IU Indianapolis ScholarWorks :: Browsing by Author "Koehler, Karl R."

Browsing by Author "Koehler, Karl R."

Now showing 1 - 10 of 18

3D mouse embryonic stem cell culture for generating inner ear organoids
(Springer Nature, 2014) Koehler, Karl R.; Hashino, Eri; Otolaryngology -- Head and Neck Surgery, School of Medicine
This protocol describes a culture system in which inner-ear sensory tissue is produced from mouse embryonic stem (ES) cells under chemically defined conditions. This model is amenable to basic and translational investigations into inner ear biology and regeneration. In this protocol, mouse ES cells are aggregated in 96-well plates in medium containing extracellular matrix proteins to promote epithelialization. During the first 14 d, a series of precisely timed protein and small-molecule treatments sequentially induce epithelia that represent the mouse embryonic non-neural ectoderm, preplacodal ectoderm and otic vesicle epithelia. Ultimately, these tissues develop into cysts with a pseudostratified epithelium containing inner ear hair cells and supporting cells after 16-20 d. Concurrently, sensory-like neurons generate synapse-like structures with the derived hair cells. We have designated the stem cell-derived epithelia harboring hair cells, supporting cells and sensory-like neurons as inner ear organoids. This method provides a reproducible and scalable means to generate inner ear sensory tissue in vitro.
A single-cell level comparison of human inner ear organoids with the human cochlea and vestibular organs
(Cell Press, 2023) van der Valk, Wouter H.; van Beelen, Edward S. A.; Steinhart, Matthew R.; Nist-Lund, Carl; Osorio, Daniel; de Groot, John C. M. J.; Sun, Liang; van Benthem, Peter Paul G.; Koehler, Karl R.; Locher, Heiko; Otolaryngology -- Head and Neck Surgery, School of Medicine
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.
Aerosol and droplet generation from orbital repair: Surgical risk in the pandemic era
(Elsevier, 2021) Ye, Michael J.; Vadhul, Raghav B.; Sharma, Dhruv; Campiti, Vincent J.; Burgin, Sarah J.; Illing, Elisa A.; Ting, Jonathan Y.; Hong Park, Jae; Koehler, Karl R.; Lee, Hui Bae; Vernon, Dominic J.; Johnson, Jeffrey D.; Nesemeier, B. Ryan; Shipchandler, Taha Z.; Otolaryngology -- Head and Neck Surgery, School of Medicine
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.
Building inner ears: recent advances and future challenges for in vitro organoid systems
(Springer Nature, 2021-01) van der Valk, Wouter H.; Steinhart, Matthew R.; Zhang, Jingyuan; Koehler, Karl R.; Otolaryngology -- Head and Neck Surgery, School of Medicine
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.
Defective Tmprss3-Associated Hair Cell Degeneration in Inner Ear Organoids
(Elsevier, 2019-07-09) Tang, Pei-Ciao; Alex, Alpha L.; Nie, Jing; Lee, Jiyoon; Roth, Adam A.; Booth, Kevin T.; Koehler, Karl R.; Hashino, Eri; Nelson, Rick F.; Otolaryngology, IU School of Medicine
Mutations in the gene encoding the type II transmembrane protease 3 (TMPRSS3) cause human hearing loss, although the underlying mechanisms that result in TMPRSS3-related hearing loss are still unclear. We combined the use of stem cell-derived inner ear organoids with single-cell RNA sequencing to investigate the role of TMPRSS3. Defective Tmprss3 leads to hair cell apoptosis without altering the development of hair cells and the formation of the mechanotransduction apparatus. Prior to degeneration, Tmprss3-KO hair cells demonstrate reduced numbers of BK channels and lower expressions of genes encoding calcium ion-binding proteins, suggesting a disruption in intracellular homeostasis. A proteolytically active TMPRSS3 was detected on cell membranes in addition to ER of cells in inner ear organoids. Our in vitro model recapitulated salient features of genetically associated inner ear abnormalities and will serve as a powerful tool for studying inner ear disorders.
Defining Inner Ear Cell Type Specification at Single-Cell Resolution in a Model of Human Cranial Development
(2022-07) Steinhart, Matthew Reed; Meyer, Jason S.; Koehler, Karl R.; Herbert, Brittney-Shea; Landreth, Gary E.; Shearer, A. Eliot; Yates, Charles W.
Inner ear development requires the complex interaction of numerous cell types arising from multiple embryologic origins. Current knowledge of inner ear organogenesis is limited primarily to animal models. Although most mechanisms of cellular development show conservation between vertebrate species, there are uniquely human aspects of inner ear development which remain unknown. Our group recently described a model of in vitro human inner ear organogenesis using pluripotent stem cells in a 3D organoid culture system. This method promotes the formation of an entire sensorineural circuit, including hair cells, inner ear neurons, and Schwann cells. Our past work has characterized certain aspects of this culture system, however we have yet to fully define all the cell types which contribute to inner ear organoid assembly. Here, our goal was to reconstruct a time-based map of in vitro development during inner ear organoid induction to understand the developmental elements captured in this system. We analyzed inner ear organoid development using single-cell RNA sequencing at ten time points during the first 36 days of induction. We reconstructed the on-target progression of undifferentiated pluripotent stem cells to surface ectoderm, pre-placodal, and otic epithelial cells, including supporting cells, hair cells, and neurons, following treatment with FGF, BMP, and WNT signaling modulators. Our data revealed endogenous signaling pathwayrelated gene expression that may influence the course of on-target differentiation. In addition, we classified a diverse array of off-target ectodermal cell types encompassing the neuroectoderm, neural crest, and mesenchymal lineages. Our work establishes the Inner ear Organoid Developmental Atlas (IODA), which can provide insights needed for understanding human biology and refining the guided differentiation of in vitro inner ear tissue.
Directed Differentiation of Mouse Embryonic Stem Cells Into Inner Ear Sensory Epithelia in 3D Culture
(Springer Nature, 2017) Nie, Jing; Koehler, Karl R.; Hashino, Eri; Otolaryngology -- Head and Neck Surgery, School of Medicine
The inner ear sensory epithelium harbors mechanosensory hair cells responsible for detecting sound and maintaining balance. This protocol describes a three-dimensional (3D) culture system that efficiently generates inner ear sensory epithelia from aggregates of mouse embryonic stem (mES) cells. By mimicking the activations and repressions of key signaling pathways during in vivo inner ear development, mES cell aggregates are sequentially treated with recombinant proteins and small molecule inhibitors for activating or inhibiting the Bmp, TGFβ, Fgf, and Wnt signaling pathways. These stepwise treatments promote mES cells to sequentially differentiate into epithelia representing the non-neural ectoderm, preplacodal ectoderm, otic placodal ectoderm, and ultimately, the hair cell-containing sensory epithelia. The derived hair cells are surrounded by a layer of supporting cells and are innervated by sensory neurons. This in vitro inner ear organoid culture system may serve as a valuable tool in developmental and physiological research, disease modeling, drug testing, and potential cell-based therapies.
Dynamic Click Hydrogels for Xeno-Free Culture of Induced Pluripotent Stem Cells
(Wiley, 2020-11) Arkenberg, Matthew R.; Dimmitt, Nathan H.; Johnson, Hunter C.; Koehler, Karl R.; Lin, Chien-Chi; Biomedical Engineering, School of Engineering and Technology
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.
Early Wnt Signaling Activation Promotes Inner Ear Differentiation via Cell Caudalization in Mouse Stem Cell-Derived Organoids
(Oxford University Press, 2023) Tang, Pei-Ciao; Chen, Li; Singh, Sunita; Groves, Andrew K.; Koehler, Karl R.; Liu, Xue Zhong; Nelson, Rick F.; Otolaryngology -- Head and Neck Surgery, School of Medicine
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.
Functional development of mechanosensitive hair cells in stem cell-derived organoids parallels native vestibular hair cells
(Nature Publishing Group, 2016-05-24) Liu, Xiao-Ping; Koehler, Karl R.; Mikosz, Andrew M.; Hashino, Eri; Holt, Jeffrey R.; Department of Otolaryngology—Head & Neck Surgery, IU School of Medicine
Inner ear sensory epithelia contain mechanosensitive hair cells that transmit information to the brain through innervation with bipolar neurons. Mammalian hair cells do not regenerate and are limited in number. Here we investigate the potential to generate mechanosensitive hair cells from mouse embryonic stem cells in a three-dimensional (3D) culture system. The system faithfully recapitulates mouse inner ear induction followed by self-guided development into organoids that morphologically resemble inner ear vestibular organs. We find that organoid hair cells acquire mechanosensitivity equivalent to functionally mature hair cells in postnatal mice. The organoid hair cells also progress through a similar dynamic developmental pattern of ion channel expression, reminiscent of two subtypes of native vestibular hair cells. We conclude that our 3D culture system can generate large numbers of fully functional sensory cells which could be used to investigate mechanisms of inner ear development and disease as well as regenerative mechanisms for inner ear repair.

Browsing by Author "Koehler, Karl R."

Results Per Page

Sort Options