Hierarchal single-cell lineage tracing reveals differential fate commitment of CD8 T-cell clones in response to acute infection.

Generating balanced populations of CD8 effector and memory T cells is necessary for immediate and durable immunity to infections and cancer. Yet, a definitive understanding of CD8 differentiation remains unclear. We used CARLIN, a processive lineage recording mouse model with single-cell RNA-seq and TCR-seq to track endogenous antigen-specific CD8 T cells during acute viral infection. We identified a diverse repertoire of expanded T-cell clones represented by seven transcriptional states. TCR enrichment analysis revealed differential memory- or effector-fate biases within clonal populations. Shared Vb segments and amino acid motifs were found within biased categories despite high TCR diversity. Using single-cell CARLIN barcode-seq we tracked multi-generational clones and found that unlike unbiased or memory-biased clones, which stably retain their fate profiles, effector-biased clones could adopt memory- or effector-bias within subclones. Collectively, our study demonstrates that a heterogenous T-cell repertoire specific for a shared antigen is composed of clones with distinct TCR-intrinsic fate-biases.

Skin Sensitivity Assessment Using Smartphone Haptic Feedback.

Goal: This work presents a smartphone application to assess cutaneous sensory perception by establishing Vibrational Perception Thresholds (VPTs). Cutaneous sensory perception diagnostics allow for the early detection and symptom tracking of tactile dysfunction. However, lack of access to healthcare and the limited frequency of current screening tools can leave skin sensation impairments undiscovered or unmonitored. Methods: A 23-participant cross-sectional study in subjects with a range of finger sensation tests Smartphone Established VPTs (SE-VPTs) by varying device vibrational intensity. These are compared against monofilament test scores, a clinical measure of skin sensitivity. Results: We find a strong positive correlation between SE-VPTs and monofilament scores ([Formula: see text] = 0.86, p = 1.65e-07). Conclusions: These results demonstrate the feasibility of using a smartphone as a skin sensation screening tool.

A Wearable Testbed for Studying Variable Transmission in Body-Powered Prosthetic Gripping.

For those with upper limb absence, body-powered prostheses continue to be popular for many activities despite being an old technology; these devices can provide both inherent haptic feedback and mechanical robustness. Yet, they can also result in strain and fatigue. Body-powered prosthetic graspers typically consist of a simple lever providing a relatively constant transmission ratio between the input forces from the user's shoulder harness and the grip force of their prosthetic prehensor. In the field of robotic hand design, new continuously varying transmissions demonstrate particular promise in generating a wide range of grasping speeds without sacrificing grip strength. These benefits, if applied to shoulder-driven prosthetic grippers, have the potential to both reduce shoulder exertion and fatigue. This work presents the integration of a continuously variable transmission into a body-powered, voluntary close prosthetic testbed. We introduce the design and validate its performance in a benchtop experiment. We compare constant transmission conditions with a force-dependent, continually varying condition. The device is mounted on a prosthetic emulator for a preliminary wearable demonstration.

"Self-care selfies": Patient-uploaded videos capture meaningful changes in dexterity over 6 months.

OBJECTIVE: Upper extremity function reflects disease progression in multiple sclerosis (MS). This study evaluated the feasibility, validity, and sensitivity to change of remote dexterity assessments applying human pose estimation to patient-uploaded videos. METHODS: A discovery cohort of 50 adults with MS recorded "selfie" videos of self-care tasks at home: buttoning, brushing teeth, and eating. Kinematic data were extracted using MediaPipe Hand pose estimation software. Clinical comparison tests were grip and pinch strength, 9 hole peg test (9HPT), and vibration, and patient-reported dexterity assessments (ABILHAND). Feasibility and acceptability were evaluated (Health-ITUES framework). A validation cohort (N = 35) completed 9HPT and videos. RESULTS: The modality was feasible: 88% of the 50 enrolled participants uploaded ≥3 videos, and 74% completed the study. It was also usable: assessments easy to access (95%), platform easy to use (97%), and tasks representative of daily activities (86%). The buttoning task revealed four metrics with strong correlations with 9HPT (nondominant: r = 0.60-0.69, dominant: r = 0.51-0.57, P < 0.05) and ABILHAND (r = -0.48, P = 0.05). Retest validity at 1 week was stable (r > 0.8). Cross-sectional correlations between video metrics and 9HPT were similar at 6 months, and in the validation cohort (nondominant: r = 0.46, dominant: r = 0.45, P < 0.05). Over 6 months, pinch strength (5.8-5.0 kg/cm2 , P = 0.05) and self-reported pinch (ABILHAND) decreased marginally. While only 15% of participants worsened by 20% on 9HPT, 70% worsened in key buttoning video metrics. INTERPRETATION: Patient-uploaded videos represent a novel, patient-centered modality for capturing dexterity that appears valid and sensitive to change, enhancing its potential to be disseminated for neurological disease monitoring and treatment.

In vitro generation of mouse morula-like cells.

Generating cells with the molecular and functional properties of embryo cells and with full developmental potential is an aim with fundamental biological significance. Here we report the in vitro generation of mouse transient morula-like cells (MLCs) via the manipulation of signaling pathways. MLCs are molecularly distinct from embryonic stem cells (ESCs) and cluster instead with embryo 8- to 16-cell stage cells. A single MLC can generate a blastoid, and the efficiency increases to 80% when 8-10 MLCs are used. MLCs make embryoids directly, efficiently, and within 4 days. Transcriptomic analysis shows that day 4-5 MLC-derived embryoids contain the cell types found in natural embryos at early gastrulation. Furthermore, MLCs introduced into morulae segregate into epiblast (EPI), primitive endoderm (PrE), and trophectoderm (TE) fates in blastocyst chimeras and have a molecular signature indistinguishable from that of host embryo cells. These findings represent the generation of cells that are molecularly and functionally similar to the precursors of the first three cell lineages of the embryo.

Efficient reciprocating burrowing with anisotropic origami feet.

Origami folding is an ancient art which holds promise for creating compliant and adaptable mechanisms, but has yet to be extensively studied for granular environments. At the same time, biological systems exploit anisotropic body forces for locomotion, such as the frictional anisotropy of a snake's skin. In this work, we explore how foldable origami feet can be used to passively induce anisotropic force response in granular media, through varying their resistive plane. We present a reciprocating burrower which transfers pure symmetric linear motion into directed burrowing motion using a pair of deployable origami feet on either end. We also present an application of the reduced order model granular Resistive Force Theory to inform the design of deformable structures, and compare results with those from experiments and Discrete Element Method simulations. Through a single actuator, and without the use of advanced controllers or sensors, these origami feet enable burrowing locomotion. In this paper, we achieve burrowing translation ratios-net forward motion to overall linear actuation-over 46% by changing foot design without altering overall foot size. Specifically, anisotropic folding foot parameters should be tuned for optimal performance given a linear actuator's stroke length.

Characterizing the Force-Motion Tradeoff in Body-Powered Transmission Design.

Upper-limb prosthesis users continue to reject devices despite continued research efforts. Today, the passive topology of body-powered prehensors, which physically transmits grasp force and position data between user and device, results in improved performance over myoelectric alternatives. However, the loads and postures on the user's body also result in discomfort, fatigue, and worsened grasp force control. Despite the long history and everyday adoption of body-powered prehensors in society, the measurement of how specific body loads and postures affect grasp performance and user experience has yet to be systematically studied. In this work, we present a body-powered prosthesis emulator to independently change required input forces and motions to study the positive and negative effects provided by the inherent haptic feedback. Using a simulated grasping task, we collect functional and qualitative data from 15 participants using a shoulder harness interface. Outcomes show that lowering required input motions and forces independently reduces negative outcomes, with diminishing returns below 1:1 output mappings. Given the tradeoff between force and motion in traditional body-powered transmissions, a transmission ratio of 1:1 balances both requirements. The purpose of this study is to inform future prehensor designs that leverage the transparency of body-power to deliver high functionality while mitigating user discomfort.

Cellular population dynamics shape the route to human pluripotency.

Human cellular reprogramming to induced pluripotency is still an inefficient process, which has hindered studying the role of critical intermediate stages. Here we take advantage of high efficiency reprogramming in microfluidics and temporal multi-omics to identify and resolve distinct sub-populations and their interactions. We perform secretome analysis and single-cell transcriptomics to show functional extrinsic pathways of protein communication between reprogramming sub-populations and the re-shaping of a permissive extracellular environment. We pinpoint the HGF/MET/STAT3 axis as a potent enhancer of reprogramming, which acts via HGF accumulation within the confined system of microfluidics, and in conventional dishes needs to be supplied exogenously to enhance efficiency. Our data suggest that human cellular reprogramming is a transcription factor-driven process that it is deeply dependent on extracellular context and cell population determinants.

Developmental cell fate choice in neural tube progenitors employs two distinct cis-regulatory strategies.

In many developing tissues, the patterns of gene expression that assign cell fate are organized by graded secreted signals. Cis-regulatory elements (CREs) interpret these signals to control gene expression, but how this is accomplished remains poorly understood. In the neural tube, a gradient of the morphogen sonic hedgehog (Shh) patterns neural progenitors. We identify two distinct ways in which CREs translate graded Shh into differential gene expression in mouse neural progenitors. In most progenitors, a common set of CREs control gene activity by integrating cell-type-specific inputs. By contrast, the most ventral progenitors use a unique set of CREs, established by the pioneer factor FOXA2. This parallels the role of FOXA2 in endoderm, where FOXA2 binds some of the same sites. Together, the data identify distinct cis-regulatory strategies for the interpretation of morphogen signaling and raise the possibility of an evolutionarily conserved role for FOXA2 across tissues.

3D ECM-rich environment sustains the identity of naive human iPSCs.

The establishment of in vitro naive human pluripotent stem cell cultures opened new perspectives for the study of early events in human development. The role of several transcription factors and signaling pathways have been characterized during maintenance of human naive pluripotency. However, little is known about the role exerted by the extracellular matrix (ECM) and its three-dimensional (3D) organization. Here, using an unbiased and integrated approach combining microfluidic cultures with transcriptional, proteomic, and secretome analyses, we found that naive, but not primed, hiPSC colonies are characterized by a self-organized ECM-rich microenvironment. Based on this, we developed a 3D culture system that supports robust long-term feeder-free self-renewal of naive hiPSCs and also allows direct and timely developmental morphogenesis simply by modulating the signaling environment. Our study opens new perspectives for future applications of naive hiPSCs to study critical stages of human development in 3D starting from a single cell.

Mole crab-inspired vertical self-burrowing.

We present EMBUR-EMerita BUrrowing Robot-the first legged robot inspired by the Pacific mole crab, Emerita analoga, capable of burrowing vertically downward. We choose Emerita analoga as a model organism for its rapid downward burrowing behaviors, as it is four times as fast as the most rapid bivalve mollusk. Vertical burrowing in granular media is a challenging endeavor due to the tendency for the media to create upwards resistive forces on an intruder, even during purely horizontal motions. Our robot is capable of vertically burrowing its body in granular substrate primarily through excavation using two leg pairs, which are functionally analogous to groupings of leg pairs of the mole crab. We implement a novel leg mechanism with a sweeping trajectory, using compliant fabric to enable an anisotropic force response. The maximum resistive force during the power stroke is 6.4 times that of the return stroke. We compare robot body pitch and spatial trajectories with results from biomechanical studies of the mole crabs. We characterize the sensitivity of the robot to initial depth, body pitch and leg pose, and propose bounds on initial conditions which predict various burrowing failure modes. Parametric studies utilizing Granular Resistive Force Theory inform our understanding of robot behavior in response to leg phasing and orientation. Not only does this robotic platform represent the first robophysical model of vertical mole crab-inspired burrowing, it is also one of the first legged, primarily excavative small-scale burrowing agents.

Effect of variable transmission on body-powered prosthetic grasping.

Body-powered upper-limb prostheses remain a popular option for those with limb absence due to their passive nature. These devices typically feature a constant transmission ratio between the forces input by the user and the grasp forces output by the prosthetic gripper. Work incorporating continuously variable transmissions into robotic hands has demonstrated a number of benefits in terms of their motion and forces. In this work, we use a custom prosthesis emulator to evaluate the viability of applying variable transmissions to a body-powered prosthetic context. With this haptics test bed, we measured user performance during a grasping and lift task under a variety of transmission ratio conditions and with three different test objects. Results indicate that use of a variable transmission leads to the successful manipulation of a wider variety of objects than the constant transmission ratio systems, while requiring less shoulder motion. Analysis also shows a potential tendency for users to apply higher grasp forces than necessary, when compared to constant transmission conditions. These findings suggest a multifaceted effect on grasp performance with both benefits and drawbacks when considering a variable approach that supports the continued study of variable transmissions in assisted grasping.

Human Pluripotent Stem Cell-Derived Micropatterned Ectoderm Allows Cell Sorting of Meso-Endoderm Lineages.

The human developmental processes during the early post-implantation stage instruct the specification and organization of the lineage progenitors into a body plan. These processes, which include patterning, cell sorting, and establishment of the three germ layers, have been classically studied in non-human model organisms and only recently, through micropatterning technology, in a human-specific context. Micropatterning technology has unveiled mechanisms during patterning and germ layer specification; however, cell sorting and their segregation in specific germ layer combinations have not been investigated yet in a human-specific in vitro system. Here, we developed an in vitro model of human ectodermal patterning, in which human pluripotent stem cells (hPSCs) self-organize to form a radially regionalized neural and non-central nervous system (CNS) ectoderm. We showed that by using micropatterning technology and by modulating BMP and WNT signals, we can regulate the appearance and spatial distribution of the different ectodermal populations. This pre-patterned ectoderm can be used to investigate the cell sorting behavior of hPSC-derived meso-endoderm cells, with an endoderm that segregates from the neural ectoderm. Thus, the combination of micro-technology with germ layer cross-mixing enables the study of cell sorting of different germ layers in a human context.

SARS-CoV-2 infection and replication in human gastric organoids.

COVID-19 typically manifests as a respiratory illness, but several clinical reports have described gastrointestinal symptoms. This is particularly true in children in whom gastrointestinal symptoms are frequent and viral shedding outlasts viral clearance from the respiratory system. These observations raise the question of whether the virus can replicate within the stomach. Here we generate gastric organoids from fetal, pediatric, and adult biopsies as in vitro models of SARS-CoV-2 infection. To facilitate infection, we induce reverse polarity in the gastric organoids. We find that the pediatric and late fetal gastric organoids are susceptible to infection with SARS-CoV-2, while viral replication is significantly lower in undifferentiated organoids of early fetal and adult origin. We demonstrate that adult gastric organoids are more susceptible to infection following differentiation. We perform transcriptomic analysis to reveal a moderate innate antiviral response and a lack of differentially expressed genes belonging to the interferon family. Collectively, we show that the virus can efficiently infect the gastric epithelium, suggesting that the stomach might have an active role in fecal-oral SARS-CoV-2 transmission.

StemBond hydrogels control the mechanical microenvironment for pluripotent stem cells.

Studies of mechanical signalling are typically performed by comparing cells cultured on soft and stiff hydrogel-based substrates. However, it is challenging to independently and robustly control both substrate stiffness and extracellular matrix tethering to substrates, making matrix tethering a potentially confounding variable in mechanical signalling investigations. Moreover, unstable matrix tethering can lead to poor cell attachment and weak engagement of cell adhesions. To address this, we developed StemBond hydrogels, a hydrogel in which matrix tethering is robust and can be varied independently of stiffness. We validate StemBond hydrogels by showing that they provide an optimal system for culturing mouse and human pluripotent stem cells. We further show how soft StemBond hydrogels modulate stem cell function, partly through stiffness-sensitive ERK signalling. Our findings underline how substrate mechanics impact mechanosensitive signalling pathways regulating self-renewal and differentiation, indicating that optimising the complete mechanical microenvironment will offer greater control over stem cell fate specification.

Dynamic transcriptional reprogramming leads to immunotherapeutic vulnerabilities in myeloma.

While there is extensive evidence for genetic variation as a basis for treatment resistance, other sources of variation result from cellular plasticity. Using multiple myeloma as an example of an incurable lymphoid malignancy, we show how cancer cells modulate lineage restriction, adapt their enhancer usage and employ cell-intrinsic diversity for survival and treatment escape. By using single-cell transcriptome and chromatin accessibility profiling, we show that distinct transcriptional states co-exist in individual cancer cells and that differential transcriptional regulon usage and enhancer rewiring underlie these alternative transcriptional states. We demonstrate that exposure to standard treatment further promotes transcriptional reprogramming and differential enhancer recruitment while simultaneously reducing developmental potential. Importantly, treatment generates a distinct complement of actionable immunotherapy targets, such as CXCR4, which can be exploited to overcome treatment resistance. Our studies therefore delineate how to transform the cellular plasticity that underlies drug resistance into immuno-oncologic therapeutic opportunities.

Single-Cell Profiling Reveals Metabolic Reprogramming as a Resistance Mechanism in BRAF-Mutated Multiple Myeloma.

PURPOSE: Although remarkably effective in some patients, precision medicine typically induces only transient responses despite initial absence of resistance-conferring mutations. Using BRAF-mutated myeloma as a model for resistance to precision medicine we investigated if BRAF-mutated cancer cells have the ability to ensure their survival by rapidly adapting to BRAF inhibitor treatment. EXPERIMENTAL DESIGN: Full-length single-cell RNA (scRNA) sequencing (scRNA-seq) was conducted on 3 patients with BRAF-mutated myeloma and 1 healthy donor. We sequenced 1,495 cells before, after 1 week, and at clinical relapse to BRAF/MEK inhibitor treatment. We developed an in vitro model of dabrafenib resistance using genetically homogeneous single-cell clones from two cell lines with established BRAF mutations (U266, DP6). Transcriptional and epigenetic adaptation in resistant cells were defined by RNA-seq and H3K27ac chromatin immunoprecipitation sequencing (ChIP-seq). Mitochondrial metabolism was characterized by metabolic flux analysis. RESULTS: Profiling by scRNA-seq revealed rapid cellular state changes in response to BRAF/MEK inhibition in patients with myeloma and cell lines. Transcriptional adaptation preceded detectable outgrowth of genetically discernible drug-resistant clones and was associated with widespread enhancer remodeling. As a dominant vulnerability, dependency on oxidative phosphorylation (OxPhos) was induced. In treated individuals, OxPhos was activated at the time of relapse and showed inverse correlation to MAPK activation. Metabolic flux analysis confirmed OxPhos as a preferential energetic resource of drug-persistent myeloma cells. CONCLUSIONS: This study demonstrates that cancer cells have the ability to rapidly adapt to precision treatments through transcriptional state changes, epigenetic adaptation, and metabolic rewiring, thus facilitating the development of refractory disease while simultaneously exposing novel vulnerabilities.

Sox2 modulation increases naïve pluripotency plasticity.

Induced pluripotency provides a tool to explore mechanisms underlying establishment, maintenance, and differentiation of naive pluripotent stem cells (nPSCs). Here, we report that self-renewal of nPSCs requires minimal Sox2 expression (Sox2-low). Sox2-low nPSCs do not show impaired neuroectoderm specification and differentiate efficiently in vitro into all embryonic germ lineages. Strikingly, upon the removal of self-renewing cues Sox2-low nPSCs differentiate into both embryonic and extraembryonic cell fates in vitro and in vivo. This differs from previous studies which only identified conditions that allowed cells to differentiate to one fate or the other. At the single-cell level self-renewing Sox2-low nPSCs exhibit a naive molecular signature. However, they display a nearer trophoblast identity than controls and decreased ability of Oct4 to bind naïve-associated regulatory sequences. In sum, this work defines wild-type levels of Sox2 as a restrictor of developmental potential and suggests perturbation of naive network as a mechanism to increase cell plasticity.

OCT4 induces embryonic pluripotency via STAT3 signaling and metabolic mechanisms.

OCT4 is a fundamental component of the molecular circuitry governing pluripotency in vivo and in vitro. To determine how OCT4 establishes and protects the pluripotent lineage in the embryo, we used comparative single-cell transcriptomics and quantitative immunofluorescence on control and OCT4 null blastocyst inner cell masses at two developmental stages. Surprisingly, activation of most pluripotency-associated transcription factors in the early mouse embryo occurs independently of OCT4, with the exception of the JAK/STAT signaling machinery. Concurrently, OCT4 null inner cell masses ectopically activate a subset of trophectoderm-associated genes. Inspection of metabolic pathways implicates the regulation of rate-limiting glycolytic enzymes by OCT4, consistent with a role in sustaining glycolysis. Furthermore, up-regulation of the lysosomal pathway was specifically detected in OCT4 null embryos. This finding implicates a requirement for OCT4 in the production of normal trophectoderm. Collectively, our findings uncover regulation of cellular metabolism and biophysical properties as mechanisms by which OCT4 instructs pluripotency.

Motor-Augmented Wrist-Driven Orthosis: Flexible Grasp Assistance for People with Spinal Cord Injury.

This paper presents the design of a motor-augmented wrist-driven orthosis (MWDO) for improved grasp articulation for people with C6-C7 spinal cord injuries. Based on the traditional passive, wrist-driven orthotic (WDO) mechanism, the MWDO allows for both body-powered and motorized actuation of the grasping output thus enabling more flexible and dexterous operation. Here, the associated control scheme enables active decoupling of wrist and finger articulation, which can be useful during certain phases of manipulation tasks. An additional modification to the traditional WDO is the integration of a magnetic latch at the Distal Interphalangeal (DIP) joint allowing for improved pinching. These abilities are demonstrated with common activities of daily living (ADL).