On the path to predicting immune responses in the lung: Modeling the pulmonary innate immune system at the air-liquid interface (ALI).

Chronic respiratory diseases and infections are among the largest contributors to death globally, many of which still have no cure, including chronic obstructive pulmonary disorder, idiopathic pulmonary fibrosis, and respiratory syncytial virus among others. Pulmonary therapeutics afford untapped potential for treating lung infection and disease through direct delivery to the site of action. However, the ability to innovate new therapeutic paradigms for respiratory diseases will rely on modeling the human lung microenvironment and including key cellular interactions that drive disease. One key feature of the lung microenvironment is the air-liquid interface (ALI). ALI interface modeling techniques, using cell-culture inserts, organoids, microfluidics, and precision lung slices (PCLS), are rapidly developing; however, one major component of these models is lacking-innate immune cell populations. Macrophages, neutrophils, and dendritic cells, among others, represent key lung cell populations, acting as the first responders during lung infection or injury. Innate immune cells respond to and modulate stromal cells and bridge the gap between the innate and adaptive immune system, controlling the bodies response to foreign pathogens and debris. In this article, we review the current state of ALI culture systems with a focus on innate immune cells and suggest ways to build on current models to add complexity and relevant immune cell populations.

Cell therapy biomanufacturing: integrating biomaterial and flow-based membrane technologies for production of engineered T-cells.

Adoptive T-cell therapies (ATCTs) are increasingly important for the treatment of cancer, where patient immune cells are engineered to target and eradicate diseased cells. The biomanufacturing of ATCTs involves a series of time-intensive, lab-scale steps, including isolation, activation, genetic modification, and expansion of a patient's T-cells prior to achieving a final product. Innovative modular technologies are needed to produce cell therapies at improved scale and enhanced efficacy. In this work, well-defined, bioinspired soft materials were integrated within flow-based membrane devices for improving the activation and transduction of T cells. Hydrogel coated membranes (HCM) functionalized with cell-activating antibodies were produced as a tunable biomaterial for the activation of primary human T-cells. T-cell activation utilizing HCMs led to highly proliferative T-cells that expressed a memory phenotype. Further, transduction efficiency was improved by several fold over static conditions by using a tangential flow filtration (TFF) flow-cell, commonly used in the production of protein therapeutics, to transduce T-cells under flow. The combination of HCMs and TFF technology led to increased cell activation, proliferation, and transduction compared to current industrial biomanufacturing processes. The combined power of biomaterials with scalable flow-through transduction techniques provides future opportunities for improving the biomanufacturing of ATCTs.

Effects of solvent conditions on the self-assembly of heterotrimeric collagen-like peptide (CLP) triple helices: a coarse-grained simulation study.

We perform coarse-grained (CG) molecular dynamics (MD) simulations to investigate the self-assembly of collagen-like peptide (CLP) triple helices into fibrillar structures and percolated networks as a function of solvent quality. The focus of this study is on CLP triple helices whose strands are different lengths (i.e., heterotrimers), leading to dangling 'sticky ends'. These 'sticky ends' are segments of the CLP strands that have unbonded hydrogen-bonding donor/acceptor sites that drive heterotrimeric CLP triple helices to physically associate with one another, leading to assembly into higher-order structures. We use a validated CG model for CLP in implicit solvent and capture varying solvent quality through changing strength of attraction between CG beads representing the amino acids in the CLP strands. Our CG MD simulations show that, at lower CLP concentrations, CLP heterotrimers assemble into fibrils and, at higher CLP concentrations, into percolated networks. At higher concentrations, decreasing solvent quality causes (i) the formation of heterogeneous network structures with a lower degree of branching at network junctions and (ii) increases in the diameter of network strands and pore sizes. We also observe a nonmonotonic effect of solvent quality on distances between network junctions due to the balance between heterotrimer end-end associations driven by hydrogen bonding and side-side associations driven by worsening solvent quality. Below the percolation threshold, we observe that decreasing solvent quality leads to the formation of fibrils composed of multiple aligned CLP triple helices, while the number of 'sticky ends' governs the spatial extent (radius of gyration) of the assembled fibrils.

Systematic d-Amino Acid Substitutions to Control Peptide and Hydrogel Degradation in Cellular Microenvironments.

Enzymatically degradable peptides are commonly used as linkers within hydrogels for biological applications; however, controlling the degradation of these engineered peptides with different contexts and cell types can prove challenging. In this work, we systematically examined the substitution of d-amino acids (D-AAs) for different l-amino acids in a peptide sequence commonly utilized in enzymatically degradable hydrogels (VPMS↓MRGG) to create peptide linkers with a range of different degradation times, in solution and in hydrogels, and investigated the cytocompatibility of these materials. We found that increasing the number of D-AA substitutions increased the resistance to enzymatic degradation both for free peptide and peptide-linked hydrogels; yet, this trend also was accompanied by increased cytotoxicity in cell culture. This work demonstrates the utility of D-AA-modified peptide sequences to create tunable biomaterials platforms tempered by considerations of cytotoxicity, where careful selection and optimization of different peptide designs is needed for specific biological applications.

Dynamic bioinspired coculture model for probing ER+ breast cancer dormancy in the bone marrow niche.

Late recurrences of breast cancer are hypothesized to arise from disseminated tumor cells (DTCs) that reactivate after dormancy and occur most frequently with estrogen receptor-positive (ER+) breast cancer cells (BCCs) in bone marrow (BM). Interactions between the BM niche and BCCs are thought to play a pivotal role in recurrence, and relevant model systems are needed for mechanistic insights and improved treatments. We examined dormant DTCs in vivo and observed DTCs near bone lining cells and exhibiting autophagy. To study underlying cell-cell interactions, we established a well-defined, bioinspired dynamic indirect coculture model of ER+ BCCs with BM niche cells, human mesenchymal stem cells (hMSCs) and fetal osteoblasts (hFOBs). hMSCs promoted BCC growth, whereas hFOBs promoted dormancy and autophagy, regulated in part by tumor necrosis factor-α and monocyte chemoattractant protein 1 receptor signaling. This dormancy was reversible by dynamically changing the microenvironment or inhibiting autophagy, presenting further opportunities for mechanistic and targeting studies to prevent late recurrence.

Impact of collagen-like peptide (CLP) heterotrimeric triple helix design on helical thermal stability and hierarchical assembly: a coarse-grained molecular dynamics simulation study.

Collagen-like peptides (CLP) are multifunctional materials garnering a lot of recent interest from the biomaterials community due to their hierarchical assembly and tunable physicochemical properties. In this work, we present a computational study that links the design of CLP heterotrimers to the thermal stability of the triple helix and their self-assembly into fibrillar aggregates and percolated networks. Unlike homotrimeric helices, the CLP heterotrimeric triple helices in this study are made of CLP strands of different chain lengths that result in 'sticky' ends with available hydrogen bonding groups. These 'sticky' ends at one end or both ends of the CLP heterotrimer then facilitate inter-helix hydrogen bonding leading to self-assembly into fibrils (clusters) and percolated networks. We consider the cases of three sticky end lengths - two, four, and six repeat units - present entirely on one end or split between two ends of the CLP heterotrimer. We observe in CLP heterotrimer melting curves generated using coarse grained Langevin dynamics simulations at low CLP concentration that increasing sticky end length results in lower melting temperatures for both one and two sticky ended CLP designs. At higher CLP concentrations, we observe non-monotonic trends in cluster sizes with increasing sticky end length with one sticky end but not for two sticky ends with the same number of available hydrogen bonding groups as the one sticky end; this nonmonotonicity stems from the formation of turn structures stabilized by hydrogen bonds at the single, sticky end for sticky end lengths greater than four repeat units. With increasing CLP concentration, heterotrimers also form percolated networks with increasing sticky end length with a minimum sticky end length of four repeat units required to observe percolation. Overall, this work informs the design of thermoresponsive, peptide-based biomaterials with desired morphologies using strand length and dispersity as a handle for tuning thermal stability and formation of supramolecular structures.

Rational Design of Hydrogel Networks with Dynamic Mechanical Properties to Mimic Matrix Remodeling.

Engineered hydrogels are increasingly used as extracellular matrix (ECM) surrogates for probing cell function in response to ECM remodeling events related to injury or disease (e.g., degradation followed by deposition/crosslinking). Inspired by these events, this work establishes an approach for pseudo-reversible mechanical property modulation in synthetic hydrogels by integrating orthogonal, enzymatically triggered crosslink degradation, and light-triggered photopolymerization stiffening. Hydrogels are formed by a photo-initiated thiol-ene reaction between multiarm polyethylene glycol and a dually enzymatically degradable peptide linker, which incorporates a thrombin-degradable sequence for triggered softening and a matrix metalloproteinase (MMP)-degradable sequence for cell-driven remodeling. Hydrogels are stiffened by photopolymerization using a flexible, MMP-degradable polymer-peptide conjugate and multiarm macromers, increasing the synthetic matrix crosslink density while retaining degradability. Integration of these tools enables sequential softening and stiffening inspired by matrix remodeling events within loose connective tissues (Young's modulus (E) ≈5 to 1.5 to 6 kPa with >3x ΔE). The cytocompatibility and utility of this approach is examined with breast cancer cells, where cell proliferation shows a dependence on the timing of triggered softening. This work provides innovative tools for 3D dynamic property modulation that are synthetically accessible and cell compatible.

Fatty acid synthase as a feasible biomarker for triple negative breast cancer stem cell subpopulation cultured on electrospun scaffolds.

There is no targeted therapy for triple negative breast cancer (TNBC), which presents an aggressive profile and poor prognosis. Recent studies noticed the feasibility of breast cancer stem cells (BCSCs), a small population responsible for tumor initiation and relapse, to become a novel target for TNBC treatments. However, new cell culture supports need to be standardized since traditional two-dimensional (2D) surfaces do not maintain the stemness state of cells. Hence, three-dimensional (3D) scaffolds represent an alternative to study in vitro cell behavior without inducing cell differentiation. In this work, electrospun polycaprolactone scaffolds were used to enrich BCSC subpopulation of MDA-MB-231 and MDA-MB-468 TNBC cells, confirmed by the upregulation of several stemness markers and the existence of an epithelial-to-mesenchymal transition within 3D culture. Moreover, 3D-cultured cells displayed a shift from MAPK to PI3K/AKT/mTOR signaling pathways, accompanied by an enhanced EGFR and HER2 activation, especially at early cell culture times. Lastly, the fatty acid synthase (FASN), a lipogenic enzyme overexpressed in several carcinomas, was found to be hyperactivated in stemness-enriched samples. Its pharmacological inhibition led to stemness diminishment, overcoming the BCSC expansion achieved in 3D culture. Therefore, FASN may represent a novel target for BCSC niche in TNBC samples.

Rapid Production of Multifunctional Self-Assembling Peptides for Incorporation and Visualization within Hydrogel Biomaterials.

Peptides are of continued interest for therapeutic applications, from soluble and immobilized ligands that promote desired binding or uptake to self-assembled supramolecular structures that serve as scaffolds in vitro and in vivo. These applications require efficient and scalable synthetic approaches because of the large amounts of material that often are needed for studies of bulk material properties and their translation. In this work, we establish new methods for the synthesis, purification, and visualization of assembling peptides, with a focus on multifunctional collagen mimetic peptides (mfCMPs) relevant for formation and integration within hydrogel-based biomaterials. First, a methodical approach useful for the microwave-assisted synthesis of assembling peptide sequences prone to deletions was established, beginning with the identification of the deleted residues and their locations and followed by targeted use of dual chemistry couplings for those specific residues. Second, purification techniques that integrate the principles of heating and ion displacement with traditional chromatography and dialysis were implemented to improve separation and isolation of the desired multifunctional peptide product, which contained blocks for thermoresponsiveness and ionic interactions. Third, an approach for fluorescent labeling of these mfCMPs, which is orthogonal to their assembly and their covalent incorporation into a bulk hydrogel material, was established, allowing visualization of the resulting hierarchical fibrillar structures in three dimensions within hydrogels using confocal microscopy. The methods presented in this work allow the production of multifunctional peptides in scalable quantities and with minimal deletions, enabling future studies for better understanding of composition-structure-property relationships and for translating these biomaterials into a range of applications. Although mfCMPs are the focus of this work, the methods demonstrated could prove useful for other assembling peptide systems and for the production of peptides more broadly for therapeutic applications.

Combining simulations and experiments for the molecular engineering of multifunctional collagen mimetic peptide-based materials.

Assembling peptides allow the creation of structurally complex materials, where amino acid selection influences resulting properties. We present a synergistic approach of experiments and simulations for examining the influence of natural and non-natural amino acid substitutions via incorporation of charged residues and a reactive handle on the thermal stability and assembly of multifunctional collagen mimetic peptides (CMPs). Experimentally, we observed inclusion of charged residues significantly decreased the melting temperature of CMP triple helices with further destabilization upon inclusion of the reactive handle. Atomistic simulations of a single CMP triple helix in explicit water showed increased residue-level and helical structural fluctuations caused by the inclusion of the reactive handle; however, these atomistic simulations cannot be used to predict changes in CMP melting transition. Coarse-grained (CG) simulations of CMPs at experimentally relevant solution conditions, showed, qualitatively, the same trends as experiments in CMP melting transition temperature with CMP design. These simulations show that when charged residues are included electrostatic repulsions significantly destabilize the CMP triple helix and that an additional inclusion of a reactive handle does not significantly change the melting transition. Based on findings from both experiments and simulations, the sequence design was refined for increased CMP triple helix thermal stability, and the reactive handle was utilized for the incorporation of the assembled CMPs within covalently crosslinked hydrogels. Overall, a unique approach was established for predicting stability of CMP triple helices for various sequences prior to synthesis, providing molecular insights for sequence design towards the creation of bulk nanostructured soft biomaterials.

Understanding ER+ Breast Cancer Dormancy Using Bioinspired Synthetic Matrices for Long-Term 3D Culture and Insights into Late Recurrence.

Late recurrences of breast cancer are hypothesized to originate from disseminated tumor cells that re-activate after a long period of dormancy, ≥5 years for estrogen-receptor positive (ER+) tumors. An outstanding question remains as to what the key microenvironment interactions are that regulate this complex process, and well-defined human model systems are needed for probing this. Here, a robust, bioinspired 3D ER+ dormancy culture model is established and utilized to probe the effects of matrix properties for common sites of late recurrence on breast cancer cell dormancy. Formation of dormant micrometastases over several weeks is examined for ER+ cells (T47D, BT474), where the timing of entry into dormancy versus persistent growth depends on matrix composition and cell type. In contrast, triple negative cells (MDA-MB-231), associated with early recurrence, are not observed to undergo long-term dormancy. Bioinformatic analyses quantitatively support an increased "dormancy score" gene signature for ER+ cells (T47D) and reveal differential expression of genes associated with different biological processes based on matrix composition. Further, these analyses support a link between dormancy and autophagy, a potential survival mechanism. This robust model system will allow systematic investigations of other cell-microenvironment interactions in dormancy and evaluation of therapeutics for preventing late recurrence.

Tunable hydrogels for mesenchymal stem cell delivery: Integrin-induced transcriptome alterations and hydrogel optimization for human wound healing.

Therapeutic applications for mesenchymal stem/stromal cells (MSCs) are growing; however, the successful implementation of these therapies requires the development of appropriate MSC delivery systems. Hydrogels are ideally suited to cultivate MSCs but tuning hydrogel properties to match their specific in vivo applications remains a challenge. Thus, further characterization of how hydrogel-based delivery vehicles broadly influence MSC function and fate will help lead to the next generation of more intelligently designed delivery vehicles. To date, few attempts have been made to comprehensively characterize hydrogel impact on the MSC transcriptome. Herein, we have synthesized cell-degradable hydrogels based on bio-inert poly(ethylene glycol) tethered with specific integrin-binding small molecules and have characterized their resulting effect on the MSC transcriptome when compared with 2D cultured and untethered 3D hydrogel cultured MSCs. The 3D culture systems resulted in alterations in the MSC transcriptome, as is evident by the differential expression of genes related to extracellular matrix production, glycosylation, metabolism, signal transduction, gene epigenetic regulation, and development. For example, genes important for osteogenic differentiation were upregulated in 3D hydrogel cultures, and the expression of these genes could be partially suppressed by tethering an integrin-binding RGD peptide within the hydrogel. Highlighting the utility of tunable hydrogels, when applied to ex vivo human wounds the RGD-tethered hydrogel was able to support wound re-epithelialization, possibly due to its ability to increase PDGF expression and decrease IL-6 expression. These results will aid in future hydrogel design for a broad range of applications.

Hierarchically structured hydrogels utilizing multifunctional assembling peptides for 3D cell culture.

Approaches for the creation of soft materials, particularly hydrogels, with hierarchical structure are of interest in a variety of applications owing to their unique properties. In the context of tissue mimics, hydrogels with multiscale structures more accurately capture the complexities of tissues within the body (e.g., fibrous collagen-rich microenvironments). However, cytocompatible fabrication of such materials with hierarchical structures and independent control of mechanical and biochemical properties remains challenging and is needed for probing and directing cell-microenvironment interactions for three-dimensional (3D) cell encapsulation and culture applications. To address this, we have designed innovative multifunctional assembling peptides: these unique peptides contain a core block that mimics the structure of collagen for achieving relevant melting temperatures; 'sticky' ends to promote assembly of long fibrils; and a biocompatible reactive handle that is orthogonal to assembly to allow the formation of desired multiscale structures and their subsequent rapid, light-triggered integration within covalently crosslinked synthetic hydrogels. Nano- to micro-fibrils were observed to form in physiologically-relevant aqueous solutions, where both underlying peptide chemical structure and assembly conditions were observed to impact the resulting fibril sizes. These assembled structures were 'locked' into place and integrated as linkers within cell-degradable, bioactive hydrogels formed with photoinitiated thiol-ene 'click' chemistry. Hydrogel compositions were identified for achieving robust mechanical properties like those of soft tissues while also retaining higher ordered structures after photopolymerization. The utility of these innovative materials for 3D cell culture was demonstrated with human mesenchymal stem cells, where cell morphologies reminiscent of responses to assembled native collagen were observed now with a fully synthetic material. Using a bottom-up approach, a new materials platform has been established that combines the advantageous properties of covalent and assembling chemistries for the creation of synthetic hydrogels with controllable nanostructure, mechanical properties, and biochemical content.

Tunable synthetic extracellular matrices to investigate breast cancer response to biophysical and biochemical cues.

The extracellular matrix (ECM) is thought to play a critical role in the progression of breast cancer. In this work, we have designed a photopolymerizable, biomimetic synthetic matrix for the controlled, 3D culture of breast cancer cells and, in combination with imaging and bioinformatics tools, utilized this system to investigate the breast cancer cell response to different matrix cues. Specifically, hydrogel-based matrices of different densities and modified with receptor-binding peptides derived from ECM proteins [fibronectin/vitronectin (RGDS), collagen (GFOGER), and laminin (IKVAV)] were synthesized to mimic key aspects of the ECM of different soft tissue sites. To assess the breast cancer cell response, the morphology and growth of breast cancer cells (MDA-MB-231 and T47D) were monitored in three dimensions over time, and differences in their transcriptome were assayed using next generation sequencing. We observed increased growth in response to GFOGER and RGDS, whether individually or in combination with IKVAV, where binding of integrin ß1 was key. Importantly, in matrices with GFOGER, increased growth was observed with increasing matrix density for MDA-MB-231s. Further, transcriptomic analyses revealed increased gene expression and enrichment of biological processes associated with cell-matrix interactions, proliferation, and motility in matrices rich in GFOGER relative to IKVAV. In sum, a new approach for investigating breast cancer cell-matrix interactions was established with insights into how microenvironments rich in collagen promote breast cancer growth, a hallmark of disease progression in vivo, with opportunities for future investigations that harness the multidimensional property control afforded by this photopolymerizable system.

Immunofunctional photodegradable poly(ethylene glycol) hydrogel surfaces for the capture and release of rare cells.

Circulating tumor cells (CTCs) play a central role in cancer metastasis and represent a rich source of data for cancer prognostics and therapeutic guidance. Reliable CTC recovery from whole blood therefore promises a less invasive and more sensitive approach to cancer diagnosis and progression tracking. CTCs, however, are exceedingly rare in whole blood, making their quantitative recovery challenging. Several techniques capable of isolating these rare cells have been introduced and validated, yet most suffer from low CTC purity or viability, both of which are essential to develop a clinically viable CTC isolation platform. To address these limitations, we introduce a patterned, immunofunctional, photodegradable poly(ethylene glycol) (PEG) hydrogel capture surface for the isolation and selective release of rare cell populations. Flat and herringbone capture surfaces were successfully patterned via PDMS micromolding and photopolymerization of photolabile PEG hydrogels. Patterned herringbone surfaces, designed to convectively transport cells to the capture surface, exhibited improved capture density relative to flat surfaces for target cell capture from buffer, buffy coat, and whole blood. Uniquely, captured cells were released for collection by degrading the hydrogel capture surface with either bulk or targeted irradiation with cytocompatible doses of long wavelength UV light. Recovered cells remained viable following capture and release and exhibited similar growth rates as untreated control cells. The implementation of molded photodegradable PEG hydrogels as a CTC capture surface provides a micropatternable, cytocompatible platform that imparts the unique ability to recover pure, viable CTC samples by selectively releasing target cells.

Manipulation of Glutathione-Mediated Degradation of Thiol-Maleimide Conjugates.

The retro Michael-type addition and thiol exchange of thioether succinimide click linkages in response to thiol-containing environments offers a novel strategy for the design of glutathione-sensitive degradable hydrogels for controlled drug delivery. Here we characterize the kinetics and extent of the retro Michael-type addition and thiol exchange with changes in both the p Ka of the thiols and the identity of N-substituents of maleimides. A series of N-substituted thioether succinimides were prepared through typical Michael-type addition. Model studies (1H NMR, HPLC) of 4-mercaptophenylacetic acid (MPA, p Ka 6.6) conjugated to N-ethyl maleimide (NEM), N-phenyl maleimide (NPM), or N-aminoethyl maleimide (NAEM) and then incubated with glutathione showed half-lives of conversion from 3.1 to 18 h, with extents of conversion from approximately 12% to 90%. The variations in the rates of exchange and hydrolytic ring opening appear to be mediated by resonance effects, electron-withdrawing capacity of the N-substituted moiety, as well as the potential for intramolecular catalytic hydrogen bonding of amine substituents with water (particularly in the case of ring opening). Further model studies of 4-mercaptohydrocinnamic acid (MPP, p Ka 7.0) and N-acetyl-l-cysteine (NAC, p Ka 9.5) conjugated to selected N-substituted maleimides and then incubated with glutathione showed half-lives of conversion from 3.6 to 258 h, with extents of conversion from approximately 1% to 90%. A higher p Ka of the thiol decreased the rate of the exchange reaction and limited the impact of other electronic effects of N-substituents on the extents of conversion. Additional factors affecting the conversion kinetics were studied on NEM conjugates. The kinetics of the retro Michael-type addition and exchange reaction were not hindered by thiol traps of lower p Ka, but were retarded in conditions of lower pH. These studies shed light into details of thiol and maleimide design that could be used to tune the rates of degradation of drug and polymer conjugates for a variety of applications.

Design of synthetic extracellular matrices for probing breast cancer cell growth using robust cyctocompatible nucleophilic thiol-yne addition chemistry.

Controlled, three-dimensional (3D) cell culture systems are of growing interest for both tissue regeneration and disease, including cancer, enabling hypothesis testing about the effects of microenvironment cues on a variety of cellular processes, including aspects of disease progression. In this work, we encapsulate and culture in three dimensions different cancer cell lines in a synthetic extracellular matrix (ECM), using mild and efficient chemistry. Specifically, harnessing the nucleophilic addition of thiols to activated alkynes, we have created hydrogel-based materials with multifunctional poly(ethylene glycol) (PEG) and select biomimetic peptides. These materials have definable, controlled mechanical properties (G' = 4-10 kPa) and enable facile incorporation of pendant peptides for cell adhesion, relevant for mimicking soft tissues, where polymer architecture allows tuning of matrix degradation. These matrices rapidly formed in the presence of sensitive breast cancer cells (MCF-7) for successful encapsulation with high cell viability, greatly improved relative to that observed with the more widely used radically-initiated thiol-ene crosslinking chemistry. Furthermore, controlled matrix degradation by both bulk and local mechanisms, ester hydrolysis of the polymer network and cell-driven enzymatic hydrolysis of cell-degradable peptide, allowed cell proliferation and the formation of cell clusters within these thiol-yne hydrogels. These studies demonstrate the importance of chemistry in ECM mimics and the potential thiol-yne chemistry has as a crosslinking reaction for the encapsulation and culture of cells, including those sensitive to radical crosslinking pathways.

Designing well-defined photopolymerized synthetic matrices for three-dimensional culture and differentiation of induced pluripotent stem cells.

Induced pluripotent stem cells (iPSCs) are of interest for the study of disease, where these cells can be derived from patients and have the potential to be differentiated into any cell type; however, three-dimensional (3D) culture and differentiation of iPSCs within well-defined synthetic matrices for these applications remains limited. Here, we aimed to establish synthetic cell-degradable hydrogels that allow precise presentation of specific biochemical cues for 3D culture of iPSCs with relevance for hypothesis testing and lineage-specific differentiation. We synthesized poly(ethylene glycol)-(PEG)-peptide-based hydrogels by photoinitiated step growth polymerization and used them to test the hypothesis that the viability of iPSCs within these matrices could be rescued with appropriate biochemical cues inspired by proteins and integrins important for iPSC culture on Matrigel. Specifically, we selected a range of motifs inspired by iPSC binding to Matrigel, including laminin-derived IKVAV and YIGSR, α5ß1-binding PHSRNG10RGDS, αvß5-binding KKQRFRHRNRKG, and RGDS that is known to bind a variety of integrins for generally promoting cell adhesion. YIGSR and PHSRNG10RGDS resulted in the highest iPSC viability, where binding of ß1 integrin was key, and these permissive compositions also allowed iPSC differentiation into neural progenitor cells (NPCs) (decreased oct4 expression and increased pax6 expression) in response to soluble factors. The resulting NPCs formed clusters of different sizes in response to each peptide, suggesting that matrix biochemical cues affect iPSC proliferation and clustering in 3D culture. In summary, we have established photopolymerizable synthetic matrices for the encapsulation, culture, and differentiation of iPSCs for studies of cell-matrix interactions and deployment in disease models.

Fast, irreversible modification of cysteines through strain releasing conjugate additions of cyclopropenyl ketones.

A method of cysteine alkylation using cyclopropenyl ketones is described. Due to the significant release of cyclopropene strain energy, reactions of thiols with cyclopropenyl ketones are both fast and irreversible and give rise to stable conjugate addition adducts. The resulting cyclopropenyl ketones have a low molecular weight and allow for simple attachment of amides via N-hydroxysuccinimide (NHS)-esters. While cyclopropenyl ketones do display slow background reactivity toward water, labeling by thiols is much more rapid. The reaction of a cyclopropenyl ketone with glutathione (GSH) proceeds with a rate of 595 M-1 s-1 in PBS at pH 7.4, which is considerably faster than α-halocarbonyl labeling reagents, and competitive with maleimide/thiol couplings. The method has been demonstrated in protein conjugation, and an arylthiolate conjugate was shown to be stable upon prolonged incubation in either GSH or human plasma. Finally, cyclopropenyl ketones were used to create PEG-based hydrogels that are stable to prolonged incubation in a reducing environment.

Isolation and Identification of Proteins Secreted by Cells Cultured within Synthetic Hydrogel-Based Matrices.

Cells interact with and remodel their microenvironment, degrading large extracellular matrix (ECM) proteins (e.g., fibronectin, collagens) and secreting new ECM proteins and small soluble factors (e.g., growth factors, cytokines). Synthetic mimics of the ECM have been developed as controlled cell culture platforms for use in both fundamental and applied studies. However, how cells broadly remodel these initially well-defined matrices remains poorly understood and difficult to probe. In this work, we have established methods for widely examining both large and small proteins that are secreted by cells within synthetic matrices. Specifically, human mesenchymal stem cells (hMSCs), a model primary cell type, were cultured within well-defined poly(ethylene glycol) (PEG)-peptide hydrogels, and these cell-matrix constructs were decellularized and degraded for subsequent isolation and analysis of deposited proteins. Shotgun proteomics using liquid chromatography and mass spectrometry identified a variety of proteins, including the large ECM proteins fibronectin and collagen VI. Immunostaining and confocal imaging confirmed these results and provided visualization of protein organization within the synthetic matrices. Additionally, culture medium was collected from the encapsulated hMSCs, and a Luminex assay was performed to identify secreted soluble factors, including vascular endothelial growth factor (VEGF), endothelial growth factor (EGF), basic fibroblast growth factor (FGF-2), interleukin 8 (IL-8), and tumor necrosis factor alpha (TNF-α). Together, these methods provide a unique approach for studying dynamic reciprocity between cells and synthetic microenvironments and have the potential to provide new biological insights into cell responses during three-dimensional (3D) controlled cell culture.