Human placental organoids as a model to probe early gestation maternal immune dynamics.

During pregnancy, the human placenta establishes tolerance toward fetal allogeneic tissue, where specialized trophoblast subtypes play a complex role in local and peripheral immunomodulation. However, due to inadequate models to study the early gestation of the human placenta, each trophoblast subtype's role in modulating the maternal immune response has remained elusive. Here, we derived human placental organoids from early gestation trophoblast stem cells to (1) identify patterns of immunomodulatory protein expression by trophoblast subtype and (2) evaluate the effects of the placental organoid secretome on immune cell activation and regulation. We show that the three primary trophoblast phenotypes had distinct influences on immune cell phenotype and activation and that three-dimensional culture significantly alters trophoblast immunomodulation relative to traditional two-dimensional trophoblast culture.

Engineered 3D Hydrogel Matrices to Modulate Trophoblast Stem Cell-Derived Placental Organoid Phenotype.

Placental organoid models are a promising platform to study human placental development and function. Organoid systems typically use naturally derived hydrogel extracellular matrices (ECM), resulting in batch-to-batch variability that limits experimental reproducibility. As an alternative, synthetic ECM-mimicking hydrogel matrices offer greater consistency and control over environmental cues. Here, we generated trophoblast stem cell-derived placental organoids using poly(ethylene glycol) (PEG) hydrogels with tunable degradability and placenta-derived ECM cues to evaluate trophoblast differentiation relative to Matrigel and two-dimensional (2D) culture controls. Our data demonstrate that PEG hydrogels support trophoblast viability and metabolic function comparable to gold standard Matrigel. Additionally, phenotypic characterization via proteomic analysis revealed that PEG and Matrigel matrices drive syncytiotrophoblast and extravillous trophoblast-dominant placental organoid phenotypes, respectively. Further, three-dimensional (3D) environments promoted greater integrin expression and ECM production than 2D culture. This study demonstrates that engineered 3D culture environments can be used to reliably generate placental organoids and guide trophoblast differentiation.

Controlling placental spheroid growth and phenotype using engineered synthetic hydrogel matrices.

The human placenta is a complex organ comprised of multiple trophoblast subtypes, and inadequate models to study the human placenta in vitro limit the current understanding of human placental behavior and development. Common in vitro placental models rely on two-dimensional culture of cell lines and primary cells, which do not replicate the native tissue microenvironment, or poorly defined three-dimensional hydrogel matrices such as Matrigel™ that provide limited environmental control and suffer from high batch-to-batch variability. Here, we employ a highly defined, synthetic poly(ethylene glycol)-based hydrogel system with tunable degradability and presentation of extracellular matrix-derived adhesive ligands native to the placenta microenvironment to generate placental spheroids. We evaluate the capacity of a hydrogel library to support the viability, function, and phenotypic protein expression of three human trophoblast cell lines modeling varied trophoblast phenotypes and find that degradable synthetic hydrogels support the greatest degree of placental spheroid viability, proliferation, and function relative to standard Matrigel controls. Finally, we show that trophoblast culture conditions modulate cell functional phenotype as measured by proteomics analysis and functional secretion assays. Engineering precise control of placental spheroid development in vitro may provide an important new tool for the study of early placental behavior and development.

Lipid-Polymer Hybrid Nanoparticles Utilize B Cells and Dendritic Cells to Elicit Distinct Antigen-Specific CD4+ and CD8+ T Cell Responses.

Antigen-presenting cells (APCs) are widely studied for treating immune-mediated diseases, and dendritic cells (DCs) are potent APCs that uptake and present antigens (Ags). However, DCs face several challenges that hinder their clinical translation due to their inability to control Ag dosing and low abundance in peripheral blood. B cells are a potential alternative to DCs, but their poor nonspecific Ag uptake capabilities compromise controllable priming of T cells. Here, we developed phospholipid-conjugated Ags (L-Ags) and lipid-polymer hybrid nanoparticles (L/P-Ag NPs) as delivery platforms to expand the range of accessible APCs for use in T cell priming. These delivery platforms were evaluated using DCs, CD40-activated B cells, and resting B cells to understand the impacts of various Ag delivery mechanisms for generation of Ag-specific T cell responses. L-Ag delivery (termed depoting) of MHC class I- and II-restricted Ags successfully loaded all APC types in a tunable manner and primed both Ag-specific CD8+ and CD4+ T cells, respectively. Incorporating L-Ags and polymer-conjugated Ags (P-Ag) into NPs can direct Ags to different uptake pathways to engineer the dynamics of presentation and shape T cell responses. DCs were capable of processing and presenting Ag delivered from both L- and P-Ag NPs, yet B cells could only utilize Ag delivered from L-Ag NPs, which led to differential cytokine secretion profiles in coculture studies. Altogether, we show that L-Ags and P-Ags can be rationally paired within a single NP to leverage distinct delivery mechanisms to access multiple Ag processing pathways in two APC types, offering a modular delivery platform for engineering Ag-specific immunotherapies.

Hydrogel Injection Molding to Generate Complex Cell Encapsulation Geometries.

Biofabrication methods capable of generating complex, three-dimensional, cell-laden hydrogel geometries are often challenging technologies to implement in the clinic and scaled manufacturing processes. Hydrogel injection molding capitalizes on the reproducibility, efficiency, and scalability of the injection molding process, and we adapt this technique to biofabrication using a library of natural and synthetic hydrogels with varied crosslinking chemistries and kinetics. We use computational modeling to evaluate hydrogel library fluid dynamics within the injection molds in order to predict molding feasibility and cytocompatibility. We evaluate the reproducibility of hydrogel construct molding and extraction and establish criteria for the selection of hydrogels suitable for injection molding. We demonstrate that hydrogel injection molding is capable of generating complex three-dimensional cell-laden construct geometries using diverse hydrogel materials and that this platform is compatible with primary human islet encapsulation. These results highlight the versatility and feasibility of hydrogel injection molding as a biofabrication technique with potential applications in the clinic and biomanufacturing.

A Method for Organoid Transplantation and Whole-Mount Visualization of Post-Engraftment Vascularization.

As the field of organoid development matures, the need to transplant organoids to evaluate and characterize their functionality grows. Decades of research developing islet organoid transplantation for the treatment of type 1 diabetes can contribute substantially to accelerating diverse tissue organoid transplantation. Biomaterials-based organoid delivery methods offer the potential to maximize organoid survival and engraftment. In this protocol, we describe a vasculogenic degradable hydrogel vehicle and a method to deliver organoids to intraperitoneal tissue. Further, we describe a method to fluorescently label and image functional vasculature within the graft as a tool to investigate organoid engraftment.

Biomaterial-based approaches to engineering immune tolerance.

The development of biomaterial-based therapeutics to induce immune tolerance holds great promise for the treatment of autoimmune diseases, allergy, and graft rejection in transplantation. Historical approaches to treat these immunological challenges have primarily relied on systemic delivery of broadly-acting immunosuppressive agents that confer undesirable, off-target effects. The evolution and expansion of biomaterial platforms has proven to be a powerful tool in engineering immunotherapeutics and enabled a great diversity of novel and targeted approaches in engineering immune tolerance, with the potential to eliminate side effects associated with systemic, non-specific immunosuppressive approaches. In this review, we summarize the technological advances within three broad biomaterials-based strategies to engineering immune tolerance: nonspecific tolerogenic agent delivery, antigen-specific tolerogenic therapy, and the emergent area of tolerogenic cell therapy.

Lipid-Mediated Insertion of Toll-Like Receptor (TLR) Ligands for Facile Immune Cell Engineering.

Cell-based immunotherapies have tremendous potential to treat many diseases, such as activating immunity in cancer or suppressing it in autoimmune diseases. Most cell-based cancer immunotherapies in the clinic provide adjuvant signals through genetic engineering to enhance T cell functions. However, genetically encoded signals have minimal control over dosing and persist for the life of a cell lineage. These properties make it difficult to balance increasing therapeutic efficacy with reducing toxicities. Here, we demonstrated the potential of phospholipid-coupled ligands as a non-genetic system for immune cell engineering. This system provides simple, controlled, non-genetic adjuvant delivery to immune cells via lipid-mediated insertion into plasma membranes. Lipid-mediated insertion (termed depoting) successfully delivered Toll-like receptor (TLR) ligands intracellularly and onto cell surfaces of diverse immune cells. These ligands depoted into immune cells in a dose-controlled fashion and did not compete during multiplex pairwise loading. Immune cell activation could be enhanced by autocrine and paracrine mechanisms depending on the biology of the TLR ligand tested. Depoted ligands functionally persisted on plasma membranes for up to 4 days in naïve and activated T cells, enhancing their activation, proliferation, and skewing cytokine secretion. Our data showed that depoted ligands provided a persistent yet non-permanent adjuvant signal to immune cells that may minimize the intensity and duration of toxicities compared to permanent genetic delivery. Altogether, these findings demonstrate potential for lipid-mediated depoting as a universal cell engineering approach with unique, complementary advantages to other cell engineering methods.

Yap suppresses T-cell function and infiltration in the tumor microenvironment.

A major challenge for cancer immunotherapy is sustaining T-cell activation and recruitment in immunosuppressive solid tumors. Here, we report that the levels of the Hippo pathway effector Yes-associated protein (Yap) are sharply induced upon the activation of cluster of differentiation 4 (CD4)-positive and cluster of differentiation 8 (CD8)-positive T cells and that Yap functions as an immunosuppressive factor and inhibitor of effector differentiation. Loss of Yap in T cells results in enhanced T-cell activation, differentiation, and function, which translates in vivo to an improved ability for T cells to infiltrate and repress tumors. Gene expression analyses of tumor-infiltrating T cells following Yap deletion implicates Yap as a mediator of global T-cell responses in the tumor microenvironment and as a negative regulator of T-cell tumor infiltration and patient survival in diverse human cancers. Collectively, our results indicate that Yap plays critical roles in T-cell biology and suggest that Yap inhibition improves T-cell responses in cancer.