Project description:Human induced pluripotent stem cells (hiPSCs) have emerged as a promising in vitro model system for studying neurodevelopment. However, current models remain limited in their ability to incorporate tunable biochemical and biomechanical signaling cues imparted by the neural extracellular matrix (ECM). The native brain ECM is viscoelastic and stress-relaxing, exhibiting a time-dependent response to an applied force. To recapitulate the remodelability of the neural ECM, we developed a family of protein-engineered hydrogels crosslinked with either static or dynamic covalent bonds that exhibit tunable stress relaxation rates. hiPSC-derived neural progenitor cells (NPCs) encapsulated within these gels underwent relaxation rate-dependent maturation. Specifically, NPCs within hydrogels with faster stress relaxation rates extended longer, more complex neuritic projections, exhibited decreased metabolic activity, and expressed higher levels of genes associated with neural maturation. By inhibiting actin polymerization, we observed decreased neuritic projections and a concomitant decrease in the expression of neural maturation genes. Taken together, these results suggest that microenvironmental viscoelasticity is sufficient to bias human NPC maturation.
Project description:Biomaterial scaffolds have the potential to enhance neuronal development and regeneration. Understanding the genetic responses of astrocytes and neurons to biomaterials could facilitate the development of synthetic environments that enable the specification of neural tissue organization with engineered scaffolds. In this study, we used high throughput transcriptomic and imaging methods to determine the impact of a hydrogel with no known bioactive epitopes, Puramatrix™, on human glial cells in vitro. Parallel studies were undertaken with cells grown in a monolayer environment on tissue culture polystyrene. When the Normal Human Astrocyte (NHA) cell line is grown in a hydrogel matrix environment, the glial cells adopt a structural organization that resembles that of neuronal-glial cocultures, where neurons form clusters that are distinct from the surrounding glia. Statistical analysis of next generation RNA sequencing data uncovered a set of genes that are differentially expressed in the monolayer and matrix hydrogel environments. Functional analysis demonstrated that hydrogel-upregulated genes can be grouped into three broad categories: neuronal differentiation and/or neural plasticity, response to neural insult, and sensory perception. Our results demonstrate that hydrogel biomaterials have the potential to transform human glial cell identity, and may have applications in the repair of damaged brain tissue
Project description:Meaningful models of human neural development and neurodegeneration are extremely important when exploring stem-cell-based regenerative therapies. However, existing 3D cultures fall short of being highly defined, modular, and controllable. Adapting a glycosaminoglycan-based, cell-responsive hydrogel platform, we stimulated primary and induced human neural stem cells (NSCs) to manifest neurogenic plasticity and form extensive neuronal networks in vitro. The 3D cultures exhibited neurotransmitter responsiveness, electrophysiological activity, and tissue-specific extracellular matrix (ECM) deposition. By whole transcriptome sequencing, we identified that 3D cultures express mature neuronal markers, and reflect the in vivo genetic program of mature cortical neurons compared to 2D cultures. Thus, our data suggest that our established 3D hydrogel culture supports the tissue-mimetic maturation of human neurons in an unprecedented manner. We modeled neurodegenerative conditions by treating the cultures with A?42 peptide and observed the known human pathological effects of Alzheimer?s disease including reduced NSC proliferation, impaired neuronal network formation, synaptic loss and failure in ECM deposition as well as elevated Tau hyperphosphorylation and formation of neurofibrillary tangles. We also determined the changes in transcriptomes of primary and induced NSC-derived neurons after A?42, providing a useful resource for further studies. Thus, our hydrogel-based human cortical 3D cell culture is a powerful platform for studying various aspects of neural development and neurodegeneration, as exemplified for A?42 toxicity and neurogenic stem cell plasticity.
Project description:Increased extracellular matrix (ECM) stiffness has been implicated in esophageal adenocarcinoma (EAC) progression, metastasis, and resistance to therapy. However, the underlying pro-tumorigenic pathways are yet to be defined. Additional work is needed to develop physiologically relevant in vitro 3D culture models that better recapitulate the human tumor microenvironment and can be used to dissect the contributions of matrix stiffness to EAC pathogenesis. Here, we describe a modular, tumor ECM-mimetic hydrogel platform with tunable mechanical properties, defined presentation of cell-adhesive ligands, and protease-dependent degradation that supports robust in vitro growth and expansion of patient-derived EAC 3D organoids (EAC PDOs). Hydrogel mechanical properties control EAC PDO formation, growth, proliferation, and activation of tumor-associated pathways that elicit stem-like properties in the cancer cells, as highlighted through in vitro and in vivo environments.
Project description:DNA barcodes can be used to identify single cells in a sequencing data space while optical codes can be used to track single live cells in an image data space. We have developed dual image and DNA (ID)-coding, which identifies individual single cells in both live image and sequencing data spaces. Samples provided here are relevant to proof-of-concept studies of ID-coding presented in the associated publication. DNA barcoded micro-particles were encapsulated in hydrogel droplets with or without single cells. The hydrogel droplets were then subjected to “single-droplet sequencing” where whole polyA-bearing nucleic acid components within a hydrogel droplet (i.e. mRNA from cells and synthetic DNA on beads) were concatenated by the same cell barcodes.
Project description:Increased extracellular matrix (ECM) stiffness has been implicated in esophageal adenocarcinoma (EAC) progression, metastasis, and resistance to therapy. However, the underlying pro-tumorigenic pathways are yet to be defined. Additional work is needed to develop physiologically relevant in vitro 3D culture models that better recapitulate the human tumor microenvironment and can be used to dissect the contributions of matrix stiffness to EAC pathogenesis. Here, we describe a modular, tumor ECM-mimetic hydrogel platform with tunable mechanical properties, defined presentation of cell-adhesive ligands, and protease-dependent degradation that supports robust in vitro growth and expansion of patient-derived EAC 3D organoids (EAC PDOs). Hydrogel mechanical properties control EAC PDO formation, growth, proliferation, and activation of tumor-associated pathways that elicit stem-like properties in the cancer cells, as highlighted through in vitro and in vivo environments. We also demonstrate that the engineered hydrogel serves as a platform to identify potential therapeutic targets to disrupt the contribution of pro-tumorigenic matrix mechanics in EAC. Together, these studies show that an engineered PDO culture platform can be used to elucidate underlying matrix-mediated mechanisms of EAC, and inform the development of therapeutics that target ECM stiffness in EAC.
Project description:Explore the role of these hydrogels in wound healing, this study assessed the effects of both, Dersani Hydrogel with Alginate (DHA) and Dersani Hydrogel (DH), in human skin keratinocytes and fibroblasts gene expression profiles in a wound healing context. Sodium alginate (SA) and culture medium were also included as controls.
Project description:Delivery of therapeutic stem cells to treat bone tissue damage is a promising strategy that faces many hurdles to clinical translation. Among them is the design of a delivery vehicle which promotes desired cell behavior for new bone formation. In this work, we describe the use of an injectable microporous hydrogel, made of crosslinked gelatin microgels, for the encapsulation and delivery of human mesenchymal stem cells (MSCs) and compared it to a traditional nonporous injectable hydrogel. MSCs encapsulated in the microporous hydrogel showed rapid cell spreading with direct cell-cell connections whereas the MSCs in the nonporous hydrogel were entrapped by the surrounding polymer mesh and isolated from each other. Microporous hydrogel induced more robust osteogenic differentiation of MSCs and calcium mineral deposition than the nonporous hydrogel confirmed by alkaline phosphatase (ALP) assay and calcium assay. RNA-seq confirmed the upregulation of the genes and pathways that are associated with cell spreading and cell-cell connections, as well as the osteogenesis in the microporous hydrogel. These results demonstrate that the microgel-based injectable hydrogels can be useful tools for therapeutic cell delivery for bone tissue repair.