Engineered 3D Immuno-Glial-Neurovascular Human Brain Model.

Patient-specific, human-based cellular models that integrate biomimetic BBB, immune, and myelinated neuron components are critically needed to enable translationally relevant and accelerated discovery of neurological disease mechanisms and interventions. By engineering a brain-mimicking 3D hydrogel and co-culturing all six major brain cell types derived from patient iPSCs, we have constructed, characterized, and utilized a multicellular integrated brain (miBrain) immuno-glial-neurovascular model with in vivo- like hallmarks. As proof of principle, here we utilized the miBrain to model Alzheimer's Disease pathologies associated with APOE4 genetic risk. APOE4 miBrains differentially exhibit amyloid aggregation, tau phosphorylation, and astrocytic GFAP. Unlike the co-emergent fate specification of glia and neurons in organoids, miBrains integrate independently differentiated cell types in a modular system with unique utility for elucidating cell-type specific contributions to pathogenesis. We here harness this feature to identify that risk factor APOE4 in astrocytes promotes tau pathogenesis and neuronal dysregulation through crosstalk with microglia. One-Sentence Summary: A novel patient-specific brain model with BBB, neuronal, immune, and glial components was developed, characterized, and harnessed to model Alzheimer's Disease-associated pathologies and APOE4 genetic risk.

Engineering multicellular living systems-a Keystone Symposia report.

The ability to engineer complex multicellular systems has enormous potential to inform our understanding of biological processes and disease and alter the drug development process. Engineering living systems to emulate natural processes or to incorporate new functions relies on a detailed understanding of the biochemical, mechanical, and other cues between cells and between cells and their environment that result in the coordinated action of multicellular systems. On April 3-6, 2022, experts in the field met at the Keystone symposium "Engineering Multicellular Living Systems" to discuss recent advances in understanding how cells cooperate within a multicellular system, as well as recent efforts to engineer systems like organ-on-a-chip models, biological robots, and organoids. Given the similarities and common themes, this meeting was held in conjunction with the symposium "Organoids as Tools for Fundamental Discovery and Translation".

Aligned Gelatin Microribbon Scaffolds with Hydroxyapatite Gradient for Engineering the Bone-Tendon Interface.

Injuries of the bone-to-tendon interface, such as rotator cuff and anterior cruciate ligament tears, are prevalent musculoskeletal injuries, yet effective methods for repair remain elusive. Tissue engineering approaches that use cells and biomaterials offer a promising potential solution for engineering the bone-tendon interface, but previous strategies require seeding multiple cell types and use of multiphasic scaffolds to achieve zonal-specific tissue phenotype. Furthermore, mimicking the aligned tissue morphology present in native bone-tendon interface in three-dimensional (3D) remains challenging. To facilitate clinical translation, engineering bone-tendon interface using a single cell source and one continuous scaffold with alignment cues would be more attractive but has not been achieved before. To address these unmet needs, in this study, we develop an aligned gelatin microribbon (µRB) hydrogel scaffold with hydroxyapatite nanoparticle (HA-np) gradient for guiding zonal-specific differentiation of human mesenchymal stem cell (hMSC) to mimic the bone-tendon interface. We demonstrate that aligned µRBs led to cell alignment in 3D, and HA gradient induced zonal-specific differentiation of mesenchymal stem cells that resemble the transition at the bone-tendon interface. Short chondrogenic priming before exposure to osteogenic factors further enhanced the mimicry of bone-cartilage-tendon transition with significantly improved tensile moduli of the resulting tissues. In summary, aligned gelatin µRBs with HA gradient coupled with optimized soluble factors may offer a promising strategy for engineering bone-tendon interface using a single cell source. Impact statement Our 3D macroporous microribbon hydrogel platform with alignment cues zonally integrated with hydroxyapatite nanoparticles enables differentiation across the bone-tendon interface within a continuous scaffold. While most interfacial scaffolds heretofore rely on composites and multilayer approaches, we present a continuous scaffold utilizing a single cell source. The synergy of niche cues with human mesenchymal stem cell (hMSC) culture leads to an over 45-fold enhancement in tensile modulus in culture. We further demonstrate that priming hMSCs towards the chondrogenic lineage can enhance the differential osteogenesis. Relying on a single cell source could enhance zone integration and scaffold integrity, along with practical benefits.

Negative Transpulmonary Pressure Disrupts Airway Morphogenesis by Suppressing Fgf10.

Mechanical forces are increasingly recognized as important determinants of cell and tissue phenotype and also appear to play a critical role in organ development. During the fetal stages of lung morphogenesis, the pressure of the fluid within the lumen of the airways is higher than that within the chest cavity, resulting in a positive transpulmonary pressure. Several congenital defects decrease or reverse transpulmonary pressure across the developing airways and are associated with a reduced number of branches and a correspondingly underdeveloped lung that is insufficient for gas exchange after birth. The small size of the early pseudoglandular stage lung and its relative inaccessibility in utero have precluded experimental investigation of the effects of transpulmonary pressure on early branching morphogenesis. Here, we present a simple culture model to explore the effects of negative transpulmonary pressure on development of the embryonic airways. We found that negative transpulmonary pressure decreases branching, and that it does so in part by altering the expression of fibroblast growth factor 10 (Fgf10). The morphogenesis of lungs maintained under negative transpulmonary pressure can be rescued by supplementing the culture medium with exogenous FGF10. These data suggest that Fgf10 expression is regulated by mechanical stress in the developing airways. Understanding the mechanical signaling pathways that connect transpulmonary pressure to FGF10 can lead to the establishment of novel non-surgical approaches for ameliorating congenital lung defects.

Varying solvent type modulates collagen coating and stem cell mechanotransduction on hydrogel substrates.

Type I collagen is the most abundant extracellular matrix protein in the human body and is commonly used as a biochemical ligand for hydrogel substrates to support cell adhesion in mechanotransduction studies. Previous protocols for conjugating collagen I have used different solvents; yet, how varying solvent pH and composition impacts the efficiency and distribution of these collagen I coatings remains unknown. Here, we examine the effect of varying solvent pH and type on the efficiency and distribution of collagen I coatings on polyacrylamide hydrogels. We further evaluate the effects of varying solvent on mechanotransduction of human mesenchymal stem cells (MSCs) by characterizing cell spreading and localization of Yes-Associated Protein (YAP), a key transcriptional regulator of mechanotransduction. Increasing solvent pH to 5.2 and above increased the heterogeneity of coating with collagen bundle formation. Collagen I coating highly depends on the solvent type, with acetic acid leading to the highest conjugation efficiency and most homogeneous coating. Compared to HEPES or phosphate-buffered saline buffer, acetic acid-dissolved collagen I coatings substantially enhance MSC adhesion and spreading on both glass and polyacrylamide hydrogel substrates. When acetic acid was used for collagen coatings, even the low collagen concentration (1 µg/ml) induced robust MSC spreading and nuclear YAP localization on both soft (3 kPa) and stiff (38 kPa) substrates. Depending on the solvent type, stiffness-dependent nuclear YAP translocation occurs at a different collagen concentration. Together, the results from this study validate the solvent type as an important parameter to consider when using collagen I as the biochemical ligand to support cell adhesion.

Extracellular matrix type modulates mechanotransduction of stem cells.

Extracellular matrix (ECM) is comprised of different types of proteins, which change in composition and ratios during morphogenesis and disease progression. ECM proteins provide cell adhesion and impart mechanical cues to the cells. Increasing substrate stiffness has been shown to induce Yes-associated protein (YAP) translocation from the cytoplasm to the nucleus, yet these mechanistic studies used fibronectin only as the biochemical cue. How varying the types of ECM modulates mechanotransduction of stem cells remains largely unknown. Using polyacrylamide hydrogels with tunable stiffness as substrates, here we conjugated four major ECM proteins commonly used for cell adhesion: fibronectin, collagen I, collagen IV and laminin, and assessed the effects of varying ECM type and density on YAP translocation in human mesenchymal stem cells (hMSCs). For all four ECM types, increasing ECM ligand density alone can induce YAP nuclear translocation without changing substrate stiffness. The ligand threshold for such biochemical ligand-induced YAP translocation differs across ECM types. While stiffness-dependent YAP translocation can be induced by all four ECM types, each ECM requires a different optimized ligand density for this to occur. Using antibody blocking, we further identified integrin subunits specifically involved in mechanotransduction of different ECM types. Finally, we demonstrated that altering ECM type further modulates hMSC osteogenesis without changing substrate stiffness. These findings highlight the important role of ECM type in modulating mechanotransduction and differentiation of stem cells, and call for future mechanistic studies to further elucidate the role of changes in ECM compositions in mediating mechanotransduction during morphogenesis and disease progression. STATEMENT OF SIGNIFICANCE: Our study addresses a critical gap of knowledge in mechanobiology. Increasing substrate stiffness has been shown to induce nuclear YAP translocation, yet only on fibronectin-coated substrates. However, extracellular matrix (ECM) is comprised of different protein types. How varying the type of ECM modulates stem cell mechanotransduction remains largely unknown. We here reveal that the choice of ECM type can directly modulate stem cell mechanotransduction, filling this critical gap. This work has broad impacts in mechanobiology and biomaterials, as it provides the first evidence that varying ECM type can impact YAP translocation independent of substrate stiffness, opening doors for a more rational biomaterials design tuning ECM properties to control cell fate for promoting normal development and for preventing disease progression.

Hydrogels with enhanced protein conjugation efficiency reveal stiffness-induced YAP localization in stem cells depends on biochemical cues.

Polyacrylamide hydrogels have been widely used in stem cell mechanotransduction studies. Conventional conjugation methods of biochemical cues to polyacrylamide hydrogels suffer from low conjugation efficiency, which leads to poor attachment of human pluripotent stem cells (hPSCs) on soft substrates. In addition, while it is well-established that stiffness-dependent regulation of stem cell fate requires cytoskeletal tension, and is mediated through nuclear translocation of transcription regulator, Yes-associated protein (YAP), the role of biochemical cues in stiffness-dependent YAP regulation remains largely unknown. Here we report a method that enhances the conjugation efficiency of biochemical cues on polyacrylamide hydrogels compared to conventional methods. This modified method enables robust hPSC attachment, proliferation and maintenance of pluripotency across varying substrate stiffness (3 kPa-38 kPa). Using this hydrogel platform, we demonstrate that varying the types of biochemical cues (Matrigel, laminin, GAG-peptide) or density of Matrigel can alter stiffness-dependent YAP localization in hPSCs. In particular, we show that stiffness-dependent YAP localization is overridden at low or high density of Matrigel. Furthermore, human mesenchymal stem cells display stiffness-dependent YAP localization only at intermediate fibronectin density. The hydrogel platform with enhanced conjugation efficiency of biochemical cues provides a powerful tool for uncovering the role of biochemical cues in regulating mechanotransduction of various stem cell types.

Biochemical Ligand Density Regulates Yes-Associated Protein Translocation in Stem Cells through Cytoskeletal Tension and Integrins.

Different tissue types are characterized by varying stiffness and biochemical ligands. Increasing substrate stiffness has been shown to trigger Yes-associated protein (YAP) translocation from the cytoplasm to the nucleus, yet the role of ligand density in modulating mechanotransduction and stem cell fate remains largely unexplored. Using polyacrylamide hydrogels coated with fibronectin as a model platform, we showed that stiffness-induced YAP translocation occurs only at intermediate ligand densities. At low or high ligand densities, YAP localization is dominated by ligand density independent of substrate stiffness. We further showed that ligand density-induced YAP translocation requires cytoskeleton tension and αVß3-integrin binding. Finally, we demonstrate that increasing ligand density alone can enhance osteogenic differentiation regardless of matrix stiffness. Together, the findings from the present study establish ligand density as an important parameter for modulating stem cell mechanotransduction and differentiation, which is mediated by integrin clustering, focal adhesion, and cytoskeletal tension.