Role Of Notch Pathway In Differentiation Of Human Embryonic Stem Cells Into FunctionalHair-cell-like Cells In The Absence Of Stromal Cells

Hearing loss has become one of the most common disabilities in the world, and affects almost every age group. Although hearing loss may not be life threatening, it can greatly influence quality of life and social interactions, and has a significant financial impact on society. The inner ear is a highly specialized sensory organ with a complex structure, and has been referred to as the labyrinth. It contains hair cells arranged in a highly organized pattern, and is innervated by sensory neurons. Hair cells are sensory receptors located in the inner ear. In the human cochlea, hair cells die due to variety of reasons such as age-related deafness (presbycusis), high doses of ototoxic drugs (gentamycin, cisplatin, aminoglycosides, etc.), genetic disorders, infectious diseases, and exposure to high levels of noise. The loss of hair cells in higher vertebrates appears to be non-reversible and leads to permanent hearing loss. Therefore, restoration of mammalian hearing requires the regeneration of inner ear hair cells for restoring the function of the inner ear. In the past few years, stem cell-based therapeutic strategies for curing hearing loss have been explored. The generation of new hair cells from a renewable source of progenitors is the principal requirement for the development of a cell-based therapy within this sensory organ.Human embryonic stem cells (hESCs) are the important seed cells for inner ear hair cell regeneration. It has been shown that hESCs can self-renew and have pluripotency that can be induced to differentiate into hair cells. However, there are still problems in studies about hESCs and hair cells as follows:(1) It is necessary to develop a culture method that otic progenitors can be induced directly as monolayers. (2) Several attempts to develop hair cells by using chicken utricle stromal cells as feeder cells have resulted in phenotypic conversion of stem cells into inner ear hair-cell-like cells. Co-culture with stromal cells, however, may be problematic for subsequent examination of the induced hair-cell-like cells. (3) Notch signaling pathway plays an important role in otic neurogenesis by regulating the differentiation of hair cells. Objective to investigate the differentiation of hair-cell-like cells in vitro, however, how does Notch signaling play its role? Therefore, the first two parts of this study (Chapter 2 and Chapter 3) are focused on development of a culture method that may promote the differentiation of OEPs into the functional hair-cell-like cells with the expression of marker genes and the morphology of hair bundles under differentiation conditions in the absence of stromal cells. The last part (Chapter 4) is focused on the effects of Notch ligands or the Notch receptor on differentiation of hESCs into hair cells.Part 1:For the differentiation of otic progenitors, hESC colonies were dissociated, plated as a monolayer in the laminin-coated dish or plated as embryoid bodies in the suspension tissue culture plate. Gene expression of cells differentiated under each of induction methods was investigated respectively. Results showed that the expression of marker specific for hESCs decreased, and the expression of markers specific for otic progenitors increased. Immunofluorescence of cells differentiated under each of the induction methods showed that some cells expressed otic progenitor-specific markers Pax2 and Pax8. These results revealed the potential of hESCs to differentiate into otic progenitors. hESCs could be induced into progenitor cells on the laminin substratum. This is a simple, time-saving method. Following the differentiation of hESCs into otic progenitors induced by using this method, OEPs separated from otic progenitors were induced for differentiation into hair cells.Part 2:We induced the differentiation of otic epithelial progenitors (OEPs) into hair-cell-like cells using different substrates. Our results showed that OEPs cultured on the chicken utricle stromal cells with the induction medium could differentiate into hair-cell-like cells with stereociliary bundles. Co-culture with stromal cells, however, may be problematic for subsequent examination of the induced hair-cell-like cells. In order to avoid the interference from stromal cells, we cultured OEPs on laminin with different induction media and examined the effects of the induction medium on the differentiation potentials of OEPs into hair-cell-like cells. The results revealed that the culture of OEPs on laminin with the conditioned medium from chicken utricle stromal cells supplemented with EGF and all-trans retinoic acid (RA) could promote the organization of cells into epithelial clusters displaying hair-cell-like cells with stereociliary bundles. These cells also displayed the expected electrophysiological properties. The results indicated that hESCs could be induced to differentiate into the functional mature hair cells under differentiation conditions in the absence of stromal cells.Part3:Notch signaling pathway plays an important role in otic neurogenesis by regulating the differentiation of hair cells. In this part, our objective is to investigate whether Notch signaling plays the similar role or not in the differentiation of hair-cell-like cells in vitro. Identification and regulation of Notch signaling during the development of inner ear hair cells is helpful to control differentiation of hair cells. Based on the culture method that could promote the differentiation of hESCs into the functional hair-cell-like cells under differentiation conditions in the absence of stromal cells, we investigated what role Notch play during differentiation in vitro, and the effect of Notch ligands or the Notch receptor. Frist, we selected investigate the temporal expression pottern of Notch ligands or the Notch receptor in the stages of progenitor cell and the hair cell differentiation. Based on the temporal expression of Notch ligands, then, pAJ-U6-shRNA-CMV-Puro/GFP recombinant lentiviral vector encoding short hairpin RNAs was used to silence Jag-1 gene by transfecting the otic progenitor cells differentiated for 6 days and 12 days. The hair cells differentiated for 5 days were infected with lentiviral constructs encoding shRNAs to silence Jag-2 gene. Recombinant lentivirus Dll-1 shRNA vector was used to transfect the hair cells differentiated for 5 days to silence Dll-1gene. The effects of each ligand on the in vitro differentiation of hair cells were examined by RT-PCR, immunofluorescence and SEM. Results showed that Jag-1 ligand played an important role in regulating the differentiation of otic progenitor cells. The individual deletion of Jag2 or Dll1 function resulted in a mild increase of hair cells. However, reductions in both Dll1 and Jag2 gene dosage display clear loss of supporting cells and a more robust increase of hair cells. These results will be the theoretical basis for studying the Notch mechanism for in vitro differentiation of hair cells.