To manipulate GAG expression and deposition in tumor microenvironments could represent a novel potential therapeutic strategy.

Summary: Decorin (DCN) is an important small leucine-rich proteoglycan present in the extracellular matrix (ECM) of many organs and tissues. Endothelial progenitor cells (EPCs) are able to interact with the surrounding ECM and bind to molecules such as DCN. Here, we recombinantly produced full-length human DCN under good laboratory practice (GLP) conditions, and after detailed immunological characterization, we investigated its potential to attract murine and human EPCs (mEPCs and hECFCs). Electrospun polymeric scaffolds were coated with DCN or stromal cell-derived factor-1 (SDF-1α) and were then dynamically cultured with both cell types. Cell viability was assessed via imaging flow cytometry. The number of captured cells was counted and compared with the non-coated controls. To characterize cell-scaffold interactions, immunofluorescence staining and scanning electron microscopy analyses were performed. We identified that DCN reduced T cell responses and attracted innate immune cells, which are responsible for ECM remodeling. A significantly higher number of EPCs attached on DCN- and SDF-1α-coated scaffolds, when compared with the uncoated controls. Interestingly, DCN showed a higher attractant effect on hECFCs than SDF-1α. Here, we successfully demonstrated DCN as promising EPC-attracting coating, which is particularily interesting when aiming to generate off-the-shelf biomaterials with the potential of in vivo cell seeding.

Summary: Bioreactors are essential cell and tissue culture tools that allow the introduction of biophysical signals into in vitro cultures. One major limitation is the need to interrupt experiments and sacrifice samples at certain time points for analyses. To address this issue, we designed a bioreactor that combines high-resolution contact-free imaging and continuous flow in a closed system that is compatible with various types of microscopes. The high throughput fluid flow bioreactor was combined with two-photon fluorescence lifetime imaging microscopy (2P-FLIM) and validated. The hydrodynamics of the bioreactor chamber were characterized using COMSOL. The simulation of shear stress indicated that the bioreactor system provides homogeneous and reproducible flow conditions. The designed bioreactor was used to investigate the effects of low shear stress on human umbilical vein endothelial cells (HUVECs). In a scratch assay, we observed decreased migration of HUVECs under shear stress conditions. Furthermore, metabolic activity shifts from glycolysis to oxidative phosphorylation-dependent mechanisms in HUVECs cultured under low shear stress conditions were detected using 2P-FLIM. Future applications for this bioreactor range from observing cell fate development in real-time to monitoring the environmental effects on cells or metabolic changes due to drug applications.

Award summary: MIT Technology Review, as a rule, focuses on the technology first—the breakthrough, the surprise, the accidental discovery with the potential to upend the way we live. Our annual look at 35 outstanding innovators under 35 is a reminder that behind all those innovations are people with dreams, fears, and ambitions. Sometimes they hack away at a problem for years before figuring out a way forward. Sometimes they stumble on a solution they didn’t know they were searching for. We hope these portraits offer a sense of the variety of work being done in technology, and a sense of what’s coming next. The Inventors: Creating the breakthroughs that will make everything from AI to solar power to heart valves more practical and essential. Svenja Hinderer: A design for a heart valve that’s biodegradable—potentially eliminating the need for repeat surgeries.

Innovation Grants in the Sciences and Life Sciences make it possible to test the viability of possible applications arising from the results of basic research. Junior researchers (postdocs who have recently completed a PhD) will be given the chance to turn results attained from their PhD studies into usable products. To be eligible for funding, projects must demonstrate a reasonable prospect of becoming commercially viable or of leading to collaboration with non-University institutions. Funding: Innovation Grants may be used to finance the applicant’s position, one other position and material expenses. Innovation Grants last for one year with an option to extend for a further year, by which time they must either be completed or have secure external funding. Project details cannot be disclosed due to pending IP processes.

The Athene Program of the University of Tübingen aims to attract highly promising female early career researchers to an academic career by offering them support for their academic work, individual career planning and opportunities for gaining overarching professional competences.

Award summary: Many newborns have heart problems. But therapy is difficult because artificial heart valves do not grow. So the doctors have to do several surgeries - or wait for the children to be old enough for an adult valve. Svenja Hinderer has developed implants that stimulate the development of a natural heart valve in the body. They consist of a protein-coated polymer, to which stem cells can dock and form a new tissue.

Svenja was also nominated as a finalist for the European award for the top innovators under 35, which will be awarded later this year in Paris, France.

Summary: Cardiovascular disease remains a leading cause of mortality and morbidity worldwide. Embryonic stem cell-derived cardiomyocytes (ESC-CMs) may offer significant advances in creating in vitro cardiac tissues for disease modeling, drug testing, and elucidating developmental processes; however, the induction of ESCs to a more adult-like CM phenotype remains challenging. In this study, we developed a bioreactor system to employ pulsatile flow (1.48 mL/min), cyclic strain (5%), and extended culture time to improve the maturation of murine and human ESC-CMs. Dynamically-cultured ESC-CMs showed an increased expression of cardiac-associated proteins and genes, cardiac ion channel genes, as well as increased SERCA activity and a Raman fingerprint with the presence of maturation-associated peaks similar to primary CMs. We present a bioreactor platform that can serve as a foundation for the development of human-based cardiac in vitro models to verify drug candidates, and facilitates the study of cardiovascular development and disease.

Abstract: Myocardial infarction results in debilitating remodeling of the myocardial extracellular matrix. In this proof-of-principle study it was sought to modulate this aggressive remodeling by injecting a hyaluronic acid-based reservoir delivering exogenous microRNA-29B (miR-29B). This proof-of-principal study was executed whereby myocardial ischemia/reperfusion was performed on C57BL/6 mice for 45 minutes after which five 10 µl boluses of a hydrogel composed of thiolated hyaluronic acid crosslinked with poly (ethylene glycol) diacrylate, containing exogenous miR-29B as an active therapy, were injected into the borderzone of the infarcted myocardium. Following surgery, the myocardial function of the animals was monitored up to five weeks. Delivering miR-29B locally using an injectable hyaluronan-based hydrogel resulted in a maintenance of myocardial function at two and five weeks following myocardial infarction in this proof-of-principle study. Additionally, while animals treated with the control of a non-targeting microRNA delivered using the hyaluronan-based hydrogel had a significant deterioration of myocardial function; those treated with miR-29B did not. Histological analysis revealed a significantly decreased presence of elastin and significantly less immature/newly deposited collagen fibers at the borderzone of the infarct. Increased vascularity of the myocardial scar was also detected and Raman microspectroscopy discovered significantly altered extracellular matrix-specific biochemical signals at the borderzone of the infarct. This preclinical proof-of-principle study demonstrates that an injectable hyaluronic acid hydrogel system could be capable of delivering miR-29B towards maintaining cardiac function following myocardial infarction. Additionally, Raman microspectroscopy revealed subtle, yet significant changes in ECM organization and maturity. These findings have great potential with regard to using injectable biomaterials as a local treatment for ischemic tissue and exogenous microRNAs to modulate tissue remodeling.

The Handelsblatt is one of Germany's top business and financial newspapers, similiar to the Wall Street Journal. In their article, "Germany's 100 Smartest Heads", they selected Katja for her work in tissue engineering and regenerative medicine. The three-quarters of a page article discusses her work and hopes for the future of medicine.

The Pact for Research and Innovation strengthens the scientific system in Germany and contributes to its competitiveness and visibility. On 14 December, the five pact organizations - the German Research Association, the Fraunhofer Society, the Helmholtz Association, the Leibniz Association and the Max Planck Society - evaluated the pact together with the German federal and state governments in the Leibniz Community House. The conference "10 Years Pact for Research and Innovation" was opened by a keynote on the topic of "Innovation - the Right Framework", by Andreas Busch, a member of the Executive Committee of the Pharmaceuticals Division and Head of Drug Discovery at Bayer AG. An interview with Johanna Wanka, Federal Minister for Education and Research and Chairwoman of the Joint Science Conference (GWK), Eva Quante-Brandt, Senator for Science, Health and Consumer Protection of the Free Hanseatic City of Bremen, and deputy chairman of the GWK, Katja Schenke-Layland, Managing Director of the Fraunhofer Institute for Interfacial Engineering and Biotechnology IGB and Head of the Department of Cell and Tissue Engineering at the IGB, Andreas Busch and Leibniz President Matthias Kleiner. Afterwards, scientists from the five pact organizations discussed the successes and perspectives of research.

Abstract: Fibrillin microfibrils and elastic fibers are critical determinants of elastic tissues where they define as tissue-specific architectures vital mechanical properties such as pliability and elastic recoil. Fibrillin microfibrils also facilitate elastic fiber formation and support the association of epithelial cells with the interstitial matrix. Mutations in fibrillin-1 (FBN1) are causative for the Marfan syndrome, a congenital multisystem disorder characterized by progressive deterioration of the fibrillin microfibril/ elastic fiber architecture in the cardiovascular, musculoskeletal, ocular, and dermal system. In this study, we utilized Raman microspectroscopy in combination with principal component analysis (PCA) to analyze the molecular consequences of fibrillin-1 deficiency in skin of a mouse model (GT8) of Marfan syndrome. In addition, full-thickness skin models incorporating murine wild-type and Fbn1GT8/GT8 fibroblasts as well as human HaCaT keratinocytes were generated and analyzed. Skin models containing GT8 fibroblasts showed an altered epidermal morphology when compared to wild-type models indicating a new role for fibrillin-1 in dermal-epidermal crosstalk. Obtained Raman spectra together with PCA allowed to discriminate between healthy and deficient microfibrillar networks in murine dermis and skin models. Interestingly, results obtained from GT8 dermis and skin models showed similar alterations in molecular signatures triggered by fibrillin-1 deficiency such as amide III vibrations and decreased levels of glycan vibrations. Overall, this study indicates that Raman microspectroscopy has the potential to analyze subtle changes in fibrillin-1 microfibrils and elastic fiber networks. Therefore Raman microspectroscopy may be utilized as a non-invasive and sensitive diagnostic tool to identify connective tissue disorders and monitor their disease progression.

Abstract: Actinic Keratosis’ (AKs) are small skin lesions that are related to a prolonged sun-damage, which can develop into invasive squamous cell carcinoma (SCC) when left untreated. Effective, specific and well tolerable therapies to cure AKs are still of great interest. Diclofenac (DCF) is the current gold standard for the local treatment of AKs in terms of costs, effectiveness, side effects and tolerability. In this work, an electrospun polylactic acid (PLA) scaffold loaded with a synthetic DCF prodrug was developed and characterized. Specifically, the prodrug was successfully synthetized by binding DCF to a glycine residue via solid phase peptide synthesis (SPPS) and then incorporated in an electrospun PLA scaffold. The drug encapsulation was verified using multiphoton microscopy (MPM) and its scaffold release was spectrophotometrically monitored and confirmed with MPM. The scaffold was further characterized with scanning electron microscopy (SEM), tensile testing and contact angle measurements. Its biocompatibility was verified by performing a cell proliferation assay and compared to PLA scaffolds containing the same amount of DCF sodium salt (DCFONa). Finally, the effect of the electrospun scaffolds on human dermal fibroblasts (HDFs) morphology and metabolism was investigated by combining MPM with fluorescence lifetime imaging microscopy (FLIM). The obtained results suggest that the obtained scaffold could be suitable for the controlled and targeted delivery of the synthesized prodrug for the treatment of AKs.

Melanie Maurer was awarded the very presitigous Fulbright scholarship to continue her research at the Schenke-Layland Lab for one year. Appointed by the President of the United States, the 12-member J. William Fulbright Foreign Scholarship Board was established by Congress to supervise the global Fulbright Program as authorized by the Fulbright-Hays Act of 1961. Representing diverse facets of American society, Board members select students, scholars, teachers, and others from the United States and abroad to participate in Fulbright exchanges. In close relationship with the U.S. Department of State’s Bureau of Educational and Cultural Affairs, U.S. embassies, bilateral Fulbright Commissions in host countries and the U.S. Department of Education’s International and Foreign Language Office of Postsecondary Education, the Board prepares an annual report and promotes the Fulbright Program to audiences around the world. The Board also meets quarterly to establish policies for Fulbright selection and operating procedures.

Pirmin Lakner was awarded with the Guestfalen prize for outstanding research for his diploma thesis about “A phasor approach analysis of multiphoton FLIM measurements of three-dimensional cell culture models” on 24th of June 2016. His physics diploma thesis was supervised by Prof. Dr. Katja Schenke-Layland, Prof. Dr. Tilman Schäffer and Dr. Michael Monaghan. Pirmin Lakner developed a customised Matlab code for Phasor FLIM analysis and did interdisciplinary research from cell culture over microscopy to data analysis.

The Guestfalen price for outstanding research was awarded this year for the fourth time. It is an advancement award for young researchers who have produced excellent bachelor, master, diploma, doctoral or postdoctoral theses that are written within the last year. The prize is awarded by AV Guestfalia Tübingen and the Felix-Porsch-Stiftung once per year and is endowed with EUR 10,000. This year the prize was split and awarded to Raziye Karapinar and Pirmin Lakner. Raziye Karapinar was awarded for her Bachelor thesis about “Elektrophysiologische Charakterisierung des retinalen Photopigments Opn4 in HEK TRPC3 Zellen“.

Abstract: Nonlinear microscopy, namely multiphoton imaging and second harmonic generation (SHG), is an established noninvasive technique useful for the imaging of extracellular matrix (ECM). Typically, measurements are performed in vivo on freshly excised tissues or biopsies. In this article, we describe the effect of rehydrating paraffin-embedded sections on multiphoton and SHG emission signals and the acquisition of nonlinear images from hematoxylin and eosin (H&E)-stained sections before and after a destaining protocol. Our results reveal that bringing tissue sections to a physiological state yields a significant improvement in nonlinear signals, particularly in SHG. Additionally, the destaining of sections previously processed with H&E staining significantly improves their SHG emission signals during imaging, thereby allowing sufficient analysis of collagen in these sections. These results are important for researchers and pathologists to obtain additional information from paraffin-embedded tissues and archived samples to perform retrospective analysis of the ECM or gain additional information from rare samples.

Abstract: 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.

Abstract: One major obstacle to the application of stem cell-derived cardiomyocytes (CMs) for disease modeling and clinical therapies is the inability to identify the developmental stage of these cells without the need for genetic manipulation or utilization of exogenous markers. In this study, we demonstrate that Raman microspectroscopy can non-invasively identify embryonic stem cell (ESC)-derived chamber-specific CMs and monitor cell maturation. Using this marker-free approach, Raman peaks were identified for atrial and ventricular CMs, ESCs were successfully discriminated from their cardiac derivatives, a distinct phenotypic spectrum for ESC-derived CMs was confirmed, and unique spectral differences between fetal versus adult CMs were detected. The real-time identification and characterization of CMs, their progenitors, and subpopulations by Raman microspectroscopy strongly correlated to the phenotypical features of these cells. Due to its high molecular resolution, Raman microspectroscopy offers distinct analytical characterization for differentiating cardiovascular cell populations.

Abstract: Elucidation of mechanisms in semilunar valve development may enable the development of new therapies. Here, we found differences in proliferation-associated genes and genes repressed by vascular endothelial growth factor between human semilunar valves from first and second trimester valve leaflets. The proliferation of valve interstitial cells and ventricular valve endothelial cells (VECs) and cellular density declined from the first to the second trimester. Cytoplasmic expression of nuclear factor of activated T-cells cytoplasmic 1 (NFATc-1) in VECs (4 weeks), and later cells in the leaflet/annulus junction mesenchyme expressing inactive NFATc-1 (5.5-9 weeks) were detected, indicative of EndMT in valvulogenesis. At this leaflet/annulus junction CD44+ cells clustered during elongation (11 weeks), extending toward the tip along the fibrosal layer in second trimester leaflets. Differing patterns of maturation in the fibrosa and ventricularis were detected via increased fibrosal periostin content, which tracked the presence of the CD44+ cells in the second trimester. We revealed that spatiotemporal NFATc-1 expression actively regulates EndMT during human valvulogenesis, as early as 4 weeks. Additionally, CD44+ cells play a role in leaflet maturation toward the trilaminar structure, possibly via migration of VECs undergoing EndMT, which subsequently ascend from the leaflet/annulus junction.

Abstract: Regenerative strategies such as stem cell-based therapies and tissue engineering applications are being developed with the aim to replace, remodel, regenerate or support damaged tissues and organs. In addition to careful cell type selection, the design of appropriate three-dimensional (3D) scaffolds is essential for the generation of bio-inspired replacement tissues. Such scaffolds are usually made of degradable or non-degradable biomaterials and can serve as cell or drug carriers. The development of more effective and efficient drug carrier systems is also highly relevant for novel cancer treatment strategies. In this review, we provide a summary of current approaches that employ ECM and ECM-like materials, or ECM-synthetic polymer hybrids, as biomaterials in the field of regenerative medicine. We further discuss the utilization of such materials for cell and drug delivery, and highlight strategies for their use as vehicles for cancer therapy.

For her doctoral thesis "Electrospinning – a suitable method to generate scaffolds for regenerative medicine applications", the former doctoral student from Stuttgart University's Institute of Interfacial Prcoess Engineering and Plasma Technology (IGPV) was given the German Students Award by the Körber Foundation for her work on a cell-free heart valve replacement, which was performed at the Fraunhofer IGB in the Department of Cell and Tissue Engineering headed by Prof. Dr. Katja Schenke-Layland.

The panel nominated 27 finalists from 418 young scientists in the areas of "humanities", "social science" and "natural sciences and engineering”. Dr. Svenja Hinderer, who completed her doctorate in July 2014 , was chosen as the winner in the area of “natural sciences and engineering”. The award from each area is endowed with a EUR 25,000 prize. The selection criteria for the award were based on the scientific excellence of the doctoral work as well as its potential impact on society. The overall benefit of the work towards the advancement of scientific knowledge was valued over potential economic gain.

Dr. Michael Monaghan was awarded the European Society for Biomaterials-European Doctoral Award for his research in biomaterials. The award is given annually by the European Society for Biomaterials (ESB) and confers added value to the doctoral degrees of the recipients. The award serves to recognise a European or international dimension of the researchers’ work. Dr. Monaghan qualified for the award by fulfilling a number of requirements, including studying abroad during his doctoral research, publishing in high-impact scientific journals and presenting at international conferences. The Julia Polak European Doctorate Award award is established to recognise, encourage and stimulate outstanding translational research contributions to the field of biomaterials. The award is presented annually during the Biomaterials conference of the Society, and the winner is given a certificate and an honorarium of 300 Euro.