Photo-click hydrogels for 3D in situ differentiation of pancreatic progenitors from induced pluripotent stem cells.

Background: Induced pluripotent stem cells (iPSC) can be differentiated to cells in all three germ layers, as well as cells in the extraembryonic tissues. Efforts in iPSC differentiation into pancreatic progenitors in vitro have largely been focused on optimizing soluble growth cues in conventional two-dimensional (2D) culture, whereas the impact of three-dimensional (3D) matrix properties on the morphogenesis of iPSC remains elusive.

Methods: In this work, we employ gelatin-based thiol-norbornene photo-click hydrogels for in situ 3D differentiation of human iPSCs into pancreatic progenitors (PP).

Generating high-fidelity cochlear organoids from human pluripotent stem cells.

Mechanosensitive hair cells in the cochlea are responsible for hearing but are vulnerable to damage by genetic mutations and environmental insults. The paucity of human cochlear tissues makes it difficult to study cochlear hair cells. Organoids offer a compelling platform to study scarce tissues in vitro; however, derivation of cochlear cell types has proven non-trivial.

Photo-click hydrogels for 3D in situ differentiation of pancreatic progenitors from induced pluripotent stem cells.

Induced pluripotent stem cells (iPSC) can be differentiated to cells in all three germ layers, as well as cells in the extraembryonic tissues. Efforts in iPSC differentiation into pancreatic progenitors have largely been focused on optimizing soluble growth cues in conventional two-dimensional (2D) culture, whereas the impact of three-dimensional (3D) matrix properties on the morphogenesis of iPSC remains elusive. In this work, we employ gelatin-based thiol-norbornene photo-click hydrogels for 3D differentiation of human iPSCs into pancreatic progenitors (PP).

CHD7 regulates otic lineage specification and hair cell differentiation in human inner ear organoids.

Mutations in CHD7 cause CHARGE syndrome, affecting multiple organs including the inner ear in humans. We investigate how CHD7 mutations affect inner ear development using human pluripotent stem cell-derived organoids as a model system. We find that loss of CHD7 or its chromatin remodeling activity leads to complete absence of hair cells and supporting cells, which can be explained by dysregulation of key otic development-associated genes in mutant otic progenitors.

Directed Differentiation of Human Pluripotent Stem Cells into Inner Ear Organoids.

The sensory epithelia of the inner ear contain mechanosensitive hair cells that detect sound and head acceleration. This protocol details a 3D differentiation method to generate inner ear organoids containing sensory epithelia with hair cells. Human pluripotent stem cells are aggregated in low-binding 96-well plates and treated in chemically defined media with extracellular matrix to promote epithelialization.

Deafness-in-a-dish: modeling hereditary deafness with inner ear organoids.

Sensorineural hearing loss (SNHL) is a major cause of functional disability in both the developed and developing world. While hearing aids and cochlear implants provide significant benefit to many with SNHL, neither targets the cellular and molecular dysfunction that ultimately underlies SNHL. The successful development of more targeted approaches, such as growth factor, stem cell, and gene therapies, will require a yet deeper understanding of the underlying molecular mechanisms of human hearing and deafness.

Generation of inner ear organoids from human pluripotent stem cells.

The sensory epithelia of the inner ear contain mechanosensitive hair cells that transmit sound, gravity and head motion signals. This protocol describes an in vitro 3D differentiation method, by which the inner ear sensory epithelium harboring hair cells are derived from human pluripotent stem cells (hPSCs). To begin the differentiation, hPSCs are aggregated in low-binding 96-well plates and treated with extracellular matrix proteins to promote epithelialization.

Progress in Modeling and Targeting Inner Ear Disorders with Pluripotent Stem Cells.

Sensorineural hearing loss and vestibular dysfunction are caused by damage to neurons and mechanosensitive hair cells, which do not regenerate to any clinically relevant extent in humans. Several protocols have been devised to direct pluripotent stem cells (PSCs) into inner ear hair cells and neurons, which display many properties of their native counterparts. The efficiency, reproducibility, and scalability of these protocols are enhanced by incorporating knowledge of inner ear development.

Defective Tmprss3-Associated Hair Cell Degeneration in Inner Ear Organoids.

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.

The histone demethylase LSD1 regulates inner ear progenitor differentiation through interactions with Pax2 and the NuRD repressor complex.

The histone demethylase LSD1 plays a pivotal role in cellular differentiation, particularly in silencing lineage-specific genes. However, little is known about how LSD1 regulates neurosensory differentiation in the inner ear. Here we show that LSD1 interacts directly with the transcription factor Pax2 to form the NuRD co-repressor complex at the Pax2 target gene loci in a mouse otic neuronal progenitor cell line (VOT-N33).

Generation of inner ear organoids containing functional hair cells from human pluripotent stem cells.

The derivation of human inner ear tissue from pluripotent stem cells would enable in vitro screening of drug candidates for the treatment of hearing and balance dysfunction and may provide a source of cells for cell-based therapies of the inner ear. Here we report a method for differentiating human pluripotent stem cells to inner ear organoids that harbor functional hair cells. Using a three-dimensional culture system, we modulate TGF, BMP, FGF, and WNT signaling to generate multiple otic-vesicle-like structures from a single stem-cell aggregate.

Directed Differentiation of Mouse Embryonic Stem Cells Into Inner Ear Sensory Epithelia in 3D Culture.

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.

Organoid technologies meet genome engineering.

Three-dimensional (3D) stem cell differentiation cultures recently emerged as a novel model system for investigating human embryonic development and disease progression , complementing existing animal and two-dimensional (2D) cell culture models. Organoids, the 3D self-organizing structures derived from pluripotent or somatic stem cells, can recapitulate many aspects of structural organization and functionality of their organ counterparts, thus holding great promise for biomedical research and translational applications. Importantly, faithful recapitulation of disease and development processes relies on the ability to modify the genomic contents in organoid cells.

Modulation of Wnt Signaling Enhances Inner Ear Organoid Development in 3D Culture.

Stem cell-derived inner ear sensory epithelia are a promising source of tissues for treating patients with hearing loss and dizziness. We recently demonstrated how to generate inner ear sensory epithelia, designated as inner ear organoids, from mouse embryonic stem cells (ESCs) in a self-organizing 3D culture. Here we improve the efficiency of this culture system by elucidating how Wnt signaling activity can drive the induction of otic tissue.

Functional development of mechanosensitive hair cells in stem cell-derived organoids parallels native vestibular hair cells.

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.

Tlx3 promotes glutamatergic neuronal subtype specification through direct interactions with the chromatin modifier CBP.

Nervous system development relies on the generation of precise numbers of excitatory and inhibitory neurons. The homeodomain transcription factor, T-cell leukemia 3 (Tlx3), functions as the master neuronal fate regulator by instructively promoting the specification of glutamatergic excitatory neurons and suppressing the specification of gamma-aminobutyric acid (GABAergic) neurons. However, how Tlx3 promotes glutamatergic neuronal subtype specification is poorly understood.

Generating Inner Ear Organoids from Mouse Embryonic Stem Cells.

This protocol describes a three-dimensional culture method for generating inner ear sensory epithelia, which comprises sensory hair cells and a concurrently arising neuronal population. Mouse embryonic stem cells are initially plated in 96-well plates with differentiation media; following aggregation, Matrigel is added in order to promote epithelialization. A series of small molecule applications is then used over the first 14 days of culture to guide differentiation towards an otic lineage.

3D mouse embryonic stem cell culture for generating inner ear organoids.

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.

Generation of inner ear sensory epithelia from pluripotent stem cells in 3D culture.

The inner ear contains sensory epithelia that detect head movements, gravity and sound. It is unclear how to develop these sensory epithelia from pluripotent stem cells, a process that will be critical for modelling inner ear disorders or developing cell-based therapies for profound hearing loss and balance disorders. So far, attempts to derive inner ear mechanosensitive hair cells and sensory neurons have resulted in inefficient or incomplete phenotypic conversion of stem cells into inner-ear-like cells.

Retinoic acid deficiency impairs the vestibular function.

The retinaldehyde dehydrogenase 3 (Raldh3) gene encodes a major retinoic acid synthesizing enzyme and is highly expressed in the inner ear during embryogenesis. We found that mice deficient in Raldh3 bear severe impairment in vestibular functions. These mutant mice exhibited spontaneous circling/tilted behaviors and performed poorly in several vestibular-motor function tests.

ROCK1 functions as a critical regulator of stress erythropoiesis and survival by regulating p53.

Erythropoiesis is a dynamic, multistep process whereby hematopoietic stem cells differentiate toward a progressively committed erythroid lineage through intermediate progenitors. Although several downstream signaling molecules have been identified that regulate steady-state erythropoiesis, the major regulators under conditions of stress remain poorly defined. Rho kinases (ROCKs) belong to a family of serine/threonine kinases.

Class I(A) PI3Kinase regulatory subunit, p85α, mediates mast cell development through regulation of growth and survival related genes.

Stem cell factor (SCF) mediated KIT receptor activation plays a pivotal role in mast cell growth, maturation and survival. However, the signaling events downstream from KIT are poorly understood. Mast cells express multiple regulatory subunits of class 1(A) PI3Kinase (PI3K) including p85α, p85β, p50α, and p55α.

Extended passaging increases the efficiency of neural differentiation from induced pluripotent stem cells.

Background: The use of induced pluripotent stem cells (iPSCs) for the functional replacement of damaged neurons and in vitro disease modeling is of great clinical relevance. Unfortunately, the capacity of iPSC lines to differentiate into neurons is highly variable, prompting the need for a reliable means of assessing the differentiation capacity of newly derived iPSC cell lines. Extended passaging is emerging as a method of ensuring faithful reprogramming.