Isolation, culture and characterization of human olfactory stem cells and investigation of their effect on regeneration of damaged rat facial nerves

Facial paralysis is a disorder that is seen in our country and worldwide, has an incidence of 30/100.000 and can result from congenital, neurological, metabolic, toxic, neoplastic, infectious, traumatic, iatrogenic and idiopathic reasons. The disorder, as well as affecting human health seriously, creates esthetic and psychological problems. Although several approaches are in application in order to treat this disorder, recovery rate and sequels related to recovery rate are grand obstacles against treatment. For that matter, development of new approaches is required to remove pathologies related to damaged nerves. Progresses in stem cell research and their application for treatment are also very promising for treatment of the damages in nervous tissue. Although fat, bone marrow and hippocampal neural stem cells have been shown to have affirmative consequences on peripheral nerve damages in some experimental studies, a precise solution has not been shown yet. Latest studies state that in order to succeed in stem cell treatments, the micro-environment of the damaged area and the stem cell source have to be coherent. Studies in the literature related to this topic are numerically few, but they progressively gain importance. Besides, until now, no study has been conducted in this area to show the effect on regeneration of damaged facial nerves. Hereunder, the aim of this thesis study is stem cell isolation from human olfactory mucous biopsies, continuous culture, characterization and cryopreservation of these cells, development of a new approach based on niche for regeneration of damaged facial nerves (of rats) for the first time and generation of an olfactory stem cell cryobank for the use of cellular treatment and tissue engineering studies. For this purpose, olfactory mucous biopsy samples were obtained from three patients with consent prior to a septoplasty operation in Istanbul Education and Research Hospital, Department of Otolaryngology Head and Neck Surgery and brought to Yildiz Technical University, Department of Bioengineering in Cell Culture and Tissue Engineering Laboratory for isolation, culture, characterization and cryopreservation. Continuously cultured cells were morphologically investigated. Cells that were obtained from biopsy samples were induced with adipogenic, osteogenic and neurogenic differentiation kits for stem cell characterization, immunophenotypically characterized by flow cytometry and obtained stem cells were cryopreserved. Olfactory stem cells were stained with CM-DiI to be used in in vivo studies and a multi-layered cell mass was obtained after passaging the cells 9 times. Obtained multi-layered cell mass was cultured in vitro to determine viability. In vivo studies to investigate the effect of CM-DiI labeled olfactory stem cell mass on peripheral nerve regeneration of experimentally face paralyzed rats have been conducted in two steps. In the preliminary examination, 7 Wistar Hannover (350-380 gr) female rats were used for a facial cut and inoculation of stem cells in the cut area using 4 different methods in Bezm-i Alem University Experimental Animal Laboratory. In this study, 500.000 CMD-DiI labeled olfactory stem cells in 10µl PBS were inoculated in damaged area of rat 1, 500.000 CMD-DiI labeled olfactory stem cells in 10µl PBS were inoculated in damaged area of rat 2 and the inoculation area was covered with approximately 1mm3 fat tissue (fascia) from the same rat, 500.000 CMD-DiI labeled olfactory stem cells in 10µl PBS were inoculated in damaged area of rat 3 and 4 and fibrin glue was applied to inoculation area, CM-DiI labeled multi-layered cell mass was inoculated on damaged area of rats 5 and 6 and as a control group rat 7 was given 10µl PBS into damaged area. In order to determine facial paralysis before the experiment and recovery rate after the experiment; whisker movements were scored on Weeks 2, 4, 6 and 8 before and after the full-thickness cut. The results were calculated using Mann Whitney U statistics. Considering the results of preliminary experiments; on the second step of in vivo studies, 27 rats were divided into 3 groups named M, T and K and for each group rat facial nerves on the left side were cut and nerve ends were anastomosed into neurotube poliglycolic acid conduit. Multi-layered cell pellet was added into conduit for Group M and PBS was added into conduit for Group T. Group K was not supplemented additionally since it was the control group. Recovery in rats was monitored physiologically, electrophysiologically and with whisker movements on weeks 2, 4, 6, 8 and 10. At the end of Week 10, rats were sacrificed and preparations were taken from damage area for histological examination. Preparations were stained with hematoxylene, eosin, S100, Toluidine Blue and observed under fluorescent microscope. The results were statistically evaluated using Mann-Whitney U and Annova. According to the results, the cells obtained from olfactory tissue had stem cell characteristics in terms of morphology, immunophenotype and differentiation features and the cells were able to remain 91% viable after 24 months after cryopreservation. It was determined that the olfactory stem cells were labeled with CM-DiI stain and the multi-layered cell mass obtained from passaging the cells 9 times was able to preserve viability in the culture media. According to the whisker scores, among the four different methods the highest recovery rate was observed in the group that was applied with multi-layered cell mass prepared from olfactory stem cells. Physiological and electrophysiological results obtained from in vivo studies on 27 rats show that nerve regeneration in facial nerve reconstruction was faster in groups applied with olfactory stem cells (p=0,030. It was shown using histological evaluation with S100 staining that stem cells applied to the damage area in Group M were differentiated into Schwann-like cells. This study, supported by 2012-07-04 KAP02 numbered project and Yıldız Technical University Scientific Research Projects Coordination Department, aims to fulfill the statement published on 2003-2023 Technology Foresight Study by TÜBİTAK that highlights the importance of "development of stem cell technologies for scientific studies to be conducted in Turkey and their availability especially in regenerative medicine" by isolating, characterizing and cryopreserving of olfactory stem cells which are the only available neural stem cells for the first time in our country, by creating a cryobank, by generating a multi-layered cell mass to be used in tissue engineering and by pointing to a new cellular treatment method that accelerates nerve regeneration of facial nerve damages