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. Modeling human diseases in vitro through genetic manipulation of PSCs is already feasible, thereby permitting the elucidation of mechanistic understandings of a wide array of disease etiologies. Early studies on transplantation of PSC-derived otic progenitors have been successful in certain animal models, yet restoration of function and long-term cell survival remain unrealized. Through further research, PSC-based approaches will continue to revolutionize our understanding of inner ear biology and contribute to the development of therapeutic treatments for inner ear disorders.

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. We found that a carefully timed treatment with the potent Wnt agonist CHIR99021 promotes induction of otic vesicles-a process that was previously self-organized by unknown mechanisms. The resulting otic-like vesicles have a larger lumen size and contain a greater number of Pax8/Pax2-positive otic progenitor cells than organoids derived without the Wnt agonist. Additionally, these otic-like vesicles give rise to large inner ear organoids with hair cells whose morphological, biochemical and functional properties are indistinguishable from those of vestibular hair cells in the postnatal mouse inner ear. We conclude that Wnt signaling plays a similar role during inner ear organoid formation as it does during inner ear development in the embryo.

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. In this study, we found that Tlx3 directly interacts with the epigenetic co-activator cyclic adenosine monophosphate (cAMP)-response element-binding protein (CREB)-binding protein (CBP) and that the Tlx3 homeodomain is essential for this interaction. The interaction between Tlx3 and CBP was enhanced by the three amino acid loop extension (TALE)-class homeodomain transcription factor, pre-B-cell leukemia transcription factor 3 (Pbx3). Using mouse embryonic stem (ES) cells stably expressing Tlx3, we found that the interaction between Tlx3 and CBP became detectable only after these Tlx3-expressing ES cells were committed to a neural lineage, which coincided with increased Pbx3 expression during neural differentiation from ES cells. Forced expression of mutated Tlx3 lacking the homeodomain in ES cells undergoing neural differentiation resulted in significantly reduced expression of glutamatergic neuronal subtype markers, but had little effect on the expression on pan neural markers. Collectively, our results strongly suggest that functional interplay between Tlx3 and CBP plays a critical role in neuronal subtype specification, providing novel insights into the epigenetic regulatory mechanism that modulates the transcriptional efficacy of a selective set of neuronal subtype-specific genes during differentiation.

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. 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.

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. A key insight lacking from these previous studies is the importance of the non-neural and preplacodal ectoderm, two critical precursors during inner ear development. Here we report the stepwise differentiation of inner ear sensory epithelia from mouse embryonic stem cells (ESCs) in three-dimensional culture. We show that by recapitulating in vivo development with precise temporal control of signalling pathways, ESC aggregates transform sequentially into non-neural, preplacodal and otic-placode-like epithelia. Notably, in a self-organized process that mimics normal development, vesicles containing prosensory cells emerge from the presumptive otic placodes and give rise to hair cells bearing stereocilia bundles and a kinocilium. Moreover, these stem-cell-derived hair cells exhibit functional properties of native mechanosensitive hair cells and form specialized synapses with sensory neurons that have also arisen from ESCs in the culture. Finally, we demonstrate how these vesicles are structurally and biochemically comparable to developing vestibular end organs. Our data thus establish a new in vitro model of inner ear differentiation that can be used to gain deeper insight into inner ear development and disorder.

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. Using gene-targeted ROCK1-deficient mice, we show that lack of ROCK1 in phenylhydrazine-induced oxidative stress model results in enhanced recovery from hemolytic anemia as well as enhanced splenic stress erythropoiesis compared with control mice. Deficiency of ROCK1 also results in enhanced survival, whereas wild-type mice die rapidly in response to stress. Enhanced survivability of ROCK1-deficient mice is associated with reduced level of reactive oxygen species. BM transplantation studies revealed that enhanced stress erythropoiesis in ROCK1-deficient mice is stem cell autonomous. We show that ROCK1 binds to p53 and regulates its stability and expression. In the absence of ROCK1, p53 phosphorylation and expression is significantly reduced. Our findings reveal that ROCK1 functions as a physiologic regulator of p53 under conditions of erythroid stress. These findings are expected to offer new perspectives on stress erythropoiesis and may provide a potential therapeutic target in human disease characterized by anemia.

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, p85ß, p50α, and p55α. While it is known that PI3K plays an essential role in mast cells; the precise mechanism by which these regulatory subunits impact specific mast cell functions including growth, survival and cycling are not known. We show that loss of p85α impairs the growth, survival and cycling of mast cell progenitors (MCp). To delineate the molecular mechanism (s) by which p85α regulates mast cell growth, survival and cycling, we performed microarray analyses to compare the gene expression profile of MCps derived from WT and p85α-deficient mice in response to SCF stimulation. We identified 151 unique genes exhibiting altered expression in p85α-deficient cells in response to SCF stimulation compared to WT cells. Functional categorization based on DAVID bioinformatics tool and Ingenuity Pathway Analysis (IPA) software relates the altered genes due to lack of p85α to transcription, cell cycle, cell survival, cell adhesion, cell differentiation, and signal transduction. Our results suggest that p85α is involved in mast cell development through regulation of expression of growth, survival and cell cycle related genes.

Balanced interactions between Lyn, the p85alpha regulatory subunit of class I(A) phosphatidylinositol-3-kinase, and SHIP are essential for mast cell growth and maturation.

The growth and maturation of bone marrow-derived mast cells (BMMCs) from precursors are regulated by coordinated signals from multiple cytokine receptors, including KIT. While studies conducted using mutant forms of these receptors lacking the binding sites for Src family kinases (SFKs) and phosphatidylinositol-3-kinase (PI3K) suggest a role for these signaling molecules in regulating growth and survival, how complete loss of these molecules in early BMMC progenitors (MCps) impacts maturation and growth during all phases of mast cell development is not fully understood. We show that the Lyn SFK and the p85α subunit of class I(A) PI3K play opposing roles in regulating the growth and maturation of BMMCs in part by regulating the level of PI3K. Loss of Lyn in BMMCs results in elevated PI3K activity and hyperactivation of AKT, which accelerates the rate of BMMC maturation due in part to impaired binding and phosphorylation of SHIP via Lyn's unique domain. In the absence of Lyn's unique domain, BMMCs behave in a manner similar to that of Lyn- or SHIP-deficient BMMCs. Importantly, loss of p85α in Lyn-deficient BMMCs not only represses the hyperproliferation associated with the loss of Lyn but also represses their accelerated maturation. The accelerated maturation of BMMCs due to loss of Lyn is associated with increased expression of microphthalmia-associated transcription factor (Mitf), which is repressed in MCps deficient in the expression of both Lyn and p85α relative to controls. Our results demonstrate a crucial interplay of Lyn, SHIP, and p85α in regulating the normal growth and maturation of BMMCs, in part by regulating the activation of AKT and the expression of Mitf.

Wnt signaling promotes neuronal differentiation from mesenchymal stem cells through activation of Tlx3.

Wnt/ß-catenin signaling promotes neural differentiation by activation of the neuron-specific transcription factors, Neurogenin1 (Ngn1), NeuroD, and Brn3a, in the nervous system. As neurons in cranial sensory ganglia and dorsal root ganglia transiently express Ngn1, NeuroD, and Brn3a during embryonic development, we hypothesized that Wnt proteins could instructively promote a sensory neuronal fate from mesenchymal stem cells (MSCs) directed to differentiate into neurons. Consistent with our hypothesis, Wnt1 induced expression of sensory neuron markers including Ngn1, NeuroD, and Brn3a, as well as glutamatergic markers in neurally induced MSCs in vitro and promoted engraftment of transplanted MSCs in the inner ear bearing selective loss of sensory neurons in vivo. Given the consensus function of T-cell leukemia 3 (Tlx3), as a glutamatergic selector gene, we postulated that the effects of canonical Wnt signaling on sensory neuron and glutamatergic marker gene expression in MSCs may be mediated by Tlx3. We first confirmed that Wnt1 indeed upregulates Tlx3 expression, which can be suppressed by canonical Wnt inhibitors. Next, our chromatin immunoprecipitation assays revealed that T-cell factor 3/4, Wnt-activated DNA binding proteins, interact with a regulatory region of Tlx3 in MSCs after neural induction. Furthermore, we demonstrated that forced expression of Tlx3 in MSCs induced sensory and glutamatergic neuron markers after neural induction. Together, these results identify Tlx3 as a novel target for canonical Wnt signaling that confers somatic stem cells with a sensory neuron phenotype upon neural induction.

Increased c-Jun expression and reduced GATA2 expression promote aberrant monocytic differentiation induced by activating PTPN11 mutants.

Juvenile myelomonocytic leukemia (JMML) is characterized by myelomonocytic cell overproduction and commonly bears activating mutations in PTPN11. Murine hematopoietic progenitors expressing activating Shp2 undergo myelomonocytic differentiation, despite being subjected to conditions that normally support only mast cells. Evaluation of hematopoietic-specific transcription factor expression indicates reduced GATA2 and elevated c-Jun in mutant Shp2-expressing progenitors. We hypothesized that mutant Shp2-induced Ras hyperactivation promotes c-Jun phosphorylation and constitutive c-Jun expression, permitting, as a coactivator of PU.1, excessive monocytic differentiation and reduced GATA2. Hematopoietic progenitors expressing activating Shp2 demonstrate enhanced macrophage CFU (CFU-M) compared to that of wild-type Shp2-expressing cells. Treatment with the JNK inhibitor SP600125 or cotransduction with GATA2 normalizes activating Shp2-generated CFU-M. However, cotransduction of DeltaGATA2 (lacking the C-terminal zinc finger, needed to bind PU.1) fails to normalize CFU-M. NIH 3T3 cells expressing Shp2E76K produce higher levels of luciferase expression directed by the macrophage colony-stimulating factor receptor (MCSFR) promoter, which utilizes c-Jun as a coactivator of PU.1. Coimmunoprecipitation demonstrates increased c-Jun-PU.1 complexes in mutant Shp2-expressing hematopoietic progenitors, while chromatin immunoprecipitation demonstrates increased c-Jun binding to the c-Jun promoter and an increased c-Jun-PU.1 complex at the Mcsfr promoter. Furthermore, JMML progenitors express higher levels of c-JUN than healthy controls, substantiating the disease relevance of these mechanistic findings.

Tlx3 exerts context-dependent transcriptional regulation and promotes neuronal differentiation from embryonic stem cells.

The T cell leukemia 3 (Tlx3) gene has been implicated in specification of glutamatergic sensory neurons in the spinal cord. In cranial sensory ganglia, Tlx3 is highly expressed in differentiating neurons during early embryogenesis. To study a role of Tlx3 during neural differentiation, mouse embryonic stem (ES) cells were transfected with a Tlx3 expression vector. ES cells stably expressing Tlx3 were grown in the presence or absence of a neural induction medium. In undifferentiated ES cells, there was no significant difference in gene expression in the presence or absence of Tlx3, even after ES cells were cultured for an extensive time period. In contrast, expression levels of Mash1, Ngn1, and NeuroD were significantly higher in Tlx3-expressing cells after neural induction for 4 days compared with those in cells expressing the control vector. At 7 days after neural induction, whereas expression of the proneural genes was down-regulated, VGLUT2, GluR2, and GluR4 were significantly increased in ES cell-derived neurons expressing Tlx3. The sequential and coordinated expression of the proneural and neuronal subtype-specific genes identifies Tlx3 as a selector gene in ES cells undergoing neural differentiation. In addition, the differential effects of Tlx3 overexpression in undifferentiated ES cells compared with ES cell-derived neurons suggest that Tlx3 exerts context-dependent transcriptional signals on its downstream target genes. The context-dependent function of Tlx3 as a selector gene may be used to establish a novel strategy to conditionally generate excitatory glutamatergic neurons from ES cells to cure various types of neurodegenerative disorders.

Enhanced survival of bone-marrow-derived pluripotent stem cells in an animal model of auditory neuropathy.

OBJECTIVE: The loss of spiral ganglion neurons (SGNs) is one of the major causes of profound sensorineural hearing loss (SNHL). Stem cell replacement therapy, which is still in its infancy, has the potential to treat or cure those who suffer from an array of illnesses and degenerative neurologic disorders, including sensorineural deafness (SNHL). Little is known about the potentials of mesenchymal stem cells (MSCs) and their ability to take on properties of SGNs. The two main purposes of this study were to evaluate the survival of mouse MSCs transplanted into normal and ouabain-treated gerbil cochleae and to determine the migratory patterns of MSCs with two differing injection methods. SUBJECTS: Thirty-two Mongolian gerbils, 3 to 4 months old, were used as recipients, and four 6-week-old TgN(ACTbEGFP) mice that ubiquitously express green fluorescent protein (GFP) were used as donors. DESIGN: The animals were deafened by ouabain, which damaged SGNs while leaving hair cell systems intact. After 4 weeks of recovery, the animals received an intraperilymphatic transplantation of 1.0x10(6) GFP-positive undifferentiated MSCs via two different injection methods: scala tympani injection and modiolar injection. Seven days after the transplantation, the survival of MSCs was evaluated by microscopic examination of frozen sections cut through the cochleae of the recipient animals. The number of profiles was counted on the five most central modiolar sections. One-way analyses of variance (ANOVA) were used to determine any significantdifferences among mean profile counts across the experimental conditions. RESULTS: Our findings indicated that undifferentiated MSCs were able to survive in the modiolus both in the control and the ouabain-treated cochleae. The average number of profiles found in the modiolus was greater in the ouabain-treated cochleae than in the control cochleae. This difference was statistically significant (P<.01) as determined using a one-way ANOVA and an ad hoc Tukey-Kramer's test. With the scala tympani injection, there were no profiles found in the modiolus either in the control or ouabain-treated cochleae. This finding may indicate that donor MSCs need to be directly injected into the modiolus to replace injured SGNs. Finally, there was no evidence of hyperacute rejection in any of the gerbils despite the use of xenotransplantation. CONCLUSIONS: These findings may have important clinical implications as a means of delivering MSCs in the cochlea for stem-cell replacement therapy. Survival of transplanted MSCs into the modiolus of the cochlea may result in regeneration of damaged SGNs.

In vivo and in vitro characterization of bone marrow-derived stem cells in the cochlea.

OBJECTIVES/HYPOTHESIS: Stem cell replacement therapy has the potential to treat or cure an array of degenerative neurologic disorders, including sensorineural deafness. However, little is known about the potential for marrow-derived stem cells (MSCs) to take on properties of spiral ganglion neurons. The main purpose of this prospective animal study was to evaluate the survival of MSCs transplanted into the gerbil cochlea. METHODS: Eight 3- to 4-month-old Mongolian gerbils were used as recipients. The animals received an intraperilymphatic transplantation of 100,000 green fluorescent protein (GFP)-positive MSCs with scala tympani injection and modiolar injection. Seven days after transplantation, MSC survival was evaluated by microscopic examination of frozen sections cut through the cochleae of the recipient animals. RESULTS: MSCs isolated from the TgN (ACTbEGFP) mouse line used in this study exhibited bright green florescence after five to seven passages in vitro. Seven days after postoperatively, most transplanted MSCs were found in the scala tympani and scala vestibule and only a small number located in the scala media in animals that received both forms of injection. There were no GFP-positive MSCs in the modiolus in animals with scala tympani injection. In contrast, the mean profile count in animals with modiolar injection was 28, which was the highest in all regions. Although MSCs have the potential to migrate, the anatomic barrier between the perilymphatic space and the modiolus might account for the absence of GFP-positive MSCs in this region. CONCLUSION: These findings may have important clinical implications as a means of delivering MSCs in the cochlea for cell replacement therapy.

Skeletal muscle-derived progenitor cells exhibit neural competence.

Skeletal muscle contains heterogenous progenitor cells that give rise to muscle, hematopoietic cells and bone. The exact phenotypic definition of skeletal muscle progenitor cells has not been fully elucidated nor the potential of these cells to differentiate into neurons. Here, we demonstrate that phenotypically homogenous skeletal muscle progenitor cells defined as Lin-CD45-CD117-CD90+ cells express neural stem cell markers and are responsive to neural induction signals. When exposed to neural induction medium containing basic fibroblast growth factor and brain-derived neurotrophic factor, skeletal muscle progenitor cells dramatically changed their cell morphology, became postmitotic and began expressing neuronal markers. These results reveal unexpected potentials of muscle progenitor cells and suggest that these cells may potentially be used in cell-based therapies to replace damaged neurons.

Sonic hedgehog and retinoic acid synergistically promote sensory fate specification from bone marrow-derived pluripotent stem cells.

Recent studies demonstrated that stromal cells isolated from adult bone marrow have the competence of differentiating into neuronal cells in vitro and in vivo. However, the capacity of marrow stromal cells or mesenchymal stem cells (MSCs) to differentiate into diverse neuronal cell populations and the identity of molecular factors that confer marrow stromal cells with the competence of a neuronal subtype have yet to be elucidated. Here, we show that Sonic hedgehog (Shh) and retinoic acid (RA), signaling molecules secreted from tissues in the vicinity of peripheral sensory ganglia during embryogenesis, exert synergistic effects on neural-competent MSCs to express a comprehensive set of glutamatergic sensory neuron markers. Application of Shh or RA alone had little or no effect on the expression of these neuronal subtype markers. In addition, incubation of MSCs with embryonic hindbrain/somite/otocyst conditioned medium or prenatal cochlea explants promoted up-regulation of additional sensory neuron markers and process outgrowth. These results identify Shh and RA as sensory competence factors for adult pluripotent cells and establish the importance of interactions between adult pluripotent cells and the host microenvironment in neuronal subtype specification.