Adenoviral Mediated Gene Delivery to Human Umbilical Cord Mesenchymal Stromal Cells for Inner Ear Hair Cell Differentiation

Hearing is one of our main sensory systems and having a hearing disorder, although not life threatening, can have a disturbing impact in an individual's quality of life. Approximately 49 million Americans suffer from some form of hearing loss. Sensory neural hearing loss (SNHL) is the most common form, which results from degeneration of inner ear sensory hair cells and auditory neurons in the cochlea. In recent years, there has been an increasing interest in gene delivery to mesenchymal stem cells. Gene delivery approaches to stem cells can provide an opportunity to engineer a variety of specialized cell types. The objective of this thesis was to evaluate the potential of human umbilical cord mesenchymal stromal cells (hUCMSCs) as a possible source for regenerating inner ear hair cells. This thesis was successful in developing an adenoviral mediated gene delivery approach to deliver the Math1 gene to hUCMSCs. The expression of Math1 induced the differentiation of hUCMSCs into cells that resembled inner ear hair cells morphologically and immunocytochemically, evidenced by the expression of hair cell-specific and glial cell markers. The results obtained in this thesis demonstrated for the first time that hUCMSCs can differentiate into hair cell-like cells, thus introducing a potential tissue engineering and cell transplantation approach for the treatment of hearing loss

Cochlear hair cell fate determination and differentiation in vitro

Mammalian cochlear hair cells are relatively inaccessible and few in number. This hampers any research on their fate determination and differentiation. The production of conditionally immortal cell lines from the H2KbtsA58 transgenic mouse should overcome these difficulties. The aims of the present study were threefold. Firstly, to establish that the cell lines provide a viable in vitro system, by examining the pattern of molecular expression in the cochlear hair cell line UB/OC-1. Secondly, to examine differentiation by using clonal derivatives from the heterogeneous cell line UB/OC-l. Thirdly, to explore the process of lateral specification in the determination of cell fate and to explain the differentiation of hair cells and supporting cells from a common precursor. The methods used were cell culture, immunocytochemistry, reverse transcription polymerase chain reaction and western blot. The results demonstrated that firstly; the temporal expression pattern of Brn3.1, an essential transcription factor required for hair cell differentiation, and the a9 subunit of the nicotinic acetylcholine receptor, followed a similar pattern to that during normal development. Secondly, epithelial cell markers such as, vimentin, cytokeratin, actin and cadherin, and specific hair cell markers such as myosinVIIA and fimbrin were expressed when the hair cells differentiated. The pattern of expression suggested parallel pathways of gene expression during differentiation of hair cells. Thirdly, from the expression of Numblike, Notch 1, Jagged I and Jagged2, factors which are known to be involved in lateral specification, a model is proposed to explain hair cell fate determination. The results also demonstrate the much greater experimental flexibility offered by cell lines in understanding hair cell development. Future studies will focus on functional experiments that alter hair cell fate

EXPRESSION AND MISEXPRESSION OF THE MIR-183 FAMILY IN THE DEVELOPING HEARING ORGAN OF THE CHICKEN

The miR-183 family consists of 3 related microRNAs (miR-183, miR-96 and miR-182) that are required for the proper maturation of primary sensory cells in both the inner ear and the retina in mammals. The miR-183 family shows dynamic longitudinal and radial gradients in the developing mouse cochlea, which raises a question whether the microRNA levels play a role in specifying hair cell phenotypes. To answer this question, I used the chicken inner ear to study expression and misexpression of the miR-183 family. In this study, I reported the differential gene expression of the miR-183 family through development in the embryonic chicken inner ear by in situ hybridization. The spatiotemporal expression patterns of all three miRNAs were similar. At E7, labeled hair cells were present in several vestibular sensory organs. At the same age, expression was detectable in the apex of the basilar papilla with differentiated hair cells, and a weak radial gradient was seen with the highest expression on the superior side in the base of the basilar papillae with undifferentiated precursors. At E12-E18, the higher packing density of tall hair cells located on the superior basilar papilla suggested the persistence of a radial gradient from the surface view. However, sections through the basilar papillae suggested that the miRNA levels appeared to be similar on the superior and inferior sides. On the other hand, a longitudinal gradient was observed at E16-E18: levels were higher in the apex than the base. The functional role of the expression gradients in the basilar papillae was tested by overexpression of the miR-183 family using Tol2 transposase-mediated stable expression of the miRNAs. Electroporation of plasmids into the E2-E3 otocyst did not affect hair cell morphologies along the longitudinal axis 11-14 days later, nor did it affect the differentiation of tall versus short hair cells across the radial axis. Instead, midway along the longitudinal axis, there appeared to be a higher incidence of electroporated hair cells relative to supporting cells, indicating a slight bias toward a hair cell fate. Therefore, the manipulation of the miR-183 family could influence cell lineage decisions, but it was insufficient to direct the differentiation of hair cells towards specific radial or longitudinal phenotypes. As a first step toward cataloging potential downstream genes regulated by members of this hair-cell-enriched miRNA family, I performed luciferase assays in vitro and verified 14 human gene targets of miR-182

The clusterin gene in mouse inner ear development: expression analysis and generation of reporter constructs

Clusterin has previously been identified as a gene potentially involved in development of the cochlear sensory epithelium. In order to be able to predict the cellular roles that clusterin may play in the development of this organ, an understanding of the spatiotemporal expression pattern is required. Therefore, clusterin gene expression during mouse inner ear development was studied using riboprobes from the mouse gene. Clusterin mRNA demonstrates a dynamic expression pattern within the developing cochlear sensory epithelium. Clusterin mRNA expression is initiated at 12.5dpc (days post coitum) along the entire length of the cochlear sensory epithelium. Throughout in utero development, expression is maintained but becomes progressively restricted in this sensory epithelium. Postnataly expression resolves to specific cellular regions, but clusterin expression ceases at some time point between P2-P17. The analysis of clusterin protein expression revealed this was not restricted to the developing sensory epithelium alone, but also was detected transiently in the periotic mesenchye, otic capsule, as well as Reissner’s membrane, a non-sensory epithelium. The detailed localisation of clusterin was compared to other inner ear markers. Using α and β tectorin mRNA markers, clusterin mRNA was shown to localise to the developing inner and outer sulcus and spiral prominence. Clusterin expression also overlaps with both Myosin VIIA and Prox1, markers for hair cells and supporting cells respectively. Clusterin mRNA and protein was absent from the developing mouse vestibular system. In order to study the regulatory mechanisms underlying inner ear clusterin expression, a clusterin BAC was modified by insertion of ZsGreen reporter gene into clusterin genomic regions using recombineering technique. The pronuclear injection of this construct has not been successful so far although these studies are ongoing. Finally in order to determine the fate of clusterin expressing cells after the expression is ceased in the inner ear, clusterin BAC was modified by insertion of Cre recombinant gene at the same location as ZsGreen gene for the generation of Cre transgenic mouse. This transgenic mouse will be crossed with a silent reporter mouse for future clusterin fate mapping studies

Lineage Analysis of the Late Otocyst Stage Mouse Inner Ear by Transuterine Microinjection of A Retroviral Vector Encoding Alkaline Phosphatase and an Oligonucleotide Library

The mammalian inner ear subserves the special senses of hearing and balance. The auditory and vestibular sensory epithelia consist of mechanically sensitive hair cells and associated supporting cells. Hearing loss and balance dysfunction are most frequently caused by compromise of hair cells and/or their innervating neurons. The development of gene- and cell-based therapeutics will benefit from a thorough understanding of the molecular basis of patterning and cell fate specification in the mammalian inner ear. This includes analyses of cell lineages and cell dispersals across anatomical boundaries (such as sensory versus nonsensory territories). The goal of this study was to conduct retroviral lineage analysis of the embryonic day 11.5(E11.5) mouse otic vesicle. A replication-defective retrovirus encoding human placental alkaline phosphatase (PLAP) and a variable 24-bp oligonucleotide tag was microinjected into the E11.5 mouse otocyst. PLAP-positive cells were microdissected from cryostat sections of the postnatal inner ear and subjected to nested PCR. PLAP-positive cells sharing the same sequence tag were assumed to have arisen from a common progenitor and are clonally related. Thirty five multicellular clones consisting of an average of 3.4 cells per clone were identified in the auditory and vestibular sensory epithelia, ganglia, spiral limbus, and stria vascularis. Vestibular hair cells in the posterior crista were related to one another, their supporting cells, and nonsensory epithelial cells lining the ampulla. In the organ of Corti, outer hair cells were related to a supporting cell type and were tightly clustered. By contrast, spiral ganglion neurons, interdental cells, and Claudius' cells were related to cells of the same type and could be dispersed over hundreds of microns. These data contribute new information about the developmental potential of mammalian otic precursors in vivo

Progressive leukoencephalopathy impairs neurobehavioral development in sialin-deficient mice

Slc17a5−/− mice represent an animal model for the infantile form of sialic acid storage disease (SASD). We analyzed genetic and histological time-course expression of myelin and oligodendrocyte (OL) lineage markers in different parts of the CNS, and related this to postnatal neurobehavioral development in these mice. Sialin-deficient mice display a distinct spatiotemporal pattern of sialic acid storage, CNS hypomyelination and leukoencephalopathy. Whereas few genes are differentially expressed in the perinatal stage (p0), microarray analysis revealed increased differential gene expression in later postnatal stages (p10–p18). This included progressive upregulation of neuroinflammatory genes, as well as continuous down-regulation of genes that encode myelin constituents and typical OL lineage markers. Age-related histopathological analysis indicates that initial myelination occurs normally in hindbrain regions, but progression to more frontal areas is affected in Slc17a5−/− mice. This course of progressive leukoencephalopathy and CNS hypomyelination delays neurobehavioral development in sialin-deficient mice. Slc17a5−/− mice successfully achieve early neurobehavioral milestones, but exhibit progressive delay of later-stage sensory and motor milestones. The present findings may contribute to further understanding of the processes of CNS myelination as well as help to develop therapeutic strategies for SASD and other myelination disorders

Distinct enhancers of ptf1a mediate specification and expansion of ventral pancreas in zebrafish

AbstractDevelopment of the pancreas and cerebellum require Pancreas-specific transcription factor-1a (Ptf1a), which encodes a subunit of the transcription factor complex PTF1. Ptf1a is required in succession for specification of the pancreas, proper allocation of pancreatic progenitors to endocrine and exocrine fates, and the production of digestive enzymes from the exocrine acini. In several neuronal structures, including the cerebellum, hindbrain, retina and spinal cord, Ptf1a is transiently expressed and promotes inhibitory neuron fates at the expense of excitatory fates. Transcription of Ptf1a in mouse is maintained in part by PTF1 acting on an upstream autoregulatory enhancer. However, the transcription factors and enhancers that initially activate Ptf1a expression in the pancreas and in certain structures of the nervous system have not yet been identified. Here we describe a zebrafish autoregulatory element, conserved among teleosts, with activity similar to that described in mouse. In addition, we performed a comprehensive survey of all non-coding sequences in a 67kb interval encompassing zebrafish ptf1a, and identified several neuronal enhancers, and an enhancer active in the ventral pancreas prior to activation of the autoregulatory enhancer. To test the requirement for autoregulatory control during pancreatic development, we restored ptf1a function through BAC transgenesis in ptf1a morphants, either with an intact BAC or one lacking the autoregulatory enhancer. We find that ptf1a autoregulation is required for development of the exocrine pancreas and full rescue of the ptf1a morphant phenotype. Similarly, we demonstrate that a ptf1a locus lacking the early enhancer region is also capable of rescue, but only supports formation of a hypoplastic exocrine pancreas. Through our dissection of the complex regulatory control of ptf1a, we identified separate cis-regulatory elements that underlie different aspects of its expression and function, and further demonstrated the requirement of maintained ptf1a expression for normal pancreatic morphogenesis. We also identified a novel enhancer that mediates initiation of ptf1a expression in the pancreas, through which the signals that specify the ventral pancreas are expected to exert their action

Induced Pluripotent Stem Cells: Advances and Applications in Regenerative Medicine

Reprogramming adult somatic cells into induced pluripotent stem cells (iPSCs) through the ectopic expression of reprogramming factors offers truly personalized cell-based therapy options for numerous human diseases. The iPSC technology also provides a platform for disease modeling and new drug discoveries. Similar to embryonic stem cells, iPSCs can give rise to any cell type in the body and are amenable to genetic correction. These properties of iPSCs allow for the development of permanent corrective therapies for many currently incurable disorders. In this chapter, we summarize recent progress in the iPSC field with a focus on potential clinical applications of these cells

Tbx1 and Bmp2 in the development of the ear, neural crest and pharyngeal system in mice

Dissertation presented to obtain the Ph.D degree in BiologyDevelopment of the vertebrate head involves complex interactions between tissues derived from the three germ layers. Significantly, alterations in the development of this region of the embryo are often associated with a wide variety of human congenital birth defects. Some of them are inherited disorders such as Treacher Collins, Branchio-oto-renal and DiGeorge syndromes. During embryonic development, morphogenesis of the craniofacial area occurs sequentially with the formation of a variety of transient structures, which then undergo complex sequential reorganization to form the adult structures.(...