Incorporation, polarization and maturation of human photoreceptor transplants in the mouse retina

Photoreceptors are highly specialized neurons within the eye and the key retinal cells sensing light. They are indispensable for our visual perception and loss of photoreceptors consequently leads to loss of vision, a sense that alone is responsible for more than 30% of the input to our brain. Vision impairment and blindness is a leading cause of disability in the industrialized world and is in many cases ultimately due to a degeneration of the photoreceptors, which cannot be halted or reversed. Retinal degenerative diseases encompass a heterogeneous group of etiologies, mainly caused by various mutations in a plethora of proteins involved in the visual process. Currently, several therapeutic options are being explored, with so far one gene therapy for a rare inherited blinding condition being clinically approved. However, the gene therapy approach requires not only the presence of remaining photoreceptors but the tailoring of the therapy to each individual mutation. An alternative, more generally applicable approach is to restore vision through photoreceptor replacement therapy. As such, research on mouse-to-mouse photoreceptor transplantations has been carried out for many years, though with mixed results. In the last decade, it has however also become possible to generate large quantities of human photoreceptors through retinal organoid technology, allowing to instead transplant human cells. While promising, this field is still in development and principal conditions for successful photoreceptor transplantation have yet to be defined. Here, human-to-mouse photoreceptor transplantations were performed and assessed with the aim to receive insights into retinal cell replacement technology with specific focus on photoreceptor maturation, polarization and functional integration. Using a cone-degeneration host line, large-scale incorporation of human photoreceptor grafts into the murine retina was shown for the first time. It was found that for human photoreceptors, the choice of developmental stage strongly affects incorporation and maturation capacity. Furthermore, the results demonstrate the necessity of adequate graft-host interaction for successful transplant maturation and function, suggesting that photoreceptor replacement strategies might benefit from transplantation in earlier rather than late stages of retinal degeneration. Taken together, this thesis lays important groundwork for the further development of human photoreceptor replacement strategies to treat retinal degenerative disease.:ACKNOWLEDGEMENTS I ABSTRACT III ZUSAMMENFASSUNG V PUBLICATIONS VII TABLE OF CONTENTS IX LIST OF FIGURES XIII LIST OF TABLES XIV GENERAL ABBREVIATIONS XV GENE AND PROTEIN ABBREVIATIONS XVII 1 INTRODUCTION 1 1.1 THE RETINA AND LIGHT PERCEPTION 1 1.1.1 General structure of the eye 1 1.1.2 General structure of the retina 1 1.1.3 General photoreceptor structure 3 1.1.4 Phototransduction 4 1.1.5 Signal transmission to the brain 6 1.1.6 Major differences between rods and cones 7 1.1.7 The role of Müller glia in photoreceptor support and light perception 9 1.2 RETINAL DEGENERATION DISEASES AND TREATMENT OPTIONS 11 1.2.1 Retinal degeneration diseases 11 1.2.2 Therapeutic approaches to treat retinal degeneration diseases 12 1.3 CELL REPLACEMENT AS TREATMENT APPROACH FOR RETINOPATHIES 14 1.3.1 Transplantations of rodent retinal tissue and cells 14 1.3.2 Transplantations of human retinal tissue and cells 17 1.4 AIM OF THIS THESIS 22 2 CHARACTERIZATION OF CRX-MCHERRY HUMAN RETINAL ORGANOIDS AS PHOTORECEPTOR CELL SOURCE 23 2.1 AIMS 23 2.2 CHARACTERIZATION OF CRX-MCHERRY REPORTER-EXPRESSING CELLS 23 2.2.1 Crx-mCherry expression overlaps with endogenous CRX expression and increases over time 23 2.2.2 Crx-mCherry organoids contain an outer and an inner nuclear layer 24 2.2.3 Crx-mCherry+ cells express early and mature rod and cone markers 25 2.2.4 Crx-mCherry+ cells do not express proliferation markers 27 2.3 ENRICHMENT AND CHARACTERIZATION OF CRX-MCHERRY+ DONOR CELLS 28 2.3.1 Enrichment of Crx-mCherry+ cells by FACS 28 2.3.2 Characterization of Crx-mCherry enriched cells by single cell sequencing 29 2.3.3 Characterization of D200 Crx-mCherry-enriched cells by immunocytochemistry 30 2.4 SUMMARY 31 3 TRANSPLANTATION OF HUMAN CRX-MCHERRY+ GRAFTS AGED D100, D200 AND D300 INTO CPFL1 MICE 33 3.1 AIMS 33 3.2 CRX-MCHERRY+ CELLS OF ALL AGES CAN BE TRANSPLANTED AND SURVIVE IN THE MURINE RETINA 33 3.2.1 Human grafts can be identified by RCVRN staining 34 3.2.2 D100 Crx-mCherry+ transplants are larger than D200 and D300 grafts 34 3.2.3 Graft volume increase over time is not due to in vivo proliferation 36 3.3 GRAFT MORPHOLOGY DIFFERS WITH DONOR AGES 37 3.3.1 Human grafts can adopt an intraretinal position 37 3.3.2 Graft positioning changes over time 37 3.3.3 Qualitative differences in graft morphology between donor ages 38 3.4 GRAFT MATURATION 41 3.4.1 D200 but not D100 or D300 grafts develop large quantities of inner segments 41 3.4.2 Inner segment development is associated with close proximity to the host retina 42 3.5 HUMAN IDENTITY OF INTRARETINAL GRAFTS 43 3.5.1 Intraretinal Crx-mCherry+ grafts are largely a result of true morphological incorporation 43 3.5.2 Rare indications of potential human-to-mouse material transfer 45 3.6 SUMMARY 47 4 IN DEPTH CHARACTERIZATION OF TRANSPLANTED D200 CRX-MCHERRY+ CELLS 49 4.1 AIMS 49 4.2 EARLY POST TRANSPLANTATION DYNAMICS IN GRAFT POSITIONING AND GRAFT-HOST INTERACTIONS 49 4.2.1 Intraretinal and proximal D200 grafts interact with the host retina while isolated and distal clusters show only little interaction 49 4.2.2 Incorporation of D200 grafts is first evident at 8 weeks post transplantation 50 4.2.3 Host Müller glia extend processes into the graft before host bipolar cells 51 4.2.4 MG staining in D200 grafts originates from host MG 51 4.3 INCORPORATING D200 GRAFTS POLARIZE AND FORM STRUCTURES OF MATURE PHOTORECEPTORS 53 4.3.1 Grafts and host form an outer limiting membrane (OLM)-like structure 53 4.3.2 Inner segment formation occurs where an OLM is formed 54 4.3.3 Incorporating grafts form outer segment-like structures 55 4.3.4 Incorporating grafts form synaptic structures 57 4.3.5 Transplanted Crx-mCherry+ cells become enriched for cones 58 4.3.6 Higher levels of mature photoreceptor markers in ex vivo compared to in vitro cones 60 4.4 INCORPORATION AND MATURATION CAPACITY DEPEND ON THE HOST ENVIRONMENT 63 4.4.1 Graft morphology and maturation in C57BL/6JRj recipients resembles that in Cpfl1 hosts 63 4.4.2 Graft morphology and maturation in highly degenerated rd1 and tgCR host lines differs strongly from the outcome in models with an ONL 63 4.5 SUMMARY 67 5 FUNCTIONAL ASSESSMENT OF TRANSPLANTED CRX-MCHERRY+ CELLS 69 5.1 AIMS 69 5.2 HIGH-LEVEL FUNCTION 69 5.2.1 Light-Dark Box 69 5.3 TISSUE-LEVEL FUNCTION 71 5.3.1 Multi-electrode array assessment of D200+26w grafts in Cpfl1 mice 71 5.3.2 Isolation of cone-mediated RGC response through photopic light stimulation and L-AP4 addition 71 5.3.3 Graft-containing retinal portions exhibit cone-mediated light responses 72 5.4 SUMMARY 74 6 DISCUSSION AND FUTURE PERSPECTIVES 75 6.1 HUMAN GRAFTS CAN MORPHOLOGICALLY INCORPORATE INTO THE MODERATELY DEGENERATED MOUSE RETINA 75 6.2 INTRARETINAL GRAFTS MOSTLY REPRESENT TRUE INCORPORATION EVENTS, NOT MATERIAL TRANSFER 76 6.3 GRAFT MATURATION DEPENDS ON GRAFT-HOST INTERACTION 77 6.4 ESTABLISHMENT OF GRAFT-HOST INTERACTION AND GRAFT INCORPORATION 78 6.5 D200 CRX-MCHERRY+ CELLS ARE THE PREFERABLE DONOR POPULATION COMPARED TO D100 AND D300 80 6.6 CONES SHOW PREFERENTIAL SURVIVAL POST GRAFTING 81 6.7 FUNCTIONAL ANALYSES OF TRANSPLANTED ANIMALS 82 6.8 FUTURE CLINICAL TRANSLATION 85 6.9 MAJOR CONTRIBUTION TO OTHER WORK 88 7 FINAL CONCLUSION 89 8 MATERIALS AND METHODS 91 8.1 STUDY APPROVAL 91 8.2 MATERIALS 91 8.2.1 Materials and Chemicals 91 8.2.2 Cell Line 92 8.2.3 Mouse Lines 92 8.2.4 Antibodies 93 8.3 METHODS 95 8.3.1 Cell culture 95 8.3.2 Transplantations 96 8.3.3 Functional analyses 98 8.3.4 Immunohistochemistry and Immunocytochemistry 100 8.3.5 Imaging and image processing 103 8.3.6 Statistics 106 8.3.7 Single cell sequencing 107 8.3.8 Bioinformatic analysis 108 9 BIBLIOGRAPHY 111 10 APPENDIX 128 10.1 APPENDIX 1: ERKLÄRUNGEN ZUR ERÖFFNUNG DES PROMOTIONSVERFAHRENS 128 10.2 APPENDIX 2: BESTÄTIGUNG ÜBER EINHALTUNG DER AKTUELLEN GESETZLICHEN VORGABEN 12

Excavations in the Archaic Civic Buildings at Azoria in 2005-2006

Continuing excavation on the South Acropolis at Azoria in northeastern Crete has exposed buildings of Archaic date (7th–early 5th century b.c.) that served communal or public functions. Work conducted in 2005 and 2006 completed the exploration of Late Archaic levels within the Communal Dining Building (putative andreion complex), the Monumental Civic Building, and the adjacent Service Building. These contexts and their assemblages, especially the animal and plant remains, permit the characterization of diverse dining practices and the interpretation of patterns of food production and consumption. Both the Communal Dining Building and the Monumental Civic Building show extensive evidence of communal feasting and the integration of cult

Effet de densité initiale de mise en charge sur la survie et la croissance des larves d’Heterobranchus longifilis (Valenciennes, 1840) élevées en bassins fertilisés

Objectif : La présente étude a consisté à évaluer l’effet de la densité initiale de mise en charge sur la survie et la croissance des larves d’Heterobranchus longifilis (Valenciennes, 1840) en bassins  fertilisés.Méthodologie et résultats : L'expérience a été conduite en un replicat dans 08 bassins en béton remplis avec 200 litres d’eau et fertilisés avec de la fiente de volaille sèche (dose : 600 g/m3), puis ensemencés avec 83 ± 13 ind/L de zooplancton. Six jours après l’ensemencement du zooplancton, les larves de H. longifilis âgées de 2 jours (Pmi = 2,8 ± 0,1 mg) ont été directement introduites dans les bassins. La mise en charge est réalisée avec 4 densités (D300 = 300 individus/m3, D400 = 400 individus/m3, D500 = 500 individus/m3 et D600 = 600 individus/m3). Ces larves se sont servies exclusivement du zooplancton pendant les 7 premiers jours d’élevage. A partir du 8ème jour, elles sont nourries à l’aliment Coppens jusqu’à 30 jours d’âge. A la fin de l’expérience, la densité D300 a donné les meilleurs taux de survie (58,33 ± 0,52 % ; p < 0,05) et de conversion alimentaire (0,28 ± 0,02 ; p < 0,05). Les poids moyens finaux sont élevés à toutes les densités et ne sont pas significativement différents. Par ailleurs, les meilleurs taux de croissance spécifique sont obtenus aux densités D300, D500 et D600.Conclusion et application des résultats : La densité de 300 individus/m3 constitue la meilleure densité initiale de mise en charge des larves qui donne de meilleures performances de survie et de croissance pour les larves en bassins fertilisés. Ainsi, avec cette densité de mise en charge, ce système offre de réelles perspectives moins onéreuses de production d’alevins de H. longifilis en milieu paysan.Mots clés : Heterobranchus longifilis, bassins fertilisés, densité initiale, survie, croissance

Effets De La Digestion Gastrique Sur Les Propriétés Anthelminthiques De Zanthoxylum Zanthoxyloides (Lam.) Zepernick & Timlerto Et De Newbouldia Laevis (P.Beauv.) Sur Haemonchus Contortus

Several recent studies have shown that medicinal plants Zanthoxylum zanthoxyloides (Fagara) and Newbouldia laevis possess anthelmintic activities in vitro on different stages of development of gastrointestinal nematodes. The objective of this study was to evaluate the in vitro anthelmintic properties of residues from digestion in the rumen of leaf powders of both plants on the migration of the 3rd-stage larvae L3s of Haemonchus contortus. Residues obtained after incubation at 0 h, 24 h and 96 h kinetic points of the leaf powders of both plants in the rumen of sheep with fistulae were used for the assay. The larval migration inhibition test evaluated the anthelmintic properties of the methanolic extracts of residues of the two plants. The effect of plant extracts on larval migration was notdose- dependent (p> 0.05) but was a function of plant incubation time (p 0.05). N. Laevis seems to have retained his anthelmintic property after incubation in sacco in the rumen. On the other hand, Fagara seems to lose its effectiveness as it stays in the rumen. Findings obtained on these plants confirm their traditional use in veterinary medicine especially in the control of helminthiasis

Photoperiod effects on circadian rhythms and puberty onset in African catfish Clarias gariepinus

ABSTRACT Photoperiod manipulation is routinely used in the aquaculture industry with the aim to enhance growth by manipulating the timing of reproduction in several commercially important temperate fish species. However, there are clear gaps in our understanding of how photoperiod is perceived by the circadian axis and transmitted to the brain to alter reproduction. Furthermore, due to the wide range of environments inhabited by fish, it is unlikely that one single organization exists. It is therefore believed that comparative studies of temperate species “models” with tropical species such as the African catfish (Clarias gariepinus) that adapted to different environments characterized by weaker light signals can help in such an aim. A number of studies were therefore performed in this PhD project to expand our knowledge on circadian biology and environmental physiological effects in African catfish. The first aim was to characterize the circadian melatonin system in this species (chapter 3). Results clearly showed that the control of melatonin production by the pineal gland was very different in the African catfish as compared to temperate species such as salmon and trout. Indeed, melatonin production appeared to mainly depend on light stimuli perceived by the eyes as opposed to salmonids where light directly perceived by the pineal gland regulates its own melatonin production within photoreceptors. The main evidence was obtained in ophthalmectomised fish that were unable to synthesize and release melatonin into the blood circulation during the dark period. This was the first time that such a decentralized organisation, similar in a way to the mammalian system, was found in any teleost species. In vitro results also supported such findings as African catfish pineal glands in isolation were not able to normally produce melatonin at night as usually seen in all other fish species studied so far. This indirectly suggested that pineal gland photo-sensitivity might be different in this tropical species. Further studies were performed to better determine the amount of light that can be perceived by the African catfish pineal gland depending on light transmittance though the skull (where the pineal gland is located). Surprisingly, it appeared that catfish cranium act as a stronger light filter than in other species resulting in lower light irradiance of the pineal gland. This could explain, although it still needs to be further confirmed, why African catfish photic control of melatonin produced by the pineal would have evolved differently than in temperate species. The work then focused on better characterizing diel melatonin production and endogenous entrainment through exposure to continuous photic regimes (continuous light, LL or darkness, DD) (chapter 4). Daily melatonin profiles of fish exposed to 12L:12D photoperiod (routinely used in indoor systems) confirmed low melatonin production at day (<10 pg/ml) and increase at night (50 pg/ml) as reported in most vertebrate species studied to date. Interestingly, results also showed that melatonin production or suppression can anticipate the change from night to day with basal melatonin levels observed 45 mins prior to the switch on of the light. These observations clearly suggest the involvement of a clock-controlled system of melatonin secretion that is capable of anticipating the next photophase period. Furthermore, when constant light (LL) was applied, day/night melatonin rhythms were abolished as expected due to the constant photic inhibition of AANAT activity (e.g. one of the enzyme responsible for the conversion of serotonin into melatonin). However when fish were exposed to constant darkness (DD), a strong endogenous melatonin rhythm (maintained for at least 4 days and 18 days in catfish and Nile tilapia respectively) was found, demonstrating once again the presence of robust circadian oscillators in this species. The next aim of the doctoral project was then to investigate circadian behaviour of catfish through locomotor activity studies (Chapter 5). African catfish is again a very interesting “model” due to its reported nocturnal activity rhythmicity as compared to most other teleosts species. Locomotor activity is considered as a very useful tool to elucidate the mechanisms of circadian organization in both invertebrates and vertebrates circadian. Results first confirmed the nocturnal activity rhythms in the species. Furthermore, clear circadian endogenous rhythms were observed under constant light (LL) or darkness (DD) during several days before losing rhythmicity. Interestingly, the activity levels varied depending on the stocking density. Finally, the last aim of this project was to test the effects of a range of photoperiodic manipulations on growth performances, sexual development and reproductive performances in African catfish reared from eggs to puberty. Results did not show any differences at the early sages (up to 90 days post hatching) in growth performances nor mortality (high) between control 12L:12D and LL treatments. In contrast, during the juvenile-adult period (from 120 to 360 DPH), significant growth effects were observed, as previously reported in other catfish species, with fish under LL displaying lower growth rate, food consumption and feed conversion efficiency in comparison to most other treatments (12:12, LL, 6:6, 6:18, 12-LL and LL-12) especially 12l:12D. However, no major effects of the photoperiodic treatments were observed with all fish recruited into puberty and developing gonads although differences in the timing of gametogenesis could be observed, especially a delay (circa 2 months) in females exposed to short daylength (6L:18D and 6L:6D). As for egg quality, egg diameter was the only parameter to differ between treatments (slightly larger in egg batch from LL treated females). Overall, none of the photoperiodic regime suppressed maturation in African catfish as opposed to some temperate species. The work carried out during this PhD project clearly advanced our understanding of circadian rhythmicity, light perception and effects of photoperiod on physiology in a tropical species. Future studies are now required to further characterise the circadian system and link it to evolutionary trends within vertebrates

Enhancement of c-Myc degradation by BLM helicase leads to delayed tumor initiation

The spectrum of tumors that arise owing to the overexpression of c-Myc and loss of BLM is very similar. Hence, it was hypothesized that the presence of BLM negatively regulates c-Myc functions. By using multiple isogenic cell lines, we observed that the decrease of endogenous c-Myc levels that occurs in the presence of BLM is reversed when the cells are treated with proteasome inhibitors, indicating that BLM enhances c-Myc turnover. Whereas the N-terminal region of BLM interacts with c-Myc, the rest of the helicase interacts with the c-Myc E3 ligase Fbw7. The two BLM domains act as ‘clamp and/or adaptor’, enhancing the binding of c-Myc to Fbw7. BLM promotes Fbw7-dependent K48-linked c-Myc ubiquitylation and its subsequent degradation in a helicase-independent manner. A subset of BLM-regulated genes that are also targets of c-Myc were determined and validated at both RNA and protein levels. To obtain an in vivo validation of the effect of BLM on c-Myc-mediated tumor initiation, isogenic cells from colon cancer cells that either do or do not express BLM had been manipulated to block c-Myc expression in a controlled manner. By using these cell lines, the metastatic potential and rate of initiation of tumors in nude mice were determined. The presence of BLM decreases c-Myc-mediated invasiveness and delays tumor initiation in a mouse xenograft model. Consequently, in tumors that express BLM but not c-Myc, we observed a decreased ratio of proliferation to apoptosis together with a suppressed expression of the angiogenesis marker CD31. Hence, partly owing to its regulation of c-Myc stability, BLM acts as a ‘caretaker tumor suppressor’

Influence of testing procedure on evaluation of white clover (Trifolium repens L.)

peer-reviewedThis study examined data sets derived from the white clover cultivar evaluation programmes of AFBI (N. Ireland), and DAFF (Republic of Ireland) to determine whether elite performing genotypes are identifiable, independent of test procedure and leaf size factors. Genetic variation in yield and persistency, independent of the leaf size continuum effect, was observed. Identification of elite cultivars by breeders or testers therefore required readjustment of assessment standards to account for the mostly curvilinear relationships between performance and leaf size. The different testing procedures, involving cutting or grazing at different heights, frequencies and nitrogen rates changed the relative performances between the cultivars, making it difficult to predict performance potential beyond specific test conditions. The underlying causes for these changes in rankings was considered, including sensitivity to season and location, the antagonistic affects of defoliation pressure and companion grass competition, the independence of different seasonal profiles and the probable role of other morphological characteristics. In is concluded that testing authorities must calculate the management by leaf size relationships to adjust pass/fail standards if elite performing cultivars are to be correctly indentified