Hearing loss affects more than 40 million people in the United States alone. Sensorineural hearing loss (SNHL) commonly involves damage to cochlear hair cells (HCs), and since mammalian HCs are incapable of regeneration, most forms of SNHL are permanent. Recent work has demonstrated the ability to isolate, clonally expand, and differentiate neonatal murine Lgr5+ HC progenitors (LCPs) in high yield. These LCPs are found in both the cochlear medial compartment (MC) with inner hair cells (IHCs) and the lateral compartment (LC) with outer hair cells (OHCs). Following in vitro expansion and differentiation in 3D Matrigel, organoids derived from the clonal expansion of single LCPs contain cells expressing either IHC markers or OHC markers, but both HC markers are never found within the same organoid. Currently, it is not known what drives LCPs within clonally derived organoids toward IHC or OHC fate. However, since uniform differentiation conditions give rise to both IHC and OHC positive organoids, LCP fate may be pre-determined based on the compartmental origin of the original cell. However, LCPs still may retain sufficient plasticity to differentiate into either IHCs or OHCs regardless of whether they possess existing fate preferences. I hypothesize that LCP IHC and OHC fate is pre-determined based on the medial or lateral compartmental origin of individual cells. I further hypothesize that LCPs retain sufficient plasticity to enable them to adopt either IHC or OHC fate through manipulation of Wnt3a and Bmp4 signaling. By utilizing two transgenic reporter mouse lines (Glast- CreER;tdTomato and FgfR3-CreER;tdTomato) which respectively highlight the MC and LC of the postnatal murine cochlea, we aim to perform in vitro lineage tracing experiments to examine the role of LCP compartmental origin in determining HC fate following differentiation. Additionally, we aim to examine the plasticity of LCPs by studying the effects of Wnt3a and Bmp4 on LCP fate determination in 3D culture. Finally, we aim to investigate the importance of morphogen gradient signaling in the patterning of LCPs in 2D culture using a simple but physiologically relevant silk fibroin film delivery system. This work aims to provide insight into the determinants of LCP IHC and OHC fate specification with respect to both the pre-programmed preferences of these cells and their competence to be driven towards either IHC or OHC fate regardless of these preferences upon exposure to morphogens which pattern the pro-sensory domain during development. Together, this project will provide me with comprehensive cochlear developmental biology, regenerative medicine, and biomaterials engineering training, equipping me with the necessary skills to achieve my long-term career goals of becoming an otolaryngologist working to translate basic hearing loss research to the clinic.
Hearing loss affects more that 40 million people in the United States alone, resulting in a tremendous socioeconomic burden on society and a decreased quality of life for afflicted patients. Recent work has demonstrated the ability to generate populations of inner and outer hair cells in vitro from Lgr5+ cochlear progenitor cells isolated from the postnatal murine cochlea, and the experiments described herein aim to study the fate determinants and plasticity of these cells using in vitro lineage tracing and morphogen supplemented 2D and 3D culture systems. Together, this project will provide me with comprehensive cochlear developmental biology, regenerative medicine, and biomaterials engineering training, equipping me with the necessary skills to achieve my long-term career goals of becoming an otolaryngologist working to translate basic hearing loss research to the clinic.
