Defining the Teratoma as a Model for Multi-lineage Human Development.

We propose that the teratoma, a recognized standard for validating pluripotency in stem cells, could be a promising platform for studying human developmental processes. Performing single-cell RNA sequencing (RNA-seq) of 179,632 cells across 23 teratomas from 4 cell lines, we found that teratomas reproducibly contain approximately 20 cell types across all 3 germ layers, that inter-teratoma cell type heterogeneity is comparable with organoid systems, and teratoma gut and brain cell types correspond well to similar fetal cell types. Furthermore, cellular barcoding confirmed that injected stem cells robustly engraft and contribute to all lineages. Using pooled CRISPR-Cas9 knockout screens, we showed that teratomas can enable simultaneous assaying of the effects of genetic perturbations across all germ layers. Additionally, we demonstrated that teratomas can be sculpted molecularly via microRNA (miRNA)-regulated suicide gene expression to enrich for specific tissues. Taken together, teratomas are a promising platform for modeling multi-lineage development, pan-tissue functional genetic screening, and tissue engineering.

A general platform for targeting MHC-II antigens via a single loop.

Class-II major histocompatibility complexes (MHC-IIs) are central to the communications between CD4+ T cells and antigen presenting cells (APCs), but intrinsic structural features associated with MHC-II make it difficult to develop a general targeting system with high affinity and antigen specificity. Here, we introduce a protein platform, Targeted Recognition of Antigen-MHC Complex Reporter for MHC-II (TRACeR-II), to enable the rapid development of peptide-specific MHC-II binders. TRACeR-II has a small helical bundle scaffold and uses an unconventional mechanism to recognize antigens via a single loop. This unique antigen-recognition mechanism renders this platform highly versatile and amenable to direct structural modeling of the interactions with the antigen. We demonstrate that TRACeR-II binders can be rapidly evolved across multiple alleles, while computational protein design can produce specific binding sequences for a SARS-CoV-2 peptide of unknown complex structure. TRACeR-II sheds light on a simple and straightforward approach to address the MHC peptide targeting challenge, without relying on combinatorial selection on complementarity determining region (CDR) loops. It presents a promising basis for further exploration in immune response modulation as well as a broad range of theragnostic applications.

Programmatic introduction of parenchymal cell types into blood vessel organoids.

Pluripotent stem cell-derived organoids have transformed our ability to recreate complex three-dimensional models of human tissue. However, the directed differentiation methods used to create them do not afford the ability to introduce cross-germ-layer cell types. Here, we present a bottom-up engineering approach to building vascularized human tissue by combining genetic reprogramming with chemically directed organoid differentiation. As a proof of concept, we created neuro-vascular and myo-vascular organoids via transcription factor overexpression in vascular organoids. We comprehensively characterized neuro-vascular organoids in terms of marker gene expression and composition, and demonstrated that the organoids maintain neural and vascular function for at least 45 days in culture. Finally, we demonstrated chronic electrical stimulation of myo-vascular organoid aggregates as a potential path toward engineering mature and large-scale vascularized skeletal muscle tissue from organoids. Our approach offers a roadmap to build diverse vascularized tissues of any type derived entirely from pluripotent stem cells.

Charting oncogenicity of genes and variants across lineages via multiplexed screens in teratomas.

Deconstructing tissue-specific effects of genes and variants on proliferation is critical to understanding cellular transformation and systematically selecting cancer therapeutics. This requires scalable methods for multiplexed genetic screens tracking fitness across time, across lineages, and in a suitable niche, since physiological cues influence functional differences. Towards this, we present an approach, coupling single-cell cancer driver screens in teratomas with hit enrichment by serial teratoma reinjection, to simultaneously screen drivers across multiple lineages in vivo. Using this system, we analyzed population shifts and lineage-specific enrichment for 51 cancer associated genes and variants, profiling over 100,000 cells spanning over 20 lineages, across two rounds of serial reinjection. We confirmed that c-MYC alone or combined with myristoylated AKT1 potently drives proliferation in progenitor neural lineages, demonstrating signatures of malignancy. Additionally, mutant MEK1 S218D/S222D provides a proliferative advantage in mesenchymal lineages like fibroblasts. Our method provides a powerful platform for multi-lineage longitudinal study of oncogenesis.

Long-lasting analgesia via targeted in situ repression of NaV1.7 in mice.

Current treatments for chronic pain rely largely on opioids despite their substantial side effects and risk of addiction. Genetic studies have identified in humans key targets pivotal to nociceptive processing. In particular, a hereditary loss-of-function mutation in NaV1.7, a sodium channel protein associated with signaling in nociceptive sensory afferents, leads to insensitivity to pain without other neurodevelopmental alterations. However, the high sequence and structural similarity between NaV subtypes has frustrated efforts to develop selective inhibitors. Here, we investigated targeted epigenetic repression of NaV1.7 in primary afferents via epigenome engineering approaches based on clustered regularly interspaced short palindromic repeats (CRISPR)-dCas9 and zinc finger proteins at the spinal level as a potential treatment for chronic pain. Toward this end, we first optimized the efficiency of NaV1.7 repression in vitro in Neuro2A cells and then, by the lumbar intrathecal route, delivered both epigenome engineering platforms via adeno-associated viruses (AAVs) to assess their effects in three mouse models of pain: carrageenan-induced inflammatory pain, paclitaxel-induced neuropathic pain, and BzATP-induced pain. Our results show effective repression of NaV1.7 in lumbar dorsal root ganglia, reduced thermal hyperalgesia in the inflammatory state, decreased tactile allodynia in the neuropathic state, and no changes in normal motor function in mice. We anticipate that this long-lasting analgesia via targeted in vivo epigenetic repression of NaV1.7 methodology we dub pain LATER, might have therapeutic potential in management of persistent pain states.

A Platform to Study the Effects of Electrical Stimulation on Immune Cell Activation During Wound Healing.

Wound healing is a complex process involving diverse changes in multiple cell types where the application of electric fields has been shown to accelerate wound closure. To define the efficacy of therapies based on electric fields, it would be valuable to have a platform to systematically study the effects of electrical stimulation (ES) upon the inflammation phase and the activation of signaling mediators. Here, an in vivo ES model in which flexible electrodes are applied to an animal model for monitoring inflammation in a wound is reported on. Subcutaneous implants of polyvinyl alcohol sponges elicit inflammation response as defined by the infiltration of leukocytes. The wound site is subjected to electric fields using two types of additively fabricated flexible electrode arrays. The sponges are then harvested for flow cytometry analysis to identify changes in the phosphorylation state of intracellular targets. This platform enables studies of molecular mechanisms, as it shows that an application of low-frequency ES ≤0.5 Hz increases phosphorylation of Erk proteins in recruited leukocytes, identifying a signaling pathway that is activated during the healing process.

Mapping Cellular Reprogramming via Pooled Overexpression Screens with Paired Fitness and Single-Cell RNA-Sequencing Readout.

Understanding the effects of genetic perturbations on the cellular state has been challenging using traditional pooled screens, which typically rely on the delivery of a single perturbation per cell and unidimensional phenotypic readouts. Here, we use barcoded open reading frame overexpression libraries coupled with single-cell RNA sequencing to assay cell state and fitness, a technique we call SEUSS (scalable functional screening by sequencing). Using SEUSS, we perturbed hPSCs with a library of developmentally critical transcription factors (TFs) and assayed the impact of TF overexpression on fitness and transcriptomic states. We further leveraged the versatility of the ORF library approach to assay mutant genes and whole gene families. From the transcriptomic responses, we built genetic co-regulatory networks to identify altered gene modules and found that KLF4 and SNAI2 drive opposing effects along the epithelial-mesenchymal transition axis. From the fitness responses, we identified ETV2 as a driver of reprogramming toward an endothelial-like state.

Facile Engineering of Long-Term Culturable Ex Vivo Vascularized Tissues Using Biologically Derived Matrices.

Recent advances in tissue engineering and 3D bioprinting have enabled construction of cell-laden scaffolds containing perfusable vascular networks. Although these methods partially address the nutrient-diffusion limitations present in engineered tissues, they are still restricted in both their viable vascular geometries and matrix material compatibility. To address this, tissue constructs are engineered via encapsulation of 3D printed, evacuable, free standing scaffolds of poly(vinyl alcohol) (PVA) in biologically derived matrices. The ease of printability and water-soluble nature of PVA grant compatibility with biologically relevant matrix materials and allow for easily repeatable generation of complex vascular patterns. This study confirms the ability of this approach to produce perfusable vascularized matrices capable of sustaining both cocultures of multiple cell types and excised tumor fragments ex vivo over multiple weeks. The study further demonstrates the ability of the approach to produce hybrid patterns allowing for coculture of vasculature and epithelial cell-lined lumens in close proximity, thereby enabling ex vivo recapitulation of gut-like systems. Taken together, the methodology is versatile, broadly applicable, and importantly, simple to use, enabling ready applicability in many research settings. It is believed that this technique has the potential to significantly accelerate progress in engineering and study of ex vivo organotypic tissue constructs.

Stretchable Dry Electrodes with Concentric Ring Geometry for Enhancing Spatial Resolution in Electrophysiology.

The multichannel concentric-ring electrodes are stencil printed on stretchable elastomers modified to improve adhesion to skin and minimize motion artifacts for electrophysiological recordings of electroencephalography, electromyography, and electrocardiography. These dry electrodes with a poly(3,4-ethylenedioxythiophene) polystyrene sulfonate interface layer are optimized to show lower noise level than that of commercial gel disc electrodes. The concentric ring geometry enables Laplacian filtering to pinpoint the bioelectric potential source with spatial resolution determined by the ring distance. This work shows a new fabrication approach to integrate and create designs that enhance spatial resolution for high-quality electrophysiology monitoring devices.