STEM CELL IN-VITRO STRATEGIES FOR THE INDUCTION OF SENSORY NEURONS FOR INNER EAR CELL THERAPY

The cochlea, a part of the auditory system, is a complex structure consisting of several different types of cells including hair cells and spiral ganglion neurons. In mammals, the regenerative potential of hair cells and spiral ganglion neurons is lost soon after the birth and damage to any of these cells causes sensorineural hearing loss. Cochlear implant is presently the only available treatment for sensorineuronal hearing loss, bypassing the malfunctioning hair cells and directly simulating the spiral ganglion neurons. Cochlear implant functionality depends on the activation of remaining spiral ganglion neurons. A possibility for a stem cell approach replacing auditory neurons in the cochlea has attracted great interest. The aim of this thesis was to identify methods to induce progenitors for sensory neurons from human pluripotent stem cells, with the prospective use in a cell therapy for the inner ear. Paper I focused on differentiation of sensory neural types from precursors at different stages of neural rosette formation derived from human embryonic stem cells (day 4, 7, or 11rosettes). Neural and sensory neural phenotype differentiation was examined by immunocytochemistry. Cells positive for NESTIN and TUJ1 were present at all tested time points, indicating presence of the earliest stages of neural differentiation. Starting from the 11-days neural rosettes resulted in decreased potential for neural differentiation, compared to starting from the 4-and 7-days protocols which more effectively could be driven into differentiation towards cells with sensory neuron marker phenotype. In paper II an approach using the SB431542 small molecule for blocking of the TGFs/Activin/Nodal signaling pathway was evaluated for effects on sensory neural differentiation, examined by gene expression and immunocytochemistry. Blocking of this pathway significantly facilitated the induction of markers present on sensory neurons; NGN1, NEUROD1 and BRN3A. Consecutive treatment with bFGF further enhanced the expression of these markers, as well as an increase in expression of GATA3. This protocol also enhanced the presence of colonies positive for TUJ1 as well as BRN3A or ISL1. Notably, addition of a third culture step, with exposure to the neurotrophic factors BDNF + NT3 resulted in > 90% presence of colonies with a phenotype compatible with sensory neurons. In paper III, we evaluated the capacity of additional small molecules (Isoxazole-9 and Metformin) to induced sensory neurons, starting from long-term neuroepithelial stem (ltNES) cells. Here, Isoxazole 9 (ISX9), but not Metformin or SB431542, significantly induced sensory neural genes (GATA3, BRN3A and PERIPHERIN) after a 4-day treatment. Exposure to ISX9 significantly increased the number of BRN3A/TUJ1 positive cells. Further treatment with BDNF7NT3 resulted in increased levels of cells with GATA3 expression and also BRN3A/TUJ1 positive cells in ISX9-exposed populations. In conclusion, this thesis has explored and demonstrated cultural conditions for in vitro induction of sensory neural phenotypes, starting from in vitro cultured human pluripotent stem cells or lt-NES cells. The presented approaches may provide appropriate strategies to develop an effective treatment for sensory neural hearing loss. LIST OF SCIENTIFIC PAPERS I. Rouknuddin Qasim Ali, Yen-Fu Cheng, Beata Kostyszyn, Lars AhrlundRichter, Mats Ulfendahl. Differentiation of human embryonic stem cells towards a sensory neural phenotype. Advances in stem cells. Vol. 2014. Article ID 252491, Page 1-12, (2014). II. Rouknuddin Qasim Ali, Lars Ahrlund-Richter, Mats Ulfendahl. The TGFs/Activin/Nodal pathway inhibitor SB431542 is a potent inducer of sensory neural markers when applied to neural differentiation from HS181 embryonic stem cells. Manuscript. III. Rouknuddin Qasim Ali, Evelina Blomberg, Anna Falk, Lars AhrlundRichter, Mats Ulfendahl. Induction of sensory neurons from neuroepithelial stem cells by the ISX9 small molecule. American journal of stem cells. Accepted for publication (2015).