Cardiovascular disease remains one of the leading causes of mortality in the Western world. Congenital heart disease affects nearly 1 % of newborns, with approximately one-fourth requiring reconstructive surgery during their lifetime. Current cardiovascular replacement options have significant limitations. Their inability to grow poses particular challenges for pediatric patients. Tissue Engineered Matrix (TEM)-based in situ constructs, with their self-repair and growth potential, offer a promising solution to overcome the limitations of current clinically used replacement options. Various functionalization strategies, involving the integration of biomechanical or biochemical components to enhance biocompatibility, have been developed for Tissue Engineered Vascular Grafts (TEVG) and Tissue Engineered Heart Valves (TEHV) to foster their capacity for in vivo remodeling. In this review, we present the current state of clinical translation for TEVG and TEHV, and provide a comprehensive overview of biomechanical and biochemical functionalization strategies for TEVG and TEHV. We discuss the rationale for functionalization, the implementation of functionalization cues in TEM-based TEVG and TEHV, and the interrelatedness of biomechanical and biochemical cues in the in vivo response. Finally, we address the challenges associated with functionalization and discuss how interdisciplinary research, especially when combined with in silico models, could enhance the translation of these strategies into clinical applications. STATEMENT OF SIGNIFICANCE: Cardiovascular disease remains one of the leading causes of mortality, with current replacements being unable to grow and regenerate. In this review, we present the current state of clinical translation for tissue engineered vascular grafts (TEVG) and heart valves (TEHV). Particularly, we discuss the rationale and implementation for functionalization cues in tissue engineered matrix-based TEVGs and TEHVs, and for the first time we introduce the interrelatedness of biomechanical and biochemical cues in the in-vivo response. These insights pave the way for next-generation cardiovascular implants that promise better durability, biocompatibility, and growth potential. Finally, we address the challenges associated with functionalization and discuss how interdisciplinary research, especially when combined with in silico models, could enhance the translation of these strategies into clinical applications .

Upon implanting tissue-engineered heart valves (TEHVs), blood-derived macrophages are believed to orchestrate the remodeling process. They initiate the immune response and mediate the remodeling of the TEHV, essential for the valve’s functionality. The exact role of another macrophage type, the tissue-resident macrophages (TRMs), has not been yet elucidated even though they maintain the homeostasis of native tissues. Here, we characterized the response of hTRM-like cells in contact with a human tissue engineered matrix (hTEM). HTEMs comprised intracellular peptides with potentially immunogenic properties in their ECM proteome. Human iPSC-derived macrophages (iMφs) could represent hTRM-like cells in vitro and circumvent the scarcity of human donor material. iMφs were derived and after stimulation they demonstrated polarization towards non-/inflammatory states. Next, they responded with increased IL-6/IL-1β secretion in separate 3/7-day cultures with longer production-time-hTEMs. We demonstrated that iMφs are a potential model for TRM-like cells for the assessment of hTEM immunocompatibility. They adopt distinct pro- and anti-inflammatory phenotypes, and both IL-6 and IL-1β secretion depends on hTEM composition. IL-6 provided the highest sensitivity to measure iMφs pro-inflammatory response. This platform could facilitate the in vitro immunocompatibility assessment of hTEMs and thereby showcase a potential way to achieve safer clinical translation of TEHVs.

The reprogramming of somatic cells into induced pluripotent stem cells using Yamanaka factors has revolutionized biomedical research. In the past few years, substantial advances have been made in differentiating them into a variety of different cell types, including neurons, cardiomyocytes, megakaryocytes, endothelial cells, β-pancreatic cells and more. Today, these cells are ready to be used for clinical purposes in regenerative medicine and disease modelling to explore and pinpoint new therapeutic approaches. Nonetheless, before these therapies are ready for practical application, we must ensure the quality and integrity of the cells employed in various uses. Thus, it is essential to possess well-characterized and high-quality purified cell populations. In this Review, we discuss the latest applicable cell purification methods for clinical preparations, encompassing both current technologies that have been used in clinical settings and emerging state-of-the-art approaches that promise even greater potential. This Review explores the principles and applications of cell purification technologies, with a particular emphasis on their clinical relevance for pluripotent stem-cell-based therapies. The authors discuss current technologies and emerging state-of-the-art approaches that promise even greater potential.

Journal Article Intracoronary delivery of extracellular vesicles from human cardiac progenitor cells reduces infarct size in porcine acute myocardial infarction Get access Maximilian Y Emmert, Maximilian Y Emmert Department of Cardiothoracic and Vascular Surgery, Deutsches Herzzentrum der Charite (DHZC), Augustenburger Platz 1, 13353 Berlin, GermanyCharité-Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin and Humboldt-Universität zu Berlin, Augustenburger Platz 1, 13353 Berlin, GermanyBIH Center for Regenerative Therapies (BCRT), Berlin Institute of Health at Charité-Universitätsmedizin Berlin, Augustenburger Platz 1, 13353 Berlin, GermanyInstitute for Regenerative Medicine (IREM), University of Zurich, Wagistrasse 12, 8952 Schlieren, Switzerland Corresponding authors. Tel: +49 30 4593 2030, Email: maximilian.emmert@dhzc-charite.de (M.Y.E); Tel: +41 91 811 7115, Email: giuseppe.vassalli@eoc.ch (G.V.) https://orcid.org/0000-0002-8837-1716 Search for other works by this author on: Oxford Academic PubMed Google Scholar Jacopo Burrello, Jacopo Burrello Department of Medical Sciences, University of Turin, Via Giuseppe Verdi 8, 10124 Turin, Italy https://orcid.org/0000-0001-7884-7314 Search for other works by this author on: Oxford Academic PubMed Google Scholar Petra Wolint, Petra Wolint Division of Surgical Research, University Hospital Zurich, University of Zurich, Sternwartstrasse 14, 8091 Zurich, Switzerland https://orcid.org/0000-0002-9978-1438 Search for other works by this author on: Oxford Academic PubMed Google Scholar Monika Hilbe, Monika Hilbe Institute of Veterinary Pathology, Vetsuisse Faculty, University of Zurich, Winterthurerstrasse 268, 8057 Zurich, Switzerland https://orcid.org/0000-0002-4133-2402 Search for other works by this author on: Oxford Academic PubMed Google Scholar Gabriella Andriolo, Gabriella Andriolo Lugano Cell Factory, Istituto Cardiocentro Ticino, Ente Ospedaliero Cantonale, Via Tesserete 48, 6900 Lugano, Switzerland Search for other works by this author on: Oxford Academic PubMed Google Scholar Carolina Balbi, Carolina Balbi Laboratory of Cellular and Molecular Cardiology, Istituto Cardiocentro Ticino, Ente Ospedaliero Cantonale, Via Tesserete 48, 6900 Lugano, SwitzerlandCenter for Molecular Cardiology, University of Zurich, Wagistrasse 12, 8952 Schlieren, Switzerland https://orcid.org/0000-0002-7394-1173 Search for other works by this author on: Oxford Academic PubMed Google Scholar Elena Provasi, Elena Provasi Lugano Cell Factory, Istituto Cardiocentro Ticino, Ente Ospedaliero Cantonale, Via Tesserete 48, 6900 Lugano, Switzerland https://orcid.org/0000-0003-4073-1564 Search for other works by this author on: Oxford Academic PubMed Google Scholar Lucia Turchetto, Lucia Turchetto Lugano Cell Factory, Istituto Cardiocentro Ticino, Ente Ospedaliero Cantonale, Via Tesserete 48, 6900 Lugano, Switzerland Search for other works by this author on: Oxford Academic PubMed Google Scholar Marina Radrizzani, Marina Radrizzani Lugano Cell Factory, Istituto Cardiocentro Ticino, Ente Ospedaliero Cantonale, Via Tesserete 48, 6900 Lugano, Switzerland Search for other works by this author on: Oxford Academic PubMed Google Scholar Timo Z Nazari-Shafti, Timo Z Nazari-Shafti Department of Cardiothoracic and Vascular Surgery, Deutsches Herzzentrum der Charite (DHZC), Augustenburger Platz 1, 13353 Berlin, GermanyCharité-Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin and Humboldt-Universität zu Berlin, Augustenburger Platz 1, 13353 Berlin, GermanyBIH Center for Regenerative Therapies (BCRT), Berlin Institute of Health at Charité-Universitätsmedizin Berlin, Augustenburger Platz 1, 13353 Berlin, Germany https://orcid.org/0000-0002-9055-6891 Search for other works by this author on: Oxford Academic PubMed Google Scholar ... Show more Nikola Cesarovic, Nikola Cesarovic Department of Cardiothoracic and Vascular Surgery, Deutsches Herzzentrum der Charite (DHZC), Augustenburger Platz 1, 13353 Berlin, GermanyCharité-Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin and Humboldt-Universität zu Berlin, Augustenburger Platz 1, 13353 Berlin, GermanyBIH Center for Regenerative Therapies (BCRT), Berlin Institute of Health at Charité-Universitätsmedizin Berlin, Augustenburger Platz 1, 13353 Berlin, GermanyDepartment of Health Sciences and Technology, ETH Zurich, Rämistrasse 101, 8092 Zurich, Switzerland https://orcid.org/0000-0001-6744-2928 Search for other works by this author on: Oxford Academic PubMed Google Scholar Sebastian Neuber, Sebastian Neuber Department of Cardiothoracic and Vascular Surgery, Deutsches Herzzentrum der Charite (DHZC), Augustenburger Platz 1, 13353 Berlin, GermanyCharité-Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin and Humboldt-Universität zu Berlin, Augustenburger Platz 1, 13353 Berlin, GermanyBIH Center for Regenerative Therapies (BCRT), Berlin Institute of Health at Charité-Universitätsmedizin Berlin, Augustenburger Platz 1, 13353 Berlin, Germany https://orcid.org/0000-0003-1833-8440 Search for other works by this author on: Oxford Academic PubMed Google Scholar Volkmar Falk, Volkmar Falk Department of Cardiothoracic and Vascular Surgery, Deutsches Herzzentrum der Charite (DHZC), Augustenburger Platz 1, 13353 Berlin, GermanyCharité-Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin and Humboldt-Universität zu Berlin, Augustenburger Platz 1, 13353 Berlin, GermanyDepartment of Health Sciences and Technology, ETH Zurich, Rämistrasse 101, 8092 Zurich, Switzerland https://orcid.org/0000-0002-7911-8620 Search for other works by this author on: Oxford Academic PubMed Google Scholar Simon P Hoerstrup, Simon P Hoerstrup Institute for Regenerative Medicine (IREM), University of Zurich, Wagistrasse 12, 8952 Schlieren, Switzerland Search for other works by this author on: Oxford Academic PubMed Google Scholar Rayyan Hemetsberger, Rayyan Hemetsberger Department of Cardiology, Medical University of Vienna, Spitalgasse 23, 1090 Vienna, Austria Search for other works by this author on: Oxford Academic PubMed Google Scholar Mariann Gyöngyösi, Mariann Gyöngyösi Department of Cardiology, Medical University of Vienna, Spitalgasse 23, 1090 Vienna, Austria https://orcid.org/0000-0002-7083-2107 Search for other works by this author on: Oxford Academic PubMed Google Scholar Lucio Barile, Lucio Barile Laboratory for Cardiovascular Theranostics, Istituto Cardiocentro Ticino, Ente Ospedaliero Cantonale, Via Tesserete 48, 6900 Lugano, SwitzerlandFaculty of Biomedical Sciences, Università della Svizzera italiana (USI), Via Buffi 13, 6900 Lugano, Switzerland https://orcid.org/0000-0002-5827-0439 Search for other works by this author on: Oxford Academic PubMed Google Scholar Giuseppe Vassalli Giuseppe Vassalli Laboratory of Cellular and Molecular Cardiology, Istituto Cardiocentro Ticino, Ente Ospedaliero Cantonale, Via Tesserete 48, 6900 Lugano, SwitzerlandCenter for Molecular Cardiology, University of Zurich, Wagistrasse 12, 8952 Schlieren, SwitzerlandFaculty of Biomedical Sciences, Università della Svizzera italiana (USI), Via Buffi 13, 6900 Lugano, Switzerland Corresponding authors. Tel: +49 30 4593 2030, Email: maximilian.emmert@dhzc-charite.de (M.Y.E); Tel: +41 91 811 7115, Email: giuseppe.vassalli@eoc.ch (G.V.) https://orcid.org/0000-0002-6378-5883 Search for other works by this author on: Oxford Academic PubMed Google Scholar European Heart Journal, ehad636, https://doi.org/10.1093/eurheartj/ehad636 Published: 03 October 2023 Article history Received: 12 December 2022 Revision received: 03 July 2023 Accepted: 11 September 2023 Published: 03 October 2023

Background: Heart valves are living structures whose sophisticated functions are mediated by a specialized population of valvular interstitial cells (VICs). Given their central role in valve homeostasis, VICs represent a promising cell population for studying valve diseases and developing novel therapies to treat them. Herein, we describe the generation of VIC-like cells from human pluripotent stem cells (hPSCs). Methods and Results: Using a previously established protocol, we first generated endocardial cells from cardiovascular mesoderm. Within this endocardial population, we identified a subset of cells — marked by the expression of PDGFRβ — that express valvular endocardial cell (VEC) markers and demonstrate VEC-like functional properties, namely the ability to generate calcium transients in response to ATP and the ability to undergo endothelial-to-mesenchymal transition (EndoMT). Through stage-specific manipulation of developmental signaling pathways, we established a protocol that promotes the development of VIC-like cells from these hPSC-derived VEC progenitors (Figure 1A). The cells thus generated transcriptionally matched primary human fetal VICs by scRNAseq. When embedded in a tissue-engineered scaffold, hPSC-VICs secreted collagen and glycosaminoglycans, and demonstrated compact tissue organization analogous to that of native valve leaflets (Figure 1B, C). Finally, we show that VICs generated from an hPSC line with a Noonan syndrome mutation displayed excessive endocardial cell proliferation, diminished EndoMT, and dysregulated pERK activity, recapitulating key aspects of the disease. Conclusions: Together, the findings presented in this report provide a reproducible method for the scaled generation of bona fide VICs from hPSCs. The generation of hPSC-VICs addresses an important gap in the field and provides a platform to study valvulogenesis and heart valve disease, as well as a novel avenue for heart valve tissue engineering.

Human tissue-engineered matrices (hTEMs) have been proposed as a promising approach for in situ tissue engineered heart valves (TEHVs). However, there is still a limited understanding on how ECM composition in hTEMs develops over tissue culture time. Therefore, we performed a longitudinal hTEM assessment by 1) multiscale evaluation of hTEM composition during culture time (2, 4, 6-weeks), using (immuno)histology, biochemical assays, and mass spectrometry (LC-MS/MS); 2) analysis of protein pathways involved in ECM development using gene set enrichment analysis (GSEA); and 3) assessment of hTEM mechanical characterization using uniaxial tensile testing. Finally, as a proof-of-concept, TEHVs manufactured using 6-weeks hTEM samples were tested in a pulse duplicator. LC-MS/MS confirmed the tissue culture time-dependent increase in ECM proteins observed in histology and biochemical assays, revealing the most abundant collagens (COL6, COL12), proteoglycans (HSPG2, VCAN), and glycoproteins (FN, TNC). GSEA identified the most represented protein pathways in the hTEM at 2-weeks (mRNA metabolic processes), 4-weeks (ECM production), and 6-weeks (ECM organization and maturation). Uniaxial mechanical testing showed increased stiffness and stress at failure, and reduction in strain over tissue culture time. hTEM-based TEHVs demonstrated promising in vitro performance at both pulmonary and aortic pressure conditions, with symmetric leaflet coaptation and no stenosis. In conclusion, ECM protein abundance and maturation increased over tissue culture time, with consequent improvement of hTEM mechanical characteristics. These findings suggest that longer tissue culture impacts tissue organization, leading to an hTEM that may be suitable for high-pressure applications. STATEMENT OF SIGNIFICANCE: It is believed that the composition of the extracellular matrix (ECM) in the human tissue engineered matrices (hTEM) may favor tissue engineered heart valve (TEHV) remodeling upon implantation. However, the exact protein composition of the hTEM, and how this impacts tissue mechanical properties, remains unclear. Hence, we developed a reproducible rotation-based tissue culture method to produce hTEM samples. We performed a longitudinal assessment using different analytical techniques and mass spectrometry. Our data provided an in-depth characterization of the hTEM proteome with focus on ECM components, their development, and how they may impact the mechanical properties. Based on these results, we manufactured functional hTEM-based TEHVs at aortic-like condition in vitro. These outcomes pose an important step in translating hTEM-based TEHVs into clinics and in predicting their remodeling potential upon implantation.

Cardiovascular disease is a major cause of morbidity and mortality worldwide and, to date, the clinically available prostheses still present several limitations. The design of next-generation regenerative replacements either based on cellular or extracellular matrix technologies can address these shortcomings. Therefore, tissue engineered constructs could potentially become a promising alterative to the current therapeutic options for patients with cardiovascular diseases. In this review, we selectively present an overview of the current tissue engineering tools such as induced pluripotent stem cells, biomimetic materials, computational modeling, and additive manufacturing technologies, with a focus on their application to translational cardiovascular therapies. We discuss how these advanced technologies can help the development of biomimetic tissue engineered constructs and we finally summarize the latest clinical evidence for their use, and their potential therapeutic outcome.

The development of next‐generation biomimetic cardiovascular implants using tissue engineering concepts can address the existing shortcomings of the clinically available prostheses, offering the possibility to generate life‐long, native‐analogous constructs with self‐remodeling and regenerative capacities. Scaffolds for tissue‐engineered cardiovascular prostheses can be obtained from allogenic cell sources, that can then produce human tissue‐engineered matrices (hTEMs) in vitro. Traditionally, fetal bovine serum (FBS) is used as a universal cell growth supplement. However, concerns regarding its biosafety remain a challenge for clinical translation. The aim of this study is to develop a novel xenogeneic serum‐free approach for the manufacturing of clinical grade hTEMs. To achieve this, decellularized hTEMs are generated under xenogeneic serum‐free conditions and have subsequently demonstrated hTEMs perform similarly to the FBS‐supplemented control group in terms of extracellular matrix (ECM) composition, hemocompatibility, thrombogenicity, and calcification potential. Finally, the xenogeneic serum‐free protocol is successfully adapted to the development of hTEM‐based tissue‐engineered heart valves for the systemic circulation, showing proof‐of‐concept functionality in vitro. Overall, the data suggest the effectiveness of xenogeneic serum‐free culture method as a valid alternative to FBS for the production of hTEM for cardiovascular applications.

Pediatric heart valve disease affects children worldwide and necessitates valve replacements that remodel and grow with the patient. Current valve manufacturing technologies struggle to create valves that facilitate native tissue remodeling for permanent replacements. Here, we present focused rotary jet spinning (FRJS) for implantable medical devices, such as heart valves, to address this challenge. Combining RJS and a focused air stream, FRJS prints FibraValves, micro- and nanofibrous heart valves, in minutes. The micro- and nanoscale features provide structural cues to orient cells at the biotic-abiotic interface, while the centimeter-scale valve shape regulates cardiac flow. We built valves using poly(L-lactide-co-Ɛ-caprolactone) fiber scaffolds, which supported rapid cellular infiltration and displayed native valve-like mechanical properties. Evaluating clinical translatability, we assessed acute performance in a large animal model using a transcatheter delivery approach. These tests indicate that FRJS is a viable method for manufacturing heart valves and future medical implants.

Recent preclinical investigations and clinical trials with stem cells mostly studied bone-marrow-derived mononuclear cells (BM-MNCs), which so far failed to meet clinically significant functional study endpoints. BM-MNCs containing small proportions of stem cells provide little regenerative potential, while mesenchymal stem cells (MSCs) promise effective therapy via paracrine impact. Genetic engineering for rationally enhancing paracrine effects of implanted stem cells is an attractive option for further development of therapeutic cardiac repair strategies. Non-viral, efficient transfection methods promise improved clinical translation, longevity and a high level of gene delivery. Hypoxia-induced factor 1α is responsible for pro-angiogenic, anti-apoptotic and anti-remodeling mechanisms. Here we aimed to apply a cellular gene therapy model in chronic ischemic heart failure in pigs. A non-viral circular minicircle DNA vector (MiCi) was used for in vitro transfection of porcine MSCs (pMSC) with HIF1α (pMSC-MiCi-HIF-1α). pMSCs-MiCi-HIF-1α were injected endomyocardially into the border zone of an anterior myocardial infarction one month post-reperfused-infarct. Cell injection was guided via 3D-guided NOGA electro-magnetic catheter delivery system. pMSC-MiCi-HIF-1α delivery improved cardiac output and reduced myocardial scar size. Abundances of pro-angiogenic proteins were analyzed 12, 24 h and 1 month after the delivery of the regenerative substances. In a protein array, the significantly increased angiogenesis proteins were Activin A, Angiopoietin, Artemin, Endothelin-1, MCP-1; and remodeling factors ADAMTS1, FGFs, TGFb1, MMPs, and Serpins. In a qPCR analysis, increased levels of angiopeptin, CXCL12, HIF-1α and miR-132 were found 24 h after cell-based gene delivery, compared to those in untreated animals with infarction and in control animals. Expression of angiopeptin increased already 12 h after treatment, and miR-1 expression was reduced at that time point. In total, pMSC overexpressing HIF-1α showed beneficial effects for treatment of ischemic injury, mediated by stimulation of angiogenesis.

Background Currently, there is no regenerative therapy for patients with neurological and neurodegenerative disorders. Cell-therapies have emerged as a potential treatment for numerous brain diseases. Despite recent advances in stem cell technology, major concerns have been raised regarding the feasibility and safety of cell therapies for clinical applications. Methods We generated good manufacturing practice (GMP)-compatible neural progenitor cells (NPCs) from transgene- and xeno-free induced pluripotent stem cells (iPSCs) that can be smoothly adapted for clinical applications. NPCs were characterized in vitro for their differentiation potential and in vivo after transplantation into wild type as well as genetically immunosuppressed mice. Results Generated NPCs had a stable gene-expression over at least 15 passages and could be scaled for up to 10 18 cells per initially seeded 10 6 cells. After withdrawal of growth factors in vitro, cells adapted a neural fate and mainly differentiated into active neurons. To ensure a pure NPC population for in vivo applications, we reduced the risk of iPSC contamination by applying micro RNA-switch technology as a safety checkpoint. Using lentiviral transduction with a fluorescent and bioluminescent dual-reporter construct, combined with non-invasive in vivo bioluminescent imaging, we longitudinally tracked the grafted cells in healthy wild-type and genetically immunosuppressed mice as well as in a mouse model of ischemic stroke. Long term in-depth characterization revealed that transplanted NPCs have the capability to survive and spontaneously differentiate into functional and mature neurons throughout a time course of a month, while no residual pluripotent cells were detectable. Conclusion We describe the generation of transgene- and xeno-free NPCs. This simple differentiation protocol combined with the ability of in vivo cell tracking presents a valuable tool to develop safe and effective cell therapies for various brain injuries.

Since early 2020 the COVID-19 pandemic has paralyzed the world, resulting in more than half a billion infections and over 6 million deaths within a 28-month period. Knowledge about the disease remains largely disjointed, especially when considering the molecular mechanisms driving the diversity of clinical manifestations and symptoms. Despite the recent availability of vaccines, there remains an urgent need to develop effective treatments for cases of severe disease, especially in the face of novel virus variants. The complexity of the situation is exacerbated by the emergence of COVID-19 as a complex and multifaceted systemic disease affecting independent tissues and organs throughout the body. The development of effective treatment strategies is therefore predicated on an integrated understanding of the underlying disease mechanisms and their potentially causative link to the diversity of observed clinical phenotypes. To address this need, we utilized a computational technology (the Dataome platform) to build an integrated clinico-molecular view on the most important COVID-19 clinical phenotypes. Our results provide the first integrated, whole-patient model of COVID-19 symptomatology that connects the molecular lifecycle of SARS-CoV-2 with microvesicle-mediated intercellular communication and the contact activation and kallikrein-kinin systems. The model not only explains the clinical pleiotropy of COVID-19, but also provides an evidence-driven framework for drug development/repurposing and the identification of critical risk factors. The associated knowledge is provided in the form of the open source COVID-19 Explorer (https://covid19.molecularhealth.com), enabling the global community to explore and analyze the key molecular features of systemic COVID-19 and associated implications for research priorities and therapeutic strategies. Our work suggests that knowledge modeling solutions may offer important utility in expediting the global response to future health emergencies.

Hemocompatibility of cardiovascular implants represents a major clinical challenge and, to date, optimal antithrombotic properties are lacking. Next-generation tissue-engineered heart valves (TEHVs) made from human-cell-derived tissue-engineered extracellular matrices (hTEMs) demonstrated their recellularization capacity in vivo and may represent promising candidates to avoid antithrombotic therapy. To further enhance their hemocompatibility, we tested hTEMs pre-endothelialization potential using human-blood-derived endothelial-colony-forming cells (ECFCs) and umbilical vein cells (control), cultured under static and dynamic orbital conditions, with either FBS or hPL. ECFCs performance was assessed via scratch assay, thereby recapitulating the surface damages occurring in transcatheter valves during crimping procedures. Our study demonstrated: feasibility to form a confluent and functional endothelium on hTEMs with expression of endothelium-specific markers; ECFCs migration and confluency restoration after crimping tests; hPL-induced formation of neo-microvessel-like structures; feasibility to pre-endothelialize hTEMs-based TEHVs and ECFCs retention on their surface after crimping. Our findings may stimulate new avenues towards next-generation pre-endothelialized implants with enhanced hemocompatibility, being beneficial for selected high-risk patients.

The outbreak of COVID-19 has become a serious public health emergency. The virus targets cells by binding the ACE2 receptor. After infection, the virus triggers in some humans an immune storm containing the release of proinflammatory cytokines and chemokines followed by multiple organ failure. Several vaccines are enrolled, but an effective treatment is still missing. Mesenchymal stem cells (MSCs) have shown to secrete immunomodulatory factors that suppress this cytokine storm. Therefore, MSCs have been suggested as a potential treatment option for COVID-19. We report here that the ACE2 expression is minimal or nonexistent in MSC derived from three different human tissue sources (adipose tissue, umbilical cord Wharton`s jelly and bone marrow). In contrast, TMPRSS2 that is implicated in SARS-CoV-2 entry has been detected in all MSC samples. These results are of particular importance for future MSC-based cell therapies to treat severe cases after COVID-19 infection.

Atherosclerosis is an arterial disease characterized by intravascular plaques. Disease hallmarks are vessel stenosis and hyperplasia, eventually escalating into plaque rupture and acute clinical presentations. Innate immune cells and local flow variations are core players in the pathology, but their combined effects have never been investigated before in human vessel replicas due to the lack of modeling systems with adequate degree of complexity. Here, we combined computational fluid dynamics and tissue-engineering to achieve full human atherosclerotic plaque development on-a-chip. Our model incorporates induced pluripotent stem cell-derived populations into small-caliber arteries that are cultured in atheroprone conditions. Using machine-learning-aided immunophenotyping, as well as molecular and nanoprobe-based tensile analyses, we found that immune cells and extracellular matrix were comparable between in vitro and ex vivo human plaques. Our results provide further insights into the relation between disturbed flow dynamics and vascular inflammation, introducing a versatile, scalable modeling tool to study atherosclerosis onset and progression.

Background aims: Cultured patient-specific keratinocyte sheets have been used clinically since the 1970s for the treatment of large severe burns. However, despite significant developments in recent years, successful and sustainable treatment is still a challenge. Reliable, high-quality grafts with faster availability and a flexible time window for transplantation are required to improve clinical outcomes. Methods: Keratinocytes are usually grown in vitro at 37 degrees C. Given the large temperature differences in native skin tissue, the aim of the authors' study was to investigate thermal conditioning of keratinocyte sheet production. Therefore, the influence of 31 degrees C, 33 degrees C and 37 degrees C on cell expansion and differentiation in terms of proliferation and sheet formation efficacy was investigated. In addition, the thermal effect on the biological status and thus the quality of the graft was assessed on the basis of the release of wound healing-related biofactors in various stages of graft development. Results: The authors demonstrated that temperature is a decisive factor in the production of human keratinocyte sheets. By using specific temperature ranges, the authors have succeeded in optimizing the individual manufacturing steps. During the cell expansion phase, cultivation at 37 degrees C was most effective. After 6 days of culture at 37 degrees C, three times and six times higher numbers of viable cells were obtained compared with 33 degrees C and 31 degrees C. During the cell differentiation and sheet formation phase, however, the cells benefited from a mildly hypothermic temperature of 33 degrees C. Keratinocytes showed increased differentiation potential and formed better epidermal structures, which led to faster biomechanical sheet stability at day 18. In addition, a cultivation temperature of 33 degrees C resulted in a longer lasting and higher secretion of the investigated immunomodulatory, anti-inflammatory, angiogenic and pro-inflammatory biofactors. Conclusions: These results show that by using specific temperature ranges, it is possible to accelerate the large-scale production of cultivated keratinocyte sheets while at the same time improving quality. Cultivated keratinocyte sheets are available as early as 18 days post-biopsy and at any time for 7 days thereafter, which increases the flexibility of the process for surgeons and patients alike. These findings will help to provide better clinical outcomes, with an increased take rate in severe burn patients. (c) 2021 International Society for Cell & Gene Therapy. Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/)

Induced pluripotent stem cells (iPSCs) originate from the reprogramming of adult somatic cells using four Yamanaka transcription factors. Since their discovery, the stem cell (SC) field achieved significant milestones and opened several gateways in the area of disease modeling, drug discovery, and regenerative medicine. In parallel, the emergence of clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (CRISPR-Cas9) revolutionized the field of genome engineering, allowing the generation of genetically modified cell lines and achieving a precise genome recombination or random insertions/deletions, usefully translated for wider applications. Cardiovascular diseases represent a constantly increasing societal concern, with limited understanding of the underlying cellular and molecular mechanisms. The ability of iPSCs to differentiate into multiple cell types combined with CRISPR-Cas9 technology could enable the systematic investigation of pathophysiological mechanisms or drug screening for potential therapeutics. Furthermore, these technologies can provide a cellular platform for cardiovascular tissue engineering (TE) approaches by modulating the expression or inhibition of targeted proteins, thereby creating the possibility to engineer new cell lines and/or fine-tune biomimetic scaffolds. This review will focus on the application of iPSCs, CRISPR-Cas9, and a combination thereof to the field of cardiovascular TE. In particular, the clinical translatability of such technologies will be discussed ranging from disease modeling to drug screening and TE applications.

Bone regeneration is a complex process and the clinical translation of tissue engineered constructs (TECs) remains a challenge. The combination of biomaterials and mesenchymal stem cells (MSCs) may enhance the healing process through paracrine effects. Here, we investigated the influence of cell format in combination with a collagen scaffold on key factors in bone healing process, such as mineralization, cell infiltration, vascularization, and ECM production. MSCs as single cells (2D-SCs), assembled into microtissues (3D-MTs) or their corresponding secretomes were combined with a collagen scaffold and incubated on the chicken embryo chorioallantoic membrane (CAM) for 7 days. A comprehensive quantitative analysis was performed on a cellular level by histology and by microcomputed tomography (microCT). In all experimental groups, accumulation of collagen and glycosaminoglycan within the scaffold was observed over time. A pronounced cell infiltration and vascularization from the interface to the surface region of the CAM was detected. The 3D-MT secretome showed a significant mineralization of the biomaterial using microCT compared to all other conditions. Furthermore, it revealed a homogeneous distribution pattern of mineralization deposits in contrast to the cell-based scaffolds, where mineralization was only at the surface. Therefore, the secretome of MSCs assembled into 3D-MTs may represent an interesting therapeutic strategy for a next-generation bone healing concept.