Macrophages enhance contractile force in iPSC-derived human engineered cardiac tissue.

Resident cardiac macrophages are critical mediators of cardiac function. Despite their known importance to cardiac electrophysiology and tissue maintenance, there are currently no stem-cell-derived models of human engineered cardiac tissues (hECTs) that include resident macrophages. In this study, we made an induced pluripotent stem cell (iPSC)-derived hECT model with a resident population of macrophages (iM0) to better recapitulate the native myocardium and characterized their impact on tissue function. Macrophage retention within the hECTs was confirmed via immunofluorescence after 28 days of cultivation. The inclusion of iM0s significantly impacted hECT function, increasing contractile force production. A potential mechanism underlying these changes was revealed by the interrogation of calcium signaling, which demonstrated the modulation of ß-adrenergic signaling in +iM0 hECTs. Collectively, these findings demonstrate that macrophages significantly enhance cardiac function in iPSC-derived hECT models, emphasizing the need to further explore their contributions not only in healthy hECT models but also in the contexts of disease and injury.

An engineered human cardiac tissue model reveals contributions of systemic lupus erythematosus autoantibodies to myocardial injury.

Systemic lupus erythematosus (SLE) is a highly heterogenous autoimmune disease that affects multiple organs, including the heart. The mechanisms by which myocardial injury develops in SLE, however, remain poorly understood. Here we engineered human cardiac tissues and cultured them with IgG fractions containing autoantibodies from SLE patients with and without myocardial involvement. We observed unique binding patterns of IgG from two patient subgroups: (i) patients with severe myocardial inflammation exhibited enhanced binding to apoptotic cells within cardiac tissues subjected to stress, and (ii) patients with systolic dysfunction exhibited enhanced binding to the surfaces of viable cardiomyocytes. Functional assays and RNA sequencing (RNA-seq) revealed that IgGs from patients with systolic dysfunction exerted direct effects on engineered tissues in the absence of immune cells, altering tissue cellular composition, respiration and calcium handling. Autoantibody target characterization by phage immunoprecipitation sequencing (PhIP-seq) confirmed distinctive IgG profiles between patient subgroups. By coupling IgG profiling with cell surface protein analyses, we identified four pathogenic autoantibody candidates that may directly alter the function of cells within the myocardium. Taken together, these observations provide insights into the cellular processes of myocardial injury in SLE that have the potential to improve patient risk stratification and inform the development of novel therapeutic strategies.

Lung-Mimetic Hydrofoam Sealant to Treat Pulmonary Air Leak.

Pulmonary air leak is the most common complication of lung surgery, contributing to post-operative morbidity in up to 60% of patients; yet, there is no reliable treatment. Available surgical sealants do not match the demanding deformation mechanics of lung tissue; and therefore, fail to seal air leak. To address this therapeutic gap, a sealant with structural and mechanical similarity to subpleural lung is designed, developed, and systematically evaluated. This "lung-mimetic" sealant is a hydrofoam material that has alveolar-like porous ultrastructure, lung-like viscoelastic properties (adhesive, compressive, tensile), and lung extracellular matrix-derived signals (matrikines) to support tissue repair. In biocompatibility testing, the lung-mimetic sealant shows minimal cytotoxicity and immunogenicity in vitro. Human primary monocytes exposed to sealant matrikines in vitro upregulate key genes (MARCO, PDGFB, VEGF) known to correlate with pleural wound healing and tissue repair in vivo. In rat and swine models of pulmonary air leak, this lung-mimetic sealant rapidly seals air leak and restores baseline lung mechanics. Altogether, these data indicate that the lung-mimetic sealant can effectively seal pulmonary air leak and promote a favorable cellular response in vitro.

Modeling and countering the effects of cosmic radiation using bioengineered human tissues.

Cosmic radiation is the most serious risk that will be encountered during the planned missions to the Moon and Mars. There is a compelling need to understand the effects, safety thresholds, and mechanisms of radiation damage in human tissues, in order to develop measures for radiation protection during extended space travel. As animal models fail to recapitulate the molecular changes in astronauts, engineered human tissues and "organs-on-chips" are valuable tools for studying effects of radiation in vitro. We have developed a bioengineered tissue platform for studying radiation damage in individualized settings. To demonstrate its utility, we determined the effects of radiation using engineered models of two human tissues known to be radiosensitive: engineered cardiac tissues (eCT, a target of chronic radiation damage) and engineered bone marrow (eBM, a target of acute radiation damage). We report the effects of high-dose neutrons, a proxy for simulated galactic cosmic rays, on the expression of key genes implicated in tissue responses to ionizing radiation, phenotypic and functional changes in both tissues, and proof-of-principle application of radioprotective agents. We further determined the extent of inflammatory, oxidative stress, and matrix remodeling gene expression changes, and found that these changes were associated with an early hypertrophic phenotype in eCT and myeloid skewing in eBM. We propose that individualized models of human tissues have potential to provide insights into the effects and mechanisms of radiation during deep-space missions and allow testing of radioprotective measures.

Engineering early memory B-cell-like phenotype in hydrogel-based immune organoids.

Memory B cells originate in response to antigenic stimulation in B-cell follicles of secondary lymphoid organs where naive B cells undergo maturation within a subanatomical microenvironment, the germinal centers. The understanding of memory B-cell immunology and its regulation is based primarily on sophisticated experiments that involve mouse models. To date, limited evidence exists on whether memory B cells can be successfully engineered ex vivo, specifically using biomaterials-based platforms that support the growth and differentiation of B cells. Here, we report the characterization of a recently reported maleimide-functionalized poly(ethylene glycol) (PEG) hydrogels as immune organoids towards the development of early memory B-cell phenotype and germinal center-like B cells. We demonstrate that the use of interleukin 9 (IL9), IL21, and bacterial antigen presentation as outer membrane-bound fragments drives the conversion of naive, primary murine B cells to early memory phenotype in ex vivo immune organoids. These findings describe the induction of early memory B-cell-like phenotype in immune organoids and highlight the potential of synthetic organoids as a platform for the future development of antigen-specific bona fide memory B cells for the study of the immune system and generation of therapeutic antibodies.

Engineering complexity in human tissue models of cancer.

Major progress in the understanding and treatment of cancer have tremendously improved our knowledge of this complex disease and improved the length and quality of patients' lives. Still, major challenges remain, in particular with respect to cancer metastasis which still escapes effective treatment and remains responsible for 90% of cancer related deaths. In recent years, the advances in cancer cell biology, oncology and tissue engineering converged into the engineered human tissue models of cancer that are increasingly recapitulating many aspects of cancer progression and response to drugs, in a patient-specific context. The complexity and biological fidelity of these models, as well as the specific questions they aim to investigate, vary in a very broad range. When selecting and designing these experimental models, the fundamental question is "how simple is complex enough" to accomplish a specific goal of cancer research. Here we review the state of the art in developing and using the human tissue models in cancer research and developmental drug screening. We describe the main classes of models providing different levels of biological fidelity and complexity, discuss their advantages and limitations, and propose a framework for designing an appropriate model for a given study. We close by outlining some of the current needs, opportunities and challenges in this rapidly evolving field.

Emerging Trajectories for Next Generation Tissue Engineers.

The field of tissue engineering has evolved from its early days of engineering tissue substitutes to current efforts at building human tissues for regenerative medicine and mechanistic studies of tissue disease, injury, and regeneration. Advances in bioengineering, material science, and stem cell biology have enabled major developments in the field. In this perspective, we reflect on the September 2021 virtual Next Generation Tissue Engineering symposium and trainee workshop, as well as our projections for the field over the next 15 years.

Engineered models of tumor metastasis with immune cell contributions.

Most cancer deaths are due to tumor metastasis rather than the primary tumor. Metastasis is a highly complex and dynamic process that requires orchestration of signaling between the tumor, its local environment, distant tissue sites, and immune system. Animal models of cancer metastasis provide the necessary systemic environment but lack control over factors that regulate cancer progression and often do not recapitulate the properties of human cancers. Bioengineered "organs-on-a-chip" that incorporate the primary tumor, metastatic tissue targets, and microfluidic perfusion are now emerging as quantitative human models of tumor metastasis. The ability of these systems to model tumor metastasis in individualized, patient-specific settings makes them uniquely suitable for studies of cancer biology and developmental testing of new treatments. In this review, we focus on human multi-organ platforms that incorporate circulating and tissue-resident immune cells in studies of tumor metastasis.

Organoid Polymer Functionality and Mode of Klebsiella Pneumoniae Membrane Antigen Presentation Regulates Ex Vivo Germinal Center Epigenetics in Young and Aged B Cells.

Antibiotic-resistant bacteria are a major global health threat that continues to rise due to a lack of effective vaccines. Of concern are Klebsiella pneumoniae that fail to induce in vivo germinal center B cell responses, which facilitate antibody production to fight infection. Immunotherapies using antibodies targeting antibiotic-resistant bacteria are emerging as promising alternatives, however, they cannot be efficiently derived ex vivo, necessitating the need for immune technologies to develop therapeutics. Here, PEG-based immune organoids were developed to elucidate the effects of polymer end-point chemistry, integrin ligands, and mode of K. pneumoniae antigen presentation on germinal center-like B cell phenotype and epigenetics, to better define the lymph node microenvironment factors regulating ex vivo germinal center dynamics. Notably, PEG vinyl sulfone or acrylate failed to sustain primary immune cells, but functionalization with maleimide (PEG-4MAL) led to B cell expansion and germinal center-like induction. RNA sequencing analysis of lymph node stromal and germinal center B cells showed niche associated heterogeneity of integrin-related genes. Incorporation of niche-mimicking peptides revealed that collagen-1 promoted germinal center-like dynamics and epigenetics. PEG-4MAL organoids elucidated the impact of K. pneumoniae outer membrane-embedded protein antigen versus soluble antigen presentation on germinal centers and preserved the response across young and aged mice.

Multiscale engineering of immune cells and lymphoid organs.

Immunoengineering applies quantitative and materials-based approaches for the investigation of the immune system and for the development of therapeutic solutions for various diseases, such as infection, cancer, inflammatory diseases and age-related malfunctions. The design of immunomodulatory and cell therapies requires the precise understanding of immune cell formation and activation in primary, secondary and ectopic tertiary immune organs. However, the study of the immune system has long been limited to in vivo approaches, which often do not allow multidimensional control of intracellular and extracellular processes, and to 2D in vitro models, which lack physiological relevance. 3D models built with synthetic and natural materials enable the structural and functional recreation of immune tissues. These models are being explored for the investigation of immune function and dysfunction at the cell, tissue and organ levels. In this Review, we discuss 2D and 3D approaches for the engineering of primary, secondary and tertiary immune structures at multiple scales. We highlight important insights gained using these models and examine multiscale engineering strategies for the design and development of immunotherapies. Finally, dynamic 4D materials are investigated for their potential to provide stimuli-dependent and context-dependent scaffolds for the generation of immune organ models.

In Vitro Model of Macrophage-Biomaterial Interactions.

Tissue engineering and regenerative medicine, facilitated by biomaterial-based therapies, hold promise for the repair, replacement, or regeneration of damaged tissue. The success or failure of all implanted biomaterials, ranging from stainless steel total joint replacements to naturally or synthetically derived skin grafts, is predominantly mediated by macrophages, the primary cell of the innate immune system. In an effort to better assess safety and efficacy of novel biomaterials, evaluating and understanding macrophage-biomaterial interactions is a necessary first step. Here, we describe the culture of macrophages on 3D biomaterials, such as decellularized human cortical bone or commercially available wound matrices, and subsequent analysis using gene expression and protein secretion to help understand how biomaterial properties may influence macrophage phenotype in vitro.

Biomaterials and Bioactive Factor Delivery Systems for the Control of Macrophage Activation in Regenerative Medicine.

Macrophages play an important role in tissue repair, regeneration, and the ability of biomaterials to mediate these processes. Macrophages are highly plastic cells that exhibit altered behavior in response to changes in the microenvironment. With the growing knowledge of the roles that different macrophage phenotypes play in specific pathologies and/or injuries, researchers are now focusing on designing biomaterials to actively control macrophage behavior and promote healing outcomes. In this review, we highlight a variety of biomaterial strategies for controlling macrophage phenotype in chronic wounds, tissue defects, and inflammatory conditions, although these strategies can be applied to many other applications. In particular, we highlight the different situations in which biomaterials should inhibit or promote M1 or M2 activation, or both, for therapeutic outcomes.

In vitro response of macrophages to ceramic scaffolds used for bone regeneration.

Macrophages, the primary cells of the inflammatory response, are major regulators of healing, and mediate both bone fracture healing and the inflammatory response to implanted biomaterials. However, their phenotypic contributions to biomaterial-mediated bone repair are incompletely understood. Therefore, we used gene expression and protein secretion analysis to investigate the interactions in vitro between primary human monocyte-derived macrophages and ceramic scaffolds that have been shown to have varying degrees of success in promoting bone regeneration in vivo Specifically, baghdadite (Ca3ZrSi2O9) and strontium-hardystonite-gahnite (Sr-Ca2ZnSi2O7-ZnAl2O4) scaffolds were chosen as two materials that enhanced bone regeneration in vivo in large defects under load compared with clinically used tricalcium phosphate-hydroxyapatite (TCP-HA). Principal component analysis revealed that the scaffolds differentially regulated macrophage phenotype. Temporal changes in gene expression included shifts in markers of pro-inflammatory M1, anti-inflammatory M2a and pro-remodelling M2c macrophage phenotypes. Of note, TCP-HA scaffolds promoted upregulation of many M1-related genes and downregulation of many M2a- and M2c-related genes. Effects of the scaffolds on macrophages were attributed primarily to direct cell-scaffold interactions because of only minor changes observed in transwell culture. Ultimately, elucidating macrophage-biomaterial interactions will facilitate the design of immunomodulatory biomaterials for bone repair.

Response of human macrophages to wound matrices in vitro.

Chronic wounds remain a major burden to the global healthcare system. Myriad wound matrices are commercially available but their mechanisms of action are poorly understood. Recent studies have shown that macrophages are highly influenced by their microenvironment, but it is not known how different biomaterials affect this interaction. Here, it was hypothesized that human macrophages respond differently to changes in biomaterial properties in vitro with respect to phenotype, including pro-inflammatory M1, anti-inflammatory M2a, known for facilitating extracellular matrix deposition and proliferation, and M2c, which has recently been associated with tissue remodeling. Using multiple donors, it was found that collagen scaffolds cross-linked with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide (EDC/NHS) promoted the least inflammatory phenotype in primary human macrophages compared with scaffolds cross-linked with formaldehyde or glutaraldehyde. Importantly, gene expression analysis trends were largely conserved between donors, especially TNFa (M1), CCL22 (M2a), and MRC1 (M2a). Then the response of primary and THP1 monocyte-derived macrophages to four commercially available wound matrices were compared-Integra Dermal Regeneration Template (Integra), PriMatrix Dermal Repair Scaffold (PriMatrix), AlloMend Acellular Dermal Matrix (AlloMend), and Oasis Wound Matrix (Oasis). Gene expression trends were different between primary and THP1 monocyte-derived macrophages for all six genes analyzed in this study. Finally, the behavior of primary macrophages cultured onto the wound matrices over time was analyzed. Integra and Oasis caused down-regulation of M2a markers CCL22 and TIMP3. PriMatrix caused up-regulation of TNFa (M1) and CD163 (M2c) and down-regulation of CCL22 and TIMP3 (both M2a). AlloMend caused up-regulation in CD163 (M2c). Lastly, Oasis promoted the largest increase in the combinatorial M1/M2 score, defined as the sum of M1 genes divided by the sum of M2 genes. This preliminary study suggested that biomaterials influenced the wound microenvironment to affect macrophage phenotype.

Differential gene expression in human, murine, and cell line-derived macrophages upon polarization.

The mechanisms by which macrophages control the inflammatory response, wound healing, biomaterial-interactions, and tissue regeneration appear to be related to their activation/differentiation states. Studies of macrophage behavior in vitro can be useful for elucidating their mechanisms of action, but it is not clear to what extent the source of macrophages affects their apparent behavior, potentially affecting interpretation of results. Although comparative studies of macrophage behavior with respect to cell source have been conducted, there has been no direct comparison of the three most commonly used cell sources: murine bone marrow, human monocytes from peripheral blood (PB), and the human leukemic monocytic cell line THP-1, across multiple macrophage phenotypes. In this study, we used multivariate discriminant analysis to compare the in vitro expression of genes commonly chosen to assess macrophage phenotype across all three sources of macrophages, as well as those derived from induced pluripotent stem cells (iPSCs), that were polarized towards four distinct phenotypes using the same differentiation protocols: M(LPS,IFN) (aka M1), M(IL4,IL13) (aka M2a), M(IL10) (aka M2c), and M(-) (aka M0) used as control. Several differences in gene expression trends were found among the sources of macrophages, especially between murine bone marrow-derived and human blood-derived M(LPS,IFN) and M(IL4,IL13) macrophages with respect to commonly used phenotype markers like CCR7 and genes associated with angiogenesis and tissue regeneration like FGF2 and MMP9. We found that the genes with the most similar patterns of expression among all sources were CXCL-10 and CXCL-11 for M(LPS,IFN) and CCL17 and CCL22 for M(IL4,IL13). Human PB-derived macrophages and human iPSC-derived macrophages showed similar gene expression patterns among the groups and genes studied here, suggesting that iPSC-derived monocytes have the potential to be used as a reliable cell source of human macrophages for in vitro studies. These findings could help select appropriate markers when testing macrophage behavior in vitro and highlight those markers that may confuse interpretation of results from experiments employing macrophages from different sources.