Architectural protein subclasses shape 3D organization of genomes during lineage commitment.

Understanding the topological configurations of chromatin may reveal valuable insights into how the genome and epigenome act in concert to control cell fate during development. Here, we generate high-resolution architecture maps across seven genomic loci in embryonic stem cells and neural progenitor cells. We observe a hierarchy of 3D interactions that undergo marked reorganization at the submegabase scale during differentiation. Distinct combinations of CCCTC-binding factor (CTCF), Mediator, and cohesin show widespread enrichment in chromatin interactions at different length scales. CTCF/cohesin anchor long-range constitutive interactions that might form the topological basis for invariant subdomains. Conversely, Mediator/cohesin bridge short-range enhancer-promoter interactions within and between larger subdomains. Knockdown of Smc1 or Med12 in embryonic stem cells results in disruption of spatial architecture and downregulation of genes found in cohesin-mediated interactions. We conclude that cell-type-specific chromatin organization occurs at the submegabase scale and that architectural proteins shape the genome in hierarchical length scales.

Differentiation of V2a interneurons from human pluripotent stem cells.

The spinal cord consists of multiple neuronal cell types that are critical to motor control and arise from distinct progenitor domains in the developing neural tube. Excitatory V2a interneurons in particular are an integral component of central pattern generators that control respiration and locomotion; however, the lack of a robust source of human V2a interneurons limits the ability to molecularly profile these cells and examine their therapeutic potential to treat spinal cord injury (SCI). Here, we report the directed differentiation of CHX10+ V2a interneurons from human pluripotent stem cells (hPSCs). Signaling pathways (retinoic acid, sonic hedgehog, and Notch) that pattern the neural tube were sequentially perturbed to identify an optimized combination of small molecules that yielded ∼25% CHX10+ cells in four hPSC lines. Differentiated cultures expressed much higher levels of V2a phenotypic markers (CHX10 and SOX14) than other neural lineage markers. Over time, CHX10+ cells expressed neuronal markers [neurofilament, NeuN, and vesicular glutamate transporter 2 (VGlut2)], and cultures exhibited increased action potential frequency. Single-cell RNAseq analysis confirmed CHX10+ cells within the differentiated population, which consisted primarily of neurons with some glial and neural progenitor cells. At 2 wk after transplantation into the spinal cord of mice, hPSC-derived V2a cultures survived at the site of injection, coexpressed NeuN and VGlut2, extended neurites >5 mm, and formed putative synapses with host neurons. These results provide a description of V2a interneurons differentiated from hPSCs that may be used to model central nervous system development and serve as a potential cell therapy for SCI.

Aggregate formation and suspension culture of human pluripotent stem cells and differentiated progeny.

Culture of human pluripotent stem cells (hPSC) as in vitro multicellular aggregates has been increasingly used as a method to model early embryonic development. Three-dimensional assemblies of hPSCs facilitate interactions between cells and their microenvironment to promote morphogenesis, analogous to the multicellular organization that accompanies embryogenesis. In this paper, we describe a method for reproducibly generating and maintaining populations of homogeneous three-dimensional hPSC aggregates using forced aggregation and rotary orbital suspension culture. We propose solutions to several challenges associated with the consistent formation and extended culture of cell spheroids generated from hPSCs and their differentiated progeny. Further, we provide examples to demonstrate how aggregation can be used as a tool to select specific subpopulations of cells to create homotypic spheroids, or as a means to introduce multiple cell types to create heterotypic tissue constructs. Finally, we demonstrate that the aggregation and rotary suspension method can be used to support culture and maintenance of hPSC-derived cell populations representing each of the three germ layers, underscoring the utility of this platform for culturing many different cell types.

Engineered Cardiac Microtissue Biomanufacturing Using Human Induced Pluripotent Stem Cell Derived Epicardial Cells.

Epicardial cells are a crucial component in constructing in vitro 3D tissue models of the human heart, contributing to the ECM environment and the resident mesenchymal cell population. Studying the human epicardium and its development from the proepicardial organ is difficult, but induced pluripotent stem cells can provide a source of human epicardial cells for developmental modeling and for biomanufacturing heterotypic cardiac tissues. This study shows that a robust population of epicardial cells (approx. 87.7% WT1+) can be obtained by small molecule modulation of the Wnt signaling pathway. The population maintains WT1 expression and characteristic epithelial morphology over successive passaging, but increases in size and decreases in cell number, suggesting a limit to their expandability in vitro. Further, low passage number epicardial cells formed into more robust 3D microtissues compared to their higher passage counterparts, suggesting that the ideal time frame for use of these epicardial cells for tissue engineering and modeling purposes is early on in their differentiated state. Additionally, the differentiated epicardial cells displayed two distinct morphologic sub populations with a subset of larger, more migratory cells which led expansion of the epicardial cells across various extracellular matrix environments. When incorporated into a mixed 3D co-culture with cardiomyocytes, epicardial cells promoted greater remodeling and migration without impairing cardiomyocyte function. This study provides an important characterization of stem cell-derived epicardial cells, identifying key characteristics that influence their ability to fabricate consistent engineered cardiac tissues.

Ultraviolet light induces mechanical and structural changes in full thickness human skin.

While the detrimental health effects of prolonged ultraviolet (UV) irradiation on skin health have been widely accepted, the biomechanical process by which photoaging occurs and the relative effects of irradiation with different UV ranges on skin biomechanics have remained relatively unexplored. In this study, the effects of UV-induced photoageing are explored by quantifying the changes in the mechanical properties of full-thickness human skin irradiated with UVA and UVB light for incident dosages up to 1600 J/cm2. Mechanical testing of skin samples excised parallel and perpendicular to the predominant collagen fiber orientation show a rise in the fractional relative difference of elastic modulus, fracture stress, and toughness with increased UV irradiation. These changes become significant with UVA incident dosages of 1200 J/cm2 for samples excised both parallel and perpendicular to the dominant collagen fiber orientation. However, while mechanical changes occur in samples aligned with the collagen orientation at UVB dosages of 1200 J/cm2, statistical differences in samples perpendicular to the collagen orientation emerge only for UVB dosages of 1600 J/cm2. No notable or consistent trend is observed for the fracture strain. Analyses of toughness changes with maximum absorbed dosage reveals that no one UV range is more impactful in inducing mechanical property changes, but rather these changes scale with maximum absorbed energy. Evaluation of the structural characteristics of collagen further reveals an increase in collagen fiber bundle density with UV irradiation, but not collagen tortuosity, potentially linking mechanical changes to altered microstructure.

Single-Cell Determination of Cardiac Microtissue Structure and Function Using Light Sheet Microscopy.

Native cardiac tissue is composed of heterogeneous cell populations that work cooperatively for proper tissue function; thus, engineered tissue models have moved toward incorporating multiple cardiac cell types in an effort to recapitulate native multicellular composition and organization. Cardiac tissue models composed of stem cell-derived cardiomyocytes (CMs) require inclusion of non-myocytes to promote stable tissue formation, yet the specific contributions of the supporting non-myocyte population on the parenchymal CMs and cardiac microtissues have to be fully dissected. This gap can be partly attributed to limitations in technologies able to accurately study the individual cellular structure and function that comprise intact three-dimensional (3D) tissues. The ability to interrogate the cell-cell interactions in 3D tissue constructs has been restricted by conventional optical imaging techniques that fail to adequately penetrate multicellular microtissues with sufficient spatial resolution. Light sheet fluorescence microscopy (LSFM) overcomes these constraints to enable single-cell resolution structural and functional imaging of intact cardiac microtissues. Multicellular spatial distribution analysis of heterotypic cardiac cell populations revealed that CMs and cardiac fibroblasts were randomly distributed throughout 3D microtissues. Furthermore, calcium imaging of live cardiac microtissues enabled single-cell detection of CM calcium activity, which showed that functional heterogeneity correlated with spatial location within the tissues. This study demonstrates that LSFM can be utilized to determine single-cell spatial and functional interactions of multiple cell types within intact 3D engineered microtissues, thereby facilitating the determination of structure-function relationships at both tissue-level and single-cell resolution. Impact statement The ability to achieve single-cell resolution by advanced three-dimensional light imaging techniques enables exquisite new investigation of multicellular analyses in native and engineered tissues. In this study, light sheet fluorescence microscopy was used to define structure-function relationships of distinct cell types in engineered cardiac microtissues by determining heterotypic cell distributions and interactions throughout the tissues as well as by assessing regional differences in calcium handing functional properties at the individual cardiomyocyte level.

Phenotypic Variation Between Stromal Cells Differentially Impacts Engineered Cardiac Tissue Function.

IMPACT STATEMENT: Understanding the relationship between parenchymal and supporting cell populations is paramount to recapitulate the multicellular complexity of native tissues. Incorporation of stromal cells is widely recognized to be necessary for the stable formation of stem cell-derived cardiac tissues; yet, the types of stromal cells used have varied widely. This study systematically characterized several stromal populations and found that stromal phenotype and morphology was highly variable depending on cell source and exerted differential impacts on cardiac tissue function and induced pluripotent stem cell-cardiomyocyte phenotype. Therefore, the choice of supporting stromal population can differentially impact the phenotypic or functional performance of engineered cardiac tissues.

Engineering Biology by Controlling Tissue Folding.

Achieving complex self-organization in vitro has remained a fundamental challenge in tissue engineering. A recent study in Developmental Cell by Hughes and colleagues uses computational and experimental approaches to understand and control the morphogenic process of tissue folding. These approaches provide an engineering framework to reproducibly control tissue shape.

Fabrication of Custom Agarose Wells for Cell Seeding and Tissue Ring Self-assembly Using 3D-Printed Molds.

Engineered tissues are being used clinically for tissue repair and replacement, and are being developed as tools for drug screening and human disease modeling. Self-assembled tissues offer advantages over scaffold-based tissue engineering, such as enhanced matrix deposition, strength, and function. However, there are few available methods for fabricating 3D tissues without seeding cells on or within a supporting scaffold. Previously, we developed a system for fabricating self-assembled tissue rings by seeding cells into non-adhesive agarose wells. A polydimethylsiloxane (PDMS) negative was first cast in a machined polycarbonate mold, and then agarose was gelled in the PDMS negative to create ring-shaped cell seeding wells. However, the versatility of this approach was limited by the resolution of the tools available for machining the polycarbonate mold. Here, we demonstrate that 3D-printed plastic can be used as an alternative to machined polycarbonate for fabricating PDMS negatives. The 3D-printed mold and revised mold design is simpler to use, inexpensive to produce, and requires significantly less agarose and PDMS per cell seeding well. We have demonstrated that the resulting agarose wells can be used to create self-assembled tissue rings with customized diameters from a variety of different cell types. Rings can then be used for mechanical, functional, and histological analysis, or for fabricating larger and more complex tubular tissues.

Assembly of Tissue-Engineered Blood Vessels with Spatially Controlled Heterogeneities.

Tissue-engineered human blood vessels may enable in vitro disease modeling and drug screening to accelerate advances in vascular medicine. Existing methods for tissue-engineered blood vessel (TEBV) fabrication create homogenous tubes not conducive to modeling the focal pathologies characteristic of certain vascular diseases. We developed a system for generating self-assembled human smooth muscle cell (SMC) ring units, which were fused together into TEBVs. The goal of this study was to assess the feasibility of modular assembly and fusion of ring building units to fabricate spatially controlled, heterogeneous tissue tubes. We first aimed to enhance fusion and reduce total culture time, and determined that reducing ring preculture duration improved tube fusion. Next, we incorporated electrospun polymer ring units onto tube ends as reinforced extensions, which allowed us to cannulate tubes after only 7 days of fusion, and culture tubes with luminal flow in a custom bioreactor. To create focal heterogeneities, we incorporated gelatin microspheres into select ring units during self-assembly, and fused these rings between ring units without microspheres. Cells within rings maintained their spatial position along tissue tubes after fusion. Because tubes fabricated from primary SMCs did not express contractile proteins, we also fabricated tubes from human mesenchymal stem cells, which expressed smooth muscle alpha actin and SM22-α. This work describes a platform approach for creating modular TEBVs with spatially defined structural heterogeneities, which may ultimately be applied to mimic focal diseases such as intimal hyperplasia or aneurysm.

Functionalization of microparticles with mineral coatings enhances non-viral transfection of primary human cells.

Gene delivery to primary human cells is a technology of critical interest to both life science research and therapeutic applications. However, poor efficiencies in gene transfer and undesirable safety profiles remain key limitations in advancing this technology. Here, we describe a materials-based approach whereby application of a bioresorbable mineral coating improves microparticle-based transfection of plasmid DNA lipoplexes in several primary human cell types. In the presence of these mineral-coated microparticles (MCMs), we observed up to 4-fold increases in transfection efficiency with simultaneous reductions in cytotoxicity. We identified mechanisms by which MCMs improve transfection, as well as coating compositions that improve transfection in three-dimensional cell constructs. The approach afforded efficient transfection in primary human fibroblasts as well as mesenchymal and embryonic stem cells for both two- and three-dimensional transfection strategies. This MCM-based transfection is an advancement in gene delivery technology, as it represents a non-viral approach that enables highly efficient, localized transfection and allows for transfection of three-dimensional cell constructs.

Design Principles for Engineering of Tissues from Human Pluripotent Stem Cells.

Recent advances in human pluripotent stem cell (hPSC) technologies have enabled the engineering of human tissue constructs for developmental studies, disease modeling, and drug screening platforms. In vitro tissue formation can be generally described at three levels of cellular organization. Multicellular hPSC constructs are initially formed either with polymeric scaffold materials or simply via self-assembly, adhesive mechanisms. Heterotypic interactions within hPSC tissue constructs can be achieved by physically mixing independently differentiated cell populations or coaxed to simultaneously co-emerge from a common population of undifferentiated cells. Higher order tissue architecture can be engineered by imposing external spatial constraints, such as molds and scaffolds, or depend upon cell-driven organization that exploits endogenous innate developmental mechanisms. The multicellular, heterogeneous, and highly organized structure of hPSC constructs ultimately dictates the resulting form and function of in vitro engineered human tissue models.

A Multi-Modality CMOS Sensor Array for Cell-Based Assay and Drug Screening.

In this paper, we present a fully integrated multi-modality CMOS cellular sensor array with four sensing modalities to characterize different cell physiological responses, including extracellular voltage recording, cellular impedance mapping, optical detection with shadow imaging and bioluminescence sensing, and thermal monitoring. The sensor array consists of nine parallel pixel groups and nine corresponding signal conditioning blocks. Each pixel group comprises one temperature sensor and 16 tri-modality sensor pixels, while each tri-modality sensor pixel can be independently configured for extracellular voltage recording, cellular impedance measurement (voltage excitation/current sensing), and optical detection. This sensor array supports multi-modality cellular sensing at the pixel level, which enables holistic cell characterization and joint-modality physiological monitoring on the same cellular sample with a pixel resolution of 80 µm × 100 µm. Comprehensive biological experiments with different living cell samples demonstrate the functionality and benefit of the proposed multi-modality sensing in cell-based assay and drug screening.

Engineering three-dimensional stem cell morphogenesis for the development of tissue models and scalable regenerative therapeutics.

The physiochemical stem cell microenvironment regulates the delicate balance between self-renewal and differentiation. The three-dimensional assembly of stem cells facilitates cellular interactions that promote morphogenesis, analogous to the multicellular, heterotypic tissue organization that accompanies embryogenesis. Therefore, expansion and differentiation of stem cells as multicellular aggregates provides a controlled platform for studying the biological and engineering principles underlying spatiotemporal morphogenesis and tissue patterning. Moreover, three-dimensional stem cell cultures are amenable to translational screening applications and therapies, which underscores the broad utility of scalable suspension cultures across laboratory and clinical scales. In this review, we discuss stem cell morphogenesis in the context of fundamental biophysical principles, including the three-dimensional modulation of adhesions, mechanics, and molecular transport and highlight the opportunities to employ stem cell spheroids for tissue modeling, bioprocessing, and regenerative therapies.

Microscale generation of cardiospheres promotes robust enrichment of cardiomyocytes derived from human pluripotent stem cells.

Cardiomyocytes derived from human pluripotent stem cells (hPSCs) are a promising cell source for regenerative medicine, disease modeling, and drug discovery, all of which require enriched cardiomyocytes, ideally ones with mature phenotypes. However, current methods are typically performed in 2D environments that produce immature cardiomyocytes within heterogeneous populations. Here, we generated 3D aggregates of cardiomyocytes (cardiospheres) from 2D differentiation cultures of hPSCs using microscale technology and rotary orbital suspension culture. Nearly 100% of the cardiospheres showed spontaneous contractility and synchronous intracellular calcium transients. Strikingly, from starting heterogeneous populations containing ∼10%-40% cardiomyocytes, the cell population within the generated cardiospheres featured ∼80%-100% cardiomyocytes, corresponding to an enrichment factor of up to 7-fold. Furthermore, cardiomyocytes from cardiospheres exhibited enhanced structural maturation in comparison with those from a parallel 2D culture. Thus, generation of cardiospheres represents a simple and robust method for enrichment of cardiomyocytes in microtissues that have the potential use in regenerative medicine as well as other applications.

Self-assembled smooth muscle cell tissue rings exhibit greater tensile strength than cell-seeded fibrin or collagen gel rings.

In this study, we created self-assembled smooth muscle cell (SMC) tissue rings (comprised entirely of cells and cell-derived matrix; CDM) and compared their structure and material properties with tissue rings created from SMC-seeded fibrin or collagen gels. All tissue rings were cultured statically for 7 days in supplemented growth medium (with ε-amino caproic acid, ascorbic acid, and insulin-transferrin-selenium), prior to uniaxial tensile testing and histology. Self-assembled CDM rings exhibited ultimate tensile strength and stiffness values that were two-fold higher than fibrin gel and collagen gel rings. Tensile testing of CDM, fibrin gel and collagen gel rings treated with deionized water to lyse cells showed little to no change in mechanical properties relative to untreated ring samples, indicating that the ECM dominates the measured ring mechanics. In addition, CDM rings cultured in supplemented growth medium were significantly stronger than CDM rings cultured in standard, unsupplemented growth medium. These results illustrate the potential utility of self-assembled cell rings as model CDM constructs for tissue engineering and biomechanical analysis of ECM material properties.