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Modulation of DNA repair pathway choice by a potent inhibitor of 53BP1 improves the efficiency of homology-dependent genome editing in human and mouse cells. Programmable nucleases, such as Cas9, are used for precise genome editing by homology-dependent repair (HDR) 1 , 2 , 3 . However, HDR efficiency is constrained by competition from other double-strand break (DSB) repair pathways, including non-homologous end-joining (NHEJ) 4 . We report the discovery of a genetically encoded inhibitor of 53BP1 that increases the efficiency of HDR-dependent genome editing in human and mouse cells. 53BP1 is a key regulator of DSB repair pathway choice in eukaryotic cells 4 , 5 and functions to favor NHEJ over HDR by suppressing end resection, which is the rate-limiting step in the initiation of HDR. We screened an existing combinatorial library of engineered ubiquitin variants 6 for inhibitors of 53BP1. Expression of one variant, named i53 (inhibitor of 53BP1), in human and mouse cells, blocked accumulation of 53BP1 at sites of DNA damage and improved gene targeting and chromosomal gene conversion with either double-stranded DNA or single-stranded oligonucleotide donors by up to 5.6-fold. Inhibition of 53BP1 is a robust method to increase efficiency of HDR-based precise genome editing.

Traumatic joint injuries often result in elevated proinflammatory cytokine (such as IL-1β) levels in the joint cavity, which can increase the catabolic activities of chondrocytes and damage cartilage. This study investigated the early genetic responses of healthy in situ chondrocytes under IL-1β attack with a focus on cell cycle and calcium signaling pathways. RNA sequencing analysis identified 2,232 significantly changed genes by IL-1β, with 1,259 upregulated and 973 downregulated genes. Catabolic genes related to ECM degeneration were promoted by IL-1β, consistent with our observations of matrix protein loss and mechanical property decrease during 24-day in vitro culture of cartilage explants. IL-1β altered the cell cycle (108 genes) and Rho GTPases signaling (72 genes) in chondrocytes, while chondrocyte phenotypic shift was observed with histology, cell volume measurement, and MTT assay. IL-1β inhibited the spontaneous calcium signaling in chondrocytes, a fundamental signaling event in chondrocyte metabolic activities. The expression of 24 genes from 6 calcium-signaling related pathways were changed by IL-1β exposure. This study provided a comprehensive list of differentially expressed genes of healthy in situ chondrocytes in response to IL-1β attack, which represents a useful reference to verify and guide future cartilage studies related to the acute inflammation after joint trauma.

The KEOPS complex, which is conserved across archaea and eukaryotes, is composed of four core subunits; Pcc1, Kae1, Bud32 and Cgi121. KEOPS is crucial for the fitness of all organisms examined. In humans, pathogenic mutations in KEOPS genes lead to Galloway–Mowat syndrome, an autosomal-recessive disease causing childhood lethality. Kae1 catalyzes the universal and essential tRNA modification N 6 -threonylcarbamoyl adenosine, but the precise roles of all other KEOPS subunits remain an enigma. Here we show using structure-guided studies that Cgi121 recruits tRNA to KEOPS by binding to its 3’ CCA tail. A composite model of KEOPS bound to tRNA reveals that all KEOPS subunits form an extended tRNA-binding surface that we have validated in vitro and in vivo to mediate the interaction with the tRNA substrate and its modification. These findings provide a framework for understanding the inner workings of KEOPS and delineate why all KEOPS subunits are essential. KEOPS is an evolutionary conserved complex with a core of four core subunits—Pcc1, Kae1, Bud32 and Cgi121—that catalyzes the universal and essential tRNA modification N 6 -threonylcarbamoyl adenosine (t 6 A). Here the authors describe a Cgi121-tRNA crystal structure and new composite model of the KEOPS holo-enzyme-substrate complex that shed light on the t 6 A catalytic cycle and its regulation.

Our understanding of the left–right (LR) asymmetry of embryonic development, in particular the contribution of intrinsic handedness of the cell or cell chirality, is limited due to the confounding systematic and environmental factors during morphogenesis and a ack of physiologically relevant in vitro 3D platforms. Here we report an efficient two-layered biomaterial platform for determining the chirality of individual cells, cell aggregates, and self-organized hollow epithelial spheroids. This bioengineered niche provides a uniform defined axis allowing for cells to rotate spontaneously with a directional bias toward either clockwise or counterclockwise directions. Mechanistic studies reveal an actin-dependent, cell-intrinsic property of 3D chirality that can be mediated by actin cross-linking via α-actinin-1. Our findings suggest that the gradient of extracellular matrix is an important biophysicochemical cue influencing cell polarity and chirality. Engineered biomaterial systems can serve as an effective platform for studying developmental asymmetry and screening for environmental factors causing birth defects.

The vertebrate body plan is overall symmetrical but left–right (LR) asymmetric in the shape and positioning of internal organs. Although several theories have been proposed, the biophysical mechanisms underlying LR asymmetry are still unclear, especially the role of cell chirality, the LR asymmetry at the cellular level, on organ asymmetry. Here with developing chicken embryos, we examine whether intrinsic cell chirality or handedness regulates cardiac C looping. Using a recently established biomaterial-based 3D culture platform, we demonstrate that chick cardiac cells before and during C looping are intrinsically chiral and exhibit dominant clockwise rotation in vitro. We further show that cells in the developing myocardium are chiral as evident by a rightward bias of cell alignment and a rightward polarization of the Golgi complex, correlating with the direction of cardiac tube rotation. In addition, there is an LR polarized distribution of N-cadherin and myosin II in the myocardium before the onset of cardiac looping. More interestingly, the reversal of cell chirality via activation of the protein kinase C signaling pathway reverses the directionality of cardiac looping, accompanied by a reversal in cellular biases on the cardiac tube. Our results suggest that myocardial cell chirality regulates cellular LR symmetry breaking in the heart tube and the resultant directionality of cardiac looping. Our study provides evidence of an intrinsic cellular chiral bias leading to LR symmetry breaking during directional tissue rotation in vertebrate development.

The efforts to grow mechanically functional cartilage from human mesenchymal stem cells have not been successful. We report that clinically sized pieces of human cartilage with physiologic stratification and biomechanics can be grown in vitro by recapitulating some aspects of the developmental process of mesenchymal condensation. By exposure to transforming growth factor-β, mesenchymal stem cells were induced to condense into cellular bodies, undergo chondrogenic differentiation, and form cartilagenous tissue, in a process designed to mimic mesenchymal condensation leading into chondrogenesis. We discovered that the condensed mesenchymal cell bodies (CMBs) formed in vitro set an outer boundary after 5 d of culture, as indicated by the expression of mesenchymal condensation genes and deposition of tenascin. Before setting of boundaries, the CMBs could be fused into homogenous cellular aggregates giving rise to well-differentiated and mechanically functional cartilage. We used the mesenchymal condensation and fusion of CMBs to grow centimeter-sized, anatomically shaped pieces of human articular cartilage over 5 wk of culture. For the first time to our knowledge biomechanical properties of cartilage derived from human mesenchymal cells were comparable to native cartilage, with the Young’s modulus of >800 kPa and equilibrium friction coeffcient of <0.3. We also demonstrate that CMBs have capability to form mechanically strong cartilage–cartilage interface in an in vitro cartilage defect model. The CMBs, which acted as “lego-like” blocks of neocartilage, were capable of assembling into human cartilage with physiologic-like structure and mechanical properties.

Left-right (LR) asymmetry (handedness, chirality) is a well-conserved biological property of critical importance to normal development. Changes in orientation of the LR axis due to genetic or environmental factors can lead to malformations and disease. While the LR asymmetry of organs and whole organisms has been extensively studied, little is known about the LR asymmetry at cellular and multicellular levels. Here we show that the cultivation of cell populations on micropatterns with defined boundaries reveals intrinsic cell chirality that can be readily determined by image analysis of cell alignment and directional motion. By patterning 11 different types of cells on ring-shaped micropatterns of various sizes, we found that each cell type exhibited definite LR asymmetry (p value down to 10⁻¹⁸⁵) that was different between normal and cancer cells of the same type, and not dependent on surface chemistry, protein coating, or the orientation of the gravitational field. Interestingly, drugs interfering with actin but not microtubule function reversed the LR asymmetry in some cell types. Our results show that micropatterned cell populations exhibit phenotype-specific LR asymmetry that is dependent on the functionality of the actin cytoskeleton. We propose that micropatterning could potentially be used as an effective in vitro tool to study the initiation of LR asymmetry in cell populations, to diagnose disease, and to study factors involved with birth defects in laterality.

Congenital heart defects are associated with significant health challenges, demanding a deep understanding of the underlying biological mechanisms and, thus, better devices or platforms that can recapitulate human cardiac development. The discovery of human pluripotent stem cells has substantially reduced the dependence on animal models. Recent advances in stem cell biology, genetic editing, omics, microfluidics, and sensor technologies have further enabled remarkable progress in the development of in vitro platforms with increased fidelity and efficiency. In this review, we provide an overview of advancements in in vitro cardiac development platforms, with a particular focus on technological innovation. We categorize these platforms into four areas: two-dimensional solid substrate cultures, engineered substrate architectures that enhance cellular functions, cardiac organoids, and embryos/explants-on-chip models. We conclude by addressing current limitations and presenting future perspectives.

The ability to engineer anatomically correct pieces of viable and functional human bone would have tremendous potential for bone reconstructions after congenital defects, cancer resections, and trauma. We report that clinically sized, anatomically shaped, viable human bone grafts can be engineered by using human mesenchymal stem cells (hMSCs) and a \"biomimetic\" scaffold-bioreactor system. We selected the temporomandibular joint (TMJ) condylar bone as our tissue model, because of its clinical importance and the challenges associated with its complex shape. Anatomically shaped scaffolds were generated from fully decellularized trabecular bone by using digitized clinical images, seeded with hMSCs, and cultured with interstitial flow of culture medium. A bioreactor with a chamber in the exact shape of a human TMJ was designed for controllable perfusion throughout the engineered construct. By 5 weeks of cultivation, tissue growth was evidenced by the formation of confluent layers of lamellar bone (by scanning electron microscopy), markedly increased volume of mineralized matrix (by quantitative microcomputer tomography), and the formation of osteoids (histologically). Within bone grafts of this size and complexity cells were fully viable at a physiologic density, likely an important factor of graft function. Moreover, the density and architecture of bone matrix correlated with the intensity and pattern of the interstitial flow, as determined in experimental and modeling studies. This approach has potential to overcome a critical hurdle--in vitro cultivation of viable bone grafts of complex geometries--to provide patient-specific bone grafts for craniofacial and orthopedic reconstructions.