Stepwise Stiffening/Softening of and Cell Recovery from Reversibly Formulated Hydrogel Double Networks.

Biomechanical contributions of the ECM underpin cell growth and proliferation, differentiation, signal transduction, and other fate decisions. As such, biomaterials whose mechanics can be spatiotemporally altered - particularly in a reversible manner - are extremely valuable for studying these mechanobiological phenomena. Herein, we introduce a poly(ethylene glycol) (PEG)-based hydrogel model consisting of two interpenetrating step-growth networks that are independently formed via largely orthogonal bioorthogonal chemistries and sequentially degraded with distinct bacterial transpeptidases, affording reversibly tunable stiffness ranges that span healthy and diseased soft tissues (e.g., 500 Pa - 6 kPa) alongside terminal cell recovery for pooled and/or single-cell analysis in a near "biologically invisible" manner. Spatiotemporal control of gelation within the primary supporting network was achieved via mask-based and two-photon lithography; these stiffened patterned regions could be subsequently returned to the original soft state following sortase-based secondary network degradation. Using this approach, we investigated the effects of 4D-triggered network mechanical changes on human mesenchymal stem cell (hMSC) morphology and Hippo signaling, as well as Caco-2 colorectal cancer cell mechanomemory at the global transcriptome level via RNAseq. We expect this platform to be of broad utility for studying and directing mechanobiological phenomena, patterned cell fate, as well as disease resolution in softer matrices.

Single-cell census of human tooth development enables generation of human enamel.

Tooth enamel secreted by ameloblasts (AMs) is the hardest material in the human body, acting as a shield to protect the teeth. However, the enamel is gradually damaged or partially lost in over 90% of adults and cannot be regenerated due to a lack of ameloblasts in erupted teeth. Here, we use single-cell combinatorial indexing RNA sequencing (sci-RNA-seq) to establish a spatiotemporal single-cell census for the developing human tooth and identify regulatory mechanisms controlling the differentiation process of human ameloblasts. We identify key signaling pathways involved between the support cells and ameloblasts during fetal development and recapitulate those findings in human ameloblast in vitro differentiation from induced pluripotent stem cells (iPSCs). We furthermore develop a disease model of amelogenesis imperfecta in a three-dimensional (3D) organoid system and show AM maturation to mineralized structure in vivo. These studies pave the way for future regenerative dentistry.

Stromal FOXF2 suppresses prostate cancer progression and metastasis by enhancing antitumor immunity.

Cancer-associated fibroblasts (CAFs) mediate an immunosuppressive effect, but the underlying mechanism remains incompletely defined. Here we show that increasing prostatic stromal Foxf2 suppresses the growth and progression of both syngeneic and autochthonous mouse prostate cancer models in an immunocompetent context. Mechanistically, Foxf2 moderately attenuates the CAF phenotype and transcriptionally downregulates Cxcl5, which diminish the immunosuppressive myeloid cells and enhance T cell cytotoxicity. Increasing prostatic stromal Foxf2 sensitizes prostate cancer to the immune checkpoint blockade therapies. Augmenting lung stromal Foxf2 also mediates an immunosuppressive milieu and inhibits lung colonization of prostate cancer. FOXF2 is expressed higher in the stroma of human transition zone (TZ) than peripheral zone (PZ) prostate. The stromal FOXF2 expression level in primary prostate cancers inversely correlates with the Gleason grade. Our study establishes Foxf2 as a stromal transcription factor modulating the tumor immune microenvironment and potentially explains why cancers are relatively rare and indolent in the TZ prostate.

Embryo-scale, single-cell spatial transcriptomics.

Spatial patterns of gene expression manifest at scales ranging from local (e.g., cell-cell interactions) to global (e.g., body axis patterning). However, current spatial transcriptomics methods either average local contexts or are restricted to limited fields of view. Here, we introduce sci-Space, which retains single-cell resolution while resolving spatial heterogeneity at larger scales. Applying sci-Space to developing mouse embryos, we captured approximate spatial coordinates and whole transcriptomes of about 120,000 nuclei. We identify thousands of genes exhibiting anatomically patterned expression, leverage spatial information to annotate cellular subtypes, show that cell types vary substantially in their extent of spatial patterning, and reveal correlations between pseudotime and the migratory patterns of differentiating neurons. Looking forward, we anticipate that sci-Space will facilitate the construction of spatially resolved single-cell atlases of mammalian development.

Thermofluidic heat exchangers for actuation of transcription in artificial tissues.

Spatial patterns of gene expression in living organisms orchestrate cell decisions in development, homeostasis, and disease. However, most methods for reconstructing gene patterning in 3D cell culture and artificial tissues are restricted by patterning depth and scale. We introduce a depth- and scale-flexible method to direct volumetric gene expression patterning in 3D artificial tissues, which we call "heat exchangers for actuation of transcription" (HEAT). This approach leverages fluid-based heat transfer from printed networks in the tissues to activate heat-inducible transgenes expressed by embedded cells. We show that gene expression patterning can be tuned both spatially and dynamically by varying channel network architecture, fluid temperature, fluid flow direction, and stimulation timing in a user-defined manner and maintained in vivo. We apply this approach to activate the 3D positional expression of Wnt ligands and Wnt/ß-catenin pathway regulators, which are major regulators of development, homeostasis, regeneration, and cancer throughout the animal kingdom.

A Rainbow Reporter Tracks Single Cells and Reveals Heterogeneous Cellular Dynamics among Pluripotent Stem Cells and Their Differentiated Derivatives.

Single-cell transcriptomic approaches have found molecular heterogeneities within populations of pluripotent stem cells (PSCs). A tool that tracks single-cell lineages and their phenotypes longitudinally would reveal whether heterogeneity extends beyond molecular identity. Hence, we generated a stable Cre-inducible rainbow reporter human PSC line that provides up to 18 unique membrane-targeted fluorescent barcodes. These barcodes enable repeated assessments of single cells as they clonally expand, change morphology, and migrate. Owing to the cellular resolution of this reporter, we identified subsets of PSCs with enhanced clonal expansion, synchronized cell divisions, and persistent localization to colony edges. Reporter expression was stably maintained throughout directed differentiation into cardiac myocytes, cortical neurons, and hepatoblasts. Repeated examination of neural differentiation revealed self-assembled cortical tissues derive from clonally dominant progenitors. Collectively, these findings demonstrate the broad utility and easy implementation of this reporter line for tracking single-cell behavior.

Spatial presentation of biological molecules to cells by localized diffusive transfer.

Cellular decisions in human development, homeostasis, regeneration, and disease are coordinated in large part by signals that are spatially localized in tissues. These signals are often soluble, such that biomolecules produced by one cell diffuse to receiving cells. To recapitulate soluble factor patterning in vitro, several microscale strategies have been developed. However, these techniques often introduce new variables into cell culture experiments (e.g., fluid flow) or are limited in their ability to pattern diverse solutes in a user-defined manner. To address these challenges, we developed an adaptable method that facilitates spatial presentation of biomolecules across cells in traditional open cultures in vitro. This technique employs device inserts that are placed in standard culture wells, which support localized diffusive pattern transmission through microscale spaces between device features and adherent cells. Devices can be removed and cultures can be returned to standard media following patterning. We use this method to spatially control cell labeling with pattern features ranging in scale from several hundred microns to millimeters and with sequential application of multiple patterns. To better understand the method we investigate relationships between pattern fidelity, device geometry, and consumption and diffusion kinetics using finite element modeling. We then apply the method to spatially defining reporter cell heterogeneity by patterning a small molecule modulator of genetic recombination with the requisite sustained exposure. Finally, we demonstrate use of this method for patterning larger and more slowly diffusing particles by creating focal sites of gene delivery and infection with adenoviral, lentiviral, and Zika virus particles. Thus, our method leverages devices that interface with standard culture vessels to pattern diverse diffusible factors, geometries, exposure dynamics, and recipient cell types, making it well poised for adoption by researchers across various fields of biological research.

User-defined morphogen patterning for directing human cell fate stratification.

Concentration gradients of biochemical stimuli such as morphogens play a critical role in directing cell fate patterning across species and throughout development but are not commonly recapitulated in vitro. While in vitro biomolecule gradients have been generated using customized microfluidic platforms, broad implementation has been limited because these platforms introduce new variables to cell culture such as externally driven flow, culture in a specialized matrix, or extended time for in situ long range diffusion. Here we introduce a method that enables preforming and then transferring user-controlled gradients to cells in standard "open" cultures. Our gradient patterning devices are modular and decoupled from the culture substrate. We find that gradient generation and transfer are predictable by finite element modeling and that device and loading parameters can be used to tune the stimulus pattern. Furthermore, we demonstrate use of these devices to spatially define morphogen signal gradients and direct peri-gastrulation fate stratification of human pluripotent stem cells. This method for extrinsic application of biochemical signal gradients can thus be used to spatially influence cellular fate decisions in a user-controlled manner.

The Influence of Biomaterials on Cytokine Production in 3D Cultures.

As a result of improved relevance to in vivo physiology, in vitro studies are increasingly performed in diverse, three-dimensional (3D) biomaterials. However, material-cell type pairing effects on cytokine availability remain unclear. We cultured five cell types in agarose, alginate, collagen, Matrigel, or RGD-functionalized polyethylene glycol (PEG) hydrogels. We measured 21 cytokines in the conditioned media, and we identified differences in measured cytokine levels that were cell-type- or material-dependent. We further evaluated our data using principal component analysis. Interestingly, component one identified two classes of biomaterials with characteristic cytokine expression levels. Component two identified cell-type-dependent differences in cytokines related to the wound response. Although elements of soluble cytokine availability are shared despite parameter differences, material and cellular properties variably influenced cytokine levels, underlining the influence of biomaterial-cell type pairings on in vitro assay outcomes. Relationships between material properties, cellular responses, and cytokine availability in 3D in vitro models warrant further investigation.

Magnetic System for Automated Manipulation of Paramagnetic Particles.

The simple, rapid magnetic manipulation of paramagnetic particles (PMPs) paired with the wide range of available surface chemistries has strongly positioned PMPs in the field of analyte isolation. One recent technology, sliding lid for immobilized droplet extractions (SLIDE), presents a simple, rapid alternative to traditional PMP isolation protocols. Rather than remove fluid from PMP-bound analyte, SLIDE directly removes the PMPs from the fluid. SLIDE collects the PMPs on a hydrophobic, removable surface, which allows PMPs to be captured from one well and then transferred and released into a second well. Despite several key advantages, SLIDE remains limited by its passive magnetic manipulation that only allows for a one-time capture-and-release of PMPs, preventing wash steps and limiting purity. Furthermore, the strategy employed by SLIDE constrains the position of the wells, thereby limiting throughput and integration into automated systems. Here, we introduce a new, mechanically and operationally simplistic magnetic manipulation system for integration with the SLIDE technology to overcome the previously stated limitations. This magnetic system is compatible with nearly any plate design, can be integrated into automated workflows, enables high-throughput formats, simplifies mechanical requirements, and is amenable to a range of analytes. Using this magnetic system, PMPs can be collected, released, and resuspended throughout multiple wells regardless of proximity. We demonstrate this system's capabilities to isolate whole cells, mRNA, and DNA, demonstrating up to a 28-fold improvement of purity via the multiwash protocols enabled by this magnetic technology.

Transitions from mono- to co- to tri-culture uniquely affect gene expression in breast cancer, stromal, and immune compartments.

Heterotypic interactions in cancer microenvironments play important roles in disease initiation, progression, and spread. Co-culture is the predominant approach used in dissecting paracrine interactions between tumor and stromal cells, but functional results from simple co-cultures frequently fail to correlate to in vivo conditions. Though complex heterotypic in vitro models have improved functional relevance, there is little systematic knowledge of how multi-culture parameters influence this recapitulation. We therefore have employed a more iterative approach to investigate the influence of increasing model complexity; increased heterotypic complexity specifically. Here we describe how the compartmentalized and microscale elements of our multi-culture device allowed us to obtain gene expression data from one cell type at a time in a heterotypic culture where cells communicated through paracrine interactions. With our device we generated a large dataset comprised of cell type specific gene-expression patterns for cultures of increasing complexity (three cell types in mono-, co-, or tri-culture) not readily accessible in other systems. Principal component analysis indicated that gene expression was changed in co-culture but was often more strongly altered in tri-culture as compared to mono-culture. Our analysis revealed that cell type identity and the complexity around it (mono-, co-, or tri-culture) influence gene regulation. We also observed evidence of complementary regulation between cell types in the same heterotypic culture. Here we demonstrate the utility of our platform in providing insight into how tumor and stromal cells respond to microenvironments of varying complexities highlighting the expanding importance of heterotypic cultures that go beyond conventional co-culture.

Progress towards understanding heterotypic interactions in multi-culture models of breast cancer.

Microenvironments in primary tumors and metastases include multiple cell types whose dynamic and reciprocal interactions are central to progression of the disease. However, the literature involving breast cancer studied in vitro is dominated by cancer cells in mono-culture or co-cultured with one other cell type. For in vitro studies of breast cancer the inclusion of multiple cell types has led to models that are more representative of in vivo behaviors and functions as compared to more traditional monoculture. Here, we review foundational co-culture techniques and their adaptation to multi-culture (including three or more cell types). Additionally, while macroscale methods involving conditioned media, direct contact, and indirect interactions have been informative, we examined many advances that have been made more recently using microscale systems with increased control over cellular and structural complexity. Throughout this discussion we consider the benefits and limitations of current multi-culture methods and the significant results they have produced.

Fabrication and characterization of DNA-loaded zein nanospheres.

BACKGROUND: Particulates incorporating DNA are promising vehicles for gene delivery, with the ability to protect DNA and provide for controlled, localized, and sustained release and transfection. Zein, a hydrophobic protein from corn, is biocompatible and has properties that make it a promising candidate material for particulate delivery, including its ability to form nanospheres through coacervation and its insolubility under physiological conditions, making it capable of sustained release of encapsulated compounds. Due to the promise of this natural biomaterial for drug delivery, the objective of this study was to formulate zein nanospheres encapsulating DNA as the therapeutic compound, and to characterize size, charge, sustained release, cell cytotoxicity and cellular internalization of these particles. RESULTS: Zein nanospheres encapsulating DNA were fabricated using a coacervation technique, without the use of harsh solvents or temperatures, resulting in the preservation of DNA integrity and particles with diameters that ranged from 157.8 ± 3.9 nm to 396.8 ± 16.1 nm, depending on zein to DNA ratio. DNA encapsulation efficiencies were maximized to 65.3 ± 1.9% with a maximum loading of 6.1 ± 0.2 mg DNA/g zein. The spheres protected encapsulated DNA from DNase I degradation and exhibited sustained plasmid release for at least 7 days, with minimal burst during the initial phase of release. Zein/DNA nanospheres demonstrated robust biocompatibility, cellular association, and internalization. CONCLUSIONS: This study represents the first report on the formation of zein particles encapsulating plasmid DNA, using simple fabrication techniques resulting in preservation of plasmid integrity and tunable sizes. DNA encapsulation efficiencies were maximized to acceptable levels at higher zein to DNA ratios, while loading was comparable to that of other hydrophilic compounds encapsulated in zein and that of DNA incorporated into PLGA nano- and microspheres. The hydrophobic nature of zein resulted in spheres capable of sustained release of plasmid DNA. Zein particles may be an excellent potential tool for the delivery of DNA with the ability to be fine-tuned for specific applications including oral gene delivery, intramuscular delivery, and in the fabrication of tissue engineering scaffolds.