Olfactory placode organoids from human induced pluripotent stem cells

Abstract

The olfactory sense is one of the oldest and most fundamental senses we have, serving a multitude of purposes like partner choice and risk avoidance. Additionally, olfactory decompensation is an early symptom of many neuro-degenerative diseases and the olfactory epithelium – as a potential entry point to the central nervous system for unknown agents – is possibly even at the center of their pathophysiology. The olfactory epithelium is also one of the few tissues capable of adult neurogenesis and neuroregeneration, poorly under-stood processes with great therapeutic potential in an aging and chronically ill population. Nevertheless, olfaction has long remained in the offsite of biomed-ical research and profound interest in its function and development only arose over the last couple of decades. The developmental mechanisms of the human olfactory epithelium are poorly understood, and any insight is mostly based on model organism research like mice and xenopus laevis. To study the development of the human olfactory epithelium, as well as to generate a three-dimensional model system for neu-rodegenerative disease and drug testing, I have established a differentiation protocol of human induced pluripotent stem cell derived olfactory placode or-ganoids. In this thesis, I describe and characterize the differentiation of human olfactory placode organoids. For this purpose, I have first established a developmental timeline of the organoid development from day 4 to day 31 of the differentia-tion, using morphology and immunofluorescence staining of key markers of ectoderm and olfactory development SIX1, TFAP2A and E-CADHERIN. The olfactory placode organoids are composed of a non-neural surface ectoderm containing placodal patches on the outside, neural tubuli representing the de-veloping forebrain on the inside and a head mesenchyme in between, mimick-ing the developmental situation in vivo. I have further characterized the discov-ered features on a protein level and analyzed bulk RNA data from whole olfac-tory placode organoids. To improve maturation of the organoids and their dis-tinctive features, the differentiation protocol was adapted based on brain or-ganoid and other organoid techniques. The preliminary result of the modified protocol shows a high level of maturation and organization, but further efforts are needed to generate a mature human olfactory epithelium in vitro. This work shows for the first time the generation of an olfactory placode organ-oid derived from human induced pluripotent stem cells, exhibiting patches of developing olfactory tissue in a three-dimensional in vitro system. Furthermore, these olfactory placode organoids show an outside-out orientation incorporat-ing at least four different tissues. The surface epithelium as well as the neural tubuli are anchored on their own basement membrane providing stability and orientation. These features of high-level organization closely resemble the em-bryonic head and have – to my best knowledge – not been achieved in a three-dimensional organoid model before. This thesis sets the groundwork for the generation of a fully functional human olfactory epithelium model in a three-dimensional in vitro organoid and gives first insight on human olfactory development. In the future, the obtained results could contribute to the generation of a powerful model system for neurosci-ence and developmental biology.