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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., (© 2024. The Author(s).)

The adult human heart cannot regain complete cardiac function following tissue injury, making cardiac regeneration a current clinical unmet need. There are a number of clinical procedures aimed at reducing ischemic damage following injury; however, it has not yet been possible to stimulate adult cardiomyocytes to recover and proliferate. The emergence of pluripotent stem cell technologies and 3D culture systems has revolutionized the field. Specifically, 3D culture systems have enhanced precision medicine through obtaining a more accurate human microenvironmental condition to model disease and/or drug interactions in vitro. In this study, we cover current advances and limitations in stem cell-based cardiac regenerative medicine. Specifically, we discuss the clinical implementation and limitations of stem cell-based technologies and ongoing clinical trials. We then address the advent of 3D culture systems to produce cardiac organoids that may better represent the human heart microenvironment for disease modeling and genetic screening. Finally, we delve into the insights gained from cardiac organoids in relation to cardiac regeneration and further discuss the implications for clinical translation.

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 Author(s) 2023. Published by Oxford University Press.)

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., Competing Interests: Declaration of Competing Interest S.P.H. is a shareholder at Xeltis BV and LifeMatrix AG. M.Y.E. is a shareholder at LifeMatrix AG. All other authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Infection with SARS-CoV-2 coronavirus causes systemic, multi-faceted COVID-19 disease. However, knowledge connecting its intricate clinical manifestations with molecular mechanisms remains fragmented. Deciphering the molecular basis of COVID-19 at the whole-patient level is paramount to the development of effective therapeutic approaches. With this goal in mind, we followed an iterative, expert-driven process to compile data published prior to and during the early stages of the pandemic into a comprehensive COVID-19 knowledge model. Recent updates to this model have also validated multiple earlier predictions, suggesting the importance of such knowledge frameworks in hypothesis generation and testing. Overall, our findings suggest that SARS-CoV-2 perturbs several specific mechanisms, unleashing a pathogenesis spectrum, ranging from "a perfect storm" triggered by acute hyper-inflammation, to accelerated aging in protracted "long COVID-19" syndromes. In this work, we shortly report on these findings that we share with the community via 1) a synopsis of key evidence associating COVID-19 symptoms and plausible mechanisms, with details presented within 2) the accompanying "COVID-19 Explorer" webserver, developed specifically for this purpose (found at https://covid19.molecularhealth.com). We anticipate that our model will continue to facilitate clinico-molecular insights across organ systems together with hypothesis generation for the testing of potential repurposing drug candidates, new pharmacological targets and clinically relevant biomarkers. Our work suggests that whole patient knowledge models of human disease can potentially expedite the development of new therapeutic strategies and support evidence-driven clinical hypothesis generation and decision making., Competing Interests: Authors SB, DJ, TS, KH, AS, and FD were employed by Molecular Health GmbH. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Brock, Jackson, Soldatos, Hornischer, Schäfer, Diella, Emmert and Hoerstrup.)

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Brock, Soldatos, Jackson, Diella, Hornischer, Schäfer, Hoerstrup and Emmert.)

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., (© 2022. The Author(s).)

In situ heart valve tissue engineering approaches have been proposed as promising strategies to overcome the limitations of current heart valve replacements. Tissue engineered heart valves (TEHVs) generated from in vitro grown tissue engineered matrices (TEMs) aim at mimicking the microenvironmental cues from the extracellular matrix (ECM) to favor integration and remodeling of the implant. A key role of the ECM is to provide mechanical support to and attract host cells into the construct. Additionally, each ECM component plays a critical role in regulating cell adhesion, growth, migration, and differentiation potential. Importantly, the immune response to the implanted TEHV is also modulated biophysically via macrophage-ECM protein interactions. Therefore, the aim of this review is to summarize what is currently known about the interactions and signaling networks occurring between ECM proteins and macrophages, and how these interactions may impact the long-term in situ remodeling outcomes of TEMs. First, we provide an overview of in situ tissue engineering approaches and their clinical relevance, followed by a discussion on the fundamentals of the remodeling cascades. We then focus on the role of circulation-derived and resident tissue macrophages, with particular emphasis on the ramifications that ECM proteins and peptides may have in regulating the host immune response. Finally, the relevance of these findings for heart valve tissue engineering applications is discussed., Competing Interests: Author SH was a shareholder at Xeltis BV and LifeMatrix AG. Author ME was a shareholder at LifeMatrix AG. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Poulis, Martin, Hoerstrup, Emmert and Fioretta.)

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Gara, Ong, Winkler, Zlabinger, Lukovic, Merkely, Emmert, Wolint, Hoerstrup, Gyöngyösi, Wu and Pavo.)

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., Competing Interests: SH is shareholder at Xeltis BV and LifeMatrix AG. ME is a shareholder at LifeMatrix AG. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Motta, Zaytseva, Fioretta, Lintas, Breymann, Hoerstrup and Emmert.)

Background and Aims: Autologous keratinocyte sheets constitute an important component of the burn wound treatment toolbox available to a surgeon and can be considered a life-saving procedure for patients with severe burns over 50% of their total body surface area. Large-scale keratinocyte sheet cultivation still fundamentally relies on the use of animal components such as inactivated murine 3T3 fibroblasts as feeders, animal-derived enzymes such as trypsin, as well as media components such as fetal bovine serum (FBS). This study was therefore aimed to optimize autologous keratinocyte sheets by comparing various alternatives to critical components in their production., Methods: Human skin samples were retrieved from remnant operative tissues. Cell isolation efficiency and viability were investigated by comparing the efficacy of porcine-derived trypsin and animal-free enzymes (Accutase and TrypLESelect). The subsequent expansion of the cells and the keratinocyte sheet formation was analyzed, comparing various cell culture substrates (inactivated murine 3T3 fibroblasts, inactivated human fibroblasts, Collagen I or plain tissue culture plastic), as well as media containing serum or chemically defined animal-free media., Results: The cell isolation step showed clear cell yield advantages when using porcine-derived trypsin, compared to animal-free alternatives. The keratinocyte sheets produced using animal-free serum were similar to those produced using 3T3 feeder layer and FBS-containing medium, particularly in mechanical integrity as all grafts were liftable. In addition, sheets grown on collagen in an animal-free medium showed indications of advantages in homogeneity, speed, reduced variability, and differentiation status compared to the other growth conditions investigated. Most importantly, the procedure was compatible with the up-scaling requirements of major burn wound treatments., Conclusion: This study demonstrated that animal-free components could be used successfully to reduce the risk profile of large-scale autologous keratinocyte sheet production, and thereby increase clinical accessibility., Competing Interests: The authors have declared that there is no conflict of interest. All authors have read and approved the final version of the manuscript. Dr. med. Maurizio Calcagni had full access to all of the data in this study and has taken complete responsibility for the integrity of the data and the accuracy of the data analysis., (© 2022 The Authors. Health Science Reports published by Wiley Periodicals LLC.)

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., (© 2021 The Authors. Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)