Indiana University-Purdue University Indianapolis (IUPUI) / Loss of the finite cochlear hair cells of the inner ear results in sensorineural deafness. Human cochlear hair cells do not regenerate, and there is no cure for deafness. Our laboratory has established a three-dimensional culture system for deriving functional sensory hair cells from human pluripotent stem cells. A major limitation of this approach is that derived hair cells exhibit a morphological and gene expression phenotype reflective of native vestibular hair cells. Previous studies have shown that establishment of localized domains of gene expression along the dorso-ventral axis of the developing otic vesicle is necessary for proper morphogenesis of both auditory and vestibular inner ear structures. Sonic hedgehog (SHH) signaling has been shown to play a key role in specification of the ventral otic vesicle and subsequent cochlear development. Here, SHH treatment was pursued as a potential strategy for inducing a patterning phenotype permissive to cochlear induction in vitro. Single-cell RNAsequencing analysis revealed that while treatment with the SHH pathway agonist Purmorphamine reduced expression of markers for the vestibular-yielding dorsal otic vesicle, upregulation of ventral otic marker genes was modest. More strikingly, the number of otic progenitors exhibiting a neuroprogenitor phenotype increased in response to Purmorphamine treatment. These results suggest that SHH pathway modulation in early-stage inner ear organoids may bias their differentiation toward a neural lineage at the expense of an epithelial lineage. The present study is the first to evaluate the patterning phenotype of human stem cell derived otic progenitors, and sheds light on the transcriptomic profile at this critical point of inner ear development. This study may also cultivate future efforts to derive cochlear cell types as well as inner ear neural cell types from human pluripotent stem cells, and contribute to the establishment of a more complete in vitro model of inner ear development. / 2021-08-21

Indiana University-Purdue University Indianapolis (IUPUI) / Retinal ganglion cells (RGCs) serve as a vital connection between the eye and the brain with damage to their axons resulting in loss of vision and/or blindness. Reti- nal organoids are three-dimensional structures derived from human pluripotent stem cells (hPSCs) which recapitulate the spatial and temporal differentiation of the retina, providing a valuable model of RGC development in vitro. The working hypothesis of these studies is that hPSC-derived RGCs are capable of extensive outgrowth and display target specificity and pathfinding abilities. Initial efforts focused on charac- terizing RGC differentiation throughout early stages of organoid development, with a clearly defined RGC layer developing in a temporally-appropriate manner express- ing a compliment of RGC-associated markers. Beyond studies of RGC development, retinal organoids may also prove useful to investigate and model the extensive axonal outgrowth necessary to reach post-synaptic targets. As such, additional efforts aimed to elucidate factors promoting axonal outgrowth. Results demonstrated significant enhancement of axonal outgrowth through modulation of both substrate composi- tion and growth factor signaling. Furthermore, RGCs possessed guidance receptors that are essential in influencing outgrowth and pathfinding. Subsequently, to de- termine target specificity, aggregates of hPSC-derived RGCs were co-cultured with explants of mouse lateral geniculate nucleus (LGN), the primary post-synaptic target of RGCs. Axonal outgrowth was enhanced in the presence of LGN, and RGCs dis- played recognition of appropriate targets, with the longest neurites projecting towards LGN explants compared to control explants or RGCs grown alone. Generated from xvii the fusion of regionally-patterned organoids, assembloids model projections between distinct regions of the nervous system. Therefore, final efforts of these studies focused upon the generation of retinocortical assembloids in order to model the long-distance outgrowth characteristic of RGCs. RGCs displayed extensive axonal outgrowth into cortical organoids, with the ability to respond to environmental cues. Collectively, these results establish retinal organoids as a valuable tool for studies of RGC develop- ment, and demonstrate the utility of organoid-derived RGCs as an effective platform to study factors influencing outgrowth as well as modeling long-distance projections and pathfinding abilities.

京都大学 / 新制・課程博士 / 博士(理学) / 甲第24458号 / 理博第4957号 / 新制||理||1708(附属図書館) / 京都大学大学院理学研究科生物科学専攻 / (主査)教授 高橋 淑子, 准教授 佐藤 ゆたか, 教授 中務 真人 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DGAM

京都大学 / 新制・課程博士 / 博士(医学) / 甲第24502号 / 医博第4944号 / 新制||医||1064(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 長船 健二, 教授 朝長 啓造, 教授 伊達 洋至 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

The epithelial layer of the gastrointestinal (GI) tract provides a barrier between the environment and the body. Dysfunction of the epithelium, including changes of the innate immune response facilitated by pattern recognition receptors (PRRs), plays a major role in the development of GI disorders. However, the organization of innate immune sensing, the expression and activity of PRRs and the factors contri¬buting to such possible organization along the GI tract are unclear. In recent years, stem cell-derived organoids gained increasing attention as promising tissue models. Here, a biobank of human and murine organoids comprising three lines from each GI segment; corpus, pylorus, duodenum, jejunum, ileum, colon was generated. RNA sequencing of 42 lines confirmed the preservation of tissue identity and revealed an extensive organization of innate immune signaling components along the cephalocaudal axis, giving each segment a specific innate immune profile. Comple-menting the region-specific expression analysis, several PRRs in human and murine organoids showed region- and species-specific function. To investigate the factors contributing to the patterning of innate immunity in the GI tract, the impact of microbial components was analyzed using murine embryo-derived, never colonized gastric and proximal intestinal organoids. Transcriptional profiling of embryo-derived organoids showed that while expression of some PRRs may depend on environmental cues as expected, an unexpectedly large part of segment-specific expression of PRR signaling components is independent of prior contact with microbial products. Further, analysis of published RNA-seq data as well as in vitro experiments using directed differentiation of organoids into specific cell types showed that expression of innate immune gene also depended on cellular differentiation along the crypt-villus axis. This underlined the importance of cellular differentiation rather than contact to microbial compounds for expression of PRRs. Lastly, analysis of published datasets of RNA-seq and ATAC-seq after knockout of the intestinal transcription factor Cdx2 demonstrated that Cdx2 is likely important for the expression of Nlrp6 and Naip1 in the murine intestine. Future experiments have to support these preliminary findings. Taken together, the expression of a large part of epithelial innate immunity is develop¬mentally defined and conserved in tissue-resident stem cells. The identification of mechanisms governing expression of genes related to immunity will provide further insights into the mechanisms that play a role in the progress of inflammatory diseases. / Das Epithel des gastrointestinalen (GI) Traktes fungiert als Barriere zwischen der Umwelt und dem Körperinneren. Störungen des Epithels, darunter Veränderungen in der angeborenen Immunantwort, welche über „Pattern Recognition Receptors“(PRRs) ermöglicht wird, spielen eine bedeutende Rolle in der Entstehung gastrointestinaler Krankheiten. Auf welche Weise die angeborene Immunantwort im gastrointestinalen Trakt zwischen symbiotischen und schädlichen Mikroben unterscheidet, wie die Expression und Aktivität von PRRs organisiert sind, und die Faktoren die zu einer möglichen Organization beitragen sind bisher allerdings nicht bekannt. In den letzten Jahren haben aus Stammzellen gewonnene Organoide als vielversprechende Gewebemodelle steigende Aufmerksamkeit erregt. In dieser Arbeit wurde eine „Biobank“ humaner und muriner Organoide, jeweils bestehend aus 3 Linien jedes der gastrointestinalen Segmente Korpus, Pylorus, Duodenum, Jejunum, Ileum und Kolon generiert. Die RNA Sequenzierung von 42 Linien bestätigte den Erhalt der Gewebsidentität und zeigte eine umfangreiche Organization innerhalb der Signalkomponenten des angeborenen Immunsystems entlang der kraniokaudalen Achse, wodurch jedes Segment ein spezifisches Immunprofil erhält. Ergänzend zur regions-spezifischen Expressionsanalyse zeigten einige PRRs sowohl in humanen als auch in murinen Organoiden eine regions- und spezies-spezifische Funktion. Zur Untersuchung der Faktoren, die zur Strukturierung des angeborenen Immunsystems im GI-Trakt beitragen, wurde der Einfluss mikrobieller Komponenten untersucht. Hierfür wurden aus embryonalem Gewebe gewonnene Organoide des Magens und des proximalen Dünndarms verwendet, welche noch nicht mit dem Mikrobiom in Kontakt waren. Transkriptionsprofile embryonaler Organoide zeigten, dass die Expression einiger PRRs wie erwartet wahrscheinlich von Umweltfaktoren abhängt, dass ein unerwartet großer Anteil der segment-spezifischen Expression von Komponenten der PRR-induzierten Signalwege sich allerdings unabhängig vom Kontakt mit mikrobiellen Komponenten entwickelt. Des Weiteren zeigte die Analyse von bereits publizierten RNA Sequenzierungsdaten und in vitro Experimenten bei denen durch gezielte Differenzierung von Organoiden spezifische Zelltypen generiert wurden, dass die Expression von Genen des angeborenen Immunsystems auch von der zellulären Differenzierung entlang der Krypten-Zotten-Achse abhängt. Dies unterstreicht die Bedeutung von zellulärer Differenzierung für die Expression von PRRs, anstelle des Kontaktes zu mikrobiellen Komponenten. Auch konnte durch die Analyse bereits publizierter RNA- und ATAC-Sequenzierungsdaten nach knockout des im Dünndarm exprimierten Transkriptionsfaktors Cdx2 nachgewiesen werden, dass Cdx2 mit hoher Wahrscheinlichkeit wichtig für die Expression von Nlrp6 und Naip1 im murinen Dünndarm ist. Diese Erkenntnisse müssen in zukünftigen Experimenten validiert werden. Zusammengenommen zeigen die Ergebnisse, dass die Expression eines Großteils der angeborenen Immunität entwicklungsbiologisch festgelegt und in gewebe-spezifischen Stammzellen konserviert ist. Die zukünftige Identifikation von Mechanismen die die Expression von zur Immunität zugehörigen Genen steuern, wird weitere Erkenntnisse über die Mechanismen die eine Rolle in der Entwicklung entzündlicher Erkrankungen spielen bringen.

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京都大学 / 新制・課程博士 / 博士(医学) / 甲第25169号 / 医博第5055号 / 新制||医||1071(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 尾野 亘, 教授 羽賀 博典, 教授 後藤 慎平 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

京都大学 / 新制・課程博士 / 博士(医学) / 甲第25495号 / 医博第5095号 / 新制||医||1073(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 溝脇 尚志, 教授 伊達 洋至, 教授 黒田 知宏 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Human organoids have potential to revolutionize in vitro disease modeling by providing multicellular architecture and functional that are similar to those in vivo. Nonetheless, organoid-based, high-throughput screening (HTS) of compounds is challenged by lack of easy-to-use fluidic systems that are compatible with relatively large organoids. Therefore, we first fabricated a pillar plate, which was coupled with a complementary deep well plate and a perfusion well plate for static and dynamic culture via injection molding. We established various cell loading methods in hydrogels on the pillar plate. In addition, we investigated the effect of flow on the necrotic core of spheroids in the pillar/perfusion plate. Finally, we developed microarray three-dimensional (3D) bioprinting technology using the pillar and perfusion plates for human organoid culture and analysis. High-precision, high-throughput stem cell printing and encapsulation techniques were demonstrated on a pillar plate, which was coupled with a complementary deep well plate and a perfusion well plate for static and dynamic organoid culture. Bioprinted cells and spheroids in hydrogels were differentiated into organoids for in situ functional assays. The pillar/perfusion plates are compatible with standard 384-well plates and HTS equipment, and thus may be easily adopted in current drug discovery efforts.