Tracy A. Hookway

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.

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

Summary 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.

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.

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.

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 and 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.

Differentiation of V2a interneurons from human pluripotent stem cells

Significance Spinal cord injury (SCI) significantly disrupts normal neural circuitry, leading to severe degradation of motor and sensory function. Excitatory interneurons that relay signals from the brain to neural networks throughout the spinal cord, including glutamatergic V2a interneurons that coordinate respiration and locomotion, are lost after SCI. Thus, transplantation of V2a interneurons after SCI could provide a novel therapy to restore functional connections between the brain and spared downstream neurons. This study describes the generation of V2a interneurons from human pluripotent stem cells, using developmentally relevant morphogenic signaling pathways. This work provides initial insight into the development of excitatory human interneurons and enables the examination of their therapeutic efficacy for SCI repair. 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.

11.7 A multimodality CMOS sensor array for cell-based assay and drug screening

Cell-based assays are powerful tools to characterize cell- or tissue-specific physiological behaviors under external biochemical stimuli. External biochemical stimuli trigger endogenous cellular mechanisms that produce a cascade of physiological changes, resulting in easily measurable signals. Cell-based assays are widely used for large-scale drug screening in the pharmaceutical industry, where in vitro cultured cells are used to characterize the potency and toxicity of thousands of chemicals, leading to new drug development. This is particularly relevant in individualized medicine as patient-derived cells can test personalized drug responses. However, most current cell-based assays are conducted on single-modality sensors (electrical or optical only), which cannot capture the complexity of multi-parameter physiological responses. Sequentially transporting cell samples through different sensor platforms results in low throughput and potential abrogation of cell functions, while parallel monitoring of multiple samples with different modalities is subject to cell-to-cell variation even in a homogeneous cell population.

Fabrication of and cell growth on ‘silicon membranes’ with high density TSVs for bio-sensing applications

A silicon membrane acting as an interface layer between live cells and the sensing electronics enabling low-cost, high-throughput bio-sensing is proposed; the interface is capable of supporting high pixel density allowing accurate image mapping. Cell attachment and growth was carried out on five different silicon based surfaces and compared to the standard Tissue Culture Polystyrene (TCPS) surface in order to optimize the selection process of the interface material; cell imaging is performed after 48 hours to study the effect of different surface variations (different materials on silicon, presence of metal, increased surface roughness due to CMP, etc.) on cell adhesion and growth. The results show that cells successfully adhere and grow onto the sensing electrodes for all the surfaces under test after the first 48 hours. Key enabling technologies for high pixel density membranes are also presented.

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.