Auditory organotypic cultures and progenitor cell implantation

According to the WHO, about 5 % of the world’s population suffers from disabling hearing loss. In people above the age of 65 the number is more than 30 %. Common effects of this condition are poor speech discrimination and impaired communication abilities. Patient surveys show that hearing loss often leads to a diminished quality of life. The work presented in this thesis aims to evaluate and improve the performance of transplanted cells in vitro. Ultimately, we want to facilitate new connections between neurons of the brainstem (BS) and inner ear structures, or a hearing aid, with the help of transplanted cells. We predict that this will prove to be a feasible path to enhance the rehabilitation results for a selected group of patients with severe hearing disability. We have developed an organotypic auditory cell culture model for the study of cell performance in vitro to help enhance cell transplantation performance in vivo. This model utilizes a BS slice including the cochlear nucleus to simulate an auditory target organ for transplanted cells. Here, we use this model to investigate new ways to improve existing transplantation protocols.

Paper I reports the effects of enriching a mouse boundary cap (BC) progenitor cell culture with the trophic factors inherent to a rat auditory BS slice conditioned medium (CM). The BS CM proved to have positive effects on the survival and differentiation of mouse BC cells in culture. We also report the specific brain derived neurotrophic factor (BDNF) and glial-cell derived neurotrophic factor (GDNF) contents of BS CM and how this content fluctuates over time. Paper II reports the effects on human neural progenitor cells (HNPCs) from the addition of a surface substrate presenting bioactive molecules and a specific neurotrophin content. The commercially available Corning® Matrigel® tested here, proved to enhance the survival of seeded dissociated HN-PCs when evaluated after three weeks in culture. We also report on the differentiation characteristics of HNPCs and their axon forming capacity.

In Paper III we investigate the survival rate in vitro of progenitor cells transplanted at different differentiation time points. Here, neuroepithelial-like stem (NES) cells derived from human induced pluripotent stem cells (hiPSCs) was cultured in three groups, with cells at separate differentiation states (NES cells, neuroblasts and neurons of various maturity (NVM)). These specimens were cultured as a monoculture or in co-culture with a mouse auditory BS slice. Cells seeded as a monoculture, without a BS slice, displayed a considerable lower survival rate as compared to the co-cultured cells. We conclude that the seeded NES were quite dependent of the trophic support from the co-cultured BS slice. We speculate that NES cells are quite vulnerable to the trauma and stress involved in the seeding process, and particularly so when cultured in a more differentiated state. We also report on the differentiation characteristics of cells after culture as revealed by immunocytochemical staining, as well as their tendency to migrate towards the BS slice in co-culture. In Paper IV, we present the evaluation of a method to perform an electrophysiological activity assay of tissue specimens including the cochlear nucleus, cultured ad modum Stoppini. Here, we utilize the microelectrode array (MEA) platform with a modified protocol, to successfully record the endogenous neural activity of auditory BS slices. Our results concur with the basic characteristics of previously reported electrical activity in brain tissue.

In conclusion, these studies report novel information that could not practically have been gathered in vivo without utilizing a significantly larger number of experimental animals. We present promising results on the utilization of CM and cell matrix additives, the possible improvement of time schemes for cell transplantation, extensive characterization of cells in differentiation and a new model for future functional evaluation of cells in interface-culture. These results help us to better comprehend the innate obstacles encountered when searching for a successful in vivo cell transplantation paradigm to the impaired auditory nervous system.

List of scientific papers

I. Kaiser A, Kale A, Novozhilova E, Siratirakun P, Aquino JB, Thonabulsombat C, Ernfors P, Olivius P. 2014. Brain stem slice conditioned medium contains endogenous BDNF and GDNF that affect neural crest boundary cap cells in co-culture. Brain Res. 1566:12-23.
https://doi.org/10.1016/j.brainres.2014.04.006

II. Kaiser A, Kale A, Novozhilova E, Olivius P. The effects of Matrigel® on the survival and differentiation of a human neural progenitor dissociated sphere culture. [Accepted]
https://doi.org/10.1002/ar.24131

III. Novozhilova E, Kaiser A, Falk A, Olivius P. 2018. The differentiation potential of human induced pluripotent cell-derived neuroepithelial-like stem cells in a mouse auditory brainstem milieu. [Submitted]

IV. Novozhilova E, Kaiser A, Olivius P. 2018. The recordings of electrophysiological activity in an interface-cultured organotypic mouse brainstem slice. [Manuscript]