Development of an iPSC-derived tissue-resident macrophage-based platform for the in vitro immunocompatibility assessment of human tissue engineered matrices.

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-1ß 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-1ß 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.

Advances in 3D Organoid Models for Stem Cell-Based Cardiac Regeneration.

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.

Combining Cell Technologies With Biomimetic Tissue Engineering Applications: A New Paradigm for Translational Cardiovascular Therapies.

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.

Macrophage-extracellular matrix interactions: Perspectives for tissue engineered heart valve remodeling.

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.

Xeno-free induced pluripotent stem cell-derived neural progenitor cells for in vivo applications.

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 1018 cells per initially seeded 106 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.

Cell-Based HIF1α Gene Therapy Reduces Myocardial Scar and Enhances Angiopoietic Proteome, Transcriptomic and miRNA Expression in Experimental Chronic Left Ventricular Dysfunction.

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.

Endothelial Progenitor Cell-Based in vitro Pre-Endothelialization of Human Cell-Derived Biomimetic Regenerative Matrices for Next-Generation Transcatheter Heart Valves Applications.

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.

Heterogeneous expression of ACE2 and TMPRRS2 in mesenchymal stromal cells.

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.

Human iPSCs and Genome Editing Technologies for Precision Cardiovascular Tissue Engineering.

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.

Both Specific Endothelial and Proximal Tubular Adam17 Deletion Protect against Diabetic Nephropathy.

ADAM17 is a disintegrin and metalloproteinase capable of cleaving the ectodomains of a diverse variety of molecules including TNF-α, TGF-α, L-selectin, and ACE2. We have previously demonstrated that renal ADAM17 is upregulated in diabetic mice. The role of endothelial (eAdam17) and proximal tubular (tAdam17) Adam17 deletion in renal histology, modulation of the renin angiotensin system (RAS), renal inflammation, and fibrosis was studied in a mouse model of type 1 Diabetes Mellitus. Moreover, the effect of Adam17 deletion in an in vitro 3D cell culture from human proximal tubular cells under high glucose conditions was evaluated. eAdam17 deletion attenuates renal fibrosis and inflammation, whereas tAdam17 deletion decreases podocyte loss, attenuates the RAS, and decreases macrophage infiltration, α-SMA and collagen accumulation. The 3D in vitro cell culture reinforced the findings obtained in tAdam17KO mice with decreased fibrosis in the Adam17 knockout spheroids. In conclusion, Adam17 deletion either in the endothelial or the tubular cells mitigates kidney injury in the diabetic mice by targeting different pathways. The manipulation of Adam17 should be considered as a therapeutic strategy for treating DN.

3D-microtissue derived secretome as a cell-free approach for enhanced mineralization of scaffolds in the chorioallantoic membrane model.

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.

Thermal conditioning improves quality and speed of keratinocyte sheet production for burn wound treatment.

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°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°C, 33°C and 37°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°C was most effective. After 6 days of culture at 37°C, three times and six times higher numbers of viable cells were obtained compared with 33°C and 31°C. During the cell differentiation and sheet formation phase, however, the cells benefited from a mildly hypothermic temperature of 33°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°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.

Next-generation tissue-engineered heart valves with repair, remodelling and regeneration capacity.

Valvular heart disease is a major cause of morbidity and mortality worldwide. Surgical valve repair or replacement has been the standard of care for patients with valvular heart disease for many decades, but transcatheter heart valve therapy has revolutionized the field in the past 15 years. However, despite the tremendous technical evolution of transcatheter heart valves, to date, the clinically available heart valve prostheses for surgical and transcatheter replacement have considerable limitations. The design of next-generation tissue-engineered heart valves (TEHVs) with repair, remodelling and regenerative capacity can address these limitations, and TEHVs could become a promising therapeutic alternative for patients with valvular disease. In this Review, we present a comprehensive overview of current clinically adopted heart valve replacement options, with a focus on transcatheter prostheses. We discuss the various concepts of heart valve tissue engineering underlying the design of next-generation TEHVs, focusing on off-the-shelf technologies. We also summarize the latest preclinical and clinical evidence for the use of these TEHVs and describe the current scientific, regulatory and clinical challenges associated with the safe and broad clinical translation of this technology.

Geometry influences inflammatory host cell response and remodeling in tissue-engineered heart valves in-vivo.

Regenerative tissue-engineered matrix-based heart valves (TEM-based TEHVs) may become an alternative to currently-used bioprostheses for transcatheter valve replacement. We recently identified TEM-based TEHVs-geometry as one key-factor guiding their remodeling towards successful long-term performance or failure. While our first-generation TEHVs, with a simple, non-physiological valve-geometry, failed over time due to leaflet-wall fusion phenomena, our second-generation TEHVs, with a computational modeling-inspired design, showed native-like remodeling resulting in long-term performance. However, a thorough understanding on how TEHV-geometry impacts the underlying host cell response, which in return determines tissue remodeling, is not yet fully understood. To assess that, we here present a comparative samples evaluation derived from our first- and second-generation TEHVs. We performed an in-depth qualitative and quantitative (immuno-)histological analysis focusing on key-players of the inflammatory and remodeling cascades (M1/M2 macrophages, α-SMA+- and endothelial cells). First-generation TEHVs were prone to chronic inflammation, showing a high presence of macrophages and α-SMA+-cells, hinge-area thickening, and delayed endothelialization. Second-generation TEHVs presented with negligible amounts of macrophages and α-SMA+-cells, absence of hinge-area thickening, and early endothelialization. Our results suggest that TEHV-geometry can significantly influence the host cell response by determining the infiltration and presence of macrophages and α-SMA+-cells, which play a crucial role in orchestrating TEHV remodeling.

Tissue engineered heart valves for transcatheter aortic valve implantation: current state, challenges, and future developments.

INTRODUCTION: The establishment of transcatheter aortic valve implantation (TAVI) has revolutionized the treatment of severe aortic stenosis. However, with TAVI being approved for low-risk patients, valve durability is becoming of central importance. Here, we summarize how tissue engineered heart valves (TEHVs) may provide a clinically-relevant durable valve replacement compatible with TAVI. AREAS COVERED: Since its introduction, TAVI prostheses have advanced in design and development. However, TAVI bioprostheses are based on fixed xenogeneic materials prone to progressive degeneration. Transcatheter TEHVs may have the potential to overcome the drawbacks of current TAVI bioprostheses, with their remodeling, self-repair, and growth capacities. So far, performance and remodeling of transcatheter TEHV with in-situ regenerative potential were demonstrated in the low-pressure system, with acute performance proved in the systemic circulation. However, several challenges remain to be solved to ensure a safe clinical translation of TEHVs for TAVI approaches. EXPERT OPINION: With TAVI rapidly evolving, the establishment of long-term valve durability represents the top priority to reduce the rate of patient re-interventions, remove the associated risks and adverse events, and improve patients' life quality worldwide. With long-term performance and remodeling proved, TEHVs may represent the next-generation technology for a life-long TAVI prosthesis.

Human Cardiac Organoids for Modeling Genetic Cardiomyopathy.

Genetic cardiomyopathies are characterized by changes in the function and structure of the myocardium. The development of a novel in vitro model could help to better emulate healthy and diseased human heart conditions and may improve the understanding of disease mechanisms. In this study, for the first time, we demonstrated the generation of cardiac organoids using a triculture approach of human induced pluripotent stem-cell-derived cardiomyocytes (hiPS-CMs)-from healthy subjects and cardiomyopathy patients-human cardiac microvascular endothelial cells (HCMECs) and human cardiac fibroblasts (HCFs). We assessed the organoids' suitability as a 3D cellular model for the representation of phenotypical features of healthy and cardiomyopathic hearts. We observed clear differences in structure and beating behavior between the organoid groups, depending on the type of hiPS-CMs (healthy versus cardiomyopathic) used. Organoids may thus prove a promising tool for the design and testing of patient-specific treatments as well as provide a platform for safer and more efficacious drug development.