Bisphenol A replacement chemicals, BPF and BPS, induce protumorigenic changes in human mammary gland organoid morphology and proteome.

SignificanceBisphenol A (BPA), found in many plastic products, has weak estrogenic effects that can be harmful to human health. Thus, structurally related replacements-bisphenol S (BPS) and bisphenol F (BPF)-are coming into wider use with very few data about their biological activities. Here, we compared the effects of BPA, BPS, and BPF on human mammary organoids established from normal breast tissue. BPS disrupted organoid architecture and induced supernumerary branching. At a proteomic level, the bisphenols altered the abundance of common targets and those that were unique to each compound. The latter included proteins linked to tumor-promoting processes. These data highlighted the importance of testing the human health effects of replacements that are structurally related to chemicals of concern.

Gender Is a Spectrum: Evaluating Current and Novel Ways to Inquire About Gender Identity in the Health Care Setting.

PURPOSE: The use of visuals to inquire about gender in the clinical setting has been rare. We developed a survey that included a visual spectrum to assess perceptions about the most and least inclusive ways of inquiring about gender in patients with gender dysphoria. METHODS: The survey included a multiple-choice question (MCQ), free-response question, and a visual spectrum on which respondents were asked to select one box that best depicts their gender. The survey was administered to all patients diagnosed with gender dysphoria at our institution between April and June 2022. RESULTS: A total of 223 of 856 patients responded. Those with more masculine gender identities selected boxes near the visual spectrum corner of "man," whereas responses were more variable for more feminine genders. The free-response question was identified by 59% of respondents as the most inclusive. The MCQ was identified as least inclusive by 70.4%. The visual spectrum was considered the most inclusive method by the majority of patients who self-identified as woman and demiwoman/demifemale. Being asked about pronouns was extremely or very important in the health care setting for 52% of respondents, but 68.6% indicated that they are rarely or sometimes asked about their pronouns in this setting. CONCLUSIONS: The traditional MCQ format for self-identifying gender may be lacking in inclusivity and fails to represent the nuances of gender identity. Free response was considered the most inclusive way to inquire about gender among our respondents. These findings highlight the importance of formatting gender identity questionnaires to foster inclusivity for transgender patients.

Microfluidics-free single-cell genomics with templated emulsification.

Current single-cell RNA-sequencing approaches have limitations that stem from the microfluidic devices or fluid handling steps required for sample processing. We develop a method that does not require specialized microfluidic devices, expertise or hardware. Our approach is based on particle-templated emulsification, which allows single-cell encapsulation and barcoding of cDNA in uniform droplet emulsions with only a vortexer. Particle-templated instant partition sequencing (PIP-seq) accommodates a wide range of emulsification formats, including microwell plates and large-volume conical tubes, enabling thousands of samples or millions of cells to be processed in minutes. We demonstrate that PIP-seq produces high-purity transcriptomes in mouse-human mixing studies, is compatible with multiomics measurements and can accurately characterize cell types in human breast tissue compared to a commercial microfluidic platform. Single-cell transcriptional profiling of mixed phenotype acute leukemia using PIP-seq reveals the emergence of heterogeneity within chemotherapy-resistant cell subsets that were hidden by standard immunophenotyping. PIP-seq is a simple, flexible and scalable next-generation workflow that extends single-cell sequencing to new applications.

Evaluating the Quality and Reliability of Gender-affirming Surgery Videos on YouTube and TikTok.

Background: Social media platforms have changed the way medical information is disseminated. Transgender patients may utilize social media to learn about gender-affirming surgery (GAS). Although videos on social media are readily accessible, their content is not verified or peer-reviewed. Therefore, this study aimed to evaluate the quality and reliability of YouTube and TikTok videos related to GAS. Methods: YouTube and TikTok were queried for gender-affirming top surgery, metoidioplasty, phalloplasty, breast augmentation, and vaginoplasty. Quality of video content was analyzed by the DISCERN scale. Quality scores were compared among the type of GAS, account user, and content category. Results: There were 275 YouTube videos and 55 TikTok videos. Most videos focused on masculinizing top surgery (P < 0.001). Overall, videos on masculinizing GAS had higher quality and reliability than videos on feminizing GAS (P < 0.001). Chest surgery videos were of higher quality than those on genital surgery (P ≤ 0.001). Videos on masculinizing top surgery had the highest quality, whereas vaginoplasty had the lowest quality and reliability (P < 0.001). Videos produced by health care professionals and academic institutions had the greatest quality and reliability, respectively (P < 0.0001), whereas videos produced by patients were the least reliable (P < 0.0001). Conclusions: Videos on GAS ranged from poor to good quality and reliability. Health care professionals, especially plastic surgeons, should create high-quality videos on social media to educate transgender patients. There should also be greater efforts in disseminating existing high-quality videos on social media. Resources posted on social media platforms can reach a wide audience through accessible means.

Mapping hormone-regulated cell-cell interaction networks in the human breast at single-cell resolution.

The rise and fall of estrogen and progesterone across menstrual cycles and during pregnancy regulates breast development and modifies cancer risk. How these hormones impact each cell type in the breast remains poorly understood because they act indirectly through paracrine networks. Using single-cell analysis of premenopausal breast tissue, we reveal a network of coordinated transcriptional programs representing the tissue-level response to changing hormone levels. Our computational approach, DECIPHER-seq, leverages person-to-person variability in breast composition and cell state to uncover programs that co-vary across individuals. We use differences in cell-type proportions to infer a subset of programs that arise from direct cell-cell interactions regulated by hormones. Further, we demonstrate that prior pregnancy and obesity modify hormone responsiveness through distinct mechanisms: obesity reduces the proportion of hormone-responsive cells, whereas pregnancy dampens the direct response of these cells to hormones. Together, these results provide a comprehensive map of the cycling human breast.

Organoid models for mammary gland dynamics and breast cancer.

The mammary gland is a highly dynamic tissue that undergoes repeated cycles of growth and involution during pregnancy and menstruation. It is also the site from which breast cancers emerge. Organoids provide an in vitro model that preserves several of the cellular, structural, and microenvironmental features that dictate mammary gland function in vivo and have greatly advanced our understanding of glandular biology. Their tractability for genetic manipulation, live imaging, and high throughput screening have facilitated investigation into the mechanisms of glandular morphogenesis, structural maintenance, tumor progression, and invasion. Opportunities remain to enhance cellular and structural complexity of mammary organoid models, including incorporating additional cell types and hormone signaling.

Biophysical and biomolecular interactions of malaria-infected erythrocytes in engineered human capillaries.

Microcirculatory obstruction is a hallmark of severe malaria, but mechanisms of parasite sequestration are only partially understood. Here, we developed a robust three-dimensional microvessel model that mimics the arteriole-capillary-venule (ACV) transition consisting of a narrow 5- to 10-µm-diameter capillary region flanked by arteriole- or venule-sized vessels. Using this platform, we investigated red blood cell (RBC) transit at the single cell and at physiological hematocrits. We showed normal RBCs deformed via in vivo-like stretching and tumbling with negligible interactions with the vessel wall. By comparison, Plasmodium falciparum-infected RBCs exhibited virtually no deformation and rapidly accumulated in the capillary-sized region. Comparison of wild-type parasites to those lacking either cytoadhesion ligands or membrane-stiffening knobs showed highly distinctive spatial and temporal kinetics of accumulation, linked to velocity transition in ACVs. Our findings shed light on mechanisms of microcirculatory obstruction in malaria and establish a new platform to study hematologic and microvascular diseases.

Human intestinal spheroids cultured using Sacrificial Micromolding as a model system for studying drug transport.

In vitro models of the small intestine are crucial tools for the prediction of drug absorption. The Caco-2 monolayer transwell model has been widely employed to assess drug absorption across the intestine. However, it is now well-established that 3D in vitro models capture tissue-specific architecture and interactions with the extracellular matrix and therefore better recapitulate the complex in vivo environment. However, these models need to be characterized for barrier properties and changes in gene expression and transporter function. Here, we report that geometrically controlled self-assembling multicellular intestinal Caco-2 spheroids cultured using Sacrificial Micromolding display reproducible intestinal features and functions that are more representative of the in vivo small intestine than the widely used 2D transwell model. We show that Caco-2 cell maturation and differentiation into the intestinal epithelial phenotype occur faster in spheroids and that they are viable for a longer period of time. Finally, we were able to invert the polarity of the spheroids by culturing them around Matrigel beads allowing superficial access to the apical membrane and making the model more physiological. This robust and reproducible in vitro intestinal model could serve as a valuable system to expedite drug screening as well as to study intestinal transporter function.

Reconstructing the Human Renal Vascular-Tubular Unit In Vitro.

Engineered human kidney-on-a-chip platforms show tremendous promise for disease modeling and drug screening. Outstanding challenges exist, however, in reconstructing the complex architecture, cellular make-up, and matrix composition necessary for the proper modeling of kidney function. Herein, the first fully tunable human kidney-on-a-chip platform is reported that allows the reconstruction of the native architecture of the renal endothelial-epithelial exchange interface using entirely cell-remodelable matrix and patient-derived kidney cells. This platform consists of a double-layer human renal vascular-tubular unit (hRVTU) enabled by a thin collagen membrane that replicates the kidney exchange interface. It is shown that endothelial and epithelial cells lining their respective lumens remodel the membrane in culture into a ≈1 µm thick exchange interface composed of native basement membrane proteins. This interface displays sufficient mechanical integrity for media flow and blood perfusion. As a proof of principle, it is demonstrated that the hRVTU performs kidney-specific functions including reabsorption of albumin and glucose from the epithelial channel. By incorporating multiple cell populations from single donors, it is demonstrated that the hRVTU may have utility for future precision medicine applications. The success of the system provides new opportunities for the next generation of organ-on-a-chip models.

Engineering a multicellular vascular niche to model hematopoietic cell trafficking.

BACKGROUND: The marrow microenvironment and vasculature plays a critical role in regulating hematopoietic cell recruitment, residence, and maturation. Extensive in vitro and in vivo studies have aimed to understand the marrow cell types that contribute to hematopoiesis and the stem cell environment. Nonetheless, in vitro models are limited by a lack of complex multicellular interactions, and cellular interactions are not easily manipulated in vivo. Here, we develop an engineered human vascular marrow niche to examine the three-dimensional cell interactions that direct hematopoietic cell trafficking. METHODS: Using soft lithography and injection molding techniques, fully endothelialized vascular networks were fabricated in type I collagen matrix, and co-cultured under flow with embedded marrow fibroblast cells in the matrix. Marrow fibroblast (mesenchymal stem cells (MSCs), HS27a, or HS5) interactions with the endothelium were imaged via confocal microscopy and altered endothelial gene expression was analyzed with RT-PCR. Monocytes, hematopoietic progenitor cells, and leukemic cells were perfused through the network and their adhesion and migration was evaluated. RESULTS: HS27a cells and MSCs interact directly with the vessel wall more than HS5 cells, which are not seen to make contact with the endothelial cells. In both HS27a and HS5 co-cultures, endothelial expression of junctional markers was reduced. HS27a co-cultures promote perfused monocytes to adhere and migrate within the vessel network. Hematopoietic progenitors rely on monocyte-fibroblast crosstalk to facilitate preferential recruitment within HS27a co-cultured vessels. In contrast, leukemic cells sense fibroblast differences and are recruited preferentially to HS5 and HS27a co-cultures, but monocytes are able to block this sensitivity. CONCLUSIONS: We demonstrate the use of a microvascular platform that incorporates a tunable, multicellular composition to examine differences in hematopoietic cell trafficking. Differential recruitment of hematopoietic cell types to distinct fibroblast microenvironments highlights the complexity of cell-cell interactions within the marrow. This system allows for step-wise incorporation of cellular components to reveal the dynamic spatial and temporal interactions between endothelial cells, marrow-derived fibroblasts, and hematopoietic cells that comprise the marrow vascular niche. Furthermore, this platform has potential for use in testing therapeutics and personalized medicine in both normal and disease contexts.

Microvasculature-directed thrombopoiesis in a 3D in vitro marrow microenvironment.

Vasculature is an interface between the circulation and the hematopoietic tissue providing the means for hundreds of billions of blood cells to enter the circulation every day in a regulated fashion. The precise mechanisms that control the interactions of hematopoietic cells with the vessel wall are largely undefined. Here, we report on the development of an in vitro 3D human marrow vascular microenvironment (VME) to study hematopoietic trafficking and the release of blood cells, specifically platelets. We show that mature megakaryocytes from aspirated marrow as well as megakaryocytes differentiated in culture from CD34+ cells can be embedded in a collagen matrix containing engineered microvessels to create a thrombopoietic VME. These megakaryocytes continue to mature, penetrate the vessel wall, and release platelets into the vessel lumen. This process can be blocked with the addition of antibodies specific for CXCR4, indicating that CXCR4 is required for megakaryocyte migration, though whether it is sufficient is unclear. The 3D marrow VME system shows considerable potential for mechanistic studies defining the role of marrow vasculature in thrombopoiesis. Through a stepwise addition or removal of individual marrow components, this model provides potential to define key pathways responsible for the release of platelets and other blood cells.

Tissue engineering toward organ-specific regeneration and disease modeling.

Tissue engineering has been recognized as a translational approach to replace damaged tissue or whole organs. Engineering tissue, however, faces an outstanding knowledge gap in the challenge to fully recapitulate complex organ-specific features. Major components, such as cells, matrix, and architecture, must each be carefully controlled to engineer tissue-specific structure and function that mimics what is found in vivo. Here we review different methods to engineer tissue, and discuss critical challenges in recapitulating the unique features and functional units in four major organs-the kidney, liver, heart, and lung, which are also the top four candidates for organ transplantation in the USA. We highlight advances in tissue engineering approaches to enable the regeneration of complex tissue and organ substitutes, and provide tissue-specific models for drug testing and disease modeling. We discuss the current challenges and future perspectives toward engineering human tissue models.

Micropatterning and Assembly of 3D Microvessels.

In vitro platforms to study endothelial cells and vascular biology are largely limited to 2D endothelial cell culture, flow chambers with polymer or glass based substrates, and hydrogel-based tube formation assays. These assays, while informative, do not recapitulate lumen geometry, proper extracellular matrix, and multi-cellular proximity, which play key roles in modulating vascular function. This manuscript describes an injection molding method to generate engineered vessels with diameters on the order of 100 µm. Microvessels are fabricated by seeding endothelial cells in a microfluidic channel embedded within a native type I collagen hydrogel. By incorporating parenchymal cells within the collagen matrix prior to channel formation, specific tissue microenvironments can be modeled and studied. Additional modulations of hydrodynamic properties and media composition allow for control of complex vascular function within the desired microenvironment. This platform allows for the study of perivascular cell recruitment, blood-endothelium interactions, flow response, and tissue-microvascular interactions. Engineered microvessels offer the ability to isolate the influence from individual components of a vascular niche and precisely control its chemical, mechanical, and biological properties to study vascular biology in both health and disease.