Rapid and robust directed differentiation of mouse epiblast stem cells into definitive endoderm and forebrain organoids.

Directed differentiation of pluripotent stem cells (PSCs) is a powerful model system for deconstructing embryonic development. Although mice are the most advanced mammalian model system for genetic studies of embryonic development, state-of-the-art protocols for directed differentiation of mouse PSCs into defined lineages require additional steps and generates target cell types with lower purity than analogous protocols for human PSCs, limiting their application as models for mechanistic studies of development. Here, we examine the potential of mouse epiblast stem cells cultured in media containing Wnt pathway inhibitors as a starting point for directed differentiation. As a proof of concept, we focused our efforts on two specific cell/tissue types that have proven difficult to generate efficiently and reproducibly from mouse embryonic stem cells: definitive endoderm and neural organoids. We present new protocols for rapid generation of nearly pure definitive endoderm and forebrain-patterned neural organoids that model the development of prethalamic and hippocampal neurons. These differentiation models present new possibilities for combining mouse genetic tools with in vitro differentiation to characterize molecular and cellular mechanisms of embryonic development.

Surgical Management of Fibroadipose Vascular Anomaly of the Lower Extremities.

BACKGROUND: Fibroadipose vascular anomaly (FAVA) is a recently-defined vascular malformation often involving the extremities and presenting in childhood. Patients may present to orthopaedic surgeons with pain, swelling, joint contractures, and leg length discrepancy. There is no established therapy or treatment paradigm. We report on outcomes following surgical excision for patients with this condition. METHODS: Between 2007 and 2016, all 35 patients that underwent excision of lower-extremity FAVA were retrospectively reviewed using a combination of medical records, radiologic findings, and telemedicine reviews. RESULTS: Mean age at initial presentation was 12.3±6.8 years. Mean follow-up from time of definitive diagnosis at our institution was 66 months (range: 12 to 161 mo). Mean follow-up after surgery was 35 months (range: 6 to 138 mo). Females were affected more than males (71% vs. 29%). The most common location of FAVA was in the calf (49%), followed by the thigh (40%). The most commonly involved muscle was gastrocnemius (29%), followed by the quadriceps (26%). At latest follow-up after surgery, there was an improvement in the proportion of patients with pain at rest (63% vs. 29%), pain with activity (100% vs. 60%), as well as analgesia use (94% vs. 37%). Fourteen patients (40%) had symptomatic residual disease or recurrence of FAVA requiring further treatment. Six patients (17%) required further surgery and 6 (17%) required further interventional radiologic procedures. Three patients (9%) required eventual amputation for intractable pain and loss of function. Lesions with direct nerve involvement were associated with persistent neuropathic symptoms at latest follow-up (P=0.002) as well as symptomatic residual disease and/or recurrence requiring further treatment (P=0.01). Seventeen patients (49%) had 19 preoperative joint contractures. Eighteen of the 19 contractures (95%) had sustained improvement at latest follow-up. CONCLUSIONS: In carefully selected patients, surgical excision of FAVA results in improvement of symptoms. However, symptomatic residual disease and/or recurrence are not uncommon. Direct nerve involvement is associated with a worse outcome. LEVEL OF EVIDENCE: Level IV-case series.

Mesenchymal stromal cell-derived exosomes improve mitochondrial health in pulmonary arterial hypertension.

Secreted exosomes are bioactive particles that elicit profound responses in target cells. Using targeted metabolomics and global microarray analysis, we identified a role of exosomes in promoting mitochondrial function in the context of pulmonary arterial hypertension (PAH). Whereas chronic hypoxia results in a glycolytic shift in pulmonary artery smooth muscle cells (PASMCs), exosomes restore energy balance and improve O2 consumption. These results were confirmed in a hypoxia-induced mouse model and a semaxanib/hypoxia rat model of PAH wherein exosomes improved the mitochondrial dysfunction associated with disease. Importantly, exosome exposure increased PASMC expression of pyruvate dehydrogenase (PDH) and glutamate dehydrogenase 1 (GLUD1), linking exosome treatment to the TCA cycle. Furthermore, we show that although prolonged hypoxia induced sirtuin 4 expression, an upstream inhibitor of both GLUD1 and PDH, exosomes reduced its expression. These data provide direct evidence of an exosome-mediated improvement in mitochondrial function and contribute new insights into the therapeutic potential of exosomes in PAH.

Multidimensional spectroscopy with a single broadband phase-shaped laser pulse.

We calculate the frequency-dispersed nonlinear transmission signal of a phase-shaped visible pulse to fourth order in the field. Two phase profiles, a phase-step and phase-pulse, are considered. Two dimensional signals obtained by varying the detected frequency and phase parameters are presented for a three electronic band model system. We demonstrate how two-photon and stimulated Raman resonances can be manipulated by the phase profile and sign, and selected quantum pathways can be suppressed.

A PLURIPOTENT STEM CELL PLATFORM FOR IN VITRO SYSTEMS GENETICS STUDIES OF MOUSE DEVELOPMENT.

The directed differentiation of pluripotent stem cells (PSCs) from panels of genetically diverse individuals is emerging as a powerful experimental system for characterizing the impact of natural genetic variation on developing cell types and tissues. Here, we establish new PSC lines and experimental approaches for modeling embryonic development in a genetically diverse, outbred mouse stock (Diversity Outbred mice). We show that a range of inbred and outbred PSC lines can be stably maintained in the primed pluripotent state (epiblast stem cells -- EpiSCs) and establish the contribution of genetic variation to phenotypic differences in gene regulation and directed differentiation. Using pooled in vitro fertilization, we generate and characterize a genetic reference panel of Diversity Outbred PSCs (n = 230). Finally, we demonstrate the feasibility of pooled culture of Diversity Outbred EpiSCs as "cell villages", which can facilitate the differentiation of large numbers of EpiSC lines for forward genetic screens. These data can complement and inform similar efforts within the stem cell biology and human genetics communities to model the impact of natural genetic variation on phenotypic variation and disease-risk.

Training Robust T1-Weighted Magnetic Resonance Imaging Liver Segmentation Models Using Ensembles of Datasets with Different Contrast Protocols and Liver Disease Etiologies.

Image segmentation of the liver is an important step in several treatments for liver cancer. However, manual segmentation at a large scale is not practical, leading to increasing reliance on deep learning models to automatically segment the liver. This manuscript develops a deep learning model to segment the liver on T1w MR images. We sought to determine the best architecture by training, validating, and testing three different deep learning architectures using a total of 819 T1w MR images gathered from six different datasets, both publicly and internally available. Our experiments compared each architecture's testing performance when trained on data from the same dataset via 5-fold cross validation to its testing performance when trained on all other datasets. Models trained using nnUNet achieved mean Dice-Sorensen similarity coefficients > 90% when tested on each of the six datasets individually. The performance of these models suggests that an nnUNet liver segmentation model trained on a large and diverse collection of T1w MR images would be robust to potential changes in contrast protocol and disease etiology.

Dynamic network-guided CRISPRi screen identifies CTCF-loop-constrained nonlinear enhancer gene regulatory activity during cell state transitions.

Comprehensive enhancer discovery is challenging because most enhancers, especially those contributing to complex diseases, have weak effects on gene expression. Our gene regulatory network modeling identified that nonlinear enhancer gene regulation during cell state transitions can be leveraged to improve the sensitivity of enhancer discovery. Using human embryonic stem cell definitive endoderm differentiation as a dynamic transition system, we conducted a mid-transition CRISPRi-based enhancer screen. We discovered a comprehensive set of enhancers for each of the core endoderm-specifying transcription factors. Many enhancers had strong effects mid-transition but weak effects post-transition, consistent with the nonlinear temporal responses to enhancer perturbation predicted by the modeling. Integrating three-dimensional genomic information, we were able to develop a CTCF-loop-constrained Interaction Activity model that can better predict functional enhancers compared to models that rely on Hi-C-based enhancer-promoter contact frequency. Our study provides generalizable strategies for sensitive and systematic enhancer discovery in both normal and pathological cell state transitions.

Dynamic network-guided CRISPRi screen reveals CTCF loop-constrained nonlinear enhancer-gene regulatory activity in cell state transitions.

Comprehensive enhancer discovery is challenging because most enhancers, especially those affected in complex diseases, have weak effects on gene expression. Our network modeling revealed that nonlinear enhancer-gene regulation during cell state transitions can be leveraged to improve the sensitivity of enhancer discovery. Utilizing hESC definitive endoderm differentiation as a dynamic transition system, we conducted a mid-transition CRISPRi-based enhancer screen. The screen discovered a comprehensive set of enhancers (4 to 9 per locus) for each of the core endoderm lineage-specifying transcription factors, and many enhancers had strong effects mid-transition but weak effects post-transition. Through integrating enhancer activity measurements and three-dimensional enhancer-promoter interaction information, we were able to develop a CTCF loop-constrained Interaction Activity (CIA) model that can better predict functional enhancers compared to models that rely on Hi-C-based enhancer-promoter contact frequency. Our study provides generalizable strategies for sensitive and more comprehensive enhancer discovery in both normal and pathological cell state transitions.

Topical Review: Bone Marrow Aspirate Concentrate and Its Clinical Use in Foot and Ankle Surgery.

Bone marrow aspirate concentrate (BMAC) is now commonly used in orthopedic surgery. Animal studies showed promising results for cartilage, bone, and soft tissue healing; however, many of these outcomes have yet to be translated to human models. While there has been an increase in the use of BMAC in foot and ankle procedures, the associated clinical evidence is limited. The purpose of this review is to analyze the existing literature in order to evaluate the safety and efficacy of BMAC in foot and ankle surgery.

Minimally Invasive Dorsal Cheilectomy and Hallux Metatarsal Phalangeal Joint Arthroscopy for the Treatment of Hallux Rigidus.

BACKGROUND: Hallux rigidus (HR) is a common source of forefoot pain and disability. For those who fail nonoperative treatment, minimally invasive dorsal cheilectomy (MIDC) is an increasingly popular alternative to the open approach with early positive results. Early failures may be due to lose bone debris from the MIDC as well as other intra-articular pathology that cannot be addressed with MIDC alone. Metatarsophalangeal (MTP) arthroscopy can be used in addition to MIDC to assess the joint after MIDC and address any intra-articular pathology while still maintaining the benefits of minimally invasive surgery. We report our clinical outcomes following MIDC combined with MTP arthroscopy. METHODS: From November 2017 to July 2020, a retrospective analysis of all MIDC cheilectomies with MTP arthroscopy performed by the 2 senior authors was done. Wound complications, infections, revision rates, need for future surgery, conversion to fusion rates, pre- and postoperative range of motion, visual analog scale (VAS) scores, time to return to normal shoe, intraoperative arthroscopic findings, and operative time were collected. Follow-up average was 16.5 months (range 3-33 months). RESULTS: A total of 20 patients were included with an average follow-up of 16.5 months. The average VAS score improved from 7.05 preoperatively to 0.75 postoperatively (P < .05). Average range of motion in dorsiflexion increased from 32 to 48 degrees (P < .05) and plantarflexion increased from 15 to 19 degrees plantarflexion (P < .05). All patients were weightbearing as tolerated immediately after surgery in a postoperative shoe and transitioned to a regular shoe at average of 2.1 weeks. We had no wound infections, wound complications, revision surgeries, tendon injuries or nerve damage. One patient required conversion to a fusion 3 years after the index procedure. Average tourniquet time was 30.39 minutes (range 17-60 minutes) and total average operating room time was 59.7 minutes (range 40-87 minutes). On arthroscopic evaluation of the MTP joint after MIDC, 100% of patients had bone debris, 100% had synovitis, 10% had loose bodies, and 30% had large cartilage flaps within the joint. CONCLUSION: MIDC and first MTP joint arthroscopy for treatment of hallux rigidus provide improved pain relief with minimal complications while still maintaining the benefits touted for minimally invasive operative procedures. Additionally, we have shown a high rate of intra-articular debris along with intra-articular pathology such as synovitis, loose chondral flaps, and loose bodies that exist after MIDC. This combined procedure has the potential for improving patient outcomes and may minimize risk of future revision surgeries compared with MIDC alone. LEVEL OF EVIDENCE: Level IV, case series study.

Correlation of the Single-Assessment Numeric Evaluation (SANE) Score With Hip-Specific Patient-Reported Outcome Measures.

PURPOSE: The purpose of this study was to determine if the Single-Assessment Numeric Evaluation (SANE) score correlates with existing validated hip-specific patient-reported outcome measures (PROMs), including the Modified Harris Hip Score (mHHS), the International Hip Outcome Tool (IHOT-33), the Hip Outcome Score, Activities of Daily Living subscale (HOS-ADL), and the Hip Outcome Score, Sport-Specific subscale (HOS-SS), for patients preparing to undergo hip arthroscopy for treatment of femoroacetabular impingement syndrome (FAIS). METHODS: A single surgeon's operative database was retrospectively reviewed to identify patients undergoing primary hip arthroscopy for treatment of FAIS from April 2018 to October 2019. Patient-specific factors including age, sex, body mass index (BMI), and duration of symptoms were collected. Preoperative SANE, mHHS, IHOT-33, HOS-ADL, and HOS-SS scores were analyzed. Statistical analysis using Pearson correlation was performed to identify the relationship between the SANE score and the mHHS, IHOT-33, HOS-ADL, and HOS-SS, preoperatively. RESULTS: 154 patients were included in the study. The mean mHHS was 54.4 ± 11.7; mean IHOT-33 score was 32.7 ± 15.0; mean HOS-SS 42.9 ± 23.7; and mean HOS-ADL was 63.3 ± 1. The mean SANE score was 36.7 ± 19.9. The Simple Hip Score was directly correlated with the mHHS (P < .01), the IHOT-33 (P < .01); the HOS-ADL (P < .01), and the HOS-SS (P < .01). The mean patient age was 35.9 years; 109 (70.8%) were female and 45 (29.2%) were male. Average patient BMI was 26.9. At the time of patient completion of the questionnaire, the majority of patients (65%) had been having symptoms for >1 year. CONCLUSION: The SANE score was strongly correlated with mHHS, IHOT-33, HOS-ADL, and HOS-SS in the preoperative setting for patients undergoing hip arthroscopy for treatment of FAIS. Given its simplicity, SANE may be a valuable tool for rapid assessment of joint function and pain in this patient population. LEVEL OF EVIDENCE: IV, therapeutic case series.

A bipartite element with allele-specific functions safeguards DNA methylation imprints at the Dlk1-Dio3 locus.

Loss of imprinting (LOI) results in severe developmental defects, but the mechanisms preventing LOI remain incompletely understood. Here, we dissect the functional components of the imprinting control region of the essential Dlk1-Dio3 locus (called IG-DMR) in pluripotent stem cells. We demonstrate that the IG-DMR consists of two antagonistic elements: a paternally methylated CpG island that prevents recruitment of TET dioxygenases and a maternally unmethylated non-canonical enhancer that ensures expression of the Gtl2 lncRNA by counteracting de novo DNA methyltransferases. Genetic or epigenetic editing of these elements leads to distinct LOI phenotypes with characteristic alternations of allele-specific gene expression, DNA methylation, and 3D chromatin topology. Although repression of the Gtl2 promoter results in dysregulated imprinting, the stability of LOI phenotypes depends on the IG-DMR, suggesting a functional hierarchy. These findings establish the IG-DMR as a bipartite control element that maintains imprinting by allele-specific restriction of the DNA (de)methylation machinery.

Factors associated with high pain catastrophizing in patients undergoing hip arthroscopy for femoroacetabular impingement syndrome.

The purpose of this study was to determine if physical, mental health and patient-specific factors are associated with increased Pain Catastrophizing in patients undergoing hip arthroscopy for femoroacetabular impingement syndrome (FAIS). Patients who underwent primary hip arthroscopy for FAIS were retrospectively analyzed. Patients were included if they completed a standard pre-operative questionnaire which included the Pain Catastrophizing Scale (PCS), VAS and 12-Item Short Form Survey (SF-12) Physical and Mental Composite Scores. Patient-specific variables including age, gender, BMI, tobacco use, number of allergies, pre-operative opioid use and diagnosis of depression or anxiety were recorded. Multiple linear regression was performed to assess for a relationship between physical and mental health scores, patient-specific variables, and a 'High Catastrophizing' PCS score. One-hundred and sixty-eight patients were included in this study. Patients with a PCS score of 22 or above were categorized as 'High Catastrophizing'. The variables included in the multiple linear regression model statistically significantly predicted high pain catastrophizing, F(10,149) = 4.75, P < 0.001, R 2 = 0.4. SF-12 Physical and Mental Composite Scores and a mental health illness diagnosis added statistically significantly to the prediction, P < 0.005. Pre-operative hip arthroscopy patients with better general physical and mental health, as measured by the SF-12, and those without mental health illness are less likely to having higher pain catastrophizing scores. Age, gender, BMI, visual analog pain scale (VAS), tobacco use, number of allergies and pre-operative opioid use were not independently associated with elevated pain catastrophizing scores. These findings may be helpful when interpreting PCS scores and counseling patients prior to arthroscopic hip surgery.

Biased agonists of the chemokine receptor CXCR3 differentially control chemotaxis and inflammation.

The chemokine receptor CXCR3 plays a central role in inflammation by mediating effector/memory T cell migration in various diseases; however, drugs targeting CXCR3 and other chemokine receptors are largely ineffective in treating inflammation. Chemokines, the endogenous peptide ligands of chemokine receptors, can exhibit so-called biased agonism by selectively activating either G protein- or ß-arrestin-mediated signaling after receptor binding. Biased agonists might be used as more targeted therapeutics to differentially regulate physiological responses, such as immune cell migration. To test whether CXCR3-mediated physiological responses could be segregated by G protein- and ß-arrestin-mediated signaling, we identified and characterized small-molecule biased agonists of the receptor. In a mouse model of T cell-mediated allergic contact hypersensitivity (CHS), topical application of a ß-arrestin-biased, but not a G protein-biased, agonist potentiated inflammation. T cell recruitment was increased by the ß-arrestin-biased agonist, and biopsies of patients with allergic CHS demonstrated coexpression of CXCR3 and ß-arrestin in T cells. In mouse and human T cells, the ß-arrestin-biased agonist was the most efficient at stimulating chemotaxis. Analysis of phosphorylated proteins in human lymphocytes showed that ß-arrestin-biased signaling activated the kinase Akt, which promoted T cell migration. This study demonstrates that biased agonists of CXCR3 produce distinct physiological effects, suggesting discrete roles for different endogenous CXCR3 ligands and providing evidence that biased signaling can affect the clinical utility of drugs targeting CXCR3 and other chemokine receptors.

A Practical Guide to Approaching Biased Agonism at G Protein Coupled Receptors.

Biased agonism, the ability of a receptor to differentially activate downstream signaling pathways depending on binding of a "biased" agonist compared to a "balanced" agonist, is a well-established paradigm for G protein-coupled receptor (GPCR) signaling. Biased agonists have the promise to act as smarter drugs by specifically targeting pathogenic or therapeutic signaling pathways while avoiding others that could lead to side effects. A number of biased agonists targeting a wide array of GPCRs have been described, primarily based on their signaling in pharmacological assays. However, with the promise of biased agonists as novel therapeutics, comes the peril of not fully characterizing and understanding the activities of these compounds. Indeed, it is likely that some of the compounds that have been described as biased, may not be if quantitative approaches for bias assessment are used. Moreover, cell specific effects can result in "system bias" that cannot be accounted by current approaches for quantifying ligand bias. Other confounding includes kinetic effects which can alter apparent bias and differential propagation of biological signal that results in different levels of amplification of reporters downstream of the same effector. Moreover, the effects of biased agonists frequently cannot be predicted from their pharmacological profiles, and must be tested in the vivo physiological context. Thus, the development of biased agonists as drugs requires a detailed pharmacological characterization, involving both qualitative and quantitative approaches, and a detailed physiological characterization. With this understanding, we stand on the edge of a new era of smarter drugs that target GPCRs.

Single Broadband Phase-Shaped Pulse Stimulated Raman Spectroscopy for Standoff Trace Explosive Detection.

Recent success with trace explosives detection based on the single ultrafast pulse excitation for remote stimulated Raman scattering (SUPER-SRS) prompts us to provide new results and a Perspective that describes the theoretical foundation of the strategy used for achieving the desired sensitivity and selectivity. SUPER-SRS provides fast and selective imaging while being blind to optical properties of the substrate such as color, texture, or laser speckle. We describe the strategy of combining coherent vibrational excitation with a reference pulse in order to detect stimulated Raman gain or loss. A theoretical model is used to reproduce experimental spectra and to determine the ideal pulse parameters for best sensitivity, selectivity, and resolution when detecting one or more compounds simultaneously.

Multiphoton excited hemoglobin fluorescence and third harmonic generation for non-invasive microscopy of stored blood.

Red blood cells (RBC) in two-photon excited fluorescence (TPEF) microscopy usually appear as dark disks because of their low fluorescent signal. Here we use 15fs 800nm pulses for TPEF, 45fs 1060nm pulses for three-photon excited fluorescence, and third harmonic generation (THG) imaging. We find sufficient fluorescent signal that we attribute to hemoglobin fluorescence after comparing time and wavelength resolved spectra of other expected RBC endogenous fluorophores: NADH, FAD, biliverdin, and bilirubin. We find that both TPEF and THG microscopy can be used to examine erythrocyte morphology non-invasively without breaching a blood storage bag.

Femtosecond Nanoplasmonic Dephasing of Individual Silver Nanoparticles and Small Clusters.

We present experimental measurements of localized surface plasmon emission from individual silver nanoparticles and small clusters via accurately delayed femtosecond laser pulses. Fourier transform analysis of the nanoplasmonic coherence oscillations reveals different frequency components and dephasing rates for each nanoparticle. We find three different types of behavior: single exponential decay, beating between two frequencies, and beating among three or more frequencies. Our results provide insight into inhomogeneous and homogeneous broadening mechanisms in nanoplasmonic spectroscopy that depend on morphology and nearby neighbors. In addition, we find the optical response of certain pairs of nanoparticles to be at least an order of magnitude more intense than the response of single particles.