Spatiotemporal modulation of growth factors directs the generation of multilineage mouse embryonic stem cell-derived mammary organoids.

Ectodermal appendages, such as the mammary gland (MG), are thought to have evolved from hair-associated apocrine glands to serve the function of milk secretion. Through the directed differentiation of mouse embryonic stem cells (mESCs), here, we report the generation of multilineage ESC-derived mammary organoids (MEMOs). We adapted the skin organoid model, inducing the dermal mesenchyme to transform into mammary-specific mesenchyme via the sequential activation of Bone Morphogenetic Protein 4 (BMP4) and Parathyroid Hormone-related Protein (PTHrP) and inhibition of hedgehog (HH) signaling. Using single-cell RNA sequencing, we identified gene expression profiles that demonstrate the presence of mammary-specific epithelial cells, fibroblasts, and adipocytes. MEMOs undergo ductal morphogenesis in Matrigel and can reconstitute the MG in vivo. Further, we demonstrate that the loss of function in placode regulators LEF1 and TBX3 in mESCs results in impaired skin and MEMO generation. In summary, our MEMO model is a robust tool for studying the development of ectodermal appendages, and it provides a foundation for regenerative medicine and disease modeling.

Mapping oto-pharyngeal development in a human inner ear organoid model.

Inner ear development requires the coordination of cell types from distinct epithelial, mesenchymal and neuronal lineages. Although we have learned much from animal models, many details about human inner ear development remain elusive. We recently developed an in vitro model of human inner ear organogenesis using pluripotent stem cells in a 3D culture, fostering the growth of a sensorineural circuit, including hair cells and neurons. Despite previously characterizing some cell types, many remain undefined. This study aimed to chart the in vitro development timeline of the inner ear organoid to understand the mechanisms at play. Using single-cell RNA sequencing at ten stages during the first 36 days of differentiation, we tracked the evolution from pluripotency to various ear cell types after exposure to specific signaling modulators. Our findings showcase gene expression that influences differentiation, identifying a plethora of ectodermal and mesenchymal cell types. We also discern aspects of the organoid model consistent with in vivo development, while highlighting potential discrepancies. Our study establishes the Inner Ear Organoid Developmental Atlas (IODA), offering deeper insights into human biology and improving inner ear tissue differentiation.

Human pluripotent stem cell-derived inner ear organoids recapitulate otic development in vitro.

Our molecular understanding of the early stages of human inner ear development has been limited by the difficulty in accessing fetal samples at early gestational stages. As an alternative, previous studies have shown that inner ear morphogenesis can be partially recapitulated using induced pluripotent stem cells directed to differentiate into inner ear organoids (IEOs). Once validated and benchmarked, these systems could represent unique tools to complement and refine our understanding of human otic differentiation and model developmental defects. Here, we provide the first direct comparisons of the early human embryonic otocyst and fetal sensory organs with human IEOs. We use multiplexed immunostaining and single-cell RNA-sequencing to characterize IEOs at three key developmental steps, providing a new and unique signature of in vitro-derived otic placode, epithelium, neuroblasts and sensory epithelia. In parallel, we evaluate the expression and localization of crucial markers at these equivalent stages in human embryos. Together, our data indicate that the current state-of-the-art protocol enables the specification of bona fide otic tissue, supporting the further application of IEOs to inform inner ear biology and disease.

Applications of Human Pluripotent Stem Cell-Derived Skin Organoids in Dermatology.

Pluripotent stem cells have the potential to become any cell type, and recently, they have been used to create organoids that can recapitulate several pertinent features of human organs. Skin organoids have been developed that possess many of the crucial accessory organs, including hair follicles, sebaceous glands, nerves, fat, and melanocytes. These skin organoids present the opportunity to study skin development and disease as well as perform screens to identify new drug candidates. In the future, skin organoids might augment clinical practice by serving as source material for transplantation to treat wounds or other conditions. Nevertheless, several limitations, such as the lengthy differentiation protocol, which can result in heterogeneous products, must first be addressed before the full potential of skin organoids can be realized. The purpose of this article is to provide a broad overview of skin organoids so that a broader audience can become familiar with this technology, which has important implications for dermatologic research and medicine.

A single-cell level comparison of human inner ear organoids with the human cochlea and vestibular organs.

Inner ear disorders are among the most common congenital abnormalities; however, current tissue culture models lack the cell type diversity to study these disorders and normal otic development. Here, we demonstrate the robustness of human pluripotent stem cell-derived inner ear organoids (IEOs) and evaluate cell type heterogeneity by single-cell transcriptomics. To validate our findings, we construct a single-cell atlas of human fetal and adult inner ear tissue. Our study identifies various cell types in the IEOs including periotic mesenchyme, type I and type II vestibular hair cells, and developing vestibular and cochlear epithelium. Many genes linked to congenital inner ear dysfunction are confirmed to be expressed in these cell types. Additional cell-cell communication analysis within IEOs and fetal tissue highlights the role of endothelial cells on the developing sensory epithelium. These findings provide insights into this organoid model and its potential applications in studying inner ear development and disorders.

Human pluripotent stem cells-derived inner ear organoids recapitulate otic development in vitro.

Our molecular understanding of the early stages of human inner ear development has been limited by the difficulty in accessing fetal samples at early gestational stages. As an alternative, previous studies have shown that inner ear morphogenesis can be partially recapitulated using induced pluripotent stem cells (iPSCs) directed to differentiate into Inner Ear Organoids (IEOs). Once validated and benchmarked, these systems could represent unique tools to complement and refine our understanding of human otic differentiation and model developmental defects. Here, we provide the first direct comparisons of the early human embryonic otocyst and human iPSC-derived IEOs. We use multiplexed immunostaining, and single-cell RNA sequencing to characterize IEOs at three key developmental steps, providing a new and unique signature of in vitro derived otic -placode, -epithelium, -neuroblasts, and -sensory epithelia. In parallel, we evaluate the expression and localization of critical markers at these equivalent stages in human embryos. We show that the placode derived in vitro (days 8-12) has similar marker expression to the developing otic placode of Carnegie Stage (CS) 11 embryos and subsequently (days 20-40) this gives rise to otic epithelia and neuroblasts comparable to the CS13 embryonic stage. Differentiation of sensory epithelia, including supporting cells and hair cells starts in vitro at days 50-60 of culture. The maturity of these cells is equivalent to vestibular sensory epithelia at week 10 or cochlear tissue at week 12 of development, before functional onset. Together, our data indicate that the current state-of-the-art protocol enables the specification of bona fide otic tissue, supporting the further application of IEOs to inform inner ear biology and disease.

Early Wnt Signaling Activation Promotes Inner Ear Differentiation via Cell Caudalization in Mouse Stem Cell-Derived Organoids.

The inner ear is derived from the otic placode, one of the numerous cranial sensory placodes that emerges from the pre-placodal ectoderm (PPE) along its anterior-posterior axis. However, the molecular dynamics underlying how the PPE is regionalized are poorly resolved. We used stem cell-derived organoids to investigate the effects of Wnt signaling on early PPE differentiation and found that modulating Wnt signaling significantly increased inner ear organoid induction efficiency and reproducibility. Alongside single-cell RNA sequencing, our data reveal that the canonical Wnt signaling pathway leads to PPE regionalization and, more specifically, medium Wnt levels during the early stage induce (1) expansion of the caudal neural plate border (NPB), which serves as a precursor for the posterior PPE, and (2) a caudal microenvironment that is required for otic specification. Our data further demonstrate Wnt-mediated induction of rostral and caudal cells in organoids and more broadly suggest that Wnt signaling is critical for anterior-posterior patterning in the PPE.

Biofabrication of Human Skin with Its Appendages.

Much effort has been made to generate human skin organ in the laboratory. Yet, the current models are limited due to the lack of many critical biological and structural features of the skin. Importantly, these in vitro models lack appendages and fail to recapitulate the whole human skin construction. Thus, engineering a human skin with the capacity to generate all components, including appendages, is a major challenge. This review intends to provide an update on the recent efforts underway to regenerate appendage-bearing skin organs based on scaffold-free and scaffold-based bioengineering approaches. Although the mouse skin equivalents containing hair follicles, sebaceous glands, and sweat glands have been established in vitro, there has been limited success in humans. A combination of biofabricated matrices and cell aggregates, such as organoids, can pave the way for generating skin substitutes with human-like biological, structural, and physical features. Accordingly, the formation of human skin organoids and reconstruction of vascularized skin equipped with immune cells prompt calls for more scientific research. The generation of appendage-bearing skin substitutes can be applied in practice for wound healing, hair restoration, and scar treatment.

Advancements in inner ear development, regeneration, and repair through otic organoids.

The vertebrate inner ear contains a diversity of unique cell types arranged in a particularly complex 3D cytoarchitecture. Both of these features are integral to the proper development, function, and maintenance of hearing and balance. Since the elucidation of the timing and delivery of signaling molecules to produce inner ear sensory cells, supporting cells, and neurons from human induced pluripotent stem cells, we have entered a revolution using organ-like 'otic organoid' cultures to explore inner ear specific genetic programs, developmental rules, and potential therapeutics. This review aims to highlight a selection of reviews and primary research papers from the past two years of particular merit that use otic organoids to investigate the broadly defined topics of cell reprogramming, regeneration, and repair.

The mechanical forces that shape our senses.

Developing organs are shaped, in part, by physical interaction with their environment in the embryo. In recent years, technical advances in live-cell imaging and material science have greatly expanded our understanding of the mechanical forces driving organ formation. Here, we provide a broad overview of the types of forces generated during embryonic development and then focus on a subset of organs underlying our senses: the eyes, inner ears, nose and skin. The epithelia in these organs emerge from a common origin: the ectoderm germ layer; yet, they arrive at unique and complex forms over developmental time. We discuss exciting recent animal studies that show a crucial role for mechanical forces in, for example, the thickening of sensory placodes, the coiling of the cochlea and the lengthening of hair. Finally, we discuss how microfabricated organoid systems can now provide unprecedented insights into the physical principles of human development.

Generation and characterization of hair-bearing skin organoids from human pluripotent stem cells.

Human skin uses millions of hairs and glands distributed across the body surface to function as an external barrier, thermoregulator and stimuli sensor. The large-scale generation of human skin with these appendages would be beneficial, but is challenging. Here, we describe a detailed protocol for generating hair-bearing skin tissue entirely from a homogeneous population of human pluripotent stem cells in a three-dimensional in vitro culture system. Defined culture conditions are used over a 2-week period to induce differentiation of pluripotent stem cells to surface ectoderm and cranial neural crest cells, which give rise to the epidermis and dermis, respectively, in each organoid unit. After 60 d of incubation, the skin organoids produce hair follicles. By day ~130, the skin organoids reach full complexity and contain stratified skin layers, pigmented hair follicles, sebaceous glands, Merkel cells and sensory neurons, recapitulating the cell composition and architecture of fetal skin tissue at week 18 of gestation. Skin organoids can be maintained in culture using this protocol for up to 150 d, enabling the organoids to be used to investigate basic skin biology, model disease and, further, reconstruct or regenerate skin tissue.

Aerosol and Droplet Generation from Open Rhinoplasty: Surgical Risk in the Pandemic Era.

Introduction: The coronavirus disease 2019 pandemic has led to concerns over transmission risk from head and neck operations including facial cosmetic surgeries. Objectives: To quantify droplet and aerosol generation from rhinoplasty techniques in a human anatomic specimen model using fluorescein staining and an optical particle sizer. Methods: Noses of human anatomic specimens were infiltrated using 0.1% fluorescein. Droplets and aerosols were measured during rhinoplasty techniques including opening the skin-soft tissue envelope, monopolar electrocautery, endonasal rasping, endonasal osteotomy, and percutaneous osteotomy. Results: No visible droplet contamination was observed for any rhinoplasty techniques investigated. Compared with the negative control of anterior rhinoscopy, total 0.300-10.000 µm aerosols were increased after monopolar electrocautery (p < 0.001) and endonasal rasp (p = 0.003). Opening the skin-soft tissue envelope, endonasal osteotomies, and percutaneous osteotomies did not generate a detectable increase in aerosols (p > 0.15). Discussion and Conclusions: In this investigation, droplets were not observed under ultraviolet light, and aerosol generation was noted only with cautery and endonasal rasping.

Review of Genotype-Phenotype Correlations in Usher Syndrome.

Usher syndrome (USH) encompasses a group of clinically and genetically heterogenous disorders defined by the triad of sensorineural hearing loss (SNHL), vestibular dysfunction, and vision loss. USH is the most common cause of deaf blindness. USH is divided clinically into three subtypes-USH1, USH2, and USH3-based on symptom severity, progression, and age of onset. The underlying genetics of these USH forms are, however, significantly more complex, with over a dozen genes linked to the three primary clinical subtypes and other atypical USH phenotypes. Several of these genes are associated with other deaf-blindness syndromes that share significant clinical overlap with USH, pointing to the limits of a clinically based classification system. The genotype-phenotype relationships among USH forms also may vary significantly based on the location and type of mutation in the gene of interest. Understanding these genotype-phenotype relationships and associated natural disease histories is necessary for the successful development and application of gene-based therapies and precision medicine approaches to USH. Currently, the state of knowledge varies widely depending on the gene of interest. Recent studies utilizing next-generation sequencing technology have expanded the list of known pathogenic mutations in USH genes, identified new genes associated with USH-like phenotypes, and proposed algorithms to predict the phenotypic effects of specific categories of allelic variants. Further work is required to validate USH gene causality, and better define USH genotype-phenotype relationships and disease natural histories-particularly for rare mutations-to lay the groundwork for the future of USH treatment.

Aerosol and droplet generation from orbital repair: Surgical risk in the pandemic era.

INTRODUCTION: The highly contagious COVID-19 has resulted in millions of deaths worldwide. Physicians performing orbital procedures may be at increased risk of occupational exposure to the virus due to exposure to secretions. The goal of this study is to measure the droplet and aerosol production during repair of the inferior orbital rim and trial a smoke-evacuating electrocautery handpiece as a mitigation device. MATERIAL AND METHODS: The inferior rim of 6 cadaveric orbits was approached transconjunctivally using either standard or smoke-evacuator electrocautery and plated using a high-speed drill. Following fluorescein inoculation, droplet generation was measured by counting under ultraviolet-A (UV-A) light against a blue background. Aerosol generation from 0.300-10.000 µm was measured using an optical particle sizer. Droplet and aerosol generation was compared against retraction of the orbital soft tissue as a negative control. RESULTS: No droplets were observed following the orbital approach using electrocautery. Visible droplets were observed after plating with a high-speed drill for 3 of 6 orbits. Total aerosol generation was significantly higher than negative control following the use of standard electrocautery. Use of smoke-evacuator electrocautery was associated with significantly lower aerosol generation in 2 of 3 size groups and in total. There was no significant increase in total aerosols associated with high-speed drilling. DISCUSSION AND CONCLUSIONS: Droplet generation for orbital repair was present only following plating with high-speed drill. Aerosol generation during standard electrocautery was significantly reduced using a smoke-evacuating electrocautery handpiece. Aerosols were not significantly increased by high-speed drilling.

Skin organoids: A new human model for developmental and translational research.

Culturing skin cells outside of the body has been a cornerstone of dermatological investigation for many years; however, human skin equivalent systems typically lack the full complexity of native skin. Notably, skin appendages, such as hair follicles and sweat glands, remain a challenge to generate or maintain in cell cultures and reconstruct in damaged skin. Recent work from our lab has demonstrated methods for generating appendage-bearing skin tissue-known as skin organoids-from pluripotent stem cells. Here, we will summarize this work and other related works, and then discuss the potential future applications of skin organoids in dermatological research.

Building inner ears: recent advances and future challenges for in vitro organoid systems.

While inner ear disorders are common, our ability to intervene and recover their sensory function is limited. In vitro models of the inner ear, like the organoid system, could aid in identifying new regenerative drugs and gene therapies. Here, we provide a perspective on the status of in vitro inner ear models and guidance on how to improve their applicability in translational research. We highlight the generation of inner ear cell types from pluripotent stem cells as a particularly promising focus of research. Several exciting recent studies have shown how the developmental signaling cues of embryonic and fetal development can be mimicked to differentiate stem cells into "inner ear organoids" containing otic progenitor cells, hair cells, and neurons. However, current differentiation protocols and our knowledge of embryonic and fetal inner ear development in general, have a bias toward the sensory epithelia of the inner ear. We propose that a more holistic view is needed to better model the inner ear in vitro. Moving forward, attention should be made to the broader diversity of neuroglial and mesenchymal cell types of the inner ear, and how they interact in space or time during development. With improved control of epithelial, neuroglial, and mesenchymal cell fate specification, inner ear organoids would have the ability to truly recapitulate neurosensory function and dysfunction. We conclude by discussing how single-cell atlases of the developing inner ear and technical innovations will be critical tools to advance inner ear organoid platforms for future pre-clinical applications.

Dynamic Click Hydrogels for Xeno-Free Culture of Induced Pluripotent Stem Cells.

Xeno-free, chemically defined poly(ethylene glycol) (PEG)-based hydrogels are being increasingly used for in vitro culture and differentiation of human induced pluripotent stem cells (hiPSCs). These synthetic matrices provide tunable gelation and adaptable material properties crucial for guiding stem cell fate. Here, sequential norbornene-click chemistries are integrated to form synthetic, dynamically tunable PEG-peptide hydrogels for hiPSCs culture and differentiation. Specifically, hiPSCs are photoencapsulated in thiol-norbornene hydrogels crosslinked by multiarm PEG-norbornene (PEG-NB) and proteaselabile crosslinkers. These matrices are used to evaluate hiPSC growth under the influence of extracellular matrix properties. Tetrazine-norbornene (Tz-NB) click reaction is then employed to dynamically stiffen the cell-laden hydrogels. Fast reactive Tz and its stable derivative methyltetrazine (mTz) are tethered to multiarm PEG, yielding mono-functionalized PEG-Tz, PEG-mTz, and dualfunctionalized PEG-Tz/mTz that react with PEG-NB to form additional crosslinks in the cell-laden hydrogels. The versatility of Tz-NB stiffening is demonstrated with different Tz-modified macromers or by intermittent incubation of PEG-Tz for temporal stiffening. Finally, the Tz-NB-mediated dynamic stiffening is explored for 4D culture and definitive endoderm differentiation of hiPSCs. Overall, this dynamic hydrogel platform affords exquisite controls of hydrogel crosslinking for serving as a xeno-free and dynamic stem cell niche.

Hair-bearing human skin generated entirely from pluripotent stem cells.

The skin is a multilayered organ, equipped with appendages (that is, follicles and glands), that is critical for regulating body temperature and the retention of bodily fluids, guarding against external stresses and mediating the sensation of touch and pain1,2. Reconstructing appendage-bearing skin in cultures and in bioengineered grafts is a biomedical challenge that has yet to be met3-9. Here we report an organoid culture system that generates complex skin from human pluripotent stem cells. We use stepwise modulation of the transforming growth factor ß (TGFß) and fibroblast growth factor (FGF) signalling pathways to co-induce cranial epithelial cells and neural crest cells within a spherical cell aggregate. During an incubation period of 4-5 months, we observe the emergence of a cyst-like skin organoid composed of stratified epidermis, fat-rich dermis and pigmented hair follicles that are equipped with sebaceous glands. A network of sensory neurons and Schwann cells form nerve-like bundles that target Merkel cells in organoid hair follicles, mimicking the neural circuitry associated with human touch. Single-cell RNA sequencing and direct comparison to fetal specimens suggest that the skin organoids are equivalent to the facial skin of human fetuses in the second trimester of development. Moreover, we show that skin organoids form planar hair-bearing skin when grafted onto nude mice. Together, our results demonstrate that nearly complete skin can self-assemble in vitro and be used to reconstitute skin in vivo. We anticipate that our skin organoids will provide a foundation for future studies of human skin development, disease modelling and reconstructive surgery.