Probing the mechanism for hydrogel-based stasis induction in human pluripotent stem cells: is the chemical functionality of the hydrogel important?

It is well-known that pluripotent human embryonic stem cells (hPSC) can differentiate into any cell type. Recently, we reported that hPSC colonies enter stasis when immersed in an extremely soft hydrogel comprising hydroxyl-functional block copolymer worms (I. Canton, N. J. Warren, A. Chahal, K. Amps, A. Wood, R. Weightman, E. Wang, H. Moore and S. P. Armes, ACS Centr. Sci., 2016, 2, 65-74). The gel modulus and chemical structure of this synthetic hydrogel are similar to that of natural mucins, which are implicated in the mechanism of diapause for mammalian embryos. Does stasis induction occur merely because of the very soft nature of such hydrogels or does chemical functionality also play a role? Herein, we address this key question by designing a new hydrogel of comparable softness in which the PGMA stabilizer chains are replaced with non-hydroxylated poly(ethylene glycol) [PEG]. Immunolabeling studies confirm that hPSC colonies immersed in such PEG-based hydrogels do not enter stasis but instead proliferate (and differentiate if no adhesion substrate is present). However, pluripotency is retained if an appropriate adhesion substrate is provided. Thus, the chemical functionality of the hydrogel clearly plays a decisive role in the stasis induction mechanism.

A model for mammalian cochlear hair cell differentiation in vitro: effects of retinoic acid on cytoskeletal proteins and potassium conductances.

We have established a model for the in-vitro differentiation of mouse cochlear hair cells and have used it to explore the influence of retinoic acid on proliferation, cytoskeletal proteins and voltage-gated potassium conductances. The model is based on the conditionally immortal cell line University of Sheffield/ventral otocyst-epithelial cell line clone 36 (US/VOT-E36), derived from ventral otic epithelial cells of the mouse at embryonic day 10.5 and transfected with a reporter for myosin VIIa. Retinoic acid did not increase cell proliferation but led to up-regulation of myosin VIIa and formation of prominent actin rings that gave rise to numerous large, linear actin bundles. Cells expressing myosin VIIa had larger potassium conductances and did not express the cyclin-dependent kinase inhibitor p27(kip1). US/VOT-E36 endogenously expressed the voltage-gated potassium channel alpha-subunits Kv1.3 and Kv2.1, which we subsequently identified in embryonic and neonatal hair cells in both auditory and vestibular sensory epithelia in vivo. These subunits could underlie the embryonic and neonatal delayed-rectifiers recorded in nascent hair cells in vivo. Kv2.1 was particularly prominent on the basolateral membrane of cochlear inner hair cells. Kv1.3 was distributed throughout all hair cells but tended to be localized to the cuticular plates. US/VOT-E36 recapitulates a coherent pattern of cell differentiation under the influence of retinoic acid and will provide a convenient model for screening the effects of other extrinsic factors on the differentiation of cochlear epithelial cell types in vitro.

Differentiation of an auditory neuronal cell line suitable for cell transplantation.

The auditory neuroblast cell line US/VOT-N33 (N33), which is conditionally immortal, was studied as an in vitro model for the differentiation of spiral ganglion neurons (SGNs) and as a candidate for cell transplantation in rodents. It expresses numerous molecular markers characteristic of auditory neuroblasts, including the transcription factors GATA3, NeuroD, Brn3a and Islet1, as well as the neuronal cytoskeletal protein beta3-tubulin. It displays active migratory behaviour in vitro and in vivo. In the presence of the fibroblast growth factors FGF1 or FGF2 it differentiates bipolar morphologies similar to those of native SGNs. In coculture with neonatal cochlear tissue it is repelled from epithelial surfaces but not from native SGNs, alongside which it extends parallel neuronal processes. When injected into the retina in vivo, EGFP-labelled N33 cells were traced for 1-2 weeks and migrated rapidly within the subretinal space. Cells that found their way into the retinal ganglion cell layer extended multiple processes but did not express beta3-tubulin. The ability of N33 to migrate, to differentiate, to localize with native SGNs in vitro and to survive in vivo suggests that they provide an effective model for SGN differentiation and for cell transplantation into the ear.

Ventral otic cell lines as developmental models of auditory epithelial and neural precursors.

Conditionally immortal cell lines were established from the ventral otocyst of the Immortomouse at embryonic day 10.5 and selected to represent precursors of auditory sensory neural and epithelial cells. Selection was based upon dissection, tissue-specific markers, and expression of the transcription factor GATA3. Two cell lines expressed GATA3 but possessed intrinsically different genetic programs under differentiating conditions. US/VOT-E36 represented epithelial progenitors with potential to differentiate into sensory and nonsensory epithelial cells. US/VOT-N33 represented migrating neuroblasts. Under differentiating conditions in vitro the cell lines expressed very different gene expression profiles. Expression of several cell- and tissue-specific markers, including the transcription factors Pax2, GATA3, and NeuroD, differed between the cell lines in a pattern consistent with that observed between their counterparts in vivo. We suggest that these and other conditionally immortal cell lines can be used to study transient events in development against different backgrounds of cell competence.

Notch signaling and the emergence of auditory hair cells.

OBJECTIVE: Recent insights into the mechanisms that determine a hair cell's fate have emerged from studies on invertebrate sensory organs and the avian inner ear. These mechanisms have important implications for our understanding of the possible therapeutic management of sensorineural deafness. This article reviews the current state of our knowledge regarding mammalian auditory hair cell fate specification. DESIGN: Data were obtained from the MEDLINE database and data presented at the Molecular Biology of Hearing and Deafness Meeting (Bethesda, Md, October 1998). Articles reporting information about cell fate specification and Notch and its ligands were selected. MAIN OUTCOME MEASURES: Data pertaining to cell fate mechanisms, Notch and its ligands, and application to hearing were extracted. RESULTS: The Notch/ligand mechanism is responsible for the specification of the hair cell phenotype. CONCLUSIONS: Major progress has been made in understanding this fundamental process, and its application to hair cell determination is only now being realized. Possible applications could involve the "switching" of supporting cells to hair cells, thus replenishing those hair cells damaged in sensorineural hearing loss.

Calcium signalling mediated by the 9 acetylcholine receptor in a cochlear cell line from the immortomouse.

1. We have investigated the characteristics of the alpha9 acetylcholine receptor (alpha9AChR) expressed in hair cell precursors in an immortalized cell line UB/OC-2 developed from the organ of Corti of the transgenic H-2Kb-tsA58 mouse (the Immortomouse) using both calcium imaging and whole-cell recording. 2. Ratiometric measurements of fura-2 fluorescence revealed an increase of intracellular calcium concentration in cells when challenged with 10 microM ACh. The calcium increase was seen in 66 % of the cells grown at 39 degrees C in differentiated conditions. A sm aller fraction (34%) of cells grown at 33 degrees C in proliferative con ditions responded. 3. Caffeine (10mM) elevated cell calcium. In the ab sence of caffeine, the majority of imaged cells responded only once to A Ch presentations. Pretreatment with caffeine ingibited all calcium respo nses to ACh. 4. In whole-cell tight-seal recordings 10 microM ACh activa ted inward current was dependent on the extracellular calcium concentrat ion with an estimated PCa/PNa of 80 for the alpha9 receptor at physiological calcium levels. 5 . The data indicate that ACh activates a calcium-permeable channel alpha 9AChR in UB/OC-2 cells and that the channel has a significantly higher c alcium permeability than other AChRs. The results indicate that the alp ha9AChR may be able to elevate intracellular calcium levels in hair cell s both directly and via store release.

Differentiation of mammalian vestibular hair cells from conditionally immortal, postnatal supporting cells.

We provide evidence from a newly established, conditionally immortal cell line (UB/UE-1) that vestibular supporting cells from the mammalian inner ear can differentiate postnatally into more than one variant of hair cell. A clonal supporting cell line was established from pure utricular sensory epithelia of H2k(b)tsA58 transgenic mice 2 d after birth. Cell proliferation was dependent on conditional expression of the immortalizing gene, the "T" antigen from the SV40 virus. Proliferating cells expressed cytokeratins, and patch-clamp recordings revealed that they all expressed small membrane currents with little time-dependence. They stopped dividing within 2 d of being transferred to differentiating conditions, and within a week they formed three defined populations expressing membrane currents characteristic of supporting cells and two kinds of neonatal hair cell. The cells expressed several characteristic features of normal hair cells, including the transcription factor Brn3.1, a functional acetylcholine receptor composed of alpha9 subunits, and the cytoskeletal proteins myosin VI, myosin VIIa, and fimbrin. Immunofluorescence labeling and electron microscopy showed that the cells formed complex cytoskeletal arrays on their upper surfaces with structural features resembling those at the apices of normal hair cells. The cell line UB/UE-1 provides a valuable in vitro preparation in which the expression of numerous structural and physiological components can be initiated or upregulated during early stages of mammalian hair cell commitment and differentiation.

Auditory hair cell precursors immortalized from the mammalian inner ear.

Mammalian auditory hair cells are few in number, experimentally inaccessible, and do not proliferate postnatally or in vitro. Immortal cell lines with the potential to differentiate into auditory hair cells would substantially facilitate auditory research, drug development, and the isolation of critical molecules involved in hair cell biology. We have established two conditionally immortal cell lines that express at least five characteristic hair cell markers. These markers are the transcription factor Brn3.1, the alpha 9 subunit of the acetylcholine receptor, the stereociliary protein fimbrin and the myosins VI and VIIA. These hair cell precursors permit functional studies of cochlear genes and in the longer term they will provide the means to explore therapeutic methods of stimulating auditory hair cell regeneration.

Timed markers for the differentiation of the cuticular plate and stereocilia in hair cells from the mouse inner ear.

The differentiation of the cuticular plate and stereocilia in cochleovestibular hair cells from the mouse was traced with monoclonal antibodies raised by in vitro immunization. The cuticular plate is detected first from embryonic days 14-15 (E14-E15), before cell differentiation is apparent, either with scanning electron microscopy or with actin filament labeling. A flat disc of material forms beneath the apical membrane and subsequently expands, forming a fully shaped cuticular plate at postnatal stages 3-5 (P3-P5). A second antibody labels stereocilia from stage E16 to E18. In the cochlea, the label initially appears as a punctate disc on the cell apex and then follows the development of the stereocilia until the adult shape of the bundle forms at P4-P6. Additional antibodies label stereocilia from P4 to P6 and are apparently specific for the inner ear. They do not label the cuticular plate at any stage and do not cross react with tissues of muscle, kidney, eye, tongue, gut, skin, or brain. At stage P12-P14, coinciding with the functional maturity of the ear, they label the apical regions of Deiter's cells. The temporally overlapping sequence of antibody labeling sheds new light on the development of the hair cell apex and allows us to monitor the differentiation of hair cells from their last mitotic division to the initiation of organ function, a period of over 2 weeks.

GATA3 is downregulated during hair cell differentiation in the mouse cochlea.

GATA3 is a transcription factor expressed in the inner ear during the early stages of development. A monoclonal antibody revealed that it is expressed in spiral ganglion cells and in all cells of the developing auditory sensory epithelium in the mouse before the hair cells differentiate at embryonic days 14-16. Expression decreases selectively in the hair cells as they differentiate progressively from the base to the apex of the developing organ of Corti. GATA3 subsequently decreases in the supporting cells and cannot be detected by immunofluorescence in any cell of the adult sensory epithelium. It is not expressed in the vestibular sensory epithelia or surrounding tissues from embryonic day 14. We suggest that GATA3 could act as a repressor of critical genes involved in cell differentiation in the organ of Corti, enabling a progressive formation of the adult cellular pattern.

Transcription factors in the ear: molecular switches for development and differentiation.

In order to understand the molecular events underlying differentiation and development in the inner ear, we need to identify and characterize the molecular 'switches' involved in the regulation of gene expression in the system. The most important molecular regulators are represented by a family of proteins generically called transcription factors. This article reviews our current knowledge of the expression of several transcription factors in the ear and their implications for both development and homeostasis in the auditory organs.

A novel zinc finger gene preferentially expressed in the retina and the organ of Corti localizes to human chromosome 12q24.3.

A cDNA encoding a novel member of the zinc finger gene family, designated zfOC1, has been cloned from the organ of Corti. This is the first transcriptional regulator cloned from this sensory epithelium. This transcript encodes a peculiar protein composed of 9 zinc finger domains and a few additional amino acids. The deduced polypeptide shares 66% amino acid similarity with MOK-2, another protein of only zinc finger motifs and preferentially expressed in transformed cell lines. Northern blot hybridization analysis reveals that zfOC1 transcripts are predominantly expressed in the retina and the organ of Corti and at lower levels in the stria vascularis, auditory nerve, tongue, cerebellum, small intestine and kidney. The human gene was mapped, using a human x hamster somatic cell hybrid panel and fluorescent in situ hybridization, to chromosome 12q24.3. Because of its relative abundance in sensorineural structures (retina and organ of Corti), this regulatory gene should be considered a candidate for hereditary disorders involving hearing and visual impairments that link to 12q24.3.

A soluble motor from the alga Nitella supports fast movement of actin filaments in vitro.

In the streaming cytoplasm of the Characean algae cell, the movement of organelles along actin bundles occurs at a striking rate of up to 60 microns s-1. To further characterize the molecular mechanisms responsible for this phenomenon, we have reconstituted the movement of actin filaments in vitro using defined biochemical components. We report that only a soluble cytoplasmic fraction devoid of organelles and filamentous material supports the movement of fluorescent-labeled actin filaments on glass at a rate of up to 60 microns s-1. This fraction also contains the K(+)-EDTA ATPase and the actin-activated Mg2+ ATPase activities characteristic of myosin proteins. Therefore, on the basis of these observations, we conclude that Nitella cells have a soluble pool of non-filamentous myosin molecules with the mechanochemical properties expected for a motor responsible for cytoplasmic streaming in vivo. The preparation and conditions described here should be useful for the purification of this translocator.

Cloning of an organ of Corti anion exchanger 2 isoform with a truncated C-terminal domain.

We have isolated a cDNA clone from a guinea pig organ of Corti library encoding a new isoform of the Anion Exchanger 2 (AE2) protein. This cDNA clone shows an 83 bp deletion in the region that encodes the membrane domain of AE2. Analysis of the overlapping regions of genomic and cDNA clones indicates that the missing portion does not correspond exactly to a constitutive exon. The alternate splicing process that generates this transcript involves internal donor and acceptor sites which introduces a shift in the open reading frame. The resulting polypeptide has a conserved cytoplasmic N-terminal domain but the membrane C-terminal domain has only two of the fourteen membrane spanning regions. An affinity-purified antipeptide antibody to the novel C-terminus detects an 89 kDa polypeptide which agrees with the molecular mass predicted from the cDNA.

G protein Gi2 alpha in the cochlea: cloning and selective occurrence in receptor cells.

A cDNA library was made from the mouse cochlea and screened with a G protein-cDNA like molecule obtained from cochlear tissue by polymerase chain reaction. The nucleotide sequence of a clone, named cochlear Gi2 alpha, had 99.2% identity to mouse macrophage Gi2 alpha. Using an antibody which is selective for Gi2 alpha, expression of the cochlear Gi2 alpha was localized in outer and inner hair cells of the organ of Corti. Possible functional roles of this G protein in hair cells are discussed.