Generation of an induced pluripotent stem cell line (ESi107-A) from a transthyretin amyloid cardiomyopathy (ATTR-CM) patient carrying a p.Ser43Asn mutation in the TTR gene.

Transthyretin (TTR) amyloid cardiomyopathy (ATTR-CM) is a life-threatening disease caused by the abnormal production of misfolded TTR protein by liver cells, which is then released systemically. Its amyloid deposition in the heart is linked to cardiac toxicity and progression toward heart failure. A human induced pluripotent stem cell (iPSC) line was generated from peripheral blood mononuclear cells (PBMCs) from a patient suffering familial transthyretin amyloid cardiomyopathy carrying a c.128G>A (p.Ser43Asn) mutation in the TTR gene. This iPSC line offers a useful resource to study the disease pathophysiology and a cell-based model for therapeutic discovery.

Three-Dimensional Bioprinting in Cardiovascular Disease: Current Status and Future Directions.

Three-dimensional (3D) printing plays an important role in cardiovascular disease through the use of personalised models that replicate the normal anatomy and its pathology with high accuracy and reliability. While 3D printed heart and vascular models have been shown to improve medical education, preoperative planning and simulation of cardiac procedures, as well as to enhance communication with patients, 3D bioprinting represents a potential advancement of 3D printing technology by allowing the printing of cellular or biological components, functional tissues and organs that can be used in a variety of applications in cardiovascular disease. Recent advances in bioprinting technology have shown the ability to support vascularisation of large-scale constructs with enhanced biocompatibility and structural stability, thus creating opportunities to replace damaged tissues or organs. In this review, we provide an overview of the use of 3D bioprinting in cardiovascular disease with a focus on technologies and applications in cardiac tissues, vascular constructs and grafts, heart valves and myocardium. Limitations and future research directions are highlighted.

Fabrication of human myocardium using multidimensional modelling of engineered tissues.

Biofabrication of human tissues has seen a meteoric growth triggered by recent technical advancements such as human induced pluripotent stem cells (hiPSCs) and additive manufacturing. However, generation of cardiac tissue is still hampered by lack of adequate mechanical properties and crucially by the often unpredictable post-fabrication evolution of biological components. In this study we employ melt electrowriting (MEW) and hiPSC-derived cardiac cells to generate fibre-reinforced human cardiac minitissues. These are thoroughly characterized in order to build computational models and simulations able to predict their post-fabrication evolution. Our results show that MEW-based human minitissues display advanced maturation 28 post-generation, with a significant increase in the expression of cardiac genes such as MYL2, GJA5, SCN5A and the MYH7/MYH6 and MYL2/MYL7 ratios. Human iPSC-cardiomyocytes are significantly more aligned within the MEW-based 3D tissues, as compared to conventional 2D controls, and also display greater expression of C×43. These are also correlated with a more mature functionality in the form of faster conduction velocity. We used these data to develop simulations capable of accurately reproducing the experimental performance. In-depth gauging of the structural disposition (cellular alignment) and intercellular connectivity (C×43) allowed us to develop an improved computational model able to predict the relationship between cardiac cell alignment and functional performance. This study lays down the path for advancing in the development ofin silicotools to predict cardiac biofabricated tissue evolution after generation, and maps the route towards more accurate and biomimetic tissue manufacture.

Engineering a Humanised Niche to Support Human Haematopoiesis in Mice: Novel Opportunities in Modelling Cancer.

Despite the bone marrow microenvironment being widely recognised as a key player in cancer research, the current animal models that represent a human haematopoietic system lack the contribution of the humanised marrow microenvironment. Here we describe a murine model that relies on the combination of an orthotopic humanised tissue-engineered bone construct (ohTEBC) with patient-specific bone marrow (BM) cells to create a humanised bone marrow (hBM) niche capable of supporting the engraftment of human haematopoietic cells. Results showed that this model supports the engraftment of human CD34+ cells from a healthy BM with human haematopoietic cells migrating into the mouse BM, human BM compartment, spleen and peripheral blood. We compared these results with the engraftment capacity of human CD34+ cells obtained from patients with multiple myeloma (MM). We demonstrated that CD34+ cells derived from a diseased BM had a reduced engraftment potential compared to healthy patients and that a higher cell dose is required to achieve engraftment of human haematopoietic cells in peripheral blood. Finally, we observed that hematopoietic cells obtained from the mobilised peripheral blood of patients yields a higher number of CD34+, overcoming this problem. In conclusion, this humanised mouse model has potential as a unique and patient-specific pre-clinical platform for the study of tumour-microenvironment interactions, including human bone and haematopoietic cells, and could, in the future, serve as a drug testing platform.

Cells, Materials, and Fabrication Processes for Cardiac Tissue Engineering.

Cardiovascular disease is the number one killer worldwide, with myocardial infarction (MI) responsible for approximately 1 in 6 deaths. The lack of endogenous regenerative capacity, added to the deleterious remodelling programme set into motion by myocardial necrosis, turns MI into a progressively debilitating disease, which current pharmacological therapy cannot halt. The advent of Regenerative Therapies over 2 decades ago kick-started a whole new scientific field whose aim was to prevent or even reverse the pathological processes of MI. As a highly dynamic organ, the heart displays a tight association between 3D structure and function, with the non-cellular components, mainly the cardiac extracellular matrix (ECM), playing both fundamental active and passive roles. Tissue engineering aims to reproduce this tissue architecture and function in order to fabricate replicas able to mimic or even substitute damaged organs. Recent advances in cell reprogramming and refinement of methods for additive manufacturing have played a critical role in the development of clinically relevant engineered cardiovascular tissues. This review focuses on the generation of human cardiac tissues for therapy, paying special attention to human pluripotent stem cells and their derivatives. We provide a perspective on progress in regenerative medicine from the early stages of cell therapy to the present day, as well as an overview of cellular processes, materials and fabrication strategies currently under investigation. Finally, we summarise current clinical applications and reflect on the most urgent needs and gaps to be filled for efficient translation to the clinical arena.

Anisotropic Cryostructured Collagen Scaffolds for Efficient Delivery of RhBMP-2 and Enhanced Bone Regeneration.

In the treatment of bone non-unions, an alternative to bone autografts is the use of bone morphogenetic proteins (BMPs), e.g., BMP-2, BMP-7, with powerful osteoinductive and osteogenic properties. In clinical settings, these osteogenic factors are applied using absorbable collagen sponges for local controlled delivery. Major side effects of this strategy are derived from the supraphysiological doses of BMPs needed, which may induce ectopic bone formation, chronic inflammation, and excessive bone resorption. In order to increase the efficiency of the delivered BMPs, we designed cryostructured collagen scaffolds functionalized with hydroxyapatite, mimicking the structure of cortical bone (aligned porosity, anisotropic) or trabecular bone (random distributed porosity, isotropic). We hypothesize that an anisotropic structure would enhance the osteoconductive properties of the scaffolds by increasing the regenerative performance of the provided rhBMP-2. In vitro, both scaffolds presented similar mechanical properties, rhBMP-2 retention and delivery capacity, as well as scaffold degradation time. In vivo, anisotropic scaffolds demonstrated better bone regeneration capabilities in a rat femoral critical-size defect model by increasing the defect bridging. In conclusion, anisotropic cryostructured collagen scaffolds improve bone regeneration by increasing the efficiency of rhBMP-2 mediated bone healing.

Periosteum-derived mesenchymal progenitor cells in engineered implants promote fracture healing in a critical-size defect rat model.

An attractive alternative to bone autografts is the use of autologous mesenchymal progenitor cells (MSCs) in combination with biomaterials. We compared the therapeutic potential of different sources of mesenchymal stem cells in combination with biomaterials in a bone nonunion model. A critical-size defect was created in Sprague-Dawley rats. Animals were divided into six groups, depending on the treatment to be applied: bone defect was left empty (CTL); treated with live bone allograft (LBA); hrBMP-2 in collagen scaffold (CSBMP2 ); acellular polycaprolactone scaffold (PCL group); PCL scaffold containing periosteum-derived MSCs (PCLPMSCs ) and PCL containing bone marrow-derived MSCs (PCLBMSCs ). To facilitate cell tracking, both MSCs and bone graft were isolated from green fluorescent protein (GFP)-transgenic rats. CTL group did not show any signs of healing during the radiological follow-up (n = 6). In the LBA group, all the animals showed bone bridging (n = 6) whereas in the CSBMP2 group, four out of six animals demonstrated healing. In PCL and PCLPMSCs groups, a reduced number of animals showed radiological healing, whereas no healing was detected in the PCLBMSCs group. Using microcomputed tomography, the bone volume filling the defect was quantified, showing significant new bone formation in the LBA, CSBMP2 , and PCLPMSCs groups when compared with the CTL group. At 10 weeks, GFP positive cells were detected only in the LBA group and restricted to the outer cortical bone in close contact with the periosteum. Tracking of cellular implants demonstrated significant survival of the PMSCs when compared with BMSCs. In conclusion, PMSCs improve bone regeneration being suitable for mimetic autograft design.

Tissue engineered human prostate microtissues reveal key role of mast cell-derived tryptase in potentiating cancer-associated fibroblast (CAF)-induced morphometric transition in vitro.

The tumour microenvironment plays a vital role in the development of solid malignancies. Here we describe an in vitro human prostate cancer microtissue model that facilitates the incorporation and interrogation of key elements of the local prostatic tumour microenvironment. Primary patient-derived cancer-associated fibroblasts (CAFs) were cultured in three-dimensional (3D) melt electrowritten scaffolds where they deposited extensive extracellular matrix (ECM) and promoted significant changes in prostate epithelial morphology, when compared to matched non-malignant prostatic fibroblasts (NPFs). The addition of mast cells, a resident prostatic immune population that is expanded during early malignancy, enhanced the morphometric transition of benign epithelia via a tryptase-mediated mechanism. Our patient-specific 3D microtissues reveal a cascade of interactions between prostatic CAFs, their native ECM and mast cell-derived tryptase, rendering them important microenvironmental drivers of prostate cancer progression.

Melt Electrospinning Writing of Poly-Hydroxymethylglycolide-co-ε-Caprolactone-Based Scaffolds for Cardiac Tissue Engineering.

Current limitations in cardiac tissue engineering revolve around the inability to fully recapitulate the structural organization and mechanical environment of native cardiac tissue. This study aims at developing organized ultrafine fiber scaffolds with improved biocompatibility and architecture in comparison to the traditional fiber scaffolds obtained by solution electrospinning. This is achieved by combining the additive manufacturing of a hydroxyl-functionalized polyester, (poly(hydroxymethylglycolide-co-ε-caprolactone) (pHMGCL), with melt electrospinning writing (MEW). The use of pHMGCL with MEW vastly improves the cellular response to the mechanical anisotropy. Cardiac progenitor cells (CPCs) are able to align more efficiently along the preferential direction of the melt electrospun pHMGCL fiber scaffolds in comparison to electrospun poly(ε-caprolactone)-based scaffolds. Overall, this study describes for the first time that highly ordered microfiber (4.0-7.0 µm) scaffolds based on pHMGCL can be reproducibly generated with MEW and that these scaffolds can support and guide the growth of CPCs and thereby potentially enhance their therapeutic potential.