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.

The impact of living with transfeminine vocal gender dysphoria: Health utility outcomes assessment.

Background: The voice signals a tremendous amount of gender cues. Transfeminine individuals report debilitating quality-of-life deficits as a result of their vocal gender dysphoria.Aims: We aimed to quantify the potential impact of this dysphoria experienced with quality-adjusted life years (QALYs), as well as associated treatments, through validated health utility measures. Methods: Peri-operative phonometric audio recordings of a consented transfeminine patient volunteer with a history of vocal gender dysphoria aided in the description of two transfeminine health states, pre- and post-vocal feminization gender dysphoria; monocular and binocular blindness were health state controls. Survey responses from general population adults rate these four health states via visual analogue scale (VAS), standard gamble (SG), and time tradeoff (TTO). Results: Survey respondents totaled 206 with a mean age of 35.8 years. Through VAS measures, these general adult respondents on average perceived a year of life with transfeminine vocal gender dysphoria as approximately three-quarters of a life-year of perfect health. Respondents also on average would have risked a 15%-20% chance of death on SG analysis and would have sacrificed 10 years of their remaining life on TTO measures to cure the condition. The QALY scores for the post-gender affirming treatments for vocal gender dysphoria (+0.09 VAS, p < 0.01) were significantly higher compared to the pretreatment state. There were no differences in the severity of these QALY scores by survey respondent's political affiliation or gender identity. Conclusions: To our knowledge, this study is the first to quantify how the general population perceives the health burden of vocal gender dysphoria experienced by transfeminine patients. Feminization treatments including voice therapy with feminization laryngoplasty appear to significantly increase health utility scores.

Social Perception of External Laryngeal Anatomy Related to Gender Expression in a Web-based Survey.

OBJECTIVE(S): To quantify the effect of laryngeal prominence size on socially perceived attributes relating to gender expression. Chondrolaryngoplasty ("tracheal shave") is a common procedure performed for transgender women to feminize neck appearance. The extent of thyroid cartilage resection needed to convey socially-perceived feminine gender expression without destabilizing the voice is incompletely understood. METHODS: Cross-sectional evaluation of a randomized allocation of images of varying laryngeal prominence to a non-repeated, random sample from November 2021 to December 2021. Photos of laryngeal prominence were isolated against a constant neck baseline with lateral, oblique, and frontal views. The images were embedded into a web-based survey with visual analog scales to capture perceived scaled gender expression (masculinity, femininity) and social traits (e.g., attractiveness, friendliness, leadership). We performed bivariate and multivariate analyses relating the laryngeal prominence to perceived gender expression and social traits. RESULTS: The analytic sample included 1,026 respondents. Laryngeal grades similar to the demonstrated "grade M" in this study and smaller demonstrated similar perceptions of increased femininity and decreased masculinity. Grades larger than M demonstrate significantly increased perceived masculinity and significantly decreased perceived femininity. The lateral and oblique views of the neck appear to be the most gender-informative. CONCLUSION: This crowd-sourced analysis of external laryngeal anatomy by a large population of observers provides clear, reproducible insights into social perceptions of gender identity and specifically femininity. These data will meaningfully inform patient counseling and surgical planning for gender-affirming interventions by establishing normative data representing the general public's perceptions. LEVEL OF EVIDENCE: NA Laryngoscope, 133:2292-2300, 2023.

Integrated genome and tissue engineering enables screening of cancer vulnerabilities in physiologically relevant perfusable ex vivo cultures.

Genetic screens are powerful tools for both resolving biological function and identifying potential therapeutic targets, but require physiologically accurate systems to glean biologically useful information. Here, we enable genetic screens in physiologically relevant ex vivo cancer tissue models by integrating CRISPR-Cas-based genome engineering and biofabrication technologies. We first present a novel method for generating perfusable tissue constructs, and validate its functionality by using it to generate three-dimensional perfusable dense cultures of cancer cell lines and sustain otherwise ex vivo unculturable patient-derived xenografts. Using this system we enable large-scale CRISPR screens in perfused tissue cultures, as well as emulate a novel point-of-care diagnostics scenario of a clinically actionable CRISPR knockout (CRISPRko) screen of genes with FDA-approved drug treatments in ex vivo PDX cell cultures. Our results reveal differences across in vitro and in vivo cancer model systems, and highlight the utility of programmable tissue engineered models for screening therapeutically relevant cancer vulnerabilities.

The Health Burden of Transfeminine Facial Gender Dysphoria: An Analysis of Public Perception.

Objectives/Hypothesis: The face is a major communicator of gender identity. Transfeminine individuals report debilitating quality-of-life deficits as a result of their gender dysphoria, which may be addressed with feminizing therapies. We aimed to quantify the potential impact of facial gender dysphoria experienced by transfeminine patients, as well as associated treatments, including feminizing facial gender surgery, through validated health state utility measures. Methods: A transfeminine patient volunteer who underwent head and neck gender-affirming treatments was consented for research use of perioperative photographs. These media aided in the description of two transfeminine health states, pre- and postfacial feminization facial gender dysphoria. Monocular blindness and binocular blindness were two health state controls. General population adults rated these four health states through visual analog scale (VAS), standard gamble (SG), and time trade-off (TTO), which were used to calculate the quality-adjusted life years (QALYs). Results: Survey respondents totaled 206 with a mean (standard deviation [SD]) age of 35.8 (11.9) years. Mean (SD) health utility measures included 0.75 (0.22) QALYs for VAS, 0.82 (0.19) QALYS for SG, 0.79 (0.21) QALYS for TTO for pretreatment facial gender dysphoria, and 0.81 (0.21), 0.86 (0.19), 0.83 (0.20) QALYS for postgender-affirming treatments for facial gender dysphoria. The health utility scores for the postgender-affirming treatments for facial gender dysphoria (+0.06 VAS, p = 0.005) were significantly improved compared with the pretreatment state. Conclusions: To our knowledge, this study is the first to examine how the general population perceives the health burden of facial gender dysphoria experienced by transfeminine patients. Facial gender dysphoria is perceived to have a negative impact on health states, not dissimilar to monocular blindness in our sample. Feminizing facial gender surgery appears to significantly increase health utility measures.

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.

Functional Genomics via CRISPR-Cas.

RNA-guided CRISPR (clustered regularly interspaced short palindromic repeat)-associated Cas proteins have recently emerged as versatile tools to investigate and engineer the genome. The programmability of CRISPR-Cas has proven especially useful for probing genomic function in high-throughput. Facile single-guide RNA library synthesis allows CRISPR-Cas screening to rapidly investigate the functional consequences of genomic, transcriptomic, and epigenomic perturbations. Furthermore, by combining CRISPR-Cas perturbations with downstream single-cell analyses (flow cytometry, expression profiling, etc.), forward screens can generate robust data sets linking genotypes to complex cellular phenotypes. In the following review, we highlight recent advances in CRISPR-Cas genomic screening while outlining protocols and pitfalls associated with screen implementation. Finally, we describe current challenges limiting the utility of CRISPR-Cas screening as well as future research needed to resolve these impediments. As CRISPR-Cas technologies develop, so too will their clinical applications. Looking ahead, patient centric functional screening in primary cells will likely play a greater role in disease management and therapeutic development.

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.