CAR T cells for treating autoimmune diseases.

Autoimmune disorders occur when immune cells go wrong and attack the body's own tissues. Currently, autoimmune disorders are largely treated by broad immunosuppressive agents and blocking antibodies, which can manage the diseases but often are not curative. Thus, there is an urgent need for advanced therapies for patients suffering from severe and refractory autoimmune diseases, and researchers have considered cell therapy as potentially curative approach for several decades. In the wake of its success in cancer therapy, adoptive transfer of engineered T cells modified with chimeric antigen receptors (CAR) for target recognition could now become a therapeutic option for some autoimmune diseases. Here, we review the ongoing developments with CAR T cells in the field of autoimmune disorders. We will cover first clinical results of applying anti-CD19 and anti-B cell maturation antigen CAR T cells for B cell elimination in systemic lupus erythematosus, refractory antisynthetase syndrome and myasthenia gravis, respectively. Furthermore, in preclinical models, researchers have also developed chimeric autoantibody receptor T cells that can eliminate individual B cell clones producing specific autoantibodies, and regulatory CAR T cells that do not eliminate autoreactive immune cells but dampen their wrong activation. Finally, we will address safety and manufacturing aspects for CAR T cells and discuss mRNA technologies and automation concepts for ensuring the future availability of safe and efficient CAR T cell products.

Engineered hydrogels for mechanobiology.

Cells' local mechanical environment can be as important in guiding cellular responses as many well-characterized biochemical cues. Hydrogels that mimic the native extracellular matrix can provide these mechanical cues to encapsulated cells, allowing for the study of their impact on cellular behaviours. Moreover, by harnessing cellular responses to mechanical cues, hydrogels can be used to create tissues in vitro for regenerative medicine applications and for disease modelling. This Primer outlines the importance and challenges of creating hydrogels that mimic the mechanical and biological properties of the native extracellular matrix. The design of hydrogels for mechanobiology studies is discussed, including appropriate choice of cross-linking chemistry and strategies to tailor hydrogel mechanical cues. Techniques for characterizing hydrogels are explained, highlighting methods used to analyze cell behaviour. Example applications for studying fundamental mechanobiological processes and regenerative therapies are provided, along with a discussion of the limitations of hydrogels as mimetics of the native extracellular matrix. The article ends with an outlook for the field, focusing on emerging technologies that will enable new insights into mechanobiology and its role in tissue homeostasis and disease.

Extrinsic Macrophages Protect While Tendon Progenitors Degrade: Insights from a Tissue Engineered Model of Tendon Compartmental Crosstalk.

Tendons are among the most mechanically stressed tissues of the body, with a functional core of type-I collagen fibers maintained by embedded stromal fibroblasts known as tenocytes. The intrinsic load-bearing core compartment of tendon is surrounded, nourished, and repaired by the extrinsic peritendon, a synovial-like tissue compartment with access to tendon stem/progenitor cells as well as blood monocytes. In vitro tendon model systems generally lack this important feature of tissue compartmentalization, while in vivo models are cumbersome when isolating multicellular mechanisms. To bridge this gap, an improved in vitro model of explanted tendon core stromal tissue (mouse tail tendon fascicles) surrounded by cell-laden collagen hydrogels that mimic extrinsic tissue compartments is suggested. Using this model, CD146+ tendon stem/progenitor cell and CD45+ F4/80+ bone-marrow derived macrophage activity within a tendon injury-like niche are recapitulated. It is found that extrinsic stromal progenitors recruit to the damaged core, contribute to an overall increase in catabolic ECM gene expression, and accelerate the decrease in mechanical properties. Conversely, it is found that extrinsic bone-marrow derived macrophages in these conditions adopt a proresolution phenotype that mitigates rapid tissue breakdown by outwardly migrated tenocytes and F4/80+ "tenophages" from the intrinsic tissue core.

From Single Batch to Mass Production-Automated Platform Design Concept for a Phase II Clinical Trial Tissue Engineered Cartilage Product.

Advanced Therapy Medicinal Products (ATMP) provide promising treatment options particularly for unmet clinical needs, such as progressive and chronic diseases where currently no satisfying treatment exists. Especially from the ATMP subclass of Tissue Engineered Products (TEPs), only a few have yet been translated from an academic setting to clinic and beyond. A reason for low numbers of TEPs in current clinical trials and one main key hurdle for TEPs is the cost and labor-intensive manufacturing process. Manual production steps require experienced personnel, are challenging to standardize and to scale up. Automated manufacturing has the potential to overcome these challenges, toward an increasing cost-effectiveness. One major obstacle for automation is the control and risk prevention of cross contaminations, especially when handling parallel production lines of different patient material. These critical steps necessitate validated effective and efficient cleaning procedures in an automated system. In this perspective, possible technologies, concepts and solutions to existing ATMP manufacturing hurdles are discussed on the example of a late clinical phase II trial TEP. In compliance to Good Manufacturing Practice (GMP) guidelines, we propose a dual arm robot based isolator approach. Our novel concept enables complete process automation for adherent cell culture, and the translation of all manual process steps with standard laboratory equipment. Moreover, we discuss novel solutions for automated cleaning, without the need for human intervention. Consequently, our automation concept offers the unique chance to scale up production while becoming more cost-effective, which will ultimately increase TEP availability to a broader number of patients.

Shear-stress sensing by PIEZO1 regulates tendon stiffness in rodents and influences jumping performance in humans.

Athletic performance relies on tendons, which enable movement by transferring forces from muscles to the skeleton. Yet, how load-bearing structures in tendons sense and adapt to physical demands is not understood. Here, by performing calcium (Ca2+) imaging in mechanically loaded tendon explants from rats and in primary tendon cells from rats and humans, we show that tenocytes detect mechanical forces through the mechanosensitive ion channel PIEZO1, which senses shear stresses induced by collagen-fibre sliding. Through tenocyte-targeted loss-of-function and gain-of-function experiments in rodents, we show that reduced PIEZO1 activity decreased tendon stiffness and that elevated PIEZO1 mechanosignalling increased tendon stiffness and strength, seemingly through upregulated collagen cross-linking. We also show that humans carrying the PIEZO1 E756del gain-of-function mutation display a 13.2% average increase in normalized jumping height, presumably due to a higher rate of force generation or to the release of a larger amount of stored elastic energy. Further understanding of the PIEZO1-mediated mechanoregulation of tendon stiffness should aid research on musculoskeletal medicine and on sports performance.

Advanced Flow Cytometry Assays for Immune Monitoring of CAR-T Cell Applications.

Adoptive immunotherapy using chimeric antigen receptor (CAR)-T cells has achieved successful remissions in refractory B-cell leukemia and B-cell lymphomas. In order to estimate both success and severe side effects of CAR-T cell therapies, longitudinal monitoring of the patient's immune system including CAR-T cells is desirable to accompany clinical staging. To conduct research on the fate and immunological impact of infused CAR-T cells, we established standardized 13-colour/15-parameter flow cytometry assays that are suitable to characterize immune cell subpopulations in the peripheral blood during CAR-T cell treatment. The respective staining technology is based on pre-formulated dry antibody panels in a uniform format. Additionally, further antibodies of choice can be added to address specific clinical or research questions. We designed panels for the anti-CD19 CAR-T therapy and, as a proof of concept, we assessed a healthy individual and three B-cell lymphoma patients treated with anti-CD19 CAR-T cells. We analyzed the presence of anti-CD19 CAR-T cells as well as residual CD19+ B cells, the activation status of the T-cell compartment, the expression of co-stimulatory signaling molecules and cytotoxic agents such as perforin and granzyme B. In summary, this work introduces standardized and modular flow cytometry assays for CAR-T cell clinical research, which could also be adapted in the future as quality controls during the CAR-T cell manufacturing process.

Inhibition of ERK 1/2 kinases prevents tendon matrix breakdown.

Tendon extracellular matrix (ECM) mechanical unloading results in tissue degradation and breakdown, with niche-dependent cellular stress directing proteolytic degradation of tendon. Here, we show that the extracellular-signal regulated kinase (ERK) pathway is central in tendon degradation of load-deprived tissue explants. We show that ERK 1/2 are highly phosphorylated in mechanically unloaded tendon fascicles in a vascular niche-dependent manner. Pharmacological inhibition of ERK 1/2 abolishes the induction of ECM catabolic gene expression (MMPs) and fully prevents loss of mechanical properties. Moreover, ERK 1/2 inhibition in unloaded tendon fascicles suppresses features of pathological tissue remodeling such as collagen type 3 matrix switch and the induction of the pro-fibrotic cytokine interleukin 11. This work demonstrates ERK signaling as a central checkpoint to trigger tendon matrix degradation and remodeling using load-deprived tissue explants.

Smart Hydrogels for the Augmentation of Bone Regeneration by Endogenous Mesenchymal Progenitor Cell Recruitment.

The treatment of bone defects with recombinant bone morphogenetic protein-2 (BMP-2) requires high doses precluding broad clinical application. Here, a bioengineering approach is presented that strongly improves low-dose BMP-2-based bone regeneration by mobilizing healing-associated mesenchymal progenitor cells (MPCs). Smart synthetic hydrogels are used to trap and study endogenous MPCs trafficking to bone defects. Hydrogel-trapped and prospectively isolated MPCs differentiate into multiple lineages in vitro and form bone in vivo. In vitro screenings reveal that platelet-derived growth factor BB (PDGF-BB) strongly recruits prospective MPCs making it a promising candidate for the engineering of hydrogels that enrich endogenous MPCs in vivo. However, PDGF-BB inhibits BMP-2-mediated osteogenesis both in vitro and in vivo. In contrast, smart two-way dynamic release hydrogels with fast-release of PDGF-BB and sustained delivery of BMP-2 beneficially promote the healing of bone defects. Collectively, it is shown that modulating the dynamics of endogenous progenitor cells in vivo by smart synthetic hydrogels significantly improves bone healing and holds great potential for other advanced applications in regenerative medicine.

Extracellular Matrix Production by Mesenchymal Stromal Cells in Hydrogels Facilitates Cell Spreading and Is Inhibited by FGF-2.

In native tissues, the interaction between cells and the surrounding extracellular matrix (ECM) is reciprocal, as cells not only receive signals from the ECM but also actively remodel it through secretion of cell-derived ECM. However, very little is known about the reciprocal interaction between cells and their secreted ECM within synthetic biomaterials that mimic the ECM for use in engineering of tissues for regenerative medicine or as tissue models. Here, poly(ethylene glycol) (PEG) hydrogels with fully defined biomaterial properties are used to investigate the emerging role of cell-derived ECM on culture outcomes. It is shown that human mesenchymal stromal cells (MSCs) secrete ECM proteins into the pericellular space early after encapsulation and that, even in the absence of material-presented cell adhesion motifs, cell-derived fibronectin enables cell spreading. Then, it is investigated how different culture conditions influence MSC ECM expression in hydrogels. Most strikingly, it is found by RNA sequencing that the fibroblast growth factor 2 (FGF-2) changes ECM gene expression and, in particular, decreases the expression of structural ECM components including fibrillar collagens. In summary, this work shows that cell-derived ECM is a guiding cue in 3D hydrogels and that FGF-2 is a potentially important ECM regulator within bioengineered cell and tissue systems.

Tendon explant models for physiologically relevant invitro study of tissue biology - a perspective.

Background: Tendon disorders increasingly afflict our aging society but we lack the scientific understanding to clinically address them. Clinically relevant models of tendon disease are urgently needed as established small animal models of tendinopathy fail to capture essential aspects of the disease. Two-dimensional and three-dimensional cell and tissue culture models are similarly limited, lacking many physiological extracellular matrix cues required to maintain tissue homeostasis or guide matrix remodeling. These cues reflect the biochemical and biomechanical status of the tissue, and encode information regarding the mechanical and metabolic competence of the tissue. Tendon explants overcome some of these limitations and have thus emerged as a valuable tool for the discovery and study of mechanisms associated with tendon homeostasis and pathophysiology. Tendon explants retain native cell-cell and cell-matrix connections, while allowing highly reproducible experimental control over extrinsic factors like mechanical loading and nutritional availability. In this sense tendon explant models can deliver insights that are otherwise impossible to obtain from in vivo animal or in vitro cell culture models. Purpose: In this review, we aimed to provide an overview of tissue explant models used in tendon research, with a specific focus on the value of explant culture systems for the controlled study of the tendon core tissue. We discuss their advantages, limitations and potential future utility. We include suggestions and technical recommendations for the successful use of tendon explant cultures and conclude with an outlook on how explant models may be leveraged with state-of-the-art biotechnologies to propel our understanding of tendon physiology and pathology.

Tendon response to matrix unloading is determined by the patho-physiological niche.

Although the molecular mechanisms behind tendon disease remain obscure, aberrant stromal matrix turnover and tissue hypervascularity are known hallmarks of advanced tendinopathy. We harness a tendon explant model to unwind complex cross-talk between the stromal and vascular tissue compartments. We identify the hypervascular tendon niche as a state-switch that gates degenerative matrix remodeling within the tissue stroma. Here pathological conditions resembling hypervascular tendon disease provoke rapid cell-mediated tissue breakdown upon mechanical unloading, in contrast to unloaded tendons that remain functionally stable in physiological low-oxygen/-temperature niches. Analyses of the stromal tissue transcriptome and secretome reveal that a stromal niche with elevated tissue oxygenation and temperature drives a ROS mediated cellular stress response that leads to adoption of an immune-modulatory phenotype within the degrading stromal tissue. Degradomic analysis further reveals a surprisingly rich set of active matrix proteases behind the progressive loss of tissue mechanics. We conclude that the tendon stromal compartment responds to aberrant mechanical unloading in a manner that is highly dependent on the vascular niche, with ROS gating a complex proteolytic breakdown of the functional collagen backbone.

Tendon tissue microdamage and the limits of intrinsic repair.

The transmission of mechanical muscle force to bone for musculoskeletal stability and movement is one of the most important functions of tendon. The load-bearing tendon core is composed of highly aligned collagen-rich fascicles interspersed with stromal cells (tenocytes). Despite being built to bear very high mechanical stresses, supra-physiological/repetitive mechanical overloading leads to tendon microdamage in fascicles, and potentially to tendon disease and rupture. To date, it is unclear to what extent intrinsic healing mechanisms of the tendon core compartment can repair microdamage. In the present study, we investigated the healing capacity of the tendon core compartment in an ex vivo tissue explant model. To do so, we isolated rat tail tendon fascicles, damaged them by applying a single stretch to various degrees of sub-rupture damage and longitudinally assessed downstream functional and structural changes over a period of several days. Functional damage was assessed by changes in the elastic modulus of the material stress-strain curves, and biological viability of the resident tenocytes. Structural damage was quantified using a fluorescent collagen hybridizing peptide (CHP) to label mechanically disrupted collagen structures. While we observed functional mechanical damage for strains above 2% of the initial fascicle length, structural collagen damage was only detectable for 6% strain and beyond. Minimally loaded/damaged fascicles (2-4% strain) progressively lost elastic modulus over the course of tissue culture, despite their collagen structures remaining intact with high degree of maintained cell viability. In contrast, more severely overloaded fascicles (6-8% strain) with damage at the molecular/collagen level showed no further loss of the elastic modulus but markedly decreased cell viability. Surprisingly, in these heavily damaged fascicles the elastic modulus partially recovered, an effect also seen in further experiments on devitalized fascicles, implying the possibility of a non-cellular but matrix-driven mechanism of molecular repair. Overall, our findings indicate that the tendon core has very little capacity for self-repair of microdamage. We conclude that stromal tenocytes likely do not play a major role in anabolic repair of tendon matrix microdamage, but rather mediate catabolic matrix breakdown and communication with extrinsic cells that are able to effect tissue repair.

Mesenchymal stromal cell activation by breast cancer secretomes in bioengineered 3D microenvironments.

Mesenchymal stromal cells (MSCs) are key contributors of the tumour microenvironment and are known to promote cancer progression through reciprocal communication with cancer cells, but how they become activated is not fully understood. Here, we investigate how breast cancer cells from different stages of the metastatic cascade convert MSCs into tumour-associated MSCs (TA-MSCs) using unbiased, global approaches. Using mass spectrometry, we compared the secretomes of MCF-7 cells, invasive MDA-MB-231 cells, and sublines isolated from bone, lung, and brain metastases and identified ECM and exosome components associated with invasion and organ-specific metastasis. Next, we used synthetic hydrogels to investigate how these different secretomes activate MSCs in bioengineered 3D microenvironments. Using kinase activity profiling and RNA sequencing, we found that only MDA-MB-231 breast cancer secretomes convert MSCs into TA-MSCs, resulting in an immunomodulatory phenotype that was particularly prominent in response to bone-tropic cancer cells. We have investigated paracrine signalling from breast cancer cells to TA-MSCs in 3D, which may highlight new potential targets for anticancer therapy approaches aimed at targeting tumour stroma.

Inspired by Nature: Hydrogels as Versatile Tools for Vascular Engineering.

Significance: Diseases related to vascular malfunction, hyper-vascularization, or lack of vascularization are among the leading causes of morbidity and mortality. Engineered, vascularized tissues as well as angiogenic growth factor-releasing hydrogels could replace defective tissues. Further, treatments and testing of novel vascular therapeutics will benefit significantly from models that allow for the study of vascularized tissues under physiological relevant in vitro conditions. Recent Advances: Inspired by fibrin, the provisional matrix during wound healing, naturally derived and synthetic hydrogel scaffolds have been developed for vascular engineering. Today, engineers and biologists use commercially available hydrogels to pre-vascularize tissues, to control the delivery of angiogenic growth factors, and to establish vascular diseases models. Critical Issue: For clinical translation, pre-vascularized tissue constructs must be sufficiently large and stable to substitute function-relevant tissue defects and integrate with host vascular perfusion. Moreover, the continuous integration of knowhow from basic vascular biology with innovative, tailorable materials and advanced manufacturing technologies is key to achieving near-physiological tissue models and new treatments to control vascularization. Future Directions: For transplantation, engineered tissues must comprise hierarchically organized vascular trees of different caliber and function. The development of novel vascularization-promoting or -inhibiting therapeutics will benefit from physiologically relevant vessel models. In addition, tissue models representing treatment-relevant vascular tissue functions will increase the capacity to screen for therapeutic compounds and will significantly reduce the need for animals for their validation.

Notch-inducing hydrogels reveal a perivascular switch of mesenchymal stem cell fate.

The fate of mesenchymal stem cells (MSCs) in the perivascular niche, as well as factors controlling their fate, is poorly understood. Here, we study MSCs in the perivascular microenvironment of endothelial capillaries by modifying a synthetic 3D biomimetic poly(ethylene glycol) (PEG)-hydrogel system in vitro We show that MSCs together with endothelial cells form micro-capillary networks specifically in soft PEG hydrogels. Transcriptome analysis of human MSCs isolated from engineered capillaries shows a prominent switch in extracellular matrix (ECM) production. We demonstrate that the ECM phenotypic switch of MSCs can be recapitulated in the absence of endothelial cells by functionalizing PEG hydrogels with the Notch-activator Jagged1. Moreover, transient culture of MSCs in Notch-inducing microenvironments reveals the reversibility of this ECM switch. These findings provide insight into the perivascular commitment of MSCs by use of engineered niche-mimicking synthetic hydrogels.

A dual phenotype of MDA-MB-468 cancer cells reveals mutual regulation of tensin3 and adhesion plasticity.

A change regarding the extent of adhesion - hereafter referred to as adhesion plasticity - between adhesive and less-adhesive states of mammalian cells is important for their behavior. To investigate adhesion plasticity, we have selected a stable isogenic subpopulation of human MDA-MB-468 breast carcinoma cells growing in suspension. These suspension cells are unable to re-adhere to various matrices or to contract three-dimensional collagen lattices. By using transcriptome analysis, we identified the focal adhesion protein tensin3 (Tns3) as a determinant of adhesion plasticity. Tns3 is strongly reduced at mRNA and protein levels in suspension cells. Furthermore, by transiently challenging breast cancer cells to grow under non-adherent conditions markedly reduces Tns3 protein expression, which is regained upon re-adhesion. Stable knockdown of Tns3 in parental MDA-MB-468 cells results in defective adhesion, spreading and migration. Tns3-knockdown cells display impaired structure and dynamics of focal adhesion complexes as determined by immunostaining. Restoration of Tns3 protein expression in suspension cells partially rescues adhesion and focal contact composition. Our work identifies Tns3 as a crucial focal adhesion component regulated by, and functionally contributing to, the switch between adhesive and non-adhesive states in MDA-MB-468 cancer cells.

Cell-Mediated Proteolytic Release of Growth Factors from Poly(Ethylene Glycol) Matrices.

Engineering in vitro tissue mimetics that resemble the corresponding living tissues requires the 3D arrangement of tissue progenitor cells and their differentiation by localized growth factor (GF) signaling cues. Recent technological advances open a large field of possibilities for the creation of complex GF arrangements. Additionally, cell-instructive biomaterials, which bind GFs by various mechanisms and release them with different kinetics depending on binding affinity, have become available. This paper describes the development of a matrix metalloproteinase (MMP)-degradable streptavidin-based linker module, which allows the release of immobilized GFs from synthetic biomimetic poly(ethylene glycol) hydrogels independently of the hydrogel degradation. The MMP-sensitive streptavidin linker is shown to efficiently bind biotinylated molecules, and as proof of concept, bone morphogenetic protein-2 (BMP-2) delivery via the MMP-degradable linker is used to induce osteogenic differentiation in C2C12 cells and mesenchymal stem cells. The results show a significantly increased net effect of proteolytically releasable BMP-2 in comparison to stably immobilized and soluble BMP-2. This study indicates that a GF delivery system directly responsive to cellular activity can have important implications for the synthesis of tissue mimetics and regenerative medicine, as it can influence the availability, the localization of effects, as well as efficacy of employed GFs.

Dual Role of Mesenchymal Stem Cells Allows for Microvascularized Bone Tissue-Like Environments in PEG Hydrogels.

In vitro engineered tissues which recapitulate functional and morphological properties of bone marrow and bone tissue will be desirable to study bone regeneration under fully controlled conditions. Among the key players in the initial phase of bone regeneration are mesenchymal stem cells (MSCs) and endothelial cells (ECs) that are in close contact in many tissues. Additionally, the generation of tissue constructs for in vivo transplantations has included the use of ECs since insufficient vascularization is one of the bottlenecks in (bone) tissue engineering. Here, 3D cocultures of human bone marrow derived MSCs (hBM-MSCs) and human umbilical vein endothelial cells (HUVECs) in synthetic biomimetic poly(ethylene glycol) (PEG)-based matrices are directed toward vascularized bone mimicking tissue constructs. In this environment, bone morphogenetic protein-2 (BMP-2) or fibroblast growth factor-2 (FGF-2) promotes the formation of vascular networks. However, while osteogenic differentiation is achieved with BMP-2, the treatment with FGF-2 suppressed osteogenic differentiation. Thus, this study shows that cocultures of hBM-MSCs and HUVECs in biological inert PEG matrices can be directed toward bone and bone marrow-like 3D tissue constructs.